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The Tamil Nadu Dr. M. G. R. Medical University, Chennai MASTER OF ISOLATION OF A NATURAL POLYSACCHARIDE FROM TWO DIMORPHIC FUNGI AND ITS BIOLOGICAL ACTIVITIES Dissertation submitted to The Tamil Nadu Dr. M. G. R. Medical University, Chennai in partial fulfillment of the award of degree of MASTER OF PHARMACY (PHARMACEUTICAL BIOTECHNOLOGY) Submitted by ARUN RAJ T.R Under the guidance of Mrs. RM.AKILA, M. Pharm., (Ph.D.) Assistant Professor Department of Pharmaceutical Biotechnology MARCH – 2009 COLLEGE OF PHARMACY SRI RAMAKRISHNA INSTITUTE OF PARAMEDICAL SCIENCES COIMBATORE – 641 044. ISOLATION OF A NATURAL POLYSACCHARIDE FROM TWO DIMORPHIC FUNGI AND ITS BIOLOGICAL ACTIVITIES Dissertation submitted to The Tamil Nadu Dr. M. G. R. Medical University, Chennai in partial fulfillment of the award of degree of MASTER OF PHARMACY (PHARMACEUTICAL BIOTECHNOLOGY) MARCH – 2009 COLLEGE OF PHARMACY SRI RAMAKRISHNA INSTITUTE OF PARAMEDICAL SCIENCES COIMBATORE – 641 044. CERTIFICATE This is to certify that the dissertation entitled "ISOLATION OF A NATURAL POLYSACCHARIDE FROM TWO DIMORPHIC FUNGI AND ITS BIOLOGICAL ACTIVITIES." was carried out by ARUN RAJ T.R in the Department of Pharmaceutical Biotechnology, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, which is affiliated to The Tamil Nadu Dr.M.G.R. Medical university, Chennai, under my direct supervision and guidance to my fullest satisfaction. Mrs. RM. AKILA, M.Pharm., (Ph. D.) Assistant Professor, Department of Pharmaceutical Biotechnology, College of Pharmacy, SRIPMS, Coimbatore – 44 Place: Coimbatore Date: CERTIFICATE This is to certify that the dissertation entitled "ISOLATION OF A NATURAL POLYSACCHARIDE FROM TWO DIMORPHIC FUNGI AND ITS BIOLOGICAL ACTIVITIES”. was carried out by ARUN RAJ T.R, in the Department of Pharmaceutical Biotechnology, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, which is affiliated to The Tamil Nadu Dr.M.G.R. Medical University, Chennai, under supervision and direct guidance of Mrs. RM.Akila, M.Pharm.,(Ph.D.), Assistant Professor, Department of Pharmaceutical Biotechnology, College of Pharmacy, SRIPMS, Coimbatore – 44. Dr. T. K. Ravi, M. Pharm., Ph. D., FAGE., Principal, College of pharmacy, SRIPMS Coimbatore-44 Place: Coimbatore Date: CERTIFICATE This is to certify that the dissertation entitled " ISOLATION OF A NATURAL POLYSACCHARIDE FROM TWO DIMORPHIC FUNGI AND ITS BIOLOGICAL ACTIVITIES." was carried out by ARUN RAJ T.R, in the Department of Pharmaceutical Biotechnology, College of Pharmacy, Sri Ramakrishna Institute of Paramedical Sciences, Coimbatore, which is affiliated to The Tamil Nadu Dr.M.G.R. Medical University, Chennai, under supervision and direct guidance of Mrs. RM.Akila, M.Pharm.,(Ph.D.), Assistant Professor, Department of Pharmaceutical Biotechnology, College of Pharmacy, SRIPMS, Coimbatore – 44. Prof.S.Krishnan, M.Pharm., (Ph. D.), Head, Dept. of Pharmaceutical Biotechnology, College of Pharmacy, SRIPMS, Coimbatore – 44 Place: Coimbatore Date: ACKNOWLEDGEMENT I take this opportunity to render my profound sense of gratitude, indebtedness and respectful regards to my esteemed teacher and guide Mrs. RM. Akila, M. Pharm.,(Ph.D.) Assistant Professor, Department of Pharmaceutical Biotechnology for her excellent suggestions, kind encouragement and able guidance throughout the course of my work. My words would not be adequate to express my thankfulness to my teacher Prof. S. Krishnan, M.Pharm.,(Ph.D.),for his valuable suggestions and garnered blessings showered during my M.Pharm., course. I am overwhelmed by the timely guidance at each and every step of my work and enthusiastic encouragement offered by our beloved principal Dr. T. K. Ravi, M. Pharm., Ph.D., FAGE, throughout the course of investigation and the successful completion of this work. I owe my thanks to Dr.Sumita Singh, M.Sc., Ph.D., Dr.D.C.Sundaravelan, M.Phrm., Ph.D. and K.Muthusamy, M.Pharm.,(Ph.D.) assistant professors, Department of Pharmaceutical Biotechnology, for their support, timely help and suggestions. It is my privilege to express my sincere gratitude to Mrs. M. Gandhimathi, M. Pharm., PGDMM, (Ph.D.,), Department of Pharmaceutical Analysis, for lending me all the facilities to carryout the analytical work. I take this opportunity, with pride and immense pleasure in expressing my heartfelt thanks to Tamilnadu Pharmaceutical Sciences Welfare Trust for awarding scholarship for my project work. I submit my sincere thanks and respect to our Managing trustee, Sevaratna Dr. R. Venkatesalu Naidu for all the facilities provided in our institution. I would like to express my sincere thanks to Miss Sivasindhu, Mrs.Beaulah, Mrs. Karpagam and who directly or indirectly gave a helping hand to perform this study. My special thanks to the Library Staffs of SRIPMS for providing the library facilities during the literature survey. I extend my deep sense of gratitude to my friends Nishad, Munavir, Nancy, santhana Krishnan and Vanaja and my juniors for their valuable suggestions, co- operations and encouragement. A special word of thanks to my loving ‘Sree Sabari’ room mates Alan, Aneesh, Binu, Bijosh, Manoj, Mugeer and Thomas Luke for their support during my work. I feel proud to express my gratefulness to my beloved Parents, whose precious love, persistent prayers and moral supports guided me in the right path and have always been the key to accomplish the shore of success. My sincere thanks are extended to Computer Park for helping me to complete my project in right time. Above all, allow me to proclaim the over powering presence of The Almighty who is the source of all wisdom and knowledge for the successful completion of this dissertation. Arun Raj T.R CONTENTS Title Page No Abbreviations 1 SCOPE AND PLAN OF WORK 1 2 SCHEME OF THE WORK 4 3 INTRODUCTION 5 3.1 Polysaccharides 5 3.2 Dimorphism in fungi 8 3.3 Factors influencing dimorphic transition 9 3.4 Fungal cell wall: components,organisation and chemistry 11 3.4. 1 Cell wall composition,structure and organisation 11 3.5 Biosynthesis of chitosan in the fungal cell wall 14 3.6 Chitosan 16 3.7 Fungal production of chitosan 19 3.8 Physicochemical properties of chitosan 20 3.9 Applications of chitosan 23 3.10 5 Antibacterial properties of chitosan 27 3.11 Antioxidants 29 3.12. Organism profile 34 4 REVIEW OF LITERATURE 38 5 MATERIALS AND METHODS 48 5.1 Apparatus used 48 5.2 Instruments used 49 5.3 Cultivation media and conditions 50 5.4 Identification of fungi by microscopy 50 5.5 Identification of chitosan containing fungi 52 5.6 Inoculum preparation 54 5.7 Fermentation medium 54 5.8 Chitosan isolation 54 5.9. Determination of growth curve, extractable chitin and chitosan 55 5.10. Medium optimization 56 5.11. Chitosan characterization by FT-IR, 1HNMR, X-ray diffraction patterns,Viscosity, Ash content and Moisture content. 57 5.12. Antioxidant activity by DPPH method 60 5.13. Screening for antimicrobial activity 61 6 RESULTS AND DISCUSSION 65 6.1. Strain identification 65 6.2. Chitosan identification 65 6.3. Growth profile 66 6.4. Medium optimization with relation to Chitosan production 70 6.5. Antioxidant activity 77 6.6. Characterization of chitosan FT-IR, 1HNMR, X-ray diffraction patterns, Viscosity, Ash content and Moisture content. 90 6.7. Antimicrobial studies 92 7 SUMMARY AND CONCLUSION 96 8 REFERENCES ANNEXURE I III. INTRODUCTION Polysaccharide-based biomaterials are an emerging class in several biomedical fields such as tissue regeneration, particularly for cartilage, drug delivery devices and gel entrapment systems for the immobilization of cells. Important properties of the polysaccharides include controllable biological activity, biodegradability, and their ability to form hydrogels. Most of the polysaccharides used derive from natural sources; particularly, alginate and chitin, two polysaccharides which have an extensive history of use in medicine, pharmacy and basic sciences, and can be easily extracted from marine plants (algae kelp) and crab shells, respectively. The recent rediscovery of poly-saccharide based materials is also attributable to new synthetic routes for their chemical modification, with the aim of promoting new biological activities or to modify the final properties of the biomaterials for specific purposes. These synthetic strategies also involve the combination of polysaccharides with other polymers. 3.1 Polysaccharides The majority of carbohydrate materials in nature occur in the form of polysaccharides. By our definition, polysaccharides include not only those substances composed only of glycosidically linked sugar residues, but also molecules that contain polymeric saccharide structures linked via covalent bonds to amino acids, peptides, proteins, lipids and other structures. Polysaccharides, also called glycans, consist of monosaccharides and their derivatives. If a polysaccharide contains only one kind of monosaccharide molecule, it is known as a homopolysaccharide, or homoglycan, whereas those containing more than one kind of monosaccharide are heteropolysaccharides. The most common constituent of polysaccharides is D-glucose, but D-fructose, D-galactose, L-galactose, D-mannose, L- arabinose, and D-xylose are also frequent. Some monosaccharide derivatives found in polysaccharides include the amino sugars (D-glucosamine and D-galactosamine) as well as their derivatives (N-acetylneuraminic acid and N-acetylmuramic acid), and simple sugar acids (glucuronic and iduronic acids). Homopoly- saccharides are often named for the sugar unit they contain, so glucose homopolysaccharides are called glucans,
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