Table Of ContentVINCA ALKALOIDS FROM ENDOPHYTIC
FUNGI: ISOLATION, PURIFICATION,
CHARACTERIZATION AND BIOASSAYS
Thesis submitted to University of Pune
For the degree of
DOCTOR OF PHILOSOPHY
IN BIOTECHNOLOGY
BY
ASHUTOSH KUMAR
UNDER THE GUIDANCE OF
DR. ABSAR AHMAD
DIVISION OF BIOCHEMICAL SCIENCES
NATIONAL CHEMICAL LABORATORY
PUNE - 411 008,
INDIA
FEBRUARY 2011
Affectionately Dedicated
To
My Beloved Mother
Research is to see what everybody else has seen
and to think what nobody else has thought.
-Albert-Szent-Gyorgi (1893-1986) U. S. Biochemist
ACKNOWLEDGEMENT
The work presented in this thesis would not have been possible without sincere
support of my mother.
First and foremost, I would like to extend my sincere gratitude to my research guide
Dr. Absar Ahmad for introducing me to this exciting field of science and for his
dedicated help, advice, inspiration, encouragement and continuous support,
throughout my Ph.D. His enthusiasm, integral view on research and his mission for
providing high-quality work, has made a deep impression on me. During our course
of interaction during the last six years, I have learnt extensively from him, including
how to raise new possibilities, how to regard an old question from a new perspective,
how to approach a problem by systematic thinking, data-driven decision making and
exploiting serendipity. Every time, I meet him, I can feel “the fire in the belly” which
he always talks about. I owe him lots of gratitude for having me shown this way of
research. His constant guidance, cooperation, motivation and support have always
kept me going ahead. I owe a lot of gratitude to him for always being there for me
and I feel privileged to be associated with a person like him during my life. I am
really glad to be associated with a person like Dr. Absar Ahmad in my life.
My special words of thanks should also go to my research co-guide late Dr.
M.I.Khan for his continuous support, guidance, cooperation, encouragement and for
facilitating all the requirements, going out of his way.
I would like to thank our collaborator Dr. P.R.Rajmohanan, Dr. H.B.Borate and Dr.
(Mrs). Shanta Kumari for their help during the course of investigation.
I am thankful to, Dr. Vidya Gupta, Dr. C. G. Suresh and Dr. Dhiman Sarkar for their
help and cooperation during my tenure as research scholar.
My heartfelt thanks to fellow labmates, Dr.Sreekanth, Asad, Shadab ,Ravi, Imran,
Prasad, Sana, Vidyut,Vidya, Ankita,Rusha, Purva and Rasika for always standing by
my side and sharing a great relationship as compassionate friends. I will always
cherish the warmth shown by them.
I would like to express my deep felt gratitude to my friends Dr.Feroz, Dr.Atul,
Dr.Vinod , Dr.Sajid, Dr. Sarvesh, Dr.Shivshankar, Avinash, Ansary, Madhurima,
Sonali, Upasna, Sampa, Anamika, Satish, Deepak ,Yogesh, Fazal, Amol, Harish,
Manisha, Vitthal, Harish,Urvashi, Mansi, Raju and Ishwar from whom I have
received unfailing support and encouragement during my research.
A special mention of thanks to my Trainees in NCL-Vinayak, Rakesh, Leena,
Sushmita, Anushree, Swati, Rajashree, Venna, Smita, Yogendra, Sheetal, Aparna,
Vidhya, Shanawaz, Hasan, Rusha,Swati, Rajshree, Swapnil, Milind, Nirupma, Neha ,
Shubhashree , Sujeet, Varsha and many more for their constant support and
cooperation during my stay.
A special thanks to Sana, Ankita, Varsha, Shubhashree and Neha for their support
during the completion of my work.
My special regards to my teachers because of whose teaching at different stages of
education has made it possible for me to see this day. Because of their kindness I was
able to reach a stage where I could write this thesis.
I must thank Dr.Sushma Gaikwad, Dr.Sumedha Deshmukh for their kind support
throughout my tenure at NCL.
I acknowledge Indira Mohandasan, Giri jee and Trehan jee who were always ready
to give their timely help whenever required.
I owe my deepest gratitude towards my beloved MOTHER for her eternal support
and understanding of my goals and aspirations. Her infallible love and support has
always been my strength. Her patience and sacrifice will remain my inspiration
throughout my life. Without her help, I would not have been able to complete much of
what I have done and become who I am. She had taught me another aspect of life,
that, “goodness can never be defied and good human beings can never be denied”
As always it is impossible to mention everybody who had an impact to this work
however there are those whose spiritual support is even more important. I feel a deep
sense of gratitude for my Father, who formed part of my vision and taught me good
things that really matter in life.
I would be grateful to my better half Mudita, for her love and moral support. I am
thankful to my cute son Roshan for giving me happiness during the last two years of
my studies.
My heartfelt regards goes to my sisters, brothers, nieces and nephews for their love
and affection.
I am also very much grateful to all my family members for their constant inspiration
and encouragement.
I also thank Ma’am (Mrs. Absar Ahmad) for her love and care, which never made me
feel that I am away from my mother.
I gratefully acknowledge Council of Scientific and Industrial Research, for
providing me financial support and Director, NCL to allow me to carry out my
research in this esteemed laboratory.
Finally, I am thankful to library staff and administrative staff of NCL for
cooperation.
ASHUTOSH KUMAR
CERTIFICATE
Certified that the work incorporated in the thesis entitled “Vinca alkaloids from
endophytic fungi: Isolation, Purification, Characterization and Bioassays”,
submitted by Mr. Ashutosh Kumar, for the Degree of Doctor of Philosophy, was
carried out by the candidate under my supervision at Division of Biochemical
Sciences, National Chemical Laboratory, Pune - 411008, Maharashtra, India.
Date: Dr. Absar Ahmad
Place: Pune (Research Guide)
DECLARATION BY RESEARCH SCHOLAR
I hereby declare that the thesis entitled “Vinca alkaloids from endophytic fungi:
Isolation, Purification, Characterization and Bioassays”, submitted for the
Degree of Doctor of Philosophy to the University of Pune, has been carried out by
me at Division of Biochemical Sciences, National Chemical Laboratory, Pune- 411
008, Maharashtra, India, under the supervision of Dr. Absar Ahmad. The work is
original and has not been submitted in part or full by me for any other degree or
diploma to any other University.
Date: Ashutosh kumar
Place: Pune (Research Scholar)
INDEX
CHAPTER 1: General Introduction
Introduction 01-36
References 37-50
CHAPTER 2: Isolation, purification and maintenance of Endophytic fungi
associated with leaves of Catharanthus roseus.
Summary 51
Introduction 52-53
Materials and methods 54
Results 55-59
Discussion 60
References 61-64
CHAPTER 3: Screening of endophytic fungi for production of vinca
(vinblastine and vincristine) alkaloids.
Summary 65
Introduction 66-67
Materials and methods 68-69
Results 70-72
Discussion 73
References 74-75
CHAPTER 4: Cultural, morphological and molecular characterization of vinca
alkaloids producing endophytic fungi of Catharanthus roseus.
Summary 76
Introduction 77-78
Materials and methods 79-81
Results 82-93
Discussion 94-95
References 96-98
CHAPTER 5: Isolation, purification, characterization and bioassays of
vinblastine.
Summary 99
Introduction 100-101
Materials and methods 102-105
Results 106-114
Discussion 115-116
References 117-120
CHAPTER 6: Isolation, purification, characterization and bioassays of
vincristine.
Summary 120
Introduction 121
Materials and methods 122-126
Results 127-135
Discussion 136-137
References 138-141
CHAPTER 7: Fungal mediated transformations
I.Fungal mediated transformation of vinblastine to vincristine.
Summary 142
Introduction 143-144
Materials and methods 145-146
Results 147-150
Discussion 151
II. Fungal mediated transformation of catharanthine and vindoline to
vinblastine.
Summary 152
Materials and methods 153-154
Results 155-156
Discussion 157
II. Fungal mediated transformation of L-tryptophan and secologanin to
vinblastine.
Summary 158
Materials and methods 159-160
Results 161-162
Discussion 163
References 164-167
CHAPTER 8: Conjugation of nanoparticles with vinca alkaloids.
Summary 168
Introduction 169-170
Materials and methods 171
Results 172
Discussion 173
References 174-175
CHAPTER 9: General discussion and conclusion.
Discussion 176-182
Conclusion 183-184
References 185-190
Chapter 1
General Introduction
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