Study on Antimicrobial and Cytotoxicity Activity of Methanol Extract of Dracaena spicata A DISSERTATION SUBMITTED TO THE DEPARTMENT OF PHARMACY, EAST WEST UNIVERSITY IN THE PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF BACHELOR OF PHARMACY Submitted By Fatema Jannat ID: 2013-1-70-042 Department of Pharmacy East West University Department of Pharmacy East West University i Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata Declaration by the Research Candidate I, Fatema Jannat, hereby declare that the dissertation entitled “Study on Antimicrobial and Cytotoxic activity of Methanol Extract of Dracaena spicata” submitted by me to the Department of Pharmacy, East West University, in the partial fulfillment of the requirement for the award of the degree Bachelor of Pharmacy is a complete record of original research work carried out by me during 2016, under the supervision and guidance of Ms Nazia Hoque, Assistant professor, Department of Pharmacy, East West University and the thesis has not formed the basis for the award of any other degree/diploma/fellowship or other similar title to any candidate of any university. ________________________ Fatema Jannat ID# 2013-1-70-042 Department of Pharmacy East West University ii Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata Certificate by the Supervisor This is to certify that the thesis entitled “Study on Antimicrobial and Cytotoxic activity of Methanol Extract of Dracaena spicata” submitted to the Department of Pharmacy, East West University, in the partial fulfillment of the requirement for the degree of Bachelor of pharmacy was carried out by Fatema Jannat, ID# 2013-1-70-042 in 2016, under the supervision and guidance of me. The thesis has not formed the basis for the award of any other degree/diploma/fellowship or other similar title to any candidate of any university. Nazia Hoque Assistant Professor, Department of Pharmacy iii Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata East West University, Dhaka. ENDORSEMENT BY THE CHAIRPERSON This is to certify that the dissertation, entitled “Study on Antimicrobial and Cytotoxic activity of Methanol Extract of Dracaena spicata” is a bona fide research work done by Fatema Jannat (ID#2013-1-70-042), in partial fulfillment of the requirements for the degree of Bachelor of Pharmacy. --------------------------------------- Dr. Shamsun Nahar Khan Chairperson and Associate Professor Pharmacy Department iv Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata East West University Aftabnagar, Dhaka Acknowledgements All praise is for Almighty for all the bounties granted to me and only with His guidance and help this achievement has become possible. It is my pleasure and proud privilege to express my heartiest regards and gratitude to my respected teacher and supervisor Ms. Nazia Hoque, Assistant professor, Department of Pharmacy, East West University, for her expert supervision, constructive criticism, valuable advice, optimistic counseling, constant support and continuous backup and encouragement throughout every phase of the project as well as to prepare this dissertation. I put forward my most sincere regards and profound gratitude to Chairperson Dr.Shamsun Nahar Khan, Associate Professor, Department of Pharmacy, East West University, for her inspiration in my study. She also paid attention for the purpose of my research work and extending the facilities to work. I am thankful to all Lab instructors, Department of Pharmacy, East West University, for her amiability to provide me with untiring guidance, whole hearted cooperation and for her extensive knowledge about reagents that helped me in all the spheres to perform the research work. I owe special thanks to my fellow research group members Muhammedullah Rumi, Sumona Nazneen, Nusrat Jahan for their immense support and contribution in my research work. Last but not the least, I would like to thank my family, and friends for their care and encouragement during my research work. v Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata Thank you Dedication This Research Paper is dedicated to my beloved parents, they are my biggest inspirations. vi Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata ABSTRACT The purpose of the study was to evaluate the cytotoxic and antimicrobial activity of methanolic extract of Dracaena spicata of the family Asparagaceae.Dracaena spicata has been used as a medicinal plant for the general promotion of health and longevity by Asian tribal. It is used as a traditional medicine for the treatment ofvarious diseases like cough, syphilis, conjunctivitis, constipation, boils, eczema, scabies, septic abscess, itching and skin allergy, burns, chicken pox, warts and leucoderma, fungal and bacterial infections, including healing cuts and wounds has been documented by randomly interviewing Chakma, Marma and Tanchunga tribes of the hill tracts districts of Bangladesh since 1995.TheantimicrobialactivitiesofmethalonicextractofDracaenaspicataplantweretested againstthegram-positive(Bacillus sereus, Bacillus megaterium,Staphylococcus aureaus) andgram-negativebacterial(Pseudomonas aureaus,Salmonella paratyphi, Salmonella typhi, Vibrio parahemolyticus, Vibrio mimicus, Sheigella dysenteriae, Escherichia coli) strainsbyobservingthezoneofinhibition.Theantimicrobialtestwasperformedbydiscdiffus ionmethod.ThecrudemethanolextractofDracaenaspicata plant showedmoderate to strong (9mm-30mm) antimicrobial activitiesagainst themicroorganisms. ButmethanolextractofDracaenaspicatashowedstrong activityagainstPseudomonasaureaus (30mm) and the lowest activity was found against Bacillusmegaterium (9mm). However, no activity was found againstSalmonella paratyphi, Salmonella typhi, Vibrio parahemolyticus, Staphylococcus aureaus, Escherichia coli, Sheigella dysenteriae. The cytotoxic activity of the plant was done by using Artemia saline Leach. The LC50 was observed approximately as 46.4729 μg /mL with a R2 value of 0.772. Cytotoxicity of methanolic extract of Dracaena spicata was low to moderate. The obtained results provide a support for the use of this plant in traditional medicine and its further investigation Key Words Dracaena spicata, Antimicrobial, Artemia saline Leach Cytotoxicity, DMSO vii Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata List of Contents Serial No. Topics Page no. 1.1 Introduction 1 1.2 Medicinal plants 1 1.3 Medicinal plants in Oncology 2 1.4 Importance of Medicinal Plant 3-4 1.5 Historical Sources Relevant For Study of Medicinal Plants’ Use 4 1.6 Modern Medicinal Chemistry 5 1.7 Traditional medicines 6 1.8 Classification of Medicinal Plant 7-10 1.9 Plant information 11 1.9.1 Synonyms 11 1.9.2 Bengali name 11 1.9.3 Distribution 11 1.9.4 Height/Spread 11 1.9.5 Taxonomy 11 1.9.6 Description of the plant 12 1.10 Species & Cultivars 12-15 viii Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata 1.11 Aim of this experiment 15 1.11.1 Study Area: 16 1.11.2 Data Collection: 16 1.12 Approaches of drug development 17 1.12.1 Selection of plant species: 17 1.12.2 Evaluation of toxicity: 17 1.12.3 Preparation of plant sample and element analysis 17 1.12.4 In-vivo analysis 18 1.13 Antibiotics 18 1.14 Natural Antibiotics 18 1.15 Classification of antibiotics 18 1.16 Uses of antibiotic 20 1.17 Cytotoxic drugs 20 LITERATURE REVIEW 2.1: Literature review 21 2.2 Biological activity of saponins from two Dracaena species 21 2.3 Anti thrombal activity 21 2.4 Membrane Stabilizing Activity 22 2.5 Anti-ulcer activity 22 2.6 Anti-tussive activity 22 ix Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata 2.7 Antioxidant activity 22 2.8 Phytochemical evaluation: 23 2.9 Antibacterial activity: 23 MATERIALS AND METHODS 3.1 Collection of plant materials and extraction 24 3.1.1. Plant Material 24 3.2. Preparation of plant extract for experiments 24 3.2.1. Process of powdering 24 3.2.2 Extraction 25 3.2.3. Filtration 25 3.2.4. Evaporation and extract preparation 25 3.2.5 Antimicrobial screening 25 3.3 Test materials used for the study 26 3.3.1 Reagents 26 3.4 Apparatus 26 3.5 Test Organisms: 27 3.6 Preparation of the Medium: 28 3.7 Sterilization Procedure 29 3.8 Preparation of subculture 29 3.9 Preparation of the test plate 32 3.10 Preparation of disc 32 x Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata 3.11 Diffusion and incubation 33 3.12 Determination of antimicrobial activity by measuring the zone 33 of inhibition Cytotoxicity Test: 3.13. Principle 33 3.13.1 Preparation of seawater 33 3.13.2 Hatching of Brine Shrimps 34 3.13.3 Preparation of test solutions 34 3.13.4 Counting of nauplii and analysis of data 34 4.1 Results and Discussions of antimicrobial screening: 4.2 Result and discussion of Cytotoxicity test 36-37 4.3 Discussion 39-40 5. References 40-41 No. Table Names 1 Table 6 2 Medicinal Shrubs 7 3 Medicinal Herbs 8 4 Medicinal Annuals 8 5 Biennial 9 6 Tubers and Rhizomes 9 7 Biennial 9 8 Classification of Antibiotics 19-20 9 List of the test pathogenic bacteria Name of the test 27 xi Study on Antimicrobial and Cytotoxic activities of Methanol Extract of Dracaena spicata organism 10 Anti-Microbial test (result in mm) 36 11 Effect of Dracaena spicata (methanol extract) on shrimp nauplii 37 Figures Page No 1. Dracaena spicata 10 2. Dracaena cinnabari 13 3. Dracaena cochinchinensis 13 4. Dracaena fragrans 14 5. Dracaena godseffiana 14 6. Dracaena marginata 15 7. Dragon Blood 15 8. Laminar hood 28 9. test plate 8 (Staphylococcusaureaus) 38 10. test plate 11 (Pseudomonasaureaus) 38 11. test plate 10 (Sheigella dysenteriae) 38 12. test plate 7 (Vibrio mimicus) 38 13. test plate 6 (Vibrio parahemolyticus) 38 14. test plate 5 (Salmonella typhi) 39 15. test plate 4 (Salmonella paratyphi) 39 16. test plate 1 (Bacillussereus) 39 17. test plate 2 (Bacillusmegaterium) 39 18. test plate 3 (Bacillus subtilis) 39 19.