Investigation of Cytotoxic and Antibacterial Activity of Methanol Extract of Callicarpa macrophylla Leaves. A project submitted by Rubaiyat Islam Mona ID: 12146024 Session: Spring 2012 to The Department of Pharmacy in partial fulfillment of the requirements for the degree of Bachelor of Pharmacy (Hons.) Department of Pharmacy Dhaka, Bangladesh October 2016 This work is dedicated to my parents, my younger brother and my husband for their love and constant support… Certification Statement Certification Statement This is to certify that this project titled ―Investigation of Cytotoxic and Antibacterial Activity of Methanol Extract of Callicarpa macrophylla Leaves‖ submitted for the partial fulfillment of the requirements for the degree of Bachelor of Pharmacy (Hons.) from the Department of Pharmacy, BRAC University constitutes my own work under the supervision of Dr. Raushanara Akter, Assistant Professor, Department of Pharmacy, BRAC University and the contents of this dissertation, in full or in parts, have not been submitted to any other Institute or University for the award of any Degree or Diploma of Fellowship. Signed, ---------------------------------------------- Countersigned by the Supervisor, -------------------------------------------- i Acknowledgement Acknowledgement First of all, all praise and glory to almighty Allah for all the generosities granted to me and only His support and help this achievement has become possible for me. It is my pleasure and also privilege to express my heartiest regards and gratitude to my respected teacher and research supervisor, Dr. Raushanara Akter, Assistant Professor, Department of Pharmacy, BRAC University for her scholarly guidance, constant supervision and support. I also thank for her helping hands & affectionate attitude whenever I needed. It is also a great pleasure for me to offer my deepest gratitude to Dr. Eva Rahman Kabir, Associate Professor and Chairperson, Department of Pharmacy, BRAC University for giving me all the necessary support whenever I needed to conduct my project work. I am thankful to my respected teacher Dr. Mahboob Hossain, Associate Professor, Department of Mathematics and Natural Sciences (MNS), BRAC University for his continual cooperation and for giving me the guidance during the work in Microbiology Lab, BRAC University. I would like to thank Dr. Sheikh Ariful Hoque, Senior Scientist and Head, Cell and Tissue Culture Laboratory, Center for Advance Research in Sciences (CARS), University of Dhaka for his guidance, constant support during the work in CARS center. I am thankful to all teachers of Department of Pharmacy in BRAC University to get constant encouragement, support and guidance which helped me in to complete this project work. I would also like to extend my thanks to all lab officers and other staffs of the Department of Pharmacy for their help and assistance, friendly behavior and earnest co-operation which enabled me to complete my project work successfully. The success and final outcome of this project also required a lot of guidance and the assistance from many people and I am extremely fortunate to have got this all along completion of my project work. I would not forget to thank them. Finally, I would like to give thanks from my heart to my family and friends for their care and encouragement during my research work. ii Abstract Abstract Callicarpa macrophylla (family: Verbenaceae) has been commonly used in traditional medicine for a wide range of ailments related to the circulatory, digestive, endocrine, respiratory and skeletal systems as well as to infectious diseases. The purpose of the study was to evaluate the cytotoxic and antibacterial effect of Callicarpa macrophylla (C. macrophylla) leaf extract. The cytotoxic potential of the extract was examined on HeLa (a human cervical carcinoma cell line) cells using MTT assay and the antibacterial activity was assessed by disc diffusion method. The highest cytotoxic potential (92% cell growth inhibition) was evident at the concentration of 2.5 mg/mL and the lowest cytotoxic potential (7% cell growth inhibition) was found at the concentration of 0.25 mg/mL. The extract exhibited the cytotoxic activity in a concentration dependent manner. This extract showed potent cytotoxic activity with an IC50 value of 1.38 mg/mL. The antibacterial activity of methanol extract of the plant was investigated and low to moderate antibacterial activity against the tested bacterial strains was showed by the extract compared to the kanamycin that was used as a positive control. The highest antibacterial activity (zone of inhibition 22 mm) was found for Bacillus cereus at the concentration of 350 µg/disc. The results of study clearly indicated the presence of cytotoxic and antibacterial properties of the methanol extract. Phytochemical screening of this plant extract showed the presence of flavonoids, tannins, steroids and glycosides. The potent cytotoxic effect may be due to the presence of flavonoids, tannins, and glycosides and the potent antibacterial effect may be due to the presence of tannins and steroids. Thus, this study finding provide a scientific basis for the use of this plant as traditional medicine in the treatment of cancer and infectious disease and its further investigation and commercial exploitations of the plant. Future work will be focused on isolation of compounds which are responsible for cytotoxic and antibacterial activity and on new bioactivity investigation of the plant extract of C. macrophyllay. iii Table of Contents Table of contents Table of contents Page Certification statement i Acknowledgement ii Abstract iii List of contents iv-vii List of tables viii List of figures ix List of abbreviations x CHAPTER 1: INTRODUCTION 1.1 Natural Products 1 1.2 New Chemical Entities from Natural Products 2-5 1.3 Traditional Medicines 5 1.4 Medicinal Plants 6 1.5 Significance of Medicinal Plants in Drug Discovery 6-8 1.6 Scientific Basis of Herbal Drug 8 1.7 Rationale of Herbal Drug Research: Special Reference to Bangladesh 8-9 1.8 Cancer and Role of Plant Extract to Treat the Cancer Disease 9-10 1.8.1 Overview on Cancer Disease and Anticancer Drug Mechanism in Cancer 10-11 1.8.2 Anticancer Drug Isolated from Plants 11-13 1.9 Method of Cytotoxic Study 13 1.9.1 Principle of MTT Assay Method 13-14 1.10 Antibacterial Effect of Plant 14-17 1.11 Principle of Disc Diffusion Method 17-18 1.12 The Plant Callicarpa macrophylla 18 1.12.1 Taxonomic Hierarchy of Callicarpa macrophylla 18 1.12.2 Description of the Plant 18-19 1.12.3 Medicinal Uses 19 1.12.4 Chemical Constituents 19 1.12.5 Common Names of the Plant 20 1.12.6 Distribution/Habitat of the Plant 20 iv Table of Contents 1.13 Rationale of the Study 20-21 1.14 Aim of the project 21 1.15 Objectives of the project 21 CHAPTER 2: LITERATURE REVIEW 2.1 Pharmacological Activity Previously Studied 22 2.1.1 Antibacterial Activity 22 2.1.2 Antidiabetic Activity 22 2.1.3 Analgesic and Antipyretic Activity 22 2.1.4 Antifungal Activity 23 2.1.5 Anti-inflammatory Activity 23 2.1.6 Anti-arthritic Activity 23 2.2 Previously Isolated Photochemical Compounds of C. macrophylla 23-25 CHAPTER 3: METHODOLOGY 3.1 Collection and Authentication of Plant Material 26 3.2 Extraction Procedure 26 3.2.1 Plant Material Preparation and Drying 27 3.2.2 Extraction Procedure 27 3.2.2.1 Size Reduction 27 3.2.2.2 Extraction 27 3.2.2.3 Filtration 27-28 3.2.2.4 Concentration 29 3.2.2.5 Drying 29 3.3 Phytochemical Screening 29 3.3.1 Detection of Alkaloid 29 3.3.1.1 Hager’s Test 29 3.3.1.2 Mayer’s Test 30 3.3.1.3 Wagner’s Test 30 3.3.2 Detection of Flavonoids 30 3.3.2.1 Lead Acetate Test 30 3.3.2.2 Zinc Ribbon Test 30 3.3.3 Detection of Saponins 30-31 v Table of Contents 3.3.4 Detection of Phytosterols 31 3.3.5 Detection of Steroid 31 3.3.6 Detection of Tannins 31 3.3.6.1 Lead Acetate Test 31 3.3.6.2 Ferric Chloride Test 31 3.3.7 Detection of Glycosides 31-32 3.3.8 Detection of Phenols/ Phenolic compounds 32 3.4 In-vitro Cytotoxic Activities 32 3.4.1 Preparation of Sample from Plant Extracts 32 3.4.2 Instruments Used in this Study 32 3.4.3 Materials Used 32 3.4.4 Procedure 33 3.5 Antibacterial Activity by Disc Diffusion Method 33 3.5.1 Apparatus & Reagents 33 3.5.2 Test Sample of C. macrophylla 34 3.5.3 Test Organisms 34 3.5.4 Procedure 34 3.5.4.1 Preparation of the Medium 34 3.5.4.2 Sterilization Procedure 34 3.5.4.3 Preparation of the Test Plate 35 3.5.4.4 Preparation of Discs 35 3.5.4.5 Preparation of Test Sample 35 3.5.4.6 Application of Test Samples 35-36 3.5.4.7 Diffusion & Incubation 36 3.5.4.8 Determination of Antibacterial Activity by Measuring the Zone of Inhibition 36 CHAPTER 4: RESULTS AND DISCUSSION 4.1 Result of Phytochemical Screening of C. macrophylla 37 4.1.2 Discussion of Phytochemical Screening of C. macrophylla 37-38 4.2 Result of Cytotoxic Activity Test 38 4.2.1 Percentage of Survival Cells and the Inhibition of Cell Growth 38-39 vi Table of Contents 4.2.2 Graphical Representation of Cytotoxic Activity 39 4.2.3 Discussion 40-41 4.3 Results of Antibacterial Activity Test 41-42 4.3.1 Zone of Inhibition of Methanol Extract of C. macrophylla 42-43 4.3.2 Graphical Representation of Antibacterial Activity Test 44 4.3.3 Discussion 44-45 CHAPTER 5: CONCLUTION 46 REFERENCES 47-51 vii List of Tables List of Tables Table no.