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Antimicrobial Evaluation of Extracts of Psidium Guajva and Syzgium Cumini Against Oral Pathogens

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Antimicrobial Evaluation of Extracts of Psidium Guajva and Syzgium Cumini Against Oral Pathogens Antimicrobial evaluation of extracts of Psidium guajva and Syzgium cumini against oral pathogens By Lubna Tahir Department of Microbiology Faculty of Biological Sciences Quaid-i-Azam University Islamabad 2015 Antimicrobial evaluation of extracts of Psidium guajva and Syzgium cumini against oral pathogens A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY In Microbiology By Lubna Tahir Department of Microbiology Faculty of Biological Sciences Quaid-i-Azam University Islamabad. Pakistan 2015 I In the name of Allah, Most gracious, most merciful "..........let them devote themselves to studies in religion and admonish their comrades when they return to them so that they may guard themselves against evil." (Sura 9, Verse 121) II CERTIFICATE This thesis submitted by Lubna Tahir is accepted in the present form by Department of Microbiology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, as satisfying the dissertation requirements for the degree of Doctor of Philosophy in Microbiology. External Examiner I ________________________________ External Examiner II __________________________________ Supervisor: ___________________________________ (Prof. Dr. Safia Ahmed) Chairperson: _____________________________________ (Dr. Fariha Hasan) Dated:----------------------------------------------- III LIST OF EXTERNAL EXAMINERS 1. Dr. John McCall Professor Robert Gordon University Riverside East Garthdee Road Aberdeen, AB107GJ United Kingdom Tel: +441224262473 Email: [email protected] 2. Dr. Ananda M. Chakrabarty Professor College of Medicine Department of microbiology and Immunology (MC 790) E-704 Medical Sciences Building 835 South Wolcott Avenue Chicago, Illinois 60612-7344 Email: [email protected] 3. Dr. Albert La Spada Professor Institute of Genomic Medicine 2880 Torrey Pines science Drive Sanford Consortium for regenerative Medicine MC 0642, Room 3804 University of California, San Diego California 92037-0642 Email: [email protected] IV DEDICATED To my loving parents & FAMILY V DECLARATION The experimental work described in this thesis was carried out in the Department of Microbiology, Quaid-i-Azam University, Islamabad, Pakistan; Department of Applied Chemistry, Pakistan Council of Scientific and Industrial Research, Lahore, Pakistan and Department of Biochemistry, University of Maryland, College Park, USA. I have not presented any part of this work for any other degree. All the assistance and help received during the course of research have been duly acknowledged. Lubna Tahir VI List of Tables TABLE OF CONTENTS List of Contents DEDICATED ..................................................................................................................V DECLARATION .......................................................................................................... VI TABLE OF CONTENTS ............................................................................................. VII LIST OF TABLES ....................................................................................................... XII LIST OF FIGURES.....................................................................................................XIV ABBREVIATIONS................................................................................................... XVII ACKNOWLEDGMENTS...........................................................................................XIX ABSTRACT ................................................................................................................. XX 1. INTRODUCTION ...................................................................................................... 1 1.1. Aims and Objective .............................................................................................. 6 2. REVIEW OF LITERATURE ..................................................................................... 7 2.1. Medicinal Plants and Their Importance ............................................................... 7 2.2. Oral Health and Global Concerns ........................................................................ 9 2.3. Oral Health of People of Pakistan ...................................................................... 10 2.4. Dental Caries ...................................................................................................... 11 2.5. From Synthetic to Herbal Products .................................................................... 14 VII List of Tables 2.6. The Ethnobotanical Importance, Bioassay and Phytochemical Review of Selected Plants of the Present Study ............................................................................. 17 2.6.1. Syzygium cumuni ethanobotanical importance .......................................... 17 2.6.2. Psidium guajava ethanobotanical importance............................................. 18 2.6.3. Morus nigra ethanobotanical importance.................................................... 19 2.6.4. Phoenix dactylifera ethanobotanical importance ........................................ 21 2.6.5. Mangifera indica ethanobotanical importance ............................................ 22 2.6.6. Vitis vinifera ethanobotanical importance .................................................. 23 2.6.7. Dispyros blancoi ethanobotanical importance ............................................ 24 2.6.8. Litchi chinensis ethanobotanical importance .............................................. 25 3. MATERIALS AND METHODS.............................................................................. 27 3.1. Collection of Plant Material ............................................................................... 27 3.2. Drying and Extraction ........................................................................................ 27 3.3. Fractionation....................................................................................................... 27 3.4. Phytochemical Analysis ..................................................................................... 33 3.4.1. Test for Alkaloids........................................................................................ 33 3.4.2. Test for Saponins ........................................................................................ 33 3.4.3. Test for Tannins .......................................................................................... 33 3.4.4. Test for Flavonoids ..................................................................................... 34 VIII List of Tables 3.5. Antibacterial Sensitivity of Crude Extracts against Dental Caries Causing Pathogens (in vitro). ...................................................................................................... 34 3.5.1. Test Microorganisms................................................................................... 34 3.5.2. Antibacterial Assay of crude extract ........................................................... 34 3.5.3. Minimum Inhibitory Concentration Determination .................................... 35 3.6. Formulation of Guava & Jaman Tablets ............................................................ 35 3.7. In-vitro Antibacterial Sensitivity of Syzigium cumunii:Psidinm guajava Tablet against Bacterial Strains ................................................................................................ 36 3.8. Evaluation of Physical Properties of Syzygium cumuni:Psidinm guajava Tablet 36 3.9. Determination of Bactericidal Activity of Syzygium cumuni: Psidium guajava Chewable Tablet............................................................................................................ 37 3.10. In-vivo Study of Syzygium cumuni : Psidinm guajava Chewable Tablet ....... 37 3.11. Isolation of Bioactive Fractions ...................................................................... 37 3.11.1. Flash chromatography ............................................................................. 38 3.11.2. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) 45 Sample Preparation ................................................................................................... 45 3.11.3. Flash Chromatography (FC).................................................................... 45 3.11.4. Preparative Thin Layer Chromatography................................................ 46 3.12. Bio-assay of Collected Fractions .................................................................... 46 IX List of Tables 3.13. Detection of Biofilm Formation in Streptococci and Biofilm Inhibition Assay 47 3.14. Scanning Electronic Microscopy (SEM) of the Bacterial Cells after Treatment with Bioactive Components of the Extracts .................................................................. 48 3.15. Analysis of Bioactive Fractions ...................................................................... 48 3.15.1. Mass Spectroscopy .................................................................................. 49 3.15.2. FT-IR analysis ......................................................................................... 49 3.15.3. NMR analysis .......................................................................................... 49 4. RESULTS ................................................................................................................. 50 4.1. Screening for Antibacterial activity ................................................................... 50 4.2. Preliminary Phytochemical Analysis ................................................................
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