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JULY, 2012 Dillenia Pentagyna 2 Dillenia pentagyna 1 IN VITRO ANTI-MICROBIAL EVALUATION OF DIFFERENT CRUDE EXTRACTS FROM DILLENIA PENTAGYNA LEAF AND STEM Dissertation Submitted in partial fulfillment of the requirements for the degree of Bachelor of Pharmacy Submitted by RIDWANA AKHAND ID: 2008-1-70-022 Department of Pharmacy East West University, Dhaka JULY, 2012 Dillenia pentagyna 2 DECLARATION BY THE CANDIDATE I hereby declare that this dissertation, entitled ‘In Vitro Anti-Microbial Assessment of Different Crude Extracts from Dillenia pentagyna Leaf and Stem’ is an authentic and genuine research work carried out by me under the guidance of Ms. Nazia Hoque, lecturer, Department of Pharmacy, East West University, Dhaka. Ridwana Akhand ID: 2008-1-70-022 Department of Pharmacy East West University, Dhaka. Dillenia pentagyna 3 CERTIFICATE BY THE INVIGILATOR This is certify that the dissertation, entitled In Vitro Anti-Microbial Assessment of Different Crude Extracts from Dillenia pentagyna Leaf and Stem’ is a bonafide research work done by Ridwana Akhand, in partial fulfillment of the requirements for the Degree of Bachelor of Pharmacy. Ms. Nazia Hoque Lecturer Department of Pharmacy East West University, Dhaka. Dillenia pentagyna 4 ENDORSEMENT BY THE CHAIRPERSON This is certify that the dissertation, entitled ‘In Vitro Anti-Microbial Assessment of Different Crude Extracts from Dillenia pentagyna Leaf and Stem’ is a bonafide research work done by Ridwana Akhand, under the guidance of Ms. Nazia Hoque, lecturer, Department of Pharmacy, East West University, Dhaka. Dr. Sufia Islam Chairperson and Associate Professor Department of Pharmacy East West University, Dhaka. Dillenia pentagyna 5 ACKNOWLEDGEMENTS I express my gratitude to Allah almighty for giving me the opportunity to complete my research. I feel obliged to my supervisor Ms. Nazia Hoque, Department of Pharmacy, East West University, Dhaka, for her keen interest, kindness and her valuable views and experience. I am also grateful to Mr. Razibul Habib, lecturer, Department of Pharmacy, East West University, for his valueable suggestion regarding my research. I would like to thanks Department of Pharmacy, East West University for timely providing me all the necessary facilities and administrative support. Moreover, I also would like to express my gratitude towards Mr. Ajoy Roy, Ms. Razia Sultana and Ms. Tania whom has helped me directly during plant verification process in my project. In last I am greatly thankful to my parents who provide me all support, inspiration and appreciation. Dillenia pentagyna 6 TABLE OF CONTENTS Contents Page CHAPTER 1 – INTRODUCTION 1 1.1 PREFACE 2 1.2 ANTIBIOTIC RESISTANCE BY MICROORGANIS 3 1.3 ANTIMICROBIAL PROPERTIES OF MEDICINAL PLANTS 3 1.4 PLANT PROFILE: DILLENIA PENTAGYNA 4 1.4.1 Scientific Classification of Dillenia Pentagyna Roxb. 4 1.4.2 Vernacular Names 4 1.4.3 Geographical Distribution 5 1.4.4 Plant Description 6 1.4.5 Medicinal Uses 8 1.5 ANTIMICROBIAL SUSCEPTIBILITY TEST 8 1.5.1Disk Diffusion Method 8 1.6 OBJECTIVE OF THE RESEARCH 8 CHAPTER 2 – REVIEW OF LITERATURE 9 2.1 ETHNOMEDICINAL STUDY 9 2.2 PHYTOCHEMICAL STUDY 11 2.3 PHARMACOLOGICAL STUDY 13 CHAPTER 3- MATERIALS & METHODS 14 3.1 MATERIALS 15 3.1.1 Plant Sample 15 3.1.2 Strains of Microorganism 15 Dillenia pentagyna 7 Contents Page 3.1.3 Culture Media 15 3.1.4 Reagents & Solvents 15 3.1.5 Apparatus & Equipments 15 3.2 METHODS 17 3.2.1 Collection of Crude Extract and Preparation of Dosage Form 17 3.2.2 Preparation of Bacterial Culture 17 3.2.3 In Vitro Antimicrobial Screening of Plant Extract 20 CHAPTER 4- RESULTS & DISCUSSION 25 4.1 RESULTS 25 4.2 DISCUSSION 33 CONCLUSION 40 REFERENCES 41 List of Tables Table No. and Title Page Table 4. 1: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna stem at a concentration of 800μg/disc 25 Table 4. 2: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna stem at a concentration of 400μg/disc 26 Dillenia pentagyna 8 Table No. and Title Page Table 4. 3: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna leaf at a concentration of 800μg/disc 29 Table 4. 4: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna leaf at a concentration of 400μg/disc 31 List of Figures Figure No. and Title Page Figure 1.1: Geographical Distribution of Dillenia Pentagyna 5 Figure 1.2: Dillenia Pentagyna Plant 6 Figure 1.3: Dillenia Pentagyna Leaf 6 Figure 1.4: Dillenia Pentagyna Flower 7 Figure 1.5: Dillenia Pentagyna Fruit 7 Figure 2.1: Rhamnetin 3-glucoside 11 Figure 2.2: Taxifolin 5-galactoside 12 Figure 2.3: 5,7,4'-Trihydroxyflavanone 7-O-galactosylglucoside 12 Figure 2.4: Lupeol, Betulin and Betulinic acid 12 Dillenia pentagyna 9 Figure No. and Title Page Figure 3.1: Nutrient Agar in Culture Bottle 17 Figure 3.2: Autoclave machine (HIRAYAMA, Japan) & Hot air oven (FN-500, Niive) 18 Figure 3.3: Steps of Inoculation 19 Figure 3.4: Incubator 20 Figure 3.5: Filter paper discs 20 Figure 3.6: Eppendrof Tubes & Vortex Mixer 21 Figure 3.7: Inoculation Process 22 Figure 3.8: Eppendrof Tube & Tips 23 Figure 4.1: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna stem at a concentration of 800μg/disc 26 Figure 4.2: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna stem at a concentration of 400μg/disc 28 Figure 4.3: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna leaf at a concentration of 800μg/disc 30 Figure 4.4: Antimicrobial activity of different solvent extracts of at a Dillenia pentagyna leaf at a concentration of 400μg/disc 32 Figure 4.5: Zone of inhibition produced by the antibiotic on the growth of microorganism 34 Figure 4.6: Zone of inhibition produced by the antibiotic on the growth of microorganism 35 Dillenia pentagyna 10 Figure No. and Title Page Figure 4.7: Zone of inhibition produced by the extract on the growth of microorganism 36 Figure 4.8: Zone of inhibition produced by the extract on the growth of microorganism 37 Figure 4.9: Zone of inhibition produced by the extract on the growth of microorganism 38 Figure 4.10: Zone of inhibition produced by the extract on the growth of microorganism 39 Dillenia pentagyna 11 ABSTRACT Background: Dillenia pentagyna is widely distributed in tropical regions of Asia. The traditional use of this plant suggests its possible antimicrobial properties, but its efficacy has not been examined yet. Objective: The purpose of this research was to evaluate the antimicrobial efficacy of crude extracts of Dillenia pentagyna leaf and stem against pathogenic microorganism using the disk diffusion method. In this study, the crude ethyl acetate, ethanol & water extract of both leaf and stem was assessed. Materials and methods: Two different concentrations of 800µg/disc and 400µg/disc for crude leaf and stem extracts of Dillenia pentagyna were tested for antimicrobial efficacy against five Gram-positive (Bacillus sereus, Bacillus subtilis, Bacillius megaterium, Staphylococcus aureus & Sarcina lutea), eight Gram negative bacteria( Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi, Shigella dysenteriae, Shigella boydii, Vibrio mimicus & Vibrio parahemolyticus) and two fungi (Candida albicans & Saccharomyces cerevisiae). The antimicrobial activity of the extracts was observed against the standard antimicrobial agent Cephradine of 30 µg/disc. The sensitivity of plant was tested using the disk diffusion method by observing and measuring the zone of inhibition for each sample against each microorganism. Results: Maximum activity was revealed by ethyl acetate extract of the stem followed by the ethanol extract. The ethyl acetate and ethanol extract of leaf showed more or less sensitivity almost against all pathogens. The water extracts revealed very less activity against the microorganisms. Conclusion: This plant may be effective for treatment of different pathogenic diseases. Keywords: Dillenia pentagyna, antimicrobial activity, microorganism, disk diffusion, zone of inhibition. Dillenia pentagyna 12 Chapter One Introduction INTRODUCTION Dillenia pentagyna 13 1.1 PREFACE Nature has been a source of medicinal agents for thousands of years and an impressive number of modern drugs have been isolated from natural sources. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases. In recent years, multiple drug resistance in human pathogenic microorganisms has developed due to indiscriminate use of commercial antimicrobial drugs commonly used in the treatment of infectious diseases. One way to prevent antimicrobial resistance of pathogenic species is by using new compounds that are not based on existing synthetic antimicrobial agents [1]. Clinical microbiologists have great interest in screening of medicinal plants for antimicrobial activities and phytochemicals as potential new therapeutics. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties. Naturally occurring combinations of these compounds can be synergistic and often results in crude extracts having greater antimicrobial activity than the purified individual constituents. For these reasons, medicinal plants are important substances for the study of their traditional uses through the verification of pharmacological effects and can be natural composite sources that act as new ant infectious agents [2]. The plant chosen for the present study is Dillenia Pentagyna belonging to the family Dilleniaceae which is a deciduous tree found mainly in rain forests, thickets and hills below 400 m. Traditionally the plant was patronized for its unique property of healing all type of cuts, wounds and boils in very less time and with less pain. According to Ayurveda, the plant is used in anal fistula, wounds, diabetes, diabetic carbuncle, neuritis, pleurisy, pneumonia, and burning sensation [3]. The tribal and folk communities also use various parts of it in different ailments and diseases like delivery, bone fracture, body pain, piles, diarrhoea and dysentery.
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