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Title
DECLARATION
CERTIFICATE
ACKNOWLEDGEMENT
CONTENTS
ABBREVIATIONS
1. Introduction and review of literature
INTRODUCTION
REVIEW OF LITERATURE
I. Molecular biology of cancer
A. Genes and Cancer
1. Proto-oncogenes
2. Tumor suppressor genes
3. Repair genes
4. Chromosomal aberrations
B. Cancer and cell growth regulatory pathways
1. Growth factors (GPs)
2. Cell cycle check points.
II. Etiology of Cancer
A. Carcinogenesis
1) Physical insults (Radiation)
a) Ultraviolet radiation
b) Ionizing Radiation
2) Biological insults (Viral carcinogenesis)
3) Chemical insults (Chemical carcinogenesis)
a) Organic carcinogens
i) Alkylating agents
ii) Aralkylating agents
iii) Arylhydroxylamines
b) Inorganic compounds
(i) Tumor initiation
ii) Tumor promotion
iii) Tumor progression
III. Free radicals, Antioxidants and Cancer
IV. Cancer Therapy
a) Surgery
b) Radiation Therapy
c) Chemotherapy
Chemotherapeutic agents
1) Non-plant derived anticancer agents
a) Alkylating agents
b) Antimetabolites
c) Antitumor antibiotics
d) Enzymes
f) Cisplatin
g) Procarbazine
h) Hydroxyurea
i) Amsacrine
2) Plant Derived Anticancer Agents
1. Vinca alkaloids
2. Homoharringonin (BHT)
3. Podophyllotoxins
4. Maytansine
5. Taxanes
6. Camptothecin
V. Production of anti-cancer secondary metabolites through tissue culture
2. Materials and methods
2.1. Materials
2.1.1. Plant Materials
2.1.2. Chemicals
2.1.3. Instruments
2.1.4. Cell lines
2.1.5. Animals
2.2. Methods
2.2.1. Explants and surface sterilization
2.2.2. Preparation of nutrient media
2.2.3. Culture Environment
2.2.4. Culture initiation and callus culture establishment
2.2.5. Callus growth measurement
2.2.6. Preparation of the plant extract
2.2.7. Preparation of the callus extracts
2.2.8 Preparation and application of spray reagents
2.2.9. Extraction of crude camptothecin (CPT)
2.2.10. Isolation of CPT
2.2.11. Crystallization of CPT
2.2.12. TLC analysis
2.2.13. Melting point
2.2.14. UV-visible absorption spectra
2.2.15. IR-spectra
2.2.16. High performance liquid chromatography (HPLC)
2.2.17. Electron Spray Mass Spectrometry (ESMS)
2.2.18. H-nuclear magnetic resonance
2.2.19. Maintenance of Experimental animals
2.2.20. Maintenance of tumour cells in mice
2.2.21. Maintenance of cell lines in tissue culture
2.3. Anti tumor studies
2.3.1 Short term in vitro cytotoxocity
2.3.2 Long term chemosensitivity
2.3.3 MTT assay
2.3.4 3H-thymidine incorporation assay
2.3.5 Ascites tumor model
2.3.6 Solid tumor model
2.4 Antioxidant property in vitro
2.4.1 Superoxide scavenging activity
2.4.2 Hydroxyl radical scavenging activity
2.4.3 Lipid peroxide inhibition activity
2.4.4 Anti-inflammatory activity
2.4.5 Evaluation of anti carcinogenic activity of the extracts
2.4.6. Histopathology
2.5. Statistical analysis
3. Isolation and quantification of camptothecin from.Nothapodytes foetida
3.1 Introduction
3.2 Materials and methods
3.2.1 Plant material
3.2.2 Preparation of the extract
Fig: 3.1. Nothapodyes foetida (White) Slummer.
3.2.3 Crystallization of CPT
3.2.4 Determination of CPT melting point
3.2.5 uv-visible spectrum
3.2.6 HPLC analysis and quantification
3.3 Results and discussion
Fig 3.2 Schematic representation tor the isolation of CPT from plantparts of Nothapodytes foetida
4. Camptothecin from callus cultures of Nothapodytes foetida
4.1 Introduction
4.2 Materials and methods
4.2.1 Explants
4.2.2 Surface sterilization
4.2.3 Callus subculture
4.2.4 Callus growth measurement
4.2.5 Extraction of CPT and HPLC analysis
4.2.6 Purification and characterization of CPT
Fig 4.1 Camptothecin extraction from callus
4.2.7 Long term Cytotoxicity of the isolated CPT
4.3 Results
4.3.1 Callus induction and callus growth
Fig 4.2
(A) An Immature embryo of N.foietida
(B) 90 day old callus shows leafy out growth in the modified MS medium supplemented with NAA 2mg/I and KN 0.5 mg/I
(C, D, E) Plantlets grown in the MS medium supplemented with NAA 2 mg/I and BA 0.5 mg/I
4.3.2 Thin layer chromatography
4.3.3 uv absorption
4.3.4 HPLC analysis
4.3.5 IR spectra of purified CPT
4.3.6 Mass spectra
4.3.7 H-NMR NMR spectra
4.3.8 Cytotoxicity
4.4 Discussion
5. Camptothecin from tissue cultures of Ervatamia heyneana
5.1 Introduction
5.2 Materials and Methods
5.2.1 Plants
5.2.2 Surface sterilization of the explant and culture
5.2.3 Callus growth measurement
5.2.4 Callus subculture
5.2.5 Callus extract preparation
5.2.6 Determination of chemosensitivity of the extract on L929 cells
5.2.7 Anti-tumor activity of iu vitro derived root extract
5.2.8 TLC analysis
5.2.9 uv visible absorption spectra
5.2.10 HPLC analysis
5.3 Results
5.3.1 Surface sterilization
5.3.2 Culture initiation
5.3.3 Influence of auxins
5.3.4 Influence of auxins and cytokinins
Fig: 5.1 Ervatamia heyneana (Wall) Cook
Fig 5.2
(A) Internode-derived callus in MS medium containing 2, 4-D2 mg/l and KN 0.5 mg/l.
(B) Rhizogenesis in seed embryo cultured in MS medium Supplemented with NAA 4 mg/l and KN 0.5m g/l
(C) Plantlets in seed internode callus cultured tn NAA 2mg/l and BA 0.5 mg/l
(D) Plantlets and rhizognesis in MS medium supplemented with NAA 2mg/l and BA 0.5 mg/l
(E) Rhizognesis in seed embryo cultured in MS liquid medium Supplemented with NAA 4mg/l and KN 0.5 mg/I
(F) Somatic embryoids in seed embryo cultured in MS medium containing 2, 4-D 3 mg/l and KN 0.5 mg/l
Fig 5.3 Leaf disc explant after 30 dav culture in MS medium supplemented with 2, 4-D 2mg/I & BN 0.5 mg/I
5.3.5 Callus growth
5.3.6 Anticancer activity of the extracts on L929 cells
5.3.7 in vivo anti-tumor propeq of the extract
5.3.8 TLC analysis
5.3.9 uv-visible absorption spectra
5.3.10 HPLC analysis
5.4 Discussion
6. Study on antioxidant and anticancer activity of Emilia sonchifolia
6.1 Introduction
6.2 Materials and methods
6.2.1 Preparation of crude extract from plant
6.2.3 Animals
6.2.4 Superoxide scavenging activity
6.2.5 Hydroxyl radical scavenging activity
6.2.6 Anti-inflammatory activity
6.2.7 In vitro cytotoxicity of the extract
6.2.8 Effect of methanollic extract on ascites tumor in mice
6.2.9 Effect of methanolic extract on solid tumor development
6.2.10 Effect of methanolic extract on DNA synthesis
6.2.11 Partial purification of E.sonchifolia extract by XAD amberlite column
6.2.12 Phytochemical analysis of the active fraction
6.2.13 uv visible absorption of the active fraction
6.3 Results
6.3.1 Antioxidant and anti-inflammatory activity of the methanolic extract of E. sonchifolia
6.3.2 Cytotoxic and antitumor property
6.3.3 Effect of methanolic extract on DNA synthesis
6.3.4 Antioxidant and anticarcinogenic activity of partially purified extract
6.4 Discussion
7. In vitro antioxidant and cytotoxic Properties of Callus and plantlet cultures of Emilia sonchifolia
7.1 Introduction
7.2 Materials and methods
7.2.1 Surface sterilization and culture medium
7.2.2 Callus culture
7.2.3 Rooting of regenerated shoots
7.2.4 Establishment of regenerated plantlets
7.2.5 Preparation of the extracts
7.3 Antioxidant property
7.3.1 Superoxide scavenging activity
7.3.2 Hydroxyl radical scavenging activity
7.3.3 Lipid peroxidation inhibition
7.4 In vitro cytotoxicity of the extracts.
7.5 TLC analysis of the extracts
7.6 Results
Fig 7.1 Shoot multiplication in E.sonchifolia cultured in MS medium supplemented with IAA 2 mg/l and BA mg/l
Fig 7.2 Leaf disc derived callus in MS medium supplemented with 2, 4-D 2 mg/l and KN 0.5 mg/l
DISCUSSION
8. Summary and Conclusion
APPENDIX
References
Papers published