extraction of bioactive compounds from whole red cabbage
TRANSCRIPT
University of Nebraska - Lincoln University of Nebraska - Lincoln
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Dissertations, Theses, & Student Research in Food Science and Technology Food Science and Technology Department
5-2011
Extraction of Bioactive Compounds from Whole Red Cabbage and Extraction of Bioactive Compounds from Whole Red Cabbage and
Beetroot using Pulsed Electric Fields and Evaluation of their Beetroot using Pulsed Electric Fields and Evaluation of their
Functionality Functionality
Valli Kannan University of Nebraska-Lincoln, [email protected]
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EXTRACTION OF BIOACTIVE COMPOUNDS FROM WHOLE RED CABBAGE
AND BEETROOT USING PULSED ELECTRIC FIELDS AND EVALUATION OF
THEIR FUNCTIONALITY
by
Valli Kannan
A THESIS
Presented to the Faculty of
The Graduate College at the University of Nebraska
In Partial Fulfillment of Requirements
For the Degree of Master of Science
Major: Food Science & Technology
Under the Supervision of Professor Harshavardhan Thippareddi
Lincoln, Nebraska
May, 201l
ABSTRACT
EXTRACTION OF BIOACTIVE COMPOUNDS FROM WHOLE RED
CABBAGE AND BEETROOT USING PULSED ELECTRIC FIELDS AND
EVALUATION OF THEIR FUNCTIONALITY
Valli Kannan, MS
University of Nebraska, 2011
Adviser: Harshavardhan Thippareddi
Application of high voltage pulsed electric fields (PEF) to plant tissues increases
porosity of the plant cells, enhancing juice extraction from fruits and vegetables. Red
cabbage and beetroot are rich sources of bioactive compounds anthocyanins and
betalains, respectively. Pulsed electric field (red cabbage: 1 kV/cm electric field strength,
0.66 µF and 20 pulses; red beets: 1.5 kV/cm electric field strength, 0.66 µF and 20
pulses) was applied to whole red cabbage and beetroot to enhance juice extraction. Total
polyphenols, anthocyanins (red cabbage), betalains (beetroot) and total antioxidant
activity (ABTS and DPPH) of the extracted juice was determined. The bioactivity of the
juices on colon cancer cell line HCT-116 (p53 +/+ and p53 -/-) were analyzed. The anti-
proliferative activity, apoptosis and cytotoxicity were determined. Pulsed electric field
treatment increased (p 0.01) juice extraction, total phenolics, anthocyanins (red
cabbage), betalains (beetroots) and antioxidant activity of both red cabbage and beetroot
juice compared to non-PEF extracted samples. The juices reduced colon cancer cell
proliferation, increased apoptosis and did not exhibit cytotoxicity. There was no
difference between the PEF extracted and non-PEF extracted juice with respect to its
bioactivity on cancer cells.
Keywords: pulsed electric field, extraction, red cabbage, beetroot, bioactivity
Acknowledgements
I would like to thank my adviser Dr. Harshavardhan Thippareddi, for giving me an
opportunity to work in this project and for his patience and confidence in my work.
I thank Dr. Jeyamkondan Subbiah, for helping me with the pulsed electric field system.I
would like to thank my other committee members Dr. Milford Hanna, Dr. Randy
Wehling and Dr. Jairam Vanamala for their invaluable inputs to my research. A special
thanks to Dr. Yixiang Xu for helping me with the analytical methods. And also I would
like to thank Tanya Gachovska for her help with the pulsed electric field system. I thank
Robert Weber, and Courtney from Dr. Milford Hanna‘s lab for helping me with the
instron.
I take it as an honor, working with Dr. Jairam Vanamala and his lab members in
Colorado State University. I thank him for giving me an opportunity like this. Especially,
I would like to thank Dr. Lavanya Reddivari for teaching me the cell culture experiments
and helping me out with my data analysis. My lab mates in CSU, Sridhar Radhakrishnan,
Gaurav Madiwale, Twila Henley and Andy Kester, a special thanks to all of them for
making my stay in Fort Collins special and unforgettable.
I want to thank all my lab mates and friends specially, KN, Krish, SG, Carol, Mauricio,
Aikansh, Dr. Rao, Dr. Birla and Alex. You all made sure that I never missed home and I
really enjoyed the time we spent together.
And finally a special thanks to my parents, my sister and all my family members, for their
encouragement and patience. Without their support, I could not have done so much.
i
Table of Contents
CHAPTER 1 ....................................................................................................................... 1
INTRODUCTION .............................................................................................................. 1
1.1 References ................................................................................................................. 5
CHAPTER 2 ....................................................................................................................... 9
LITERATURE REVIEW ................................................................................................... 9
2.1 Phenolics ................................................................................................................... 9
2.2 Flavonoids ............................................................................................................... 12
2.2.1 Distribution of Flavonoids in Plants ................................................................. 12
2.2.2 Functions of Flavonoids in Nature ................................................................... 12
2.2.3 Human Uses of Flavonoids............................................................................... 13
2.3 Anthocyanins ........................................................................................................... 14
2.3.1 Introduction ...................................................................................................... 14
2.3.2 History .............................................................................................................. 14
2.3.3 Evolution of Anthocyanins ............................................................................... 15
2.3.4 Anthocyanin Structure ...................................................................................... 16
2.3.5 The Biosynthesis of Anthocyanins ................................................................... 17
2.3.6 Anthocyanins Sources ...................................................................................... 20
2.3.7 Anthocyanins as a Food Colorant ..................................................................... 20
2.3.7 The Properties and Reactions of Anthocyanins ................................................ 22
2.3.8 Anthocyanins and Health.................................................................................. 26
2.3.9 Anthocyanin Absorption and Metabolism ........................................................ 35
2.3.10 Extraction of Anthocyanins ............................................................................ 38
2.4 Pulsed Electric Field................................................................................................ 41
2.4.1 History .............................................................................................................. 41
2.4.2 Fundamental Aspects of Pulsed Electric Field Treatment ................................ 42
2.4.3 Mechanisms of Membrane and Cell Damage in External Electric Fields ........ 45
2.4.4 Applications ...................................................................................................... 48
2.5 References ............................................................................................................... 51
ii
CHAPTER 3 ..................................................................................................................... 67
EXTRACTION OF JUICE FROM WHOLE RED CABBAGE BY PULSED ELECTRIC
FIELD AND DETERMINING THEIR BIOACTIVITY ................................................. 67
Abstract ......................................................................................................................... 67
3.1. Introduction ............................................................................................................ 68
3.2. Materials and Methods ........................................................................................... 70
3.2.1 Total Phenolics ................................................................................................. 72
3.2.2 Total Monomeric Anthocyanins by pH-differential Method ........................... 73
3.2.3 Total Antioxidant Activity by ABTS Assay..................................................... 74
3.2.4 Total Antioxidant Activity by DPPH Assay..................................................... 75
3.2.5 Cell Lines .......................................................................................................... 75
3.2.6 Anti-Proliferation Assay ................................................................................... 76
3.2.7 Pro-Apoptotic Assay......................................................................................... 76
3.2.8 Lactate Dehydrogenase (LDH) Enzyme Assay (Cytotoxicity) ........................ 77
3.2.9 Experimental Design ........................................................................................ 78
3.2.10 Statistical Analyses ......................................................................................... 78
3.3. Results and Discussion ........................................................................................... 79
3.3.1 Amount of Juice................................................................................................ 79
3.3.2 Total Phenolics ................................................................................................. 80
3.3.3 Total Monomeric Anthocyanins ....................................................................... 81
3.3.4 Total Antioxidant Activity: ABTS and DPPH Assays ..................................... 82
3.3.5 Anti-Proliferation Activity ............................................................................... 83
3.3.6 Pro-Apoptotic Activity ..................................................................................... 85
3.3.7 Lactate Dehydrogenase (LDH) Enzyme Assay (Cytotoxicity) ........................ 86
3.4. Conclusions ............................................................................................................ 86
3.5 References ............................................................................................................... 87
3.6 Legend to Figures .................................................................................................... 94
CHAPTER 4 ................................................................................................................... 104
EXTRACTION OF BETALAINS FROM BEETROOTS USING PULSED ELECTRIC
FIELD AND DETERMINING THEIR BIOACTIVITY ............................................... 104
Abstract ....................................................................................................................... 104
iii
4.1. Introduction .......................................................................................................... 105
4.1.1 Biosynthesis of Betacyanins and Betaxanthins .............................................. 106
4.1.2 Health Benefits of Betalains ........................................................................... 107
4.1.3 Juice Extraction .............................................................................................. 108
4.2 Materials and Methods .......................................................................................... 109
4.2.1 Total Phenolics ............................................................................................... 111
4.2.2 Betalain Analysis ............................................................................................ 111
4.2.3 Total Antioxidant Activity by ABTS Assay................................................... 112
4.2.4 Total Antioxidant Activityby DPPH Assay.................................................... 113
4.2.5 Cell Lines ........................................................................................................ 113
4.2.6 Anti- Proliferation Assay ................................................................................ 114
4.2.7 Pro-Apoptotic Assay....................................................................................... 114
4.2.8 Lactate Dehydrogenase (LDH) Enzyme Assay (Cytotoxicity) ...................... 115
4.2.9 Experimental Design ...................................................................................... 116
4.2.10 Statistical Analyses ....................................................................................... 116
4.3 Results and Discussion .......................................................................................... 117
4.3.1 Amount of Juice.............................................................................................. 117
4.3.2 Total Phenolics ............................................................................................... 117
4.3.3 Betacyanins and Betaxanthins ........................................................................ 118
4.3.4 Total Antioxidant Activity: ABTS and DPPH Assays ................................... 119
4.3.5 Anti-Proliferation Activity ............................................................................. 120
4.3.6 Pro-Apoptotic Activity ................................................................................... 121
4.3.7 Lactate Dehydrogenase (LDH) Enzyme Activity (Cytotoxicity) ................... 122
4.4 Conclusion ............................................................................................................. 122
4.5 References ............................................................................................................. 123
4.6 Legend to Figures .................................................................................................. 129
CHAPTER 5 ................................................................................................................... 137
SUMMARY AND CONCLUSION ............................................................................... 137
Reccomendations for Future Research ........................................................................... 139
Appendix ......................................................................................................................... 140
Procedures ....................................................................................................................... 140
iv
A.1 Total Phenolics ................................................................................................. 140
A.2 Total Monomeric Anthocyanin Concentration ................................................. 142
A.3 Betalain Concentration ..................................................................................... 143
A.5 Total Antioxidant Activity: DPPH Assay ......................................................... 145
A.6 Anti-Proliferation Assay: WST-1 Assay .......................................................... 146
A.7 Anti-Proliferation Assay: Cell Count ............................................................... 146
A.8 Pro-Apoptotic Assay: Caspase-Glo .................................................................. 147
A.9 Cytotoxicity: LDH Assay ................................................................................. 147
v
List of Figures
Chapter 1
Figure 1. 1 Acquired capabilities of Cancer ....................................................................... 1
Figure 1. 2 Flowchart for juice extraction using conventional method and method
implemented in this study. ............................................................................... 3
CHAPTER 2
Figure 2. 1. Basic structure and numbering system of flavonoid ..................................... 10
Figure 2. 2 Classification of anthocyanins. ....................................................................... 17
Figure 2. 3 Biosynthesis of anthocyanins ........................................................................ 19
Figure 2. 4 Thermal degradation anthocyanins................................................................. 25
Figure 2. 5 Potential mechanisms of cancer prevention by anthocyanins ........................ 30
CHAPTER 3
Figure 3. 1 Experimental set-up. ....................................................................................... 96
Figure 3. 2 Measurement of impedance to determine pulse number. ............................... 97
Figure 3. 3 Red cabbage after PEF treatment. .................................................................. 98
Figure 3. 4 Effect of pulsed electric field treatment of whole red cabbage on the
extraction (juice) volume, total phenolics and anthocyanin concentration in the
juice and antioxidant activity (ABTS and DPPH assay) of the extracted juice.
......................................................................................................................... 99
Figure 3. 5 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the anti-proliferative activity of colon cancer cell line HCT-
116 (p53 +/+ and p53 -/-) determined using cell counter. ............................ 100
Figure 3. 6 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the anti-proliferative activity of colon cancer cell line HCT-
116 (p53 +/+ and p53 -/-) determined using WST-1 assay. .......................... 101
vi
Figure 3. 7 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the pro-apoptotic activity of colon cancer cell line HCT-116
(p53 +/+ and p53 -/-). .................................................................................... 102
Figure 3. 8 Cytotoxicity (%) of juice extracted from red cabbage treated with pulsed
electric field treated and non-pulsed electric field on colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-). ................................................................... 103
CHAPTER 4
Figure 4. 1 Structure of betalamic acid, betacyanin (betanidin) and betaxanthin
(indicaxanthin). ............................................................................................. 105
Figure 4. 2 Biosynthesis of betacyanin (betanidin) and betaxanthin (indicaxanthin) ..... 107
Figure 4. 3 Measurement of impedance to determine pulse number. ............................. 130
Figure 4. 4 Effect of pulsed electric field treatment of whole beetroot on the extraction
(juice) volume, total phenolics concentration in the juice and antioxidant
activity (ABTS and DPPH assay) of the extracted juice. ............................. 131
Figure 4. 5 Effect of pulsed electric field treatment of whole beetroot on the extraction of
betalains (betacyanins and betaxanthins) of the extracted juice. ................. 132
Figure 4. 6 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the anti-proliferative activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-) determined using cell counter. ................... 133
Figure 4. 7 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the anti-proliferative activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-) determined using WST-1 assay. ................ 134
Figure 4. 8 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the pro-apoptotic activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-). ................................................................... 135
Figure 4. 9 Cytotoxicity (%) of juice extracted from whole beetroot treated with pulsed
electric field treated and non-pulsed electric field on colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-). ................................................................... 136
vii
List of Tables
CHAPTER 2
Table 2. 1. Classification of flavonoids. ........................................................................... 11
Table 2. 2 Common sources of anthocyanins. .................................................................. 21
1
CHAPTER 1
INTRODUCTION
Cancer is one of the leading causes of death all over the world ranked after
cardiovascular disease. Cancer is caused both by internal and external factors. The
internal factors include inherited mutations, hormones, immune conditions and mutations
due to metabolism. The external factors are tobacco, infectious organisms, chemicals and
radiation (Cancer facts and figures, 2009). In any cancer type, six essential alterations
occur in cells that lead to malignant growth (Figure 1.1; Hanahan, 2000). The six
alterations are:
i. Self-sufficiency in growth signals.
ii. Insensitivity to growth-inhibitory (antigrowth) signals.
iii. Evasion of programmed cell death (apoptosis).
iv. Limitless replicative potential.
v. Sustained angiogenesis, and
vi. Tissue invasion and metastasis.
Figure 1. 1 Acquired capabilities of Cancer (Hanahan, 2000).
2
Many epidemiological studies have consistently shown that a high dietary intake
of fruits, vegetables and whole grains is strongly associated with reduced risk of
developing chronic diseases like cancer and cardiovascular diseases. The National
Academy of Sciences in a report on diet and health recommended that consuming five or
more servings of fruits and vegetables for reducing the risk of both cancerand heart
disease (National Academy of Sciences Report, 1989). Phytochemicals are nonnutrient
plant compounds derived from fruits, vegetables, grains, and other plant foods that have
been linked to reducing the risk of major chronic diseases. Phytochemicals can be
classified as carotenoids, phenolics, alkaloids, nitrogen-containing compounds, and
organosulfur compounds (Liu, 2004).
Anthocyanins, a group of polyphenols present in fruits and vegetables, are the
most abundant antioxidants in our diet (Scalbert, 2000; Francis, 1989). The interest in
anthocyanins increased because of the recognition of their potential health benefits (He,
2010). Several epidemiological studies have suggested associations between the
consumption of anthocyanin-rich foods and prevention of chronic diseases like, cancer
(Reddivari et al. 2007; Zhao et al., 2004), cardiovascular disease (Gracia et al., 1997),
and others. Red cabbage is a rich source of acylated anthocyanins which are stable to pH,
temperature, light and acidic gastric digestion conditions (Tanchev, 1969; Dyrby et al.,
2001; Walkowiak-Tomczak, 2007; McDougall et al., 2007). Red cabbage color has been
found to suppress colorectal cancer in mice (Hagiwara et al., 2001).
Betalains are a class of water-soluble pigments which is classified into
betacyanins and betaxanthins. Beetroots are a rich source of betalains and beetroot red is
a widely permitted natural food colorant (E162; Azeredo et al., 2009; Henry, 1996).
3
Betalains contribute to the antioxidant activity of beetroots, thus preventing against
cancer (Cao et al., 1996; Kapadia et al., 2003).
Studies have shown that anthocyanins and betalains are extracted using an organic
solvent or water (Metivier et al., 1980; Kanner et al., 2001). Several novel extraction
technologies have also been used to extract anthocyanins (Corrales, 2008; Sun et al.,
2007). Pulsed electric field is a non-thermal processing technology used to extract juice
and other value-added components from fruits and vegetables (Bouzrara, 2000;
Gachovska et al., 2010). To extract juice or intracellular components from a plant cell, a
sample preparation step such as homogenizing is required. We implemented a method,
where PEF pre-treatment is given to red cabbages and beetroots without any sample
preparation step.
Fruit/Vegetable
Mashing
Treatment
Pressing
Juice/Analysis
Fruit/Vegetable
Treatment
Pressing
Juice/Analysis
4
Figure 1. 2 Flowchart for juice extraction using conventional method and method
implemented in this study.
The objective of this study was to:
i. Extract juice from whole red cabbage and beetroot using pulsed electric field
without a sample preparation step.
ii. Quantify the bioactive compounds in the extracted juice and
iii. Evaluate the anti-carcinogenic activity of the extracted juice using colon cancer
cell lines HCT-116 (p53+/+ and p53 -/-).
5
1.1 References
1. Azeredo, H. M. C. (2009). Betalains: properties, sources, applications, and
stability: A review. International Journal of Food Science & Technology, 44(12),
2365-2376.
2. Bouzrara, H., Vorobiev, E. (2000). Beet juice extraction by pressing and pulsed
electric fields. International Sugar Journal, 102(1216), 194-200.
3. Cancer facts and figures 2009. our.cancer.org/downloads/STT/500809web.pdf.
4. Cao, G., Sofic, E., & Prior, R. L. (1996). Antioxidant capacity of tea and common
vegetables. Journal of Agricultural and Food Chemistry, 44(11), 3426-3431.
5. Corrales, M., Toepfl, S., Butz, P., Knorr, D., & Tauscher, B. (2008). Extraction of
anthocyanins from grape by-products assisted by ultrasonics, high hydrostatic
pressure or pulsed electric fields: A comparison. Innovative Food Science &
Emerging Technologies, 9(1), 85-91.
6. Dyrby, M., Westergaard, N., & Stapelfeldt, H. (2001). Light and heat sensitivity
of red cabbage extract in soft drink model systems. Food Chemistry, 72(4), 431-
437.
7. Francisa, F. J. & Pericles C. (1989). Food Colorants: Anthocyanins. Critical
reviews in food science and nutrition, 28(4), 273.
8. Gachovska, T., Cassada, D., Subbiah, J., Hanna, M., Thippareddi, H., & Snow, D.
(2010). Enhanced anthocyanin extraction from red cabbage using pulsed electric
field processing. Journal of Food Science, 75(6), E323-9.
9. Hagiwara, A., Yoshino, H., Ichihara, T., Kawabe, M., Tamano, S., Aoki, H.,
Koda, T., Nakamura, M., Imaida, K., Ito, N., & Shirai, T. (2002). Prevention by
6
natural food anthocyanins, purple sweet potato color and red cabbage color, of 2-
amino-1-methyl-6-phenylimidazo[4,5-b]Pyridine (PhIP)-associated colorectal
carcinogenesis in rats initiated with 1,2-dimethylhydrazine. The Journal of
toxicological sciences, 27(1), 57-68.
10. Hanahan, D. (2000). The hallmarks of cancer. Cell, 100(1), 57-70.
11. He, J. & Giusti, M. M. (2010). Anthocyanins: Natural colorants with health-
promoting properties. Annual Review of Food Science and Technology, 1(1), 163-
187.
12. Henry BS (1996). Natural Food Colours. In: Hendry GAF,
Houghton JD (Eds) Natural Food Colourants (pp. 40-79). London: Chapman &
Hall.
13. Kanner, J., Harel, S., & Granit, R. (2001). Betalains: A new class of dietary
cationized antioxidants. Journal of Agricultural and Food Chemistry, 49(11),
5178-5185.
14. Kapadia, G. J., Tokuda, H., Konoshima, T., & Nishino, H. (1996).
Chemoprevention of lung and skin cancer by Beta vulgaris (beet) root extract.
Cancer letters, 100(1-2), 211-214.
15. Liu, R. H. (2004). Potential synergy of phytochemicals in cancer prevention:
Mechanism of action. The Journal of nutrition, 134(12), 3479S-3485S.
16. McDougall, G. J., Fyffe, S., Dobson, P., & Stewart, D. (2007). Anthocyanins
from red cabbage-Stability to simulated gastrointestinal digestion.
Phytochemistry, 68(9), 1285-1294.
7
17. Metivier, R. P., Francis, F. J., & Clydesdale, F. M. (1980). Solvent extraction of
anthocyanins from wine pomace. Journal of Food Science, 45(4), 1099-1100.
18. National Academy of Sciences, Committee on Diet and Health, National
Research Council (1989) Diet and Health: Implications for reducing chronic
disease risk. National Academy Press, Washington, DC. .
19. Reddivari, L., Vanamala, J., Chintharlapalli, S., Safe, S. H., & Miller, J. C.
(2007). Anthocyanin fraction from potato extracts is cytotoxic to prostate cancer
cells through activation of caspase-dependent and caspase-independent pathways.
Carcinogenesis, 28(10), 2227-2235.
20. Satue-Gracia, M., Heinonen, M., & Frankel, E. N. (1997). Anthocyanins as
antioxidants on human low-density lipoprotein and lecithin-liposome systems.
Journal of Agricultural and Food Chemistry, 45(9), 3362-3367.
21. Scalbert, A. & Williamson, G. (2000). Dietary intake and bioavailability of
polyphenols. The Journal of nutrition, 130(8), 2073S-2085S.
22. Tanchev, S. S. & Timberlake, C. F. (1969). The anthocyanins of red cabbage
(Brassica oleracea). Phytochemistry, 8(9), 1825-1827.
23. Walkowiak-Tomczak, D. & Czapski, J. (2007). Colour changes of a preparation
from red cabbage during storage in a model system. Food Chemistry, 104(2), 709-
714.
24. Yangzhao Sun, Xiaojun Liao, Zhengfu Wang, Xiaosong Hu and Fang Chen
(2007). Optimization of microwave-assisted extraction of anthocyanins in red
raspberries and identification of anthocyanin of extracts using high-performance
8
liquid chromatography – mass spectrometry . European Food Research and
Technology, 225(3-4), 511-523.
25. Zhao, C., Giusti, M. M., Malik, M., Moyer, M. P., & Magnuson, B. A. (2004).
Effects of commercial anthocyanin-rich extracts on colonic cancer and
nontumorigenic colonic cell growth. Journal of Agricultural and Food Chemistry,
52(20), 6122-6128.
9
CHAPTER 2
LITERATURE REVIEW
2.1 Phenolics
Plants synthesize a variety of compounds for growth and are classified as primary
and secondary metabolites. The primary metabolites include carbohydrates, proteins and
lipids, which play an important role in the basic life functions such as cell division and
growth, respiration, storage and reproduction. The secondary metabolites are synthesized
from lipid precursors and aromatic amino acids and play an important role in the
adaptation of plants to their environment (Bourgaud, 2001).
Based on their biosynthetic pathways, the plant secondary metabolites are
classified into three large families (Bourgaud, 2001): (i) phenolics, (ii) terpenes and (iii)
steroids. Within the three families, the phenolic compounds or polyphenols are the most
numerous with more than 8,000 phenolic structures currently identified (Harborne, 1986).
Natural polyphenols have a wide variety of structures. They range from simple
compounds containing an aromatic ring with one or more hydroxyl moieties to highly
complex polymeric substances such as tannins (Dragsted, 1993; Harborne, 1994). Most
of the polyphenols are conjugated with one or more sugar residues. Commonly, glucose
or rutinose is attached, although other sugars galactose, rhamnose and arabinose are also
found. Conjugation with organic acids, lipids and other phenols are also observed (Bravo,
1998).
10
The polyphenols can be classified into ten classes depending on their basic
chemical structure (Table 2.1; Harborne, 1989). Flavonoids constitute the most common
and widely distributed group of plant phenolics and have a skeleton of diphenylpropanes
(C6-C3-C6) and consist of two benzene rings (A and B) linked through three carbons
forming a closed pyran ring (C) with the benzene A ring (Figure 2.1). Biosynthesis of the
flavonoids starts with the A ring derived from a molecule of resorcinol or phloroglucinol
synthesized in the acetate/malonate pathway and the B ring derived from the carbons of
phenylalanine in the Shikimate pathway (Figure 2.1; Bravo, 1998).
Flavonoids are further subdivided into 13 classes, with more than 5,000
compounds. Most of the flavonoids are low molecular weight and are soluble depending
on their polarity and chemical structure (Table 2.1; degree of hydroxylation,
glycosylation, acylation, etc.; Bravo, 1998).
Figure 2. 1. Basic structure and
numbering system of flavonoid
11
Table 2. 1. Classification of flavonoids (Bravo, 1998).
Chalcones
Dihydrochalcones
Aurones
Flavones
Flavonols
Dihydroflavonol
Flavanones
Flavanol
Flavandiol or leucoanthocyanidin
Anthocyanidin
Isoflavonoids
Biflavonoids
Proanthocyanidins or condensed
tannins
12
2.2 Flavonoids
2.2.1 Distribution of Flavonoids in Plants
Flavonoids are present in various plant groups and are more abundant in the
angiosperms. Animals and fungi except marine coral lack the flavonoids. The most
primitive plant species, in which the flavonoids have been found, is green algae
(Markham, 1969). They are present universally in leaves and petals and also frequently
found in other organs: fruits, roots, sepals, etc. Flavonoids also occur in leaf waxes, bud
excretions and in heartwood of trees (Harborne, 1977). Specifically, flavonoids are
present in the vacuoles of guard cells, epidermal cells as well as sub-epidermal cells and
shoots of plants (Hutzler, 1998).
2.2.2 Functions of Flavonoids in Nature
The main function of flavonoids in plants is to attract insects to help in pollination
and in seed dispersion because of their bright color and patterns. Many of the scientific
breakthroughs in the past 150 years have used flavonoids as an important factor. Besides
providing pigmentation in flowers, fruits, seeds, and leaves, flavonoids have key roles in
signaling between plants (allelopathy), and microbes, in male fertility of some species,
in
defense as antimicrobial agents (phytoalexins) and feeding deterrents, and in UV
protection (Shirley, 2001). The epidermal layer of plants absorbs 90-99% of the incident
ultraviolet radiation (Robberecht, 1978; Caldwell, 1983), acting as a shield against the
potentially harmful UV-A and UV-B radiation (Schnabl, 1986; Hutzler, 1998).
13
2.2.3 Human Uses of Flavonoids
Flavonoids have applications in food industry as well as cut flower industry.
Flavonoids are generally responsible for color, taste, prevention of fat oxidation and
protection of vitamins and enzymes in foods (Yao, 2004).
The flavonoids are considered as bioactive compounds, the ―extra-nutritional‖
constituents that are typically naturally occurring in small quantities in plant products and
lipid rich foods. The bioactive compounds of plant origin include phenolic compounds
(flavonoids, resveratrol and phytoestrogens) and are present in tea, red wine; lycopene;
organosulfur compounds; plant sterols; dietary fibers; isothiocyanates; and monoterpenes.
The bioactive compounds have beneficial health effects (Kris-Etherton, 2002), and
contribute to the antioxidant properties of green vegetables, fruits, olive and soybean oils,
red wine, chocolate and tea. Some flavonoids are antiallergic, anti-inflammatory,
antiviral, antidiabetic, antiproliferative, anticarcinogenic and have effects on mammalian
metabolism. Flavanoids act as antioxidants in the prevention of cancer and cardiovascular
diseases. They also protect from ulcers, allergies, vascular fragility and viral and bacterial
infections (Yao, 2004).
Several epidemiological studies have reported an inverse association between
flavonoid intake and the risk of coronary disease and cancer.
14
2.3 Anthocyanins
2.3.1 Introduction
Anthocyanins constitute the most conspicuous group of flavonoids. Anthocyanins
(from the Greek anthos, a flower; and kyanos, dark blue), best known of the natural
pigment are responsible for the blue, purple, violet, magenta, red and orange colors of a
majority of plant species and their products (Delgardo-Vargas, 2000; Mazza, 1993).
Anthocyanins are found in flowers, leaves, stems, fruits, seeds, and the root tissue of
plants.
2.3.2 History
The name anthocyanin was first coined by Marquart in 1835 and is still retained
to the present day. Vegetable pigments served as a matter for investigation dating back to
1664. In ―Experiments and Considerations Touching Colours‖ by Robert Boyle discusses
the red and green color the plant pigments produce when treated with an acid or a base. In
1849, Morot isolated a blue pigment from cornflower and found that it contained carbon,
hydrogen and oxygen. Wigand in 1862, suggested that anthocyanin ―arises by the
oxidation of a colorless tannin-like chromogen, a substance widely distributed in plants‖.
As to the chemical processes involved in anthocyanin formation, Palladin in 1905, put
forward a hypothesis that ―anthocyanins are formed from a flavone by the action of an
oxidising enzyme or oxidase‖.
15
Willstatter (1913) stated that ―all natural anthocyanins are present in the plant in
the condition of glucosides, and that many of them are very unstable in water solution,
and readily change in these circumstances to a colorless isomer‖. The change can be
prevented by adding certain neutral salts, and also acids, to the pigment solution. The
explanation, according to Willstatter, of these phenomena lies in the fact that anthocyanin
is an oxonium compound having a quinonoid structure and containing tetravalent oxygen.
The quinonoid structure is rendered stable by the formation of oxonium salts with acids
or with neutral salts, such as sodium nitrate or chloride. The pigment itself, in the neutral
state, is purple in color, and has the structure of an inner oxonium salt. With acids the
pigment forms red oxonium salt, and with alkaline blue salts, the position of the metal
being undetermined.
2.3.3 Evolution of Anthocyanins
The anthocyanins pelargonidin and delphinidin types occur more frequently in
advanced plants, replacing cyanidin which is considered to be the simplest and most
primitive pigments. This trend was found to be related to natural selection for bright
orange and scarlet colors (pelargonidin) in tropical climates where humming birds
pollinate the flowers and for deep blue colors (delphinidin) in temperate climates where
bees are the major pollinators (Harborne, 1977).
The anthocyanins are widespread in higher plants, except for the 9 caryophylalles
families, i.e., Aizoaceae, Amarathaceae, Basellaceae, Cactaceae, Chenopodiaceae,
Didiereaceae, Nyctaginaceae, Phytolaccaceae and Portulacaceae except Caryophyllaceae
16
and Molluginaceae. Instead, the members of these families synthesize the betalain
pigments (Piattelli, 1964).
2.3.4 Anthocyanin Structure
Anthocyanins consist of an aglycone or anthocyanidin bound to one or more sugar
moieties (glycosylated derivatives of 3, 5, 7, 3‘-tetrahydroxy-flavylium cation). While the
sugar at position-3 is always present, additional sugars at positions-5 and 7 are often
attached. Free anthocyanidins are rarely formed in plants as the electron deficiency of the
flavylium cation makes the free anthocyanidins highly reactive and thus, the molecule is
very unstable. Since sugar stabilizes the anthocyanin molecule, glycosidic structures are
more stable than the anthocyanidins. The sugar moieties can be a mono or disaccharide
unit or acylated with a phenolic or aliphatic acid but the sugar molecules are commonly
glucose, rhamnose, galactose or arabinose. Further, modification of the glycosides
through acylation and through complexation with non-cyanic flavonoids and metal ions
can also occur. Subtle differences in cell pH, along with the possibilities of
copigmentation and ionic complexation lead to a virtually unlimited degree of color
variation.
17
Figure 2. 2 Classification of anthocyanins.
There are a total of 539 anthocyanins reported to be isolated from plants
(Andersen, 2005) and all of this variation is based on a limited number of fundamental
flavonoids. The commonly occurring anthocyanidins in foodstuffs are cyanidin (Cy;
30%), delphinidin (Dp; 22%), pelaragonidin (Pg; 18%), peonidin (Pn; 7.5%), malvidin
(Mv; 7.5%) and petunidin (Pt; 5%; Nyman 2001; Francis 1999; Figure 2.2). Cyanidin,
delphinidin and pelaragonidin are non-methylated anthocyanidins and are widespread in
nature compared to the three methylated anthocyanidins, peonidon, malvidin and
petunidin.
2.3.5 The Biosynthesis of Anthocyanins
There are several points in the flavonoid pathway where the product of one
reaction provides the substrate for two or more subsequent steps (Figure 2.3). The first
18
step in the biosynthesis of flavonoids is the formation of chalcones. The enzyme chalcone
synthase (CHS) catalyses the reaction between p-coumaroyl CoA and malonyl CoA to
yield chalcone. P-coumaroyl CoA and malonyl CoA is synthesized from L-phenylalanine
in the phenyalanine/hydroxycinnamate pathway (Strack, 1993) catalysed by
phenylalanineammonia-lyase (PAL), cinnamic acid 4-hydroxylase (CY4H), and
hydroxycinnamatecoenzyme A ligase (C4L; Hahlbrock, 1989).
Chalcone isomerase (CHI), the second enzyme in the flavonoid pathway catalyzes the
cyclization of a chalcone to the corresponding flavanone isomers (Naringenin). The next
enzyme is the Flavanone 3-hydroxylase (FN3H) which catalyzes the stereospecific-
flavanones to the dihydroflavonols (dihydrokaempferol).
Stafford (1982) detected the dihydroflavonol reductase (DFR) activity and
identified the product as a flavan-3, 4-diol. The reaction involves the reduction of
dihydrokaempferol to flavan-3, 4-diol. Flavan-3, 4-diols, which are also called
leucoanthocyanidins, are very unstable molecules and are likely converted into the
corresponding anthocyanidin. Adequate demonstration of this conversion in a cell-free
system has not been accomplished, although Stafford and others use the designation ANS
(anthocyain synthase) to provide a name that can identify the overall reaction (Stafford,
1990). Other workers refer to the enzyme as leucoanthocyanidin dioxygenase (LDOX;
Sparvoli, 1994). The conversion of leucoanthocyanidin to anthocyanin is likely both very
fast and closely associated with a UDP glucose flavonoid 3-glucosyltransferase (UF3GT)
that converts the newly formed anthocyanidin into the corresponding 3-O-glucoside (the
anthocyanin).
19
Figure 2. 3 Biosynthesis of anthocyanins (Zufall, 2004).
Anthocyanins are present in the vacuole, to be more specifically, in anthocyanoplasts, the
membrane bound spherical vesicles present inside the vacuole (Pecket, 1980) or in the
cytoplasm (Harborne, 1988). Anthocyanoplasts are formed during pigment synthesizes,
and are dispersed to produce a pigmented vacuole. In flowers, anthocyanins are
exclusively located in epidermal cells, and only occasionally in the mesophyll cells
(Delgado-Vargas, 2000). The flavonoids (anthocyanins) in grape berry are accumulated
20
in the central vacuole of three to six sub-epidermal cell layers at high concentrations in an
acidic environment (Moskowitz, 1981).
2.3.6 Anthocyanins Sources
Natural sources of anthocyanins include a variety of colored fruits, vegetables,
spices and nuts. These include berries, grapes, plums, peaches, pomegranate, red onion,
red cabbage, egg plant, purple corn, purple sweet potato, black bean, pistachios and dates
(Wu, 2006). The concentration and type of anthocyanins present in each fruit and
vegetable vary considerably. For example, strawberry or raspberry contains cyanidin and
pelaragonidin derivatives and red cabbage contain only cyanidin derivatives, while
grapes and blueberries have almost all the anthocyanidin derivatives. In the U.S., the
average daily intake of anthocyanins has been estimated to be 215 mg during the summer
and 180 mg during the winter (Kuhnau, 1976). The common dietary sources include a
variety of colored fruits and vegetables as well as fruit based processed foods and
beverages like jelly, juices and red wine (Table 2.2).
2.3.7 Anthocyanins as a Food Colorant
Consumers are increasingly associating natural colorants to health benefits.
Interest in the use of natural pigments in the food inplace of synthetic food dyes has
increased since the ban of FD&C Red Nos. 2 and 4 and Red No. 40 (Sapers, 1981).
Pigments from natural sources are generally considered safe, display a wide range of
colors and in addition, they possess health promoting benefits. Some of the natural colors
21
used in food include carotenoids from carrots, betalains from beets and anthocyanins
from grapes, red cabbage and other fruits and vegetables.
Table 2. 2 Common sources of anthocyanins (Clifford, 2000).
Commodity Content (mg litre−¹ or mg kg−¹)
Blackberry 1,150
Blueberry 825 – 4,200
Boysenberry 1,609
Cherry 20 – 4,500
Chokeberry 5,060 – 10,000
Cranberry 600 – 2,000
Cowberry 1,000
Currant (black) 1,300 – 4,000
Elderberry 2,000 – 10,000
Grape (red) 300 – 7,500
Loganberry 774
Orange, Blood (juice) 2,000
Plum 20 – 250
Raspberry (black) 1,700 – 4,277
Raspberry (red) 100 – 600
Raspberry (red) single strength juice 4 – 1,101
Sloe 1,600
Strawberry 150 – 350
Cabbage (red) 250
Eggplant 7,500
Onion Up to 250
Rhubarb Up to 2,000
Wines (red) 240 – 350
Wines (port) 140 – 1,100
According to the numbering system used by the Codex Alimentarius Commission,
anthocyanins (any anthocyanin-derived colorant) are listed as natural colorant by the
European Union (EU) legislation as product E163. In the U.S., the FDA (Food and Drug
Administration) has a different list of ―natural‖ colors that do not require certification
(without any FD & C numbers), and anthocyanins can be obtained either from ―grape
22
color extract‖, ―grape skin extract‖, or ―fruit juices or vegetable juices‖. In addition to
grapes, other common sources include red cabbage, blackcurrants, radishes,
elderberries, chokeberries, blackberries, black raspberries, and black currants. Espin
(2000) studied the antiradical capacity (DPPH) of anthocyanin based fruit extract (black
chokeberry, black-thorn and strawberry) and commercial colorant Ponceau 4R. The fruit
based natural color exhibited antioxidant activity comparable to that of butylated
hydroxyanisole whereas the synthetic colorant Ponceau 4R did not. Espin (2000)
suggested that natural extracts can be used as potential colorants and as antiradicals.
Enocyanin, an anthocyanin was extracted from grape skin and used as a food
colorant in Italy as early as 1879 and was initially used to color red wine. But later, it was
used as a food colorant and a number of countries started producing the colorant. Other
sources, elderberries, blueberries, cherry plums, black currants, red raspberries, red sweet
potatoes are also used for the extraction of anthocyanins to be used as food colorant.
When Imbert (1966) compared pigments of commercial blackberry extract, enocyanin
and red sweet potato, the sweet potato pigments (acylated cyanidin and peonidin based
anthocyanins) were more stable than the commercial blackberry extract and enocyanin.
2.3.7 The Properties and Reactions of Anthocyanins
pH
Anthocyanins co-exist in aqueous solution in equilibrium between four species
depending on pH: red flavylium cation, blue quinonoidal base, colorless carbinol
23
pseudobase and the colorless chalcone (Brouillard and Delaporte, 1977; Brouillard and
Dubois, 1977; Brouillard, 1990; Brouillard, 1982).
Below pH 2, anthocyanins exist predominantly in the red flavylium cation form.
As the pH increases, kinetic and thermodynamic competition occurs between the
hydration reaction of the flavylium cation and the thermodynamic proton transfer
reaction related to the acidic hydroxyl groups of the aglycone. Rapid hydration of the
flavylium cation occurs at the C-2 position to generate the colorless carbinol pseudobase
at pH values ranging from 3 to 6 which can further undergo ring opening to a chalcone
pseudo-base (Fossen, 1998). At any pH, the chalcone present is much lower than the
carbinol form. The formation of chalcone can be increased with an increase in
temperature. Deprotonation of the flavylium cation to generate the quinonoidal bases
occurs at slightly acidic to neutral condition, and this reaction is extremely fast
(Brouillard, 1977). Further, deprotonation of the quinonoidal bases takes place with the
formation of purplish resonance-stabilized quinonoid anions between pH values of 6 and
7.
The reverse transition from carbinol pseudobase to flavylium cation is almost
instant upon acidification (Brouillard, 1977). Reversion of chalcone to flavylium cation is
a very slow process and takes hours to reach completion (Francis, 1989).
Temperature
Foods containing anthocyanins are thermally processed before consumption and
this process greatly influences the anthocyanin content in the final product (Giusti, 2003).
24
Generally, temperatures higher than 70°C cause rapid degradation and discoloration of
anthocyanins (Cemeroglu, 1994; Kirca, 2003). Opening of the pyrylium ring with a loss
of the B-ring and chalcone formation which further decompose to coumarin glucoside
derivatives was considered as the first anthocyanin degradation step (Markakis, 1957;
Hrazdina, 1971). Hydrolysis of the glycosidic moiety and formation of aglycon even at
acidic pH as the initial degradation reaction, has been reported (Adams, 1973). Tanchev
(1976) detected seven compounds following thermal degradation of anthocyanins, among
which quercetin, phloroglucinaldehyde and protocatechuic acid were identified by paper
chromatography. It is believed that the thermal degradation of anthocyanins leads to two
end products: aldehyde and benzoic acid derivatives or chalcone and coumaric acid
(Markakis, 1974; Jackman, 1992; Piffaut, 1994; Seeram, 2001). In case of acylated
anthocyanins, acyl-glycoside moieties were first split off from the flavylium backbone
and finally ended up with phenolic acids and phloroglucinaldehyde (Figure 2.4; Sadilova,
2007). Anthocyanin degradation is generally correlated to a loss or change in color and a
decline in the antioxidant activity. Sadilova (2007) studied the structural changes of
anthocyanins extracted from black carrot, elderberry and strawberry heated over 95°C.
The antioxidant activity of anthocyanins from strawberry had lower antioxidant activity
compared those from black carrots had the maximum antioxidant activity compared
among the three extracts. The loss of anthocyanin bioactivity could not be compensated
by the antioxidant activity of the newly formed phenolics. The degradation of black
currant anthocyanins was studied using black currant nectar for a period of six months.
25
Iversen (1999) reported a loss of 50 % of the anthocyanins after six months stored at
20°C.
Figure 2. 4 Thermal degradation anthocyanins (Sadilova, 2007).
Light
Light is a significant factor accelerating anthocyanin degradation similar to
temperature and oxygen. While temperature effects predominate in degradation of
cranberry anthocyanins, at higher temperature (55°C), fluorescent light effects
predominate at lower temperature (40°C). Also, molecular oxygen is required for
degradation of anthocyanins by light. The degradation of anthocyanins by light follows
first order kinetics, similar to degradation at higher temperatures (Attoe, 1981).
Photodegradation of anthocyanins proceeds from flavylium cation via carbinol pseudo-
base to the chalcone (Maccarone, 1985), although direct photodegradation of the
26
flavylium cation can also occur (Furtado, 1993). Acylation of anthocyanins reduces their
susceptibility to light (Giusti, 1996).
Other factors like oxygen, hydrogen peroxide, ascorbic acid, and enzymes like
polyphenoloxidase and peroxidase can also degrade anthocyanins. Sugar degraded
anthocyanins at a faster rate compared to thermal degradation. The degradation products
of sugar - furfural and 5-hydroxymethylfurfural greatly accelerate the pigment
destruction (Daravingas, 1968).
2.3.8 Anthocyanins and Health
Interest in anthocyanins intensified after the recognition of their potential health
benefits (Scalbert, 2000). Epidemiological studies have suggested a reverse association
between consumption of polyphenols and incidence of chronic diseases cancer and
cardiovascular deiseases. Anthocyanins are among the most abundant polyphenols in
fruits and vegetables and possess potent antioxidant activity and have been incorporated
into the human diet for several centuries. The anthocyanins were components of
traditional herbal medicines used by North American Indians, the Europeans and the
Chinese and were normally derived from dried leaves, fruits (berries), storage roots or
seeds. Anthocyanin-rich mixtures and extracts (though not purified compounds) have
been used historically to treat conditions as diverse as hypertension, pyrexia, liver
disorders, dysentery and diarrhoea, urinary problems including kidney stones and urinary
tract infections, and the common cold. Consumption of anthocyanins have been reported
27
to improve vision and blood circulation (Konczak, 2004), antiulcer activity (Cristoni,
1987) and UV protection (Sharma, 2001).
Anthocyanins and Prevention of Cardiovascular Diseases
Oxidation of low-density lipoprotein (LDL) triggers accumulation of macrophage
white blood cells in the artery wall. Rupture of the plaque deposits oxidized cholesterol
onto the artery wall, leading to atherosclerosis and eventually cardiovascular diseases
(Aviram, 2000). Mortality from coronary heart disease can be reduced by moderate
consumption of red wine. The primary mechanisms for this reduced risk include reduced
platelet coagulation and higher circulatory high-density lipoprotein cholesterol (HDL).
Other effects, inhibition of lipoprotein oxidation, free-radical scavenging and modulation
of eicosanoid metabolism are presumed to play a role in the reduction of atherosclerosis
(Mazza, 2007). Gracia et al. (1997) evaluated the antioxidant activity of several
anthocyanins in vitro on human LDL and in a lecithin-liposome system and reported
inhibition of LDL oxidation increases with increase in concentration of the anthocyanin.
Malvidin exhibited greater inhibition of oxidation followed by delphinidin, cyanidin and
pelarogonidin.
Antioxidant Activity
Reactive oxygen species (ROS), including free radicals (O₂‾), singlet oxygen
(¹O₂), hydrogen peroxides (H₂O₂) and reactive nitrogen species such as nitric oxide are
generated in animals and humans as a result of metabolic reactions. The cells utilize ROS
as biological stimuli. They influence biochemical and molecular processes and cause
28
changes in cells during differentiation, aging and transformation. They influence the
expression several genes, signal transduction pathways and act as sub-cellular
messengers for certain growth factors (Allen, 2000). Thus, they are important to the
immune system, cell signaling, and several normal body functions (He, 2010). However,
over production of ROS may exceed the physiological antioxidant capacity of the
antioxidant enzymes (glutathione peroxidase, catalase, superoxide dismutase) and
antioxidant compounds (glutathione, tocopherol or ascorbic acid). This may lead to
damage and dysfunction of enzymes, cell membranes and genetic material (Stintzing,
2004). Eventually, such sustained damage can lead to degenerative diseases like
inflammation, cardiovascular disease, cancer and aging (Allen, 2000). Antioxidants can
reduce oxidative molecular and cellular damage by preventing the damage to the
biomolecules by free radicals or by interrupting the perpetuation of the free radical
species.
Anthocyanins are potent antioxidants in vitro, quench free radicals and terminate
the chain reaction that is responsible for the oxidative damage. Renis (2008)
demonstrated the antioxidant effects of anthocyanins in vitro using human colon cancer
cell line (CaCo2). Cyanidin and Cyanidin-3-glucoside treatment reduced cell growth and
cell proliferation in a dose-dependent way, decreased ROS level by any concentration of
CY and only at the lowest concentration, by CY3G and increased cell cycle/stress
proteins expression. Both the compounds affected DNA fragmentation, highlighting their
antioxidant activity. The authors concluded that anthocyanins exhibit antioxidant
properties and could therefore be useful in the treatment of pathologies where free radical
29
production plays a key role (Acquaviva, 2003). The antioxidant activity of anthocyanins
is largely because of the presence of hydroxyl groups in position 3 of ring C and also in
the 3‘, 4‘ and 5‘ positions in ring B of the molecule. Anthocyanidins have superior
antioxidant activity compared to their respective anthocyanins and the activity decreases
as the number of sugar moieties increase (Wang, 2008).
Ramirez-Toetosa (2001) investigated the antioxidant activity of anthocyanins in
vitamin-E depleted rats. The rats were fed with rations containing highly purified
anthocyanin-rich extract (3-glucopyranoside of delphinidin, cyanidin, petunidin, peonidin
and malvidin) for 12 weeks. Consumption of anthocyanin rich diet significantly improves
plasma antioxidant activity and decreases vitamin E deficiency enhanced lipid
peroxidation and DNA damage. The antioxidant activity of many anthocyanins was
comparable to the commercial antioxidants such as tert-butylhydroquinone (TBHQ),
butylated hydroytoluene (BHT), butylated hydroxyanisole (BHA) and vitamin E
(Seeram, 2001).
Anticarcinogenic Activity
The anticancer activity of anthocyanins has been established largely based on in
vitro evidence. The anticancer activities of anthocyanins are attributed to multiple
mechanisms: anti-oxidative activity, phase II enzyme activation, anti-cell proliferation,
induction of apoptosis, anti-inflammatory effects, anti-angiogenesis, anti-invasiveness,
and induction of differentiation (Figure 2.5). Several in vivo studies in animals have
shown the effect of anthocyanins in multiple cancer types. Most of the observed cancer
30
preventive effects were on gastrointestinal tract (GIT)-related organs including oral
cavity, esophagus and the colon. In the GIT lumen, anthocyanins are largely available
and are in direct contact with the epithelial layer (He, 2005), whereas their availability to
non-GIT organs requires delivery through blood (He, 2010).
Figure 2. 5 Potential mechanisms of cancer prevention by anthocyanins (Hou, 2003).
Phase II Enzymes
The human body is continually exposed to potential carcinogens in the
environment. The body deals with these compounds through a system of xenobiotic-
metabolizing enzymes called phase I and phase II enzymes. Phase I enzymes oxidize
xenochemicals into electrophilic intermediates, that can induce DNA damage and
mutations. These mutations are responsible for the carcinogenic activity of many
chemicals. Phase II detoxification enzymes are responsible for metabolizing products of
the phase I metabolic reactions and degrade reactive electrophilic intermediates through
31
conjugation or reduction reactions, protecting cells from oxidative damage (Srivastava,
2007). Activation of phase II detoxifying and antioxidant enzymes is considered a major
protective strategy against carcinogenesis. Activation of phase II detoxifying enzymes
such as UDP-glucuronyl transferase (UGT), glutathione S-transferase (GST), and
NAD(P)H:quinone oxidoreductase (QR) results in the detoxification of carcinogens and
hence reducing their anticarcinogenic effect. Anthocyanins can induce phase II
antioxidant and detoxifying enzymes in clone 9 liver cells (Shih, 2007). Among the ten
anthocyanins tested, cyanidin, kuromanin, delphinidin and malvidin showed higher
efficacy in their antioxidant capacity and induction of phase II enzymes.
Antiproliferative Activity
Anthocyanins and anthocyanin rich extracts from fruits and vegetables have
exhibited anti-proliferative activity towards multiple cancer cell types in vitro. Cell
proliferation was inhibited by the ability of anthocyanins to block various stages of the
cell cycle via effects on cell cycle regulator proteins (e.g, p53, p21, p27, cyclin D1, cyclin
A, etc.; Wang, 2008). Zhang (2005) evaluated the anti-proliferated activity of
anthocyanidins (cyanidin, delphinidin, pelargonidin, petunidin and malvidin) and
anthocyanins (cyanidin-3-glucoside, cyanidin-3-galactoside, delphinidin-3-galactoside
and pelargonidin-3-galactoside) against human cancer cell lines (stomach, colon, breast,
lung, and central nervous system). The anthocyanins did not inhibit cell proliferation
whereas the anthocyanidins showed antiproliferation activity. Malvidin showed the
highest inhibitory activity, owing to the presence of a free hydroxyl group at 3-position in
the C-ring, the hydroxyl groups at 3 and 4‘and methoxy groups at 3‘ and 5‘ positions in
32
the B ring in malvidin is responsible for its higher activity. In anthocyanins, the hydroxyl
group at 3-position is substituted by various sugar moieties and hence prevented it from
being inhibitory to cancer cell proliferation.
Apoptosis
Apoptosis or programmed cell death plays a key role in the development and
growth regulation of normal cells, and is often dys-regulated in cancer cells. Some of the
most effective chemopreventive agents are strong inducers of apoptosis in premalignant
and malignant cells (Wang, 2008). Apoptosis is characterized by chromatin condensation,
cytoplasmic blebbing, and DNA fragmentation. The cells that have undergone
apoptosis are rapidly recognized and engulfed by macrophages before cell lysis,
and subsequently removed without inducing inflammation (Hou, 2003). Several
anthocyanins, anthocyanidins and anthocyanin-rich extracts have exhibited pro-apoptotic
effects in multiple cancer cell lines in vitro. They induce apoptosis through both intrinsic
(mitochondrial) and extrinsic (FAS) pathways. Srivastava (2007) studied the effect of
anthocyanin fractions from four high bush blueberry cultivars and reported that the
anthocyanin fractions increased apoptosis by increasing the DNA fragmentation and
caspase-3 activity when compared to the control cells.
Inflammation
Inflammation is a complex biological in response to tissue injury. Several cancers
occur at sites of inflammation as inflammatory cells provide a microenvironment
favorable for tumor development (He, 2010). Abnormal up-regulation of inflammatory
proteins cyclooxygenase-2 (COX-2), which convert arachidonic acid to various
33
prostaglandins, prostacyclin, thromboxane A₂ and nuclear factor-kappa B (NF-κB) is a
common occurrence in many cancers. Anthocyanins inhibit the mRNA and/or protein
expression levels of COX-2, NF-κB and various interleukins, thus exhibiting their anti-
inflammatory effect (Wang, 2008). Seeram (2001) investigated several cherry and berry
anthocyanins for their cyclooxygenase inhibitory and antioxidant activities. The anti-
inflammatory activity of anthocyanins from raspberries and sweet cherries were
comparable to that of commercial anti-inflammatory drugs like ibuprofen and naproxen
(Seeram, 2001).
Angiogenesis
Angiogenesis is the process of new blood vessel formation from the existing
vascular network and is a key event that feeds tumor growth and cancer metastasis.
Vascular endothelial growth factors (VEGF) play a crucial role in angiogenesis, and
VEGF expression is frequently enhanced in developing tumors. Anthocyanins have
been shown to suppress angiogenesis through several mechanisms such as: inhibition of
H₂O₂ - and tumor necrosis factor alpha (TNF-α)-induced VEGF expression in
epidermal keratinocytes, and by reducing VEGF and VEGF receptor expression in
endothelial cells (Wang, 2008). Matsunaga (2009) showed that bilberry anthocyanins
(delphinidin, cyanidin and malvidin) inhibited VEGF-induced tube formation in a co-
culture of human umblical vein endothelial cells and fibroblasts in a concentration
dependent manner. Bilberry was also shown to decrease vessel proliferation in chick
chlorioallantoic membrane angiogenesis model in vivo (Ozgurtas, 2009).
34
Metastasis
The progression of a tumor from being in situ to invasive is a major prerequisite
for spread of cancer and involves the acquisition of cell motility, surface adhesion
properties and activity of extracellular proteases like serine proteinase, matrix
metalloproteinases (MMPs) and cathepsins. These proteases degrade the basement
membrane collagen and eventually result in metastasis (Chen, 2006). Mulberry
anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside exerted a dose-dependent
inhibitory effect on the migration and invasion of highly metastatic A549 human lung
carcinoma cells (Chen, 2006). The two cyanidins were shown to decrease the expression
of matrix metalloproteinase-2 (MMP-2) and urokinase-plasminogen activator (u-PA) in a
dose-dependent manner and enhance the expression of tissue inhibitor of matrix
metalloproteinase-2 (TIMP-2) and plasminogen activator inhibitor (PAI).
The number of sugar and hydroxyl groups present on the B ring plays an
important role in determining the bioactivity of anthocyanin. The ortho-dihydroxyphenyl
structure on the B-ring is also essential for the activity. Many authors who studied the
activity of cyanidin and cyanidin-glucosides or cyanidin-diglucosides report that cyanidin
showed better activity at much lower concentration compared to the antioxidant activity
of commercial antioxidants like BHA, BHT. The biological activities of anthocyanins
increase with decreasing number of sugar units and/or increasing number of hydroxyl
groups on the flavylium cation (Hou, 2003).
35
2.3.9 Anthocyanin Absorption and Metabolism
Most of the research on the health benefits of anthocyanins has been performed in
vitro. To validate the prominent health promoting benefits, it is important to consider the
bioavailability of anthocyanins in vivo. Bioavailability is generally defined as the
proportion of the nutrient that is digested, absorbed and metabolized through normal
pathways. Anthocyanins have a range of molecular structures as a result of biosynthesis
and accumulation in plant tissues. The naturally occurring anthocyanin can be modified
before consumption, particularly during the processing and storage/shelf life (McGhie,
2007). Anthocyanins experience numerous environments and physiochemical conditions
which may also modify them once they enter the body. Within the gastrointestinal tract
there are distinct environments, stomach, small intestine, and colon, is characterized by
different pH and microbial populations; both of which can modify the anthocyanins. The
pH of the stomach is very low (pH 1-2), which maintains the anthocyanins in their stable
form, the flavylium cation (McDougall, 2005). In contrast, the pH of the small and large
intestine is largely neutral, where anthocyanins are much less stable and undergo multiple
modifications (Seeram, 2001). And the microbial populations especially in the colon are
likely to modify the molecular structures of anthocyanins. Deglycosylated and
demethylated to the corresponding aglycones occurs on exposure of the anthocyanins to
gut microflora (Keppler, 2005). The aglycones are unstable at neutral pH and are rapidly
degraded to their corresponding phenolic acids, aldehydes through cleavage of the C-
ring.
36
Anthocyanins are large, highly water-soluble molecules which cannot be absorbed
by the cells or into the circulatory systems of animals and humans. Despite their high
water solubility, the anthocyanins can cross the cell membrane and enter the cell.
However, this absorption varies between different anthocyanidins and sugar moieties. For
example, comparison between delpinidin-3-gluciside and malvidin-3-glucoside showed
that malvidin had higher transport efficiency than delphidin. Malvidin has two methyl
groups which make them slightly non-polar compared to delphidin which has all the
hydroxyl groups. Similarly, a comparison between glucosides and galactosides showed
that glucosides had higher transport efficiencies. Absorption of the anthocyanins could
start from the stomach (Piskula, 1999) or the small intestine. When absorbed by stomach,
they appear rapidly in the circulatory system. Bilitranslocase have been shown to interact
with the absorbed anthocyanins and transfer it from the stomach to the liver. Some
studies also suggest that anthocyanins were significantly absorbed by jejunum and
slightly absorbed by duodenal tissue. Once absorbed by the jejunum or duodenum,
activity of aglycones from primary flavonoid-glucoside occurs due to endogenous β-
glucosidase and to a lesser extent the galactosides, xylosides and arabinosides. Free
anthocyanidins are more hydrophobic and are much smaller than their corresponding
anthocyanin, thus can easily penetrate the cell membrane passively. Intact anthocyanins
are also absorbed by the small intestine either by inefficient passive diffusion or by the
sodium-dependent glucose transporter (SGLT1). Acylated anthocyanins are generally
recognized as non-absorbable in the small intestine due to their larger molecular size and
lack of a sugar moiety for transporter binding. Non-absorbed anthocyanins and
37
anthocyanin-rhamnose and anthocyanin-rutinose travel down to the colon, where
tremendous amount of microorganisms (~10¹²/cm³) reside to provide catalytic and
hydrolytic potential (Scheline, 1973). Humans do not have endogenous esterases to
release the phenolic acids from these anthocyanidin-rhamnose and rutinose (Scalbert,
2000). So, the esterase activity of colonic microflora is required for the metabolism of
these compounds and the acylated anthocyanins (Plumb, 1999). The released
anthocyanidins undergo spontaneous ring fission to some extent to generate simple
compounds such as phenolic acids. Anthocyanidins taken up from the GIT lumen are
subsequently metabolized by phase II drug-metabolizing enzymes in the liver to
glucuronides, sulfates and methylates. These conjugated metabolites are likely to be
excreted in urine but alternatively a portion of them may re-enter the jejunum with the
bile and later either being absorbed by the colon entering the enterohepatic circulation
(Ichiyanagi et al., 2005). The absorbed intact anthocyanins and anthocyanidins are
largely excreted in urine whereas the non-absorbed anthocyanins are excreted through
feces.
Anthocyanin distribution in tissues has recently been evaluated in blackberry
extract fed rat and blueberry fed pig models. In rats, the anthocyanin fractions were found
in jejunum, stomach, kidney, liver and brain (Talavera, 2005), whereas in some, the
anthocyanins were found mainly in liver and also in eyes, cortex and cerebellum (Kalt,
2008). These results suggest that the anthocyanins cross the blood-brain barrier and the
blood-retinal barrier to potentially provide protection for brain and eye tissues.
38
2.3.10 Extraction of Anthocyanins
As anthocyanins are located inside the plant cell, organic solvents are used to
extract them from homogenized plant material. In conventional extraction procedures,
acidified organic solvents like methanol, ethanol or acetone are used. These solvents,
along with the acid can denature the cellular membrane and release of intracellular
anthocyanins into the solvent and maintain the anthocyanins in their native form. Use of
excess acid may cause hydrolysis of the sugar residues into furfural and
hydroxymethylfurfural, which have been shown to favor pigment decay. Elevated
temperatures are used for the extraction of anthocyanins because of the increased
diffusion rate and solubility of analytes in the solvent (Ju, 2003). However, temperatures
above 70°C have been shown to degrade anthocyanins at a higher rate (Markakis, 1957).
Thus, conventional extraction technologies are time consuming, require large volumes of
solvent and additional process steps to remove the solvent for useof anthocyanins as
additive in foods.
Novel extraction methods like accelerated solvent extraction, ultrasound assisted
extraction, microwave assisted extraction, high hydrostatic pressure and pulsed electric
field assisted extraction are used instead of the conventional Soxhlet extraction technique.
In accelerated solvent extraction, mostly water is used as a solvent and high temperature
and pressure gives the water a non-polar character. Moreover, the viscosity and the
surface tension of the water decreases and the diffusion rate and solubility of the target
compound increases. But as accelerated solvent extraction employs high temperature,
anthocyanins may degrade during extraction. Petersson (2010) extracted red onion
39
anthocyanins using accelerated solvent extraction at 110°C, and reported that the
extraction effect dominates at first, but soon, the degradation of anthocyanins takes over.
Ultrasound assisted extraction is another novel technique for the extraction of
bioactive compounds. Sound waves with frequencies higher than 20 kHz are involves in
expansion and compression cycles when it travels through a medium. The expansion
creates bubbles in the liquid producing negative pressure. The collapse of a bubble near a
solid surface pushes solvents into cellular materials at high speed, improving mass
transfer. When Corrales (2008) compared ultrasonics, high hydrostatic pressure and
pulsed electric field assisted extraction of anthocyanins from grape by-products, the
ultrasonics had the lowest extraction compared to the other techniques. Compared to
conventional extraction at 70°C for 1 hour, ultrasonics has two-fold more extraction. The
major disadvantage of the ultrasonics is that the waves are restricted to a zone located
near to the ultrasound emitter. And also with increase in the solid particles, the ultrasound
intensity goes down. For a uniform treatment of the sample, continuous stirring of the
sample is required (Wang, 2005). Microwave assisted extraction can be used to achieve
higher extraction rate with high temperature and short time. Water which has high
dielectric constant heats up rapidly and with increase in the temperature, the diffusion
rate increases. The plant matrix can also act as a dielectric and exposure to microwaves
results in heating the matrix, leading to expansion and rupture of the cell wall, liberating
the intracellular compounds. For extractions using microwaves, polar solvents like water
or acetone are required as non-polar solvents like hexane cannot be used. Microwave
assisted extraction is a good alternative to conventional extraction because of the low
40
consumption of solvent and shorter extraction time. Sun (2007) reported an extraction
efficiency of 98.3% of the total red pigments recovery from red raspberries using
microwaves. However, this method requires the presence of a solvent with similar
dielectric constant as water. And also an increase in temperature of the solvent is another
factor to be considered even though literature on the degradation of anthocyanins during
microwave extracted is lacking. High hydrostatic pressure works by deprotonating the
charged groups, disrupting the salt bridges and hydrophobic bonds of the proteins present
in the cell membrane. Because of these changes, the proteins lose their conformational
structure and denature. Hence, the compounds present inside the cell can easily move out
into the solvent. Corrales (2008) extracted anthocyanins from grape skins using high
hydrostatic pressure. With extraction conditions of 100% ethanol, 50°C and 600 MPa, an
improvement (23%) in extraction was reported.. When the high pressure processing and
thermal processing on total antioxidant activity, phenolic, ascorbic acid and anthocyanins
content of strawberry and blackberry purees were studied, high pressure processing did
not have any significant impact on the above properties. However, thermal processing
reduced ascorbic acid, the anthocyanin concentration went down and also the antioxidant
activity reduced when compared to the control (Patras, 2009). Extraction of bioactive
components using high pressure processing requires packaging of the product prior to
high pressure treatment. The sample has to be in vacuum packages and this limits the
amount and quantity of samples which can be treated.
Compared to other extraction technologies, pulsed electric field has been used
widely for extraction of pigments, sugar and other value added metabolites. The major
41
advantage of pulsed electric field, the temperature increase is negligible (< 1°;
Gachovska, 2010). Previous research on extraction of intracellular compounds from plant
cells using pulsed electric field employed a treatment chamber which can treat only
limited quantity of sample and also they require the presence of a solvent like water or
any organic solvent suitable for extraction. In this study, we treated the samples as a
whole without any shredding and also without any solvent.
2.4 Pulsed Electric Field
2.4.1 History
Interest in electric field application for treatment of food and agricultural raw
materials has increased over the past two decades. In 1940s, the electric fields were used
in food processing for purposes other than inactivation of microorganisms. Flaumenbaum
successfully used pulsed electric fields in a process that increases the permeability of
plant tissues, facilitating extraction of cellular fluid. Many applications of PEF focus on
increasing the efficiency of juice extraction from fruits using PEF as pretreatment.
The advantages of electrical treatment applications in the food industry are as
follows:
1. simple and does not require any complex and expensive equipment
2. short processing time
3. possible application of AC electrical fields with industrial parameters
42
4. material processing without any food quality deterioration, in particular, as
compared with traditional thermal processing methods;
5. can be easily applied in a combined mode, as supplementary to any pressing,
thermal or microwave treatment.
2.4.2 Fundamental Aspects of Pulsed Electric Field Treatment
PEF technology is based on a pulsing power delivered to the product placed
between two electrodes. The equipment consists of a high voltage pulse generator and a
treatment chamber and necessary monitoring and control devices. Food product is placed
in the treatment chamber, where two electrodes are connected together with a non-
conductive material to avoid electrical flow from one to the other. Generated high voltage
electrical pulses are applied to the electrodes, which then conduct the high intensity
electrical pulse to the product placed between the two electrodes. The food product
experiences a force per unit charge (the electric field), which is responsible for the
irreversible cell membrane breakdown (Benz, 1980).
System Components
The pulsed electric field processing system consists of a high voltage source,
capacitor bank, switch, treatment chamber, voltage and current measuring devices.
Generation of pulsed electric fields requires a fast discharge of electrical energy within a
short period of time. This is accomplished by the pulse-forming network (PFN), an
electrical circuit consisting of one or more power supplies with the ability to charge
voltages (up to 60 kV), switches (ignitron, thyratron, tetrode, spark gap, semiconductors),
43
capacitors (0.1-10 µF), inductors (30 µH), resistors (2 Ω-10 MΩ), and treatment
chambers (Gongora-Nieto, 2002).
Power Supply
High voltage pulses are supplied to the system via a high voltage pulse generator
at the required intensity, shape, and duration. The high voltage power supply for the
system can either be an ordinary source of direct current (DC) or a capacitor charging
power supply with high frequency alternating current (AC) inputs that provide a
command charge with higher repetitive rates than the DC power supply (Zhang, 1997).
The simplest PFN is an RC (resistance-capacitance) circuit in which a power supply
charges a capacitor that can deliver its stored energy to a resistive load (treatment
chamber) in a couple of microseconds, by activation of a switch (Gongoro-Nieto, 2002).
Total power of the system is limited by the number of times a capacitor can be charged
and discharged in a given time. The electrical resistance of the charging resistor and the
number and size of the capacitors determine the power required to charge the capacitor,
wherein a small capacitor will require less time and power to be charged than a larger
one. The capacitance (F) of the energy storage capacitor is given by:
.........................(2.1)
Where τ (s) is the pulse duration, R (Ω) is the resistance, σ (S/m) is the conductivity of
the food, d (m) is the treatment gap between electrodes, and A (m²) is the area of the
44
electrode surface. The energy stored in a capacitor is defined by the mathematical
expression:
.........................(2.2)
Where Q is the stored energy, is the capacitance, and V is the charge voltage.
The second component of the PFN is the high voltage switching device needed to
discharge the stored energy through the PFN circuit instantaneously. The switch plays an
important role in the efficiency of the PEF system and is selected on its ability to operate
at a high voltage and repetition rate. There are two main groups of switches currently
available: ON switches and ON/OFF switches. ON switches provide full discharging of
the capacitor but can only be turned off when discharging is completed.
For a pulse-forming network system, the relative electrical value of each
component determines the shape of the pulse. In a capacitance-resistance circuit, the
pulse generated is exponentially decaying where the voltage across the treatment
chamber as a function of time is defined as
( )
.........................(2.3)
Where is the voltage charged in the capacitor of the PFN, t is the pulse duration time,
and τ = RC is the time constant where in an RC circuit pulse duration equals
approximately five time constants (Cogdell, 1999).
45
2.4.3 Mechanisms of Membrane and Cell Damage in External Electric
Fields
The main purpose of PEF is to disrupt the plant cell membrane, to increase the
release of intracellular compounds. The PEF treatment removes the cellular turgor
component of the texture and exerts an effect on the viscoelastic properties of plant tissue
(Fincan and Dejmek, 2003; Lebovka, 2003). The damage degree of materials depends on
the electric field treatment strength, E and the treatment time. Electric field selectively
influences the structure of the biological membranes inside the solid-water saturated
biological materials. Because of the very low electrical conductivity of the membrane as
compared to the surrounding liquid inside the biological tissue, the electric field appears
to be concentrated mainly on the membranes.
For an ideal biological cell which is spherical, the transmembrane potential
depends on the angle ‗θ‘ between the external electrical field E and the radius-vector on
the membrane surface (Schwan, 1957):
.........................(2.4)
Here, is the cell diameter, f is a parameter depending on electrophysical and
dimensional properties of the membrane, cell and surrounding media. Intensity of the
electric field generated inside the membrane is and the electric field
enhancement factor may be defined as
46
.........................(2.5)
Because of the selective concentration of the electric field on membranes, the membrane
structure alteration and damage may occur. The critical transmembrane potential of the
membrane damage is of order ~ 1 V (Weaver and Chizmadzhev, 1996) which
corresponds to the critical electric field intensity generated inside the membrane of
order . The PEF treatment with short pulses may cause damage of
biological cells without noticeable heating of the media.
Some of the theories put forward to explain the selective damage of membrane is
electroporation theory, electromechanical, electro-hydrodynamical, visco-elastic,
electrothermal and electro-osmotic instability.
The electromechanical instability theory proposed by Zimmerman (1974) is
widely accepted and proposes the formation of pores in the membrane. This theory
considers the cell membrane as a capacitor that is filled with a dielectric material of a
very low dielectric constant compared to the inside the cell and the surrounding
environment. Because of the difference in the dielectric charges between cell membrane
and inside of cells, free charges accumulate on both sides of the membrane generating a
transmembrane potential of about 10 mV. A normal cell maintains this transmembrane
potential for a variety of energy-linked processes like maintainenance of intracellular pH,
generation of ATP, active transport of solutes etc. Application of an external electric field
results in accumulation of more charges causing membrane compression. The charges
attract each other and more compression takes place and the membrane thickness is
47
reduced and the viscoelastic forces oppose this electrocompression of the membrane.
When the transmembrane potential reaches approximately 1 V, the compressive force
takes over the viscoelastic properties of the membrane and causes electrical membrane
breakdown occurs.
During Joule overheating of the membrane surface, the electrical current flowing
in a conductive media generates the Joule heat and warms up the medium. Here the
membranes are the main Joule heating elements and the local temperature rise on a
membrane is very high and can cause thermal damage of the membrane. Tsong (1991)
proposed that exposure of a biological membrane to an electric field causes the formation
of hydrophobic and hydrophilic pores. Hydrophilic pores conduct electricity generating
localized Joule heating and hence the damage of the semi-permeable membrane of the
cell. The cell membrane also has protein channels, pores and pumps. Joule overheating
can also occur on these protein channels and permanently denature them resulting in the
formation of pores. Electromechanical theory is based on the calculation of balance
between the electric compressive forces and the elastic restoring forces of the membrane.
Breakdown of a membrane occurs when electric compressive forces dominate. This
model predicts thinning of the membrane by 30% of its original thickness.
Most of the theories are not yet fully understood, as numerous of discrepancies
between the theoretical and experimental results exist, partly because of the complexity
of the biological membrane.
48
2.4.4 Applications
The application of PEF for extraction of juice, pigment and other compounds
from food materials is not new. The research on extraction using PEF started in 1949
when Flaumenbaum reported an increase in juice yield from prunes, apples and grapes
using an alternating electric field of 220 V. PEF have been used as a pre-treatment step
prior to juice pressing or as an intermediate treatment after pressing the juice. PEF has
also been used for the extraction of sugar from sugar beets, anthocyanins from a large
number of fruits, vegetables and also from agricultural waste, betalains from red beets
and pectin from orange peel.
Juice Extraction
Pulsed electric field has been largely used in the extraction of juice and
anthocyanins from grapes and grape byproducts. Expression and characteristics of juice
extracted from white grapes was studied by Praporscic (2007) and reported an increase in
juice yield from 49% to 76% with a significant improvement in absorbance and turbidity,
indicating concentration. Juice extraction from Chardonnay white grape using pulsed
electric field with two pressure conditions was studied. A PEF treatment of 400 V/cm
was applied. The PEF pretreatment increased the juice yield by 67 - 75% compared to the
control sample without any treatment (Grimi, 2009).
PEF was used to increase the extraction of juice from alfalfa mash. The mash was
subjected to a PEF treatment of 200 pulses at 1 Hz in two successions. The juice yield
49
increased 38% and also the protein, mineral content and the dry matter increased
significantly compared to non-PEF treated samples (Gachovska et al., 2006).
Bouzrara and Vorobiev (2000) showed an improvement in juice yield from sugar beet
slices treated at 215, 300 and 427 V/cm at 500 pulses. The yield increased 43%, 68% and
79% for the above electric fields.
PEF was used as an intermediate in the cold juice extraction from sugar beet cossettes
using a pilot scale multi-plate and frame pressing equipment and a pulse generator. A
yield of about 80% in juice per initial mass of cossettes was achieved before washing.
Purity of juices was higher following PEF treatment compared to those juices without to
PEF treatment (96-98% and 90-93% respectively; Jemai and Vorobiev, 2004).
The influence of PEF treatment on apple mash (Royal Gala) prior to pressing was
investigated by Toepfl et al. (2005), using a field strength of 0.5 to 5 kV/cm, applying 10-
40 pulses. After a treatment at 3 kV/cm and 20 pulses a juice yield of 83.0% was
obtained after pressing in comparison to 80.1% after enzymatic maceration and 75.4% for
the untreated control.
Anthocyanin Extraction
Corrales (2008) compared extraction of anthocyanins from grape by-products
using ultrasonics, high hydrostatic pressure and pulsed electric field, and reported an
improvement of 10% in extraction compared to HHP and 17% more compared to
conventional extraction, which minimal increase was observed with ultrasonics. They
used electric field strength of 3 kV/cm and 30 pulses and further extraction was carried
50
out at 70°C using ethanol and water as solvents. Lopez (2008) studied the effect of PEF
on the extraction of phenolic compounds during the fermentation of Tempranillo grapes.
A treatment of 5 kV/cm and 10 kV/cm was used. The concentration of anthocyanin
increased 21.5% and 28.6% for 5 kV/cm and 10 kV/cm respectively compared to control
where there was no pretreatment of the must with PEF. PEF was used to extract
anthocyanins from red raspberry. The extraction of anthocyanins increased with an
increase in the number of treatment pulses (Zhang et al., 2006). Toepfl (2006) extracted
anthocyanins from red grapes using PEF at 1.3 kV/cm and 120 pulses prior to pressing.
Increased anthocyanin extraction was achieved compared to the control sample. In terms
of the phenolic content, highest recovery was obtained for PEF treated sample, 3% lower
yield was obtained for high pressure liquid extraction and 35% lower yield was obtained
for conventional extraction method. A PEF treatment of 3 kV/cm and 30 pulses increased
the anthocyanin extraction up to 10% in comparison to high pressure liquid extraction
and 30% compared to convention extraction method.
The advantage of using PEF for the extraction of juice is increase in the yield as
well as purity compared to mechanical pressing of juice. Mechanical pressing breaks the
cells with weaker cell walls and extracts only that juice, sometimes highly turbid needing
further processing such as centrifugation and filtration.
Normal juice extraction procedures do not extract all of the juice from the plant
material. A pretreatment step which will break a majority of the cells in the plant material
and results in higher juice yield is required. Pulsed electric field has been reported to
efficiently extract juice from a variety of plant materials. Previous reports on pulsed
51
electric field treat a limited quantity of sample placed in a treatment chamber at a time. In
industrial scale, treating small quantities is not feasible, more time consuming and
requires design of large treatment chambers. To avoid these problems, the samples were
treated directly by placing them between the electrodes without any pre-preparation step.
This way, more samples can be treated continuously. Many of the papers reporting higher
extraction of juice with PEF analyzed only for their total phenolics, anthocyanin or
betalain concentration and their antioxidant activity. The effect of PEF on the compounds
of the juice and if PEF affects the bioactivity against cancer cells was not tested. In this
experiment, the extracted juice was analyzed for its phenolics and anthocyanin or betalain
concentration, antioxidant activity and their bioactivity was tested against colon cancer
cell lines.
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CHAPTER 3
EXTRACTION OF JUICE FROM WHOLE RED CABBAGE BY PULSED ELECTRIC
FIELD AND DETERMINING THEIR BIOACTIVITY
Abstract
Red cabbage is a rich source of anthocyanins which have health promoting properties and
potential applications as natural food color. Pulsed electric field (PEF) technology used to
enhance juice extraction from plant material by causing irreversible breakdown of the cell
membrane. Red cabbage (whole floret) was treated without preparation (shredding or
mashing), by placing it between two electrodes and 1 kV/cm electric field of 0.66 µF
capacitance and 20 pulses were applied. The treated red cabbage was pressed to express
the juice and the juice was analyzed for total phenolics, anthocyanin concentration and
total antioxidant activity (2, 2ʹ-Azinobis-(3-ethylbenzothiazoline-6-sulphonic acid
(ABTS) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)). Anti-proliferative and pro-
apoptotic activities of the components of the extracted juice were evaluated on colon
cancer cell line HCT-116 (p 53 +/+ and p53 -/-). The juice volume, total phenolics,
anthocyanin concentration and total antioxidant activity increased (p 0.01) for the PEF
extracted red cabbage compared to non-PEF red cabbage. The red cabbage juice,
irrespective of PEF treatment, reduced (p 0.05) HCT-116 cell proliferation and
increased the pro-apoptotic activity. Application of PEF improved juice extraction, total
phenolics and anthocyanin content, and did not affect bioactivity of the extracted juice.
Keywords: pulsed electric field, extraction, red cabbage, bioactivity, cancer
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3.1. Introduction
Anthocyanins, responsible for the red, orange and purple colors of many flowers,
fruits and vegetables also provide several health-promoting benefits (Markakis, 1982).
Common sources of anthocyanins include blueberry, strawberry, grapes, sweet potato,
black beans, red radish, purple corn and red cabbage. Red cabbage is a rich source of
anthocyanins, mainly acylated anthocyanins: cyanidin 3, 5-diglucoside, cyanidin 3-
sophoroside-5-glucoside and cyanidin3-sophoroside-glucoside acylated with sinapic acid
(Tanchev, 1969).
Red cabbage belongs to the Cruciferae family, is inexpensive and is easy to grow,
harvest and store (Pliszka et al., 2009). Red cabbage color can be used as a natural food
color. In general, acylated anthocyanins contain two sugar molecules (glucose and
sophorose) and several aromatic acids (Dyrby, 2001). Other common sources of acylated
anthocyanins are red radishes, red potatoes, black carrots and purple sweet potatoes
(Giusti, 2003). While anthocyanin concentration is reduced after pancreatic digestion
(McDougall, 2007), acylated anthocyanins are stable under acidic gastric digestion
conditions. Acylated anthocyanins are very stable to pH, light and temperature as an
extract, in non-carbonated beverages and pharmaceutical formulations compared to non-
acylated anthocyanins (Dyrby et al., 2001; Walkowaik-Tomczak, 2007; Chigurupati,
2002). Red cabbage color is being marketed commercially as a solution and as a spray
dried powder for use as a food colorant. The red cabbage anthocyanins transition from
purple-red to pink-red to blue-green between pH levels of 3 and 6, respectively. This red
cabbage color can be widely used in wines, beverages, fruit sauces, candies and cakes.
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While anthocyanins have been traditionally used as food colorants, they also
possess health promoting benefits including anticarcinogenic activity. The effect of
anthocyanins on different cancers have been studied, including colon, pancreatic,
esophageal, lung and skin cancers.
Colon cancer is the third most common frequent cause of cancer-related death and
the fourth most common malignancy in the United States (WHO, 2009). Diets high in fat
are believed to cause colon cancer in humans (Chao, 2005) and diets rich in
carbohydrates, fruits, vegetables and fiber may have a protective effect on cancer
development (Terry et al., 2001). Red cabbage color available in the market was found to
suppress colorectal carcinogenesis in mice (Hagiwara et al., 2002). Red cabbage extract
has been reported to possess anti-diabetic and anti-inflammatory effects (Kataya, 2007;
Igarashi, 2000).
Juice from fruits and vegetables is normally extracted using a hydraulic press.
During this process, only a portion of the plant tissue cells are ruptured while some cells
remain intact, resulting in inefficient extraction of juice. Pre-treatment steps such as
heating, freezing/thawing, alkaline and enzymatic treatments are used to increase cellular
disruption. However, some of the pre-treatment steps may destroy the bioactivity of the
resultant juice and may affect its freshness (Chalermchat, 2004). Application of pulsed
electric irreversible damage to cell walls and other structures and improves juice yield
with minimal impact on nutritional quality (Chalermchat, 2004; Bouzrara, 2000; Bazhal,
2001; Ade-Omowaye, 2001). Gachovska (2010) used PEF treatment to enhance
anthocyanin extraction from red cabbage samples and reported a 2.15 times increase in
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anthocyanin extraction compared to non-treated red cabbage. However, the tissue
samples had to be prepared (mashed) to fit the treatment chamber. Similarly, high juice
yield from sugar beets, apple and paprika were reported (Bouzrara, 2000; Bazhal, 2001;
Ade-Omowaye, 2001). However, most of the experimental treatments were done on
small sample sizes (40 g). However, commercially it will be advantageous to have
minimal preparatory steps and scale up to a continuous process.
The objectives were to evaluate
i. juice yield, total phenolics, total monomeric anthocyanins and total
antioxidant activity of juice from PEF extracted and non-PEF extracted
red cabbage and
ii. the effect of red cabbage juice on anti-proliferative and pro-apoptotic
activities of colon cancer cell lines.
3.2. Materials and Methods
All reagents and chemicals were purchased from Fisher Scientific (Pittsburg, PA)
and Sigma-Aldrich (St. Louis, MO), unless otherwise stated. The red cabbages were
purchased from a local market and were used immediately for the experiment. The outer
soiled leaves were removed, weighed, the height and diameter across the stem were
measured. The red cabbages were then randomly assigned to control and treatment
groups based on their weights.
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The pulsed electric field treatment circuit consisted of an exponential decay pulse
generator (CF60/25-12C, Hipotronics, Inc., Brewster, NY), a capacitor (General Atomics
Electronic systems, San Diego, CA) and a spark gap switch. The generator contained a
DC power supply with a maximum voltage of 60 kV and maximum power of 10 mA. The
energy stored in the capacitor was discharged directly to the red cabbage placed between
two stainless steel electrodes. The distance between the electrodes was the height of the
red cabbage placed along its diameter (11 cm). One electrode was a circular stainless
steel plate which was placed on one end of the red cabbage (stem). The ground electrode
was a rectangular plate. Aqueous sodium chloride solution (7% w/w) was applied to the
ground electrode to improve electrical conduction. The insulation material for the two
electrodes was purchased from Deorin (American Plastics Supply & Fabrication, Omaha,
NE). PEF treatment parameters were chosen based on preliminary experiments (not
reported). The voltage applied to the sample was measured with a voltmeter (Model,
P6015 A, Tektronix, Inc., Beaverton, OR). To select the number of pulses, the electrical
impedance, an indirect measure of cell permeability was measured after each pulse
treatment of the sample.
The PEF treatment was given to whole red cabbage by placing it between the two
electrodes at different positions (diagonally not along the stem, along the stem and
cutting into two halves and treating each half separately). The contact between the
electrode and the red cabbage was better only when the red cabbage was placed along its
stem (Figure 3.1). The distance between the electrodes was 12 cm and a voltage of 12 kV
was applied (electric field strength of 1 kV/cm and capacitance 0.66µF). The impedance
72
was measured every 10 pulses, to evaluate the tissue rupture. Impedance vs. pulse number
was plotted and a pulse number of 20 were selected (Figure 3.2). The pulse width was
approximately 15 µs and the frequency of the pulse was 1 Hz. Treatment time is the
product of pulse width and the number of pulses and therefore the treatment time was 0.3
ms. Negligible (<1°C) increase in temperature was observed.
The red cabbages were shredded after treatement using a domestic food processor
(MFP 200, Minipro™, Black & Decker, Baltimore, MD) for 1 min to obtain a
homogenous mash without any addition of water.
The homogenized mash (approximately 40 g) were placed in a juice press as
reported by Gachovska et al. (2010) and pressed (3000 N) using an Instron universal
testing machine (Bluehill2 Software, Norwood, MA).
A portion (5 mL) of the extracted juice was filtered (0.22 µm) and stored at -20°C
to evaluate the bioactivity. The other part was refrigerated and used for
spectrophotometric analysis.
3.2.1 Total Phenolics
Total phenolics were determined using Folin-Ciocalteu reagent method (Singleton and
Rossi, 1965). Briefly, Folin-Ciocalteu reagent (FC; 150 µL of 0.2 M) was added to
diluted sample (35 µL). The samples were thoroughly mixed, held for 5 min at room
temperature and sodium carbonate (115 µL ; 7.5%) was added to the sample and
incubated for 30 min at room temperature (25°C). Absorbance was then measured at 765
73
nm (Synergy 2, BioTek, Winooski, VT) using gallic acid as a standard. The results were
expressed as mg GAE/100 g fresh weight. All samples were analyzed in triplicate.
3.2.2 Total Monomeric Anthocyanins by pH-differential Method
Anthocyanins undergo reversible structural transformations with pH changes,
showing a striking difference in the absorption spectra. The red colored oxonium form
and the colorless hemi-ketal form predominates at pH 1.0 and 4.5, respectively. The pH-
differential method permits accurate and rapid measurement of the total anthocyanins,
even in the presence of polymerized degraded pigments and other interfering compounds
(Giusti and Wrolstad, 2001). Degraded pigments do not change color with pH changes
and they are not included in the measurement as they absorb light at both pH values (1.0
and 4.5; Durst, 2005).
Two dilutions of the sample with potassium chloride buffer (pH 1.0; 0.025 M) and
sodium acetate buffer (pH 4.5; 0.4 M) were prepared. An aliquot (0.1 mL) of the juice
was transferred to a volumetric flask (10 mL) and made up to 10 mL with the
corresponding buffer and absorbance was measured at 520 and 700 nm using a
spectrophotometer (Shimadzu, UV-1800 Spectrophotometer, Columbia, MD). The
absorbance (A) of the sample was calculated using the following equation:
A = ( – ) – ( – ) ………………..(3.1)
The monomeric anthocyanin concentration in the original sample was calculated using
the following formula and expressed as mg Cy3gl equivalents/L:
Monomeric anthocyanin pigment (mgCy3glu eq./L) = (A × MW × DF × 1000)/(ε × l)
74
Where:
A is the absorbance calculated using the previous equation
MW is the molecular weight for cyanidin-3-O-glucoside (449.2).
DF is the dilution factor.
ε is the molar absorptivity of cyanidin-3-O-glucoside (26900).
l is the path-length (1 cm) of the cuvette.
The final results were expressed as mg cy3glu eq/100 g FW.
3.2.3 Total Antioxidant Activity by ABTS Assay
The total antioxidant capacity of the red cabbage juice was determined using 2, 2ʹ-
Azinobis-(3-ethylbenzothiazoline-6-sulphonic acid ( ) and was performed as
described by Re (1999). Pre-formed radical mono-cation was generated by
oxidation of ABTS with potassium persulfate. The working solution was
generated by mixing two stock solutions (8 mM ABTS and 3 mM potassium persulfate)
and allowed to react for at least 12 h at room temperature (25°C) in the dark. The solution
(5 mL) was then diluted with phosphate buffer (45 mL of pH 7.4) containing 150 mM
NaCl (405 mL of 0.2 M Na₂HPO₄, 95 mL of 0.2 M NaH₂PO₄ and NaCl (8.77 g) made
up to 500 mL with deionized water and pH adjusted to 7.4 with 1 M NaOH). To 10 µL of
sample (10 times diluted), 290 µL of /phosphate buffer solution was added. The
samples were mixed and allowed to react for 30 min and measured at 734 nm (Synergy 2,
BioTek, Winooski, VT), using trolox (concentration 0 µg/mL to 500 µg/mL) was used as
75
the standard. The results were expressed as µg trolox equivalents/100 g fresh weight. All
the samples were conducted in quadruplicate.
3.2.4 Total Antioxidant Activity by DPPH Assay
The total antioxidant activity of the beetroot juice was measured using the 1, 1-diphenyl-
2-picrylhydrazyl (DPPH) method described by Brand-Williams (1995). A DPPH stock
solution (24 mg DPPH in 100 mL 80% ethanol) was diluted with ethanol (80% aqueous)
to obtain an absorbance of 1.1 at 515 nm using a spectrophotometer. The diluted sample
(15 µL) was allowed to react with of the diluted DPPH (285 µL) solution for 30 min.
After 30 min, the samples were read at 515 nm (Synergy 2, BioTek, Winooski, VT),
using trolox as a standard. All samples were measured in quadruplicate. The results were
expressed as µg trolox equivalents/100 g fresh weight.
3.2.5 Cell Lines
Human colon carcinoma cells HCT-116 (p53 +/+ and p53 -/-) were grown in
McCoy‘s 5A medium containing phenol red (Sigma, St. Louis, MO) and maintained at
37°C in 5% CO₂ jacketed incubator. The media was supplemented with of bovine serum
albumin (100 mL/L; Hyclone, Fisher, Pittsburg, PA), sodium bicarbonate (2.2 g/L;
Sigma, St. Louis, MO) and streptomycin/penicillin solution (10 mL/L; Fisher, Pittsburgh,
PA).
76
3.2.6 Anti-Proliferation Assay
HCT-116 cells were seeded (6,000 cells/well) and incubated for 24 h at 37°C.The
medium was replaced with Dulbecco‘s modified Eagle‘s medium F-12 media (DMEM)
without phenol red containing 0.25% charcoal stripped serum. Filtered red cabbage juice
was added at concentrations ranging from 5 to 25 µg of GAE/mL of media. Medium
(DMEM without phenol red) and deionized sterile water (pH 4.5) were used as controls.
The cells were incubated for 48 h at 37°C and cell proliferation was measured using the
water soluble tetrazolium assay (WST-1, Roche Molecular Biochemicals, Indianapolis,
IN). WST-1 (10 µL) was added to each well and the cells were incubated for 2 h and the
absorbance of the medium was measured at 480 nm (Synergy 2, BioTek, Winooski, VT).
The HCT-116 cells were plated (5 cells/mL per well) and incubated for 24
h at 37°C. Different concentrations of the red cabbage juice were added to the cells. The
medium and deionized water pH 4.5 served as controls. The cell population was
enumerated using a Cellometer Automated Cell Counter (Nexcelom, Lawrence, MA). All
experiments were performed in triplicate and the results were expressed as means SE.
3.2.7 Pro-Apoptotic Assay
The HCT-116 cells were plated (5 cells/mL per well) incubated for 24 h at
37°C. Different concentrations of the beetroot juice were added to the cells. After 48 h of
incubation, cell number was determined using a Cellometer (Nexcelom, Lawrence, MA)
and 2 cells from each well were transferred to a 96-well opaque plate (Nunc 96
MicroWell Plate White, Thermo Scientific, Rochester, NY). The volume of each well
77
was made to 200 µL with DMEM stripped fetal bovine serum medium without phenol
red. Caspase-Glo® 3/7 reagent (Caspase-Glo® 3/7 buffer and Caspase-Glo® substrate;
100 µL; Promega, Madison, WI) was added to each well. The plate was incubated for 30
min at room temperature (25°C) and the luminescence was measured (Synergy 2,
BioTek, Winooski, VT). The samples were measured in triplicate and the results were
expressed as means ± SE.
3.2.8 Lactate Dehydrogenase (LDH) Enzyme Assay (Cytotoxicity)
The cytotoxicity of the red cabbage juice was determined using a cytotoxicity
detection kit (Roche Molecular Biochemicals, Indianapolis, IN). Cell-free supernatant
(100 µL) from treated cells were collected. DMEM medium (200 µL) containing stripped
fetal bovine serum (100 mL/L) was used as background control. Triton-X 100 (100 µL,
2%) treated cells were used as high control and supernatant from the cells treated with
medium alone was used as low control. The reaction mixture (Diaphorase/NAD⁺,
Iodotetrazolium chloride and sodium lactate) was also used as a control. The reaction
mixture (100 µL) was added to the supernatant (100 µL) and high control (100 µL) and
incubated for 30 min in dark at room temperature (25°C) and the absorbance was
measured at 492 nm. All samples were processed in triplicate. The background control
absorbance was subtracted from the absorbance value of each of the sample to obtain the
expected value. The cytotoxicity (%) was calculated as
Cytotoxicity (%) =
-
-
.........................(3.2)
The cytotoxicity (%) was expressed as mean.
78
3.2.9 Experimental Design
Red cabbages were purchased from a local market. The height and weight were
measured. Ten red cabbages were selected based on their weights. The ten red cababges
were then separated into two groups randomly. The weight in both the groups was similar
(800 g). First group containg five red cabbages were given PEF treatment. The second
group of five red cabbages was not given any treatment and was called non-PEF treated
red cabbages. For spectrophotometric analysis, juice from each red cabbage was analyzed
in triplicate (total phenolics) and quadruplicate (total antioxidant activity). For cell
culture studies, juice from one PEF-treated and non-PEF treated red cabbage was selected
randomly. The juice was analyzed at 5 µg GAE/mL (WST-1 assay) and 15 and 25 µg
GAE/mL (cell count, pro-apoptotic activity and cytotoxicity). These concentrations were
selected because this the maximum concentration at which the colon cancer cells can be
treated in vitro. The PEF-extracted and non-PEF extracted juice at each concentration
was done in duplicate (pro-apoptotic activity) and triplicate (WST-1 assay, cell count,
cytotoxicity).
3.2.10 Statistical Analyses
The amount of extracted juice, total phenolics, anthocyanins and antioxidant activity
were analyzed by independent t- test. Cell proliferation and apoptosis were analyzed by
general linear model SPSS Statistics Software (SPSS Inc., Chicago, IL).
79
3.3. Results and Discussion
3.3.1 Amount of Juice
PEF treatment of red cabbages resulted in drip of juices with brighter blue color
within 20 min compared to the control samples (Figure 3.3). PEF treatment of plant tissue
reduces turgor in the cells and the elastic modulus of the plant membrane, resulting in
breakdown of the plant membrane and dripping of water (Bazhal, 2004). Gachovska et al.
(2006) reported juice drip from PEF treated alfalfa mash within 20 min, indicating
disruption of the cells due to electroplasmolysis.
Greater juice volume and yield (2.56 times) was obtained from PEF treated red cabbage
compared to non-treated control (Figure 3.4). Geulen (1994) reported an increase in juice
yield from 30% to 70% in carrot mash with particle size 3 mm and treatment of 2.6
kV/cm and 50 pulses. Similar improvement in juice yield (29%) was observed in sugar
beets, after first pressing. A PEF treatment following the first press increased the juice
yield to 80% of sugar beets(Jemai et al., 2006). Gachovska et al. (2006) reported a 38%
increase in juice extraction for PEF treated alfalfa mash (1.5 kV/cm, 1 µF and 200 pulses)
compared to control samples. Extensive sample (fruits or vegetables) preparation was
performed for application of PEF in all previous reports. This preparation requires an
additional unit operation(s) for the extraction of juices. Treatment of whole fruit or
vegetable will simplify the process and also may reduce energy costs for juice
production.
80
3.3.2 Total Phenolics
Application of PEF to red cabbage improved the total phenolics concentration by
2.47 times compared to the control (Figure 3.4). The PEF extracted red cabbage juice had
149.21 mg GAE/100 g fresh weight (FW) while the non-PEF extracted red cabbage juice
had 60.33 mg GAE/100 g FW. Podsedek (2006) reported a total phenolics concentration
of 171.36 mg/100 g FW for Kissendrup cultivar of red cabbage and 134.73 mg/100 g FW
for the Koda variety. Singh (2006) reported a total phenolics (hydroxybenzoic,
hydrocinnamic acids and anthocyanins) concentration of 101.30 mg/100 g for red
cabbage. As we were only pressing out the juice, it was possible that some of the
phenolics were trapped in the solid material, resulting in lower amount of total phenolics.
Guyot and others (2003) reported that only 42% of the total phenolics were extracted into
the apple juice while over half of the total phenolics were trapped in the apple pomace.
The total phenolics concentration of a solvent extracted sample is higher, as solvent
extracts the majority of the components from the solid material. However, when a solvent
is used for extraction, additional steps are needed to remove the solvent, which require
additional time and energy. The method of extraction as well as the application of
pressure (constant vs. progressively increasing) affect the release of the polyphenols from
the plant tissue (fruit or vegetable; Grimi, 2009). Application of compressive force (3,000
N) for 2 min on control red cabbage resulted in extraction of 24.13 mg GAE/100 g FW of
polyphenols. The lower amount of polyphenols probably was due to the application of the
constant force, thus retaining the polyphenols in the tissue. However, in our experiment,
81
the total phenolics concentration for juice extracted from PEF treated cabbage increased
compared to control red cabbage samples.
3.3.3 Total Monomeric Anthocyanins
The total monomeric anthocyanins in the extracted red cabbage juice were
determined by a pH differential method. The major anthocyanins of red cabbage were
cyanidin based molecules and hence, cyanidin-3-glucoside was used as a standard.
Greater concentrations (1.84 times; p 0.01) of cyanidin-3-glucoside equivalents of
anthocyanins were extracted from PEF treated red cabbage (23.72 mg/100 g FW)
compared to non-treated red cabbage (12.89 mg/100 g FW; Figure 3.4). Timberlake et al.
(1988) reported 25 mg/100 g FW of total monomeric anthocyanin content in red cabbage
extracted using methanol. Mazza (1993) reported a wide range of anthocyanin
concentration in red cabbage from 25 to 495 mg/100 g FW. The variations in the
anthocyanin concentration depended largely on the cultivar selected and the extraction
technology used. For example, the Koda cultivar of red cabbage had 40.53 mg/100 g FW
whereas the Kissendrup cultivar had 76.16 mg/100 g FW of total anthocyanins
(Podsedek, 2006). Further, total phenolics were extracted using 70% methanol and the
anthocyanins concentration was determined using an HPLC, which may have resulted in
the determination of higher concentration of anthocyanins. McDougall et al. (2007)
reported that red cabbage contained 137.5 mg/100 g FW of total anthocyanins extracted
using 0.5% acetic acid in water as solvent. It is possible that the solvent used for
extraction resulted in determining different concentration of anthocyanins. Gachovska et
al. (2010) extracted anthocyanins from red cabbage mash using pulsed electric field, and
82
water extraction was used followed by HPLC to quantitate the individual anthocyanin
concentration. They reported a 2.15 times increase in anthocyanin extraction in PEF
treated compared to non-treated red cabbage. Solvents extract more anthocyanins from
solid materials than application of pressure alone. It is possible anthocyanins could have
been trapped within the solid particles, resulting in lower concentration extracted in the
juice. Metivier et al. (1980) reported that methanol extracted 20% more than ethanol and
73% more than water. When PEF is used as a pre-treatment to extract anthocyanins, use
of organic solvents is not necessary and hence, further concentration techniques are not
required. And also as red cabbage juice was further analyzed for its anti-carcinogenic
activity, the presence of a solvent may interfere with the analysis. In this case, the solvent
is removed without affecting the activity of the compounds present in the juice.
3.3.4 Total Antioxidant Activity: ABTS and DPPH Assays
The juice from PEF treated red cabbage contained 3.65 mMol Trolox/L and non-
treated red cabbage juice had 1.42 mMol Trolox/L (Figure 3.4). Pliszka (2009) reported
2.59 to 3.19 mMol Trolox/L for three cultivars of red cabbage. It is possible that other
components with antioxidant activity also were extracted and determined by ABTS*+
method. This method determines the antiradical activity of the polyphenols and other
compounds with antioxidant activity in the extract such as anthocyanin condensate
products, vitamins, amino acids, minerals and synergistic effects. Also, PEF can possibly
inactivate enzymes. That also may be the reason for higher antioxidant activity (Corrales,
2008).
83
The DPPH assay measures a decrease in absorbance which is caused by reduction in free
radicals in the presence of an antioxidant. The PEF treated red cabbage juice exhibited
greater antioxidant activity (0.96 µMol Trolox/1 g FW) compared to the juice from non-
PEF treated red cabbage (0.37 µMol trolox equivalents/1 g FW; Figure 3.4). Antioxidant
activity of red cabbage juice using DPPH method was reported to be 6.76 - 9.19 µMol
trolox/1 g of FW (Podsedek, 2006). The author reported that the red cabbage had higher
antioxidant activity and ranked several vegetables based on their antioxidant capacity as
red cabbage > Brussels sprouts > savoy cabbage > white cabbage.
3.3.5 Anti-Proliferation Activity
The anti-proliferative effect of red cabbage juice on p53 +/+ and p53 -/- HCT-116
colon cancer cell lines was investigated at two concentrations (15 and 25 µg of GAE/mL)
of the red cabbage juice. In both p53 +/+ and p53 -/-cell lines, the red cabbage juice (PEF
and non-PEF extracted) inhibited the proliferation of cells compared to controls (media
and water pH 4.5) at both concentrations. With both cell lines, 15 µg GAE/mL did not
inhibit cell proliferation (p 0.05) compared to 25 µg GAE/mL, indicating that the
inhibition was dose dependent (Figure 3.5). Anthocyanins are potential inhibitors of
cancer cell proliferation (Kang et al., 2003; Zhao et al., 2004; Yi et al., 2005; Reddivari
et al., 2007). Tart cherry anthocyanins significantly decreased the proliferation of colon
cancer cell line HT-29 and HCT 116 (Kang, 2003). Similarly, Zhao (2004) showed that
chokeberry anthocyanin rich extract inhibited HT-29 cells more effectively that grape and
bilberry anthocyanin rich extract. In both cases, cyanidin or cyanidin based derivatives
84
were more inhibitory than other anthocyanidin derivatives. Hagiwara et al. (2002)
reported the effect of red cabbage color on 2- amino-1-methyl-6-phenylimidazo [4, 5-b]
pyridine (PhiP)-associated colorectal carcinogenesis in rats initiated with 1, 2-
dimethylhydrazibe. The anthocyanins inhibited promotion of colon tumors. Roy et al.
(2006) showed that white cabbage juice had the highest anti-proliferative activity in HL
60 cells. Literature on anti-proliferative activity of red cabbage juice is lacking. As red
cabbage juice is rich in acylated cyanidin derivatives, it may possess anti-cell
proliferation properties. The PEF treatment did not affect (p ≤ 0.05) compounds in the red
cabbage, as we did not observe differences between the juice from PEF-treated and non-
PEF treated red cabbage.
The cell proliferation also was measured using a water soluble tetrazolium (WST-
1) assay to confirm the results obtained in the cell count assay. Tetrazolium (slightly red)
is cleaved to formazan (yellow) by the cellular enzymes. When the population of cells
increases, the mitochondrial dehydrogenases in the sample increase, resulting in an
increase in the amount of formazan dye. The formation of formazan is directly
proportional to the number of metabolically active cells in the culture. The juice from
PEF-treated as well as non-PEF treated red cabbages showed difference (p 0.05) in
proliferation compared to media and water. At 5 µg GAE/mL, the treatments (PEF and
control) did not show any difference in proliferation compared to controls (media and
water pH 4.5). Largest concentration evaluated (25 µg GAE/mL) showed a decrease in
cell proliferation compared to other two concentrations (5 and 15 µg GAE/mL; Figure
3.6). PEF has been reported to extract more bioactive compounds from a plant material
85
and may be responsible for the difference between the control and PEF treated samples.
The results were similar to the cell count data. Reddivari et al. (2007) reported that, when
using WST-1 assay, the purple potato showed a reduced cell proliferation and their data
were similar to that obtained from the cell count data.
3.3.6 Pro-Apoptotic Activity
A reduction in apoptosis is required for survival of the cancer cells. Caspases are
the primary enzymes involved in apoptosis as they cleave a number of intracellular
substrates that trigger cell dissolution and eventually, cell death. The juice from both
PEF-treated as well as non-PEF treated red cabbages increased (p < 0.05) apoptosis at 25
µg GAE/mL compared to 15 µg GAE/mL (Figure 3.7). We did not observe specific
differences in apoptotic activity between the red cabbage juice from PEF and non-PEF
treated red cabbage at the concentrations evaluated. Srivastava (2007) reported an
increase in caspase-3 activity when HT-29 cells were treated with anthocyanin fraction
from different blueberry cultivars. They reported that the highest activity was observed at
150 µg/mL. Apoptosis was more pronounced in the p53 +/+ cell line compared to the p53
-/- cell line, indicating that a functional p53 was required for its apoptosis enhancing
activity. Resveratrol and other bioactive compounds require a functional p53 gene to
enhance apoptosis of tumor cells (Bhat, 2001; Hastak, 2005). The cells lacking the p53
gene continued to grow without undergoing apoptosis even upon treatment with red
cabbage juice.
86
3.3.7 Lactate Dehydrogenase (LDH) Enzyme Assay (Cytotoxicity)
The LDH assay measures the cytoplasmic enzyme lactate dehydrogenase which is
rapidly released into the cell culture supernatant upon damage of the plasma membrane.
Treatment of the cells with Triton-X 100, a surfactant damages plasma membrane,
releasing LDH. The red cabbage juice, irrespective of the treatment (PEF and non-PEF),
did not show toxicity to the cells compared to the Triton-X 100 treated sample (Figure
3.8). Similarly, tart cherry anthocyanins did not exhibit cytotoxic effect on HT 29 and
HCT 116 cells even at the highest concentration of 1000 µM evaluated (Kang, 2003).
Other reports also suggest that anthocyanins from other fruits did not have cytotoxic
effect on colon cancer cells (Zhao, 2004).
3.4. Conclusions
Pulsed electric field treatment can be applied to the whole red cabbage without
pre-treatment preparation such as mashing or size reduction. The PEF treatment resulted
in an increase in juice expression (64 %), total phenolics, and anthocyanin concentrations
and had greater antioxidant activity. The red cabbage juice reduced (p 0.05) the
proliferation of human colon cancer cells and increased pro-apoptotic activity of the cells.
The red cabbage juice makes an attractive alternative to artificial food colorants used in
the food industry in addition to its potential health benefits.
87
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3.6 Legend to Figures
Figure 3.1 Experimental set-up.
Figure 3.2 Measurement of impedance to determine pulse number.
Figure 3.3 Red cabbage after PEF treatment.
Figure 3.4 Effect of pulsed electric field treatment of whole red cabbage on the extraction
(juice) volume, total phenolics and anthocyanin concentration in the juice and
antioxidant activity (ABTS and DPPH assay) of the extracted juice.
Juice volume (g), total phenolics (mg GAE/100 g FW), anthocyanins (mg
cyanidin-3-glucoside eq./100 g FW) and antioxidant activity (mg Trolox/100
g FW); *significant difference between PEF and non-PEF extracted red
cabbage juice (p ≤ 0.01).
Figure 3.5 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the anti-proliferative activity of colon cancer cell line HCT-
116 (p53 +/+ and p53 -/-) determined using cell counter.
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics; Different letters represent significant difference (p ≤ 0.05).
Figure 3.6 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the anti-proliferative activity of colon cancer cell line HCT-
116 (p53 +/+ and p53 -/-) determined using WST-1 assay.
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics; Different letters represent significant difference (p ≤ 0.05).
Figure 3.8 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the pro-apoptotic activity of colon cancer cell line HCT-116
(p53 +/+ and p53 -/-).
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics; Different letters represent significant difference (p ≤ 0.05).
Figure 3.9 Cytotoxicity (%) of juice extracted from red cabbage treated with pulsed
electric field treated and non-pulsed electric field on colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-).
95
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics.
96
Figure 3. 1 Experimental set-up.
97
Figure 3. 2 Measurement of impedance to determine pulse number.
0
1
2
3
4
5
6
0 10 20 30 40 50
Imp
eda
nce
(K
Ω)
Pulse Number
98
Figure 3. 3 Red cabbage after PEF treatment.
99
Figure 3. 4 Effect of pulsed electric field treatment of whole red cabbage on the
extraction (juice) volume, total phenolics and anthocyanin concentration in the
juice and antioxidant activity (ABTS and DPPH assay) of the extracted juice.
*
*
*
*
*
0
10
20
30
40
50
60
70
80
90
100
Juice Volume Total
Phenolics
Anthocyanins Antioxidant
Activity
(ABTS)
Antioxidant
Activity
(DPPH)
g, m
g G
AE
/10
0g,
mg
CY
-3G
LU
Eq
./1
00
g, m
g T
rolo
x/1
00 g
Non-PEF PEF
100
Figure 3. 5 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the anti-proliferative activity of colon cancer cell line HCT-
116 (p53 +/+ and p53 -/-) determined using cell counter.
a
b
c
e
c
d
w w
x
z
y z
0
100000
200000
300000
400000
500000
600000
700000
800000
900000
Media Water PEF-15 PEF-25 Non-PEF_15 Non-PEF_25
Cel
ls
p53 +/+ p53 -/-
101
Figure 3. 6 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the anti-proliferative activity of colon cancer cell line HCT-
116 (p53 +/+ and p53 -/-) determined using WST-1 assay.
a
a
b
c c
a a
b
x x
x x
y
x
x
x
0.00
0.50
1.00
1.50
2.00
2.50
Media Water PEF_5 PEF_15 PEF_25 Non-PEF_5 Non-PEF_15 Non-PEF_25
Ab
sorb
an
ce
p53 +/+ p53 -/-
102
Figure 3. 7 Effect of pulsed electric field treated and non-pulsed electric field treated red
cabbage juice on the pro-apoptotic activity of colon cancer cell line HCT-116
(p53 +/+ and p53 -/-).
a a
b
c
a
c
x x
y
z
x y
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
Media Water PEF-15 PEF-25 Non-PEF_15 Non-PEF_25
Lu
min
esce
nce
p53 +/+ p53 -/-
103
Figure 3. 8 Cytotoxicity (%) of juice extracted from red cabbage treated with pulsed
electric field treated and non-pulsed electric field on colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-).
0.0
20.0
40.0
60.0
80.0
100.0
120.0
Triton X-100 Water PEF-15 PEF-25 Non-PEF_15Non-PEF_25
Cy
toto
xic
ity (
%)
p53 +/+ p53 -/-
104
CHAPTER 4
EXTRACTION OF BETALAINS FROM BEETROOTS USING PULSED ELECTRIC
FIELD AND DETERMINING THEIR BIOACTIVITY
Abstract
Red beet color is a universally permitted food color and is a rich source of the betalains
(betacyanins and betaxanthins). Increasing consumer demand for healthy and natural
foods has resulted in searches for colorants that have health benefits and meet the
regulatory requirements. Majority of the pulsed electric field (PEF) applications for
extraction of bioactive components from plant materials require extensive preparation
such as grinding and size reduction. Whole beetroots were treated with a 1.5 kV/cm
electric field, 0.66 µF capacitance and 20 pulses. The beetroots were pressed for juice
(3,000 N) and analyzed for total phenolics, betalains (betacyanins and betaxanthins) and
total antioxidant activity (2, 2ʹ-Azinobis-(3-ethylbenzothiazoline-6-sulphonic acid
( ) and 1, 1-diphenyl-2-picrylhydrazyl (DPPH)). The juice was analyzed for its
anti-proliferative and pro-apoptotic activity on colon cancer cell line HCT-116 (p53 +/+
and p53 -/-). The juice volume, total phenolics, betacyanins, betaxanthins concentrations
and antioxidant activity were increased (p 0.01) for the juice from PEF treated
compared to non-PEF treated beetroot. The red beet juice, irrespective of treatment (PEF
and non-PEF) reduced (p 0.05) HCT-116 cell proliferation and increased apoptosis.
Keywords: pulsed electric field, extraction, beetroots, colon cancer
105
4.1. Introduction
Betalains are water-soluble pigments present in most families of the order
Caryophyllales, Ranunculaceae (Cronquist, 1988) and can be classified as yellow
betaxanthins and violet betacyanins. Similar to anthocyanins, they are present in the
vacuoles of both vegetative tissue and the reproductive organs, mostly epidermal and/or
sub-epidermal tissues (Jackman, 1996).
Betalains are derived from betalamic acid (Wohlpart and Mabry, 1968), with
tyrosine as precursor via L-DOPA. The condensation of cyclo-DOPA (derivative of L-
DOPA) and betalamic acid results in red betacyanin (betanidin) whereas the condensation
of betalamic acid with an amino acid gives yellow betaxanthin (indicaxanthin; Stafford,
1994; Fig. 4.1).
Figure 4. 1 Structure of betalamic acid, betacyanin (betanidin) and
betaxanthin (indicaxanthin).
106
4.1.1 Biosynthesis of Betacyanins and Betaxanthins
The biosynthesis of betalains starts with two molecules of tyrosine. The tyrosine
molecule is hydroxylated to L-DOPA by tyrosinase and L-DOPA is oxidized further and
rearranged to form cyclo-DOPA by the same enzyme. L-DOPA formed from the other
tyrosine undergoes a ring opening reaction at position 4 and 5 to form an unstable
product seco-DOPA which spontaneously closes itself to form the betalamic acid. This is
catalyzed by the enzyme DOPA 4, 5 dioxygenase (Fischer, 1972; Terradas, 1991 and
Schliemann, 1998). Condensation of betalamic acid with cyclo-DOPA results in
betacyanin. The betacyanin undergoes further glucosylation and acylation steps carried
out by glucosyltransferases and acyltransferases. Betaxanthins are formed by the
condensation of an amino acid to the aldehyde group of the betalamic acid to form
Schiff‘s base. Indicaxanthin is formed from the condensation of proline and betalamic
acid (Zryd, 2004). The betaxanthins has an absorption maximum at 480 nm and the
betacyanins at 538 nm, where the aromatic structure of cyclo-DOPA is responsible for
the absorbance (Wyler and Dreiding, 1961; Figure 4.2).
The edible sources of betalains are red and yellow beetroot, colored Swiss chard,
grain or leafy amaranth and cactus fruits of Opuntia and Hylodereus genera (Azeredo,
2009). The beetroot belongs to the Chenopodiaceae family of the Caryophyllales
kingdom (Moreno, 2008). The two major pigments in red beet are the red-violet betanin,
isobetaninn betanidin and isobetanidin and the yellow vulgaxanthin I and vulgaxanthin II
(Azeredo, 2009). Depending on the cultivar, the betacyanin and the betaxanthin content
of the red beetroots varies from 0.04-0.21% and 0.02-0.14%, respectively (Nilsson,
107
1970). Beetroots are cultivated widely for their betanin compound for use as a natural
food colorant (E162). Beetroot red is permitted widely in Europe and North America as a
natural food colorant in dairy products like ice cream, sherbet and yogurt, dry soft drink
mixes, confectionary and also used in soups as well as tomato and bacon products
(Henry, 1996).
Figure 4. 2 Biosynthesis of betacyanin (betanidin) and betaxanthin (indicaxanthin;
Stafford, 1994).
4.1.2 Health Benefits of Betalains
Beet root is one of the ten most potent vegetable for antioxidant activity, and
betalains are the main components contributing to the antioxidant activity (Cao, 1996;
108
Stintzing, 2004). The antiradical activity of betacyanins was greater than that of
betaxanthins, the two main components of betalains. The antioxidant activity of the
betalains increases with an increase in the number of hydroxyl/imino groups and the
position of hydroxyl groups and glycosylation of aglycones in the betalain molecules
(Cai, 2003). Acylation increase the antioxidant activity while glycosylation reduces the
activity.
The beet root extract has a greater anticancer activity compared to caspanthin,
cranberry, red onion skin and short and long red bell peppers (Kapadia, 1996). Beetroot
extracts induce phase II enzymes like quinone reductase in murine hepatoma cells in vitro
(Wettasinghe, 2002) that detoxify potential carcinogens. Beetroot products inhibit
neutrophil oxidative metabolism in a concentration-dependent manner and its pro-
apoptotic effects were observed in stimulated neutrophils (Zielinska-Przyjemska, 2009).
4.1.3 Juice Extraction
Beetroot juice is normally extracted by pressing or aqueous extraction (Herbach,
2004; Kanner, 2001). Pulsed electric field (PEF) is a non-thermal processing technology
used to enhance juice extraction from plant materials. PEF have been used to extract
bioactive added compounds from plant cells including anthocyanins (Corrales, 2008;
Tanya, 2010), sugar (Bouzrara, 2000; Belghiti, 2004) and betalains (Chalermchat, 2004;
Fincan, 2004; Lopez, 2009). PEF was used previously to extract betalains from beet
slices by subjecting them in a treatment chamber. To avoid pre-preparation steps, in this
study we subjected whole beetroots without peeling the skin to electric field.
109
The objectives of this study were to evaluate
i. juice yield, total phenolics, betalains (betacyanins and betaxanthins) and
total antioxidant activity of juice from PEF treated and non-PEF treated
red beet and
ii. The anti-proliferative and pro-apoptotic activity of PEF-treated and non-
PEF extracted beetroot juice on colon cancer cell lines.
4.2 Materials and Methods
All reagents and chemicals were purchased from Fisher Scientific (Pittsburg, PA)
and Sigma-Aldrich (St. Louis, MO). The beetroot were purchased from a local market
and were used immediately for the experiment. The beetroots were washed thoroughly to
remove the soil, weighed and the heights were measured. The beetroots were then
assigned randomly to control or treatment groups based on their weights.
Pulsed electric field treatment circuit consisted of an exponential decay pulse generator
(CF60/25-12C, Hipotronics, Inc., Brewster, NY), a capacitor (General Atomics
Electronic systems, San Diego, CA) and a spark gap switch. The generator contained a
DC power supply with a maximum voltage of 60 kV and maximum power 10 mA. The
energy stored in the capacitor was discharged directly to the red beet placed between two
stainless steel electrodes. The distance between the electrodes was the height of the
beetroot (6 cm). An electrode, a small circular stainless steel plate was placed on one end
of the beetroot and the ground electrode was a rectangular plate. Aqueous sodium
chloride solution (7% w/w) was applied to the ground electrode to improve electrical
110
conduction. The insulation material for the two electrodes was purchased from Deorin
(American Plastics Supply & Fabrication, Omaha, NE). PEF treatment parameters were
chosen based on preliminary experiments and literature review (Fincan, 2004). The
voltage applied to the sample was measured with a voltmeter (Model, P6015 A,
Tektronix, Inc., Beaverton, OR). The pulse number was selected based on measurement
of electrical impedance, an indirect measure of cell permeability (Figure 4.3).
The beetroot was placed between the two electrodes and electric field strength of
1.5 kV/cm, 0.66 µF and 20 pulses were applied. The pulse width was approximately 15
µs and the frequency of the pulse was 1 Hz. Treatment time was the product of pulse
width and the number of pulses and therefore the treatment time was 0.3 ms. Temperature
of the red beetroot after PEF treatment was measured using a temperature probe. The
temperature increase was negligible.
Subsequent to PEF treatment, both the control and treated beetroot were shredded
using a domestic food processor (MFP 200, Minipro™, Black & Decker, Baltimore, MD)
for 1 min to obtain a homogenous mash without any addition of water.
The homogenized mash (approximately 60 g) was placed in a juice press. The
press had a sample chamber, a plunger and a juice container. The juice was pressed using
an Instron universal testing machine (Bluehill 2 software, Norwood, MA). A force of
3,000 N was used to press the juice out of the beetroots.
111
A portion (5 mL) of the extracted juice was filtered (0.22 µm) and stored at -20°C
to evaluate the bioactivity. The other part was refrigerated and used for
spectrophotometric analysis.
4.2.1 Total Phenolics
Total phenolics were determined using Folin-Ciocalteu reagent method (Singleton
and Rossi, 1965). Briefly, Folin-Ciocalteu reagent (FC; 150 µL of 0.2 M) was added to
the diluted sample (35 µL). The samples were thoroughly mixed, held for 5 min at room
temperature and sodium carbonate (115 µL ; 7.5%) was added to the sample and
incubated for 30 min at room temperature (25°C) . Then, the absorbance was then
measured at 765 nm (Synergy 2, BioTek, Winooski, VT) using gallic acid as a standard.
The results were expressed as mg GAE/100 g fresh weight. All samples were analyzed in
triplicate.
4.2.2 Betalain Analysis
The betalain content was quantified by a method proposed by Nilsson (1970) with
few modifications. The red beet juice was diluted (75 times) with deionized water and the
absorbance of the diluted juice was read at 538 nm and 480 nm using a
spectrophotometer (Shimadzu, UV-1800 Spectrophotometer, Columbia, MD).The
betalain content was calculated using an equation proposed by Cai (1999)
Betalain content (mg/L) = [
] ...………………….(4.1)
112
where,
A is the final absorbance after correcting the absorbance at 700 nm
DF is the dilution factor
MW is the molecular weight. 550 g/mol for betanin and 308 g/mol for
indicaxanthin
ε is the molar extinction coefficient. 60000 l/mol for betanin and 48000 l/mol for
indicaxanthin, and
L is the pathlength of the 1-cm cuvette.
All were measured in triplicate and results were expressed as means ± SE.
4.2.3 Total Antioxidant Activity by ABTS Assay
The total antioxidant capacity of the beetroot juice was determined using 2, 2ʹ-
Azinobis-(3-ethylbenzothiazoline-6-sulphonic acid ( ) and was performed as
described by Re (1999). Pre-formed radical mono-cation was generated by
oxidation of ABTS with potassium persulfate. The working solution was
generated by mixing two stock solutions (8 mM ABTS and 3 mM potassium persulfate)
and allowing them to react for at least 12 h at room temperature (25°C) in the dark. The
solution (5 mL) was then diluted with phosphate buffer (45 mL of pH 7.4) containing
150 mM NaCl (405 mL of 0.2 M Na₂HPO₄, 95 mL of 0.2 M NaH₂PO₄ and NaCl (8.77
g) made up to 500 mL with deionized water and pH adjusted to 7.4 with 1 M NaOH). To
10 µL of sample (10 times diluted), 290 µL of /phosphate buffer solution were
113
added. The samples were mixed and allowed to react for 30 min and was measured at 734
nm (Synergy 2, BioTek, Winooski, VT) using trolox (concentration 0 µg/mL to 500
µg/mL) as standard. The results were expressed as µg trolox equivalents/100 g fresh
weight. All the samples were conducted in quadruplicate.
4.2.4 Total Antioxidant Activityby DPPH Assay
The total antioxidant activity of the beetroot juice was measured using the 1, 1-
diphenyl-2-picrylhydrazyl (DPPH) method described by Brand-Williams (1995). A
DPPH stock solution (24 mg DPPH in 100 mL 80% ethanol) was diluted with ethanol
(80% aqueous) to obtain an absorbance of 1.1 at 515 nm using a spectrophotometer. The
diluted sample (15 µL) was allowed to react with of the diluted DPPH (285 µL) solution
for 30 min. After 30 min, the samples were read at 515 nm (Synergy 2, BioTek,
Winooski, VT) using trolox was used as a standard. All samples were measured in
quadruplicate. The results were expressed as µg trolox equivalents/100 g fresh weight.
4.2.5 Cell Lines
Human colon carcinoma cells HCT-116 (p53 +/+ and p53 -/-) were grown in
McCoy‘s 5A medium containing phenol red (Sigma, St. Louis, MO) and maintained at
37°C in 5% CO₂ jacketed incubator. The medium was supplemented with of bovine
serum albumin (100 mL/L; Hyclone, Fisher, Pittsburg, PA), sodium bicarbonate (2.2 g/L;
Sigma, St. Louis, MO) and streptomycin/penicillin solution (10 mL/L; Fisher, Pittsburg,
PA).
114
4.2.6 Anti- Proliferation Assay
HCT-116 cells were seeded (6,000 cells/well) and incubated for 24 h at 37°C.The
medium was replaced with Dulbecco‘s modified Eagle‘s medium F-12 media (DMEM)
without phenol red containing 0.25% charcoal stripped serum. Filtered beetroot juice was
added at concentrations ranging from 5 to 25 µg of GAE/mL of media. Medium (DMEM
without phenol red) and deionized sterile water (pH 2.5) were used as controls. The cells
were incubated for 48 h at 37°C in an incubator and cell proliferation was measured using
the water soluble tetrazolium assay (WST-1, Roche Molecular Biochemicals,
Indianapolis, IN). WST-1(10 µL) was added to each well, incubated for 2 h and the
absorbance of the medium was measured at 480 nm (Synergy 2, BioTek, Winooski, VT).
The HCT-116 cells were plated (5 cells/mL per well) and incubated for 24
h at 37°C. Different concentrations of the beetroot juice were added to the cells. The
medium and deionized water ( pH 2.5) served as controls. The population of cell was
enumerated using a Cellometer Automated Cell Counter (Nexcelom, Lawrence, MA). All
the experiments were performed out in triplicate and the results were expressed as means
SE.
4.2.7 Pro-Apoptotic Assay
The HCT-116 cells were plated (5 cells/mL per well) incubated for 24 h at
37°C. Different concentrations of the beetroot juice were added to the cells. After 48 h of
incubation, cell number was determined using a Cellometer (Nexcelom, Lawrence, MA)
and 2 cells from each well were transferred to a 96-well opaque plate (Nunc 96
115
MicroWell Plate White, Thermo Scientific, Rochester, NY). The volume was made to
200 µL with DMEM stripped fetal bovine serum medium without phenol red. Caspase-
Glo® 3/7 reagent (Caspase-Glo® 3/7 buffer and Caspase-Glo® substrate; 100 µL;
Promega, Madison, WI) was added to each well. The plate was incubated for 30 min at
room temperature (25°C) and the luminescence was measured (Synergy 2, BioTek,
Winooski, VT). The samples were measured out in triplicate and the results were
expressed as mean ± SEs.
4.2.8 Lactate Dehydrogenase (LDH) Enzyme Assay (Cytotoxicity)
The cytotoxicity of the beetroot juice was determined using a cytotoxicity
detection kit (Roche Molecular Biochemicals, Indianapolis, IN). Cell-free supernatant
(100 µL) from treated cells were collected and transferred to a 96- well plate. DMEM
medium (200 µL) containing stripped fetal bovine serum (100 mL/L) was used as
background control. Triton-X 100 (100 µL, 2%) treated cells were used as high control
and supernatant from the cells treated with medium alone was used as low control. The
reaction mixture (Diaphorase/NAD⁺, Iodotetrazolium chloride and sodium lactate) was
also used as a control. The reaction mixture (100 µL) was added to the supernatant (100
µL) and high control (100 µL) and incubated for 30 min in the dark at room temperature
(25°C) and the absorbance was measured at 492 nm. All the samples were processed in
triplicate. The background control absorbance was subtracted from the absorbance value
of each of the sample to obtain the expected value. The cytotoxicity (%) was calculated
as:
116
Cytotoxicity (%) =
expected value - low control
high control - low control
.........................(4.2)
The cytotoxicity (%) was expressed as mean.
4.2.9 Experimental Design
Beetroots were purchased from a local market. The height and weight were measured.
Twelve beetroots were selected based on their weights. The Twelve beetroots were then
separated into two groups randomly. The weight in both the groups was similar (90-100
g). First group containg six beetroots were given PEF treatment. The second group of six
beetroots was not given any treatment and was called non-PEF treated beetroots. For
spectrophotometric analysis, juice from each beetroot was analyzed in triplicate (total
phenolics) and quadruplicate (total antioxidant activity). For cell culture studies, juice
from one PEF-treated and non-PEF treated beetroot was selected randomly. The juice
was analyzed at 5 µg GAE/mL (WST-1 assay) and 15 and 25 µg GAE/mL (cell count,
pro-apoptotic activity and cytotoxicity). These concentrations were selected because this
the maximum concentration at which the colon cancer cells can be treated in vitro. The
PEF-extracted and non-PEF extracted juice at each concentration was done in duplicate
(pro-apoptotic activity) and triplicate (WST-1 assay, cell count, cytotoxicity).
4.2.10 Statistical Analyses
The amount of extracted juice, total phenolics, anthocyanins and antioxidant activity
were analyzed by independent t- test. Cell proliferation and apoptosis were analyzed by
general linear model SPSS Statistics Software (SPSS Inc., Chicago, IL).
117
4.3 Results and Discussion
4.3.1 Amount of Juice
PEF treatment of beetroots resulted in drip of juices immediately after treatment
compared to the control samples (visual observation). Similar observations were reported
by Gachovska et al. (2006) from PEF treated alfalfa mash.
PEF has been reported to increase juice yield in several fruits and vegetables
(Bouzrara, 2000; Bazhal, 2001; Schilling, 2007; Praporscic, 2007). PEF treatment of
whole beetroots resulted in an increase (38.5 %; p ) in the juice yield compared to
the control (10%; Figure 4.4). Schilling (2007) reported an increase in apple juice yield
from 1.7 to 7.7% for an electric field treatment of 1, 3 and 5 kV/cm of apple mash.
Bouzrara (2000) reported a three fold increase in juice extraction from sugar beet
cossettes. An increase (80%) in juice yield was reported by Jemai (2005) for a treatment
which involved two initial pressing steps with an intermediate PEF treatment. In all of
these reports, extensive sample preparation steps were performed, which requires an
additional unit operation in the extraction of juices. For scaling up, treatment of whole
fruit or vegetables would speed up the process and may also reduce energy costs for juice
production.
4.3.2 Total Phenolics
The concentration of total phenolics increased (p 0.01) by 55% for the PEF
extracted sample compared to the non-PEF extracted (15%) beetroots (Figure 4.4). The
total phenolics concentrations reported in literature are phenolics extracted using an
118
organic solvent or water. Kujala (2000) reported a total phenolics concentration of 15.5
mg GAE/g of dry material. As we were only pressing out the juice, it was possible that
some of the phenolics were trapped in the solid material, resulting in lower amount of
total phenolics yields in the juice. Kujala (2002) detected three phenolics [5, 5‘, 6, 6‘-
tetrahydroxy- 3, 3‘-biindolyl, feruloylglucose and β-D-fructofuranosyl-α- D- (6- O- (E)
-feruloylglucopyranoside)], two phenolic amides (N-Trans-feruloyltyramine and N-trans-
feruloylhomovanillylamine) and four flavonoids (betagarin, betavulgarin, cochliophilin A
and dihydroisorhamnetin) in aqueous methanol (80%) extracts from beetroot. These
compounds were observed in the peel, crown and flesh of the beetroot with the flesh
containing the least amount.
4.3.3 Betacyanins and Betaxanthins
The major betacyanin pigments are betanin and isobetanin while the major
betaxanthin pigments are vulgaxanthin I and vulgaxanthin II (Azeredo, 2009). The
application of PEF to beetroots increased betacyanin content from 9.29 to 26.61 mg/100
g FW and betaxanthin content from 4.38 to 9.18 mg/100 g FW (Figure 4.5). The red-
violet betacyanin was the major pigment compared to the yellow betaxanthin. Fincan
(2003) subjected thin disks of red beetroot to 1 kV/cm electric field strength and reported
extraction of approximately 90% of total red pigment and ionic content were released
following 1 h aqueous extraction compared to the untreated beetroots (5%). Aqueous
extraction of shredded beetroots extracted 45-70% of the betalain pigments while from
PEF extracted (7 kV/cm) beetroots five-fold more betalain pigments were extracted
119
(Lopez et al., 2009). The authors also reported that PEF in combination with pressing
shortened the extraction time 18-fold.
4.3.4 Total Antioxidant Activity: ABTS and DPPH Assays
Beetroots are among the most potent vegetables for of their antioxidant activity
(Cao et al., 1996) which include garlic, kale, spinach, brussels sprouts, alfalfa sprouts and
broccoli flowers. Green pepper, spinach, purple onion, broccoli, beet and cauliflower are
the leading sources of antioxidant activities (Ou et al., 2002). In assay, the
antioxidant activity increased (p ) from 0.054 mMol trolox equivalents/100 g FW
in non-PEF extracted beetroot juice to 0.223 mMol trolox equivalents/100 g FW in PEF
extracted beetroot juice (Figure 4.4). Betalains that contain imino and hydroxyl groups as
well as phenolics contribute to the antioxidant activity (Wu et al., 2005). Escribano
(1998) reported that the antioxidant activity of betacyanins was greater than that of
betaxanthins. Wettasinghe (2002) reported 210 µg/mL antioxidant activity for beetroots,
similar to concentration observed in this study. The antioxidant activity was also
determined by DPPH assay. The antioxidant activity increased from 0.079 mMol trolox
equivalents/mL in non-PEF extracted sample to 0.293 mMol trolox equivalents/mL for
PEF extracted samples (Figure 4.4). Literature on the antioxidant activity of red beet
juice determined by DPPH assay is lacking. In this experiment, the PEF treatment of
beetroot increased the antioxidant activity of the juice determined both by ABTS and
DPPH assay. Since, more juice was extracted using PEF, it resulted in more antioxidant
activity.
120
4.3.5 Anti-Proliferation Activity
The anti-proliferative of the beetroot juice on colon cancer cell line HCT-116
(p53 +/+ and p53 -/-) was determined by counting the cells and WST-1 assay. A set of
random PEF and non-PEF extracted samples were selected and concentrations of 15 and
25 µg GAE/mL were used. The PEF extracted and the non-PEF extracted juice reduced
(p 0.05) cell proliferation compared to the controls (water and pH 2.5) using the cell
count as well as the WST-1 assays. The number of cells decreased in both p53 +/+ and
p53 -/- cell line (Figure 4.6 and 4.7).
Addition of beetroot juice from non-PEF extracted beetroot did not affect (p
0.05) p53 +/+ cell proliferation. Similarly, differences between the two concentrations of
juice used (15 and 25 µg GAE/mL) were minimal for cell line. In the WST-1 assay, there
was a difference between the PEF and non-PEF extracted juice, with reduced (p 0.05)
cell concentration in the PEF extracted juice compared to the non-PEF extracted juice.
Reduced (p 0.05) cell proliferation were observed with PEF and non-PEF
extracted beetroot juice compared to controls (media and water pH 2.5). The cell
proliferation activity were similar for PEF and non-PEF extracted juice was similar (p
0.05). Higher concentration (25 µg/mL) of beetroot juice reduced cell proliferation when
compared to 15 µg/mL in both PEF and non-PEF extracted juice. Cell proliferation
determined by WST-1 assay showed similar results with PEF extracted and non-PEF
extracted juice reducing the cell growth compared to the controls (media and water pH
2.5).
121
Literature on the anti-proliferative activity of red beet on cancer cells is not
available. However, Chavez-Santoscoy (2009) reported that prickly pear juice diminished
cell viability of four cancer cells evaluated (prostrate, colon, mammary and hepatic
cancer cell lines). An increase in the total phenolic content from 22.3 to 226.3 µg GAE/g
was reported from prickly pear. The betacyanin content ranged from 3.1 to 189 µg/g and
the betaxanthin content ranged from 1.6 to 300.5 µg/g. Similarly, Wu (2005) reported
that the peel of red pitaya prevented proliferation of melanoma cells. Red pitaya a source
of betalains (10.3 mg/100 g FW of betacyanins), had an influence on the anti-
proliferative effect. And also the flavonoids (myricetin, baicalein and gallic acid) present
in red pitaya would have had an effect on cell viability. It is suggested that the presence
of a C2-C3 double bond and ortho-dihydroxyphenyl structure in the flavonoid A- or B-
ring confer greater antiproliferative activity to the flavonoid (Martinez, 2003).
4.3.6 Pro-Apoptotic Activity
Apotox- GLO Triplex assay was used to determine the pro-apoptotic effect of red beet
juice on colon cancer cell line. The juice from PEF extracted red beet at 25 µg/mL
showed pro-apoptotic activity while the non-PEF extracted juice did not show any pro-
apoptotic activity in the p53 +/+ cell line (Figure 4.8). That showed that the beetroot juice
required a functional p53 gene to induce apoptosis. In the p53 -/- cell line, there was no
difference in apoptosis between the controls and the beetroot juice.
Betanin from Opuntia ficus-indica induced apoptosis in chronic myeloid
leukemia cells. The author showed that betanin induced apoptosis after cell entry through
122
intrinsic pathway mediated by the release of cytochrome c from mitochondria into the
cytosol and PARP cleavage (Sreekanth, 2007).
4.3.7 Lactate Dehydrogenase (LDH) Enzyme Activity (Cytotoxicity)
The LDH assay measures the cytoplasmic enzyme lactate dehydrogenase which is
rapidly released into the cell culture supernatant upon damage of the plasma membrane.
The beetroot juice tested was not cytotoxic to the cells, as indicated by the low LDH
activity (Figure 4.9). The juice inhibited cell proliferation by not being cytotoxic to the
cells.
4.4 Conclusion
Pulsed electric field treatment of whole beetroots (1.5 kV/cm) resulted in greater juice
extraction compared to non-PEF extracted beetroots. The juice from PEF extracted
beetroots contained higher concentration of polyphenols, betalains and antioxidant
activity compared to non-PEF extracted juice. The beetroot juice decreased cell
proliferation in both the cell lines (p53 +/+ and p53 -/-) and increased apoptosis in the
p53 +/+ cell line. There were no significant differences between the juices from PEF
extracted beetroot and non-PEF extracted beetroot on colon cancer cell line proliferation
and apoptosis. Thus, PEF treatment improved juice extraction from beetroots and did not
affect their functional and beneficial health promoting properties.
123
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128
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35. Wohlpart, A., & Mabry, T. J. (1968). On the light requirement for betalain
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129
4.6 Legend to Figures
Figure 4.3 Measurement of impedance to determine pulse number.
Figure 4.4 Effect of pulsed electric field treatment of whole beetroot on the extraction
(juice) volume, total phenolics concentration in the juice and antioxidant
activity (ABTS and DPPH assay) of the extracted juice.
Juice volume (g), total phenolics (mg GAE/100 g FW), and antioxidant
activity (mg Trolox/100 g FW); *significant difference between PEF and non-
PEF extracted beetroot juice (p ≤ 0.01).
Figure 4.5 Effect of pulsed electric field treatment of whole beetroot on the extraction of
betalains (betacyanins and betaxanthins) of the extracted juice.
Betalains (mg/100g FW); *significant difference between PEF and non-PEF
extracted beetroot juice (p ≤ 0.01).
Figure 4.6 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the anti-proliferative activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-) determined using cell counter.
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics; Different letters represent significant difference (p ≤ 0.05).
Figure 4.7 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the anti-proliferative activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-) determined using WST-1 assay.
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics; Different letters represent significant difference (p ≤ 0.05).
Figure 4.8 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the pro-apoptotic activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-).
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics; Different letters represent significant difference (p ≤ 0.05).
Figure 4.9 Cytotoxicity (%) of juice extracted from whole beetroot treated with pulsed
electric field treated and non-pulsed electric field on colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-).
Designations 5, 15 and 25 represent 5, 15 and 25 µg GAE/mL of total
phenolics.
130
Figure 4. 3 Measurement of impedance to determine pulse number.
0
0.5
1
1.5
2
2.5
0 10 20 30 40 50
Imp
eda
nce
(K
Ω)
Pulse Number
131
Figure 4. 4 Effect of pulsed electric field treatment of whole beetroot on the extraction
(juice) volume, total phenolics concentration in the juice and antioxidant
activity (ABTS and DPPH assay) of the extracted juice.
*
*
*
*
0
10
20
30
40
50
60
70
80
90
100
Juice Volume Total Phenolics Antioxidant
Activity (ABTS)
Antioxidant
Activity (DPPH)
g, m
g G
AE
/10
0g
, m
g
Tro
lox
/100
g
Non-PEF PEF
132
Figure 4. 5 Effect of pulsed electric field treatment of whole beetroot on the extraction of
betalains (betacyanins and betaxanthins) of the extracted juice.
*
*
0
0.1
0.2
0.3
0.4
0.5
Betacyanins Betaxanthins
mg
/10
0 g
FW
Non-PEF PEF
133
Figure 4. 6 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the anti-proliferative activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-) determined using cell counter.
a a
b b
b b x x
y
z y
z
0
200000
400000
600000
800000
1000000
Media Water PEF-15 PEF-25 Non-PEF_15Non-PEF_25
Cel
ls
p53 +/+ p53 -/-
134
Figure 4. 7 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the anti-proliferative activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-) determined using WST-1 assay.
a
ab
e
e e
d
cd bc
y y
z z z z z
z
0.00
0.50
1.00
1.50
2.00
2.50
Media Water PEF-5 PEF-15 PEF-25 Non-PEF_5 Non-PEF_15 Non-PEF_25
Ab
sorb
an
ce
p53 +/+ p53 -/-
135
Figure 4. 8 Effect of pulsed electric field treated and non-pulsed electric field treated
whole beetroot juice on the pro-apoptotic activity of colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-).
bc
a
cd
d
ab ab
x xy
y xy
z
xy
0
500
1000
1500
2000
2500
3000
Media Water PEF-15 PEF-25 Non-PEF_15 Non-PEF_25
Lu
min
esce
nce
p53 +/+ p53 -/-
136
Figure 4. 9 Cytotoxicity (%) of juice extracted from whole beetroot treated with pulsed
electric field treated and non-pulsed electric field on colon cancer cell line
HCT-116 (p53 +/+ and p53 -/-).
0
20
40
60
80
100
120
Triton X-100 Water PEF-15 PEF-25 Non-PEF_15 Non-PEF_25
Cy
toto
xic
ity (
%)
p53 +/+ p53 -/-
137
CHAPTER 5
SUMMARY AND CONCLUSION
Pulsed electric field was used to treat red cabbages and beetroots for juice
extraction without any sample preparation step. The PEF treatment increased (p 0.01)
the juice volume for both red cabbage and beetroot compared to non-PEF extracted
vegetables. The PEF- treated juice was evaluated for total phenolics, anthocyanin (red
cabbage), betalains (beetroot) and antioxidant activity. For the red cabbage juice, the total
phenolics and anthocyanin concentration increased thus increasing the antioxidant
activity of the juice. Similarly, for the beetroot juice, the antioxidant activity increased
with increase in the total phenolics and betalains concentration. The juices were tested for
their activity on colon cancer cells (HCT 116: p53 +/+ and p53 -/-). The juices reduced
cancer cell growth and increased apoptosis in the cancer cells. The juices showed
increased apoptosis in the p53 +/+ cell line compared to the p53-/- cell line. This finding
suggests that the red cabbage anthocyanins and beetroot betalains require a functional
p53 gene to induce apoptosis. Otherwise, the PEF extracted and non-PEF extracted juice
did not have any difference with respect to their activity on cancer cell growth and
apoptosis. This suggests that PEF did not affect the compounds present in the juice.
Overall, PEF resulted in better extraction of juice and the PEF-treated and non-PEF
extracted juices showed anti-carcinogenic activity on colon cancer cells. Higher yield
obtained with pulsed electric field is of major interest from industrial point of view, since
solvent amounts might be reduced and extraction times shortened. We have shown that
138
PEF can be applied to whole fruits and vegetables, this reduces the overall extraction
time.
139
Reccomendations for Future Research
Recommendations for the future of this study includes, developing a continuous process,
where the fruits and vegetables are treated with pulsed electric fields and pressed for
juice. This requires designing electrodes appropriately so that any size of fruit and
vegetable can be treated. The ground electode can be designed to hold the fruit/vegetable
and the positive electrode can be designed to have a spring mechanism to adjust the
height according to the fruit/vegetable. This process if made automatic may not need any
trained personnel.
Since both red cabbage and beetroot juice showed anticarcinogenic activity, further
experiments are needed to evaluate their performance in vivo. First, the mechanism by
which the juices increases apoptosis needs to be studied. As apoptosis increased in
p53+/+ cell line, it suggests that the juices need a functional p53 gene. So when a p53
inhibitor such as α-pithifrin is added to the cells treated with juice, we can see how the
p53 +/+ cells behave in the presence of both the juice and the p53 inhibitor. If the
apoptosis is reduced in this case, we can confirm that the juices require a functional p53
gene for apoptosis. Further experiments can be designed to carry out in animal models. A
diet can be designed which will contain the red cabbage and beetroot juice and fed to
animals having colon cancer and studied for the anticarcinogenic activity of the juice.
As PEF parameters for whole vegetable treatment has been optimized on a batch-
scale, the focus should be on making this a continuous process. And also, based on the in
vitro cell culture experiments, further research on animal models is required to evaluate
the anti-carcinogenic activity of red cabbage and beetroot juice in vivo.
140
Appendix
Procedures
A.1 Total Phenolics
Preparation of Gallic acid Standards
Accurately prepare fresh gallic acid stock standard of ~1mg/mL in 80%
acetone. Prepare 25mg in 25mL volumetric flask for best accuracy.
Record exact weight and adjust standard curve accordingly.
Dilute stock standard to appropriate range for the types of samples being
assayed.
Standard: For samples with higher TP concentrations, use less dilute standards.
7.0 mL stock GA + 3.0 mL 80% acetone. Then prepare GA standard curve
dilutions using volumes as follows for the best accuracy.
µg/mL in assay µL of Std µL of distilled water
0 0 350
40 20 330
80 40 310
120 60 290
160 80 270
200 100 250
141
On plate, set up standard curve as above. In triplicate, pipette 35µL of each standard
concentration into the well.
Assay Procedure
1. Parameters used
End point assay with 10 second mixing
Wavelength – 765nm
Incubator - 45°C
Standard curve constructed
Concentrations and statistics reported
2. Dilute 100µL juice with 900µL distilled water and then vortex. In triplicate
microplate wells, pipette 35µL of dilute sample. (Note: Use different pipette
tips for different samples)
3. Add 150µL 0.2M Folin-Ciocalteu (FC) reagent (freshly dilute strength
SIGMA reagent 1/10 with distilled water) to all wells. 2.0mL FC Reagent +
18mL distilled water is sufficient for one 96-well microplate).
4. Mix plate on a platform vortex. Shake at 400rpm for 30 seconds then hold for
exactly 5 minutes at room temperature.
5. After 5 minutes, add 115µL 7.5% (w/v) Na2CO3 (7.5g/100mL distilled water)
to all wells.
6. Mix again as above on a platform vortex.
142
7. Place the microplate in an incubator at 45°C for exactly 30 minutes. Cool to
room temperature for 1 hour.
8. Read plate absorbance at 765nm.
Results
1. Use absorbance values form the microplate reader at concentrations expresses
as µg GAE/mL in assay for calculations.
2. When calculating linear equation for GA standard curve, force line through
zero (0mg/ml std is ―blank‖ in TP microplate protocol).
A.2 Total Monomeric Anthocyanin Concentration
Prepare pH 1.0 and 4.5 buffer solutions.
Potassium chloride buffer, 0.025 M, pH 1.0: Mix 1.86 g KCl and 980 ml of
distilled water in a beaker. Measure the pH and adjust to 1.0 with concentrated
HCl. Transfer to a 1 liter volumetric flask and fill to 1 liter with distilled water.
Sodium acetate buffer, 0.4 M, pH 4.5: Mix 54.43 g CH 3 CO 2 Na⋅3 H 2 O and
∼960 ml distilled water in a beaker. Measure the pH and adjust to 4.5 with
concentrated HCl. Transfer to a 1 liter volumetric flask and fill to 1 liter with
distilled water.
1. Take 0.1 mL of juice and prepare two dilutions of the sample, one with 10 mL of
potassium chloride buffer, pH 1.0, and the other with 10 mL of sodium acetate
buffer, pH 4.5.
143
2. Calculate the absorbance of the diluted sample (A) as follows:
A = (A520–A700) pH1.0
– (A520 –A700)pH4.5
3. Calculate the monomeric anthocyanin pigment concentration in the original
sample using the following formula:
Monomeric anthocyanin pigment (mgCy3glu eq./L) = (A × MW × DF × 1000)/(ε × l)
Where:
A is the absorbance calculated using the previous equation
MW is the molecular weight for cyanidin-3-O-glucoside (449.2).
DF is the dilution factor.
ε is the molar absorptivity of cyanidin-3-O-glucoside (26900).
l is the path-length (1 cm) of the cuvette.
A.3 Betalain Concentration
1. Dilute the juice 75 times.
2. Mix the diluted juice with 10 mL of water at pH 6.5.
3. Measure the absorbance at 438 nm, 510 nm and 700 nm using a
spectrophotometer.
4. Calculate the concentration of betacyanins and betaxanthins using:
Betalain content (mg/L) = [A×DF×MW×1000
ε×L]
where,
A is the final absorbance after correcting the absorbance at 700 nm
144
DF is the dilution factor
MW is the molecular weight. 550 g/mol for betanin and 308 g/mol for
indicaxanthin
ε is the molar extinction coefficient. 60000 l/mol for betanin and 48000 l/mol for
indicaxanthin, and
L is the pathlength of the 1-cm cuvette.
A.4 Total Antioxidant Activity: ABTS Assay
1. Mother Solution: Prepare 8 mM of ABTS (44 mg/10 ml) and 3 mM of potassium
persulfate K2S2O8 (8 mg/10 ml) solutions using distilled deionized water. Mix
equal volumes of the 2 and let react in the dark for atleast 12 hr at room
temperature.
2. Working Solution: Add 5 ml of Mother Solution to 145 ml of phosphate buffer
solution.
Prepare 500 mL of phosphate buffer containing 0.2 M Na2HPO4 (12.1 g)
and 0.2 M NaH2PO4.H2O (2.62 g). 8.77 g of NaCl was added and the
pH was adjusted to 7.4 using 1 Mol NaOH.
3. Trolox standard: Add 290 µl of the working solution to 10 µl serial trolox
dilutions (0 µg/ ml, 50 µg/ ml, 100 µg ml, 200 µg/ml, 300 µg/ml, 400 µg/ ml, 500
µg/ ml), shake and let react for 15 min. Measure absorbance at 734 nm.
Absorbance readings should fall between 0.1-1.6. R² should be atleast 0.995.
Trolox:
145
Standard solution:10000 µg/ml, which is 10 mg ml. ake 100mg of trolox
and add 20 ml 200 proof ethanol. Now the concentration is 5 mg/ ml. but a
working solution of 1 mg/ ml is required.
Working solution: from the 5 mg/ml standard solution, take 1 ml and
dilute with 4 ml of 200 proof ethanol. So this makes 1 mg/ ml.
Trolox dilutions: 0 µg/ ml: 1000 µl 10% ethanol
50 µg/ ml + 950 µl 10% ethanol
100 µg/ ml + 900 µl 10% ethanol
200 µg/ ml + 800 µl 10% ethanol
300 µg/ ml + 700 µl 10% ethanol
4. Sample Analysis: add 290 µl working solution to 10 µl sample extract (dilute 10
times with deionized water), shake and let react for exactly 30 min. Measure
absorbance at 734 nm.
A.5 Total Antioxidant Activity: DPPH Assay
1. Prepare DPPH solution by dissolving 24 mg DPPH in 100 ml 80% ethanol.
2. Dilute this stock solution ~10:55 with 80% ethanol until the spectrophotometer
reads 1.1 at 515 nm.
3. Pipette out juice of 15 l in to a scintillation vial and add 285 l of diluted DPPH
solution.
4. Allow the reaction for 30 min. after 30 min measure the absorbance at 517 nm
using a spectrophotometer.
146
5. Prepare a standard curve of known concentrations of trolox. Use regression
equation to convert the antioxidant activity into equivalents of trolox.
A.6 Anti-Proliferation Assay: WST-1 Assay
1. Seed 6,000 cells /well in microplates to a final volume of 200 µL culture
medium and incubate in a humidified atmosphere (37°C, 5% CO₂).
2. After 24 h, replace the medium in the cells with fresh DMEM medium
containing 0.25% stripped serum. Add 5, 15 and 25 µg GAE / mL of juice to
the cells. Incubate at 37°C for 24 h.
3. Add 10 µL/ well WST-1 reagent after 24 h.
4. Incubate the cells for 2 h in a humidified atmosphere (37°C, 5% CO₂).
5. Measure the absorbance using a microplate reader at 480 nm.
A.7 Anti-Proliferation Assay: Cell Count
1. Seed 5× 10⁴ cells in 12-well plates. Allow the cells to grow for 24 h in a
humidified atmosphere (37°C, 5% CO₂).
2. After 24 h, replace the medium with fresh DMEM medium containing 0.25%
stripped serum and add 15 and 25 µg GAE / mL of juice to the cells. Let the cells
to react for 24 h at 37°C in an incubator.
3. 24 h later, count the cells using a cell counter. Add 20 µL of the trypsinized cells
from the plate to a disposable cell counting slide. The cell counter gives the
results as cells /mL.
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A.8 Pro-Apoptotic Assay: Caspase-Glo
1. Seed 5× 10⁴ cells in 12-well plates. Allow the cells to grow for 24 h in a
humidified atmosphere (37°C, 5% CO₂).
2. After 24 h, replace the medium with fresh DMEM medium containing 0.25%
stripped serum and add 15 and 25 µg GAE / mL of juice to the cells. Let the cells
to react for 24 h at 37°C in an incubator.
3. 24 h later, count the cells using a cell counter. Transfer 2× 10⁴ cells to a opaque
microplate used for reading luminescence and make up the volume to 200 µL
using DMEM medium containing 0.25% charcoal stripped serum.
4. Add 100 µL of Caspase-Glo 3/7 reagent to all wells and briefly mix by shaking.
5. Incubate for 30 min at room temperature.
6. Measure the luminescence.
A.9 Cytotoxicity: LDH Assay
1. Seed 6,000 cells /well in microplates to a final volume of 200 µL culture medium
and incubate in a humidified atmosphere (37°C, 5% CO₂).
2. After 24 h, replace the medium in the cells with fresh DMEM medium containing
0.25% stripped serum. Add 5, 15 and 25 µg GAE / mL of juice to the cells.
Incubate at 37°C for 24 h.
3. Next day, transfer 100 µL of supernatant from the cells to a 96-well microplate.
Remove the supernatant without disturbing the cells. Supernatant is taken before
WST-1 assay or cell count is done.
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4. 100 µL of the LDH reagent was added and was incubated for 30 min in dark at
room temperature.
5. After 30 min, the absorbance was measured at 492 nm.
6. Background control: LDH reagent.
Low control: supernatant from cells not treated with juice.
High control: cells treated with Triton-X 100.
7. Cytotoxicity (%) was calculated by using the following equation. The absorbance
value was subtracted from its background control to obtain the expected value.
Cytotoxicity (%) = expected value - low control
high control - low control