RELATIONSHIPS AMONG ANTIOXIDANTS, PHENOLICS, AND SPECIFIC
GRAVITY IN POTATO CULTIVARS, AND EVALUATION OF WILD POTATO
SPECIES FOR ANTIOXIDANTS, GLYCOALKALOIDS, AND ANTI-CANCER
ACTIVITY ON HUMAN PROSTATE AND COLON CANCER CELLS IN VITRO
A Dissertation
by
MAGNIFIQUE NDAMBE NZARAMBA
Submitted to the Office of Graduate Studies of Texas A&M University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
December 2008
Major Subject: Plant Breeding
RELATIONSHIPS AMONG ANTIOXIDANTS, PHENOLICS, AND SPECIFIC
GRAVITY IN POTATO CULTIVARS, AND EVALUATION OF WILD POTATO
SPECIES FOR ANTIOXIDANTS, GLYCOALKALOIDS, AND ANTI-CANCER
ACTIVITY ON HUMAN PROSTATE AND COLON CANCER CELLS IN VITRO
A Dissertation
by
MAGNIFIQUE NDAMBE NZARAMBA
Submitted to the Office of Graduate Studies of Texas A&M University
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Approved by:
Chair of Committee, J. Creighton Miller Jr. Committee Members, Jeffrey D. Hart
R. Daniel Lineberger William L. Rooney Head of Department, Tim D. Davis
December 2008
Major Subject: Plant Breeding
iii
ABSTRACT
Relationships among Antioxidants, Phenolics, and Specific Gravity in Potato Cultivars,
and Evaluation of Wild Potato Species for Antioxidants, Glycoalkaloids, and Anti-
cancer Activity on Human Prostate and Colon Cancer Cells In Vitro. (December 2008)
Magnifique Ndambe Nzaramba, B.S., Makerere University, Kampala, Uganda;
M.S., Texas A&M University, College Station, TX
Chair of Advisory Committee: Dr. J. Creighton Miller Jr.
Understanding the influence of environment and correlation/relationships among
traits is necessary in selection for crop quality improvement. Therefore, correlations
among antioxidant activity (AOA), total phenolics (TP), phenolic composition, and
specific gravity (SPG) in four potato (Solanum tuberosum, L.) cultivars (Atlantic, Red
La Soda, Russet Norkotah, and Yukon Gold) grown in nine states (California, Idaho,
Michigan, Minnesota, New Jersey, North Carolina, Oregon, Texas, and Wisconsin) for
three years, and in 15 advanced selections grown in Texas were investigated. Cultivars
within and between locations were significantly different in AOA, TP, and SPG.
Significant effects of cultivar, year, location and their interactions on AOA, TP, and SPG
were observed. There were significant positive correlations among the four cultivars
between AOA and TP, and negative correlations between AOA and SPG, and between
TP and SPG. However, correlations between AOA and SPG, and between TP and SPG,
in the advanced selections were not significant.
Some tuber-bearing wild potato species were higher in AOA and TP than the
commercial cultivars; therefore, they could be used as parental material in breeding for
iv
high AOA and TP. However, use of wild species that might be higher in total
glycoalkaloids (TGA) than cultivars could result in progenies with high TGA if the traits
are positively correlated. To elucidate the relationships among AOA, TP and TGA,
accessions of Solanum jamesii and S. microdontum from the US Potato Genebank were
screened for these traits and their correlations determined. Also, anti-proliferative and
cytotoxic effects of 15 S. jamesii tuber extracts (5 and 10 μg/ml) on human prostate
(LNCaP) and colon (HT-29) cancer cells was determined in vitro.
Alpha-solanine and α-chaconine were found in both species, while tomatine and
dehydrotomatine were quantified in some S. microdontum accessions. Both species were
higher in all above traits than the Atlantic, Red La Soda, and Yukon Gold cultivars.
More than 90% of S. jamesii accessions had TGA levels < 20 mg/100g fresh weight,
while only two accessions of S. microdontum, P1 500041 and PI 473171, exhibited TGA
< 20 mg/100g. Neither AOA nor TP was significantly correlated with TGA in both
species. Also, individual phenolics were not correlated with TGA. Solanum jamesii
accessions significantly reduced proliferation of HT-29 (5 and 10µg/ml) and LNCaP
(10µg/ml) cells and were not cytotoxic compared to the control (DMSO). Therefore,
since AOA and TP were not found to be correlated with TGA, using wild accessions in
breeding for increased health promoting compounds would not necessarily increase
glycoalkaloids in newly developed potato cultivars.
v
DEDICATION
This dissertation is dedicated to my parents the late Rev. Canon Martin Blaise
Nzaramba and Espérence Mutamba Nzaramba, and my brothers and sisters who have
been the pillar of support throughout my academic endeavor.
vi
ACKNOWLEDGEMENTS
I would like to sincerely thank my advisor, Dr. J. Creighton Miller, Jr. for the
opportunity to pursue PhD studies. His guidance, advice, encouragement, and support
during my graduate studies will always be treasured. Many thanks go to members of my
advisory committee: Dr. R. Daniel Lineberger, Dr. Jeffrey D. Hart and Dr. William L.
Rooney for their advice and constructive suggestions. Also, thanks are extended to Dr.
Javier Betran for his constructive comments during the preparation of the research
proposal.
Special thanks are due to Dr. Jeannie Miller, for her assistance in proofreading
and editing this dissertation. Sincere appreciation goes to Dr. Lavanya Reddivari for her
constant advice and encouragement. She was always helpful to me in finding solutions
to many research challenges.
I wish to acknowledge the support and advice from Dr. John Bamberg and for
providing me with wild potato materials. Heartfelt thanks go to Douglas Scheuring for
his tremendous technical help in carrying out laboratory experiments. He ensured that
the equipment and supplies were available and crowned it all with humor to boost
morale in the lab. Thanks to Jeff Koym and to Dr. Harry Carlson, Dr. David Douches,
Dr. Dan Hane, Dr. Jeff Miller, Dr. Mel Henninger, Mr. Steven James, Mr. Charles
Kostichka, Dr. Rich Novy, Dr. Rikki Sterrett, and Dr. Craig Yencho for providing potato
cultivar samples for three years as part of this research.
Many thanks to Dr. Scott Senseman for letting me use the equipment in his lab
and to Dr. Sarah Lancaster, Dr. Katherine Carson, Armando del Follo, and Gabriela
vii
Angel for their assistance in using the HPLC. I also would like to thank student workers
Kristen, Robin, Brian, Shara, Clint, and Sarah for their help. I sincerely acknowledge the
help and good academic environment provided to me by the faculty and staff of the
Department of Horticultural Sciences.
With immense appreciation and love, I acknowledge the encouragement, help
and prayers from my parents, the late Rev. Canon Martin Nzaramba and Espérence
Nzaramba, and from brothers and sisters. Their belief in me is the motivation in
everything I do.
viii
TABLE OF CONTENTS
Page
ABSTRACT ..................................................................................................................... iii
DEDICATION ...................................................................................................................v
ACKNOWLEDGEMENTS ..............................................................................................vi
TABLE OF CONTENTS ............................................................................................... viii
LIST OF FIGURES............................................................................................................x
LIST OF TABLES ...........................................................................................................xii
CHAPTER
I INTRODUCTION ..........................................................................................1 Significance of the Research ................................................................4 Objectives.............................................................................................6
II LITERATURE REVIEW ...............................................................................8 Origin of the Potato ..............................................................................8 Nutritional Value of the Potato ..........................................................11 Tuber Specific Gravity .......................................................................12 Antioxidant Activity...........................................................................13 Polyphenols ........................................................................................16 Antioxidants and Phenolics in Human Health ...................................19 Glycoalkaloids....................................................................................23 Cancer and Carcinogenesis ................................................................26 Cell Proliferation ................................................................................31 Apoptosis............................................................................................33
III RELATIONSHIPS AMONG ANTIOXIDANT ACTIVITY, PHENOLICS, AND SPECIFIC GRAVITY IN POTATO (SOLANUM TUBEROSUM L.) CULTIVARS GROWN IN DIFFERENT ENVIRONMENTS.......................................................................................36
Introduction ........................................................................................36 Materials and Methods .......................................................................39 Results ................................................................................................44 Discussion ..........................................................................................56
ix
CHAPTER Page
IV TOTAL GLYCOALKALOIDS, ANTIOXIDANT ACTIVITY, AND PHENOLIC LEVELS IN SOLANUM MICRODONTUM AND SOLANUM JAMESII ACCESSIONS...........................................................60
Introduction ........................................................................................60 Materials and Methods .......................................................................62 Results ................................................................................................69 Discussion ..........................................................................................88
V ANTI-PROLIFERATIVE ACTIVITY AND CYTOTOXICITY OF SOLANUM JAMESII TUBER EXTRACTS TO HUMAN COLON AND PROSTATE CANCER CELLS IN VITRO.........................................91
Introduction ........................................................................................91 Materials and Methods .......................................................................96 Results ..............................................................................................102 Discussion ........................................................................................117
VI CONCLUSIONS ........................................................................................121
LITERATURE CITED ..................................................................................................125
VITA ....................................................................................................................147
x
LIST OF FIGURES FIGURE Page
2.1 Chemical structure of four important phenolic acids in plants........................17
2.2 Chemical structure of the two major glycoalkaloids in potato tubers .............24
3.1 Regression analysis and correlation coefficients among antioxidant activity (DPPH and ABTS assays), phenolic content, and specific gravity of four potato cultivars grown over three years at nine locations. ......52
3.2 A biplot of genotypes-by-trait in potato advanced selections grown near Springlake, TX, in the 2005 growing season. Traits are in red and upper case and accessions are in blue and lower case. Traits are abbreviated as SPG- specific gravity, DPPH and ABTS- antioxidant activity, TP- total phenolics, CGA- chlorogenic acid, CA- caffeic acid, SA- sinapic acid, RH- rutin hydrate, and MYC myricetin...........................................................57
4.1 Typical chromatographs from HPLC analysis of glycoalkaloids in S. jamesii and S. microdontum tuber extracts. .....................................................78
4.2 A biplot of principal component 1 (PC1) vs. principal component 2 (PC2) demonstrating interrelationships among traits; antioxidant activity (ABTS & DPPH assays), total phenolic content (TP), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MYC), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in S. jamesii accessions. Traits are in red and upper case while accessions are in blue and lower case................................................................................83
4.3 A biplot of principal component 1 (PC1) vs. principal component 2 (PC2) demonstrating interrelationships among traits; antioxidant activity (ABTS & DPPH assays), total phenolic content (TP), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MYC), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in S. microdontum accessions. Traits are in red and upper case while accessions are in blue and lower case..............................................................84
5.1 Cell proliferation of HT-29 colon cancer cells measured after 24, 48, and 72 h of incubation with 5 and 10 μg/ml of tuber extracts from 15 S. jamesii accessions. Results are presented as means ± SE of three experiments....................................................................................................104
xi
FIGURE Page
5.2 Cell proliferation of LNCaP prostate cancer cells evaluated after 24, 48, and 72 h of incubation with 5 and 10 μg/ml of tuber extracts from 15 S. jamesii accessions. Results are presented as means ± SE of three experiments. Significantly lower values than the DMSO control (LSD at p < 0.05) are indicated by an asterisk. ...........................................................105
5.3 Cytotoxicity of tuber extracts from 15 S. jamesii accessions (5 and 10 μg/ml) to HT-29 human colon cancer cells expressed as percentage of lactate dehydrogenase enzyme (LDH) released from the cells after 24 hours of incubation. Results are presented as means ± SE of three experiments. Significantly lower values than the DMSO control (LSD at p < 0.05) are indicated by an asterisk. ...........................................................108
5.4 Cytotoxicity of tuber extracts from 15 S. jamesii accessions (5 and 10 μg/ml) to LNCaP human prostate cancer cells expressed as percentage of lactate dehydrogenase enzyme (LDH) released from the cells after 24 hours of incubation. Results are presented as means ± SE of three experiments. Significantly lower values than the DMSO are indicated by an asterisk, and values significantly higher (LSD at p < 0.05) than the DMSO control are indicated by a symbol ε...................................................109
xii
LIST OF TABLES
TABLE Page
3.1 Analysis of variance mean squares and significance of cultivar, location, year, and interaction effects for antioxidant activity, phenolic content, and specific gravity of four potato cultivars grown in five locations during the 2005, 2006, and 2007 growing seasons..........................................46
3.2 Percentage of total observed variability in antioxidant activity, total phenolics, and specific gravity contributed by each variance component- cultivar, location, year, and interactions. ........................................................47
3.3 Ratios of environmental (σ2e) to genetic (cultivar) (σ2
g) variance components and genetic to genotype-by environment (σ2
gxe) interaction effects for antioxidant activity, total phenolics, and specific gravity of four potato cultivars grown in five states for three seasons.............................49
3.4 Mean values of antioxidant activity (DPPH and ABTS), total phenolics, and specific gravity over three years for four potato cultivars grown at nine locations (States)......................................................................................50
3.5 Mean values of antioxidant activity, phenolic content, specific gravity, and individual phenolic compounds of potato advanced selections grown near Spring Lake, TX in the 2005 growing season. .............................54
3.6 Correlation analysis among antioxidant activity (DPPH and ABTS assays), total phenolics (TP), specific gravity, and individual phenolic compounds of potato advanced selections grown near Spring Lake, TX in the 2005 growing season. ............................................................................55
4.1 Mean values of antioxidant activity (DPPH and ABTS assays), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), total glycoalkaloids (TGA), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MY), and ratio of solanine to chaconine (S:C) in S. jamesii accessions (ACCESS). ................................................................70
xiii
TABLE Page
4.2 Mean values of antioxidant activity (DPPH and ABTS assays), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), dehydrotomatine (DTO), tomatine (TOM), total glycoalkaloids (TGA), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MY), and ratio of solanine to chaconine (S:C) in S. microdontum accessions (ACCESS). ..............................................................74
4.3 Range of antioxidant activity (AOA), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), and total gylcoalkaloids (TGA) in S. jamesii and S. microdontum, and means of three commercial cultivars, Atlantic, Red La Soda, and Yukon Gold. ........................................81
4.4 Correlation analysis of antioxidant activity (AOA), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in S. jamesii and S. microdontum accessions. .............86
4.5 Correlation analysis of individual phenolic compounds [chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH) and myricetin (MYC)], total phenolic content (TP), antioxidant activity (DPPH and ABTS), individual glycoalkaloids [α-solanine (SOL) and α-chaconine (CHA)], and total glycoalkaloids (TGA) in S. jamesii and S. microdontum accessions. .................................................................................87
5.1 Correlation analysis of antioxidant activity (DPPH and ABTS), total phenolics (TP), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in Solanum jamesii accessions, and inhibition of HT-29 colon cancer cell proliferation............................................................111
5.2 Correlation analysis of antioxidant activity (DPPH and ABTS), total phenolics (TP), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in Solanum jamesii accessions, and inhibition of LNCaP prostate cancer cell proliferation. .....................................................114
5.3 Correlation analysis of antioxidant activity (DPPH and ABTS), total phenolics (TP), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in Solanum jamesii accessions, and cytotoxicity to HT-29 colon cancer and LNCaP prostate cancer cell lines. ......................116
1
CHAPTER I
INTRODUCTION
Crop plants have long been known as a source of essential nutrients such as
proteins, carbohydrates, vitamins, and lipids, which are required for human
development, growth, and survival. These nutrients, in addition to producer-oriented
traits like pest and disease resistance and drought tolerance, have been the focus of crop
improvement initiatives for generations.
In addition to proteins, carbohydrates, vitamins, and lipids, crop plants provide
bioactive compounds that play a significant role in disease prevention and health
promotion. The bioactive non-nutrients from plant foods, also referred to as
phytochemicals, are numerous, and more than 5000 have been identified, but several
more are still unknown (Shahidi and Naczk, 1995a).
The bioactive phytochemicals are still referred to as non-nutrients, implying that
they are not yet qualified to be in the same category as proteins, carbohydrates, fats and
vitamins. Duyff (2002) referred to these compounds as phytonutrients, meaning plant
chemicals, and categorized them differently from vitamins and minerals. As more
research findings support and confirm their ultimate necessity in the diets of animals and
humans, they will be upgraded to the level of essential nutrients.
The format and style of this dissertation follows that of the Journal of the American Society for Horticultural Science.
2
The prominence of phytochemicals comes from several epidemiological studies
that have shown consumption of fruits, vegetables, and grains to be associated with
reduced risk of chronic diseases such as cardiovascular disease, cancer, diabetes,
Alzheimer’s, cataracts, and other age-related ailments (Arai et al., 2000; Joshipura et al.,
2001; Liu, 2003). It is established that fruits and vegetables are rich in phytochemicals
such as phenolic acids, flavonoids, anthocyanins, and carotenoids.
Currently, research has intensified on investigating the health benefits of
phytonutrients. Several research reports have indicated that the benefit of plant foods is
due not only to levels of vitamins, proteins, lipids or carbohydrates they provide, but also
to activity of the non-nutritive factors found in plants. Many of these plant secondary
components are antioxidants (Riedl et al., 2002). With the discovery of health benefits
from certain phytochemicals, i.e. antioxidant capabilities, the meaning of a balanced diet
is changing from provision of sufficient amounts of carbohydrates, proteins, fats and
vitamins, to inclusion of such compounds as carotenoids, anthraquinones, flavonoids
etc., that are believed to possess antioxidant activity (Nzaramba, 2004).
Antioxidants are compounds that can quench free radicals (oxidants) thereby
delaying or inhibiting oxidation of molecules and protect biological systems against
potential harmful effects of free radical (Arnao, 2000; Morello et al., 2002). Oxidative
stress induced by free radicals and other external agents can damage DNA and other
molecules, and if not repaired may set off a cascade of events such as mutations, DNA
strand breakage, and chromosomal breakage and rearrangement resulting in disease risks
like cancer.
3
Humans and animals are exposed to various disease-causing agents, ranging from
external agents such as bacteria, fungi, viruses, radiation, chemical agents etc, and
internally generated agents like reactive oxygen (ROS) and reactive nitrogen species
(NOS) from body metabolic activities. Therefore, protection against these agents is
paramount. Reactive oxygen and nitrogen radicals act as oxidants, thereby causing
oxidative stress within the body. It is therefore, necessary to keep a balance between
oxidants and antioxidants to maintain healthy physiologic conditions. Phytochemicals
such as phenolics and carotenoids in plant foods have antioxidant capabilities that help.
to protect cellular systems from oxidative damage (Chu et al., 2002; Eberhardt et al.,
2000; Liu, 2003).
Several studies have reported that phytochemicals, especially antioxidants from
plants, can inhibit metagenesis and carcinogenesis, and reduce cancer risks by
scavenging oxidative radicals (Boyle et al., 2000; Giovannelli et al., 2000; Rodriguez et
al., 2007; Shahidi, 2002), modulation of detoxifying enzymes, stimulation of the
immune system, regulation of cell proliferation and apoptosis (Kern et al., 2007; Kim et
al., 2006; Reddivari et al., 2007b), and antiviral and antibacterial effects (Friedman et al.,
2006).
Le Marchand et al. (2000) indicated that consumption of quercetin from onions
and apples was inversely associated with lung cancer risk in Hawaii. Similarly,
Giovannelli et al. (2000) demonstrated that polyphenols from wine significantly
decreased DNA oxidative damage in rat colon mucosal cells, and concluded that dietary
4
polyphenols can modulate in vivo oxidative damage in the gastrointestinal tract of
rodents.
Other studies have shown a link between intake of dietary phytochemicals and
reduced risk of cardiovascular disease. Ridker et al. (2002) stated the inflammation is a
critical factor in cardiovascular disease. Inflammation promotes initiation and
development of atherosclerosis. Since phenolic compounds exhibit anti-inflammatory
activity (Dai et al., 2007), they play a role in cardiovascular disease prevention.
Joshipura et al. (2001) reported that high fruit and vegetable intake is associated with
decreased risk of coronary artery disease. A study in Japan indicated that intake of
flavonoids was inversely correlated with the amount of total cholesterol and low-density
lipoprotein (LDL) in plasma (Arai et al., 2000).
The importance of antioxidants in preventing diseases and maintenance of health
has raised interest among scientists, food producers/manufacturers, and consumers
towards functional foods (Robards et al., 1999; Velioglu et al., 1998). The Food and
Nutrition Board of the National Academy of Sciences (FNB/NAS, 1994) defined
functional foods as any “food or food ingredient that may provide a health benefit
beyond the traditional nutrients it contains”. Several authors (Al-Saikhan et al., 1995;
Hale, 2004; Kanatt et al., 2005; Kawakami et al., 2000) have suggested that potato is a
functional food due to presence of antioxidant compounds in potato tubers.
Significance of the Research
Given the importance of antioxidants in disease prevention, plant breeders need
to develop cultivars with substantial amounts of antioxidants to complement medical and
5
social activities in preventing diseases. However, in developing high antioxidant
cultivars, other traits such as high specific gravity have to be maintained if not increased.
Therefore, breeders need to known the relationships among traits, information which
helps in understanding how selection for one trait would affect others. Ascertaining the
effect of other factors – genotype, environment, and genotype x environment, on traits of
interest is also helpful.
Potato cultivars and breeding lines exhibit varying amounts of phenolic
compounds and antioxidants (Kawakami et al., 2000; Reddivari et al., 2007a).
Furthermore, identification of related wild species with desirable nutritional benefits
would provide parental material in breeding improved cultivars with enhanced health
benefits. Several wild species have been screened for antioxidant activity and some were
reported to possess more antioxidant activity than currently grown potato cultivars.
Species identified as containing high antioxidant activity were S. jamesii, S.
pinnatisectum, S. megistacrolobum, and S. microdontum (Hale, 2004; Nzaramba et al.,
2007). In the above studies, only a few accessions of each species in the mini-core
collection were screened.
Having identified some species as containing more antioxidant activity than
cultivated varieties, it was important to screen all populations of these species to identify
specific accessions that are the highest in antioxidant activity and phenolic compounds.
However, it should be noted that breeding with wild Solanum species can result in toxic
levels of glycoalkaloids in new progenies (Laurila et al., 2001). Glycoalkaloids are
known to be toxic to humans by acting as cholinesterase inhibitors, and also interact
6
synergistically in destabilizing cell membranes (Smith et al., 2001). Therefore,
glycoalkaloid accumulation affects food quality and safety, and the accepted level in
tubers is < 20 mg/100g fresh weight (Papathanasiou et al., 1998). Yet, wild potato
species are believed to contain amount of glycoalkaloids above this level.
Given that high glycoalkaloids levels, and in some cases very high amounts of
antioxidants and phenolics are undesirable, wild potato species need to be evaluated for
cytotoxicity before their introduction into breeding programs. Also, tuber extracts from
wild potato species may contain other unknown cytotoxic compounds that might be
undesirable for human consumption.
Objectives
One of the objectives of the present study was to investigate the relative
importance of cultivars, environment, seasons, and their interaction on antioxidant
activity, total phenolic content, and specific gravity in potato cultivars grown under
widely diverse environmental conditions (nine states) for three years (2005, 2006, and
2007 seasons), and also to determine the correlations among these traits to ascertain how
selection for any of the traits would affect others.
In addition, ninety-two wild accessions of S. jamesii and eighty-six of S.
microdontum species in the US Potato Genebank, Sturgeon Bay, WI., were fine-
screened for antioxidant activity, total phenolic content, and total glycoalkaloid levels.
Also, the linear correlations among these traits were investigated. This information is
necessary in selecting accessions to use in introgressing desirable traits into cultivated
7
potato varieties, while avoiding introducing or increasing levels of undesirable
compounds such as glycoalkaloids.
Finally, anti-proliferative activity and cytotoxicity potential of tuber extracts
from 15 S. jamesii accessions, representing the whole range of glycoalkaloid content in
this species, was investigated using human prostate (LNCaP) cells and colon (HT-29)
cancer cell lines in vitro.
8
CHAPTER II
LITERATURE REVIEW
Origin of the Potato
The word “potato” commonly refers to the potato of commerce belonging to the
species Solanum tuberosum L. and other cultivated tuber-bearing species found in South
America. These plants belong to the family Solanaceae, genus Solanum, section Petota.
Most species in section Petota possess underground stolons bearing potato tubers at their
tips, but some species lack these characteristic structures. Therefore, section Petota was
divided into two subsections; subsection Potatoe containing both cultivated and wild
tuber-bearing species, and subsection Estolonifera that contains non-tuber-bearing series
(Hawkes, 1992). The tuber is the edible part of the potato, which is a part of the stem
that stores food and plays a role in propagation. The tuber is also regarded as an enlarged
stolon. Stolons are formed from lateral buds at the bottom of the stem (Beukema and van
der Zaag, 1990).
Potatoes originated in many countries of South America: Peru, Ecuador, Chile,
Colombia, and Bolivia (Harris, 1978; Hawkes, 1978a). According to Correll (1962) and
Hawkes (1992), the potato was cultivated in South America long before the arrival of
Europeans. Hawkes (1978a) stated that the cultivated potato was derived from one of the
many wild species found in South America, more specifically in the Andes of Peru and
Bolivia. He reported that the introduction of the potato into Europe was first into Spain
in about 1570, then into England between 1588 and 1593, later spreading to almost
9
every part of the world. From Spain, it diffused into continental Europe, and from
England it spread to Ireland, Scotland, and British overseas colonies, including the US.
The documented number of potato species has been increasing as more plant
collection excursions are undertaken. Hawkes (1978b) indicated that there were seven
cultivated species and 154 wild species, whereas Horton (1987) stated that eight
cultivated and 200 wild species were known. Miller, Jr. (1992) estimated that more than
2,000 species of potato exist, about 200 of which are tuber bearing. According to
Spooner and Hijmans (2001), Solanum section Petota contains about 200 wild species
distributed from the southwestern United States to central Argentina and Chile, with a
secondary center of diversity in the central Mexican highlands. Spooner et al. (2004)
provide a summary of recent morphological and molecular studies on interrelationships
among potato species in North and Central America. They recognized twenty-five
species and four nothospecies which they assigned to eleven informal species.
According to Huamán and Spooner (2002), all landrace populations of cultivated
potatoes are a single species, S. tuberosum, with eight cultivar groups. The landrace
potato cultivars are highly diverse, containing diploids (2n = 2x = 24), triploids (2n = 3x
= 36), tetraploids (2n = 4x = 48), and pentaploids (2n = 5x = 60). The tetraploids are the
highest yielding and they are the sole cytotype of modern cultivars (Ames and Spooner,
2008).
The taxonomy of cultivated potatoes has been controversial with anywhere from
one to 20 species recognized (Huaman and Spooner, 2002). Spooner et al. (2005)
reported that all landraces of cultivated potato form a common gene pool and have a
10
monophyletic origin from Andean and Chilean landrace complex. Using simple
sequence repeat (SSR) genotyping in combination with morphological analysis, (2007)
suggested classifying the cultivated potatoes into four species; S. tuberosum, S.
ajanhuiri, S. juzepczukii, and S. curtilobum.
Potatoes are among the most widely-grown crop plants in the world, giving good
yield under various soil and weather conditions (Lisinska and Leszcynski, 1989). Potato
has been ranked as the fourth important food crop worldwide after wheat, rice and corn,
and one of the main vegetables consumed in European diets (Tudela et al., 2002). More
recently, potato has been ranked third by the FAO. According to Lachman et al. (2001),
annual world-wide production of potatoes is approximately 350 million tons (771,618
million lbs). The US potato production was about 44 billion lbs (0.02 billion tons) in
2006 (USDA, 2007). The world average per capita consumption in 2005 was estimated
at 33.7 kg (74.3 lbs) (FAO, 2007), while the US per capita consumption of potatoes is
about 57kg (126 lbs) (National Potato Council, 2008). Highest potato consumption is in
Europe with a per capita consumption of about 96 kg, followed by North America at 57
kg. Per capita consumption is low in Africa and Latin America, but is increasing (FAO,
2007).
The high consumption rate of potatoes is attributed to both their palatability and
high nutritive value (Rytel et al., 2005). Potatoes serve as a major food source, as well as
an inexpensive source of energy and good quality protein (Lachman et al., 2001).
11
Nutritional Value of the Potato
Potato tubers are important sources of vitamins and minerals such as calcium,
potassium, and phosphorous, and their value in the human diet is often understated or
ignored, particularly as a source of ascorbic acid (Dale et al., 2003). The potato is a very
low fat food, and is an important source of vitamins C, B, and A (Dale et al., 2003;
Kolasa, 1993; Lachman et al., 2000). According to Kolasa (1993), the potato’s
contribution of nutrients to diet or its role in human nutrition is actually greater than it
appears on the nutrition label, because of the volume of potatoes consumed in the U.S.
Therefore, the potato plays a more important role in nutrition than might be expected
based on its absolute nutrient values.
Kant and Block (1990) stated that potatoes are the third largest source of vitamin
B6 for adults 19-74 years of age. They also reported that potatoes were the second most
important contributor of vitamin B6 for the elderly, who are especially at risk of chronic
disease. Vitamin B6 is involved in amino acid, nucleic acid, glycogen, and lipid
metabolism. It influences hormone modulation, erythrocyte production, and immune and
nervous system functions. It is also proposed to play a role in the etiology and /or
treatment of various chronic diseases such as sickle cell anemia, asthma, and cancer
(Kolasa, 1993).
Potato tubers contain several minerals that are important in diet, including
phosphorous, calcium, zinc, potassium, and iron (Andre et al., 2007; Yilmaz et al.,
2005). Potatoes are also a good source of high-quality protein such as lysine (Friedman,
12
2004). They also contain significant levels of functional compounds such as antioxidants
and polyphenols (Breithaupt and Bamedi, 2002; Kanatt et al., 2005; Reyes et al., 2005).
Tuber Specific Gravity
Specific gravity is an important quality attribute and is one of the properties of
potato that could be used as a basis for nondestructive quality evaluation especially in
processing. The relationship between specific gravity and cooking quality of potato is
well known (Gould, 1999; Komiyama et al., 2007), and the potato processing industry
needs cultivars with high tuber specific gravity and acceptable color of processed
products (Haynes, 2001). Processers usually pay less for tubers with low specific
gravity. According to Gould (1999) potatoes with higher specific gravity are well
formed, smooth, and firm, and every 0.005 increase in specific gravity results in an
increase in the number of chips that can be processed from 100 pounds of raw potatoes
by one pound.
Specific gravity and solids-content have also been shown to have an effect on fat
uptake into french fries. Potatoes with a high specific gravity (>1.090) have been shown
to produce a high yield of French fries with a lower fat content than lower specific
gravity potatoes (Lulai and Orr, 1979). Hagenimana et al. (1998) reported a linear
relationship between dry matter content and fat uptake in thin sliced sweet potato crisps,
with fat uptake decreasing as dry matter increased.
13
Antioxidant Activity
Antioxidants are compounds which, when present in low concentrations
compared to oxidizable substrates, can quench free radicals and significantly delay or
inhibit oxidation of the substrate and protect biological systems against potential harmful
effects of free radicals (Arnao, 2000; Diplock et al., 1998).
Antioxidants are categorized as synthetic or natural. Synthetic antioxidants are
compounds with phenolic structures of varying degrees of alkyl substitution, such as
butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT). Their usage is
being restricted, as they are suspected to cause negative health effects such as
carcinogenicity (Barlow, 1990; Ito et al., 1983), and there is increasing interest in
replacing synthetic antioxidants with naturally occurring antioxidants (Chang et al.,
2002; Koleva et al., 2002).
Antioxidants can also be categorized as either free radical scavengers (non-
enzymatic) that trap or decompose free radicals, or cellular and extracellular enzymes
(enzymatic) that inhibit peroxidase reactions involved in the production of free radicals.
Free radical scavengers or non-enzymatic antioxidants include ascorbate (vit. C) (Kojo,
2004; Suh et al., 2003), tocopherols (vit. E) (Pryor, 2000), carotenoids (El-Agamey et
al., 2004; Mortensen et al., 2001; Niles, 2004), flavonoids and polyphenols (Arts and
Hollman, 2005; Aviram et al., 2005; Scalbert et al., 2005), α-lipoic acid (Holmquist et
al., 2007; Smith et al., 2004) and glutathione (Giustarini et al., 2008; Jones et al., 2000;
Masella et al., 2005). Antioxidant enzymes include glutathione peroxidase, superoxide
dismutase, and catalase. Enzymatic antioxidants are important for intracellular defenses,
14
while non-enzymatic antioxidants are the major defense mechanism against extracellular
oxidants.
Natural antioxidants can be phenolic compounds (tocopherols, flavonoids,
anthocyanins, and phenolic acids), nitrogen compounds (alkaloids, chlorophyll
derivatives, amino acids, and amines), or carotenoids, as well as vitamins C and E, and
phospholipids (Hudson, 1990; Shahidi, 2002). Most of these antioxidant compounds are
present in foods as endogenous constituents and are referred to as dietary antioxidants
(Siddhuraju et al., 2002). The Food and Nutrition Board of the National Academy of
Sciences (National Academy of Science, 1998) defined a dietary antioxidant as a
substrate in foods that significantly decreases the adverse effects of free radicals such as
reactive oxygen species (ROS), reactive nitrogen species (RNS), or both on normal
physiological function in humans.
Free radicals are molecules or molecular fragments containing one or more
unpaired electrons. The presence of unpaired electrons confers a considerable degree of
reactivity to free radicals (Valko et al., 2004). Free radicals are ubiquitous in the body
and can be generated by normal physiological processes, including aerobic metabolism
and inflammatory responses, to eliminate invading pathogenic microorganisms (Hussain
et al., 2003). Reactive oxygen species can be produced from endogenous sources such as
mitochondria, cytochrome P450 metabolism, peroxisomes, and inflammatory cell
activation (Inoue et al., 2003).
Troszyńska et al. (2002) reported that imbalance between ROS/RNS and
antioxidant defense systems may lead to chemical modification of biologically relevant
15
macromolecules like DNA, proteins, carbohydrates or lipids. To avoid such
modifications, antioxidants inhibit oxidation of these molecules and prevent initiation of
oxidizing chain reactions (Klein and Kurilich, 2000; Velioglu et al., 1998). They
scavenge free radicals by donation of an electron or hydrogen atom, or by deactivation
of prooxidant metal ions and singlet oxygen (Shahidi, 2002).
Antioxidants exert their effects through different mechanisms and functions;
therefore, it is essential to clarify which function is being measured when analyzing
samples (Niki and Noguchi, 2000). A wide array of assays has been suggested to
measure antioxidant activity. Modes of antioxidant action are grouped into two
categories based on the chemical reactions involved: hydrogen atom transfer reaction-
based assays and single electron transfer reaction-based assays. Hydrogen atom transfer
reaction-based assays include total radical trapping antioxidant parameter (TRAP),
oxygen radical absorbance capacity (ORAC), and crocin bleaching assays. Electron
transfer reaction-based assays include trolox equivalence antioxidant capacity (TEAC),
Cu (II) complex antioxidant potential, ferric ion reducing antioxidant power (FRAP),
2,2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid (ABTS), and 2,2-Diphenyl-1-
picrylhydrazyl (DPPH) assays. Other methods used to measure antioxidant activity are
superoxide, hydrogen peroxide, the hydroxyl radical, singlet oxygen and peroxynitrite
scavenging capacity assays (Huang et al., 2005). The most commonly used assays are
DPPH· and ABTS+· radicals because of their ease, speed, and sensitivity. The DPPH
radical is only soluble in organic solvents, but ABTS+· is soluble in both hydrophilic and
16
lipophilic media and can be used over a wide range of pH (Arnao, 2000; Arnao et al.,
1999; Lemanska et al., 2001).
Polyphenols
Polyphenolic compounds constitute one of the most commonly occurring
ubiquitous groups of secondary plant metabolites and represent an integral part of the
human diet (Rice-Evans et al., 1996). There are more than 8,000 known phenolic
structures (Bravo, 1998; Harborne, 1998). Polyphenols range from simple molecules like
phenolic acids (Fig. 2.1) to highly polymerized compounds such as tannins. Polyphenols
are synthesized through two main pathways in plants: the shikimate pathway and the
acetate pathway (Bravo, 1998).
The common structural feature of polyphenolic compounds is the
diphenylpropane moiety that consists of two aromatic rings linked through three carbon
atoms, where together usually form an oxygenated heterocycle (Rodriguez et al., 2007;
Sekher Pannala et al., 2001; Teixeira et al., 2005). Polyphenols usually occur as
conjugates with one or more sugars, attached either to the hydroxyl group or to an
aromatic carbon atom. The attached sugar can be a mono, di or an oligosaccharide, with
glucose as the most common type.
Phenolic compounds are essential for plant growth and reproduction. They act as
anti-feedants, anti-pathogens, and also aid in recognition of symbionts (Shahidi and
Naczk, 1995b). In live plants, phenolic compounds provide protection against oxidative
stress and attack by herbivores, and act as UV filters and healing agents.
17
Fig. 2.1. Chemical structure of four important phenolic acids in plants.
18
Many properties of plant products are associated with the presence and content of
polyphenolic compounds. Phenolics and anthocyanins have been reported to possess a
very high capacity to quench free radicals (Chu et al., 2000; Kalt et al., 2001). Studies
have shown that polyphenols in plants such as flavonols, flavonoids (Comis, 2000;
McBride, 1999), anthraquinones (Yen et al., 2000), xanthones and proanthocyanidins
(Minami et al., 1994), and zeaxanthin (Stelljes, 2001) act as antioxidants or agents of
mechanisms that exhibit cardioprotective or anti-carcinogenic effects.
Phenolic compounds acting as antioxidants may function as terminators of free
radicals and as chelators of redox-active metal ions that are capable of catalyzing lipid
peroxidation (Schroeter et al., 2002). According to Milde et al. (2007), phenolics
together with carotenoids protect low-density lipoproteins (LDL) from oxidation.
Oxidation of LDL is believed to lead to development of atherosclerosis and
accompanying disorders. Phenolic antioxidants interfere with the oxidation of lipids and
other molecules by donation of hydrogen atoms to radicals. The phenoxyl radical
intermediates are relatively stable so they do not initiate further radical reactions. The
key factors affecting the biological activity of polyphenols are the extent, nature, and
position of the substituents and the number of hydroxyl groups (Schroeter et al., 2002).
In vitro and in vivo studies have shown that polyphenols induce responses consistent
with the protective effects of diets rich in fruits and vegetables against degenerative
conditions like cardiovascular diseases and carcinogenesis (Chung et al., 2003; Manach
et al., 2005).
19
Interest in phenolic compounds has increased recently owing to their antioxidant
capacity and their possible beneficial effects on human health. These include the
treatment and prevention of cancer, cardiovascular disease, and other pathological
disorders (Babich et al., 2007; Damianaki et al., 2000; Polovka et al., 2003; Rice-Evans,
2001; Seeram et al., 2005; Sharma et al., 2007). Regular intake of polyphenols has been
linked to lower rates of stomach, pancreatic, lung, and breast cancer (Damianaki et al.,
2000).
Kim et al. (2006) reported that these polyphenols induced cell death in SNU-C4
human colon cancer cells in a dose-dependent manner. They observed that polyphenol
treatment of cells resulted in the regulation of the expression of apoptotic-regulating
genes, decreased expression of the Bcl-2 gene, and increased expression of both the Bax
gene and Caspase-3 activity. Friedman et al. (2006) reported that flavonoids in green tea
exhibited antimicrobial activities at nanomolar levels and that most compounds were
more active than medicinal antibiotics, such as tetracycline or vancomycin, at
comparable concentrations.
Antioxidants and Phenolics in Human Health
Antioxidants play a role in balancing the effect of reactive oxygen and nitrogen
species and other free radicals to protect biological sites (Valko et al., 2006).
Antioxidants quench free radicals, chelate redox metals, and interact with other
antioxidants within the antioxidant network, thereby enabling living organisms to
overcome the deleterious effects of free radicals while maintaining the beneficial effects
of free radicals (Morello et al., 2002; Valko et al., 2007). Enzymatic and non-enzymatic
20
antioxidants protect living organisms from various oxidative stresses by controlling the
redox status and maintaining the redox homeostasis in vivo (Droge, 2002).
Antioxidants help maintain or restore cell integrity by preventing reactive oxygen
species from damaging cell structures, nucleic acids, lipids, protein, and DNA.
Permanent modification of genetic material resulting from oxidative damage represents
the initial stages of mutagenesis, carcinogenesis, and ageing (Valko et al., 2007). Several
studies have implicated oxidative stress in various pathological conditions, including
cardiovascular disease, cancer, neurological disorders, diabetes, and ageing (Dalle-
Donne et al., 2006; Makazan et al., 2007; Tappia et al., 2006).
In addition to scavenging deleterious free radicals and maintaining cell integrity,
antioxidants modulate cell-signaling pathways (Mates et al., 1999). According to Valko
et al. (2007), modulation of cell signaling pathways by antioxidants could help prevent
cancer by preserving normal cell cycle regulation, inhibiting proliferation and inducing
apoptosis, inhibiting tumor invasion and angiogenesis, suppressing inflammation, and
stimulating phase II detoxification enzyme activity.
Enzymatic antioxidants such as L-cysteine, N-acetyl cysteine, and non-enzymatic
antioxidants such as polyphenols and vitamin E can block activation of nuclear
transcription factor κB (NF-κB). The NF-κB regulates several genes involved in cell
transformation, proliferation, and angiogenesis (Thannickal and Fanburg, 2000), and its
activation has been linked to the carcinogenesis process (Leonard et al., 2004).
Kaneto et al. (1999) reported that antioxidants can help in diabetes prevention.
They observed that antioxidant treatment preserved the amounts of insulin content and
21
insulin mRNA, and also resulted in increased expression of pancreatic and duodenal
homeobox factor-1, a β-cell-specific transcription factor. Valko et al. (2007) also
reported that antioxidant treatment can exert beneficial effects in diabetes by preserving
in vivo β-cell function. They noted that antioxidant treatment suppresses apoptosis in β-
cells without changing the rate of β-cell proliferation.
Several studies have indicated that consumption of fruits and vegetables helps
prevent a wide range of diseases. These observations are attributed to presence of
polyphenolic compounds in these fruit and vegetable products. It is believed that the
beneficial effects derived from fruits and vegetables include antioxidant nutrients such as
vitamins C and E, carotenoids, and phenolics that are thought to be involved in the
pathophysiology of many chronic diseases (Stanner et al., 2004).
Epidemiological data have shown that people with a high consumption of fruits
and vegetables are at a lower risk of developing several types of cancer (Riboli and
Norat, 2003), and cardiovascular disease and stroke (Hu, 2003) than those with low fruit
and vegetable consumption. In a study aimed at assessing the relationship between
overall mortality in Spanish adults and consumptions of fruit and vegetables, Agudo et
al. (2007) reported that a reduction in mortality was associated with increased intake of
fresh fruits and vegetables. They also observed that a lower risk of death seemed to be
associated with high intakes of vitamin C, provitamin A, carotenoids, and lycopene.
They concluded that antioxidant capacity could explain the potential effect of ascorbic
acid and provitamin A. Similar results were observed in cohort studies in Greece
(Trichopoulou et al., 2003) and in the United States (Steffen et al., 2003).
22
Hwang and Yen (2008) reported that citrus flavanones, hesperidin, hesperetin,
and neohesperidin, which exhibit antioxidant activities have neuroprotective effects
against H2O2-induced cytotoxicity in the rat pheochromocytoma PC12 cell line. They
concluded that these dietary antioxidants are potential candidates for use in intervention
for neurodegenerative diseases.
Antioxidants in the diet contribute or exhibit antibacterial, antiviral, anti-
inflammatory, and antiallergic actions (Cook and Samman, 1996). Plants vary in
composition of phytochemicals with protective functions; therefore, to attain maximum
health benefits, sufficient amounts of phytochemicals from a variety of sources such as
vegetables, fruits and grains are necessary (Adom and Liu, 2002).
Health benefits of individual phenolic compounds have been investigated.
Caffeic acid increased the sensitivity of tumor cells to chemotherapeutic agents in vitro
(Ahn et al., 1997). Shimizu et al. (1999) and Matsunaga et al. (2002) observed that
chlorogenic acid reduced the number of tumors in both colon and stomach at initiation
and post initiation stages in F344 rats. Gallic acid from grape seed extract inhibited cell
proliferation and induced apoptotic death in DU145 human prostate carcinoma cells. It
activated caspase-3 and caspase-9, and cleavage of PARP (Veluri et al., 2006). However,
most studies have indicated that complex mixtures of phytochemicals in foods provide
better protective benefits than single phytochemicals through additive and/or synergistic
effects (Eberhardt et al., 2000).
23
Glycoalkaloids
Glycoalkaloids are steroidal nitrogen–containing metabolites found in potatoes
and many solanaceous plants (McCue et al., 2007). Steroidal glycoalkaloids are found in
almost all parts of the potato, with the highest concentrations associated with tissues that
are undergoing high metabolic activity (Jadhav et al., 1973). These include flowers,
unripe berries, young leaves, sprouts, peels, and the area around the eyes. Small
immature tubers are normally high in glycoalkaloids since they are still metabolically
active (Papathanasiou et al., 1998). Glycoalkaloids are concentrated in a 1.5 to 3.0 mm
layer immediately under the skin in normal tubers (Pęksa et al., 2006).
The two major glycoalkaloids in potatoes are α-solanine and α-chaconine (Fig.
2.2), which together comprise approximately 95 % of the total glycoalkaloids in the
plant (Edwards and Cobb, 1999). The ratio of α-solanine to α-chaconine differs
depending on the anatomical part of the potato plant or its variety, and ranges from 1:2
to 1:7 (Bejarano et al., 2000). The other glycoalkaloids found in cultivated potatoes are
β- and γ-solanines and chaconines, α- and β-solamarines, demissidine, and 5-β-
solanidan-3-a-ol, and in wild potatoes leptines, commersonine, demissine, and tomatine
(Lachman et al., 2001).
24
α-solanine
α-chaconine
Fig. 2.2. Chemical structure of the two major glycoalkaloids in potato tubers.
25
Various factors influence the concentration of glycoalkaloids in tubers - physical
injury due to pest or mechanical injury during harvesting and handling, fungal attack,
climate, growing environment, and poor storage conditions. Light exposure during
growth, harvesting, and storage is the most important factor influencing the amount of
glycoalkaloids in potato tubers (Sengul et al., 2004). Also, breeding with wild Solanum
species can also result in high glycoalkaloid levels in the new progenies (Laurila et al.,
2001).
Steroidal glycoalkaloids are involved in defense against microbial and insect
pests (Hollister et al., 2001). However, they are undesirable when present in large
amounts in potato tubers. Glycoalkaloids are known to be toxic by acting as
cholinesterase inhibitors, causing sporadic out-breaks of poisoning in humans (Smith et
al., 1996), and also interact synergistically in destabilizing cell membranes (Smith et al.,
2001). Due to their poisonous nature, potato glycoalkaloids have been of major concern
and investigated since their discovery in 1820 by the pharmacist Desfosses (Bergers,
1980).
Safety of gylcoalkaloids for humans is still being debated (Friedman et al., 2003;
Korpan et al., 2004; Rietjens et al., 2005). Most potato cultivars for human consumption
have about 7.5 mg/100g of both α-solanine and α-chaconine (Lachman et al., 2001).
Tubers with glycoalkaloid levels greater than 14 mg/100g are bitter in taste, and those
with more than 20 mg/100g cause a burning sensation in the throat and mouth. The
permitted level of glycoalkaloids in tubers is 20 mg/100g fresh weight (Papathanasiou et
26
al., 1998). Therefore, new varieties must contain less than 20 mg/100g fresh weight, and
varieties containing 2 to 13 mg/100g fresh weight are preferred (Smith et al., 1996).
Wild Solanum species are commonly used in potato breeding as a source of
valuable germplasm. They are often used to introduce pest and disease resistance into
cultivated potato. However, some of these species have high levels of glycoalkaloids
such that, together with desirable characteristics, toxic glycoalkaloids may be transferred
to potato cultivars (Laurila et al., 2001). Therefore, screening of wild species for
glycoalkaloid content is important to determine their suitability as potential parental
material in breeding programs.
Cancer and Carcinogenesis
According to the American Cancer Society (2008), cancer refers to a group of
diseases characterized by uncontrolled growth and spread of abnormal cells. Abnormal
or cancerous cells are caused by both external factors such as chemical toxins, tobacco,
radiation, and infectious organisms, and internal factors such as hormones, immune
conditions, and mutations from metabolism or inherited mutations. The causal factors
may act together or in sequence to initiate and promote carcinogenesis.
Oxidative stress caused by free radicals has been implicated in oncogenic
stimulation by inducing cellular redox imbalance. Elevated levels of cellular oxidative
stress might result in permanent modification of genetic material (DNA), RNA, proteins,
and lipids which normally represent the initial steps involved in mutagenesis and
carcinogenesis (Marnett, 2000; Valko et al., 2007).
27
In addition to causing mutations in cancer-related genes or post-translational
modification of proteins, free radicals can also modulate cell growth and tumor
promotion by activating signal-transduction pathways that results in the transcriptional
induction of proto-oncogenes, including c-FOS, c-JUN, and c-MYC, involved in
stimulating growth (Hussain et al., 2003; Vogelstein and Kinzler, 2004). Proteins such as
DNA-repair enzymes, those involved in signal transduction, apoptotic modulators, and
the p53 protein can be modified both structurally and functionally when exposed to free
radicals (Hussain et al., 2003).
Reactive oxygen species can structurally and functionally modify DNA, resulting
in single- or double-stranded DNA breaks, purine, pyrimidine, or deoxyribose
modifications, arrest or induction of transcription and signal transduction pathways,
replication errors, and genomic instability (Marnett, 2000; Poli et al., 2004). Reactive
nitrogen species (RNS) such as peroxynitrites and nitrogen oxides have also been
implicated in DNA damage. In addition, various redox metals with the ability to generate
free radicals, and non-redox metals with the ability to bind to critical thiols, have been
implicated in the mechanism of carcinogenesis (Leonard et al., 2004; Roy et al., 2002;
Valko et al., 2005; Waalkes et al., 2004). Valko et al (2001) reported that iron-induced
stress is considered to be a principal determinant of human colorectal cancer.
Carcinogenesis is a complex multi-sequence/stage process leading a cell from a
healthy to a precancerous state and finally to an early stage of cancer (Klaunig and
Kamendulis, 2004; Trueba et al., 2004). The process of cancer development involves
initiation, promotion, and progression stages occurring in a single cell. The initiation
28
stage involves non-lethal mutation of DNA that produces an altered cell followed by at
least one round of DNA synthesis to fix the damage that occurred during initiation (Loft
and Poulsen, 1996). The promotion stage is characterized by clonal expansion of
initiated cells by the induction of cell proliferation and/or inhibition of programmed cell
death (apoptosis). This stage requires a continuous presence of the tumor promoting
stimulus and is therefore reversible by eliminating the stimuli. The progression stage
involves cellular and molecular changes that occur from preneoplastic to neoplastic
states. This stage is irreversible and involves additional genetic damage, genetic
instability, and disruption of chromosome integrity resulting in transition of the cell from
benign to malignant. Because tumor promotion may be the only reversible event during
cancer development, its suppression is regarded as an effective way to inhibit
carcinogenesis (Friedman et al., 2007).
Two mechanisms have been proposed for the induction of cancer. One suggests
that an increase in DNA synthesis and mitosis by nongenotoxic carcinogens may induce
mutations in dividing cells through misrepair. These mutations may then clonally expand
from an initiated preneoplastic cell state to a neoplastic cell state (Ames and Gold, 1990;
Guyton and Kensler, 1993). The other mechanism stipulates that a breakdown of
equilibrium between cell proliferation and cell death induces cancer. Therefore,
carcinogenesis can be described as an imbalance between cell proliferation and cell
death shifted towards cell proliferation (Valko et al., 2006).
During cell proliferation, protein p53 plays a primordial role, checking the
integrity of DNA (Oren, 2003; Zurer et al., 2004). It triggers mechanisms that eliminate
29
the oxidized DNA bases that cause mutations. And when cell damage is great, p53
triggers cell death by apoptosis. According to Hussain et al. (2003), uncontrolled
apoptosis can be harmful to an organism, leading to destruction of healthy cells. Hence,
there is a regulatory system consisting of pro-apoptotic factors such as p53 and anti-
apoptotic factors. Most cancers have defects in upstream or downstream genes of p53
function.
Colon Cancer
Colorectal cancer is the third most common cancer in both men and women in
the U.S. (American Cancer Society, 2008). It accounted for about 10% of cancer
mortality in the United States, and caused about 57,000 deaths in 2004 (Jemal et al.,
2004). It is estimated that colon and rectal cancer will account for 9% of all cancer
deaths in 2008 (American Cancer Society, 2008).
Colon cancer development is often characterized in an early stage by a hyper-
proliferation of the epithelium leading to the formation of adenomas. Colon
carcinogenesis is a multi-step process, and early intervention should target inhibition of
enhanced cell proliferation in transformed cells by induction of the apoptotic pathway to
delete cells carrying mutations (Hawk et al., 2005).
Diet and lifestyle are thought to be major risk factors for developing colorectal
cancer (Bray et al., 2002). Other studies have associated the risk of developing colorectal
cancer to inflammatory bowel disease (IBD) (Munkholm, 2003; Podolsky, 2002).
30
Prostate Cancer
The prostate is a small sex accessory gland surrounding the urethra at the base of
the bladder and consists of epithelial and stromal cells (Cunha et al., 2004). A normal
human prostate is divided into three regions according to their position in relation to the
urethra – the transition the transition zone comprising 5%–10%, the central zone
comprising approximately 25%, and the peripheral zone which makes the bulk (70%) of
the prostate glandular tissue (Dehm and Tindall, 2006). The cells within these zones vary
significantly in their contribution to the prevalence of prostate cancer (Che and Grignon,
2002).
The American Cancer Society (2006) reported that prostate cancer is the most
frequently diagnosed cancer and the third leading cause of cancer death among men in
the US. At the time, it was estimated that 27,350 deaths would occur due to prostate
cancer. Current estimates have placed prostate cancer as the second leading cause of
cancer death in men, with about 28,660 deaths expected to occur in 2008 (American
Cancer Society, 2008).
Prostate cancer initially develops as a high-grade intraepithelial neoplasia
(HGPIN) in the peripheral and transition zones of the prostate gland. The HGPIN
eventually becomes a latent carcinoma, which may subsequently progress to a large,
higher grade, metastasizing carcinoma (Abate-Shen and Shen, 2000; Bosland et al.,
1991; Shukla and Gupta, 2005). Promotion and progression stages are controlled by
signal transduction molecules triggered by hormones such as androgens (Giovannucci,
1999; Shukla and Gupta, 2005). Androgen receptor (AR) signaling, cell proliferation and
31
cell death play a critical role in regulating the growth and differentiation of epithelial
cells in the normal prostate (Cunha et al., 2004).
Occurrence of prostate cancer is influenced by both genetic and non-genetic
factors. About 43% of cancer cases are attributed to genetic factors and these factors are
important at younger ages. Aging increases the risk of prostate cancer development
(Brothman, 2002).
Prostate cancer is classified as androgen-dependent or androgen-independent
(Dehm and Tindall, 2006; Eder et al., 2000; Haag et al., 2005). In androgen-dependent
prostate cancers, the cells depend on androgens for their growth and survival and can be
treated by either blocking the androgen pathway or using anti-androgens. Androgen
ablation initially inhibits androgen receptors and reduces prostate specific antigen (PSA).
Androgen-independent type of cancer appears in later stages of cancer reoccurrence and
is resistant to hormonal treatment (Roy-Burman et al., 2005).
Cell Proliferation
Cell proliferation is the increase in number of cells as a result of cell growth and
division. Cancerous cells are characterized by uncontrolled increase in cell numbers.
Cell proliferation is balanced by programmed cell death in normal organs, while mutated
cells gain a proliferative advantage resulting in excessive growth (Denmeade et al.,
1996; Magi-Galluzzi et al., 1998).
According to Vogelstein and Kinzler (2004) cancer-gene mutations enhance net
cell growth or proliferation, and they suggested that there are fewer pathways than genes
involved in carcinogenesis. In normal cells, the cell cycle is regulated at two check
32
points; G1–S and G2–M phases. Most of the cancer genes control transitions from the
resting stage (G1) to a replicating phase (S) of the cycle. Some of the products of these
genes include proteins such as kinases and cyclins.
Studies in human tumors have shown that some of the molecules often altered in
cancer are those involved in the control of the G1–S transition of the cell cycle,
particularly the cyclin-dependent kinase (CDK) and CDK inhibitors. These cell cycle
regulators have been found to be altered in more than 80% of human neoplasias, either
by mutations within the genes encoding these proteins or in their upstream regulators
(Ortega et al., 2002).
Mutations in tumor-suppressor genes encoding CDK inhibitors such as p16
(Ortega et al., 2002) and in genes encoding transcription factors such p53 (Oren, 2003)
result in enhanced cell proliferation. Expression of the nuclear transcription factor kappa
B (NF-κB) has been shown to promote cell proliferation, while inhibition of NF-κB
activation blocks cell proliferation. Several studies reported that tumor cells from colon,
breast, and pancreas cell lines expressed activated NF-κB (Storz, 2005; Valko et al.,
2006).
Several studies have also shown that mitogen-activated protein kinase (MAPK)
signaling pathways also play a critical role in both cell proliferation and apoptosis. The
three sub-groups of MAPKs in mammalian cells are extracellular signal-regulated kinase
(ERK), the c-Jun NH2-terminal Kinase (JNK), and the p38 MAPK (Kyriakis and
Avruch, 2001; Zhao et al., 2006). The extracellular signal-regulated kinase (ERK)
pathway is activated by growth factors and JNK by a variety of environmental stressors.
33
These kinases can induce both survival and apoptotic responses in cells depending on
cell type and environment (Lu and Xu, 2006). Valko et al (2007) reported that the
balance between ERK and JNK activation is important for cell survival since both a
decrease in ERK and an increase in JNK are required for the induction of apoptosis.
There are several assays for measuring cell proliferation in vitro by using
colorimetric methods such as the tetrazolium salt assay (Lawnicka et al., 2004). The
number of cells in vitro can be counted using a haemocytometer or coulter counter. Cell
proliferation can also be measured in vivo by tumor volume (Nakanishi et al., 2003).
Apoptosis
Apoptosis is an evolutionarily conserved form of programmed cell death that
requires a specialized mechanism to get rid of excess or potentially dangerous cells
(Thornberry and Lazebnik, 1998). Programmed cell death (apoptosis) is required for
proper development and to destroy cells that represent a threat to the integrity of the
organism. According to Hengartner (2000), apoptosis is as important as cell division and
cell migration, since regulated cell death allows the organism to tightly control cell
numbers and tissue size, and to protect itself from rogue cells that threaten homeostasis.
Apoptosis is not random but normally occurs in cells with damaged DNA. When
a cell becomes mutated and does not repair itself, apoptosis selectively eliminates the
altered cells. Programmed cell death results in morphological changes in cells such as
shrinkage, development of blebs, chromatin condensation, and biochemical changes
such as DNA fragmentation (Chaudhary et al., 1999).
34
According to Hale et al. (1996), there are three mechanisms by which a cell
commits suicide by apoptosis: one triggered by an internal signal (the intrinsic or
mitochondrial pathway), another triggered by an external signal (extrinsic or death
receptor pathway), and a third by apoptosis inducing factor (AIF). The major component
of the apoptotic machinery is a proteolytic system involving a family of cysteine
proteases called caspases (Thornberry and Lazebnik, 1998). Caspases are considered the
central executioners of the apoptotic pathway and over a dozen of them have been
identified in humans (Hengartner, 2000).
Valko et al (2007) stated that the intrinsic or mitochondrial pathway is
represented by intracellular damage of the cell causing Bc1-2 protein in the outer
membranes of mitochondria to activate Bax that causes cytochrome c to release from the
mitochondria. This pathway can be caspase-dependent or caspase-independent. In the
released caspase-dependent pathway, cytochrome c binds to apoptotic protease
activating factor-1 (APAF-1) forming apoptosomes. The apoptosome complex binds to
and activates caspase-9. Cleaved caspase-9 activates other caspases (3 and 7) leading to
digestion of structural proteins in the cytoplasm, degradation of DNA, and phagocytosis
of the cell. The caspase-independent pathway involves activation of apoptosis inducing
factor (AIF) or endonuclease G through translocation from mitochondria to nucleus
(Mohamad et al., 2005)
The transmembrane pathway of apoptosis involves the tumor necrosis factor
(TNF) ligand and receptor superfamily members (TNFα, Fas ligand and TNF-related
apoptosis-inducing ligand; TRAIL). Apoptosis pathways can start at the plasma
35
membrane by death receptor ligation (transmembrane or Fas-ligand dependent pathway)
or at the mitochondria (mitochondrial or Fas-ligand independent pathway) (Delmas et
al., 2003; Fulda and Debatin, 2006; Huang et al., 2006).
36
CHAPTER III
RELATIONSHIPS AMONG ANTIOXIDANT ACTIVITY, PHENOLICS, AND
SPECIFIC GRAVITY IN POTATO (SOLANUM TUBEROSUM L.) CULTIVARS
GROWN IN DIFFERENT ENVIRONMENTS
Introduction
Antioxidant compounds are present in foods as endogenous constituents or
phytochemicals (Siddhuraju et al., 2002) and efforts are underway to extract them from
plant sources. Some of the phytochemicals present in plants are polyphenols, and these
compounds, such as flavonols, flavonoids (Comis, 2000; McBride, 1999),
anthraquinones (Yen et al., 2000), xanthones and proanthocyanidins (Minami et al.,
1994) and zeaxanthin (Stelljes, 2001) act as antioxidants and agents of mechanisms that
exhibit cardioprotective or anticarcinogenic effects. Phenolics and anthocyanins have
been reported to possess a very high capacity to quench free radicals (Chu et al., 2000;
Kalt et al., 2001), hence attracting scientists to investigate fruits and vegetables for their
antioxidant properties.
Plants vary in composition of phytochemicals with protective functions;
therefore, to attain maximum health benefits, sufficient amounts of phytochemicals from
a variety of sources such as vegetables, fruits and grains are necessary (Adom and Liu,
2002). Previous studies have also indicated that complex mixtures of phytochemicals in
foods provide better protective benefits than single phytochemicals through additive
and/or synergistic effects (Eberhardt et al., 2000).
37
Tuber specific gravity is an important quality factor and is one of the properties
of potato tubers used as a measure of tuber quality. Processors usually pay less for tubers
with low specific gravity. Environmental factors influence tuber specific gravity
(Davenport, 2000; Sterrett et al., 2003). According to Gould (1999) potatoes with higher
specific gravity are well formed, smooth, and firm, and every 0.005 increase in specific
gravity results in increase of the number of potato chips that can be processed from 100
pounds of raw potatoes by one pound.
Specific gravity and solids-content have also been shown to have an effect on fat
uptake into French fries. Potatoes with a high specific gravity (>1.090) have been shown
to produce a high yield of French fries with a lower fat content than lower specific
gravity potatoes (Lulai and Orr 1979). Hagenimana et al. (1998) reported that there is a
linear relationship between dry matter content and fat uptake in thin sliced sweet potato
crisps, with fat uptake decreasing as dry matter increased.
Environmental conditions influence crop productivity and quality, including
phytonutrient levels. Crops perform differently under different environments, thereby
exhibiting genotype-by-environment interaction. Genotype-environment interaction in
crops is the differential response of genotypes to changing environmental conditions.
Such interactions complicate testing and selection in breeding programs and result in
reduced overall genetic gain (Goncalves et al., 2003). Differential performance of
genotypes due to genotype-by-environment interaction results in yield and quality
parameter instability in crops. Peterson et al. (1992) suggested that for breeders, stability
is important in terms of changing ranks of genotypes across environments and affects
38
selection efficiency. For end-users, such as wheat millers and bakers, consistency in
quality characteristics of cultivars is very important regardless of changing cultivar ranks
(Rharrabti et al., 2003a).
Becker and Léon (1988) suggested that a stable genotype is one with an
unchanged performance in various environments, i.e. static stability concept. According
to Rharrabti et al. (2003a) stability of quality parameters in crop products is an important
requirement in product development and may result into economic instability for end-
users. Economic instability is commonly caused by both environment and genotype-by-
environment interaction effects. Grausgruber et al. (2000) stated that the quality of a
genotype usually reacts like other quantitative characters to changing environmental
conditions. Therefore, a genotype is considered economically stable if its contribution to
G x E interaction is low.
Several studies have investigated effects of genotype and environment on yield,
nutrients and antioxidant activity in potato (Dale et al., 2003; Nzaramba et al., 2006),
oats (Emmons and Peterson, 2001), wheat (del Moral et al., 2003; Grausgruber et al.,
2000; Graybosch et al., 2004), barley (Atlin et al., 2000), and maize (Epinat-Le Signor et
al., 2001). All these studies aimed at gaining an understanding of genotype x
environment interactions. Producers experience local and annual variations in crop yield
and quality, yet the industry and consumers demand a constant quality of crop products.
Breeding programs normally select for local adaptation in order to exploit
genotype x environment interactions. Before new cultivars are released, they have to be
tested at several locations and for many years. Multi-environment trials encounter
39
problems with genotype x environment interactions, especially differential genotypic
responses to environmental condition which limit identification of superior and stable
genotypes (Epinat-Le Signor et al., 2001). However, in so doing, stable genotypes for
broad adaption and unstable ones for local adaption are identified.
Given the importance of antioxidants in disease prevention, developing potato
cultivars with high antioxidant levels would complement physical, medical, and social
activities in preventing diseases. Also, to ensure tuber quality, high specific gravity in
newly developed varieties should be maintained. However, to accomplish this there is
need to understand the relative importance of cultivar, environment, and their interaction
on antioxidant activity, phenolic compounds, and specific gravity, and also understand
the relationship among these traits. Relationships among traits provide information on
how selection for one trait would affect other traits. Therefore, the objective of this study
was to investigate the relative importance of cultivar, environment and season, and their
interaction on antioxidant activity, total phenolic content, and specific gravity in potato
cultivars grown under widely diverse environmental conditions (nine states) for three
years (2005, 2006, and 2007 seasons). Also, correlations among antioxidant activity,
total phenolics, individual phenolic compounds, and specific gravity were investigated.
Materials and Methods Plant Materials
Four popular potato cultivars representing different market classes, Yukon Gold
(fresh, yellow flesh), Atlantic (chipper), Red La Soda (fresh, red) , and Russet Norkotah
(fresh, russet), were selected for this study. These cultivars were grown in nine states
40
(locations): Springlake, TX., Tulelake, CA., Becker, MN., Plymouth, NC., Aberdeen,
ID., Powell Butte, OR., East Lansing, MI., Rhinelander, WI., and Pitstown, NJ.
Agronomic practices at all locations were assumed to be typical for potato production in
these areas. Potato samples were collected in the 2005, 2006, and 2007 growing seasons.
Fifteen advanced selections from the Texas A&M University Potato Improvement
Program grown near Springlake, TX were also analyzed.
Chemicals
DPPH (2,2-Diphenyl-1-picrylhydrazyl), Trolox (6-Hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt), potassium persulfate, monobasic sodium phosphate
(NaHPO4), dibasic sodium phosphate (Na2HPO4), sodium chloride, sodium carbonate,
and Folin-Ciocalteu reagent were purchased from Fisher Scientific (Pittsburgh, PA).
Methanol and acetonitrile were obtained from VWR International (Suwanee, GA). Pure
phenolic compounds (chlorogenic acid, rutin hydrate, caffeic acid, myricetin, and sinapic
acid) were obtained from Acros Organics (Pittsburgh, PA).
Specific Gravity Measurement
Specific gravity of each cultivar from every location was measured by weighing
ten tubers in air and then when immersed in water. Specific gravity was estimated as the
quotient of weight in air and the difference between weight in air and weight in water.
Sample Extraction
Three samples were randomly selected from tubers whose specific gravity had
been measured. Each sample contained three tubers. The tubers in each sample were
41
diced together, and five grams of diced material were placed in Corning centrifuge tubes,
and 15 ml of HPLC-grade methanol was added. Samples were hom*ogenized with an
IKA Utra-turrax tissuemizer for 3 min. Tuber extract was centrifuged at 31,000 g for 20
min. with a Beckman model J2-21 refrigerated centrifuge. One and one half ml of
supernatant was collected into microcentrifuge tubes for antioxidant and total phenolic
content analysis. Seven ml of supernatant were collected in glass vials for individual
phenolics compound analysis with HPLC. Sample extracts were stored at -20o C until
analysis.
Antioxidant Analysis
DPPH assay
Antioxidant activity in tuber extracts was estimated using the DPPH (2,2-
Diphenyl-1-picrylhydrazyl) method (Brand-Williams et al., 1995). One-hundred-fifty μl
of extract was placed in a scintillation vial, 2,850 μl of DPPH methanol solution was
added, and the mixture was placed on a shaker for 15 min. The mixture was transferred
to UV-cuvettes and absorbance recorded using a Shimadzu BioSpec-1601
spectrophotometer at 515 nm. Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid), a synthetic antioxidant, was used as a standard, and total AOA was
expressed as micrograms of trolox equivalents per gram of tuber fresh weight (μg
TE/gfw).
ABTS assay
Radical scavenging capacity of potato methanolic extracts was measured against
the ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt)
42
radical (Awika et al., 2003). Seventy-five µl of methanol was added to 25 µl of potato
extract to make 100 µl of diluted sample extract. Two-thousand-nine-hundred µl of the
working solution was added to the diluted sample extract and reacted for 30 min. on a
shaker. Absorbance of the solution was measured at 734 nm with a Shimadzu BioSpec-
1601 spectrophotometer. The working solution composed of a mixture of 5 ml of mother
solution [mixture of equal volumes of 8 mM of ABTS and 3 mM of potassium persulfate
solutions] and 145 ml of phosphate buffer solution pH 7.4 [40.5 ml of 0.2 M Na2HPO4
(dibasic), 9.5 ml of 0.2 M NaHPO4 (monobasic) and 150 mM NaCl. Trolox (6-Hydroxy-
2,5,7,8-tetramethylchroman-2-carboxylic acid), a synthetic antioxidant, was used as a
standard, and total AOA was expressed as micrograms of trolox equivalents per gram of
potato fresh weight (μg TE/gfw).
Total Phenolic Analysis
Total phenolic content was determined following the method of Singleton et al.
(1999). One-hundred-fifty µl of sample extract was pipetted into scintillation vials, and
2.4 ml of nanopure water was added. One-hundred-fifty µl of 0.25 N Folin-Ciocalteu
reagent was added and, after 3 min of reaction, 0.3 ml of 1N Na2CO3 reagent was added
and allowed to react for 2 hours. The spectrophotometer (Shimadzu BioSpec-1601) was
zeroed with a blank (0.150 ml methanol, 2.4 ml H2O, 150 µl of 0.25 N Folin, and 0.3 ml
1 N Na2CO2) before sample analysis. Absorbance of sample extracts was read at 725
nm. Chlorogenic acid was used as a standard, and total phenolic content expressed as
milligrams of chlorogenic acid equivalents per 100 grams of potato fresh weight (mg
CGA/100gfw).
43
Phenolic Composition
Composition of tuber methanolic extracts was determined using a HPLC system.
Samples were concentrated before analysis by drying 5 ml of potato extract with a speed
vac concentrator, and then re-dissolved in 1 ml of methanol. The concentrated extracts
were filtered through 0.45 µm syringe filters. The phenolic acid composition of extracts
was analyzed using an Atlantis C-18 reverse-phase column (4.6 x 150 mm, 5 µm) from
Waters, Milford, MA, maintained at 400 C. The HPLC system consisted of a binary
pump system (Waters 515), autoinjector (Waters 717 plus), a photodiode array (PDA)
detector (Waters 996), and column heater (SpectraPhysics SP8792). Separation of
phenolic acids was done using a linear gradient elution with mobile phase solvents A
(acetonitrile) and B (water/acetic acid, pH 2.3). Solvent flow rate was set at 1 ml/min
and gradient 15 to 100% in 35 min. Pure phenolic compounds were used as standards for
HPLC analysis: chlorogenic acid, rutin hydrate, caffeic acid, myricetin, and sinapic acid.
Identification and quantification of phenolic acids present in the potato extracts was
done by comparing retention time and area of the peaks in the extracts with that of the
standard compounds. Quantities of the phenolic acids were expressed as µg/g of fresh
weight.
Statistical Analysis
Data from each location in each year were analyzed separately using analysis of
variance (ANOVA). In some locations not all four cultivars were grown in all years.
Therefore, cultivar, location, year, and their interaction effects were determined
following a factorial design using data from locations in which all cultivars were grown
44
in all years. A combined analysis of variance for all the factors (cultivars, locations, and
years) was performed using the following factorial general linear model (GLM); Yijkl = μ
+ αi + βi + γk + (αβ)ij + (αγ)jk + (βγ)ik + (αβγ)ijk + εijkl , where Yijkl is the ith value of the lth
variety in jth location in the kth year. The four terms, μ, αi, βi, and γk are the mean and the
main effects of cultivars, locations, and years, respectively. The terms, αβij, αγjk, and βγik
are first order interactions, and αβγijk is a second order interaction involving all three
factors, and the environmental deviation within locations and years was denoted as εijkl
(Lentner and Bishop, 1993). The model was a mixed effects model with cultivars and
locations considered as fixed effects while years were random.
Mean separation was by least squares analysis. Phenotypic correlations between
traits were computed following Pearson’s correlation method. Principal component
analysis (PCA) was performed on the mean data of all replicates. General linear
regression was performed with SAS software version 9.1 (SAS, 2002), while principal
component analysis was performed with GGEbiplot software version 5.2 (Yan, 2001).
Results
Location, Year, Cultivar, and Interaction Effects
The combined analysis of variance showed a strong influence of the main effects
of cultivar, location, and year on antioxidant activity, total phenolic content, and specific
gravity (Table 3.1). Two-way interaction effects of location x year and cultivar x
location were significant for all measured traits. Interaction effect between cultivar and
year was significant for antioxidant activity (both DPPH and ABTS assays) and specific
45
gravity but not significant for total phenolic content. The three-way interaction effect
was also significant for all parameters.
Growing location accounted for 16% of the total observed variability in
antioxidant activity measured by the DPPH assay (Table 3.2). This percentage was equal
to variability due to cultivar differences, and also close to that due to seasons (year).
More than 50% of the observed variability in antioxidant activity measured by the ABTS
assay, total phenolic content, and specific gravity was attributed to the cultivar main
effect.
The location main effect accounted for 10.3, 11.8, and 20% of the variability in
the ABTS assay, total phenolics, and specific gravity, respectively (Table 3.2). All
interaction effects accounted for less than 10% of the variability in ABTS assay, total
phenolics, and specific gravity. However, interaction between location and year
(Location x Year) was responsible for 29.8% of the observed variability for antioxidant
activity measured by the DPPH assay.
According to Rharrabti et al. (2003b), the ratio of variances associated with
environmental effects (σ2e) to variances associated with genotypes (σ2
g) shows the
relative influences of genotype and environment on traits of interest. In this study, the
environmental variance component was considered as that due to location, year and their
effects (σ2e = σ2
l + σ2y + σ2
lxy), and the genotype x environment component was the sum
of cultivar x location, cultivar x year, and their three-way interaction effects (σ2gxe = σ2
gxl
+ σ2gxy + σ2
gxyxl).
46Table 3.1. Analysis of variance mean squares and significance of cultivar, location, year, and interaction effects for antioxidant
activity, phenolic content, and specific gravity of four potato cultivars grown in five locations during the 2005, 2006, and 2007 growing seasons.
Mean squares
Antioxidant activity
Source of variation df DPPH Assay ABTS Assay Total phenolics Specific gravity
Location 4 108097.7** 2319774.1** 2078.0** 0.00148**
Year 2 204787.4** 1596437.3** 577.1** 0.00029**
Location x Year 8 98080.3** 278589.1** 724.9** 0.00016**
Rep (Location x Year) 30 1230.5 86266.3 53.8 0.00001
Cultivar 3 143602.1** 18053028.2** 14588.4** 0.00519**
Cultivar x Year 6 19758.5** 476911.0** 88.5 0.00014**
Cultivar x Location 12 9441.9** 207621.9** 158.9** 0.00015**
Cultivar x Year x Location 24 6375.8** 230659.7** 119.0** 0.00008**
Residual 90 1675.2 85260.2 48.4 0.00001 *significance at p-value <0.05 **significant at p-value <0.01
47Table 3.2. Percentage of total observed variability in antioxidant activity, total phenolics, and specific gravity contributed by
each variance component- cultivar, location, year, and interactions.
Antioxidant activity
Source of variation DPPH Assay ABTS Assay Total phenolics Specific gravity
Location 16.5 10.3 11.8 20.0
Year 15.6 3.5 1.6 1.9
Cultivar 16.4 60.2 62.3 52.8
Location x Year 29.8 2.5 8.2 4.4
Cultivar x Year 4.5 3.2 0.8 2.8
Cultivar x Location 4.3 2.8 2.7 6.0
Cultivar x Year x Location 5.8 6.2 4.1 6.4
48
The ratio σ2e ⁄ σ
2g was greater than 1 for the DPPH assay (Table 3.3), which
implies that environmental effects were greater than genetic effects. Ratios less than 1
were observed for the ABTS assay, total phenolics, and specific gravity, indicating more
genetic influence than environmental effects on these traits. All traits, antioxidant
activity (both DPPH and ABTS assays), total phenolic content, and specific gravity
exhibited a ratio σ2g ⁄ σ
2gxe greater than 1 (Table 3.3). This demonstrates that genetic
effects (cultivar differences) were greater than variability due to interaction of genotype
and environment.
Table 3.4 shows actual values of cultivar performances in each location. Russet
Norkotah exhibited the highest amount of antioxidants (DPPH and ABTS assays) and
total phenolics, whereas Atlantic had the lowest antioxidants and phenolics in all
locations. Rankings of Red La Soda and Yukon Gold for antioxidants and phenolic
content changed over locations, with one performing better than the other in some states
and worse in other states. As for specific gravity, Atlantic consistently exhibited highest
specific gravity, while Russet Norkotah, Red La Soda, and Yukon Gold switched
rankings from state to state.
49 Table 3.3. Ratios of environmental (σ2
e) to genetic (cultivar) (σ2g) variance components and genetic to genotype-by
environment (σ2gxe) interaction effects for antioxidant activity, total phenolics, and specific gravity of four potato cultivars
grown in five states for three seasons.
Antioxidant activity
Ratio DPPH Assay ABTS Assay Total phenolics Specific gravity
σ2e ⁄σ2
g 2.9 0.2 0.2 0.4
σ2g ⁄σ2
gxe 4.0 19.7 39.8 14.3
50Table 3.4. Mean values of antioxidant activity (DPPH and ABTS), total phenolics, and specific gravity over three years for
four potato cultivars grown at nine locations (States). Locations
Parameter Cultivar CA ID MI MN NJ NC OR TX WI LSDa
Russet Norkotah 315 379 303 144 217 257 171 241 254 201DPPH Red La Soda 274 332 193 171 148 242 168 Yukon Gold 226 264 170 153 171 212 149 239 236 100 Atlantic 168 177 155 90 90 129 82 133 130 111 LSDb 92 154 199 70 53 79 42 77 105 Russet Norkotah 2875 2587 3009 2656 2023 2501 2087 2410 2565 544ABTS Red La Soda 2605 2033 1811 1911 1651 2157 638 Yukon Gold 2454 1748 2157 2199 1509 1887 1676 1816 1971 606 Atlantic 1214 929 1046 944 898 1010 1028 799 1033 369 LSDb 408 402 577 353 180 768 439 342 289 Russet Norkotah 91 90 91 77 81 92 71 80 78 21Total phenolics Yukon Gold 83 69 80 67 64 63 53 70 62 15 Red La Soda 80 70 56 61 60 70 21 Atlantic 46 40 44 34 35 40 34 38 37 10 LSDb 10 10 11 14 8 34 8 15 6 Atlantic 1.107 1.082 1.089 1.071 1.086 1.089 1.088 1.089 1.084 0.013Specific gravity Yukon Gold 1.088 1.083 1.059 1.061 1.079 1.077 1.083 1.077 1.074 0.011 Red La Soda 1.078 1.065 1.066 1.068 1.067 1.057 0.008 Russet Norkotah 1.074 1.064 1.066 1.047 1.075 1.079 1.073 1.073 1.063 0.013 LSDb 0.005 0.009 0.014 0.035 0.005 0.009 0.006 0.005 0.005
a LSD among location means of a cultivar trait; bLSD among cultivars within a location
51
Relationships among Antioxidant Activity, Phenolic Content, and Specific Gravity
Regression analysis revealed significant relationships among antioxidant activity,
total phenolics, and specific gravity (Fig. 3.1). Correlation coefficient between
antioxidant activity (AOA) measured by the DPPH assay and AOA measured by the
ABTS assay was significant (p-value <0.01) with a value of r = 0.508. Antioxidant
activity (measured by both DPPH and ABTS) and total phenolic content were
significantly correlated with correlation coefficients (r) = 0.579 and 0.876, respectively.
Similar results showing significant correlation between AOA and total phenolic content
in the potato (Reddivari et al 2007) and sweet potatoes (Huang et al., 2004) were
reported. Negative relationships between antioxidant activity (DPPH and ABTS assays)
and specific gravity, and between total phenolic content and specific gravity were
observed (Fig. 3.1). The correlation coefficient between AOA (DPPH assay) and
specific gravity was -0.232 and significant at p-value = 0.01. Relationship between the
ABTS assay and specific gravity was also significant (p-value = 0.01) with a correlation
coefficient of -0.494. Likewise, total phenolic content was negatively correlated (r = -
0.452) with specific gravity. These results indicate that breeding for high antioxidants
and phenolic content may result in reduced specific gravity of the tubers.
52
Fig. 3.1. Regression analysis and correlation coefficients among antioxidant activity (DPPH and ABTS assays), phenolic content, and specific gravity of four potato cultivars grown over three years at nine locations.
53
This study used cultivars that are commonly grown in the United States or grown
by most potato breeders as standard checks. However, given the small sample of
cultivars used, correlations of antioxidant activity and phenolic content with specific
gravity may have been biased due to sampling size. Also, the cultivar Atlantic was
consistently the lowest in antioxidant activity and total phenolic content, and the highest
in specific gravity, thereby behaving as an influential outlier. Therefore, several
advanced selections from the Texas A&M University Potato Improvement Program,
College Station, were used to confirm the observed correlation analysis results above.
Tuber specific gravity, antioxidant activity, total phenolic content, and individual
phenolic compounds in the breeding lines are shown in Table 3.5. Phenolic compounds
quantified with HPLC analysis were chlorogenic acid, rutin hydrate, caffeic acid,
myricetin, and sinapic acid. Results showed no significant linear relationship between
antioxidant activity and specificity gravity in potato breeding lines. Also there was no
significant correlation between total phenolic content and specific gravity, or between all
individual phenolic compounds and specific gravity (Table 3.6). .
54Table 3.5. Mean values of antioxidant activity, phenolic content, specific gravity, and individual phenolic compounds of potato
advanced selections grown near Spring Lake, TX in the 2005 growing season.
Antioxidant activity Phenolic composition
Genotype DPPH ABTS Total phenolics Specific gravity Sinapic Rutin Myricetin Caffeic Chlorogenic
A833501-9R 59.7 2273.3 65.6 1.066 3.1 14.6 12.0 29.8 60.9 Atlantic 190.7 713.4 44.8 1.086 3.4 7.7 10.4 29.6 109.2 ATTX961014-1AR/Y 96.4 2339.9 66.6 1.062 7.9 6.5 10.9 29.6 123.4 ATTX98444-16R/Y 225.7 2620.3 89.0 1.074 2.6 25.4 11.2 36.2 276.5 ATTX98462-9R/Y 169.5 2478.8 71.9 1.071 11.3 7.1 10.7 30.0 215.7 ATTX98468-3R/Y 296.2 3448.2 92.5 1.070 4.2 5.8 11.2 33.8 344.4 ATTX98493-2P/P 422.1 3037.5 96.4 1.067 10.3 14.3 11.8 31.8 441.0 ATTX98510-1R/Y 336.7 3999.0 104.6 1.063 2.9 7.2 10.4 32.9 363.0 COTX01403-4R/Y 204.2 2602.8 80.5 1.061 7.4 8.6 10.7 31.7 250.2 NDTX4271-5R 122.5 2389.2 68.8 1.058 2.8 4.1 10.8 30.8 104.8 NDTX4304-1R 138.6 2478.4 69.6 1.058 5.0 8.9 11.2 29.2 61.3 NDTX4756-1R/Y 359.2 3684.7 101.6 1.062 8.4 7.1 11.4 31.9 315.2 NDTX731-1R 113.0 2133.1 66.2 1.058 4.1 9.7 10.9 30.1 142.9 POTX03PG19-1Pu/YR 394.2 2731.9 92.1 1.082 5.5 7.3 10.7 31.8 542.7 PORTX03PG25-2R/P 471.1 3558.7 106.1 1.057 22.7 22.8 11.5 42.3 659.1 Russet Norkotah 201.9 2103.1 86.5 1.068 6.0 10.6 11.3 31.6 231.6 TX1674-W/Y 196.8 2884.0 86.3 1.080 4.2 5.2 11.1 30.0 179.3 Yukon Gold 268.3 1682.6 76.0 1.072 10.1 6.8 11.7 30.1 47.4
LSD 86.3 816.5 13.2 0.005 4.5 12.5 1.2 4.5 134.2
55
Table 3.6. Correlation analysis among antioxidant activity (DPPH and ABTS assays), total phenolics (TP), specific gravity, and individual phenolic compounds of potato advanced selections grown near Spring Lake, TX in the 2005 growing season.
ABTS TP Sinapic Rutin hydrate Myricetin Caffeic Chlorogenic Specific gravity
DPPH 0.58* 0.80** 0.55* 0.27 0.14 0.65** 0.88** 0.09
ABTS 0.88** 0.26 0.14 0.11 0.52* 0.65** -0.39
TP 0.38 0.29 0.23 0.65** 0.79** -0.18
Sinapic 0.36 0.32 0.60** 0.54* -0.28
Rutin hydrate 0.43 0.71** 0.40 -0.11
Myricetin 0.18 0.02 -0.20
Caffeic 0.77** -0.20
Chlorogenic 0.00
*significance at p-value <0.05 **significant at p-value <0.01
56
Despite the lack of a significant relationship between specific gravity and any of
the measured traits, principal component analysis was done to further elucidate the
particularity of the relationships (Yan and Hunt, 2001). The first two principal
components (PC1 and PC2) were used to construct a biplot consisting of the measured
traits and the potato genotypes (Fig. 3.2). The two principal components accounted for
66.8% of the total variance: 50.6 and 16.2% for PC1 and PC2, respectively. The first PC
axis (PC1) separated specific gravity from all other traits. Specific gravity was placed on
the negative direction of the PC1 axis, whereas antioxidant activity, total phenolic
content, and individual phenolic compounds were all on the positive side of the axis
(Fig. 3.2). This clearly demonstrates that there is no positive relationship between
specific gravity and any of these traits. The biplot (Fig. 3.2) also shows that, among the
individual phenolic compounds quantified, chlorogenic acid was the most closely
associated with antioxidant activity and total phenolic content followed by caffeic acid.
Discussion
This study demonstrated the influence of genotype and environment on
antioxidant activity, total phenolic content, and specific gravity, and also elucidated the
relationships among these traits in potato tubers. Results from this investigation showed
that antioxidant activity measured by the ABTS assay, total phenolic content, and
specific gravity are mostly governed by genotype. This is so because more than 50% of
the observed variability in the ABTS assay, total phenolic content, and specific gravity
was attributed to the cultivar main effect (Table 3.2). However, for DPPH assay,
location, season and cultivar main effects were equally influential to antioxidant activity.
57
Fig. 3.2. A biplot of genotypes-by-trait in potato advanced selections grown near Springlake, TX, in the 2005 growing season. Traits are in red and upper case and accessions are in blue and lower case. Traits are abbreviated as SPG- specific gravity, DPPH and ABTS- antioxidant activity, TP- total phenolics, CGA- chlorogenic acid, CA- caffeic acid, SA- sinapic acid, RH- rutin hydrate, and MYC myricetin.
58
Relationships among traits are of interest in plant breeding as they influence
strategies employed by breeders in improving crops. A significant positive correlation
between desirable traits makes breeding for one or both traits easier, while a negative
correlation poses challenges, as increasing one trait results in reduced value of the other.
However, if one trait is desirable and the other undesirable, a negative correlation makes
breeding easier, while a positive correlation makes trait improvement difficult.
Observations from this study indicated that antioxidant activity and phenolic
content in potato tubers have a significant positive linear relationship. These results are
in agreement with previous studies in potato tubers (Kanatt et al., 2005; Reddivari et al.,
2007a), bamboo extracts (Kweon et al., 2001), fruits (Kim et al., 2003), and vegetables
(Troszynska et al., 2002). Therefore, breeding for high antioxidant activity in potatoes
can be achieved by increasing the amount of phenolic compounds available in potato
tubers.
Specific gravity is the solids content of potato tubers and is an important quality
factor for processing. It is one of the properties of potato tubers that could be used as a
basis for nondestructive quality evaluation (Chen et al., 2005). Correlation analysis of
data from the four common cultivars showed a significant negative relationship between
antioxidant activity (DPPH and ABTS assays) and specific gravity, and between total
phenolic content and specific gravity. However, correlation analysis of data from
advanced selections indicated no significant relationship between antioxidant and
specific gravity or between total phenolic content and specific gravity.
59
The difference in observed results from the two analyses was due to sample
sizes. Only cultivars, which could be obtained from breeding programs in the nine states,
were used. Therefore, the small sample, in addition to cultivar Atlantic being
consistently the lowest in antioxidant activity and total phenolic content and the highest
in high specific gravity, could have resulted in biased correlation results. With a larger
sample (15 advanced selections and 3 cultivars) with distributed trait values, there was
no significant relationship/correlation between antioxidant activity and specific gravity
or between total phenolic content and specific gravity. Also, none of the individual
phenolic compounds, caffeic acid, chlorogenic acid, myricetin, rutin hydrate and sinapic
acid were significantly correlated with specific gravity.
The important finding of the investigation is that there is no significant
relationship between antioxidant activity and specific gravity. And also there were no
relationships observed between total phenolic content and specific gravity, and between
individual phenolic compounds and specific gravity. Therefore, breeding for high
antioxidants and phenolic compounds in potato tubers would increase their nutritional
value without compromising tuber quality in terms of specific gravity. However,
significant genotype-by-environment interactions may hinder rapid progress.
60
CHAPTER IV
TOTAL GLYCOALKALOIDS, ANTIOXIDANT ACTIVITY, AND PHENOLIC
LEVELS IN SOLANUM MICRODONTUM AND SOLANUM JAMESII
ACCESSIONS
Introduction
Research has intensified on investigating the health benefits of phytonutrients
(Duyff, 2002), and several research reports have indicated that the benefit of plant foods
is due not only to levels of vitamins or other nutritive compounds they provide, but also
to activity of the non-nutritive factors they contain. Many of these plant secondary
components are antioxidants (Riedl et al., 2002). Food-derived antioxidants, such as
vitamins and phytochemicals, are receiving much attention for their function as
chemopreventive agents against oxidative damage (Hwang and Yen, 2008).
The importance of antioxidants in preventing diseases and maintenance of health
has raised interest among scientists, food producers/manufacturers, and consumers, as
the trend of the future is towards functional foods (Robards et al., 1999; Velioglu et al.,
1998). Many authors (Al-Saikhan et al., 1995; Hale, 2004; Kanatt et al., 2005;
Kawakami et al., 2000) have reported presence of antioxidant compounds in potatoes.
In order to further improve the nutritional value of the potato, plant breeders need
to develop cultivars with substantial amounts of antioxidants. Kolasa (1993) proposed
analyzing the nutrient value of potatoes not commonly grown in the U.S. to determine if
there are significant quantities of antioxidants, like vitamin E and beta-carotene, or other
cancer preventing phytochemicals, such as butyric acid, and suggested enhancing the
61
contribution of potato to human nutrition through education, marketing, breeding, field
management, and preparation for consumption. Also, related wild species with desirable
nutritional benefits can be used as parental material in developing improved varieties
with enhanced health benefits. Several wild species were analyzed for antioxidant
activity and total phenolic content by the Potato Improvement Program at Texas A&M
University, and many were reported to possess more antioxidant activity and phenolic
content than currently grown cultivars. Some of the species identified as containing high
antioxidant activity were Solanum jamesii, S. pinnatisectum, S. megistacrolobum, and S.
microdontum (Hale, 2004; Nzaramba et al., 2007). However, in the above studies, only a
few accessions of each species from a “mini-core” collection were analyzed. The mini-
core collection was assembled by Dr. John Bamberg, Curator, US Potato Genebank to
represent the gene bank’s diversity as well as facilitate pioneering research and
preliminary evaluation of the germplasm for various traits, given the large number of
populations and species held in the gene bank.
Having identified some species as containing more antioxidant activity than
cultivated varieties, it was decided to screen all populations of these species to identify
specific accessions that are the highest in antioxidant activity and phenolic content.
However, many wild potato species are reported to contain high levels (>20 mg/100g) of
glycoalkaloids which are toxic to humans.
Therefore, the objective of this study was to screen all accessions of S. jamesii
and S. microdontum species in the US Potato GeneBank for antioxidant activity, total
phenolic content, and total glycoalkaloid levels. Also, linear correlations among
62
glycoalkaloid (TGA) levels, antioxidant activity, and total phenolic content were
investigated. Solanum jamesii and S. microdontum species were selected for this study
because they are known to be efficient in tuber calcium accumulation. Also, S.
microdontum is easily crossable to varieties of tetraploid S. tuberosum, while S. jamesii
is native to southern US and northern Mexico therefore would easily be adaptable to the
North American climate. The information obtained from this study would be helpful in
selecting accessions to use in introgressing desirable traits into cultivated potato
varieties, while avoiding introducing or increasing levels of undesirable compounds such
as glycoalkaloids.
Materials and Methods
Plant Material
Ninety-two accessions of S. jamesii and 86 accessions of S. microdontum were
obtained from the US Potato Genebank, Sturgeon Bay, WI. Tubers analyzed were
obtained from seedlings transplanted into 10 cm - 400ml pots filled with commercial
soilless potting medium in the greenhouse. The seedlings were watered as needed with 1
g/L 20-20-20 fertilizer with micronutrients. Supplementary lighting was provided with
400 W alternating sodium and metal halide lamps 2.5 m apart and 1.5 m above the
benchtops for 16 h days. Temperatures were maintained at 22o C day and 13o C night
until harvest. Triplicates of five grams of fresh tubers from each accession were washed
with water, diced and placed in 50 ml falcon tubes. Samples were store at -20o C until
extraction of phytochemicals.
63
Chemicals
DPPH (2,2-Diphenyl-1-picrylhydrazyl), Trolox (6-Hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), ABTS (2,2’-azinobis(3-ethylbenzothiazoline-6-
sulfonic acid) diammonium salt), potassium persulfate, dibasic sodium phosphate,
monobasic sodium phosphate, sodium chloride, Folin-Ciocalteu reagent, ammonium
phosphate, and sodium carbonate were purchased from Fisher Scientific (Pittsburgh,
PA). Methanol, acetonitrile, acetone, and chloroform were obtained from VWR
International (Suwanee, GA). Chlorogenic acid, rutin hydrate, caffeic acid, myricetin, α-
chaconine and ammonium hydroxide were purchased from Sigma-Adrich (St. Louis,
MO). Alpha-solanine and tomatine were obtained from MP Biomedicals (Solon, OH).
Sample Extraction
Five grams of diced tubers were extracted with 20 ml of HPLC-grade methanol.
The samples were hom*ogenized with an IKA Utra-turrax tissuemizer for 3 min. The
extract was centrifuged at 31,000 g for 20 min. with a Beckman model J2-21 refrigerated
centrifuge. Two ml of supernatant was collected into microcentrifuge tubes for AOA
determination and total phenolic content analysis, and 5 ml of supernatant was collected
in glass vials for individual phenolic compound analysis with HPLC. Sample extracts
were stored at -20o C until analysis.
Antioxidant Analysis
Antioxidant activity was measured using two assays, the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay (Brand-Williams et al., 1995) and the 2,2-azinobis (3-
ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS) assay (Awika et al.,
64
2003; Miller and Rice-Evans, 1997). Intense sample color interferes with the DPPH
assay estimates; therefore the ABTS assay was used to confirm the results.
DPPH assay:
Antioxidant activity in extracts was estimated using the DPPH (2,2-Diphenyl-1-
picrylhydrazyl) method (Brand-Williams et al., 1995). The DPPH method is commonly
used to determine antioxidant activity of pure compounds as well as natural plant
extracts. DPPH. is a stable free radical that absorbs at 515 nm, and when reduced by an
antioxidant its absorbance is lost and the change in absorbance is determined
spectrophotometrically (Brand-Williams et al., 1995; f*ckumoto and Mazza, 2000;
Mahinda and Shahidi, 2000). The method is commonly used due to its good repeatability
but has little relevance to biological systems. Samples that contain anthocyanins may
lead to color interference of DPPH., resulting in underestimation of antioxidant activity
(Arnao, 2000). A 150 μl aliquot was placed into a scintillation vial, 2,850 μl of DPPH
methanol solution was added, and the mixture was placed on a shaker for 15 min. The
mixture was transferred to UV-cuvettes and its absorbance recorded using a Shimadzu
BioSpec-1601 spectrophotometer at 515 nm. Trolox (6-Hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid), a synthetic antioxidant, was used as a standard
to generate a standard curve, and antioxidant activity was expressed as micrograms of
trolox equivalents per gram of tuber fresh weight (μg TE/gfw).
ABTS assay:
The ABTS assay measures the relative ability of antioxidants to scavenge the
ABTS.+ radicals generated in an aqueous phase, as compared to a trolox standard which
65
is a water soluble vitamin E analogue. The ABTS.+ is generated by reacting a strong
oxidizing agent (potassium permanganate or potassium persulfate) with ABTS salt. The
reduction of the blue-green ABTS.+ radical by a hydrogen-donating antioxidant is
measured by the suppression of its characteristic long-wave absorption spectrum (Miller
and Rice-Evans, 1997). It is a rapid method and can be used over a wide range of pH
values (Arnao et al., 1999; Lemanska et al., 2001) in both aqueous and organic solvent
systems. The assay has good repeatability and is simple to perform. Radical scavenging
capacities of potato methanolic extracts were measured against the ABTS radical (Awika
et al., 2003). Seventy-five µl of methanol was added to 25 µl of potato extract to make
100 µl of diluted sample extract. Two-thousand-nine hundred µl of the working solution
was added to the diluted sample extract and reacted for 30 min. on a shaker. The
working solution was composed of a mixture of 5 ml of mother solution [mixture of
equal volumes of 8 mM of ABTS and 3 mM of potassium persulfate solutions] and 145
ml of phosphate buffer solution pH 7.4 [40.5 ml of 0.2 M Na2HPO4 dibasic, 9.5 ml of
0.2 M NaHPO4 monobasic and 150 mM NaCl]. Absorbance of the solution was
measured at 734 nm with a Shimadzu BioSpec-1601 spectrophotometer. Trolox, a
synthetic antioxidant, was used as a standard, and total antioxidant activity was
expressed as micrograms of trolox equivalents per gram of potato tuber fresh weight (μg
TE/gfw).
Total Phenolic Analysis
Phenolic content was determined following the method of Singleton et al. (1999).
One-hundred-fifty µl of tuber extract were pipetted into scintillation vials, and 2.4 ml of
66
nanopure water was added. One-hundred-fifty µl of 0.25 N Folin-Ciocalteu reagent was
added, and after 3 min of reaction 0.3 ml of 1N Na2CO3 reagent was added and allowed
to react for 2 hours. The spectrophotometer (Shimadzu BioSpec-1601) was zeroed with a
blank (0.150 ml methanol, 2.4 ml H2O, 150 µl of 0.25 N Folin-Ciocalteu, and 0.3 ml 1 N
Na2CO2) before sample analysis. Absorbance of tuber extracts was read at 725 nm.
Chlorogenic acid was used as a standard, and total phenolic content was expressed as
milligrams of chlorogenic acid equivalents per 100 grams of potato tuber fresh weight
(mg CGA/100gfw).
Phenolic Composition
Five ml of tuber extract was concentrated before analysis by drying the extract
with a SpeedVac concentrator, and re-dissolved in 1 ml of aqueous methanol (50:50
v/v). The concentrated extracts were filtered through 0.45 µm syringe filters and injected
into the HPLC system. The HPLC system consisted of a binary pump system (Waters
515), an auto-injector (Waters 717 plus), a photodiode array (PDA) detector (Waters
996), and a column heater (SpectraPhysics SP8792). An Atlantis C-18 reverse-phase
column (4.6 x 150 mm, 5 µm) (Waters, Milford, MA.) maintained at 400 C was used to
separate phenolic acids in the sample extracts. Twenty μl of extract was injected into the
system, and the mobile phase used consisted of two solvents: solvent A acetonitrile and
B nano-pure water adjusted to pH 2.3 with acetic acid. Solvent flow rate was set at 1
ml/min with a gradient of 0/85, 6-35/85-0, 36-45/85 (min/%A). Pure phenolic
compounds - chlorogenic acid, rutin hydrate, caffeic acid and myricetin, previously
reported in potato tubers were used as standards to identify and quantify some of the
67
phenolic compounds present in the sample extracts. Identification of phenolic acids
present in the potato extracts was done by comparing retention time and spectra of peaks
detected at 280 nm in the extracts with retention times and spectra of peaks of the
standard compounds. Quantification of individual phenolic acids in the sample extract
was done by comparing peak area of a known concentration of standards, and results
were expressed as µg/g of tuber fresh weight (µg/gfw).
Total Glycoalkaloid Extraction
Extraction of glycoalkaloids followed the method of (Rodriguez-Saona et al.,
1999). Five g of fresh tubers was hom*ogenized with 10 ml of acetone to a uniform
consistency. The extract was centrifuged at 13,000 g for 15 min., and the clear
supernatant collected into a falcon tube. The residue was re-extracted with 10 ml of
aqueous acetone (acetone:water 30:70 v/v). The extract was centrifuged and the
supernatant combined with the first extract. Chloroform was added to the acetone extract
(2 volumes of chloroform for each volume of acetone extract), thoroughly mixed by
shaking the tubes and stored overnight at 1o C. The top aqueous portion was collected
into glass vials and concentrated in a rotovapor SpeedVac at 40o C until all residue
acetone was evaporated. The extract was brought to a known volume with nano-pure
water and analyzed for glycoalkaloids.
Glycoalkaloid Analysis
The sample extracts and solvents were filtered through 0.45 µm filters.
Glycoalkaloid analysis with a high performance liquid chromatography (HPLC) system
followed the method of Sotelo and Serrano (2000) with some modifications. A Waters
68
HPLC with an Atlantis C-18 reverse-phase column (4.6 x 150 mm, 5 µm) from Waters
(Milford, MA.) maintained at 350 C was used. The mobile phase used for glycoalkaloid
elution was (35:65 v/v) acetonitrile : 0.05 M monobasic ammonium phosphate buffer
((NH4)H2PO4), adjusted with NH4OH to pH 6.5. The solvent flow was isocratic at a rate
of 1 ml/min and the UV absorbance detector set at 200 nm with 5% AUFS sensitivity.
Amount of sample extract injected was 20 µl. Different concentrations of commercially
obtained α-solanine, α-chaconine, and tomatine were injected into the HPLC system and
their peaks used to identify glycoalkaloids in the tuber extracts by comparing peak
retention times and spectra detected at 200 nm in the extracts with retention times and
spectra of peaks of the commercial standard compounds. Also, standard curves prepared
by regressing known concentrations to their corresponding peak areas were used to
quantify amounts of glycoalkaloids in the extracts.
Statistical Analysis
Analysis of variance (ANOVA) was performed using SAS version 9.1 software
(SAS, 2002) to determine the variability of the measured parameters in the potato
accessions. Mean separation was by least squares analysis. Phenotypic correlations
between traits were computed following Pearson’s correlation method and principal
component analysis was performed by GGEBiplot software version 5.2 (Yan, 2001).
69
Results
Antioxidant Activity
Antioxidant activity values determined with the DPPH and ABTS assays were
widely variable in both S. jamesii (Table 4.1) and S. microdontum (Table 4.2)
accessions. Antioxidant activity values measured by ABTS were greater than those
measured by DPPH. This may be due to differences in the absorption maxima of the two
radicals. The DPPH maximum absorption wavelength (515 nm) is in the visible region,
and the interference due to sample color is much more pronounced in this region as
compared to the ABTS maximum absorption wavelength (725 nm), which is not in the
visible region (Kanatt et al., 2005). However, consistency in relative ranking is probably
more important than consistency in absolute numerical scores.
The DPPH values in S. jamesii ranged from 173 (PI 592408) to 961 μg TE/gfw
(PI 620875), while values from the ABTS assay ranged from 1,383 (PI 592408) to 3,513
(PI 275172) μg TE/gfw. Analysis of variance also showed significant differences (p-
value <0.01) in AOA (DPPH and the ABTS assays) among S. jamesii accessions (Table
4.1). The DPPH values in S. microdontum ranged from 202 (PI 558097) to 1,535 (PI
498127) μg TE/gfw, while the ABTS values ranged from 1,084 (PI 558097) to 6,288 (PI
498127) μg TE/gfw (Table 4.2), and analysis of variance showed significant differences
among accessions.
Total Phenolic Content
Significant differences among S. jamesii and S. microdontum accessions in total
phenolic content were revealed by analysis of variance. Wide variation in TP was also
70
Table 4.1. Mean values of antioxidant activity (DPPH and ABTS assays), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), total glycoalkaloids (TGA), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MY), and ratio of solanine to chaconine (S:C) in S. jamesii accessions (ACCESS).
ACCESS DPPH ABTS TP SOL CHA TGA CGA CA RH MY S:C PI 275172 784 3513 151 2.2 4.0 6.3 432 282 24 2 0.6 PI 564047 476 1502 87 4.2 5.6 9.8 197 32 12 1 0.8 PI 564048 618 2412 111 4.6 4.5 9.2 282 133 22 2 1.0 PI 564049 647 2618 128 9.3 8.3 17.5 270 132 29 3 1.1 PI 564051 626 1920 128 6.7 7.4 14.2 344 72 51 3 0.9 PI 564052 653 1939 128 6.3 7.4 13.7 344 159 53 2 0.8 PI 564053 711 2145 129 5.1 7.2 12.3 346 138 63 2 0.7 PI 564054 744 2530 140 5.8 6.5 12.3 521 311 54 3 0.9 PI 564055 572 2139 130 6.7 6.6 13.2 397 122 58 2 1.0 PI 564056 820 2279 139 7.9 6.3 14.2 409 223 30 4 1.2 PI 564057 695 2770 131 7.4 8.7 16.0 250 235 27 3 0.8 PI 578236 656 2075 121 5.6 7.5 13.1 308 132 54 5 0.7 PI 578237 636 2245 123 6.5 5.2 11.7 344 121 51 2 1.3 PI 578238 465 1769 92 7.7 6.6 14.3 207 80 28 2 1.2 PI 585116 827 2467 137 20.0 16.8 36.8 292 101 33 3 1.2 PI 585118 397 1474 97 6.2 6.8 13.0 146 123 25 4 0.9 PI 585119 627 1884 125 10.0 6.1 16.1 381 186 34 4 1.6 PI 592397 321 1987 88 12.5 8.6 21.1 178 247 15 4 1.5 PI 592398 589 2051 118 12.9 6.5 19.4 222 254 24 5 2.0 PI 592407 925 2897 145 6.7 9.1 15.8 217 180 34 4 0.7 PI 592408 173 1383 50 9.6 7.2 16.8 123 76 9 2 1.3 PI 592410 682 2449 122 12.7 7.1 19.8 239 129 23 4 1.8 PI 592411 607 2121 124 5.5 6.0 11.4 339 76 44 6 0.9 PI 592413 503 2136 113 8.0 13.6 21.5 213 156 50 5 0.6 PI 592414 289 2122 81 5.5 5.7 11.2 133 124 7 4 1.0 PI 592416 598 2074 107 4.3 6.0 10.3 280 231 41 4 0.7 PI 592417 709 2281 124 11.6 7.4 18.9 309 273 28 8 1.6 PI 592418 458 2074 96 7.0 5.8 12.9 172 231 27 6 1.2 PI 592419 608 2222 119 4.2 6.4 10.6 341 172 60 6 0.7 PI 592422 752 2515 139 5.9 5.9 11.8 393 159 72 3 1.0 PI 592423 846 2372 141 8.5 5.4 13.8 504 339 36 4 1.6 PI 595775 879 2607 161 5.0 6.4 11.3 368 136 71 15 0.8 PI 595777 719 2001 126 8.5 4.3 12.8 440 110 21 4 2.0 PI 595778 829 2497 140 13.1 7.5 20.6 403 167 48 12 1.8 PI 595780 731 2449 129 9.0 5.7 14.7 330 131 31 6 1.6
71
Table 4.1. Continued..
ACCESS DPPH ABTS TP SOL CHA TGA CGA CA RH MY S:C PI 595782 556 2273 113 11.9 5.8 17.6 227 148 39 9 2.1 PI 595783 644 2647 125 9.8 4.8 14.7 320 186 27 2 2.0 PI 595784 789 2641 137 11.0 5.6 16.7 503 117 40 3 2.0 PI 595785 540 2320 113 9.4 4.6 14.0 319 133 35 3 2.1 PI 595786 588 2064 114 9.1 4.6 13.7 342 63 33 2 2.0 PI 595787 793 3037 140 5.1 6.6 11.7 468 347 42 3 0.8 PI 595788 531 1927 109 6.0 7.4 13.4 226 191 43 3 0.8 PI 596519 707 2444 114 5.1 7.0 12.1 318 156 14 4 0.7 PI 603051 758 2904 129 4.9 6.5 11.4 282 209 32 5 0.8 PI 603052 672 2146 121 2.9 4.6 7.5 289 205 29 4 0.6 PI 603053 572 2401 154 5.3 10.9 16.2 440 314 65 3 0.5 PI 603054 783 2206 140 13.6 6.4 20.0 466 205 31 3 2.1 PI 603055 684 1981 119 6.3 6.3 12.6 349 73 26 2 1.0 PI 603056 599 2021 110 7.1 6.9 14.0 277 113 24 3 1.0 PI 603057 620 2276 123 7.0 6.1 13.1 322 265 31 2 1.2 PI 603058 525 1802 102 6.0 5.9 12.0 270 102 21 3 1.0 PI 605357 875 2573 144 11.5 6.2 17.8 585 140 55 3 1.9 PI 605358 703 2078 126 10.0 5.1 15.1 309 327 32 4 1.9 PI 605359 735 2172 128 8.4 6.1 14.6 353 359 33 4 1.4 PI 605360 624 1858 114 7.0 3.7 10.8 334 202 23 5 1.9 PI 605361 813 2406 132 7.2 4.5 11.7 323 242 40 7 1.6 PI 605362 783 2678 130 3.7 3.2 6.9 350 267 39 6 1.1 PI 605363 622 2502 115 6.5 3.5 10.0 266 221 25 3 1.8 PI 605364 958 2947 140 4.1 4.5 8.6 413 154 42 3 0.9 PI 605365 667 2399 108 2.8 3.4 6.1 189 142 18 4 0.8 PI 605366 703 2161 120 4.9 3.8 8.7 260 114 26 3 1.3 PI 605367 564 1922 102 2.4 3.6 5.9 223 119 24 4 0.7 PI 605368 560 1857 99 4.8 5.1 9.9 217 157 23 3 1.0 PI 605369 750 2163 124 4.2 4.4 8.7 348 278 18 4 0.9 PI 605370 714 2201 118 8.5 4.9 13.5 263 123 34 3 1.7 PI 605371 610 2965 145 7.7 12.2 19.9 297 297 62 6 0.6 PI 605372 687 2687 128 4.3 5.1 9.4 363 254 34 3 0.8 PI 612450 648 2237 112 8.9 6.2 15.0 306 213 21 5 1.4 PI 612451 685 2257 120 6.4 6.6 13.0 356 127 29 3 1.0 PI 612452 531 1807 113 5.9 6.1 12.0 242 242 35 3 1.0 PI 612453 814 1998 127 4.1 4.6 8.7 370 211 31 3 0.9 PI 612454 620 2429 126 3.1 5.0 8.2 359 218 34 5 0.6 PI 612455 662 2461 118 7.0 6.5 13.4 325 173 26 4 1.1
72
Table 4.1. Continued..
ACCESS DPPH ABTS TP SOL CHA TGA CGA CA RH MY S:C PI 612456 585 2312 112 4.4 4.9 9.3 238 197 15 4 0.9 PI 620869 604 2098 109 3.4 4.3 7.7 248 168 14 4 0.8 PI 620870 895 2727 141 13.3 6.6 19.9 351 221 36 7 2.0 PI 620872 832 2628 125 4.9 5.9 10.8 342 41 33 3 0.8 PI 620875 961 2786 136 5.0 6.3 11.3 347 261 27 3 0.8 PI 620876 652 2000 104 6.3 5.9 12.2 254 106 26 3 1.1 PI 620877 833 2420 124 9.4 5.6 15.0 301 354 30 5 1.7 PI 620878 631 2293 124 10.5 6.0 16.4 372 309 39 7 1.8 PI 632322 713 2555 125 7.1 6.4 13.5 311 108 42 2 1.1 PI 632323 313 2737 87 6.8 9.0 15.8 234 36 25 3 0.7 PI 632324 797 2537 126 6.7 6.7 13.4 363 91 27 3 1.0 PI 632325 416 1906 92 4.1 4.7 8.9 164 44 16 2 0.9 PI 632326 659 2088 115 5.1 6.1 11.2 289 82 20 3 0.8 PI 632329 738 3034 124 3.2 4.9 8.0 205 83 21 4 0.7 PI 632331 627 2534 113 5.8 6.6 12.3 163 48 13 5 0.9 PI 634361 752 2017 112 8.7 9.8 18.5 308 187 27 2 0.9 PI 634362 787 2684 121 6.3 7.5 13.8 372 55 29 2 0.8 PI 634363 847 2843 137 7.7 7.7 15.5 457 272 41 4 1.0 PI 634364 680 2679 118 5.7 7.0 12.7 296 268 25 6 0.8 Mean 665 2311 121 7.1 6.3 13.4 313 175 33 4 LSD 53 212 3 1.2 0.9 1.6 37 29 5 1
73
observed in accessions of both species. Total phenolic content in S. jamesii accessions
ranged from 50 (PI 592408) to 161 (PI 595775) mg CGA/100gfw. The range of TP
values in S. microdontum was from 51 (PI 558097) to 269 (PI 498127) mg
CGA/100gfw. Generally, values from S. microdontum were higher than those of S.
jamesii accessions (Tables 4.1 and 4.2).
Phenolic Composition
Identification of individual phenolics present in the sample extract was
accomplished by comparing retention times and spectra of peaks in tuber sample extracts
detected at 280 nm with retention times and peaks’ spectra of the standard compounds.
Quantification was done by comparing peak area of a known concentration of standard
with peak areas of the sample extract. Four compounds (chlorogenic acid, caffeic acid,
rutin hydrate, and myricetin) were observed in all accessions of S. jamesii and S.
microdontum (Tables 4.1 and 4.2). The most abundant phenolic compounds in all
accessions were chlorogenic and caffeic acids.
Relative amounts of these acids varied from one accession to another, with some
accessions containing more chlorogenic than caffeic acid, and others containing more
caffeic than chlorogenic acid. Estimates of chlorogenic acid in S. jamesii ranged from
123 (PI 592408) to 585 (PI 605357) μg/gfw, caffeic acid values ranged from 32 (PI
564047) to 359 (PI 605359) μg/gfw, rutin hydrate from 7 (592414) to 72 (PI 592422)
μg/gfw, and myricetin ranged from 1 (PI 564047) to 15 (PI 595775) μg/gfw. Similarly,
amounts of these phenolic compounds were widely variable in S. microdontum.
Chlorogenic acid values ranged from 21 (PI 473362) to 147 (PI 545902) μg/gfw and
74
Table 4.2. Mean values of antioxidant activity (DPPH and ABTS assays), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), dehydrotomatine (DTO), tomatine (TOM), total glycoalkaloids (TGA), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MY), and ratio of solanine to chaconine (S:C) in S. microdontum accessions (ACCESS).
ACCESS DPPH ABTS TP SOL CHA DTO TOM TGA CGA CA RH MY S:C PI 195185 543 2340 117 65.5 52.1 3.9 2.7 121.9 115 96 2.7 6.3 1.3 PI 208866 738 2536 133 43.3 38.0 81.2 74 164 3.8 11.3 1.1 PI 218222 968 3145 155 72.0 77.6 12.0 4.3 155.0 79 136 3.2 6.6 0.9 PI 218223 798 2944 134 73.9 71.3 6.2 3.3 148.3 76 108 2.3 5.2 1.0 PI 218226 999 3174 151 57.5 57.6 1.2 3.9 118.5 62 109 2.8 6.5 1.0 PI 265575 683 2792 120 66.4 58.9 15.3 4.8 138.7 52 124 3.1 4.8 1.1 PI 265881 980 3289 157 91.6 63.4 155.1 104 85 5.2 3.3 1.4 PI 275150 357 2078 86 52.3 41.9 9.0 5.4 105.1 69 77 6.2 2.7 1.2 PI 310979 884 3028 150 109.8 114.3 24.7 19.4 268.0 51 140 6.9 3.9 1.0 PI 320304 572 2181 106 38.2 44.3 1.4 3.6 84.1 69 35 1.1 3.0 0.9 PI 320305 255 1225 60 37.8 41.1 0.8 0.7 79.4 52 57 1.3 2.4 0.9 PI 320306 1004 3809 151 81.5 82.1 69.9 27.4 260.8 79 70 2.3 4.5 1.0 PI 320307 408 1988 91 99.8 85.1 15.6 3.2 203.8 60 76 1.7 2.6 1.2 PI 320309 735 2859 132 74.8 71.3 146.1 72 47 2.3 4.4 1.0 PI 320310 569 2290 114 52.2 44.2 5.6 98.2 38 100 2.1 4.4 1.2 PI 320311 982 3001 150 100.8 114.0 27.5 13.7 256.0 47 138 1.2 2.3 0.9 PI 320312 985 2850 160 90.1 91.6 6.6 3.2 191.6 53 51 2.3 3.2 1.0 PI 320313 422 1463 81 34.1 39.9 0.2 0.4 74.1 46 65 2.4 4.5 0.9 PI 320315 545 2345 110 81.6 75.8 2.9 4.5 160.8 58 99 1.1 4.0 1.1 PI 320316 384 1819 86 52.8 50.7 7.0 4.7 109.8 29 29 1.3 2.6 1.0 PI 320319 1201 3789 197 79.7 61.6 5.9 2.8 146.2 122 176 6.9 4.7 1.3 PI 320320 268 1872 70 80.2 54.4 2.9 136.5 54 57 2.8 3.7 1.5 PI 458353 738 2524 126 57.4 84.3 32.5 50.5 224.6 28 192 2.3 5.9 0.7 PI 458354 1047 3698 173 104.1 98.6 4.4 1.6 206.7 119 147 5.0 6.6 1.1 PI 458355 1499 4099 237 93.1 84.6 16.8 7.7 185.9 111 70 1.4 12.0 1.1 PI 458356 1419 3622 219 97.9 61.6 4.0 162.2 71 127 4.0 7.0 1.6 PI 458357 639 2206 119 83.8 55.1 20.6 145.8 97 49 2.1 4.2 1.5 PI 458358 257 2009 77 24.3 14.5 46.9 19.0 104.8 49 50 2.8 3.6 1.7 PI 473166 866 2367 139 49.9 67.4 11.9 5.4 134.5 75 41 3.0 6.9 0.7 PI 473167 939 2863 150 122.7 98.3 28.5 6.1 232.6 93 96 1.6 5.3 1.2 PI 473168 309 1741 73 11.5 8.8 83.2 9.8 113.4 79 65 3.6 3.9 1.3 PI 473169 682 2341 118 77.0 63.9 6.3 1.6 143.5 81 49 2.3 6.5 1.2 PI 473170 329 1874 81 33.6 32.0 65.6 48 30 2.8 3.2 1.0 PI 473171 498 2038 94 8.0 5.2 13.2 67 50 2.3 3.7 1.5
75
Table 4.2. Continued…
ACCESS DPPH ABTS TP SOL CHA DTO TOM TGA CGA CA RH MY S:C
PI 473172 505 2227 100 60.0 72.1 132.2 75 57 2.7 5.3 0.8 PI 473173 383 2037 98 52.4 54.0 106.4 58 95 2.3 3.9 1.0 PI 473174 643 2540 128 64.8 66.2 131.0 68 104 1.7 8.3 1.0 PI 473175 425 2152 91 84.1 92.8 9.3 4.3 186.0 78 61 2.2 5.4 0.9 PI 473176 759 2365 123 52.2 53.8 106.0 64 70 1.9 3.5 1.0 PI 473177 465 2436 104 92.3 86.3 7.5 181.0 84 69 2.9 3.9 1.1 PI 473178 987 2821 154 82.4 108.1 3.7 2.4 193.3 62 106 1.8 3.2 0.8 PI 473179 637 2420 122 131.5 144.2 13.4 280.1 131 76 3.8 10.5 0.9 PI 473180 993 2997 170 72.3 66.3 0.2 2.0 139.3 79 106 5.2 7.9 1.1 PI 473362 984 4843 181 151.0 148.0 67.7 321.6 21 65 3.2 7.8 1.0 PI 473363 505 1995 107 140.7 139.4 42.5 294.2 35 60 2.6 6.5 1.0 PI 473525 291 1567 68 108.3 105.6 214.0 47 57 1.6 3.2 1.0 PI 498121 978 3478 173 121.7 109.6 36.8 12.0 280.0 69 141 3.1 6.5 1.1 PI 498123 1171 4236 208 120.8 125.1 206.0 110.2 562.2 36 29 2.1 8.2 1.0 PI 498124 1398 3175 223 190.0 134.3 27.4 8.8 360.5 72 106 2.6 8.1 1.4 PI 498125 1005 4767 175 130.6 115.6 16.6 7.5 264.8 79 76 7.2 13.2 1.1 PI 498126 995 4738 176 115.1 89.3 1.3 1.0 205.6 100 131 19.8 12.8 1.3 PI 498127 1535 6288 269 176.2 191.0 1.6 367.8 91 127 4.3 5.8 0.9 PI 498128 995 3773 170 85.9 72.4 12.7 162.6 79 130 9.5 12.0 1.2 PI 500032 426 2171 84 8.2 7.9 33.9 13.7 63.8 69 56 6.8 7.5 1.0 PI 500033 602 2599 108 62.3 61.5 15.7 5.1 137.7 80 57 4.1 9.0 1.0 PI 500034 523 2008 101 39.0 34.7 73.7 87 97 2.8 7.6 1.1 PI 500035 773 2324 123 43.1 32.1 454.5 196.8 726.6 78 53 1.4 8.5 1.3 PI 500036 321 1783 77 38.5 36.9 1.2 5.1 81.6 53 35 1.4 3.4 1.0 PI 500037 545 2155 102 51.3 45.5 96.7 81 125 1.4 9.3 1.1 PI 500038 379 1845 83 4.8 5.5 136.6 62.4 209.4 77 70 2.4 7.3 0.9 PI 500039 358 1972 84 18.3 18.8 402.3 334.4 773.7 88 63 3.4 7.8 1.0 PI 500040 478 2207 94 15.8 17.7 19.9 14.9 68.2 53 39 1.5 5.9 0.9 PI 500041 770 3026 130 9.3 6.5 15.9 89 51 5.9 10.2 1.4 PI 500044 371 2430 83 27.8 35.7 40.5 18.2 96.5 55 37 2.1 9.1 0.8 PI 500064 413 1472 82 92.6 87.1 5.7 181.5 73 28 1.0 9.9 1.1 PI 545901 763 2919 130 64.6 62.0 8.0 4.5 133.4 121 109 7.2 6.7 1.0 PI 545902 995 4639 176 109.4 95.4 69.6 16.9 291.2 147 153 7.7 11.7 1.1 PI 545904 803 2013 133 109.9 129.5 10.9 243.0 35 32 1.4 6.6 0.8 PI 545905 977 2967 161 115.6 108.0 53.2 21.3 298.0 62 120 4.3 7.6 1.1 PI 558097 202 1084 51 67.6 57.9 16.0 7.9 133.5 59 45 1.1 5.2 1.2 PI 558098 263 1501 62 46.2 39.9 86.1 101 43 1.2 5.2 1.2 PI 558099 1000 3712 172 69.3 71.0 5.1 4.2 143.4 63 32 1.9 10.0 1.0
76
Table 4.2. Continued…
ACCESS DPPH ABTS TP SOL CHA DTO TOM TGA CGA CA RH MY S:C
PI 558100 770 2969 144 133.0 104.5 9.3 15.1 250.7 97 73 2.5 5.7 1.3 PI 558101 566 2322 109 33.8 26.1 59.9 82 78 1.2 5.5 1.3 PI 558218 432 2710 101 22.4 24.4 46.8 75 68 4.0 8.4 0.9 PI 565075 331 1696 69 8.3 6.8 304.8 228.1 548.1 57 82 3.3 3.4 1.2 PI 595505 1003 3477 171 89.1 63.9 43.7 18.4 215.0 26 89 2.2 4.2 1.4 PI 595506 722 2337 139 105.3 98.7 10.6 207.5 49 41 1.0 4.8 1.1 PI 595508 416 1843 93 90.9 72.2 13.7 9.4 186.1 88 76 2.0 14.1 1.3 PI 595509 319 1255 72 44.4 34.9 79.3 91 29 1.8 8.1 1.3 PI 595510 445 1684 98 115.4 126.0 99.5 41.8 382.6 55 30 1.9 12.6 0.9 PI 595511 941 2789 157 100.2 95.2 447.9 164.1 807.3 29 45 2.6 5.2 1.1 PI 597756 829 2290 143 135.9 131.3 33.5 10.6 311.4 42 35 1.5 6.4 1.0 PI 597757 652 2187 135 200.3 180.9 19.7 387.7 70 63 1.9 11.0 1.1 PI 631211 862 2641 137 45.9 38.4 84.2 59 55 2.2 3.8 1.2 Mean 699 2636 127 75.9 71.0 52.0 27.1 198.6 71 79 3.1 6.4 LSD 22 194 4 9.4 8.1 19.5 14 16 0.8 1.2
77
caffeic acid ranged from 28 (PI 500064) to 192 (PI 458353) μg/gfw. Rutin hydrate
ranged from 1 (PI 500064) to 20 (PI 498126) μg/gfw, and myricetin ranged from 2 (PI
320311) to 14 (PI 595508) μg/gfw. The analysis of variance for these compounds
showed significant differences (p-value <0.01) among accessions of both S. jamesii
(Table 4.1) and S. microdontum (Table 4.2).
Glycoalkaloid Composition
The main glycoalkaloids in potato tubers are α-solanine and α-chaconine, and they
comprise more than 95 % of all glycoalkaloids in the potato plant. Pure compounds of α-
solanine, α-chaconine, and tomatine were used as standards in HPLC analysis of
glycoalkaloids present in the tuber extracts. Figure 4.1 shows examples of typical
chromatographs obtained from HPLC glycoalkaloid analysis of S. jamesii accession PI
593408 and S. microdontum PI 498123, respectively. High amounts of α-solanine and α-
chaconine were found in all S. jamesii and S. microdontum accessions. Tomatine and
dehydrotomatine were identified and quantified in several S. microdontum accessions
but not in S. jamesii (Table 4.2). Total glycoalkaloid values for S. microdontum
accessions reported in Table 4.2 are sums of all glycoalkaloids quantified, including
tomatine and dehydrotomatine for the accessions that exhibited it.
Generally, the amount of glycoalkaloids in S. microdontum was higher than the
levels in S. jamesii accessions. The amount of α-solanine was significantly different (p-
value <0.01) in S. jamesii, with values ranging from 2.3 (PI 275172) to 20 (PI 585116)
mg/100g fresh weight. Alpha-solanine in S. microdontum ranged from 4.8 (PI 500038)
to 200.3 (PI 597757) mg/100gfw. Also, α-chaconine was appreciably variable in both
78
Fig. 4.1. Typical chromatographs from HPLC analysis of glycoalkaloids in S. jamesii and S. microdontum tuber extracts.
S. jamesii accession (PI 592408)
Retention Time (min)2 4 6 8 10
Abs
orba
nce
0.0
0.4
0.8
1.2
1.6
2.0
2.4
α-so
lani
ne
α-c h
acon
ine
S. microdontum accession (PI 498123)
Retention Time (min)
2 4 6 8 10
Abs
orba
nce
0.0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
α-so
lani
ne
α-ch
acon
ine
dehy
drot
omat
ine
tom
atin
e
79
species, ranging from 3.2 (PI 605362) to 16.8 (PI 585116) mg/100gfw and from 5.2 (PI
473171) to 191 (PI 498127) mg/100gfw in S. jamesii and S. microdontum, respectively.
Ratios between α-solanine and α-chaconine varied among accessions, with values
ranging from 0.5 to 2.1 in S. jamesii (Table 4.1) and from 0.7 to 1.7 in S. microdontum
(Table 4.2). Several accessions of S. microdontum exhibited high amounts of tomatine
and dehydrotomatine. Some accessions such as PIs 500039, 565075, 500035, 595511,
498123, 500038, 500032, and 473168 exhibited more dehydrotomatine and tomatine
than α-solanine and α-chaconine (Table 4.2).
The reported safety level of potato tuber total glycoalkaloids (TGA) for human
consumption is 20 mg/100gfw (Friedman et al., 2003). Therefore, any variety to be
released must contain less than 20 mg/100gfw of total glycoalkaloids.
To avoid high levels of glycoalkaloids in progenies, accessions to be used in any
breeding programs should contain less than 20 mg/10gfw TGA. Results from this study
show that only eight of the 92 S. jamesii accessions screened (PI 585116, PI 592413, PI
592397, PI 595778, PI 605371, PI 620870, PI 592410, and PI 603054) contain total
glycoalkaloid levels close to or greater than the safety limit (20 mg/100gfw). Only two
accessions (PI 473171 and PI 500041) of S. microdontum exhibited total glycoalkaloid
levels less than 20 mg/100gfw. Therefore, most S. jamesii accessions and the two
accessions of S. microdontum can potentially be used in breeding for traits of interest
without increasing amounts of glycoalkaloids in the progenies, since they contain low
levels.
80
Comparison of Wild Accessions with Common Cultivars
Antioxidant activity, total phenolics and glycoalkaloids in S. jamesii and S.
microdontum accessions and in three cultivars, Atlantic, Yukon Gold, and Red La Soda
were compared (Table 4.3). This was done to determine whether the two wild species
contain some accessions that are lower in total glycoalkaloids and higher in antioxidant
activity than popular commercial cultivars. Such accessions would be potential
candidates as parental material for breeding for high antioxidant activity and phenolic
compounds in new cultivars. Results in Table 4.3 show that the common cultivars are at
the lower end distribution of the traits of interest in wild species. This implies that most
accessions of both species exhibit higher levels of these traits than the cultivars.
Therefore, those accessions with higher values of the desirable traits can be used as
parents in breeding of new cultivars.
Relationships among Antioxidant Activity, Phenolics, and Glycoalkaloid Content
Accessions screened contained high levels of both desirable (antioxidants and
phenolics) and undesirable (glycoalkaloids) compounds. Hence, there may be a risk of
increasing glycoalkaloids in progenies if these wild species are used as parental material
for breeding. This is of much concern in instances where there are linear positive
relationships between antioxidant activity and total glycoalkaoids, and between
phenolics and glycoalkaloids, implying that increasing antioxidant and/or phenolics
might result in increased levels of glycoalkaloids.
81
Table 4.3. Range of antioxidant activity (AOA), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), and total gylcoalkaloids (TGA) in S. jamesii and S. microdontum, and means of three commercial cultivars, Atlantic, Red La Soda, and Yukon Gold.
Wild species Cultivars
S. jamesii S. microdontum Atlantic Red La Soda Yukon Gold AOA (DPPH) 173 - 961 202 - 1535 48 96 133 AOA (ABTS) 1383 - 3513 1084 - 6288 819 1405 1674 TP 50 - 161 51 - 269 27 39 52 SOL 2.2 – 20.0 4.8 – 200.3 3.1 2.3 1.3 CHA 3.2 – 16.8 5.2 – 191.0 7.8 6.4 4.6 TGA 5.9 – 36.8 13.2 – 807.3 10.9 8.7 5.9
82
Principal component analysis (PCA) was used to investigate relationships among
antioxidants, phenolics, and glycoalkaloids (Yan and Hunt, 2001). PCA is a technique
used to reduce multidimensional data sets to lower dimensions for analysis. It is mostly
used as a tool in exploratory data analysis and for making predictive models. Results
from PCA are shown as plots of primary principal component PC1 versus PC2 (Figs 4.2
and 4.3). On the plots, accessions are in blue and lower case, traits are in red and upper
case, and the two principal components explained at least 60% of the variations in traits
in both S. jamesii accessions (Fig. 4.2) and S. microdontum accessions (Fig. 4.3). These
plots show relationships among traits and the performance of each accession.
Relationships among traits are indicated by the angle between trait vectors. These angles
show the extent of the correlations among traits, acute angles indicate positive
correlation, obtuse angles indicate negative correlation, and a right angle means that
there is no correlation between the traits of interest. Strongly correlated traits are
normally grouped together in the biplot.
Figures 4.2 and 4.3 show that the traits are grouped in two; one group consists of
glycoalkaloids and the other contains phenolic compounds and antioxidant activity. The
observation that glycoalkaloids are grouped separately from antioxidant activity and
phenolics suggests that there is no linear relationship between glycoalkaloids and
antioxidant activity, or between glycoalkaloids and phenolic content.
83
Fig. 4.2. A biplot of principal component 1 (PC1) vs. principal component 2 (PC2) demonstrating interrelationships among traits; antioxidant activity (ABTS & DPPH assays), total phenolic content (TP), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MYC), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in S. jamesii accessions. Traits are in red and upper case while accessions are in blue and lower case.
84
Fig. 4.3. A biplot of principal component 1 (PC1) vs. principal component 2 (PC2) demonstrating interrelationships among traits; antioxidant activity (ABTS & DPPH assays), total phenolic content (TP), chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate (RH), myricetin (MYC), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in S. microdontum accessions. Traits are in red and upper case while accessions are in blue and lower case.
85
The significance of the relationships observed from PCA results were confirmed
by correlation analysis shown in Table 4.4. Results show that there were significant
correlations (p-value <0.01) between antioxidant activity (DPPH) and total phenolics,
with correlation coefficients (r) = 0.83 and 0.98 in S. jamesii and S. microdontum,
respectively. Also, correlations between antioxidant activity (ABTS) and total phenolics
were highly significant with values of r = 0.64 and 0.89 in S. jamesii and S.
microdontum, respectively. Similar results showing significant correlation between
antioxidant activity and total phenolic content were previously reported in potatoes
(Reddivari et al., 2007a; Reyes et al., 2005) and sweet potatoes (Huang et al., 2004).
Glycoalkaloids were not significantly correlated to antioxidant activity or total phenolic
content in S. jamesii. However, in S. microdontum, α-solanine and α-chaconine were
significantly correlated with antioxidant activity and total phenolic content, but there
was no significant correlation between total glycoalkaloids and antioxidant activity, or
between total glycoalkaloids and total phenolic content (r = 0.27). Individual phenolic
compounds analyzed with HPLC showed no significant correlation with glycoalkaloids
in either S. jamesii or S. microdontum accessions (Table 4.5).
Results from this study indicate that antioxidant activity and total phenolic
content are not correlated with total glycoalkaloids. Also, there was no significant
correlation between individual phenolic compounds and glycoalkaloids. Therefore, using
wild accessions in breeding for high antioxidant activity and total phenolics would not
necessarily increase glycoalkaloids in the developed potato progenies.
86
Table 4.4. Correlation analysis of antioxidant activity (AOA), total phenolic content (TP), α-solanine (SOL), α-chaconine (CHA), and total glycoalkaloids (TGA) in S. jamesii and S. microdontum accessions.
S. jamesii
AOA (ABTS) TP SOL CHA TGA
AOA (DPPH) 0.599** 0.832** 0.069 -0.047 0.026
AOA (ABTS) 0.642** -0.048 0.086 0.007
TP 0.105 0.124 0.131
SOL 0.462** 0.909**
CHA 0.789**
S. microdontum
AOA (ABTS) TP SOL CHA TGA
AOA (DPPH) 0.847** 0.982** 0.553** 0.517** 0.260
AOA (ABTS) 0.888** 0.491** 0.469** 0.212
TP 0.610** 0.561** 0.279
SOL 0.951** 0.396**
CHA 0.404** ** refers to significant values at p-value < 0.01
87Table 4.5. Correlation analysis of individual phenolic compounds [chlorogenic acid (CGA), caffeic acid (CA), rutin hydrate
(RH) and myricetin (MYC)], total phenolic content (TP), antioxidant activity (DPPH and ABTS), individual glycoalkaloids [α-solanine (SOL) and α-chaconine (CHA)], and total glycoalkaloids (TGA) in S. jamesii and S. microdontum accessions.
S. jamesii
CA RH MYC DPPH ABTS TP SOL CHA TGA
CGA 0.328** 0.526** 0.016 0.652** 0.391** 0.749** 0.115 -0.057 0.055
CA 0.177 0.239* 0.283** 0.275** 0.401** 0.069 -0.006 0.046
RH 0.285** 0.315** 0.213* 0.612** 0.043 0.274* 0.158
MYC 0.187 0.138 0.269** 0.184 0.032 0.139
S. microdontum
CA RH MYC DPPH ABTS TP SOL CHA TGA
CGA 0.288** 0.423** 0.389** 0.185 0.246* 0.188 0.033 -0.051 -0.102
CA 0.418** 0.108 0.462** 0.462** 0.456** 0.197 0.173 -0.006
RH 0.272* 0.246* 0.459** 0.271* 0.031 -0.003 0.016
MYC 0.238* 0.313** 0.279** 0.239* 0.207 0.154
* refers to significant values at p-value < 0.05 ** refers to significant values at p-value < 0.01
88
Discussion
Glycoalkaloids have several roles both in plant and in humans. They provide a
defense mechanism for potato plants against different pests and pathogens, such as
insects, viruses, bacteria, and fungi (Friedman, 2006; Lachman et al., 2001). However,
glycoalkaloids are known to be toxic to humans by acting as cholinesterase inhibitors
and cause sporadic out-breaks of poisoning (Mensinga et al., 2005; Smith et al., 1996),
and may also affect reproduction in animals (Wang et al., 2005). Despite their toxic
nature to humans and animals, recent studies have suggested that glycoalkaloids do have
health promoting effects in humans. Studies by Friedman et al (2005), Yang et al.
(2006), and Reddivari et al. (2007b) reported that glycoalkaloids exhibit anticancer
properties by inhibiting proliferation of various cancer cell lines through induction of
apoptosis.
The multi-effect of glycoalkaloids makes determining their necessity in plants
and humans difficult. Hence, their safety to humans is still being debated (Korpan et al.,
2004; Rietjens et al., 2005). Since the beginning of the 20th century, the acceptable level
of glycoalkaloids in the potato of commerce has been 20 mg/100g tuber weight
(Papathanasiou et al., 1998; Smith et al., 1996). This threshold value has largely
remained unchanged to date. Therefore, care is always taken in breeding new varieties to
ensure that they do not contain glycoalkaloid levels above 20 mg/100g fresh weight.
Most potato varieties released contain less than 10 mg/100gfw of both α-solanine and α-
chaconine (Lachman et al., 2001).
89
Wild Solanum species are commonly used in potato breeding as a source of
valuable germplasm. They are often used to introduce pest and disease resistance into
cultivated potato. Some of the wild species contain more health-benefiting
phytochemicals such as antioxidants and phenolics than currently grown popular
cultivars (Hale, 2004; Nzaramba et al., 2007). Therefore, not only could wild species
provide genes for pest and disease resistance, but also genes for high antioxidants and
phenolic compounds. However, some of these species have high levels of glycoalkaloids
such that, together with desirable characteristics, toxic glycoalkaloids might be
transferred to potato cultivars.
Much as there is need to develop new potato varieties resistant to pests and
diseases, and which can provide more health-benefiting phytochemicals like
antioxidants, prior screening of wild species for glycoalkaloid content is important to
ascertain their suitability as potential parental material in breeding programs. In this
study, accessions from two wild species (S. jamesii and S. microdontum) previously
observed to contain high levels of antioxidants were fine-screened for antioxidant
activity, total phenolics, and total glycoalkaloid content.
Results from this study showed that most accessions of both species exhibited
higher levels of both desirable (antioxidants and phenolics) and undesirable
(glycoalkaloids) traits than three important common cultivars (Table 4.3). Most (95%) of
the S. jamesii accessions exhibited glycoalkaloid levels less than 20 mg/100gfw
compared to only two accessions of S. microdontum that were below this value.
90
Therefore, accessions with low glycoalkaloid values and higher antioxidants could be
used as parents in breeding for high antioxidant and phenolic content.
If there is a relationship between antioxidant activity and glycoalkaloids or
between phenolic content and glycoalkaloids, the use of wild species in breeding
becomes challenging depending on the nature of the correlations among the traits.
According to Falconer and Mackay (1996), different traits can be correlated if the same
loci affect the traits (pleiotropy), different loci affect the traits but these loci are linked
together - linkage in coupling causing positive correlation and in repulsion negative
correlation, or the environment may affect the traits in the same way creating correlation.
To elucidate the relationships among these traits principal component analysis was
carried out on their values.
Principal component analysis results illustrating genetic phenotypic correlations
among traits in both species are shown in Figs 4.2 and 4.3. The figures show two
groupings of traits, one consisting of glycoalkaloids and the other phenolic compounds
and antioxidant activity. This suggests that no linear relationship exist between
glycoalkaloids antioxidant activity or between glycoalkaloids and phenolic content.
Therefore, there is no correlation between either glycoalkaloids and antioxidant activity
or glycoalkaloids and phenolics. Therefore using wild accessions in breeding for high
antioxidant activity and total phenolics would not necessarily increase glycoalkaloids in
the developed potato progenies, if the selected parental materials (accessions) are low in
glycoalkaloids.
91
CHAPTER V
ANTI-PROLIFERATIVE ACTIVITY AND CYTOTOXICITY OF SOLANUM
JAMESII TUBER EXTRACTS TO HUMAN COLON AND PROSTATE
CANCER CELLS IN VITRO
Introduction
According to the American Cancer Society (2008), colorectal cancer is the third
most common cancer in both men and women in the United States. It accounted for
about 10% of cancer mortality in the US, and caused about 57,000 deaths in 2004 (Jemal
et al., 2004). It is estimated that about 49,960 deaths from colon and rectal cancer will
occur in 2008, accounting for 9% of all cancer deaths.
Prostate cancer is the most frequently diagnosed cancer and is the leading cause
of cancer death in men. The American Cancer Society (2006) reported that prostate
cancer is the third leading cause of cancer death among men in the US. At the time, it
was estimated that 27,350 deaths would occur due to prostate cancer. Current estimates
(American Cancer Society, 2008) of about 28,660 deaths expected to occur in 2008, has
placed prostate cancer as the second leading cause of cancer death in men.
Cancer development or carcinogenesis is a complex, multi-sequence/stage
process that leads a normal cell into a precancerous state and finally to an early stage of
cancer (Klaunig and Kamendulis, 2004; Trueba et al., 2004). Carcinogenesis involves
initiation, promotion, and progression stages. According to Friedman et al. (2007) tumor
promotion is the only reversible event during cancer development. Therefore, Hawk et
92
al. (2005) and Friedman et al. (2007) suggested that early intervention should target
inhibition of cell proliferation and induction of apoptotic pathways in cancerous cells.
Cancer is characterized by uncontrolled growth of abnormal cells. The abnormal
or cancerous cells are caused by both external factors such as chemical toxins, tobacco,
radiation, and infectious organisms, and internal factors such as inherited mutations or
mutations from metabolism, hormones, and immune conditions. The causal factors may
act together or in sequence to initiate and promote carcinogenesis (American Cancer
Society, 2008).
Endogenous cellular processes such as metabolism as well as external factors like
chemical toxins and radiation generate free radicals known as reactive oxygen (ROS)
and reactive nitrogen (RNS) species (Inoue et al., 2003). The free radicals create an
oxidative or nitrosative stress within cells that may result in oxidative damage of DNA,
lipids, and proteins (Chu et al., 2002; Hussain et al., 2003; Kovacic and Jacintho, 2001).
High levels of cellular oxidative stress might result in permanent modification of
genetic material which normally represents the initial steps involved in mutagenesis,
carcinogenesis, and aging. Elevated levels of oxidative DNA lesions have been noted in
various tumors, strongly implicating such damage in the etiology of cancer (Valko et al.,
2007). Several studies have implicated oxidative stress in initiation of various diseases
like cardiovascular disease, cancer, neurological disorders, diabetes, and aging (Dalle-
Donne et al., 2006; Makazan et al., 2007; Tappia et al., 2006). Cellular oxidative stress is
assuaged by enzymatic and non-enzymatic antioxidants that maintain cellular
93
homeostasis. A chronic shift in the maintenance of cellular homeostasis can lead to
permanent changes associated with carcinogenesis (Droge, 2002; Hussain et al., 2003).
Antioxidants derived from fruits and vegetables complement enzymatic
antioxidants in reducing oxidative stress, and thereby help boost defensive mechanisms
against the risk of chronic diseases. Some of the beneficial phytochemicals from fruits
and vegetables are vitamins C and E, carotenoids, and phenolics, and are thought to be
involved in the pathophysiology of many chronic diseases (Stanner et al., 2004; Steffen
et al., 2003; Trichopoulou et al., 2003).
Onset of colon cancer is characterized by hyperproliferation of the epithelial cells
resulting in formation of adenomas (Hawk et al., 2005). Prostate cancer initially
develops as a high-grade intraepithelial neoplasia (HGPIN) in the peripheral and
transition zones of the prostate gland. The HGPIN eventually becomes a latent
carcinoma, which may subsequently progress to a large, higher grade, metastasizing
carcinoma (Abate-Shen and Shen, 2000; Bosland et al., 1991; Shukla and Gupta, 2005).
Promotion and progression stages are controlled by signal transduction molecules which
are triggered by hormones such as androgens (Giovannucci, 1999; Shukla and Gupta,
2005; Thompson, 1990). Androgen receptor (AR) signaling, cell proliferation and cell
death play a critical role in regulating the growth and differentiation of epithelial cells in
the normal prostate (Cunha et al., 2004).
Several studies have reported that phytochemicals present in fruits and
vegetables are important in prevention of chronic diseases, such as cancer,
cardiovascular diseases, and diabetes, (Chu et al., 2002; Hu, 2003; Liu, 2004; Riboli and
94
Norat, 2003). Benefits derived from phytochemicals in regard to disease prevention are
attributed to their antioxidant capabilities (Agudo et al., 2007; Stanner et al., 2004).
Several anti-inflammatory, anti-necrotic, and neuroprotective drugs have an antioxidant
and/or radical scavenging mechanism as part of their activity (Liu et al., 2004; Perry et
al., 1999; Repetto and Llesuy, 2002).
Juan et al. (2006) reported that olive fruit extract inhibited proliferation of HT-29
human colon cancer cells by inducing apoptosis. Crude extracts from sweet potato
(Ipomoea batatas) inhibited proliferation of the human leukemia NB4 cell line in vitro
(Huang et al., 2004). Romero et al. (2002) observed that polyphenols in red wine inhibit
proliferation and induce apoptosis in prostate LNCaP cancer cells. Several studies have
demonstrated that tea extracts exhibit anticancer properties on breast (MCF-7), liver
(HepG2), colon (HT-29) (Friedman et al., 2007), lung (Yang et al., 2005), stomach (Mu
et al., 2005), prostate (PC-3) (Bettuzzi et al., 2006; Friedman et al., 2007), and skin
(Camouse et al., 2005).
Potato tuber extracts have also been tested on several cancer types. Chu et al.
(2002) observed minor anti-proliferative activity of potato tuber extract to HepG2 human
liver cancer cells in vitro. Reddivari et al. (2007b) reported that whole potato tuber
extract and anthocyanin fractions inhibited proliferation and induced apoptosis in both
LNCaP and PC-3 prostate cancer cells. Glycoalkaloids from commercial potato cultivars
have also been reported to inhibit growth of human colon (HT-29), liver (HepG2),
cervical (HeLa), lymphoma (U937), stomach (AGS and KATO III) (Friedman et al.,
95
2005; Lee et al., 2004), and both LNCaP and PC-3 human prostate (Reddivari et al.,
2007b) cancer cell lines.
Most recent research is focusing on increasing the amount of beneficial
phytochemicals in food crops and also increasing the variety of plant products consumed
by introducing exotic and wild fruits and vegetables (Nzaramba et al., 2006), spices and
herbs (Ko et al., 2007; Siddhuraju et al., 2002). Some of the wild and exotic products
contain natural toxicants at levels that might cause health problems. Development of pest
resistant varieties or changes in methods of cultivation, storage, and preparation can
change the balance between beneficial and toxic compounds in staple foods, with
significant consequences to human health (Phillips et al., 1996). This has been especially
observed in the potato of commerce as regards glycoalkaoids (Abreu et al., 2007;
Griffiths and Dale, 2001; Laurila et al., 2001; Pęksa et al., 2002; Rytel et al., 2005).
Plant products contain a mixture of complex compounds with both toxic and
beneficial effects. Also, compounds that are toxic at high concentrations may be
beneficial at lower concentrations; therefore, it is appropriate to test whole plant
products. In vitro techniques such as mammalian cell culture were developed to screen
complex materials like plant extracts for inhibition of diseased cell proliferation and
toxicity to normal cells and organs (Hostanska et al., 2007; Phillips, 1996).
Several in vitro assays have been developed and vary in sensitivity to different
types of toxins. Potato extracts were reported to be more toxic in the LDH (Lactate
dehydrogenase) release assay than in the protein synthesis assay (Phillips, 1996). Despite
96
having little value per se, in vitro techniques of measuring cytotoxicity are useful in
initial screening of exotic plant materials.
Wild potato species have been reported to contain higher amounts of antioxidants
and polyphenolic compounds (Nzaramba et al., 2006) and glycoalkaloids (Laurila et al.,
2001) than the potato of commerce. Given that high levels of glycoalkaloids in tubers are
undesirable, wild potato species should be screened for cytotoxicity before their
introduction in breeding programs. Tuber extracts from wild potato species may also
contain other unknown compounds that may be toxic to humans.
Therefore, the objective of this study was to investigate anti-proliferative activity
and cytotoxicity potential of tuber extracts from Solanum jamesii accessions with
different antioxidant, phenolic, and glycoalkaloid contents on human prostate (LNCaP)
and colon (HT-29) cancer cell lines in vitro. Also, the study sought to determine
correlations among antioxidant activity, anti-proliferative activity and cytotoxicity, and
among glycoalkaloids, anti-proliferative activity and cytotoxicity.
Materials and Methods
Plant Material
Ninety-two accessions of S. jamesii were obtained from the US Potato Genebank,
Sturgeon Bay, WI. Fifteen accessions, representing the range of total glycoalkaloids in
the 92 accessions, were selected for evaluation of anti-proliferative effects and
cytotoxicity on human prostate (HT-29) and colon (LNCaP) cancer cells lines in vitro.
97
Chemicals
The DPPH (2,2-Diphenyl-1-picrylhydrazyl), Folin-Ciocalteu reagent, Trolox (6-
Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), ABTS (2,2’-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt), potassium persulfate, Na2HPO4
(dibasic), NaHPO4 (monobasic), NaCl, ammonium phosphate, and Na2CO3 were
purchased from Fisher Scientific (Pittsburgh, PA). Methanol, dimethly sulfoxide
(DMSO), and acetonitrile were obtained from VWR International (Suwanee, GA).
Alpha-chaconine and ammonium hydroxide were purchased from Sigma-Adrich (St.
Louis, MO). Alpha-solanine and tomatine were obtained from MP Biomedicals (Solon,
OH). The cell proliferation reagent WST-1 [4-(3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-
5-tetrazolio)-1,3-benzene disulfonate] and cytotoxicity detection kit were obtained from
Roche Applied Sciences (Indianapolis, IN).
Cell Lines
Human prostate cancer LNCaP (androgen-dependent) cells and HT-29 colon
cancer cells were obtained from the American Type Culture Collection (Manassas, VA).
The cells were maintained at 370 C in 5% CO2 jacketed incubator in RPMI 1640 (Sigma;
St. Louis, MO) supplemented with 2.38 g/L HEPES 2.0 g/L sodium bicarbonate, 0.11
g/L sodium pyruvate, 4.5 g/L glucose, 100 ml/L FBS, and 10 mL/L antibiotic
antimycotic solution (Sigma).
Sample Extraction
Five grams of diced tubers were extracted with 20 ml of HPLC-grade methanol.
The samples were hom*ogenized with an IKA Utra-turrax tissuemizer for 3 min. The
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tuber extract was centrifuged at 31,000 g for 20 min. with a Beckman model J2-21
refrigerated centrifuge. Five ml of supernatant was collected in glass vials, and the
methanol evaporated using a Speed Vac. The dried extract was re-dissolved in DMSO
and filtered through 0.45 µm syringe filters. Sample extracts were stored at -20o C until
analysis of antioxidant activity (AOA), phenolics and glycoalkaloids.
Antioxidant Activity Analysis
Total antioxidant activity was estimated using both the DPPH (2,2-Diphenyl-1-
picrylhydrazyl) assay (Brand-Williams et al., 1995) and ABTS [2,2’-azinobis(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt] assay (Miller and Rice-Evans,
1997).
DPPH assay:
A 150 μl aliquot was placed into scintillation vials, 2,850 μl of DPPH methanol
solution was added, and the mixture was placed on a shaker for 15 min. The mixture was
transferred to UV-cuvettes and its absorbance recorded using a Shimadzu BioSpec-1601
spectrophotometer at 515 nm. Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid), a synthetic antioxidant, was used as a standard to generate a standard
curve (Figure 1), and total AOA in tuber extracts was expressed as micrograms of
Trolox equivalents per gram of potato tuber fresh weight (μg TE/gfw).
ABTS assay:
The ABTS.+ radical was generated by reacting potassium persulfate with ABTS
salt [2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt]. A
working solution composed of a mixture of 5 ml of mother solution and 145 ml of
99
phosphate buffer was prepared. The mother solution contained equal volumes of 8 mM
of ABTS and 3 mM of potassium persulfate solutions, and the phosphate buffer solution
pH 7.4 was composed of 40.5 ml of 0.2 M Na2HPO4 dibasic, 9.5 ml of 0.2 M NaHPO4
monobasic and 150 mM NaCl. One-hundred µl of tuber extract were used for analysis.
Two-thousand-nine hundred µl of the working solution was added to tuber extracts and
reacted for 30 min on a shaker. Absorbance of the solution was measured at 734 nm with
a Shimadzu BioSpec-1601 spectrophotometer. Trolox was used as a standard, and total
AOA was expressed as micrograms of Trolox equivalents per gram of tuber fresh weight
(μg TE/gfw).
Total Phenolic Analysis
Total phenolic content was determined following the method of Singleton et al.
(1999). One-hundred-fifty µl of tuber extract was pipetted into scintillation vials, and 2.4
ml of nanopure water was added. One-hundred-fifty µl of 0.25 N Folin-Ciocalteu
reagent was added, and after 3 min of reaction 0.3 ml of 1 N Na2CO3 reagent was added
and allowed to react for two hours. The spectrophotometer (Shimadzu BioSpec-1601)
was zeroed with a blank (150 µl methanol, 2.4 ml H2O, 150 µl of 0.25 N Folin-
Ciocalteu, and 0.3 ml 1 N Na2CO2) before sample analysis. Absorbance of tuber extracts
was read at 725 nm. Chlorogenic acid was used as a standard, and total phenolic content
was expressed as milligrams of chlorogenic acid equivalents per 100 grams of potato
tuber fresh weight (mg CGAequ/100gfw).
100
Determination of Glycoalkaloids
Glycoalkaloids were analyzed with a high performance liquid chromatography
(HPLC) system following the method of Sotelo and Serrano (2000). An HPLC system
(Waters, Milford, MA) and Atlantis C-18 reverse-phase columns (4.6 x 150 mm, 5 µm)
were used for glycoalkaloid analysis. The mobile phase used for separating
glycoalkaloids was (35:65 v/v) acetonitrile:0.05 M monobasic ammonium phosphate
buffer ((NH4)H2PO4), adjusted to pH 6.5 with NH4OH. The solvent flow was isocratic at
a rate of 1 ml/min, with the UV absorbance detector set at 200 nm with 5% AUFS
sensitivity. The amount of extract sample injected was 20 µl. Different concentrations of
pure α-solanine, α-chaconine, and tomatine standards were used to identify
glycoalkaloids in the tuber extracts by comparing peak retention times and spectra
detected at 280 nm. Standard curves were prepared by regressing known concentrations
of glycoalkaloid standards to their corresponding peak areas, and these curves were used
to quantify amounts of glycoalkaloids in the tuber extracts.
Cell Proliferation
Cells were plated at a density of 1x104 /well in 96 well plates. They were allowed
to attach to the plate for 24 h. After 24 h, media was replaced with DMEM F-12 media
containing 2.5% charcoal-stripped serum and tuber extracts. Two concentrations (5 and
10 μl/ml) of tuber extract were tested. After every 24 h, cell proliferation was measured
using the WST assay. The assay required pre-incubation of cells in media with the
tetrazolium salt WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-
benzene disulfonate) (10 μl/well) for 4 h, followed by measuring absorbance at 450 nm
101
with the ELISA plate reader. The cell proliferation assay was repeated at 48 and 72 h of
incubation with potato extracts. Percent cell proliferation due to each tuber extract
treatment was calculated based on control (DMSO) absorbance (100%) after each
incubation period. All extracts were tested in triplicate.
Cytotoxicity Analysis
Cytotoxicity of tuber extracts to cancer cells was determined by measuring the
amount of lactate dehydrogenase (LDH) enzyme leaked from the cytosol of damaged
cells into the medium (Phillips, 1996) after exposure of the cells to the extracts for 24 h.
The LDH release represents necrosis as opposed to apoptosis. Lactate dehydrogenase in
the supernatant was measured using the Cytotoxicity Detection Kit (LDH) (Roche
Applied Science, Mannheim, Germany) following the manufacturer’s protocol. One-
hundred μl of the supernatant from the cells was placed in a 96, well plate and 100 μl of
LDH assay solution [mixture of catalyst lyophilisate (catalyst, diaphoraase/NAD+,
lyophilizate) and dye solution (iodotetrazolium chloride and sodium lactate)] were added
to each well and incubated for 30 min in the dark. Absorbance of the mixture was read
with an ELISA plate reader at 490 nm. Extract cytotoxicity was calculated as a percent
of the control (DMSO) absorbance (100%). All samples were analyzed in triplicate.
Statistical Analysis
Results for each treatment were expressed as means ± standard error. Analysis of
variance (ANOVA) was performed to determine the variability of anti-proliferative
activity and cytotoxicity of the accessions’ tuber extracts. Mean separation procedure
(LSD) was done to compare accessions for each measured variable. Correlations among
102
antioxidant activity, total phenolics, and glycoalkaloid content were computed following
Pearson’s correlation method. All statistical analyses were done using SAS Version 9.1
software (SAS, 2002).
Results
Effect of Tuber Extract on Cell Proliferation
Analysis of variance results for anti-proliferative activity exhibited by 15 tuber
extracts from S. jamesii accessions on human colon (HT-29) and prostate (LNCaP)
cancer cells in vitro are presented in Figures 5.1 and 5.2, respectively. The tuber extracts
decreased proliferation of HT-29 colon cancer and LNCaP prostate cancer cells in a dose
and time-dependent manner. Proliferation of both HT-29 colon cancer cells and LNCaP
prostate cancer cells decreased with increased time of incubation with extracts. Cell
proliferation was least after 72 h of incubation. Also, accessions exhibited varying
degrees of cell proliferation inhibition at each incubation period, and all accessions
showed more anti-proliferative activity with longer times of incubation.
A significant reduction in proliferation of HT-29 colon cancer cells by all
extracts was observed. All accession extracts at concentrations of 5 and 10 μg/ml
significantly reduced proliferation of HT-29 cells compared to the DMSO control (Fig.
5.1). Cell proliferation was less than 60 % of the control (DMSO) after 24 h of cell
incubation with tuber extracts of either 5 or 10 μg/ml concentration (Fig. 5.1A and D).
103
After 48 and 72 h of incubation with any of the extracts (5 and 10 μg/ml), HT-29 cell
proliferation was less than 40% of the DMSO control (Fig. 5.1B, E, C and F).
Prostate (LNCaP) cancer cells were not as responsive to tuber extract treatment
as were the HT-29 colon cancer cells. At the 5 μg/ml extract concentration, only three
accessions (PI 595784, PI 592411, and PI 620870) significantly reduced LNCaP cell
proliferation more than the control (DMSO) after 24 and 48 h of incubation (Fig 5.2A
and B). However, after 72 h of incubation, seven accessions in the following order- PI
620870 > PI 595784 > PI 592411 > PI 603054 > PI 605372 > PI 592398 > PI 564049
significantly inhibited LNCaP cell proliferation compared to the DMSO control (Fig.
5.2C). With a higher extract concentration (10 μg/ml) all accessions exhibited significant
inhibition of LNCaP cell proliferation compared to the DMSO control after 24, 48, and
72 h of incubation (Fig. 5.2D, E and F). After 72 h of incubation with 10 μg/ml extracts,
all accessions reduced cell proliferation by about 60% of the DMSO control (Fig. 5.2F).
104
A
Fig. 5.1. Cell proliferation of HT-29 colon cancer cells measured after 24, 48, and 72 h of incubation with 5 and 10 μg/ml of tuber extracts from 15 S. jamesii accessions. Results are presented as means ± SE of three experiments.
B
D
E
FC
105
Cytotoxicity of Tuber Extract
A
B
D
E
F C
Fig. 5.2. Cell proliferation of LNCaP prostate cancer cells evaluated after 24, 48, and 72 h of incubation with 5 and 10 μg/ml of tuber extracts from 15 S. jamesii accessions. Results are presented as means ± SE of three experiments. Significantly lower values than the DMSO control (LSD at p < 0.05) are indicated by an asterisk.
* * *
***
***
*
***
106
Cytotoxicity of tuber extracts to cancer cells in vitro due to necrosis was
determined by measuring the amount of lactate dehydrogenase (LDH) enzyme leaked
from the damaged cells into the medium (supernatant) after exposure of the cells to the
extracts for 24 h. Two concentrations (5 and 10 μg/ml) of the extracts were used and the
amount of LDH released was expressed as percent of the control (DMSO). Results are
reported as mean ± standard error of three replicated analyses. Accessions PI 595784
and PI 620870 at a concentration of 5 μg/ml caused slightly more but not significant
LDH leakage from HT-29 cells than the DMSO control. Only PI 592398, PI 592411, PI
603051, PI 603054, and PI 632325 caused significantly less LDH leakage than the
control (Fig . 5.3A). At a higher concentration of extract (10 μg/ml), nine accessions- PI
564049, PI 592411, PI 595784, PI 603051, PI 603054, PI 605364, PI 605368, PI
605372, and PI 612453 were significantly less toxic than the control (Fig. 5.3B). All
other accessions were not significantly different from the control in cytotoxicity to HT-
29 cells.
Lactate dehydrogenase released by LNCaP prostate cells after treatment with 5
μg/ml of tuber extracts of PI 564056, PI 595775, PI 595784, PI 605368, and PI 605372
accessions was significantly lower than that of the control (DMSO) (Fig. 5.4A). Five
μg/ml of tuber extracts from PI 592398, PI 612450, and PI 620870 caused more LDH
leakage than the control, but they were not significantly different. At twice the
concentration (10μg/ml), PI 612453 caused significantly less LDH leakage from LNCaP
prostate cells than the DMSO control. Two accessions (PI 595784 and PI 620870) at
10μg/ml concentration caused significantly higher LDH leakage than the control (Fig.
107
5.4B). It appears that at high concentrations, the two accessions, PI 595784 and PI
620870, might be toxic to LNCaP cells. The other accessions tested were not
significantly different from the DMSO control.
In general, all accessions tested exhibited as much as or significantly lower LDH
leakage than the control in both HT-29 (Fig. 5.3) and LNCaP cells (Fig. 5.4). Only two
accessions (PI 595784 and PI 620870) at a high concentration (10μg/ml) showed
significantly higher LDH leakage than the control in LNCaP cells.
The accessions of S. jamesii tested for cytotoxicity were not necessarily toxic to
HT-29 colon cancer and LNCaP cancer cell lines, since the amount of LDH released
after cell incubation with tuber extracts was not significantly different from cells
incubated without extracts (only DMSO). Therefore, the observed reduction in
proliferation of HT-29 and LNCaP cancer cells after incubation with tuber extracts of S.
jamesii accessions was not due to necrosis but rather to enhanced apoptosis. A previous
study (Reddivari et al., 2007b) reported that tuber extracts from speciality potato
cultivars contain phytochemicals that can inhibit LNCaP and PC-3 cell growth and
induce apoptosis.
108
Fig. 5.3. Cytotoxicity of tuber extracts from 15 S. jamesii accessions (5 and 10 μg/ml) to HT-29 human colon cancer cells expressed as percentage of lactate dehydrogenase enzyme (LDH) released from the cells after 24 hours of incubation. Results are presented as means ± SE of three experiments. Significantly lower values than the DMSO control (LSD at p < 0.05) are indicated by an asterisk.
A
B
* * *
*
**
** * * * ** *
109
Fig. 5.4. Cytotoxicity of tuber extracts from 15 S. jamesii accessions (5 and 10 μg/ml) to LNCaP human prostate cancer cells expressed as percentage of lactate dehydrogenase enzyme (LDH) released from the cells after 24 hours of incubation. Results are presented as means ± SE of three experiments. Significantly lower values than the DMSO are indicated by an asterisk, and values significantly higher (LSD at p < 0.05) than the DMSO control are indicated by a symbol ε.
A
B
* * * * *
*
ε ε
110
Correlations among Antioxidants, Phenolics, Glycoalkaloids and Anti-proliferative
Activity
Several studies have associated consumption of foods rich in antioxidants and
polyphenols with decrease in prevalence of degenerative diseases such as cancer (Lui et
al., 2002). Other studies have investigated polyphenols extracted from plants for their
potential effect in curing colon (Juan et al., 2008; Kim et al., 2006; McCann et al., 2007)
and prostate cancers (Bettuzzi et al., 2006; Reddivari et al., 2007b; Romero et al., 2002).
Glycoalkaloids have also been reported to play a role in reducing cancer cell
proliferation (Friedman et al., 2005; Lee et al., 2004; Reddivari et al., 2007b) by up-
regulating apoptosis in these cells. Therefore, relationships among antioxidant activity,
phenolic and glycoalkaloid content in tuber extracts, and their anti-proliferative activity
in HT-29 colon and LNCaP prostate cancer cell lines were also investigated.
Results from correlation analysis among antioxidant activity, total phenolics,
glycoalkaloids, and anti-proliferation activity on HT-29 colon cancer cells show
inconsistent relationships (Table 5.1). At 5 μg/ml of tuber extract concentration, the
correlation between inhibition of HT-29 cell proliferation after 24 h of incubation and
antioxidant activity measured by the DPPH and ABTS assays was positive with
correlation coefficients r = 0.749 and r = 0.389, respectively. Also, correlation between
inhibition of HT-29 cell proliferation and total phenolics after 24 h of incubation with 5
μg/ml of tuber extract was significant (r = 0.81). These results suggest that cell
proliferation was inhibited by increasing the amount of antioxidants and phenolics after
24 h of incubation. However, after 48 and 72 h of incubation, there were no significant
111Table 5.1. Correlation analysis of antioxidant activity (DPPH and ABTS), total phenolics (TP), α-solanine (SOL), α-chaconine
(CHA), and total glycoalkaloids (TGA) in Solanum jamesii accessions, and inhibition of HT-29 colon cancer cell proliferation.
Inhibition of cell proliferation
5 μg/ml 10 μg/ml
ABTS TP SOL CHA TGA 24 h 48 h 72 h 24 h 48 h 72 h
DPPH 0.647** 0.887** 0.144 -0.047 0.109 0.749** -0.072 0.090 0.407** 0.579** 0.089
ABTS 0.657** -0.025 0.142 0.013 0.398* -0.104 0.044 0.184 0.496** -0.269
TP 0.338* 0.225 0.337* 0.810** 0.092 0.128 0.378* 0.675** -0.081
SOL 0.600** 0.981** 0.255 0.346* 0.344* 0.278 0.009 -0.184
CHA 0.744** -0.058 0.253 0.423 0.142 0.009 -0.274
TGA 0.198 0.350* 0.389* 0.267 0.009 -0.220
* refers to significant values at p-value < 0.05 ** refers to significant values at p-value < 0.01
112
correlations between inhibition of cell proliferation and antioxidant activity or total
phenolic content (Table 5.1).
Individual glycoalkaloids and total glycoalkaloids showed no significant
correlation with inhibition of HT-29 cell proliferation after 24 h. But after 48 and 72 h α-
solanine showed a significant correlation with cell proliferation inhibition, r = 0.346 and
r = 0.344, respectively. Similarly, total glycoalkaloid was significantly correlated with
inhibition of cell proliferation after 48 h (r = 0.35) and 72 h (r = 0.389) of incubation.
Actual proliferation values (Fig 5.1) show that proliferation of HT-29 cells was
significantly inhibited by treatment with tuber extracts even after 24 h of incubation.
Therefore, correlation results, together with cell proliferation data, suggest that not a
single but a combination of compounds (acting together) are responsible for inhibiting
cell proliferation.
With a higher concentration of tuber extract (10 μg/ml), antioxidant activity
(DPPH) and inhibition of cell proliferation were significantly correlated after 24 h (r =
0.407) and 48 h (r = 0.579) of incubation. The ABTS assay was correlated with cell
proliferation inhibition only after 48 h (r = 0.496) of incubation, and total phenolic
content was significantly correlated with inhibition of cell proliferation after 24 h (r =
0.378) and 48 h (r = 0.675) of incubation. There were no significant correlations
between either antioxidant activity (DPPH and ABTS assays) and inhibition of HT-29
cell proliferation or total phenolic content and inhibition of HT-29 cell proliferation after
72 h of incubation. Also α-solinine, α-chaconine, and total glycoalkaloids showed no
113
significant correlation with inhibition of HT-29 cell proliferation after 24, 48, and 72 h
of incubation.
Generally, correlation analysis indicated that LNCaP prostate cancer cell
proliferation was significantly reduced with increasing amounts of antioxidants, total
phenolics, and glycoalkaloids in tuber extracts. After 24 h of incubation with 5 μg/ml of
tuber extract, total phenolic content and α-solanine exhibited a significant negative
correlation with inhibition of LNCaP cell proliferation with correlation coefficients (r) of
-0.301 and -0.293 (p-value <0.05), respectively (Table 5.2). At 72 h of incubation, α-
solanine (r = -0.411) and total glycoalkaloid (r = -0.386) were negatively and
significantly correlated with inhibition of LNCaP prostate cancer cell proliferation.
Antioxidant activity measured by ABTS was negatively and significantly
correlated with inhibition of LNCaP cell proliferation after 24 h (r = -0.358) and 72 h (r
= -0.343) of incubation with 10 μg/ml of tuber extract. Also, after 72 h of incubation
with 10 μg/ml of extract, α-solanine showed a significantly negative correlation with
inhibition of LNCaP prostate cancer cell proliferation, while total glycoalkaloid were
positively correlated with inhibition of LNCaP prostate cancer cell proliferation (Table
5.2).
114Table 5.2. Correlation analysis of antioxidant activity (DPPH and ABTS), total phenolics (TP), α-solanine (SOL), α-
chaconine (CHA), and total glycoalkaloids (TGA) in Solanum jamesii accessions, and inhibition of LNCaP prostate cancer cell proliferation.
Inhibition of cell proliferation
5 μg/ml 10 μg/ml
ABTS TP SOL CHA TGA 24 h 48 h 72 h 24 h 48 h 72 h
DPPH 0.647** 0.887** 0.144 -0.047 0.109 -0.113 -0.019 -0.033 -0.259 -0.121 -0.083
ABTS 0.657** -0.025 0.142 0.013 -0.236 -0.151 -0.238 -0.358* -0.031 -0.343*
TP 0.338* 0.225 0.337* -0.301* -0.173 -0.276 -0.226 -0.216 0.018
SOL 0.600** 0.981** -0.293* -0.205 -0.411** 0.245 0.147 -0.346*
CHA 0.744** -0.093 -0.011 -0.173 -0.189 -0.232 0.178
TGA -0.268 -0.174 -0.386* 0.159 0.067 0.332*
* refers to significant values at p-value < 0.05 ** refers to significant values at p-value < 0.01
115
Correlations among Antioxidants, Phenolics, Glycoalkaloids and Cytotoxicity
Induction of cell death in cancerous cells may be due to induction of apoptosis
(programmed cell death) or necrosis. Necrosis can be determined by measuring the
amount of lactate dehydrogenase (LDH) enzyme released from cells into the culture
medium. Results of cytotoxicity analysis using the LDH assay exhibited significant
positive correlation between antioxidant activity measured with the DPPH assay and the
amount of LDH released (r = 0.381), and between the ABTS assay and amount of LDH
released (r = 0.471) from HT-29 colon cancer cells (Table 5.3) after incubation with 5
μg/ml of tuber extract. Total phenolics and glycoalkaloids showed no significant
correlation with LDH released from HT-29 colon cancer cells at the 5 μg/ml
concentration. At 10 μg/ml of extract, there was no significant correlation between LDH
released from HT-29 cells and antioxidant activity, total phenolics or glycoalkaloids.
Lactate dehydrogenase released from LNCaP prostate cancer cells was positively
correlated with α-solanine (r = 0.369) and total glycoalkaloids (r = 0.356) after
incubation with 5 μg/ml tuber extract (Table 5.3). However, no significant correlation
was observed between antioxidant activity and LDH, between total phenolics and LDH,
and between glycoalkaloids and LDH in LNCaP prostate cancer cells after incubation
with 10 μg/ml tuber extract (Table 5.3).
116Table 5.3. Correlation analysis of antioxidant activity (DPPH and ABTS), total phenolics (TP), α-solanine (SOL), α-
chaconine (CHA), and total glycoalkaloids (TGA) in Solanum jamesii accessions, and cytotoxicity to HT-29 colon cancer and LNCaP prostate cancer cell lines.
%LDH
HT-29 LNCaP
ABTS TP SOL CHA TGA 5 μg/ml 10 μg/ml 5 μg/ml 10 μg/ml
DPPH 0.647** 0.887** 0.144 -0.047 0.109 0.381* 0.084 0.054 0.012
ABTS 0.657** -0.025 0.142 0.013 0.471** 0.012 -0.002 0.269
TP 0.338* 0.225 0.337* 0.247 0.052 0.106 0.074
SOL 0.600** 0.981** -0.023 0.246 0.369* 0.264
CHA 0.744** -0.136 0.263 0.195 0.003
TGA -0.052 0.269 0.356* 0.222
117
Discussion
Wild potato accessions contain higher amounts of beneficial phytochemicals,
such as antioxidants and polyphenols, than the potato of commerce, and are therefore
potential sources of parental material in breeding for these phytochemicals. However,
wild species are also known to contain higher amounts of toxic compounds such as
glycoalkaloids that are considered a health hazard for human consumption.
Solanum jamesii accessions significantly inhibited HT-29 and LNCaP cell
proliferation. However, the important finding of this study is that the cytotoxicity of
many of these accessions is not necessarily due to necrosis. Therefore, these accessions
might not pose a health problem if used as parental material in improving the nutritional
value of potato cultivars.
Tuber extracts from 15 accessions of S. jamesii, representing the range of total
glycoalkaloids in S. jamesii, inhibited proliferation of colon (HT-29) and prostate
(LNCaP) cancer cell lines. The amount of LDH released from cells incubated with tuber
extracts was not significantly different from amounts of LDH released from cells
incubated without tuber extracts (DMSO as a control). This implies that the cytotoxic
effects of the tuber extracts were not due to necrosis, but probably to induction of
apoptosis.
Colon and prostate cancer cells responded differently to tuber extract treatments.
Colon (HT-29) cancer cell lines seemed more responsive to tuber extract treatment than
prostate (LNCaP) cancer cell lines. Proliferation of colon (HT-29) cancer cells was
significantly reduced by all extracts at 5μg/ml (Fig. 5.1), yet a higher concentration
118
(10μg/ml) of extract was required for all accessions to inhibit proliferation of LNCaP
prostate cancer cells (Fig. 5.2). These results agree with Kim et al. (2006) who reported
that polyphenol concentrations required for anti-cancer effects depend on the type of
cancer cell line. Friedman et al. (2005) and Lee et al. (2004) came to a similar
conclusion while investigating anti-carcinogenic effects of glycoalkaloids against
cervical, liver, lymphoma, and stomach cancer cells.
Correlation analysis results from this study suggest that compounds, other than
those evaluated in this investigation, may also be contributing to anti-proliferative
effects of potato tuber extracts. Antioxidants, phenolics, and glycoalkaloids, together
with other compounds present in tuber extracts may be acting competitively, additively,
and/or antagonistically to inhibit proliferation of colon and prostate cancer cells. This
may be the reason why correlations between anti-proliferation and levels of antioxidants,
phenolics, and glycoalkaloids in the tuber extracts were not consistent. Chu et al. (2002)
reported that correlations between phytochemical contents of five vegetables and anti-
proliferative activity of HepG2 human liver cells were not significant. They observed that
inhibition of human liver cancer cells by vegetables does not solely depend on their
phenolic content, but that other chemicals in the vegetables were also responsible for
anti-proliferative activities. The above observations support the idea proposed by Liu
(2004), Cirico and Omaye (2006), and Milde et al. (2007) that combinations of different
phytochemicals synergistically confer more health benefits than individual chemicals.
It has been reported that antioxidants or phytochemicals with antioxidant
capacity can become pro-oxidants depending on their concentration and the environment
119
in which they act (Mortensen et al., 2001). Therefore, a network of phytochemicals is
necessary in promoting health. This may further explain the inconsistencies observed in
correlation analysis between single chemicals in tuber extracts and inhibition of cell
proliferation. These results suggest that not only concentration of phytochemicals is
important in inhibiting cell proliferation, but also moderate combination of diverse
phytochemicals. In fact, very high levels of phenolic compounds and certainly
glycoalkaloids are toxic for human consumption.
Other studies have explained why no single antioxidant can replace the
combination of natural phytochemicals in fruits and vegetables in achieving greater
health benefits. This was based on observations that combinations of extracts from
different fruits resulted in greater antioxidant activity that was additive and synergistic
(Eberhardt et al., 2000; Liu, 2003; 2004; Sun et al., 2002) than individual extracts.
Friedman et al. (2005) demonstrated that certain combinations of the two major potato
glycoalkaloids (α-solanine and α-chaconine) act synergistically in inhibiting cell
proliferations of several human cancer cell lines. Likewise, Rayburn et al. (1995) and
Smith et al. (2001) reported that combinations of α-solanine and α-chaconine acted
synergistically to cause cytotoxicity and disruption of cell membranes.
Phytochemicals in foods differ in molecular size, polarity, and solubility, and
these differences may affect the bioavailability and distribution of each phytochemical in
different macromolecules, organelles, cells, organs, and tissues (Liu, 2003). This implies
that biological effects of phytochemical mixtures are greater than the expected additive
effects of individual compounds. Currently it is not clear how single nutrients and
120
combinations of nutrients affect one’s risk of specific cancers. Many questions remain
unanswered until more is known about the specific components of diet that influence
cancer risk. Presently the best advice is to consume wholesome foods in a balanced diet
(American Cancer Society, 2008).
The complexities of cancer research make it difficult to translate in vitro cell
assay results to in vivo applications. But since previous studies have shown that several
plant extracts inhibit cancer cell proliferation, more in vivo, i.e. animal experiments, are
necessary to confirm the in vitro observations and design more and probably better
chemotherapeutic compounds.
Dietary constituents in the relevant foods must be sufficient to attain the cellular
concentrations that display sufficient bioactivity and chemopreventive capacity (Juan et
al., 2008). Presence of high amounts of chemopreventive compounds in plant foods such
as the potato of commerce would increase bioavailability of the bioactive
phytochemicals. Therefore, crop improvement or breeding to increase health-promoting
phytochemicals in plant foods is important.
121
CHAPTER VI
CONCLUSIONS
Results from this investigation show that antioxidant activity measured by the
ABTS assay, total phenolic content, and specific gravity are governed more by genetic
factors than environmental conditions. More than 50% of the variability in antioxidant
activity (ABTS assay), total phenolic content, and specific gravity was attributed to the
cultivar main effect. As for antioxidant activity (DPPH assay), location, season, and
cultivar main effects are equally influential in variability of antioxidant activity.
Interactions of cultivar, location, and season effects were also significant, and these may
obscure progress in breeding for high antioxidants.
There was no significant relationship between antioxidant activity and specific
gravity, or between total phenolic content and specific gravity. Also, there was no
significant correlation between any of the individual phenolic compounds and specific
gravity. Therefore, breeding for high antioxidants and phenolic compounds in potato
tubers would not compromise tuber quality in terms of specific gravity.
Accessions of S. jamesii and S. microdontum species exhibited higher levels of
antioxidants, phenolics, and glycoalkaloids than the commonly grown cultivars.
Antioxidant activity in S. jamesii accessions ranged from 173 (PI 592408) to 961 μg
TE/gfw (PI 620875), and 1,383 (PI 592408) to 3,513 (PI 275172) μg TE/gfw, for the
DPPH and ABTS assays, respectively. Values in S. microdontum ranged from 202 (PI
558097) to 1,535 (PI 498127) μg TE/gfw, and from 1,084 (PI 558097) to 6,288 (PI
498127) μg TE/gfw, for the DPPH and ABTS assays, respectively. Total phenolic
122
content in S. jamesii accessions ranged from 49.7 (PI 592408) to 161 (PI 595775) mg
CGA/100gfw, and in S. microdontum from 51 (PI 558097) to 269 (PI 498127) mg
CGA/100gfw.
High amounts of α-solanine and α-chaconine were found in S. jamesii and S.
microdontum accessions. Tomatine and dehydrotomatine were identified and quantified
only in some S. microdontum accessions. Generally, amounts of glycoalkaloids in S.
microdontum were higher than in S. jamesii accessions. Most (95%) of the S. jamesii
accessions exhibited glycoalkaloid levels less than 20 mg/100g, while only two
accessions of S. microdontum were below this value. The following S. jamesii
accessions; PI 585116, PI 592413, PI 592397, PI 595778, PI 605371, PI 620870, PI
592410, and PI 603054 exhibited total glycoalkaloid levels close to or greater than the
safety limit (20 mg/100g). Only two accessions- PI 473171 and PI 500041 of S.
microdontum exhibited total glycoalkaloid levels less than 20 mg/100g. Therefore, most
S. jamesii accessions and the two accessions of S. microdontum can potentially be used
in breeding without increasing amounts of glycoalkaloids in the progenies, since they
contain low levels of glycoalkaloids.
Principal component analysis results showed that there is no significant linear
relationship between glycoalkaloids and antioxidant activity, or between glycoalkaloids
and phenolic content. Therefore, using wild accessions in breeding for high antioxidant
activity and total phenolics would not necessarily increase glycoalkaloids in the
developed potato progenies if selected parental materials (accessions) are low in
glycoalkaloids.
123
Tuber extracts from S. jamesii accessions inhibited proliferation of colon (HT-
29) and prostate (LNCaP) cancer cell lines. The anti-proliferation activity exhibited by
the tuber extracts is not due to necrosis, because the amount of LDH released from cells
incubated with tuber extracts was not significantly different from that released by cells
incubated without tuber extracts (DMSO as a control). Also, the results indicate that
accessions of S. jamesii are not necessarily cytotoxic to HT-29 colon and LNCaP
prostate cancer cell lines.
Colon (HT-29) cancer cell lines were more responsive to tuber extract treatment
than prostate (LNCaP) cancer cell lines. Proliferation of colon (HT-29) cancer cells was
significantly inhibited by all extracts at 5 μg/ml but a concentration of 10 μg/ml was
required for all accessions to inhibit proliferation of LNCaP prostate cancer cells.
Correlations between anti-proliferation and levels of antioxidants, phenolics, and
glycoalkaloids in the extracts were not significant. This suggests that compounds, other
than the ones measured, may also be contributing to anti-proliferative effects of potato
tuber extracts. Antioxidants, phenolics, and glycoalkaloids, together with other
compounds present in tuber extracts, may be acting competitively, additively, and/or
antagonistically to inhibit proliferation of colon and prostate cancer cells.
In summary, crop improvement or breeding to increase health-promoting
phytochemicals in plant foods is necessary in order to boost the bioavailability of the
active phytochemicals. However, some of the health-promoting compounds such as
glycoalkaloids are required in very low amounts as they are toxic when consumed in
124
larger quantities. Therefore breeding strategies should ensure that such phytochemicals
are not increased but maintained at the necessary low levels.
125
LITERATURE CITED
Abate-Shen, C. and M.M. Shen. 2000. Molecular genetics of prostate cancer. Genes Dev. 14: 2410-2434.
Abreu, P., A. Relva, S. Matthew, Z. Gomes, and Z. Morais. 2007. High-performance liquid chromatographic determination of glycoalkaloids in potatoes from conventional, integrated, and organic crop systems. Food Control. 18: 40-44.
Adom, K.K. and R.H. Liu. 2002. Antioxidant activity of grains. J. Agric. Food Chem. 50: 6182-6187.
Agudo, A., L. Cabrera, P. Amiano, E. Ardanaz, A. Barricarte, T. Berenguer, M.D. Chirlaque, M. Dorronsoro, P. Jakszyn, N. Larranaga, C. Martinez, C. Navarro, J.R. Quiros, M.J. Sanchez, M.J. Tormo, and C.A. Gonzalez. 2007. Fruit and vegetable intakes, dietary antioxidant nutrients, and total mortality in Spanish adults: Findings from the Spanish cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Spain). Am. J. Clin. Nutr. 85: 1634-1642.
Ahn, C.H., W.C. Choi, and J.Y. Kong. 1997. Chemosensitizing activity of caffeic acid in multidrug-resistant MCF-7/Dox human breast carcinoma cells. Anticancer Res. 17: 1913-1917.
Al-Saikhan, M.S., L.R. Howard, and J.C. Miller Jr. 1995. Antioxidant activity and total phenolics in different genotypes of potato (Solanum tuberosum, L.). J. Food Sci. 60: 341-343.
American Cancer Society. 2006. Overview: Prostate cancer. American Cancer Society, Atlanta, GA.
American Cancer Society. 2008. Cancer Facts & Figures 2008. American Cancer Society, Atlanta, GA.
Ames, B.N. and L.S. Gold. 1990. Chemical carcinogenesis: too many rodent carcinogens. PNAS. 87: 7772-7776.
Ames, M. and D.M. Spooner. 2008. DNA from herbarium specimens settles a controversy about origins of the European potato. Am. J. Bot. 95: 252-257.
Andre, C.M., M. Ghislain, P. Bertin, M. Oufir, M.D. Herrera, L. Hoffmann, J.F. Hausman, Y. Larondelle, and D. Evers. 2007. Andean potato cultivars (Solanum tuberosum L.) as a source of antioxidant and mineral micronutrients. J. Agric. Food Chem. 55: 366-378.
126
Arai, Y., S. Watanabe, M. Kimira, K. Shimoi, R. Mochizuki, and N. Kinae. 2000. Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration. J. Nutr. 130: 2243-2250.
Arnao, M.B. 2000. Some methodological problems in the determination of antioxidant activity using chromogen radicals: A practical case. Trends Food Sci. Technol. 11: 419-421.
Arnao, M.B., A. Cano, and M. Acosta. 1999. Methods to measure the antioxidant activity in plant material. A comparative discussion. Free Radic. Res. 31: S89-S96.
Arts, I.C. and P.C. Hollman. 2005. Polyphenols and disease risk in epidemiologic studies. Am. J. Clin. Nutr. 81: 317S-325S.
Atlin, G.N., K.B. McRae, and X. Lu. 2000. Genotype x region interaction for two-row barley yield in Canada. Crop Sci. 40: 1-6.
Aviram, M., M. Kaplan, M. Rosenblat, and B. Fuhrman. 2005. Dietary antioxidants and paraoxonases against LDL oxidation and atherosclerosis development. Handb. Exp. Pharmacol. 170: 263-300.
Awika, J.M., L.W. Rooney, X. Wu, R.L. Prior, and L. Cisneros-Zevallos. 2003. Screening methods to measure antioxidant activity of sorghum (Sorghum bicolor) and sorghum products. J. Agric. Food Chem. 51: 6657-6662.
Babich, H., H.L. Zuckerbraun, and S.M. Weinerman. 2007. In vitro cytotoxicity of (-)-catechin gallate, a minor polyphenol in green tea. Toxicol. Lett. 171: 171-180.
Barlow, S.M. 1990. Toxicological aspects of antioxidants used as food additives, p. 253-307. In: B.J.F. Hudson (ed.). Food antioxidants. Elsevier, London.
Becker, H.C. and J. Leon. 1988. Stability analysis in plant-breeding. Plant Breed. 101: 1-23.
Bejarano, L., E. Mignolet, A. Devaux, N. Espinola, E. Carrasco, and Y. Larondelle. 2000. Glycoalkaloids in potato tubers: The effect of variety and drought stress on the alpha-solanine and alpha-chaconine contents of potatoes. J. Sci. Food Agric. 80: 2096-2100.
Bergers, W.W.A. 1980. On the colour and reactions of potato glycoalkaloids in strong acids in the presence of paraformaldehyde. Food Chem. 6: 123-131.
127
Bettuzzi, S., M. Brausi, F. Rizzi, G. Castagnetti, G. Peracchia, and A. Corti. 2006. Chemoprevention of human prostate cancer by oral administration of green tea catechins in volunteers with high-grade prostate intraepithelial neoplasia: A preliminary report from a one-year proof-of-principle study. Cancer Res. 66: 1234-1240.
Beukema, H.P. and D.E. van der Zaag. 1990. Introduction to potato production. Pudoc, Wageningen, The Netherlands.
Bosland, M.C., H.C. Dreef-Van Der Meulen, S. Sukumar, P. Ofner, I. Leav, X. Han, and J.G. Liehr. 1991. Multistage prostate carcinogenesis: The role of hormones. Princess Takamatsu Symp. 22: 109-123.
Boyle, S.P., V.L. Dobson, S.J. Duthie, J.A. Kyle, and A.R. Collins. 2000. Absorption and DNA protective effects of flavonoid glycosides from an onion meal. Eur. J. Nutr. 39: 213-223.
Brand-Williams, W., M.E. Cuvelier, and C. Berset. 1995. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol. 28: 25-30.
Bravo, L. 1998. Polyphenols: Chemistry, dietary sources, metabolism, and nutritional significance. Nutr. Rev. 56: 317-333.
Bray, F., R. Sankila, J. Ferlay, and D.M. Parkin. 2002. Estimates of cancer incidence and mortality in Europe in 1995. Eur. J. Cancer. 38: 99-166.
Breithaupt, D.E. and A. Bamedi. 2002. Carotenoids and carotenoid esters in potatoes (Solanum tuberosum L.): New insights into an ancient vegetable. J. Agric. Food Chem. 50: 7175-7181.
Brothman, A.R. 2002. Cytogenetics and molecular genetics of cancer of the prostate. Am. J. Med. Genet. 115: 150-156.
Camouse, M.M., K.K. Hanneman, E.P. Conrad, and E.D. Baron. 2005. Protective effects of tea polyphenols and caffeine. Expert Rev. Anticancer Ther. 5: 1061-1068.
Chang, L.W., W.J. Yen, S.C. Huang, and P.D. Duh. 2002. Antioxidant activity of sesame coat. Food Chem. 78: 347-354.
Chaudhary, K.S., P.D. Abel, and E.N. Lalani. 1999. Role of the Bcl-2 gene family in prostate cancer progression and its implications for therapeutic intervention. Environ. Health Pers. 107: 49-57.
Che, M.X. and D. Grignon. 2002. Pathology of prostate cancer. Cancer Metastasis Rev. 21: 381-395.
128
Chen, J.Y., H. Zhang, Y.L. Miao, and R. Matsunaga. 2005. NIR measurement of specific gravity of potato. Food Sci. Technol. Res. 11: 26-31.
Chu, Y.F., J. Sun, X. Wu, and R.H. Liu. 2002. Antioxidant and antiproliferative activities of common vegetables. J. Agric. Food Chem. 50: 6910-6916.
Chu, Y.H., C.L. Chang, and H.F. Hsu. 2000. Flavonoid content of several vegetables and their antioxidant activity. J. Sci. Food Agric. 80: 561-566.
Chung, F.L., J. Schwartz, C.R. Herzog, and Y.M. Yang. 2003. Tea and cancer prevention: studies in animals and humans. J. Nutr. 133: 3268S-3274S.
Cirico, T.L. and S.T. Omaye. 2006. Additive or synergetic effects of phenolic compounds on human low density lipoprotein oxidation. Food Chem. Toxicol. 44: 510-516.
Comis, D. 2000. Bring you better beans. Agric. Res. Mag. 48: 14-17.
Cook, N.C. and S. Samman. 1996. Flavonoids - Chemistry, metabolism, cardioprotective effects, and dietary sources. J. Nutr. Biochem. 7: 66-76.
Correll, D.S. 1962. The potato and its relatives. Texas Research Foundation, Renner, TX.
Cunha, G.R., W. Ricke, A. Thomson, P.C. Marker, G. Risbridger, S.W. Hayward, Y.Z. Wang, A.A. Donjacour, and T. Kurita. 2004. Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development. J. Steroid Biochem. Mol. Biol. 92: 221-236.
Dai, J., J.D. Patel, and R.J. Mumper. 2007. Characterization of blackberry extract and its antiproliferative and anti-inflammatory properties. J. Med. Food. 10: 258-265.
Dale, M.F., D.W. Griffiths, and D.T. Todd. 2003. Effects of genotype, environment, and postharvest storage on the total ascorbate content of potato (Solanum tuberosum) tubers. J. Agric. Food Chem. 51: 244-248.
Dalle-Donne, I., R. Rossi, R. Colombo, D. Giustarini, and A. Milzani. 2006. Biomarkers of oxidative damage in human disease. Clin. Chem. 52: 601-623.
Damianaki, A., E. Bakogeorgou, M. Kampa, G. Notas, A. Hatzoglou, S. Panagiotou, C. Gemetzi, E. Kouroumalis, P.M. Martin, and E. Castanas. 2000. Potent inhibitory action of red wine polyphenols on human breast cancer cells. J. Cell Biochem. 78: 429-441.
Davenport, R.J. 2000. Potassium and specific gravity of potato tubers. Better Crops. 84: 14-15.
129
Dehm, S.M. and D.J. Tindall. 2006. Molecular regulation of androgen action in prostate cancer. J. Cellular Biochem. 99: 333-344.
Delmas, D., C. Rebe, S. Lacour, R. Filomenko, A. Athias, P. Gambert, M. Cherkaoui-Malki, B. Jannin, L. Dubrez-Daloz, N. Latruffe, and E. Solary. 2003. Resveratrol-induced apoptosis is associated with Fas redistribution in the rafts and the formation of a death-inducing signaling complex in colon cancer cells. J. Biol. Chem. 278: 41482-41490.
del Moral, L.F.G., Y. Rharrabti, D. Villegas, and C. Royo. 2003. Evaluation of grain yield and its components in durum wheat under Mediterranean conditions: An ontogenic approach. Agron J. 95: 266-274.
Denmeade, S.R., X.S. Lin, and J.T. Isaacs. 1996. Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer. Prostate. 28: 251-265.
Diplock, A.T., J.L. Charleux, G. Crozier-Willi, F.J. Kok, C. Rice-Evans, M. Roberfroid, W. Stahl, and J. Vina-Ribes. 1998. Functional food science and defence against reactive oxidative species. Br. J. Nutr. 80 Suppl 1: S77-112.
Droge, W. 2002. Free radicals in the physiological control of cell function. Physiol. Rev. 82: 47-95.
Duyff, R.L. 2002. American dietetic association complete food and nutrition guide. Wiley, New York.
Eberhardt, M.V., C.Y. Lee, and R.H. Lui. 2000. Antioxidant activity of fresh apples. Nature. 405: 903-904.
Eder, I.E., Z. Culig, R. Ramoner, M. Thurnher, T. Putz, C. Nessler-Menardi, M. Tiefenthaler, G. Bartsch, and H. Klocker. 2000. Inhibition of LncaP prostate cancer cells by means of androgen receptor antisense oligonucleotides. Cancer Gene Ther. 7: 997-1007.
Edwards, E.J. and A.H. Cobb. 1999. The effect of prior storage on the potential of potato tubers (Solanum tuberosum L) to accumulate glycoalkaloids and chlorophylls during light exposure, including artificial neural network modelling. J. Sci. Food Agric. 79: 1289-1297.
El-Agamey, A., G.M. Lowe, D.J. McGarvey, A. Mortensen, D.M. Phillip, T.G. Truscott, and A.J. Young. 2004. Carotenoid radical chemistry and antioxidant/pro-oxidant properties. Arch. Biochem. Biophys. 430: 37-48.
Emmons, C.L. and D.M. Peterson. 2001. Antioxidant activity and phenolic content of oat as affected by cultivar and location. Crop Sci. 41: 1676-1681.
130
Epinat-Le Signor, C., S. Dousse, J. Lorgeou, J.B. Denis, R. Bonhomme, P. Carolo, and A. Charcosset. 2001. Interpretation of genotype X environment interactions for early maize hybrids over 12 years. Crop Sci. 41: 663-669.
Falconer, D.S. and T.F.C. Mackay. 1996. Introduction to quantitative genetics. Longman, New York.
FAO. 2007. FAO Statistical Yearbook 2005-2006. FAO Statistics Division, Rome, Italy.
FNB/NAS. 1994. Opportunities in the nutrition and food sciences. National Academy of Sciences, National Academy Press, Washington, DC.
Friedman, M. 2004. Analysis of biologically active compounds in potatoes (Solanum tuberosum), tomatoes (Lycopersicon esculentum), and jimson weed (Datura stramonium) seeds. J. Chromatogr. A. 1054: 143-155.
Friedman, M. 2006. Potato glycoalkaloids and metabolites: Roles in the plant and in the diet. J. Agric. Food Chem. 54: 8655-8681.
Friedman, M., P.R. Henika, C.E. Levin, R.E. Mandrell, and N. Kozukue. 2006. Antimicrobial activities of tea catechins and theaflavins and tea extracts against Bacillus cereus. J. Food Prot. 69: 354-361.
Friedman, M., P.R. Henika, and B.E. Mackey. 2003. Effect of feeding solanidine, solasodine and tomatidine to non-pregnant and pregnant mice. Food Chem. Toxicol. 41: 61-71.
Friedman, M., K.R. Lee, H.J. Kim, I.S. Lee, and N. Kozukue. 2005. Anticarcinogenic effects of glycoalkaloids from potatoes against human cervical, liver, lymphoma, and stomach cancer cells. J. Agric. Food Chem. 53: 6162-6169.
Friedman, M., B.E. Mackey, H.J. Kim, I.S. Lee, K.R. Lee, S.U. Lee, E. Kozukue, and N. Kozukue. 2007. Structure-activity relationships of tea compounds against human cancer cells. J. Agric. Food Chem. 55: 243-253.
f*ckumoto, L.R. and G. Mazza. 2000. Assessing antioxidant and prooxidant activities of phenolic compounds. J. Agric. Food Chem. 48: 3597-3604.
Fulda, S. and K.M. Debatin. 2006. Modulation of apoptosis signaling for cancer therapy. Arch. Immunol. Ther. Exp. 54: 173-175.
Giovannelli, L., G. Testa, C. De Filippo, V. Cheynier, M.N. Clifford, and P. Dolara. 2000. Effect of complex polyphenols and tannins from red wine on DNA oxidative damage of rat colon mucosa in vivo. Eur. J. Nutr. 39: 207-212.
131
Giovannucci, E. 1999. Insulin-like growth factor-1 and binding protein-3 and risk of cancer. Horm. Res. 51: 34-41.
Giustarini, D., A. Milzani, I. Dalle-Donne, and R. Rossi. 2008. Red blood cells as a physiological source of glutathione for extracellular fluids. Blood Cells Mol. Dis. 40: 174-179.
Goncalves, P.D.S., N. Bortoletto, A.L.M. Martins, R.B. da Costa, and P.B. Gallo. 2003. Genotype-environment interaction and phenotypic stability for girth growth and rubber yield of Hevea clones in Sao Paulo State, Brazil. Genet. Mol. Biol. 26: 441-448.
Gould, W.A. 1999. Potato production, processing, and technology. CTI Publications, Baltimore, MD
Grausgruber, H., M. Oberforster, M. Werteker, P. Ruckenbauer, and J. Vollmann. 2000. Stability of quality traits in Austrian-grown winter wheats. Field Crop Res. 66: 257-267.
Graybosch, R., N. Ames, P.S. Baenziger, and C.J. Peterson. 2004. Genotypic and environmental modification of Asian noodle quality of hard winter wheats. Cereal Chem. 81: 19-25.
Griffiths, D.W. and M.F.B. Dale. 2001. Effect of light exposure on the glycoalkaloid content of Solanum phureja tubers. J. Agric. Food Chem. 49: 5223-5227.
Guyton, K.Z. and T.W. Kensler. 1993. Oxidative mechanisms in carcinogenesis. Br. Med. Bull. 49: 523-544.
Haag, P., J. Bektic, G. Bartsch, H. Klocker, and I.E. Eder. 2005. Androgen receptor down regulation by small interference RNA induces cell growth inhibition in androgen sensitive as well as in androgen independent prostate cancer cells. J. Steroid Biochem. Mol. Biol. 96: 251-258.
Hagenimana, V., E.G. Karuri, and M.A. Oyunga. 1998. Oil content in fried processed sweetpotato products. J. Food Process. Preserv. 22: 123-137.
Hale, A.J., C.A. Smith, L.C. Sutherland, V.E. Stoneman, V. Longthorne, A.C. Culhane, and G.T. Williams. 1996. Apoptosis: molecular regulation of cell death. Eur. J. Biochem. 237: 884.
Hale, A.L. 2004. Screening potato genotypes for antioxidant activity, identification of the responsible compounds and differentiating russet norkotah strains using AFLP and microsatellite marker analysis. PhD dissertation, Texas A&M University, College Station.
132
Harborne, J.B. 1998. The flavonoids, advances in research since 1986. Chapman and Hall, London.
Harris, P.M. 1978. The potato crop. Halsted Press, New York.
Hawk, E.T., A. Umar, E. Richmond, and J.L. Viner. 2005. Prevention and therapy of colorectal cancer. Med. Clinic N. Amer. 89: 85-110.
Hawkes, J.G. 1978a. History of the potato, p. 1-13. In: P.M. Harris (ed.). The potato crop. Halsted, New York.
Hawkes, J.G. 1978b. Systematic notes on solanaceae. Bot. J. Linn. Soc. 76: 287-295.
Hawkes, J.G. 1992. Biosystematics of the potato, p. 13-64. In: P.M. Harris (ed.). The potato crop: The scientific basis for improvement. Chapman & Hall, London.
Haynes, K.G. 2001. Variance components for yield and specific gravity in a diploid potato population after two cycles of recurrent selection. Am. J. Potato Res. 78: 69-75.
Hengartner, M.O. 2000. The biochemistry of apoptosis. Nature. 407: 770-776.
Hollister, B., J.C. Dickens, F. Perez, and K.L. Deahl. 2001. Differential neurosensory responses of adult Colorado potato beetle, Leptinotarsa decemlineata, to glycoalkaloids. J. Chem. Ecol. 27: 1105-1118.
Holmquist, L., G. Stuchbury, K. Berbaum, S. Muscat, S. Young, K. Hager, J. Engel, and G. Munch. 2007. Lipoic acid as a novel treatment for Alzheimer's disease and related dementias. Pharmacol. Ther. 113: 154-164.
Horton, D. 1987. Potato production, marketing and programs for developing countries. Westview Press, Boulder, CO.
Hostanska, K., G. Jurgenliemk, G. Abel, A. Nahrstedt, and R. Saller. 2007. Willow bark extract (BNO1455) and its fractions suppress growth and induce apoptosis in human colon and lung cancer cells. Cancer Detect. Prev. 31: 129-139.
Hu, F.B. 2003. Plant-based foods and prevention of cardiovascular disease: An overview. Am. J. Clin. Nutr. 78: 544S-551S.
Huaman, Z. and D.M. Spooner. 2002. Reclassification of landrace populations of cultivated potatoes (Solanum sect. Petota). Am. J. Bot. 89: 947-965.
Huang, D.J., C.D. Lin, H.J. Chen, and Y.H. Lin. 2004. Antioxidant and antiproliferative activities of sweet potato (Ipomoea batatas [L.] Lam 'Tainong 57) constituents. Bot. Bull. Acad. Sinica. 45: 179-186.
133
Huang, D.J., B.X. Ou, and R.L. Prior. 2005. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 53: 1841-1856.
Huang, R.H., J.Y. Chai, and A.S. Tarnawski. 2006. Identification of specific genes and pathways involved in NSAIDs-induced apoptosis of human colon cancer cells. World J. Gastroenterol. 12: 6446-6452.
Hudson, B.J.F. 1990. Food antioxidants. Elsevier, London.
Hussain, S.P., L.J. Hofseth, and C.C. Harris. 2003. Radical causes of cancer. Nat. Rev. Cancer. 3: 276-285.
Hwang, S.L. and G.C. Yen. 2008. Neuroprotective effects of the citrus flavanones against H2O2-induced cytotoxicity in PC12 cells. J. Agric. Food Chem. 56: 859-864.
Inoue, M., E.F. Sato, M. Nishikawa, A.M. Park, Y. Kira, I. Imada, and K. Utsumi. 2003. Mitochondrial generation of reactive oxygen species and its role in aerobic life. Curr. Med. Chem. 10: 2495-2505.
Ito, N., S. f*ckushima, A. Hagiwara, M. Shibata, and T. Ogiso. 1983. Carcinogenicity of butylated hydroxyanisole in F344 Rats. J. Natl. Cancer Inst. 70: 343-352.
Jadhav, S.J., D.K. Salunkhe, R.E. Wyse, and R.R. Dalvi. 1973. Solanum alkaloids - biosynthesis and inhibition by chemicals. J. Food Sci. 38: 453-455.
Jemal, A., R.C. Tiwari, T. Murray, A. Ghafoor, A. Samuels, E. Ward, E.J. Feuer, and M.J. Thun. 2004. Cancer statistics, 2004. CA Cancer J. Clin. 54: 8-29.
Jones, D.P., J.L. Carlson, V.C. Mody, J. Cai, M.J. Lynn, and P. Sternberg. 2000. Redox state of glutathione in human plasma. Free Radic. Biol. Med. 28: 625-635.
Joshipura, K.J., F.B. Hu, J.E. Manson, M.J. Stampfer, E.B. Rimm, F.E. Speizer, G. Colditz, A. Ascherio, B. Rosner, D. Spiegelman, and W.C. Willett. 2001. The effect of fruit and vegetable intake on risk for coronary heart disease. Ann. Intern. Med. 134: 1106-1114.
Juan, M.E., J.M. Planas, V. Ruiz-Gutierrez, H. Daniel, and U. Wenzel. 2008. Antiproliferative and apoptosis-inducing effects of maslinic and oleanolic acids, two pentacyclic triterpenes from olives, on HT-29 colon cancer cells. Br. J. Nutr.: 1-8.
Juan, M.E., U. Wenzel, V. Ruiz-Gutierrez, H. Daniel, and J.M. Planas. 2006. Olive fruit extracts inhibit proliferation and induce apoptosis in HT-29 human colon cancer cells. J. Nutr. 136: 2553-2557.
134
Kalt, W., D.A.J. Ryan, J.C. Duy, R.L. Prior, M.K. Ehlenfeldt, and S.P. Vander Kloet. 2001. Interspecific variation in anthocyanins, phenolics, and antioxidant capacity among genotypes of highbush and lowbush blueberries (Vaccinium section cyanococcus spp.). J. Agric. Food Chem. 49: 4761-4767.
Kanatt, S.R., R. Chander, P. Radhakrishna, and A. Sharma. 2005. Potato peel extract - a natural antioxidant for retarding lipid peroxidation in radiation processed lamb meat. J. Agric. Food Chem. 53: 1499-1504.
Kaneto, H., Y. Kajimoto, J. Miyagawa, T. Matsuoka, Y. Fujitani, Y. Umayahara, T. Hanafusa, Y. Matsuzawa, Y. Yamasaki, and M. Hori. 1999. Beneficial effects of antioxidants in diabetes: Possible protection of pancreatic beta-cells against glucose toxicity. Diabetes. 48: 2398-2406.
Kant, A.K. and G. Block. 1990. Dietary vitamin-B6 intake and food sources in the United-States population - Nhanes Ii, 1976-1980. Am. J. Clin. Nutr. 52: 707-716.
Kawakami, S., M. Mizuno, and H. Tsuchida. 2000. Comparison of antioxidant enzyme activities between Solanum tuberosum L. cultivars Danshaku and Kitaakari during low-temperature storage. J. Agric. Food Chem. 48: 2117-2121.
Kern, M., G. Pahlke, K.K. Balavenkatraman, F.D. Bohmer, and D. Marko. 2007. Apple polyphenols affect protein kinase C activity and the onset of apoptosis in human colon carcinoma cells. J. Agric. Food Chem. 55: 4999-5006.
Kim, D.O., O.K. Chun, Y.J. Kim, H.Y. Moon, and C.Y. Lee. 2003. Quantification of polyphenolics and their antioxidant capacity in fresh plums. J. Agric. Food Chem. 51: 6509-6515.
Kim, M.J., Y.J. Kim, H.J. Park, J.H. Chung, K.H. Leem, and H.K. Kim. 2006. Apoptotic effect of red wine polyphenols on human colon cancer SNU-C4 cells. Food Chem. Toxicol. 44: 898-902.
Klaunig, J.E. and L.M. Kamendulis. 2004. The role of oxidative stress in carcinogenesis. Annu. Rev. Pharmacol. Toxicol. 44: 239-267.
Klein, B.P. and A.C. Kurilich. 2000. Processing effects on dietary antioxidants from plant foods. HortScience. 35: 580-584.
Ko, J.K., W.C. Leung, W.K. Ho, and P. Chiu. 2007. Herbal diterpenoids induce growth arrest and apoptosis in colon cancer cells with increased expression of the nonsteroidal anti-inflammatory drug-activated gene. Eur. J. Pharmacol. 559: 1-13.
Kojo, S. 2004. Vitamin C: Basic metabolism and its function as an index of oxidative stress. Curr. Med. Chem. 11: 1041-1064.
135
Kolasa, K.M. 1993. The potato and human nutrition. Am. Potato J. 70: 375-384.
Koleva, I.I., T.A. van Beek, J.P.H. Linssen, A. de Groot, and L.N. Evstatieva. 2002. Screening of plant extracts for antioxidant activity: A comparative study on three testing methods. Phytochem. Anal. 13: 8-17.
Komiyama, S., J. Kato, H. Honda, and K. Matsushima. 2007. Development of sorting system based on potato starch content using visible and near-infrared spectroscopy. J. Jpn. Soc. Food. Sci. Technol. 54: 304-309.
Korpan, Y.I., E.A. Nazarenko, I.V. Skryshevskaya, C. Martelet, N. Jaffrezic-Renault, and A.V. El'skaya. 2004. Potato glycoalkaloids: True safety or false sense of security? Trends Biotechnol. 22: 147-151.
Kovacic, P. and J.D. Jacintho. 2001. Mechanisms of carcinogenesis: Focus on oxidative stress and electron transfer. Curr. Med. Chem. 8: 773-796.
Kweon, M.H., H.J. Hwang, and H.C. Sung. 2001. Identification and antioxidant activity of novel chlorogenic acid derivatives from bamboo (Phyllostachys edulis). J. Agric. Food Chem. 49: 4646-4655.
Kyriakis, J.M. and J. Avruch. 2001. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol. Rev. 81: 807-869.
Lachman, J., K. Hamouz, M. Orsak, and V. Pivec. 2000. Potato tubers as a significant source of antioxidants in human nutrition. Rost. Vyroba. 46: 231-236.
Lachman, J., K. Hamouz, M. Orsak, and V. Pivec. 2001. Potato glycoalkaloids and their significance in plant protection and nutrition. Rost. Vyroba. 47: 181-1912.
Laurila, J., I. Laakso, J. Larkka, T. Gavrilenko, V.M. Rokka, and E. Pehu. 2001. The proportions of glycoalkaloid aglycones are dependent on the genome constitutions of interspecific hybrids between two Solanum species (S. brevidens and S. tuberosum). Plant Sci. 161: 677-683.
Lawnicka, H., A.M. Potocka, A. Juzala, M.C. Fournie-Zaluski, and M. Pawlikowski. 2004. Angiotensin II and its fragments (angiotensins III and IV) decrease the growth of DU-145 prostate cancer in vitro. Med. Sci. Monit. 10: BR410-413.
Le Marchand, L., S.P. Murphy, J.H. Hankin, L.R. Wilkens, and L.N. Kolonel. 2000. Intake of flavonoids and lung cancer. J. Natl. Cancer Inst. 92: 154-160.
136
Lee, K.R., N. Kozukue, J.S. Han, J.H. Park, E.Y. Chang, E.J. Baek, J.S. Chang, and M. Friedman. 2004. Glycoalkaloids and metabolites inhibit the growth of human colon (HT29) and liver (HepG2) cancer cells. J. Agric. Food Chem. 52: 2832-2839.
Lemanska, K., H. Szymusiak, B. Tyrakowska, R. Zielinski, A.E.M.F. Soffers, and I.M.C.M. Rietjens. 2001. The influence of pH on antioxidant properties and the mechanism of antioxidant action of hydroxyflavones. Free Radic. Biol. Med. 31: 869-881.
Lentner, M. and T. Bishop. 1993. Experimental design and analysis. Valley Book Company, Blacksburg, VA.
Leonard, S.S., G.K. Harris, and X. Shi. 2004. Metal-induced oxidative stress and signal transduction. Free Radic. Biol. Med. 37: 1921-1942.
Lisinska, G. and W. Leszcynski. 1989. Potato science and technology. Elsevier, London.
Liu, J.J., T.S. Huang, M.L. Hsu, C.C. Chen, W.S. Lin, F.J. Lu, and W.H. Chang. 2004. Antitumor effects of the partially purified polysaccharides from Antrodia camphorata and the mechanism of its action. Toxicol. Appl. Pharmacol. 201: 186-193.
Liu, R.H. 2003. Health benefits of fruit and vegetables are from additive and synergistic combinations of phytochemicals. Am. J. Clin. Nutr. 78: 517S-520S.
Liu, R.H. 2004. Potential synergy of phytochemicals in cancer prevention: Mechanism of action. J. Nutr. 134: 3479S-3485S.
Loft, S. and H.E. Poulsen. 1996. Cancer risk and oxidative DNA damage in man. J. Mol. Med. 74: 297-312.
Lu, Z. and S. Xu. 2006. ERK1/2 MAP kinases in cell survival and apoptosis. IUBMB Life. 58: 621-631.
Lui, M., X.Q. Li, C. Weber, C.Y. Lee, J. Brown, and R.H. Lui. 2002. Antioxidant and antiproliferative activities of raspberries. J. Agric. Food Chem. 50: 2926-2930.
Lulai, E.C. and P.H. Orr. 1979. Influence of potato specific gravity on yield and oil content of chips. Am. Potato J. 56: 379-390.
Magi-Galluzzi, C., M. Murphy, M.G. Cangi, and M. Loda. 1998. Proliferation, apoptosis and cell cycle regulation in prostatic carcinogenesis. Anal. Quant. Cytol. Histol. 20: 343-350.
137
Mahinda, W. and F. Shahidi. 2000. Scavenging of reactive-oxygen species and DPPH free radicals by extracts of borage and evening primrose meals. Food Chem. 70: 17-26.
Makazan, Z., H.K. Saini, and N.S. Dhalla. 2007. Role of oxidative stress in alterations of mitochondrial function in ischemic-reperfused hearts. Am. J. Physiol. Heart Circ. Physiol. 292: H1986-1994.
Manach, C., A. Mazur, and A. Scalbert. 2005. Polyphenols and prevention of cardiovascular diseases. Curr. Opin. Lipidol. 16: 77-84.
Marnett, L.J. 2000. Oxyradicals and DNA damage. Carcinogenesis. 21: 361-370.
Masella, R., R. Di Benedetto, R. Vari, C. Filesi, and C. Giovannini. 2005. Novel mechanisms of natural antioxidant compounds in biological systems: Involvement of glutathione and glutathione-related enzymes. J. Nutr. Biochem. 16: 577-586.
Mates, J.M., C. Perez-Gomez, and I. Nunez de Castro. 1999. Antioxidant enzymes and human diseases. Clin. Biochem. 32: 595-603.
Matsunaga, K., M. Katayama, K. Sakata, T. Kuno, K. Yoshida, Y. Yamada, Y. Hirose, N. Yoshimi, and H. Mori. 2002. Inhibitory effects of chlorogenic acid on azoxymethane-induced colon carcinogenesis in male F344 Rats. Asian Pac. J. Cancer Prev. 3: 163-166.
McBride, J. 1999. Can foods forestall aging. Agric. Res. Mag. 47: 14-17.
McCann, M.J., C.I. Gill, O.B. G, J.R. Rao, W.C. McRoberts, P. Hughes, R. McEntee, and I.R. Rowland. 2007. Anti-cancer properties of phenolics from apple waste on colon carcinogenesis in vitro. Food Chem. Toxicol. 45: 1224-1230.
McCue, K.F., P.V. Allen, L.V.T. Shepherd, A. Blake, M.M. Maccree, D.R. Rockhold, R.G. Novy, D. Stewart, H.V. Davies, and W.R. Belknap. 2007. Potato glycosterol rhamnosyltransferase, the terminal step in triose side-chain biosynthesis. Phytochemistry. 68: 327-334.
Mensinga, T.T., A.J.A.M. Sips, C.J.M. Rompelberg, K. van Twillert, J. Meulenbelt, H.J. van den Top, and H.P. van Egmond. 2005. Potato glycoalkaloids and adverse effects in humans: An ascending dose study. Regul. Toxicol. Pharm. 41: 66-72.
Milde, J., E.F. Elstner, and J. Grassmann. 2007. Synergistic effects of phenolics and carotenoids on human low-density lipoprotein oxidation. Mol. Nutr. Food Res. 51: 956-961.
138
Miller Jr, J.C. 1992. Potato, Irish. The McGraw-Hill encyclopedia of science and technology. Vol. 14, McGraw-Hill, New York.
Miller, N.J. and C.A. Rice-Evans. 1997. Factors influencing the antioxidant activity determined by the ABTS.+ radical cation assay. Free Radic. Res. 26: 195-199.
Minami, H., M. Kinosh*ta, Y. f*ckuyama, M. Kodama, T. Yoshizawa, M. Sugiura, K. Nakagawa, and H. Tago. 1994. Antioxidant xanthones from Garcinia-Subelliptica. Phytochemistry. 36: 501-506.
Mohamad, N., A. Gutierrez, M. Nunez, C. Cocca, G. Martin, G. Cricco, V. Medina, E. Rivera, and R. Bergoc. 2005. Mitochondrial apoptotic pathways. Biocell. 29: 149-161.
Morello, M.J., F. Shahidi, and T.C. Ho. 2002. Free radicals in foods: Chemistry, nutrition, and health effects. ACS Symposium Series 807, Washington, DC.
Mortensen, A., L.H. Skibsted, and T.G. Truscott. 2001. The interaction of dietary carotenoids with radical species. Arch. Biochem. Biophys. 385: 13-19.
Mu, L.N., Q.Y. Lu, S.Z. Yu, Q.W. Jiang, W. Cao, N.C. You, V.W. Setiawan, X.F. Zhou, B.G. Ding, R.H. Wang, J. Zhao, L. Cai, J.Y. Rao, D. Heber, and Z.F. Zhang. 2005. Green tea drinking and multigenetic index on the risk of stomach cancer in a Chinese population. Int. J. Cancer. 116: 972-983.
Munkholm, P. 2003. Review article: The incidence and prevalence of colorectal cancer in inflammatory bowel disease. Aliment. Pharmacol. Ther. 18 Suppl 2: 1-5.
Nakanishi, H., O. Mazda, E. Satoh, H. Asada, H. Morioka, T. Kishida, M. Nakao, Y. Mizutani, A. Kawauchi, M. Kita, J. Imanishi, and T. Miki. 2003. Nonviral genetic transfer of Fas ligand induced significant growth suppression and apoptotic tumor cell death in prostate cancer in vivo. Gene Ther. 10: 434-442.
National Academy of Science. 1998. Dietary reference intakes: Proposed definition and plan for review of dietary antioxidants and related compounds. National Academy Press, Washington, DC.
National Potato Council. 2008. Potato statistical year book. National Potato Council, Washington, DC.
Niki, E. and N. Noguchi. 2000. Evaluation of antioxidant capacity: What capacity is being measured by which method? IUBMB Life. 50: 323-329.
Niles, R.M. 2004. Signaling pathways in retinoid chemoprevention and treatment of cancer. Mutat. Res. 555: 81-96.
139
Nzaramba, M.N. 2004. Inheritance of antioxidant activity and its association with seed coat color in cowpea (Vigna unguiculata L. Walp). M.S Thesis, Texas A&M University, College Station.
Nzaramba, M.N., J. Bamberg, and J.C. Miller Jr. 2006. Antioxidant activity in Solanum species as influenced by seed type and growing location. Am. J. Potato Res. 83: 127.(Abstr.).
Nzaramba, M.N., J.B. Bamberg, and J.C. Miller. 2007. Effect of propagule type and growing environment on antioxidant activity and total phenolic content in potato germplasm. Am. J. Potato Res. 84: 323-330.
Oren, M. 2003. Decision making by p53: Life, death and cancer. Cell Death Differ. 10: 431-442.
Ortega, S., M. Malumbres, and M. Barbacid. 2002. Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim. Biophys. Acta. 1602: 73-87.
Papathanasiou, F., S.H. Mitchell, and B.M.R. Harvey. 1998. Glycoalkaloid accumulation during tuber development of early potato cultivars. Potato Res. 41: 117-125.
Pęksa, A., G. Golubowska, K. Aniolowski, G. Lisińska, and E. Rytel. 2006. Changes of glycoalkaloids and nitrate contents in potatoes during chip processing. Food Chem. 97: 151-156.
Pęksa, A., G. Golubowska, E. Rytel, G. Lisińska, and K. Aniolowski. 2002. Influence of harvest date on glycoalkaloid contents of three potato varieties. Food Chem. 78: 313-317.
Perry, E.K., A.T. Pickering, W.W. Wang, P.J. Houghton, and N.S. Perry. 1999. Medicinal plants and Alzheimer's disease: From ethnobotany to phytotherapy. J. Pharm. Pharmacol. 51: 527-534.
Peterson, C.J., R.A. Graybosch, P.S. Baenziger, and A.W. Grombacher. 1992. Genotype and environment effects on quality characteristics of hard red winter-wheat. Crop Sci. 32: 98-103.
Phillips, B.J. 1996. Development of cell culture techniques for assessment of the toxicity of plant products. Toxicol. in Vitro. 10: 69-76.
Phillips, B.J., J.A. Hughes, J.C. Phillips, D.G. Walters, D. Anderson, and C.S.M. Tahourdin. 1996. A study of the toxic hazard that might be associated with the consumption of green potato tops. Food Chem. Toxicol. 34: 439-448.
Podolsky, D.K. 2002. Inflammatory bowel disease. N. Engl. J. Med. 347: 417-429.
140
Poli, G., G. Leonarduzzi, F. Biasi, and E. Chiarpotto. 2004. Oxidative stress and cell signalling. Curr. Med. Chem. 11: 1163-1182.
Polovka, M., V. Brezova, and A. Stasko. 2003. Antioxidant properties of tea investigated by EPR spectroscopy. Biophys. Chem. 106: 39-56.
Pryor, W.A. 2000. Vitamin E and heart disease: Basic science to clinical intervention trials. Free Radic. Biol. Med. 28: 141-164.
Rayburn, J.R., M. Friedman, and J.A. Bantle. 1995. Synergistic interaction of glycoalkaloids alpha-chaconine and alpha-solanine on developmental toxicity in Xenopus embryos. Food Chem. Toxicol. 33: 1013-1019.
Reddivari, L., A.L. Hale, and J.C. Miller. 2007a. Determination of phenolic content, composition and their contribution to antioxidant activity in specialty potato selections. Am. J. Potato Res. 84: 275-282.
Reddivari, L., J. Vanamala, S. Chintharlapalli, S.H. Safe, and J.C. Miller, Jr. 2007b. Anthocyanin fraction from potato extracts is cytotoxic to prostate cancer cells through activation of caspase-dependent and caspase-independent pathways. Carcinogenesis. 28: 2227-2235.
Repetto, M.G. and S.F. Llesuy. 2002. Antioxidant properties of natural compounds used in popular medicine for gastric ulcers. Braz. J. Med. Biol. Res. 35: 523-534.
Reyes, L.F., J.C. Miller Jr, and L. Cisneros-Zevallos. 2005. Antioxidant capacity, anthocyanins and total phenolics in purple- and red-fleshed potato (Solanum tuberosum L.) genotypes. Am. J. Potato Res. 82: 271-277.
Rharrabti, Y., L.F.G. del Moral, D. Villegas, and C. Royo. 2003a. Durum wheat quality in Mediterranean environments III. Stability and comparative methods in analysing GxE interaction. Field Crop Res. 80: 141-146.
Rharrabti, Y., D. Villegas, C. Royo, V. Martos-Nunez, and L.F.G. del Moral. 2003b. Durum wheat quality in Mediterranean environments II. Influence of climatic variables and relationships between quality parameters. Field Crop Res. 80: 133-140.
Riboli, E. and T. Norat. 2003. Epidemiologic evidence of the protective effect of fruit and vegetables on cancer risk. Am. J. Clin. Nutr. 78: 559S-569S.
Rice-Evans, C. 2001. Flavonoid antioxidants. Curr. Med. Chem. 8: 797-807.
Rice-Evans, C.A., N.J. Miller, and G. Paganga. 1996. Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radic. Biol. Med. 20: 933-956.
141
Ridker, P.M., N. Rifai, L. Rose, J.E. Buring, and N.R. Cook. 2002. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N. Engl. J. Med. 347: 1557-1565.
Riedl, K.M., S. Carando, M.H. Alessio, M. McCarty, and E.A. Hagerman. 2002. Antioxidant activity of tannins and tannin-protein complexes: Assessment of in vitro and in vivo, p. 188-200. In: M.J. Morello, F. Shahidi, and T.C. Ho (eds.). Free radicals in foods: Chemistry, nutrition , and health effects. American Chemical Society, Washington, DC.
Rietjens, I.M.C.M., M.J. Martena, M.G. Boersma, W. Spiegelenberg, and G.M. Alink. 2005. Molecular mechanisms of toxicity of important food-borne phytotoxins. Mol. Nutr. Food Res. 49: 131-158.
Robards, K., P.D. Prenzler, G. Tucker, P. Swatsitang, and W. Glover. 1999. Phenolic compounds and their role in oxidative processes in fruits. Food Chem. 66: 401-436.
Rodriguez, J., C. Olea-Azar, C. Cavieres, E. Norambuena, T. Delgado-Castro, J. Soto-Delgado, and R. Araya-Maturana. 2007. Antioxidant properties and free radical-scavenging reactivity of a family of hydroxynaphthalenones and dihydroxyanthracenones. Bioorg. Med. Chem. 15: 7058-7065.
Rodriguez-Saona, L.E., R.E. Wrolstad, and C. Pereira. 1999. Glycoalkaloid content and anthocyanin stability to alkaline treatment of red-fleshed potato extracts. J. Food Sci. 64: 445-450.
Romero, I., A. Paez, A. Ferruelo, M. Lujan, and A. Berenguer. 2002. Polyphenols in red wine inhibit the proliferation and induce apoptosis of LNCaP cells. BJU Int. 89: 950-954.
Roy, K., A. Saha, K. De, and C. Sengupta. 2002. Evaluation of probucol as suppressor of ceftizoxime induced lipid peroxidation. Acta Pol. Pharm. 59: 231-234.
Roy-Burman, P., D.J. Tindall, D.M. Robins, N.M. Greenberg, M.J. Hendrix, S. Mohla, R.H. Getzenberg, J.T. Isaacs, and K.J. Pienta. 2005. Androgens and prostate cancer: Are the descriptors valid? Cancer Biol. Ther. 4: 4-5.
Rytel, E., G. Golubowska, G. Lisińska, A. Pęksa, and K. Aniolowski. 2005. Changes in glycoalkaloid and nitrate contents in potatoes during French fries processing. J. Sci. Food Agric. 85: 879-882.
SAS. 2002. SAS software version 9.1, SAS Institute, Inc., Cary, NC.
Scalbert, A., I.T. Johnson, and M. Saltmarsh. 2005. Polyphenols: Antioxidants and beyond. Am. J. Clin. Nutr. 81: 215S-217S.
142
Schroeter, H., C. Boyd, J.P. Spencer, R.J. Williams, E. Cadenas, and C. Rice-Evans. 2002. MAPK signaling in neurodegeneration: Influences of flavonoids and of nitric oxide. Neurobiol. Aging. 23: 861-880.
Seeram, N.P., L.S. Adams, S.M. Henning, Y. Niu, Y. Zhang, M.G. Nair, and D. Heber. 2005. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. J. Nutr. Biochem. 16: 360-367.
Sekher Pannala, A., T.S. Chan, P.J. O'Brien, and C.A. Rice-Evans. 2001. Flavonoid B-ring chemistry and antioxidant activity: Fast reaction kinetics. Biochem. Biophys. Res. Commun. 282: 1161-1168.
Sengul, M., F. Keles, and M.S. Keles. 2004. The effect of storage conditions (temperature, light, time) and variety on the glycoalkaloid content of potato tubers and sprouts. Food Control. 15: 281-286.
Shahidi, F. 2002. Antioxidants in plants and oleaginous seeds, p. 162-175. In: M.J. Morello, F. Shahidi, and T.C. Ho (eds.). Free radicals in food: Chemistry, nutrition, and health effects. American Chemical Society, Washington, DC.
Shahidi, F. and M. Naczk. 1995a. Food phenolic: An overview, p. 1-5. In: F. Shahidi and M. Naczk (eds.). Food phenolics: Sources, chemistry, effects, applications. Technomic Publishing Co., Lancaster, PA.
Shahidi, F. and M. Naczk. 1995b. Phenolic compounds in grains, p. 30-39. In: F. Shahidi and M. Naczk (eds.). Food phenolics: Sources, chemistry, effects, and applications. Technomic Publishing Co., Lancaster, PA.
Sharma, S.D., S.M. Meeran, and S.K. Katiyar. 2007. Dietary grape seed proanthocyanidins inhibit UVB-induced oxidative stress and activation of mitogen-activated protein kinases and nuclear factor-kappaB signaling in in vivo SKH-1 hairless mice. Mol. Cancer Ther. 6: 995-1005.
Shimizu, M., N. Yoshimi, Y. Yamada, K. Matsunaga, K. Kawabata, A. Hara, H. Moriwaki, and H. Mori. 1999. Suppressive effects of chlorogenic acid on N-methyl-N-nitrosourea-induced glandular stomach carcinogenesis in male F344 rats. J. Toxicol. Sci. 24: 433-439.
Shukla, S. and S. Gupta. 2005. Dietary agents in the chemoprevention of prostate cancer. Nutr. Cancer. 53: 18-32.
Siddhuraju, P., P.S. Mohan, and K. Becker. 2002. Studies on the antioxidant activity of Indian Laburnum (Cassia fistula L.): A preliminary assessment of crude extracts from stem bark, leaves, flowers and fruit pulp. Food Chem. 79: 61-67.
143
Singleton, V.L., R. Orthofer, and R.M. Lamuela-Raventos. 1999. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 299: 152-178.
Smith, A.R., S.V. Shenvi, M. Widlansky, J.H. Suh, and T.M. Hagen. 2004. Lipoic acid as a potential therapy for chronic diseases associated with oxidative stress. Curr. Med. Chem. 11: 1135-1146.
Smith, D.B., J.G. Roddick, and J.L. Jones. 1996. Potato glycoalkaloids: Some unanswered questions. Trends Food Sci. Technol. 7: 126-131.
Smith, D.B., J.G. Roddick, and J.L. Jones. 2001. Synergism between the potato glycoalkaloids alpha-chaconine and alpha-solanine in inhibition of snail feeding. Phytochemistry. 57: 229-234.
Sotelo, A. and B. Serrano. 2000. High-performance liquid chromatographic determination of the glycoalkaloids alpha-solanine and alpha-chaconine in 12 commercial varieties of Mexican potato. J. Agric. Food Chem. 48: 2472-2475.
Spooner, D.M. and R.J. Hijmans. 2001. Potato systematics and germplasm collecting, 1989-2000. Am. J. Potato Res. 78: 237-268.
Spooner, D.M., K. McLean, G. Ramsay, R. Waugh, and G.J. Bryan. 2005. A single domestication for potato based on multilocus amplified fragment length polymorphism genotyping. PNAS. 102: 14694-14699.
Spooner, D.M., J. Nunez, G. Trujillo, M.D. Herrera, F. Guzman, and M. Ghislain. 2007. Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification. PNAS. 104: 19398-19403.
Spooner, D.M., R.G. van den Berg, A. Rodriguez, J. Bamberg, R.J. Hijmans, and L. Cabrera. 2004. Wild potato (Solanum section Petota; Solanaceae) of north and central America. In: C. Anderson (Ed., Systematic botany monographs Vol. 68, Ann Arbor, MI.
Stanner, S.A., J. Hughes, C.N. Kelly, and J. Buttriss. 2004. A review of the epidemiological evidence for the 'antioxidant hypothesis'. Pub. Health Nutr. 7: 407-422.
Steffen, L.M., D.R. Jacobs, Jr., J. Stevens, E. Shahar, T. Carithers, and A.R. Folsom. 2003. Associations of whole-grain, refined-grain, and fruit and vegetable consumption with risks of all-cause mortality and incident coronary artery disease and ischemic stroke: The Atherosclerosis Risk in Communities (ARIC) Study. Am. J. Clin. Nutr. 78: 383-390.
144
Stelljes, K.B. 2001. Colorful potatoes offer nutrition, variety. Agric. Res. Mag. 49: 6.
Sterrett, S.B., M.R. Henninger, G.C. Yencho, W. Lu, B.T. Vinyard, and K.G. Haynes. 2003. Stability of internal heat necrosis and specific gravity in tetraploid x diploid potatoes. Crop Sci. 43: 790-796.
Storz, P. 2005. Reactive oxygen species in tumor progression. Front. Biosci. 10: 1881-1896.
Suh, J., B.Z. Zhu, and B. Frei. 2003. Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide. Free Radic. Biol. Med. 34: 1306-1314.
Sun, J., Y.F. Chu, X.Z. Wu, and R.H. Liu. 2002. Antioxidant and anti proliferative activities of common fruits. J. Agric. Food Chem. 50: 7449-7454.
Tappia, P.S., M.R. Dent, and N.S. Dhalla. 2006. Oxidative stress and redox regulation of phospholipase D in myocardial disease. Free Radic. Biol. Med. 41: 349-361.
Teixeira, S., C. Siquet, C. Alves, I. Boal, M.P. Marques, F. Borges, J.L. Lima, and S. Reis. 2005. Structure-property studies on the antioxidant activity of flavonoids present in diet. Free Radic. Biol. Med. 39: 1099-1108.
Thannickal, V.J. and B.L. Fanburg. 2000. Reactive oxygen species in cell signaling. Am. J. Physiol. Lung Cell Mol. Physiol. 279: L1005-1028.
Thompson, T.C. 1990. Growth factors and oncogenes in prostate cancer. Cancer Cells. 2: 345-354.
Thornberry, N.A. and Y. Lazebnik. 1998. Caspases: Enemies within. Science. 281: 1312-1316.
Trichopoulou, A., T. Costacou, C. Bamia, and D. Trichopoulos. 2003. Adherence to a Mediterranean diet and survival in a Greek population. N. Engl. J. Med. 348: 2599-2608.
Troszynska, A., I. Estrella, M.L. Lopez-Amores, and T. Hernandez. 2002. Antioxidant activity of pea (Pisum sativum L.) seed coat acetone extract. Lebensm-Wiss. Technol. 35: 158-164.
Trueba, G.P., G.M. Sanchez, and A. Giuliani. 2004. Oxygen free radical and antioxidant defense mechanism in cancer. Front. Biosci. 9: 2029-2044.
Tudela, J.A., E. Cantos, J.C. Espin, F.A. Tomas-Barberan, and M.I. Gil. 2002. Induction of antioxidant flavonol biosynthesis in fresh-cut potatoes. Effect of domestic cooking. J. Agric. Food Chem. 50: 5925-5931.
145
USDA. 2007. Potato 2006 Summary, National Agricultural Statistics Services, USDA, Washington, DC.
Valko, M., M. Izakovic, M. Mazur, C.J. Rhodes, and J. Telser. 2004. Role of oxygen radicals in DNA damage and cancer incidence. Mol. Cell. Biochem. 266: 37-56.
Valko, M., D. Leibfritz, J. Moncol, M.T. Cronin, M. Mazur, and J. Telser. 2007. Free radicals and antioxidants in normal physiological functions and human disease. Int. J. Biochem. Cell Biol. 39: 44-84.
Valko, M., H. Morris, and M.T. Cronin. 2005. Metals, toxicity and oxidative stress. Curr. Med. Chem. 12: 1161-1208.
Valko, M., H. Morris, M. Mazur, P. Rapta, and R.F. Bilton. 2001. Oxygen free radical generating mechanisms in the colon: Do the semiquinones of vitamin K play a role in the aetiology of colon cancer? Biochim. Biophys. Acta. 1527: 161-166.
Valko, M., C.J. Rhodes, J. Moncol, M. Izakovic, and M. Mazur. 2006. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 160: 1-40.
Velioglu, Y.S., G. Mazza, L. Gao, and B.D. Oomah. 1998. Antioxidant activity and total phenolics in selected fruits, vegetables, and grain products. J. Agric. Food Chem. 46: 4113-4117.
Veluri, R., R.P. Singh, Z. Liu, J.A. Thompson, R. Agarwal, and C. Agarwal. 2006. Fractionation of grape seed extract and identification of gallic acid as one of the major active constituents causing growth inhibition and apoptotic death of DU145 human prostate carcinoma cells. Carcinogenesis. 27: 1445-1453.
Vogelstein, B. and K.W. Kinzler. 2004. Cancer genes and the pathways they control. Nature Med. 10: 789-799.
Waalkes, M.P., J. Liu, J.M. Ward, and B.A. Diwan. 2004. Mechanisms underlying arsenic carcinogenesis: Hypersensitivity of mice exposed to inorganic arsenic during gestation. Toxicology. 198: 31-38.
Wang, S., K.E. Panter, W. Gaffield, R.C. Evans, and T.D. Bunch. 2005. Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development. Anim. Reprod. Sci. 85: 243-250.
Yan, W.K. 2001. GGEbiplot - A windows application for graphical analysis of multienvironment trial data and other types of two-way data. Agron. J. 93: 1111-1118.
146
Yan, W.K. and L.A. Hunt. 2001. Interpretation of genotype x environment interaction for winter wheat yield in Ontario. Crop Sci. 41: 19-25.
Yang, C.S., J. Liao, G.Y. Yang, and G. Lu. 2005. Inhibition of lung tumorigenesis by tea. Exp. Lung Res. 31: 135-144.
Yang, S.A., S.H. Paek, N. Kozukue, K.R. Lee, and J.A. Kim. 2006. Alpha-chaconine, a potato glycoalkaloid, induces apoptosis of HT-29 human colon cancer cells through caspase-3 activation and inhibition of ERK 1/2 phosphorylation. Food Chem. Toxicol. 44: 839-846.
Yen, G.C., P.D. Duh, and D.Y. Chuang. 2000. Antioxidant activity of anthraquinones and anthrone. Food Chem. 70: 437-441.
Yilmaz, G., M. Tuzen, N. Kandemir, D. Mendil, and H. Sari. 2005. Trace metal levels in some modern cultivars and Turkish landraces of potato. Asian J. Chem. 17: 79-84.
Zhao, Y., S. Shen, J. Guo, H. Chen, D.Y. Greenblatt, J. Kleeff, Q. Liao, G. Chen, H. Friess, and P.S. Leung. 2006. Mitogen-activated protein kinases and chemoresistance in pancreatic cancer cells. J. Surg. Res. 136: 325-335.
Zurer, I., L.J. Hofseth, Y. Cohen, M. Xu-Welliver, S.P. Hussain, C.C. Harris, and V. Rotter. 2004. The role of p53 in base excision repair following genotoxic stress. Carcinogenesis. 25: 11-19.
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VITA Name: Magnifique Ndambe Nzaramba Address:
Department of Horticultural Sciences Texas A&M University, College Station, TX 77843-2133
Education:
Ph.D. (Plant Breeding) 2008, Texas A&M University, College Station M.S. (Horticulture) 2004, Texas A&M University, College Station B.S. (Agriculture) 1998, Makerere University, Kampala-Uganda
Professional Background:
Teaching Assistant, Department of Horticultural Sciences, TAMU Research Assistant, Department of Horticultural Sciences, TAMU Research Scientist, ISAR, Butare, Rwanda Station Head, Ruhande Agroforestry Station, ISAR, Butare, Rwanda
Awards and Honors: • First Place, Graduate Student Paper Competition, 92nd Annual Meeting of Potato
Association of America, Buffalo, NY, August 12, 2008 • National Potato Council Scholarship, NPC, Washington, DC, August 2007 • Tom Slick Senior Graduate Research Fellow, College of Agriculture and Life
Sciences, Texas A&M University, 2006 • Student Scholarship Award, Phi Beta Delta, Alpha Eta Chapter, Honor Society for
International Scholars, Spring 2006 • Texas A&M University Faculty Senate Aggie Spirit Award, May 10, 2004 Publication: Nzaramba, M.N., J.B. Bamberg, and J.C. Miller, Jr. 2007. Effect of propagule type
and growing environment on antioxidant activity and total phenolic content in potato germplasm. Am. J. Potato Res. 84:323-330
Hale, A.L., M.W. Farnham, M.N. Nzaramba, and C.A. Kimbeng. 2007. Heterosis for horticultural traits in broccoli. Theor. Appl. Genet. 115:351-360.
Blessington, T., J.C. Miller, Jr., M.N. Nzaramba, A.L. Hale, L. Reddivari, D.C. Scheuring, and G.J. Hallman. 2007. The effect of low-dose gamma irradiation and storage time on carotenoids, antioxidant activity, and phenolics in potato cultivar Atlantic. Am. J. Potato Res. 84:125-131.
Nzaramba M.N, D.C. Scheuring, and J.C. Miller, Jr. 2007. The influence of production environments on antioxidant activity, phenolics, and specific gravity in potato (Solanum tuberosum L.). Am. J. Potato Res. 84:107. (Abstr.).
Nzaramba, M.N, A.L. Hale, D.C. Scheuring, and J.C. Miller, Jr. 2005. Inheritance of antioxidant activity and its association with seed coat color in cowpea (Vigna unguiculata (L.) Walp.). J. Amer. Soc. Hort. Sci. 130:386-391.