Preparation and Characterization of Blackberry Extracts and Their

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University of Kentucky Doctoral Dissertations Graduate School
2009
PREPARATION AND CHARACTERIZATION OF BLACKBERRY

EXTRACTS AND THEIR ANTICANCER AND ANTI-INFLAMMATORY
PROPERTIES
Jin Dai
University of Kentucky, jdai2@uky.edu
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ABSTRACT OF DISSERTATION
Jin Dai
The Graduate School
2009
PREPARATION AND CHARACTERIZATION OF BLACKBERRY EXTRACTS
AND THEIR ANTICANCER AND ANTI-INFLAMMATORY PROPERTIES
_______________________________
_______________________________
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 © 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
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Name Date
DISSERTATION
Jin Dai
The Graduate School
2009
PREPARATION AND CHARACTERIZATION OF BLACKBERRY EXTRACTS AND
THEIR ANTICANCER AND ANTI-INFLAMMATORY PROPERTIES
_________________________________________________
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

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.
iii
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
2.1. Preparation and characterization of anthocyanin-containing extracts (ACEs)
from blackberries ............................................................................................................ 5
2.2. Anticancer and anti-inflammatory properties of blackberry extracts ................. 5
2.3. Antioxidant and antiproliferative properties of blackberry extracts versus their
phytochemical constituents in vitro ................................................................................ 6
Chapter 3: Background ....................................................................................................... 7
3.1. Preparation and characterization of phenolic extracts ........................................ 7
3.1.1. An introduction to natural phenolics........................................................... 7
3.1.2. Extraction of phenolics ............................................................................. 12
3.1.3. Purification and fractionation of phenolics ............................................... 16
3.1.4. Analysis and quantification of phenolics .................................................. 18
3.2. Antioxidant properties of phenolic compounds ................................................ 23
3.2.1. Phenolics as free radical scavengers and metal chelators ......................... 24
3.2.2. Prooxidant activity of phenolic compounds ............................................. 26
3.2.3. Determination of total antioxidant capacity (TAC) of phenolic extracts . 27
3.3. Natural phenolics and cancer ............................................................................ 29
3.3.1. In vitro effects of phenolics ...................................................................... 30
3.3.2. In vivo effects of phenolics ....................................................................... 31
3.3.3. Human intervention studies using phenolics ............................................ 32
3.3.4. Mechanism of Action of phenolics ........................................................... 34
3.3.4.1. Modification of the redox status of phenolics................................... 35
3.3.4.2. Interference of basic cellular functions by phenolics ....................... 36
Chapter 4: Preparation and Characterization of Anthocyanin-containing Extracts from
Blackberries ...................................................................................................................... 40
iv
4.1. Summary ........................................................................................................... 40
4.2. Introduction ....................................................................................................... 41
4.3. Materials and methods ...................................................................................... 43
4.4. Results ............................................................................................................... 48
4.5. Discussion ......................................................................................................... 52
Chapter 5: In vitro Anticancer and Anti-inflammatory Effects of Blackberry Extracts and
Possible Mechanisms of Action ........................................................................................ 63
5.1. Summary ........................................................................................................... 63
5.2. Introduction ....................................................................................................... 64
5.4. Results ............................................................................................................... 70
5.5. Discussion ......................................................................................................... 74
Chapter 6: Correlation of the In Vitro Antioxidant and Antiproliferative Properties of
Blackberry Extracts to Their Phytochemical Constituents ............................................... 86
6.1. Summary ........................................................................................................... 86
6.2. Introduction ....................................................................................................... 87
6.4. 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
v
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
vi
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 ºC.................................................................................................................................. 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
vii
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-electrospray/MS..................................................................................... 56
Figure 4. 2.The 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 °C and 37 °C. .......... 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 J774A.1 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
intracellular ROS level in HL-60 cells............................................................................ 100
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
viii
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 °C over 2 months. ............................................................................ 126
ix
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 FDA's 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
1
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 and/or 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
2
development of Botanical Drug Products from extracts derived from blackberries and
other fruits.
3
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 2.1 to 2.3 was
carried out.
4
2.1. Preparation and characterization of anthocyanin-containing extracts (ACEs)
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.
2.2. 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
5
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, J774A.1
was examined and compared.
2.3. 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.
6
Chapter 3
Background
3.1. Preparation and characterization of phenolic extracts

3.1.1. 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 3.1) and the less
common stilbenes and lignans (Figure 3.2). 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
7
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
8
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.
9
Figure 3. 1. Structures of flavonoids, phenolic acids and tannins
10
Figure 3. 2. Structures of stilbenes and lignan
11
3.1.2. 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 (e.g.,
phenolic acids, anthocyanins) to highly polymerized substances (e.g., 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
12
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 0.5-
3.0% of trifluoroacetic acid and < 1.0% 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 °C [28], because temperatures >70 °C have been shown to cause
rapid anthocyanin degradation [32]. Therefore, it is of critical importance to select
efficient extraction procedure/method 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,
13
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 (3.3-20.3 MPa) and temperatures (40-200 °C) provides
faster extraction processes that require small amounts of solvents (e.g., 20 min using 10–
50 ml of solvent in PLE can be compared with a traditional extraction step in which 10–
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
14
water as an extraction solvent in PLE is the so-called subcritical water extraction (SWE).
In SWE, water is heated up to 200 °C 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 °C is equal to 36 which is close to methanol [44]. Ju et
al. showed that PLE (80 – 100 °C) 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
(e.g., PLE), allowing extraction performed with nonpolluting, nontoxic solvents, such as
water (e.g., SWE), supercritical CO2 fluid (e.g., 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 (e.g., 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 °C for 20
min [62]. Phenolic compounds having a higher number of hydroxyl-type substituents
(e.g., tannins) and those that are sensitive to elevated temperature (e.g., anthocyanins)
15
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].
3.1.3. Purification and fractionation of phenolics

Plant crude extract usually contain large amount of carbohydrates and/or 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 and/or 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
16
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 7.0 and eluted
phenolic acids with water in the first fraction [75]. Following this step, the C18 cartridge
was acidified with 0.01 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),
17
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 ether/n-butanol/acetonitrile/water (2:2:1:5, v/v/v/v) 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 ether/acetonitrile/0.1% aqueous TFA (2:2:3,
v/v/v) [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.
3.1.4. 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
18
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
phosphomolybdic/phosphotungstic 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
and/or 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
19
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 4.5 (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 “real” 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
20
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 (95:5, v/v) 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
21
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
“state of art” 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
22
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 ultraviolet/visible (UV/VIS)
region. Thus, the most common means of detection, coupled to LC, are UV/VIS,
photodiode array (PDA), and UV-fluorescence detectors. PDA is the most prevalent
method since it allows for scanning real time UV/VIS 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].
3.2. 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 and/or nitrogen species (ROS/RNS, e.g., 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].
23
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 ROS/RNS; (2) suppressing ROS/RNS formation by
inhibiting some enzymes or chelating trace metals involved in free radical production; (3)
up-regulating or protecting antioxidant defense [141].
3.2.1. 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 → 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· → 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 –CH=CH–COOH linked to the phenyl ring by resonance [138].
(2) For flavonoids, the major factors that determine the radical-scavenging capability
24
[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 ROS/RNS 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 3’,4’-o-diphenolic groups in the B ring, 3,4 or 3,5-o-diphenolic groups, and the
ketol structures 4-keto,3-hydroxy or 4-keto,5-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 catechol/gallol groups [147, 149].
H2O2 + Cu+ or Fe2+ → Cu2+ or Fe3+ + OH + ·OH- (3)
25
(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.
3.2.2. 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 → 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 → Cu+ or Fe2+ + PO· + H+ (6)
PO· + RH → POH + R· (7)
R· + O2 → ROO· (8)
ROO· + RH → ROOH + R· (9)
ROOH + Cu+ or Fe2+ → Cu2+ or Fe3+ + RO· + OH- (10)
26
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.
3.2.3. 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
oxidation/reduction 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].
27
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 (e.g., fluorescein) to compete with sample
antioxidant for peroxyl radicals generated by decomposition of 2,2’-azobis(2-
amidinopropane) dihydrochloride (AAPH). The fluorescence intensity is measured every
minute at ambient conditions (pH 7.4, 37 °C) 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 (e.g., 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 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation
(ABTS· +
) in TEAC, Fe3+(2,4,6-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 3.6
[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
28
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
“total antioxidant capacity” 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 ROS/RNS such as O2· -, HO·, and ONOO- to fully elucidate a
full profile of antioxidant capacity [159].
3.3. Natural phenolics and cancer

Cancer is a multi-step disease incorporated environmental, chemical, physical,
metabolic, and genetic factors which play a direct and/or 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.
29
3.3.1. 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
30
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].
3.3.2. 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 (e.g., 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 7,12-
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 (3.5 µM, topical application, twice/week) decreased the number of
tumors per mouse at all exposure times. After 20 weeks of TPA promotion, a greater than
31
53% inhibition of papillomagenesis by C3G was observed. After 22 weeks, there were
four tumors greater than 4–5 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.
3.3.3. 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-2’-deoxyguanosine (8-OHdG)
concentration, etc.. Improvement of antioxidant status and/or protection against oxidative
stress was observed in short term intervention studies (1 dose) with various polyphenol-
32
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 Barrett’s 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 6.5 versus pH 3.5 [202].
A recent study evaluated the effects of anthocyanin/polyphenolic-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
ml/day 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 < 0.0001), protein and lipid
peroxidation (P < 0.0001 and P < 0.001, respectively), and NF-κB binding activity (P <
0.01), and an increase of glutathione level and status (both P < 0.0001) during juice
33
uptake. The authors attributed this reduction in oxidative (cell) damage in hemodialysis
patients to the especially high anthocyanin/polyphenol 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].
3.3.4. 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,
infection/inflammation, and nutrition/diet [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].
34
3.3.4.1. Modification of the redox status of phenolics
ROS/RNS 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 carcinogen/toxin-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
35
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 (<40 mm Hg). It is not clear whether a similar
mechanism could also occur in vivo.
3.3.4.2. 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 and/or 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].
36
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 G1/G0 and G2/M 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 and/or inhibition of proliferation/survival 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.
37
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 (e.g., 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 (J774.1)
[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-
38
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.
39
Chapter 4
Preparation and Characterization of Anthocyanin-containing Extracts from

Blackberries
4.1. 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 50/50 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.
40
4.2. 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 4.5 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
41
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].
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. 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 and/or 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.
42
4.3. Materials and methods
Materials
Hull, Chester, and Black Satin blackberries were grown at WindStone Farms (Paris,
KY). 2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS),
Trolox (6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid), potassium persulfate,
formic acid (ACS, >96%), Folin-Ciocalteu phenol reagent, gallic acid (98% purity) were
purchased from Sigma (St. Louis, MO). Hydrogen chloride ~1.25 mol/l 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, 230/460 voltmeter. Whole
blackberries were passed through the Lagsenkamp pulper at a rate of 50–75 gallons/min
to produce a homogeneous blackberry puree free of skin and seeds. The blackberry puree
was stored frozen at -20°C 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 0.01% HCl (v/v). The supernatants were
collected after filtration and dried by rotary evaporation at 40 °C. The dried extract was
resuspended in deionized water and filtered through a 20-25 µm filter paper and
lyophilized to obtain dried ACE. Dried ACE was then redissolved in deionized water as a
43
stock ACE solution (140 mg/ml) and stored at -80 °C for further characterization and
cell-based studies.
Black Satin puree was used to prepare various ACEs with different solvent
combinations of water and ethanol/0.01% 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 ethanol/0.01% HCl (v/v) ratios were as follows: 0/100,
10/90, 25/75, 50/50, 75/25, 90/10, and 100/0. 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 449.2 g/mol 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 (0.1 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].
ABTS•+ was produced by reacting 7 mM ABTS with 2.5 mM potassium persulfate for 16
44
h in the dark at room temperature. The ABTS•+ solution was diluted with ethanol or water
to an absorbance of 0.70 (± 0.02) at 734 nm and equilibrated at 30 ºC. Twenty (20) µl of
ACE samples or Trolox standards in ethanol were added to 980 µl of diluted ABTS•+
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
ºC. 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-Bridge™ C18 column (250
mm × 4.6 mm, particle size 5 µm) (Waters, Milford, MA) equipped with an X-Bridge™
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: 0–45 min, linear gradient from
1% to 16% B (v/v); 46–50 min, linear gradient from 16% to 100% B; and 51–60 min,
100% B; post-time 5 min with 1% B; flow rate 1 ml/min. The UV-visible detection
wavelength was 524 nm, and the injection volume was 50 µl of ACE stock prepared from
powder of Hull blackberries (2005). The MS instrument was operated at the following
settings: ESP+ mode; capillary voltage, 3.0 kV; cone voltage, 35 V; desolvation
temperature, 300 °C; source temperature, 100 °C; scan range, 100–1,000 m/z.
HPLC assay for anthocyanins

HPLC analysis was performed using a X-BridgeTM C18 column (250 mm × 4.6 mm,
5 µm) (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 1.37), and Solvent B, 100%
acetonitrile. The elution profile was 100% to 91% A for 0.5 min, 91% A (isocratic) for
0.5-4 min, 91% A to 87% A (linear gradient) for 4-10 min, 87% A to 70% A (linear
45
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 1.0 ml/min, 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 (1.5 ml) and kept in the following
controlled conditions: -80 °C freezer, 2-8 °C stability chamber, 25 °C 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
°C, 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 °C and 37 °C

ACE stock solution derived from powder of Hull blackberries (2005) was diluted to a
final concentration of 2 mg/ml with (1) pH 1.0 buffer (214.6 mM NaCl, 8.7 mM KCl);
(2) pH 7.4 phosphate buffered saline (PBS: 30.3 mM Na2HPO4, 8.7 mM KH2PO4, 0.9%
NaCl); (3) pH 7.4 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 ± 0.5 °C. Another sample of ACE in buffer (3) and (4) were kept
in 37 ± 0.5 °C. 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 7.4) was
stopped by adding 36.5% hydrochloric acid (1/100, v/v). 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 °C for 0.5 h. Then samples were centrifuged at 13,000 rpm for
46
15 min at 4 °C 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 ± standard error (SE). Statistical analysis was performed using one
way ANOVA followed by Dunnett’s Multiple Comparison test (α ≤ 0.05) with GraphPad
5.0 (GraphPad Software Inc.; San Diego, CA).
47
4.4. 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 4.1. 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 = 0.9832). 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% (w/w) water, the actual extraction solvent was
not 100% ethanol/0.01% HCl but 73.5% ethanol/0.01% 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 1–6 (Figure 4.1). 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 4.2). It is notable that other small
peaks as shown in the chromatogram in Figure 4.1 were not identifiable by MS, and their
identities remain unknown. Cyanidin-3-glucoside (peak 1) was the main component
(71.0%) in HBE with the respective parent and daughter ion pairs (m/z 449/287). The
other three major peaks (peak 4, 12.4%; peak 5, 3.5%; peak 6, 11.6%) revealed the m/z
values of 419/287, 535/287, and 593/287, which were identified as cyanidin-3-xyloside,
48
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 (m/z 435/303), 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 4.2, 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 = 0.8057) as well as between
TAC and total anthocyanin content (r2 = 0.8746). Total anthocyanin content was also
found to be negatively correlated with the percentage of polymeric color (r2 = 0.9646).
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 4.3). 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%
49
of the total anthocyanin) in ACEs. The HPLC method was validated with external
standard cyanidin 3-glucoside and demonstrated linearity within the range of 4.7-14.2
mg/l (r2 = 0.9984 and an RSD = 1.15%). The intra and inter-day variations were 1.0 and
2.0%, respectively. The recovery percentage was in the range of 98.6-100.4%.
The change of cyanidin 3-glucoside concentration in ACE at different storage
conditions over 90 days is shown in Figure. 4.4. No significant change of cyanidin 3-
glucoside concentration was observed at –80 °C over 90 days. However, with increasing
storage temperature (4 °C and 25 °C), 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) > cyanidin-3-xyloside (peak 3) = cyanidin-3-
dioxalylglucoside (peak 5) > 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 1.95 ± 0.01 to
2.04 ± 0.01, 2.12 ± 0.00, 2.23 ± 0.01 when stored at –80 °C, 4 °C and 25 °C, 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 4.3, 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 °C stability chamber and the temperature-controlled room (22-28 °C,
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
50
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 °C

The degradation profiles of ACE anthocyanins and phenolics in biological buffers at
25 °C and 37 °C are shown in Figure 4.5. The final pH of after adding ACE (2 mg/ml) to
all media and buffers was pH 7.2, except for the pH 1.0 buffer which was maintained at
pH 1.0. As expected, elevated pH and/or temperature accelerated anthocyanins
degradation (Figure 4.5 A). Total phenolics content was also decreased with increasing
temperature (Figure 4.5 B). As a control, ACE was diluted in pH 1.0 buffer and it did not
show any significant loss of total anthocyanin and phenolics content at 25 °C 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 °C. The lowest amount of total
phenolics content retained after 48 h was in samples with RPMI 1640 medium at 37 °C,
about 53.8% 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 °C, 10% FBS in PBS at 37 °C, and RPMI 1640 medium at 37 °C were 32.7,
12.6, 5.0 and 6.2 h with r2 of 0.9757, 0.9780, 0.9907 and 0.9850, respectively.
51
4.5. 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 (> 50 °C) 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% w/w 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 1:2.5. 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
52
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 ethanol/0.01%
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 and/or 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 (e.g., 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
53
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 °C was 63.8 days (r2 = 0.9949).
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
4.5A). 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 2.6-fold shorter than that with PBS at 25 °C, although the mechanism behind
it remains unclear. When the temperature was increased from 25 °C to 37 °C, the half life
of total anthocyanin in samples with 10% FBS in PBS decreased by 2.5-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.
54
Table 4. 1. Composition and Characterization of Blackberry Extracts a
Level (mg/g of DBE)
Raw Polymeric color TAC
Cultivar (year) Total
Material Total Phenolics c (%) (μmol TE/g DBE)
anthocyanin b
Hull (2005) 5.34 ± 0.19 24.07 ± 2.70 12.99 ± 2.50 171.43 ± 15.67
Hull (2006) 7.54 ± 0.14 25.78 ± 0.56 7.20 ± 0.28 186.65 ± 0.63
Puree
Chester (2006) 7.95 ± 0.15 25.31 ± 1.80 4.89 ± 0.33 186.75 ± 10.49
Black Satin (2006) 7.16 ± 0.22 22.91 ± 1.24 3.27 ± 0.15 158.52 ± 6.90
Hull (2005) 4.51 ± 0.50 12.00 ± 0.77 1.97 ± 1.97 64.40 ± 0.60
Powder Chester (2006) 7.88 ± 0.23 14.61 ± 1.48 0.94 ± 0.94 78.02 ± 6.07
Black Satin (2006) 7.67 ± 0.51 14.81 ± 1.59 1.11 ± 0.75 71.68 ± 9.18
a
The extraction was repeated at least two times. All assays were carried out in triplicate.
Data are expressed as the mean ± SE (N = 2 or 3).
b
Total anthocyanin are expressed as cyanidin 3-glucoside equivalent.
c
Total phenolics are expressed as gallic acid equivalent.
55
Figure 4. 1. HPLC chromatogram of anthocyanins in HBE identified by liquid
chromatography-electrospray/MS: (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.
56
Table 4. 2. Peak Assignments, Retention Time, and Mass Special Data of Blackberry
Anthocyanins Detected by HPLC and Electrospray Ionization-MS
Retention time
Peak [M+] Aglycon Sugar moiety Peak identification
(minutes)
1 20.1 449/287 Cyanidin Hexose Cyanidin-3-glucoside
2 21.5 419/287 Cyanidin Pentose Cyanidin-3-arabinoside
3 26.4 435/303 Delphinidin Pentose Delphinidin-3-xyloside
4 28.7 419/287 Cyanidin Pentose Cyanidin-3-xyloside
5 31.3 535/287 Cyanidin Malonyl-hexose Cyanidin-3-malonyl-glucoside
6 34.9 593/287 Cyanidin Dioxalyl-hexose Cyanidin-3-dioxalyl-glucoside
57
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
ethanol/0.01% HCl ratios (v/v) were as follows: 0/100, 10/90, 25/75, 50/50, 75/25, 90/10,
and 100/0. 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% ethanol/0.01% HCl. Data are presented as the mean ± SE of three
independent extractions.
58
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).
59
Figure 4. 4. Changes in cyanidin 3-glucoside concentration in powder-derived ACE
from Hull blackberry under different storage conditions over 90 days: –80 °C (■); 2-
8 °C (∆); temperature-controlled room (22-28 °C) with white light (▼); 25 °C (◊).
60
Table 4. 3. Effect of Storage Conditions on Several Parameters of the Hull
Blackberry Extract after 90 Days a
Day 90
Day 0
Parameters -80 °C 4 °C 25 °C
Level / (% of Retention)
600 ± 4.3 589 ± 8.2 539 ± 3.8 230 ± 0.9
Cyanidin 3-glucoside (mg/liter)
(100%) (98.3%) (89.8%) (38.4%)
1899 ± 18.1 1680 ± 6.9 1696 ± 6.8 1537 ± 16.2
Total Phenolics (mg/liter) b
(100%) (88.5%) (89.3%) (80.1%)
710 ± 22.3 684 ± 15.8 621 ± 8.3 261 ± 4.7
Total anthocyanin (mg/liter) c
(100%) (96.3%) (87.5%) (36.8%)
0.12 ± 0.12 1.43 ± 0.11 2.61 ± 0.88 13.96 ± 1.11
Polymeric Color (%)
NA NA NA NA
8.9 ± 0.13 8.0 ± 0.27 7.8 ± 0.11 7.3 ± 0.09
TAC (µmol/liter) d
(100%) (89.2%) (86.4%) (81.2%)
a
Data are expressed as mean ± SE (n = 3).
b
Total anthocyanin are expressed as mg of cyanidin 3-glucoside equivalent/l of extract.
c
Total phenolics are expressed as mg of gallic acid equivalent/liter of extract.
d
TAC are expressed as µmol of trolox equivalent/liter of extract.
% of Retention was calculated based on the day 0 concentration as 100%.
NA: Not applicable.
61
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 °C
and 37 °C. ACE stock solutions were diluted to a final concentration of 2 mg/ml in (1)
pH 1.0 buffer at 25 °C (●); (2) pH 7.4 PBS at 25 °C (□); (3) pH 7.4 PBS with 10% FBS
at 25 °C (▲); (4) pH 7.4 PBS with 10% FBS at 37 °C (○); (4) RPMI 1640 medium
supplemented with 10% FBS at 37 °C (▼) 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 ± SE of three independent
experiments.
62
Chapter 5
In vitro Anticancer and Anti-inflammatory Effects of Blackberry Extracts and

Possible Mechanisms of Action
5.1. 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 0.80 to 11.2 mg/ml 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 J774A.1 murine macrophages. It was found
using concentrations ranging from 0.15 to 1.55 mg/ml, 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 µg/ml) and low-dose (0.1 µg/ml) 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.
63
5.2. 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, and/or 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
64
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, J774A.1. 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.
65
Materials
5-(and-6)-carboxy-2´,7´-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-(4,5-dimethyl-thiazol-2-yl)-2,5-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
J774A.1, were purchased from American Type Cell Culture Collection (ATCC,
Rockville, MD). HT-29 cells were grown in modified McCoy’s 5A medium (ATCC,
Rockville, MD). All other cells were grown in RPMI-1640 medium (Invitrogen;
Carlsbad, CA). All media were supplemented with 100 U/ml penicillin, 100 µg/ml
streptomycin and 10% FBS. Cells were maintained at 37 ºC in a humidified 5% CO2
incubator.
Bone marrow cells were obtained by flushing the femurs of BALB/c mice (Harlan
Sprague-Dawley Laboratories, Indianapolis, IN) with 1× Hanks’ balanced salt solution.
Cells were cultured in 100-mm bacteriological Petri dishes at 2 × 105 cells/ml in 10 ml of
complete RPMI 1640 medium (supplemented with 10% heat-inactivated fetal calf serum,
1 mmol/l HEPES, 2 µmol/l L-glutamine, 10 U/ml penicillin, 100 U/ml streptomycin, and
50 µmol/l 2-mercaptoethanol) containing 20–25 ng/ml granulocyte-macrophage colony-
stimulating factor (GM-CSF) at 37°C in 7% CO2. The cells were supplemented with an
additional 10 ml of complete RPMI 1640 with 20–25 ng/ml 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 20–25 ng/ml 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.
66
Cell viability assay
The adherent HT-29, MCF-7 and the suspension HL-60 cells were seeded at an initial
concentration of 3×104 cells/well in 96-well plates. After 24 h, cells were treated with the
ACE stock solution (140 mg/ml) with final doses ranging from 0.084 to 11.2 mg/ml.
Vehicle controls were the normal media with the corresponding pH adjusted with 2.5 N
HCl for each treated group. HL-60 cells were also treated with cyanidin 3-glucoside (in
0.1% DMSO) with final doses ranging from 10.4 to 62.2 µg/ml. 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 (0.5 mg/ml) in fresh medium in the dark at 37 °C
for 4 h. Next, supernatant was removed and 200 µl 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 × 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 (3×104 cells/well) were
loaded with 100 µM CH2DCFDA (dissolved in DMSO) for 30 min at 37 °C. 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 ± 20 nm, emission 528 ± 20 nm with the microplate reader
every 10 min for 1.5 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
67
of catalase (100 U/ml) 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 µM.
Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α) stimulation studies

J774A.1 cells were plated in 400 µl complete RPMI 1640 medium at a density of 4 ×
105 cells per well in 48-well tissue culture plates (Costar, Corning, NY) at 37°C in 5%
CO2 overnight. J774A.1 cells were treated with 1 µg/ml LPS for 30 min, followed by
ACEs addition at final concentrations ranging from 0.31 to 1.55 mg/ml medium.
Supernatants were collected after 5.5 h and stored at -80 °C 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 (mono/mono) ELISA kit from BD Biosciences (San
Diego, CA) according to the instructions from the manufacturer. Briefly, 96-well plates
were coated with 100 µl of either IL-6 capture antibody (1:250 dilution of stock solution
in 0.1 M sodium carbonate, pH 9.5) or TNF-α capture antibody (1:250 dilution of stock
solution in 0.1 M sodium phosphate, pH 6.5) overnight at 4 °C. The plates were blocked
for 1 h at room temperature (RT) with 200 µl assay diluents (10% FBS in 0.01 M
phosphate buffered saline, pH 7.4). After washing with wash buffer (PBS with 0.05%
Tween 20), the plates were incubated with 100 µl sample dilutions, or standard
recombinant (IL-6 or TNF-α in a range of 15.6-500 pg/ml) for 2 h at RT. The plates were
washed with wash buffer and incubated with 100 µl 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 µl of
tetramethylbenzidine (TMB) substrate and incubated for 30 min at RT. The color
development was stopped by the addition of 50 µl of 2 M H2SO4 and the absorbance at
68
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 µl of
complete RPMI 1640 containing 20–25 ng/ml GM-CSF at 4 × 105 cells per well in 48-
well tissue culture plates (Costar, Corning, NY) at 37 °C 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 µg/ml) or low-dose (0.1 µg/ml) 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 °C 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 ± standard error (SE). Statistical analysis was performed
using one way ANOVA followed by Dunnett’s or Bonferroni’s Multiple Comparison test
(α ≤ 0.05) with GraphPad 5.0 (GraphPad Software Inc.; San Diego, CA).
69
5.4. 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 5.1A), ovarian cancer (MCF-
7, Figure 5.1B) and leukemia (HL-60, Figure 5.1C) 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 5.1D. The cell viability of HL-60 cells was decreased from
99.5% to 45.9% in a dose-dependent manner after 48 h of exposure with cyanidin 3-
glucoside in a concentration range of 10.4-62.2 µg/ml.
H2O2 generation by ACEs and cyanidin 3-glucoside in RPMI 1640 medium

H2O2 production by ACEs at 1.4 mg/ml with or without catalase in RPMI 1640
medium with 10% FBS over time is illustrated in Figure 5.2. 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 5.1. 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 U/ml) 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 0.7 mg/ml of puree-derived ACE but not until 48 h with 1.4 mg/ml of puree-
derived ACE in RPMI1640 medium with 10% FBS. Since the pH of the medium
decreased from pH 7.2 to 6.5 with increasing concentration of ACE from 0.14 to 7
mg/ml, 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,
70
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 1.95 and 2.46-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 µM) 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 1.4 mg/ml, the concentration of H2O2 at 2 h in the
medium was 6.3-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
(< 10 µM) 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 5.3. 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 1.5 h (data not shown). As shown in Figure 5.3. , the
baseline intracellular ROS level in HL-60 cells (Veh.) was increased by 71.8 ± 2.7%
(mean ± SE) as compared to non-CDCFDA loaded cells. Authentic H2O2 (98 µM)
significantly increased the fluorescence intensity by about 1104.4%. As expected,
71
catalase (500 U/ml) decreased the increase of fluorescence intensity caused by H2O2 (98
µM) to the baseline level. Puree-derived ACE increased the intracellular ROS level dose-
dependently from 69.3 ± 16.2% to 322.2 ± 23.1% in concentrations ranging from 0.14 to
2.80 mg/ml. 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 µg/ml) 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 5.4). Authentic
H2O2 (98 µM) reduced HL-60 cell viability to 8.8 ± 0.5%. Catalase (500 U/ml) rescued
the H2O2-induced cell death to 92.1 ± 1.3%. 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 48.7 ± 4.0% to
77.2 ± 1.8%, and 28.8 ± 2.7% to 52.3 ± 1.5% at ACE concentrations of 2.1 and 2.8
mg/ml, respectively. However, at high concentration (5.6 mg/ml), 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 J774A.1 cells

To investigate whether ACEs exhibit immune-modulation activity on macrophages,
J774A.1 cells were treated with various concentrations of ACEs 30 min after LPS
72
stimulation (Figure 6.2). Exposure of LPS (1µg/ml) for 6 h induced TNF-α and IL-6
secretion significantly in J774A.1 cells. For all concentrations of powder-derived ACEs
added, both TNF-α and IL-6 were reduced in a concentration-dependent manner (Figure
6.2A). 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 2.5 to 3.5 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 6.2B). 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 J774A.1 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 6.1A, baseline
release of IL-12 from nonstimulated DCs was low, with an IL-12 concentration of only
1.46 ng/ml. However, for all concentrations of ACE added, the IL-12 release was reduced
in a concentration-dependent manner with only 0.37 ng of IL-12/ml secreted using a
concentration of 5.25 mg/ml of medium. As expected, both high-dose (10 µg/ml) and
low-dose (0.1 µg/ml) Lipid A resulted in very high release of IL-12 from DCs of 624
ng/ml and 468 ng/ml, respectively (Figure 6.1B). 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 ng/ml when ACE was added in the
range from 0.7 to 5.25 mg/ml. 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 ng/ml when
ACE was added in the range from 0.7 to 5.25 mg/ml. 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.
73
5.5. 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
5.1A, 5.1B, 5.1C). 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 5.7 and 3.3-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 5.1. Our results showed that both puree and powder-derived
ACEs produced levels of H2O2 (>10 µM) 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 and/or 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 5.3) and H2O2
concentration in medium did not exceed 10 µM in the first 6 h of incubation (Table 5.1),
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
74
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 µM 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 G2/M 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 5.1D) as well as its
ability to spontaneously generate H2O2 in RPMI 1640 medium (Table 5.1) and
intracellular ROS in HL-60 cells (Figure 5.2). It was found that cyanidin 3-glucoside
exhibited antiproliferative activity in a concentration range of 10.4-62.2 µg/ml. 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 3.6 and 14.5 µg/ml, 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
75
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.
76
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 J774A.1 macrophages. The inhibition
potency was comparable among cultivars. However, it is worth noting that in another
experiment, we pretreated J774A.1 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
264.7 macrophages were pretreated with anthocyanins and berry extracts followed by
LPS/IFN-γ stimulation. It was found that blackberry extract has little or mild induction
effect on TNF-α production in RAW 264.7 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.
77
*
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.
78
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 ± SE (n = 3). Curves were fit by nonlinear regression using
GraphPad Prism. Half maximum effective concentration (EC50) is reported as the mean ±
SE (n = 3) in the figure legend.
79
Figure 5. 2. H2O2 generation by Black Satin ACEs in RPMI 1640 medium with 10%
FBS over time. Authentic H2O2 (50 µM), puree and powder-derived ACE (1.4 mg/ml)
with or without catalase (100 U/ml) were incubated with RPMI 1640 medium with 10%
FBS at 37ºC for 48 h. At each time point, an aliquot was taken out and measured for
H2O2 concentration. Data are presented as the mean ± SE (n = 6).
80
Table 5. 1. H2O2 Generation by ACEs and Cyanidin 3-glucoside (C-3-G) in Medium
at 37 ºC a
H2O2 level (µM) at various times (h)
Medium Concentration
1 2 6 24 48
0.14 5.33 ± 0.12 5.09 ± 1.00 4.42 ± 0.40 6.57 ± 0.25 6.74 ± 0.37
0.70 8.98 ± 0.24 27.45 ± 0.83 20.85 ± 0.61 11.82 ± 0.26 9.97 ± 0.37
RPMI 1640
10% FBS
11.48 ± 0.28 33.00 ± 1.76 41.97 ± 1.89 63.70 ± 2.89 61.13 ± 1.20
1.4
(ND)# (ND)# (ND)# (ND)# (ND)#
7.0 9.06 ± 0.22 21.98 ± 1.56 52.74 ± 2.00 145.41 ± 3.98 171.91 ± 1.15
ACEpuree
(mg/ml)
2.33 ± 0.19 4.24 ± 0.22 3.10 ± 0.79
0.70
(ND)# (ND)# (ND)#
RPMI 1640
1.96 ± 0.23 5.22 ± 0.20 4.32 ± 0.24
10% FBS 1.4
(ND)# (ND)# (ND)#
HL-60 cells
3.26 ± 0.25 6.54 ± 0.18 ND
2.8
(ND)# (ND)# (ND)#
RPMI 1640 1.4 81.70 ± 2.66 150.46 ± 1.22
0.14 1.30 ± 0.25 1.09 ± 0.30 1.50 ± 0.40 2.05 ± 0.31 3.01 ± 0.29
0.70 ND 1.92 ± 0.26 4.93 ± 0.73 6.09 ± 0.41 10.92 ± 0.39

RPMI 1640
10% FBS
ND 3.63 ± 0.29 8.59 ± 1.12 15.18 ± 0.63 19.39 ± 1.21
1.4
(ND)# (ND)# (ND)# (ND)# (ND)#
ACEpowder
7.0 ND 3.42 ± 0.21 6.74 ± 0.52 11.28 ± 0.65 28.69 ± 1.09
(mg/ml)
1.40 ± 0.10 1.54 ± 0.17 ND

1.4
RPMI 1640 (ND)# (ND)# (ND)#
10% FBS
HL-60 cells 2.02 ± 0.28 1.68 ± 0.17 1.43 ± 0.14
2.8
(ND)# (ND)# (ND)#
RPMI 1640 1.4 9.68 ± 0.36 33.36 ± 1.20
20.7 ND ND ND
RPMI 1640
10% FBS
62.2 2.97 ± 0.16 2.73 ± 0.14 1.90 ± 0.28
20.7 1.26 ± 0.10 5.90 ± 0.15 12.79 ± 0.31

C-3-G
RPMI 1640
(µg/ml)
62.2 3.09 ± 0.18 9.04 ± 0.18 19.39 ± 0.32
20.7 ND
RPMI 1640
10% FBS
HL-60 cells
62.2 ND
RPMI 1640 46.26 ± 1.16 44.24 ± 1.92 35.29 ± 0.90 17.65 ± 0.33 12.49 ± 0.25
50
10% FBS (ND)# (ND)# (ND)# (ND)# (ND)#
H2O2 (µM)
RPMI 1640
10% FBS 100 ND
HL-60 cells
a
Data are expressed as mean ± SE (n = 6).
#
With catalase (100 U/ml).
ND: Not detected.
81
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 µM), H2O2 (98 µM) plus catalase (500 U/ml) 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 ± SE of three independent experiments. b P ≤ 0.01, c
P ≤ 0.001 compared to the vehicle group.
82
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 U/ml) (■) or
PBS (□), and concomitantly with H2O2 (98 µM) or puree and powder-derived ACEs from
Black Satin for 48 h and cell viability was measured. Data are presented as the mean ±
SE of three independent experiments. b P ≤ 0.01, c P ≤ 0.001 compared to the non-catalase
treatment group with corresponding H2O2 or ACE concentration.
83
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 ± SE (n = 3) for each concentration. (A): * P ≤ 0.05, ** P ≤
0.01 compared to the vehicle control without ACE and Lipid A treatment. (B): **
P≤
0.01, *** P ≤ 0.001 compared to the vehicle control with low-dose Lipid A treatment. # P
≤ 0.01, ## P ≤ 0.001 compared to the vehicle control with high-dose Lipid A treatment.
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 J774A.1 macrophages. Cells were treated
with LPS (1 µg/ml) for 30 min, followed by puree derived (A) or powder-derived (B)
ACE treatment. Supernatants were collected after 5.5 h and assayed by using specific
ELISA. Results are represented as mean ± SE (n = 3). a P ≤ 0.05, b P ≤ 0.01, c P ≤ 0.001
compared to LPS controls.
85
Chapter 6
Correlation of the In Vitro Antioxidant and Antiproliferative Properties of

Blackberry Extracts to Their Phytochemical Constituents
6.1. 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.
86
6.2. 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
87
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.
88
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 U/ml penicillin, 100
µg/ml streptomycin and 10% FBS. Cells were maintained at 37 ºC in a humidified 5%
CO2 incubator.
Methanol, ethyl acetate and hydrochloric acid (36.5%) were purchased from Fisher
Scientific (Fair Lawn, NJ). 5-(and-6)-carboxy-2´,7´-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
2.8) or powder-derived (pH 2.0) ACEs stock solution (140 mg/ml) 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 °C 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.
89
Total phenolic measurement
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.
Trolox equivalent antioxidant capacity (TEAC) assay

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.
Cell viability assay

The suspension HL-60 cells were seeded at an initial concentration of 3×104
cells/well 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.
Determination of intracellular ROS in HL-60 cells

used as an indicator of intracellular ROS generation. HL-60 cells (3×104 cells/well) were
loaded with 100 μM CH2DCFDA (dissolved in DMSO) for 30 min at 37 °C. 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 ± 20 nm, emission 528 ± 20 nm with the microplate reader
for 1 h. Data are reported as percentage increase of DCF fluorescence associated with the
90
CH2DCFDA-loaded cells compared to that of corresponding non-CH2DCFDA-loaded
cells (negative controls).
Caspase 3-like activity

The HL-60 cells were seeded at an initial concentration of 3×104 cells/well 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 3/7 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).
Data were given as the mean ± standard error (SE). Statistical analysis was performed
using one way ANOVA followed by Dunnett’s or Bonferroni’s Multiple Comparison test
(α ≤ 0.05) with GraphPad 5.0 (GraphPad Software Inc.; San Diego, CA).
91
6.4. 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 6.1, the WF contained the majority of the total
substance in the Hull puree ACE (953.07 ± 9.86 mg out of 989.59 ± 11.32 mg of total
solid recovered after separation) with an insignificant level of total phenolics (1.93 ± 0.31
mg/g of solid in WF). Most phenolics were concentrated in the EAF and MF with the
majority of the anthocyanin obtained in the MF (97.33 ± 3.16% of yield).
Correspondingly, MF and EAF possessed significantly higher TAC than WF as well as
the original puree ACE (Table 6.1). For example, the total anthocyanin, total phenolics
and TAC levels of MF were 32.92, 21.51 and 24.06-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 6.2). 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
92
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 6.1). 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 6.1, the potencies of antiproliferative activity were in the order of: MF >
EAF > ACE > WF for ACE and corresponding fractions derived from both puree (Figure
6.1 A) and powder (Figure 6.1B). 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 49.2 ± 7.9 μg/ml), which was comparable with
that of the pure cyanidin 3-glucoside determined in our previous studies (cell viability
was decreased to 45.9% at 62.2 µg/ml of cyanidin 3-glucoside) [307]. Since the total
anthocyanin in MF was about 16.7%, 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,
93
783.8 μg/ml ACE contained 21.7 μg/ml MF, 6.3 μg/ml EAF and 755.5 μg/ml 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
(6.3 μg/ml) and WF (755.5 μg/ml) 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) and/or 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 6.2) 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 μg/ml) and powder (292 μg/ml) were comparable with that
induced by 49 μM of H2O2 (about 342.0%). 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 6.3, NAC (4 mM)
rescued the H2O2 (98 μM)-induced cell death from 8.8 ± 0.8 to 93.3 ± 0.9%. 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 μg/ml, puree-derived MF dose-dependently decreased cell viability from 85.5 ±
2.3% to 10.2 ± 0.8%. 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 6.3). On the other hand,
NAC (4 mM) significantly rescued the cell death induced by powder-derived MF at
concentrations ranging from 146 to 292 μg/ml. For example, the cell viability was
increased from 31.7 ± 3.5% to 80.3 ± 3.2% when cells were incubated with NAC
together with powder-derived MF (292 μg/ml) (Figure 6.3). These results suggested that
the antiproliferative effects of MF and ACE derived from puree were ROS-independent
94
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 6.4). As a positive control, H2O2 (49 μM)-induced caspase 3-
like activity (2.72-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 (2.23-fold of vehicle control,
146 μg/ml; 3.64-fold of vehicle control, 292 μg/ml) was reduced to the baseline level
(0.87-fold of vehicle control, 146 μg/ml; 0.99-fold of vehicle control, 292 μg/ml) 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
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-
95
like activity. Furthermore, our studies 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.
96
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 953.07 ± 9.86 8.06 ± 0.50 28.46 ± 0.63 989.59 ± 11.32
(mg)
Total anthocyanin in recovered ND 0.12 ± 0.01 4.90 ± 0.16 5.02 ± 0.17
fraction (mg)
Anthocyanin level in recovered ND 14.77 ± 0.75 165.59 ± 5.07 -
fraction (mg/g solid)
Yield (%) # - 2.53 ± 0.28 97.33 ± 3.16 99.86 ± 3.40
Fold concentrated in recovered - 2.94 32.92 -

fraction*
Total Phenolics c
(mg)
Total phenolics in recovered 1.93 ± 0.31 440.35 ± 27.92 512.83 ± 4.22 -
fraction (mg)
Phenolics level in recovered 1.85 ± 0.42 3.79 ± 0.46 14.91 ± 0.62 20.55 ± 0.96
Yield (%) # 9.19 ± 2.10 18.87 ± 2.29 74.20 ± 3.09 102.67 ± 4.78
Fold concentrated in recovered 0.09 21.89 25.51 -

fraction *
TAC
TAC Level in recovered fraction 14.98 ± 3.12 2734.61 ± 96.77 4838.20 ± 236.96 -
(μmol TE/g solid)
fraction *
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 ± SE (N = 3).
b
c
#
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.
97
Table 6. 2. Composition and Characterization of Fractions Separated from Hull
Powder ACE by SPE a
WF EAF MF Total
Total Anthocyanin b
(mg)
Total anthocyanin in recovered ND 5.65 ± 1.36 305.74 ± 33.56 -
fraction (mg)
Anthocyanin level in recovered ND 0.036 ± 0.006 3.57 ± 0.38 3.60 ± 0.37
Yield (%) # - 0.77 ± 0.13 76.02 ± 8.03 76.79 ± 7.91
Fold concentrated in recovered - 1.19 65.18 -

fraction*
Total Phenolics c
(mg)
Total phenolics in recovered 0.95 ± 0.053 253.29 ± 33.59 455.01 ± 27.92 -
fraction (mg)
Phenolics level in recovered 0.64 ± 0.32 1.67 ± 0.22 5.32 ± 0.52 7.63 ± 0.37
Yield (%) # 4.50 ± 3.67 16.91 ± 2.27 53.87 ± 5.30 77.22 ± 4.57

fraction *
TAC
TAC Level in recovered fraction 16.61 ± 5.60 1614.26 ± 303.38 4739.90 ± 624.78 -
(μmol TE/g solid)
Fold increase in recovered 0.22 21.61 63.45 -
fraction *
a
The SPE separation was repeated three times with the same ACE sample. The volume of Hull powder 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 ± SE (N = 3).
b
c
#
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.
98
A MFpuree: 49.2 ± 7.9 µg/ml
EAFpuree: 115.2 ± 7.8 µg/ml
WFpuree: 3746 ± 325.2 µg/ml
ACEpuree: 783.8 ± 49.6 µg/ml
120
Cell Viability (% Control)

100
80
60
40
20
0
0 100 200 300 400 500 1000 2000 3000 4000
C (µg/ml)
B MFpowder: 78.8 ± 4.8 µg/ml

EAFpowder: 183.2 ± 35.4 µg/ml
WFpowder: 4013 ± 219 µg/ml
ACEpowder: 2491 ± 335 µg/ml
120
Cell Viability (% Control)
100
80
60
40
20
0
0 100 200 300 400 500 2000 4000 6000 8000
C (µg/ml)
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 ±
SE (n = 3). Curves were fit by nonlinear regression using GraphPad Prism. Half
maximum effective concentration (EC50) is reported as the mean ± SE (n = 3) in the
figure legend.
99
Figure 6. 2. The effects of Hull ACE (derived from puree) and methanol fractions on
the intracellular ROS level in HL-60 cells. CH2DCFDA-loaded and non-CH2DCFDA
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 ±
SE (n = 3). c P ≤ 0.001 compared to the vehicle group.
100
Figure 6. 3. The effects of NAC on cytotoxicity induced by puree-derived ACE and
methanol fractions of ACEs from Hull blackberries. Cells were treated with NAC (4
mM) (■) or PBS (□), and concomitantly with H2O2 (98 µM), 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 ± SE
(n = 3). c P ≤ 0.001 compared to the corresponding non-NAC treatment group.
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. Cells were
treated with NAC (4 mM) (■) or PBS (□), and concomitantly with H2O2 (49 µM), or
puree-derived ACE or methanol fractions of ACEs from Hull for 24 h and caspase 3-like
activity was measured by Caspase-Glo 3/7assay. Data are presented as mean ± SE (n =
3). c P ≤ 0.001, NAC-treated groups were compared to the corresponding NAC-untreated
groups.
102
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 50/50 greatly decreased the
103
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 0.80 to 11.2 mg/ml 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 10.4-62.2 µg/ml. 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 (>10 µM) 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
104
cells and subsequently induced cytotoxicity by generating ROS inside the cells and/or by
other mechanisms. However, as for powder-derived ACE, its growth inhibition activity
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 J774A.1 macrophages and the
inhibition potencies were comparable among cultivars. However, it is worth noting that in
another experiment, we pretreated the J774A.1 macrophage with ACEs followed by LPS
stimulation. 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 mechanism studies are essential to understand the possible role of ACEs in
inflammatory diseases or host defense system.
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
105
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
106
therapy of cancer and inflammatory diseases may be achieved by carefully selecting
administration route and formulation development.
107
Appendices
This section contains the following information and additional experiments:
• Appendix A: An in vitro investigation of the effects of the blackberry extracts on

UVR-induced cell death on human epidermal keratinocytes (HEK)
• Appendix B: Antimicrobial and anti-viral properties of blackberry extract
• 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)
• Appendix D: Preparation of Vanishing Cream Containing Blackberry ACE
• Appendix E: Preparation of Mucoadhesive Gel Containing Freeze Dried Black
Raspberry (FDBR) Powder
• Appendix F: Stabilization of Anthocyanin in Dried Blackberry ACE by Mannitol
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)
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 – 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 70.5 ± 4.4% to 35.7 ± 2.0% in a dosing range of 30-60
mJ/cm2 (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 μg/ml 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.
109
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
– 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 ± SE (n = 3).
110
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 1.31 mg/ml of extract inhibited virus-induced CPE by 50% (EC50).
Acknowledgements
I would like to thank Dr. Miller’s lab in the University of Kentucky for performing
these studies.
111
100
80 A. actino.
S. gordonii
% Inhibition
60
40
20
0
0.000 0.003 0.005 0.011 0.022 0.044 0.088 0.175 0.350 0.700 1.400
Extract (mg/mL)
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.
112
120
% Reduction in CPE
100
80
60 EC 50
40
20
0
14 7 3.5 1.75 0.875
Extract (mg/mL)
Figure B 2. Inhibition of Herpes Virus (HSV-1)-induced cytopathic effect (CPE)

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.
113
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) ~1.25 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 2.19 ml Hydrogen
chloride ~1.25 M in ethanol to 7.81 ml ethyl alcohol and mix well.
2. Prepare 25 ml ethanol containing 0.01% HCl. Dilute 250 µl 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 0.01% 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 °C.
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 1.4 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 °C (preferred) or -20 oC until assayed.
114
C2. Total anthocyanin measurement (pH Differential Method)
Materials
Blackberry extract (in water).
Potassium chloride, Sigma, Cat. No. P3911
Sodium acetate, Sigma, Cat. No. 236500
Concentrated hydrogen chloride (HCl), 36.5%-38.5%, Sigma, Cat. No. 320331
1 liter glass bottles
Procedure
1. Prepare 0.025 M potassium chloride buffer, pH 1.0. Mix 1.86 g KCl and 980 ml of
distilled water in a beaker. Measure the pH and adjust to 1.0 with concentrated HCl.
Transfer to a 1 liter volumetric flask and fill to 1 liter with distilled water.
2. Prepare 0.4 M sodium acetate buffer, pH 4.5. Mix 54.43 g CH3CO2Na·3 H2O and
~960 ml distilled water in a beaker. Measure the pH and adjust to 4.5 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 0.025 M
potassium chloride buffer, pH 1.0 until the absorbance of the sample at the λvis-max
(510 nm) is within the linear range of the spectrophotometer (i.e., for most
spectrophotometers the absorbance should be less than 1.2). 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 1.0, and
the other with sodium acetate buffer, pH 4.5, 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 = (A510– A700) pH 1.0 – (A510– A700) pH 4.5
7. Calculate the monomeric anthocyanin pigment concentration in the original sample
using the following formula:
C (mg/L) = (A × MW × DF × 1000)/( ε × 1)
115
(MW = 449.2, ε = 26900 for cyanidin-3-glucoside)
8. To calculate “Total Anthocyanins (TA)” per gram of “Blackberry Concentrate” (BC),
complete the following calculation:
TA (mg/g BC) = C (mg/liter) × 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 3.4-5.2
mg/gram Blackberry Concentrate with a target concentration of 4.3 mg/gram.
Notes
1. When diluting sample with buffers, in order to not exceed the buffer's 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.
116
C3. Total phenolics measurement (Folin-Ciocalteu Method)
Materials
Blackberry extract (in water)
Folin-Ciocalteu’s 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 0.500 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 mg/liter gallic acid, the effective range of the assay.
4. From each gallic acid working solution, sample, or blank, pipet 20 µl into separate
cuvettes, and to each add 1.58 ml distilled water, and then add 100 µl of the Folin-
Ciocalteu phenol reagent, and mix well. Wait for between 30 sec and 8 min, and then
add 300 µl of the sodium carbonate solution, and shake to mix.
5. Leave the solutions at 20 °C 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:
117
Concentration (mg GAE/liter) = (A765 nm– Intercept)/Slope
8. To calculate “Total phenol (TP)” per gram of “Blackberry Concentrate” (BC),
complete the following calculation:
TP (mg GAE/g BC) = C (mg GAE/liter) × 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 7.0-10.6 mg/gram
Blackberry Concentrate with a target concentration of 8.8 mg/gram.
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 mg/ml may be diluted 2.5-5
times with water before the assay.
118
C4. Total antioxidant capacity measurement (Trolox Antioxidant Equivalent Assay)
Materials
Blackberry extract (in water)
2,2′-Azinobis(3-ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS), Sigma,
Cat. No. 11557
6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox), Sigma, Cat. No.
391913
Potassium persulfate, Sigma, Cat. No. 216224
Ethyl alcohol (200 proof, absolute, anhydrous, ACS-USP grade), Pharmco-AAPER.
20 ml-glass vials
50 ml-plastic centrifuge tubes
Procedure
1. Prepare ABTS•+ stock solution. Add 38.4 mg ABTS to 10 ml distilled water in 20 ml-
glass vial, then add 6.62 mg potassium persulfate to the solution and store it for 16 h
in the dark at room temperature.
2. Prepare 2.5 mM Trolox stock solution. Add 12.52 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 ABTS•+ working solution. Dilute the ABTS•+ stock solution with distilled
water to an absorbance of 0.70 ± 0.02 in a 50 ml-plastic centrifuge tube and
equilibrate at 30 °C. 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 mg/ml, the dilution
factor is around 20-50.
7. Add 980 µl of ABTS•+ working solution into the cuvette and measure the absorbance
at 734 nm (A 0 min). From each Trolox working solution and sample, pipet 20 µl into
980 µl of ABTS•+ working solution in separate cuvettes. Mix well and measure the
119
absorbance at 734 nm at 30 °C 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 min– A 7 min) × 100/ A 0 min
9. Plot % of inhibition vs. concentration of the ABTS•+ 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 (µM TE) = (% of inhibition – Intercept)/Slope
11. To calculate “Total Antioxidant Capacity (TAC)” per gram of “Blackberry
Concentrate” (BC), complete the following calculation:
TAC (µmol TE/g BC) = C (µM TE) × 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 micromole/gram Blackberry Concentrate with a target
concentration of 50 micromole/gram.
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. 26:1231-1237 (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 ABTS•+ working solution.
120
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 °C. After
the ingredients of the oil phase were melted at about 60 °C, the oil phase was mixed with
the water phase at 70 °C for 0.5 h at 1,000 rpm. Then, the combined phases were allowed
to mix at room temperature for another 2 h at 1,000 rpm and left to sit at room
temperature overnight. To prepare cream containing 10% blackberry ACE, 90% (w/w)
placebo cream was mixed with 10% (w/w) blackberry ACE. 2.5 N sodium hydroxide
(NaOH) was used to adjust pH to 3.5-4.0.
121
Table D 1. Formula for Placebo Vanishing Cream
Ingredients % (w/w)
Water 79.8
Propylene Glycol 3.0
Sorbitol, 70% 2.0
Sorbic Acid 0.2
Butylated Hydroxytoluene 0.1
Simethicone 0.1
Sub-total Water Phase 85.2
Petrolatum 5.6
Cetostearyl Alcohol 4.4
Brij 58 4.0
Glyceryl Monostearate 0.2
PEG 400 Monostearate 0.6
Sub-total Oil Phase 14.8
Total 100
122
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 1,025 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% w/w. Either 2.5 N sodium hydroxide (NaOH) or
2.33 N HCl was added to adjust to the desired pH. Deionized water was then added to
q.s. 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.
123
Table E 1. Formula of Mucoadhesive Gels Containing Freeze-Dried Black Raspberries (FDBR) Powder
Ingredient 10% FDBR Gel 10% FDBR Gel 10% FDBR Gel 10% FDBR Gel 10% FDBR Gel
pH 3.0, %(w/w) pH 3.5, %(w/w) pH 4.0, %(w/w) with 0.1% AA with 0.1% AA
pH 3.0, %(w/w) pH 3.5, %(w/w)
Water 83.925% 83.975% 83.79% 83.82% 83.875%
Noveon AA1, NF 1.35% 1.35% 1.35% 1.35% 1.35%
Carbopol 971P, NF 1.575% 1.575% 1.575% 1.575% 1.575%
Glycerin, USP 1.0% 1.0% 1.0% 1.0% 1.0%
EDTA, USP 0.1% 0.1% 0.1% 0.1% 0.1%
2-phenoxyethanol 1.0% 1.0% 1.0% 1.0% 1.0%
Benzyl alcohol, USP 1.0% 1.0% 1.0% 1.0% 1.0%
2.5N NaOH N N ～0.185％ N N
2.33N HCl ～0.05% N N ～0.055％ N
Ascorbic Acid N N N 0.1% 0.1%
FBDR 10% 10% 10% 10% 10%
124
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 ~1:10 w/w
corresponding to 7.7 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 2.5-2.6. 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 ºC
and 25 ºC, and 3) dried berry extract with mannitol stored at 4 ºC and 25 ºC. 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.
125
Cn-3-glucoside
Cn-3-arabinoside
100
Cn-3-xyloside
Cn-3-malonyl-glucoside
Retention of Average
75
% Anthocyanin
50
25
0
0 1 2 0 1 2
Dried Berry Extract Dried Berry Extract

Months at 25oC with Mannitol
Fig. 4: Dried berry extract with mannitol shows enhanced

Figure Fretention
1. Dried blackberry extractatwith
of anthocyanins 25 Cmannitol
o showed enhanced retention of
over 2 months.
anthocyanins at 25 °C 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.).
126
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148
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 “The effect of SIPI5052, a
new polyamine site NMDA receptor antagonist, on ischemia”. 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.
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University of Kentucky

University of Kentucky Doctoral Dissertations Graduate School

PREPARATION AND CHARACTERIZATION OF BLACKBERRY

The Graduate School

A dissertation submitted in partial fulfillment

Co-Directors: Dr. Russell Mumper, Professor of Molecular Pharmaceutics, University of

Copyright © Jin Dai 2009

PREPARATION AND CHARACTERIZATION OF BLACKBERRY EXTRACTS

Blackberries are rich in polyphenols including anthocyanins. Polyphenols are

KEYWORDS: Blackberry extracts, Extraction, Stability, Cancer, Inflammation

July 25, 2009

Dr. Russell J. Mumper

The Graduate School

A dissertation submitted in partial fulfillment of the

Co-Directors: Dr. Russell Mumper, Professor of Molecular Pharmaceutics, University of

Copyright © Jin Dai 2009

Table 4. 1. Composition and Characterization of Blackberry Extracts ............................ 55

Figure 3. 1. Structures of flavonoids, phenolic acids and tannins ................................... 10

Introduction and Statement of Problem

Copyright © Jin Dai 2009

Hypothesis 1. Extracts which contain high amount of anthocyanins and polyphenols

Hypothesis 2. Blackberry extracts will possess anticancer and anti-inflammatory

2.2. Anticancer and anti-inflammatory properties of blackberry extracts

2.3. Antioxidant and antiproliferative properties of blackberry extracts versus

Copyright © Jin Dai 2009

3.1. Preparation and characterization of phenolic extracts

3.1.3. Purification and fractionation of phenolics

3.1.4. Analysis and quantification of phenolics

3.2. Antioxidant properties of phenolic compounds

3.2.1. Phenolics as free radical scavengers and metal chelators

3.2.2. Prooxidant activity of phenolic compounds

3.2.3. Determination of total antioxidant capacity (TAC) of phenolic extracts

3.3. Natural phenolics and cancer

3.3.2. In vivo effects of phenolics

3.3.3. Human intervention studies using phenolics

3.3.4. Mechanism of action of phenolics

3.3.4.2. Interference of basic cellular functions by phenolics

Copyright © Jin Dai 2009

Preparation and Characterization of Anthocyanin-containing Extracts from

Preparation of anthocyanin-containing extracts (ACE)

Monomeric anthocyanins and polymeric color measurement

Total phenolic measurement

Trolox equivalent antioxidant capacity (TEAC) assay

HPLC-UV-mass spectrometry (MS) analysis of anthocyanins

HPLC assay for anthocyanins

90 day stability study of ACE under different storage conditions

48 h stability study of ACE in biologically relevant buffers at 25 °C and 37 °C

Stability of ACE under various storage conditions

Stability of ACE in biologically relevant buffers at 25 and 37 °C

1 20.1 449/287 Cyanidin Hexose Cyanidin-3-glucoside

2 21.5 419/287 Cyanidin Pentose Cyanidin-3-arabinoside

3 26.4 435/303 Delphinidin Pentose Delphinidin-3-xyloside

4 28.7 419/287 Cyanidin Pentose Cyanidin-3-xyloside

5 31.3 535/287 Cyanidin Malonyl-hexose Cyanidin-3-malonyl-glucoside

6 34.9 593/287 Cyanidin Dioxalyl-hexose Cyanidin-3-dioxalyl-glucoside

Copyright © Jin Dai 2009

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

Determination of intracellular ROS in HL-60 cells

H2O2 generation in medium

Interleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α) stimulation studies

Interleukin-12 (IL-12) release assay

H2O2 generation by ACEs and cyanidin 3-glucoside in RPMI 1640 medium

Effect of H2O2 produced by ACEs in the medium on cytotoxic properties of ACEs in