Phytopharmaceutical Studies of Some Selected Medicinal Plants Locally

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Phytopharmaceutical Studies of Some Selected Medicinal Plants Locally ADDIS ABABA UNIVERSITY SCHOOL OF GRADUATE STUDIES PHYTOPHARMACEUTICAL STUDIES OF SOME SELECTED MEDICINAL PLANTS LOCALLY USED IN THE TREATMENT OF SKIN DISORDERS BY HAILU TADEG JANUARY 2004 PHYTOPHARMACEUTICAL STUDIES OF SOME SELECTED MEDICINAL PLANTS LOCALLY USED IN THE TREATMENT OF SKIN DISORDERS A thesis submitted to the School of Graduate Studies of the Addis Ababa University in partial fulfillment of the requirements for the Degree of Master of Science in Pharmaceutics in the Department of Pharmaceutics, School of Pharmacy By Hailu Tadeg (B. Pharm) January 2004 ACKNOWLEDGEMENTS First of all I would like to express my sincere gratitude to my advisors Prof. Tsige Gebre- Mariam and Dr. Kaleab Asres and my Co-advisor Ato Endris Seid for their valuable advice and follow up throughout the course of my work. My deepest gratitude also goes to all the staff members of Departments of Bacteriology and Drug Research (EHNRI), for their assistance in their areas of specialty and for allowing me to use their laboratory facilities that enabled me carry out my study. I am also grateful to the departments of Drug Quality Control and Toxicology (DACA) for providing me the standard drugs, Quality Control (EPHARM) for allowing me to use their facilities and National Herbarium (Department of Biology, Science Faculty) for identifying my plant specimens. My special appreciation also goes to the staff members of departments of Pharmaceutics, Pharmaceutical Chemistry, Pharmacognosy and Pharmacology (School of Pharmacy) for their respective support during my entire work. I am very much thankful to Ato Workalemahu Mikre and my family for their pleasant companionship and encouragement until the completion of my study. Last but not least, my acknowledgement goes to Addis Ababa University for sponsoring my study. i TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i TABLE OF CONTENTS ii LIST OF TABLES vi LIST OF FIGURES viii ACRONYMS ix ABSTRACT xi 1. INTRODUCTION 1 1.1 Herbal Drugs in Medical Health Practices 2 1.1.1 Historical Perspectives 2 1.1.2 Current Status of Herbal Drugs: Global Perspective 4 1.1.3 Potential of Herbal Remedies as Sources of New Drugs 6 1.1.4 Herbal Products as Antimicrobial/Antiinfective Agents 8 1.2 Common Dermatological Disorders and Their Prevalence 10 1.2.1 Infectious Skin Disorders 10 1.2.1.1 Bacterial Infections 10 1.2.1.2 Fungal Infections 11 1.2.1.3 Viral Infections 12 1.2.2 Inflammatory and Allergic Skin Disorders 12 1.2.3 HIV-Associated Skin Disorders 13 1.2.4 Prevalence of Skin Disorders 13 ii 1.3 Treatment of Skin Disorders 15 1.3.1 Conventional Approaches 16 1.3.1.1 Treatment of Infectious Skin Disorders 16 1.3.1.2 Treatment of Inflammatory and Allergic Skin Disorders 17 1.3.1.3 Limitations of Conventional Approaches 19 1.3.2 Phytopharmaceutical Approaches 21 1.3.2.1 Herbal Drugs Commonly Used in the Treatment of Dermatological Disorders 21 1.3.2.2 Scientific Studies Made on Topically Used Herbal Drugs 23 1.4 Review of Some Topically Used Herbs Included in this Study 26 1.4.1 Acokanthera schimperi (Apocynaceae) 26 1.4.2 Calpurnia aurea (Fabaceae, Leguminosae) 27 1.4.3 Kalanchoe petitiana (Crassulaceae) 28 1.4.4 Lippia adoensis (Verbenaceae) 29 1.4.5 Malva parviflora (Malvaceae) 30 1.4.6 Olinia rochetiana (Oliniaceae) 31 1.4.7 Phytolacca dodecandra (Phytolaccaceae) 31 1.4.8 Verbascum sinaiticum (Scrophulariaceae) 32 1.5 Performance Evaluation of Topical Formulations 33 1.6 Objectives 38 1.6.1 General Objective 38 1.6.2 Specific Objectives 38 iii 2. EXPERIMENTAL 39 2.1 Materials and Methods 40 2.1.1 Materials 40 2.1.2 Methods 42 2.1.2.1 Collection and Preparation of the Plant Material 42 2.1.2.2 Extraction 43 2.1.2.2.1 Preparation of Crude Extract 43 2.1.2.2.2 Preparation of the Fractional Extracts 43 2.1.2.3 Antimicrobial Screening of the Crude Extracts and Fractions 43 2.1.2.3.1 Screening for Antibacterial Activity 44 2.1.2.3.2 Screening for Antifungal Activity 45 2.1.2.3.3 Determination of MIC 45 2.1.2.4 Screening for Anti-inflammatory Activity 46 2.1.2.5 Phytochemical Screening 47 2.1.2.6 Formulation of the Herbal Drugs and In Vitro Evaluation of Their Performance 50 2.1.2.6.1 Preparation of the Bases 52 2.1.2.6.2 Preparation of the Topical Formulations 52 2.1.2.6.3 Performance Evaluation of the Topical Formulations 52 2.1.2.7 Preliminary Standardization 55 2.1.2.7.1 Determination of Ash Values 55 2.1.2.7.2 Determination of Extractable Matter 56 2.1.2.7.3 Determination of Moisture Content 56 2.1.2.7.4 TLC Finger Print Analysis 57 iv 3. RESULTS AND DISCUSSION 59 3.1 Yields of the Crude (80% Methanol) Extracts 60 3.2 Antimicrobial Activities 61 3.2.1 Antimicrobial Activities of the Crude Extracts 61 3.2.2 Antimicrobial Activities of the Fractions 65 3.2.3 Minimum Inhibitory Concentrations (MIC) of the Crude Extracts 68 3.3 Anti-inflammatory Activities of the Crude Extracts 71 3.4 Phytochemical Constituents of the Herbal Drugs 75 3.5 Performance Profiles of Topical Formulations of the Herbal Drugs 82 3.6 Preliminary Standardization of the Herbal Drugs 92 4. CONCLUSIONS 105 SUGGESTIONS FOR FURTHER WORK 107 REFERENCES 109 v LIST OF TABLES Page Table 2.1: List of Formulation Bases and their Compositions Used as Vehicles for the Preparation of Topical Formulations of L. adoensis and O. rochetiana . 51 Table 2.2: List of Topical Antibacterial and Antifungal Products Used as Positive Controls During the Performance Evaluation of Topical Formulations. 54 Table 3.1: Percentage Yields of 80% Methanol Extracts of the Dried and Powdered Plant Materials (n = 3). 60 Table 3.2: Antimicrobial Activities of the Crude Extracts against Different Strains of Bacteria and Fungi. 62 Table 3.3: Percentage Yields of Different Fractions of L. adoensis and O. rochetiana 66 Table 3.4: Antimicrobial Activities of Different Fractions of L. adoensis and O. rochetiana against Selected Strains of Bacteria and Fungi. 67 Table 3.5: Minimum Inhibitory Concentration (MIC) Values of the Crude Extracts of L. adoensis and O. rochetiana on the Tested Strains. 70 Table 3.6: Anti-inflammatory Activity of the Crude Extract of L. adoensis on Rat Hind- Paw Oedema Induced by Carrageenin. 72 Table 3.7: Anti-inflammatory Activity of the Crude Extract of O. rochetiana on Rat Hind-Paw Oedema Induced by Carrageenin. 73 Table 3.8: Results of Preliminary Phytochemical Investigation of the Leaves of L. adoensis and O. rochetiana Using Chemical Test Methods. 76 Table 3.9: Results of Preliminary Phytochemical Investigation of the Leaves of L. adoensis and O. rochetiana Using Chromatographic Methods. 78 vi Table 3.10: Antimicrobial Activity Profiles of Topical Formulations of L. adoensis & O. rochetiana and the Formulation Bases (Negative Controls) 84 Table 3.11: Antibacterial Activity Profiles of the Marketed Topical Antibacterial Agents in Comparison with Some Formulations of the Crude Extracts. 89 Table 3.12: Antifungal Activity Profiles of the Marketed Topical Antifungal Agents in Comparison with Some Formulations of the Crude Extracts. 90 Table 3.13: Ash Values of the Dried and Powdered Leaves of L. adoensis and O. rochetiana . 96 Table 3.14: Solvent Extractable Values of the Dried and Powdered Leaves of L. adoensis and O. rochetiana . 97 Table 3.15: Loss on Drying of the Dried and Powdered Leaves of L. adoensis and O. rochetiana . 97 Table 3.16: TLC Fingerprints of the Crude (80% Methanol) Extract and Various Fractions of L. adoensis 100 Table 3.17: TLC Fingerprints of the Crude (80% Methanol) Extract and Various Fractions of O. rochetiana 101 vii LIST OF FIGURES Page Figure 3.1: Chromatogram of the Crude (80% Methanol) Extracts of L. adoensis and O. rochetiana 102 Figure 3.2: Chromatogram of Methanol and Acetone Fractions of L. adoensis and O. rochetiana 103 Figure 3.3: Chromatogram of Chloroform and Petroleum Ether Fractions of L. adoensis and O. rochetiana 104 viii ACRONYMS AIDS: Acquired Immunodeficiency Virus ATCC: American Type Culture Collection BP: British Pharmacopoeia DACA: Drug Administration and Control Authority DNA: Deoxyribonucleic acid DPPH: Diphenylpicrylhydrazyl EHNRI: Ethiopian Health and Nutrition Research Institute EI: Edema Inhibition EV: Edema Volume FDA: Food and Drug Administration GC: Gas Chromatography HIV: Human Immunodeficiency Virus HPLC: High Pressure Liquid Chromatography MIC: Minimum Inhibitory Concentration MS: Mass Spectrometry NDA: New Drug Application PEG: Poly Ethylene Glycol Rf: Retention Factor SD: Standard Deviation SEM: Standard Error of the Mean SSTI: Skin and Soft Tissue Infections TLC: Thin Layer Chromatography ix TM: Traditional Medicine TS: Test Solution TSY: Tryptone Soya USD: United States Dollar USP: United States Pharmacopoeia UV: Ultra Violet WHO: World Health Organization x ABSTRACT In this study, eight species of traditionally used medicinal plants namely Acokanthera schimperi (Apocynaceae), Calpurnia aurea (Fabaceae, Leguminosae), Kalanchoe petitiana (Crassulaceae), Lippia adoensis (Verbenaceae), Malva parviflora (Malvaceae) Olinia rochetiana (Oliniaceae), Phytolacca dodecandra (Phytolaccaceae) and Verbascum sinaiticum (Scrophulariaceae), were screened for antimicrobial activity against different strains of bacteria and fungi which are known to cause various types of skin infections. Among these plants, L. adoensis and O. rochetiana , which showed better antimicrobial activity in the initial screening test, were selected for further investigations. Fractionation and antimicrobial activity tests of the fractions, anti-inflammatory activity tests, phytochemical screening, evaluation of topical formulations, and preliminary standardization studies were carried out on the two species of plants. The results of the initial antimicrobial screening test indicated the potential of these herbal drugs in treating bacterial and fungal infections of the skin.
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