i ANTIHYPERLIPIDEMIC AND ANTIOXIDANT EFFECTS OF PHASEOLUS VULGARIS LINN IN WISTAR RATS BY AHANTE EJIROGHENE PG/MSC/13/66902 A RESEARCH PROJECT SUBMITTED TO DEPARTMENT OF PHARMACOLOGY & TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA SUPERVISOR: PROF. P.A. AKAH & DR. C.S. NWORU AUGUST, 2015 i ii ANTIHYPERLIIDEMIC AND ANTIOXIDANT EFFECTS OF PHASEOLUS VULGARIS LINN IN WISTAR RATS BY AHANTE EJIROGHENE PG/MSc./13/66902 A RESEARCH PROJECT SUBMITTED TO DEPARTMENT OF PHARMACOLOGY & TOXICOLOGY FACULTY OF PHARMACEUTICAL SCIENCES UNIVERSITY OF NIGERIA, NSUKKA IN PARTIAL FULFILMENT FOR THE AWARD OF MASTER OF SCIENCE (MSC) IN PHARMACOLOGY & TOXICOLOGY AUGUST, 2015 ii iii CERTIFICATION This research, antihyperlipidemic and antioxidant effects of Phaseolus vulgaris L. in Wistar rats, has been read and approved as having met the requirements of the Department of Pharmacology&Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, for award of degree of Master of Science in Pharmacology and Toxicology. ........................................... Ahante Ejiroghene ….………………………. ..……………………... Prof. P.A. Akah Dr. C.S. Nworu (Supervisor I) (Supervisor II) iii iv DEDICATION This project is dedicated: To: The Almighty God Now unto The King Eternal, Most Gracious God And Merciful God Be The Honour And Glory Forever And Ever, Amen. iv v ACKNOWLEDGEMENT I wish to acknowledge God Almighty who in his infinite mercies granted me the strength and patience, ordering my steps in everything to the successful completion of this project, May His name be glorified. I wish to express my profound gratitude to my supervisors; Prof P.A. Akah and Dr C.S. Nworu for their guidance, supervision, and candid assistance in making this project a successful one and also for making it achieve an acceptable standard. I also thank all the members of staff of Pharmacology and Toxicology, I pray God continue to increase every one of you in all aspect of your life. To my parents Mr and Mrs E.M. Ahante and my siblings (Mr Emena, Miss Gare and Barr. Oke) whose love, support, advice and friendship have kept me, I pray that God will shower you with His infinite Favour and abundant Grace in Jesus name Amen. Finally, my gratitude goes to my wonderful and special friend Osifo Itohan Mercy whose life has inspired me to be a better person in all I do, and also to all my colleagues at the department of Pharmacology and Toxicology for always raising the bar higher and pushing me to work harder, may God reward you all To everyone who wished me well, may God continue to bless and enrich you in love. Ahante Ejiroghene August 2015 v vi Table of Contents Title page ---------------------------------------------------------------------------------------- i Table of Contents ------------------------------------------------------------------------------ vi Lists of Tables ---------------------------------------------------------------------------------- ix Lists of Figures --------------------------------------------------------------------------------- x Abstract ------------------------------------------------------------------------------------------ xi CHAPTER ONE 1.0 Introduction ------------------------------------------------------------------------------- 1 1.1Review on Phaseolus vulgaris -------------------------------------------------------- 3 1.2Hyperlipidemia --------------------------------------------------------------------------- 6 1.2.1Lipoproteins------------------------------------------------------------------------------- 6 1.2.2 Classification of lipoprotein ----------------------------------------------------------- 8 1.2.3Lipoproteins metabolism --------------------------------------------------------------- 10 1.2.4 Classification of hyperlipidemia ------------------------------------------------------ 13 1.2.5 Management of hyperlipidemia-------------------------------------------------------- 18 1.2.5.1 Non-Pharmacological interventions------------------------------------------------ 18 1.2.5.2Pharmacological interventions ------------------------------------------------------ 19 1.3 Antioxidants --------------------------------------------------------------------------- 26 1.3.1 Classification of antioxidants-------------------------------------------------------- 27 1.3.2 Types of antioxidants ----------------------------------------------------------------- 28 1.3.3 Oxidative Stress ------------------------------------------------------------------------ 30 1.3.4 Antioxidants and Cardiovascular diseases ----------------------------------------- 30 1.4 Induction of hyperlipidemia---------------------------------------------------------- 31 vi vii 1.4.1Carbon Tetrachloride (CCl4) --------------------------------------------------------- 32 1.5 Statement of the problem ------------------------------------------------------------- 33 1.6 Aim of Study --------------------------------------------------------------------------- 33 1.7 Objectives of Study ------------------------------------------------------------------- 34 1.8Significance of Study ----------------------------------------------------------------- 34 1.9 Justification of Study------------------------------------------------------------------ 34 CHAPTER TWO 2.0 Materials and Method------------------------------------------------------------------ 35 2.1Experimental animals ----------------------------------------------------------------- 35 2.2 Plant material --------------------------------------------------------------------------- 35 2.3 Phytochemical screening-------------------------------------------------------------- 36 2.4 Hplc fingerprinting --------------------------------------------------------------------- 36 2.5Acute toxicity (LD50) ------------------------------------------------------------------ 37 2.6Hypolipidemicstudies --------------------------------------------------------------- 37 2.6.1Experimental Design ------------------------------------------------------------------ 37 2.6.2Acute Study of the effects of extract and fractions of PVE on lipid profile- 38 2.6.3Sub-acute Study of the effects of extract and fractions of PVE on lipid profile- 38 2.6.4Chronic Study of the effects of extract and fractions of PVE on lipid profile----- 39 2.6.5BiochemicalDetermination of Lipid profile parameters ----------------------- ----- 40 2.6.5.1 Determination of Total Cholesterol Level (TC) --------------------------------- 40 2.6.5.2 Determination of serum Triglycerides levels (TRIGS) ------------------------- 42 2.6.5.3 Determination of serum High Density Lipoproteins Cholesterol (HDL-C) --- 42 2.6.5.4 Determination of serum Low Density Lipoproteins Cholesterol (LDL-C) ----- 43 2.6.5.5 Determination of serum Very Low Density Lipoproteins Cholesterol (VLDL-C) -- 44 vii viii 2.7Determination of Body weight ------------------------------------------------------ 44 2.8 Determination of Atherogenic Index--------------------------------------------------- 44 2.9Studies on Antioxidants effects of extract and fractions of PVE --------------- 44 2.9.1DPPH scavenging assay of extract and fractions of PVE------------------------ 44 2.9.2Nitric oxide scavenging assay of extract and fractions of PVE----------------------- 45 2.9.3In-vivo Study --------------------------------------------------------------------------------- 45 2.9.4Estimation of Catalase activity ---------------------------------------------------------- 45 2.9.5 Estimation of Lipid-peroxidation activity ------------------------------------------- 46 2.9.6Estimation of Glutathione Peroxidase activity---------------------------------------- 46 2.10Method of data analyses ------------------------------------------------------------------ 47 CHAPTER THREE 3.0 Results --------------------------------------------------------------------------------------- 48 CHAPTER FOUR 4.0 Discussion, Conclusion and Recommendations ----------------------------------------- 90 4.1 Discussion ----------------------------------------------------------------------------------- 90 4.2 Conclusion ------------------------------------------------------------------------------- 94 4.3References ------------------------------------------------------------------------------- 95 APPENDICES ----------------------------------------------------------------------------------- 108 viii ix List of Tables Table Page 1 Fredrickson Hyperlipidemia classification ------------------------------------------- 15 2Phytochemical screening of all fractions of Phaseolus vulgaris L. -------------- 49 3 LD50of Phaseolus vulgaris L. --------------------------------------------------------- 50 4 The effects of crude of Phaseolus vulgaris L.on antioxidants enzyme ------- 88 5 The effects of various extract of Phaseolus vulgaris L. --------------------------- 89 ix x List of Figures Figures Pages 1. Showing structure of Phaseolus vulgaris ------------------------------------------- 3 2. Structure of Lipoproteins---------------------------------------------------------------- 7 3. Showing diagram of lipoproteins metabolism---------------------------------------- 12 4. Antioxidants and cardiovascular system --------------------------------------------- 31 5. HPLC analysis studies ------------------------------------------------------------------- 51 6. The effect of crude extract of Phaseolus vulgaris L. on lipid profile parameters (acute study)------------------------------------------------------------------ 55 7. The effect of crude extract