studies on antioxidants and antimetastatic components from spices against cancer

238 Pages·2010·5.08 MB·English

by  Harish Nayaka

STUDIES ON ANTIOXIDANTS AND
ANTIMETASTATIC COMPONENTS FROM
SPICES AGAINST CANCER

A thesis submitted to the

University of Mysore

for the award of the Degree of

Doctor of Philosophy

in

Biochemistry

by

M.A. Harish Nayaka, M.Sc.

Department of Biochemistry and Nutrition
Central Food Technological Research Institute
Mysore – 570 020, India

December 2007
DECLARATION

I hereby declare that the thesis entitled “STUDIES ON
ANTIOXIDANTS AND ANTIMETASTATIC COMPONENTS FROM SPICES
AGAINST CANCER” submitted to the University of Mysore for the award
of degree of Doctor of Philosophy in Biochemistry, is the result of
research work carried out by me under the guidance of Dr. Shylaja M
Dharmesh, Scientist, Department of Biochemistry and Nutrition, Central
Food Technological Research Institute (CFTRI), Mysore - 570020, India,
during the period 2000-07. I further declare that the results presented in
this thesis have not been submitted for the award of any other degree or
fellowship.

Date:
Place: Mysore

(M.A. Harish Nayaka)
Dr. Shylaja M Dharmesh, PhD
Scientist,
Department of Biochemistry and Nutrition

CERTIFICATE

This is to certify that the thesis entitled “STUDIES ON
ANTIOXIDANTS AND ANTIMETASTATIC COMPONENTS FROM SPICES
AGAINST CANCER” submitted by Mr. M. A. Harish Nayaka, for the award
of Doctor of Philosophy in Biochemistry to the University of Mysore is
the result of research work carried out by him in the Department of
Biochemistry and Nutrition, Central Food Technological Research Institute,
Mysore, under my guidance during the period 2000-07.

Date:
Place: Mysore

(Shylaja M Dharmesh)
(Guide)
DDeeddiiccaatteedd ttoo

all the DDeevvootteedd SScciieennttiissttss across time who have
helped us to realize life

and to mmyy bbeelloovveedd tteeaacchheerrss..
Acknowledgements

My heartfelt gratitude to my research supervisor Dr. Shylaja M
Dharmesh for her guidance, constant supervision and support. I am grateful
to her for having believed in my research capabilities and providing me with
the utmost independence at work during my research program. I cherish all
her discussions and advice which strengthened my confidence.

I wish to express my sincere thanks to Dr. V. Prakash, Director, CFTRI,
Mysore for providing me an opportunity to work in the institute and also for
his keen interest and guidance in my research program.

I extend my warm gratitude to Dr. P. V. Salimath, Head, Department of
Biochemistry and Nutrition and former Head, Dr. S. G. Bhat for their concern
towards students and also for extending support to carry out my research
work.

I owe my thanks to Special Cell for SC/ST, University of Mysore for
providing me Teacher Research Fellowship to complete my PhD work. Also,
my thanks go to The Principal, Yuvaraja’s College, University of Mysore for
relieving me from my duties to carry out my PhD program.

I thank Dr. P. Saibaba, Animal House Incharge, Dept. of Biochemistry
and Nutrition, CFTRI, Mysore for his timely help in conducting animal
experiments.

I appreciate the help and support of my lab mates Yogendra Nayak,
Siddaraju, Smitha, Sathisha and Srikanta.
I thank my seniors starting from Venkatesh L Hegde, Yashoda, Saby
John, Kempaiah, Jagadish, Subba Rao, Rachappaji, Thippeswamy, Suresh
Kumar and many more for their help to the maximum.

My special thanks go to CFTRI cricket and volley ball team friends, and
swimming instructor Mr. Gopal for making my stay at CFTRI memorable.

I am highly indebted to Dr. R. L. Jagadish, Senior Lecturer, Dept of
Polymer Science, University of Mysore and Mr. M. P. Manohar, Lecturer, Dept.
of Sugar Technology, University of Mysore for their invaluable help and moral
support.

Finally, I thank my parents S. Annaiah and B. D. Kamalamma, Brother
M. A. Mahesh Nayaka, Sister M. A. Prema, Brother-in-law, B. D. Mahadeva,
Mother-in-law Nanjammani and my wife Suma for their support.
Contents
Topics Page No.
Abbreviations and Units -- i
Abstract -- vi
Synopsis -- viii
Chapter 1. General Introduction -- 01

Chapter 2. Isolation and characterization of newer
antioxidants from black cumin and swallow root

2.1. Hypothesis -- 47
2.2. Work Concept -- 48
2.3. Introduction -- 49
2.4. Materials and Methods -- 55
2.5. Results
2.5.1 Total Phenolic content in Black Cumin and
Swallow root extracts. -- 64
2.5.2 Effect of Black Cumin extracts in inhibiting
LDL oxidation. -- 66
2.5.3 Effect of Black Cumin extracts on soybean
lipoxygenase induced linoleic acid peroxidation. -- 67
2.5.4. DPPH radical Scavenging ability. -- 68
2.5.5. Reducing power ability. -- 69
2.5.6. Cytoprotective effect. -- 71
2.5.7. DNA Protective effect. -- 73
2.5.8. Identification of phenolic acids in Black cumin
and Swallow root extracts. -- 74
2.5.9. Characterization of the major antioxidant
compounds of Black cumin and Swallow root extracts. -- 77
2.5.10. Contribution from phenolic acids in Black cumin
and Swallow root antioxidant activity. -- 80
2.6. Discussion -- 83
Summary and conclusion -- 91

Chapter 3. Isolation and characterization of
antimetastatic components from spices

3.1. Hypothesis -- 92
3.2. Work Concept -- 93
3.3. Introduction -- 94
3.4. Materials and Methods -- 96
3.5. Results
3.5.1. Extraction of pectic polysaccharide from
Black cumin and Swallow root -- 104
3.5.2. Antimetastatic property of pectic
polysaccharides of black cumin (BCPP) and
swallow root (SRPP) -- 104
3.5.3 Purification of SRPP -- 105
3.5.4. Determination of purity of active SRPP
fraction – 0.15 M by HPLC. -- 106
3.5.5. Sugar composition analysis of SRPP and
DEAE fractions of SRPP -- 107
3.5.6. Determination of molecular size of active
0.15 M pectic polysaccharide fraction of SRPP -- 108
3.5.7. Determination of NMR analysis of active
0.15 M pectic polysaccharide fraction of SRPP. -- 110
3.6. Discussion -- 112
Summary and conclusion -- 115
Chapter 4. Determination of efficacy andmechanism
of action of antioxidants and antimetastatic components
using in vitro biochemical and cell culture assay
model systems

4.1. Hypothesis -- 116
4.2. Work Concept -- 118
4.3. Introduction -- 119
4.4. Materials and Methods -- 121
4.5. Results
4.5.1 HSA-phenolics interaction studies -- 124
4.5.2 DNA – phenolics interaction studies -- 133
4.5.3 Antimetastatic components-cell interaction studies -- 138
4.6. Discussion -- 141
Summary and conclusion -- 146

Chapter 5. Efficacy of combinational supplementation
of antioxidants and antimetastatic components against
cancer employing in vivo cancer animal models

5.1. Hypothesis -- 147
5.2. Work Concept -- 148
5.3. Introduction -- 149
5.4. Materials and Methods -- 151
5.5. Results
5.5.1. Effect of antioxidants in aqueous extracts of
black cumin and swallow root on oxidative stress
induced hepatotoxicity by CCl in albino rat model. -- 162
4
5.5.2. Effect of Antioxidants in aqueous extracts of
black cumin and swallow root on 20-methylcholanthrene
induced cervical cancer in albino mice model. -- 166
5.5.3. Effect of antioxidants of swallow root-HMBA
and galectin-3 inhibitory polysaccharides-SRPP on
B16F10 mouse melanoma cells induced metastasis. -- 170
5.6. Discussion -- 179
Summary and conclusion -- 187

References -- 188
Publications -- 209