The Antifungal Effect of Valeriana Officinalis Mother Tincture and Herbal Extract on the Growth of Trichophyton Rubrum and Microsporum Canis in Vitro

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How to cite this thesis

Surname, Initial(s). (2012). Title of the thesis or dissertation (Doctoral Thesis / Master’s Dissertation). Johannesburg: University of Johannesburg. Available from: http://hdl.handle.net/102000/0002 (Accessed: 22 August 2017). The Antifungal Effect of Valeriana officinalis Mother Tincture and Herbal Extract on the Growth of Trichophyton rubrum and Microsporum canis in vitro

A research dissertation submitted to the Faculty of Health Sciences, University of Johannesburg, as partial fulfilment for the admission to the degree of Master of Technology in Homoeopathy

by Yara Bartolomeu (Student number: 201004230)

Supervisor: Date: 9 Dr Tsele-Tebakang M-Tech Hom (UJ)

Co-supervisor: Date: ______Dr Thierry Fonkui D-Tech Biotech (UJ)

DECLARATION

I, Yara Bartolomeu, declare that this research dissertation is my own, unaided work. It is being submitted for the degree of Master of Technology, at the University of Johannesburg, Department of Homoeopathy. It has not been submitted previously to this or any other institution for the purpose of obtaining a qualification.

______Signature of candidate

The ______day of______2019

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ABSTRACT

The Trichophyton rubrum (T. rubrum) and Microsporum canis (M. canis) species are the leading causative agents for superficial fungal infections worldwide (Richardson & Warnock, 2012; Havlickova et al., 2008). When individuals with compromised immune system come into contact with these pathogenic agents, they may experience invasive lesions, which in turn, increases their risk for mortality (Richardson & Warnock, 2012). Due to antifungal resistance continually increasing globally, this is aggressively threatening the success of antifungal treatments (Perlin et al., 2017).

The aim of this in vitro study was to determine the antifungal effect of Valeriana officinalis mother tincture and herbal extract on the growth of T. rubrum and M. canis using the Kirby-Bauer disc diffusion susceptibility test.

The study was undertaken by preparing fresh fungal spores of T. rubrum and M. canis, each standardized to 1.5 x 105 CFU/ml using a hemocytometer. Plates were subdivided into five sections and further labelled with the organism’s name and medications tested. The plates were streaked with 200 μl of each organisms, respectively, and spread in a 90° clockwise direction, using a sterile plastic scraper, over a 90 mm petri dish containing 20 ml of solidified Saboraud dextrose agar (SDA), to completely cover its entire surface (Mamba et al., 2010).

The species were tested against the Valeriana officinalis homoeopathic mother tincture (Ø), Valeriana officinalis herbal extract (HE), Terbinafine (positive control) and 45% and 67% ethanol (negative control) on SDA plates. Five sterile blank test discs were impregnated with 20 μl of each medication. The plates were allowed to dry at room temperature for 5 minutes. They were then incubated at 30°C for three, six and nine days. The zones of inhibition were measured in millimeters (mm) with a ruler and recorded (Mamba et al., 2010).

The results obtained were statistically analyzed using non-parametric tests – Kruskal-Wallis, Mann-Whitney and Friedman (Pallant, 2007). The results showed that the Valeriana officinalis HE had antifungal activity against both T. rubrum and M. canis however the Ø had antifungal activity against T. rubrum only.

To conclude, it is recommended that the plant extracts (Ø and HE) be tested at various concentration levels for optimal results, and to empirically determine the relationship between the level of concentration of the plant extracts and the range of the zones of inhibition.

DEDICATION

This research is dedicated to our Father, our Lord and Saviour, Jesus Christ, and the Holy Spirit, for they have walked with me through all the challenges and abounded me with faith, hope and love in this journey. I also dedicate this research to my wonderful family for their tremendous love and support.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank our Father, our Lord and Saviour, Jesus Christ, and the Holy Spirit for the strength to persevere and the courage to overcome challenges to complete my studies.

I would like to extend my utmost gratitude and appreciation to the following individuals for their amazing assistance and contribution towards the completion of my research dissertation:

 My supervisor, Dr Tebogo Tsele-Tebakang, for her encouragement, valuable guidance, contribution and support;  My co-supervisor, Dr Thierry Fonkui Youmbi, for keeping me on track and helping me to stay positive throughout;  My head of department, Dr Radmilla Razlog, for her encouragement and support;  All the lectures in the Department of Homoeopathy; for providing me with knowledge and discernment in the field of Homoeopathy;  My statistician, Mrs. Jaclyn de Klerk, for her assistance and statistical knowledge;  My mother, Ms. Maria da Conceição, for her constant prayers, strength, patience, perseverance, support, wisdom and love, for carrying me through every step of the way;  My father, Mr. Paulo Bartolomeu, for his support, encouragement and love;  My sister, Ms. Paula Bartolomeu, for her unconditional support throughout, her love, strength, positivity, and constantly reminding me who I am and what I can be;  My brothers, Mr. Sandro Bartolomeu & Juelsio Bartolomeu, for their love, patience, support, humor, prayer and encouragement;  My sister-in-law, Mrs. Andrea Rosa Bartolomeu, for her wise words, prayers, love and encouragement.

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TABLE OF CONTENTS

AFFIDAVIT...... II DECLARATION...... III ABSTRACT...... IV DEDICATION...... VI ACKNOWLEDGEMENTS...... VII LIST OF FIGURES...... XII LIST OF TABLES...... XIII LIST OF SYMBOLS...... XIV LIST OF ABBREVIATIONS...... XIV

CHAPTER ONE: INTRODUCTION

1.1 Problem Statement...... 1 1.2 Aim of Study...... 2 1.3 Objective of Study...... 2 1.4 Hypothesis...... 2 1.5 Null Hypothesis...... 2

CHAPTER TWO: LITERATURE REVIEW

2.1 Introduction...... 3 2.2 Ecological Classification of Dermatophytes...... 5 2.3 Identification and Morphology of Trichophyton...... 6 2.3.1 Trichophyton rubrum...... 7 2.4 Identification and Morphology of Microsporum canis...... 7 2.4.1 Microsporum canis...... 8 2.5 Pathogenesis of Trichophyton and Microsporum...... 9 2.6 Geographic Distribution of Dermatophytes...... 9

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2.7 Infections in Humans...... 10 2.7.1 Tinea pedis...... 10 2.7.2 Tinea capitis...... 11 2.7.3 Tinea corporis...... 11 2.7.4 Tinea cruris...... 12 2.7.5 Tinea unguium...... 12 2.7.6 Tinea manuum...... 12 2.8 Immunology of Dermatophytosis...... 13 2.8.1 Innate immune response...... 13 2.8.2 Adaptive immune response...... 13 2.8.3 Nonspecific response...... 14 2.8.4 Dermatophytes in immune-compromised...... 14 2.9 Diagnosis of Dermatophytes...... 15 2.10 Prevention of Dermatophytosis...... 17 2.11 Common Conventional Treatments...... 18 2.11.1 Side effects of conventional treatments...... 20 2.12 Antimicrobial Resistance...... 20 2.13 Complementary Medicine...... 21 2.14 Homoeopathy...... 22 2.14.1 History of Homoeopathy...... 22 2.14.2 The Law of Similars...... 22 2.14.3 The Minimum Dose...... 22 2.15 Valeriana officinalis...... 24 2.15.1 Valeriana officinalis herbal extract...... 26 2.15.2 Valeriana officinalis homoeopathic mother tincture...... 26 2.15.3 Homoeopathic symptom picture of Valeriana officinalis...... 27 2.16 Related Research...... 27

CHAPTER THREE: METHODOLOGY

3.1 Experimental Design...... 29 3.2 Preparation of Valeriana officinalis...... 29

3.3 Microorganisms...... 30 3.4 Positive Control...... 30 3.5 Negative Controls...... 30 3.6 Kirby-Bauer Disc Diffusion Method...... 30 3.6.1 Antifungal tests...... 31 3.7 Data Collection...... 32 3.8 Data Analysis...... 33

CHAPTER FOUR: RESULTS

4.1 Introduction...... 34 4.2 Control Plates...... 34 4.2.1 Negative controls...... 34 4.2.2 Positive control...... 34 4.3 Antifungal Experimental Results...... 35 4.3.1 Trichophyton rubrum...... 35 4.3.2 Microsporum canis...... 40 4.4 Interpretation of Output from Nonparametric Tests...... 46 4.4.1 The Kruskal-Wallis Test...... 47 4.4.2 The Mann-Whitney Test...... 50 4.4.3 The Friedman Test...... 55 4.5 Interpretation of Output from Boxplots...... 56

CHAPTER FIVE: DISCUSSION OF RESULTS

5.1 Discussion of Experimental Results...... 60 5.1.1 Discussion of experimental results of T. rubrum...... 60 5.1.2 Discussion of experimental results of M. canis...... 63 5.1.3 Discussion of the non-parametric tests...... 64 5.2 Conclusion...... 65

CHAPTER SIX: CONCLUSION & RECOMMENDATIONS

6.1 Conclusion...... 66 6.2 Limitation...... 67 6.3 Recommendations...... 68

REFERENCES...... 69

APPENDICES...... 80

1. Appendix A...... 80 1.1 Faculty of Health Sciences Higher Degrees Committee...... 80 2. Appendix B...... 81 2.1 Faculty of Health Sciences Research Ethics Committee...... 81 3. Appendix C...... 82 3.1 Department of Biotechnology & Food Technology...... 82

LIST OF FIGURES

Figure 2.1: Conidia of Trichophyton species...... 6 Figure 2.2: Culture morphology of T. rubrum on a SDA media...... 7 Figure 2.3: Conidia of Microsporum species...... 8 Figure 2.4: Culture morphology of M. canis on a SDA media...... 9 Figure 2.5: Process of Potentization...... 24 Figure 2.6: Valeriana officinalis plant...... 25 Figure 3.1: An illustration of the subdivision of the plates...... 31 Figure 3.2: Measuring the zone of inhibition...... 32 Figure 4.1: T. rubrum streaked plate No.1 after three days...... 36 Figure 4.2: T. rubrum streaked plate No.2 after three days...... 36 Figure 4.3: T. rubrum streaked plate No.3 after three days...... 37 Figure 4.4: T. rubrum streaked plate No.1 after six days...... 37

Figure 4.5: T. rubrum streaked plate No.2 after six days...... 38 Figure 4.6: T. rubrum streaked plate No.3 after six days...... 38 Figure 4.7: T. rubrum streaked plate No.1 after nine days...... 39 Figure 4.8: T. rubrum streaked plate No.2 after nine days...... 39 Figure 4.9: T. rubrum streaked plate No.3 after nine days...... 40 Figure 4.10: M. canis streaked plate No.1 after three days...... 42 Figure 4.11: M. canis streaked plate No.2 after three days...... 42 Figure 4.12: M. canis streaked plate No.3 after three days...... 43 Figure 4.13: M. canis streaked plate No.1 after six days...... 43 Figure 4.14: M. canis streaked plate No.2 after six days...... 44 Figure 4.15: M. canis streaked plate No.3 after six days...... 44 Figure 4.16: M. canis streaked plate No.1 after nine days...... 45 Figure 4.17: M. canis streaked plate No.2 after nine days...... 45 Figure 4.18: M. canis streaked plate No.3 after nine days...... 46 Figure 4.19: Boxplots of results of T. rubrum after three days...... 57 Figure 4.20: Boxplots of results of M. canis after three days...... 57 Figure 4.21: Boxplots of results of T. rubrum after six days...... 58 Figure 4.22: Boxplots of results of M. canis after six days...... 58 Figure 4.23: Boxplots of results of T. rubrum after nine days...... 59 Figure 4.24: Boxplots of results of M. canis after nine days...... 59

LIST OF TABLES

Table 2.1: Classification of antifungal therapy based on their structure...... 19 Table 4.1: Mean zones of inhibition of T. rubrum: after three days...... 35 Table 4.2: Mean zones of inhibition of T. rubrum: after six days...... 35 Table 4.3: Mean zones of inhibition of T. rubrum: after nine days...... 36 Table 4.4: Mean zones of inhibition of M. canis: after three days...... 41 Table 4.5: Mean zones of inhibition of M. canis: after six days...... 41 Table 4.6: Mean zones of inhibition of M. canis: after nine days...... 41 Table 4.7: The Kruskal-Wallis Test on T. rubrum...... 48 Table 4.8: The Kruskal-Wallis Test on M. canis...... 49

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Table 4.9: Significance Test for the Kruskal-Wallis Test...... 49 Table 4.10: The Mann-Whitney Test of Pair 1 – HE and Ø...... 51 Table 4.11: Significance Test for the Mann-Whitney Test of Pair 1 – HE and Ø...... 51 Table 4.12: The Mann-Whitney Test of Pair 2 – HE and Terbinafine...... 52 Table 4.13: Significance Test for the Mann-Whitney Test of Pair 2 – HE and Terbinafine...... 53 Table 4.14: The Mann-Whitney Test of Pair 3 – Ø and Terbinafine...... 54 Table 4.15: Significance Test for the Mann-Whitney Test of Pair 3 – Ø and Terbinafine...... 55 Table 4.16: Comparison overtime for each fungus and medication...... 56

LIST OF SYMBOLS

Ø: Homoeopathic mother tincture ˂: Smaller than ≥: Larger than and equal to N: Overall data set p: Probability value α: Alpha

LIST OF ABBREVIATIONS

AMR: Antimicrobial resistance CD4: Cluster differentiation 4 CMV: Cytomegalovirus DMSO: Dimethyl sulfoxide DTH: Delayed type hypersensitivity GC: Gas chromatography GC-MS: Gas chromatography-mass spectrometry GVHO: Graft-versus-host disease

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HE: Herbal extract HIV: Human immunodeficiency virus IFIs: Invasive fungal infections IgE: Immunoglobulin E IgG: Immunoglobulin G IH: Immediate hypersensitivity IL: Interleukin KOH: Potassium hydroxide MIC: Minimum inhibitory concentration PAS: Periodic acid-Schiff PCR: Polymerase chain reaction SDA: Sabouraud dextrose agar Sig: Significance Th: T-helper cell TLR: Toll-like receptor TNF: Tumor necrosis factor TTO: Tree tea oil UV: Ultraviolet ZI: Zone of inhibition

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CHAPTER ONE INTRODUCTION

1.1 Problem Statement

Trichophyton rubrum (T. rubrum) and Microsporum canis (M. canis), known as dermatophytes, are the most common fungal agents to cause dermatophytosis (Richardson & Warnock, 2012) and their distributions are worldwide (Havlickova et al., 2008).

The dermatophytes, T. rubrum and M. canis may cause tinea pedis, tinea capitis, tinea corporis, tinea cruris, tinea manuum, tinea barbae and tinea unguium (Richardson & Warnock, 2012). It is known that more than 20 – 25% of the world’s population has dermatophytosis making infections caused by these agents very common and frequent (Havlickova et al., 2008). In 2005, it was noted in sub- Saharan Africa, an estimation of 78 million cases of dermatophytosis (Alemayehu et al., 2016).

These dermatophytes are significant because of their ability to also cause invasive lesions in immuno-compromised people (Richardson & Warnock, 2012). Such invasive lesions include: majocchi’s granuloma, localized or subcutaneous lesions and/or the spreading to bones, lymph nodes, liver and nervous system (Liu, 2011; Calonje et al., 2011).

Antifungal resistance is a global problem as all serious fungal infections need the appropriate administrations in order for treatments to show successful outcomes. Unfortunately, there are only a certain amount of antifungal therapies available in the population therefore the emergence of resistance highly hinders patient management (Perlin et al., 2017).

Complementary medicine has increased its popularity mainly due to ineffective conventional therapies in treating chronic diseases, the high cases of antifungal

resistance occurring worldwide, the attitude towards health in general and the wanting of control over personal health and well-being (McFadden et al., 2010; Perlin et al., 2017). It is believed that around 60 – 80% of the population in the world still depends on complementary medicine to treat common diseases (Mekonnen et al., 2016).

1.2 Aim of the Study

The aim of this study was to determine the antifungal effect of Valeriana officinalis mother tincture and herbal extract on the growth of Trichophyton rubrum and Microsporum canis in vitro, using the Kirby-Bauer disk diffusion susceptibility test.

1.3 Objective of the Study

The main objective of this study was to expose T. rubrum and M. canis to mother tincture and herbal extract of Valeriana officinalis.

1.4 Hypothesis

It was hypothesized that both the mother tincture and the herbal extract of Valeriana officinalis would indicate antifungal effects against T. rubrum and M. canis species by illustrating clear zones of inhibition.

1.5 Null Hypothesis

It was hypothesized that both the mother tincture and the herbal extract of Valeriana officinalis would not indicate antifungal effects against T. rubrum and M. canis.

CHAPTER TWO LITERATURE REVIEW

2.1 Introduction

Infectious diseases are caused by pathogenic microbes or micro-organisms (bacteria, viruses, parasites or fungi) (World Health Organization, 2018). Infectious diseases caused by fungi have shown their ability to increase in the population and become a serious threat to the health of mankind (Mekonnen et al., 2016). There are more than 100 000 known species of fungi, but only a few are capable of causing infection (Richardson & Warnock, 2012).

Dermatophytosis, also known as “ringworm” or “tinea”, is a superficial infection affecting the keratinized tissue of the skin, hair and nails caused by mold-like filamentous fungi, called dermatophytes (Sharma et al., 2017). A “dermatophyte”, is the term used to describe a pathogenic fungus that grows on skin, hair and nails (Bottone, 2006).They fall into three types of genera: Trichophyton, Microsporum and Epidermophyton (Sharma et al., 2017) – which when combined, contain a sum of 40 or more species, of which 11 affect mainly humans (Bottone, 2006).

If an infection occurs in an immuno-competent individual, it will remain superficial and not have the ability to penetrate into deeper tissues or organs (Sharma et al., 2017). Immuno-compromised individuals are susceptible to invasive lesions or invasive fungal infections (IFIs) caused by these species (Richardson & Warnock, 2012). Although there are new forms of diagnosing and preventing IFIs, immuno- compromised individuals have the highest incidences of diseases, failure of conventional treatments and as well as mortality rates. Studies demonstrate crude mortality is as high as 35% in a 3 months period. Additionally, the infection and conventional treatment failure rates are even higher in patients that have persistent neutropenia and graft-versus-host disease (GVHD) (Pound et al., 2011). GVHD is a possible fatal condition in which the cells from the donor destroys the recipient’s

normal and healthy cells causing a variety of complications in the body (Smith, 2017).

In 2016, a clinical study compared the efficacy of a complementary medicine with a conventional medicine for oral fungal infection. The complementary medicine used in the study was tea tree oil (TTO) and the conventional medicine was clotrimazole. There were 36 participants and each individual were randomly put into groups. The first group received the TTO 0.25% rinse (diluting 5 ml oil/ 50 ml water – concentration 0.10%) to be done three times a day. The second group applied clotrimazole ointment three times a day. The study’s results indicated that group one (TTO) was more efficient than group two (clotrimazole) concluding that TTO, as a natural product, is a better alternative due to being a nontoxic agent compared to clotrimazole in the treatment of oral fungal infections (Maghu et al., 2016).

The need for complementary treatments has increased due to the inefficacy of conventional treatments in treating acute and chronic infections. Consequently, many effective botanical treatments have gained popularity for the eradication of fungal infections (McFadden et al., 2010). According to Pintas & Lio (2018), such botanical treatments (Melaleuca alternifolia, Quassia amara L., Azadirachta indica, Cassia alata, Aloe vera, Anethum graveolens, Artemisia sieberi, Cymbopogon citratus, Ageratina pichinchensis and Allium sativum) have experienced clinical trials and shown antifungal properties for skin conditions such as seborrheic dermatitis, tinea versicolor, tinea pedis and tinea unguium.

However, although the interest is growing in using complementary or natural products, there are not enough research on plant extracts that display antifungal properties therefore future research on plant extracts should be conducted to examine their safety and effectiveness in the treatment of fungal infections (Pintas & Lio, 2018). Hence the exploration of the plant, Valeriana officinalis, in this research study. Valeriana officinalis is a plant that is commonly used for treating conditions associated with the nervous system (Khare, 2004) but there are studies

indicating its antifungal properties against Candida albicans (C. albicans) (Wang et al., 2010). However there are no studies conducted as a homoeopathic mother tincture and herbal extract against species caused by the three types of genera stated.

2.2 Ecological Classification of Dermatophytes

Dermatophytes’ host specificity is attributed to the amount of keratin available. Dermatophytes are dependent on host specificity for classification. Classifications are based on three ecological groups: anthropophiles (human), zoophiles (animals) and geophiles (soil) (Lakshmipathy & Kannabiran, 2010).

The anthropophilic species affects mainly human but may cause infections in animals. The main form of transmitting the infection is human to human. Examples of anthropophilic species are: Trichophyton rubrum (T. rubrum), Trichophyton kanei (T. kanei), Trichophyton schoenleini (T. schoenleini), Trichophyton concentricum (T. concentricum), Trichophyton tonsurans (T. tonsurans), Microsporum gypseum (M. gypseum), Microsporum audouinii (M. audouinii), Microsporum ferrugineum (M. ferrugineum) and Epidermophyton floccosum (E. floccosum) (Lakshmipathy & Kannabiran, 2010).

The zoophilic species have only one animal host and develop as saprophytes but may infect humans. The transmission of infection is from animal to human. Examples of zoophilic species are: Trichophyton simii (T. simii) (monkeys), Trichophyton mentagrophytes (T. mentagrophytes) (rats), Trichophyton equinum (T. equinum) (horses), Microsporum canis (M. canis) (cats) and Microsporum nannum (M. nannum) (pigs) (Lakshmipathy & Kannabiran, 2010).

Species that are geophilic are closely related to keratinous hair and feathery elements and they are rare to elicit an infection (Liu, 2011) in either animals or humans. Examples of geophilic species are: T. ajelloi, T. terrestre, M. fulvum, M. gypseum, M. cookie and E. stockdaleae (Lakshmipathy & Kannabiran, 2010).

2.3 Identification and Morphology of Trichophyton

There are 24 recognized species in Trichophyton (Lakshimipathy & Kannabiran, 2010). The colonies of Trichophyton have a powdery, velvety or waxy pigmentation appearance with septate hyphae and rough surfaces. They consist of conidia which is used to identify species and groups. Microconidia are produced more than macroconidia (Kokare, 2008; Malcom et al., 2009). Microconidia grow singularly and laterally to the hyphae or in clusters of a grape-like appearance (Phat, 2016). Macroconidia’s shape is similar to a cigar or cylinder, with sizes ranging from 8 – 50 μm x 4 – 8 μm (Kokare, 2008). Figure 2.1 illustrates the conidia of Trichophyton species (Phat, 2016).

The walls of Trichophyton tend to be smoother and are either thick or thin. Their spores form in a chain-like appearance and can only be located inside hair shafts (Kokare, 2008). Majority of the species of Trichophyton require nitrogen as nutrition. Whilst other species such as T. tonsurans needs ornithine, citrul-line and Arginine and, T. mentagrophytes needs methionine. Acquiring knowledge on nutritional specificity helps to identify different species of Trichophyton (Lakshimipathy & Kannabiran, 2010).

Figure 2.1: Conidia of Trichophyton species (Phat, 2016).

2.3.1 Trichophyton rubrum

T. rubrum can survive outside the human body for a limited period only. This fungus is limited to the stratum corneum of the skin. It typically causes a chronic, relatively non-inflammatory disease in areas of the foot, mainly because palms and soles do not make antimicrobial substances. This increases the risk of acquiring a chronic infection in these areas. A glycoprotein called Mannan is found in the cell walls of T. rubrum and known to suppress cell-mediated immune function (in vitro) and associated to chronic clinical presentations (Dismukes et al., 2003). Figure 2.2 illustrates culture morphology of T. rubrum on a Sabouraud dextrose agar (SDA) media (Ellis & Kidd, 2016).

Figure 2.2: Culture morphology of T. rubrum on a SDA media (Ellis & Kidd, 2016).

2.4 Identification and Morphology of Microsporum

There are 16 recognized species in Microsporum (Lakshimipathy & Kannabiran, 2010). The colonies of Microsporum have a cottony, velvety or powdery appearance. Their pigmentation are of white to brown colours. They are spindle- shaped with thick, roughened walls and with several macroconidia and scanty microconidia. The macroconidia are divided into 5 – 10 cells with a length extending to 100 μm with a width of 6 – 8 μm (Kokare, 2008). The microconidia

grow singularly on the hyphae with a pear-shaped appearance (Phat, 2016). It is less likely for some Microsporum species to not produce either microconidia or macroconidia and nutritional specificity does not apply in this genus (Lakshimipathy & Kannabiran, 2010). Figure 2.3 illustrates the conidia of Microsporum species (Phat, 2016).

Figure 2.3: Conidia of Microsporum species (Phat, 2016).

2.4.1 Microsporum canis

The M. canis species grow quickly. They have a granular appearance with a yellow- orange to orange-brown tinge. In rare occasions, sporulation does not occur or they are atypical and further tests are needed for identification (Dismukes et al., 2003). A zoophilic infection caused by M. canis can stimulate a strong immune reaction in humans and therefore diagnosis has to be accurate in order for the appropriate form of treatment to take place (Amano et al., 2013). Figure 2.4 illustrates culture morphology of M. canis on SDA media (Ellis & Kidd, 2016).

Figure 2.4: Culture morphology of M. canis on a SDA media (Ellis & Kidd, 2016).

2.5 Pathogenesis of Trichophyton and Microsporum

The most common route for dermatophytes to enter the host is through injured skin, scars and burns. Arthrospores or conidia is what causes the infection in the host although hair does not provide nutrition that is needed for the dermatophyte to develop. However, the organism penetrates the superficial, non-living, keratinized tissue layer of the skin called the stratum corneum. It produces an enzyme called exo-enzyme keratinase and elicits inflammation at the location of the infection. The cardinal signs of inflammation at the infected site includes redness, swelling, warmth and hair loss. Normally, when an inflammatory response occurs, it causes for the organism to relocate to another site which then causes the classical sign of a lesion with a ring appearance (Lakshimipathy & Kannabiran, 2010).

2.6 Geographic Distribution of Dermatophytes

Dermatophytes are distributed throughout all continents, however, dermatophytosis is commonly found in developing countries. Regardless of dermatophytes being distributed worldwide, each species various in their geographic distribution and self-virulence (Alemayehu et al., 2016). Regions that are tropical and subtropical

(warm and humid environments) tends to favour the growth of these species (The Center for Food Security & Public Health, 2018) and therefore explains its high prevalence in Africa (El, 2010).

Due to the easy access and increase in mobility of people from continent to continent, it has prevented diseases caused by these species to be considered geographically limited to an area. Manifestations of diseases in countries that previously were not found, has been noted more frequently, and therefore it is important to acquire updated knowledge of the geographical distribution of dermatophytic agents for the correct diagnosis to be made and to understand possible risk factors (El, 2010).

2.7 Infections in Humans

Trichophyton and Microsporum species cause various forms of clinical manifestations of dermatophytosis such as tinea pedis, tinea capitis, tinea corporis, tinea cruris, tinea unguium and tinea manuum (Richardson & Warnock, 2012). They are depended on the causative agent, the location affected on the body and the immune status of the host (Liu, 2011). In humans, according to The Center for Food Security & Public Health (2018), the incubation period is 1 to 2 weeks.

2.7.1 Tinea pedis

Tinea pedis, also known as Athlete’s foot, is mostly common amongst athletes and favourable in humid or warm environments. The predisposing factors to acquire such condition are humid or warm environment in shoes combined with lack of hygiene, hyperhidrosis, age and acrocyanosis. Due to the organisms being anthropophilic (affecting humans), any person in contact with it will have it in their system for a while. There are three types of tinea pedis that describes its manifestations: chronic interdigital scaling, hyperkeratotic (thickening of the outer layer of skin) and dyshidrotic (pruritic vesicular eruptions) (Braun Falco et al., 2000).

2.7.2 Tinea capitis

Tinea capitis is the infection of the scalp commonly amongst school children and in males but disappears during puberty. The origin of the infection may be from a human or animal host. If an adult individual acquires the disease from an animal it is self-limited (it will disappear on its own). The Microsporum species only produce ectothrix infections where the dermatophyte is confined to the surface of the hair and grows down the hair follicle until the zone of keratinization is reached whilst Trichophyton species produce both ectothrix and endothrix infections. Endothrix infections cause the invasion of the hair shaft and grows internally in the hair cells (Braun Falco et al., 2000).

These infections (ectothrix and endothrix) also known as the Adamson’s fringe, do not involve non-keratinized hairs and therefore the fungi grows within the hair making it weak. Soon after, the hair falls out and a black oval pigmentation (also known as the “black dot”) is observed at the site. There are three types of tinea capitis: inflammatory, non-inflammatory and the “black dot” (Braun Falco et al., 2000).

2.7.3 Tinea corporis

Tinea corporis, is infection of the skin of the trunk and extremities (excluding hair, nails, palms, soles and groin). It is commonly in countries with a tropical-type weather. An individual can acquire the infection through human-human, animal- human or soil-human interactions. Predisposing factors are occupational or recreational exposure e.g. gymnasiums, locker rooms, military housing, contaminated clothing and immuno-compromised individuals. Lesions vary in appearance e.g. circular, arcuate, oval, vesicular, granulomatous or verrucous (Bolognia et al., 2012).

2.7.4 Tinea cruris

Tinea cruris, is an infection of the crotch or groins, commonly known as “crotch itch”. The first sign of infection is redness and itchiness in the fold between the inner thighs and scrotum. The lesions may appear circular and later serpiginous. The infection can either affect one or both sides of the thighs. Usually the scrotum is not affected but if it is involved and erosions or satellite pustules are present, cutaneous candidiasis is a possibility (Bolognia et al., 2012).

2.7.5 Tinea unguium

Tinea unguium, also known as Onychomycosis, is an infection of the nails. Firstly, tinea pedis and possibly tinea manuum, are present before tinea unguium manifests. The fungi tends to grow below the nail plate and then consumes the entire nail bed. Predisposing factors are decreased circulation, diabetes mellitus, neuropathies and immuno-compromised individuals. Toenails are mostly affected than fingernails and occasionally only a few nails are affected. There are five types of tinea unguium: distal and proximal subungual onychomycosis, white superficial onychomycosis, dystrophic onychomycosis and dermatophytoma (Braun Falco et al., 2000).

2.7.6 Tinea manuum

Tinea manuum is the infection of the hand and usually secondary to tinea pedis. Primary tinea manuum may arise due to being exposed at one’s occupation and this occurs in rarest occasions. Its clinical manifestations are similar to that of tinea pedis but most common manifestation is the hyperkeratotic form. The hands look and feel very dry and rough and should not be mistaken for chronic dermatitis (Braun Falco et al., 2000).

2.8 Immunology of Dermatophytosis

A host infected by dermatophytes undergoes immune responses that are either innate, adaptive (humoral and cell-mediated immunity) and/or nonspecific response mechanisms. The immune response which is considered most currently accurate is the cell-mediated immune response in order to control dermatophytosis (Mahajan & Sahoo, 2016).

2.8.1 Innate immune response

The beta-glucan are carbohydrate molecules located in the cell wall of dermatophytes that are recognized by the innate immune mechanisms (Dectin-1 and Dectin-2) which in turn activates receptors (TLR-2 and TLR-4). Dectin-1 intensifies the production of tumor necrosis factor-alpha (TNF-α) and IL-17, IL-6 and IL-10, which in turn, all will stimulate the adaptive immune mechanisms. In the presence of dermatophyte antigens, the keratinocytes such as trichophytin, will release a strong neutrophilic chemo-attractant called trichophytin (Mahajan & Sahoo, 2016).

2.8.2 Adaptive immune response

 Humoral immunity: This immunity is not considered to provide protection against dermatophytes. The high levels of specific IgE and IgG4 are observed in individuals who have chronic dermatophytosis and this is responsible for the positive result in an immediate hypersensitivity (IH) skin test to Trichophyton. On the contrary, low levels of Ig is found in individuals with a positive delayed type hypersensitivity (DTH) skin test. Serum IgE and IgG (mainly IgG4) are present and are associated with the IH skin test for Trichophyton antigens therefore assuring a T-helper cell 2 (Th2) which is also known as cluster of differentiation 4 T-cells (CD4 T-cells) reaction. The Th2 cells produces IL-4 which then causes an antibody isotype to switch to IgG4 and IgE (Mahajan & Sahoo, 2016; Koga, 2009).

 Cell-mediated immunity: It has been observed by multiple studies that the DTH skin test mediates dermatophytosis. The Th1 and Th2 cells determines the result of the infection. A positive DTH skin test to trichophytin is associated with an acute inflammation and the clearing up of the infection whereas in a chronic case, there are high levels in an IH test and low levels in a DTH skin test (Mahajan & Sahoo, 2016; Koga 2009).

2.8.3 Nonspecific response

 Unsaturated transferrin causes inhibition to dermatophytes when attached to its hyphae. Lipolysis is assisted by the commensal pityrosporum which then further causes for the increase in fatty acid for the inhibition of fungal growth (Mahajan & Sahoo, 2016).

2.8.4 Dermatophytes in immuno-compromised

Immuno-compromised patients who have disrupted cell-mediated immunity (e.g. human immunodeficiency virus (HIV) patients, organ transplant recipients, long- term use of medications such as corticosteroids, asplenia, innate immunodeficiency, and other infections such as cytomegalovirus (CMV)) (Saleh, 2017) may cause dermatophytes to cause further invasive lesions, this is according to The Center for Food Security & Public Health, 2018.

It is not very common of Trichophyton and Microsporum species to cause aggressive invasive lesions but T. rubrum and M. canis, specifically, may cause majocchi’s granuloma which is the localized dermal/ subcutaneous infection where the hair follicles are disrupted and the release of fungi into the skin causes a granulomatous inflammation but in the immune-compromised the infection is limited only to the extremities. It may also cause localized/subcutaneous lesions but with no hair follicles involved, and additionally, the spreading to bones, lymph nodes, liver and the nervous system (Liu, 2011; Calonje et al., 2011). The spreading to other organs may result in abscess, exophytic nodules and pseudomycetomas

(although it can occur in non-compromised individuals as well) (The Center for Food Security & Public Health, 2018).

2.9 Diagnosis of Dermatophytosis

According to The Center for Food Security & Public Health (2018), for a diagnosis to take place, the following should be adhered to:

 History taking from the patient  Physical examination of the patient  Microscopic examination o Infected hairs and skin are usually scraped for microscopic examinations. It is believed that when a hyphae is developing into an arthroconidia, it becomes a diagnostic trait, however, if the hyphae is singular therefore the aetiology could be due to other fungi. Arthroconidia are mainly located internal (endothrix) or external (ectothrix) aspects of the hair shaft. o The process of skin scrapings requires the edge of the lesion to be considered and hairs should be plucked out and not shaved or cut out in this area of the skin. o The ideal hairs to consider are the ones that are broken or have scales and that fluoresce using the Wood’s lamp. o Nail scrapings requires the nail bed or deeper parts of the nail if the infection is no longer superficial. o Mainly potassium hydroxide (KOH) clears the samples (skin, hair and nail) in order for visualization of the dermatophyte. Stains such as chlorazol black E, Parker blue-black ink, Swartz-Lamkin stain or Congo red are an addition to the KOH.  Fungal cultures o Fungal cultures are usually performed if the diagnosis is not certain or if the infection is not reacting to standardized treatment.

o Hair, skin and nail are the samples as mentioned under microscopic examination. o There are cases where different techniques are required if infections are located in sensitive areas or for identifying asymptomatic carriers: brushing of the hair, adhesive tape for collecting the samples, rubbing the site with a sterile toothbrush, and as well as, the use of a sterile cotton swab. o The timeframe for colonies to appear depends on the dermatophyte; may occur within 5 days to 4 weeks. The colony morphology arises in various forms depending on the medium. o SDA and other fungal culture media provides clear descriptions of the dermatophyte. o For identification of the dermatophyte: colony morphology, appearance of microconidia, macroconidia and other microscopic structures, urease production and nutritional recommendations. o Bromocresol purple-milk solids glucose, as a differential media, to assist in the diagnosis.  Special investigations/ tests o Some dermatophytes have the ability to fluoresce when undergoing the technique of Wood’s lamp due to the ultraviolet (UV) light that is used in this technique. M. canis, a zoophilic dermatophyte, exhibits fluorescence traits. It is known that certain topical antifungal agents may hide the fluorescence whilst alcohol suppresses or causes a non-specific fluorescence. o Fluorescence microscopy may use calcofluor white or other stains for visualization of structures of dermatophytes. o Special tests that have the ability to penetrate hairs in vitro or mating tests are rarely used.  Histological tests o These tests are extremely useful in deep-seated infections, especially of the nails. Visualizations is best with the periodic acid-Schiff (PAS) staining and hematoxylin-eosin staining.

o Polymerase chain reaction (PCR) tests are used for broad spectrum of microorganisms and its molecular methods of diagnosis has a high possibility to be more frequent. According to Mahajan & Sahoo (2016), the following tests helps in the fast detection and diagnosis of infection and also assists in determining any drug resistance: . Uniplex PCR is for the direct detection of dermatophyte in clinical samples: available as PCR-ELISA assay which allows for the identification of many species of dermatophytes. . Multiplex PCR is for the detection of fungi in dermatophytes: the test is available commercially and permits 21 dermatomycotic pathogen with subsequent DNA to be amplified using the agarose gel electrophoresis.

2.10 Prevention of Dermatophytosis

The Center for Food Security & Public Health (2018) stated that prevention of dermatophytosis in humans from a zoophilic cause requires the controlling of dermatophytes in animals. Animals that are infected should be treated appropriately and disinfection of infected sites should be adhered to. The amount of contact with animals that are infected should be decreased and safety-wear (e.g. gloves, protective clothing) should always be used when handling the animal. The following prevention factors should be noted:

Prevention factors:  Improved surveillance  Better living conditions  Adequate treatments  Hygiene  No contact with infected animals  Moisture control

2.11 Common Conventional Treatments

Common conventional antifungal agents, in the form of topical or oral administrations, are used to treat dermatophytosis. The common ingredients found in these antifungal agents are Terbinafine, Naftifine, Griseofulvin, Itraconazole, Clotrimazole, Ketoconazole, Fluconazole (Soares et al., 2013; Marks & Miller, 2017), and Amphotericin B (Hospenthal & Rinaldi, 2015). These antifungal agents are mainly safe and effective, however, occasional inefficacy does occur due to resistance mechanisms and as well as the high probability of recurrence of the superficial skin infections (Pintas & Lio, 2018).

According to Mahajan & Sahoo (2016), the following is the summary of how topical antifungal agents are managed for the treatment of tinea pedis, tinea corporis and tinea cruris and Table 2.1 illustrates the classification of antifungal therapy based on their structure:

 Imidazoles, prepared in a cream or lotion, for the treatment of tinea corporis, tinea cruris and tinea pedis, should be applied once or twice a day for approximately up to 4 weeks.  Triazoles, prepared in a solution, for the treatment of tinea pedis, should be applied once a day, for up to 52 weeks in co-existing tinea unguium.  Allylamines, prepared in a cream or powder, for the treatment of tinea corporis, tinea cruris, tinea pedis and tinea manuum, should be applied once to twice a day, for up to 2 to 4 weeks.  Others (Amolorfine and Amphotericin B), prepared in a cream and lipid gel based respectively, for the treatment of tinea corporis only, should be applied twice a day, for 4 weeks (Amolorfine) and 2 weeks (Amphotericin B).

Table 2.1: Classification of antifungal therapy based on their structure. Antifungal class Examples Antibiotics  Polyenes Amphotericin B, nystatin, natamycin  Heterocyclic Griseofulvin benzofuran Antimetabolite Flucytosine Azoles  Imidazoles Topical – clotrimazole, econazole, miconazole, bifonazole, fenticonazole, oxiconazole, tioconazole, sertaconazole, berconazole, luliconazole, eberconazole

Systemic – ketoconazole  Triazoles Itraconazole, fluconazole (also topical), voriconazole, posaconazole, isavuconazole, posoconazole, ravuconazole, pramiconazole, albaconazol Allylamines Terbinafine, butenafine, naftifine Echinocandins Caspofungin, anidulafungin, micafungin, aminocandin Sordarin derivatives GR135402, GM237354 Cell wall antagonist Caspofungin, micafungin Other agents Tolnaftate, ciclopirox, amorolfine, undecylenic acid, buclosamide, Whitefield’s ointment, benzoyl peroxide, zinc pyrithione, selenium sulfide, azelaic acid etc., nikkomycins, icofungipen Newer and potential therapies Demcidin, macrocarpal C

Terbinafine is the leading antifungal agent for the treatment of dermatophytosis due to its ability to inhibit fungal growth by causing a disruption in sterol biosynthesis

(Abdel-Rahman & Newland, 2009). However, its biochemical composition has side effects such as loss of taste of all major qualities (sour, sweet, bitter and salty), and mild to severe skin disorders such as macular exanthems, erythema multiforme and toxic epidermal necrolysis. The use of this drug in children is questionable as it has demonstrated side effects in clinical trial studies, such as headache, gastrointestinal complaints (altered eating habits, loss of appetite, stomach pain and diarrhoea), and a decrease in neutrophil counts. It also has the potential for long-lasting drug-drug interactions (Aronson, 2008).

2.11.1 Side effects of conventional treatments

The common side effects observed in the azole class are the following (Ghannoum & Perfect, 2016):  Gastrointestinal – nausea, abdominal pain, vomiting and diarrhoea  Hepatic dysfunction – hepatitis, cholestasis and fulminant hepatic failure  Adrenal insufficiency  Foetal abnormalities

In addition to the class, the triazoles have characteristic side effects that are discussed below (Ghannoum & Perfect, 2016):

 Fluconazole – alopecia and Stevens-Johnson syndrome  Itraconazole – hypertension, hypokalemia, peripheral oedema and heart failure  Voriconazole – photophobia, colour changes, blurred vision, photopsia, visual hallucinations, pancreatitis, hypoglycaemia and rash.

2.12 Antimicrobial Resistance

Antimicrobial resistance (AMR) occurs when pathogenic microorganisms (fungi, bacteria, parasites and viruses) undergo a change when antimicrobial agents (antifungals, antibiotics, antivirals, antimalarials and anthelmintics) are exposed to

them. Therefore, the medication given has no effect and the infection has a longer duration in the host which increases the risk to spread to the population (World Health Organization, 2018).

According to the World Health Organization (2018), AMR has the ability to threaten the effectiveness in prevention and treatment of a vast range of infections caused by pathogenic microorganisms. Due to the increase in new resistance mechanisms globally, is threatening the ability for successful treatments of common infectious diseases to have a longer duration of illness, disability and death. Ineffective conventional antimicrobial agents may further result medical measures (organ transplantation, cancer, management of diabetes, cancer chemotherapy and other important surgery) to be a high risk. The healthcare needed for individuals with resistant infections is highly expensive due to the illness’ duration, further tests needed and expensive medication.

2.13 Complementary Medicine

The General Regulations to the Medicines Act of South Africa defines complementary medicine as “any substance or mixture of substance that (i) originates from plants, minerals or animals; (ii) is used or intended to be used for, or manufactured or sold for use in assisting the innate healing power of a human being or animal to mitigate, modify, alleviate or prevent illness or the symptoms thereof or abnormal physical or mental state; and (iii) is used in accordance with the practice of the professions regulated under the Allied Health Professions Act, 1982 (Act No. 63 of 1982)” (Fourie et al., 2017). Homoeopathy is one of the professions that is part of the body of the Allied Health Professions Council of South Africa (Hanekom, 2011).

2.14 Homoeopathy 2.14.1 History of Homoeopathy

The word “Homoeopathy” (“homoios” means similar, “pathos” means suffering in Greek) was created in 1796, by the German founder, Doctor Samuel Hahnemann (1755 – 1843) (Reddy, 2018). Homoeopathic Medicine adheres to a holistic and natural approach in the treatment of illnesses (Grams, 2019). The treatment consists in taking the whole individual’s state and resulting in the eradication of their symptoms (Oftedal, 2009). The fundamental concept of Homoeopathy is based on the law of similars or “like cures like”, that was formerly presented by Hippocrates (known as the Father of Medicine) and Paracelsus (Reddy, 2018). This means that the homoeopathic medication or remedy that is administered, is like the condition or disease that is experienced or expressed by the individual, in their totality, and not like a disease classification (Grams, 2019).

2.14.2 The Law of Similars

The concept of “let like cures like”, similia similibus curentur, resulted in Dr. Samuel Hahnemann to codify this first principle and insert it into a medical system. He began with trials called “provings” on himself and later on administering medicinal substances to healthy volunteers called “provers”. The symptoms that were expressed by him and the prover while taking the medicinal substance provided the “homoeopathic picture” and the clinical indications for that specific medication (Grams, 2019).

2.14.3 The Minimum Dose

The term “potentization” was developed due to side effects Hahnemann observed in his provers, when he administered the medicinal substances in their crude form, although his provers showed signs of amelioration. Hahnemann minimized these side effects by potentization – series of dilutions and the process of vigorous agitation or “succussion” of the medicinal substances until traceable toxicity in the

substance were absent. This further developed the term “homoeopathic remedy”. Additionally, the more the substance is diluted, the more potent or dynamic the homoeopathic remedy is. Therefore, the “minimum dose”, is considered to have the maximum therapeutic effect with little to no side effects (Owen, 2007).

2.14.3.1 Potentization The starting material of a homoeopathic remedy is called a mother tincture. The mother tincture is further potentized and gives rise to numerous amount of other remedies. As mentioned in 2.14.3, the process of “potentization” or “dynamization” in Homoeopathy, is based on using high dilutions of medicinal substances which go further than Avogadro’s number. These high dilutions are termed “potencies”. Potentization comprises the process of succussion whereby mechanical, vigorous agitation is either performed by hand or by a machine (Sukul & Sukul, 2005).

There are homoeopathic remedies that are not highly diluted and contain an abundance of material from the mother tincture whilst the ones that are highly diluted only contain a few particles, and the remedies that are far too diluted may contain a single particle of the mother tincture. For example, a C1 potency is a 1:100 dilution of the mother tincture however a C10 potency dilution indicates 1 part of mother tincture in 100 000 000 000 000 000 000 parts of diluent. Figure 2.5 illustrates how the mother tincture (natural substance) is potentized further (Ernst, 2016).

Figure 2.5: Process of Potentization. (Ernst, 2016).

2.15 Valeriana officinalis

The use of medicinal plants dates back for many centuries. Parts of the plants (leaves, flower, roots, stems and fruits) used by many, provides beneficial therapeutic effects for healing of diseases or ailments (Komolafe, 2014). The dried rhizomes and roots of Valeriana officinalis are the most frequent used parts of the plant (Khare, 2004).

Classification and Nomenclature (McKenna et al., 2011):

 Scientific name: Valeriana officinalis L.  Family name: Valerianaceae  Genus: Valeriana  Common names: valerian, vandalroot, tobacco root, great wild valerian, garden heliotrope, allheal, dysentery root, fragrant valerian, German valerian and English Valerian.

Valeriana officinalis is a medicinal plant that thrives in a humid environment (McKenna et al., 2011) but commonly found in Europe and North Asia (White,

2009). They consist of simple rhizomes that are short and stoloniferous. Their stems may expand to 30 – 150 cm and occasionally to 240 cm. They are normally observed singularly and strong consisting of leaves that are pinnate with 3 – 25 leaflets. The leaves are either straight, narrow-oval or oval shaped. Valeriana officinalis is asexual and is a pink or white flower with its compound inflorescence. The corolla tube expands approximately to 2.5 – 5 mm and the fruit is 2 – 5 mm long with hairs or glabrous (McKenna et al., 2011). Figure 2.6 illustrates the Valeriana officinalis plant (Eisenberg, 2009).

Figure 2.6: Valeriana officinalis plant (Eisenberg, 2009).

Depending on the type of extraction method used, the biochemical composition of Valeriana officinalis’ root contains the following: mono-terpenes (bornyl esters, camphene and pinenes), sesquiterpenes (valerenal and valeranone), the less volatile sesquiterpene carboxylic acids (valerenic acid and derivatives), as well as amino acids (gamma-aminobutyric acid, glutamine and arginine, alkaloids, flavonoids and lignans). The valepotriates (nonglycosidic iridoid esters) are known to be unstable and less likely to have its traces in the finished the product. The volatile oil’s constituents varies due to factors such as genetics and environment (sowing techniques and harvest time) (European Medicines Agency, 2007).

The antimicrobial activity of Valeriana officinalis are attributed to the mono- terpenes and sesquiterpenes. Sesquiterpenes are C15-terpenoids most commonly found in higher plants, marine organisms and fungi. They occur naturally as hydrocarbons or in oxygenated derivatives such as lactones, alcohols, acids, aldehydes and ketones (Awouafack et al., 2013).

It has been shown that the Valeriana officinalis essential oil have antifungal properties against Candida albicans growth in vitro (Wang et al., 2010).

2.15.1 Valeriana officinalis herbal extract

To extract is to remove a substance by effort or force by a special method (Stevenson, 2010). Percolation is a widely used method for liquid extraction of herbs. In this method, the herb is moistened with a solvent (water, glycerin or ethanol) (Ross, 2007) and placed in one of a sequence of percolation chambers. The herb or plant material is frequently washed with the solvent of choice until all of its active ingredients has been extracted (Bones, 2016). Furthermore, a cold percolation method consists of a solvent being passed through a powdered plant material where it will absorb the extract, which then leaks out at the bottom end of the container (Raaman, 2006). The liquid herbal extract of Valeriana officinalis was prepared in a 1:2 cold percolation using 45% ethanol. This method has shown effectiveness in extracting the full phytochemical profile as well as preventing loss of or destruction to the constituent of the plant (Bones, 2016).

2.15.2 Valeriana officinalis homoeopathic mother tincture

A homoeopathic mother tincture is a remedy that has been pharmaceutically made from a vegetable or animal kingdom using a potent ethanol as a solvent by processes called maceration or percolation (Mandal & Mandal, 2001). The production by maceration consists of mixing a dried plant with ethanol in a sealed container and let to stand for a specific period. The residue is thereafter separated from the ethanol and pressed out. However, in the production by percolation, the herbal/ ethanol

mixture is transferred into a percolator, allowing the percolate to flow slowly, at room temperature, in order for the dried plant to be completely covered with the ethanol. The residue is further pressed out and the liquid is further added to the percolate (European Pharmacopoeia, 2016). Valeriana officinalis Ø was prepared according to the HAB 4A method consisting of 1 part of the dried root of Valeriana officinalis plant and 10 parts 67% ethanol (German Homoeopathic Pharmacopoeia, 1993). The mother tinctures are mainly administered directly to the patient at small doses (10 to 20 drops) added to a little of water. Mother tinctures are denoted as “M.T” or with the suffix “Ø” (Sukul & Sukul, 2005).

2.15.3 Homoeopathic symptom picture of Valeriana officinalis

Valeriana officinalis is commonly indicated for its effect upon the nervous system with disorders such as anxiety, insomnia, menopause-related conditions, restless- leg syndrome (White, 2009), irritability and neuralgia. Symptoms and signs of dermatophytosis will be related to the skin picture of Valeriana officinalis which consists of red parts of the skin becomes white in appearance with painful eruptions. The skin is very dry and warm to the touch with smarting as if excoriated in spots that could be covered with the tip of the finger (Vermuelen, 2015).

2.16 Related Research

Currently, there are no studies conducted on Valeriana officinalis homoeopathic mother tincture (Ø) and herbal extract (HE) against the species of T. rubrum and M. canis. However, similar studies have been conducted on Valeriana officinalis plant on T. rubrum and/or M. canis microorganisms.

In 2010, a research study investigated the chemical analysis and biological activity of the essential oils of two valerianaceous species from China: Nardostachys chinensis and Valeriana officinalis. They were screened for their antimicrobial and antioxidant activity by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The positive control was Streptomycin Sulfate. The

essential oils were shown to have broad spectrum antibacterial activity and moderate antifungal activity to Candida albicans (C. albicans) and Magnaporthe oryzae however the antibacterial activity of both oils was still weaker than that of the positive control (Wang et al., 2010).

An in vitro study on the antimicrobial activity of the water-ethanol extract of Valeriana officinalis was conducted in 2001 on five bacterial strains and three fungal strains. Rice, roots of Valeriana officinalis and table-cloths were the materials used to produce the herbal drugs. The study demonstrated positive antimicrobial activity on the following microorganisms: Escherichia coli, Bacillus subtilis, Proteus mirabilis, Enterobacter sp., T. mentagrophytes, Epidermophyton floccosum and M. canis (Stevic et al., 2001).

In 2019, a research study investigated the antimicrobial properties of Valeriana officinalis, Satureja bachtiarica and Thymus daenensis methanolic extracts against Helicobacter pylori (H. pylori) using the disk diffusion test – Kirby Bauer. Ten clinical isolates of H. pylori were obtained from patients with gastrointestinal conditions in Tehran, Iran. There were four positive controls in the experiment (Tetracycline, Ampicillin, Metronidazole and Clarithromycin). The results indicated that the clinical strains had less resistance to tetracycline (10%) compared to the other antibiotics. The zone of inhibition diameter was the highest at 10% concentration of Valeriana officinalis, Satureja bachtiarica and Thymus daenensis. However, Satureja bachtiarica methanolic extract had a stronger antimicrobial effect than the others (Khademian et al., 2019).

CHAPTER THREE METHODOLOGY

3.1 Experimental Design

This quantitative in vitro descriptive experimental study was conducted at the Biotechnology and Food Technology Department, Faculty of Science, at the University of Johannesburg, Doornfontein campus, under the supervision of a qualified laboratory technician with the necessary permission granted. The permission letter from the laboratory is added as an appendix (Appendix C).

3.2 Preparation of Valeriana officinalis

Valeriana officinalis herbal extract (HE) was obtained from CoMed Health, the supplier of MediHerb. The preparation was according to the cold percolation liquid extraction method with a ratio of herbal drug to drug preparation of 1:2 and the vehicle used was 45% ethanol complying with the British Pharmacopoeia. The certificate of analysis of the HE, states the following (Bones, 2016):

 Part of the plant used: Root  Batch number: 19237  Temperature stored: 30°C.

Valeriana officinalis homoeopathic mother tincture (Ø) was obtained from Fusion Homoeopathics cc. The preparation was according to the German Homeopathic Pharmacopoeia HAB 4A method with 67% ethanol (German Homeopathic

Pharmacopoeia, 1993). The certificate of analysis of the Ø, states the following (Fusion Homoeopathics, 2012):

 Part of the plant used: Dried root  Batch number: C18047  Temperature stored: Below 25°C

3.3 Microorganisms

The fungal culture of T. rubrum (ATCC28188) and M. canis (ATCC36299) were purchased as lyophilized freeze-dried culture strains from Davies Diagnostics (Pty) Ltd and used to evaluate the possible antifungal activity of Valeriana officinalis HE and Ø. The fungal strains were aseptically opened and grown on SDA medium plates for ten days in a 30°C incubator.

3.4 Positive Control

The standard antifungal drug Terbinafine was the positive control and obtained as a standard powdered form (250 mg) from a local pharmacy. It was further dissolved in distilled water and dimethyl sulfoxide (DMSO).

3.5 Negative Controls

Ethanol of 45% and 67% were the negative controls and were provided by the Biotechnology and Food Technology Department, Faculty of Science, at the University of Johannesburg, Doornfontein campus. They were the vehicle of the HE and Ø, respectively, and therefore the reason they were used as the negative control.

3.6 Kirby-Bauer Disc Diffusion Method

The antifungal activity of Valeriana officinalis was determined using the Kirby- Bauer disc diffusion method in accordance with the Clinical and Laboratory Standard Institute (Cockerill et al., 2012).

3.6.1 Antifungal tests

Fungal spores of T. rubrum (ATCC2818) and M. canis (ATCC36299) were harvested from a ten-day culture and further pipetted from its SDA plate and dispersed into a ringer solution. Morphological features of T. rubrum and M. canis species were identified microscopically and a hemocytometer was used to standardize the spore suspension to 1.5 x 105 CFU/ml (Mamba et al., 2010).

New plates were subdivided into five sections and further labelled with the organism’s name (T. rubrum and M. canis) and medication tested (Ø, HE, Terbinafine, 45% and 67% ethanol). Figure 3.1 illustrates the subdivision of the plates (Sutherland, 2000). The plates were streaked with 200 μl of each organisms, respectively, and spread in a 90° clockwise direction, using a sterile plastic scraper, over a 90 mm petri dish containing 20 ml of solidified SDA, to completely cover its entire surface (Mamba et al., 2010). .

HE Ø

45% Terbinafine 67%

Figure 3.1: An illustration of the subdivision of the plates (Sutherland, 2000).

Five sterile blank test discs (6 mm in diameter) were impregnated with 20 μl of the Ø, HE, Terbinafine, 45% and 67% ethanol respectively. The negative control (45% and 67% ethanol) as well as the positive control (Terbinafine) were used to ensure the reliability and trustworthiness of the method. Additionally, each medicated disc

was individually placed, using sterile fine-pointed forceps, on the surface of each labelled section of the plate respectively (Mamba et al., 2010).

The plates were allowed to dry at room temperature for 5 minutes. They were then incubated at 30°C for three, six and nine days (Mamba et al., 2010). The reason for the incubation periods (three, six and nine) was to observe the effectiveness of each medication overtime and also to explore the possibility of drug resistance. The zones of inhibition were measured in millimeters with a ruler and recorded. The test was conducted in triplicate for repeatability and reproducibility of the experiment. Therefore a total of six plates were tested. The results were further statistically analyzed using non-parametric tests – Kruskal-Wallis, Mann-Whitney and Friedman (Pallant, 2007).

3.7 Data Collection

A zone of inhibition is the transparent circular area that surrounds the medicated disc on the media plate (Bhargav et al., 2016). The zones of inhibition around each plate were measured and the results observed were recorded in mm. Figure 3.2 illustrates how the zone of inhibition were measured and held at least 30 cm from the naked eye (Mathur & Bargotya, 2015).

Figure 3.2: Measuring the zone of inhibition (Mathur & Bargotya, 2015).

3.8 Data Analysis

The analysis of the data collected was analyzed by the statistician, using non- parametric tests – Kruskal-Wallis, Mann-Whitney and Friedman (Pallant, 2007). The Kruskal-Wallis tests whether the mean ranks are the same and continuous throughout all the groups (McDonald, 2015). The Mann-Whitney test is used for data that does not have a normal distribution and therefore tests for whether one variable tends to have higher values than the other variable (Hart, 2001). The Friedman tests for the differences between groups when the dependent variable has the potential to be continuous (Schenkelberg, 2018).

CHAPTER FOUR RESULTS

4.1 Introduction

The aim of the study was to determine the antifungal effect of Valeriana officinalis Ø and HE on the growth of T. rubrum and M. canis. Sabouraud dextrose agar (SDA) was the medium of choice to grow the two species. Valeriana officinalis HE showed antifungal activity against T. rubrum and M. canis compared to Valeriana officinalis Ø that showed antifungal activity against T. rubrum only. Both tests were statistically analysed using non-parametric tests – Kruskal-Wallis, Mann-Whitney and Friedman (Pallant, 2007) and they will be further explained individually below. The antifungal tests were conducted by the Biotechnology and Food Department.

4.2 Control Plates 4.2.1 Negative controls

The negative controls used in this experiment were 45% ethanol for the HE and 67% ethanol for the Ø. The negative controls were used in order to validate whether the anticipated inhibition of either the HE or Ø is not attributed to ethanol as both of these plants used ethanol as a vehicle. As observed, the negative controls showed no inhibition in the growth of T. rubrum and M. canis.

4.2.2 Positive control

The positive control used in this experiment was Terbinafine for T. rubrum and M. canis. The positive control was used to evaluate the suitability of the environment for the experiment. As observed, the positive control inhibited the growth of both species, T. rubrum and M. canis; therefore, the environment was conducive for the experiment.

4.3 Antifungal Experimental Results 4.3.1 Trichophyton rubrum

Plates containing SDA were streaked with T. rubrum. The plates were subdivided into five sections. The organism was tested against HE, Ø, Terbinafine, 45% and 67% ethanol. Each plate was labelled, respectively, with the organism’s name, HE, Ø, Terbinafine, 45% and 67% ethanol. Each plate contained five discs. Disc one contained the HE. Disc two contained the Ø. Disc three contained the positive control (Terbinafine). Disc four and five contained the negative controls (45% and 67%) respectively. Each disc was placed in the centre of their respective subdivision. The experiment was conducted in triplicate. The plates were observed after three, six and nine days. Table 4.1 to Table 4.3 show the recorded mean zones of inhibition of each medication on T. rubrum. Figure 4.1 to Figure 4.9 shows the plates streaked with T. rubrum with each medicated disc.

Table 4.1: Mean zones of inhibition of T. rubrum: after three days. DISCS Diameter of inhibition MEAN zone in (mm) for the (mm) different plates No.1 No.2 No.3 HE 26 23 19 22.67 Ø 17 10 15 14.00 Terbinafine 14 10 18 14.00 45% ethanol 0 0 0 0.00 67% ethanol 0 0 0 0.00

Table 4.2: Mean zones of inhibition of T. rubrum: after six days. DISCS Diameter of inhibition MEAN zone in (mm) for the (mm) different plates No.1 No.2 No.3 HE 19 18 18 18.33 Ø 0 0 8 2.67 Terbinafine 12 8 16 12.00 45% ethanol 0 0 0 0.00 67% ethanol 0 0 0 0.00

Table 4.3: Mean zones of inhibition of T. rubrum: after nine days. DISCS Diameter of inhibition MEAN zone in (mm) for the (mm) different plates No.1 No.2 No.3 HE 14 14 17 15.00 Ø 0 0 8 2.67 Terbinafine 11 8 13 10.67 45% ethanol 0 0 0 0.00 67% ethanol 0 0 0 0.00

Figure 4.1: T. rubrum streaked plate No.1 after three days.

Figure 4.2: T. rubrum streaked plate No.2 after three days.

Figure 4.3: T. rubrum streaked plate No.3 after three days.

Figure 4.4: T. rubrum streaked plate No.1 after six days.

Figure 4.5: T. rubrum streaked plate No.2 after six days.

Figure 4.6: T. rubrum streaked plate No.3 after six days.

Figure 4.7: T. rubrum streaked plate No.1 after nine days.

Figure 4.8: T. rubrum streaked plate No.2 after nine days.

Figure 4.9: T. rubrum streaked plate No.3 after nine days.

4.3.2 Microsporum canis

Plates containing SDA were spread with M. canis. The plates were subdivided into five sections. The organism was tested against HE, Ø, Terbinafine, 45% and 67% ethanol. Each plate was labelled, respectively, with the organism’s name, HE, Ø, Terbinafine, 45% and 67% ethanol. Each plate contained five discs. Disc one contained the HE. Disc two contained the Ø. Disc three contained the positive control (Terbinafine). Disc four and five contained the negative controls (45% and 67%) respectively. Each disc was placed in the centre of their respective subdivision. The experiment was conducted in triplicate. The plates were observed after three, six and nine days. Table 4.4 to Table 4.6 show the recorded mean zones of inhibition of the HE and Ø on M. canis. Figure 4.10 to Figure 4.18 shows the plates streaked with M. canis with the HE and Ø.

Table 4.4: Mean zones of inhibition of M. canis: after three days. DISCS Diameter of inhibition MEAN zone in (mm) for the (mm) different plates No.1 No.2 No.3 HE 17 14 12 14.33 Ø 0 0 0 0.00 Terbinafine 22 18 16 18.67 45% ethanol 0 0 0 0.00 67% ethanol 0 0 0 0.00

Table 4.5: Mean zones of inhibition of M. canis: after six days. DISCS Diameter of inhibition MEAN zone in (mm) for the (mm) different plates No.1 No.2 No.3 HE 26 20 20 22.00 Ø 0 0 0 0.00 Terbinafine 22 19 19 20.00 45% ethanol 0 0 0 0.00 67% ethanol 0 0 0 0.00

Table 4.6: Mean zones of inhibition of M. canis: after nine days. DISCS Diameter of inhibition MEAN zone in (mm) for the (mm) different plates No.1 No.2 No.3 HE 21 14 13 16.00 Ø 0 0 0 0.00 Terbinafine 24 18 18 20.00 45% ethanol 0 0 0 0.00 67% ethanol 0 0 0 0.00

Figure 4.10: M. canis streaked plate No.1 after three days.

Figure 4.11: M. canis streaked plate No.2 after three days.

Figure 4.12: M. canis streaked plate No.3 after three days.

Figure 4.13: M. canis streaked plate No.1 after six days.

Figure 4.14: M. canis streaked plate No.2 after six days.

Figure 4.15: M. canis streaked plate No.3 after six days.

Figure 4.16: M. canis streaked plate No.1 after nine days.

Figure 4.17: M. canis streaked plate No.2 after nine days.

Figure 4.18: M. canis streaked plate No.3 after nine days.

4.4 Interpretation of Output from Non-parametric Tests

Exploring non-parametric tests for a small sample size, as in this study, was strictly needed in order for variables to be properly evaluated. The testing of distribution and normality of the study were not explored further due to only three replications per medication, making it too small to test for normality and distribution. The non- parametric tests were further defined and outlined by Pallant (2007) and Gonzalez (2008).

 Descriptive statistics: The basic structure of the data are described and provides with the summarization of the statistics of the descriptive information on all of the quantitative variables.  Kruskal Wallis test: Allows for the comparisons of scores on quantitative variable for three or more groups. The scores are converted to ranks, and additionally, the mean rank of each group is compared.  Mann-Whitney test: The test is performed if the Kruskal-Wallis test confirms to be statistically significant and therefore aims at analysing

medians. There is the conversion of the scores of the continuous variable to ranks between the groups, additionally, evaluating whether the ranks between the groups have any significant differences.  Friedman test: This technique tests for the same sample group and they are measured at three or more points in time i.e. over time or under different conditions.  Tests of significance: a technology that uses statistical data in order to determine the probability of the data for assuming real effect i.e. the association between variables or to show the effectiveness of a new treatment. Results are considered statistically significant if the probability of the data is small enough and has a p-value smaller than, the threshold of 5% (sig< 0.05) i.e. there is difference between groups/ overtime. However, results larger than, or equal to, the selected threshold (sig≥ 0.05) are considered non-significant i.e. there is no difference between groups/ overtime.

4.4.1 The Kruskal-Wallis test

The descriptive statistics provides the overall data set (N) i.e. the number or replication, the mean scores, standard deviation, mean rank, median and the zone of inhibition (ZI) (Pallant 2007; Gonzalez, 2008). The Kruskal-Wallis test provides an indication of the influence of the microorganisms with the medication over several days. Table 4.7 to Table 4.8 show the Kruskal Wallis test and its descriptive statistics of the results of the experiment conducted on each microorganism. Table 4.9 shows the test of significance for the Kruskal-Wallis Test.

Table 4.7: The Kruskal-Wallis Test on T. rubrum. ZI_DAYS DISCS N MEAN STANDARD MEAN MEDIAN (overall DEVIATION RANK data set) 3 Days HE 3 22.67 3.512 14.00 23.00 Ø 3 14.00 3.606 9.50 15.00 Terbinafine 3 14.00 4.000 9.50 14.00 45% 3 0.00 0.000 3.50 0.00 ethanol 67% 3 0.00 0.000 3.50 0.00 ethanol

6 Days HE 3 18.33 0.577 14.00 18.00 Ø 3 2.67 4.619 6.17 0.00 Terbinafine 3 12.00 4.000 10.83 12.00 45% 3 0.00 0.000 4.50 0.00 ethanol 67% 3 0.00 0.000 4.50 0.00 ethanol

9 Days HE 3 15.00 1.732 14.00 14.00 Ø 3 2.67 4.619 6.17 0.00 Terbinafine 3 10.67 2.517 10.83 11.00 45% 3 0.00 0.000 4.50 0.00 ethanol 67% 3 0.00 0.000 4.50 0.00 ethanol

Table 4.8: The Kruskal-Wallis Test on M. canis. ZI_DAYS DISCS N MEAN STANDARD MEAN MEDIAN (overall DEVIATION RANK data set) 3 Days HE 3 14.33 4.509 12.00 14.00 Ø 3 0.00 0.000 5.00 0.00 Terbinafine 3 18.67 1.155 13.00 18.00 45% 3 0.00 0.000 5.00 0.00 ethanol 67% 3 0.00 0.000 5.00 0.00 ethanol

6 Days HE 3 22.00 3.464 12.00 20.00 Ø 3 0.00 0.000 5.00 0.00 Terbinafine 3 20.00 0.577 13.00 18.00 45% 3 0.00 0.000 5.00 0.00 ethanol 67% 3 0.00 0.000 5.00 0.00 ethanol

9 Days HE 3 16.00 4.359 12.00 13.00 Ø 3 0.00 0.000 5.00 0.00 Terbinafine 3 20.00 1.528 13.00 18.00 45% 3 0.00 0.000 5.00 0.00 ethanol 67% 3 0.00 0.000 5.00 0.00 ethanol

Table 4.9 Significance Test for the Kruskal-Wallis Test. TEST STATISTICSa,b FUNGI T. rubrum M. canis Kruskal- DEGREES P- Kruskal DEGREES P- Wallis OF VALUE Wallis OF VALUE FREEDOM FREEDOM ZI_3 12.985 4 0.011 13.011 4 0.011 days ZI_6 12.740 4 0.013 13.041 4 0.011 days ZI_9 12.740 4 0.013 12.982 4 0.011 days

a) Kruskal-Wallis Test b) Grouping Variable: Medication

4.4.1.1 Interpretation of output from Kruskal-Wallis Test The Kruskal-Wallis test revealed a statistically significant difference in optimism levels across five different medication groups overtime. The probability values (p), for both microorganisms, over the three, six and nine days are ˂ 0.05. For additional specific interpretation of the Kruskal-Wallis test, an inspection of the mean ranks for the groups suggests that the HE had the highest optimism scores, with the Ø reporting the lowest between the two.

4.4.1.2 Effect size In order to know which of the groups are statistically significantly different from one another, a follow-up with the Mann-Whitney test between pairs of groups is performed.

4.4.2 The Mann-Whitney Test

The pairs considered significantly important in the study were: Pair 1 – HE and Ø; Pair 2 - HE and Terbinafine and Pair 3 – Ø and Terbinafine. The 45% and 67% ethanol were disregarded as there were no further significant data to analyse. Table 4.10 to Table 4.15 show the pairs of groups and their respective tests of significance.

Table 4.10: The Mann-Whitney Test of Pair 1 – HE and Ø. RANKS FUNGI ZI_DAYS MEDICATION N MEAN SUM OF (overall RANK RANKS data set) T. 3 Days HE 3 5.00 15.00 rubrum Ø 3 2.00 6.00 Total 6

6 Days HE 3 5.00 15.00 Ø 3 2.00 6.00 Total 6

9 Days HE 3 5.00 15.00 Ø 3 2.00 6.00 Total 6

M. canis 3 Days HE 3 5.00 15.00 Ø 3 2.00 6.00 Total 6

6 Days HE 3 5.00 15.00 Ø 3 2.00 6.00 Total 6

9 Days HE 3 5.00 15.00 Ø 3 2.00 6.00 Total 6

Table 4.11: Significance Test for the Mann-Whitney Test of Pair 1 – HE and Ø. TEST STATISTICSa,b FUNGI T. rubrum M. canis Mann- Z P- Mann- Z P- Whitney VALUE Whitney VALUE ZI_3 0.000 -1.964 0.050 0.000 -2.087 0.037 days ZI_6 0.000 -2.023 0.043 0.000 -2.121 0.034 days ZI_9 0.000 -2.023 0.043 0.000 -2.087 0.037 days

a) Grouping Variable: Medication b) Not corrected for ties.

4.4.2.1 Interpretation of output from Mann-Whitney Test on Pair 1 The Mann-Whitney test revealed a statistically significant difference in optimism levels across the two medication overtime for both organisms.

Table 4.12: The Mann-Whitney Test of Pair 2 – HE and Terbinafine. RANKS FUNGI ZI_DAYS MEDICATION N MEAN SUM OF (overall RANK RANKS data set) T. 3 Days HE 3 5.00 15.00 rubrum Terbinafine 3 2.00 6.00 Total 6

6 Days HE 3 5.00 15.00 Terbinafine 3 2.00 6.00 Total 6

9 Days HE 3 5.00 15.00 Terbinafine 3 2.00 6.00 Total 6

M. canis 3 Days HE 3 3.00 9.00 Terbinafine 3 4.00 12.00 Total 6

6 Days HE 3 3.00 9.00 Terbinafine 3 4.00 12.00 Total 6

9 Days HE 3 3.00 9.00 Terbinafine 3 4.00 12.00 Total 6

Table 4.13: Significance Test of Mann-Whitney Test of Pair 2 – HE and Terbinafine. TEST STATISTICSa,b FUNGI T. rubrum M. canis Mann- Z P- Mann- Z P- Whitney VALUE Whitne VALUE y ZI_3 0.000 -1.964 0.050 3.000 -0.664 0.507 days ZI_6 0.000 -1.993 0.046 3.000 -0.674 0.500 days ZI_9 0.000 -1.993 0.046 3.000 -0.655 0.513 days

a) Grouping Variable: Medication b) Not corrected for ties.

4.4.2.2 Interpretation of output from Mann-Whitney Test on Pair 2 The Mann-Whitney test revealed a statistically significant difference in optimism levels across the two medication overtime for T. rubrum. However, the probability values (p) of M. canis were more than 0.05 (> 0.05), so the result is considered statistically insignificant and therefore no difference between the HE and Terbinafine overtime.

Table 4.14: The Mann-Whitney Test of Pair 3 – Ø and Terbinafine. RANKS FUNGI ZI_DAYS MEDICATION N MEAN SUM OF (overall RANK RANKS data set) T. 3 Days Ø 3 3.50 10.50 rubrum Terbinafine 3 3.50 10.50 Total 6

6 Days Ø 3 2.17 6.50 Terbinafine 3 4.83 14.50 Total 6

9 Days Ø 3 2.17 6.50 Terbinafine 3 4.83 14.50 Total 6

M. canis 3 Days Ø 3 2.00 6.00 Terbinafine 3 5.00 15.00 Total 6

6 Days Ø 3 2.00 6.00 Terbinafine 3 5.00 15.00 Total 6

9 Days Ø 3 2.00 6.00 Terbinafine 3 5.00 15.00 Total 6

Table 4.15: Significance Test for the Mann-Whitney Test of Pair 3 – Ø and Terbinafine. TEST STATISTICSa,b FUNGI T. rubrum M. canis Mann- Z P- Mann- Z P- Whitney VALUE Whitney VALUE ZI_3 4.500 0.000 1.000 0.000 -2.121 0.034 days ZI_6 0.500 -1.798 0.072 0.000 -2.121 0.034 days ZI_9 0.500 -1.798 0.072 0.000 -2.087 0.037 days

a) Grouping Variable: Medication b) Not corrected for ties.

4.4.2.3 Interpretation of output from Mann-Whitney Test on Pair 3 The Mann-Whitney test revealed a statistically insignificant difference in optimism levels across the two medication overtime for T. rubrum. However, there was a statistically significant difference in optimism levels for M. canis.

4.4.3 The Friedman Test

The Friedman test provides statistics based on the overall on how the medications worked overtime for each fungus. Table 4.16 shows their correlations after three, six and nine days using mean ranks.

Table 4.16: Comparison overtime for each fungus and medication separately using mean ranks. FUNGI MEDICATION MEAN RANK T. rubrum HE ZI_3 days 3.00 ZI_6 days 2.00 ZI_9 days 1.00 Ø ZI_3 days 3.00 ZI_6 days 1.50 ZI_9 days 1.50 Terbinafine ZI_3 days 3.00 ZI_6 days 1.83 ZI_9 days 1.17

M. canis HE ZI_3 days 3.00 ZI_6 days 1.83 ZI_9 days 1.17 Ø ZI_3 days 0.00 ZI_6 days 0.00 ZI_9 days 0.00 Terbinafine ZI_3 days 3.00 ZI_6 days 1.67 ZI_9 days 1.33

According to Table 4.16 the HE appears to have a higher mean rank after six and nine days than the Ø, however, the Ø on M. canis did not rank. The HE and Terbinafine, both indicate slightly different mean ranks overtime for both microorganisms. The ethanol (45% and 67%) showed no significant statistics and therefore were not ranked.

4.5 Interpretation of output from Boxplots

Boxplots are useful tools to illustrate the comparison of the distribution of scores on variables. Figure 4.19 to Figure 4.24 below allowed us to visually inspect the relationship between the HE, Ø and Terbinafine and the displayed zones of inhibitions for each microorganism overtime.

Figure 4.19: Boxplots of results of T. rubrum after three days.

Figure 4.20: Boxplots of results of M. canis after three days.

Figure 4.21: Boxplots of results of T. rubrum after six days.

Figure 4.22: Boxplots of results of M. canis after six days.

Figure 4.23: Boxplots of results of T. rubrum after nine days.

Figure 4.24: Boxplots of results of M. canis after nine days.

CHAPTER FIVE DISCUSSION OF RESULTS

5.1 Discussion of Experimental Results 5.1.1 Discussion of experimental results of T. rubrum 5.1.1.1 Discs medicated with the herbal extract (HE) The HE showed strong antifungal properties as the inhibition on the growth of the fungi occurred with significant mean zones of inhibition. Plates one, two and three after day three, indicated that the HE (22.67 mm) had the highest mean zones of inhibition compared to the Ø (14.00 mm) and the antifungal agent, Terbinafine (14.00 mm). This indicated that the HE had a stronger antifungal effect against T. rubrum.

A possible explanation for the occurrence of the zones of inhibition surrounding the discs, indicating antifungal activity, can be understood by the interaction between the HE’s biochemical composition and the morphological alteration of the organism. As mentioned in 2.15, the antimicrobial activity of Valeriana officinalis is attributed to its polar compounds, mono-terpenes and sesquiterpenes, either in the natural form of hydrocarbons or in oxygenated derivatives such as lactones, alcohols, acids, aldehydes and ketones (Awouafack et al., 2013).

As stated by a similar literature, the antifungal property can account for the polar compounds found in Valeriana officinalis HE causing an inhibition of the extracellular enzymes synthesis and disruption of the Mannan found in the cell wall structure of T. rubrum. As a result, the cytoplasm lacks, the integrity of the membrane is compromised, and eventually, the death of the mycelia occurs (Pereira et al., 2011; Dismukes et al., 2003). According to Wang et al (2010), their study concluded that Valeriana officinalis essential oil extract had antimicrobial activity due to the abundance of sesquiterpenoids and monoterpenoids found in the rhizomes and root of the plant.

However, on day six and nine, the organism showed mild resistance to the HE and therefore decreased mean zones of inhibition (18.33 to 15.00 mm) were observed around the discs. As a possible reason for resistance occurring, according to literatures, is due to long exposure (six and nine days) and therefore reduced the concentration of the HE that was needed to inhibit or kill the organism leading to decreased diameters of zones of inhibition (Axelsen, 2002).

Moreover, Martinez-Rossi et al in 2018 reported on T. rubrum being constantly exposed to sub-lethal doses of antifungal agents, in an in vitro environment, led to strains developing survival traits to higher concentrations due to drug pressure. As in this study, the HE (including the Ø and Terbinafine) are at its highest concentration and therefore could account for the organism’s resistance on day six and nine. These types of strains that can survive higher concentrations are classified as tolerant, have the ability to survive transient exposure or resistant and/or acquired mutations that provides the ability to survive higher concentrations. Unfortunately, there are no additional literature on herbal extracts on T. rubrum that explains why resistance occurred on day six and nine besides the ones stated previously.

Additionally, the zones of inhibition were observed until the ninth day due to a study reporting on the best incubation time for reproducible inhibition zones and possible observation of antifungal activities of the medications. In 2010, Nweze et al discussed the optimal incubation time for reproducible inhibition zones, using the disc diffusion method, for T. rubrum, is ≥ 7 days.

5.1.1.2 Discs medicated with the mother tincture (Ø) The Ø showed mild antifungal properties with significant mean zones of inhibition. Plates one, two and three after day three, showed that the Ø (14.00 mm) had the lowest mean zones of inhibition. The explanation mentioned in 5.1.1.1 can be used in the case of the Ø regarding the occurrence of the zones of inhibition. However, the main difference between the biochemical composition of the Ø and the HE, is that the Ø was prepared using dried plant tissue in accordance with the German Homoeopathic Pharmacopoeia standards, in a 1:10 ratio (German Homoeopathic

Pharmacopoeia, 1993), whilst the HE was prepared using the cold percolation method with a ratio of 1:2 complying with the British Pharmacopoeia (British Pharmacopoeia, 2016). Consequently, the Valeriana officinalis Ø = 1D or 1X i.e. has only 10% strength (Razlog et al., 2012).

However, on day six and nine, the organism had a stronger resistance against the Ø than it had with the HE and Terbinafine, and therefore decreased mean zones of inhibition around the discs were 2.67 mm. The reason for resistance that occurred on day six and nine is a possible explanation as the one mentioned in 5.1.1.1. There are no further literature on mother tinctures on T. rubrum explaining the possible reasons for resistance on day six and nine.

5.1.1.3 Discs medicated with Terbinafine Terbinafine showed moderate antifungal properties with significant zones of inhibition. Plates one, two, three after day three, showed that Terbinafine (14.00 mm) had moderate mean zones of inhibition compared to the HE (22.67 mm) but the same as the Ø (14.00 mm). However, on day six and nine, the organism had a moderate resistance against Terbinafine and therefore decreased mean zones of inhibition were reasonable around the discs (12.00 to 10.67 mm). The resistance on day six and nine can account for the capacity of T. rubrum to develop resistance towards conventional antifungal agents after prolonged exposure, according to an in vitro study (Hryncewicz-Gwóźdź et al., 2013; Mukherjee et al., 2003).

Terbinafine is an investigated antifungal agent and it was expected that it would inhibit the growth of T. rubrum. According to an in vitro study conducted by Diogo et al (2010), concluded that Terbinafine is highly effective in the treatment of dermatophytosis caused by T. rubrum (including M. canis). Another research conducted in Spain, which aimed for the characterization of the antimicrobial susceptibility of fungi responsible for onychomycosis, mentioned that Terbinafine was highly active against all dermatophytes in the study (Zalacain et al., 2011).

5.1.1.4 Discs medicated with Ethanol The ethanol (45% and 67%) showed no antifungal properties with no significant zones of inhibition present with all plates after three, six and nine days. A study on the degradation of ethanol by two species of dermatophytes (including Trichophyton spp.) concluded that these species tend to grow faster when low concentrations of ethanol is present and this is due to the absence of carbon (Janabi, 2009). This accounts for one of the explanations as to why the ethanol (45% and 67%), which were of low concentrations, were unable to inhibit the growth of T. rubrum.

5.1.2 Discussion of experimental results of M. canis 5.1.2.1 Discs medicated with the herbal extract (HE) The HE showed moderate antifungal properties against M. canis, with significant mean zones of inhibition. Plates one, two and three after day three, indicated that the HE (14.33 mm) had moderate mean zones of inhibition compared to Terbinafine (18.67 mm) and significantly higher than the Ø (0.00 mm). As mentioned in 5.1.1.1, M. canis, experienced similar morphological alterations as T. rubrum when the HE’s polar compounds interacted with the organism. However, in addition to the above-mentioned, according to Otang et al (2011), possible morphological alterations such as the shrinkage and partial distortion of the conidia may have caused zones of inhibition to occur.

However, on day six, the mean zones of inhibition was 22.00 mm. Nonetheless on day nine, the organism showed minor resistance to the HE and therefore decreased mean zones of inhibition of 16.00 mm was observed around the discs. This occurrence of the HE increasing in diameter of mean zones inhibition on day six could possibly be due to the HE becoming more potent and inhibiting the growth of M. canis on day six. However, the potency of the HE decreased thereafter; indicating that the incubation period possibly affected its potency and was not persistent and therefore depreciated in its toxicity, as mentioned in 5.1.1.1, on day nine (VI & AO, 2018).

5.1.2.2 Discs medicated with the mother tincture (Ø) The Ø showed no antifungal properties with no significant mean zones of inhibition present with all plates after three, six and nine days. There are no literature that proves that homeopathic mother tinctures have antifungal activity on M. canis.

5.1.2.3 Discs medicated with Terbinafine As mentioned in 5.1.1.3, Terbinafine was expected to show antifungal properties. It showed the strongest antifungal properties with significant zones of inhibition. Plates one, two, three after day three, showed that Terbinafine (18.67 mm) had the highest mean zones of inhibition compared to the HE (14.33 mm) and Ø (0.00 mm). However, on day six and nine, the mean zones of inhibition was 20.00 mm. There are no literatures explaining this particular occurrence on M. canis.

5.1.2.4 Discs medicated with ethanol Ethanol (45% and 67%) showed no antifungal properties with no significant zones of inhibition present with all plates after three, six and nine days. The explanations mentioned in 5.1.1.4 is applicable here as well.

5.1.3 Discussion of the non-parametric tests

The Kruskal-Wallis test, as mentioned in 4.4, provides indications of the influence of the microorganisms with the medication overtime. This test revealed statistically and practically significant difference in levels across the five medication groups overtime (p = ˂ 0.05).

The Mann-Whitney test is of beneficial as it assists the Kruskal-Wallis test (after it has revealed statistical significance) in evaluating and comparing the effect of two medicines and whether they are considered to be the same or different. Additionally, it is also used to obtain information on whether the two medicines can cause a cure or not (Statistics Solutions, 2019). In summary, pair 1 revealed that the HE had a stronger antifungal effect than the Ø for both microorganisms. Pair 2 revealed that the HE had a stronger antifungal effect than Terbinafine for T. rubrum

however Terbinafine had a stronger antifungal effect than the HE for M. canis. Pair 3 revealed that the Terbinafine had a stronger antifungal effect than the Ø for both microorganisms.

The Friedman test provides statistics based on the overall on how the medications worked overtime for each fungus. In order to do this test, the data in the study passed the following assumptions (i) the one group is tested on three or more different scenarios or occasions (ii) random sample group (iii) the dependent variables are continuous and (iv) samples do not require normal distribution (Laerd Statistics, 2019). The test then revealed that with time the medications on the growth of T. rubrum and M. canis decreased in its efficacy.

These three tests were beneficial for this study as it indicated that both extracts (HE and Ø) had antifungal activities and additionally, demonstrated which had the strongest antifungal properties against each microorganism overtime.

5.2 Conclusion

We can conclude that the Valeriana officinalis HE can be an alternative agent to treat infections in humans caused by T. rubrum and M. canis however the Valeriana officinalis Ø can be an alternative agent to treat infections in humans caused by T. rubrum only.

CHAPTER SIX CONCLUSION & RECOMMENDATIONS

6.1 Conclusion

The Kirby-Bauer disc diffusion method in accordance with the Clinical and Laboratory Standard Institute was used to ensure that the preparation of the organisms were consistent. The experiments were conducted in triplicate for repeatability and reproducibility and analysed after three, six and nine days. All the experiment had the same positive and negative controls.

There are no in vitro studies on the antifungal properties of Valeriana officinalis herbal extract and homoeopathic mother tincture on T. rubrum and M. canis. Therefore, in these regards, an undeviating comparison of results cannot be formulated.

Nonetheless, as stated in chapter 2, there are similar in vitro studies that have been conducted on the Valeriana officinalis plant on other pathogenic microorganisms and therefore the results of this experiment could not be compared to these studies due to the different experimental methods.

In this in vitro study, the findings clearly indicated that the Valeriana officinalis herbal extract did have antifungal effects on T. rubrum and M. canis, however, the Valeriana officinalis homoeopathic mother tincture only had antifungal effect on T. rubrum and did not have any antifungal effect on M. canis.

Valeriana officinalis herbal extract showed a great consistent size of clear zones of inhibition on both T. rubrum and M. canis. We can conclude that this plant as a herbal extract possesses great antifungal properties against T. rubrum and M. canis despite, there are no other in vitro studies to compare it to. The homoeopathic mother tincture showed smaller sizes of clear zones of inhibition on T. rubrum only and no inhibition for M. canis therefore we can conclude that this plant as a

homoeopathic mother tincture does possess substantial antifungal properties against T. rubrum. Additionally, the zones of inhibition for both herbal extract and homoeopathic mother tincture decreased overtime (after three, six and nine days) due to an increase in resistance from the organisms and the decrease in the concentration of the medications due to long exposure (six and nine days). . Therefore, more research should be conducted in order to determine if further dilutions of the Valeriana officinalis herbal extract will still indicate antifungal properties against T. rubrum and M. canis.

In the case of the herbal extract on T. rubrum, the plant produced a wider zones of inhibition compared to Terbinafine, which is the preferred conventional antifungal drug for the treatment of dermatophytosis caused by T. rubrum. Further research should be conducted in order to confirm that Valeriana officinalis herbal extract possesses greater antifungal properties against T. rubrum than Terbinafine to support the notion of using the herbal extract as an alternative medication in the treatment of dermatophytosis caused by this specie.

The negative control, ethanol (45% and 67%), did not indicate any zones of inhibition and therefore, we can construe that the antifungal effects of Valeriana officinalis herbal extract and homoeopathic mother tincture is not attributed to its presence but rather to their active ingredients such as the sesquiterpenes (including mono-terpenes) hydrocarbons and oxygenated derivatives.

6.2 Limitation

The main limitation experienced during the conduction of this experimental study was mainly due to being a small group sample and therefore various non-parametric tests were used to analyse the data which may lead to biased interpretation of results.

6.3 Recommendations

Below are some recommendations:  Valeriana officinalis herbal extract and homoeopathic mother tincture could be tested against other fungi such as C. albicans, M. oryzae, T. mentagrophytes and E. floccosum.  Valeriana officinalis herbal extract and homeopathic mother tincture could be tested against bacteria such as E. coli, B. subtilis, P. mirabilis and Enterobacter sp.  Different forms of concentration of the herbal extract, could be tested against T. rubrum and M. canis.  Valeriana officinalis antifungal properties could be determined using other forms of experimental methods such as the broth dilution method.  The active compounds of Valeriana officinalis could be identified and tested prior to the experimental testing.  The active compounds of Valeriana officinalis could be identified and tested on the microorganisms listed above.  The experiments could be repeated on different days.  The experiments could be repeated using a bigger sample group.

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APPENDIX A

APPENDIX B

APPENDIX C