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THE EFFECT OF ENVIRONMENTAL CONDITIONS ON THE ANTIBACTERIAL PROPERTIES OF CALENDULA OFFICINALIS A Dissertation submitted to The Faculty of Health Sciences, University of Johannesburg, as partial fulfilment of the Master’s Degree in Technology: Homoeopathy by: Jan-Nita Wilken (Student number 920200244) Supervisor: ________________________ ___________________ Dr R. Razlog Date M.Tech Hom (TWR) BMPD (TWR) Co-supervisor: ________________________ ____________________ Dr N. Niemann Date Ph.D Botany (UJ) DECLARATION I hereby declare that this dissertation is my own, unaided work. It is being submitted for the Master’s Degree of Technology: Homoeopathy at the University of Johannesburg, Johannesburg. It has not been submitted before for any degree or examination at any other university. _________________________ Signature of Jan-Nita Wilken ______ day of _________________ 20____ i ABSTRACT When a health practitioner prescribes medication to a patient, one of the concerns is that the quality of the medication taken by the patient remains unaltered throughout the treatment period. Calendula officinalis is a well-researched herb, known for its antimicrobial efficacy and was therefore used in this study to establish whether its properties changed when exposed to different environmental conditions. An herbal extract from Mediherb and a homeopathic mother tincture were used in this study. Although both samples were prepared from Calendula officinalis, they were extracted differently. The Mediherb herbal extract was a 1:2 dilution of the plant material (flowers) with a cold percolation method with 90% ethanol; whereas the homeopathic mother tincture was extracted by mincing the fresh over-ground parts of the plant during flowering time and adding 62% ethanol to the plant material. It was then stored for 10 days and filtered to obtain the homeopathic mother tincture. The stock samples and dilutions of the samples were then exposed to different environmental conditions (Control – UJ Dispensary, Direct Sunlight, Kitchen Scenario, Office Scenario and Decreased Temperature) and the antibacterial properties were evaluated with the Kirkby Bauer Disc Diffusion Method and the broth dilution minimum inhibitory concentration test. Some samples were also separated and analysed with Gas Chromatography Mass Spectrometry. Statistical analysis of the Kirkby Bauer Disc Diffusion results was done and compared accordingly with the Mann Whitney U-Test and the kurtosis and skewness values. A comparison of the results for the broth dilution minimum inhibitory concentration test and the Gas Chromatography Mass Spectrometry was also done and reported on. The test results showed that the samples exposed to the different environmental conditions changed in their activity as well as their composition. The testing on the diluted samples were halted due to the inconsistency of the results and a suggestion was made to allow further research on this topic. Amber glass bottles showed overall better protection against environmental conditions than blue glass bottles. The samples in the control environment, the UJ Dispensary, with temperature constant at an average of 21ºC and with minimal exposure to sunlight, external lighting and electromagnetic waves and radiation showed stability and would be proposed as the best storage environment for herbal extracts and homeopathic mother tinctures. ii Dedicated in memory of the late Dr Bernard Coetzee, my parents, family and friends for all their love, support and care. iii ACKNOWLEDGEMENTS I would like to thank the following individuals for their assistance, support, guidance and care throughout this research study: Dr Radmila Razlog for being my supervisor and overlooking the whole process. Thank you for all your guidance, motivation, care and support. Thank you for always being willing to help even while you were on leave. Dr Nicolette Niemann for taking on the responsibility of overseeing the laboratory aspects of the study. Thank you for all your time, guidance, support and patience during this time. Thank you for all the input into the study. Dr Derek Ndinteh for your input and assistance with the Gas Chromatography Mass Spectrometry. The staff members and lab assistants from the Biotechnology Department for all your help and guidance. Thank you to my parents, family, friends and work colleagues for their motivation, love and care during my years of study, and especially the final stretch with the research. iv TABLE OF CONTENTS DECLARATION . ............................................................................................................... I ABSTRACT ......................................................................................................................... II DEDICATION ..................................................................................................................... III ACKNOWLEDGEMENTS ................................................................................................ IV TABLE OF CONTENTS .................................................................................................... V LIST OF TABLES AND FIGURES .................................................................................. VIII LIST OF APPENDICES ..................................................................................................... XI CHAPTER 1: INTRODUCTION ...................................................................................... 1 1.1 Problem statement ......................................................................................................... 1 1.2 Aim .................................................................................................................................. 1 1.3 Importance of study ...................................................................................................... 1 1.4 Expected outcomes of study .......................................................................................... 2 CHAPTER 2: LITERATURE REVIEW .......................................................................... 4 2.1 Calendula officinalis ...................................................................................................... 4 2.1.1 C. officinalis active compounds .................................................................... 5 2.1.1.1 Carotenoids ..................................................................................... 5 2.1.1.2 Phenolic compounds ....................................................................... 6 2.1.1.3 Saponins ........................................................................................... 6 2.1.2 Medicinal action of C. officinalis ................................................................. 6 2.1.3 C.officinalis Extract ...................................................................................... 7 2.1.4 C. officinalis Mother Tincture ..................................................................... 7 2.1.5 C. officinalis 3:7 Dilution ............................................................................. 8 2.1.6 C. officinalis 1:10 Dilution ........................................................................... 8 2.1.7 Antimicrobial Properties of C. officinalis ................................................... 8 2.2 Staphylococcus aureus (S. aureus) ................................................................................ 9 2.3 Kirkby Bauer Disc Diffusion Method .......................................................................... 10 2.4 Minimum Inhibitory Concentration (MIC) ................................................................ 11 2.5 Gas Chromatography Mass Spectrometry (GC/MS) ................................................. 12 2.6 Storage of Remedies ...................................................................................................... 13 v CHAPTER 3: METHODOLOGY 3.1 Facilities Utilised ............................................................................................................ 15 3.2 Procurement ................................................................................................................... 15 3.3 Staphylococcus aureus ................................................................................................... 16 3.4 Research Design ............................................................................................................. 16 3.5 Sample Preparation ....................................................................................................... 16 3.5.1 Mediherb Extract ........................................................................................... 16 3.5.2 Homeopathic Mother