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Exploration of Antioxidant and Anticancer Potential of Essential Oil of Salvia Plebeia Rbr

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Exploration of Antioxidant and Anticancer Potential of Essential Oil of Salvia Plebeia Rbr EXPLORATION OF ANTIOXIDANT AND ANTICANCER POTENTIAL OF ESSENTIAL OIL OF SALVIA PLEBEIA RBR. A Dissertation submitted to the Central University of Punjab For the Award of Master of Philosophy in Environmental Science and Technology BY Nandini Gautam Supervisor: Dr. Sunil Mittal Centre for Environmental Science and Technology School of Environment and Earth Sciences Central University of Punjab, Bathinda September, 2012 CERTIFICATE I declare that the dissertation entitled “EXPLORATION OF ANTIOXIDANT AND ANTICANCER POTENTIAL OF ESSENTIAL OIL OF SALVIA PLEBEIA RBR.” has been prepared by me under the guidance of Dr. Sunil Mittal, Assistant Professor, Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab. No part of this dissertation has formed the basis for the award of any degree or fellowship previously. (Nandini Gautam) Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab, Bathinda - 151001. Date: i ACKNOWLEDGEMENT Accomplishment of this work would not have been possible without the blessings of almighty God. I pay Him tribute whose blissful blessings enabled me to scale every problem that came in my path. I would like to thank my Supervisor, Dr. Sunil Mittal, Assistant Professor, Centre of Environmental Science and Technology, Central University of Punjab, Bathinda, under whose guidance the present work was carried out. I am grateful for his guidance, cooperation, immense support and valuable suggestions during the research work. I am highly obliged for the efforts of Dr. Sandeep Singh, who was there at every step to help, guide and critically analyse the difficulties encountered at every step of the accomplishment of this work. I would like to thank him from the core of my heart for teaching tissue culture techniques. Also, I would like to express my heartiest gratitude to Dr. Ajit Vikram who helped me in my work. I express my deep gratitude to Prof. P. Rama Rao, Dean Academic Affairs, Central University of Punjab, Bathinda for solving my queries, consistent support and guidance and for providing excellent laboratory facilities. I am highly thankful to Prof. R. G. Saini, COC Biosciences, Central University of Punjab, Bathinda for his immense support and guidance. I acknowledge Dr. Punita Pandy, Dr. Dhanya, Dr. Yogalakhshami, Dr. Nageder Babu, Dr. Vikas Jaitak and Dr. Raj Kumar for their encouragement and helping nature. I am highly grateful to Dr. Sanjeev Kumar Thakur, Assistant Professor, for his continuous support and helpful attitude while working in Tissue culture laboratory. I would like to acknowledge Dr. Bikram Singh, Head of the Natural Plant Products Division, IHBT, Palampur (H.P), for helping me in the GC-MS analysis of the essential oil. I am thankful to my seniors Mr. Gajendra Singh Vishwkarma, Mr.Prateek, Mr.Pushpender, Ms.Anamika, Ms.Upma and Mrs. Manohari for their immense support and help in day-to-day work. I have no words to thank my friends Shilpa, Gurpreet Kaur, Jaskiran and all others for their cooperation and support. I am thankful to all the non-teaching staff that helped me at various stages during this project but may not be listed here. I also offer big thanks to Mr. Gopal, Ms. Sona and Mr. Ashwani for their kind cooperation and support in the hour of need. I pay my sincere thanks to my parents, Mandakini Di, Ashu and beloved ones for their unconditional love, support, encouragement, trust and moral support. This work is dedicated to my sweet parents. (Nandini Gautam) iii CERTIFICATE I certify that NANDINI GAUTAM has prepared her dissertation entitled “EXPLORATION OF ANTIOXIDANT AND ANTICANCER POTENTIAL OF ESSENTIAL OIL OF SALVIA PLEBEIA RBR.” for the award of M.Phil. degree of the Central University of Punjab, under my guidance. She has carried out this work at the Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab, Bathinda. (Dr. Sunil Mittal) Assistant Professor Centre for Environmental Science and Technology, School of Environment and Earth Sciences, Central University of Punjab, Bathinda - 151001. Date: ii ABSTRACT “Exploration of Antioxidant and Anticancer Potential of Essential Oil of Salvia plebeia RBr.” Name of student : Nandini Gautam Registration Number : CUP/MPh-PhD/SEES/EVS/2010-2011/04 Degree for which submitted : Master of Philosophy Name of Supervisor : Dr. Sunil Mittal Centre : Centre for Environmental Science and Technology School of Studies : School of Environment and Earth Sciences Cancer; Anticancer Agents; Essential Oil; Salvia Key words : plebeia; Caryophyllene Cancer is one of the major problems that accounts for millions of death worldwide per year. Breast cancer is the most commonly encountered cancer in the females. Chemotherapy and radiotherapy are the standard methods used in the treatment of cancer but these have various side effects also. Other complementary treatments based on plants and its products are used to lessen the side effects. Some essential oils are the potent source of the novel molecules and have been reported to have anticancer effect. In the present study, essential oil from Salvia plebeia has been screened for cytotoxic activity in vitro using MTT assay and has been observed to show inhibitory effect on the cancer cells with the increase in concentration against breast cancer cell lines, MCF-7 and MDAMB-231. The essential oil was observed to show IC50 at concentration 30µg/ml for MCF-7 cells and in MDAMB-231, IC50 was calculated to be 65µg/ml for 24 hours treatment. Essential oil was found to be effective against highly metatstatic MDAMB-231cells Exposure time effect was studied to know whether it has sustained effect or not and it was observed that essential oil had sustained effect at higher concentrations and longer exposure time was required to show sustained effect. Single cell gel electrophoresis (Comet assay) was used to find out its mode of action and it was observed to induce DNA damage in the cancer cells that might have resulted in cell death at various concentrations. Essential oil was observed to have antioxidant activity, which was studied using DPPH and hydroxyl radical scavenging assay. Various compounds like cayophyllene, alpha-humulene and cayophllene oxide were determined by GC-MS analysis. Cayophyllene was the major compound (46%). Some of the constituents might be responsible for anticancer activity of the oil. It has been concluded from the study that novel essential oil is very effective against breast cancer cells. This oil can be screened further against other cell lines and can be used as nutritional supplement. (Nandini Gautam) (Dr. Sunil Mittal) vii TABLE OF CONTENTS Sr. No. Contents Page No. 1. Introduction (Chapter-1) 1-5 2. Review of Literature (Chapter-2) 6-18 3. Objectives (Chapter-3) 19 4. Materials and Methods (Chapter-4) 20-26 5. Results and Discussion (Chapter-5) 27-42 6. Conclusion and Future Prospect 43-44 7. Summary 45-46 8. References 47-55 iv LIST OF TABLES Table Table Description Page No. No. 1.1 Distribution and characteristics of the plant and its essential 4 oil 2.1 Studies related to various species of Salvia indicating its 9-11 anticancer properties 2.2 Essential oils as anticancer agents 14-15 4.1 Instruments used and the company 20 4.2 Treatments of the essential oil 24 5.1 Major compounds of essential oil of Salvia plebeia RBr. and 28 the data obtained by GC-MS analysis with their classification v LIST OF FIGURES Figure Description of Figure Page No. No. GC chromatogram of the essential oil obtained on the 5.2.1 27 DB-5 column DPPH radical scavenging ability of the essential oil of 5.3.1 29 Salvia plebeia Effect of essential oil Salvia plebeia on hydroxyl radical 5.3.2 30 scavenging Effect of essential oil of Salvia plebeia on MCF-7 cell 5.4.1 31 line for 12 and 24 hours treatments Effect of essential oil of Salvia plebeia on MDAMB-231 5.4.2 32 cell line for 12 and 18 hours Essential oil of Salvia plebeia showing significant 5.4.3 33 decrease in the viability of MCF-7 cells for 24 hours Effect of 24 hours treatment of essential oil of Salvia 33 5.4.4 plebeia on MBAMB-231 cells Effect of essential oil of Salvia plebeia on MCF-7 cells 5.4.5 34 with different exposure and chase times Effect of essential oil of Salvia plebeia on MDAMB-231 5.4.6 35 cells with different exposure and chase time Effect of the essential oil of Salvia plebeia on the individual MCF-7 cells showing the formation of the 5.4.7 36 comets in the treatment groups compared to control group observed at 40X of the fluorescent microscope Histograms (A-F) showing the DNA damage induced by the different concentration determined by parameters 5.4.8 37 such as Head Intensity, Tail Intensity, % DNA in Head and Tail, Head diameter and Tail Length vi CHAPTER-1 INTRODUCTION Cancer has emerged as one of the most dreadful diseases in the last few decades throughout the world. It is a multifactorial disease leading to uncontrolled growth and invasion of the abnormal cells leading to the formation of tumour. The steep rise in the number of cancer cases may be attributed to change in food habits, use of tobacco and alcohol, chronic infections, exposure to harmful radiations and environmental pollution or more widely we can say due to change in lifestyle and environmental pollution (NCBI, 2010). As per International Agency for Research on Cancer, there were approximately 12 million cancer cases and these had accounted for 7.6 million deaths worldwide (around 13% of all deaths) in the year 2008. Moreover, cancer cases have been projected to increase over 13.1 million by 2030.
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