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ANTIDIABETIC POTENTIALS of LIPOSPHERES ENCAPSULATING Anogeissus Leiocarpus DC Guill & Perr ROOT BARK METHANOL EXTRACT

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ANTIDIABETIC POTENTIALS of LIPOSPHERES ENCAPSULATING Anogeissus Leiocarpus DC Guill & Perr ROOT BARK METHANOL EXTRACT ANTIDIABETIC POTENTIALS OF LIPOSPHERES ENCAPSULATING Anogeissus leiocarpus DC Guill & Perr ROOT BARK METHANOL EXTRACT BY UCHECHI OKORO PG/M.PHARM/10/52392 A PROJECT PRESENTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER OF PHARMACY (M.PHARM) IN PHYSICAL PHARMACEUTICS OF THE UNIVERSITY OF NIGERIA NSUKKA SUPERVISOR: PROF. A. A. ATTAMA DEPARTMENT OF PHARMACEUTICS, FACULTY OF PHARMACEUTICAL SCIENCES, UNIVERSITY OF NIGERIA, NSUKKA APRIL, 2014 i TITLE ANTIDIABETIC POTENTIALS OF LIPOSPHERES ENCAPSULATING Anogeissus leiocarpus DC Guill & Perr ROOT BARK METHANOL EXTRACT ii CERTIFICATION This is to certify that Uchechi Okoro, a postgraduate student in the Department of Pharmaceutics, with the registration number PG/M.Pharm./10/52392, has satisfactorily completed the requirements for the award of Master of Pharmacy (M. Pharm) degree in Physical Pharmaceutics. The work embodied in this project is original and has not been submitted in part or full for any other diploma or degree of this or any other University. Supervisor: Prof. A. A. Attama Head of Department: Prof. K.C. Oforkansi ………………………………. ………………………………….. Sign/Date Sign/Date iii DEDICATION This work is dedicated to God Almighty for all His mercies and grace; and to my parents for their continued support and love. iv ACKNOWLEDGMENT To God be the glory for all His wonderful works, His goodness, grace and mercies. My immense gratitude goes to my supervisor, Prof. A. A. Attama, one of a kind, for all his support, encouragement and guidance. I sincerely thank him for providing most of the materials and equipment used for this research work. I also, do really thank him for opening my eyes to the opportunities in the field of research. I pray I would use all I learnt from him. My profound gratitude goes to Dr. Omeje who introduced and provided the plant material used for this research work. I also appreciate him for all his advice and assistance in and outside this research work. I remain grateful. To my wonderful parents, Mr. and Mrs. L. Okoro and my siblings, Dr. Chizoba, Chinwe and Engr. Ihechi, who continuously encouraged me till the end of this work. May God richly reward all their efforts. The best is yet to come. I sincerely appreciate all the staff of the Department of Pharmaceutics, University of Nigeria Nsukka, especially, Prof. E. C. Ibezim, Prof. V. C. Okorie, Dr. Momoh, Dr. Akpa, Mr. Dave Okechukwu, Pharm. John Ogbonna, Pharm. Kenechukwu for all their assistance and encouragement. I would not fail to appreciate, Pharm. Salome, Pharm. Richard, Pharm. Ngozi, for all their encouragement and care. The non-academic staff of Pharmaceutics Department are appreciated for all their kindness and assistance in the course of this research work. My gratitude goes to the staff at Energy Center, UNN and Home Science and Nutrition Department for all their assistance. May God richly reward all of them. Pharm. Uchechi Okoro April, 2014 v TABLE OF CONTENTS Title . i Certification . ii Dedication . iii Acknowledgement . iv Table of contents . vi List of tables and figures . xii Abstract . xiv CHAPTER ONE: INTRODUCTION . 1 1.2 Drug delivery systems . 1 1.2.1 Drug delivery carriers . 2 1.2.1.0 Particle parameters . 3 1.2.1.1 Size . 3 1.2.1.2 Surface chemistry . 4 1.2.1.3 Shape . 5 1.2.1.4 Mechanical flexibility . 6 1.2.2 Lipids as carriers . 7 1.2.3 Advantages of lipid based delivery system s . 8 1.3 Lipospheres as drug delivery systems . 9 1.3.1 Advantages of lipospheres . 11 1.3.2 Disadvantage of lipospheres . 11 1.3.3 Formulation of lipospheres . 12 1.3.4 Preparation of lipospheres . 13 vi 1.3.5 Homogenisation method . 14 1.3.5.1 High shear homogenisation method . 14 1.3.5.2 High pressure homogenisation (HPH) method . 14 1.3.5.3 Hot homogenisation method . 14 1.3.5.4 Cold homogenisation method . 15 1.3.6 Solvent emulsification/evaporation method . 15 1.3.7 Supercritical method . 16 1.3.8 Sonication method . 16 1.3.9 Rotoevaporation method . 16 1.3.10 Sterilization of lipospheres . 17 1.4 In vitro characterization of lipospheres . 17 1.4.1 Liposphere morphology . 17 1.4.2 Structure . 18 1.4.3 Entrapment efficiency . 18 1.4.4 In vitro drug release . 19 1.5 Types of lipospheres . 20 1.5.1 Solid lipid microparticles (SLMs) . 20 1.5.2 Solid lipid nanoparticles (SLNs) . 21 1.5.3 Nanostructured lipid carriers (NLCs) . 21 1.5.4 Lipid drug conjugate (LDC) nanoparticles . 22 1.6 Materials used in the formulation . 22 1.6 Phospholipids . 22 1.6.1 (Phospholipon® 90H) . 24 vii 1.7 Beeswax . 24 1.8 Polaxamer . 25 1.8.1 Polaxamer as pharmaceutical excipients . 26 1.9 Sorbic acid . 27 1.9.1 Applications of sorbic acid . 27 1.10 Diabetes mellitus . 28 1.10.1 Diagnosis of diabetes mellitus . 29 1.10.1.1 Prediabetes . 29 1.10.1.2 Type 2 diabetes . 29 1.10.2 Type 1 diabetes. 32 1.10.3 Type 2 diabetes mellitus . 32 1.10.4 Pathophysiology . 34 1.10.4.1 Type 1 diabetes mellitus. 34 1.10.5 Other forms of diabetes . 35 1.10.6 Prevention of type 1 and type 2 diabetes mellitus . 36 1.10.7 Treatment . 37 1.10.7.1 Medical nutrition therapy . 37 1.10.8 Insulin therapy . 38 1.10.9 Oral antidiabetic agents. 39 1.10.9.1 Sulfonylureas . 39 1.10.9.2 Glinides . 39 1.10.9.3 Biguanides . 40 1.10.9.4 α- Glucosidase inhibitors . 40 viii 1.10.9.5 Thiazolinediones . 41 1.10.9.6 GLP-1 agonists . 41 1.10.9.7 Gliptins (DPP-4-Inhibitors) . 41 1.10.9.8 Amylin agonist . 42 1.10.9.9 Bile acid sequestrant . 42 1.10.10 Pancreas and islets cell transplant . 42 1.11 Plant materials used in the treatment of diabetes. 44 1.12. Recent studies on plants with antidiabetic potential . 45 1.12.1 Costus igneus . 45 1.12.2. Dendrophthoe pentandra (L) Miq. 46 1.12.3. Symplocos racemosa . 46 1.12.4. Annona reticulate L . 47 1.12.5. Carissa carandas L . 47 1.12.6. Elaeodendron glaucum Pers. 48 1.12.7 Zingiber officinale roscoe . 48 1.12.8. Cocculus hirsutus. 48 1.12.9. Oxalis corniculata . 48 1.12.10. Basella rubra . 48 1.13. Anogeissus leiocarpus . 50 1.13.1 Description . 52 1.13.2. Uses . 52 1.13.3. Distribution and habitat . 53 1.13.4. Botanical description . 53 ix 1.14 Formulation of plant extract into dosage form . 53 1.14.1 Problems encountered in formulation of plant extracts into dosage forms . 54 1.15 Rational and objective of the present study. 54 CHAPTER TWO: MATERIALS AND METHODS . 56 2.1 Materials . 56 2.2 Methods . 56 2.2.1 Extraction of Anoqeissus leiocarpus DC.Guill and Perr root bark . 56 2.2.2 Phytochemical analysis of Anogeissus leiocarpus root bark extracts. 56 2.2.2.1 Test for carbohydrates . 56 2.2.2.2 Detection of alkaloids. 57 2.2.2.3 Detection of glycosides . 57 2.2.2.4 Detection of saponins . 57 2.2.2.5 Detection of flavonoids . 57 2.2.3 In vivo antidiabetic evaluation of the extract . 58 2.2.3.1 Preparation of experimental rats . 58 2.2.3.2 Induction of diabetes mellitus . 58 2.2.3.3 Antidiabetic evaluation . 59 2.2.4 Preparation of physiological fluids (SIF and SGF) . 59 2.2.5 Establishment of spectral characteristics . 59 2.2.6 Beer-Lambert′s plot for Anogeissus leiocarpus DC.Guill and Perr methanol extract in water, SIF and SGF . 60 2.2.7 Preparation of lipid matrix. 60 2.2.8 Preparation of unloaded lipospheres . 61 x 2.2.9 Preparation of Anogeissus. L methanol extract loaded lipospheres . 61 2.2.10 Characterisation of Anogeissus L methanol extract loaded lipospheres 63 2.2.10.1 Particle size and morphology analyses . 63 2.2.10.2 pH measurement . 63 2.2.10.3 Drug encapsulation efficiency determination . 63 2.2.10.4 Loading capacity determination . 64 2.2.10.5 Drug release evaluation . 64 2.2.10.7 Determination of kinetic mechanism of release . 64 2.2.10.7 Statistical analysis . 65 CHAPTER THREE: RESULTS AND DISCUSSION . 66 3.1 Phytochemical analysis of Anogeissus leiocarpus root bark extract . 66 3.2 Spectral characterization studies . 69 3.3 Beer-Lambert’s plot . 73 3.4 pH values . 77 3.5 Particle size and morphology analyses . 79 3.6 Entrapment efficiency and loading capacity . 82 3.7 In vitro drug release . 84 3.8 Release kinetics . 88 3.9 In vivo antidiabetic studies . 94 CHAPTER FOUR: SUMMARY AND CONCLUSION . 97 4.1 Summary . 97 4.2 Conclusion . 98 REFERENCES . 99 APPENDICES . 108 xi LIST OF TABLES AND FIGURES Tables Table 1. Formulae used in preparation of the lipospheres. 62 Table 2. Phytochemical analysis of Anogeissus leiocarpus. 67 Table 3. Calibration plot results. 74 Table 4. Average particle size of the drug-loaded and plain lipospheres. 78 Table 5. kinetics of drug release from the lipospheres in SGF and SIF. 93 Figures Figure1. Size- dependent processes related to particle transport in the body. 4 Figure 2. Scanning electron micrograph and actin staining confirm time-lapse video microscopy observations. 6 Figure 3. Structure of lipospheres showing fat core stabilized by monolayer of phospholipids. 10 Figure 4. Schematic representation for drug incorporation models. 19 Figure 5. Molecular formular of poloxamer. 25 Figure 6. Structure of sorbic acid. 27 Figure 7. Anogeissus leiocarpus tree showing the trunk and part of the root.
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