Kwame Nkrumah University of Science and Technology, Kumasi COLLEGE OF SCIENCE DEPARTMENT OF CHEMISTRY The Antimicrobial Activities of the Stem Extract of Strophanthus gratus, Apocynaceae DANIEL HENNEH JUNE, 2013 THE ANTIMICROBIAL ACTIVITIES OF THE STEM EXTRACT OF STROPHANTHUS GRATUS, APOCYNACEAE By Daniel Henneh BPharm(Hons), KNUST, Kumasi A Thesis submitted to the Department of Chemistry, Kwame Nkrumah University of Science and Technology in partial fulfillment of the requirements for the award of the degree of MASTER OF SCIENCE (ORGANIC CHEMISTRY) College of Science OCTOBER, 2013 CERTIFICATION I hereby declare that this submission is my own work towards the Master of Science degree and that to the best of my knowledge, it neither contains material previously published by another person nor material which has been accepted for the award of any degree of the university, except where due acknowledgement has been made in the text. Daniel Henneh (PG2851708) …………………… …………………….. Candidate Signature Date Certified by: Dr. Sylvester K. Twumasi …………………… …………………….. Supervisor Signature Date Certified by: Mr. R. B. Voegborlo …………………… …………………….. Head of Department Signature Date DEDICATION This thesis is dedicated to my wife, Janet Henneh, and my elder brother, Hon. Kwasi Ameyaw- Cheremeh, MP (Sunyani East Constituency) whose unflinching encouragement helped me to enrol in the MSc program and also to be able to complete the program. Thank you for your invaluable assistance. ACKNOWLEDGEMENT First, I thank God for helping, strengthening and guiding me throughout the program. Whenever the challenges abounded, you were there to take me through. You sent men to encourage and strengthen me. I am grateful to you. Second, to my dynamic supervisor, Dr. S. K. Twumasi, I say a big thank you for your commitment and support. You were always available to help, guide and advise. Without your unreserved assistance, I would never have completed my MSc work. I specially thank Dr. S. Osafo Acquaah of the Chemistry Department, College of Science, KNUST. Your encouragement always echoed in my mind and produced desire and energy for the completion of this work. I thank all the lecturers of the Department of Chemistry, College of Science, KNUST, for their variety of assistance to me. I also, thank Mr Adu and all lecturers and technicians of the Microbiology Department, Faculty of Pharmacy, KNUST for their assistance in my laboratory work. Lastly, I thank my course mates, especially David and Maxwell for their co-operation. ABSTRACT The ethanolic and aqueous extracts of the stem of Strophanthus gratus from the Botanical Gardens, KNUST, were tested for in vitro antimicrobial activities. Traditional herbal practitioners use the decoctions of the stem of this plant to treat gonorrhoea and syphilis. Phytochemical tests on the extracts showed that they contained saponins, flavonoids, steroids, alkaloids, anthraquinone glycosides, cyanogenetic glycosides, cardiac glycosides and tannins. Test organisms used included E. faecalis, Pr. vulgaris, Staph aureus, B. subtilis, B. thuringiensis, S. typhi, Ps aeruginosa and Neisseria gonorrhoeae. The results showed that both the ethanolic and aqueous extracts were active against the test organisms. However, the ethanolic extract recorded lower Minimum Inhibitory Concentrations against the organisms when compared with the aqueous extract. When the activities of the extracts were compared with those of ciprofloxacin under the same experimental conditions, it was realized that the extracts were more active than ciprofloxacin against the test organisms with the exceptions of Neisseria gonorrhoeae, S. typhi and E. coli. Auxiliary tests on the ethanolic extract of the plant showed that the plant has some antioxidant activity in addition to its antimicrobial activity. TABLE OF CONTENTS TITLE PAGE CERTIFICATION……………………………………………………………………….ii DEDICATION……………………..……………………………………………………iii ACKNOWLEDGEMENT………….…………………………………………………..iv ABSTRACT……...……………………………………………………………………….v TABLE OF CONTENTS……………………………….………………………………vi LIST OF TABLES……………………………………………….……………………....x LIST OF FIGURES……………………………………………………………………...x 1 INTRODUCTION……………………………………………………………………....1 1.1 BACKGROUND……………….…………………………………………….…….1 1.2 STATEMENT OF THE PROBLEM…………….………………………………....2 1.3 OBJECTIVES…………………………………………………………………...….3 1.4 JUSTIFICATION OF THE PROJECT………………………………………..……4 2 LITERATURE REVIEW …………………………………………………………...….7 2.1 PHYTOCHEMICAL PRINCIPLES IN PLANTS……………………………...….7 2.1.1 Alkaloids …………………………………………………………...……….7 2.1.2 Flavonoids ……………………………………………………………...…...9 2.1.3 Tannins ……………………………………………………………...……..10 2.1.4 Terpenoids ……………………………………………………………....…11 2.1.5 Steroids from plants……………………………………………………..…11 2.1.6 Anthracene and anthraquinone derivatives…………………………..….…12 2.1.7 Cyanogenetic glycosides ……………………………………………….….12 2.2 STROPHANTHUS …………………………………………………………….…12 2.2.1 Uses of strophanthus gratus ……………………………...……………….13 2.2.2 Phytochemicals from Strophanthus gratus ………………………………..13 2.3 THE PRINCIPLE OF SOXHLET EXTRACTION ……..…………………….….15 2.4 DISEASE CAUSING BACTERIA …………………………………..………..…16 2.4.1 Staphylococci……………………………………...……………………….16 2.4.2 Neisseria ……………………………………………………………….......16 2.4.3 Bacillus.………………………………………………...……………….…17 2.4.4 Pseudomonas …………………………………………………..………….17 2.4.5 Escherichia………………………………………………………….…...…17 2.4.6 Salmonella …………………………….……………………………..……18 2.4.7 Proteus ……………………………………………………..……….….….18 2.4.8 Klebsiella …………………………………………………...………….….18 2.5 ANTIBIOTICS ………………………………………………………………......18 2.5.1 Mechanisms of action of antimicrobial agents ……………………..……..21 2.5.2 Assay of antibiotics……………………………………………….…….…21 2.6 ANTIOXIDANT ACTIVITY OF PLANTS ………………………………….….22 3 MATERIALS AND METHODS ……………………………………………………..25 3.1 Materials ……………………………………………………………………….....25 3.1.1 Collection of plant material …………………………………………….…25 3.1.2 Chemicals…………………………………………………………………..25 3.1.3 General cleaning and sterilization of glassware ……………………….…..26 3.2 METHODS ……………………………………………………………………….....26 3.2.1 Preparation of extracts…………………………………………………..…26 3.2.2 Phytochemical screening……………………………………………….….27 3.2.2.1 Test for saponins ………………………………………………...27 3.2.2.2 Test for general glycosides …………………………………...…27 3.2.2.3 Test for flavonoids ………………………………………………28 3.2.2.4 Test for terpenoids and steroids …………………………………28 3.2.2.5 Test for carotenoids …………………………………………..….28 3.2.2.6 Test for coumarins …………………………………………...….29 3.2.2.7 Test for alkaloids ………………………………………………...29 3.2.2.8 Test for anthraquinones………………………………….…….....29 3.2.2.9 Test for anthraquinone glycosides ……………………………....30 3.2.2.10 Test for cyanogenetic glycoside …………………..……………30 3.2.2.11 Test for cardiac glycosides………………………….…………..30 3.2.2.12 Test for tannins ……………………………………..……….…31 3.2.3 THIN LAYER CHROMATOGRAPHY ….…………………….….…...31 3.2.4 IR METHODOLOGY ……………………………..……………….…...31 3.2.5 PREPARATION OF SOLUTIONS OF EXTRACT ………………..…..32 3.2.6 PREPARATION OF MEDIA …………………………….…………..…32 3.2.6.1 Sterile Distilled Water …………………….……………………..32 3.2.6.2 Nutrient Agar …………………………………………….……...32 3.2.6.3 Nutrient Broth ……………….………………………………..…32 3.2.7 ANTIMICROBIAL ACTIVITY TESTS …………………….……….…33 3.2.7.1 Preparation of broth culture …………………………….…….…33 3.2.7.2 Preparation of nutrient agar culture ……………………………..33 3.2.8 ANTIOXIDANT ACTIVITY TESTS ……………………………….…....34 3.2.8.1 Preparation of solutions………………………………………….34 3.2.8.1 Total Phenol Assay …………………………………………...…35 3.2.8.2 Total Antioxidant Capacity Assay ………………………………35 3.2.8.3 Reducing Power …………………………………………………36 4. RESULTS AND DISCUSSION ……………………………………………...…38 4.1 RESULTS …………..…………………………………………………………...38 4.1.1 Results for phytochemical screening………………………………………38 4.1.2 Results for extraction………………………………………………………39 4.1.3 Results for thin layer chromatography……………………………………..40 4.1.4 Results for antibiotic activity tests………………………………………....40 4.1.5 Results for antioxidant activity tests…………………………………….…47 4.1.6 Results for the IR spectrophotometry…………………………………...…51 4.2 GENERAL DISCUSSIONS ………………………..…………………………...54 4.3 ANTIMICROBIAL ACTIVITY ……………………..…………………………56 4.4 ANTIOXIDANT ACTIVITY ……………………..……………………………60 5. CONCLUSIONS AND RECOMMENDATIONS………………………………62 5.1 CONCLUSIONS………………………………………………………………...62 5.2 RECOMMENDATIONS………………………………………………...…………62 References ……………………………………………………………………………....63 Appendix 1: Anova: Two-Factor Without Replication……………………………….…68 Appendix 2: MIC Exploratory plots……………………………………………………..69 Appendix 3: t-Tests……………………………………………………………………....70 Appendix 4: Correlation of MICs between test drugs……………….....………………..71 LIST OF TABLES Table 4.1.1: Results for phytochemical screening……………………………………….39 Table 4.1.2 Extraction yields………………………………………………………….…39 Table 4.1.3: Results for thin layer chromatography…………………………………….40 Table 4.1.4a: Antibiotic activity test results for ethanol extract…………………………41 Table 4.1.4b: Antibiotic activity test results for water extract………………………...…41 Table 4.1.4c: Antibiotic activity test results for ciprofloxacin…………………………..42 Table 4.1.4e: Minimum Inhibitory Concentrations (MIC) and Ratios…………………..46 Table 4.1.4f: Minimum Inhibitory Concentrations and Ratios…………………………..46 Table 4.1.5a: Total Phenolic Content test results………………………………………..47 Table 4.1.5b: Total antioxidant capacity test results………………………………….…48 Table 4.1.5c: Reducing Power test results for S. gratus and n-propylgallate…………....50 LIST OF FIGURES Figure 2.1: Chemical structures of some useful alkaloids ………………………………..8 Figure 2.2: Basic flavonoid rings