The Effect of Environmental Conditions on the Antibacterial Properties of Calendula Officinalis

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How to cite this thesis

Surname, Initial(s). (2012) Title of the thesis or dissertation. PhD. (Chemistry)/ M.Sc. (Physics)/ M.A. (Philosophy)/M.Com. (Finance) etc. [Unpublished]: University of Johannesburg. Retrieved from: https://ujcontent.uj.ac.za/vital/access/manager/Index?site_name=Research%20Output (Accessed: Date). 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____

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.

Dedicated in memory of the late Dr Bernard Coetzee, my parents, family and friends for all their love, support and care.

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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.

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

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 Tincture...... 16 3.5.3 Dilutions ...... 16 3.5.3.1 D1 Dilutions ...... 16 3.5.3.2 1:10 Dilutions ...... 17 3.6 Petri dishes, paper discs and bacterial smear preparation ...... 17 3.7 Measurements of Zones of Inhibition ...... 17 3.8 Exposure Factors ...... 18 3.8.1 UJ Dispensary ...... 18 3.8.2 Direct Sunlight ...... 19 3.8.3 Kitchen Scenario ...... 19 3.8.4 Office Scenario ...... 19 3.8.5 Decreased Temperature ...... 19 3.9 Further procedures on diluted samples ...... 19 3.10 Minimum Inhibitory Concentration (MIC) ...... 20 3.11 Gas Chromatography Mass Spectrometry (GC/MS) ...... 21 3.12 Data and Statistical Ananlysis ...... 22 3.12.1 Kirkby Bauer Disc Diffusion Test ...... 22 3.12.2 Minimum Inhibitory Concentration ...... 22 3.12.3 Gas Chromatography Mass Spectrometry ...... 23

CHAPTER 4: RESULTS 4.1 Overview ...... 24 4.2 Representation of agar plates ...... 24 4.3 Zone of Inhibition of Mediherb Herbal Extract in blue bottles ...... 26 4.4 Zone of Inhibition of Mediherb Herbal Extract in amber bottles ...... 32 4.5 Zone of Inhibition of Homeopathic Mother Tincture in blue bottles ...... 37 4.6 Zone of Inhibition of Homeopathic Mother Tincture in amber bottles ...... 43 4.7 Zones of Inhibition comparison of the Mediherb Herbal Extract ...... 48 vi

4.8 Zones of Inhibition comparison of the Homeopathic Mother Tincture ...... 51 4.9 Minimum Inhibitory Concentration (MIC) ...... 55 4.10 Gas Chromatography Mass Spectrometry (GC/MS) ...... 57

CHAPTER 5: DISCUSSION 5.1 Overview ...... 62 5.2 Zones of Inhibition (Kirkby Bauer Method) ...... 62 5.2.1 Zones of Inhibition of Herbal Extract in blue bottles ...... 62 5.2.2 Zones of Inhibition of Herbal Extract in amber bottles ...... 64 5.2.3 Zones of Inhibition of Mother Tincture in blue bottles ...... 64 5.2.4 Zones of Inhibition of Mother Tincture in amber bottles ...... 65 5.2.5 Herbal Extracts Zones of Inhibition Comparison ...... 66 5.2.6 Mother Tinctures Zones of Inhibition Comparison ...... 67 5.2.7 Dilutions and further testing ...... 68 5.3 Minimum Inhibitory Concentration (MIC) ...... 68 5.3.1 MIC Comparison of the Mediherb Herbal Extract ...... 69 5.3.2 MIC Comparison for Homeopathic Mother Tincture ...... 70 5.4 Correlation between Kirkby Bauer Disc Method and MIC ...... 71 5.5 Gas Chromatography Mass Spectrometry (GC/MS) ...... 71

CHAPTER 6: CONCLUSION ...... 74 6.1 Conclusion ...... 74 6.2 Recommendations ...... 75

REFERENCES ...... 76

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LIST OF TABLES AND FIGURES

TABLES 4.1 Comparison of the median inhibition zones for each exposure group in blue bottles .... 27 4.2 Normality of data for each extract exposure group in blue bottles ...... 27 4.3 Comparison of the median inhibition zones for each exposure group in amber bottles . 32 4.4 Normality of data for each extract exposure group in amber bottles ...... 33 4.5 Comparison of the median inhibition zones for each exposure group in blue bottles .... 37 4.6 Normality of data for each mother tincture exposure group in blue bottles ...... 38 4.7 Comparison of the median inhibition zones for each exposure group in amber bottles . 43 4.8 Normality of data for each mother tincture exposure group in amber bottles ...... 44 4.9 Results for MIC for samples tested ...... 56 4.10 Comparison of the compounds separated on the GC/MS for the herbal extract and the homeopathic mother tincture ...... 59 4.11 Comparison of the diluted (3:7) extract samples with different solvents ...... 60 4.12 Comparison of the compounds of the exposed extract samples in blue bottles with the unexposed extract as control...... 61

FIGURES 2.1 Schematic drawing of Calendula officinalis ...... 4 2.2 Gram-positive Staphylococcus aureus under a light microscope ...... 9 2.3 Electron micrograph of Staphylococcus aureus ...... 10 2.4 Schematic representation of how measurements are done with the Kirkby Bauer Disc Diffusion method ...... 11 2.5 96 well microtitre plate used during broth dilution MIC testing ...... 12 3.1 Microscope slide to confirm S. aureus for each new bacteria stock solution ...... 18 3.2 Dilutions of samples (1:1) with Mueller Hinton Nutrient Broth ...... 20 4.1 Herbal extract sample agar plate for a Mediherb herbal extract ...... 25 4.2 Herbal extract sample diluted 3:7 with distilled water ...... 25 4.3 Herbal extract sample diluted 3:7 with DMSO ...... 26 4.4 Mean inhibition diameters for the extract samples (blue bottles) ...... 28 4.5 Inhibition diameters for extract samples in blue bottles (UJ Dispensary) ...... 29 4.6 Inhibition diameters for extract samples in blue bottles (Direct Sunlight)...... 29 4.7 Inhibition diameters for extract samples in blue bottles (Kitchen Scenario) ...... 30 viii

4.8 Inhibition diameters for extract samples in blue bottles (Office Scenario) ...... 31 4.9 Inhibition diameters for extract samples in blue bottles (Decreased Temperature) ...... 31 4.10 Mean inhibition diameters for the extract sample (amber bottles) ...... 33 4.11 Inhibition diameters for extract samples in amber bottles (UJ Dispensary)...... 34 4.12 Inhibition diameters for extract samples in amber bottles (Direct Sunlight) ...... 35 4.13 Inhibition diameters for extract samples in amber bottles (Kitchen Scenario) ...... 35 4.14 Inhibition diameters for extract samples in amber bottles (Office Scenario) ...... 36 4.15 Inhibition diameters for extract samples in amber bottles (Decreased Temperature) ... 37 4.16 Mean inhibition diameters for the mother tincture samples (blue bottles) ...... 39 4.17 Inhibition diameters for mother tincture samples in blue bottles (UJ Dispensary) ...... 40 4.18 Inhibition diameters for mother tincture samples in blue bottles (Direct Sunlight) ...... 40 4.19 Inhibition diameters for mother tincture samples in blue bottles (Kitchen Scenario) ... 41 4.20 Inhibition diameters for mother tincture samples in blue bottles (Office Scenario) ..... 42 4.21 Inhibition diameters for mother tincture samples in blue bottles (Decreased Temperature) ...... 42 4.22 Mean inhibition diameters for the mother tincture samples (amber bottles) ...... 44 4.23 Inhibition diameters for mother tincture samples in amber bottles (UJ Dispensary) .... 45 4.24 Inhibition diameters for mother tincture samples in amber bottles (Direct Sunlight) ... 46 4.25 Inhibition diameters for mother tincture samples in amber bottles (Kitchen Scenario) 46 4.26 Inhibition diameters for mother tincture samples in amber bottles (Office Scenario) .. 47 4.27 Inhibition diameters for mother tincture samples in amber bottles (Decreased Temperature) ...... 48 4.28 Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (UJ Dispensary) ...... 48 4.29 Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Direct Sunlight) ...... 49 4.30 Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Kitchen Scenario) ...... 49 4.31 Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Office Scenario) ...... 50 4.32 Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Decreased Temperature) ...... 51 4.33 Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (UJ Dispensary) ...... 51 4.34 Mean inhibition diameters BE and AE for mother tincture samples in blue and amber ix

bottles (Direct Sunlight) ...... 52

4.35 Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (Kitchen Scenario) ...... 53 4.36 Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (Office Scenario) ...... 53 4.37 Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (Decreased Temperature) ...... 54 4.38 Microtiter Plate number 3 ...... 55 4.39 GC/MS graph for the separation of a Mediherb herbal extract sample that was not exposed to any environmental conditions (Control)...... 57 4.40 GC/MS graph for the separation of a homeopathic mother tincture sample that was not exposed to any environmental conditions (Control)...... 58

LIST OF APPENDICES

APPENDIX 1: Permission letter ...... 82 APPENDIX 2: Certificate of Analysis: Nutrient Agar ...... 83 APPENDIX 3: Certificate of Analysis: Nutrient Broth ...... 85 APPENDIX 4: Certificate of Analysis: Mediherb Herbal Extract ...... 88 APPENDIX 5: Certificate of Analysis: Homeopathic Mother Tincture ...... 89 APPENDIX 6: Breakdown of samples ...... 90 APPENDIX 7: Breakdown of samples ...... 94 APPENDIX 8: Table used for noting measurements ...... 95 APPENDIX 9: Pilot Study 1 and 2 ...... 96 APPENDIX 10: Exposure factor times and details ...... 100 APPENDIX 11: MIC sample size and details ...... 102 APPENDIX 12: GC/MS sample size and details ...... 103 APPENDIX 13: Raw data for all tests ...... 104

CHAPTER 1: INTRODUCTION

1.1 Problem statement When homeopathic medication is dispensed to a patient, the practitioner’s expectation is that the patient will have the full benefit of high quality medication. There is, however, currently no clear evidence of the change in the quality of herbal extracts, homeopathic mother tinctures and low potency remedies when stored, dispensed to patients or when exposed to changes in environmental conditions. The preservative properties of ethanol when diluted for homeopathic use is also a concern. Typical environmental changes include the timeframe from when the remedy is prepared to when the patient takes the remedy; storage of the remedy, such as a patient carrying the remedy in a handbag for 6 weeks or other different possible degrading factors such as sunlight, radiation and temperature changes while the remedy is stored in a dispensary or by the patient. There are a number of preventative measures in place to avoid degradation of the remedy but little associated research of the remedies’ resultant efficacy. These preventative measures include using amber glass bottles for storage and warnings not to expose the remedy to strong odours, sunlight, radiation, dust, smoke and moisture. With regards to the preparation of dilutions from extracts and mother tinctures to homeopathic first dilutions, there is little consistency with the methods used amongst homeopaths and this may impact on the efficacy of the remedies.

1.2 Aim The aim of this study was to determine the effect of environmental conditions on the antibacterial properties of various Calendula officinalis (C. officinalis) preparations (namely the herbal extract, homeopathic mother tincture, D1 homeopathic preparation of both the herbal extract and mother tincture, and a 1:10 dilution of both the herbal extract and homeopathic mother tincture), by using the Kirby-Bauer Disc Diffusion Susceptibility Test. Subsequently, after the results were collected for the study, it was decided to use Gas Chromatography Mass Spectrometry and Minimum Inhibitory Concentration testing to confirm the findings and give a possible explanation for the results obtained.

1.3 Importance of the study Based on the outcomes of this study, guidelines can be formulated on how to store various complementary medicinal preparations including homeopathic remedies as well as herbal extracts, in order to prevent degradation of the efficacy of the medication. It can also be established whether exposure to environmental conditions compromises the quality of the remedy. The use of different solvents and concentrations of ethanol for dilutions, as often used by homeopaths, is also under

1 review. This will allow for a better understanding when compounding and dispensing remedies and mixtures for patients.

A number of experimental studies have been conducted on herbal extracts using direct extraction processes to ensure that all the active ingredients are extracted from the plant material. With homeopathic mother tinctures though, a different technique is used, and not the same quality and volume of extraction of the active ingredients is obtained and maintained as a standardised process of extraction.

In this study, C. officinalis homeopathic mother tincture and the herbal extract were exposed to some tests, for comparison. The homeopathic mother tincture was compared to the herbal extract in order to establish the degree of difference in the active ingredients and antibacterial properties that may exist.

The separation of C. officinalis samples on a two dimensional gas chromatography along with structure elucidation via mass spectrophotometry determined compounds that might not have been evident with standard separation techniques, and allowed for comparisons to be made between the herbal extract and the homeopathic mother tincture.

The Minimum Inhibitory Concentration test for the exposed samples, allowed confirmation of the results obtained during the Kirkby Bauer Disc Diffusion testing.

1.4 Expected outcomes of study Literature suggests that environmental conditions, especially direct sunlight and temperature changes can denature medication. Therefore it is expected that the antibacterial properties of C. officinalis with exposure to environmental conditions would either decrease or remain the same over time.

Although there are guidelines in the various Homeopathic Pharmacopoeia on how to dilute herbal extracts and homeopathic mother tinctures, the science behind it is not clear or consistent. Some clarity should be obtained about diluting herbal extracts and mother tinctures with different ethanol concentrations, polar and nonpolar solvents that could aid in the formulation of guidelines to homeopaths and phytotherapists alike on the compounding and dispensing of medication and creating awareness of precautionary measures when compounding their own mixtures (Texas Tech University Health Sciences Center, 2011). 2

Due to the expected higher concentrations of active ingredients of the C. officinalis herbal extract against the expected lower concentration of active ingredients of the homeopathic mother tincture, it can be assumed that the antibacterial properties of the herbal extract would be higher than the homeopathic mother tincture. These differences are due to the different extraction methods (Roopashree et al., 2008).

The results from the two dimensional gas chromatography with mass spectrophotometry would give some insight in the differences between the herbal extract and the mother tincture. Possible changes in the active compounds before and after exposure should also give some insight on the antibacterial properties before and after exposure to the different environmental conditions.

CHAPTER 2: LITERATURE REVIEW

2.1 Calendula officinalis C. officinalis (common name marigold) is a member of the Asteraceae (also referred to as the Compositae) family (Efstratiou et al., 2012). The marigold is identified by its broadly lance-shaped leaves and 4-7cm wide flower heads (Figure 2.1 below). The flower heads contains orange-yellow florets that can take a tubular form (Czygan et al., 2004) and its stems can reach up to 60cm (Ehrlich, 2013). The plant flowers from spring to winter. C. officinalis have been used since the 12th century (Ehrlich, 2013). During the Middle Ages it was grown and harvested for its medicinal properties as well as a food source. Its flower petals were also used as a colour dye for fabrics, foods and cosmetics (Efstratiou et al., 2012). The common and ordinary deep orange flowered type of C. officinalis is considered to have the highest medicinal implication (Bissa and Bohra 2011). Although it is native to the Mediterranean countries, it is now found grown worldwide. The type of marigold growing outside the Mediterranean countries is not the C. officinalis that is used for medicinal properties (Ehrlich, 2013). The ornamental marigolds which are usually planted in vegetable gardens are from the Tagets genus (Medline Plus, 2015).

Figure 2.1: Schematic drawing of Calendula officinalis (Whelan, 2011)

2.1.1 C. officinalis active compounds C. officinalis has various active compounds including carotenoids (lycopene, lutein, flavoxanthin, ribixanthin, β-carotene and γ-carotene); phenolic compounds (flavonoids and phenolic acids); saponins and in lesser concentrations, proteins, amino acids, saturated hydrocarbons, vitamin C, mineral substances and soluble poly-carbohydrates that play a vital role in the healing of tissues (Efstratiou et al., 2012; Butnariu and Coradini, 2012). When the C. officinalis flowers are picked, a sticky substance is present due to the resinous bracts that form the base of the flower head. Resin is an important compound for the healing and antimicrobial properties of C. officinalis. (Blankespoor, 2012).

Triterpenic alcohols and polyunsaturated fatty acids such as calendic acid (which is a polyunsaturated fatty acid) is also contained in C. officinalis. These specifically allow for the anti- inflammatory properties of C. officinalis. The soluble poly-carbohydrates play a vital role in the healing of tissues. Of lesser content are the proteins, amino acids, saturated hydrocarbons, vitamin C and mineral substances (Butnariu and Coradini, 2012).

A study done by Roopashree et al. (2008) showed that different extraction solvents yield different active ingredients. An ethanol extraction of the flowers of C. officinalis showed glycosides, saponins, triterpenes, diterpenes and flavonoids. The extraction was done by powdering the flowers of C. officinalis extracted in a soxhlet apparatus. Different solvents such as ether and methanol were also used and compared with the ethanol-extracted C. officinalis. Mathur and Goyal (2011) also extracted C. officinalis with different solvents, but showed the different active ingredients according to what was found in the leaves, stem, flowers and roots respectively. They found that saponins, terpenoids, flavonoids, tritepenoids and steroids were found in all parts. Alkaloids, glycosides, phenols and tannins were found in lesser parts of the plant.

2.1.1.1 Carotenoids Carotenoids are pigments that are synthesized by plants, algae or photosynthetic bacteria. These pigments are the source of the colour of the plant or flowers; including yellow, orange and red colours. Some carotenoids such as the α-carotenoids, β-carotenoids and β-cryptoxanthin can be converted to vitamin A by the human body, by converting the carotenoid into retinol. Carotenoids can be divided into two main classes: carotenes that include α-carotene, β-carotene and lycopene; and xanthophylls that include β-cryptoxanthin, lutein and zeaxanthin. Carotenoids also play a role in the immune system of the human body where it stimulates natural killer cells (Higdon et al.,

2009). When carotenoids are converted to active Vitamin A, it is a good antioxidant and antimicrobial substance (Efstratiou et al., 2012)

2.1.1.2 Phenolic compounds Flavonoids are divided in six subtypes including chalcones, flavones, isoflavonoids, flavanones, anthoxanthins and anthocyanins. Flavonoids, like carotenoids, allow for the colour pigment of the plant or flower. They do, however, have a more important role where they are required for ultra violet filtration, cell cycle inhibition and chemical messengers. Flavonoids are antioxidants and exhibit antiviral, anticancer, anti-inflammatory and anti-allergic functions. They are also suggested to have a positive impact on cardiac health, including lowering cholesterol (Robertson, 2014).

Phenolic acids can play a role against oxidative damages as in cardiovascular diseases and cancers. In the plant, phenolic compounds are needed for reproduction and growth as well as defending the plant against pathogens (Sahelian, 2014).

A number of phenolic and/or flavonoid compounds have been identified by High Performance Liquid Chromatography (HPLC) in the leaves and flowers of C. officinalis. In the leaves gallic acid, scopoletin-7-O-glucoside and quercetin-3-O-glucoside have been identified. In the flowers, gallic acid, quercetin-3-O-glucoside, rutin and isorhamnetin-3-O-glucoside have been identified. During the same study it was concluded that the flavonoids in the C. officinalis allow for antimicrobial action (Rigane et al., 2013).

2.1.1.3 Saponins Saponins have strong surfactant properties that give them a soapy character. They can play a role in haemolytic, expectorate, anti-inflammatory and immune-stimulating activities when taken internally (Sahelian, 2014).

2.1.2 Medicinal actions of C. officinalis The actions of C. officinalis are documented to range from healing, anti-inflammatory, haemostatic to an antimicrobial, antiviral and antifungal (Bone, 2003). Murphy (2010) refers to C. officinalis as the “great herbal anti-septic” as it protects wounds from putrefaction. It does not cause any irritation or sensitivity to the wound or surrounding skin and assists with local pain management (Bone, 2003). The possible mode of action to assist with wound healing is to increase the blood flow to the area, allowing higher oxygen supply to the affected tissues (Ehrlich, 2013).

C. officinalis can be used for various clinical indications following the evaluation of the patient’s symptoms and signs that they present with which include: inflammation of the oral and pharyngeal mucosa, gastric and duodenal ulcers (promoting healthy mucosal linings in the upper gastrointestinal tract), inflamed lymph nodes (promoting healthy lymphatic draining system), dysmenorrhoea or other spasmodic conditions, topical treatment of burns supporting tissue repair), leg ulcers, poor venous circulation (also promoting vascular integrity and function), haemorrhoids, controlling bleeding, eczema, acne, vaginal discharges (Bone, 2003; Mediherb, 2013), treatment of constipation and assisting with detoxification of the gallbladder (Butnariu and Coradini, 2012). Some studies have been done on breast cancer patients where C. officinalis assists with the prevention and treatment of dermatitis and related skin conditions during chemotherapy while treating breast cancer (Ehrlich, 2013).

2.1.3 C. officinalis extract Van Wyk and Wink (2004) define a liquid herbal extract as a mixture of chemical compounds extracted from plant material by using an organic solvent such as ethanol or water. The extract used in this study was a 1:2 dilution of the plant material, extracted by using a cold percolation method with 90% ethanol. Ethanol is used to ensure that the full phytochemical range of the plant is extracted. Ethanol was used for the extraction as it is relatively safe for humans, as the human liver is conditioned against the small amounts of ethanol found in fruits and naturally fermented foods (Mediherb, 2013).

2.1.4 C. officinalis mother tincture Homeopathic practitioners usually make use of the mother tincture of a plant instead of a herbal extract. Depending on the company manufacturing these, it is mainly extracted according to the method defined in the German Homeopathic Pharmacopoeia or the French Homeopathic Pharmacopoeia. For the purpose of this study, the mother tincture obtained from CoMed Health was prepared according to the German Homeopathic Pharmacopoeia, HAB method 3A. In this method, the plant materials are minced and added to 62% ethanol. It is then stored in airtight containers for 10 days while swirled at regular intervals. The temperature during this period may not exceed 20ºC. The mixture is then filtered, and the liquid portion is termed the mother tincture of the plant. For identification processes, the C. officinalis mother tincture is a yellow-green to brown-green liquid and it has a slight aromatic odour and mild spicy taste (German Homeopathic Pharmacopoeia, 2001).

2.1.5 C. officinalis 3:7 dilution (D1 according to German Homeopathic Pharmacopoeia) The mother tincture can then be diluted to a D1 (first decimal dilution), which would be the first homeopathic dilution (“potency”) by adding three parts of the mother tincture to 7 parts 62% ethanol, and succussing it ten times (German Homeopathic Pharmacopoeia, 2001). Succussion is a technique where the bottle with its contents is powerfully struck down on a surface, such as on a leather-bound book. This mechanical process is used in the “potentization” of remedies which refers to the different strengths (potencies) of a remedy (Banerjee, 2011). For the purpose of this study, the C. officinalis extract was also diluted according to this method.

2.1.6 C. officinalis 1:10 dilution (for purposes of the study) A 1:10 dilution is usually prepared by homeopaths, by adding 1 part of the extract or the mother tincture to 9 parts of ethanol (20% and 62% respectively). When preparing higher potencies from mother tinctures, these dilutions are not succussed. This method, however, is not documented in the pharmacopoeia and used incorrectly by homeopaths for ease of preparation of higher potencies. Therefore efficacy of the preparations may be compromised (German Homeopathic Pharmacopoeia: 2001).

2.1.7 Antimicrobial properties of C. officinalis In a study done by Mathur and Goyal (2011), a high focus was placed on the comparison of the antibacterial properties of the different parts of the C. officinalis plant; namely the leaves, stems, roots and flowers. This was done by extracting the C. officinalis samples of the different parts of the plant with 70% ethanol for 24 hours by using the soxhlet method. The soxhlet extraction method consists of a flask and condenser device to allow continuous extraction of alcohol and ether soluble materials (Parker, 2003). Different microbial organisms, bacterial and fungal, were used in the study including Staphylococcus aureus. All the different C. officinalis samples inhibited microbial properties on all the organisms. It was concluded that the leaves, stems, roots and flowers have variable antibacterial properties, with the flowers and stems being more effective than the roots and leaves (Mathur and Goyal, 2011).

A study to compare the antibacterial activities of C. officinalis with two other herbal extracts, showed that C. officinalis exhibited antibacterial properties against Staphylococcus aureus. The study used C. officinalis dried flowers extracted with ethanol, methanol, petroleum-ether and distilled water. The extractions with distilled water showed better antibacterial efficacy than the extractions with the other solutions. Samples of the mentioned plants were all extracted to a concentration of 20mg/ml. When the minimum inhibitory concentration (MIC) was tested, it 8 showed that a minimum concentration of 32mg/ml to 64mg/ml was needed to have an inhibitory effect on Staphylococcus aureus when extracted with ethanol (Roopashree et al., 2008).

In a further study, the flowers of the C. officinalis plant were used and the antibacterial efficacy was tested on a number of clinical bacteria, including Staphylococcus aureus. The disc diffusion method was used to measure the antibacterial efficacy. Ciprofloxacin was compared with the ethanol and methanol extractions. It was concluded that both methanol and ethanol extractions possessed antibacterial properties towards various bacteria (Efstratiou et al., 2012). Chakraborthy (2008) showed in his study on the antimicrobial activity of the leaves of the C. officinalis plant that C. officinalis leaves shows antimicrobial activity against Gram-positive and Gram-negative bacteria.

2.2 Staphylococcus aureus (S. aureus) S. aureus, which is a Gram-positive bacteria, is mainly found in the nasal passages of the human body, but can also be found on the skin, oral cavity and the gastrointestinal tract. It can cause serious conditions such as pneumonia, endocarditis and osteomyelitis. Abscess formation is also common with this bacterial infection and is often implicated in infections of wounds (Todar, 2012). S. aureus infections have become more common and serious in recent years; a reason to support this might be the continued increase in antibiotic resistance (Ganesh et al., 2008).

When inoculated on an enriched medium, S. aureus forms yellow colonies that grow at temperatures between 15-45ºC. S. aureus grows easily by aerobic respiration or by fermentation and is easily identifiable (Todar, 2012).

Figure 2.2: Gram-positive Staphylococcus aureus under a light microscope (Batzing, 2002).

Figure 2.3: Electron micrograph of Staphylococcus aureus (Batzing, 2002).

S. aureus, staphylococci bacteria, meaning they are in the shape of round spherical cells that clusters together to take on clumps of grape-like formations (Figures 2.2 and 2.3 above). These formations are irregular and three dimensional. Being a Gram-positive bacteria, the cells have a thick peptidoglycan layer that makes it easy to be treated with antibiotics as the antibiotics interfere with the peptidoglycan biosynthesis. The peptidoglycan layer is close to the cell surface making treatment successful. Gram-negative bacteria on the other hand have a thinner peptidoglycan layer that lies under the outer membrane and acts as a barrier to prevent the antibiotic treatment from reaching the inner part of the cell (Batzing, 2002).

S. aureus is resilient and easily found on the skin and nasal passages of the human body. They can easily be grown on a nutrient medium and can be easily identified by microscopic and staining techniques (Todar, 2012). Therefore S. aureus was used in this study.

2.3 Kirkby-Bauer Disc Diffusion Method The aim of the Kirby-Bauer Disc Diffusion method is to determine the sensitivity of bacteria to a substance. The microorganism is inoculated on a Mueller-Hinton agar plate in the presence of the testing substance impregnated filter paper discs. A positive result for this test is a rounded area of nonbacterial growth around the impregnated discs (as in Figure 2.4 below) showing antimicrobial activity. The non-bacterial growth area around the paper disc is referred to as the zone of inhibition (Hudzicki, 2009).

Figure 2.4: Schematic representation of how measurements are done with the Kirkby Bauer Disc Diffusion method (Marshall, 2011).

Although the Kirkby-Bauer Disc Diffusion Susceptibility test is a standardised test, the results still depend on the type of organism used, the type of antibacterial agent, a confluent growth of bacteria and Mueller Hinton agar being from a reliable source. It is also important to take the minimum inhibition concentration of the antibacterial agent into consideration as a false negative can be obtained if the incorrect concentration of the antibacterial agent is used. Another critical aspect is that the bacterial species chosen is one that grows easily and in a short duration (S. aureus is one of those species). The species should also not need a special nutrient base and should not require carbon dioxide or anaerobic incubation (Cheesbrough, 2006).

A study done by Roopashree et al. in 2008 showed that a C. officinalis extract had to have a minimum inhibition concentration of 32mg/ml to show antibacterial efficacy. In the study, C. officinalis flowers were extracted with ethanol and tested on S. aureus. In another study done by Chakraborthy in 2008, a minimum inhibition concentration of 13mg/ml was found when extracting dried leaves of C. officinalis with ethanol and testing it on S. aureus. The C. officinalis extract that is used during this experimental study has a concentration of 500mg/ml and is extracted from the flowers of the plant with 90% ethanol (Mediherb, 2013).

2.4 Minimum Inhibitory Concentration (MIC) MIC is the lowest concentration of a substance that will show antimicrobial action by showing the inhibition of visible growth of an organism after it has been incubated overnight. This method has an important use to determine the susceptibility of organisms to antimicrobial substances. It is also used to compare the susceptibility testing of the performance of other tests. In medicinal 11 laboratories, it is also used to distinguish between unusual resistances to a substance or to test drug-resistant bacteria (Andrews, 2001).

There are two methods in determining MIC: the first being agar dilution and the second broth dilution. With agar dilution, different concentrations of an antimicrobial substance is added to nutrient agar followed by the application of a standardized number of organisms to the surface of an agar plate. With a broth dilution, as done in this test, bacteria is inoculated into a liquid medium nutrient broth and added to different concentrations of the antimicrobial agent. Growth is allowed overnight and the MIC value is noted. The broth dilution is often done in a 96-well microtiter plate (Figure 2.5 below) and is only suitable for aerobic bacteria; in this study, S. aureus is used (Wiegand et al., 2008).

Figure 2.5: 96-well microtiter plate used during broth dilution MIC testing (Universal Medical Inc., 2015)

2.5 Gas Chromatography Mass Spectrometry (GC/MS) Chromatography is an analytical technique used to separate molecules based on the difference in their structure and composition. It mainly involves allowing the testing sample to move through a stationary phase. Due to the different affinities and interactions between the sample and the stationary phase, the molecules separate according to the interaction they have with the stationary phase. Samples with a strong interaction with the stationary phase will move slower through the phase than those components with a weaker interaction (Kupiec, 2004).

Gas chromatography (GC) is a very accurate and precise technique that is widely used. Usually the sample is introduced as a vapour onto the column. The solubility is dependent on the vapour 12 pressure of the component, which would in turn determine the affinity between the stationary phase and the compound. Due to the differences in the vapour pressures, the molecules continuously move between the mobile gas phase and the stationary phase. As soon as a molecule enters the gas phase, it is moved to the detector, therefore different molecules with different physical and chemical properties will arrive at the detector at different times. These different times are referred to as residence times (Prichard, 2003).

Mass Spectrometry (MS) electrically charges molecules, then send them through a magnetic field where the molecules are broken into charged particles with different charges. It then identifies the substances accordingly. Therefore, the GC separates the particles and the MS identifies them, making the combination of the two instruments of great scientific value (Douglas, 2010).

2.6 Storage of Remedies New, well cleansed or sterilised, colourless, neutral flint glass containers should be used to store extracts, mother tinctures and dilutions in a cool dry place. Mother tinctures may not undergo extreme temperature changes as this may cause sediment to form. The same applies to homeopathic remedies (Mandel and Mandel, 2002). Preservation of different homeopathic remedies is set out in the different Homeopathic Pharmacopoeias according to the reactivity of the remedies, but general rules have been set to standardise the preservation of homeopathic remedies. It is recommended that remedies are stored in well stoppered bottles, which are kept in boxes or drawers, protecting them from direct light. Remedies should not be exposed to extreme temperatures, and stored in a cool dry place. Remedies should not be kept close to anything that can affect the purity of the remedies, such as dust, strong odours, smoke, moisture, radiation, strong light or sunlight (Banerjee, 2011).

The Texas Tech University Health Sciences Center (2011) explains that when conventional chemically based medication is exposed to conditions such as heat, humidity and light, the medication may change chemical composition and may become ineffective. With high importance medications for life-threatening diseases such as diabetes, asthma or heart conditions, it is recommended that medications be kept at the recommended temperatures and conditions to retain maximum efficacy. These recommendations include keeping medication at room temperature (or in the fridge in the case of certain chemical antibiotics) and away from direct heat, light and moisture. With chemical or conventional medication this is important due to the composition and manufacturing of the chemicals.

Radiation by definition is the direct transmission of electromagnetic energy through a medium. The spectrum of energy waves is mainly divided into a non-ionising and an ionising radiation division, where non-ionising radiation is usually referred to as less-harmful and includes radio- waves, microwaves and infrared waves. Examples of ionising radiation includes ultraviolet rays, x-rays and gamma rays that would damage the DNA of cells (United States Environmental Protection Agency, 2013).

CHAPTER 3: METHODOLOGY

3.1 Facilities utilised The experimental part of the study, where the zones of inhibition were measured with relation to the antibacterial properties of C. officinalis and the minimum inhibition concentration (MIC) of the experimental samples were done under the supervision of Dr Nicolette Niemann (PhD Botany) from the Biotechnology and Food Technology Department, Faculty of Health Sciences, University of Johannesburg (Permission to use facilities in Appendix 1).

The GC/MS section of the experimental study was done under the guidance of Dr Derek Ndinteh (PhD Organic Chemistry / Medicinal Chemistry) from the Department of Applied Chemistry, Faculty of Sciences, University of Johannesburg.

3.2 Procurement Blank paper discs were purchased from Davies Diagnostics (Pty) Ltd (situated in Randburg, Johannesburg). The discs (6mm in diameter) were prepared in quantities of 50 discs per cartridge, placed in a sterile container. The discs were dispensed from the cartridge in a sterile manner with a forceps onto the agar plates. The 95% ethanol was purchased from Associated Chemical Enterprises (Pty) Ltd (situated in Crown Mines, Johannesburg).

Clinical Sciences Diagnostics CC (based in Glenvista, Johannesburg) supplied 65mm diameter sterile petri dishes and the nutrient agar (Appendix 2 – Certificate of Analysis) that is compatible with S. aureus. The nutrient agar has a reaction of 2.8% w/v aqueous solution at 25ºC and pH range of 7.20 to 7.60. Cultural characteristics are observed after an incubation at 35-37ºC for 18- 48 hours.

The Mueller Hinton (MH) nutrient broth was obtained from Merck (Pty) Ltd. The MH broth has a pH range of 7.3 +/- 0.1 at 25ºC and contains 300.0g/l dehydrated beef infusion, 17.5g/l casein hydrolysate and 1.5g/l starch.

Merck (Pty) Ltd supplied the nutrient broth (Appendix 3 – Certificate of Analysis). The composition of the broth was 1.0g/l, 2.0g/l, 5.0g/l and 8.0g/l. The certified pH of the broth is 7.1 (+/- 0.2) after autoclaving at 25ºC.

C. officinalis extract was purchased from CoMed Health, who is the South African distributor for Mediherb Pharmaceutics (Appendix 4 – Certificate of Analysis). The C. officinalis homeopathic mother tincture was obtained from Fusion Homeopathics who is a distributor of homeopathic mother tinctures (mostly obtained from Germany) in South Africa (Appendix 5 – Certificate of Analysis).

3.3 Staphylococcus aureus Staphylococcus aureus (ATCC 25923) stock was purchased from Davies Diagnostics (Pty) Ltd. Bacteria from the stock count was suspended into nutrient broth that was autoclaved before the addition of the bacteria. A smear of the liquid bacteria was then prepared, Gram staining was done and viewed under a microscope to identify the bacteria and ensure the purity thereof.

3.4 Study Design This research study utilised an experimental quantitative design.

3.5 Sample Preparation Each environmental exposure factor had four bottles in the group. Two bottles contained the herbal extract in a blue and an amber glass bottle, and the other two bottles contained the mother tincture in a blue and an amber glass bottle.

3.5.1 Mediherb Extract All the Mediherb C. officinalis samples (ten) were directly decanted from the Mediherb C. officinalis stock supply into the respective blue and amber coloured glass bottles. The samples were marked and stored in a polystyrene box.

3.5.2 Homeopathic Mother Tincture All the C. officinalis mother tincture samples (ten) were directly decanted from the C. officinalis mother tincture stock supply into the respective blue and amber coloured glass bottles. The samples were marked and stored in a polystyrene box.

3.5.3 Dilutions 3.5.3.1 D1 Dilutions The D1 dilutions were done with the Mediherb C. officinalis extract as well as with the C. officinalis mother tincture. The 62% ethanol, 20% ethanol and distilled water were used as solutions for the dilutions. Three parts of the Mediherb C. officinalis extract and the C. officinalis 16 mother tincture were used respectively, each with seven parts of the different dilution solutions (62% ethanol, 20% ethanol and distilled water).

3.5.3.2 1:10 Dilutions The 1:10 dilutions were done with the Mediherb C. officinalis extract as well as with the C. officinalis mother tincture. The 62% ethanol, 20% ethanol and distilled water were used as solutions for the dilutions. One part of the Mediherb C .officinalis extract and C. officinalis mother tincture was used respectively, each with nine parts of the different dilution solutions (62% ethanol, 20% ethanol and distilled water).

3.6 Petri dishes, paper discs and bacteria smear preparation A mixture of nutrient agar and nutrient broth was prepared as per the instructions set out by the manufacturers. 25ml of the mixture was pipetted into individual McCartney flasks and autoclaved for sterilisation. After autoclaving, the agar-broth mix in each flask was decanted into individual petri dishes. This was done to ensure that the depth of the agar was the same in all petri dishes, as inconsistency could cause deviation in results. Agar plates were left to cool and solidify, and stored in a cool dry place for no longer than 24 hours.

100µl of the liquid bacteria was pipetted onto each agar plate and spread using sterile techniques. Six plates were done at a time. Paper discs were placed on the bacteria-spread plates by using sterile techniques (five paper discs per petri dish). 7.5µl of the C. officinalis sample was impregnated on each individual paper disc, starting with petri dish one and ending with the sixth (therefore 30 discs). Each paper disc was left for 3 minutes, and then another 7.5µl was impregnated again on each disc. The petri dishes was then sealed with polyfilm and incubated for 24 hours at 37ºC (Appendix 6 shows original breakdown of samples and Appendix 7 shows the final breakdown of samples after the first two groups of diluted samples showed inconsistent results).

3.7 Measurements of Zones of Inhibition After the incubation period, the agar plates were removed and placed on a dark surface. The diameters of the zones of inhibition were measured by taking a horizontal and vertical measurement of each paper disc.

All the C. officinalis samples were then exposed to the different environmental conditions as indicated in section 3.7 below; after the exposure period, the same procedure (as set out in 3.5 and

3.6) was repeated. Measurements were taken and the “before” and “after” exposure zones of inhibition measurements were noted for comparison (Appendix 8 – sheet used for noting of results).

A microscope slide was prepared and the organism was identified after each new batch of S. aureus to ensure that there was no contamination by other organisms. Samples from the bacteria stock solution as well as bacteria from the agar plates that were incubated, were tested.

Figure 3.1: Microscope slide to confirm S. aureus for each new bacteria stock solution.

Two pilot studies were done prior to the study to ensure that the techniques utilised were appropriate and accurate (Appendix 9).

3.8 Exposure factors 3.8.1 UJ Dispensary The UJ Dispensary acted as the control exposure factor. Temperature was kept constant at an average of 21ºC. The samples were placed in a drawer away from any strong odours and direct light and exposure to radiation levels were minimal. The temperature inside the cupboard and the dispensary was noted for seven days during the exposure time (Appendix 10). The bottles remained in the same location for seven days and were not moved or shaken up.

3.8.2 Direct Sunlight The direct sunlight group samples were placed in direct sunlight for seven consecutive days. Daily exposure time was done from 8:00 to 17:00. A thermometer was placed in the same position as the samples and the temperatures of the day were noted (Appendix 10) as well as the date of the exposure (as reference to seasonal factors). When the samples were not exposed to direct sunlight (i.e. 17:00 to 8:00 the next morning), they were stored in a polystyrene cooler box to ensure minimum exposure to other sources.

3.8.3 Kitchen Scenario The samples that were placed in the kitchen scenario were placed next to a microwave oven that was intermittently active for one hour a day (between 8:00 and 17:00). The exposure times were noted (Appendix 10). From 17:00 to 8:00 the next morning, the samples were stored in a polystyrene cooler box to ensure minimum exposure to other sources.

3.8.4 Office Scenario The samples in the office scenario group were placed next to a Wi-Fi router that established constant connections with three laptops and two portable devices. The one computer screen also emitted light onto the sample during this time. No sunlight exposure or external synthetic lighting was present. The exposure was done for seven consecutive days for eight hours on each day. The temperature of the office was measured hourly and noted (Appendix 10). After the exposure time for the day, the samples were stored in a polystyrene cooler box to ensure minimum exposure to other sources.

3.8.5 Decreased Temperature For this group, the samples were placed in a household fridge for seven consecutive days and the temperature was measured hourly during the day (Appendix 10). Samples remained in the fridge overnight. No strong odours were present in the fridge. The average temperature in the fridge was 4ºC.

3.9 Further procedures on diluted samples With the C. officinalis samples that were diluted with the different solvents (distilled water, 20% ethanol and 62% ethanol), different zones of inhibition presented. The zones were not completely clear and the measurements were inconsistent. This raised a need for further investigation.

Because water and ethanol are considered polar solvents, DMSO (dimethylsulfoxide), which is a non-polar solvent was used in a further dilution. The same procedure as in 3.5 and 3.6 was conducted and the zones of inhibition were measured.

Following these results, it was decided to make use of Gas Chromatography Mass Spectrometry to establish whether there was a change in the active ingredients of C. officinalis extracts when dissolved with different solvents.

3.10 Minimum Inhibitory Concentration (MIC) The samples that were used to establish the zones of inhibition before exposure (BE) and after exposure (AE), were also used to establish whether there was a difference in the MIC after exposure. As a control, one sample of the herbal extract and mother tincture which had not been exposed to any environmental conditions, was used for comparison. The sample size comprised of the 20 samples (as used in the disc diffusion test) and two control samples (unexposed herbal extract and mother tincture) and details thereof contained in Appendix 11.

Each sample was diluted five times from the crude sample. Each dilution was done 1:1 with Mueller Hinton broth, thereby concluding six concentrations for each sample. The Mueller Hinton broth and the McCartney flasks used for the dilutions, were autoclaved to ensure sterility. The dilutions and filling of the microtiter plates were done in a sterile laminar flow cabinet.

Figure 3.2: Dilutions of samples (1:1) with Mueller Hinton Nutrient Broth

Sterile 96-well microtiter plates (12 wells horizontally and 8 wells vertically) were used for the test. Each sample (six concentrations) was tested in a five-fold replication; therefore two samples 20 were tested on one microtiter plate, marking them from the left to the right side of the plate (as per Appendix 11). The wells around the border of the plates were filled with 200µl sterile distilled water. This allowed for ten wells horizontally and six wells vertically.

The wells were loaded with the samples starting on the left of the microtiter plate, for the first five wells (five-fold replication), the first dilution of the sample was loaded. The second dilution, in the first five wells of the second row, was loaded. The subsequent dilutions were loaded in the remaining rows of wells below. The second set of samples were loaded in the wells on the right side, following the above pattern. 100µl of the sample was added to 100µl of S. aureus in Mueller Hinton broth (0.5 McFarland standard concentration). This changed the dilutions as in Figure 3.1; the crude solution in the first row changed to 50%, the 50% dilution to a 25% dilution, the 25% dilution to a 12.50% dilution, the 12.50% dilution to a 6.25% dilution, the 6.25% dilution to a 3.125% dilution and then the 3.125% dilution to a 1.5625% dilution.

The mother tincture’s concentration was not determined by the suppliers, therefore results was set as a percentage with the crude sample starting at 100% (50% after the bacteria was added). The Mediherb herbal extract was certified to have a concentration of 500mg/ml (Mediherb; 2013), therefore the first dilution in the microtiter plate was 250mg/ml.

After all the wells were filled, the microtiter plates were covered and incubated at 37ºC overnight. The next day the microtiter plates were taken out of the incubator and 10µl of a Resazurin sodium salt solution was added to each well, excluding those filled with distilled water. The plates were covered again and incubated for two hours at 37ºC.

After two hours the plates were removed and colour changes were noted and photographed.

3.11 Gas Chromatography Mass Spectrometry (GC/MS) For the GC/MS, 100µl of each sample (breakdown of samples in Appendix 12) was placed in individual vials; samples were dried and 1ml of 99% ethanol was added to the dried samples. Residue from the drying process were dissolved in the ethanol by rotation to have the vials ready for the GC/MS separation and analysis.

The GC/MS instrument used for the separation of the different C. officinalis samples was a GC- TOFMS system (Pegasus 4D, Leco Corp., St. Joseph, MI) and the extraction was done with chloroform (analytical grade). The GC/MS was equipped with an Rxi-5Sil (1,4-

21 bis(dimethylsiloxy)phenylene dimethyl polysiloxane; 30m x 200µm Restek (Bellefonte, PA) column with film thickness of 0.18µm. The oven program started off with a temperature of 50ºC for 30 seconds and then increased to 310ºC in increments of 10ºC per minute, and finally held at 300ºC for two minutes. The injection port’s temperature was set at 225ºC and the detector at 280ºC. A pulse split injection mode was used with a 10:1 split at 30 seconds. The carrier gas flow rate was 1ml.min-1. The signal-to-noise (S/N) was set at 100 and a baseline was obtained just above the level. The peaks obtained in the study were identified by comparison with the NIST 08 Mass Spectral Library. Peaks below 700 (as set out on the ChromaTOF program), was noted as unknown. The identification of the compounds were done by matching them with the MS library and they have not been confirmed by co-injection with authentic standards. A scan range of m/z 25 to 500 at the rate of 200Hz was used to confirm the mass fragment of the derivatives. The mass fragmentation patterns were identified by using ChromaTOF software, Version 4.22 (LECO Corp.). The GC/MS procedure was done in triplicate for all samples and the data noted was the average from the three independent tests.

3.12 Data and Statistical Analysis 3.12.1 Kirkby Bauer Disc Diffusion Test Horizontal and vertical diameter measurements were taken for each zone of inhibition. An average diameter (zone of inhibition) was calculated for each disc. Each sample had 30 discs (five discs on each petri dish, and six petri dishes for each sample). The results were statistically analysed according to the ANOVA two-way test (Van Staden, 2014). The range of the results were plotted on box-and-whisker graphs to visualise the range of the results and to visually compare the difference between the “Before Exposure” measurements and the “After Exposure” measurements. Bar graphs were used to visually determine the changes between the samples in the blue glass and amber glass bottles as well as between the exposure types.

The Mann-Whitney U-Test was used to establish whether the differences in the zones of inhibition were statistically significant by grading the p-value for each set of results. The skewness and kurtosis values were used to establish whether the results were evenly spread (Van Staden, 2014).

3.12.2 Minimum Inhibitory Concentration The Mediherb herbal extract MIC’s were compared with each other and the mother tincture MIC’s were compared with each other to establish the effect of the different environmental conditions and storage in the blue glass and amber glass bottles. The Mediherb herbal extract was certified to have a concentration of 500mg/ml, therefore the MIC’s could be given as specific percentages. 22

The mother tincture had no certified concentration, therefore the MIC’s were set as percentages, which was adequate for this study as the aim of the study was to establish whether there was any change after exposure to the different environmental conditions.

The control samples (both unexposed Mediherb herbal extract and mother tincture) were also compared to establish whether the type of extract and part of the plant used would have a different MIC.

3.12.3 Gas Chromatography Mass Spectrometry The five samples that were exposed to different environmental conditions were separated on the GC/MS and compared with the separated herbal extract that was not exposed. The unexposed herbal extract was used as a control and only the most abundant 25 compounds were used for comparison. The other samples were also compared to the control. The main aim of the GC/MS results was to establish the extent of the change on the compounds of the samples after dilution with different solvents and after exposure to different environmental conditions.

CHAPTER 4: RESULTS

4.1 Overview C. officinalis samples (the herbal extracts, homeopathic mother tinctures, 3:7 dilutions (D1) and 1:10 dilutions respectively) were impregnated on paper discs and placed on agar plates spread with S. aureus bacteria that was suspended in liquid-medium broth. The dilutions were done with distilled water, 20% ethanol and 62% ethanol. The zones of inhibition were measured as indicated in the Kirkby Bauer Disc Diffusion Method. The zones of inhibition give an indication of the antibacterial properties of the samples. The samples were then exposed to different environmental conditions and the same process repeated as before the environmental exposure. The different zones of inhibitions were noted. Observation of the findings of the diluted samples resulted in the need to do further investigation. Samples were then prepared with distilled water, 20% ethanol, 62% ethanol and a nonpolar solvent called DMSO (Dimethyl sulfoxide). The zones of inhibition of the samples were compared with each other and with the zones of inhibitions of the solvents used for the dilutions. The samples were also investigated on a Gas Chromatography with Mass Spectrophotometry (GC/MS) to establish possible changes in the active ingredients of the samples. Minimum Inhibitory Concentration (MIC) was done on all 20 samples that were exposed to the different environmental conditions as used with the Kirkby Bauer Disc Diffusion Test. A mother tincture and herbal extract sample that was not exposed to different environmental conditions were used as control to compare MIC of the exposed samples. Appendix 13 shows all raw results.

The measurements for the zones of inhibition were collected and a statistical analysis was done using the Mann-Whitney U-Test to establish a statistical significance value for the difference in the inhibition zones for each set of samples. The Mann-Whitney U-Test compares differences between two dependent groups. This is used when the dependent variable is not normally distributed but is ordinal or continuous (Pallant, 2007). The GC/MS separation analysis identified a number of compounds that were compared with the different samples. The MIC values were compared with each other and changes between the groups were noted.

4.2 Representation of agar plates Photographs were taken of all the agar plates to confirm measurements of the horizontal and vertical diameters. This included the herbal extract and homeopathic mother tincture samples before and after exposure.

In Figure 4.1, the agar plate for the herbal extract sample (specifically the third in the series) was exposed to direct sunlight while kept in a blue glass bottle. A clear zone of inhibition can be seen around the paper disc. The cloudy areas around the zones of inhibition are the S. aureus growing on the agar plate.

Figure 4.1: Herbal extract sample agar plate for a Mediherb herbal extract

Figure 4.2: Herbal extract sample diluted 3:7 with distilled water

Figure 4.2 shows the agar plate for a herbal extract that was diluted as a D1 (3:7) with distilled water. Although zones of inhibition can be seen, it is cloudy and not consistent in diameter. It was

25 due to this reason that further testing on the diluted samples were stopped in order to investigate other options.

Figure 4.3: Herbal extract sample diluted 3:7 with DMSO

When comparing the agar plate in Figure 4.3 with the plate in Figure 4.2, the herbal extract was diluted 3:7 in both, but the zones of inhibition were different. In Figure 4.3, the extract was diluted with DMSO, a polar solvent, whereas in Figure 4.2 the herbal extract was diluted with distilled water, a non-polar solvent. Although DMSO is toxic to humans, it is not bactericidal and therefore has no effect on the zones of inhibition, but stabilises the herbal extract to show clearer zones of inhibition.

4.3 Zones of inhibition of Mediherb Herbal Extract in blue bottles The horizontal and vertical diameters of the zones of inhibition for each sample was measured and a mean and median diameter was calculated for each set of samples stored in blue bottles according to the different exposure factors. The results show the difference in zone of inhibition, i.e. antibacterial properties for each set of sample according to the exposure.

Table 4.1: Comparison of the median inhibition zones for each exposure group in blue bottles.

Median Diameter Median Diameter Significance level Exposure Type Before Exposure After Exposure (mm) (mm) (p-Value) Control (UJ Dispensary) 8.500 8.750 0.054

Direct Sunlight 8.500 8.500 0.050

Kitchen Scenario 8.000 8.500 0.001

Office Scenario 8.000 9.000 <0.001

Decreased Temperature 8.000 9.000 <0.001

In Table 4.1 above, the difference in the zones of inhibition before and after exposure to Direct Sunlight, the Kitchen Scenario, Office Scenario and Decreased Temperature were all statistically relevant, as the p-value was less than 0.05 according to the Mann-Whitney U Test. The difference in the zones of inhibition for the samples exposed in the UJ Dispensary were not statistically relevant as the p-value was more than 0.05 (p = 0.054).

Table 4.2: Normality of data for each extract exposure group in blue bottles.

Exposure Type Skewness Skewness Kurtosis Kurtosis Before After Before After Exposure Exposure Exposure Exposure Control (UJ Dispensary) -0.240 -0.312 -0.427 0.013

Direct Sunlight -0.422 0.505 0.042 0.158

Kitchen Scenario 0.613 0.707 0.755 0.308

Office Scenario -0.210 -0.029 -0.234 -0.947

Decreased Temperature -3.495 0.272 12.514 1.076

Normality of the data can be represented by the skewness and kurtosis values of the collective results. When the skewness and kurtosis values are close to zero it shows that there is a normal distribution of the results. The range between -1 and 1 is acceptable. The results, as in Table 4.2 above, for the samples in the Decreased Temperature group does not show normal distribution of the results, as 3 out of the 4 values exceeds the -1 to 1 mark. The results for the samples exposed

27 to the UJ Dispensary, Kitchen Scenario, Office Scenario and Direct Sunlight show normal distribution.

9,5

8,5

Before Exposure (BE) 8 After Exposure (AE) Mean Diameter (mm) Diameter Mean

7,5

7 UJ Direct Kitchen Office Decreased Dispensary Sunlight Scenario Scenario Temperature

Figure 4.4: Mean inhibition diameters for the extract samples (blue bottles)

A general increase in the diameter of the zones of inhibition was reported for all the herbal extract samples exposed to the environmental conditions in blue glass bottles. It was expected that all the zones of inhibition for the samples before exposure to different environmental conditions would be the same (to be set as a baseline), but it differed as seen in Figure 4.4.

An increase in the diameter of the zones of inhibition can be noted when referring to Figure 4.5 below. The median diameter for the zone of inhibition for the “Before Exposure” (BE) values was 8.5mm and “After Exposure” (AE) values was 8.75mm. For the BE measurements, the lowest measurement was 7.5mm and the highest measurement was 9mm. The lower or first quartile of the measurements was 8mm and the third quartile or upper quartile was 8.5mm, equivalent to the median value. The interquartile range was between 8mm and 8.5mm. The lowest measurement for the AE group was 7mm and the highest measurement was 10mm. The interquartile range was between 8.5mm and 9.5mm; and the median was 8.75mm. The interquartile range refers to the average set of measurements excluding the highest and lowest measurements.

Extract (Blue Bottle) in UJ Dispensary

9 Median = 8.75 Median = 8.5

8 Diameter (mm) Diameter

6 Before Exposure After Exposure

Figure 4.5: Inhibition diameters for extract samples in blue bottles (UJ Dispensary)

Extract (Blue Bottle) in Direct Sunlight

10 M e d i 9 a n Median = 8.5 Median = 8.5 = 8 8 . 7 Diameter (mm) Diameter 5 7

6 Median = 8.5 Before Exposure After Exposure

Figure 4.6: Inhibition diameters for extract samples in blue bottles (Direct Sunlight)

The median diameter of the zones of inhibition for the samples exposed to Direct Sunlight remained the same, however, the range of the measurements differed. The average set of measurements for the BE measurements ranged from 8mm to 8.5mm, with the median at 8.5mm. The lowest measurement for the BE group was 7mm and the highest measurement was 9.5mm. 29

The range of the average measurements for the AE measurements was greater than the BE set of measurements. It ranged from 8mm to 9mm, with the median also at 8.5mm.

In the following graph (Figure 4.7), the diameters of the zones of inhibition for the samples exposed to the Kitchen Scenario are depicted. The median diameter for the zone of inhibition increased from 8mm to 8.5mm from the BE values to the AE values. The average set of measurements for the BE values was from 8mm to 8.5mm and the average set of measurements for the AE values was from 8.5mm to 9mm. The highest measurement for the BE set of measurements was 9.5mm whereas the highest measurement for the AE set of measurements was 10mm. The lowest measurement also increased from the BE values of 7.5mm to the AE values of 8mm.

Extract (Blue Bottle) in Kitchen

Median = 8.5 Diameter (mm) Diameter Median = 8 8

7 Before Exposure After Exposure

Figure 4.7: Inhibition diameters for extract samples in blue bottles (Kitchen Scenario)

Figure 4.8 below shows the BE and AE zones of inhibition values for the herbal extract group that was exposed in the Office Scenario in blue glass bottles. There is a 1mm difference in the median values between the BE and AE groups. The median value for the BE group was 8mm and the AE group was 9mm. The lowest value for the BE group was 7.5mm and the AE group was 8mm. The highest value for the BE group was 9mm and for the AE group was 10mm. The range for the average set of values for the BE group differ by 0.5mm (from 8mm to 8.5mm) and the range of the average set of values for the AE values is 1.25mm (from 8.625mm to 9.875mm).

Extract (Blue Bottle) in Office

Median = 9 9 Diameter (mm) Diameter Median = 8 8

7 Before Exposure After Exposure

Figure 4.8: Inhibition diameters for extract samples in blue bottles (Office Scenario)

Extract (Blue Bottle) at decreased temperature

10 Median = 9

Diameter (mm) Diameter 8 Median = 8

6 Before Exposure After Exposure

Figure 4.9: Inhibition diameters for extract samples in blue bottles (Decreased Temperature)

Measurements of the zones of inhibition for the samples that were exposed to Decreased Temperature in blue glass bottles, as illustrated in the figure above, show that the median measurement increased by 1mm from the BE values to the AE values (from 8mm to 9mm). The BE values were all close together, with the lowest value at 7mm and the highest value at 8mm. The range of the average set of measurements were all at 8mm. For the AE measurements, the lowest measurement was 7.5mm and the highest 11mm; with the average set of measurements ranging from 9mm to 10mm.

4.4 Zones of inhibition of Mediherb Herbal Extract in amber bottles The horizontal and vertical diameters of the zones of inhibition for each sample were measured and a mean and median diameter was calculated for each set of samples stored in amber bottles according to the different exposure factors. The results show the difference in zone of inhibition, i.e. antibacterial properties for each set of sample according to the exposure.

Table 4.3: Comparison of the median inhibition zones for each exposure group in amber bottles.

Median Diameter Median Diameter Significance level Exposure Type Before Exposure After Exposure (p-Value) (mm) (mm) Control (UJ Dispensary) 8.500 8.250 0.167

Direct Sunlight 8.500 8.500 0.705

Kitchen Scenario 8.500 9.250 <0.001

Office Scenario 8.000 9.000 <0.001

Decreased Temperature 8.000 9.000 <0.001

The level of significance for the samples exposed in the UJ Dispensary and Direct Sunlight do not show statistical relevance due to the p-value being greater than 0.05, according to the Mann- Whitney U Test. The samples in the other three groups: Kitchen Scenario, Office Scenario and Decreased Temperature, all show statistical relevance (p < 0.05).

Table 4.4: Normality of data for each extract exposure group in amber glass bottles Skewness Skewness Kurtosis Kurtosis Exposure Type Before After Before After Exposure Exposure Exposure Exposure Control (UJ Dispensary) 0.294 0.232 -1.108 -1.374

Direct Sunlight -0.040 0.662 -1.485 -0.026

Kitchen Scenario -0.830 -0.562 2.762 -0.622

Office Scenario -0.315 -0.539 1.285 0.391

Decreased Temperature 0.897 -1.005 0.769 3.322

The skewness values for the samples are within the acceptable range (-1 to 1), but 60% of the kurtosis values are above the acceptable range. Only the the values for the BE of the samples exposed to Decreased Temperature and the AE kurtosis values for the samples exposed to Direct Sunlight, Kitchen Scenario and Office Scenario are in acceptable data normality ranges.

9,4

9,2

8,8

8,6

8,4 Before Exposure (BE) 8,2 After Exposure (AE)

Mean Diameter (mm) Diameter Mean 8

7,8

7,6

7,4 Control (UJ Direct Kitchen Office Decreased Dispensary) Sunlight Scenario Scenario Temperature

Figure 4.10: Mean inhibition diameters for each extract sample (amber bottles)

Figure 4.10 shows a significant increase in the AE results for the extract samples exposed to the Kitchen Scenario, Office Scenario and Decreased Temperature. The samples exposed to Direct Sunlight had a slight decline in the diameters of zones of inhibition and the samples in the UJ dispensary had a slightly bigger difference in the diameters for the zones of inhibition.

Extract (Amber Bottle) in UJ Dispensary

Median = 8.5 Median = 8.25

Diameter (mm) Diameter 8

7 Before Exposure After Exposure

Figure 4.11: Inhibition diameters for extract samples in amber bottles (UJ Dispensary)

The ranges of the average sets of results for both BE and AE are the same for the samples in the UJ Dispensary (Figure 4.11). The ranges are between 8mm and 9mm, but with median diameter values at 8.5mm and 8.25 mm respectively for the BE and AE set of values. The BE set of values had a higher value of 9.5mm that fell out of the average set of measurements. The AE set of results had a high value of 9.5mm and a low value of 7.5mm that fell out of the average set of measurements.

The samples exposed to Direct Sunlight had the same median diameter zone of inhibition measurement, 8.5mm (Figure 4.12 below). The range of the average measurements for the BE set of values was between 8mm and 9mm and for the AE set of values it was between 8mm and 8.5mm. The BE set of values had a low value of 7.5mm that lie out of the average set of values and the AE set of values had a higher value of 9.5mm that lie out of the average set of values.

Extract (Amber Bottle) in Direct Sunlight

Median = 8.5 Median = 8.5

8 Diameter (mm) Diameter

6 Before Exposure After Exposure

Figure 4.12: Inhibition diameters for extract samples in amber bottles (Direct Sunlight)

Extract (Amber Bottle) in Kitchen

Median = 9.25 9

8 Median = 8.5 Diameter (mm) Diameter

6 Before Exposure After Exposure

Figure 4.13: Inhibition diameters for extract samples in amber bottles (Kitchen Scenario)

The median diameter for the zones of inhibition for the samples exposed in the Kitchen Scenario significantly increased from BE of the samples to AE of the samples. The median diameter increased from 8.5mm to 9.25mm. The ranges of the average set of values for both the BE and AE values differed significantly. The BE range was from 8mm to 8.5mm and the AE range was from 35

9mm to 10mm. The BE set of values had a high and low outlier measurement of 7mm and 9mm, whereas the AE set of values only had a low value outlier of 8mm.

For the samples exposed in the Office Scenario in amber glass bottles (Figure 4.14 below), the median diameter of zone of inhibition increased from 8mm to 9mm. The range of the average measurements for the BE set of values was between 8mm and 8.5mm, whereas the AE set of values increased with a range of 8.5mm to 9mm. Both the BE and AE sets of values had a high and low outlier; the BE set of values had a low outlier of 7mm and a high outlier of 9mm. The AE set of values had a low outlier of 7.5mm and a high outlier of 10mm.

Extract (Amber Bottle) in Office

9 Median = 9

Median = 8 8 Diameter (mm) Diameter

6 Before Exposure After Exposure

Figure 4.14: Inhibition diameters for extract samples in amber bottles (Office Scenario)

In Figure 4.15 below, where the samples in the amber glass bottles were exposed to decreased temperatures, it is shown that the median diameter of the zone of inhibition increased from 8mm in the BE set of values to 9mm in the AE set of values. The range of average values also increased from 8mm to 8.5mm in the BE set of values to 8.5mm to 9mm in the AE set of values. There was a negative outlier in the BE set of values of 7.5mm and a positive outlier of 9mm. With the AE set of values there was a negative outlier of 6.5mm and a positive outlier of 10mm.

Extract (Amber Bottle) at decreased temperature

9 Median = 9

Median = 8 8 Diameter (mm) Diameter

6 Before Exposure After Exposure

Figure 4.15: Inhibition diameters for extract samples in amber bottles (Decreased Temperature)

4.5 Zones of inhibition of Homeopathic Mother Tincture in blue bottles The horizontal and vertical diameters of the zones of inhibition for each sample were measured and a mean and median diameter was calculated for each set of samples, according to the different exposure factors when stored in blue bottles. The results show the difference in zone of inhibition, i.e. antibacterial properties for each set of sample according to the exposure.

Table 4.5: Comparison of the median inhibition zones for each exposure group in blue bottles

Median Diameter Median Diameter Significance level Exposure Type Before Exposure After Exposure (p-Value) (mm) (mm) Control (UJ 6.750 6.500 0.394 Dispensary) Direct Sunlight 6.500 6.500 0.005

Kitchen Scenario 6.500 6.500 <0.001

Office Scenario 6.500 6.500 <0.001

Decreased Temperature 6.250 7.500 <0.001

The difference in the zones of inhibition for the samples exposed in the UJ Dispensary shows a significance level of 0.394, which is higher than 0.05. According to the Mann-Whitney test, it shows that this set of results is therefore not statistically relevant. All the other samples (exposure to Direct Sunlight, Kitchen Scenario, Office Scenario and Decreased Temperature) show a significance level of less than 0.05; therefore these sets of results are statistically relevant.

Table 4.6: Normality of data for each mother tincture exposure group in blue bottles Skewness Skewness Kurtosis Kurtosis Exposure Type Before After Before After Exposure Exposure Exposure Exposure Control (UJ Dispensary) 0.692 0.406 1.781 0.148

Direct Sunlight 0.086 0.702 -0.357 1.314

Kitchen Scenario 0.484 1.477 -0.620 2.910

Office Scenario -0.430 -0.151 -1.950 -0.398

Decreased Temperature 0.456 -0.014 -0.748 -0.535

The skewness and kurtosis values refers to the normal distribution of the results. A value between -1 and 1, closest to zero, is considered a normal distribution of results. From the values set out in Table 4.6, most sets of results are normally distributed, however, the after exposure results for the samples exposed to the Kitchen Scenario are not within the acceptable range. The BE kurtosis values for the samples exposed in the UJ Dispensary and the Office Scenario also show values higher than the acceptable range. Samples exposed in direct sunlight have a higher than normal AE kurtosis value.

7,6

7,4

7,2

6,8

6,6 Before Exposure (BE) 6,4 After Exposure (AE)

Mean Diameter (mm) Diameter Mean 6,2

5,8

5,6 Control (UJ Direct Kitchen Office Decreased Dispensary) Sunlight Scenario Scenario Temperature

Figure 4.16: Mean inhibition diameters for the mother tincture samples (blue bottles)

All the samples’ zone of inhibition measurements, except for those exposed in the UJ Dispensary, increased after exposure to the different environmental conditions. The BE measurements are also in the same range of between 6.2mm and 6.4mm. The measurements for the samples exposed in the UJ Dispensary had higher BE measurements than the other four sets of samples and the AE measurements showed a slight decline in the diameter of the zones of inhibition. The samples that were exposed to Decreased Temperature showed a substantial increase in the diameter for the zones of inhibition after exposure.

As seen in Figure 4.17 below, there was a slight decline in the diameter in the zones of inhibition for these samples after the exposure to the UJ Dispensary environment in the blue glass bottles. The median diameter decreased from 6.75mm to 6.5mm. The lowest measurement was the same for the before and after exposure measurements. The BE set of values’ highest measurement was 8mm, whereas the AE set of values’ highest measurement was 7.5mm. The range of the average set of data for both the BE and AE sets of measurements were the same at between 6.5mm and 7mm.

Mother Tincture (Blue Bottle) in UJ Dispensary

7 Median = 6.75 Median = 6.5 Diameter (mm) Diameter

5 Before Exposure After Exposure

Figure 4.17: Inhibition diameters for mother tincture samples in blue bottles (UJ Dispensary)

Mother Tincture (Blue Bottle) in Direct Sunlight

Median = 6.5 Median = 6.5

Diameter (mm) Diameter 6

5 Before Exposure After Exposure

Figure 4.18: Inhibition diameters for mother tincture samples in blue bottles (Direct Sunlight)

In Figure 4.18, the median for both sets of values remained at 6.5mm, however, the average set of measurements were different. The BE set of values ranged from 6mm to 6.5mm, whereas the AE set of values ranged from 6.5mm to 6.875mm. The lowest measurement for both BE and AE measurements was 6mm, and the highest measurement for the BE values was 7mm and the AE values was 8.5mm.

Mother Tincture (Blue Bottle) in Kitchen

Median = 6.5 Median = 6.5 Diameter (mm) Diameter

5 Before Exposure After Exposure

Figure 4.19: Inhibition diameters for mother tincture samples in blue bottles (Kitchen Scenario)

The median diameter for both the BE and AE sets of values was 6.5mm (Figure 4.19 above). The lowest measurement for the BE set of values was 6mm and for the AE set of values it was 6.5mm. The highest values were 7mm and 8mm respectively for the BE and AE set of values. There was an increase in diameter from the BE and AE sets of values when the average set of values were considered. The BE values ranged from 6mm to 6.5mm and the AE values ranged from 6.5mm to 7mm.

For the samples that were exposed in the Office Scenario (Figure 4.20 below), the BE set of values formed part of the average set of values, there were no measurements that fell out of the range between 6mm and 6.5mm. The median measurement for both the BE and AE set of values were 6.5mm. The AE set of values showed a range of the average set of values of 6.5mm to 7mm. There was a lower and higher value that did not fit in with the average set of data of 6mm and 7.5mm respectively.

Mother Tincture (Blue Bottle) in Office

Median = 6.5 Median = 6.5

Diameter (mm) Diameter 6

5 Before Exposure After Exposure

Figure 4.20: Inhibition diameters for mother tincture samples in blue bottles (Office Scenario)

Mother Tincture (Blue Bottle) at decreased temperature

Median = 7.5

Diameter (mm) Diameter Median = 6.25 6

5 Before Exposure After Exposure

Figure 4.21: Inhibition diameters for mother tincture samples in blue bottles (Decreased Temperature)

In Figure 4.21, there was a substantial change in the measurements between the BE and AE sets of results. The median measurement increased from 6.25mm in the BE set of values to 7.5mm in the AE set of values. The range of average values for the BE set of values was between 6mm and 6.5mm whereas the range for the AE set of results was from 7mm to 7.5mm, with a lowest value at 6.5mm and a highest value of 8mm. The BE set of values had a lowest value of 6mm, which formed part of the average set of values and a highest value of 7mm.

4.6 Zones of inhibition of Homeopathic Mother Tincture in amber bottles The horizontal and vertical diameters of the zones of inhibition for each sample were measured and a mean and median diameter was calculated for each set of samples stored in amber bottles according to the different exposure factors. The results show the difference in zone of inhibition, i.e. antibacterial properties for each set of sample according to the exposure.

Table 4.7: Comparison of the median inhibition zones for each exposure group in amber bottles.

Median Diameter Median Diameter Significance level Exposure Type Before Exposure After Exposure (mm) (mm) (p-Value) Control (UJ Dispensary) 6.500 6.500 0.973

Direct Sunlight 6.500 7.000 <0.001

Kitchen Scenario 6.250 6.500 <0.001

Office Scenario 6.000 6.750 <0.001

Decreased Temperature 6.500 6.500 0.007

The zones of inhibition for the samples exposed to Direct Sunlight, Kitchen Scenario, Office Scenario and Decreased Temperature showed statistical significance, with the p-values less than 0.05. The zones of inhibition for the samples exposed in the UJ Dispensary had a significance level of 0.973, which is greater than the 0.05 for statistical relevance as set out by the Mann-Whitney U Test.

Table 4.8: Normality of data for each mother tincture exposure group in amber bottles Skewness Skewness Kurtosis Kurtosis Exposure Type Before After Before After Exposure Exposure Exposure Exposure Control (UJ Dispensary) 0.323 -1.328 -0.722 -0.257

Direct Sunlight 0.484 0.555 -0.620 -0.577

Kitchen Scenario 0.000 0.504 -2.148 0.320

Office Scenario 0.732 0.889 -0.429 1.456

Decreased Temperature 0.040 0.549 -0.082 0.382

Although the overall skewness and kurtosis values for the samples as set out in Table 4.8 is within the normal distribution of data, the skewness AE for the UJ Dispensary group, the kurtosis BE for the kitchen scenario group and the kurtosis AE for the office scenario group were not in normal ranges.

6,8

6,6

6,4 Before Exposure (BE) After Exposure (AE) 6,2 Mean Diameter Mean Diameter (mm)

5,8 Control (UJ Direct Sunlight Kitchen Office Scenario Decreased Dispensary) Scenario Temperature

Figure 4.22: Mean inhibition diameters for the mother tincture samples (amber bottles)

The mean diameter zone of inhibition for the samples BE and AE exposed in the UJ Dispensary were the same as seen in Figure 4.22 above. There were significant increases in the mean diameter zone of inhibition for the samples exposed to the different environmental conditions.

Mother Tincture (Amber Bottle) in UJ Dispensary

Median = 6.5 Median = 6.5

Diameter (mm) Diameter 6

5 Before Exposure After Exposure

Figure 4.23: Inhibition diameters for mother tincture samples in amber bottles (UJ Dispensary)

The median diameter zone of inhibition for the BE and AE set of values as in Figure 4.23 above, remained the same at 6.5mm. This was for the mother tincture samples in amber glass bottles exposed in the UJ Dispensary. The BE set of values had an average range of 6mm to 6.5mm whereas the AE set of values had an average of 6.5mm with a high outlier of 7mm and a low outlier of 6mm. The BE set of values, however, only had a high outlier of 7.5mm.

In Figure 4.24 below, where mother tincture samples in amber glass bottles were exposed to direct sunlight, the median diameter for the zones of inhibition increased from 6.5mm to 7mm. The range of the average measurements also increased from the BE set of values, 6mm to 6.5mm, and 6.5mm to 7.325mm with the AE set of values. The BE set of values had a high measurement of 7mm that falls out of the average set of values and the AE set of values has a low measurement of 6mm and a high value of 8mm that fall out of the average set of measurements.

Mother Tincture (Amber Bottle) in Direct Sunlight

7 Median = 7 Median = 6.5 Diameter (mm) Diameter

5 Before Exposure After Exposure

Figure 4.24: Inhibition diameters for mother tincture samples in amber bottles (Direct Sunlight)

Mother Tincture (Amber Bottle) in Kitchen

Median = 6.5 Median = 6.25

Diameter (mm) Diameter 6

5 Before Exposure After Exposure

Figure 4.25: Inhibition diameters for mother tincture samples in amber bottles (Kitchen Scenario)

The median diameter zone of inhibition for the mother tincture samples exposed in the kitchen scenario in amber glass bottles increased from 6.25mm before exposure to 6.5mm after exposure. The range of the average values for the homeopathic mother tincture samples in amber glass bottles

46 also increased from 6mm to 6.5mm before exposure of the samples to 6.5mm to 7mm after exposure. Only the set of values for AE had a positive and negative outlier of 7.5mm and 6mm.

The median diameter zone of inhibition increased in Figure 4.26 below, from 6mm to 6.75mm. This was for the mother tincture samples in amber glass bottles exposed to the Office Scenario. The range of the average measurements also increased from BE to AE. The BE range is from 6mm to 6.5mm and the AE range is from 6.5mm to 7mm. The lowest measurement for the AE set of values was 6mm and the highest measurement was 8mm. With the BE set of values, however, there is only a positive outlier of 7mm.

Mother Tincture (Amber Bottle) in Office

7 Median = 6.75 Diameter (mm) Diameter Median = 6 6

5 Before Exposure After Exposure

Figure 4.26: Inhibition diameters for mother tincture samples in amber bottles (Office Scenario)

Although the median diameter zone of inhibition remained constant BE and AE for the mother tincture samples in amber glass bottle exposed to Decreased Temperature, the range of the average values increased from the BE set of values to the AE set of values (Figure 4.27 below). The range of values in the BE set of values is between 6.125 and 6.5, whereas the range of values in the AE set of values is 6.5mm to 7mm. Both sets of values have a positive and negative outlier, the negative ones being both at 6mm and the positive ones at 7mm and 7.5mm respectively for the BE set of values and the AE set of values.

Mother Tincture (Amber Bottle) at decreased temperature

Median = 6.5 Median = 6.5

Diameter (mm) Diameter 6

5 Before Exposure After Exposure

Figure 4.27: Inhibition diameters for mother tincture samples in amber bottles (Decreased Temperature)

4.7 Zones of inhibition comparison of the Mediherb Herbal Extract The results below shows a comparison of the zones of inhibition for the Mediherb herbal extract between the blue and amber bottles according to the different exposure factors.

Exposure: UJ Dispensary 8,8

8,7

8,6

8,5 Blue Bottle 8,4 Amber Bottle

Mean Diameter (mm) Diameter Mean 8,3

8,2

8,1 Before Exposure After Exposure

Figure 4.28: Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (UJ Dispensary) 48

There is a definite increase in the mean diameter of the zones of inhibition for the extract samples in the blue glass bottles, and a slight decrease in the mean diameter of the zones of inhibition for the extract samples in the amber glass bottles.

Exposure: Direct Sunlight 8,6 8,55 8,5 8,45 8,4 8,35 Blue Bottle Amber Bottle 8,3

Mean Diameter (mm) Diameter Mean 8,25 8,2 8,15 8,1 Before Exposure After Exposure

Figure 4.29: Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Direct Sunlight)

Exposure: Kitchen 9,4

9,2

8,8

8,6 Blue Bottle 8,4 Amber Bottle 8,2 Mean Diameter (mm) Diameter Mean 8

7,8

7,6 Before Exposure After Exposure

Figure 4.30: Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Kitchen Scenario)

Figure 4.29 shows extract samples exposed to Direct Sunlight. Those in the blue bottles show an increase in the diameter of the zones of inhibition and, in comparison, a slight decrease in the diameter of the zones of inhibition for the samples in the amber glass bottles.

The mean diameter for the zones of inhibition for the extract samples exposed to the Kitchen Scenario increased for both the samples in the blue glass and in the amber glass bottles. A higher increase, however, is seen with the amber glass bottles.

Exposure: Office 9,2

8,8

8,6

8,4 Blue Bottle 8,2 Amber Bottle 8 Mean Diameter (mm) Diameter Mean 7,8

7,6

7,4 Before Exposure After Exposure

Figure 4.31: Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Office Scenario)

According to the figure above (Figure 4.31), both the mean diameters for the samples in the blue and amber glass bottles increased AE to the Office Scenario. The samples in the blue glass bottle had a higher increase than the amber glass bottle samples.

In the figure below (Figure 4.32), both the samples in the blue and amber glass bottles had an increase in zone of inhibition diameters AE to Decreased Temperature. The samples in the blue glass bottles showed a higher increase than the samples in the amber glass bottles.

Exposure: Decreased Temperature 9,5

8,5 Blue Bottle Amber Bottle 8 Mean Diameter (mm) Diameter Mean

7,5

7 Before Exposure After Exposure

Figure 4.32: Mean inhibition diameters BE and AE for extract samples in blue and amber bottles (Decreased Temperature)

4.8 Zones of inhibition comparison for the Homeopathic Mother Tincture The results below shows a comparison of the zones of inhibition for the homeopathic mother tincture between the blue and amber bottles according to the different exposure factors.

Exposure: UJ Dispensary

6,8

6,7

6,6

6,5 Blue Bottle 6,4 Amber Bottle

Mean Diameter (mm) Diameter Mean 6,3

6,2

6,1 Before Exposure After Exposure

Figure 4.33: Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (UJ Dispensary) 51

When the mother tincture samples were exposed in the UJ Dispensary, both sets of samples in the blue and amber glass bottles remained more or less constant. The samples in the blue glass bottles had a slight decrease in diameter length of the zones of inhibition after exposure, but the samples in the amber glass bottles remained constant (Figure 4.33).

Exposure: Direct Sunlight

7 6,9 6,8 6,7 6,6 6,5 Blue Bottle Amber Bottle 6,4

Mean Diameter (mm) Diameter Mean 6,3 6,2 6,1 6 Before Exposure After Exposure

Figure 4.34: Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (Direct Sunlight)

With exposure to Direct Sunlight, both mother tincture samples’ diameter of the zones of inhibition increased; the samples in the amber glass increased by a bigger increment than the samples in the blue bottles.

Below in Figure 4.35, where the mother tincture samples were exposed to the Kitchen Scenario, both the blue and amber glass bottles’ mean diameter of zone of inhibition increased AE. The blue and amber glass bottle samples increased by the same increment.

In Figure 4.36, exposure of mother tincture samples to the Office Scenario, both sets increased diameters of the zones of inhibition. Amber glass bottle samples had a bigger increase in diameter length than the blue bottle samples.

Exposure: Kitchen

6,9 6,8 6,7 6,6 6,5 6,4 Blue Bottle Amber Bottle 6,3 6,2 Mean Diameter (mm) Diameter Mean 6,1 6 5,9 Before Exposure After Exposure

Figure 4.35: Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (Kitchen Scenario)

Exposure: Office

6,9 6,8 6,7 6,6 6,5 6,4 Blue Bottle Amber Bottle 6,3

Mean Diameter (mm) Diameter Mean 6,2 6,1 6 5,9 Before Exposure After Exposure

Figure 4.36: Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (Office Scenario)

Exposure: Decreased Temperature

7,6 7,4 7,2 7 6,8 6,6 Blue Bottle Amber Bottle 6,4

Mean Diameter (mm) Diameter Mean 6,2 6 5,8 5,6 Before Exposure After Exposure

Figure 4.37: Mean inhibition diameters BE and AE for mother tincture samples in blue and amber bottles (Decreased Temperature)

Figure 4.37 above shows that the samples in the blue glass bottles had a much higher increase in mean diameter (6.267mm to 7.333mm) than the samples in the amber glass bottles (6.417mm to 6.683mm).

4.9 Minimum Inhibitory Concentration (MIC) The MIC was tested for all the samples that were tested with the Kirkby Bauer method. The importance of these results is to compare the MIC of the different herbal extract samples with each other; the different homeopathic mother tincture samples with each other and also the control herbal extract with the control homeopathic mother tincture.

Figure 4.38: Microtiter Plate number 3.

In Figure 4.38 above, the pink coloured wells show that bacteria are still alive because they have metabolised the colour salt; and the blue coloured wells show that the bacteria has died, therefore remaining blue like the original colouring salt. Due to the dark colour of the C. officinalis, the first dilution did not change colour as well as the preceding dilutions.

Table 4.9 below compares the MIC percentage for each sample. With the results tabulated, the individual samples’ results can be compared with the control extract and mother tincture that were not exposed to the different environmental conditions. There was a change in the MIC for the amber glass extract samples that were exposed to the Kitchen Scenario as well as to Direct Sunlight and Decreased Temperature. The extracts in the blue glass bottles showed a change in the MIC for the samples exposed to Direct Sunlight, Office Scenario and to Decreased Temperature. The mother tincture samples in the amber glass bottles’ MIC all remained the same as the control mother tincture. The MIC’s for the mother tincture samples in blue glass bottles showed a change in the samples that were exposed to the Kitchen Scenario and to Decreased Temperature.

Table 4.9: Results for MIC for samples tested

The herbal extract had a known initial concentration of 500mg/ml, but the initial concentration for the homeopathic mother tincture was unknown.

Plate Side Contents Exposure Bottle MIC % MIC Value 1 Left Extract None (Control) Stock 6.25 31.25mg/ml 1 Right Mother tincture None (Control) Stock 12.50 Unknown 2 Left Extract UJ Dispensary Amber 6.25 31.25mg/ml 2 Right Extract Direct Sunlight Amber 12.50 62.50mg/ml 3 Left Extract Kitchen Amber 12.50 62.50mg/ml 3 Right Extract Office Amber 6.25 31.25mg/ml 4 Left Extract Decreased Temp Amber 12.50 62.50mg/ml 4 Right Extract UJ Dispensary Blue 6.25 31.25mg/ml 5 Left Extract Direct Sunlight Blue 12.50 62.50mg/ml 5 Right Extract Kitchen Blue 6.25 31.25mg/ml 6 Left Extract Office Blue 12.50 62.50mg/ml 6 Right Extract Decreased Temp Blue 12.50 62.50mg/ml 7 Left Mother tincture UJ Dispensary Amber 12.50 Unknown 7 Right Mother tincture Direct Sunlight Amber 12.50 Unknown 8 Left Mother tincture Kitchen Amber 12.50 Unknown 8 Right Mother tincture Office Amber 12.50 Unknown 9 Left Mother tincture Decreased Temp Amber 12.50 Unknown 9 Right Mother tincture UJ Dispensary Blue 12.50 Unknown 10 Left Mother tincture Direct Sunlight Blue 12.50 Unknown 10 Right Mother tincture Kitchen Blue 25.00 Unknown 11 Left Mother tincture Office Blue 12.50 Unknown 11 Right Mother tincture Decreased Temp Blue 25.00 Unknown

4.10 Gas Chromatography Mass Spectrometry (GC/MS) The Gas Chromatography Mass Spectrometry (GC/MS) was decided on due to the inconsistent results that were obtained with the initial dilution of the herbal extracts and homeopathic mother tincture. An interest arose to establish whether the compounds of the sample changed with the exposure to the environmental conditions since in most cases the antibacterial properties increased. Unfortunately, due to time and budget constraints with the GC/MS analysis, only a few samples could be separated and analysed (Appendix 12).

It was decided in consultation with the supervisors of the laboratory study to analyse the five herbal extracts that was exposed to the different environmental conditions in blue bottles because this group of results showed the biggest differences in the diameters of the zones of inhibition. Furthermore an unexposed herbal extract sample was used as a control sample, an unexposed homeopathic mother tincture sample (for comparison between the different extraction methods of an extract) and three dilutions of an unexposed herbal extract with 90% ethanol, 20% ethanol and DMSO in a 3:7 ratio were also analysed. A comparison of the different compounds separated by the GC/MS showed some differences in the amount and composition of the compounds.

5e+006

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC Figure 4.39: The GC/MS graph for the separation of a Mediherb Herbal extract sample that was not exposed to any environmental conditions (Control).

5e+006

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC Figure 4.40: The GC/MS graph for the separation of a homeopathic mother tincture sample that was not exposed to any environmental conditions (Control).

Figures 4.39 and 4.40 show the difference in compounds when C. officinalis is extracted with different extraction methods. The two GC/MS graphs show a clear difference between the Mediherb herbal extract and the homeopathic mother tincture. The number of compounds within the herbal extract are more than those in the mother tincture (Appendix 13).

In Table 4.10 below, the compounds identified with the separation of the herbal extract and homeopathic mother tincture on the GC/MS are compared. The compounds in the table are only the biological organic compounds from the C. officinalis plant. For the herbal extract only the first 25 compounds that have the highest percentage and, are therefore the most abundant was noted. The mother tincture only showed 12 biological organic compounds. The amount of time used for a solute to travel through the GC column is referred to as the retention time and the %Area refers to the concentration of the compound (Parker, 2003).

Table 4.10: Comparison of the compounds separated on the GC/MS for the herbal extract and homeopathic mother tincture

Mediherb Extract Homeopathic mother tincture Retention Retention Compound % Area Compound % Area Time (s) Time (s) Amphetamine 897676316 122.46 Glycine 44663721 242.22 Furfural 124743530 243.28 Isovaleric acid 11114946 246.26 Alpha Linolenic á-copaene 35830163 950.42 6067480 1220.08 acid n-Hexadecanoic à-Cadinol 27286604 922.66 3104691 1099.92 acid 5- Phytol 2617796 1190.00 Hydroxymethylfurfural 26182444 583.10 à-ylangene 25227094 1630.40 à-Cadinol 2096069 921.86 Alpha Linolenic acid 23913123 1205.34 Undecane 1965452 468.72 Isovaleric acid 14544350 248.00 Undecanoic acid 1665925 1120.68 Alloaromadendrene 12984012 809.42 Dihydroxyacetone 1112535 282.78 Decanoic acid 10153466 1121.62 Diethylpropion 487538 931.52 á-Amyrin 8830840 1883.90 Hexadecane 474919 1123.42 Hexanoic acid 8815337 352.46 Ethyl á-d-riboside 451508 894.04 à-Cadinol 7960166 913.64 à-Farnesene 7933332 1795.54 Undecanoic acid 5341787 977.58 Malic Acid 5273894 618.28 D-Allose 4927702 798.94 2-Methoxy-4- 4842344 660.92 vinylphenol Octadecanoic acid 4783457 1214.28 Methyl glyoxal 4192672 222.60 Viridiflorol 4046691 1424.54 Benzaldehyde 3817064 348.96 5-methyl-2-Furancar- 3817064 349.64 boxaldehyde Andrographolide 3344321 1842.80 4(1H)-Pyridone 2997321 377.54

Table 4.11: Comparison of the diluted (3:7) extract samples with different solvents

Extract 90% Ethanol 20% Ethanol DMSO Compound % Area % Area % Area % Area Amphetamine 897676316 Furfural 124743530 85470844 á-copaene 35830163 à-Cadinol 27286604 5313103 8196340 5-Hydroxymethylfurfural 26182444 3331843 2943112 à-ylangene 25227094 Alpha Linolenic acid 23913123 12621031 7138387 Isovaleric acid 14544350 13442903 13763972 Alloaromadendrene 12984012 7056347 Decanoic acid 10153466 2569832 3823214 4762588 á-Amyrin 8830840 2840152 Hexanoic acid 8815337 à-Farnesene 7933332 Undecanoic acid 5341787 Malic Acid 5273894 130575 D-Allose 4927702 2-Methoxy-4-vinylphenol 4842344 Octadecanoic acid 4783457 1328299 Methyl glyoxal 4192672 Viridiflorol 4046691 4699624 Benzaldehyde 3817064 1060102 5-methyl-2-Furancar- 3817064 boxaldehyde Andrographolide 3344321 4(1H)-Pyridone 2997321

The comparison between the compound distribution by the GC/MS shows that the dilution with 90% ethanol yields more compounds than the control (undiluted, unexposed) extract. The diluted sample with 20% ethanol yields less separated components and with a non-polar solvent such as DMSO, even less compounds. The breakdown as illustrated in Table 4.10 does not show all the compounds; newly formed compounds are produced due to the exposure and preparation of the sample (Appendix 13).

Table 4.12: Comparison of the compounds of the exposed extract samples in blue bottles with the unexposed extract as control

% Area % Area % Area % Area % Area % Area Compound ↓ Temp Unexposed Dispen- Sunlight Kitchen Office sary Amphetamine 897676316 Furfural 124743530 á-copaene 35830163 à-Cadinol 27286604 35869084 29621570 29934330 35148001 36011847 5-Hydroxy- methylfurfural 26182444 2094963 10812868 6990469 5910163 à-ylangene 25227094 Alpha Linolenic acid 23913123 45035656 30712660 38102475 41972563 40667459 Isovaleric acid 14544350 26019868 6767860 473194 1880674 Alloaromadendrene 12984012 32437759 2821236 4072881 4548407 Decanoic acid 10153466 17959295 20014260 3599458 á-Amyrin 8830840 14316840 11031290 14892086 20559963 16606029 Hexanoic acid 8815337 à-Farnesene 7933332 Undecanoic acid 5341787 30756936 5382238 28111618 26165721 661188 Malic Acid 5273894 1169833 510131 4478643 153624 D-Allose 4927702 4557821 9528652 2-Methoxy-4- vinylphenol 4842344 Octadecanoic acid 4783457 8335183 Methyl glyoxal 4192672 Viridiflorol 4046691 32278457 7046179 3822765 9936178 1734077 Benzaldehyde 3817064 3431409 237950 1826592 5-methyl-2-Furancar- 3817064 boxaldehyde Andrographolide 3344321 6552722 2178774 4(1H)-Pyridone 2997321

The extract samples that were exposed to different environmental conditions do not have the same compounds as the control extract (unexposed) (Newly formed compounds can be seen in the raw data in Appendix 13).

CHAPTER 5: DISCUSSION 5.1 Overview C. officinalis herbal extract and homeopathic mother tincture samples were used in the first part of the study where the zones of inhibition were tested by using the Kirkby Bauer Disc Diffusion method. The samples were exposed to different environmental conditions and the zones of inhibition were measured before exposure (BE) and after exposure (AE) to the different environmental conditions. As an added test the Minimum Inhibitory Concentration (MIC) was tested for all the samples AE and compared to an unexposed control sample.

As part of the Kirkby Bauer Disc Diffusion test dilutions of the samples with different concentrations of ethanol, distilled water and DMSO were done. The measurements from these samples were inconsistent and an alternative testing method/technique was needed. Gas Chromatography Mass Spectroscopy was suggested and some of the samples were analysed to establish a possible change in active compounds.

5.2 Zones of Inhibition (Kirkby Bauer Method) 5.2.1 Zones of Inhibition of Herbal Extract in blue bottles The measured zones of inhibition is an illustration of the antibacterial properties the sample exhibits against S. aureus. Due to the vast number of measurements the median measurement was considered rather than the mean measurement. The difference between the BE and AE measurements were statistically significant for all herbal extract samples exposed to the different environmental conditions in blue glass bottles, except the measurements for the group that was exposed to the UJ Dispensary. If the significance level (p-value) is less than, or equal to 0.05, the Mann-Whitney U Test then refers to the results as being statistically relevant (Pallant, 2007).

The samples in the UJ Dispensary showed an increase in the antibacterial measurement (median diameter from 8.500mm BE to 8.750mm AE), but results are not statistically significant because p > 0.05. The samples exposed to Direct Sunlight in blue glass bottles remained the same (median diameter was 8.500mm), no change in the antibacterial measurements was noted (p = 0.05). The samples exposed to the Kitchen Scenario (diameter changed from 8.000mm to 8.5000mm; p = 0,001), Office Scenario (diameter changed from 8.000mm to 9.000mm; p = 0.000) and Decreased Temperature (diameter changed from 8.000mm to 9.000mm; p = 0.000), showed an increase in the antibacterial measurement and, is statistically relevant.

The normal spread of data can also be seen with the skewness and kurtosis values as depicted in Table 4.2. Most of the values are between the range of -1 and 1, showing that the measurements are spread at a normal bell curve, indicating normality of results. The measurements before exposure for the samples exposed to decreased temperatures were not in the range of -1 to 1, showing some irregularity of the spread of the measurements.

Ideally all the BE samples should start off with closely related measurements as all the samples are decanted from the stock solution that was not exposed to any environmental conditions. It can be seen with Figure 4.4 that there is an overall 1mm difference in the inhibition zone measurement from the highest to the lowest BE measurement (highest value = 8.450mm (UJ Dispensary group) and the lowest value = 7.933mm (Decreased Temperature group)). The samples exposed to a decreased temperature (average 4ºC) showed the highest difference in the inhibition zone measurement (BE = 7.933mm; AE = 9.283mm; p = 0.000). It is for this reason that the BE antibacterial measurements were taken to ensure that the difference before and after exposure is as accurate as possible.

The box and whisker graphs (Figure 4.5 to 4.9) show the spread of the measurements. The samples exposed in the UJ Dispensary in the blue glass bottles, BE, show that the average set of measurements are within 0.5mm from each other with a high and low outlier of 0.5mm from the average set of results. The average set of measurements for the AE measurements were 0.875mm with a negative outlier of 1.5mm less than the average measurements and a positive outlier of 0.625mm more than the average set of measurements. From a visual perspective, the antibacterial measurement has increased in the AE set of values, although the negative outlier is lower than the negative outlier of the BE set of values.

For the other four exposure groups, where the samples were exposed to Direct Sunlight, Kitchen Scenario, Office Scenario and Decreased Temperature, all showed an increase in the antibacterial measurements when referring to the box and whisker graphs which corresponds to the statistical values. For the samples that were exposed in the UJ Dispensary there was no difference in the antibacterial measurement.

There was an overall increase in the antibacterial measurements for the extract samples that were exposed to different environmental conditions while in blue glass bottles, except for the samples exposed in the UJ Dispensary (control). It was anticipated before the study that the zones of inhibition for the samples exposed to the UJ Dispensary would stay the same as the UJ Dispensary 63 was chosen as the control environment and the samples should remain the same in the controlled environment with little change in temperature and exposure to external factors.

5.2.2 Zones of Inhibition of Herbal Extract in amber bottles Statistically, there was a difference in the groups exposed to the Kitchen Scenario (p = 0.000), Office Scenario (p = 0.000) and to Decreased Temperature (p = 0.000). This can be concluded by looking at the p-values being less than 0.05 according to the Mann-Whitney U Test. The groups exposed in the UJ Dispensary (p = 0.167) and in the direct sunlight (p = 0.705) showed no statistically significant change.

When referring to Figure 4.10, it can be visualised that there is a substantial change from the BE to the AE measurements for the groups exposed to the Kitchen Scenario, Office Scenario and the Decreased Temperature, whereas in the groups exposed to the UJ Dispensary (mean BE = 8.500mm, mean AE = 8.383mm, p = 0.167) and to Direct Sunlight (mean BE = 8.450mm, mean AE = 8.433mm, p = 0.705), a very small, insignificant change can be seen. The BE values for the three groups that changed were more or less constant, with the highest difference in the AE group for the group exposed to the Kitchen Scenario (mean BE = 8.267mm, mean AE = 9.268mm, p = 0.000), then the Office Scenario (mean BE = 8.083mm, mean AE = 8.767mm, p = 0.000) and then the group exposed to Decreased Temperature (mean BE = 8.183mm, mean AE = 8.767mm, p = 0.000).

Even with the box and whisker graphs (Figure 4.11 to 4.15), it can be established that there was almost no change in the antibacterial measurements for the groups exposed in the UJ Dispensary and to Direct Sunlight. The other groups showed significant changes, where the antibacterial measurements increased. The group of samples that were exposed to the Decreased Temperature, had a higher negative outlier in the AE part than in the BE part. This can also be seen with the skewness and kurtosis values for the AE group being higher than the suggested range of -1 to 1.

5.2.3 Zones of Inhibition of Mother Tincture in blue bottles When referring to the box and whisker graphs (Figure 4.17 to 4.21), it shows that all the sample groups, except for the samples exposed in the UJ Dispensary, showed an increase in the antibacterial measurements. The group of samples that were exposed in the UJ Dispensary displayed no difference in the pattern of measurements, only a higher positive outlier for the BE group, but the average set of measurements are the same, with a slight difference of 0.25mm in the median measurement from the BE group to the AE group. 64

Statistically, it shows that there is no difference between the BE and AE measurements for the samples that were exposed in the UJ Dispensary. The p-value for the comparison between the BE and AE measurements for this group is higher than 0.05 (value is 0.394), therefore showing no difference in the measurements statistically.

The other groups of samples (those exposed to Direct Sunlight, Kitchen Scenario, Office Scenario and Decreased Temperature), showed an increase in the antibacterial measurements when referring to the box and whisker graphs as well as the statistical values. The p-values for all the groups are less than 0.05 (according to the Mann-Whitney U Test), showing statistical significance.

According to Figure 4.16, the increase in antibacterial measurements can be seen for all the groups except for the group exposed in the UJ Dispensary (p = 0.394). For the four groups that had an increase, it can be seen that the BE measurements for each group had an average range of 6.2mm and 6.4mm. It then shows that the biggest difference was with the samples exposed to the Decreased Temperature (mean BE = 6.267mm, mean AE = 7.333mm, p = 0.000); followed by the samples exposed in the Kitchen Scenario (mean BE = 6.333mm, mean AE = 6.783mm, p = 0.000), then by the samples in the Office Scenario (mean BE = 6.300mm, mean AE = 6.667mm, p = 0.000) and finally the ones exposed to Direct Sunlight (mean BE = 6.367mm, mean AE = 6.617mm, p = 0.005). The samples in the UJ Dispensary group, remained the same between 6.7mm and 6.8mm (mean), therefore showing no difference (p = 0.394).

5.2.4 Zones of Inhibition of Mother Tincture in amber bottles The p-values for this group of measurements were all below 0.05 except for the group exposed in the UJ Dispensary. This shows that there was not statistical difference between the BE and AE for the samples exposed in the UJ Dispensary, but there was, however, a change in the antibacterial measurements for the samples exposed to Direct Sunlight, Decreased Temperature and in the Kitchen and Office Scenarios.

The bar graph (Figure 4.22) that shows the antibacterial measurements comparison between the BE and AE groups, shows no difference in the measurements for the samples exposed in the UJ Dispensary. All the box and whisker graphs for the other sets of measurements (samples exposed to Direct Sunlight, Kitchen Scenario, Office Scenario and Decreased Temperature) show an increase in the antibacterial measurements. The highest difference was for the samples exposed to Direct Sunlight (mean BE = 6.333mm, mean AE = 6.933mm, p = 0.000), followed by the samples 65 in the Office Scenario (mean BE = 6.233mm, mean AE = 6.800mm, p = 0.000), then the Kitchen Scenario (mean BE = 6.250mm, mean AE = 6.683mm, p = 0.000) and finally the samples exposed to Decreased Temperature (mean BE = 6.417mm, mean AE = 6.683mm, p = 0.007).

5.2.5 Herbal Extracts Zones of Inhibition Comparison The herbal extract samples in the blue and amber glass bottles exposed to the same environmental conditions were compared. The samples in the blue bottles exposed in the UJ Dispensary showed a greater difference between the BE and AE (mean BE = 8.450mm; mean AE = 8.767mm; difference = 0.317mm) set of values than the samples in the amber glass bottles (mean BE = 8.500mm; mean AE = 8.383mm; difference = -0.117mm), showing more stability in the samples in the amber glass bottles. Previous results and statistics showed that there was not a difference in the antibacterial measurement for the samples exposed in the UJ Dispensary. This was anticipated at the onset of the study as the UJ Dispensary was set as the control environment with the best possible storage conditions.

When comparing the samples exposed to Direct Sunlight, more stability is shown when the samples are stored in amber glass bottles. The difference in the antibacterial measurements for the samples in the blue bottles (mean BE = 8.267mm; mean AE = 8.533mm; difference = 0.266mm) were higher than the difference for the samples in the amber glass bottles (mean BE = 8.450mm; mean AE = 8.433mm; difference = -0.017). This shows that amber glass protects the contents of the bottle more against Direct Sunlight. By comparing the BE and AE measurements for the samples in the Direct Sunlight with the following environmental conditions, it can be seen that Direct Sunlight does not affect the samples as much as some of the other exposures.

The samples exposed to the Kitchen Scenario both showed an increase in the antibacterial measurements, although the samples in the blue glass bottles (mean BE = 8.217mm; mean AE = 8.800mm; difference = 0.583mm) showed a lower increase than the samples that were kept in the amber glass bottles (mean BE = 8.267mm; mean AE = 9.267mm; difference = 1.00mm).

With the Office Scenario exposure, the amber glass bottle samples (mean BE = 8.083mm; mean AE = 8.767mm; difference = 0.684mm) had a lower increase than the blue glass bottle samples (mean BE = 8.200mm; mean AE = 9.100mm; difference = 0.900mm); this was the same for the samples exposed to Decreased Temperature; samples in blue glass bottles (mean BE = 7.933mm; mean AE = 9.267mm; difference = 1.334mm) versus samples in amber glass bottles (mean BE = 8.183mm; mean AE = 8.767mm; difference = 0.584mm). The differences in the antibacterial 66 measurements differed by a higher increment than the samples exposed to the UJ Dispensary and Direct Sunlight. This shows that electromagnetic fields and radiation do have an effect on herbal extracts.

5.2.6 Mother Tinctures Zones of Inhibition Comparison The mother tincture samples in the blue and amber glass bottles exposed to the same environmental conditions were compared. With the samples exposed in the UJ Dispensary there were very slight change in the zone of inhibition measurements in the blue bottle samples (mean BE = 6.767mm; mean AE = 6.700mm; difference = -0.067mm) and no change in the amber glass bottle samples (mean BE = 6.383mm; mean AE = 6.383mm; difference = 0.000mm).

The samples exposed to Direct Sunlight, showed that both the blue (mean BE = 6.367mm; mean AE = 6.617mm; difference = 0.250mm) and the amber (mean BE = 6.333mm; mean AE = 6.933mm; difference = 0.600mm) glass sets of samples had an increased in antibacterial measurement. The blue glass bottle samples, however, had less of an increase than the amber glass bottle samples. This, however, differs from what was seen with the herbal extract in the blue and amber glass bottles (as discussed in 5.2.5) – with the herbal extract the amber glass bottle showed less change than the blue glass bottle showing more stability is obtained when stored in an amber glass bottle. With the mother tincture samples, it is the opposite, possibly showing that the type of extract or contents is also a factor, some mixtures might be better protected in different colour glass containers.

For the Kitchen Scenario, the increases were more or less the same for both sets of samples. The samples stored in the blue glass showed a difference of 0.450mm (mean BE = 6.333mm; mean AE = 6.783mm) and the samples stored in the amber glass bottles had a difference in zone of inhibition measurement of 0.433mm (mean BE = 6.250mm; mean AE = 6.683mm). The Office Scenario samples also showed an increase in the antibacterial measurements, but there was a higher increase for the samples in the amber glass bottles (mean BE = 6.233mm; mean AE = 6.800mm; difference = 0.567mm) than with the blue glass bottles (mean BE = 6.300mm; mean AE = 6.667mm; difference = 0.367mm).

The samples that were exposed to Decreased Temperatures also showed an increase in the antibacterial measurements, but the blue glass bottle (mean BE = 6.267mm; mean AE = 7.333mm; difference = 1.066mm) increase was higher than that of the amber glass bottle samples (mean BE = 6.417mm; mean AE = 6.683mm; difference = 0.266mm). 67

5.2.7 Dilutions and further testing The original protocol for this study was to dilute each sample with different ethanol concentrations including 20% ethanol, 62% ethanol and distilled water. The proposed dilutions were a 1:10 dilution (general dilution) and a 3:7 dilution. The reason for this decision was that in practise a number of these variations are readily performed by practitioners. A 3:7 dilution is for the first three dilutions from the mother tincture as stipulated in the German Homeopathic Pharmacopoeia (2001). In practise, 20% ethanol is often used as this is what is usually available in a homeopathic dispensary.

The groups of samples exposed in the UJ Dispensary and to the Direct Sunlight were tested BE and AE. Due to the inconsistency of the diluted samples as well as the cloudiness of their zones of inhibition, it was decided to stop with the testing of the dilutions. This brought about the decision to experiment with a non-polar solvent such as DMSO (Dimethyl sulphoxide). Discs were prepared with autoclaved DMSO and no zones of inhibition were seen. When DMSO was used as a solvent to dilute the C. officinalis, zones of inhibition were noted. These zones were clear whereas the diluted samples with the polar solvents (ethanol and distilled water) were cloudy. Due to the cloudiness of the zones of inhibition when the samples were diluted with a polar solvent, a diluted sample was tested on GC/MS to establish any change in active ingredients.

C. officinalis contains resin that is important for the healing and antimicrobial properties of the plant. The resin is found at the base of the flower heads and mainly protects the plant from insects and invading organisms (Blankespoor, 2012). Resins are generally not soluble in water and readily soluble in ethanol due to the consistency of the resin (Coppen, 1995). This might lead to the explanation for the dilutions with water that turns to a milky consistency delivering inconsistent zones of inhibition. Further research in this field is necessary.

This can bring difficulties for those mixing different herbal extracts and/or mother tinctures with each other, as the efficacy might be compromised. This can also pose a challenge when administering a herbal extract or mother tincture to a patient, as it is usually suggested to add a few drops of the extract or mother tincture to water before taken by the patient. This might be different for other plant extracts, therefore further research needs to be done.

5.3 Minimum Inhibitory Concentration (MIC) The herbal extract and the mother tincture has different extraction methods. The herbal extract obtained from Mediherb was extracted by a cold percolation procedure with 90% ethanol yielding a 1:2 dilution of the plant material. Only the flowers were used in this extract, after they were dried (Mediherb, 2013). The homeopathic mother tincture, extracted according to HAB method 3A as per the German Homeopathic Pharmacopoeia, was extracted in 62% ethanol, but with no force used. With this method, the fresh aerial parts of the plant are used for the extraction. The plant material is minced and kept in 62% ethanol for 10 days while swirling the mixture from time to time. The mixture is then filtered and the liquid part is then the homeopathic mother tincture (German Homeopathic Pharmacopoeia, 2001). Due to the different extraction methods used as well as the different plant parts that were used, it can be expected that the two samples would not have the exact same properties and strengths. The Mediherb herbal extract was certified as having a concentration of 500mg/ml, but the concentration of the homeopathic mother tincture was not known, therefore the MIC can only be compared as percentages.

The two control samples, as in plate 1 (as per table 4.9), were not exposed to any environmental conditions. The MIC for the control herbal extract was 6.25% and for the mother tincture it was 12.5%. This means that a minimum of only 6.25% of the herbal extract was needed to elicit an antibacterial (S. aureus) inhibitory response and a higher concentration of 12.5% of the mother tincture was needed to show the same response. Therefore it shows that the herbal extract has a stronger antibacterial inhibitory effect towards S. aureus than the mother tincture.

Roopashree et al. (2003) did a study on the antibacterial properties of a few herbal extracts including C. officinalis, also extracted with different solvents. The MIC was tested for all their samples, and it was reported that the C. officinalis extracted with ethanol had a MIC of 32- 64mg/ml. The dried flowers of the C. officinalis plant was used during the study. According to the results of the MIC on the control herbal extract, a 6.25% concentration is needed. When converting this to the actual concentration (stock concentration being 500mg/ml), it shows that the extract had a MIC of 31.25mg/ml, being well in the same region as the study done by Roopashree et al. (2003).

5.3.1 MIC Comparison of the Mediherb herbal extract Two herbal extract samples were exposed to each environmental condition; one sample in a blue glass bottle and one in an amber glass bottle. Both blue and amber samples that were exposed to a “best possible” environment, being in the UJ Dispensary, where the temperature was kept constant between 20ºC and 22ºC. No artificial lighting or direct sunlight and no influence of 69 electromagnetic waves, showed that the same concentration of the extract was needed to elicit the same response as the control sample, the control concentration was at 6.25% and both the UJ dispensary samples needed a concentration of 6.25%.

Both the samples in the blue and amber glass bottles, exposed to Direct Sunlight, showed that an increased concentration of the extract was needed to elicit the same MIC as the control extract. The control sample had a MIC of 6.25% whereas both samples exposed to Direct Sunlight had a MIC of 12.5%. This shows that the antibacterial properties of the samples exposed to Direct Sunlight decreased, possibly due to degradation of the extract in the sun. During the exposure of these samples, the temperature also increased, thereby the exposure factor was not just direct sunlight but also increased temperature.

The sample in the blue bottle exposed to the Kitchen Scenario (microwave/electromagnetic waves and a slight increase in temperature) had the same MIC as the control sample. The sample in the amber bottle, however, needed a higher concentration of the extract to elicit the same response. Therefore the MIC increased to 12.5%, where the control and the sample in the blue bottle had a MIC of 6.25%. This shows that the blue bottle showed more protection against electromagnetic waves from a microwave than the amber bottle. Some suggestions have been made that amber glass prevents shorter wavelengths of light to pass through, including ultraviolet light. It is suggested that other colours of glass do not to offer the same protection (Wrap, 2015). It is also dependent on what is stored in the glass container as some compounds would absorb light and others wouldn’t. Because there is no definitive research on the protection of different glass colours, further research should be done to confirm these findings.

The samples exposed to the Office Scenario also had a difference. The amber glass bottle showed more protection against the electromagnetic waves and radiation elicited from the Wi-Fi Router cell phones and computer devices. The sample in the blue bottle had a higher MIC (12.5%) than the sample in the amber bottle and the control (6.25%).

Both samples (in the blue and amber glass bottles) that were exposed to Decreased Temperature averaging at 4ºC had a higher MIC (12.5%) than the control (6.25%). This shows that the antibacterial properties of the samples exposed to a low temperature decreased, possibly due to degradation of the extract in the colder environment.

5.3.2 MIC Comparison for Homeopathic Mother Tincture The control homeopathic mother tincture had a MIC of 12.5%. The samples that were exposed in amber glass bottles all had a MIC of 12.5% AE in the UJ Dispensary, Direct Sunlight, Kitchen Scenario, Office Scenario, as well as the Decreased Temperature. This shows an overall stability of the MIC of the mother tincture when exposed to different environmental conditions in amber glass bottles.

The samples exposed to different environmental conditions in blue glass bottles showed different results when compared to the samples in the amber glass bottles. The samples exposed to the UJ Dispensary, Direct Sunlight and to the Office Scenario remained the same as the control mother tincture sample (MIC 12.5%). The samples exposed to the Kitchen Scenario and Decreased Temperature (averaging at 4ºC), however, had an increased MIC of 25%, showing that a higher concentration of the mother tincture was needed to elicit the same antibacterial action.

5.4 Correlation between Kirkby Bauer Disc method and MIC The Kirkby Bauer Disc Diffusion test is a relatively inexpensive and easy test, but it is also time consuming and labour intensive. The results of the disc diffusion test, however, are limited as it can only determine the resistance of bacterial growth caused by the antibacterial agent that correlates to the diameter of the zones of inhibition. Therefore the MIC cannot be established with the Kirkby Bauer Disc Diffusion method; it can only demonstrate that the antibacterial agent (in this case C. officinalis) was effective against the bacteria (in this case S. aureus) (Wilkins & Thiel, 1973; Klancnik et al., 2010).

Inconsistent results were obtained with the samples that were diluted with different ethanol concentrations and distilled water when tested with the Kirkby Bauer Disc Diffusion test. This might have been due to the change in the compounds of the samples, therefore further research is warranted to establish this. Diluted samples should be tested with the broth diluted MIC test to establish whether the MIC changes when diluted with different solvents.

The broth diluted MIC test is a quantitative test, whereas the Kirkby Bauer Disc Diffusion test is considered a qualitative test. With the broth dilution MIC test, being a quantitative test, the actual minimum inhibitory concentration can be established. This test is also less labour intensive, less amount of the sample is needed and the susceptibility of multiple antimicrobial agents can be tested simultaneously (Wiegand et al., 2008). This is an advantage above the agar based tests that should be considered when further research is done in this field. 71

5.5 Gas Chromatography Mass Spectrometry (GC/MS) The main aim of the GC/MS separation test was to establish a possible reason why the antibacterial measurements increased after exposure to different environmental conditions. Extract samples from the stock solution that were not exposed was used as a control for comparison purposes. An unexposed mother tincture sample was also tested. This allowed for the herbal extract and mother tincture to be compared, as both samples were the same species of C. officinalis, but extracted using a very different extraction method, showing that the extraction method is very important as is the parts of the plants that are used. When a sample is a specific herbal preparation, care should be taken about making assumptions about the herb’s action as this can largely depend on the type of extraction method used and the parts of the plant that was used for the extraction.

After the separation, a very long list of compounds were identified (Appendix 13). For the purpose and aim of this study, the highest 25 compounds of the herbal extract were listed – these compounds included some organic biological compounds of the C. officinalis and not compounds from the solvent used to extract the plant. It was compared (in Table 4.10) with the first 25 organic biological compounds of the mother tincture. There were insufficient compounds to compare with the extract. This also shows again that the type of extract is very important. Because of the higher amount of compounds in the herbal extract, it shows a general higher antibacterial action against S. aureus than the mother tincture. This can be compared with the results in the first part of the study, where the median zone of inhibition for the extract was 8.5mm and the median zone of inhibition for the mother tincture was 6.5mm. The compound Furfural is the main compound that contributes to Garcinia indica extract’s antibacterial action when tested in a study by Sutar et al. (2012). Furfural was found in the Mediherb herbal extract and not in the homeopathic mother tincture. The following compounds are some of the additional compounds that were found only in the Mediherb herbal extract, they also exhibit antibacterial actions: à-Copaene (Olivia et al., 2005), Hydroxyfurfural (Nafea et al., 2011), Isovaleric acid (Hayashida et al., 2008), Linoleic acid (Agoramoorthy et al., 2007) and Alpha Linolenic acid (Lee et al., 2002) to highlight a few; these were the compounds found with high concentrations. When compared to the mother tincture’s compounds, Isovaleric acid and Alpha Linolenic acid is also present, but the other main ones as found in the herbal extract are absent. The mother tincture contains Glycine, a well-known amino acid with antibacterial properties, mostly against Gram-negative bacteria though (Ilic et al., 2013). Phytol is also an important compound in the mother tincture; although it is antibacterial, it is not very effective against S.aureus (Ghaneian et al., 2015).

At the start of the study, when dilutions with 20% ethanol, 62% ethanol and distilled water were performed, it was found that the samples became milky and it was decided to exclude these samples from the Kirkby Bauer testing. With the GC/MS test, an extract sample (from the stock solution) was diluted with 90% ethanol (concentration of ethanol by which extraction took place), 20% ethanol and DMSO (non-polar solvent). The dilution was a 3:7 dilution, and the samples were separated on the GC/MS. The diluted samples were compared with each other and with the unexposed extract sample (stock solution - control). More compounds corresponded between the control sample and the sample that was diluted with the 90% ethanol. DMSO is toxic to humans, but it is a good solvent to be used in testing as it does not degrade samples. But the results (as seen in Table 4.11) showed it is best to dilute a sample with the same concentration ethanol / solvent that the extract was extracted with.

The last comparison was between the exposed extract samples that were exposed to the different environmental conditions in blue glass bottles. When referring to Table 4.12, it can be seen that a number of the compounds that were set out as the top 25 compounds (highest concentrations) were not present in the separation list of the samples that were exposed. The compound à-Cadinol has antibacterial properties; in Table 4.11, it can be seen that the % Area of à-Cadinol increased in the samples that were exposed to the different environmental conditions (Salbulal et al., 2006). À- Amyrin also has antibacterial properties (Goyal and Achla, 1987) and there was also an increase of à-Amyrin in the samples that were exposed to different environmental conditions. Due to the increased antibacterial compounds when exposed to the different environmental conditions, it might give an indication why the zones of inhibition increased in the Kirkby Bauer Disc Diffusion testing. Further research should be done, however, in the separation and analysis of compounds when exposed to environmental conditions.

CHAPTER 6: CONCLUSION 6.1 Conclusion It can be concluded from the Kirkby Bauer tests and the MIC tests that the UJ Dispensary is a good environment to keep herbal extracts and mother tinctures, as there was no or very minimal change to the zones of inhibition in the samples exposed in the UJ Dispensary. In the UJ Dispensary the samples were kept in a drawer away from direct sunlight with minimal exposure to external lighting. The temperature was also kept at an average of 21ºC. There was also no electromagnetic waves present in the dispensary. Therefore, any herbal extract or mother tincture will remain constant with minimal degradation or change when kept in a similar environment as the UJ Dispensary.

With the Kirkby Bauer tests it was proposed before the study that the antibacterial properties of the samples should either remain the same (no effect from environmental conditions) or become less (degrading). With initial analysis it was thought that the antibacterial properties were enhanced but, when looking at the further testing, it was actually determined that the samples became unstable, rather than enhanced.

The herbal extract and mother tincture samples that were stored in the amber glass bottles showed better resistance to the Direct Sunlight than the blue glass bottles. The herbal extract samples in both the blue and amber glass bottles, however, showed better stability than the mother tincture samples. Therefore it can also be concluded that different herbal materials will react slightly differently towards environmental conditions – a possible reason for this would be the active compounds in the extract.

In the Kitchen and Office Scenarios definite changes took place regardless of bottle colour. It can be concluded that an increased temperature above an average of 21ºC with electromagnetic waves present degrades herbal extracts and mother tinctures. A decreased temperature of average 4ºC also has a degrading effect on herbal extracts and mother tinctures.

An overall conclusion can be made that amber glass bottles offer more protection than blue glass bottles.

It can also be concluded that when one would like to dilute a herbal extract or mother tincture, the same solvent that was used during the extraction, should be used as this can also have an impact on the efficacy of the herbal extract or mother tincture. 74

The question of whether blue or amber glass offers the best protection for medicine also depends largely on the type of medicine. Therefore, further research is needed to bring more clarity to the matter. Regardless of blue or amber glass containers, the best environment to keep medication is: at an average of 21ºC, away from direct light, sunlight and electromagnetic waves.

6.2 Recommendations  More research should be done on different herbal extracts and mother tinctures when diluting them with different solvents. This would aid to establish whether it is safe for practitioners to mix herbal preparations, dilute them or for administration to their patients. By comparing two different botanical herbs and extract preparations with similar testing as in this study, a better understanding could be obtained.  An investigation into the combination of herbal extracts is also important. Research should be done by combining herbal extracts and establishing whether the combination is safe for human consumption and whether it still offers the same function as the individual extracts.  A similar test, with exposure to different environmental conditions, should be done with a different herb to establish whether the type of compounds that a herbal extract or mother tincture contain, have an effect on the way it protects itself against environmental conditions.  More samples should be separated on GC/MS or other separation techniques after exposure to environmental conditions to establish whether new compounds are formed when exposed to different environmental conditions.  A study to research the light passing through different colour glass and thickness of glass should be done.

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APPENDIX 1: Permission letter

UNIVERSITY OF JOHANNESBURG

25 July 2014 RE: Permission for Ms J Wilken to work in JOB2313 laboratory

To whom it may concern:

I herewith give MS Jan-Nita Wilken (student number 920200244) permission to use the laboratory facilities in JOB2313 in order to conduct experiments for her M Tech qualification with the title THE EFFECT OF ENVIRONMENTAL CONDITIONS ON THE ANTIBACTERIAL PROPERTIES OF CALENDULA OFFICINALIS. She will be performing her experiments under my supervision.

Regards

Dr Nicolette Niemann Postdoctoral Fellow Lecturer: Department of Biotechnology and Food Technology Doornfontein Campus John Orr Building Room 2210 Tel +27 11 559 6102 e-mail: [email protected]

APPENDIX 2: Certificate of Analysis: Nutrient Agar

APPENDIX 3: Certificate of Analysis: Nutrient Broth

APPENDIX 4: Certificate of Analysis: Mediherb Herbal Extract

APPENDIX 5: Certificate of Analysis: Homeopathic Mother Tincture

APPENDIX 6: Breakdown of samples

Group Bottle Bottle Number Number Contents Type Exposure Type 1 Extract Blue Control - UJ Dispensary 2 Ext 1:10 (20%) Blue Control - UJ Dispensary 3 Ext 1:10 (62%) Blue Control - UJ Dispensary 4 Ext 1:10 (Dist) Blue Control - UJ Dispensary 5 Ext D1 (20%) Blue Control - UJ Dispensary 6 Ext D1 (62%) Blue Control - UJ Dispensary 7 Ext D1 (Dist) Blue Control - UJ Dispensary 1 8 Mother Tincture Blue Control - UJ Dispensary 9 MT 1:10 (20%) Blue Control - UJ Dispensary 10 MT 1:10 (62%) Blue Control - UJ Dispensary 11 MT 1:10 (Dist) Blue Control - UJ Dispensary 12 MT D1 (20%) Blue Control - UJ Dispensary 13 MT D1 (62%) Blue Control - UJ Dispensary 14 MT D1 (Dist) Blue Control - UJ Dispensary 15 Extract Amber Control - UJ Dispensary 16 Ext 1:10 (20%) Amber Control - UJ Dispensary 17 Ext 1:10 (62%) Amber Control - UJ Dispensary 18 Ext 1:10 (Dist) Amber Control - UJ Dispensary 19 Ext D1 (20%) Amber Control - UJ Dispensary 20 Ext D1 (62%) Amber Control - UJ Dispensary 21 Ext D1 (Dist) Amber Control - UJ Dispensary 2 22 Mother Tincture Amber Control - UJ Dispensary 23 MT 1:10 (20%) Amber Control - UJ Dispensary 24 MT 1:10 (62%) Amber Control - UJ Dispensary 25 MT 1:10 (Dist) Amber Control - UJ Dispensary 26 MT D1 (20%) Amber Control - UJ Dispensary 27 MT D1 (62%) Amber Control - UJ Dispensary 28 MT D1 (Dist) Amber Control - UJ Dispensary 29 Extract Blue Direct Sunlight 30 Ext 1:10 (20%) Blue Direct Sunlight 31 Ext 1:10 (62%) Blue Direct Sunlight 32 Ext 1:10 (Dist) Blue Direct Sunlight 33 Ext D1 (20%) Blue Direct Sunlight 34 Ext D1 (62%) Blue Direct Sunlight 3 35 Ext D1 (Dist) Blue Direct Sunlight 36 Mother Tincture Blue Direct Sunlight 37 MT 1:10 (20%) Blue Direct Sunlight 38 MT 1:10 (62%) Blue Direct Sunlight 39 MT 1:10 (Dist) Blue Direct Sunlight 40 MT D1 (20%) Blue Direct Sunlight 90

41 MT D1 (62%) Blue Direct Sunlight 42 MT D1 (Dist) Blue Direct Sunlight 43 Extract Amber Direct Sunlight 44 Ext 1:10 (20%) Amber Direct Sunlight 45 Ext 1:10 (62%) Amber Direct Sunlight 46 Ext 1:10 (Dist) Amber Direct Sunlight 47 Ext D1 (20%) Amber Direct Sunlight 48 Ext D1 (62%) Amber Direct Sunlight 49 Ext D1 (Dist) Amber Direct Sunlight 4 50 Mother Tincture Amber Direct Sunlight 51 MT 1:10 (20%) Amber Direct Sunlight 52 MT 1:10 (62%) Amber Direct Sunlight 53 MT 1:10 (Dist) Amber Direct Sunlight 54 MT D1 (20%) Amber Direct Sunlight 55 MT D1 (62%) Amber Direct Sunlight 56 MT D1 (Dist) Amber Direct Sunlight 57 Extract Blue Microwave - Kitchen Scenario 58 Ext 1:10 (20%) Blue Microwave - Kitchen Scenario 59 Ext 1:10 (62%) Blue Microwave - Kitchen Scenario 60 Ext 1:10 (Dist) Blue Microwave - Kitchen Scenario 61 Ext D1 (20%) Blue Microwave - Kitchen Scenario 62 Ext D1 (62%) Blue Microwave - Kitchen Scenario 63 Ext D1 (Dist) Blue Microwave - Kitchen Scenario 5 64 Mother Tincture Blue Microwave - Kitchen Scenario 65 MT 1:10 (20%) Blue Microwave - Kitchen Scenario 66 MT 1:10 (62%) Blue Microwave - Kitchen Scenario 67 MT 1:10 (Dist) Blue Microwave - Kitchen Scenario 68 MT D1 (20%) Blue Microwave - Kitchen Scenario 69 MT D1 (62%) Blue Microwave - Kitchen Scenario 70 MT D1 (Dist) Blue Microwave - Kitchen Scenario 71 Extract Amber Microwave - Kitchen Scenario 72 Ext 1:10 (20%) Amber Microwave - Kitchen Scenario 73 Ext 1:10 (62%) Amber Microwave - Kitchen Scenario 74 Ext 1:10 (Dist) Amber Microwave - Kitchen Scenario 75 Ext D1 (20%) Amber Microwave - Kitchen Scenario 76 Ext D1 (62%) Amber Microwave - Kitchen Scenario 6 77 Ext D1 (Dist) Amber Microwave - Kitchen Scenario 78 Mother Tincture Amber Microwave - Kitchen Scenario 79 MT 1:10 (20%) Amber Microwave - Kitchen Scenario 80 MT 1:10 (62%) Amber Microwave - Kitchen Scenario 81 MT 1:10 (Dist) Amber Microwave - Kitchen Scenario 82 MT D1 (20%) Amber Microwave - Kitchen Scenario 83 MT D1 (62%) Amber Microwave - Kitchen Scenario 91

84 MT D1 (Dist) Amber Microwave - Kitchen Scenario 85 Extract Blue Office Scenario 86 Ext 1:10 (20%) Blue Office Scenario 87 Ext 1:10 (62%) Blue Office Scenario 88 Ext 1:10 (Dist) Blue Office Scenario 89 Ext D1 (20%) Blue Office Scenario 90 Ext D1 (62%) Blue Office Scenario 91 Ext D1 (Dist) Blue Office Scenario 7 92 Mother Tincture Blue Office Scenario 93 MT 1:10 (20%) Blue Office Scenario 94 MT 1:10 (62%) Blue Office Scenario 95 MT 1:10 (Dist) Blue Office Scenario 96 MT D1 (20%) Blue Office Scenario 97 MT D1 (62%) Blue Office Scenario 98 MT D1 (Dist) Blue Office Scenario 99 Extract Amber Office Scenario 100 Ext 1:10 (20%) Amber Office Scenario 101 Ext 1:10 (62%) Amber Office Scenario 102 Ext 1:10 (Dist) Amber Office Scenario 103 Ext D1 (20%) Amber Office Scenario 104 Ext D1 (62%) Amber Office Scenario 105 Ext D1 (Dist) Amber Office Scenario 8 106 Mother Tincture Amber Office Scenario 107 MT 1:10 (20%) Amber Office Scenario 108 MT 1:10 (62%) Amber Office Scenario 109 MT 1:10 (Dist) Amber Office Scenario 110 MT D1 (20%) Amber Office Scenario 111 MT D1 (62%) Amber Office Scenario 112 MT D1 (Dist) Amber Office Scenario 113 Extract Blue Decreased Temperature 114 Ext 1:10 (20%) Blue Decreased Temperature 115 Ext 1:10 (62%) Blue Decreased Temperature 116 Ext 1:10 (Dist) Blue Decreased Temperature 117 Ext D1 (20%) Blue Decreased Temperature 118 Ext D1 (62%) Blue Decreased Temperature 119 Ext D1 (Dist) Blue Decreased Temperature 9 120 Mother Tincture Blue Decreased Temperature 121 MT 1:10 (20%) Blue Decreased Temperature 122 MT 1:10 (62%) Blue Decreased Temperature 123 MT 1:10 (Dist) Blue Decreased Temperature 124 MT D1 (20%) Blue Decreased Temperature 125 MT D1 (62%) Blue Decreased Temperature 126 MT D1 (Dist) Blue Decreased Temperature 92

127 Extract Amber Decreased Temperature 128 Ext 1:10 (20%) Amber Decreased Temperature 129 Ext 1:10 (62%) Amber Decreased Temperature 130 Ext 1:10 (Dist) Amber Decreased Temperature 131 Ext D1 (20%) Amber Decreased Temperature 132 Ext D1 (62%) Amber Decreased Temperature 133 Ext D1 (Dist) Amber Decreased Temperature 10 134 Mother Tincture Amber Decreased Temperature 135 MT 1:10 (20%) Amber Decreased Temperature 136 MT 1:10 (62%) Amber Decreased Temperature 137 MT 1:10 (Dist) Amber Decreased Temperature 138 MT D1 (20%) Amber Decreased Temperature 139 MT D1 (62%) Amber Decreased Temperature 140 MT D1 (Dist) Amber Decreased Temperature

APPENDIX 7: Breakdown of samples

Group Bottle Bottle Number Number Contents Type Exposure Type 1 Extract Blue Control - UJ Dispensary 1 2 Mother Tincture Blue Control - UJ Dispensary 3 Extract Amber Control - UJ Dispensary 2 4 Mother Tincture Amber Control - UJ Dispensary 5 Extract Blue Direct Sunlight 3 6 Mother Tincture Blue Direct Sunlight 7 Extract Amber Direct Sunlight 4 8 Mother Tincture Amber Direct Sunlight 9 Extract Blue Microwave - Kitchen Scenario 5 10 Mother Tincture Blue Microwave - Kitchen Scenario 11 Extract Amber Microwave - Kitchen Scenario 6 12 Mother Tincture Amber Microwave - Kitchen Scenario 13 Extract Blue Office Scenario 7 14 Mother Tincture Blue Office Scenario 15 Extract Amber Office Scenario 8 16 Mother Tincture Amber Office Scenario 17 Extract Blue Decreased Temperature 9 18 Mother Tincture Blue Decreased Temperature 19 Extract Amber Decreased Temperature 10 20 Mother Tincture Amber Decreased Temperature

APPENDIX 8: Table used for noting measurements

APPENDIX 9: Pilot Study 1 and 2

Pilot Study 1

Pilot study done 2 -4 July 2014 Aim To establish the efficacy of the Kirkby -Bauer Disc Diffusion Method when testing the antibacterial action of Calendula officinalis on Staphylococcus aureus.

Preparation of Nutrient Agar (250ml) 3.25g Nutrient Broth (13g/L) plus 3.75g Agarose (1.5%) plus 250ml distilled water Dissolve in 500ml Glass bottle

Heat mixture to ensure Nutrient Broth and Agar dissolve properly

Autoclave Nutrient Agar to sterilise and pour into petri dishes (x11) Wait for dishes to cool and Nutrient Agar to solidify

Preparation of S.aureus liquid medium Add 0.65g Nutrient Broth to 50ml distilled water Dissolve in 500ml Glass bottle

Autoclave mixture for 1 hour

Add prepacked S.aureus to 10ml of the nutrient broth Incubate and swirl mixture for 24 hours at 37⁰C

Bacteria Innoculation and Calendula disc impregnation Spread 100µl of liquid S.aureus on each petri dish

Place 5 paper discs (6mm in diameter) on each bacteria spread petri dish and impregnate each disc with 15µl fluid as follows: ● 15 discs (3 petri dishes) with Calendula officinalis extract as supplied by Mediherb ● 15 discs (3 petri dishes) with a D1 Homoeopathic dilution of the Mediherb Calendula officinalis extract (3 parts extract to 7 parts 68% ethanol as per method HAB3A) ● 2 petri dishes with no discs (Control) ● 5 discs (1 petri dish) with 68% ethanol (Control) ● 5 discs (1 petri dish) with distilled water (Control) ● 5 discs (1 petri dish) with no fluid impregnation (Control)

Incubate petri dishes with lids facing upwards (error) for 20 hours at 37⁰C

Measure and note the inhibition areas

Summary of Results Both C. officinalis extract and D1 dilution showed antibacterial zones of inhibition

To consider with next experiment When incubating petri dishes, the lids should be facing down and not up. (Corrected by Dr Niemann as per standard procedure for incubation) Impregnation of 15µl of fluid seems to be too much as it looks like the bacteria is washed away from the disc; 15µl is the suggested volume as per research done. Another pilot study to be done with varying impregnation volumes. This can be established from the control discs impregnated with 68% Ethanol and distilled water.

Pilot study 2

Pilot study done 9-11 July 2014 Aim To establish whether better results would be obtained when decreasing the volume of the liquid that is impregnated on the paper discs (follow up from pilot study done 2-4 July)

Preparation of Nutrient Agar (150ml) 1.95g Nutrient Broth (13g/L) plus 2.25g Agarose (1.5%) plus 150ml distilled water Dissolve in 500ml Glass bottle

Heat mixture to ensure Nutrient Broth and Agar dissolve properly

Autoclave Nutrient Agar to sterilise and pour into petri dishes (x7) Wait for dishes to cool and Nutrient Agar to solidify

Preparation of S.aureus liquid medium Add 0.65g Nutrient Broth to 50ml distilled water Dissolve in 500ml Glass bottle

Autoclave mixture for 1 hour

Add prepacked S.aureus to 10ml of the nutrient broth Incubate and swirl mixture for 24 hours at 37⁰C

Bacteria Innoculation and Calendula disc impregnation Spread 100µl of liquid S.aureus on each petri dish

Place 5 paper discs (6mm in diameter) on each bacteria spread petri dish and impregnate each disc with the Calendula officinalis extract as follows: ● Petri Dish 1: All 5 discs to be impregnated with 7.5µl, wait for 5 minutes and impregnate with another 7.5µl ● Petri Dish 2: All 5 discs to be impregnated with 7.5µl, wait for 3 minutes and impregnate with another 7.5µl Petri Dish 3: All 5 discs to be impregnated with ● 15µl Petri Dish 4: All 5 discs to be impregnated with ● 12µl Petri Dish 5: All 5 discs to be impregnated with ● 10µl ● Petri Dish 6: All 5 discs to be impregnated with 8µl ● Petri Dish 7: Control - no impregnation of discs

Incubate petri dishes with lids facing downwards for 24 hours at 37⁰C

Measure and note the inhibition areas

Conclusion With this study, the petri dishes were incubated with the lids facing downward; this might have been the cause why someof the discs moved. I do believe that 15µl (as per prevvious research) does yield the best results, but the 5 minute interval between the half volumes, the discs do not move.

APPENDIX 10: Exposure factor times and details

1. UJ Dispensary (Control) Date Room Temperature (ºC) Cupboard Temperature (ºC) 6/11/2014 20 21 7/11/2014 20 22 8/11/2014 20 21 9/11/2014 21 21 10/11/2014 20 21 11/11/2014 20 21 12/11/2014 21 21

2. Direct Sunlight (Specific temperature at specific time of the day during exposure) Time 6/11/14 7/11/14 8/11/14 9/11/14 10/11/14 11/11/14 12/11/14 9:00 30ºC 36ºC 32ºC 31ºC 30ºC 31ºC 30ºC 10:00 33ºC 36ºC 36ºC 34ºC 32ºC 32ºC 33ºC 11:00 32ºC 32ºC 34ºC 35ºC 33ºC 34ºC 32ºC 12:00 32ºC 29ºC 38ºC 36ºC 33ºC 34ºC 32ºC 13:00 36ºC 27ºC 35ºC 36ºC 36ºC 34ºC 36ºC 14:00 33ºC 29ºC 35ºC 34ºC 33ºC 32ºC 33ºC 15:00 29ºC 34ºC 34ºC 32ºC 30ºC 30ºC 29ºC 16:00 30ºC 31ºC 30ºC 30ºC 30ºC 28ºC 29ºC 17:00 27ºC 27ºC 27ºC 28ºC 27ºC 26ºC 27ºC

3. Kitchen Scenario (Exposure times and temperature of kitchen) Date Exposure Times 6/11/14 8:35-8:55 10:23-10:28 14:02-14:32 15:16-15:21 7/11/14 8:03-8:33 13:40-13:55 16:02-16:17 8/11/14 9:26-9:36 11:13-11:23 13:50-14:10 15:12-15:22 16:30-16:40 9/11/14 10:40-11:00 12:46-13:01 15:12-15:27 16:40-17:00 10/11/14 9:36-9:56 12:25-12:30 13:01-13:31 16:16-16:21 11/11/14 9:03-9:33 12:39-12:54 15:02-15:17 12/11/14 8:25-8:35 12:14-11:24 12:50-13:10 14:12-14:22 15:30-15:40

Temperature of kitchen (ºC) Date 9:00 12:00 16:00 6/11/14 22 23 25 7/11/14 24 26 25 8/11/14 24 26 26 9/11/14 23 25 25 10/11/14 22 24 24 11/11/14 22 23 24 12/11/14 23 23 24

100

4. Office Scenario (Specific temperature at specific time of the day during exposure) Time 6/11/14 7/11/14 8/11/14 9/11/14 10/11/14 11/11/14 12/11/14 9:00 22ºC 24ºC 24ºC 23ºC 22ºC 22ºC 23ºC 10:00 22ºC 25ºC 25ºC 24ºC 23ºC 22ºC 23ºC 11:00 22ºC 25ºC 26ºC 24ºC 23ºC 22ºC 23ºC 12:00 23ºC 26ºC 26ºC 25ºC 24ºC 23ºC 23ºC 13:00 23ºC 25ºC 25ºC 25ºC 25ºC 24ºC 23ºC 14:00 23ºC 25ºC 25ºC 25ºC 34ºC 24ºC 24ºC 15:00 23ºC 25ºC 25ºC 25ºC 24ºC 23ºC 24ºC 16:00 24ºC 25ºC 25ºC 25ºC 24ºC 24ºC 24ºC 17:00 24ºC 24ºC 25ºC 24ºC 24ºC 23ºC 23ºC

5. Decreased Temperature Time 6/11/14 7/11/14 8/11/14 9/11/14 10/11/14 11/11/14 12/11/14 9:00 4ºC 4ºC 4ºC 3ºC 4ºC 4ºC 4ºC 10:00 4ºC 5ºC 4ºC 4ºC 4ºC 4ºC 4ºC 11:00 5ºC 4ºC 4ºC 4ºC 4ºC 4ºC 4ºC 12:00 4ºC 4ºC 4ºC 4ºC 4ºC 4ºC 3ºC 13:00 4ºC 4ºC 4ºC 4ºC 4ºC 4ºC 3ºC 14:00 3ºC 3ºC 4ºC 4ºC 4ºC 4ºC 4ºC 15:00 4ºC 4ºC 5ºC 4ºC 5ºC 4ºC 4ºC 16:00 4ºC 4ºC 4ºC 4ºC 4ºC 4ºC 4ºC 17:00 4ºC 4ºC 4ºC 4ºC 4ºC 4ºC 4ºC

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APPENDIX 11: MIC sample size and details

Sample Contents Bottle Colour Exposure Plate 1 Herbal extract Stock bottle (Control) None 1 Left 2 Mother tincture Stock bottle (Control) None 1 Right 3 Herbal extract UJ Dispensary Blue 2 Left 4 Herbal extract Direct Sunlight Blue 2 Right 5 Herbal extract Kitchen Scenario Blue 3 Left 6 Herbal extract Office Scenario Blue 3 Right 7 Herbal extract Decreased Temperature Blue 4 Left 8 Herbal extract UJ Dispensary Amber 4 Right 9 Herbal extract Direct Sunlight Amber 5 Left 10 Herbal extract Kitchen Scenario Amber 5 Right 11 Herbal extract Office Scenario Amber 6 Left 12 Herbal extract Decreased Temperature Amber 6 Right 13 Mother tincture UJ Dispensary Blue 7 Left 14 Mother tincture Direct Sunlight Blue 7 Right 15 Mother tincture Kitchen Scenario Blue 8 Left 16 Mother tincture Office Scenario Blue 8 Right 17 Mother tincture Decreased Temperature Blue 9 Left 18 Mother tincture UJ Dispensary Amber 9 Right 19 Mother tincture Direct Sunlight Amber 10 Left 20 Mother tincture Kitchen Scenario Amber 10 Right 21 Mother tincture Office Scenario Amber 11 Left 22 Mother tincture Decreased Temperature Amber 11 Right

102

APPENDIX 12: GC/MS sample size and details

1. Extract – no exposure 2. Mother tincture – no exposure 3. Extract 3:7 dilution with 90% ethanol 4. Extract 3:7 dilution with 20% ethanol 5. Extract 3:7 dilution with DMSO 6. Extract exposed in the UJ Dispensary in a blue bottle 7. Extract exposed to direct sunlight in a blue bottle 8. Extract exposed in the microwave scenario 9. Extract exposed in the office scenario 10. Extract exposed to decreased temperature

103

APPENDIX 13: Raw data for all tests

Before Exposure After Exposure Bottle Contents Exposure Statistic Std. Error Statistic Std. Error Mean 8.4500 .0733 Mean 8.7667 0.1453 95% Confidence Lower Bound 8.3001 95% Confidence Lower Bound 8.4695 Interval for Mean Upper Bound 8.5999 Interval for Mean Upper Bound 9.0638 5% Trimmed Mean 8.4630 5% Trimmed Mean 8.7963 Median 8.5000 Median 8.7500 Variance 0.1612 Variance 0.6333

Std. Deviation 0.4015 Std. Deviation 0.7958 Blue

Extract Minimum 7.5000 Minimum 7.0000

UJ Dispensary UJDispensary Maximum 9.0000 Maximum 10.0000 Range 1.5000 Range 3.0000 Interquartile Range 0.6250 Interquartile Range 1.1250 Skewness -0.2397 .4269 Skewness -0.3124 0.4269 Kurtosis -0.4266 .8327 Kurtosis 0.0133 0.8327 Mean 6.7667 .0748 Mean 6.7000 0.0567 95% Confidence Lower Bound 6.6137 95% Confidence Lower Bound 6.5840 Interval for Mean Upper Bound 6.9196 Interval for Mean Upper Bound 6.8160 5% Trimmed Mean 6.7500 5% Trimmed Mean 6.6944 Median 6.7500 Median 6.5000 Variance 0.1678 Variance 0.0966

Std. Deviation 0.4097 Std. Deviation 0.3107 Blue Minimum 6.0000 Minimum 6.0000

UJ Dispensary UJDispensary Maximum 8.0000 Maximum 7.5000 Mother Tincture Mother Range 2.0000 Range 1.5000 Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.6921 .4269 Skewness 0.4064 0.4269 Kurtosis 1.7813 .8327 Kurtosis 0.1483 0.8327 Mean 8.2667 .0920 Mean 8.5333 0.1015 95% Confidence Lower Bound 8.0785 95% Confidence Lower Bound 8.3257 Interval for Mean Upper Bound 8.4549 Interval for Mean Upper Bound 8.7410 5% Trimmed Mean 8.2870 5% Trimmed Mean 8.5093 Median 8.5000 Median 8.5000 Variance 0.2540 Variance 0.3092

Std. Deviation 0.5040 Std. Deviation 0.5561 Blue

Extract Minimum 7.0000 Minimum 7.5000

Direct Sunlight Direct Maximum 9.0000 Maximum 10.0000 Range 2.0000 Range 2.5000 Interquartile Range 0.5000 Interquartile Range 1.0000 Skewness -0.4220 .4269 Skewness 0.5055 0.4269 Kurtosis 0.0416 .8327 Kurtosis 0.1576 0.8327 Mean 6.3667 .0532 Mean 6.6167 0.0572 95% Confidence Lower Bound 6.2578 95% Confidence Lower Bound 6.4998 Interval for Mean Upper Bound 6.4756 Interval for Mean Upper Bound 6.7336 5% Trimmed Mean 6.3519 5% Trimmed Mean 6.6111 Median 6.5000 Median 6.5000 Variance 0.0851 Variance 0.0980

Std. Deviation 0.2916 Std. Deviation 0.3130 Blue Minimum 6.0000 Minimum 6.0000

Direct Sunlight Direct Maximum 7.0000 Maximum 7.5000 Mother Tincture Mother Range 1.0000 Range 1.5000 Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.0861 .4269 Skewness 0.7019 0.4269 Kurtosis -0.3575 .8327 Kurtosis 1.3143 0.8327

104

Mean 8.2167 .0854 Mean 8.8000 0.0947 95% Confidence Lower Bound 8.0421 95% Confidence Lower Bound 8.6063 Interval for Mean Upper Bound 8.3913 Interval for Mean Upper Bound 8.9937 5% Trimmed Mean 8.1944 5% Trimmed Mean 8.7778 Median 8.0000 Median 8.5000 Variance 0.2187 Variance 0.2690

Std. Deviation 0.4676 Std. Deviation 0.5186 Blue

Extract Minimum 7.5000 Minimum 8.0000

Maximum 9.5000 Maximum 10.0000 Kitchen Scenario Kitchen Range 2.0000 Range 2.0000 Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.6134 .4269 Skewness 0.7072 0.4269 Kurtosis 0.7554 .8327 Kurtosis 0.3076 0.8327 Mean 6.3333 .0603 Mean 6.7833 0.0665 95% Confidence Lower Bound 6.2099 95% Confidence Lower Bound 6.6474 Interval for Mean Upper Bound 6.4567 Interval for Mean Upper Bound 6.9192 5% Trimmed Mean 6.3148 5% Trimmed Mean 6.7407 Median 6.5000 Median 6.5000 Variance 0.1092 Variance 0.1325

Std. Deviation 0.3304 Std. Deviation 0.3640 Blue Minimum 6.0000 Minimum 6.5000

Maximum 7.0000 Maximum 8.0000

Mother Tincture Mother Kitchen Scenario Kitchen Range 1.0000 Range 1.5000 Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.4835 .4269 Skewness 1.4768 0.4269 Kurtosis -0.6197 .8327 Kurtosis 2.9102 0.8327 Mean 8.2000 .0661 Mean 9.1000 0.1232 95% Confidence Lower Bound 8.0648 95% Confidence Lower Bound 8.8481 Interval for Mean Upper Bound 8.3352 Interval for Mean Upper Bound 9.3519 5% Trimmed Mean 8.2037 5% Trimmed Mean 9.1111 Median 8.0000 Median 9.0000 Variance 0.1310 Variance 0.4552

Std. Deviation 0.3620 Std. Deviation 0.6747 Blue

Extract Minimum 7.5000 Minimum 8.0000 Maximum Maximum

Office Scenario Office 9.0000 10.0000 Range 1.5000 Range 2.0000 Interquartile Range 0.5000 Interquartile Range 1.5000 Skewness -0.2103 .4269 Skewness -0.0289 0.4269 Kurtosis -0.2343 .8327 Kurtosis -0.9467 0.8327 Mean 6.3000 .0455 Mean 6.6667 0.0692 95% Confidence Lower Bound 6.2070 95% Confidence Lower Bound 6.5251 Interval for Mean Upper Bound 6.3930 Interval for Mean Upper Bound 6.8082 5% Trimmed Mean 6.3056 5% Trimmed Mean 6.6667 Median 6.5000 Median 6.5000 Variance 0.0621 Variance 0.1437

Std. Deviation 0.2491 Std. Deviation 0.3790 Blue Minimum 6.0000 Minimum 6.0000 Maximum Maximum

Office Scenario Office 6.5000 7.5000 Mother Tincture Mother Range 0.5000 Range 1.5000 Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness -0.4301 .4269 Skewness -0.1508 0.4269 Kurtosis -1.9500 .8327 Kurtosis -0.3977 0.8327

105

Mean 7.9333 .0396 Mean 9.2833 0.1261 95% Confidence Lower Bound 7.8523 95% Confidence Lower Bound 9.0254 Interval for Mean Upper Bound 8.0144 Interval for Mean Upper Bound 9.5413 5% Trimmed Mean 7.9722 5% Trimmed Mean 9.2778 Median 8.0000 Median 9.0000 Variance 0.0471 Variance 0.4773

Std. Deviation 0.2171 Std. Deviation 0.6909 Blue

Extract Minimum 7.0000 Minimum 7.5000 Maximum 8.0000 Maximum 11.0000

Range 1.0000 Range 3.5000 Decreased Temperature Decreased Interquartile Range 0.0000 Interquartile Range 1.0000 Skewness -3.4950 .4269 Skewness 0.2720 0.4269 Kurtosis 12.5136 .8327 Kurtosis 1.0759 0.8327 Mean 6.2667 .0522 Mean 7.3333 0.0771 95% Confidence Lower Bound 6.1600 95% Confidence Lower Bound 7.1757 Interval for Mean Upper Bound 6.3733 Interval for Mean Upper Bound 7.4909 5% Trimmed Mean 6.2500 5% Trimmed Mean 7.3426 Median 6.2500 Median 7.5000 Variance 0.0816 Variance 0.1782

Std. Deviation 0.2857 Std. Deviation 0.4221 Blue Minimum 6.0000 Minimum 6.5000

Maximum 7.0000 Maximum 8.0000 Mother Tincture Mother

Range 1.0000 Range 1.5000 Decreased Temperature Decreased Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.4561 .4269 Skewness -0.0136 0.4269 Kurtosis -0.7479 .8327 Kurtosis -0.5346 0.8327 Mean 8.5000 .0830 Mean 8.3833 0.0854 95% Confidence Lower Bound 8.3302 95% Confidence Lower Bound 8.2087 Interval for Mean Upper Bound 8.6698 Interval for Mean Upper Bound 8.5579 5% Trimmed Mean 8.4815 5% Trimmed Mean 8.3889 Median 8.5000 Median 8.2500 Variance 0.2069 Variance 0.2187

Std. Deviation 0.4549 Std. Deviation 0.4676 Extract Amber Minimum 8.0000 Minimum 7.5000

UJ Dispensary UJDispensary Maximum 9.5000 Maximum 9.0000 Range 1.5000 Range 1.5000 Interquartile Range 1.0000 Interquartile Range 1.0000 Skewness 0.2944 .4269 Skewness 0.2320 0.4269 Kurtosis -1.1076 .8327 Kurtosis -1.3736 0.8327 Mean 6.3833 .0620 Mean 6.3833 0.0393 95% Confidence Lower Bound 6.2566 95% Confidence Lower Bound 6.3030 Interval for Mean Upper Bound 6.5101 Interval for Mean Upper Bound 6.4636 5% Trimmed Mean 6.3704 5% Trimmed Mean 6.3981 Median 6.5000 Median 6.5000 Variance 0.1152 Variance 0.0463 Std. Deviation 0.3395 Std. Deviation 0.2151

Amber Minimum 6.0000 Minimum 6.0000

UJ Dispensary UJDispensary Maximum 7.0000 Maximum 6.5000 Mother Tincture Mother Range 1.0000 Range 0.5000 Interquartile Range 0.5000 Interquartile Range 0.1250 Skewness 0.3232 .4269 Skewness -1.3283 0.4269 Kurtosis -0.7216 .8327 Kurtosis -0.2573 0.8327

106

Mean 8.4500 .0876 Mean 8.4333 0.0748 95% Confidence Lower Bound 8.2709 95% Confidence Lower Bound 8.2804 Interval for Mean Upper Bound 8.6291 Interval for Mean Upper Bound 8.5863 5% Trimmed Mean 8.4630 5% Trimmed Mean 8.4074 Median 8.5000 Median 8.5000 Variance 0.2302 Variance 0.1678

Std. Deviation 0.4798 Std. Deviation 0.4097 Amber Extract Minimum 7.5000 Minimum 8.0000

Direct Sunlight Direct Maximum 9.0000 Maximum 9.5000 Range 1.5000 Range 1.5000 Interquartile Range 1.0000 Interquartile Range 0.5000 Skewness -0.0401 .4269 Skewness 0.6622 0.4269 Kurtosis -1.4852 .8327 Kurtosis -0.0262 0.8327 Mean 6.3333 .0603 Mean 6.9333 0.1065 95% Confidence Lower Bound 6.2099 95% Confidence Lower Bound 6.7155 Interval for Mean Upper Bound 6.4567 Interval for Mean Upper Bound 7.1511 5% Trimmed Mean 6.3148 5% Trimmed Mean 6.9259 Median 6.5000 Median 7.0000 Variance 0.1092 Variance 0.3402 Std. Deviation 0.3304 Std. Deviation 0.5833

Amber Minimum 6.0000 Minimum 6.0000

Direct Sunlight Direct Maximum 7.0000 Maximum 8.0000 Mother Tincture Mother Range 1.0000 Range 2.0000 Interquartile Range 0.5000 Interquartile Range 1.0000 Skewness 0.4835 .4269 Skewness 0.5552 0.4269 Kurtosis -0.6197 .8327 Kurtosis -0.5773 0.8327 Mean 8.2667 .0708 Mean 9.2667 0.1240 95% Confidence Lower Bound 8.1218 95% Confidence Lower Bound 9.0132 Interval for Mean Upper Bound 8.4116 Interval for Mean Upper Bound 9.5202 5% Trimmed Mean 8.2778 5% Trimmed Mean 9.2963 Median 8.5000 Median 9.2500 Variance 0.1506 Variance 0.4609

Std. Deviation 0.3880 Std. Deviation 0.6789 Extract Amber Minimum 7.0000 Minimum 8.0000

Maximum 9.0000 Maximum 10.0000 Kitchen Scenario Kitchen Range 2.0000 Range 2.0000 Interquartile Range 0.5000 Interquartile Range 1.0000 Skewness -0.8297 .4269 Skewness -0.5623 0.4269 Kurtosis 2.7617 .8327 Kurtosis -0.6218 0.8327 Mean 6.2500 .0464 Mean 6.6833 0.0656 95% Confidence Lower Bound 6.1551 95% Confidence Lower Bound 6.5492 Interval for Mean Upper Bound 6.3449 Interval for Mean Upper Bound 6.8175 5% Trimmed Mean 6.2500 5% Trimmed Mean 6.6759 Median 6.2500 Median 6.5000 Variance 0.0647 Variance 0.1290 Std. Deviation 0.2543 Std. Deviation 0.3592

Amber Minimum 6.0000 Minimum 6.0000

Maximum 6.5000 Maximum 7.5000

Mother Tincture Mother Kitchen Scenario Kitchen Range 0.5000 Range 1.5000 Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.0000 .4269 Skewness 0.5040 0.4269 Kurtosis -2.1481 .8327 Kurtosis 0.3204 0.8327

107

Mean 8.0833 .0723 Mean 8.7667 0.0981 95% Confidence Lower Bound 7.9356 95% Confidence Lower Bound 8.5661 Interval for Mean Upper Bound 8.2311 Interval for Mean Upper Bound 8.9672 5% Trimmed Mean 8.0926 5% Trimmed Mean 8.7685 Median 8.0000 Median 9.0000 Variance 0.1566 Variance 0.2885

Std. Deviation 0.3957 Std. Deviation 0.5371 Extract Amber Minimum 7.0000 Minimum 7.5000 Maximum Maximum

Office Scenario Office 9.0000 10.0000 Range 2.0000 Range 2.5000 Interquartile Range 0.5000 Interquartile Range 0.6250 Skewness -0.3146 .4269 Skewness -0.5393 0.4269 Kurtosis 1.2853 .8327 Kurtosis 0.3914 0.8327 Mean 6.2333 .0522 Mean 6.8000 0.0743 95% Confidence Lower Bound 6.1267 95% Confidence Lower Bound 6.6481 Interval for Mean Upper Bound 6.3400 Interval for Mean Upper Bound 6.9519 5% Trimmed Mean 6.2130 5% Trimmed Mean 6.7778 Median 6.0000 Median 6.7500 Variance 0.0816 Variance 0.1655 Std. Deviation 0.2857 Std. Deviation 0.4068

Amber Minimum 6.0000 Minimum 6.0000 Maximum Maximum

Office Scenario Office 7.0000 8.0000 Mother Tincture Mother Range 1.0000 Range 2.0000 Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.7325 .4269 Skewness 0.8888 0.4269 Kurtosis -0.4294 .8327 Kurtosis 1.4565 0.8327 Mean 8.1833 .0610 Mean 8.7667 0.1240 95% Confidence Lower Bound 8.0585 95% Confidence Lower Bound 8.5132 Interval for Mean Upper Bound 8.3082 Interval for Mean Upper Bound 9.0202 5% Trimmed Mean 8.1667 5% Trimmed Mean 8.7963 Median 8.0000 Median 9.0000 Variance 0.1118 Variance 0.4609

Std. Deviation 0.3343 Std. Deviation 0.6789 Extract Amber Minimum 7.5000 Minimum 6.5000 Maximum 9.0000 Maximum 10.0000

Range 1.5000 Range 3.5000 Decreased Temperature Decreased Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.8969 .4269 Skewness -1.0050 0.4269 Kurtosis 0.7690 .8327 Kurtosis 3.3217 0.8327 Mean 6.4167 .0541 Mean 6.6833 0.0561 95% Confidence Lower Bound 6.3061 95% Confidence Lower Bound 6.5685 Interval for Mean Upper Bound 6.5272 Interval for Mean Upper Bound 6.7981 5% Trimmed Mean 6.4074 5% Trimmed Mean 6.6759 Median 6.5000 Median 6.5000 Variance 0.0876 Variance 0.0945 Std. Deviation 0.2960 Std. Deviation 0.3075

Amber Minimum 6.0000 Minimum 6.0000

Maximum 7.0000 Maximum 7.5000 Mother Tincture Mother

Range 1.0000 Range 1.5000 Decreased Temperature Decreased Interquartile Range 0.5000 Interquartile Range 0.5000 Skewness 0.0396 .4269 Skewness 0.5493 0.4269 Kurtosis -0.0824 .8327 Kurtosis 0.3824 0.8327

108

Gas Chromatography Mass Spectrometry

5e+006 Sample 1 4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

5e+006 Sample 2

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

5e+006

Sample 3 4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

109

5e+006

4e+006 Sample 4

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

1.4e+007

1.2e+007 Sample 5 1e+007

8e+006

6e+006

4e+006

2e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

6e+006

5e+006 Sample 6

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

110

6e+006

5e+006 Sample 7

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

6e+006

5e+006 Sample 8

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

6e+006

5e+006 Sample 9

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

111

6e+006

Sample 10 5e+006

4e+006

3e+006

2e+006

1e+006

0 Time (s) 250 500 750 1000 1250 1500 1750 2000 TIC

112