effect of wheatgrass on alcohol and ∆pufa induced hepatotoxicity PDF
Preview effect of wheatgrass on alcohol and ∆pufa induced hepatotoxicity
EFFECT OF WHEATGRASS ON ALCOHOL AND ∆PUFA INDUCED HEPATOTOXICITY Thesis submitted to the Pondicherry University for the award of the degree of DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY AND MOLECULAR BIOLOGY By D.VARALAKSHMI, M.Sc., M.Phil Assistant Professor, Pondicherry University Community College Under the Supervision of Dr. R. RUKKUMANI M.Sc., M.Phil., Ph.D., Assistant Professor Department of Biochemistry and Molecular Biology Pondicherry University DEPARTMENT OF BIOCHEMISTRY AND MOLECULAR BIOLOGY SCHOOL OF LIFE SCIENCES PONDICHERRY UNIVERSITY PONDICHERRY - 605 014, INDIA FEBRUARY 2015 Dr. R. Rukkumani M. Sc., M.Phil., Ph. D., Assistant Professor Department of Biochemistry and Molecular Biology Pondicherry University Pondicherry 605 014, India. Phone: 91-413-2654537(Off); 91-967-7847337(Cell) Email [email protected] CCEERRTTIIFFIICCAATTEE This is to certify that the thesis entitled "EFFECT OF WHEATGRASS ON ALCOHOL AND ∆PUFA INDUCED HEPATOTOXICITY" submitted by Mrs. D.VARALAKSHMI for the degree of DOCTOR OF PHILOSOPHY IN BIOCHEMISTRY AND MOLECULAR BIOLOGY is a record of research work done by her and that it has not previously formed the basis for the award of any degree, diploma, associateship, fellowship or any other similar title. This is also to certify that the thesis represents the independent work of the candidate. Place: Puducherry [Dr. R. RUKKUMANI] Date: 11.02.2015 Research Guide DECLARATION The thesis entitled “EFFECT OF WHEATGRASS ON ALCOHOL AND ∆PUFA INDUCED HEPATOTOXICITY” submitted by me for the Degree of Doctor of Philosophy in Biochemistry and Molecular Biology to Pondicherry University is a record of research work done by me during 2010-2015 and that the thesis is the original work which has not formed the basis for award of any Degree, Diploma, Associateship, Fellowship of this University or any other University at previous occasions. Place: Puducherry Date: 11.02.2015 D. VARALAKSHMI M.Sc., M.Phil., (Part-Time Ph.D-Internal Candidates) Assistant Professor Department of Biochemistry Pondicherry University Community College, Lawspet, puducherry- 605 008 ACKNOWLEDGEMENT First and foremost, I thank my research supervisor. It is my proud privilege to express my heartfelt gratitude to my beloved and respected Dr. R. Rukkumani, Assistant Professor, Department of Biochemistry and Molecular Biology, Pondicherry University for her infallible, magnanimous understanding, meticulous editorial care, scientific freedom, precious counsel, steadfast encouragement, enthusiastic support, untiring efforts and guidance in planning and execution of my research work. Without her assistance and dedicated involvement in every step throughout the process, this thesis would have never been accomplished. It is a good fortune to thank her for her remarkable help and care and friendly supervision which will remain in my memory for the years to come. Prof. J.A.K Tareen and Prof. Balasubramanian were the sources of inspiration and have taught me many things. The impact of their words on my career is obvious through this dissertation. I thank my DC members Dr. N. Arumugam, Dr. C. Thirunavukkarasu for their suggestions and comments during my progress report presentations. Their encouragement, insightful comments, and hard questions helped me in shaping up my research. I would like to thank them very much for their support and understanding over these past four years. I am deeply indebted to Prof. Dr. Anisa Basheer Khan, Dean, School of Life Sciences, for her kind help and support. I wish to express my deep sense of gratitude to Prof. Dr. K. Jeevaratnam, Head, Department of Biochemistry and Molecular Biology, for his unhesitating help and assistance. I express my deep sense of gratitude to Dr.P.P.Mathur, Professor, Department of Biochemistry and Molecular Biology, School of Life Sciences, at present Vice Chancellor, KIIT University, Bhuvaneshwar, India. I heartily express my sincere thanks to all the Teaching Staff of our Department for their kind support. I would also like to thank the University and all the people who provided me the facilities required for my research work. I am also extremely thankful to Central Instrumentation Lab, PU for providing me necessary infrastructure and resources to accomplish my research work. I wish to record my gratitude to University Grants Commission, India, for the financial and equipment support from DST-FIST and DBT-IPLS. My sincere thanks to Mr. Sivakumar, and Mr. Adhisivan, lab assistant, Biochemistry and Molecular Biology, Mr. Yuvaraj and Mr. Subramanian, animal house, for their willing and untiring help during blood collection and animal maintenance and sacrifice and in carrying out lab work. A million thanks would not be enough for me to convey my gratitude to my labmates Garima Shakya, Sankar Pajaniradje, Kumaravel Mohankumar Muddasarul Hoda, and Shamim A. Sufi for their unwavering support towards me throughout my study period and valuable constructive criticism and friendly discussion during the work. I am sincerely grateful to them for sharing their truthful and illuminating views on a number of issues related to the project. Certain results described in this thesis would not have been obtained without a close collaboration with PCBS laboratory. I thank them for helping me to carry out part of my research work at their institute. I express my heartfelt thanks to Dr. Hairul Islam, PCBS, Puducherry, for spending his valuable time and his kind help in carrying out the molecular work. A very big “THANKS” goes to my students D Amali, S Vijayalakshmy, R Srinivasu and R Madhu, Ms. Raddika and my friend Dr. T. Radhiga Idaya College and her husband Dr. A. Sudarasan, Sri Vinaya College. They have substantially contributed and devoted much of their time for helping me in the development of this work. It is a great pleasure to record my sincere thanks to Dr. K. S. Kumari, Principal, PUCC and I heartily express my thanks to my college colleagues Dr. R. Premalatha, Dr. M. Tharaheswari, Mr. John Mathew and Mr. Barath kumar for their wholehearted support, kind help and unfailing encouragement throughout my studies. Most importantly, none of this could have happened without my family. Words seem to be inadequate to express the sense of gratitude surging on me towards my most affectionate, lovable family members for their extreme affection, encouragement and enthusiastic support for crafting and making my studies a great success. I convey special acknowledgement to my husband Mr. S. Venkatarengan. During the inevitable ups and downs of my research journey, he often reminded me life’s true priorities and emphasized what could be the influence of the loving God. This helped me a lot to work for hours together tirelessly. I doubt that I will ever be able to convey my appreciation fully, but I owe him my eternal gratitude. I dedicate this Ph.D. thesis to my two lovely children, S.V. Jeyasri and S.V. Abinayaashri who are the pride and joy of my life. I love them more than anything and I appreciate all their patience and support during their mummy’s Ph.D. studies. I would like to pay high regards to the people those who have knowingly and unknowingly helped me in the successful completion of this project. D. Varalakshmi CONTENTS CHAPTER TITLE PAGE NO. 1 INTRODUCTION 01 2 OBJECTIVES OF THE SUDY 30 3 MATERIALS & METHODS 33 4 RESULTS & DISCUSSION 56 5 SUMMARY & CONCLUSION 128 BIBLIOGRAPHY 131 Annexure I - REAGENTS LIST Annexure II LIST OF PUBLICATIONS Annexure III REPRINTS LIST OF FIGURES FIGURE PAGE CAPTIONS NO. NO. 1 Anatomy of the liver 1 2 Oxidative metabolism of ethanol 5 Lipid peroxidation process 3 11 4 Mechanism of oxidative damage to DNA 12 5 Progression of alcoholic liver disease 19 6 Inflammatory cytokines in alcoholic liver disease 23 7 Mechanism of apoptosis 25 8 Strucutre of Wheatgrass 28 9a Ferric reducing power activity of WG aqueous extract 63 9b Metal chelating activity of WG aqueous extract 63 9c Hydroxyl radical scavenging activity of WG aqueous 64 extract 9d Hydrogen peroxide radical scavenging activity of WG 64 aqueous extract 9e Nitric oxide radical scavenging activity of WG extract 65 9f Total anti-oxidant activity of WG aqueous extract 65 10 Effect of WG on the activities of AST, ALT, ALP and 67 GGT in the plasma 11 Effect of WG on the level of TBARS in the plasma, liver, 68 kidney and heart 12 Effect of WG on the level of HP in the plasma, liver, 69 kidney and heart 13 Effect of WG on the activities of SOD in the hemolysate, 71 liver, kidney and heart 14 Effect of WG on the activities of catalase in the 72 hemolysate, liver, kidney and heart Effect of WG on the activities of glutathione peroxidase in 15 73 the hemolysate, liver, kidney and heart 16 Effect of WG on the levels of vitamin C and vitamin E in 74 the plasma, liver and kidney 17 Histopathology of liver (20X) 75 18 Effect of WG on the activities of GGT, ALP, AST and 79 ALT 19 Effect of WG on the levels of TBRAS in the plasma, liver, 82 kidney and heart 20 Effect of WG on the levels of HP in the plasma, liver, 83 kidney and heart 21 Effect of WG on the level of reduced glutathione in the 84 plasma, liver, kidney and heart 22 Effect of WG on the levels of vitamin C and vitamin E in 85 the plasma, liver, kidney 23 Effect of WG on the activities of SOD in the hemolysate, 86 liver, kidney and heart 24 Effect of WG on the activities of catalase in the 87 hemolysate, liver, kidney and heart Effect of WG on the activities of glutathione peroxidase in 25 88 the hemolysate, liver, kidney and heart 26 Effect of WG on the levels of cholesterol in the plasma, 92 liver, kidney and heart 27 Effect of WG on the levels of triglycerides in the plasma, 93 liver, kidney and heart 28 Effect of WG on the levels of free fatty acid (FFA) in the 94 plasma, liver, kidney and heart 29 Effect of WG on the levels of phospholipids (PL) in the 95 plasma, liver, kidney and heart 30 Effect of WG on the levels of phospholipases A and C in 98 liver 31 Effect of WG on the levels of phospholipid fatty acid 100 composition in liver 32 Histopathology of Liver (20X) 103 33 Histopathology of Kidney (20X) 104 34 Effect of WG on collagen in Liver 105 35 Effect of WG aqueous extract on multiwell zymogram in 106 the liver 36 Effect of WG on MMP zymogram activity in the liver 106 tissue 37 Effect of WG on the levels of TIMP-1 and TIMP-2 107 38 Effect of WG on IL-1, TNF-α, TGF- β and IL-6 in serum 110 39 Effect of WG on DNA damage by comet assay in the 112 hepatocytes.
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