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Chemical and Molecular Characterization of Marigold

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Chemical and Molecular Characterization of Marigold Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan By Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD PhD Thesis In Pharmacy COMSATS Institute of Information Technology Abbottabad - Pakistan Fall, 2015 COMSATS Institute of Information Technology Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan A Thesis Presented to COMSATS Institute of Information Technology, Abbottabad In partial fulfillment of the requirements for the degree of PhD (Pharmacy) By Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD Fall, 2015 ii Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan A Post Graduate Thesis submitted to the Department of Pharmacy as partial fulfillment of the requirement for the award of Degree of PhD in Pharmacy. Name Registration Number Muhammad Hassham Hassan CIIT/FA11-R60-003/ATD Bin Asad Supervisor Dr. Izhar Hussain Professor Department of Pharmacy Abbottabad Campus. COMSATS Institute of Information Technology (CIIT) Abbottabad Campus. December, 2015 iii Final Approval This thesis titled Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan By Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD Has been approved For the COMSATS Institute of Information Technology, Abbottabad External Examiner: __________________________________________ Dr………… ……… External Examiner: __________________________________________ Dr………… ……… Supervisor: ________________________________________________ Prof. Dr. Izhar Hussain Department of Pharmacy, Abbottabad HoD/ Chairperson: ____________________________________________________ Prof. Dr. Nisar-ur-Rehman Department of Pharmacy, Abbottabad Dean, Faculty of Sciences: _______________________________________________ Prof. Dr. Arshad Saleem Bhatt iv Declaration I, Muhammad Hassham Hassan Bin Asad, CIIT/FA11-R60-003/ATD hereby declare that I have produced the work presented in this thesis, during the scheduled period of study. I also declare that I have not taken any material from any source except referred to wherever due, and that the amount of plagiarism is within acceptable range. If a violation of rules on research has occurred in this thesis, I shall be liable to punishable action under the plagiarism rules of the HEC. Signature of the student: Date: ______________ ________________________ Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD v Certificate It is certified that Muhammad Hassham Hassan Bin Asad, CIIT/FA11-R60-003/ATD has carried out all the work related to this thesis under my supervision at the Department of Pharmacy, COMSATS Institute of Informational Technology, Abbottabad and the work fulfills the requirements for award of PhD degree. Date: ______________________ Supervisor: _________________________________ Prof. Dr. Izhar Hussain Department of Pharmacy CIIT, Abbottabad Head of Department: ________________________________ Prof. Dr. Nisar-ur-Rehman Department of Pharmacy CIIT, Abbottabad vi DEDICATION This thesis is dedicated to my parents Mrs. Tasneem Habib & Mr. Allah Dad Asad Awan Who realized me the power of knowledge vii ACKNOWLEDGEMENTS This thesis would not have been concluded without inspiring and untiring efforts of many individuals who contributed and extended their guidelines in completion of this work. First and foremost I would like to express my deep gratitude to my PhD supervisor, Prof. Dr. Izhar Hussain, Department of Pharmacy, COMSATS Institute of Information Technology (CIIT), Abbottabd, Pakistan for his constant motivation, illumination of ideas and valuable advices throughout the research work. I also indebted to Dr. Ghulam Murtaza, Assistant Professor, Department of Pharmacy, CIIT, Abbottabad for his care, collaboration and sincere efforts. I owe a great debt of appreciation to the Dr. Durr-e-Sabih, Director, Multan Institute of Nuclear Medicine and radiotherapy (MINAR), Nishtar Hospital Multan, Pakistan for his sympathetic and scholarly approach. Moreover I would like to thanks to the Dr. Khan Muhammad Sajid (Deputy Chief Scientist), Dr. Muhammad Saqib Khan (Senior Radiologist), Israr Ahmad (Radio Pharmacist), Rubada Mehmood (Clinical Biochemist) and other lab members for all the assistance they have done during my stay at MINAR, Nishtar Hospital Multan, Pakistan. During my stay at Copenhagen I highly acknowledge my supervisors Dr. Anna Katherine Jager (Head, Natural Product Research, University of Copenhagen, Denmark) and Dr. Dan Streak (Professor of NMR, University of Copenhagen, Denmark) for their useful guidelines, sympathetic approach and enlightening ideas throughout my research. At the end I would like to thanks from core of heart to my mentor at National University of Singapore (NUS) Prof. Dr. R. M. Kini (Department of Biological Sciences, National University of Singapore, Singapore) for his educated, valuable and critical guidelines towards completion of my snake venom Ph.D research project. Muhammad Hassham Hassan Bin Asad CIIT/FA11-R60-003/ATD viii ABSTRACT Biochemical and Radio Labeling Studies of Venom Naja naja karachiensis with its Neutralization by Medicinal Plants of Pakistan Background Snake bite envenomation is one of the vivid examples of neglected occupational hazards that accounts for tens of thousands of deaths all over the world. One of such instance is Naja naja karachisis bite, a nightmare for the inhibitants of Southern Punjab (Paksitan), often endup with countless deaths and sequela. To address this problem present study was designed to highlight scientific grounds for Naja naja karachisis envenomation and to rationalize folklore claimed Pakistani medicinal plants as a first aid treatment before proper hospitalization. Methods Proteomic characterization of Naja naja karchiensis venom was carried out with electrophoresis (SDS-PAGE) and HPLC (SEC & RP-HPLC) coupled LC-MS/MS whereas inorganic constituents were quantified with ICP-OES technique. Bio distribution and kinetic profile of venom was monitored with short lived radiotracer (99mTc) via direct radio isotopic binding technique. Lethal biological effects of crude venom were examined in terms of its LD50, hemolytic and anticoagulant behavior while toxic biochemical parameters (in vivo), towards liver (AST & ALT), heart (CK-MB & LDH) and kidneys (urea & creatinine) damage were investigated by following the recommendations of DGKC and IFCC methods. Venom was analyzed for different enzymatic activities (PLA2, ALPase, 5ʹ-ND, hyaluronidase and protease) by adopting conventional biochemical assays (in vitro). Twenty eight medicinal plants of Pakistan were extracted with methanol by simple maceration process and thereafter used to reverse deleterious actions of cobra venom. RP-HPLC coupled bioassay guided fractionation technique was used to characterize bioactive constituent/metabolite(s), responsible for anti-PLA2 activity in Bauhinia variegata L extract. ix Results SDS-PAGE indicated Naja naja karachiensis venom as a concoction of proteins, which ranges in molecular weight from 6 KDa to 200 KDa. Proteinous bands ranges from 50 KDa to 90 KDa were found homologous, however, after venom reduction the bands (15 KDa to 36 KDa) were not in homology (rather appreared at 9 KDa to 12 KDa) suggesting protein complex(s) in this venom. Total protein content was found to be 188 ± 0.011 µg per 200 µg of dry weight, which constitutes overall 94% proteineous stuff in cobra venom. LC-MS/MS analysis of protein component further identified and sequenced 3FTXs (58%), PLA2 (19%), SVMPs (5%), LAAO (5%), helvepryn (3%), vespryn (2%), CVF (2%), 5ʹ-ND (2%), vNGF (2%) and kunitz type serine protease inhibitor (2%). Among 3FTXs the identified components included CTXs (32%), WNTX (24%), LNTX (24%), SNTX (8%), MTLP (8%) and post synaptic-NTX (4%). MTLP-3 was found unique among 3FTXs due to 78% homology in amino acids sequence with novel haditoxin. Majority of the RP-HPLC fractions were heterogeneous while some were homogeneous. ICP-OES analysis revealed that other than protenous stuff metallic (95%) and non-metallic constituents were also present in cobra venom. Among metallic ions sodium (4519 ± 2 µg/g, 30%), potassium (2013 ± 5.5 µg/g, 13%), zinc (3473 ± 28 µg/g, 23%), magnesium (3047 ± 31 µg/g, 20%), calcium (1442 ± 19 µg/g, 9%), manganese (6.5 ± 0.65 µg/g, 0.05%) and copper (0.6 ± 0.09, 0.003%) were quantified, however, phosphorus (718 ± 8.5 µg/g, 5%) was the only detectable non-metallic component in this venom. This is the first report about divalent copper ion that has not been previously detected in other elapid venom. Venom from Naja naja karachiensis was labeled (97.7%) successfully for the first time with technetium-99m. Its labeling yield was higher than some previously reported toxins. Venom was found stable in vivo (96%) and in vitro (saline 94.3% & serum 94.1%) more 99m than one t1⁄2 of Tc (4 h). Venom (0.5mCi, 0.25 mg) was evenly distributed (R/L ratio=1) in the middle compartment and completely excreted from blood pool within 24 h of injection in rabbits. Intravenous dose of venom was distributed in all parts of the rabbit such as, stomach (0.05% ID/g), brain (0.14% ID/g), skeleton muscles (0.3% ID/g), intestines (0.35% ID/g), skin (0.45% ID/g), blood (0.56% ID/g), heart (0.8% ID/g), bones (1.38% ID/g), liver (4.3% ID/g), lungs (14.4% ID/g), urinary bladder (23.7% ID/g) and x kidneys
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