PREPARATION AND CHARACTERIZATION OF BLACKBERRY …

University of Kentucky University of Kentucky

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University of Kentucky Doctoral Dissertations Graduate School

2009

PREPARATION AND CHARACTERIZATION OF BLACKBERRY PREPARATION AND CHARACTERIZATION OF BLACKBERRY

EXTRACTS AND THEIR ANTICANCER AND ANTI-INFLAMMATORY EXTRACTS AND THEIR ANTICANCER AND ANTI-INFLAMMATORY

PROPERTIES PROPERTIES

Jin Dai University of Kentucky jdai2ukyedu

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ABSTRACT OF DISSERTATION

Jin Dai

The Graduate School

University of Kentucky

2009

PREPARATION AND CHARACTERIZATION OF BLACKBERRY EXTRACTS AND THEIR ANTICANCER AND ANTI-INFLAMMATORY PROPERTIES

_______________________________

ABSTRACT OF DISSERTATION _______________________________

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the

College of Pharmacy at the University of Kentucky

By Jin Dai

Lexington Kentucky

Co-Directors Dr Russell Mumper Professor of Molecular Pharmaceutics University of

North Carolina at Chapel Hill North Carolina and Dr Jurgen Rohr Professor of Pharmaceutical Sciences Lexington Kentucky

Lexington Kentucky

2009

Copyright copy Jin Dai 2009



Blackberries are rich in polyphenols including anthocyanins Polyphenols are hypothesized to have biological activities that impact positively on human health The purpose of these studies was to develop phenolic extracts from selected cultivars of blackberries currently grown in Kentucky as potential Botanical Drug Products for the treatment and prevention of cancer and inflammatory diseases

An ultrasound-assisted ethanol extraction method was employed to obtain anthocyanin-containing extracts (ACEs) from puree or powder (lyophilized puree) of blackberries ACEs were analyzed for total anthocyanin and phenolics content polymeric color and total antioxidant capacity (TAC) The influence of water content in the extraction system was evaluated A 90 day stability study of the extract and a 48 h stability study of the extract in biologically relevant buffers were completed HPLC-MS results showed the anthocyanins in ACE were mainly cyanidin-based As compared to powder-derived ACEs puree-derived ACEs contained similar amounts of anthocyanins but greater levels of phenolics and increased TAC

The in vitro antiproliferative effects of ACEs were evaluated in human leukemia (HL-60) colon (HT-29) and breast (MCF-7) cancer cells The anticancer mechanism involving reactive oxygen species (ROS) generation was investigated It was found puree-derived ACEs significantly enhanced production of H2O2 and cytotoxicity in all cell lines as compared to powder-derived ACEs Cyanidin 3-glucoside exerted anticancer effect by acting synergistically or additively with other active components in the extracts Furthermore the phenolic-enriched fractions were separated from non-phenolic fractions in ACEs and found to have potent antioxidant and antiproliferative activities Puree-derived ACE and corresponding phenolic-enriched methanol fraction (MF) induced cell death through ROS-independent caspase 3 pathway whereas the cytotoxicity induced by powder-derived ACE and corresponding MF is related to ROS mechanisms

The in vitro anti-inflammatory studies showed ACEs inhibited Lipid A-induced Interleukin-12 (IL-12) release from mouse dendritic cells and modulated lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) from murine macrophages

These studies have important implications for the potential use of blackberry extracts for the treatment and prevention of cancer and inflammation diseases and provide essential information for the development of Botanical Drug Products from extracts derived from blackberries and other fruits

KEYWORDS Blackberry extracts Extraction Stability Cancer Inflammation

Jin Dai

July 25 2009


By

Jin Dai

Dr Russell J Mumper Co-Director of Dissertation

Dr Jurgen Rohr Co-Director of Dissertation

Dr Janice Buss Director of Graduate Studies

July 25 2009 Date

RULES FOR THE USE OF DISSERTATIONS

Unpublished dissertations submitted for the Doctorrsquos degree and deposited in the University of Kentucky Library are as a rule open for inspection but are to be used only with due regard to the rights of the authors Bibliographical references may be noted but quotations or summaries of parts may be published only with the permission of the author and with the usual scholarly acknowledgements Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user Name Date

DISSERTATION

Jin Dai

The Graduate School


2009


_________________________________________________

DISSERTATION _________________________________________________

A dissertation submitted in partial fulfillment of the

requirements for the degree of Doctor of Philosophy in the College of Pharmacy

at the University of Kentucky

By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009


iii

ACKNOWLEDGEMENTS

I would like to take the opportunity provided here to acknowledge the many people

for helping me during my doctoral work Without their support this dissertation would

never have been possible

First and foremost I would like to thank my research mentor Dr Russell Mumper

for his guidance encouragement and having patience over the years For many times

when I met problems in my graduate studies he kept encouraging me and helped me to

seek assistance from any possible sources to work through the difficulties He continually

stimulated my thinking and greatly assisted me with scientific writing The lessons I

have learned from his enthusiasm and passion for science will guide me throughout my

life I would like to also thank Dr Jay Dr Rohr and Dr Davies for serving as my

dissertation committee and giving me the direction I very much needed at times I would

also like to acknowledge Dr Glauert for agreeing to serve as my outside examiner

Many sincere thanks to Ms Paige Short for her encouragement support and believing

in me throughout my graduate research studies I would also like to thank WindStone

Farms in Paris KY for providing the blackberry puree used in these studies and Four

Tigers LLC and Kentucky Tobacco Research and Development Center at the University

of Kentucky for providing funding to support these studies in-part

Many thanks to all past and present members in the Mumper lab for their support

expertise and for many intriguing discussions we had on research experiments

Finally but certainly not least I would like to thank my family my parents

Shengwen Dai and Meilin Lu for the copious dedication and support that they have

given me my husband Hanjun Guan for all of the understanding support and love for

all these years and my son Benjamin for accompanying me for many nights and

weekends in the laboratory Their love enables everything I have accomplished

I extend my thanks to the graduate program in the College of Pharmacy and all of the

staff I have interacted with during my graduate school studies I owe a special note of

gratitude to Ms Catina Rossoll for all the help and assistance she provided To all these

and to all those whose names would simply not fit in this space I offer my eternal

gratitude

iv

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii

TABLE OF CONTENTS iv

LIST OF TABLES vii

LIST OF FIGURES viii

Chapter 1 Introduction and Statement of Problem 1

Chapter 2 Plan of Research 4

21 Preparation and characterization of anthocyanin-containing extracts (ACEs)

from blackberries 5

22 Anticancer and anti-inflammatory properties of blackberry extracts 5

23 Antioxidant and antiproliferative properties of blackberry extracts versus their

phytochemical constituents in vitro 6

Chapter 3 Background 7

31 Preparation and characterization of phenolic extracts 7

311 An introduction to natural phenolics 7

312 Extraction of phenolics 12

313 Purification and fractionation of phenolics 16

314 Analysis and quantification of phenolics 18

32 Antioxidant properties of phenolic compounds 23

321 Phenolics as free radical scavengers and metal chelators 24

322 Prooxidant activity of phenolic compounds 26

323 Determination of total antioxidant capacity (TAC) of phenolic extracts 27

33 Natural phenolics and cancer 29

331 In vitro effects of phenolics 30

332 In vivo effects of phenolics 31

333 Human intervention studies using phenolics 32

334 Mechanism of Action of phenolics 34

3341 Modification of the redox status of phenolics 35

3342 Interference of basic cellular functions by phenolics 36

Chapter 4 Preparation and Characterization of Anthocyanin-containing Extracts from

Blackberries 40

v

41 Summary 40

42 Introduction 41

43 Materials and methods 43

44 Results 48

45 Discussion 52

Chapter 5 In vitro Anticancer and Anti-inflammatory Effects of Blackberry Extracts and

Possible Mechanisms of Action 63

51 Summary 63

52 Introduction 64


54 Results 70

55 Discussion 74

Chapter 6 Correlation of the In Vitro Antioxidant and Antiproliferative Properties of

Blackberry Extracts to Their Phytochemical Constituents 86

61 Summary 86

62 Introduction 87


64 Results and discussion 92

Chapter 7 Summary and Conclusions 103

Appendices 108

Appendix A An In Vitro Investigation of the Effects of the Blackberry Extracts on UV

radiation-Induced Cell Death on Human Epidermal Keratinocytes (HEK) 109

Appendix B Antimicrobial and Anti-viral Properties of Blackberry Extract 111

Appendix C Standard Operating Procedures (SOPs) 114

C1 Preparation of blackberry ACE from blackberry puree or powder 114

C2 Total anthocyanin measurement (pH Differential Method) 115

C3 Total phenolics measurement (Folin-Ciocalteu Method) 117

C4 Total antioxidant capacity measurement (Trolox Antioxidant Equivalent Assay)

119

Appendix D Preparation of Vanishing Cream Containing Blackberry ACE 121

vi

Appendix E Preparation of Mucoadhesive Gel Containing Freeze-Dried Black

Raspberries (FDBR) Powder 123

Appendix F Stabilization of Anthocyanin in Dried Blackberry ACE by Mannitol 125

References 127

Vita 149

vii

LIST OF TABLES

Table 4 1 Composition and Characterization of Blackberry Extracts 55

Table 4 2 Peak Assignments Retention Time and Mass Special Data of Blackberry

Anthocyanins Detected by HPLC and Electrospray Ionization-MS 57

Table 4 3 Effect of Storage Conditions on Several Parameters of the Hull Blackberry

Extract after 90 Days 61

Table 5 1 H2O2 Generation by ACEs and Cyanidin 3-glucoside (C-3-G) in Medium at

37 ordmC 81

Table 6 1 Composition and Characterization of Fractions Separated from Hull Puree

ACE by SPE 97

Table 6 2 Composition and Characterization of Fractions Separated from Hull Powder

ACE by SPE 98

Table D 1 Formula for Placebo Vanishing Cream 122

Table E 1 Formula of Mucoadhesive Gels Containing Freeze-Dried Black Raspberries

(FDBR) Powder 124

viii

LIST OF FIGURES

Figure 3 1 Structures of flavonoids phenolic acids and tannins 10

Figure 3 2 Structures of stilbenes and lignan 11

Figure 4 1 HPLC chromatogram of anthocyanins in HBE identified by liquid

chromatography-electrosprayMS 56

Figure 4 2The water content in the extraction solvent on the properties of ACEs 58

Figure 4 3 HPLC chromatogram of anthocyanins in ACEs 59

Figure 4 4 Changes in cyanidin 3-glucoside concentration in powder-derived ACE from

Hull blackberry under different storage conditions over 90 days 60

Figure 4 5 The effects of biologically relevant buffers on the stability of anthocyanins

and phenolics in powder-derived ACE from Hull blackberry at 25 degC and 37 degC 62

Figure 5 1 The cytotoxic effects of Black Satin ACEs and Cyanidin 3-glucoside (Cn-3-

G) on human cancer cell lines 79

Figure 5 2 H2O2 generation by Black Satin ACEs in RPMI 1640 medium with 10 FBS

over time 80

Figure 5 3 The effects of Black Satin ACEs and Cyanidin 3-glucoside (Cn-3-G) on the

intracellular ROS level in HL-60 cells 82

Figure 5 4 The effects of H2O2 generated by ACEs in medium on cytotoxic properties of

ACEs in HL-60 cells 83

Figure 5 5 Effect of powder-derived ACE from Hull blackberries (2005) on Lipid A-

induced IL-12 release from murine dendritic cells 84

Figure 5 6 Effect of post-treatment of ACEs from Hull Black Satin and Chester on

LPS-induced IL-6 and TNF-α release in J774A1 macrophages 85

Figure 6 1 The cytotoxic effects of Hull ACEs and fractions on HL-60 cells 99

Figure 6 2 The effects of Hull ACE (derived from puree) and methanol fractions on the


Figure 6 3 The effects of NAC on cytotoxicity induced by puree-derived ACE and

methanol fractions of ACEs from Hull blackberries 101

Figure 6 4 The effects of NAC on the induction of caspase 3-like activity by puree-

derived ACE and methanol fractions of ACEs from Hull blackberries 102

ix

Figure A 1 Protective effects of powder-derived ACE from Hull blackberries (2007) on

UVR-induced cell death in HEK 110

Figure B 1 Antimicrobial effects of blackberry extract 112

Figure B 2 Inhibition of Herpes Virus (HSV-1)-induced cytopathic effect (CPE)

inhibition assay 113

Figure F 1 Dried blackberry extract with mannitol showed enhanced retention of

anthocyanins at 25 degC over 2 months 126

1

Chapter 1

Introduction and Statement of Problem

Fruits and vegetables are rich sources of a variety of phytochemical antioxidants and

it is believed that the protection provided against diseases by fruits and vegetables is

attributed to the various antioxidants contained in these foods [1] These phytochemical

antioxidants present in plant foods are a very promising group of compounds because of

their safety low toxicity and general acceptance Consequently the identification and

development of such agents has become in the last few years a major area of

experimental health-related research Phenolic compounds are one of the most numerous

and ubiquitous group of plant metabolites and are an integral part of the human diet In

addition to their primary potent antioxidant activity this group of compounds was found

to display a wide variety of biological functions which are mainly related to intervention

in all stages of cancer development including initiation promotion progression invasion

and metastasis [2 3] Various phenolic compounds and extracts from plants and fruits

have been shown to reduce the oxidative stress-associated inflammatory diseases and

cancer [4 5] and there may be synergistic or additive biological effects due to the unique

combination of polyphenols in the extracts prepared from different fruit samples or by

different extraction methods [6-8]

In spite of the accumulated evidence of their potential usefulness in the treatment and

prevention of oxidative stress-related diseases dietary bioactive constituents have been

promoted as health-promoting products or nutrition supplements instead of drug products

due to difficulties in fully identification and characterization of the active components

and quality control of the product However the FDAs Guidance Document on

Botanical Drug Products (June 2004) paved the way for the development of registered

drug products derived from natural sources These products may contain multiple

chemical constituents often comprising phenolic phytochemicals including phenolic

acids non-flavonoids and flavonoids including anthocyanins In many cases the active

constituents in a botanical drug product are not identified nor are their biological activity

well characterized The first FDA-registered botanical drug product VeregenTM was

2

approved in 2006 to treat genital warts Veregen is a topical cream containing a mixture

of catechins and other components from the water extract of green tea leaves There are

now many additional naturally-derived products in clinical development under the

Botanical Drug Product registration pathway to treat for examples cancer and other

inflammatory-mediated diseases As development activity increases in this area it is of

critical importance to investigate methods of extraction extract stability and factors

which may influence andor elucidate mechanisms of action

Blackberries are a species of fruit belonging to the subgenus Eubatus in the genus

Rubus and are very complex in terms of genetic background growth characteristics and

number of species Recent research has revealed that blackberries contain higher amount

of phenolic compounds such as anthocyanins and other antioxidants than other fruits [9-

11] However studies to elucidate the potential therapeutic properties of blackberries

have been limited Moreover different extraction methods may introduce different

combinations of anthocyanins and phenolics in the extract resulting in different

bioactivities Since blackberries are currently grown in large scale in Kentucky there was

an interest in both characterizing and developing a phenolic blackberry extract as

potential Botanical Drug Products for the treatment and prevention of oxidative stress-

related diseases such as cancer and inflammation Therefore the present research was

focused both on preparation and characterization of phenolic extracts from three

blackberry cultivars grown in Kentucky (Hull Black Satin and Chester) and on

investigation of their anticancer and anti-inflammatory properties The properties of the

solvent that influenced phenolics extraction from blackberries were investigated The

stability of the obtained extracts at different storage conditions was examined In

addition to evaluate their anticancer and anti-inflammatory activities in vitro possible

anticancer mechanism related to ROS generation was investigated Furthermore in an

effort to examine the bioactivity resulting from the complex mixture of a group of

phytochemicals in blackberry extracts the whole extract was fractionated and the

bioactivity of fractions and whole extract was compared These studies have important

implications for the potential use of blackberry extracts for the treatment and prevention

of cancer and inflammation diseases and provide essential information for the

3

development of Botanical Drug Products from extracts derived from blackberries and

other fruits


4

Chapter 2

Plan of Research

The overall goal of this research was to develop phenolic extracts from selected

cultivars of blackberries currently grown in Kentucky as potential Botanical Drug

Products for the treatment and prevention of cancer and inflammatory diseases This

research was guided by three main hypotheses

Hypothesis 1 Extracts which contain high amount of anthocyanins and polyphenols

with high antioxidant activity will be obtained from blackberries

Hypothesis 2 Blackberry extracts will possess anticancer and anti-inflammatory

properties The anticancer activities of the blackberry extracts will involve

mechanisms related to reactive oxygen species (ROS) The blackberry extracts will

exhibit anti-inflammatory activity by inhibiting pro-inflammatory cytokine release

from immune cells

Hypothesis 3 The phenolics but not the non-phenolic compounds in the blackberry

extract will be responsible for its bioactivities

To evaluate these hypotheses the research plan described in sections 21 to 23 was

carried out

5


from blackberries

Blackberries are rich sources of phenolics including anthocyanins which may impact

positively on human health The main objective of the section was to prepare and

characterized the anthocyanin-containing extracts (ACEs) from three blackberry cultivars

(Hull Black Satin and Chester) grown in Kentucky In the preparation procedure an

ultrasound-assisted ethanol extraction method was employed Two different blackberry

materials puree and powder (which was freeze-dried puree) were used to obtain the

ACEs The total phenolic and anthocyanin content polymeric color and total antioxidant

capacity (TAC) in the ACEs were compared The major types of anthocyanins in ACEs

were determined It was found that puree-derived ACE contained higher amount of total

phenolics than powder-derived ACEs which indicated that water content in the

extraction system influenced the yield of total phenolics in ACEs since the powder was

prepared by removing water from the puree using lyophilization Thus the effect of the

water-to-ethanol ratio of the extraction system was investigated on the properties of the

obtained ACEs Finally the stability of these extracts was investigated when they were

stored alone as a function of time temperature and light and when incubated in

biologically relevant buffers as a function of temperature

22 Anticancer and anti-inflammatory properties of blackberry extracts

The main objective of this section was to investigate the anticancer and anti-

inflammatory properties of ACEs in vitro These studies can be divided into four parts 1)

evaluation of the antiproliferative effects of ACEs on human cancer cell lines 2)

evaluation of the role of cyanidin 3-glucoside the dominant anthocyanin in the extracts

in the anticancer activity of ACEs 3) investigation of possible mechanisms of

cytotoxicity including H2O2 ROS generation and the role of copper 4) investigation of

the inhibitory effects of ACEs in pro-inflammatory cytokines release from immune cells

Firstly the antiproliferative effects of puree and powder-derived ACEs were compared in

a panel of human cancer cell lines including leukemia (HL-60) colon (HT-29) and breast

(MCF-7) Secondly the antiproliferative effect of cyanidin 3-glucoside was compared

with those of ACEs in HL-60 cells Thirdly H2O2 generation and enhancement of

6

intracellular ROS level in HL-60 cells by puree and powder-derived ACEs were

evaluated and the ability of catalase to quench these effects was investigated Finally the

ability of Hull powder ACE to inhibit Lipid A-induced Interleukin-12 release from mouse

bone marrow-derived dendritic cells was investigated Moreover the immune-modulation

activity of various ACEs on lipopolysaccharides (LPS)-induced secretion of tumor

necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) from murine macrophages J774A1

was examined and compared

23 Antioxidant and antiproliferative properties of blackberry extracts versus

their phytochemical constituents in vitro

The main objective of this section was to separate phenolic compounds from non-

phenolic compounds in Hull ACEs derived from both puree and powder and to evaluate

their antioxidant activity and antiproliferative properties on cancer cells A simple solid

phase extraction (SPE) method was developed to prepare phenolic-enriched fractions and

non-phenolic fraction The composition of each fraction was characterized by total

anthocyanin and phenolics content The TAC of each fraction was also determined The

percentages of recoveries of solid by this SPE method were evaluated for both puree and

powder ACEs The antiproliferative activities of ACEs and corresponding fractions on

leukemia (HL-60) and colon (HT-29 HCT-15) cancer cells were compared Since our

previous studies suggested that the anticancer effects of ACEs involve ROS mechanisms

the role of each fraction in ROS production was investigated In addition N-acetyl-L-

cysteine a cell permeable antioxidant was employed to investigate the influence of ROS

production on the HL-60 cell death as well as caspase 3 activation induced by ACEs and

fractions


7

Chapter 3

Background

31 Preparation and characterization of phenolic extracts

311 An introduction to natural phenolics

Phenolics are compounds possessing one or more aromatic rings with one or more

hydroxyl groups They are broadly distributed in the plant kingdom and are the most

abundant secondary metabolites of plants with more than 8000 phenolic structures

currently known ranging from simple molecules such as phenolics acids to highly

polymerized substances such as tannins Plant phenolics are generally involved in defense

against ultraviolet radiation or aggression by pathogens parasites and predators as well

as contributing colors to the plants They are ubiquitous in all plant organs and are

therefore an integral part of the human diet Phenolics are widespread constituents of

plant food (fruits vegetables cereals olive legumes chocolate etc) and beverages (tea

coffee beer wine etc) and partially responsible for overall organoleptic properties of

plant foods For example Phenolics contribute to the bitterness and astringency of fruit

and fruit juices because of the interaction between phenolics mainly procyanidin and

the glycoprotein in saliva Anthocyanins one of the six subgroups of a large group of

plant polyphenol constituents known as flavonoids are responsible for the orange red

blue and purple color of many fruits and vegetables such as apples berries beets and

onions It was considered that phenolics are the most important compounds affecting

flavor and color difference among white pink and red wines they react with oxygen and

are critical to the preservation maturation and aging of the wine

Plant phenolics include phenolics acids flavonoids tannins (Figure 31) and the less

common stilbenes and lignans (Figure 32) Flavonoids are the most abundant

polyphenols in our diets The basic flavonoid structure is the flavan nucleus containing

15 carbon atoms arranged in three rings (C6-C3-C6) which are labeled as A B and C

Flavonoid are themselves divided into 6 subgroups flavones flavonols flavanols

flavanones isoflavones and anthocyanins according to the oxidation state of the central

C ring Their structural variation in each subgroup is partly due to the degree and pattern

8

of hydroxylation methoxylation prenylation or glycosylation Some of the most

common flavonoids include quercetin a flavonol abundant in onion broccoli and apple

catechin a flavanol found in tea and several fruits naringenin the main flavanone in

grapefruit cyanidin-glycoside an anthocyanin abundant in berry fruits (blackcurrant

raspberry strawberry etc) and daidzein genistein and glycitein the main isoflavones in

soybean [1]

Phenolic acids can be divided into two classes derivatives of benzoic acid such as

gallic acid and derivatives of cinnamic acid such as coumaric caffeic and ferulic acid

Caffeic acid is the most abundant phenolic acid in many fruits and vegetables most often

esterified with quinic acid as in chlorogenic acid which is the major phenolic compound

in coffee Another common phenolic acid is ferulic acid which is present in cereals and

is esterified to hemicelluloses in the cell wall [12]

Tannins are another major group of polyphenols in our diets and usually subdivided

into two groups (1) hydrolysable tannins and (2) condensed tannins Hydrolysable

tannins are compounds containing a central core of glucose or another polyol esterified

with gallic acid also called gallotannins or with hexahydroxydiphenic acid also called

ellagitannins The great variety in the structure of these compounds is due to the many

possibilities in forming oxidative linkage Intermolecular oxidation reactions give rise to

many oligomeric compounds having a molecular weight between 2000 and 5000 Daltons

[13] Condensed tannins are oligomers or polymers of flavan-3-ol linked through an

interflavan carbon bond They are also referred to as proanthocyanidins because they are

decomposed to anthocyanidins through acid-catalyzed oxidation reaction upon heating in

acidic alcohol solutions The structure diversity is a result of the variation in

hydroxylation pattern stereochemistry at the three chiral centers and the location and

type of interflavan linkage as well as the degree and pattern of methoxylation

glycosylation and galloylation [14]

Despite their wide distribution the health effects of dietary polyphenols have come to

attention of nutritionists only in recent years The main reason for the understudy on

polyphenols is the variety and complexity of their chemical structure However over the

past 15 years researchers and food manufacturers have become more and more interested

in polyphenols due to their potent antioxidant properties their abundance in the diet and

9

their credible effects in the prevention of various oxidative stress associated diseases The

preventive effects of these second plant metabolites in terms of cardiovascular

neurodegenerative diseases and cancer are deduced from epidemiologic data as well as in

vitro and in vivo [15-18] and result in respective nutritional recommendations

Furthermore polyphenols were found to modulate the activity of a wide range of enzyme

and cell receptors In this way in addition to having antioxidant properties polyphenols

have several other specific biological actions in preventing and or treating diseases

10

Figure 3 1 Structures of flavonoids phenolic acids and tannins

11

Figure 3 2 Structures of stilbenes and lignan

12

312 Extraction of phenolics

The extraction of bioactive compounds from plant materials is the first step in the

utilization of phytochemicals in the preparation of dietary supplements or nutraceuticals

functional food ingredients and additives to food pharmaceutical and cosmetic

products Phenolics can be extracted from fresh frozen or dried plant samples Usually

before extraction plant samples are treated by milling grinding and homogenization

which may be preceded by air-drying or freeze-drying Generally freeze-drying retains

higher levels of phenolics content in plant samples than air-drying [19] For example

Asami et al showed freeze-dried Marion berries strawberries and corn consistently had a

higher level of total phenolic content compared with those air-dried [20] However the

drying process including freeze-drying could cause undesirable effect on the constituent

profiles of plant samples Therefore caution should be taken when planning and

analyzing research studies on the medicinal properties of plants [19]

Solvent extraction is the most commonly used procedure to prepare extracts from

plant materials due to their ease of use efficiency and wide applicability It is generally

known that the yield of chemical extraction depends on the type of solvents with varying

polarities extraction time and temperature sample-to-solvent ratio as well as on the

chemical compositions and physical characteristics of the samples Solubility of

phenolics is governed by the chemical nature of the plant sample as well as the polarity of

the solvents used Plant materials may contain phenolics varying from simple (eg

phenolic acids anthocyanins) to highly polymerized substances (eg tannins) with

different quantities Moreover phenolics may also be associated with other plant

components such as carbohydrates and proteins Therefore there is no universal

extraction procedure suitable for extraction of all plant phenolics Depending on the

solvent system used during exaction a mixture of phenolics soluble in the solvent will be

extracted from plant materials It may also contain some non-phenolic substances such as

sugar organic acids and fats As a result additional steps may be required to remove

those unwanted components

Solvents such as methanol ethanol acetone ethyl acetate and their combinations

have been used for the extraction of phenolics from plant materials often with different

proportions of water Selecting the right solvent affects the amount and rate of

13

polyphenols extracted [21] In particular methanol has been generally found to be more

efficient in extraction of lower molecular weight polyphenols while the higher molecular

weight flavanols are better extracted with aqueous acetone [22-25] Ethanol is another

good solvent for polyphenol extraction and is safe for human consumption [26] In

preparing anthocyanin-rich phenolic extracts from plant materials an acidified organic

solvent most commonly methanol or ethanol is used This solvent system denatures the

cell membranes simultaneously dissolves the anthocyanins and stabilizes them

However care should be taken to avoid addition of excess acid which can hydrolyze

labile acyl and sugar residues during concentration steps To obtain the best yield of

anthocyanins extraction weak organic acids such as formic acid acetic acid citric acid

tartaric acid and phosphoric acid and low concentrations of strong acids such as 05-

30 of trifluoroacetic acid and lt 10 of hydrochloric acid are recommended [27-29]

In addition sulfured water has also been used as extraction solvent in seeking a reduction

of the use of organic solvents as well as the cost of extraction [30]

The recovery of phenolic compounds from plant materials is also influenced by the

extraction time and temperature which reflects the conflicting actions of solubilization

and analyte degradation by oxidation [31] The increase in the extraction temperature can

promote higher analyte solubility by increasing both solubility and mass transfer rate In

addition the viscosity and the surface tension of the solvents are decreased at higher

temperature which helps the solvents to reach the sample matrices improving the

extraction rate However many phenolic compounds are easily hydrolyzed and oxidized

Long extraction times and high temperature increase the chance of oxidation of phenolics

which decrease the yield of phenolics in the extracts For example conventional

extraction and concentration of anthocyanins is typically conducted at temperatures

ranging from 20 to 50 degC [28] because temperatures gt70 degC have been shown to cause

rapid anthocyanin degradation [32] Therefore it is of critical importance to select

efficient extraction proceduremethod and maintain the stability of phenolic compounds

The conventional extraction methods such as maceration and soxhlet extraction have

shown low efficiency and environmental pollution due to large volumes of organic

solvent used and long extraction time required in those methods A number of methods

have been developed in recent years such as microwave ultrasound-assisted extractions

14

and techniques based on use of compressed fluids as extracting agents such as subcritical

water extraction (SWE) supercritical fluid extraction (SFE) pressurized fluid extraction

(PFE) or accelerated solvent extraction (ASE) were also applied in the extraction of

phenolic compounds from plant materials

Ultrasound-assisted extraction (UAE) is a potential technology as it does not require

complex instruments and is relatively low-cost It can be used both on a small and large

scale in the phytopharmaceutical extraction industry [33] The mechanism for ultrasonic

enhancement involves the shear force created by implosion of cavitation bubbles upon

the propagation of the acoustic waves in the kHz range [34] Collapse of bubbles can

produce physical chemical and mechanical effects [35] which resulted in the disruption

of biological membranes to facilitate the release of extractable compounds and enhance

penetration of solvent into cellular materials and improve mass transfer [33 36]

Recently UAE has been widely used in the extraction of various phenolic compounds

from different parts of plants such as leaves [37] stalks [38] fruits [39 40] and plant

seeds [41] A comparison study showed that UAE caused less degradation of phenolics

and was a much faster extraction process in extraction of phenolic compounds from

strawberries compared with other extraction methods including solid-liquid subcritical

water and microwave-assisted method [42]

Pressurized liquid extraction (PLE) also known under the trade name of accelerated

solvent extraction (ASE) is a relative new technology for extraction of phytochemicals

under high temperature and pressure Benthin et al (1999) [43] were among the first to

conduct a comprehensive study on the feasibility of applying PLE to medicinal herbs

after its emergence in the mid-1990s In PLE pressure is applied to allow the use as

extraction solvents of liquids at temperatures greater than their normal boiling point The

combined use of high pressures (33-203 MPa) and temperatures (40-200 degC) provides

faster extraction processes that require small amounts of solvents (eg 20 min using 10ndash

50 ml of solvent in PLE can be compared with a traditional extraction step in which 10ndash

48 h and up to 200 ml are required) [44] High temperature and pressure improves analyte

solubility and the desorption kinetics from the matrices [45] Hence extraction solvents

including water which show low efficiency in extracting phytochemicals at low

temperatures may be much more efficient at elevated PLE temperatures The use of

15

water as an extraction solvent in PLE is the so-called subcritical water extraction (SWE)

In SWE water is heated up to 200 degC and the change in the dielectric constant of the

water with the temperature leads water to behave like an organic solvent for example the

dielectric constant of water at 200 degC is equal to 36 which is close to methanol [44] Ju et

al showed that PLE (80 ndash 100 degC) using acidified water was as effective as acidified

60 methanol in extracting anthocyanins from grape skins [46] However phenolic

compounds are easily oxidized at high temperature so it is very important to prove that

they will not degrade under the proposed PLE conditions [47] In recent years PLE has

been successfully applied to the extraction of phenolic compounds from different plant

materials such as grape seeds and skin [46 48 49] apples [50] spinach [51] eggplants

[52] and barley flours [53] Another technology using carbon dioxide as compressed fluid

as extraction solvent is called supercritical and subcritical fluid extraction Organic

modifiers were added to increase the polarity of the fluid for extraction of phenolic

compounds [54-56] SFE is performed in the absence of both light and air degradation

and oxidation processes are significantly reduced in comparison with other extraction

techniques In general all these compressed fluid-base extraction techniques are more

environmental friendly procedures than other methods in reducing use of organic solvents

(eg PLE) allowing extraction performed with nonpolluting nontoxic solvents such as

water (eg SWE) supercritical CO2 fluid (eg SFE) However due to the application of

high pressure in these techniques the requirements of instrumentation are high and the

cost of these methods on the industrial scale is high which often outweigh the technical

benefits

Microwave-assisted extraction (MAE) is a process utilizing microwave energy to

facilitate partition analytes from the sample matrix into the solvent The main advantage

of this technique is the reduced extraction time and solvent volume as compared to

conventional extraction techniques [57] It has been used for the extractions of some

small-molecule phenolic compounds such as phenolic acids (eg gallic acid ellagic acid)

[58] quercetin [59] isoflavone [60] and trans-resveratrol [61] which were proved to be

stable under microwave-assisted heating conditions at temperature up to 100 degC for 20

min [62] Phenolic compounds having a higher number of hydroxyl-type substituents

(eg tannins) and those that are sensitive to elevated temperature (eg anthocyanins)

16

may not suitable to be extracted by MAE due to degradation under MAE extraction

conditions [62]

The extraction of phenolic compounds from plant material may also be influenced by

other factors such as solvent-to-solid ratio and the particle size of the sample Increasing

solvent-to-solid ratio was found to work positively for enhancing phenol yields [63 64]

However an equilibrium between the use of high and low solvent-to-solid ratios

involving a balance between high costs and solvent wastes and avoidance of saturation

effects respectively has to be found to obtain an optimized value [65] Lowering particle

size also enhances the yield of phenolic compounds [66 67] To increase the release of

bound phenolics a number of enzymatic procedures involving the use of various mixed

pectinolytic and cell wall polysaccharide degrading enzyme preparation in phenolic

extraction have been described [68 69] The particle size of the mashed samples was

found to be a main factor to increase the enzyme action and extraction efficiency of

phenolic compounds from samples in these enzyme-assisted extractions [70]

313 Purification and fractionation of phenolics

Plant crude extract usually contain large amount of carbohydrates andor lipoidal

material and the concentration of the phenolics in the crude extract may be low To

concentrate and obtain polyphenol-rich fractions before analysis strategies including

sequential extraction or liquid-liquid partitioning andor solid phase extraction (SPE)

based on polarity and acidity have been commonly used In general elimination of

lipoidal material can be achieved by washing the crude extract with non-polar solvents

such as hexane [71] dichloromethane [72] or chloroform [73] To remove polar non-

phenolic compounds such us sugars organic acids a SPE process are usually carried out

SPE is becoming popular since it is rapid economical and sensitive and because

different cartridges with a great variety of sorbents can be used In addition it can now be

automated reducing processing time C18 cartridges have been the most widely used in

phenolic compound separation After the aqueous sample was passed the preconditioned

C18 cartridges the cartridges were washed with acidified water to remove sugar organic

acids and other water-soluble constituents The polyphenols were eluted with absolute

methanol [74] or aqueous acetone [71] Further separation of phenolic compounds can be

17

achieved by adjusting the pH of the sample as well as the pH and polarity of eluents For

example Pinelo et al adjusted the pH of dealcoholic wine sample to 70 and eluted

phenolic acids with water in the first fraction [75] Following this step the C18 cartridge

was acidified with 001 M HCl and nonpolymeric phenols such as catechins

anthocyanins and flavonols were eluted with ethyl acetate Finally a mixture of water

acetone and methanol was used to elute the polymeric phenols Other sorbents such as

Amberlite XAD-2 [76] XAD-7 [77 78] XAD-16 [73] Oasis HLB [79 80] have also

successfully been used to purify phenolic compounds in crude extracts or wine samples

A comparison of several SPE cartridge including Amberlite silica-based C8 copolymer-

based HLB PH ENV+ and MCX with silica-based C18 for the isolation of phenolic

compounds in wine at low concentration showed that the proposed SPE method with

HLB cartridge has a higher sensitivity reproducibility and loading capacity than with

C18 cartridge and HLB cartridge may be a good alternative for the C18 cartridge for the

isolation of wine phenolic compounds [81]

Column chromatography has been also employed for fractionation of phenolic

extracts Although this method is often labor-intensive and solvent-consuming it ensures

that greater amounts of fractions can be obtained for use in subsequent isolation and

identification of pure substances Typically-utilize column sorbents are RP-C18 [82]

Toyopearl [83 84] LH-20 [83 84] and to a less extent polyamide resin [85] Ethanol

methanol acetone and water and their combinations are commonly used as eluents In

particular the isolation of proanthocyanidins (condensed tannins) is routinely carried out

by employing Sephadex LH-20 column chromatography [86 87] The crude extract was

applied to the column which was washed with ethanol to elute the non-tannin substances

followed by elution with acetone-water or alcohol-water to obtain proanthocyanidins In

some cases preparative-scale HPLC has also been used in polyphenol sample

purification [88 89]

The classical liquid-liquid extraction procedure has been less commonly used because

it is a tedious highly time-consuming process with high solvent costs and low recoveries

[90] An example of sequential extraction was provided by extraction of phenolic

compounds from tissues of cider apples [91] The freeze-dried apple tissue powder was

extracted sequentially with hexane (to remove lipids carotenoids and chlorophyll)

18

methanol (sugars organic acids and phenolic compounds with low molecular weight) and

aqueous acetone (polymerized polyphenols) As an alternative to liquid chromatography

Countercurrent Chromatography (CCC) has been developed as an effective techniques

for fractionation of various classes of phenolic compounds CCC is a preparative all-

liquid chromatographic technique based on partitioning of compounds between two

immiscible liquid phases a liquid stationary phase and a liquid mobile phase Solutes are

separated according to their partition coefficients between the two solvent phases based

on their hydrophobicity The big advantage of CCC is that it uses no solid matrix and the

role of two liquid phases namely liquid stationary phase and mobile phase can be

switched during a run Thus there is no irreversible sample adsorption and the recovery

is 100 [92] Degenhardt et al used high-speed countercurrent chromatography

(HSCCC) for separation of anthocyanins in the pigment mixtures extracted from red

cabbage black current black chokeberry and roselle [93] Anthocyanins were

successfully fractionated based on their polarities into the biphasic mixture of tert-butyl

methyl ethern-butanolacetonitrilewater (2215 vvvv) acidified with trifluoroacetic

acid (TFA) Yanagida et al demonstrated that HSCCC could be used for isolation of tea

catechins and other food-related polyphenols such as procyanidins phenolic acids and

flavonol glycosides using tert-butyl methyl etheracetonitrile01 aqueous TFA (223

vvv) [94] In addition Krafczyk and Glomb employed Multilayer Countercurrent

Chromatography (MLCCC) coupled with preparative High-Performance Liquid

Chromatography (HPLC) to obtain pure flavonoids from Rooibos tea [95] This method

was able to isolate up to gram of material and to verify known polyphenol structures and

discover previously not published ones

314 Analysis and quantification of phenolics

Natural phenolics are of interest from many viewpoints (antioxidants astringency

bitterness browning reactions color etc) Selection of the proper analytical strategy for

studying phenolics in plant materials depends on the purpose of the study as well as the

nature of the sample and the analyte [31] The assays used for the analysis of phenolics

can be classified as either those which determine total phenolics content or those

quantifying a specific group or class of phenolic compounds Quantification of phenolic

19

compounds in plant extract is influenced by the chemical nature of the analyte as well as

assay method selection of standards and presence of interfering substances [96]

Because of the heterogeneity of natural phenolics and the possibility of interference

from other readily oxidized substances in the plant materials it is not surprising that

several methods have been used for total phenolics determination and none are perfect

Among such methods are the Folin-Denis method (FD) Folin-Ciocalteu method (FC)

permanganate titration colorimetry with iron salts and ultraviolet absorbance In most

cases FC has been found preferable as compared to the other methods [97] The FC

assay relies on the transfer of electrons in alkaline medium from phenolic compounds to

phosphomolybdicphosphotungstic acid complexes to form blue complexes (possibly

(PMoW11O40)4-) that are determined spectroscopically at approximately 760 nm [97 98]

Gallic acid is widely used as the comparison standard and values are usually compared as

milligram of gallic acid equivalent per liter of extract among samples Owing to the

general nature of the FC chemistry it is indeed a measure of total phenolics and other

oxidation substrates The other oxidation substrate present in a given extract sample can

interfere the total phenolics measurement in an inhibitory additive or enhancing manner

[97 98] The inhibitory effects could be due to the oxidants competing with FC reagent

andor air oxidation after the sample is made alkaline For this reason the FC reagent is

added ahead of alkali [97] Additive effects occur from unanticipated phenols aromatic

amines high sugar levels or ascorbic acid in the samples The additive effects can be

measured before adding the alkali or by a more specific assay of a known interference

and then subtracted from the FC value [97] Sulfites and sulfur dioxide which is a

common additive for wine can cause enhancing effect [97] Singleton et al [97]

discussed the effects of potential interference compounds and methods for correcting

these factors However despite these disadvantages the FC assay is simple and

reproducible and has been widely used for quantification of phenolic compounds in plant

materials and extracts

Anthocyanins are one of the six subgroups of the large and widespread group of plant

phenolics known as flavonoids While there are six common anthocyanidins more than

540 anthocyanin pigments have been identified in nature [99] The simplest assay for the

quantification of anthocyanins as a group is based on the measurement of absorption at a

20

wavelength between 490 nm and 550 nm where all anthocyanins show a maximum This

band is far from the absorption bands of other phenolics which have spectral maxima in

the UV range [100] However by this method anthocyanin polymerized degradation

products produced by browning reactions are co-determined and lead to an

overestimation of anthocyanin content Therefore an approach that differentiates

anthocyanins from their degradation products is preferable The pH differential method

takes the advantage of the structural transformations of anthocyanin chromophore as a

function of pH By this method the absorption of the sample is measured at pH 1

(anthocyanins as colored oxonium salts) as well as at pH 45 (anthocyanins as colorless

hemiketals) The anthocyanin degradation pigments do not exhibit reversible behavior

with pH and are thus excluded from the absorbance calculation [101] In this method

calculation of monomeric anthocyanin concentration is usually based on the molecular

weight (MW) and the molar extinction coefficient (ε) of either the main anthocyanin in

the sample or cyanidin-3-glucoside the most common anthocyanin in nature For all

quantification the MW and ε underlying the calculation should be given because the

differences in the MW of the anthocyanins and the influence of the solvent on ε

considerably distort the results [102] For example quantification as cyanidin-3-

glucoside equivalents gave markedly lower results for berries containing mainly

delphinidin and malvidin glycosides as compared with ldquorealrdquo values quantified based on

corresponding standard compounds [78]

In a study of 20 food supplements containing extracts of blueberry elderberry

cranberry and chokeberry the total anthocyanin content (as determined as the cyanidin-3-

glucoside equivalent) obtained with pH differential method were in good agreement with

those obtained with an HPLC method [103] In addition a collaborative study where 11

collaborators representing academic government and industrial laboratories analyzed 7

fruit juice beverage natural colorants and wine sample demonstrated that total

anthocyanin content can be measured with excellent agreement between laboratories

using the pH differential method and the method has been approved as a First Action

Official Method [104]

Anthocyanins are labile compounds and easily oxidized and condensed with other

phenolics to form brown polymeric pigments Somers and Evans developed a method

21

based on the use of sodium sulfite a bleaching reagent to determine the polymeric color

and browning in wines [103] Monomeric anthocyanin will combine with bisulfite to

form a colorless sulfonic acid addition adduct while the polymeric anthocyanin

degradation products are resistant to bleaching by bisulfite as the 4-position is not

available being covalently linked to another phenolic compounds This method has been

applied to a variety of anthocyanin-rich products and found be extremely useful for

monitoring the anthocyanin degradation and browning during processing and storage

[102]

Different colorimetric methods are used to measure total proanthocyanidin

(condensed tannin) content in plant samples The proanthocyanidin assay is carried out in

a butanol and concentrated hydrochloric acid (955 vv) solution where

proanthocyanidins are autoxidized and cleaved to colored anthocyanidin monomer [105]

In the vanillin assay condensation of resorcin- or phloroglucin- partial structure of

flavonols with vanillin in acidic medium leads to the formation of colored carbonium

ions [106] Catechin a monomeric flavanol is often used as a standard The same

reaction mechanism as in the vanillin assay is used in the dimethylamino-

cinnamaldehyde (DMCA) assay in which only the terminal units of the

proanthocyanidins react with DMCA [107] These methods of quantification are

susceptible to the structure of the analytes as well as various external factors such as

temperature concomitant substances solvent presence of oxidants etc [108] Thus

adaptation and validation of methods for different sample material are required In

addition purification of proanthocyanidins before quantification has proven to be very

supportive to minimize the interference and obtain reproducible results [108 109]

Hydrolysable tannins can be quantified by a number of approaches including the

potassium iodate method rhodanine method and sodium nitrite method Of these the

potassium iodate method is most widely used It is based on the reaction of methyl

gallate formed upon methanolysis of hydrolysable tannins in the presence of strong

acids with potassium iodate to produce a red chromophore with a maximum absorbance

between 500 nm and 550 nm [110] Similar as assays for proanthocyanidin

quantification the yield of this reaction also influenced by a number of factors such as

the structure of the hydrolysable tannins reaction time temperature other phenolics

22

present in the sample etc The rhodanine method can be used for estimation of

gallotannins and is based on determination of gallic acid in a sample subject to acid

hydrolysis under conditions that must be anaerobic to avoid oxidation of the product

[111] On the other hand the sodium nitrite assay is developed for quantification of

ellagic acid in sample hydrolysate [112] However this assay requires large quantities of

pyridine as a solvent which introduces high toxicity risk in analysis procedure

In general traditional spectrophotometric assays provide simple and fast screening

methods to quantify classes of phenolic compounds in crude plant samples However due

to the complexity of the plant phenolics and different reactivity of phenols toward assay

reagents a broad spectrum of methods is used for assay of the constituents leading to

differing incomparable results In addition to that the methods are quite prone to be

interfered and consequently result in over- or underestimation of the contents Modern

high-performance chromatographic techniques combined with instrument analysis are the

ldquostate of artrdquo for the profiling and quantification of phenolic compounds Gas

chromatographic (GC) techniques have been widely used especially for separation and

quantification of phenolic acids and flavonoids The major concern with this technique is

the low volatility of phenolic compounds Prior to chromatography phenolics are usually

transformed into more volatile derivatives by methylation conversion into trimethylsilyl

derivatives etc A detailed discussion on application of GC on analysis of phenolic acids

and flavonoids was provided by Stalicas [113]

HPLC currently represents the most popular and reliable technique for analysis of

phenolic compounds Various supports and mobile phases are available for the analysis

of phenolics including anthocyanins proanthocyanidins hydrolysable tannins flavonols

flavan-3-ols flavanones flavones and phenolic acids in different plant extract and food

samples [23 114-123] Moreover HPLC techniques offer a unique chance to analyze

simultaneously all components of interest together with their possible derivatives or

degradation products [124 125] The introduction of reversed-phase (RP) columns has

considerably enhanced HPLC separation of different classes of phenolic compounds and

RP C18 columns are almost exclusively employed It was found that column temperature

may affect the separation of phenolics such as individual anthocyanin [126] and constant

column temperature is recommended for reproducibility [113] Acetonitrile and methanol

23

are the most commonly used organic modifiers In many cases the mobile phase was

acidified with a modifier such as acetic formic and phosphoric acid to minimize peak

tailing Both isocratic and gradient elution are applied to separate phenolic compounds

The choice depends on the number and type of the analyte and the nature of the matrix

Several reviews have been published on application of HPLC methodologies for the

analysis of phenolics [113 127-129]

Given the intrinsic existence of conjugated double and aromatic bonds every phenol

exhibits a higher or lower absorption in ultraviolet (UV) or ultravioletvisible (UVVIS)

region Thus the most common means of detection coupled to LC are UVVIS

photodiode array (PDA) and UV-fluorescence detectors PDA is the most prevalent

method since it allows for scanning real time UVVIS spectra of all solutes passing

through the detector giving more information of compounds in complex mixtures such as

a plant crude extract Other methods employed for detection of phenolic compounds

include electrochemical detection (ECD) [130] voltammetry technique [131] on-line

connected PDA and electro-array detection [132] chemical reaction detection techniques

[133] mass spectrometric (MS) [120 126 134] and nuclear magnetic resonance (NMR)

detection [123 135] MS and NMR detections are more of structure confirmation means

than quantification methods

Electromigration techniques including capillary electrophoresis (CE) capillary zone

electrophoresis (CZE) and micellar electrokinetic chromatography coupled with UV and

to a less extent EC and MS detection are also employed for phenolics analysis [136]

32 Antioxidant properties of phenolic compounds

Antioxidants are termed as the compounds that can delay inhibit or prevent the

oxidation of oxidizable materials by scavenging free radicals and diminishing oxidative

stress Oxidative stress is an imbalanced state where excessive quantities of reactive

oxygen andor nitrogen species (ROSRNS eg superoxide anion hydrogen peroxide

hydroxyl radical peroxynitrite) overcome endogenous antioxidant capacity leading to

oxidation of a varieties of biomacromolecules such as enzymes proteins DNA and

lipids Oxidative stress is important in the development of chronic degenerative diseases

including coronary heart disease cancer and aging [1]

24

Recently phenolics have been considered powerful antioxidants in vitro and proved

to be more potent antioxidants than Vitamin C and E and carotenoids [137 138] The

inverse relationship between fruit and vegetable intake and the risk of oxidative stress

associated diseases such as cardiovascular diseases cancer or osteoporosis has been

partially ascribed to phenolics [139 140] It has been proposed that the antioxidant

properties of phenolic compounds can be mediated by the following mechanisms (1)

scavenging radical species such as ROSRNS (2) suppressing ROSRNS formation by

inhibiting some enzymes or chelating trace metals involved in free radical production (3)

up-regulating or protecting antioxidant defense [141]

321 Phenolics as free radical scavengers and metal chelators

Phenolic compounds (POH) act as free radical acceptors and chain breakers They

interfere with the oxidation of lipids and other molecules by rapid donation of a hydrogen

atom to radicals (R)

R + POH rarr RH + PO (1)

The phenoxy radical intermediates (PO) are relatively stable due to resonance and

therefore a new chain reaction is not easily initiated Moreover the phenoxy radical

intermediates also act as terminators of propagation route by reacting with other free

radicals

PO + R rarr POR (2)

Phenolic compounds possess ideal structure chemistry for free radical scavenging

activities because they have (1) phenolic hydroxyl groups that are prone to donate a

hydrogen atom or an electron to a free radical (2) extended conjugated aromatic system

to delocalize an unpaired electron Several relationships between structure and reduction

potential have been established as follows

(1) For phenolic acids and their esters the reduction activity depends on the number of

free hydroxyl groups in the molecule which would be strengthened by steric

hindrance [142] Hydroxycinnamic acids were found to be more effective than their

hydroxybenzoic acid counterparts possibly due to the aryloxy-radical stabilizing

effect of the ndashCH=CHndashCOOH linked to the phenyl ring by resonance [138]

(2) For flavonoids the major factors that determine the radical-scavenging capability

25

[143 144] are

(i) The ortho-dihydroxy structure on the B ring which has the best electron-donating

properties and confers higher stability to the radical form and participates in electron

delocalization

(ii) The 2 3-double bond in conjugation with a 4-oxo function in the C ring is

responsible for electron delocalization from the B ring The antioxidant potency is

related to the structure in terms of electron delocalization of aromatic nucleus

(iii) The 3- and 5-hydroxyl groups with the 4-oxo function in A and C rings are essential

for maximum radical scavenging potential

(iv) The 3-hydroxyl group is important for antioxidant activity The 3-glycosylation

reduces their activity when compared with corresponding aglycones

Quercetin is a flavonol that fulfills these criteria Anthocyanins are particularly

reactive toward ROSRNS because of their peculiar chemical structure of electron

deficiency

As an alternative antioxidant property some phenolic compounds with dihydroxy

groups can conjugate transition metals preventing metal-induced free radical formation

The redox active metal ions such as Cu+ or Fe2+ interact with hydrogen peroxide (H2O2)

through Fenton chemistry (as shown in reaction 3 below) to form hydroxyl radicals

(OH) which is the most reactive ROS known being able to initiate free radical chain

reactions by abstracting hydrogen from almost any molecule Phenolic compounds with

catecholate and gallate groups can inhibit metal-induced oxygen radical formation either

by coordination with Fe2+ and enhancing autoxidation of Fe2+ (as shown in reaction 4

below) or the formation of inactive complex with Cu2+ Fe2+ or Cu+ with relatively

weaker interaction [145 146] The attachment of metal ions to the flavonoid molecule

can be 3rsquo4rsquo-o-diphenolic groups in the B ring 34 or 35-o-diphenolic groups and the

ketol structures 4-keto3-hydroxy or 4-keto5-hydroxy groups in the C ring [147 148] It

was also proposed that optimum metal-binding and antioxidant activity is associated with

the structures which contain hydroxy-keto group (a 3-OH or 5-OH plus a 4-C=O) as well

as a large number of catecholgallol groups [147 149]

H2O2 + Cu+ or Fe2+ rarr Cu2+ or Fe3+ + OH + OH- (3)

26

(4) [146]

In theory these two antioxidant actions can result in a reduction of the steady state

concentrations of free radicals and oxidant species diminishing the subsequent oxidation

of target molecules such as lipids proteins and nucleic acids Based on these potential

capacities extensive studies have demonstrated the antioxidant activities of natural

phenolics in general in a myriad of biochemical and ex vivo systems [150] for example

in isolated low density lipoproteins (LDL) synthetic membrane ex vivo human plasma

and cells in culture In addition mutual synergistic effects were also observed between

different phenolic compounds or with other non-phenolic antioxidants [151] and it is

generally accepted that a combination of phenolic or other antioxidants exert better

antioxidant effect than pure individual compound

322 Prooxidant activity of phenolic compounds

It is worth noting that some phenolic antioxidants can initiate an autoxidation process

and behave like prooxidants [143] under certain conditions Instead of terminating a free

radical chain reaction by reacting with a second radical the phenoxy radical may also

interact with oxygen and produce quinones (P=O) and superoxide anion (O2 -) as shown

below [141]

PO + O2 rarr P=O + O2 - (5)

Nevertheless transition metal ions could also induce prooxidant activity of phenolic

antioxidants as demonstrated by the following reactions [152]

Cu2+ or Fe3+ + POH rarr Cu+ or Fe2+ + PO + H+ (6)

PO + RH rarr POH + R (7)

R + O2 rarr ROO (8)

ROO + RH rarr ROOH + R (9)

ROOH + Cu+ or Fe2+ rarr Cu2+ or Fe3+ + RO + OH- (10)

27

It was found that phenolic antioxidants intend to behave like prooxidant under the

conditions favor their autoxidation for example at high pH with high concentrations of

transition metal ions and oxygen molecule present Small phenolics which are easily

oxidized such as quercetin gallic acid possess prooxidant activity while high molecular

weight phenolics such as condensed and hydrolysable tannins have little or no

prooxidant activity [153] It is necessary to consider the possible prooxidant effects of

phenolics for in vitro antioxidant tests where great care should be taken in the design of

experimental conditions Moreover because the biological conditions in vivo may differ

dramatically from in vitro experiment great caution must be taken when interpreting in

vitro results and extrapolating to in vivo conditions

323 Determination of total antioxidant capacity (TAC) of phenolic extracts

Due to the chemical diversity of phenolic compounds and the complexity of

composition in plant samples it is costly and inefficient to separate each phenolic

antioxidant and study it individually Moreover an integrated total antioxidant power of a

complex sample is often more meaningful to evaluate the health benefits because of the

cooperative action of antioxidants Therefore it is desirable to establish convenient

screening methods for quick quantification of antioxidant effectiveness of phenolic

extract samples A variety of antioxidant assays such as Trolox equivalent antioxidant

capacity (TEAC) oxygen radical absorbance capacity (ORAC) total radical-trapping

antioxidant parameter (TRAP) ferric ion reducing antioxidant power (FRAP) and cupric

ion reducing antioxidant capacity (CUPRAC) assays have been widely used for

quantification of antioxidant capacity of phenolic samples from fruits and vegetables The

Folin-Ciocalteu antioxidant capacity assay (F-C assay or total phenolics assay) is also

considered as another antioxidant capacity assay because its basic mechanism is as

oxidationreduction reaction although it have been used as a measurement of total

phenolics content for many years On the basis of the chemical reaction involved major

antioxidant assays can be roughly classified as hydrogen atom transfer (HAT) and

electron transfer (ET) reaction based assays although these two reaction mechanisms can

be difficult to distinguish in some cases [152]

28

The HAT-based assays include ORAC and TRAP assays These assays measure the

capacity of an antioxidant to quench free radicals by hydrogen atom donation The

majority of HAT-based assays involve a competitive reaction scheme in which

antioxidant and substrate compete for thermally generated peroxyl radicals through the

decomposition of azo compounds [152] As an example of HAT-based assays ORAC

assay [154] employed a fluorescent probe (eg fluorescein) to compete with sample

antioxidant for peroxyl radicals generated by decomposition of 22rsquo-azobis(2-

amidinopropane) dihydrochloride (AAPH) The fluorescence intensity is measured every

minute at ambient conditions (pH 74 37 degC) to obtain a kinetic curve of fluorescence

decay The net area under the curve (AUC) calculated by subtracting the AUC of blank

from that of the sample or standard (eg Trolox) and the TAC of sample was calculated

as Trolox equivalent based on a standard curve [152] The ORAC method is reported to

mimic antioxidant activity of phenols in biological systems better than other methods

since it uses biologically relevant free radicals and integrates both time and degree of

activity of antioxidants [155] However the method often requires the use of expensive

equipment and it is usually a time-consuming process

TEAC F-C FRAP and CUPRAC assay are the ET-based assays These assays

measure the capacity of an antioxidant in reduction of an oxidant probe which changes

color when reduced [152] The reaction end point is reached when color change stopped

The degree of color change is proportional to the antioxidant concentration The oxidant

probes used are 22rsquo-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation

(ABTS +) in TEAC Fe3+(246-tripyridyl-s-triazine)2Cl3 in FRAP and

bis(neocuproine)Cu2+Cl2 in CUPRAC assays respectively TEAC method was reported

to be operationally simple reproducible and cost effective [156] Most importantly it

can be applied in multiple media to determine both hydrophilic and hydrophobic

antioxidant capacity of plant extracts since the reagent is soluble in both aqueous and

organic solvent media [157] As opposed to TEAC assay FRAP assay measures ferric-

to-ferrous reduction capacity of water-soluble antioxidants in acidic pH such as pH 36

[158]

It was proposed that procedures and applications for three assays namely ORAC F-

C and TEAC be considered for standardization at the First International Congress on

29

Antioxidant Methods held in Orlando FL in June 2004 [159] It must be emphasized that

these antioxidant assays measure the capacity of a sample only under defined conditions

prescribed by the given method and strictly based on the chemical reaction in vitro so the

bioactivity of a sample cannot be reflected solely by these assays In another words the

ldquototal antioxidant capacityrdquo of a particular sample cannot be truly measured by any of the

assays because of the complexity of the chemistry of antioxidant compounds For

example the total antioxidant capacity has to be able to reflect both lipophilic and

hydrophilic capacity and to reflect and distinguish hydrogen atom transfer electron

transfer as well as transition metal chelation [159] It is also very important to develop

methods specific for each radical source for evaluating effectiveness of antioxidant

compounds against various ROSRNS such as O2 - HO and ONOO- to fully elucidate a

full profile of antioxidant capacity [159]

33 Natural phenolics and cancer

Cancer is a multi-step disease incorporated environmental chemical physical

metabolic and genetic factors which play a direct andor indirect role in the induction

and deterioration of cancers Strong and consistent epidemiology evidence indicates a

diet with high consumption of antioxidant-rich fruits and vegetables significantly reduces

the risk of many cancers suggesting that certain dietary antioxidants could be effective

agents for the prevention of cancer incidence and mortality These agents present in the

diet are a very promising group of compounds on account of their safety low toxicity

and general acceptance Consequently the identification and development of such agents

has become in the last few years a major area of experimental cancer research Phenolic

compounds constitute one of the most numerous and ubiquitous group of plant

metabolites and are an integral part of the human diet This group of compounds was

found to display a wide variety of biological functions in addition to their primary

antioxidant activity which are mainly related to modulation of carcinogenesis Various in

vitro and in vivo systems have been employed to determine the anticarcinogenic and

anticancer potential of these natural phenolic compounds or extracts

30

331 In vitro effects of phenolics

Phenolic extracts or isolated polyphenols from different plant food have been studied

in a number of cancer cell lines representing different evolutionary stages of cancer For

example berry extracts prepared from blackberry raspberry blueberry cranberry

strawberry and the isolated polyphenols from strawberry including anthocyanins

kaempferol quercetin esters of coumaric acid and ellagic acid were shown to inhibit the

growth of human oral (KB CAL-27) breast (MCF-7) colon (HT-29 HCT-116) and

prostate (LNCaP DU-145) tumor cell lines in a dose-dependent manner with different

sensitivity between cell lines [73 160] Katsube et al compared the antiproliferative

activity of the ethanol extracts of 10 edible berries on HL-60 human leukemia cells and

HCT-116 cells and showed bilberry extract was the most effective one [161] Ross et al

showed that the antiproliferative activity of raspberry extract in human cervical cancer

(Hela) cells was predominantly associated with ellagitannins [162] By comparing the

phytochemical diversity of the berry extracts with their antiproliferative effectiveness

McDougall et al suggested that the key component that related to the inhibition of cancer

cell growth could be ellagitannins for the Rubus family (raspberry arctic bramble and

cloudberry) and strawberry whereas the antiproliferative activity of lingonberry was

caused predominantly by procyanidins [163] Similar results have also been reported in

several cell system with wine extracts and isolated polyphenols (resveratrol quercetin

catechin and epicatechin) [164 165] tea extract and major green tea polyphenols

(epicatechin epigallocatechin epicatechin-3-gallate and epigallocatechin-gallate) [4

166 167] although the effective concentrations depend on the system and the tested

substances Other phenolic extracts or compounds intensely studies are from olives

legumes citrus apples and also curcumin from spice turmeric For example soy

isoflavone genistein can inhibit the growth of various cancer cell lines including

leukemia lymphoma prostate breast lung and head and neck cancer cells [168] Citrus

flavonoids strongly inhibit the growth of HL-60 leukemia cells [169] McCann et al

utilized established cell models of genotoxicity (HT-29) invasion and metastatic

potential (HT-115) and colonic barrier function (CaCo-2) to examine the effect of apple

phenolic extract on key stages of colorectal carcinogenesis and found apple extract exert

beneficial influence on all three carcinogenesis stages [170] In addition growth

31

inhibitory effects of a number of polyphenols such as flavones (apigenin baicalein

luteolin and rutin) flavanones (hesperidin and naringin) and sesame lignans (sesaminol

sesamin and episesamin) which are not so extensively studied previously have been

examined in different cancer cell lines including colon [171] prostate [172 173]

leukemia [174] liver [175] stomach cervix pancreas and breast [176]

332 In vivo effects of phenolics

In addition to in vitro studies on cancer cell lines numerous in vivo experiments have

also been performed to verify the antitumor efficacy of plant food-derived phenolic

extracts or compounds with tumor incidence and multiplicity (eg number of tumors per

animal) as endpoints (reviewed in [177-180]) The animal models commonly employed

care either chemically genetically or ultraviolet light-induced tumor as well as

xenograft models including colon lung breast liver prostate stomach esophagus

small intestine pancreas mammary gland and skin tumors As an example Lala et al

investigated the chemoprotective activity of anthocyanin-rich extracts (AREs) from

bilberry chokeberry and grape in Fischer 344 male rats treated with a colon carcinogen

azoxymethane (AOM) [181] After 14 weeks rats on ARE diets had significantly fewer

colonic aberrant crypt foci (ACF) when compared with the control group Moreover rats

fed bilberry ARE had 70 fewer large ACF compared with rats fed the control diet

indicating significant chemoprevention Chokeberry-fed rats had a 59 reduction in large

ACF whereas the reduction was only 27 in rats fed grape ARE The authors concluded

that AREs from bilberry chokeberry and grape significantly inhibited ACF formation

induced by AOM

In another study by Ding et al [182] cyanidin-3-glucoside (C3G) the major

anthocyanin in blackberry was investigated for the potential ability to inhibit 712-

dimethylbenz[a]anthracene (DMBA)-12-O-tetradecanolyphorbol-13-acetate (TPA)-

induced skin papillomas in animal skin model Fourteen days following DMBA

initiation the dorsal skin of the mice was exposed to TPA in the presence or absence of

C3G twice per week to cause promotion The results showed that treatment of the

animals with C3G (35 microM topical application twiceweek) decreased the number of

tumors per mouse at all exposure times After 20 weeks of TPA promotion a greater than

32

53 inhibition of papillomagenesis by C3G was observed After 22 weeks there were

four tumors greater than 4ndash5 mm in diameter in the TPA-treated group whereas no large

tumors were found in the C3G plus TPA-treated group In addition they also tested the

effects of C3G on human lung carcinoma (A549) xenograft growth and metastasis in

athymic male nude mice The results showed that C3G reduced the size of A549 tumor

xenograft growth and significantly inhibited metastasis in nude mice The authors

concluded that C3G exhibits chemoprevention and chemotherapeutic activities by

inhibiting tumor promoter-induced carcinogenesis and tumor metastasis in vivo

The inhibition of tumorigenesis by tea preparations and its polyphenol constituents

such as epigallocatechin-gallate (EGCG) and theaflavin have also been demonstrated in

various animal models However caution must be taken when attributed the tumor

inhibitory effect of tea to tea polyphenols in some animal models For example caffeine

a nonphenol constituent of tea was found to contribute to the inhibitory effects of green

and black tea on UVB-induced complete carcinogenesis [183] as well as the inhibition

effects of black tea on lung tumorigenesis in F344 rats [184] to a significant extent

It is worth noting that the effectiveness of a phenolic extract in different organs is also

dependent on the amount of its active constituents that can reach the target tissue

Therefore the administration route and bioavailability factors of these extract

constituents should be carefully considered when comparing their inhibition efficacy in

different tumors

333 Human intervention studies using phenolics

Human intervention studies on potential health promoting or cancer preventive

activity of polyphenol-rich food or food preparations have been conducted in healthy

volunteers or individuals at high risk of developing cancer Most studies employed

biomarkers reflecting antioxidant status or oxidative stress as endpoints for example

plasma or serum antioxidant capacity plasma malondialdehyde concentration

glutathione status oxidative DNA damage in mononuclear blood cells (MNBCs) urinary

8-epi-prostaglandin F2α (8-Iso-PGF2) and 8-hydroxy-2rsquo-deoxyguanosine (8-OHdG)

concentration etc Improvement of antioxidant status andor protection against oxidative

stress was observed in short term intervention studies (1 dose) with various polyphenol-

33

rich food including fruit juices [185-189] red wines [190 191] chocolates [192-194] and

fruits such as strawberries [190] as well as food preparations such as lyophilized

blueberry powder [195] black current anthocyanin concentrate [196] grape seed

concentrate [197] dealcoholized [198] and lyophilized [190 199] red wines

In a 6-month chemopreventive pilot study conducted by researchers from the Ohio

State University patients with Barrettrsquos esophagus (BE) were treated with 32 or 45 g

(female and male respectively) of lyophilized black raspberries (LBRs) [200] BE is a

premalignant esophageal condition in which the normal stratified squamous epithelium

changes to a metaplastic columnar-lined epithelium and is underscored by the fact that it

increases the risk for the development of esophageal adenocarcinoma a rapidly

increasing and extremely deadly malignancy by 30- to 40-fold [201] Their results

suggested that daily consumption of LBRs reduced the urinary excretion of 8-Iso-PGF2

and 8-OHdG among patients with BE indicating reduced oxidative stress [200] The

same group of researchers also investigated the formulation for local delivery of LBRs to

human oral mucosal tissues [202] The results indicated that a novel gel formulation is

well-suited for absorption and penetration of anthocyanins into the target oral mucosal

tissue site as evidenced by detectable blood levels within 5 min after gel application and

the greater penetration of anthocyanins into tissue explants was observed in berry gels

with a final pH of 65 versus pH 35 [202]

A recent study evaluated the effects of anthocyaninpolyphenolic-rich fruit juice

consumption on antioxidant status in hemodialysis patients who is facing an elevated

risk of cancer arteriosclerosis and other diseases ascribed in part to increased oxidative

stress [203] In this pilot intervention study 21 hemodialysis patients consumed 200

mlday of red fruit juice (3-week run-in 4-week juice uptake 3-week wash-out) Weekly

blood sampling was done to monitor DNA damage (comet assay +-

formamidopyrimidine-DNA glycosylase enzyme) glutathione malondialdehyde protein

carbonyls Trolox equivalent antioxidant capacity triglycerides and DNA binding

capacity of the transcription factor nuclear factor-kappa B (NF-κB) Results show a

significant decrease of DNA oxidation damage (P lt 00001) protein and lipid

peroxidation (P lt 00001 and P lt 0001 respectively) and NF-κB binding activity (P lt

001) and an increase of glutathione level and status (both P lt 00001) during juice

34

uptake The authors attributed this reduction in oxidative (cell) damage in hemodialysis

patients to the especially high anthocyaninpolyphenol content of the juice The authors

concluded that consumption of antioxidant berry juices appears to be a promising

preventive measure to reduce chronic diseases such as cancer and cardiovascular disease

in population subgroups exposed to enhanced oxidative stress like hemodialysis patients

[203]

334 Mechanism of action of phenolics

Cancer development is a multistage process that involves a series of individual steps

including initiation promotion progression invasion and metastasis Tumor initiation

begins when DNA in a cell or population of cells is damaged by exposure to

carcinogens which are derived from three major sources cigarette smoking

infectioninflammation and nutritiondiet [204] If the DNA damage escapes repair it

can lead to genetic mutation The resulting somatic mutation in a damaged cell can be

reproduced during mitosis which given rise to a clone of mutated cells Tumor

promotion is a selective clonal expansion of the initiated cells to form an actively

proliferating multi-cellular premalignant tumor cell population It is an interruptible or

reversible and long term process During progression premalignant cells developed into

tumors through a process of clonal expansion In the late stages of cancer development

invasion and metastasis happens where tumor cells detach from the primary tumor mass

migrate through surrounding tissues toward blood vessels or lymphatic vessels and

create a second lesion Metastasis is the major cause of cancer mortality It is widely

accepted that human cancer development does not occur through these discrete phases in

a predictable manner rather it is best characterized as an accumulation of alteration in

cancer regulating genes [205] such as oncogenes tumor suppressor genes resulting in

altered cellular processes namely decreased apoptosis increased proliferation and cell

maturation and differentiation The inhibitory effect of natural phenolics in

carcinogenesis and tumor growth may be through two main mechanisms to modify the

redox status and to interfere with basic cellular functions (cell cycle apoptosis

inflammation angiogenesis invasion and metastasis) [2]

35

3341 Modification of the redox status of phenolics

ROSRNS are constantly produced during normal cellular metabolism or by other

exogenous means including the metabolism of environmental toxins or carcinogens by

ionizing radiation and by phagocytic cells involved in the inflammatory response When

the cellular concentration of oxidant species is highly increased the endogenous

antioxidant defense may be overcome In such cases oxidative stress occurs leading to

lipid protein and DNA damage In addition ROS particularly H2O2 are potent

regulators of cell replication and play an important role in signal transduction Hence

oxidative damage is considered a main factor contributing to carcinogenesis and

evolution of cancer Due to their ability to scavenge and reduce the production of free

radicals and act as transition metal chelators natural phenolic compounds can exert a

major chemopreventive activity Indeed it has been shown that natural polyphenols can

inhibit carcinogentoxin-induced cellular oxidative damage For example in nicotine-

treated rat peripheral blood lymphocytes ellagic acid effectively restored the antioxidant

status and reduced DNA damage as well as lipid peroxidation [206] A phenolic apple

juice extract as well as its reconstituted polyphenol mixture (rutin phloridzin

chlorogenic acid caffeic acid and epicatechin) were shown to effectively reduce

menadione-induced oxidative DNA damage and increasing of cellular ROS level [207]

Tea polyphenols [208] and other extensively studied polyphenols such as resveratrol

[164 209] quercetin [210-212] were also showed to exert protective effects against

cellular oxidative damage in different human cell lines

UV radiation-induced ROS and oxidative stress is capable of oxidizing lipids

proteins or DNA leading to the formation of oxidized products such as lipid

hydroperoxides protein carbonyls or 8-OHdG which have been implicated in the onset

of skin diseases including skin cancers [213-215] Phenolic extracts such as

pomegranate-derived extracts [216] tea [217] and wine [218] extracts have been shown

to reduce the oxidative damage of UV light in skin Purified phenolic compounds such as

anthocyanins [219] proanthocyanidin [220] and EGCG [221] were found to inhibit the

UV-radiation-induced oxidative stress and cell damage in human keratinocytes

In addition to their antioxidant activity polyphenols may exert their inhibitory effects

by acting as prooxidants on cancer cells at least in vitro It has been reported that many

36

polyphenols including flavonoids such as quercetin rutin apigenin phenolics acids such

as gallic acid tannic acid caffeic acid as well as delphinidin resveratrol curcumin

gallocatechin and EGCG can cause oxidative strand breakage in DNA in vitro [222 223]

Furthermore the cytotoxicity of quercetin and gallic acid on CaCo-2 cells and normal rat

liver epithelial cells was partially reduced by antioxidant such as catalase [224] Similar

results have also been reported in oral carcinoma cell lines with EGCG [225] These

studies suggested that the antiproliferative effects of some polyphenol antioxidants on

cancer cells are partially due to their prooxidant actions However it has been proposed

that this oxidative property depends on the amount of dissolved oxygen in the test

medium [226] The oxygen partial pressure in a cell culture system (160 mm Hg) is much

higher than that in the blood or tissues (lt40 mm Hg) It is not clear whether a similar

mechanism could also occur in vivo

3342 Interference of basic cellular functions by phenolics

Natural phenolics can affect basic cell functions that related cancer development by

many different mechanisms Firstly in the initiation stage phenolics may inhibit

activation of procarcinogens by inhibiting phase I metabolizing enzymes such as

cytochrome P450 [227] and also facilitate detoxifying and elimination of the carcinogens

by induction of phase II metabolizing enzymes such as glutathione S-transferase (GST)

NAD(P)H quinine oxidoreductase (NQO) and UDP-glucuronyltransferase (UGT) [228]

They may also limit the formation of the initiated cells by stimulating DNA repair [229

230]

Secondly phenolics may inhibit the formation and growth of tumors by induction of

cell cycle arrest and apoptosis Malignant cells are characterized by excessive

proliferation inability to terminally differentiate or perform apoptosis under normal

conditions and an extended or immortalized life span The regulation of cell cycle is

altered in these cells Thus any perturbation of cell cycle specific proteins by phenolics

can potentially affect andor block the continuous proliferation of these tumorigenic cells

Natural phenolics have been reported induce cell cycle arrest at different cell phases G1

S S-G2 and G2 by directly down-regulating cyclins and cyclins-dependent kinases

(CDKs) or indirectly inducing the expression of p21 p27 and p53 genes [3 231]

37

Moreover some studies have shown that natural phenolics exhibit differential effect in

cancer versus normal cells For example anthocyanin-rich extract from chokeberry was

found to induce cell cycle block at G1G0 and G2M phases in colon cancer HT-29 cells

but not in NCW460 normal colonic cells [232]

Apoptosis has been reported to play an important role in elimination of seriously

damaged cells or tumor cells by chemopreventive or chemotherapeutic agents [228 233]

The cells that have undergone apoptosis have typically shown chromatin condensation

and DNA fragmentation They are rapidly recognized by macrophages before cell lysis

and then can be removed without inducing inflammation Therefore apoptosis-inducing

agents are expected to be ideal anticancer drugs Polyphenols have been found to affect

cancer cell growth by inducing apoptosis in many cell lines such as the hepatoma

(HepG2) the colon (SW620 HT-29 CaCo-2 and HCT-116) the prostate (DU-145 and

LNCaP) the lung (A549) the breast (MCF-7) the melanoma (SK-MEL-28 and SK-

MEL-1) the neuroblastoma (SH-SY5Y) and the HL-60 leukemia cells [234 235] In

many cases apoptosis induced by polyphenols was caspase-3-dependent The induction

of apoptosis andor inhibition of proliferationsurvival by polyphenols has been reported

to result from a number of mechanisms including inducing cell cycle arrest blocking the

extracellular regulated kinase(ERK) c-Jun N-terminal kinase (JNK) and P38 mitogen-

activated protein kinase (MAPK) pathway inhibition of the activation of transcription

factors NF-κB and activator protein-1 (AP1) suppression of protein kinase C (PKC)

suppression of growth factor-mediated pathways [3 231] For example Afaq et al

showed that pomegranate fruit extract rich in anthocyanins and hydrolysable tannins

protected against the adverse effect of both UVB-radiation in normal human epidermal

keratinocytes in vitro [236] and 12-O-tetradecanoylphorbol-13-acetate (TPA) in CD-1

mouse skin in vivo [237] by inhibiting the activation of NF-κB and MAPK pathway In

addition green tea polyphenols was found to protect against pentachlorophenol (PCP)-

induced mouse hepatocarcinogenesis via its ability to prevent down-regulation of gap

junctional intercellular communication (GJIC) which is strongly related to cell

proliferation and differentiation [238] Pure phenolic compound such as quercetin [239]

resveratrol [240] were also found to block tumor promoter such as TPA-induced

inhibition of GJIC

38

One important aspect of carcinogenesis is recognized to be the involvement of

inflammation For instance prostaglandins are mediators of inflammation and chronic

inflammation predisposes to carcinogenesis The over-expression of inducible

cyclooxygenases (COX-2) the enzyme which catalyzes a critical step in the conversion

of arachidonic acid to prostaglandins and is induced by pro-inflammatory stimuli

including mitogens cytokines and bacterial lipopolysaccharide (LPS) is believed to be

associated with colon lung breast and prostate carcinogenesis Natural phenolics have

been reported to inhibit transcription factors closely linked to inflammation (eg NF-κB)

[241 242] pro-inflammatory cytokines release [241 243] and enzymes such as COX-2

[244 245] lipoxygenases (LOX) [246] inducible nitric oxide synthase (iNOS) [247] that

mediate inflammatory processes both in vitro and in vivo [248] In many cases

polyphenols exhibit anti-inflammatory properties through blocking MAPK-mediated

pathway Furthermore a few structure-activity studies have been conducted For

example Hou et al examined the inhibitory effects of five kinds of green tea

proanthocyanidins on cyclooxygenase-2 (COX-2) expression and PGE-2 release in LPS-

activated murine macrophage RAW-264 cells [244] It was revealed that the galloyl

moiety of proanthocyanidins appeared important to their inhibitory actions Another

study by Herath et al suggested that the double bond between carbon 2 and 3 and the

ketone group at position 4 of flavonoids are necessary for potent inhibitory effects on

LPS-induced tumor necrosis factor (TNF)-α production in mouse macrophages (J7741)

[249]

Finally natural phenolics such as green tea polyphenols (EGCG GCG) grape seeds

proanthocyanidins hydrolysable tannins genistein curcumin resveratrol and

anthocyanins were found to suppress malignant cell migration invasion and metastasis

in vitro and in vivo [250-255] The inhibition effect has been shown to be related to their

ability to down-regulate the matrix metalloproteases (MMPs) namely MMP-2 and

MMP-9 as well as urokinase-plasminogen activator (uPA) and uPA receptor (uPAR)

expression In addition to that phenolic compounds possess antiangiogenesis effect

[256] which is an important aspect in the inhibition of tumor growth invasion and

metastasis It has been reported that phenolic compounds such as ellagic acids EGCG

genistein and anthocyanin-rich berry extracts inhibit tumor angiogenesis through down-

39

regulation of vascular endothelial growth factor (VEGF) VEGF receptor-2 (VEGFR-2)

platelet-derived growth factor (PDGF) PDGF receptor (PDGFR) hypoxia-inducible

factor 1α (HIF-1α) and MMPs as well as inhibition of phosphorylation of EGFR

VEGFR and PDGFR [3]

In summary natural phenolics were found to intervene at all stages of cancer

development In addition to their antioxidant action the inhibition of cancer development

by phenolic compounds relies on a number of basic cellular mechanisms involving a

spectrum of cellular basic machinery Moreover the extensive studies of this class of

compounds will provide clues about their possible pharmaceutical exploration in the field

of oncology


40

Chapter 4

Preparation and Characterization of Anthocyanin-containing Extracts from

Blackberries

41 Summary

Blackberries are rich in polyphenols including anthocyanins Polyphenols are

hypothesized to have biological activities that may impact positively on human health In

these studies anthocyanin-containing blackberry extracts (ACEs) derived from either

puree (puree-derived) or freeze-dried puree (powder-derived) of Hull Black Satin and

Chester cultivars grown in Kentucky were obtained and characterized for total

anthocyanin and phenolic content polymeric color and total antioxidant capacity (TAC)

The influence of water content in the extraction system was evaluated A 90 day stability

study of the extract and a 48 h stability study of the extract in biologically relevant

buffers were completed Using the same extraction method it was found that the total

anthocyanin and phenolic content polymeric color and TAC were comparable between

cultivars of the same harvest year (2006) HPLC-MS results showed that the

anthocyanins in ACE derived from Hull blackberries were mainly cyanidin-based As

compared to powder-derived ACEs puree-derived ACEs contained similar amounts of

anthocyanins but greater levels of phenolics and increased TAC Extraction solvents

with water to ethanol ratios greater than 5050 greatly decreased the yield of total

anthocyanin and phenolics in the ACEs The stability studies indicated that multiple

factors such as temperature pH and time contributed to the loss of anthocyanins total

phenolics and antioxidant activity in ACE These studies provided essential information

for the development of nutritional and medicinal products from ACEs derived from

Kentucky-grown blackberries

41

42 Introduction

Fruits and vegetables are rich sources of naturally occurring antioxidants including

vitamins C and E β-carotene and phenolics Among the phenolic compounds

anthocyanins have drawn increasing attention since they possess potent antioxidant

activity A comparison of the antioxidative properties of anthocyanins to other widely

known antioxidants showed that the anthocyanins had higher antioxidative activity than

vitamin E ascorbic acid and β-carotene and were comparable to that of butylated

hydroxytoluene and butylated hydroxyanisole [137 257-259] Moreover several studies

have shown that the content and antioxidant activities of total anthocyanins and total

phenolics in various fruits are highly correlated [10 260-264] However studies using

extracts from different fruits and vegetables have suggested that there may be synergistic

or additive biological effects due to unique combinations of anthocyanins and phenolics

[8 265]

Anthocyanins are water-soluble glycosides of polyhydroxyl and polymethoxyl

derivatives of 2-phenylbenzopyrylium Anthocyanins exist at low pH as a flavylium

cation which is their naturally occurring form The flavylium cation is highly electron

deficient which leads to their potent activity toward free radicals and oxygen reactive

species The flavylium cation is intensely colored (red or orange at low pH) while the

ring-opened chalcone is uncolored at a pH of about 45 and above The pH-dependent

color difference of the anthocyanins is unique from polymerized anthocyanin-tannin or

other pigments and serves as the basis of several quantitation methods for monomeric

anthocyanins

Since antioxidants function as potential inhibitors of numerous degenerative diseases

including cancer inflammation and heart diseases various anthocyanin-containing

extracts or anthocyanin-rich fractions from fruits and vegetables have been investigated

for their therapeutic potential For example several groups have investigated the

antitumor effects of anthocyanins on human cancer cell lines [161 232 265-268] and in

animal models [269 270] Wang et al [259] reported that anthocyanin-rich extracts

inhibited the enzyme activity of cyclooxygenase-2 and acted as modulators of the

immune response in activated macrophages via inducing Tumor Necrosis Factor-α

production [271] The expression of cyclooxygenase-2 in lipopolysaccharide-stimulated

42

macrophages was also suppressed by anthocyanins at both the protein and RNA levels

[245] Studies in animal models also demonstrated the anti-inflammatory properties of

anthocyanin-containing extracts [272-275]



number of species Blackberries are among the earliest fruits used medically As early as

the 16th Century blackberry juice was used in Europe to treat infections of the mouth

and eyes Recent research has revealed that blackberries contain higher amounts of

anthocyanins and other antioxidants than other fruits [9-11] However studies to

elucidate the potential therapeutic properties of blackberries have been limited Since

blackberries are currently grown in large scale in Kentucky there was an interest in both

characterizing and developing anthocyanin-rich blackberry extract products for potential

nutritional and medical applications

It has been shown that the phenolic content andor profile as well as antioxidant

activity of blackberries varies between cultivars [276] Moreover different extraction

methods may introduce different combinations of anthocyanins and phenolics in the

extract resulting in different bioactivities The purpose of these present studies was to

prepare anthocyanin-containing extracts (ACEs) from three blackberry cultivars grown in

Kentucky (Hull Black Satin and Chester) Two different blackberry materials puree and

powder (freeze-dried puree) were used to obtain the ACEs The total phenolic and

anthocyanin content polymeric color and total antioxidant capacity (TAC) in the ACEs

were compared The major types of anthocyanins in ACEs were determined In addition

the effect of the water-to-ethanol ratio of the extraction system was investigated on the

properties of the obtained ACEs A final aim of the present studies was to investigate the

stability of these extracts stored alone as a function of time temperature and light and

when incubated in biologically relevant buffers at different temperatures

43

43 Materials and methods

Materials

Hull Chester and Black Satin blackberries were grown at WindStone Farms (Paris

KY) 22prime-Azinobis(3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS)

Trolox (6-hydroxy-2578-tetramethychroman-2-carboxylic acid) potassium persulfate

formic acid (ACS gt96) Folin-Ciocalteu phenol reagent gallic acid (98 purity) were

purchased from Sigma (St Louis MO) Hydrogen chloride ~125 moll in ethanol was

purchased from Fluka (St Gallen Switzerland) USP grade Ethanol (Absolute 200

proof) was purchased from AAPER Alcohol and Chemical Co (Shelbyville KY) HPLC

grade acetonitrile was purchased from Fisher Scientific (Fair Lawn NJ) Cyanidin 3-

glucoside (Kuromanin chloride HPLC grade) was purchased from Indofine Chemical

Company (Hillsborough NJ)

Preparation of anthocyanin-containing extracts (ACE)

The Hull blackberries were harvested at WindStone Farms in July 2005 and July

2006 and the Chester and Black Satin blackberries were harvested in July 2006 The

seeds and skin of the berries were removed using a Langsenkamp (Indianapolis IN) type

161 Colossal Pulper having two agitator arms with brushes with a stainless steel chamber

and a stainless steel catch pan with two outlets (one threaded and one with a sanitary

fitting) with a 10-horsepower three-phase 60-cycle 230460 voltmeter Whole

blackberries were passed through the Lagsenkamp pulper at a rate of 50ndash75 gallonsmin

to produce a homogeneous blackberry puree free of skin and seeds The blackberry puree

was stored frozen at -20degC until processed for these present studies

The frozen blackberry puree was lyophilized in a VirTis (Gardiner NY) model AD2

lyophilizer and ground into a free-flowing purple powder An aliquot of blackberry

powder (1 g) or blackberry puree (10 g) was treated under sonication for 30 min with 25

ml of extraction solvent of ethanol containing 001 HCl (vv) The supernatants were

collected after filtration and dried by rotary evaporation at 40 degC The dried extract was

resuspended in deionized water and filtered through a 20-25 microm filter paper and

lyophilized to obtain dried ACE Dried ACE was then redissolved in deionized water as a

44

stock ACE solution (140 mgml) and stored at -80 degC for further characterization and

cell-based studies

Black Satin puree was used to prepare various ACEs with different solvent

combinations of water and ethanol001 HCl to investigate the influence of the solvent

system on the properties of ACEs Briefly 10 g of puree was mixed with 25 ml of

extraction solvent The water to ethanol001 HCl (vv) ratios were as follows 0100

1090 2575 5050 7525 9010 and 1000 ACEs were then obtained following the

same extraction procedure as described above

Monomeric anthocyanins and polymeric color measurement

Monomeric anthocyanin content was determined using the pH-differential method of

Giusti and Wrolstad [277] Total anthocyanin content was calculated using an extinction

coefficient of 26900 l cm-1 mg-1 and a molecular weight of 4492 gmol of cyanidin 3-

glucoside (dominant anthocyanin) Color density and polymeric color were calculated

using absorption at 420 510 and 700 nm with and without bisulfite treatment The

percentage of polymeric color was determined by the ratio of polymerized color to color

density

Total phenolic measurement

Total phenolic content was estimated using the Folin-Ciocalteu method for total

phenolics [98] Briefly diluted samples were mixed with Folin-Ciocalteu reagent (01 N)

and then treated with saturated sodium carbonate to maintain the reaction pH at 10 The

absorbance was measured at 765 nm with a Beckman DU800 UV-Visible

Spectrophotometer after incubation for 2 h at room temperature Total phenolics were

calculated as gallic acid equivalent based on the standard curve with gallic acid standard

prepared in corresponding vehicles

Trolox equivalent antioxidant capacity (TEAC) assay

The TEAC assay for the extracts was carried out using a Beckman DU640B UV-Vis

Spectrophotometer in the kinetic mode following procedures described by Re et al [157]

ABTSbull+ was produced by reacting 7 mM ABTS with 25 mM potassium persulfate for 16

45

h in the dark at room temperature The ABTSbull+ solution was diluted with ethanol or water

to an absorbance of 070 (plusmn 002) at 734 nm and equilibrated at 30 ordmC Twenty (20) microl of

ACE samples or Trolox standards in ethanol were added to 980 microl of diluted ABTSbull+

solution such that each final sample produced between 20-80 inhibition of the blank

absorbance The absorbance readings were taken continuously for 7 min at 734 nm at 30

ordmC The standard curve was generated based on the percentage of inhibition of the blank

absorbance by Trolox at 7 min versus Trolox concentration The total antioxidant

capacity of samples was calculated as Trolox equivalent (TE) based on the percentage of

inhibition of the blank absorbance by samples at 7 min

HPLC-UV-mass spectrometry (MS) analysis of anthocyanins

The HPLC-UV-MS analysis was performed using a X-Bridgetrade C18 column (250

mm times 46 mm particle size 5 microm) (Waters Milford MA) equipped with an X-Bridgetrade

C18 guard column with a Waters Model 2690 separation module equipped with a model

996 photodiode array detector and coupled on-line with a Waters Micromass ZMD 4000

Mass Spectrometer The mobile phase consisted of 10 formic acid (A) and 100

acetonitrile (B) The elution conditions were as follows 0ndash45 min linear gradient from

1 to 16 B (vv) 46ndash50 min linear gradient from 16 to 100 B and 51ndash60 min

100 B post-time 5 min with 1 B flow rate 1 mlmin The UV-visible detection

wavelength was 524 nm and the injection volume was 50 microl of ACE stock prepared from

powder of Hull blackberries (2005) The MS instrument was operated at the following

settings ESP+ mode capillary voltage 30 kV cone voltage 35 V desolvation

temperature 300 degC source temperature 100 degC scan range 100ndash1000 mz

HPLC assay for anthocyanins

HPLC analysis was performed using a X-BridgeTM C18 column (250 mm times 46 mm

5 microm) (Waters Corp Milford MA) equipped with an X-BridgeTM C18 guard column and

a Thermoquest HPLC system with a UV6000LP photodiode array detector The mobile

phase was comprised of solvent A 10 formic acid (pH 137) and Solvent B 100

acetonitrile The elution profile was 100 to 91 A for 05 min 91 A (isocratic) for

05-4 min 91 A to 87 A (linear gradient) for 4-10 min 87 A to 70 A (linear

46

gradient) for 10-20 min 70 A to 0 A (linear gradient) for 20-21 min 0 A (isocratic)

for 21-24 min 0 A to 100 A (linear gradient) for 24-25 min and 100 A (isocratic)

for 25-30 min The flow rate was 10 mlmin and the detection wavelength was 524 nm

Each determination was run in triplicate Quantification of cyanidin 3-glucoside in ACEs

was carried out using the external standard method

90 day stability study of ACE under different storage conditions

A freshly prepared ACE solution derived from powder of Hull blackberries (2005)

was filled into polypropylene microcentrifuge tubes (15 ml) and kept in the following

controlled conditions -80 degC freezer 2-8 degC stability chamber 25 degC stability chamber

(60 humidity) On days 3 10 17 24 31 45 and 90 three samples at each storage

condition were withdrawn and analyzed for pH osmolality cyanidin 3-glucoside content

total anthocyanin content total phenolics and polymeric color TAC was measured at

day 24 45 and 90 The stability of ACE stored in a temperature-controlled room (22-28

degC 38 humidity) with white light (fluorescent light 1929 lx) was also investigated

using the same procedure over a period of 45 days

48 h stability study of ACE in biologically relevant buffers at 25 degC and 37 degC

ACE stock solution derived from powder of Hull blackberries (2005) was diluted to a

final concentration of 2 mgml with (1) pH 10 buffer (2146 mM NaCl 87 mM KCl)

(2) pH 74 phosphate buffered saline (PBS 303 mM Na2HPO4 87 mM KH2PO4 09

NaCl) (3) pH 74 PBS with 10 Fetal Bovine Serum (FBS) (ATCC Rockville MD) (4)

RPMI 1640 medium (Invitrogen Carlsbad CA) supplemented with 10 FBS in 50 ml

screw-capped polypropylene centrifugation tubes (25 ml each) ACE in buffer (1) (2)

and (3) were kept at 25 plusmn 05 degC Another sample of ACE in buffer (3) and (4) were kept

in 37 plusmn 05 degC At time 0 10 min 30 min 2 6 12 24 48 h after mixing with each buffer

an aliquot of sample was withdrawn and anthocyanin degradation in buffers (pH 74) was

stopped by adding 365 hydrochloric acid (1100 vv) To avoid protein interference in

the total phenolics measurement protein was removed by trichloroacetic acid

precipitation method before the Folin-Ciocalteu assay Briefly samples were mixed with

20 TCA and kept in 4 degC for 05 h Then samples were centrifuged at 13000 rpm for

47

15 min at 4 degC and the supernatant was collected for the analysis of total phenolic

content

Statistical analysis

All values of each assay were based on independent triplicate samples of ACEs and

calculated as the mean plusmn standard error (SE) Statistical analysis was performed using one

way ANOVA followed by Dunnettrsquos Multiple Comparison test (α le 005) with GraphPad

50 (GraphPad Software Inc San Diego CA)

48

44 Results

Extraction and characterization of ACEs

In these studies an ultrasound-assisted acidified ethanol extraction method was used

for the preparation of ACEs Total anthocyanin and phenolics content polymeric color

and total antioxidant activity of puree-derived or powder-derived ACE from all Hull

Chester and Black Satin cultivars are shown in Table 41 Using the same extraction

method it was found that the total anthocyanin and phenolics content polymeric color

and TAC were comparable between cultivars of the same harvest year (2006) As

compared to Hull (2006) puree-derived ACE from Hull (2005) had 27 less total

anthocyanin content and significantly increased polymeric color Interestingly using the

same extraction process ACEs derived from the puree contained about two-fold greater

amounts of phenolics than the extract derived from powder However the total

anthocyanin content in puree and powder-derived ACEs was about the same Moreover

the TAC values of puree ACEs increased by about two to three-fold as compared to those

of powder-derived ACEs A significant positive correlation was found between total

phenolics and TAC values (r2 = 09832) In addition the percentage of polymeric color in

puree ACEs was higher than that in powder ACEs indicating that more polymeric

browning products were extracted from blackberry puree than powder It must be noted

that since the puree contains around 90 (ww) water the actual extraction solvent was

not 100 ethanol001 HCl but 735 ethanol001 HCl Puree ACEs were of

interest since the time consuming and expensive lyophilization step was avoided in its

preparation

The HPLC profile of ACE from Hull blackberries showed six major peaks as

identified as 1ndash6 (Figure 41) Peak identification was carried out based on the molecular

weight and structural information obtained from their MS spectra in addition to their

retention times from HPLC-UV-visible spectra (Table 42) It is notable that other small

peaks as shown in the chromatogram in Figure 41 were not identifiable by MS and their

identities remain unknown Cyanidin-3-glucoside (peak 1) was the main component

(710) in HBE with the respective parent and daughter ion pairs (mz 449287) The

other three major peaks (peak 4 124 peak 5 35 peak 6 116) revealed the mz

values of 419287 535287 and 593287 which were identified as cyanidin-3-xyloside

49

cyanidin-3-malonylglucoside and cyanidin-3-dioxalylglucoside respectively in

accordance with data previously reported by Stintzing et al [278] Cyanidin-3-

arabinoside (peak 2) was also identified and was in agreement of the initial report by

Dugo et al [279] using blackberry extracts Another small peak (peak 3) with the

respective parent and daughter ion pairs (mz 435303) was detected in the blackberry

extract and is being reported for the first time Based on its retention time this compound

has been tentatively identified as delphinidin-3-xyloside

To investigate the effect of the extraction solvents with different water to ethanol

ratios on the properties of the extracts a series of ACEs were prepared from Black Satin

puree using a solvent system with 0 to 100 water content As shown in Figure 42 the

ACEs obtained using solvents with 0 to 50 water content contained about the same

amount of total anthocyanin and TAC An increasing trend of total phenolics and

percentage of polymeric color was observed but there was no significant difference in

this range On the other hand in ACEs prepared using solvent systems with water content

above 50 total anthocyanin total phenolics content and TAC decreased while the

percentage of polymeric color significantly increased as compared to those in ACE

prepared using solvent systems with water content below 50 A strong positive

correlation was found between TAC and total phenolics (r2 = 08057) as well as between

TAC and total anthocyanin content (r2 = 08746) Total anthocyanin content was also

found to be negatively correlated with the percentage of polymeric color (r2 = 09646)

Stability of ACE under various storage conditions

To determine the stability of anthocyanins in ACE under various storage conditions a

stability-indicating HPLC method for major anthocyanins in ACE was developed The

chromatograms resulting from the developed HPLC method showed a rapid and selective

separation of the five major blackberry anthocyanins 1-5 which account for 99 of total

anthocyanin in the ACE (Figure 43) The chromatogram fingerprint of anthocyanins was

found to be the same for the ACEs prepared from Hull Chester and Black Satin (data not

shown) In these studies powder-derived ACE from Hull blackberry was used to

investigate the influence of storage conditions on the stability of anthocyanins and

phenolics in ACEs Cyanidin 3-glucoside (peak 1) was the dominant anthocyanin (71

50

of the total anthocyanin) in ACEs The HPLC method was validated with external

standard cyanidin 3-glucoside and demonstrated linearity within the range of 47-142

mgl (r2 = 09984 and an RSD = 115) The intra and inter-day variations were 10 and

20 respectively The recovery percentage was in the range of 986-1004

The change of cyanidin 3-glucoside concentration in ACE at different storage

conditions over 90 days is shown in Figure 44 No significant change of cyanidin 3-

glucoside concentration was observed at ndash80 degC over 90 days However with increasing

storage temperature (4 degC and 25 degC) a decrease in cyanidin 3-glucoside was observed at

each time point The retention percentage of the other four major anthocyanins were also

calculated based on the percentage of peak area at each time point compared with the

corresponding peak area at day 0 in the HPLC chromatogram (data not shown) Their

degradation profiles showed the same trend as cyanidin 3-glucoside Moreover the

stability (most stable to least stable) followed as cyanidin 3-glucoside (peak 1) =

cyanidin-3-arabinoside (peak 2) gt cyanidin-3-xyloside (peak 3) = cyanidin-3-

dioxalylglucoside (peak 5) gt cyanidin-3-malonylglucoside (peak 4) The results were in

accordance with the previous report that the aliphatic acyl anthocyanins particularly

malonic acid are more labile under an acidic environment as compared to other

anthocyanins [280]

The pH of ACE samples increased slightly from day 0 to day 90 from 195 plusmn 001 to

204 plusmn 001 212 plusmn 000 223 plusmn 001 when stored at ndash80 degC 4 degC and 25 degC respectively

There was no significant change in the osmolality of the ACE samples observed in any of

the storage conditions over 90 days As shown in Table 43 with increasing storage

temperature total anthocyanin content total phenolics and TAC values decreased over

time whereas the percentage of polymeric color increased A similar trend was also

observed for all other time-points to day 90 (data not shown) Moreover it was found that

there was no significant difference for all the parameters measured between samples

stored in the 25 degC stability chamber and the temperature-controlled room (22-28 degC

38 humidity white light) over a period of 45 days (data not shown) which indicated

that white light may not have a significant effect on the degradation of anthocyanins and

phenolics in ACE It was also observed that frozen storage of ACE for up to 90 days

caused about 10 loss of total phenolics and TAC which was in accordance with the

51

previous report by Srivastava et al [281] Taken together temperature and time

contributed to the loss of anthocyanins total phenolics and antioxidant activity in ACE

while the effect of light was insignificant

Stability of ACE in biologically relevant buffers at 25 and 37 degC

The degradation profiles of ACE anthocyanins and phenolics in biological buffers at

25 degC and 37 degC are shown in Figure 45 The final pH of after adding ACE (2 mgml) to

all media and buffers was pH 72 except for the pH 10 buffer which was maintained at

pH 10 As expected elevated pH andor temperature accelerated anthocyanins

degradation (Figure 45 A) Total phenolics content was also decreased with increasing

temperature (Figure 45 B) As a control ACE was diluted in pH 10 buffer and it did not

show any significant loss of total anthocyanin and phenolics content at 25 degC over 48 h

There was no significant decrease of total phenolics content observed for up to 24 h in

samples with PBS and 10 FBS in PBS buffer at 25 degC The lowest amount of total

phenolics content retained after 48 h was in samples with RPMI 1640 medium at 37 degC

about 538 of that at time 0

Total anthocyanin content decreased much more rapidly than total phenolics content

in biological buffers The anthocyanin degradation curves were fitted using nonlinear

regression of first-order kinetics The estimated half-lives of samples with PBS 10 FBS

in PBS at 25 degC 10 FBS in PBS at 37 degC and RPMI 1640 medium at 37 degC were 327

126 50 and 62 h with r2 of 09757 09780 09907 and 09850 respectively

52

45 Discussion

In these studies we provide a comprehensive evaluation of ACEs from Kentucky-

grown blackberries including extraction methods and stability Solvent extraction of

phenolic compounds from fresh dried or freeze-dried fruit materials has been the most

common method in fruit sample preparation The solvents generally used are methanol

ethanol acetone water and their mixtures To obtain high yield of anthocyanins in the

extract solvents are usually mildly acidified to facilitate liberation and solubilization of

anthocyanins from the fruit tissue and to stabilize anthocyanins as well The relative

recovery efficiency between solvents varies with different plant materials For example

Metivier et al found that methanol was 20 more effective than ethanol and 73 more

effective than water in recovering anthocyanins from grape pomace [22] However

Giusti et al found that water was more effective in recovering anthocyanins from purple

corn waste than acidified water and ethanol [282] In addition to the solvent system other

factors such as temperature and time are important It has been reported that elevated

temperature improves extraction efficiency due to enhanced solubility and diffusion rate

of compounds into the solvent However high temperature accelerates anthocyanin

degradation in the extraction process A few extraction technologies such as pressurized

liquid extraction [46] have been developed to enable rapid extraction of anthocyanins and

other phenolics at high temperature (gt 50 degC) and were found to be successful in

retarding anthocyanin degradation during processing It was also found that ultrasound-

assisted solvent extraction was more efficient to extract anthocyanins than conventional

solvent extraction due to the strong disruption of the fruit tissue under ultrasonic acoustic

cavitation [283]

In these studies we used an ultrasound-assisted ethanol extraction method to prepare

anthocyanin-containing extract from blackberries The starting materials used for

extraction were either blackberry puree or freeze-dried puree which was ground into

powder Because the puree contains around 90 ww water 25 ml of solvent was used to

extract 10 gram of puree (corresponding ~1 gram of powder) to keep the solid-to-liquid

ratio at 125 It was found that the ACEs from Hull Black Satin and Chester contained

similar amount of total anthocyanin and total phenolics However ACEs from puree

contained 2-3 fold greater total phenolics than those from powder while the anthocyanins

53

content was about the same This suggested that the water content inside the puree

contributed to the difference of total phenolics in ACEs extracted from blackberry puree

and powder To further evaluate the influence of the water content in the extraction

system on the property of the extracts various ACEs were prepared from puree of Black

Satin cultivar with solvent systems comprising increased water content in ethanol001

HCl It was found that total anthocyanin and total phenolics contents were similar in the

ACEs obtained using the solvent systems ranging from 0 to 50 water indicating that

this range of water to ethanol was preferable A sharp increase of the percentage of

polymeric color in ACE prepared with 75 water was observed which suggested that

water content greater than 50 led to increased anthocyanin degradation and the

degradation andor condensation products contributed significantly to the percentage of

polymeric color These data may suggest that in the extraction system using high water

content some of the polyphenol oxidase activity was retained and contributed to the

condensation of anthocyanin and other phenolics during extraction

It is well known that anthocyanins and other phenolics are prone to degradation [281

284 285] Understanding the mechanism and extent of degradation are critical in the

development of Botanical Drug Products Anthocyanins are very stable under acidic

conditions however under normal processing and storage conditions readily convert to

colorless derivatives and subsequently to insoluble brown pigments (eg increased

polymeric color) The mechanisms of anthocyanins degradation involve hydrolysis

oxidation and condensation with other polyphenols For example the degradation of

cyanidin 3-glucoside in aqueous solution begins with hydrolysis of glucosidic bonds and

the opening of the pyrylium ring upon heating [286] or by anthocyanase [287] They are

further degraded and produce protocatechuic acid and phloroglucinaldehyde Cyanidin 3-

glucoside can also be co-oxidized with other phenolics such as chlorogenic acid by

polyphenol oxidase with the formation of o-quinones that generate polymerized products

by quinone-phenol reactions [288] The most important factors that influence

anthocyanin degradation are pH and heat [286] Thus the stability of anthocyanins and

total phenolics was examined in extract stock solution as well as in biologically relevant

buffers at various storage temperatures It was found that anthocyanin degradation in

aqueous solution followed first-order kinetics Anthocyanins concentrated in extract stock

54

solution exhibited relatively high stability over time The half-life of cyanidin 3-glucoside

in the extract stock solution having a pH of 2 at 25 degC was 638 days (r2 = 09949)

To access the stability of ACE in a biologically relevant environment the ACE was

diluted with biologically relevant buffers to the EC50 concentration obtained in the

cytotoxicity studies It was found that when ACE was diluted with buffers at

physiological pH the anthocyanins degradation rate increased substantially (Figure

45A) Further our results showed that FBS contributed significantly to the acceleration

of anthocyanins degradation The half-life of total anthocyanin in samples with 10 FBS

in PBS was 26-fold shorter than that with PBS at 25 degC although the mechanism behind

it remains unclear When the temperature was increased from 25 degC to 37 degC the half life

of total anthocyanin in samples with 10 FBS in PBS decreased by 25-fold In this

experiment precipitation was not observed under any condition and the percentage of

polymeric color remained the same after 48 h (data not shown)

In conclusion these studies demonstrated that ACEs from blackberry cultivars of

Hull Black Satin and Chester grown in Kentucky possess potent antioxidant properties

Using the same extraction method total anthocyanins and phenolics contents as well as

total antioxidant capacity by TEAC method were comparable in ACEs derived from these

cultivars In general puree-derived ACEs contained similar amount of total anthocyanins

but higher amount of total phenolics with higher total antioxidant capacity Extraction

solvent with different water to ethanol ratio greatly influenced the yield of total

anthocyanins and phenolics in ACEs Finally the stability studies of ACEs provide

essential information for the development of nutritional and medicinal products from

ACEs derived from Kentucky-grown blackberries

The contents of this chapter were published in Food and Chemical Toxicology Dai J

Gupte A Gates L Mumper RJ A comprehensive study on anthocyanin-containing

extracts from selected blackberry cultivars extraction methods stability anticancer

properties and mechanisms 2009 47 837-47 and in Journal of Medicinal Food Dai J

Patel J Mumper RJ Characterization of blackberry extract and its anti-proliferative and

anti-inflammatory properties 2007 10 (2) 258-65

55

Table 4 1 Composition and Characterization of Blackberry Extracts a

Raw Material Cultivar (year)

Level (mgg of DBE) Polymeric color

() TAC

(μmol TEg DBE) Total anthocyanin b Total Phenolics c

Puree

Hull (2005) 534 plusmn 019 2407 plusmn 270 1299 plusmn 250 17143 plusmn 1567


Chester (2006) 795 plusmn 015 2531 plusmn 180 489 plusmn 033 18675 plusmn 1049

Black Satin (2006) 716 plusmn 022 2291 plusmn 124 327 plusmn 015 15852 plusmn 690

Powder



Black Satin (2006) 767 plusmn 051 1481 plusmn 159 111 plusmn 075 7168 plusmn 918 a The extraction was repeated at least two times All assays were carried out in triplicate

Data are expressed as the mean plusmn SE (N = 2 or 3) b Total anthocyanin are expressed as cyanidin 3-glucoside equivalent c Total phenolics are expressed as gallic acid equivalent

56


chromatography-electrosprayMS (peak 1) cyanidin-3-glucoside (peak 2) cyanidin-3-

arabinoside (peak 3) delphinidin-3-xyloside (tentative) (peak 4) cyanidin-3-xyloside

(peak 5) cyanidin-3-malonylglucoside and (peak 6) cyanidin-3-dioxalylglucoside

57


Anthocyanins Detected by HPLC and Electrospray Ionization-MS

Peak Retention time (minutes) [M+] Aglycon Sugar moiety Peak identification

1 201 449287 Cyanidin Hexose Cyanidin-3-glucoside

2 215 419287 Cyanidin Pentose Cyanidin-3-arabinoside

3 264 435303 Delphinidin Pentose Delphinidin-3-xyloside

4 287 419287 Cyanidin Pentose Cyanidin-3-xyloside

5 313 535287 Cyanidin Malonyl-hexose Cyanidin-3-malonyl-glucoside

6 349 593287 Cyanidin Dioxalyl-hexose Cyanidin-3-dioxalyl-glucoside

58

Figure 4 2 The water content in the extraction solvent on the properties of ACEs

10 g of Black Satin puree was mixed with 25 ml of extraction solvent The water to

ethanol001 HCl ratios (vv) were as follows 0100 1090 2575 5050 7525 9010

and 1000 Various ACEs were obtained and analyzed for total anthocyanin () total

phenolics () percentage of polymeric color () and total antioxidant capacity ()

Results were normalized to the corresponding values of each parameter of ACE prepared

using 100 ethanol001 HCl Data are presented as the mean plusmn SE of three

independent extractions

59

Figure 4 3 HPLC chromatogram of anthocyanins in ACEs cyanidin 3-glucoside

(peak 1) cyanidin-3-arabinoside (peak 2) cyanidin-3-xyloside (peak 3) cyanidin-3-

malonylglucoside (peak 4) and cyanidin-3-dioxalylglucoside (peak 5)

60

Figure 4 4 Changes in cyanidin 3-glucoside concentration in powder-derived ACE

from Hull blackberry under different storage conditions over 90 days ndash80 degC () 2-

8 degC (∆) temperature-controlled room (22-28 degC) with white light () 25 degC (loz)

61

Table 4 3 Effect of Storage Conditions on Several Parameters of the Hull

Blackberry Extract after 90 Days a

Parameters Day 0 Day 90

-80 degC 4 degC 25 degC Level ( of Retention)

Cyanidin 3-glucoside (mgliter) 600 plusmn 43 (100)

589 plusmn 82 (983)

539 plusmn 38 (898)

230 plusmn 09 (384)

Total Phenolics (mgliter) b 1899 plusmn 181 (100)

1680 plusmn 69 (885)

1696 plusmn 68 (893)

1537 plusmn 162 (801)

Total anthocyanin (mgliter) c 710 plusmn 223 (100)

684 plusmn 158 (963)

621 plusmn 83 (875)

261 plusmn 47 (368)

Polymeric Color () 012 plusmn 012 NA

143 plusmn 011 NA

261 plusmn 088 NA

1396 plusmn 111 NA

TAC (micromolliter) d 89 plusmn 013 (100)

80 plusmn 027 (892)

78 plusmn 011 (864)

73 plusmn 009 (812)

a Data are expressed as mean plusmn SE (n = 3) b Total anthocyanin are expressed as mg of cyanidin 3-glucoside equivalentl of extract c Total phenolics are expressed as mg of gallic acid equivalentliter of extract d TAC are expressed as micromol of trolox equivalentliter of extract

of Retention was calculated based on the day 0 concentration as 100

NA Not applicable

62

Figure 4 5 The effects of biologically relevant buffers on the stability of

anthocyanins and phenolics in powder-derived ACE from Hull blackberry at 25 degC

and 37 degC ACE stock solutions were diluted to a final concentration of 2 mgml in (1)

pH 10 buffer at 25 degC () (2) pH 74 PBS at 25 degC () (3) pH 74 PBS with 10 FBS

at 25 degC () (4) pH 74 PBS with 10 FBS at 37 degC () (4) RPMI 1640 medium

supplemented with 10 FBS at 37 degC () and incubated for 48 h At each time point an

aliquot was withdrawn and measured for total anthocyanin content (A) and total

phenolics content (B) Data are presented as the mean plusmn SE of three independent

experiments


63

Chapter 5

In vitro Anticancer and Anti-inflammatory Effects of Blackberry Extracts and

Possible Mechanisms of Action

51 Summary

In the current studies the in vitro anticancer and anti-inflammatory properties of

blackberry anthocyanin-containing extracts (ACEs) were investigated The in vitro

studies showed that treatment with blackberry ACEs prepared from Black Satin cultivar

in concentrations ranging from 080 to 112 mgml resulted in a significant dose-

dependent reduction in cell viability versus control with human cancer cell lines

including colon (HT-29) breast (MCF-7) and leukemia (HL-60) The anticancer effects

were 1) more pronounced in leukemia cells and 2) greater with puree-derived than

powder-derived extracts in all cell lines tested Cyanidin-3-glucoside exerted anticancer

effect by acting synergistically or additively with other active components in the extracts

The anticancer mechanism studies showed that the hydrogen peroxide generated in

medium by the extracts contributed little with puree-derived extract partially with

powder-derived extract to their anticancer effects in HL-60 cells The blackberry ACEs

derived from Hull Chester and Black Satin were also evaluated for their anti-

inflammatory properties on LPS-treated J774A1 murine macrophages It was found

using concentrations ranging from 015 to 155 mgml that post-treatment with

blackberry ACEs significantly inhibited tumor necrosis factor-alpha (TNF-α) and

interleukin-6 (IL-6) release Likewise ACE derived from Hull blackberry powder

suppressed both high-dose (10 microgml) and low-dose (01 microgml) Lipid A-induced

interleukin-12 (IL-12) release from mouse bone marrow-derived dendritic cells These

data warrant further investigation into the anticancer and anti-inflammatory effects of

blackberries both in vitro and in vivo

64

52 Introduction

In addition to the protective effects of endogenous antioxidant defenses the

consumption of dietary antioxidants appears to be of great importance in counteracting

oxidative stress-associated chronic diseases Dietary phenolics have captured increasing

attention in recent years because of their known antioxidant properties [289] It has been

stated that dietary phenolics are multifunctional antioxidants either acting as reducing

agents hydrogen-donating antioxidants andor single oxygen (free-radical) quenchers

[138] Among the many phenolic compounds anthocyanins have been given a great

amount of attention since they possess potent antioxidant activity [137 257] Various

anthocyanin-containing extracts from plants and fruits have been shown to reduce the

oxidative stress-associated inflammatory diseases and cancer (reviewed in [4 290]) and

there may be synergistic or additive biological effects due to unique combination of

anthocyanins and phenolics in the extracts prepared from different fruit samples or by

different extraction methods [8 265]

It has been shown that blackberries contain higher amount of anthocyanins and other

phenolic antioxidants than other fruits [10 11] Halvorsen et al examined antioxidant

properties of 1113 food samples and blackberries was ranked number one in berry fruits

and number 19 in all food samples tested in antioxidant contents [9] Furthermore

Anthocyanins or anthocyanin-containing extracts from blackberries have showed various

bioactivities For example Serraino et al reported that blackberry juice containing

cyanindin-3-glucoside was able to scavenge peroxynitrite and protected against

endothelial dysfunction and vascular failure in vitro [291] Anthocyanin-containing

blackberry extracts could also attenuate the injury caused by LPS-induced endotoxic

shock in rats [292] and had cytotoxic effects on human oral (KB CAL-27) prostate

(LNCaP) [160] lung (A549) [293] cancer cells

Although the mechanisms behind the bioactivities of anthocyanins and phenolics

involve many pathways (reviewed in [4 294]) the most remarkable aspect of their

activities may be their ability to act as either antioxidants or prooxidants in some

biological environments The anti-inflammatory and chemopreventive properties of

dietary phenolics are generally believed to be related to their antioxidant properties For

example Elisia et al showed that a purified cyanidin 3-glucoside extract from blackberry

65

protected Caco-2 cells from peroxyl radical-induced apoptosis [295] On the other hand

dietary phenolics may be subject to redox cycling generating reactive oxygen species

(ROS) and free radicals under defined conditions [296] Importantly the prooxidant

action of phenolics may be an important mechanism for their anticancer and apoptosis-

inducing properties [297] For example cyanidin-3-rutinoside was shown to induce the

accumulation of peroxides which were involved in the induction of apoptosis in HL-60

cells [298] Dietary phenolics were also shown to cause oxidative strand breakage in

DNA in the presence of transition metal ions such as copper [299] generate substantial

amounts of H2O2 in the commonly used media [224] and subsequently exerted

antiproliferative effects on cancer cells

We have previously prepared anthocyanin-containing extracts from blackberries of

Hull Chester and Black Satin cultivars grown in Kentucky The purpose of current

studies was to investigate their anticancer and anti-inflammatory properties Our previous

studies showed that total anthocyanins and phenolics contents as well as total antioxidant

capacity by TEAC method were comparable in ACEs derived from different cultivars

However puree-derived ACEs contained similar amount of total anthocyanins but higher

amount of total phenolics with higher total antioxidant capacity Therefore in the current

studies the growth effects of the powder and puree-derived ACEs from selected cultivar

Black Satin on a panel of human cell lines including colon (HT-29) breast (MCF-7) and

leukemia (HL-60) were evaluated The antiproliferative effects of ACEs were also

compared directly to that of the dominant anthocyanin in the extracts cyanidin 3-

glucoside Possible mechanisms of cytotoxicity were evaluated including H2O2 ROS

generation and the role of copper Moreover we examined and compared the immune-

modulation activity of puree and powder-derived ACEs from different cultivars on

lipopolysaccharides (LPS)-induced secretion of Tumor Necrosis Factor-α (TNF-α) and

Interleukin-6 (IL-6) from murine macrophages J774A1 In addition the effects of

selected ACE (derived from Hull blackberry powder) on Lipid A-induced Interleukin-12

(IL-12) release from mouse bone marrow-derived dendritic cells were also investigated

66


Materials

5-(and-6)-carboxy-2acute7acute-dichlorodihydrofluorescein diacetate (CH2DCFDA) was

purchased from Invitrogen Inc (Carlsbad CA) Cyanidin 3-glucoside (Kuromanin

chloride HPLC grade) was purchased from Indofine Chemical Company (Hillsborough

NJ) Catalase 3-(45-dimethyl-thiazol-2-yl)-25-diphenyl tetrazolium bromide (MTT)

Hydrogen peroxide solution (30) dimethyl sulfoxide (DMSO) trichloroacetic acid

were purchased from Sigma (St Louis MO)

Cell culture

The human colorectal cancer cell line HT-29 human breast cancer cell lines MCF-7

(her2 negative and ER+) human leukemia cell line HL-60 and murine macrophage

J774A1 were purchased from American Type Cell Culture Collection (ATCC

Rockville MD) HT-29 cells were grown in modified McCoyrsquos 5A medium (ATCC

Rockville MD) All other cells were grown in RPMI-1640 medium (Invitrogen

Carlsbad CA) All media were supplemented with 100 Uml penicillin 100 microgml

streptomycin and 10 FBS Cells were maintained at 37 ordmC in a humidified 5 CO2

incubator

Bone marrow cells were obtained by flushing the femurs of BALBc mice (Harlan

Sprague-Dawley Laboratories Indianapolis IN) with 1times Hanksrsquo balanced salt solution

Cells were cultured in 100-mm bacteriological Petri dishes at 2 times 105 cellsml in 10 ml of

complete RPMI 1640 medium (supplemented with 10 heat-inactivated fetal calf serum

1 mmoll HEPES 2 micromoll L-glutamine 10 Uml penicillin 100 Uml streptomycin and

50 micromoll 2-mercaptoethanol) containing 20ndash25 ngml granulocyte-macrophage colony-

stimulating factor (GM-CSF) at 37degC in 7 CO2 The cells were supplemented with an

additional 10 ml of complete RPMI 1640 with 20ndash25 ngml GM-CSF on day 3 On day 6

10 ml of supernatant was removed from each plate and spun down The cells were

resuspended in fresh 10 ml of complete RPMI 1640 with 20ndash25 ngml GM-CSF and

added back to the Petri dishes Non-adherent to lightly adherent cells were harvested on

day 7 as dendritic cells (DCs) and used for the in vitro studies

67

Cell viability assay

The adherent HT-29 MCF-7 and the suspension HL-60 cells were seeded at an initial

concentration of 3times104 cellswell in 96-well plates After 24 h cells were treated with the

ACE stock solution (140 mgml) with final doses ranging from 0084 to 112 mgml

Vehicle controls were the normal media with the corresponding pH adjusted with 25 N

HCl for each treated group HL-60 cells were also treated with cyanidin 3-glucoside (in

01 DMSO) with final doses ranging from 104 to 622 microgml After 48 h of treatment

medium was removed and cell viability was measured for all cells using the MTT assay

Briefly cells were incubated with MTT (05 mgml) in fresh medium in the dark at 37 degC

for 4 h Next supernatant was removed and 200 microl of DMSO was added to each well

Plates were read at 570 nm using Biotek Synergy 2 Microplate Reader Cell viability was

calculated using Equation 1

cell viability = ABSt ABSCtrl times 100 (Equation 1)

where ABSt is the absorbance of cells treated with ACEs and ABSCtrl is the absorbance of

corresponding vehicle control

Determination of intracellular ROS in HL-60 cells


used as an indicator of intracellular ROS generation HL-60 cells (3times104 cellswell) were

loaded with 100 microM CH2DCFDA (dissolved in DMSO) for 30 min at 37 degC The cells

were washed twice with fresh PBS to remove excess CH2DCFDA Non-CH2DCFDA-

loaded cells were used as negative controls The fluorescence of control and sample wells

was recorded at excitation 485 plusmn 20 nm emission 528 plusmn 20 nm with the microplate reader

every 10 min for 15 h Data are reported as percentage increase of DCF fluorescence

associated with the CH2DCFDA-loaded cells compared to that of corresponding non-

CH2DCFDA-loaded cells (negative controls)

H2O2 generation in medium

The ability of sample to produce H2O2 was assessed in both cell culture medium and

cell culture medium with HL-60 cells The samples analyzed were cyanidin 3-glucoside

puree and powder-derived ACEs from Black Satin at various concentrations The effect

68

of catalase (100 Uml) addition as well as 10 FBS in the medium was also determined

at time points up to 48 h At each time point an aliquot was removed and H2O2

concentration was measured using a Bioxytech H2O2-560 Kit (Oxis International Foster

City CA) according to the instructions from the manufacturer Briefly 1 volume of

sample was mixed with 10 volumes of ferrous ion-xylenol orange working reagent and

the absorbance was measured at 1 h at 560 nm using the microplate reader Appropriate

controls were used to subtract the possible interference of the corresponding vehicles

H2O2 concentrations were calculated based on the standard curve of authentic H2O2 at

concentrations ranging from 0 to 50 microM

Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α) stimulation studies

J774A1 cells were plated in 400 microl complete RPMI 1640 medium at a density of 4 times

105 cells per well in 48-well tissue culture plates (Costar Corning NY) at 37degC in 5

CO2 overnight J774A1 cells were treated with 1 microgml LPS for 30 min followed by

ACEs addition at final concentrations ranging from 031 to 155 mgml medium

Supernatants were collected after 55 h and stored at -80 degC until IL-6 and TNF-α

measurement The IL-6 and TNF-α concentration in supernatant was measured using an

OptEIA Set mouse IL-6 or TNF-α (monomono) ELISA kit from BD Biosciences (San

Diego CA) according to the instructions from the manufacturer Briefly 96-well plates

were coated with 100 microl of either IL-6 capture antibody (1250 dilution of stock solution

in 01 M sodium carbonate pH 95) or TNF-α capture antibody (1250 dilution of stock

solution in 01 M sodium phosphate pH 65) overnight at 4 degC The plates were blocked

for 1 h at room temperature (RT) with 200 microl assay diluents (10 FBS in 001 M

phosphate buffered saline pH 74) After washing with wash buffer (PBS with 005

Tween 20) the plates were incubated with 100 microl sample dilutions or standard

recombinant (IL-6 or TNF-α in a range of 156-500 pgml) for 2 h at RT The plates were

washed with wash buffer and incubated with 100 microl working detector (biotinylated anti-

mouse IL-6 or TNF-α detection antibody with Enzyme concentrate streptavidin-HRP) for

1 h at RT After washing with wash buffer the plates were developed by adding 100 microl of

tetramethylbenzidine (TMB) substrate and incubated for 30 min at RT The color

development was stopped by the addition of 50 microl of 2 M H2SO4 and the absorbance at

69

450 nm was measured using a Universal Microplate Reader (Bio-Tek Instruments Inc)

IL-6 and TNF-α concentration in each sample were calculated based on the IL-6 and

TNF-α standard curve generated on each plate

Interleukin-12 (IL-12) release assay

Day 7 harvested bone marrow-derived DCs (BMDDCs) were plated in 200 microl of

complete RPMI 1640 containing 20ndash25 ngml GM-CSF at 4 times 105 cells per well in 48-

well tissue culture plates (Costar Corning NY) at 37 degC in 7 CO2 overnight The

medium was removed and replaced with fresh complete RPMI 1640 Powder-derived

ACE from Hull blackberries (2005) or vehicle (water) was added to the cells and

incubated for 30 min Then high-dose (10 microgml) or low-dose (01 microgml) Lipid A from

Salmonella minnesota R595 (Re) (List Biological Laboratories Campbell CA) was then

added to each well with or without ACE treatment After 24 h supernatant in each well

was collected and stored at -80 degC until IL-12 measurement Total IL-12 concentration in

supernatant was measured using a murine total IL-12 enzyme-linked immunosorbent

assay kit (Pierce Rockford IL) according to the instructions from the manufacturer


Data were given as the mean plusmn standard error (SE) Statistical analysis was performed

using one way ANOVA followed by Dunnettrsquos or Bonferronirsquos Multiple Comparison test

(α le 005) with GraphPad 50 (GraphPad Software Inc San Diego CA)

70

54 Results

Cytotoxic effects of ACEs and cyanidin 3-glucoside on human cancer cell lines

Powder and puree-derived ACEs from Black Satin cultivar were evaluated for their

cytotoxic properties on human colon cancer (HT-29 Figure 51A) ovarian cancer (MCF-

7 Figure 51B) and leukemia (HL-60 Figure 51C) cell lines It was found that leukemia

cells were most sensitive to ACEs among the cell lines tested On the other hand the

cytotoxic effect of puree-derived ACEs was more profound than that of powder-derived

ACEs in all three cell lines tested The cytotoxic effect of cyanidin 3-glucoside on HL-60

cells is shown in Figure 51D The cell viability of HL-60 cells was decreased from

995 to 459 in a dose-dependent manner after 48 h of exposure with cyanidin 3-

glucoside in a concentration range of 104-622 microgml

H2O2 generation by ACEs and cyanidin 3-glucoside in RPMI 1640 medium

H2O2 production by ACEs at 14 mgml with or without catalase in RPMI 1640

medium with 10 FBS over time is illustrated in Figure 52 Detailed results with respect

to cyanidin 3-glucoside ACEs at various concentrations in medium with or without 10

FBS as well as in the presence of HL-60 cells are presented in Table 51 When authentic

H2O2 was incubated with RPMI 1640 medium its concentration decreased over time

Hence the H2O2 level in the RPMI 1640 medium was determined by the rate of its

generation and decomposition Puree and powder-derived ACE as well as cyanidin 3-

glucoside produced H2O2 in a dose-dependent manner in the RPMI 1640 medium and

the puree-derived ACE generated more H2O2 than powder-derived ACE at the same

concentration Catalase (100 Uml) depleted the H2O2 in the medium at all concentrations

tested for authentic H2O2 as well as puree and powder-derived ACEs

Our results also showed that the rate of H2O2 generation by puree-derived extract was

inversely correlated to the extract concentration For example the level of H2O2 peaked

at 2 h with 07 mgml of puree-derived ACE but not until 48 h with 14 mgml of puree-

derived ACE in RPMI1640 medium with 10 FBS Since the pH of the medium

decreased from pH 72 to 65 with increasing concentration of ACE from 014 to 7

mgml it is possible that the active components in ACE that produce H2O2 was stabilized

at relatively lower pH and the rate of H2O2 generation was reduced On the other hand

71

powder-derived extract produced H2O2 at a lower rate and extent than the powder-derived

extract and it did not reach a plateau until 48 h for all concentrations tested Further

when puree-derived ACEs incubated with RPMI 1640 medium without FBS the H2O2

level was 195 and 246-fold higher than those when incubated with RPMI 1640 medium

with 10 FBS at 6 h and 48 h respectively Similar results were also found with powder-

derived ACE as well as cyanidin 3-glucoside These results indicated that FBS

significantly inhibited the generation of H2O2 by ACEs A similar phenomenon was

observed by Lapidot et al using apple extracts in medium containing fetal calf serum

and it was suggested that the H2O2 might be decomposed by the residual enzymatic

activity in the serum [300]

We also measured the H2O2 level in the medium in the presence of HL-60 cells It

was found that when authentic H2O2 (100 microM) was added to the medium with cells H2O2

was not detected in the medium at 2 h In contrast 90 of the added H2O2 was detected

when it was incubated with medium without cells These data suggested that the rate of

H2O2 diffusion into cells was much greater than its rate of decomposition in the medium

With extract derived from puree at 14 mgml the concentration of H2O2 at 2 h in the

medium was 63-fold lower in the presence of cells as compared to the absence of cells

suggesting the rapid cell entry of either the generated H2O2 or the component in the

extract responsible for the generated H2O2

In comparison to the puree derived extract a relatively small concentration of H2O2

(lt 10 microM) was detected after the addition of cyanidin 3-glucoside with HL-60 cells over

a period of 2 to 6 h

Effect of ACEs and cyanidin 3-glucoside on intracellular ROS level in HL-60 cells

Intracellular ROS level in HL-60 cells after treatment of cyanidin 3-glucoside and

ACEs for 1 h is shown on Figure 53 It was found the intracellular ROS level peaked at 1

h after treatment of puree-derived ACE but remained the same for either powder-derived

ACE or cyanidin 3-glucoside over 15 h (data not shown) As shown in Figure 53 the

baseline intracellular ROS level in HL-60 cells (Veh) was increased by 718 plusmn 27

(mean plusmn SE) as compared to non-CDCFDA loaded cells Authentic H2O2 (98 microM)

significantly increased the fluorescence intensity by about 11044 As expected

72

catalase (500 Uml) decreased the increase of fluorescence intensity caused by H2O2 (98

microM) to the baseline level Puree-derived ACE increased the intracellular ROS level dose-

dependently from 693 plusmn 162 to 3222 plusmn 231 in concentrations ranging from 014 to

280 mgml However no significant difference was found in intracellular ROS levels for

powder-derived ACE treatment groups as compared to the control group Cyanidin 3-

glucoside (20 and 60 microgml) slightly reduced the intracellular ROS level though the

difference was insignificant as compared to the control group

Effect of H2O2 produced by ACEs in the medium on cytotoxic properties of ACEs in

HL-60 cells

To evaluate the influence of H2O2 produced by ACEs in the medium on their

cytotoxic properties in HL-60 cells cells were pretreated with catalase and compared to

the ACE-induced cytotoxicity with non-catalase treatment groups (Figure 54) Authentic

H2O2 (98 microM) reduced HL-60 cell viability to 88 plusmn 05 Catalase (500 Uml) rescued

the H2O2-induced cell death to 921 plusmn 13 This demonstrated that addition of

exogenous catalase almost completely abolished H2O2-induced cytotoxicity on HL-60

cells in our experiment conditions Interestingly in spite of the fact that puree-derived

ACE produced more H2O2 in the medium than powder-derived ACE the cytotoxicity

induced by puree-derived ACE was not affected by catalase whereas catalase partially

protected the HL-60 cells from powder-derived ACE-induced cytotoxicity There was no

significant difference in cell viability between catalase and non-catalase treatment groups

for puree-derived ACE at all concentrations tested In contrast the cytotoxicity resulting

from powder-derived ACE was significantly recovered by catalase from 487 plusmn 40 to

772 plusmn 18 and 288 plusmn 27 to 523 plusmn 15 at ACE concentrations of 21 and 28

mgml respectively However at high concentration (56 mgml) there was no

significant difference in cell viability between catalase and non-catalase treatment

groups

Effect of ACEs on LPS-induced TNF-α and IL-6 release in J774A1 cells

To investigate whether ACEs exhibit immune-modulation activity on macrophages

J774A1 cells were treated with various concentrations of ACEs 30 min after LPS

73

stimulation (Figure 62) Exposure of LPS (1microgml) for 6 h induced TNF-α and IL-6

secretion significantly in J774A1 cells For all concentrations of powder-derived ACEs

added both TNF-α and IL-6 were reduced in a concentration-dependent manner (Figure

62A) For each concentration of ACEs tested a similar reduction rate between cultivars

as well as between TNF-α and IL-6 levels was observed with 25 to 35 fold of decrease

at the highest ARE concentration compared to the TNF-α and IL-6 levels of the LPS

group A similar pattern was also observed in the experiment with the puree-derived

ACEs (Figure 62B) However Puree-derived Hull and Chester ACEs were found to be

less potent than those of powder-derived ACEs It is worth noting that in the

concentration range we tested ACEs did not show any cytotoxic effects on J774A1 cells

(data not shown)

Effect of ACE on inhibition of Lipid A-induced IL-12 release from DCs

To investigate whether ACE inhibited IL-12 release from DCs DCs were exposed to

ACE with or without Lipid A induction of IL-12 As shown in Figure 61A baseline

release of IL-12 from nonstimulated DCs was low with an IL-12 concentration of only

146 ngml However for all concentrations of ACE added the IL-12 release was reduced

in a concentration-dependent manner with only 037 ng of IL-12ml secreted using a

concentration of 525 mgml of medium As expected both high-dose (10 microgml) and

low-dose (01 microgml) Lipid A resulted in very high release of IL-12 from DCs of 624

ngml and 468 ngml respectively (Figure 61B) In the low-dose Lipid A treatment

group the concentration of IL-12 in cell culture supernatant was decreased in a

concentration-dependent manner from 468 to 72 ngml when ACE was added in the

range from 07 to 525 mgml A similar pattern was observed in the high-dose Lipid A

treatment group with the reduction of IL-12 release ranging from 474 to 138 ngml when

ACE was added in the range from 07 to 525 mgml Thus ACE significantly inhibited

the release of IL-12 from murine BMDDCs with or without Lipid A treatment It is

noteworthy that no cytotoxic effects were observed on these DCs at ACE concentrations

tested These results suggested that the pigmented anthocyanin-containing extract from

blackberries may have significant anti-inflammatory properties

74

55 Discussion

Our studies demonstrated that ACEs are cytotoxic to human colon (HT-29) ovarian

(MCF-7) cancer and leukemia (HL-60) cells with different degrees of potency (Figure

51A 51B 51C) Based on the EC50 values puree-derived ACE was 4-fold more potent

than powder-derived ACE in HL-60 cells Moreover the HL-60 cells showed much

greater sensitivity to ACEs than MCF-7 and HT-29 cells since the EC50 of puree-derived

ACE was 57 and 33-fold lower for HL-60 than that for MCF-7 and HT-29 cells

respectively A number of dietary phenolics such as quercetin which is also found in

blackberries [122] have been shown to produce H2O2 in generally-used medium

including RPMI 1640 medium and contribute to the cytotoxicity of cancer cells

(reviewed in [301]) In addition H2O2 was shown to be an important regulator in HL-60

cell growth [302] Therefore we investigated the H2O2 generation by puree and powder-

derived ACEs in medium and its influence on HL-60 cell growth A detailed examination

of H2O2 production by ACEs over time in RPMI 1640 medium with or without HL-60

cells is provided in Table 51 Our results showed that both puree and powder-derived

ACEs produced levels of H2O2 (gt10 microM) which caused cytotoxicity in HL-60 cells when

incubated with RPMI 1640 medium within 48 h Interestingly the cytotoxic activity of

puree-derived ACE was not blocked by exogenous catalase On the other hand the

intracellular ROS level was also found to increase with puree-derived ACE which may

induce cytotoxicity in HL-60 cells Since it is well-known that H2O2 diffuses freely

through cell membranes while catalase is membrane-impermeable the results suggested

that the cytotoxic activity of puree-derived ACE was not due to the H2O2 generated in the

medium but due to some active components in ACE which were quickly taken up by the

cells and subsequently induced cytotoxicity by generating ROS inside the cells andor by

other mechanisms However as for powder-derived ACE its growth inhibition activity

was partially abolished by the catalase Since intracellular ROS level in HL-60 cells

remained the same after treatment of powder-derived ACE for 1 h (Figure 53) and H2O2

concentration in medium did not exceed 10 microM in the first 6 h of incubation (Table 51)

it is apparent that the cytotoxic activity through ROS mechanism by powder-derived

ACE was relatively delayed The difference of anticancer mechanism between puree and

powder-derived ACEs in HL-60 cells may be attributed to the different major active

75

components that contribute to their cytotoxic activity resulting in different anticancer

potency in HL-60 cells and similarly in HT-29 and MCF-7 cells Since we showed that

puree-derived ACE contained 2-fold greater amount of total phenolics than powder-

derived ACE it was possible that the elevated antiproliferative potency of puree-derived

ACE was due to the higher level of phenolics It is noteworthy that we also investigated

the effect of copper on the H2O2 generation by ACEs in medium It was found that H2O2

production by ACEs did not increase with increasing concentration of cupric sulfate from

0 to 100 microM in the RPMI 1640 medium (data not shown) which suggested that the H2O2

generation by ACEs was likely not copper-mediated

Cyanidin-based anthocyanins were the dominant anthocyanins in the blackberry

extract and cyanidin 3-glucoside was the major anthocyanin (71) Cyanidin 3-glucoside

has been found to possess chemopreventive and chemotherapeutic activities Chen et al

showed that cyanidin 3-glucoside purified from Black rice (Oryza sativa L indica)

inhibited human breast cancer cell HS578T growth via G2M arrest and it also improved

the cytotoxicity of doxorubicin at non-toxic concentrations [303] Ding et al showed that

cyanidin 3-glucoside not only protected JB6 cells from ultraviolet B-induced insult but

also can inhibit the proliferation migration and invasion of A549 lung tumor cells [182]

Cyanidin [304] and cyanidin-3-rutinoside [298] were also found to increase the

intracellular ROS level which may involve induction of apoptosis in HL-60 cells

Therefore we were interested in assessing the possible role of cyanidin 3-glucoside in

ACEs in the cytotoxic property of ACE in HL-60 cells The antiproliferative effect of

pure cyanidin 3-glucoside was examined on HL-60 cells (Figure 51D) as well as its

ability to spontaneously generate H2O2 in RPMI 1640 medium (Table 51) and

intracellular ROS in HL-60 cells (Figure 52) It was found that cyanidin 3-glucoside

exhibited antiproliferative activity in a concentration range of 104-622 microgml However

ACEs caused cytotoxicity in HL-60 cells with much lower anthocyanin concentrations

For example the anthocyanins concentration in powder and puree-derived ACEs at EC50

were 36 and 145 microgml respectively (of which 71 was cyanidin 3-glucoside)

However pure cyanidin 3-glucoside caused little or no cytotoxic effects on HL-60 cells

at these concentrations These data suggested that cyanidin 3-glucoside in ACEs may act

additively or synergistic with other active components in inhibition of cell growth but

76

that a significant part of the cytotoxicity could not be explained by anthocyanins alone

Moreover it was found that the concentrations of cyanidin 3-glucoside which induced

cytotoxicity in HL-60 cells produced non-toxic levels of H2O2 in the RPMI 1640 medium

with 10 FBS with little or no increase in the intracellular ROS in HL-60 cells These

results suggested that the active components that produced H2O2 increased intracellular

ROS and cytotoxicity with puree-derived ACE were components predominantly other

than cyanidin 3-glucoside Related McDougall et al compared the antiproliferative

effectiveness of a series of polyphenol-rich berry extracts on human cervical (Hela) and

colon (Caco-2) cancer cells and their polyphenol compositions and they suggested that

the antiproliferative activity of berry extracts was due to other phenolic constituents such

as ellagitannins and procyanidins more than anthocyanins [163] Importantly it has been

shown that ellagitannins are the major phenolic class than anthocyanins in blackberry

species [122] Identification of the active components in ACEs is currently ongoing in

our lab

Previous studies by Pergola et al [247] demonstrated that part of the anti-

inflammatory activity of a specific blackberry extract was due to the suppression of nitric

oxide production in J774 cells by cyanidin-3-O-glucoside Rossi et al [305] showed that

the anthocyanin fraction from blackberry extract exerted multiple protective effects in

carrageenan-induced pleurisy in rats Nevertheless most of the in vitro studies utilizing

anthocyanin-containing extracts from other fruits or vegetables have focused on the effect

of the extracts on nitric oxide synthesis and TNF-α levels in vitro using activated

macrophages [245 271 306] Other studies have assessed the effects of extracts on the

inflammation induced by hydrogen peroxide and TNF-α in human microvascular

endothelial cells [274] To our knowledge there have been few or no studies assessing

the anti-inflammatory effects of anthocyanin-containing extracts on DCs DCs are potent

antigen-presenting cells and function as initiators and modulators of the immune

response Lipid A is known to induce maturation of DCs resulting in synthesis of high

levels of pro-inflammatory IL-12 that enhances both innate (natural killer cell) and

acquired (B and T cells) immunity Our results showed that ACE derived from Hull

blackberry powder inhibited IL-12 release from DCs in both background and stimulated

levels

77

Moreover we demonstrated that puree and powder-derived ACEs Hull Chester and

Black Satin cultivars exert anti-inflammatory property by inhibiting the pro-inflammatory

cytokine IL-6 and TNF-α release in LPS-activated J774A1 macrophages The inhibition

potency was comparable among cultivars However it is worth noting that in another

experiment we pretreated J774A1 macrophages with ACEs followed by LPS

stimulation Our preliminary data showed no decrease but possible increase of TNF-α and

IL-6 secretion caused by the ACEs groups In studies by Wang et al [271 306] RAW

2647 macrophages were pretreated with anthocyanins and berry extracts followed by

LPSIFN-γ stimulation It was found that blackberry extract has little or mild induction

effect on TNF-α production in RAW 2647 macrophages which is in agreement with our

findings This may suggest that the sequence of application of ACEs before or after

macrophage activation play a critical role on the immune-modulation functions of ACEs

Further mechanistic studies are essential to understand the possible role of ACEs in

inflammatory diseases or host defense system

In conclusion these studies demonstrated that blackberry ACEs have significant

anticancer and anti-inflammatory activities In general puree-derived ACEs were more

potent in their antiproliferative activity in cancer cells as compared to powder-derived

ACEs under the concentrations investigated in these studies Cyanidin 3-glucoside itself

contributed little to the antiproliferative activity of ACEs and it suggested that cyanidin

3-glucoside may act synergistically or additively with other active components in ACE to

cause cytotoxicity in cancer cells Moreover our studies suggested that the

antiproliferative activity of puree-derived ACE was not due to the H2O2 produced in the

medium but some active components in ACE which were quickly taken up by the cells

to induce cytotoxicity in the cells before they produced toxic levels of H2O2 in the

medium On the other hand the antiproliferative activity of powder-derived ACE was

partially related to the toxicity of H2O2 generated in medium since catalase was partially

effective in reducing extract-mediated cytotoxicity Furthermore our studies showed that

blackberry exhibit anti-inflammatory activity through inhibition of pro-inflammatory

cytokines release in immune cells These findings warrant further investigation into the

anticancer and anti-inflammatory effects of blackberries both in vitro and in vivo

78







79

Figure 5 1 The cytotoxic effects of Black Satin ACEs and Cyanidin 3-glucoside

(Cn-3-G) on human cancer cell lines Cells were exposed to Black Satin puree and

powder-derived ACE (A B C) or Cn-3-G (D) for 48 h and cell viability was measured

by MTT assay Data in A-D are representative of three independent experiments and data

are presented as the mean plusmn SE (n = 3) Curves were fit by nonlinear regression using

GraphPad Prism Half maximum effective concentration (EC50) is reported as the mean plusmn

SE (n = 3) in the figure legend

80

Figure 5 2 H2O2 generation by Black Satin ACEs in RPMI 1640 medium with 10

FBS over time Authentic H2O2 (50 microM) puree and powder-derived ACE (14 mgml)

with or without catalase (100 Uml) were incubated with RPMI 1640 medium with 10

FBS at 37ordmC for 48 h At each time point an aliquot was taken out and measured for

H2O2 concentration Data are presented as the mean plusmn SE (n = 6)

81

Table 5 1 H2O2 Generation by ACEs and Cyanidin 3-glucoside (C-3-G) in Medium at 37 ordmC a

Medium Concentration H2O2 level (microM) at various times (h) 1 2 6 24 48

ACEpuree (mgml)

RPMI 1640 10 FBS

014 533 plusmn 012 509 plusmn 100 442 plusmn 040 657 plusmn 025 674 plusmn 037


14 1148 plusmn 028 (ND)

3300 plusmn 176 (ND)





RPMI 1640 10 FBS

HL-60 cells

070 233 plusmn 019 (ND)

424 plusmn 022 (ND)

310 plusmn 079 (ND)

14 196 plusmn 023 (ND)

522 plusmn 020 (ND)

432 plusmn 024 (ND)

28 326 plusmn 025 (ND)

654 plusmn 018 (ND)

ND (ND)

RPMI 1640 14 8170 plusmn 266 15046 plusmn 122

ACEpowder (mgml)

RPMI 1640 10 FBS


070 ND 192 plusmn 026 493 plusmn 073 609 plusmn 041 1092 plusmn 039

14 ND (ND)

363 plusmn 029 (ND)

859 plusmn 112 (ND)




RPMI 1640 10 FBS

HL-60 cells

14 140 plusmn 010 (ND)

154 plusmn 017 (ND)

ND (ND)

28 202 plusmn 028 (ND)

168 plusmn 017 (ND)

143 plusmn 014 (ND)


C-3-G (microgml)

RPMI 1640 10 FBS

207 ND ND ND

622 297 plusmn 016 273 plusmn 014 190 plusmn 028

RPMI 1640



RPMI 1640 10 FBS

HL-60 cells

207 ND

622 ND

H2O2 (microM)

RPMI 1640 10 FBS 50 4626 plusmn 116

(ND) 4424 plusmn 192




(ND)

RPMI 1640 10 FBS

HL-60 cells 100 ND

a Data are expressed as mean plusmn SE (n = 6) With catalase (100 Uml) ND Not detected

82

Figure 5 3 The effects of Black Satin ACEs and Cyanidin 3-glucoside (Cn-3-G) on

the intracellular ROS level in HL-60 cells CH2DCFDA-loaded and non-CH2DCFDA

loaded cells were incubated with either ACEs or Cn-3-G or media alone (vehicle) H2O2

(98 microM) H2O2 (98 microM) plus catalase (500 Uml) treatment groups were used as controls

The results are reported as the percentage increase fluorescence associated with the

CH2DCFDA-loaded cells compared to that of corresponding non-CH2DCFDA-loaded

cells Data are presented as the mean plusmn SE of three independent experiments b P le 001 c

P le 0001 compared to the vehicle group

83

Figure 5 4 The effects of H2O2 generated by ACEs in medium on cytotoxic

properties of ACEs in HL-60 cells Cells were treated with catalase (500 Uml) () or

PBS () and concomitantly with H2O2 (98 microM) or puree and powder-derived ACEs from

Black Satin for 48 h and cell viability was measured Data are presented as the mean plusmn

SE of three independent experiments b P le 001 c P le 0001 compared to the non-catalase

treatment group with corresponding H2O2 or ACE concentration

84

Figure 5 5 Effect of powder-derived ACE from Hull blackberries (2005) on Lipid

A-induced IL-12 release from murine dendritic cells DCs were exposed to ACE with

(B) or without (A) Lipid A for 24 h Inhibition of IL-12 release was observed in all cases

Results are presented as mean plusmn SE (n = 3) for each concentration (A) P le 005 P le

001 compared to the vehicle control without ACE and Lipid A treatment (B) P le

001 P le 0001 compared to the vehicle control with low-dose Lipid A treatment P

le 001 P le 0001 compared to the vehicle control with high-dose Lipid A treatment

85


LPS-induced IL-6 and TNF-α release in J774A1 macrophages Cells were treated

with LPS (1 microgml) for 30 min followed by puree derived (A) or powder-derived (B)

ACE treatment Supernatants were collected after 55 h and assayed by using specific

ELISA Results are represented as mean plusmn SE (n = 3) a P le 005 b P le 001 c P le 0001

compared to LPS controls


86

Chapter 6

Correlation of the In Vitro Antioxidant and Antiproliferative Properties of

Blackberry Extracts to Their Phytochemical Constituents

61 Summary

The aim of these studies was to separate phenolic compounds from non-phenolic

compounds in anthocyanin-containing blackberry extracts (ACEs) derived from both

puree and powder and evaluate their antioxidant activity and antiproliferative properties

on cancer cells A simple solid phase extraction (SPE) method was successfully

developed to prepare phenolics-enriched methanol fractions (MFs) and ethyl acetate

fractions (EAFs) and non-phenolic water fractions (WFs) MFs contained most of the

anthocyanins present in ACEs possess the highest antioxidant activity and most potent

antiproliferative activity on leukemia (HL-60) and colon (HT-29 HCT-15) cancer cells

among all three fractions MFs derived from both puree and powder ACEs as well as

puree-derived ACE dose-dependently enhanced intracellular ROS levels and caspase 3-

like activity However N-acetyl-L-cysteine a cell permeable antioxidant failed to block

the antiproliferative effects as well as caspase 3-like activity induced by puree MF and

ACE whereas NAC significantly rescued the cell death and decreased caspase 3-like

activity induced by powder MF These results suggested puree ACE and corresponding

MF induced cell death through ROS-independent caspase 3 pathway and the cytotoxicity

induced by MF derived from powder ACE is related to ROS mechanism

87

62 Introduction

Recent research has revealed that blackberries contain higher amount of anthocyanins

and other antioxidants than other fruits [9-11] Blackberry extracts have been shown to

have various bioactivities including protecting against endothelial dysfunction and

vascular failure in vitro [291] attenuating the injury caused by LPS-induced endotoxic

shock in rats [292] and exhibiting cytotoxic effects on human oral prostate [267] lung

[293] cancer cells The bioactivities of the blackberry extracts have been attributed to

their phenolic compounds such as anthocyanins In our previous report we showed that

blackberry anthocyanin-containing extracts (ACEs) possess significant anticancer

activities in various human cancer cell lines including colon (HT-29) breast (MCF-7)

and leukemia (HL-60) cells [307] However the nature of the compounds in the ACEs

that possesses potent antioxidant activity as well as anticancer activity has not been fully

investigated although we evaluated the role of cyanidin-3-glucoside the dominant

anthocyanin in ACEs In addition it has been well established that phytochemicals in

fruits and vegetables can have complementary and overlapping effects on oxidative stress

resulting in their synergistic or additive protection against diseases as compared to the

pure compounds However to our knowledge there have been few or no studies

evaluating the possible synergistic or additive effects of a combination of different

classes of phytochemicals from blackberries

Our preliminary data showed that the antiproliferative effects of ACEs derived from

Hull blackberries were generally more potent than those derived from Black Satin and

Chester cultivars in several human cancer cell lines including 1) colon HT-29 2) breast

BT-474 (her2 positive and ER+) and MCF-7 (her2 negative and ER+) and 3) leukemia

HL-60 (wild-type) HL-60-VCR (P-gp positive) and HL-60-ADR (MDR-1 positive)

Therefore ACEs derived from Hull blackberries were selected for the current studies In

an effort to examine the bioactivity resulting from the complex mixture of a group of

phytochemicals in blackberry ACEs the purpose of these studies was to separate the

phenolic compounds from non-phenolic compounds in ACEs prepared from Hull

blackberries and compare their antioxidant activity as well as antiproliferative properties

on cancer cells A solid phase extraction (SPE) method was developed to fractionate the

phenolic compounds from non-phenolic compounds in both puree and powder-derived

88

ACEs The obtained fractions were characterized in terms of total anthocyanin and total

phenolics content as well as total antioxidant capacity (TAC) by Trolox equivalent

antioxidant capacity (TEAC) method The efficiency of the SPE method employed was

also evaluated by the percentages of solid recoveries The antiproliferative effects of

whole ACEs versus their individual fractions on leukemia (HL-60) and colon (HT-29

HCT-15) cancer cells were evaluated and possible synergistic or additive effects were

discussed Moreover possible mechanisms of cytotoxicity of the selected bioactive

fractions in HL-60 cells were evaluated including ROS generation and caspase 3

activation Finally the ability of the antioxidant N-acetyl-L-cysteine to quench these

effects was investigated

89


Cell culture and chemicals

The human leukemia cell line HL-60 and colorectal cancer cell line HT-29 and

HCT-15 were grown in RPMI-1640 medium supplemented with 100 Uml penicillin 100

microgml streptomycin and 10 FBS Cells were maintained at 37 ordmC in a humidified 5

CO2 incubator

Methanol ethyl acetate and hydrochloric acid (365) were purchased from Fisher

Scientific (Fair Lawn NJ) 5-(and-6)-carboxy-2acute7acute-dichlorodihydrofluorescein diacetate

(CH2DCFDA) was purchased from Invitrogen Inc (Carlsbad CA) N-acetyl-L-cysteine

(NAC) Hydrogen peroxide solution (30) and dimethyl sulfoxide (DMSO) were

purchased from Sigma (St Louis MO)

Fractionation of ACEs by SPE

The method of fractionation was adjusted from Skrede et al [308] Puree-derived (pH

28) or powder-derived (pH 20) ACEs stock solution (140 mgml) from Hull

Blackberries (2007) was applied to an Discovery DSC-18 tube (Supelco Bellefonte PA)

that was preconditioned with methanol and then water acidified with hydrochloric acid to

the pH value of corresponding ACEs Then the water-soluble compounds such as sugars

in ACEs was washed out with three tube volumes of acidified water and collected as

water fraction (WF colorless) After that the cartridge was dried with a current of

nitrogen and eluted with three tube volumes of ethyl acetate to yield an orange-yellow

ethyl acetate fraction (EAF) Finally 50 aqueous methanol was used to remove the rest

of phenolic compounds and yield a red fraction (methanol faction MF) The WF was

dried in a lyophilizer and reconstituted in PBS as a stock solution The EAF and MF were

dried by a rotary evaporator under vacuum and reconstituted in DMSO as stock solutions

All fractions were stored at -80 degC for further characterization and cell-based studies

Total anthocyanin measurement

Monomeric anthocyanin contents in puree and powder-derived ACEs EAF and MF

were determined using the pH-differential method of Giusti and Wrolstad [277] as

described in the material and methods in Chapter 3

90


Total phenolic contents in puree and powder-derived ACEs WF EAF and MF were

estimated using the Folin-Ciocalteu method for total phenolics [98] as described in the

material and methods in Chapter 3


Total antioxidant capacity of puree and powder-derived ACEs WF EAF and MF was

measured by TEAC assay [157] as described in the material and methods in Chapter 3


The suspension HL-60 cells were seeded at an initial concentration of 3times104

cellswell in black-walled 96-well plates After 24 h cells were treated with the ACE

stock solutions or fractions After 48 h of treatment medium was removed and cell

viability was measured for all cells using the CellTiter-Glo Luminescent Cell Viability

assay (Promega Corp Madison WI) When added to the cells the assay reagent

generates a luminescent signal proportional to the ATP present which represents the

presence of metabolically active cells Briefly plates were equilibrated at room

temperature for 30 min Then 100 μl assay reagent was added to 100 μl medium in each

well The contents were mixed in an orbital shaker for 2 min to induce cell lysis Plates

were incubated at room temperature for another 10 min to stabilize the luminescent signal

and luminescence was read on a Biotek Synergy 2 Microplate Reader Cell viability was

calculated as percentage of the luminescence of corresponding vehicle controls




loaded with 100 μM CH2DCFDA (dissolved in DMSO) for 30 min at 37 degC The cells




for 1 h Data are reported as percentage increase of DCF fluorescence associated with the

91


cells (negative controls)

Caspase 3-like activity

The HL-60 cells were seeded at an initial concentration of 3times104 cellswell in black-

walled 96-well plates After 24 h cells were treated with the ACE stock solutions or

fractions After 24 h of treatment medium was removed and the Caspase 3-like activity

was measured using the Caspase-Glo 37 Assay (Promega Corp Madison WI) Briefly

plates were equilibrated at room temperature for 30 min Then 100 μl assay reagent was

added to 100 μl medium in each well The contents were mixed in an orbital shaker for

30 sec and incubated at room temperature for another 1 h The luminescence produced in

each well was read on a Biotek Synergy 2 Microplate Reader Data are reported as folds

of luminescence as compared to the untreated groups (controls)





92

64 Results and discussion

SPE with silica-based C18 cartridge has been widely used for isolation of phenolic

compounds in plant crude extracts especially aqueous extracts In these studies a simple

SPE method was developed to separate the phenolic components from non-phenolic

components in ACEs As shown in Table 61 the WF contained the majority of the total

substance in the Hull puree ACE (95307 plusmn 986 mg out of 98959 plusmn 1132 mg of total

solid recovered after separation) with an insignificant level of total phenolics (193 plusmn 031

mgg of solid in WF) Most phenolics were concentrated in the EAF and MF with the

majority of the anthocyanin obtained in the MF (9733 plusmn 316 of yield)

Correspondingly MF and EAF possessed significantly higher TAC than WF as well as

the original puree ACE (Table 61) For example the total anthocyanin total phenolics

and TAC levels of MF were 3292 2151 and 2406-fold greater than those in original

puree ACE In contrast the WF contained no detectable anthocyanin and the levels of

total phenolics and TAC were negligible Similar results were also found in fractions

derived from powder ACE (Table 62) Moreover the amount of total phenolics

recovered in EAF and MF derived from puree ACE was more than two-fold greater than

those derived from powder ACE whereas the total anthocyanin amounts recovered in

MFs were comparable These results correlated well with the fact that puree ACE

contained a similar amount of total anthocyanins but about two-fold greater amounts of

phenolics than the powder ACE The total recovery ( yield) of solid as well as the total

anthocyanin and total phenolics were found to be around 100 after SPE fractionation of

the puree ACE whereas the recoveries for powder ACE was lower at less than 80 This

indicated that the SPE method used was less efficient for the fractionation of ACE

derived from powder than puree Since powder ACE was obtained using a less polar

solvent system than that to obtain puree ACE it may contain more nonpolar compounds

It may be speculated that these nonpolar compounds had strong intermolecular

interactions with the nonpolar sorbent molecules in the SPE C18 cartridge as well as

other molecules in the powder ACE which prevented their complete elution under the

conditions employed in these studies

It has been showed that the predominant non-phenolic compounds such as sugars in

blackberries were mostly fructose and glucose and malic acid was the predominant

93

organic acid in blackberries [309] Anthocyanins such as cayanidin-3-glucoside and

ellagitannins such as sanguiin H-6 and lambertianin C were found to be the major

phenolic compounds in blackberries [122] Further isolation and identification may be

conducted on the three fractions of ACEs by other techniques for example column

chromatography However it was outside of the scope of the current studies

The antiproliferative effects of ACEs versus those fractions isolated from the

corresponding ACEs on HL-60 cells were determined using a highly sensitive

luminescent ATP cell viability assay (Figure 61) This assay is based on the quantitation

of the luminescent signal produced by the reaction of assay reagent with the ATP present

Therefore it is more suitable to assay samples when antioxidants or oxidants were

involved than those oxidation-reduction-based assays such as MTT and LDH assays As

shown in Figure 61 the potencies of antiproliferative activity were in the order of MF gt

EAF gt ACE gt WF for ACE and corresponding fractions derived from both puree (Figure

61 A) and powder (Figure 61B) Similar results were also found in colon cancer HT-29

and HCT-15 cells (data not shown) The observed antiproliferative activity was

significantly enhanced with phenolic compounds-enriched MFs and EAFs as compared to

the whole ACE and the non-phenolic fractions (WFs) For example the EC50 of

antiproliferative effects on HL-60 cells induced by MF was about 76-fold and 16-fold

lower than that of WF and ACE respectively derived from puree The cytotoxicity

induced by WFs at high concentrations may be attributed to its strong acidity since the

color of the medium was turned into light yellow after adding WFs at these

concentrations For all ACEs and fractions tested the anthocyanin and phenolics-

enriched fraction (MF) derived from puree ACE was found to be most potent in the

inhibition of HL-60 cells growth (EC50 492 plusmn 79 μgml) which was comparable with

that of the pure cyanidin 3-glucoside determined in our previous studies (cell viability

was decreased to 459 at 622 microgml of cyanidin 3-glucoside) [307] Since the total

anthocyanin in MF was about 167 it indicated that other components including other

phenolics than anthocyanins in MF may work additively or synergistically with

anthocyanins on the antiproliferative effects of MF Moreover synergistic and or additive

effects of three fractions in the whole ACE were also observed For example based on

the percent composition of each fraction in the whole ACE derived from puree at EC50

94

7838 μgml ACE contained 217 μgml MF 63 μgml EAF and 7555 μgml WF all of

which were less than the EC50 values obtained from corresponding individual fractions It

also suggested that the major effective components in ACE were in the MF since EAF

(63 μgml) and WF (7555 μgml) barely produced any cytotoxicity Similar results were

also found in MF derived from powder ACE

Our previous studies showed that the antiproliferative activity of ACEs may involve

ROS production intracellularly (puree-derived ACE) andor in the cell culture medium

(puree and powder-derived ACE) [307] In these studies we examined the intracellular

ROS production by ACEs prepared from Hull blackberries and their corresponding

fractions It was found that puree-derived ACE and MFs from both puree and powder

ACEs dose-dependently increased the intracellular ROS levels (Figure 62) whereas

powder-derived ACE EAFs and WFs from either powder or puree-derived ACEs did not

(data not shown) The percentage increase of fluorescence as compared vehicle induced

by MFs from puree (136 μgml) and powder (292 μgml) were comparable with that

induced by 49 μM of H2O2 (about 3420) To further elucidate the possible ROS

mechanism involved in the antiproliferative effects of these reagents in HL-60 cells we

utilized a cell-permeable antioxidant NAC to test whether their antiproliferative effects

can be blocked by inhibiting the ROS production As shown in Figure 63 NAC (4 mM)

rescued the H2O2 (98 μM)-induced cell death from 88 plusmn 08 to 933 plusmn 09 This

demonstrated that addition of NAC almost completely abolished H2O2-induced

cytotoxicity on HL-60 cells in our experiment conditions At concentrations ranging from

34 to 136 μgml puree-derived MF dose-dependently decreased cell viability from 855 plusmn

23 to 102 plusmn 08 However incubation of NAC (4 mM) with HL-60 failed to rescue

the cell death induced by the puree MF since there was no significant difference observed

between NAC-treated and untreated groups for all the concentrations tested As expected

similar results were also found in puree-derived ACE (Figure 63) On the other hand

NAC (4 mM) significantly rescued the cell death induced by powder-derived MF at

concentrations ranging from 146 to 292 μgml For example the cell viability was

increased from 317 plusmn 35 to 803 plusmn 32 when cells were incubated with NAC

together with powder-derived MF (292 μgml) (Figure 63) These results suggested that

the antiproliferative effects of MF and ACE derived from puree were ROS-independent

95

whereas the antiproliferative effects of MF derived from powder were predominantly

related to its ROS production the later of which was also correlated with our previous

studies on powder-derived ACE from Black Satin blackberries that its antiproliferative

activity was partially related to an ROS-specific mechanism [307]

Many studies have shown that plant phenolic extracts and pure phenolic compounds

induced cancer cell death through apoptotic mechanisms [160 234 304 310-312]

Caspase 3 is a frequently activated death protease in mammalian cell apoptosis [313] It

exists as an inactive pro-caspase 3 in the cytoplasm and is proteolytically activated by

multiple cleavages of pro-caspase 3 to generate the cleaved fragments in the terminal

execution phase of cell apoptosis which was induced by diverse stimuli for example

ROS In these studies we examined the effects of puree-derived ACE and MFs from both

puree and powder ACEs on caspase 3-like activity in HL-60 cells and their relationship to

ROS generation (Figure 64) As a positive control H2O2 (49 μM)-induced caspase 3-

like activity (272-fold of vehicle control) was blocked by NAC (4 mM) All the ACE

and fractions tested induced caspase 3-like activation dose-dependently in the dosing

ranges that produced cytotoxicity However NAC (4 mM) failed to block the caspase 3-

like activation by puree-derived ACE and corresponding MF whereas the increase of

caspase 3-like activity by MF derived from powder ACE (223-fold of vehicle control

146 μgml 364-fold of vehicle control 292 μgml) was reduced to the baseline level

(087-fold of vehicle control 146 μgml 099-fold of vehicle control 292 μgml) by co-

incubation with NAC These results suggested puree-derived ACE and corresponding MF

induced cell death through ROS-independent caspase 3 pathway and it further

demonstrated that the cytotoxicity induced by MF derived from powder ACE is related to

ROS mechanism

In conclusion a simple solid phase extraction (SPE) method was successfully







96

like activity Furthermore our studies suggested puree ACE and corresponding MF

induced cell death through ROS-independent caspase 3 pathway and the cytotoxicity


97

Table 6 1 Composition and Characterization of Fractions Separated from Hull

Puree ACE by SPE a WF EAF MF Total

Total Anthocyanin b

Weight of total solid recovered (mg)

95307 plusmn 986 806 plusmn 050 2846 plusmn 063 98959 plusmn 1132

Total anthocyanin in recovered fraction (mg)

ND 012 plusmn 001 490 plusmn 016 502 plusmn 017

Anthocyanin level in recovered fraction (mgg solid)

ND 1477 plusmn 075 16559 plusmn 507 -

Yield ()

- 253 plusmn 028 9733 plusmn 316 9986 plusmn 340

Fold concentrated in recovered fraction

- 294 3292 -

Total Phenolics c



Total phenolics in recovered fraction (mg)

193 plusmn 031 44035 plusmn 2792 51283 plusmn 422 -

Phenolics level in recovered fraction (mgg solid)


Yield () 919 plusmn 210 1887 plusmn 229 7420 plusmn 309 10267 plusmn 478


009 2189 2551 -

TAC TAC Level in recovered fraction

(μmol TEg solid) 1498 plusmn 312 273461 plusmn 9677 483820 plusmn 23696 -


007 1360 2406 -

a The SPE separation was repeated three times with the same ACE sample The volume of Hull puree ACE stock

solution applied to the SPE cartridge contained 1 gram of dried extract All assays were carried out in triplicate Data

are expressed as the mean plusmn SE (N = 3) b Total anthocyanin are expressed as cyanidin 3-glucoside equivalent c Total phenolics are expressed as gallic acid equivalent Calculated as the percentage of total anthocyanin (phenolics) in the Hull puree ACE added to the SPE cartridge Calculated as fold change that anthocyanin (phenolics TAC) are concentrated in the recovered solid fraction as

compared to their original level in the Hull puree ACE added to the SPE cartridge

ND not detected

98


Powder ACE by SPE a WF EAF MF Total

Total Anthocyanin b




ND 565 plusmn 136 30574 plusmn 3356 -



Yield ()



- 119 6518 -

Total Phenolics c









010 2566 4610 -



Fold increase in recovered fraction

022 2161 6345 -

a The SPE separation was repeated three times with the same ACE sample The volume of Hull powder ACE stock


are expressed as the mean plusmn SE (N = 3) b Total anthocyanin are expressed as cyanidin 3-glucoside equivalent c Total phenolics are expressed as gallic acid equivalent Calculated as the percentage of total anthocyanin (phenolics) in the Hull powder ACE added to the SPE cartridge Calculated as fold change that anthocyanin (phenolics TAC) are concentrated in the recovered solid fraction as

compared to their original level in the Hull powder ACE added to the SPE cartridge

ND not detected

99

0 100 200 300 400 5000

20

40

60

80

100

120

1000 2000 3000 4000

MFpuree 492 plusmn 79 microgmlEAFpuree 1152 plusmn 78 microgmlWFpuree 3746 plusmn 3252 microgmlACEpuree 7838 plusmn 496 microgml

A

C (microgml)

Cel

l Via

bilit

y (

Con

trol

)

0 100 200 300 400 5000

20

40

60

80

100

120

2000 4000 6000 8000

MFpowder 788 plusmn 48 microgml

WFpowder 4013 plusmn 219 microgmlACEpowder 2491 plusmn 335 microgml

BEAFpowder 1832 plusmn 354 microgml

C (microgml)

Cel

l Via

bilit

y (

Con

trol

)

Figure 6 1 The cytotoxic effects of Hull ACEs and fractions on HL-60 cells Cells

were exposed to Hull puree (A) and powder-derived (B) ACEs or fractions for 48 h and

cell viability was measured by CellTiter-Glo Luminescent Cell Viability assay Data in A

and B are representative of three independent experiments and presented as the mean plusmn

SE (n = 3) Curves were fit by nonlinear regression using GraphPad Prism Half

maximum effective concentration (EC50) is reported as the mean plusmn SE (n = 3) in the

figure legend

100

Figure 6 2 The effects of Hull ACE (derived from puree) and methanol fractions on


loaded cells were incubated with either ACE or MF or media alone (vehicle) H2O2

treatment groups were used as positive controls The results are reported as the

percentage increase fluorescence associated with the CH2DCFDA-loaded cells compared

to that of corresponding non-CH2DCFDA-loaded cells Data are presented as the mean plusmn

SE (n = 3) c P le 0001 compared to the vehicle group

101


methanol fractions of ACEs from Hull blackberries Cells were treated with NAC (4

mM) () or PBS () and concomitantly with H2O2 (98 microM) or puree-derived ACE or

methanol fractions of ACEs from Hull for 48 h and cell viability was measured by

CellTiter-Glo Luminescent Cell Viability assay The figure showed is a representative of

three independent experiments with similar results and data are presented as mean plusmn SE

(n = 3) c P le 0001 compared to the corresponding non-NAC treatment group

102


derived ACE and methanol fractions of ACEs from Hull blackberries Cells were

treated with NAC (4 mM) () or PBS () and concomitantly with H2O2 (49 microM) or

puree-derived ACE or methanol fractions of ACEs from Hull for 24 h and caspase 3-like

activity was measured by Caspase-Glo 37assay Data are presented as mean plusmn SE (n =

3) c P le 0001 NAC-treated groups were compared to the corresponding NAC-untreated

groups


103

Chapter 7

Summary and Conclusions

The purpose of this research was to develop phenolic extracts from selected cultivars

of blackberries currently grown in Kentucky as potential Botanical Drug Products for the

treatment and prevention of cancer and inflammatory diseases The hypotheses driving

this research were

1) Extracts which contain high amount of anthocyanins and polyphenols with high

antioxidant activity will be obtained from blackberries

2) Blackberry extracts will possess anticancer and anti-inflammatory properties The

anticancer activities of the blackberry extracts will involve mechanisms related to

reactive oxygen species (ROS) The blackberry extracts will exhibit anti-inflammatory

activity by inhibiting pro-inflammatory cytokine release from immune cells

3) The phenolics but not the non-phenolic compounds in the blackberry extract will

be responsible for its bioactivities

To obtain anthocyanin-containing extracts (ACEs) from blackberries an ultrasound-

assisted ethanol extraction method was employed Two different blackberry materials

puree and powder (which was freeze-dried puree) of Hull Black Satin and Chester

cultivars were used as starting materials The obtained ACEs were characterized and

compared for total anthocyanin and phenolic content polymeric color and total

antioxidant capacity The influence of water content in the extraction system was

evaluated A 90 day stability study of the extract stored alone as a function of time

temperature and light and a 48 h stability study of the extract in biologically relevant

buffers at different temperatures were completed Using the same extraction method it

was found that the total anthocyanin and phenolic content polymeric color and TAC

were comparable between cultivars of the same harvest year (2006) HPLC-MS results

showed that the anthocyanins in ACE derived from Hull blackberries were mainly

cyanidin-based As compared to powder-derived ACEs puree-derived ACEs contained

similar amounts of anthocyanins but greater levels of phenolics and increased TAC

Extraction solvents with water to ethanol ratios greater than 5050 greatly decreased the

104

yield of total anthocyanin and phenolics in the ACEs The stability studies indicated that

multiple factors such as temperature pH and time contributed to the loss of anthocyanins

total phenolics and antioxidant activity in ACE

To investigate the anticancer properties of ACEs firstly the antiproliferative effects

of puree and powder-derived ACEs were compared in a panel of human cancer cell lines

including leukemia (HL-60) colon (HT-29) and breast (MCF-7) It was shown that

treatment with blackberry ACEs prepared from Black Satin cultivar in concentrations

ranging from 080 to 112 mgml resulted in a significant dose-dependent reduction in

cell viability versus control with human cancer cell lines The anticancer effects were 1)

more pronounced in leukemia cells and 2) greater with puree-derived than powder-

derived extracts in all cell lines tested Secondly to elucidate the possible role of cyanidin

3-glucoside the dominant anthocyanin in ACEs in the anticancer properties of ACEs the

antiproliferative effect of cyanidin 3-glucoside was compared with those of ACEs in HL-

60 cells It was found that cyanidin 3-glucoside exhibited antiproliferative activity in a

concentration range of 104-622 microgml However ACEs caused cytotoxicity in HL-60

cells with much lower anthocyanin concentrations These results suggested that cyanidin

3-glucoside in ACEs may act additively or synergistic with other active components in

inhibition of cell growth but that a significant part of the cytotoxicity could not be

explained by anthocyanins alone Thirdly to investigate the possible mechanism of

cytotoxicity involving ROS production by ACEs in cancer cells hydrogen peroxide

(H2O2) generation and enhancement of intracellular ROS level in HL-60 cells by puree

and powder-derived ACEs were evaluated and the ability of catalase to quench these

effects was investigated Our results showed that both puree and powder-derived ACEs

produced levels of H2O2 (gt10 microM) which caused cytotoxicity in HL-60 cells when








105



was partially abolished by the catalase indicating that the antiproliferative activity of

powder-derived ACE was partially related to the toxicity of H2O2 generated in medium

To investigate the anti-inflammatory properties of ACEs two cell models were used

to examine the ability of ACEs to modulate pro-inflammatory cytokine release from

immune cells It was found that Hull powder ACE inhibited lipid A-induced Interleukin-

12 (IL-12) release from mouse bone marrow-derived dendritic cells In addition post-

treatment of puree and powder-derived ACEs prepared from Hull Chester and Black

Satin cultivars were showed to inhibit Interleukin-6 (IL-6) and Tumor Necrosis Factor-α

(TNF-α) release in lipopolysaccharides (LPS)-activated J774A1 macrophages and the

inhibition potencies were comparable among cultivars However it is worth noting that in

another experiment we pretreated the J774A1 macrophage with ACEs followed by LPS

stimulation This may suggest that the sequence of application of ACEs before or after


Further mechanism studies are essential to understand the possible role of ACEs in


To investigate the role of the phenolic compounds in the bioactivity of ACE the

phenolic components from non-phenolic components in ACEs derived from both puree

and powder were separated and evaluated for their antioxidant activity and

antiproliferative properties on cancer cells A simple solid phase extraction (SPE) method

was successfully developed to prepare phenolics-enriched methanol fractions (MFs) and

ethyl acetate fractions (EAFs) and non-phenolic water fractions (WFs) MFs contained

most of the anthocyanins present in ACEs possess the highest antioxidant activity and

most potent antiproliferative activity on leukemia (HL-60) and colon (HT-29 HCT-15)

cancer cells among all three fractions MFs derived from both puree and powder ACEs as

well as puree-derived ACE dose-dependently enhanced intracellular ROS levels and

caspase 3-like activity However N-acetyl-L-cysteine a cell permeable antioxidant failed

to block the antiproliferative effects as well as caspase 3-like activity induced by puree

MF and ACE whereas NAC significantly rescued the cell death and decreased caspase 3-

like activity induced by powder MF These results suggested puree ACE and

106

corresponding MF induced cell death through ROS-independent caspase 3 pathway and

the cytotoxicity induced by MF derived from powder ACE is related to ROS mechanism

In conclusion in these studies anthocyanin-containing phenolic extracts were

prepared from blackberries grown in Kentucky and were shown to possess significant

antioxidant antiproliferative and anti-inflammatory properties The phenolic-enriched

fractions were separated and were found to have potent antioxidant and antiproliferative

activities The difference of anticancer mechanism between puree and powder-derived

ACEs or their bioactive polyphenol-enriched fractions in HL-60 cells may be attributed

to the different major active components that contribute to their cytotoxic activity

resulting in different anticancer potency in HL-60 cells and similarly in HT-29 HCT-15

and MCF-7 cells Along these lines future studies would focus on further purification

and identification of principal active constituents in these fractions Their effects in

different in vitro and in vivo models which represent various cancer development stages

should be investigated Moreover their possible toxicity on normal cells should also be

evaluated In addition to the current studies on the anticancer mechanisms related to ROS

generation future studies should attempt to elucidate the important mechanisms of the

bioactive constituents that are related to their abilities to interact with cellular receptors

enzymes and transporters and influence gene expression cell signaling and cell

adhesion Furthermore the epidemiology and human studies suggested that a low risk of

cancer is more closely related to high consumption of whole foods than an individual

antioxidant It is now widely believed that the health benefit of fruits and vegetables is

attributed to the synergistic and additive effects of a combination phytochemical

antioxidants but not a single antioxidant in them The synergistic and additive effects of

different fractions separated from whole blackberry extracts presented in this dissertation

were just a beginning of the studies in this area Future studies with thorough

investigation of different individual or group of compounds in the blackberry extracts

may lead to designing of a phytochemical cocktail that contains agents with different

modes of action to provide superior anticancer efficacy and minimized toxicity In

addition as we gain more knowledge on bioavailability metabolism and tissue

disposition of phytochemical phenolics as well as their interaction with other

pharmaceutical drugs further improvements in this novel strategy in prevention and

107

therapy of cancer and inflammatory diseases may be achieved by carefully selecting

administration route and formulation development


108

Appendices

This section contains the following information and additional experiments

bull Appendix A An in vitro investigation of the effects of the blackberry extracts on

UVR-induced cell death on human epidermal keratinocytes (HEK)

bull Appendix B Antimicrobial and anti-viral properties of blackberry extract

bull Appendix C Standard Operating Procedures (SOPs)

C1 Preparation of blackberry ACEs from blackberry puree or lyophilized puree

(powder)

C2 Total anthocyanin measurement (pH Differential Method)

C3 Total phenolics measurement (Folin-Ciocalteu Method)

C4 Total antioxidant capacity measurement (Trolox Antioxidant Equivalent

Assay)

bull Appendix D Preparation of Vanishing Cream Containing Blackberry ACE

bull Appendix E Preparation of Mucoadhesive Gel Containing Freeze Dried Black

Raspberry (FDBR) Powder

bull Appendix F Stabilization of Anthocyanin in Dried Blackberry ACE by Mannitol

109

Appendix A

An In Vitro Investigation of the Effects of the Blackberry Extracts on UV radiation-

Induced Cell Death on Human Epidermal Keratinocytes (HEK)

UV radiation in the wavelength range between 290-320 nm (UVB) has proved to be

one of the major environmental factors that cause skin aging [314] cancer [315] and

immune suppression [316] UV radiation can alter cell functions via DNA damage

generation of reactive oxygen species (ROS) and activation of biochemical pathways

that related to inflammation and apoptosis Many plant phenolic extracts or compounds

such as green tea polyphenols grape seed proanthocyanidins and anthocyanins were

found to protect UVR-induced skin damage in in vitro and in vivo systems [216 219

317] In these studies blackberry ACEs were evaluated for their ability to protect UVR-

induced cell death in HEK

HEK (Invitrogen Inc Carlsbad CA) were irradiated with UVB light (280 ndash 315 nm

peak 302 nm) then treated with powder or puree-derived ACE prepared from Hull

blackberries (2007) for 24 h Cell viability was measured by a CellTiter-Glo Luminescent

Cell Viability Assay (Promega Corp Madison WI) at 24 h As expected UV radiation

decreased cell viability from 705 plusmn 44 to 357 plusmn 20 in a dosing range of 30-60

mJcm2 (Figure A1) Post-treatment of Hull powder-derived ACE dose-dependently

increased cell viability up to about 15 at a dosing range of 280-560 μgml as compared

to non-treatment groups for all the UVB doses tested Similar results also found in puree-

derived ACE (data not shown) Our preliminary results warrants further investigation of

blackberry ACEs in this area

110

Figure A 1 Protective effects of powder-derived ACE from Hull blackberries (2007)

on UV radiation-induced cell death in HEK HEK were irradiated with UVB light (280

ndash 315 nm peak 302 nm) and then treated with Hull powder extract for 24 h Cell viability

was measured by CellTiter-Glo luminescent cell viability assay at 24 h Data are

presented as mean plusmn SE (n = 3)

111

Appendix B

Antimicrobial and Anti-viral Properties of Blackberry Extract

In these studies anthocyanin-containing extract (ACE) derived from Hull

blackberries puree (2007) was used

Antimicrobial properties of blackberry extract

We have examined a number of exogenous and endogenous host factors for

antimicrobial activity These studies have included standard bacterial cultivation

assessments as well as the use of various colorimetric detection systems The results in

Figure B1 demonstrate the effects of the extract on the survival and metabolic activity of

two oral bacteria (S gordonii-gram-positive A actinomycetemcomitans-gram-negative)

during a 24 h incubation with the extract The results show a clear dose response

inhibition of bacterial growth by the extract with an apparent greater effect on the gram-

negative bacteria versus the gram-positive species that were examined

Anti-viral properties of blackberry extract

The extract was evaluated for its antiviral properties against herpes simplex virus

(HSV)-1 based on inhibition of virus-induced cytopathic effects Preliminary

cytotoxicity assay results indicated that cell viability was not adversely affected by any of

the doses of the extract (data not shown) HSV-1 was adsorbed onto Vero cells (MOI = 5)

in the presence or absence of extract for 1 h in triplicate in a 48-well plates After

removing the inoculum and rinsing the wells 3 times with PBS infections proceeded with

or without extract at 37 oC for 24 h Cell lysates were prepared by freeze-thawing 3 times

and serial dilutions were titrated onto Vero cells Results shown in Figure B2

demonstrated that 131 mgml of extract inhibited virus-induced CPE by 50 (EC50)

Acknowledgements

I would like to thank Dr Millerrsquos lab in the University of Kentucky for performing

these studies

112

0

20

40

60

80

100

0000 0003 0005 0011 0022 0044 0088 0175 0350 0700 1400

Extract (mgmL)

In

hibi

tion S gordonii

A actino

Figure B 1 Antimicrobial effects of blackberry extract Extract was added to oral

bacteria S gordonii or A actinomycetemcomitans over a 24 h interval WST-1 was then

added as a colorimetric measure of bacterial survival and metabolic activity Study was

performed by Dr Craig Miller and colleagues at the University of Kentucky

113

R

educ

tion

in C

PE

0

20

40

60

80

100

120

14 7 35 175 0875

EC50

Extract (mgmL)

R

educ

tion

in C

PE


inhibition assay Results of pharmacological dose-response performed in triplicate 48-

wells Lysates titrated onto Vero cells after freeze-thawing were evaluated in a direct

plaque assay Study was performed by Dr Craig Miller and colleagues at the University

of Kentucky

114

Appendix C

Standard Operating Procedures (SOPs)

C1 Preparation of blackberry ACE from blackberry puree or powder

Materials

Blackberry puree or lyophilized blackberry puree (powder)

Hydrogen chloride (HCl) ~125 M in ethanol Sigma Cat No 17934

Ethyl alcohol (200 proof absolute anhydrous ACS-USP grade) Pharmco-AAPER

Container for extraction glass bottle

Table top sonnicator Branson 3510

Whatman filter paper Fisher Cat No 5802-185

Procedure 1 Prepare 10 ml Stock Solution of ethanol containing 1 HCl Add 219 ml Hydrogen

chloride ~125 M in ethanol to 781 ml ethyl alcohol and mix well

2 Prepare 25 ml ethanol containing 001 HCl Dilute 250 microl of stock solution of

ethanol containing 1 HCl to 25 ml with 200 proof ethyl alcohol

3 To the glass bottle add 25 ml of ethanol containing 001 HCl and 1 gram of

blackberry powder or 10 gram of puree and shake to disperse and wet the powder or

puree

4 Ultrasound-sonicate the powder suspension in icy water for 30 min The lid of the

container should be loosened

5 Filter the powder suspension through the filter paper and collect the filtrate after

sonication

6 Evaporate the ethanol in the filtrate using a rotary evaporator under vacuum at 37 degC

7 Re-suspend the dried residue in 25 ml of distilled water and filter the suspension

through the filter paper and collect the filtrate (extract solution)

8 Remove water in the extract solution by lyophilization Accurately weigh 14 g dried

extract and add to 10 ml water Record the weight This is the weight that will be

used to normalize the TA TP and TEAC values

9 Store at -80 degC (preferred) or -20 oC until assayed

115


Materials

Blackberry extract (in water)

Potassium chloride Sigma Cat No P3911

Sodium acetate Sigma Cat No 236500

Concentrated hydrogen chloride (HCl) 365-385 Sigma Cat No 320331

1 liter glass bottles

Procedure

1 Prepare 0025 M potassium chloride buffer pH 10 Mix 186 g KCl and 980 ml of

distilled water in a beaker Measure the pH and adjust to 10 with concentrated HCl

Transfer to a 1 liter volumetric flask and fill to 1 liter with distilled water

2 Prepare 04 M sodium acetate buffer pH 45 Mix 5443 g CH3CO2Namiddot3 H2O and

~960 ml distilled water in a beaker Measure the pH and adjust to 45 with

concentrated HCl Transfer to a 1 liter volumetric flask and fill to 1 liter with distilled

water

3 Determine the appropriate dilution factor for the sample by diluting with 0025 M

potassium chloride buffer pH 10 until the absorbance of the sample at the λvis-max

(510 nm) is within the linear range of the spectrophotometer (ie for most

spectrophotometers the absorbance should be less than 12) Divide the final volume

of the sample by the initial volume to obtain the dilution factor (DF)

4 Prepare two dilutions of the sample one with potassium chloride buffer pH 10 and

the other with sodium acetate buffer pH 45 diluting each by the previously

determined dilution factor (step 3) Let these dilutions equilibrate for 15 min

5 Measure the absorbance of each dilution at the λvis-max (510 nm) and at 700 nm (to

correct for haze) against a blank cell filled with distilled water

6 Calculate the absorbance of the diluted sample (A) as follows

A = (A510ndash A700) pH 10 ndash (A510ndash A700) pH 45

7 Calculate the monomeric anthocyanin pigment concentration in the original sample

using the following formula

C (mgL) = (A times MW times DF times 1000)( ε times 1)

116

(MW = 4492 ε = 26900 for cyanidin-3-glucoside)

8 To calculate ldquoTotal Anthocyanins (TA)rdquo per gram of ldquoBlackberry Concentraterdquo (BC)

complete the following calculation

TA (mgg BC) = C (mgliter) times Volume of BC stock (liter)Weight of dried BC in the

stock (g)

9 To meet specification Total Anthocyanins (TA) should be in the range of 34-52

mggram Blackberry Concentrate with a target concentration of 43 mggram

Notes

1 When diluting sample with buffers in order to not exceed the buffers capacity the

sample should not exceed 20 of the total volume

2 All measurements should be made between 15 min and 1 h after sample preparation

since longer standing times tend to increase observed readings

3 The buffers should be stable at room temperature for a few months but the pH should

be checked and adjusted prior to use

117


Materials


Folin-Ciocalteursquos phenol reagent 2N Sigma Cat No F9252

Gallic acid Sigma Cat No G7384

Sodium carbonate anhydrous Sigma Cat No 222321

Glass or plastic vials with minimum volume of 2 ml

100-ml volumetric flasks

Glass bottles

Procedure

1 Prepare Gallic acid stock solution In a 100-ml volumetric flask dissolve 0500 g of

dry gallic acid in 10 ml of ethanol and dilute to volume with distilled water To store

keep closed in a refrigerator up to two weeks

2 Prepare saturated sodium carbonate solution Dissolve 200 g of anhydrous sodium

carbonate in 800 ml of distilled water and bring to a boil After cooling add a few

crystals of sodium carbonate and after 24 h filter and add water to 1 liter

3 Prepare gallic acid standard working solutions Add 0 4 8 10 16 and 20 ml of the

gallic acid stock solution into 100 ml volumetric flasks and then dilute to volume

with distilled water These solutions will have phenol concentrations of 0 200 400

500 800 and 1000 mgliter gallic acid the effective range of the assay

4 From each gallic acid working solution sample or blank pipet 20 microl into separate

cuvettes and to each add 158 ml distilled water and then add 100 microl of the Folin-

Ciocalteu phenol reagent and mix well Wait for between 30 sec and 8 min and then

add 300 microl of the sodium carbonate solution and shake to mix

5 Leave the solutions at 20 degC for 2 h and determine the absorbance of each solution at

765 nm against the blank (the 0 ml solution)

6 Plot absorbance vs concentration of the gallic acid working solutions to obtain a

standard curve

7 Calculate the total phenol content (gallic acid equivalent GAE) in the extract based

on the standard curve as follows

118

Concentration (mg GAEliter) = (A765 nmndash Intercept)Slope

8 To calculate ldquoTotal phenol (TP)rdquo per gram of ldquoBlackberry Concentraterdquo (BC)


TP (mg GAEg BC) = C (mg GAEliter) times Volume of BC stock (liter)Weight of

dried BC in the stock (g)

9 To meet specification Total Phenols (TP) should be in the range of 70-106 mggram

Blackberry Concentrate with a target concentration of 88 mggram

Notes

1 The extract may be diluted such that the absorbance would fit into the range of the

standard curve The extract with a concentration of 140 mgml may be diluted 25-5

times with water before the assay

119


Materials


22prime-Azinobis(3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS) Sigma

Cat No 11557

6-hydroxy-2578-tetramethychroman-2-carboxylic acid (Trolox) Sigma Cat No

391913

Potassium persulfate Sigma Cat No 216224


20 ml-glass vials

50 ml-plastic centrifuge tubes

Procedure

1 Prepare ABTSbull+ stock solution Add 384 mg ABTS to 10 ml distilled water in 20 ml-

glass vial then add 662 mg potassium persulfate to the solution and store it for 16 h

in the dark at room temperature

2 Prepare 25 mM Trolox stock solution Add 1252 mg Trolox to 20 ml of ethanol in

20 ml-glass vial

3 Prepare Trolox standard working solutions which can cause 20 to 80 of inhibition

of the blank absorbance Dilute the Trolox stock solution using distilled water with

dilution factors of 3 4 5 7 9 and 18

4 Zero the spectrophotometer with distilled water at 734 nm

5 Prepare ABTSbull+ working solution Dilute the ABTSbull+ stock solution with distilled

water to an absorbance of 070 plusmn 002 in a 50 ml-plastic centrifuge tube and

equilibrate at 30 degC The dilution factor is about 60

6 Determine the appropriate dilution factor for the sample by diluting with distilled

water For the blackberry extract with a concentration of 140 mgml the dilution

factor is around 20-50

7 Add 980 microl of ABTSbull+ working solution into the cuvette and measure the absorbance

at 734 nm (A 0 min) From each Trolox working solution and sample pipet 20 microl into

980 microl of ABTSbull+ working solution in separate cuvettes Mix well and measure the

120

absorbance at 734 nm at 30 degC for 7 min in a kinetic mode The change in absorbance

at 7 min should reach plateau Record the absorbance at 7 min (A 7 min)

8 Calculate the percentage of inhibition of absorbance of each sample as follows

of inhibition = (A 0 minndash A 7 min) times 100 A 0 min

9 Plot of inhibition vs concentration of the ABTSbull+ working solutions to obtain a

standard curve

10 Calculate the Trolox equivalent (TE) concentration of each sample based on the

standard curve using the following equation

C (microM TE) = ( of inhibition ndash Intercept)Slope

11 To calculate ldquoTotal Antioxidant Capacity (TAC)rdquo per gram of ldquoBlackberry

Concentraterdquo (BC) complete the following calculation

TAC (micromol TEg BC) = C (microM TE) times Volume of BC stock (L)Weight of dried BC

in the stock (g)

12 To meet specification Trolox Equivalent Antioxidant Capacity (TEAC) should be in

the range of 40-60 micromolegram Blackberry Concentrate with a target

concentration of 50 micromolegram

Reference

1 R Re N Pellegrini A Proteggente A Pannala M Yang and C Rice-Evans

Antioxidant activity applying an improved ABTS radical cation decolorization assay

Free Radic Biol Med 261231-1237 (1999)

Notes

1 Trolox stock and standard working solution should be prepared freshly and the standard

curve should be made up every time of measurement

2 Use Parafilm to seal the cuvette when mixing samples with ABTSbull+ working solution

121

Appendix D

Preparation of Vanishing Cream Containing Blackberry ACE

The placebo creams were prepared in stainless steel vessels using a Caframo Stirrer

Model BDC-1850 (Wiarton Ontario Canada) with attached metal blade according to the

formula given in Table D1 The ingredients of water phase were heated to 70 degC After

the ingredients of the oil phase were melted at about 60 degC the oil phase was mixed with

the water phase at 70 degC for 05 h at 1000 rpm Then the combined phases were allowed

to mix at room temperature for another 2 h at 1000 rpm and left to sit at room

temperature overnight To prepare cream containing 10 blackberry ACE 90 (ww)

placebo cream was mixed with 10 (ww) blackberry ACE 25 N sodium hydroxide

(NaOH) was used to adjust pH to 35-40

122

Table D 1 Formula for Placebo Vanishing Cream

Ingredients (ww) Water 798 Propylene Glycol 30 Sorbitol 70 20 Sorbic Acid 02 Butylated Hydroxytoluene 01 Simethicone 01

Sub-total Water Phase 852 Petrolatum 56 Cetostearyl Alcohol 44 Brij 58 40 Glyceryl Monostearate 02 PEG 400 Monostearate 06

Sub-total Oil Phase 148 Total 100

123

Appendix E

Preparation of Mucoadhesive Gel Containing Freeze-Dried Black Raspberries

(FDBR) Powder

Gels were prepared at room temperature in stainless steel vessels using a Caframo

Stirrer Model BDC-1850 (Wiarton Ontario Canada) with attached metal blade according

to the formula provided in Table E1 To the required amount of deionized water stirring

in the vessel Noveon AA1 and Carbopol 971P were added slowly and allowed to fully

hydrate for at least 1 h at 1025 rpm Next glycerin 2-phenoxyethanol and benzyl

alcohol were added followed by edetate disodium (EDTA) and the mixing speed was

reduced to 875 rpm The gel was allowed to mix for another 1 h at room temperature at

875 rpm Placebo gels were semi-transparent and homogenous in appearance Finally

powdered FBR was added at room temperature while stirring at 875 rpm to produce final

concentrations of FBR of either 10 ww Either 25 N sodium hydroxide (NaOH) or

233 N HCl was added to adjust to the desired pH Deionized water was then added to

qs the gels to weight The berry gels were mixed for an additional 15 min to produce

homogenous berry gels

These gels were evaluated for anthocyanin stability absorption and penetration into

human oral mucosa both in-vitro and in-vivo and these studies were published in

Mallery SR Stoner GD Larsen PE Fields HW Rodrigo KA Schwartz SJ Tian Q Dai

J Mumper RJ Formulation and in-vitro and in-vivo evaluation of a mucoadhesive gel

containing freeze dried black raspberries implications for oral cancer chemoprevention

Pharmaceutical Research 2007 24(4) 728-37

124

Table E 1 Formula of Mucoadhesive Gels Containing Freeze-Dried Black Raspberries (FDBR) Powder

Ingredient

10 FDBR Gel pH 30 (ww)



10 FDBR Gel with 01 AA

pH 30 (ww)


pH 35 (ww) Water 83925 83975 8379 8382 83875

Noveon AA1 NF 135 135 135 135 135 Carbopol 971P NF 1575 1575 1575 1575 1575

Glycerin USP 10 10 10 10 10 EDTA USP 01 01 01 01 01

2-phenoxyethanol 10 10 10 10 10 Benzyl alcohol USP 10 10 10 10 10

25N NaOH N N ～0185 N N 233N HCl ～005 N N ～0055 N

Ascorbic Acid N N N 01 01 FBDR 10 10 10 10 10

125

Appendix F

Stabilization of Anthocyanin in Dried Blackberry ACE by Mannitol

To develop viable commercial products we have investigated methods to stabilize the

anthocyanins preferably in a dry form and at room temperature One strategy has been to

produce a lyophilized powder blend with mannitol a known cryoprotectant Powder-

derived Hull blackberry extract was mixed with mannitol in a weight ratio of ~110 ww

corresponding to 77 mg extract and 80 mg mannitol per ml This was then lyophilized to

produce a dried berry extract with mannitol having a pH when rehydrated with water in

the range of 25-26 In addition blackberry extract alone (without mannitol) was

lyophilized to produce a dried berry extract A stability study was then performed to

ascertain the stability of anthocyanins under various storage conditions 1) whole freeze-

dried blackberry powder stored at -20 oC as a control 2) dried berry extract stored at 4 ordmC

and 25 ordmC and 3) dried berry extract with mannitol stored at 4 ordmC and 25 ordmC The data

partially summarized in Figure F1 demonstrated that mannitol significantly stabilized the

anthocyanins at room temperature for at least 2 months whereas anthocyanins in the

absence of mannitol were less stable

Acknowledgements

I would like to thank the Center of Pharmaceutical Sciences and Technology in the

University of Kentucky (now Coldstream Laboratories Inc) for their assistance in

performing these studies

126


anthocyanins at 25 degC over 2 months Studies were performed with assistance by

colleagues in the Center of Pharmaceutical Sciences and Technology in the University of

Kentucky (now Coldstream Laboratories Inc)


0

25

50

75

100

0 1 2 0 1 2

Months at 25oCDried Berry Extract

0

25

50

75

100

0 1 2 0 1 2

Dried Berry Extract with Mannitol

Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside

Cn-3-malonyl-glucoside

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Fig 4 Dried berry extract with mannitol shows enhanced retention of anthocyanins at 25oC over 2 months

127

References

1 Ames BN MK Shigenaga and TM Hagen Oxidants antioxidants and the degenerative diseases of aging Proc Natl Acad Sci U S A 1993 90(17) p 7915-22

2 Kampa M AP Nifli G Notas and E Castanas Polyphenols and cancer cell growth Rev Physiol Biochem Pharmacol 2007 159 p 79-113

3 Ramos S Cancer chemoprevention and chemotherapy dietary polyphenols and signalling pathways Mol Nutr Food Res 2008 52(5) p 507-26

4 Nichenametla SN TG Taruscio DL Barney and JH Exon A review of the effects and mechanisms of polyphenolics in cancer Crit Rev Food Sci Nutr 2006 46(2) p 161-83

5 Santangelo C R Vari B Scazzocchio R Di Benedetto C Filesi and R Masella Polyphenols intracellular signalling and inflammation Ann Ist Super Sanita 2007 43(4) p 394-405

6 de Kok TM SG van Breda and MM Manson Mechanisms of combined action of different chemopreventive dietary compounds a review Eur J Nutr 2008 47 Suppl 2 p 51-9

7 Liu RH Potential synergy of phytochemicals in cancer prevention mechanism of action J Nutr 2004 134(12 Suppl) p 3479S-3485S

8 Bagchi D CK Sen M Bagchi and M Atalay Anti-angiogenic antioxidant and anti-carcinogenic properties of a novel anthocyanin-rich berry extract formula Biochemistry (Mosc) 2004 69(1) p 75-80 1 p preceding 75

9 Halvorsen BL MH Carlsen KM Phillips SK Bohn K Holte DR Jacobs Jr and R Blomhoff Content of redox-active compounds (ie antioxidants) in foods consumed in the United States Am J Clin Nutr 2006 84(1) p 95-135

10 Moyer RA KE Hummer CE Finn B Frei and RE Wrolstad Anthocyanins phenolics and antioxidant capacity in diverse small fruits vaccinium rubus and ribes J Agric Food Chem 2002 50(3) p 519-25

11 Pantelidis GE Vasilakakis M Manganaris G A Diamantidis Gr Antioxidant capacity phenol anthocyanin and ascorbic acid contents in raspberries blackberries gooseberries and Cornelian cherries Food Chemistry 2007 102 p 777 - 783

12 DArchivio M C Filesi R Di Benedetto R Gargiulo C Giovannini and R Masella Polyphenols dietary sources and bioavailability Ann Ist Super Sanita 2007 43(4) p 348-61

13 Khanbabaee K and T van Ree Tannins classification and definition Nat Prod Rep 2001 18(6) p 641-9

14 Koleckar V K Kubikova Z Rehakova K Kuca D Jun L Jahodar and L Opletal Condensed and hydrolysable tannins as antioxidants influencing the health Mini Rev Med Chem 2008 8(5) p 436-47

15 Rasmussen SE H Frederiksen K Struntze Krogholm and L Poulsen Dietary proanthocyanidins occurrence dietary intake bioavailability and protection against cardiovascular disease Mol Nutr Food Res 2005 49(2) p 159-74

128

16 Arts IC and PC Hollman Polyphenols and disease risk in epidemiologic studies Am J Clin Nutr 2005 81(1 Suppl) p 317S-325S

17 Hertog MG EJ Feskens PC Hollman MB Katan and D Kromhout Dietary flavonoids and cancer risk in the Zutphen Elderly Study Nutr Cancer 1994 22(2) p 175-84

18 Cole GM GP Lim F Yang B Teter A Begum Q Ma ME Harris-White and SA Frautschy Prevention of Alzheimers disease Omega-3 fatty acid and phenolic anti-oxidant interventions Neurobiol Aging 2005 26 Suppl 1 p 133-6

19 Abascal K L Ganora and E Yarnell The effect of freeze-drying and its implications for botanical medicine a review Phytother Res 2005 19(8) p 655-60

20 Asami DK YJ Hong DM Barrett and AE Mitchell Comparison of the total phenolic and ascorbic acid content of freeze-dried and air-dried marionberry strawberry and corn grown using conventional organic and sustainable agricultural practices J Agric Food Chem 2003 51(5) p 1237-41

21 Xu BJ and SK Chang A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents J Food Sci 2007 72(2) p S159-66

22 Metivier RP FJ Francis and FM Clydesdale Solvent extraction of anthocyanins from wine pomace J Food Sci 1980 45 p 1099-1100

23 Prior RL SA Lazarus G Cao H Muccitelli and JF Hammerstone Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium spp) using high-performance liquid chromatographymass spectrometry J Agric Food Chem 2001 49(3) p 1270-6

24 Guyot S N Marnet and J Drilleau Thiolysis-HPLC characterization of apple procyanidins covering a large range of polymerization states J Agric Food Chem 2001 49(1) p 14-20

25 Labarbe B V Cheynier F Brossaud JM Souquet and M Moutounet Quantitative fractionation of grape proanthocyanidins according to their degree of polymerization J Agric Food Chem 1999 47(7) p 2719-23

26 Shi J H Nawaz J Pohorly G Mittal Y Kakuda and Y Jiang Extraction of polyphenolics from plant material for functional foods-engineering and technology Food Rev Int 2005 21(1) p 139-166

27 Nicoue EE S Savard and K Belkacemi Anthocyanins in wild blueberries of Quebec extraction and identification J Agric Food Chem 2007 55(14) p 5626-35

28 Jackman RL RY Yada MA Tung and RA Speers Anthocyanins as food colorants - a review J Food Biochem 1987 11(3) p 201-47

29 Revilla E J-M Ryan and G Martin-Ortega Comparison of Several Procedures Used for the Extraction of Anthocyanins from Red Grapes J Agric Food Chem 1998 46(11) p 4592-4597

30 Cacace JE and G Mazza Extraction of anthocyanins and other phenolics from black currants with sulfured water J Agric Food Chem 2002 50(21) p 5939-46

31 Robards K Strategies for the determination of bioactive phenols in plants fruit and vegetables J Chromatogr A 2003 1000(1-2) p 657-91

129

32 Havlikova L and K Mikova Heat stability of anthocyanins Z Lebensm-Unters Forsch 1985 181(5) p 427-32

33 Vinatoru M An overview of the ultrasonically assisted extraction of bioactive principles from herbs Ultrason Sonochem 2001 8(3) p 303-13

34 Laborde JL C Bouyer JP Caltagirone and A Gkard Acoustic bubble cavitation at low frequencies Ultrasonics 1998 36 p 589-594

35 Mason TJ L Paniwnyk and JP Lorimer The uses of ultrasound in food technology Ultrason Sonochem 1996 3(3) p S253-S260

36 Vinatoru M M Toma O Radu PI Filip D Lazurca and TJ Mason The use of ultrasound for the extraction of bioactive principles from plant materials Ultrason Sonochem 1997 4(2) p 135-9

37 Albu S E Joyce L Paniwnyk JP Lorimer and TJ Mason Potential for the use of ultrasound in the extraction of antioxidants from Rosmarinus officinalis for the food and pharmaceutical industry Ultrason Sonochem 2004 11(3-4) p 261-5

38 Yang Y and F Zhang Ultrasound-assisted extraction of rutin and quercetin from Euonymus alatus (Thunb) Sieb Ultrason Sonochem 2008 15(4) p 308-13

39 Herrera MC and MD Luque de Castro Ultrasound-assisted extraction for the analysis of phenolic compounds in strawberries Anal Bioanal Chem 2004 379(7-8) p 1106-12

40 Rostagno MA M Palma and CG Barroso Ultrasound-assisted extraction of soy isoflavones J Chromatogr A 2003 1012(2) p 119-28

41 Hromadkova Z Z Kostalova and A Ebringerova Comparison of conventional and ultrasound-assisted extraction of phenolics-rich heteroxylans from wheat bran Ultrason Sonochem 2008 15(6) p 1062-8

42 Herrera MC and MD de Castro Ultrasound-assisted extraction of phenolic compounds from strawberries prior to liquid chromatographic separation and photodiode array ultraviolet detection J Chromatogr A 2005 1100(1) p 1-7

43 Benthin B H Danz and M Hamburger Pressurized liquid extraction of medicinal plants J Chromatogr A 1999 837(1-2) p 211-9

44 Mendiola JA M Herrero A Cifuentes and E Ibanez Use of compressed fluids for sample preparation food applications J Chromatogr A 2007 1152(1-2) p 234-46

45 Richter BE BA Jones JL Ezzell NL Porter N Avdalovic and C Pohl Accelerated Solvent Extraction A Technique for Sample Preparation Anal Chem 1996 68(6) p 1033-9

46 Ju ZY and LR Howard Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin J Agric Food Chem 2003 51(18) p 5207-13

47 Palma M Z Pineiro and CG Barroso Stability of phenolic compounds during extraction with superheated solvents J Chromatogr A 2001 921(2) p 169-74

48 Pineiro Z M Palma and CG Barroso Determination of trans-resveratrol in grapes by pressurised liquid extraction and fast high-performance liquid chromatography J Chromatogr A 2006 1110(1-2) p 61-5

130

49 Luque-Rodriguez JM MD Luque de Castro and P Perez-Juan Dynamic superheated liquid extraction of anthocyanins and other phenolics from red grape skins of winemaking residues Bioresour Technol 2007 98(14) p 2705-13

50 Alonso-Salces RM E Korta A Barranco LA Berrueta B Gallo and F Vicente Pressurized liquid extraction for the determination of polyphenols in apple J Chromatogr A 2001 933(1-2) p 37-43

51 Howard L and N Pandjaitan Pressurized liquid extraction of flavonoids from spinach J Food Sci 2008 73(3) p C151-7

52 Luthria DL and S Mukhopadhyay Influence of sample preparation on assay of phenolic acids from eggplant J Agric Food Chem 2006 54(1) p 41-7

53 Bonoli M E Marconi and MF Caboni Free and bound phenolic compounds in barley (Hordeum vulgare L) flours evaluation of the extraction capability of different solvent mixtures and pressurized liquid methods by micellar electrokinetic chromatography and spectrophotometry J Chromatogr A 2004 1057(1-2) p 1-12

54 Palenzuela B L Arce A Macho E Munoz A Rios and M Valcarcel Bioguided extraction of polyphenols from grape marc by using an alternative supercritical-fluid extraction method based on a liquid solvent trap Anal Bioanal Chem 2004 378(8) p 2021-7

55 Wang Z M Ashraf-Khorassani and LT Taylor Feasibility study of online supercritical fluid extraction-liquid chromatography-UV absorbance-mass spectrometry for the determination of proanthocyanidins in grape seeds J Chromatogr Sci 2005 43(3) p 109-15

56 Klejdus B L Lojkova O Lapcik R Koblovska J Moravcova and V Kuban Supercritical fluid extraction of isoflavones from biological samples with ultra-fast high-performance liquid chromatographymass spectrometry J Sep Sci 2005 28(12) p 1334-46

57 Eskilsson CS and E Bjorklund Analytical-scale microwave-assisted extraction J Chromatogr A 2000 902(1) p 227-50

58 Li H B Chen L Nie and S Yao Solvent effects on focused microwave assisted extraction of polyphenolic acids from Eucommia ulmodies Phytochem Anal 2004 15(5) p 306-12

59 Huang J and Z Zhang Microwave-assisted extraction of quercetin and acid degradation of its glycosides in Psidium guajava leaves Anal Sci 2004 20(2) p 395-7

60 Rostagno MA M Palma and CG Barroso Microwave assisted extraction of soy isoflavones Anal Chim Acta 2007 588(2) p 274-82

61 Du FY XH Xiao and GK Li Application of ionic liquids in the microwave-assisted extraction of trans-resveratrol from Rhizma Polygoni Cuspidati J Chromatogr A 2007 1140(1-2) p 56-62

62 Liazid A M Palma J Brigui and CG Barroso Investigation on phenolic compounds stability during microwave-assisted extraction J Chromatogr A 2007 1140(1-2) p 29-34

63 Cacace JE and G Mazza Optimization of extraction of anthocyanins from black currants with aqueous ethanol J Food Sci 2003 68(1) p 240-248

131

64 Pinelo M M Rubilar M Jerez J Sineiro and MJ Nunez Effect of solvent temperature and solvent-to-solid ratio on the total phenolic content and antiradical activity of extracts from different components of grape pomace J Agric Food Chem 2005 53(6) p 2111-7

65 Pinelo M A Arnous and AS Meyer Upgrading of grape skins Significance of plant cell-wall structural components and extraction techniques for phenol release Trends Food Sci Technol 2006 17(11) p 579-590

66 Pinelo M P Del Fabbro L Manzocco MJ Nunez and MC Nicoli Optimization of continuous phenol extraction from Vitis vinifera byproducts Food Chem 2005 92(1) p 109-117

67 Nepote V NR Grosso and CA Guzman Optimization of extraction of phenolic antioxidants from peanut skins J Sci Food Agric 2005 85(1) p 33-38

68 Landbo A-K and AS Meyer Enzyme-Assisted Extraction of Antioxidative Phenols from Black Currant Juice Press Residues (Ribes nigrum) J Agric Food Chem 2001 49(7) p 3169-3177

69 Pinelo M B Zornoza and AS Meyer Selective release of phenols from apple skin Mass transfer kinetics during solvent and enzyme-assisted extraction Sep Purif Technol 2008 63(3) p 620-627

70 Meyer AS Enzymatic upgrading of antioxidant phenolics in berry juices and press residues Fruit Process 2005 15(6) p 382-387

71 Ramirez-Coronel MA N Marnet VS Kolli S Roussos S Guyot and C Augur Characterization and estimation of proanthocyanidins and other phenolics in coffee pulp (Coffea arabica) by thiolysis-high-performance liquid chromatography J Agric Food Chem 2004 52(5) p 1344-9

72 Neergheen VS MA Soobrattee T Bahorun and OI Aruoma Characterization of the phenolic constituents in Mauritian endemic plants as determinants of their antioxidant activities in vitro J Plant Physiol 2006 163(8) p 787-99

73 Zhang Y NP Seeram R Lee L Feng and D Heber Isolation and identification of strawberry phenolics with antioxidant and human cancer cell antiproliferative properties J Agric Food Chem 2008 56(3) p 670-5

74 Thimothe J IA Bonsi OI Padilla-Zakour and H Koo Chemical characterization of red wine grape (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans J Agric Food Chem 2007 55(25) p 10200-7

75 Pinelo M VF Laurie and AL Waterhouse A simple method to separate red wine nonpolymeric and polymeric phenols by solid-phase extraction J Agric Food Chem 2006 54(8) p 2839-44

76 Llorach R A Gil-Izquierdo F Ferreres and FA Tomas-Barberan HPLC-DAD-MSMS ESI characterization of unusual highly glycosylated acylated flavonoids from cauliflower (Brassica oleracea L var botrytis) agroindustrial byproducts J Agric Food Chem 2003 51(13) p 3895-9

77 Moreno YS GS Sanchez DR Hernandez and NR Lobato Characterization of anthocyanin extracts from maize kernels J Chromatogr Sci 2005 43(9) p 483-7

132

78 Kaehkoenen MP J Heinaemaeki V Ollilainen and M Heinonen Berry anthocyanins Isolation identification and antioxidant activities J Sci Food Agric 2003 83(14) p 1403-1411

79 George S P Brat P Alter and MJ Amiot Rapid determination of polyphenols and vitamin C in plant-derived products J Agric Food Chem 2005 53(5) p 1370-3

80 Jeffery DW MD Mercurio MJ Herderich Y Hayasaka and PA Smith Rapid isolation of red wine polymeric polyphenols by solid-phase extraction J Agric Food Chem 2008 56(8) p 2571-80

81 Perez-Magarino S M Ortega-Heras and E Cano-Mozo Optimization of a solid-phase extraction method using copolymer sorbents for isolation of phenolic compounds in red wines and quantification by HPLC J Agric Food Chem 2008 56(24) p 11560-70

82 Queiroz EF JR Ioset K Ndjoko A Guntern CM Foggin and K Hostettmann On-line identification of the bioactive compounds from Blumea gariepina by HPLC-UV-MS and HPLC-UV-NMR combined with HPLC-micro-fractionation Phytochem Anal 2005 16(3) p 166-74

83 Kandil FE MA Smith RB Rogers MF Pepin LL Song JM Pezzuto and DS Seigler Composition of a chemopreventive proanthocyanidin-rich fraction from cranberry fruits responsible for the inhibition of 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced ornithine decarboxylase (ODC) activity J Agric Food Chem 2002 50(5) p 1063-9

84 Pedreschi R and L Cisneros-Zevallos Antimutagenic and antioxidant properties of phenolic fractions from Andean purple corn (Zea mays L) J Agric Food Chem 2006 54(13) p 4557-67

85 Ranilla LG MI Genovese and FM Lajolo Polyphenols and antioxidant capacity of seed coat and cotyledon from Brazilian and Peruvian bean cultivars (Phaseolus vulgaris L) J Agric Food Chem 2007 55(1) p 90-8

86 Hagerman AE and LG Butler Condensed tannin purification and characterization of tannin-associated proteins J Agric Food Chem 1980 28(5) p 947-52

87 Asquith TN CC Izuno and LG Butler Characterization of the condensed tannin (proanthocyanidin) from a group II sorghum J Agric Food Chem 1983 31(6) p 1299-303

88 Ek S H Kartimo S Mattila and A Tolonen Characterization of phenolic compounds from lingonberry (Vaccinium vitis-idaea) J Agric Food Chem 2006 54(26) p 9834-42

89 Agarwal C R Veluri M Kaur SC Chou JA Thompson and R Agarwal Fractionation of high molecular weight tannins in grape seed extract and identification of procyanidin B2-33-di-O-gallate as a major active constituent causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells Carcinogenesis 2007 28(7) p 1478-84

90 Klejdus B and V Kuban High performance liquid chromatographic determination of phenolic compounds in seed exudates of Festuca arundinacea and F pratense Phytochem Anal 2000 11(6) p 375-379

133

91 Guyot S N Marnet D Laraba and J-F Drilleau Reversed-Phase HPLC following Thiolysis for Quantitative Estimation and Characterization of the Four Main Classes of Phenolic Compounds in Different Tissue Zones of a French Cider Apple Variety (Malus domestica Var Kermerrien) J Agric Food Chem 1998 46(5) p 1698-1705

92 Berthod A B Billardello and S Geoffroy Polyphenols in countercurrent chromatography An example of large scale separation Analusis 1999 27(9) p 750-757

93 Degenhardt A H Knapp and P Winterhalter Separation and purification of anthocyanins by high-speed countercurrent chromatography and screening for antioxidant activity J Agric Food Chem 2000 48(2) p 338-43

94 Yanagida A A Shoji Y Shibusawa H Shindo M Tagashira M Ikeda and Y Ito Analytical separation of tea catechins and food-related polyphenols by high-speed counter-current chromatography J Chromatogr A 2006 1112(1-2) p 195-201

95 Krafczyk N and MA Glomb Characterization of phenolic compounds in rooibos tea J Agric Food Chem 2008 56(9) p 3368-76

96 Naczk M and F Shahidi Phenolics in cereals fruits and vegetables occurrence extraction and analysis J Pharm Biomed Anal 2006 41(5) p 1523-42

97 Singleton VL R Orthofer and RM Lamuela-Raventos Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent Methods Enzymol 1999 299(Oxidants and Antioxidants Part A) p 152-178

98 Singleton VL and JA Rossi Jr Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents Am J Enol Vitic 1965 16(3) p 144-58

99 Andersen OM and GW Francis Techniques of pigment identification Annu Plant Rev 2004 14(Plant Pigments and Their Manipulation) p 293-341

100 Fuleki T and FJ Francis Quantitative methods for anthocyanins I Extraction and determination of total anthocyanin in cranberries J Food Sci 1968 33(1) p 72-7

101 Fuleki T and FJ Francis Quantitative methods for anthocyanins II Determination of total anthocyanin and degradation index for cranberry juice J Food Sci 1968 33(1) p 78-83

102 Giusti MM WR Characterization and measurement of anthocyanins by UV-visible spectroscopy In Current Protocols in Food Analytical Chemistry (Wrolstad RE Acree TE An H et al eds) John Wiley amp Sons New York 2001 p F121ndashF1213

103 Wrolstad RE RW Durst MM Giusti and LE Rodriguez-Saona Analysis of anthocyanins in nutraceuticals ACS Symp Ser 2002 803(Quality Management of Nutraceuticals) p 42-62

104 Lee J RW Durst and RE Wrolstad Determination of total monomeric anthocyanin pigment content of fruit juices beverages natural colorants and wines by the pH differential method Collaborative study J AOAC Int 2005 88(5) p 1269-1278

134

105 Porter LJ LN Hrstich and BG Chan The conversion of procyanidins and prodelphinidins to cyanidin and delphinidin Phytochemistry 1986 25(1) p 223-30

106 Goldstein JL and T Swain Changes in tannins in ripening fruits Phytochemistry 1963 2(4) p 371-83

107 McMurrough I and J McDowell Chromatographic separation and automated analysis of flavanols Anal Biochem 1978 91(1) p 92-100

108 Schofield P DM Mbugua and AN Pell Analysis of condensed tannins a review Anim Feed Sci Technol 2001 91(1-2) p 21-40

109 Cunningham DG S Vannozzi E OShea and R Turk Analysis and standardization of cranberry products ACS Symp Ser 2002 803(Quality Management of Nutraceuticals) p 151-166

110 Hartzfeld PW R Forkner MD Hunter and AE Hagerman Determination of hydrolyzable tannins (gallotannins and ellagitannins) after reaction with potassium iodate J Agric Food Chem 2002 50(7) p 1785-90

111 Inoue KH and AE Hagerman Determination of gallotannin with rhodanine Anal Biochem 1988 169(2) p 363-9

112 Wilson TC and AE Hagerman Quantitative determination of ellagic acid J Agric Food Chem 1990 38(8) p 1678-83

113 Stalikas CD Extraction separation and detection methods for phenolic acids and flavonoids J Sep Sci 2007 30(18) p 3268-95

114 Yanagida A T Shoji and T Kanda Characterization of polymerized polyphenols by size-exclusion HPLC Biosci Biotechnol Biochem 2002 66(9) p 1972-5

115 Vrhovsek U A Rigo D Tonon and F Mattivi Quantitation of polyphenols in different apple varieties J Agric Food Chem 2004 52(21) p 6532-8

116 Ruiz D J Egea MI Gil and FA Tomas-Barberan Characterization and quantitation of phenolic compounds in new apricot (Prunus armeniaca L) varieties J Agric Food Chem 2005 53(24) p 9544-52

117 Anttonen MJ and RO Karjalainen High-performance liquid chromatography analysis of black currant (Ribes nigrum L) fruit phenolics grown either conventionally or organically J Agric Food Chem 2006 54(20) p 7530-8

118 Lin LZ and JM Harnly A screening method for the identification of glycosylated flavonoids and other phenolic compounds using a standard analytical approach for all plant materials J Agric Food Chem 2007 55(4) p 1084-96

119 McCallum JL R Yang JC Young JN Strommer and R Tsao Improved high performance liquid chromatographic separation of anthocyanin compounds from grapes using a novel mixed-mode ion-exchange reversed-phase column J Chromatogr A 2007 1148(1) p 38-45

120 Harris CS AJ Burt A Saleem PM Le LC Martineau PS Haddad SA Bennett and JT Arnason A single HPLC-PAD-APCIMS method for the quantitative comparison of phenolic compounds found in leaf stem root and fruit extracts of Vaccinium angustifolium Phytochem Anal 2007 18(2) p 161-9

121 Rodriguez-Bernaldo de Quiros A J Lopez-Hernandez P Ferraces-Casais and MA Lage-Yusty Analysis of non-anthocyanin phenolic compounds in wine

135

samples using high performance liquid chromatography with ultraviolet and fluorescence detection J Sep Sci 2007 30(9) p 1262-6

122 Mertz C V Cheynier Z Gunata and P Brat Analysis of phenolic compounds in two blackberry species (Rubus glaucus and Rubus adenotrichus) by high-performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry J Agric Food Chem 2007 55(21) p 8616-24

123 Pawlowska AM W Oleszek and A Braca Quali-quantitative analyses of Flavonoids of Morus nigra L and Morus alba L (Moraceae) fruits J Agric Food Chem 2008 56(9) p 3377-80

124 Sakakibara H Y Honda S Nakagawa H Ashida and K Kanazawa Simultaneous determination of all polyphenols in vegetables fruits and teas J Agric Food Chem 2003 51(3) p 571-81

125 Downey MO and S Rochfort Simultaneous separation by reversed-phase high-performance liquid chromatography and mass spectral identification of anthocyanins and flavonols in Shiraz grape skin J Chromatogr A 2008 1201(1) p 43-7

126 Oh YS JH Lee SH Yoon CH Oh DS Choi E Choe and MY Jung Characterization and quantification of anthocyanins in grape juices obtained from the grapes cultivated in Korea by HPLCDAD HPLCMS and HPLCMSMS J Food Sci 2008 73(5) p C378-89

127 Naczk M and F Shahidi Extraction and analysis of phenolics in food J Chromatogr A 2004 1054(1-2) p 95-111

128 Robbins RJ Phenolic acids in foods an overview of analytical methodology J Agric Food Chem 2003 51(10) p 2866-87

129 Merken HM and GR Beecher Measurement of food flavonoids by high-performance liquid chromatography A review J Agric Food Chem 2000 48(3) p 577-99

130 Novak I P Janeiro M Seruga and AM Oliveira-Brett Ultrasound extracted flavonoids from four varieties of Portuguese red grape skins determined by reverse-phase high-performance liquid chromatography with electrochemical detection Anal Chim Acta 2008 630(2) p 107-15

131 Woodring PJ PA Edwards and MG Chisholm HPLC determination of non-flavonoid phenols in vidal blanc wine using electrochemical detection J Agric Food Chem 1990 38(3) p 729-32

132 Mattila P J Astola and J Kumpulainen Determination of flavonoids in plant material by HPLC with diode-array and electro-array detections J Agric Food Chem 2000 48(12) p 5834-41

133 de Pascual-Teresa S D Treutter JC Rivas-Gonzalo and C Santos-Buelga Analysis of Flavanols in Beverages by High-Performance Liquid Chromatography with Chemical Reaction Detection J Agric Food Chem 1998 46(10) p 4209-4213

134 Cavaliere C P Foglia R Gubbiotti P Sacchetti R Samperi and A Lagana Rapid-resolution liquid chromatographymass spectrometry for determination and quantitation of polyphenols in grape berries Rapid Commun Mass Spectrom 2008 22(20) p 3089-99

136

135 Christophoridou S and P Dais Detection and quantification of phenolic compounds in olive oil by high resolution 1H nuclear magnetic resonance spectroscopy Anal Chim Acta 2009 633(2) p 283-92

136 Jac P M Polasek and M Pospisilova Recent trends in the determination of polyphenols by electromigration methods J Pharm Biomed Anal 2006 40(4) p 805-14

137 Rice-Evans CA NJ Miller PG Bolwell PM Bramley and JB Pridham The relative antioxidant activities of plant-derived polyphenolic flavonoids Free Radic Res 1995 22(4) p 375-83

138 Rice-Evans CA NJ Miller and G Paganga Structure-antioxidant activity relationships of flavonoids and phenolic acids Free Radic Biol Med 1996 20(7) p 933-56

139 Scalbert A C Manach C Morand C Remesy and L Jimenez Dietary polyphenols and the prevention of diseases Crit Rev Food Sci Nutr 2005 45(4) p 287-306

140 Hollman PC and MB Katan Dietary flavonoids intake health effects and bioavailability Food Chem Toxicol 1999 37(9-10) p 937-42

141 Cotelle N Role of flavonoids in oxidative stress Curr Top Med Chem 2001 1(6) p 569-90

142 Dziedzic SZ and BJF Hudson Polyhydroxy chalcones and flavanones as antioxidants for edible oils Food Chem 1983 12(3) p 205-12

143 Shahidi F and PK Wanasundara Phenolic antioxidants Crit Rev Food Sci Nutr 1992 32(1) p 67-103

144 Bors W and C Michel Chemistry of the antioxidant effect of polyphenols Ann N Y Acad Sci 2002 957 p 57-69

145 Yoshino M and K Murakami Interaction of iron with polyphenolic compounds application to antioxidant characterization Anal Biochem 1998 257(1) p 40-4

146 Perron NR and JL Brumaghim A review of the antioxidant mechanisms of polyphenol compounds related to iron binding Cell Biochem Biophys 2009 53(2) p 75-100

147 Hudson BJF and JI Lewis Polyhydroxy flavonoid antioxidants for edible oils Structural criteria for activity Food Chem 1983 10(1) p 47-55

148 Rice-Evans CA NJ Miller and G Paganga Antioxidant properties of phenolic compounds Trends Plant Sci 1997 2(4) p 152-159

149 Khokhar S and RKO Apenten Iron binding characteristics of phenolic compounds Some tentative structure-activity relations Food Chem 2003 81(1) p 133-140

150 Fraga CG Plant polyphenols how to translate their in vitro antioxidant actions to in vivo conditions IUBMB Life 2007 59(4-5) p 308-15

151 Vinson JA X Su L Zubik and P Bose Phenol antioxidant quantity and quality in foods fruits J Agric Food Chem 2001 49(11) p 5315-21

152 Huang D B Ou and RL Prior The chemistry behind antioxidant capacity assays J Agric Food Chem 2005 53(6) p 1841-56

153 Hagerman AE KM Riedl GA Jones KN Sovik NT Ritchard PW Hartzfeld and TL Riechel High molecular weight plant polyphenolics (tannins) as biological antioxidants J Agric Food Chem 1998 46(5) p 1887-1892

137

154 Cao G CP Verdon AH Wu H Wang and RL Prior Automated assay of oxygen radical absorbance capacity with the COBAS FARA II Clin Chem 1995 41(12 Pt 1) p 1738-44

155 Ou B D Huang M Hampsch-Woodill JA Flanagan and EK Deemer Analysis of antioxidant activities of common vegetables employing oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays a comparative study J Agric Food Chem 2002 50(11) p 3122-8

156 Awika JM LW Rooney X Wu RL Prior and L Cisneros-Zevallos Screening methods to measure antioxidant activity of sorghum (sorghum bicolor) and sorghum products J Agric Food Chem 2003 51(23) p 6657-62

157 Re R N Pellegrini A Proteggente A Pannala M Yang and C Rice-Evans Antioxidant activity applying an improved ABTS radical cation decolorization assay Free Radic Biol Med 1999 26(9-10) p 1231-7

158 Pulido R L Bravo and F Saura-Calixto Antioxidant activity of dietary polyphenols as determined by a modified ferric reducingantioxidant power assay J Agric Food Chem 2000 48(8) p 3396-402

159 Prior RL X Wu and K Schaich Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements J Agric Food Chem 2005 53(10) p 4290-302

160 Seeram NP LS Adams Y Zhang R Lee D Sand HS Scheuller and D Heber Blackberry black raspberry blueberry cranberry red raspberry and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro J Agric Food Chem 2006 54(25) p 9329-39

161 Katsube N K Iwashita T Tsushida K Yamaki and M Kobori Induction of apoptosis in cancer cells by Bilberry (Vaccinium myrtillus) and the anthocyanins J Agric Food Chem 2003 51(1) p 68-75

162 Ross HA GJ McDougall and D Stewart Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts Phytochemistry 2007 68(2) p 218-28

163 McDougall GJ HA Ross M Ikeji and D Stewart Berry Extracts Exert Different Antiproliferative Effects against Cervical and Colon Cancer Cells Grown in Vitro J Agric Food Chem 2008 56 p 3016-3023

164 Damianaki A E Bakogeorgou M Kampa G Notas A Hatzoglou S Panagiotou C Gemetzi E Kouroumalis PM Martin and E Castanas Potent inhibitory action of red wine polyphenols on human breast cancer cells J Cell Biochem 2000 78(3) p 429-41

165 Kampa M A Hatzoglou G Notas A Damianaki E Bakogeorgou C Gemetzi E Kouroumalis PM Martin and E Castanas Wine antioxidant polyphenols inhibit the proliferation of human prostate cancer cell lines Nutr Cancer 2000 37(2) p 223-33

166 Zhang G Y Miura and K Yagasaki Effects of green oolong and black teas and related components on the proliferation and invasion of hepatoma cells in culture Cytotechnology 1999 31(1-2) p 37-44

167 Weisburg JH DB Weissman T Sedaghat and H Babich In vitro cytotoxicity of epigallocatechin gallate and tea extracts to cancerous and normal cells from the human oral cavity Basic Clin Pharmacol Toxicol 2004 95(4) p 191-200

138

168 Sarkar FH and Y Li Mechanisms of cancer chemoprevention by soy isoflavone genistein Cancer Metastasis Rev 2002 21(3-4) p 265-80

169 Manthey JA K Grohmann and N Guthrie Biological properties of citrus flavonoids pertaining to cancer and inflammation Curr Med Chem 2001 8(2) p 135-53

170 McCann MJ CI Gill OB G JR Rao WC McRoberts P Hughes R McEntee and IR Rowland Anti-cancer properties of phenolics from apple waste on colon carcinogenesis in vitro Food Chem Toxicol 2007 45(7) p 1224-30

171 Kuntz S U Wenzel and H Daniel Comparative analysis of the effects of flavonoids on proliferation cytotoxicity and apoptosis in human colon cancer cell lines Eur J Nutr 1999 38(3) p 133-42

172 Knowles LM DA Zigrossi RA Tauber C Hightower and JA Milner Flavonoids suppress androgen-independent human prostate tumor proliferation Nutr Cancer 2000 38(1) p 116-22

173 Gupta S F Afaq and H Mukhtar Selective growth-inhibitory cell-cycle deregulatory and apoptotic response of apigenin in normal versus human prostate carcinoma cells Biochem Biophys Res Commun 2001 287(4) p 914-20

174 Miyahara Y T Komiya H Katsuzaki K Imai M Nakagawa Y Ishi and H Hibasami Sesamin and episesamin induce apoptosis in human lymphoid leukemia Molt 4B cells Int J Mol Med 2000 6(1) p 43-6

175 Zhang M JP Zhang HT Ji JS Wang and DH Qian Effect of six flavonoids on proliferation of hepatic stellate cells in vitro Acta Pharmacol Sin 2000 21(3) p 253-6

176 Kanno S A Tomizawa T Hiura Y Osanai A Shouji M Ujibe T Ohtake K Kimura and M Ishikawa Inhibitory effects of naringenin on tumor growth in human cancer cell lines and sarcoma S-180-implanted mice Biol Pharm Bull 2005 28(3) p 527-30

177 Yang CS P Maliakal and X Meng Inhibition of carcinogenesis by tea Annu Rev Pharmacol Toxicol 2002 42 p 25-54

178 Lambert JD and CS Yang Cancer chemopreventive activity and bioavailability of tea and tea polyphenols Mutat Res 2003 523-524 p 201-8

179 Gerhauser C Cancer chemopreventive potential of apples apple juice and apple components Planta Med 2008 74(13) p 1608-24

180 Thomasset S N Teller H Cai D Marko DP Berry WP Steward and AJ Gescher Do anthocyanins and anthocyanidins cancer chemopreventive pigments in the diet merit development as potential drugs Cancer Chemother Pharmacol 2009

181 Lala G M Malik C Zhao J He Y Kwon MM Giusti and BA Magnuson Anthocyanin-rich extracts inhibit multiple biomarkers of colon cancer in rats Nutr Cancer 2006 54(1) p 84-93

182 Ding M R Feng SY Wang L Bowman Y Lu Y Qian V Castranova BH Jiang and X Shi Cyanidin-3-glucoside a natural product derived from blackberry exhibits chemopreventive and chemotherapeutic activity J Biol Chem 2006 281(25) p 17359-68

183 Huang MT JG Xie ZY Wang CT Ho YR Lou CX Wang GC Hard and AH Conney Effects of tea decaffeinated tea and caffeine on UVB light-

139

induced complete carcinogenesis in SKH-1 mice demonstration of caffeine as a biologically important constituent of tea Cancer Res 1997 57(13) p 2623-9

184 Chung FL M Wang A Rivenson MJ Iatropoulos JC Reinhardt B Pittman CT Ho and SG Amin Inhibition of lung carcinogenesis by black tea in Fischer rats treated with a tobacco-specific carcinogen caffeine as an important constituent Cancer Res 1998 58(18) p 4096-101

185 Netzel M G Strass C Kaul I Bitsch H Dietrich and R Bitsch In vivo antioxidative capacity of a composite berry juice Food Res Int 2002 35(23) p 213-216

186 Netzel M G Strass M Herbst H Dietrich R Bitsch I Bitsch and T Frank The excretion and biological antioxidant activity of elderberry antioxidants in healthy humans Food Res Int 2005 38(8-9) p 905-910

187 Ko SH SW Choi SK Ye BL Cho HS Kim and MH Chung Comparison of the antioxidant activities of nine different fruits in human plasma J Med Food 2005 8(1) p 41-6

188 Mertens-Talcott SU J Rios P Jilma-Stohlawetz LA Pacheco-Palencia B Meibohm ST Talcott and H Derendorf Pharmacokinetics of anthocyanins and antioxidant effects after the consumption of anthocyanin-rich acai juice and pulp (Euterpe oleracea Mart) in human healthy volunteers J Agric Food Chem 2008 56(17) p 7796-802

189 Jensen GS X Wu KM Patterson J Barnes SG Carter L Scherwitz R Beaman JR Endres and AG Schauss In vitro and in vivo antioxidant and anti-inflammatory capacities of an antioxidant-rich fruit and berry juice blend Results of a pilot and randomized double-blinded placebo-controlled crossover study J Agric Food Chem 2008 56(18) p 8326-33

190 Cao G RM Russell N Lischner and RL Prior Serum antioxidant capacity is increased by consumption of strawberries spinach red wine or vitamin C in elderly women J Nutr 1998 128(12) p 2383-90

191 Fernandez-Pachon MS D Villano AM Troncoso and MC Garcia-Parrilla Antioxidant capacity of plasma after red wine intake in human volunteers J Agric Food Chem 2005 53(12) p 5024-9

192 Rein D S Lotito RR Holt CL Keen HH Schmitz and CG Fraga Epicatechin in human plasma in vivo determination and effect of chocolate consumption on plasma oxidation status J Nutr 2000 130(8S Suppl) p 2109S-14S

193 Wang JF DD Schramm RR Holt JL Ensunsa CG Fraga HH Schmitz and CL Keen A dose-response effect from chocolate consumption on plasma epicatechin and oxidative damage J Nutr 2000 130(8S Suppl) p 2115S-9S

194 Serafini M R Bugianesi G Maiani S Valtuena S De Santis and A Crozier Plasma antioxidants from chocolate Nature 2003 424(6952) p 1013

195 Mazza G CD Kay T Cottrell and BJ Holub Absorption of anthocyanins from blueberries and serum antioxidant status in human subjects J Agric Food Chem 2002 50(26) p 7731-7

196 Matsumoto H Y Nakamura M Hirayama Y Yoshiki and K Okubo Antioxidant activity of black currant anthocyanin aglycons and their glycosides

140

measured by chemiluminescence in a neutral pH region and in human plasma J Agric Food Chem 2002 50(18) p 5034-7

197 Vinson JA J Proch and P Bose MegaNatural((R)) Gold Grapeseed Extract In Vitro Antioxidant and In Vivo Human Supplementation Studies J Med Food 2001 4(1) p 17-26

198 Serafini M G Maiani and A Ferro-Luzzi Alcohol-free red wine enhances plasma antioxidant capacity in humans J Nutr 1998 128(6) p 1003-7

199 Alberti-Fidanza A G Burini G Antonelli G Murdolo and G Perriello Acute effects of lyophilised red wine on total antioxidant capacity in healthy volunteers Diabetes Nutr Metab 2003 16(1) p 65-71

200 Kresty LA WL Frankel CD Hammond ME Baird JM Mele GD Stoner and JJ Fromkes Transitioning from preclinical to clinical chemopreventive assessments of lyophilized black raspberries interim results show berries modulate markers of oxidative stress in Barretts esophagus patients Nutr Cancer 2006 54(1) p 148-56

201 Spechler SJ Clinical practice Barretts Esophagus N Engl J Med 2002 346(11) p 836-42

202 Mallery SR GD Stoner PE Larsen HW Fields KA Rodrigo SJ Schwartz Q Tian J Dai and RJ Mumper Formulation and in-vitro and in-vivo evaluation of a mucoadhesive gel containing freeze dried black raspberries implications for oral cancer chemoprevention Pharm Res 2007 24(4) p 728-37

203 Spormann TM FW Albert T Rath H Dietrich F Will JP Stockis G Eisenbrand and C Janzowski Anthocyaninpolyphenolic-rich fruit juice reduces oxidative cell damage in an intervention study with patients on hemodialysis Cancer Epidemiol Biomarkers Prev 2008 17(12) p 3372-80

204 Doll R and R Peto The causes of cancer quantitative estimates of avoidable risks of cancer in the United States today J Natl Cancer Inst 1981 66(6) p 1191-308

205 Stanley LA Molecular aspects of chemical carcinogenesis the roles of oncogenes and tumour suppressor genes Toxicology 1995 96(3) p 173-94

206 Sudheer AR S Muthukumaran N Devipriya and VP Menon Ellagic acid a natural polyphenol protects rat peripheral blood lymphocytes against nicotine-induced cellular and DNA damage in vitro with the comparison of N-acetylcysteine Toxicology 2007 230(1) p 11-21

207 Schaefer S M Baum G Eisenbrand and C Janzowski Modulation of oxidative cell damage by reconstituted mixtures of phenolic apple juice extracts in human colon cell lines Mol Nutr Food Res 2006 50(4-5) p 413-7

208 Higdon JV and B Frei Tea catechins and polyphenols health effects metabolism and antioxidant functions Crit Rev Food Sci Nutr 2003 43(1) p 89-143

209 Burkhardt S RJ Reiter DX Tan R Hardeland J Cabrera and M Karbownik DNA oxidatively damaged by chromium(III) and H(2)O(2) is protected by the antioxidants melatonin N(1)-acetyl-N(2)-formyl-5-methoxykynuramine resveratrol and uric acid Int J Biochem Cell Biol 2001 33(8) p 775-83

141

210 Alia M R Mateos S Ramos E Lecumberri L Bravo and L Goya Influence of quercetin and rutin on growth and antioxidant defense system of a human hepatoma cell line (HepG2) Eur J Nutr 2006 45(1) p 19-28

211 Aherne SA and NM OBrien Protection by the flavonoids myricetin quercetin and rutin against hydrogen peroxide-induced DNA damage in Caco-2 and Hep G2 cells Nutr Cancer 1999 34(2) p 160-6

212 Johnson MK and G Loo Effects of epigallocatechin gallate and quercetin on oxidative damage to cellular DNA Mutat Res 2000 459(3) p 211-8

213 Danno K T Horio M Takigawa and S Imamura Role of oxygen intermediates in UV-induced epidermal cell injury J Invest Dermatol 1984 83(3) p 166-8

214 Punnonen K P Autio U Kiistala and M Ahotupa In-vivo effects of solar-simulated ultraviolet irradiation on antioxidant enzymes and lipid peroxidation in human epidermis Br J Dermatol 1991 125(1) p 18-20

215 Mitchell DL R Greinert FR de Gruijl KL Guikers EW Breitbart M Byrom MM Gallmeier MG Lowery and B Volkmer Effects of chronic low-dose ultraviolet B radiation on DNA damage and repair in mouse skin Cancer Res 1999 59(12) p 2875-84

216 Afaq F MA Zaid N Khan M Dreher and H Mukhtar Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin Exp Dermatol 2009

217 Wang ZY MT Huang YR Lou JG Xie KR Reuhl HL Newmark CT Ho CS Yang and AH Conney Inhibitory effects of black tea green tea decaffeinated black tea and decaffeinated green tea on ultraviolet B light-induced skin carcinogenesis in 712-dimethylbenz[a]anthracene-initiated SKH-1 mice Cancer Res 1994 54(13) p 3428-35

218 Tomaino A M Cristani F Cimino A Speciale D Trombetta F Bonina and A Saija In vitro protective effect of a Jacquez grapes wine extract on UVB-induced skin damage Toxicol In Vitro 2006 20(8) p 1395-402

219 Tsoyi K HB Park YM Kim JI Chung SC Shin HJ Shim WS Lee HG Seo JH Lee KC Chang and HJ Kim Protective effect of anthocyanins from black soybean seed coats on UVB-induced apoptotic cell death in vitro and in vivo J Agric Food Chem 2008 56(22) p 10600-5

220 Mantena SK and SK Katiyar Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes Free Radic Biol Med 2006 40(9) p 1603-14

221 Tobi SE M Gilbert N Paul and TJ McMillan The green tea polyphenol epigallocatechin-3-gallate protects against the oxidative cellular and genotoxic damage of UVA radiation Int J Cancer 2002 102(5) p 439-44

222 Azmi AS SH Bhat S Hanif and SM Hadi Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage a putative mechanism for anticancer properties FEBS Lett 2006 580(2) p 533-8

223 Elbling L RM Weiss O Teufelhofer M Uhl S Knasmueller R Schulte-Hermann W Berger and M Micksche Green tea extract and (-)-

142

epigallocatechin-3-gallate the major tea catechin exert oxidant but lack antioxidant activities Faseb J 2005 19(7) p 807-9

224 Lee KW HJ Hur HJ Lee and CY Lee Antiproliferative effects of dietary phenolic substances and hydrogen peroxide J Agric Food Chem 2005 53(6) p 1990-5

225 Yamamoto T J Lewis J Wataha D Dickinson B Singh WB Bollag E Ueta T Osaki M Athar G Schuster and S Hsu Roles of catalase and hydrogen peroxide in green tea polyphenol-induced chemopreventive effects J Pharmacol Exp Ther 2004 308(1) p 317-23

226 Agullo G L Gamet-Payrastre Y Fernandez N Anciaux C Demigne and C Remesy Comparative effects of flavonoids on the growth viability and metabolism of a colonic adenocarcinoma cell line (HT29 cells) Cancer Lett 1996 105(1) p 61-70

227 Hodek P P Trefil and M Stiborova Flavonoids-potent and versatile biologically active compounds interacting with cytochromes P450 Chem Biol Interact 2002 139(1) p 1-21

228 Galati G S Teng MY Moridani TS Chan and PJ OBrien Cancer chemoprevention and apoptosis mechanisms induced by dietary polyphenolics Drug Metabol Drug Interact 2000 17(1-4) p 311-49

229 Webster RP MD Gawde and RK Bhattacharya Protective effect of rutin a flavonol glycoside on the carcinogen-induced DNA damage and repair enzymes in rats Cancer Lett 1996 109(1-2) p 185-91

230 Imanishi H YF Sasaki T Ohta M Watanabe T Kato and Y Shirasu Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice Mutat Res 1991 259(1) p 79-87

231 Fresco P F Borges C Diniz and MP Marques New insights on the anticancer properties of dietary polyphenols Med Res Rev 2006 26(6) p 747-66

232 Malik M C Zhao N Schoene MM Guisti MP Moyer and BA Magnuson Anthocyanin-rich extract from Aronia meloncarpa E induces a cell cycle block in colon cancer but not normal colonic cells Nutr Cancer 2003 46(2) p 186-96

233 Thompson CB Apoptosis in the pathogenesis and treatment of disease Science 1995 267(5203) p 1456-62

234 Ramos S M Alia L Bravo and L Goya Comparative effects of food-derived polyphenols on the viability and apoptosis of a human hepatoma cell line (HepG2) J Agric Food Chem 2005 53(4) p 1271-80

235 Ramos S Effects of dietary flavonoids on apoptotic pathways related to cancer chemoprevention J Nutr Biochem 2007 18(7) p 427-42

236 Afaq F A Malik D Syed D Maes MS Matsui and H Mukhtar Pomegranate fruit extract modulates UV-B-mediated phosphorylation of mitogen-activated protein kinases and activation of nuclear factor kappa B in normal human epidermal keratinocytes paragraph sign Photochem Photobiol 2005 81(1) p 38-45

237 Afaq F M Saleem CG Krueger JD Reed and H Mukhtar Anthocyanin- and hydrolyzable tannin-rich pomegranate fruit extract modulates MAPK and NF-

143

kappaB pathways and inhibits skin tumorigenesis in CD-1 mice Int J Cancer 2005 113(3) p 423-33

238 Sai K J Kanno R Hasegawa JE Trosko and T Inoue Prevention of the down-regulation of gap junctional intercellular communication by green tea in the liver of mice fed pentachlorophenol Carcinogenesis 2000 21(9) p 1671-6

239 Chaumontet C M Suschetet E Honikman-Leban VA Krutovskikh R Berges AM Le Bon C Heberden MM Shahin H Yamasaki and P Martel Lack of tumor-promoting effects of flavonoids studies on rat liver preneoplastic foci and on in vivo and in vitro gap junctional intercellular communication Nutr Cancer 1996 26(3) p 251-63

240 Nielsen M RJ Ruch and O Vang Resveratrol reverses tumor-promoter-induced inhibition of gap-junctional intercellular communication Biochem Biophys Res Commun 2000 275(3) p 804-9

241 Karlsen A L Retterstol P Laake I Paur S Kjolsrud-Bohn L Sandvik and R Blomhoff Anthocyanins inhibit nuclear factor-kappaB activation in monocytes and reduce plasma concentrations of pro-inflammatory mediators in healthy adults J Nutr 2007 137(8) p 1951-4

242 Tsoyi K HB Park YM Kim JI Chung SC Shin WS Lee HG Seo JH Lee KC Chang and HJ Kim Anthocyanins from black soybean seed coats inhibit UVB-induced inflammatory cylooxygenase-2 gene expression and PGE2 production through regulation of the nuclear factor-kappaB and phosphatidylinositol 3-kinaseAkt pathway J Agric Food Chem 2008 56(19) p 8969-74

243 Gauliard B D Grieve R Wilson A Crozier C Jenkins WD Mullen and M Lean The effects of dietary phenolic compounds on cytokine and antioxidant production by A549 cells J Med Food 2008 11(2) p 382-4

244 Hou DX S Masuzaki F Hashimoto T Uto S Tanigawa M Fujii and Y Sakata Green tea proanthocyanidins inhibit cyclooxygenase-2 expression in LPS-activated mouse macrophages molecular mechanisms and structure-activity relationship Arch Biochem Biophys 2007 460(1) p 67-74

245 Hou DX T Yanagita T Uto S Masuzaki and M Fujii Anthocyanidins inhibit cyclooxygenase-2 expression in LPS-evoked macrophages Structure-activity relationship and molecular mechanisms involved Biochem Pharmacol 2005 70(3) p 417-25

246 Hong J TJ Smith CT Ho DA August and CS Yang Effects of purified green and black tea polyphenols on cyclooxygenase- and lipoxygenase-dependent metabolism of arachidonic acid in human colon mucosa and colon tumor tissues Biochem Pharmacol 2001 62(9) p 1175-83

247 Pergola C A Rossi P Dugo S Cuzzocrea and L Sautebin Inhibition of nitric oxide biosynthesis by anthocyanin fraction of blackberry extract Nitric Oxide 2006 15(1) p 30-9

248 Jin XH K Ohgami K Shiratori Y Suzuki Y Koyama K Yoshida I Ilieva T Tanaka K Onoe and S Ohno Effects of blue honeysuckle (Lonicera caerulea L) extract on lipopolysaccharide-induced inflammation in vitro and in vivo Exp Eye Res 2006 82(5) p 860-7

144

249 Herath HM Y Takano-Ishikawa and K Yamaki Inhibitory effect of some flavonoids on tumor necrosis factor-alpha production in lipopolysaccharide-stimulated mouse macrophage cell line J7741 J Med Food 2003 6(4) p 365-70

250 Slivova V G Zaloga SJ DeMichele P Mukerji YS Huang R Siddiqui K Harvey T Valachovicova and D Sliva Green tea polyphenols modulate secretion of urokinase plasminogen activator (uPA) and inhibit invasive behavior of breast cancer cells Nutr Cancer 2005 52(1) p 66-73

251 Tanimura S R Kadomoto T Tanaka YJ Zhang I Kouno and M Kohno Suppression of tumor cell invasiveness by hydrolyzable tannins (plant polyphenols) via the inhibition of matrix metalloproteinase-2-9 activity Biochem Biophys Res Commun 2005 330(4) p 1306-13

252 Ogasawara M T Matsunaga and H Suzuki Differential effects of antioxidants on the in vitro invasion growth and lung metastasis of murine colon cancer cells Biol Pharm Bull 2007 30(1) p 200-4

253 Bachmeier B AG Nerlich CM Iancu M Cilli E Schleicher R Vene R DellEva M Jochum A Albini and U Pfeffer The chemopreventive polyphenol Curcumin prevents hematogenous breast cancer metastases in immunodeficient mice Cell Physiol Biochem 2007 19(1-4) p 137-52

254 Chen PN WH Kuo CL Chiang HL Chiou YS Hsieh and SC Chu Black rice anthocyanins inhibit cancer cells invasion via repressions of MMPs and u-PA expression Chem Biol Interact 2006 163(3) p 218-29

255 Vayalil PK A Mittal and SK Katiyar Proanthocyanidins from grape seeds inhibit expression of matrix metalloproteinases in human prostate carcinoma cells which is associated with the inhibition of activation of MAPK and NF kappa B Carcinogenesis 2004 25(6) p 987-95

256 Mojzis J L Varinska G Mojzisova I Kostova and L Mirossay Antiangiogenic effects of flavonoids and chalcones Pharmacol Res 2008 57(4) p 259-65

257 Seeram NP and MG Nair Inhibition of lipid peroxidation and structure-activity-related studies of the dietary constituents anthocyanins anthocyanidins and catechins J Agric Food Chem 2002 50(19) p 5308-12

258 Seeram NP R Schutzki A Chandra and MG Nair Characterization quantification and bioactivities of anthocyanins in Cornus species J Agric Food Chem 2002 50(9) p 2519-23

259 Wang H MG Nair GM Strasburg YC Chang AM Booren JI Gray and DL DeWitt Antioxidant and antiinflammatory activities of anthocyanins and their aglycon cyanidin from tart cherries J Nat Prod 1999 62(5) p 802

260 Cho M L Howard R Prior and J Clark Flavonoid glycosides and antioxidant capacity of various blackberry blueberry and red grape genotypes determined by high-performance liquid chromatographymass spectrometry Journal of the Science of Food and Agriculture 2004 84 p 1771-1782

261 Chun OK DO Kim and CY Lee Superoxide radical scavenging activity of the major polyphenols in fresh plums J Agric Food Chem 2003 51(27) p 8067-72

145

262 Chun OK DO Kim HY Moon HG Kang and CY Lee Contribution of individual polyphenolics to total antioxidant capacity of plums J Agric Food Chem 2003 51(25) p 7240-5

263 Wang SY and HS Lin Antioxidant activity in fruits and leaves of blackberry raspberry and strawberry varies with cultivar and developmental stage J Agric Food Chem 2000 48(2) p 140-6

264 Sellappan S CC Akoh and G Krewer Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries J Agric Food Chem 2002 50(8) p 2432-8

265 Zhao C MM Giusti M Malik MP Moyer and BA Magnuson Effects of commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic colonic cell growth J Agric Food Chem 2004 52(20) p 6122-8

266 Olsson ME KE Gustavsson S Andersson A Nilsson and RD Duan Inhibition of cancer cell proliferation in vitro by fruit and berry extracts and correlations with antioxidant levels J Agric Food Chem 2004 52(24) p 7264-71

267 Seeram NP LS Adams ML Hardy and D Heber Total cranberry extract versus its phytochemical constituents antiproliferative and synergistic effects against human tumor cell lines J Agric Food Chem 2004 52(9) p 2512-7

268 Kang SY NP Seeram MG Nair and LD Bourquin Tart cherry anthocyanins inhibit tumor development in Apc(Min) mice and reduce proliferation of human colon cancer cells Cancer Lett 2003 194(1) p 13-9

269 Koide T Y Hashimoto H Kamei T Kojima M Hasegawa and K Terabe Antitumor effect of anthocyanin fractions extracted from red soybeans and red beans in vitro and in vivo Cancer Biother Radiopharm 1997 12(4) p 277-80

270 Harris GK A Gupta RG Nines LA Kresty SG Habib WL Frankel K LaPerle DD Gallaher SJ Schwartz and GD Stoner Effects of lyophilized black raspberries on azoxymethane-induced colon cancer and 8-hydroxy-2-deoxyguanosine levels in the Fischer 344 rat Nutr Cancer 2001 40(2) p 125-33

271 Wang J and G Mazza Effects of anthocyanins and other phenolic compounds on the production of tumor necrosis factor alpha in LPSIFN-gamma-activated RAW 2647 macrophages J Agric Food Chem 2002 50(15) p 4183-9

272 Tall JM NP Seeram C Zhao MG Nair RA Meyer and SN Raja Tart cherry anthocyanins suppress inflammation-induced pain behavior in rat Behav Brain Res 2004 153(1) p 181-8

273 Galli RL B Shukitt-Hale KA Youdim and JA Joseph Fruit polyphenolics and brain aging nutritional interventions targeting age-related neuronal and behavioral deficits Ann N Y Acad Sci 2002 959 p 128-32

274 Youdim KA J McDonald W Kalt and JA Joseph Potential role of dietary flavonoids in reducing microvascular endothelium vulnerability to oxidative and inflammatory insults ( small star filled) J Nutr Biochem 2002 13(5) p 282-288

275 Shin WH SJ Park and EJ Kim Protective effect of anthocyanins in middle cerebral artery occlusion and reperfusion model of cerebral ischemia in rats Life Sci 2006

146

276 Siriwoharn T RE Wrolstad CE Finn and CB Pereira Influence of cultivar maturity and sampling on blackberry (Rubus L Hybrids) anthocyanins polyphenolics and antioxidant properties J Agric Food Chem 2004 52(26) p 8021-30

277 Giusti MM and RE Wrolstad Characterization and measurement of anthocyanins by UV-visible spectroscopy in Current Protocols in Food Analytical Chemistry RE Wrolstad et al Editors 2001 John Wiley amp Sons NY p F121-F1213

278 Stintzing FC AS Stintzing R Carle and RE Wrolstad A novel zwitterionic anthocyanin from evergreen blackberry (Rubus laciniatus Willd) J Agric Food Chem 2002 50(2) p 396-9

279 Dugo P L Mondello G Errante G Zappia and G Dugo Identification of anthocyanins in berries by narrow-bore high-performance liquid chromatography with electrospray ionization detection J Agric Food Chem 2001 49(8) p 3987-92

280 Francis FJ Food Colorants Anthocyanins Crit Rev Food Sci Nutr 1989 28(4) p 273-314

281 Srivastava A CC Akoh W Yi J Fischer and G Krewer Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles J Agric Food Chem 2007 55(7) p 2705-13

282 Jing P and MM Giusti Effects of extraction conditions on improving the yield and quality of an anthocyanin-rich purple corn (Zea mays L) color extract J Food Sci 2007 72(7) p C363-8

283 Chen F Y Sun G Zhao X Liao X Hu J Wu and Z Wang Optimization of ultrasound-assisted extraction of anthocyanins in red raspberries and identification of anthocyanins in extract using high-performance liquid chromatography-mass spectrometry Ultrason Sonochem 2007 14(6) p 767-78

284 Matsufuji H H Kido H Misawa J Yaguchi T Otsuki M Chino M Takeda and K Yamagata Stability to light heat and hydrogen peroxide at different pH values and DPPH radical scavenging activity of acylated anthocyanins from red radish extract J Agric Food Chem 2007 55(9) p 3692-701

285 Xiong S LD Melton AJ Easteal and D Siew Stability and antioxidant activity of black currant anthocyanins in solution and encapsulated in glucan gel J Agric Food Chem 2006 54(17) p 6201-8

286 Sadilova E R Carle and FC Stintzing Thermal degradation of anthocyanins and its impact on color and in vitro antioxidant capacity Mol Nutr Food Res 2007 51(12) p 1461-71

287 Zhang Z X Pang Z Ji and Y Jiang Role of anthocyanin degradation in litchi pericarp browning Food Chemistry 2001 75 p 217-221

288 Kader F JP Haluk JP Nicolas and M Metche Degradation of Cyanidin 3-glucoside by Blueberry Polyphenol Oxidase Kinetic Studies and Mechanisms J Agric Food Chem 1998 46 p 3060-3065

289 Manach C A Scalbert C Morand C Remesy and L Jimenez Polyphenols food sources and bioavailability Am J Clin Nutr 2004 79(5) p 727-47

147

290 Prior RL and X Wu Anthocyanins structural characteristics that result in unique metabolic patterns and biological activities Free Radic Res 2006 40(10) p 1014-28

291 Serraino I L Dugo P Dugo L Mondello E Mazzon G Dugo AP Caputi and S Cuzzocrea Protective effects of cyanidin-3-O-glucoside from blackberry extract against peroxynitrite-induced endothelial dysfunction and vascular failure Life Sci 2003 73(9) p 1097-114

292 Sautebin L A Rossi I Serraino P Dugo R Di Paola L Mondello T Genovese D Britti A Peli G Dugo AP Caputi and S Cuzzocrea Effect of anthocyanins contained in a blackberry extract on the circulatory failure and multiple organ dysfunction caused by endotoxin in the rat Planta Med 2004 70(8) p 745-52

293 Feng R LL Bowman Y Lu SS Leonard X Shi BH Jiang V Castranova V Vallyathan and M Ding Blackberry extracts inhibit activating protein 1 activation and cell transformation by perturbing the mitogenic signaling pathway Nutr Cancer 2004 50(1) p 80-9

294 Hou DX Potential mechanisms of cancer chemoprevention by anthocyanins Curr Mol Med 2003 3(2) p 149-59

295 Elisia I and DD Kitts Anthocyanins inhibit peroxyl radical-induced apoptosis in Caco-2 cells Mol Cell Biochem 2008 312(1-2) p 139-45

296 Galati G and PJ OBrien Potential toxicity of flavonoids and other dietary phenolics significance for their chemopreventive and anticancer properties Free Radic Biol Med 2004 37(3) p 287-303

297 Hadi SM SF Asad S Singh and A Ahmad Putative mechanism for anticancer and apoptosis-inducing properties of plant-derived polyphenolic compounds IUBMB Life 2000 50(3) p 167-71

298 Feng R HM Ni SY Wang IL Tourkova MR Shurin H Harada and XM Yin Cyanidin-3-rutinoside a natural polyphenol antioxidant selectively kills leukemic cells by induction of oxidative stress J Biol Chem 2007 282(18) p 13468-76

299 Hadi SM SH Bhat AS Azmi S Hanif U Shamim and MF Ullah Oxidative breakage of cellular DNA by plant polyphenols a putative mechanism for anticancer properties Semin Cancer Biol 2007 17(5) p 370-6

300 Lapidot T MD Walker and J Kanner Can Apple Antioxidants Inhibit Tumor Cell Proliferation Generation of H2O2 during Interaction of Phenolic Compounds with Cell Culture Media J Agric Food Chem 2002 50(11) p 3156-3160

301 Halliwell B Are polyphenols antioxidants or pro-oxidants What do we learn from cell culture and in vivo studies Arch Biochem Biophys 2008 476 p 107-112

302 Hachiya M and M Akashi Catalase regulates cell growth in HL60 human promyelocytic cells evidence for growth regulation by H(2)O(2) Radiat Res 2005 163(3) p 271-82

303 Chen PN SC Chu HL Chiou CL Chiang SF Yang and YS Hsieh Cyanidin 3-glucoside and peonidin 3-glucoside inhibit tumor cell growth and

148

induce apoptosis in vitro and suppress tumor growth in vivo Nutr Cancer 2005 53(2) p 232-43

304 Hou DX T Ose S Lin K Harazoro I Imamura M Kubo T Uto N Terahara M Yoshimoto and M Fujii Anthocyanidins induce apoptosis in human promyelocytic leukemia cells structure-activity relationship and mechanisms involved Int J Oncol 2003 23(3) p 705-12

305 Rossi A I Serraino P Dugo R Di Paola L Mondello T Genovese D Morabito G Dugo L Sautebin AP Caputi and S Cuzzocrea Protective effects of anthocyanins from blackberry in a rat model of acute lung inflammation Free Radic Res 2003 37(8) p 891-900

306 Wang J and G Mazza Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPSIFN-gamma-activated RAW 2647 macrophages J Agric Food Chem 2002 50(4) p 850-7

307 Dai J A Gupte L Gates and RJ Mumper A comprehensive study of anthocyanin-containing extracts from selected blackberry cultivars extraction methods stability anticancer properties and mechanisms Food Chem Toxicol 2009 47(4) p 837-47

308 Skrede G RE Wrolstad and RW Durst Changes in anthocyanins and polyphenolics during juice processing of highbush blueberries (Vaccinium corymbosum L) J Food Sci 2000 65(2) p 357-364

309 Kafkas E M Kosar N Tueremis and KHC Baser Analysis of sugars organic acids and vitamin C contents of blackberry genotypes from Turkey Food Chem 2006 97(4) p 732-736

310 Yi W J Fischer and CC Akoh Study of anticancer activities of muscadine grape phenolics in vitro J Agric Food Chem 2005 53(22) p 8804-12

311 Yi W J Fischer G Krewer and CC Akoh Phenolic compounds from blueberries can inhibit colon cancer cell proliferation and induce apoptosis J Agric Food Chem 2005 53(18) p 7320-9

312 Chang YC HP Huang JD Hsu SF Yang and CJ Wang Hibiscus anthocyanins rich extract-induced apoptotic cell death in human promyelocytic leukemia cells Toxicol Appl Pharmacol 2005 205(3) p 201-12

313 Porter AG and RU Janicke Emerging roles of caspase-3 in apoptosis Cell Death Differ 1999 6(2) p 99-104

314 Rittie L and GJ Fisher UV-light-induced signal cascades and skin aging Ageing Res Rev 2002 1(4) p 705-20

315 Hussein MR Ultraviolet radiation and skin cancer molecular mechanisms J Cutan Pathol 2005 32(3) p 191-205

316 Aubin F Mechanisms involved in ultraviolet light-induced immunosuppression Eur J Dermatol 2003 13(6) p 515-23

317 Katiyar SK UV-induced immune suppression and photocarcinogenesis chemoprevention by dietary botanical agents Cancer Lett 2007 255(1) p 1-11

149

Vita

Jin Dai was born on August 4 1975 in Kaihua Zhejiang China She received her

Bachelor of Science degree in Pharmacology (1997) from the China Pharmaceutical

University graduating second in a class of 30 students She received her Master of

Science degree in Pharmacology (2000) from Shanghai Institute of Material and Medica

Chinese Academy of Sciences China under the supervision of Dr Xinzu Zhu in the

Department of Neuropharmacology and her thesis title was ldquoThe effect of SIPI5052 a

new polyamine site NMDA receptor antagonist on ischemiardquo In July of 2000 Jin

accepted a teaching position in the Pharmacology Department of Shanghai Secondary

Medical University She joined the Department of Pharmaceutical Sciences Graduate

Program at the University of Kentucky in the spring of 2004 Jin is an author and co-

author of four peer reviewed publications and one patent application

Publications

1 Dai J Gupte A Gates L Mumper RJ A comprehensive study on anthocyanin-

containing extracts from selected blackberry cultivars extraction methods stability

anticancer properties and mechanisms Food and Chemical Toxicology 2009 47 837-

47

2 Dai J Patel J Mumper RJ Characterization of blackberry extract and its anti-

proliferative and anti-inflammatory properties Journal of Medicinal Food 2007

10(2) 258-65

3 Mallery SR Stoner GD Larsen PE Fields HW Rodrigo KA Schwartz SJ Tian Q

Dai J Mumper RJ Formulation and in-vitro and in-vivo evaluation of a

mucoadhesive gel containing freeze dried black raspberries implications for oral

cancer chemoprevention Pharmaceutical Research 2007 24(4) 728-37

4 Guan HJ Dai J Zhu XZ The atypical antipsychotic effects of quetiapine fumarate in

animal models Acta Pharmacologia Sinica 2000 21(3)205-10

Patents

1 Mumper RJ Dai J and Gallicchio VS Berry Preparations and Extracts PCT Patent

Application WO2007038421 April 5 2007


Recommended Citation

COVER PAGE OF ABSTRACT

TITLE PAGE OF ABSTRACT


APPROVAL PAGE


COVER PAGE OF DISSERTATION

TITLE PAGE OF DISSERTATION

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

Chapter 1 Introduction and Statement of Problem

Chapter 2 Plan of Research

21 Preparation and characterization of anthocyanin-containing extracts (ACEs) from blackberries


23 Antioxidant and antiproliferative properties of blackberry extracts versus their phytochemical constituents in vitro

Chapter 3 Background

















Chapter 4 Preparation and Characterization of Anthocyanin-containing Extracts from Blackberries

41 Summary

42 Introduction


44 Results

45 Discussion

Chapter 5 In vitro Anticancer and Anti-inflammatory Effects of Blackberry Extracts and Possible Mechanisms of Action

51 Summary

52 Introduction


54 Results

55 Discussion

Chapter 6 Correlation of the In Vitro Antioxidant and Antiproliferative Properties of Blackberry Extracts to Their Phytochemical Constituents

61 Summary

62 Introduction



Chapter 7 Summary and Conclusions

Appendices

Appendix A An In Vitro Investigation of the Effects of the Blackberry Extracts on UV radiation-Induced Cell Death on Human Epidermal Keratinocytes (HEK)

Appendix B Antimicrobial and Anti-viral Properties of Blackberry Extract

Appendix C Standard Operating Procedures (SOPs)





Appendix D Preparation of Vanishing Cream Containing Blackberry ACE

Appendix E Preparation of Mucoadhesive Gel Containing Freeze-Dried Black Raspberries (FDBR) Powder

Appendix F Stabilization of Anthocyanin in Dried Blackberry ACE by Mannitol

References

Vita


Jin Dai

The Graduate School


2009


_______________________________




By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009










Jin Dai

July 25 2009


By

Jin Dai




July 25 2009 Date



DISSERTATION

Jin Dai

The Graduate School


2009


_________________________________________________

DISSERTATION _________________________________________________




By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009


iii

ACKNOWLEDGEMENTS





























gratitude

iv

TABLE OF CONTENTS



LIST OF TABLES vii





from blackberries 5






















Blackberries 40

v

41 Summary 40

42 Introduction 41


44 Results 48

45 Discussion 52



51 Summary 63

52 Introduction 64


54 Results 70

55 Discussion 74



61 Summary 86

62 Introduction 87




Appendices 108









119


vi




References 127

Vita 149

vii

LIST OF TABLES







37 ordmC 81


ACE by SPE 97


ACE by SPE 98



(FDBR) Powder 124

viii

LIST OF FIGURES














over time 80
















ix








1

Chapter 1





























2































3


other fruits


4

Chapter 2

Plan of Research











from immune cells




carried out

5


from blackberries





























6























fractions


7

Chapter 3

Background




























8






soybean [1]


























9








10


11


12
































13
































14
































15






















benefits










16


conditions [62]





























17
































18































19
































20
































21








[102]
























22
































23































24


















radicals

PO + R rarr POR (2)












25

[143 144] are



delocalization










deficiency

















26

(4) [146]
















below [141]

PO + O2 rarr P=O + O2 - (5)





R + O2 rarr ROO (8)



27






























28





























[158]



29





























30
































31






















induced by AOM









32




















different tumors











33
































34






[203]
























35
































36





















230]










37































inhibition of GJIC

38





















[249]











39




VEGFR and PDGFR [3]






of oncology


40

Chapter 4


Blackberries

41 Summary





















41

42 Introduction












[8 265]









anthocyanins










42



























43


Materials




























44


cell-based studies














density













45






























46






























47


content






48

44 Results





















preparation










49































50


















anthocyanins [280]














51





















52

45 Discussion






















cavitation [283]









53
































54






























55




() TAC


Puree





Powder





56





57










58









59




60




61






589 plusmn 82 (983)

539 plusmn 38 (898)

230 plusmn 09 (384)


1680 plusmn 69 (885)

1696 plusmn 68 (893)

1537 plusmn 162 (801)


684 plusmn 158 (963)

621 plusmn 83 (875)

261 plusmn 47 (368)


143 plusmn 011 NA

261 plusmn 088 NA

1396 plusmn 111 NA


80 plusmn 027 (892)

78 plusmn 011 (864)

73 plusmn 009 (812)



NA Not applicable

62









experiments


63

Chapter 5



51 Summary






















64

52 Introduction































65





























66


Materials







Cell culture








incubator













67






























68































69




















70

54 Results






























71































72









HL-60 cells

















groups




73











(data not shown)



















74

55 Discussion































75
































76














our lab

















levels

77































78







79








80






81



ACEpuree (mgml)

RPMI 1640 10 FBS



14 1148 plusmn 028 (ND)






RPMI 1640 10 FBS

HL-60 cells

070 233 plusmn 019 (ND)

424 plusmn 022 (ND)

310 plusmn 079 (ND)

14 196 plusmn 023 (ND)

522 plusmn 020 (ND)

432 plusmn 024 (ND)

28 326 plusmn 025 (ND)

654 plusmn 018 (ND)

ND (ND)


ACEpowder (mgml)

RPMI 1640 10 FBS



14 ND (ND)

363 plusmn 029 (ND)

859 plusmn 112 (ND)




RPMI 1640 10 FBS

HL-60 cells

14 140 plusmn 010 (ND)

154 plusmn 017 (ND)

ND (ND)

28 202 plusmn 028 (ND)

168 plusmn 017 (ND)

143 plusmn 014 (ND)


C-3-G (microgml)

RPMI 1640 10 FBS

207 ND ND ND


RPMI 1640



RPMI 1640 10 FBS

HL-60 cells

207 ND

622 ND

H2O2 (microM)

RPMI 1640 10 FBS 50 4626 plusmn 116





(ND)

RPMI 1640 10 FBS

HL-60 cells 100 ND


82









83







84








85








86

Chapter 6



61 Summary

















87

62 Introduction































88











89






CO2 incubator
























90





























91

















92
































93
































94
































95
























ROS mechanism








96




97



Total Anthocyanin b






ND 1477 plusmn 075 16559 plusmn 507 -

Yield ()



- 294 3292 -

Total Phenolics c









009 2189 2551 -




007 1360 2406 -





ND not detected

98



Total Anthocyanin b




ND 565 plusmn 136 30574 plusmn 3356 -



Yield ()



- 119 6518 -

Total Phenolics c









010 2566 4610 -




022 2161 6345 -





ND not detected

99

0 100 200 300 400 5000

20

40

60

80

100

120

1000 2000 3000 4000


A

C (microgml)

Cel

l Via

bilit

y (

Con

trol

)

0 100 200 300 400 5000

20

40

60

80

100

120

2000 4000 6000 8000




C (microgml)

Cel

l Via

bilit

y (

Con

trol

)







figure legend

100








101








102







groups


103

Chapter 7





this research were
























104
































105
































106
































107




108

Appendices







(powder)




Assay)





109

Appendix A






















110






111

Appendix B























Acknowledgements


these studies

112

0

20

40

60

80

100

0000 0003 0005 0011 0022 0044 0088 0175 0350 0700 1400

Extract (mgmL)

In

hibi

tion S gordonii

A actino





113

R

educ

tion

in C

PE

0

20

40

60

80

100

120

14 7 35 175 0875

EC50

Extract (mgmL)

R

educ

tion

in C

PE





of Kentucky

114

Appendix C



Materials













puree




sonication








115


Materials






Procedure







water
















116





stock (g)



Notes







117


Materials







Glass bottles

Procedure


















standard curve



118








Notes




119


Materials



Cat No 11557


391913



20 ml-glass vials


Procedure





20 ml-glass vial














120






standard curve







in the stock (g)




Reference




Notes




121

Appendix D











122





123

Appendix E


(FDBR) Powder




















124


Ingredient





pH 30 (ww)


pH 35 (ww) Water 83925 83975 8379 8382 83875






125

Appendix F
















Acknowledgements




126






0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside



127

References
















128

















129


















130
















131















132














133















134


















135















136




















137















138

















139















140















141















142
















143













144














145















146















147















148
















149

Vita












Publications




47



10(2) 258-65







Patents








APPROVAL PAGE




ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES
























41 Summary

42 Introduction


44 Results

45 Discussion


51 Summary

52 Introduction


54 Results

55 Discussion


61 Summary

62 Introduction




Appendices











References

Vita


_______________________________




By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009










Jin Dai

July 25 2009


By

Jin Dai




July 25 2009 Date



DISSERTATION

Jin Dai

The Graduate School


2009


_________________________________________________

DISSERTATION _________________________________________________




By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009


iii

ACKNOWLEDGEMENTS





























gratitude

iv

TABLE OF CONTENTS



LIST OF TABLES vii





from blackberries 5






















Blackberries 40

v

41 Summary 40

42 Introduction 41


44 Results 48

45 Discussion 52



51 Summary 63

52 Introduction 64


54 Results 70

55 Discussion 74



61 Summary 86

62 Introduction 87




Appendices 108









119


vi




References 127

Vita 149

vii

LIST OF TABLES







37 ordmC 81


ACE by SPE 97


ACE by SPE 98



(FDBR) Powder 124

viii

LIST OF FIGURES














over time 80
















ix








1

Chapter 1





























2































3


other fruits


4

Chapter 2

Plan of Research











from immune cells




carried out

5


from blackberries





























6























fractions


7

Chapter 3

Background




























8






soybean [1]


























9








10


11


12
































13
































14
































15






















benefits










16


conditions [62]





























17
































18































19
































20
































21








[102]
























22
































23































24


















radicals

PO + R rarr POR (2)












25

[143 144] are



delocalization










deficiency

















26

(4) [146]
















below [141]

PO + O2 rarr P=O + O2 - (5)





R + O2 rarr ROO (8)



27






























28





























[158]



29





























30
































31






















induced by AOM









32




















different tumors











33
































34






[203]
























35
































36





















230]










37































inhibition of GJIC

38





















[249]











39




VEGFR and PDGFR [3]






of oncology


40

Chapter 4


Blackberries

41 Summary





















41

42 Introduction












[8 265]









anthocyanins










42



























43


Materials




























44


cell-based studies














density













45






























46






























47


content






48

44 Results





















preparation










49































50


















anthocyanins [280]














51





















52

45 Discussion






















cavitation [283]









53
































54






























55




() TAC


Puree





Powder





56





57










58









59




60




61






589 plusmn 82 (983)

539 plusmn 38 (898)

230 plusmn 09 (384)


1680 plusmn 69 (885)

1696 plusmn 68 (893)

1537 plusmn 162 (801)


684 plusmn 158 (963)

621 plusmn 83 (875)

261 plusmn 47 (368)


143 plusmn 011 NA

261 plusmn 088 NA

1396 plusmn 111 NA


80 plusmn 027 (892)

78 plusmn 011 (864)

73 plusmn 009 (812)



NA Not applicable

62









experiments


63

Chapter 5



51 Summary






















64

52 Introduction































65





























66


Materials







Cell culture








incubator













67






























68































69




















70

54 Results






























71































72









HL-60 cells

















groups




73











(data not shown)



















74

55 Discussion































75
































76














our lab

















levels

77































78







79








80






81



ACEpuree (mgml)

RPMI 1640 10 FBS



14 1148 plusmn 028 (ND)






RPMI 1640 10 FBS

HL-60 cells

070 233 plusmn 019 (ND)

424 plusmn 022 (ND)

310 plusmn 079 (ND)

14 196 plusmn 023 (ND)

522 plusmn 020 (ND)

432 plusmn 024 (ND)

28 326 plusmn 025 (ND)

654 plusmn 018 (ND)

ND (ND)


ACEpowder (mgml)

RPMI 1640 10 FBS



14 ND (ND)

363 plusmn 029 (ND)

859 plusmn 112 (ND)




RPMI 1640 10 FBS

HL-60 cells

14 140 plusmn 010 (ND)

154 plusmn 017 (ND)

ND (ND)

28 202 plusmn 028 (ND)

168 plusmn 017 (ND)

143 plusmn 014 (ND)


C-3-G (microgml)

RPMI 1640 10 FBS

207 ND ND ND


RPMI 1640



RPMI 1640 10 FBS

HL-60 cells

207 ND

622 ND

H2O2 (microM)

RPMI 1640 10 FBS 50 4626 plusmn 116





(ND)

RPMI 1640 10 FBS

HL-60 cells 100 ND


82









83







84








85








86

Chapter 6



61 Summary

















87

62 Introduction































88











89






CO2 incubator
























90





























91

















92
































93
































94
































95
























ROS mechanism








96




97



Total Anthocyanin b






ND 1477 plusmn 075 16559 plusmn 507 -

Yield ()



- 294 3292 -

Total Phenolics c









009 2189 2551 -




007 1360 2406 -





ND not detected

98



Total Anthocyanin b




ND 565 plusmn 136 30574 plusmn 3356 -



Yield ()



- 119 6518 -

Total Phenolics c









010 2566 4610 -




022 2161 6345 -





ND not detected

99

0 100 200 300 400 5000

20

40

60

80

100

120

1000 2000 3000 4000


A

C (microgml)

Cel

l Via

bilit

y (

Con

trol

)

0 100 200 300 400 5000

20

40

60

80

100

120

2000 4000 6000 8000




C (microgml)

Cel

l Via

bilit

y (

Con

trol

)







figure legend

100








101








102







groups


103

Chapter 7





this research were
























104
































105
































106
































107




108

Appendices







(powder)




Assay)





109

Appendix A






















110






111

Appendix B























Acknowledgements


these studies

112

0

20

40

60

80

100

0000 0003 0005 0011 0022 0044 0088 0175 0350 0700 1400

Extract (mgmL)

In

hibi

tion S gordonii

A actino





113

R

educ

tion

in C

PE

0

20

40

60

80

100

120

14 7 35 175 0875

EC50

Extract (mgmL)

R

educ

tion

in C

PE





of Kentucky

114

Appendix C



Materials













puree




sonication








115


Materials






Procedure







water
















116





stock (g)



Notes







117


Materials







Glass bottles

Procedure


















standard curve



118








Notes




119


Materials



Cat No 11557


391913



20 ml-glass vials


Procedure





20 ml-glass vial














120






standard curve







in the stock (g)




Reference




Notes




121

Appendix D











122





123

Appendix E


(FDBR) Powder




















124


Ingredient





pH 30 (ww)


pH 35 (ww) Water 83925 83975 8379 8382 83875






125

Appendix F
















Acknowledgements




126






0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside



127

References
















128

















129


















130
















131















132














133















134


















135















136




















137















138

















139















140















141















142
















143













144














145















146















147















148
















149

Vita












Publications




47



10(2) 258-65







Patents








APPROVAL PAGE




ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES
























41 Summary

42 Introduction


44 Results

45 Discussion


51 Summary

52 Introduction


54 Results

55 Discussion


61 Summary

62 Introduction




Appendices











References

Vita









Jin Dai

July 25 2009


By

Jin Dai




July 25 2009 Date



DISSERTATION

Jin Dai

The Graduate School


2009


_________________________________________________

DISSERTATION _________________________________________________




By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009


iii

ACKNOWLEDGEMENTS





























gratitude

iv

TABLE OF CONTENTS



LIST OF TABLES vii





from blackberries 5






















Blackberries 40

v

41 Summary 40

42 Introduction 41


44 Results 48

45 Discussion 52



51 Summary 63

52 Introduction 64


54 Results 70

55 Discussion 74



61 Summary 86

62 Introduction 87




Appendices 108









119


vi




References 127

Vita 149

vii

LIST OF TABLES







37 ordmC 81


ACE by SPE 97


ACE by SPE 98



(FDBR) Powder 124

viii

LIST OF FIGURES














over time 80
















ix








1

Chapter 1





























2































3


other fruits


4

Chapter 2

Plan of Research











from immune cells




carried out

5


from blackberries





























6























fractions


7

Chapter 3

Background




























8






soybean [1]


























9








10


11


12
































13
































14
































15






















benefits










16


conditions [62]





























17
































18































19
































20
































21








[102]
























22
































23































24


















radicals

PO + R rarr POR (2)












25

[143 144] are



delocalization










deficiency

















26

(4) [146]
















below [141]

PO + O2 rarr P=O + O2 - (5)





R + O2 rarr ROO (8)



27






























28





























[158]



29





























30
































31






















induced by AOM









32




















different tumors











33
































34






[203]
























35
































36





















230]










37































inhibition of GJIC

38





















[249]











39




VEGFR and PDGFR [3]






of oncology


40

Chapter 4


Blackberries

41 Summary





















41

42 Introduction












[8 265]









anthocyanins










42



























43


Materials




























44


cell-based studies














density













45






























46






























47


content






48

44 Results





















preparation










49































50


















anthocyanins [280]














51





















52

45 Discussion






















cavitation [283]









53
































54






























55




() TAC


Puree





Powder





56





57










58









59




60




61






589 plusmn 82 (983)

539 plusmn 38 (898)

230 plusmn 09 (384)


1680 plusmn 69 (885)

1696 plusmn 68 (893)

1537 plusmn 162 (801)


684 plusmn 158 (963)

621 plusmn 83 (875)

261 plusmn 47 (368)


143 plusmn 011 NA

261 plusmn 088 NA

1396 plusmn 111 NA


80 plusmn 027 (892)

78 plusmn 011 (864)

73 plusmn 009 (812)



NA Not applicable

62









experiments


63

Chapter 5



51 Summary






















64

52 Introduction































65





























66


Materials







Cell culture








incubator













67






























68































69




















70

54 Results






























71































72









HL-60 cells

















groups




73











(data not shown)



















74

55 Discussion































75
































76














our lab

















levels

77































78







79








80






81



ACEpuree (mgml)

RPMI 1640 10 FBS



14 1148 plusmn 028 (ND)






RPMI 1640 10 FBS

HL-60 cells

070 233 plusmn 019 (ND)

424 plusmn 022 (ND)

310 plusmn 079 (ND)

14 196 plusmn 023 (ND)

522 plusmn 020 (ND)

432 plusmn 024 (ND)

28 326 plusmn 025 (ND)

654 plusmn 018 (ND)

ND (ND)


ACEpowder (mgml)

RPMI 1640 10 FBS



14 ND (ND)

363 plusmn 029 (ND)

859 plusmn 112 (ND)




RPMI 1640 10 FBS

HL-60 cells

14 140 plusmn 010 (ND)

154 plusmn 017 (ND)

ND (ND)

28 202 plusmn 028 (ND)

168 plusmn 017 (ND)

143 plusmn 014 (ND)


C-3-G (microgml)

RPMI 1640 10 FBS

207 ND ND ND


RPMI 1640



RPMI 1640 10 FBS

HL-60 cells

207 ND

622 ND

H2O2 (microM)

RPMI 1640 10 FBS 50 4626 plusmn 116





(ND)

RPMI 1640 10 FBS

HL-60 cells 100 ND


82









83







84








85








86

Chapter 6



61 Summary

















87

62 Introduction































88











89






CO2 incubator
























90





























91

















92
































93
































94
































95
























ROS mechanism








96




97



Total Anthocyanin b






ND 1477 plusmn 075 16559 plusmn 507 -

Yield ()



- 294 3292 -

Total Phenolics c









009 2189 2551 -




007 1360 2406 -





ND not detected

98



Total Anthocyanin b




ND 565 plusmn 136 30574 plusmn 3356 -



Yield ()



- 119 6518 -

Total Phenolics c









010 2566 4610 -




022 2161 6345 -





ND not detected

99

0 100 200 300 400 5000

20

40

60

80

100

120

1000 2000 3000 4000


A

C (microgml)

Cel

l Via

bilit

y (

Con

trol

)

0 100 200 300 400 5000

20

40

60

80

100

120

2000 4000 6000 8000




C (microgml)

Cel

l Via

bilit

y (

Con

trol

)







figure legend

100








101








102







groups


103

Chapter 7





this research were
























104
































105
































106
































107




108

Appendices







(powder)




Assay)





109

Appendix A






















110






111

Appendix B























Acknowledgements


these studies

112

0

20

40

60

80

100

0000 0003 0005 0011 0022 0044 0088 0175 0350 0700 1400

Extract (mgmL)

In

hibi

tion S gordonii

A actino





113

R

educ

tion

in C

PE

0

20

40

60

80

100

120

14 7 35 175 0875

EC50

Extract (mgmL)

R

educ

tion

in C

PE





of Kentucky

114

Appendix C



Materials













puree




sonication








115


Materials






Procedure







water
















116





stock (g)



Notes







117


Materials







Glass bottles

Procedure


















standard curve



118








Notes




119


Materials



Cat No 11557


391913



20 ml-glass vials


Procedure





20 ml-glass vial














120






standard curve







in the stock (g)




Reference




Notes




121

Appendix D











122





123

Appendix E


(FDBR) Powder




















124


Ingredient





pH 30 (ww)


pH 35 (ww) Water 83925 83975 8379 8382 83875






125

Appendix F
















Acknowledgements




126






0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside



127

References
















128

















129


















130
















131















132














133















134


















135















136




















137















138

















139















140















141















142
















143













144














145















146















147















148
















149

Vita












Publications




47



10(2) 258-65







Patents








APPROVAL PAGE




ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES
























41 Summary

42 Introduction


44 Results

45 Discussion


51 Summary

52 Introduction


54 Results

55 Discussion


61 Summary

62 Introduction




Appendices











References

Vita


By

Jin Dai




July 25 2009 Date



DISSERTATION

Jin Dai

The Graduate School


2009


_________________________________________________

DISSERTATION _________________________________________________




By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009


iii

ACKNOWLEDGEMENTS





























gratitude

iv

TABLE OF CONTENTS



LIST OF TABLES vii





from blackberries 5






















Blackberries 40

v

41 Summary 40

42 Introduction 41


44 Results 48

45 Discussion 52



51 Summary 63

52 Introduction 64


54 Results 70

55 Discussion 74



61 Summary 86

62 Introduction 87




Appendices 108









119


vi




References 127

Vita 149

vii

LIST OF TABLES







37 ordmC 81


ACE by SPE 97


ACE by SPE 98



(FDBR) Powder 124

viii

LIST OF FIGURES














over time 80
















ix








1

Chapter 1





























2































3


other fruits


4

Chapter 2

Plan of Research











from immune cells




carried out

5


from blackberries





























6























fractions


7

Chapter 3

Background




























8






soybean [1]


























9








10


11


12
































13
































14
































15






















benefits










16


conditions [62]





























17
































18































19
































20
































21








[102]
























22
































23































24


















radicals

PO + R rarr POR (2)












25

[143 144] are



delocalization










deficiency

















26

(4) [146]
















below [141]

PO + O2 rarr P=O + O2 - (5)





R + O2 rarr ROO (8)



27






























28





























[158]



29





























30
































31






















induced by AOM









32




















different tumors











33
































34






[203]
























35
































36





















230]










37































inhibition of GJIC

38





















[249]











39




VEGFR and PDGFR [3]






of oncology


40

Chapter 4


Blackberries

41 Summary





















41

42 Introduction












[8 265]









anthocyanins










42



























43


Materials




























44


cell-based studies














density













45






























46






























47


content






48

44 Results





















preparation










49































50


















anthocyanins [280]














51





















52

45 Discussion






















cavitation [283]









53
































54






























55




() TAC


Puree





Powder





56





57










58









59




60




61






589 plusmn 82 (983)

539 plusmn 38 (898)

230 plusmn 09 (384)


1680 plusmn 69 (885)

1696 plusmn 68 (893)

1537 plusmn 162 (801)


684 plusmn 158 (963)

621 plusmn 83 (875)

261 plusmn 47 (368)


143 plusmn 011 NA

261 plusmn 088 NA

1396 plusmn 111 NA


80 plusmn 027 (892)

78 plusmn 011 (864)

73 plusmn 009 (812)



NA Not applicable

62









experiments


63

Chapter 5



51 Summary






















64

52 Introduction































65





























66


Materials







Cell culture








incubator













67






























68































69




















70

54 Results






























71































72









HL-60 cells

















groups




73











(data not shown)



















74

55 Discussion































75
































76














our lab

















levels

77































78







79








80






81



ACEpuree (mgml)

RPMI 1640 10 FBS



14 1148 plusmn 028 (ND)






RPMI 1640 10 FBS

HL-60 cells

070 233 plusmn 019 (ND)

424 plusmn 022 (ND)

310 plusmn 079 (ND)

14 196 plusmn 023 (ND)

522 plusmn 020 (ND)

432 plusmn 024 (ND)

28 326 plusmn 025 (ND)

654 plusmn 018 (ND)

ND (ND)


ACEpowder (mgml)

RPMI 1640 10 FBS



14 ND (ND)

363 plusmn 029 (ND)

859 plusmn 112 (ND)




RPMI 1640 10 FBS

HL-60 cells

14 140 plusmn 010 (ND)

154 plusmn 017 (ND)

ND (ND)

28 202 plusmn 028 (ND)

168 plusmn 017 (ND)

143 plusmn 014 (ND)


C-3-G (microgml)

RPMI 1640 10 FBS

207 ND ND ND


RPMI 1640



RPMI 1640 10 FBS

HL-60 cells

207 ND

622 ND

H2O2 (microM)

RPMI 1640 10 FBS 50 4626 plusmn 116





(ND)

RPMI 1640 10 FBS

HL-60 cells 100 ND


82









83







84








85








86

Chapter 6



61 Summary

















87

62 Introduction































88











89






CO2 incubator
























90





























91

















92
































93
































94
































95
























ROS mechanism








96




97



Total Anthocyanin b






ND 1477 plusmn 075 16559 plusmn 507 -

Yield ()



- 294 3292 -

Total Phenolics c









009 2189 2551 -




007 1360 2406 -





ND not detected

98



Total Anthocyanin b




ND 565 plusmn 136 30574 plusmn 3356 -



Yield ()



- 119 6518 -

Total Phenolics c









010 2566 4610 -




022 2161 6345 -





ND not detected

99

0 100 200 300 400 5000

20

40

60

80

100

120

1000 2000 3000 4000


A

C (microgml)

Cel

l Via

bilit

y (

Con

trol

)

0 100 200 300 400 5000

20

40

60

80

100

120

2000 4000 6000 8000




C (microgml)

Cel

l Via

bilit

y (

Con

trol

)







figure legend

100








101








102







groups


103

Chapter 7





this research were
























104
































105
































106
































107




108

Appendices







(powder)




Assay)





109

Appendix A






















110






111

Appendix B























Acknowledgements


these studies

112

0

20

40

60

80

100

0000 0003 0005 0011 0022 0044 0088 0175 0350 0700 1400

Extract (mgmL)

In

hibi

tion S gordonii

A actino





113

R

educ

tion

in C

PE

0

20

40

60

80

100

120

14 7 35 175 0875

EC50

Extract (mgmL)

R

educ

tion

in C

PE





of Kentucky

114

Appendix C



Materials













puree




sonication








115


Materials






Procedure







water
















116





stock (g)



Notes







117


Materials







Glass bottles

Procedure


















standard curve



118








Notes




119


Materials



Cat No 11557


391913



20 ml-glass vials


Procedure





20 ml-glass vial














120






standard curve







in the stock (g)




Reference




Notes




121

Appendix D











122





123

Appendix E


(FDBR) Powder




















124


Ingredient





pH 30 (ww)


pH 35 (ww) Water 83925 83975 8379 8382 83875






125

Appendix F
















Acknowledgements




126






0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside



127

References
















128

















129


















130
















131















132














133















134


















135















136




















137















138

















139















140















141















142
















143













144














145















146















147















148
















149

Vita












Publications




47



10(2) 258-65







Patents








APPROVAL PAGE




ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES
























41 Summary

42 Introduction


44 Results

45 Discussion


51 Summary

52 Introduction


54 Results

55 Discussion


61 Summary

62 Introduction




Appendices











References

Vita



DISSERTATION

Jin Dai

The Graduate School


2009


_________________________________________________

DISSERTATION _________________________________________________




By Jin Dai

Lexington Kentucky



Lexington Kentucky

2009


iii

ACKNOWLEDGEMENTS





























gratitude

iv

TABLE OF CONTENTS



LIST OF TABLES vii





from blackberries 5






















Blackberries 40

v

41 Summary 40

42 Introduction 41


44 Results 48

45 Discussion 52



51 Summary 63

52 Introduction 64


54 Results 70

55 Discussion 74



61 Summary 86

62 Introduction 87




Appendices 108









119


vi




References 127

Vita 149

vii

LIST OF TABLES







37 ordmC 81


ACE by SPE 97


ACE by SPE 98



(FDBR) Powder 124

viii

LIST OF FIGURES














over time 80
















ix








1

Chapter 1





























2































3


other fruits


4

Chapter 2

Plan of Research











from immune cells




carried out

5


from blackberries





























6























fractions


7

Chapter 3

Background




























8






soybean [1]


























9








10


11


12
































13
































14
































15






















benefits










16


conditions [62]





























17
































18































19
































20
































21








[102]
























22
































23































24


















radicals

PO + R rarr POR (2)












25

[143 144] are



delocalization










deficiency

















26

(4) [146]
















below [141]

PO + O2 rarr P=O + O2 - (5)





R + O2 rarr ROO (8)



27






























28





























[158]



29





























30
































31






















induced by AOM









32




















different tumors











33
































34






[203]
























35
































36





















230]










37































inhibition of GJIC

38





















[249]











39




VEGFR and PDGFR [3]






of oncology


40

Chapter 4


Blackberries

41 Summary





















41

42 Introduction












[8 265]









anthocyanins










42



























43


Materials




























44


cell-based studies














density













45






























46






























47


content






48

44 Results





















preparation










49































50


















anthocyanins [280]














51





















52

45 Discussion






















cavitation [283]









53
































54






























55




() TAC


Puree





Powder





56





57










58









59




60




61






589 plusmn 82 (983)

539 plusmn 38 (898)

230 plusmn 09 (384)


1680 plusmn 69 (885)

1696 plusmn 68 (893)

1537 plusmn 162 (801)


684 plusmn 158 (963)

621 plusmn 83 (875)

261 plusmn 47 (368)


143 plusmn 011 NA

261 plusmn 088 NA

1396 plusmn 111 NA


80 plusmn 027 (892)

78 plusmn 011 (864)

73 plusmn 009 (812)



NA Not applicable

62









experiments


63

Chapter 5



51 Summary






















64

52 Introduction































65





























66


Materials







Cell culture








incubator













67






























68































69




















70

54 Results






























71































72









HL-60 cells

















groups




73











(data not shown)



















74

55 Discussion































75
































76














our lab

















levels

77































78







79








80






81



ACEpuree (mgml)

RPMI 1640 10 FBS



14 1148 plusmn 028 (ND)






RPMI 1640 10 FBS

HL-60 cells

070 233 plusmn 019 (ND)

424 plusmn 022 (ND)

310 plusmn 079 (ND)

14 196 plusmn 023 (ND)

522 plusmn 020 (ND)

432 plusmn 024 (ND)

28 326 plusmn 025 (ND)

654 plusmn 018 (ND)

ND (ND)


ACEpowder (mgml)

RPMI 1640 10 FBS



14 ND (ND)

363 plusmn 029 (ND)

859 plusmn 112 (ND)




RPMI 1640 10 FBS

HL-60 cells

14 140 plusmn 010 (ND)

154 plusmn 017 (ND)

ND (ND)

28 202 plusmn 028 (ND)

168 plusmn 017 (ND)

143 plusmn 014 (ND)


C-3-G (microgml)

RPMI 1640 10 FBS

207 ND ND ND


RPMI 1640



RPMI 1640 10 FBS

HL-60 cells

207 ND

622 ND

H2O2 (microM)

RPMI 1640 10 FBS 50 4626 plusmn 116





(ND)

RPMI 1640 10 FBS

HL-60 cells 100 ND


82









83







84








85








86

Chapter 6



61 Summary

















87

62 Introduction































88











89






CO2 incubator
























90





























91

















92
































93
































94
































95
























ROS mechanism








96




97



Total Anthocyanin b






ND 1477 plusmn 075 16559 plusmn 507 -

Yield ()



- 294 3292 -

Total Phenolics c









009 2189 2551 -




007 1360 2406 -





ND not detected

98



Total Anthocyanin b




ND 565 plusmn 136 30574 plusmn 3356 -



Yield ()



- 119 6518 -

Total Phenolics c









010 2566 4610 -




022 2161 6345 -





ND not detected

99

0 100 200 300 400 5000

20

40

60

80

100

120

1000 2000 3000 4000


A

C (microgml)

Cel

l Via

bilit

y (

Con

trol

)

0 100 200 300 400 5000

20

40

60

80

100

120

2000 4000 6000 8000




C (microgml)

Cel

l Via

bilit

y (

Con

trol

)







figure legend

100








101








102







groups


103

Chapter 7





this research were
























104
































105
































106
































107




108

Appendices







(powder)




Assay)





109

Appendix A






















110






111

Appendix B























Acknowledgements


these studies

112

0

20

40

60

80

100

0000 0003 0005 0011 0022 0044 0088 0175 0350 0700 1400

Extract (mgmL)

In

hibi

tion S gordonii

A actino





113

R

educ

tion

in C

PE

0

20

40

60

80

100

120

14 7 35 175 0875

EC50

Extract (mgmL)

R

educ

tion

in C

PE





of Kentucky

114

Appendix C



Materials













puree




sonication








115


Materials






Procedure







water
















116





stock (g)



Notes







117


Materials







Glass bottles

Procedure


















standard curve



118








Notes




119


Materials



Cat No 11557


391913



20 ml-glass vials


Procedure





20 ml-glass vial














120






standard curve







in the stock (g)




Reference




Notes




121

Appendix D











122





123

Appendix E


(FDBR) Powder




















124


Ingredient





pH 30 (ww)


pH 35 (ww) Water 83925 83975 8379 8382 83875






125

Appendix F
















Acknowledgements




126






0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

0

25

50

75

100

0 1 2 0 1 2


0

25

50

75

100

0 1 2 0 1 2


Ret

entio

n of

Ave

rage

Ant

hocy

anin

Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside


Cn-3-glucoside

Cn-3-arabinoside

Cn-3-xyloside



127

References
















128

















129


















130
















131















132














133















134


















135















136




















137















138

















139















140















141















142
















143













144














145















146















147















148
















149

Vita












Publications




47



10(2) 258-65







Patents








APPROVAL PAGE




ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES
























41 Summary

42 Introduction


44 Results

45 Discussion


51 Summary

52 Introduction


54 Results

55 Discussion


61 Summary

62 Introduction




Appendices











References

Vita

PREPARATION AND CHARACTERIZATION OF BLACKBERRY …

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