Antimicrobial and Cytotoxic Investigations Of Methanolic Extract Of Dracaena spicata A DISSERTATION SUBMITTED TO THE DEPARTMENT OF PHARMACY, EAST WEST UNIVERSITY IN THE PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF BACHELOR OF PHARMACY Submitted By Sadiya Afrin ID: 2011-3-70-010 Department of Pharmacy East West University Declaration By The Research Candidate I, Sadiya Afrin, hereby declare that the dissertation entitled“Antimicrobial and Cytotoxic Investigations of Methanolic extract of Dracaena spicata” submitted by me to the Department of Pharmacy, East West University, in the partial fulfillment of therequirement for the award of the degree Bachelor of Pharmacy is a completerecord of original research work carried out by me during 2014, under the supervision and guidance of Ms Nazia Hoque, Senior Lecturer, Department ofPharmacy, East West University and the thesis has not formed the basis for theaward of any other degree/diploma/fellowship or other similar title to anycandidate of any university. ________________________ Sadiya Afrin ID# 2011-3-70-010 Department of Pharmacy East West University Certificate By The Supervisor This is to certify that the thesis entitled “Antimicrobial and Cytotoxic Investigations of Methanolic Extract ofDracaena spicata” submitted to the Department of Pharmacy, East WestUniversity, in the partial fulfillment of the requirement for the degree ofBachelor of pharmacy was carried out by Sadiya Afrin, ID# 2011-3-70-010in 2015, under the supervision and guidance of me. The thesis has not formedthe basis for the award of any other degree/diploma/fellowship or other similartitle to any candidate of any university. Nazia Hoque Senior Lecturer, Department of Pharmacy East West University, Dhaka. Endorsement By The Chairperson This is to certify that the thesis entitled “Antimicrobial and Cytotoxic Investigations of Methanolic Extract of Dracaena spicata” submitted to the Department of Pharmacy, East WestUniversity, Dhaka, in the partial fulfillment of the requirement for the degree ofBachelor of pharmacy was carried out bySadiya Afrin, ID# 2011-3-70-010 The Chairperson Department of Pharmacy East West University, Dhaka. ACKNOWLEDGEMENTS At first, I would like to express my respect, cordial gratitude and obligation to my thesis supervisor, Naziahoque, Senior Lecturer, Department of Pharmacy, East West University. I am thankfull and gratefull to her for her valuable instructions, continuous guidance, supevision and necessary advice related to the research work and completion of the thesis paper from the very beginning to the end. I am thankfull to the Laboratory officers of the Department of Pharmacy of East West University, for their assistance in the laboratory. I am specially gratefull to my mother, father for their support, help, sacrifices, cooperation and inspirations in my work. Last but not the least, I recall my freinds, Ayesha SiddikaMonni,Raju khan and SaminYasiarOrnobfor being with me in this journey and supporting me in my research work. Thank u Sadiya Afrin ID:2011-3-70-010 Department Of Pharmacy East West University Abstract The plant Dracaena spicata has been used for the general promotion of health and longevity by Asian tribal. It is used as a traditional medicine for the treatment of various diseases like meals, fever, ulcer, stomachaches etc. The aim of the present study was to evaluate the antimicrobial activity and cytotoxic activity of methanolic extract of Dracaena spicata. The antimicrobial activities of methanolic solvent extract of plant weretested against the Gram-positive and Gram-negative bacterial strains and fungi by observing the zone of inhibition. The antimicrobial test was performed by Disc diffusion method. The crude methanolic extract of Dracaena spicataplant showed poor antimicrobial activities against Gram-negative bacteria and fungi at concentrations of 400 μg/disc. However, no activity was found against Salmonela paratyphy and Bacillus subtilis. The cytotoxic activity of the plant was done by using Artemia saline Leach. The LC50 was observed 2 approximately as 36.829μg / mL with a R value of 0.9735. Cytotoxicity of methanolic extract of Dracaena spicata was not good, LC50 value less than 5 μg / mL indicates excellent cytotoxic effect. So, further studies are needed to evaluatethe cytotoxicity of isolated pure compounds. In conclusion, furtherinvestigations are needed to identify the active constituents and the exact mechanism(s) of action responsible for the reported antimicrobial and cytotoxic properties of Dracaena spicata Key Words: Dracaena spicata, Antimicrobial, Cytotoxicity. LIST OF CONTENT SERIAL NO TOPIC PAGE NO Chapter-1: Introduction 1 General Introduction 1 1.1 Medicinal plant 1 1.1.1 Background 1 1.1.2 Importance of medicinal plant 2 1.1.2.1 Nutraceuticals and functional foods 3 1.1.2.2 Pharmaceutical compounds from plants 4 1.1.2.3 Seeking new drugs from plants 4 1.1.2.4 Economic opportunities 5 1.2 Classification of medicinal plant 6-8 1.2.1 Traditional medicine 8 1.3 Tribal medicinal plant 9 1.4 Selection and identification of plant 10 1.5 Collection of plant 11 1.6 Drying and grinding 11 1.7 Extraction procedure of medicinal plant 12 1.7.1 Meciration 12 1.7.2 Infusion 12 1.7.3 Digestion 12 1.7.4 Decoction 12 1.7.5 Percolation 12 1.7.6 Hot continuous extraction (Soxhlet) 13 1.7.7 Aqueous alcoholic extraction byfermentation 14 1.7.8 Counter-current extraction 15 1.7.9 Ultrasound extraction (Sonication) 15 1.7.10 Supercritical fluid extraction 15 1.8 Parameters for selecting an appropriate extraction method 16 1.8.1 16 Nature of constituents 1.9 Steps involved in the extraction of medicinal plant 17 1.9.1 Size reduction 17 1.9.2 Extraction 18 1.9.2.1 Cold aqueous percolation 18 1.9.2.2 Hot aqueous extraction (Decoction) 18 1.9.2.3 Filtration 18 1.9.2.4 Spray drying 18 1.9.2.5 Solvent extraction 19 1.9.2.6 Cold percolation 19 1.9.2.7 Hot percolation 20 1.9.2.8 Concentration 21 1.10 Some extraction procedure 21-23 1.11 Solvent 24 1.11 Acute oral toxicity studies 24 1.12 Plant review 25 1.12.1 Texonomy 25 1.12.2 Plant background 25 1.12.3 Introduction 26 1.12.4 Other species 26 1.12.5 General information 27 1.12.6 Description 27 1.12.7 Foliage 27 1.12.8 Flower 28 1.12.9 Fruit 28 1.12.10 Trunk and Branches 29 1.12.11 Culture 29 1.12.12 Other 29 1.13. Use 30 Chapter-2: LITERATURE REVIEW 2.1 Antimicrobial activity test 31 2.2 Thrombolytic activity 31 2.3 Membrane stabilizing activity 31 2.4 Antipyretic activity 32 2.5 Antiulcerant activity 32 Chapter-3: MATERIAL AND METHOD 3.1 Collection 33 3.1.1 Plant Material 33 3.2 Preparation of plant extract for experiments 33 3.2.1 Process of powdering 33 3.2.2 Extraction 34 3.2.3 Filtration 34 3.2.4 Evaporation and extract preparation 34 3.3 Antimicrobial screening 36 3.3.1 Test materials used for the study 36 3.3.2 Reagents 36 3.3.3 Apparatus 36 3.3.4 Test organisms 37 3.3.5. Culture medium and their composition 37 3.3.6. Preparation of the medium 38 3.3.7 Sterilization procedure 38 3.3.8 Preparation of Subculture 38 3.3.9 Preparation of the test plate 39 3.3.10 Preparation of disc 39 3.3.11 Diffusion and incubation 40 3.3.12 Determination of antimicrobial activity by measuring the zone of inhibition 40 3.3.13. Precaution 41 3.4 Cytotoxicity assay 41 3.4.1 Principle 41 3.4.2 Materials required 41 3.4.3 Procedure 41 3.4.4 Shrimp serial dilution for cytotoxicity 42 bioassay Chapter-4: RESUIT 4.1 Result of antimicrobial assay of Dracaena spicata 44 4.2 Result of cytotoxicity assay of the Dracaena spicata 44 Chapter-5: DISCUSSION 5.1. Antimicrobial assay of the methanolic extract of Dracaena spicata 46 5.2 Cytotoxicity assay of the methanolic extract of Dracaena spicata 46 Chapter-6: CONCLUSION 6.1 Conclusion 47 Chapter-7: REFFERENCE 7.1 Referrences 48-50 LIST OF TABLE SERIAL NO Table-1 Classification of medicinal plant 6-8 Plants used by the Kavirajes and tribal Table-2 medicinal practitioners of Bangladesh 9 Table-3 Common solvent for crude extraction 24 Table-4 List of test organism 37 Table-5 Ingredient of Nutrient Agar medium 37 Result of Antimicrobial assay of Table-6 Dracaena spicata 44 Table-7 Effect of Dracaena spicata on Shrimp nauplii 45 LIST OF FIGURE SERIAL NO TOPIC PAGE NO Figure-1 Parcolator 13 Figure- 2 Soxlet apparatus 14 Figure-3 Fow diagram of methanol extract 21 Figure-4 Flow diagram of oil extraction 22 Figure-5 Flow diagram of compound extraction 23 Figure-6 Dracaena spicata 27 Figure-7 Leaf of the Dracaena spicata 28 Figure-8 Flower of Dracaena spicata 28 Figure-9 Blender machine 33 Figure-10 Rotary evaporator 34 Schematic presentation of the crud Figure-11 preparation from plant 35 Figure-12 Laminar hood 38 Figure-13 Incubator 39 Figure-14 Zone of inhibition 40 Schematic representation of experimental Figure-15 procedure of the brine shrimp serial dilution for 43 cytotoxicity bioassay Figure-16 % mortality and predicted regression line of 45 Dracaena spicata Antimicrobial and Cytotoxic Investigatins of Methanolic Extract of Dracaena spicata 1.General Introduction: Natural environment has been a source of medicinal agents for thousands of years, since healing with plants dates backprobably to the evolution of Homo sapiens. Even to date, about 80% of the world‟s inhabitants relymainly on traditional medicines for their primary health care, while medicinal plants continue to play an important role in the health care systems of the remaining 20%.