The Effect of Hydrastis Canadensis and Calendula

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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 effect of Hydrastis canadensis and Calendula
officinalis Homeopathic Mother Tincture complex on
Candida albicans, in vitro
A dissertation submitted to the

Faculty of Health Sciences, at the University of Johannesburg
as partial fulfilment of a
Master’s degree of Technology in Homoeopathy
by
Ashica Subramanian
(Student number: 215071435)
Supervisor_______ _________ ___7/12/2020__________

Dr J. Pellow D Tech Hom (UJ) Date
Co-supervisor_____ _____________ ___7/12/2020_________

Dr SIK. Mieta D Tech Biomed (UJ) Date
Johannesburg 2020
DECLARATION
I, Ashica Subramanian, hereby declare that this dissertation is my own

unaided work. It is being submitted for the degree of Master of Technology
in Homoeopathy to the University of Johannesburg. It has not been
submitted to any other institution for the purpose of attaining a qualification.
___________________________
Ashica Subramanian
Date:___ 7/12/2020___________
x
ABSTRACT
Candida albicans (C. albicans) is the micro-organism responsible for

candidiasis, a fungal infection that may affect patients that are
immunocompromised. This type of infection is normally treated with
conventional anti-fungal medications such as fluconazole and clotrimazole,
however persistent infections and treatment can lead to the development of
anti-fungal resistance, Hydrastis canadensis L. (H. canadensis L.) and
Calendula officinalis L. (C. officinalis L.) are medicinal plants with known
anti-fungal effects. In vitro studies have shown that both H. canadensis
mother tincture (Ø) and C. officinalis D1 and D3 homeopathic potencies
have displayed inhibitory effects against C. albicans, however there has
been no research done on the anti-fungal effects of the combination of these
two remedies.
The aim of this study was to determine the antimicrobial effect of H.

canadensis and C. officinalis mother tincture complex on Candida albicans
(C. albicans) using the Kirby-Bauer Disc Diffusion Susceptibility and Micro-
dilution method.
The results demonstrated that the H. canadensis and C. officinalis Ø

complex inhibited the growth of C. albicans and other Candida spp., which
was confirmed by the Kirby-Bauer Disk Diffusion and Microdilution methods.
H. canadensis Ø and C. officinalis Ø on their own inhibited the growth of
C. albicans in varying degrees; however, the H. canadensis and the
C. officinalis Ø complex displayed better anti-microbial properties than the
C. officinalis Ø on its own, as well as the ethanol concentrations.
Fluconazole, however, performed significantly better than the tinctures.
xi
DEDICATION
This research is dedicated to my three daughters for their continued

support, guidance and encouragement and understanding throughout this
whole journey when everything seemed impossible.
xii
ACKNOWLEDGEMENTS
I would like to express my gratitude and appreciation to the following people

for their guidance and assistance with my dissertation:
• My supervisor Dr. Janice Pellow; for your unbelievable work ethic

and the support and guidance throughout this whole process.
• My co-supervisor Dr. Sumayya Mieta, for your support and
motivation.
• Riahaanah Paulse for assistance in the laboratory and for ensuring
all my results were up to standard.
• Dr Tsele-Tebakang for your constant support and guidance in the
clinic.
• My friends Jana Wurz , Samuel Du Toit , Celeste Vermaak , Karabo
Mapule and Aalia Ebrahim, thanks for having faith in me and for all
the support.
• My three children: Ludmila, Alyssa and Raieeka, thank you for your
continued support, and for understanding when I could not be there.
My sister Mumtaz Naidoo, thank you for being my pillar of strength.
• Dean Subramanian I am grateful for your support throughout this
journey.
xiii
TABLE OF CONTENTS
AFFIDAVIT ......................................................................................................... ix
DECLARATION .................................................................................................. x
ABSTRACT ........................................................................................................ xi
DEDICATION .................................................................................................... xii
ACKNOWLEDGEMENTS .................................................................................xiii
1. INTRODUCTION .......................................................................................... 1
1.1 Problem Statement .............................................................................. 1
1.2 Aim of the Study .................................................................................. 2
1.3 Objectives of the Study ....................................................................... 2
1.4 Hypothesis ........................................................................................... 2
1.5 Null Hypothesis ................................................................................... 2
1.6 Importance of the Study ...................................................................... 3
2. LITERATURE REVIEW ................................................................................ 4
2.1 Candida species .................................................................................. 4
2.1.1 Classification, identification, and morphology of C. albicans...... 4
2.1.2 Pathogenesis ................................................................................... 5
2.1.3 Predisposing factors for candidiasis ............................................. 7
2.1.3.1 Predisposing conditions ............................................................. 7
2.1.3.2 Medications .................................................................................. 7
2.1.3.3 Other risk factors ......................................................................... 7
2.1.4 Infections caused by the Candida Species.................................... 8
2.1.4.1 Cutaneous candidiasis ................................................................ 8
2.1.4.2 Oropharyngeal candidiasis ......................................................... 8
2.1.4.3 Vulvovaginal candidiasis ............................................................ 8
2.1.4.4 Systemic candidiasis ................................................................... 9
2.1.5 Conventional treatment of Candida infections .............................. 9
2.1.5.1 Azole anti-fungals ........................................................................ 9
2.1.5.2 Polyenes ..................................................................................... 10
2.2 Antimicrobial resistance ................................................................... 10
2.3 Homeopathy....................................................................................... 11
2.3.1 History of homeopathy.................................................................. 11
2.3.2 Principles of homeopathy ............................................................. 11
2.3.2.1 The law of similars ..................................................................... 11
2.3.2.2 Principle of the single remedy .................................................. 11
2.3.2.3 Principle of the minimum dose ................................................. 12
xiv
2.3.3 Homeopathic remedy manufacturing techniques ....................... 12
2.3.3.1 Potentisation .............................................................................. 12
2.3.4 Mother tinctures versus herbal extracts ...................................... 13
2.4 Hydrastis canadensis L..................................................................... 13
2.4.1 Hydrastis canadensis L. herbal extract ....................................... 14
2.4.1.1 Herbal extract preparation ........................................................ 14
2.4.2 Hydrastis canadensis homeopathic mother tincture .................. 15
2.4.2.1 Mother tincture preparation ...................................................... 15
2.4.2.2 Homeopathic indications .......................................................... 15
2.5 Calendula officinalis L. (C. officinalis L.) ......................................... 15
2.5.1 Calendula officinalis L. herbal extract ......................................... 16
2.5.1.1 Herbal extract preparation ........................................................ 16
2.5.2 Calendula officinalis homeopathic mother tincture .................... 17
2.5.2.1 Mother tincture preparation ...................................................... 17
2.6 Related research................................................................................ 18
3. METHODOLOGY ....................................................................................... 20
3.1 Experimental design ......................................................................... 20
3.2 Candida species ................................................................................ 20
3.3 Homeopathic remedies ..................................................................... 20
3.4 Methods used for analysis ................................................................ 20
3.4.1 Media preparation ......................................................................... 20
3.4.2 Yeast culture preparation ............................................................. 21
3.4.3 Preparation of Hydrastis canadensis and Calendula officinalis
mother tinctures ............................................................................ 21
3.4.4 Kirby Bauer Disk Diffusion method ............................................. 22
3.4.5 Microdilution method ................................................................... 24
3.5 Validity and reliability ........................................................................ 25
3.6 Data analysis ..................................................................................... 26
4. RESULTS................................................................................................... 27
4.1 Overview ............................................................................................ 27
4.2 Results for the Candida albicans strain ........................................... 28
4.2.1 Kirby-Bauer Disk Diffusion method ............................................. 28
4.2.2 Microdilution method .................................................................... 32
4.2.3 Minimum Fungicidal Concentration Method (MFC)..................... 36
4.3 Results for emerging pathogenic Candida spp. strains ................. 37
4.3.1 Kirby-Bauer Disk Diffusion method ............................................. 38
xv
4.3.1.1 C. lusitaniae............................................................................... 38
4.3.1.2 C. parapsilosis ........................................................................... 39
4.3.1.3 C. guilliermondii ......................................................................... 40
4.3.1.4 C. inconspicua/lambica ............................................................. 41
4.3.1.5 C. rugosa .................................................................................... 42
4.3.2 Microdilution method .................................................................... 44
4.3.3 Minimum fungicidal concentration (MFC) method ...................... 48
5. DISCUSSION OF RESULTS ......................................................................... 50
5.1 Discussion of experimental results for C. albicans ............................. 50
5.1.1 Kirby-Bauer Disk Diffusion Method ............................................. 50
5.1.3 Minimum Fungicidal Method (MFC) ................................................ 53
5.2 Discussion of experimental results for the Candida species ......... 53
5.2.1 Kirby-Bauer Disk Diffusion Method ............................................. 53
5.2.2.1 C. lusitaniae................................................................................ 55
5.2.2.2 C. parapsilosis ........................................................................... 56
5.2.2.3 C. rugosa, C. guilliermondii and C. inconspicua/Lambica ...... 58
5.2.3 Minimum Fungicidal Concentration (MFC) ................................. 60
5.4 Related research and discussion of results .................................... 60
5.4.1 H. canadensis Ø ............................................................................ 60
5.4.2 C. officinalis Ø............................................................................... 61
5.4.3 Ethanol concentrations ................................................................ 62
5.4.4 Fluconazole ................................................................................... 63
5.5 Limitations of this study ................................................................... 63
6. CONCLUSION & RECOMMENDATIONS .................................................. 65
6.1 Conclusion ......................................................................................... 65
6.2 Recommendations ............................................................................ 66
7. REFERENCES ........................................................................................... 67
APPENDICES ................................................................................................... 78
Appendix A ................................................................................................... 78
Appendix B ................................................................................................... 79
Appendix C ................................................................................................... 80
Appendix D ................................................................................................... 81
Appendix E ................................................................................................... 82
Appendix F ................................................................................................... 83
xvi
LIST OF TABLES
Table 3. 1 List of compounds added to 96 well plate ......................................... 24
Table 4. 1 Summary of the Kirby-Bauer Disk Diffusion susceptibility test against

C. albicans ......................................................................................... 30
Table 4. 2 Kruskal Wallis test statistics for the Kirby-Bauer Disk Diffusion test on
C. albicans ......................................................................................... 31
Table 4. 3 The Mann- Whitney U test statistics for the Kirby-Bauer Disk Diffusion
on C. albicans .................................................................................... 31
Table 4. 4 Summary of the Microdilution test against C. albicans ...................... 33
Table 4. 5 Kruskal Wallis test statistics for the Microdilution test on C. albicans 34
Table 4. 6 The percentage of dry residue concentration in each well for the
homeopathic Ø’s ............................................................................. 35
Table 4. 7 Summary of the results for the 2-fold dilution series for C. albicans .. 36
Table 4. 8 List of compounds on grid plate ........................................................ 37
Table 4. 9 Summary of the Kirby-Bauer Disk Diffusion susceptibility test against

C. albicans and other Candida spp. strains. ....................................... 43
Table 4. 10 Summary of the results for the 2-fold Microdilution method for
Candida spp. ................................................................................... 44
Table 4. 11 List of compounds on grid plate ...................................................... 49
xvii
LIST OF FIGURES
Figure 2. 1 Candida Morphology (Thompson et al., 2011)................................... 5
Figure 2. 2 Candida Biofilm Formation (Gulati and Nobile, 2016) ........................ 6
Figure 2. 3 Homeopathic Potentisation (Homeopathicassociates.com, 2020) ... 13
Figure 2. 4 Hydrastis Canadensis L. Rhizome (Phys.org, 2020). ...................... 14
Figure 2. 5 Calendula officinalis L. plant (Lim, 2014) ......................................... 16
Figure 3. 1 Fungal lawn streaking (Espinel-Ingroff, 2007) ................................. 23
Figure 3. 2 A 96 well plate template for MIC dilution experiment ....................... 25
Figure 4. 1 Disk diffusion agar of C. albicans (ATCC 90028) showing zones of

inhibition for A: Remedies (1) H. canadensis (2) C. officinalis, (3) H.
canadensis and C. officinalis complex, (4) 66% Ethanol, (5) 68%
Ethanol and (6) 70% Ethanol and B: (1) Fluconazole 25mcg and (2)
blank control. ................................................................................. 29
Figure 4. 2 96 well plate for MIC dilution of C. albicans (ATCC 90028) ............. 33
Figure 4. 3 Agar plate of MFC method for C. albicans A: Agar plate reading
showing clear growth after 24 hours B: Grid plate showing wells that
were plated on agar plate ................................................................ 37
Figure 4. 4 Disk diffusion agar of C. lusitaniae showing zones of inhibition for A:

Remedies (1) H. canadensis (2) C. officinalis, (3) H. canadensis and
C. officinalis complex , (4) 66% Ethanol, (5) 68% Ethanol and (6) 70%
Ethanol and B: (1) Fluconazole 25mcg and (2) blank control. .......... 38
Figure 4. 5 Disk diffusion agar of C. parapsilosis showing zones of inhibition for

A: Remedies (1) H. canadensis (2) C. officinalis, (3) H. canadensis
and C. officinalis complex, (4) 66% Ethanol, (5) 68% Ethanol and (6)
70% Ethanol and B: (1) Fluconazole 25mcg and (2) blank control. .. 39
Figure 4. 6 Disk diffusion agar of C. guilliermondii showing zones of inhibition for

A: Remedies (1) H. canadensis (2) C. officinalis, (3) H. canadensis
and C. officinalis complex, (4) 66% Ethanol, (5) 68% Ethanol and (6)
70% Ethanol and B: (1) Fluconazole 25mcg. ................................... 40
Figure 4. 7 Disk diffusion agar of C. inconspicua/lambica showing zones of

inhibition for A: Remedies (1) H. canadensis (2) C. officinalis, (3) H.
canadensis and C. officinalis complex, (4) 66% Ethanol, (5) 68%
Ethanol and (6) 70% Ethanol and B: (1) Fluconazole 25mcg. .......... 41
Figure 4. 8 Disk diffusion agar of C. rugosa showing zones of inhibition for A:

Remedies (1) H. canadensis (2) C. officinalis, (3) H. canadensis and
xviii
C. officinalis complex, (4) 66% Ethanol, (5) 68% Ethanol and (6) 70%
Ethanol and B: (1) Fluconazole 25mcg. ........................................... 42
Figure 4. 9 Agar plate of MFC method of Candida spp. A: Agar plate reading
showing clear growth after 24 hours B: Grid plate showing wells that
were plated on agar plate. ............................................................... 49
xix
1. INTRODUCTION
1.1 Problem Statement
Candida albicans (C. albicans) is a yeast micro-organism that forms part of

the commensal microbiota of the human host, however it can become
pathogenic when the host is immunocompromised. It has the ability to adapt
and evolve to allow for its survival (da Silva Dantas et al., 2016). This
microorganism is responsible for candidiasis, a fungal infection typically
causing oral or vaginal thrush, as well as invasive candidiasis, where it
enters the bloodstream and infects organs such as the lungs, kidneys, or
brain (Correia et al., 2019). Candidiasis has been successfully treated with
conventional anti-fungal treatments such as fluconazole and clotrimazole,
however, adverse effects and resistance to these treatments can occur
(Dadar et al., 2018). This creates the need to find alternative therapies.
Hydrastis canadensis L. (H. canadensis L.) and Calendula officinalis L.

(C. officinalis L.) are medicinal plants with known anti-fungal effects.
Homeopathically prepared mother tinctures (Ø) of these plants have also
shown potential anti-fungal effects. Although research is limited, in vitro
studies have shown that both H. canadensis Ø and C. officinalis D1 and D3
homeopathic potencies display inhibitory effects against C. albicans
(Budree, 2004; De Klerk, 1998). While homeopathic Ø’s are frequently
prescribed in combination with one another in a complex, the combined
effect of these two tinctures on C. albicans has not been previously
explored.
According to Yang et al. (2014), the synergistic therapeutic effects of herbal

medicines have been frequently reported, however the exact mechanism
by which this occurs has not been fully elucidated. The authors of this review
propose that herbal medicines used in combination may: 1) have improved
overall bioavailability; 2) co-operate together in a synergistic or antagonistic
way by means of various target pathways; 3) overcome drug resistance
1
mechanisms of microbial cells; and 4) have reduced potential for producing
adverse effects. Further research is therefore required to identify these
synergistic effects in order to discover effective therapeutic combinations.
1.2 Aim of the Study
The aim of this in vitro study was to determine the effects of a homeopathic
mother tincture complex containing H. canadensis and C. officinalis on
C. albicans and other Candida species (spp.) using the Kirby-Bauer disc
diffusion susceptibility and Microdilution method.
1.3 Objectives of the Study
• The mother tinctures and anti-fungal control were tested using the
Kirby Bauer method to determine the susceptibility of C. albicans to
these compounds.
• The minimum inhibitory concentration (MIC) for the mother tinctures

were determined using the 96 well plate serial dilution method and
the X-Mark Microplate Spectrophotometer (Bio-Rad; USA) for fungal
growth indication.
1.4 Hypothesis
It was hypothesised that the homeopathic mother tinctures of H. canadensis

and C. officinalis used in combination would show antimicrobial action
against C. albicans and other Candida spp. by demonstrating clear zones
of inhibition.
1.5 Null Hypothesis
The null hypothesis states that the homeopathic mother tincture complex of
H. canadensis and C. officinalis would not show antimicrobial action against
C. albicans and other Candida spp.
2
1.6 Importance of the Study
This organism was chosen since it is a common infection amongst women,

which they treat with over-the counter fungal medication as well as other
anti-fungals that can generate unwanted effects. C. albicans have
developed resistance to most of these medications which makes this
condition exceedingly difficult to treat.
3
2. LITERATURE REVIEW
2.1 Candida species
Candida albicans (C. albicans), Candida lusitaniae (C. lusitaniae) and

Candida parapsilosis (C. parapsilosis) as well as other Candida strains are
microorganisms belonging to the genus Candida. C. albicans and
C. parapsilosis are one of the five most common candida strains whilst
C. lusitaniae is relatively rare (Polke et al., 2015).These organisms make
up the normal flora in the body, however a weakened immune system can
result in opportunistic infections that affect the mucous membranes of the
genital, urinary, respiratory, and gastrointestinal tracts, as well as the oral
mucosa, nails, scalp, and skin. These fungal infections that are caused by
the Candida species are known as candidiasis (de Oliveira Santos et al.,
2018).
2.1.1 Classification, identification, and morphology of C. albicans
C. albicans is a unicellular oval-shaped yeast that exists on mucosal

surfaces of the body. The structure is polymorphic and asexual, and it
develops into three distinct morphologies: hyphae, pseudo hyphae, and
yeast. The yeast is oval, and the hyphae and pseudo hyphae are elongated,
as per Figure 2.1. It is difficult to distinguish between the hyphae and pseudo
hyphae as they are both filamentous forms of the fungus (da Silva Dantas
et al., 2016). C. albicans can change its morphological state according to
various environmental alterations. This characteristic allows for easy
switching between the filamentous and yeast states, which enhances its
survival (Cauchie et al., 2017).
At ambient temperature (20–22°C), the yeast reproduces by budding, but

at body temperature (36.5–37.5°C) they form hyphae. Pseudo hyphae form
by budding but do not attach to adjacent tissue, thus they remain elongated.
Alterations in the cell wall architecture and composition transform the
4
pathogen’s responsiveness to anti-fungal medication (da Silva Dantas et
al., 2016).
Figure 2. 1 Candida Morphology (Thompson et al., 2011)
2.1.2 Pathogenesis
The vast majority of persistent Candida infections are associated with

biofilm development. A biofilm is defined as a microbial community that is
embedded in a self-produced matrix of extracellular polymeric substances
(Rafii, 2015). With biofilm development, a thin layer of microbes attaches to
the surface of a structure and are enclosed and surrounded by an
extracellular matrix of mucilage, enabling the microorganisms to be
protected (Ganguly and Mitchell, 2011). As per Figure 2.2, a biofilm
formation has four stages of growth and reaches maturation within 24 hours.
While biofilms promote the survival of the microorganism, they are however
responsible for persistent infections that occur in the human body, because
of the inherent resistance of these structures to antibiotics and host defence
systems (Kumar et al., 2017). Biofilms increase further systemic infections
and elevate patient mortality (Rafii, 2015). Virulence factors possessed
5
by C. albicans are known to aid its biofilm-forming ability and lead to tissue
damage and tenacity within the host (Gulati and Nobile, 2016).
Figure 2. 2 Candida Biofilm Formation (Gulati and Nobile, 2016)
C. albicans can transform from having a symbiotic relationship with the host
to being a pathogenic infection due to its morphological switching capability
between yeast and hyphal forms. This switching is key in its formation of
biofilms. Morphological switching is an easily distinguishable morphological
state of C. albicans that controls phases of colonisation, flourishing, and
distribution. During morphogenesis, an organism changes its shape,
triggered by factors such as temperature, serum, amino acid availability, pH
level, and CO2 presence (Tsui et al., 2016). The yeast form has been
associated with both initial attachment and dispersal, while the filamentous
hyphal form enables C. albicans to invade host tissue and form a mature
biofilm (da Silva et al., 2016).
6
2.1.3 Predisposing factors for candidiasis
There are several factors that predispose an individual to developing

Candida infections.
2.1.3.1 Predisposing conditions
Candida infections may be triggered by underlying conditions that affect

immune system functioning, such as the human immunodeficiency virus
(HIV), acquired immunodeficiency syndrome (AIDS), Epstein-Barr virus,
diabetes mellitus, and nutritional deficiencies (Pérez-García et al., 2017).
2.1.3.2 Medications
Antibiotics, oral glucocorticoids, and inhaled corticosteroids also put

patients at a higher risk (Dadar et al., 2018). Corticosteroid treatment
supresses the immune system and may decrease B-cell numbers and
certain antibody responses (Fedor and Rubinstein, 2006). Antibiotic
treatment eliminates the yeast's natural competitors for resources in the oral
and intestinal flora, which increases the intensity of the condition. Hormone
replacement therapy, infertility treatments, and cancer therapies may also
increase the risk of contracting such infections, as do pregnancy and the
use of oral contraceptives (Dadar et al., 2018).
2.1.3.3 Other risk factors
In women, the use of detergents, douches, and the presence of hormonal

or physiological imbalances can result in disruption of the normal vaginal
flora, causing Candida spp. overgrowth (Dadar et al., 2018). In men, penile
candidiasis is caused by sexual intercourse with an infected partner,
although male genital yeast infections are less common (Achkar and Fries,
2010).
7
2.1.4 Infections caused by the Candida Species
2.1.4.1 Cutaneous candidiasis
Infections of the skin and nails present with redness, swelling, itching, and
white exudates in the skin folds. Nail conditions include nail paronychia,
which results in lifting of the nail and redness and swelling around the skin.
Superficial candidiasis in babies presents as dermatitis and swelling,
typically affecting the genital area (Aslan Kayıran et al., 2019)
2.1.4.2 Oropharyngeal candidiasis
This type of infection is prevalent in HIV patients. Symptoms include a sore

mouth, a red, burning tongue and an altered sense of taste. There are four
types of oral candidiasis: oral thrush, erythematous candidiasis,
hyperplastic candidiasis, and denture-induced stomatitis. Oral thrush
presents with white, curd-like, distinct plaques on an erythematous base,
which is revealed after the removal of the plaque. The infection can be found
on the linings of the cheek, throat, tongue, or gums (Millsop and Fazel,
2016). Erythematous candidiasis presents as smooth, red patches on the
hard or soft palate, the back of the tongue, or the lining of the cheek.
Hyperplastic candidiasis consists of white, firm patches or plaques that
cannot be removed; these usually spread bilaterally on the lining of the
cheek, tongue, or the hard or soft palate. Denture-induced stomatitis
presents as either a smooth or a granular rash limited to the denture-bearing
region of the hard palate, and is often linked to angular cheilitis, which
presents with red, fissured lesions in the corners of the mouth (Pankhurst,
2009).
2.1.4.3 Vulvovaginal candidiasis
Vulvovaginitis occurs when the Candida spp. infiltrate the mucosal lining of
the vagina and cause inflammation and swelling. The predominant
inflammatory cells are mostly leukocytes and macrophages. Patients may
8
have a thick and sticky discharge. They can also present with burning or
painful urination, vaginal itching, vaginal burning, dyspareunia, and redness
and swelling of the vagina (Jeanmonod and Jeanmonod, 2019).
2.1.4.4 Systemic candidiasis
This is a serious infection that can affect the blood, brain, heart, and other
parts of the body. The most common form of systemic candidiasis is when
the blood is infected, and this type of infection is called candidaemia.
Patients with candidaemia present with fever and chills and do not improve
on antibiotic treatment. Other presenting symptoms will depend on which
organ has been infected. For example, if the heart is affected, patients could
develop dyspnoea, and if the eyes are affected, this could result in blurry
vision. Patients may also present with a sore throat if the oesophagus has
been affected (Pappas et al., 2018). Systemic or invasive candidiasis is
normally present when a patient is seriously ill and immunocompromised,
hence some symptoms may be masked by these conditions (Nett, 2018).
2.1.5 Conventional treatment of Candida infections
The following anti-fungal medications are commonly used to treat

candidiasis: azoles, which include clotrimazole and fluconazole, and
polyenes, such as nystatin (Spampinato and Leonardi, 2013).
2.1.5.1 Azole anti-fungals
The azoles are the largest group of anti-fungal drugs. They consist of
midazoles (miconazole, econazole, clotrimazole, ketoconazole), triazoles
(fluconazole, itraconazole), voriconazole (second-generation, pseudo-
triazole derivative of fluconazole), and posaconazole (a hydroxylated
analogue of itraconazole). Azoles are easily absorbed and usually well-
tolerated by patients (Cowen et al., 2014). These anti-fungals interfere with
the fungal cell membrane by restricting the activity of lanosterol 14-
demethylase, which is an enzyme involved in the production of
9
ergosterol. Ergosterol is the biggest sterol portion of the fungal cell
membrane (Spampinato and Leonardi, 2013). Fluconazole is administered
orally and has been linked to hepatotoxicity, which is directly associated
with dosage and duration of use, or the presence of an underlying illness.
Other side effects of this medication may include pruritus, dry skin, and
gastrointestinal disturbances (Kumar and Jha, 2016). Clotrimazole is
normally administrated topically and side effects may include irritation and
burning of the skin and, in some cases, allergic dermatitis. Re-occurrence
of infection is possible if the course of treatment is not completed (Ahangari
et al., 2019).
2.1.5.2 Polyenes
Other fungal medications such as nystatin and amphotericin B, which are

both polyenes, bind ergosterol. This process disturbs the vital lipidic part of
the fungal cell membrane, resulting in the manufacture of aqueous pores.
This then leads to cellular permeability and leakage of cytosolic elements,
resulting in fungal death (Spampinato and Leonardi, 2013). Nystatin can be
applied topically or taken orally and is generally well-tolerated. Side effects
could include pruritis, burning of the mucous membranes, a rash, or
dyspnoea (Mazu et al., 2016). In contrast, amphotericin B is not well-
tolerated when taken orally, and is therefore usually administered
intravenously. It also has a high rate of nephrotoxicity (Ellis, 2002).
2.2 Antimicrobial resistance
Drug resistance of Candida infections is on the increase and is a

consequence of the widespread use of anti-fungal agents. Anti-fungal
resistance can also occur due to decreased drug intracellular acquisition,
reduced affinity for the drug and counteraction of the drug effect (Perlin et
al., 2017). Biofilm production is also related to a high level of anti-fungal
resistance, as it affects the efficacy of the antifungal medication, forming a
physical barrier that inhibits drugs from acting on the cell (Silva et al., 2017;
Li et al., 2018). Other factors such as efflux pumps, which are protein
10
channels that prevents the cell from accepting anti-fungal agents, add to
biofilm anti-fungal resistance (Borghi et al., 2016). There is therefore a need
for safer and more effective treatment options. Several medicinal plants
have been shown to have anti-fungal properties and may provide an
alternative treatment option for C. albicans and other Candida spp.
infections (Soliman et al., 2017).
2.3 Homeopathy
2.3.1 History of homeopathy
Homeopathy as a discipline was founded by Dr Samuel Hahnemann in the

19th century and is based on the principle of “like cures like.” Homeopathy
is an alternative approach to healthcare, which looks at the patient
holistically rather than just the symptoms with which they present (Chauhan
and Gupta, 2012).
2.3.2 Principles of homeopathy
2.3.2.1 The law of similars
The law of similars, or like cures like, is the primary law of homeopathy. It
specifies that the symptoms of the patient do not occur because of the
disease itself but are rather reactions of the body to that disease. Therefore,
these symptoms need to be matched to a remedy that has been shown to
produce a similar symptom picture in experiments called provings, which
are conducted in healthy patients (Chauhan and Gupta, 2012).
2.3.2.2 Principle of the single remedy
The principle of a single remedy states that only one remedy should be
given at a time as remedies are proven individually. The remedy should be
given based on its similarity to the patients’ symptoms and this remedy is
known as the similimum (Chauhan and Gupta, 2012).
11
2.3.2.3 Principle of the minimum dose
According to Hahnemann’s Organon, in which he described these

fundamental principles of homeopathy, the patient should be given the
smallest dose possible to initiate a healing response, reduce the risk of
aggravation or harmful effects, and restore health (Chauhan and Gupta,
2012).
2.3.3 Homeopathic remedy manufacturing techniques
Homeopathic remedies are made from substances derived from plant,

mineral, and animal sources. A mother tincture (Ø) is a liquid preparation of
a crude substance, prepared according to a standardised method as
determined by the homeopathic pharmacopoeia (Banerjee, 1996). The
crude material used for remedies are chopped up, diluted in distilled water
or alcohol and left to stand for several days or weeks. Thereafter, the
mixture is filtered to extract the liquid, which is known as the Ø. Insoluble
crude materials are first combined with lactose and ground repeatedly to
form a powder that is then soluble in water. This process is known as
trituration (Malik, 2013).
2.3.3.1 Potentisation
Potentisation is a process of serial dilution with succussion. The succussion

method is when the remedy is vigorously shaken to elicit its biological
activity. To make a centesimal potency (cH), one drop of the mother tincture
is added to 99 drops of alcohol and distilled water and succussed, and for
decimal potencies (D or X), one drop of the mother tincture is added to nine
drops of diluent and succussed. Subsequent potencies are made in a
similar manner (Figure 2.3) (Lockie, 2000).
12
Figure 2. 3 Homeopathic Potentisation (Homeopathicassociates.com,
2020)
2.3.4 Mother tinctures versus herbal extracts
Herbal extracts are prepared according to methods laid out as monographs

found in the herbal pharmacopoeia (Vlietinck et al., 2009); these can
include: infusions, decoctions, and percolation. The solvent-to-herb ratio
varies according to the type of herb and the method used for extraction. The
ratio of herb to diluent is typically 1:5, 1:10, or 1:2 (Mohammad Azmin et al.,
2016). Mother tinctures are prepared according to methods laid out in the
homeopathic pharmacopoeia, and are in a 1:10 dilution (Lockie, 2000).
2.4 Hydrastis canadensis L.
Hydrastis canadensis L. (H. canadensis L.), commonly known as goldenseal,

is shown in Figure 2.4. It is part of the Ranunculaceae family and is a
perennial herb (Christensen and Gorchov, 2010).The root and the rhizomes
are used in the herbal preparation for medicinal use. The main chemical
components of H. canadensis L. are berberine, canadine, hydrastine, and
isoquinoline alkaloids (Mandal et al., 2020). It is indicated to alleviate minor
inﬂammatory conditions of the mouth, skin, and eyes as well as to treat
diarrhoea, fever, and inflammation of vaginal and urethral mucous
13
membranes. It can also be used for urinary tract infections, liver disorders
and pneumonia (Villinski et al., 2003).
The main active constituent of H. canadensis L. is berberine, which is

responsible for many of its therapeutic properties. Even though berberine
can be obtained from other plant species such as the Berberis spp., the
added alkaloids hydrastine and canadine in H. canadensis L. are also
thought to be medically significant (Wallace et al., 2018).
Figure 2. 4 Hydrastis Canadensis L. Rhizome (Phys.org, 2020).
2.4.1 Hydrastis canadensis L. herbal extract
2.4.1.1 Herbal extract preparation
The herbal pharmacopoeia determines what the extraction, maceration, and

herb-to-solvent ratio should be when making herbal tinctures. Herbal
extracts of H. canadensis L. are generally prepared (British Pharmacopoeia,
2018).
Ultra-performance liquid chromatography (UPLC) and high-performance

liquid chromatography (HPLC) methods are used for authentication and
recognition of possible adulteration of H. canadensis L. and affiliated genus
14
species. These tests are performed to ensure that the plant samples contain
the major alkaloids hydrastine and berberine (Wallace et al., 2018).
2.4.2 Hydrastis canadensis homeopathic mother tincture
2.4.2.1 Mother tincture preparation
The homeopathic preparation of H. canadensis L. is known as

H. canadensis to distinguish it from the herbal preparation. The
homeopathic pharmacopoeia specifies the exact method required in the
preparation of homeopathic remedies, including the collection, preparation,
preservation, and standards thereof. Different countries use different
pharmacopoeias. In South Africa, homeopathic remedies are usually
prepared according to guidelines laid out in the German Homeopathic
Pharmacopoeia (Deutscher Apotheker Verlag, 2013).
H. canadensis mother tincture is made from the rhizome of the herb (Figure
2.4) and prepared using the HAB4A method (Appendix B). For the
preparation of the mother tincture, the rhizome is cleaned and finely cut.
The cut particles are then soaked in alcohol for a few days or weeks, then
strained and diluted (Sarembaud, 2019).
2.4.2.2 Homeopathic indications
In homeopathy, H. canadensis mother tincture is used as an antimicrobial,

and it has an affinity for the mucous membranes of the body, particularly
the throat, stomach, uterus, and urethra. It is indicated for infectious
conditions that produce a thick, yellowish, ropy secretion, especially in the
upper respiratory tract. It is also used to treat bacterial and fungal vaginal
infections (Vermeulen, 2011).
2.5 Calendula officinalis L. (C. officinalis L.)
C. officinalis L., commonly known as marigold (see Figure 2.5), is a

perennial herb and is part of the Asteraceae family (Shankar et al., 2017).
15
The main chemical components of this plant are carotenoids, terpenoids,
flavonoids, coumarins, quinones, and amino acids. The carotenoids in the
plant comprise twelve compounds, predominantly beta-carotene, lutein,
and violaxanthin. Of these compounds, beta-carotene is the most bioactive
(Olennikov and Kashchenko, 2014).
This plant has been used for many years as an anti-fungal, antibacterial,
anti-inflammatory, and antiseptic agent, useful in the treatment of wounds,
burns, and inflammation (Abudunia et al., 2017). C. officinalis L. is used to
treat inflamed internal organs, gastrointestinal ulcers, and dysmenorrhea. It
is also useful as a diuretic and as a diaphoretic in seizures (Muley et al.,
2009). Other uses are for inflammation of the oral and pharyngeal mucosa,
and for wounds and burns (Arora et al., 2013). In addition, the herb can be
used as a detoxification agent and as a cleanser (Arora et al., 2013). The
outcome of antimicrobial in vitro tests has shown that the Calendula genus
displays inhibitory effects on certain fungi (Efstratiou et al., 2012).
Figure 2. 5 Calendula officinalis L. plant (Lim, 2014)
2.5.1 Calendula officinalis L. herbal extract
2.5.1.1 Herbal extract preparation
C. officinalis L. is recorded in the German Commission E, European

Scientific Co‐operative on Phytotherapy, British Herbal Pharmacopoeia,
and World Health Organization monographs for its wound healing and anti‐
16
inflammatory actions (Arora et al., 2013). The flowers and leaves of the plant
are used in the herbal preparation for medicinal use (Olennikov and
Kashchenko, 2014); the flavonoids, glycosides, and tannins as well as other
antioxidant compounds are responsible for its antimicrobial effects. The
action of the tannins has been shown to oppose yeasts by crossing the cell
wall and affecting the membrane. It then binds to the surface, which inhibits
cell growth (Efstratiou et al., 2012)
2.5.2 Calendula officinalis homeopathic mother tincture
2.5.2.1 Mother tincture preparation
Prepared C. officinalis L. in homeopathy is named C. officinalis. C. officinalis

mother tincture is prepared according to the German Homeopathic
Pharmacopoeia HAB3A method (Appendix B) (Deutscher Apotheker
Verlag, 2013). It is made from the fresh flowers and leaves of the plant, that
are finely chopped, soaked in alcohol for days or weeks, and then strained
(Arora et al., 2013).
2.5.2.2 Homeopathic indications
C. officinalis mother tincture is useful in the treatment of wounds, burns, and

septic infections of the skin and mucous membranes. It is also known as a
homeopathic antiseptic for wound healing and prevents uncontrolled
bleeding. It is suitable for all instances of injury where the skin is damaged,
and it contributes to the rapid healing of wounds (Vermeulen, 2011).
C. officinalis can be applied to affected areas to treat burns, skin conditions

such as eczema, yeast infections, skin irritations and sunburn, and to treat
other conditions such as varicose ulcers, postoperative wounds, and
ruptured muscles or tendons. It is also used to treat torn perineal tissues
following childbirth as well as joint conditions where there is loss of synovial
fluid (Khairnar et al., 2013).
17
2.6 Related research
While there is limited research on the effects of these two medicinal plants
on C. albicans, there is currently no research done on the anti-fungal effects
of the combination of these two mother tinctures. With regards to the anti-
fungal effects of H. canadensis L., the majority of research has been
conducted on its active constituent berberine. Berberine is a protoberberine-
type isoquinoline alkaloid that has been shown to exhibit antibacterial and
anti-fungal activity (Bone and Mills, 2013). In a study conducted by Xie et
al. (2020) on the in vitro anti-fungal effects of berberine against Candida
spp. in planktonic and biofilm conditions, the berberine alkaloid showed
remarkable anti-fungal action and restrictive influence against biofilm
formation.
In a study by da Silva et al. (2016), the anti-fungal effect of berberine against

multi-drug resistant Candida spp. was demonstrated. The study showed
that berberine caused alterations to the integrity of the plasma and
mitochondrial membranes, as well as DNA damage, which resulted in cell
death. Berberine was also shown to produce a significant reduction in
biofilm activity.
In a study done by Wei et al. (2011) on the in vitro synergism between

berberine and miconazole against planktonic and biofilm Candida cultures,
berberine showed significant inhibition effects on the candida strains,
including C. albicans, but the most effective inhibition was on C. krusei,
which is a non-albicans species.
In terms of homeopathic research, H. canadensis mother tincture was

shown to have inhibitory effects against the in vitro growth of C. albicans
using the disc diffusion and broth dilution methods (Budree, 2004).
Saffari et al. (2016) compared the effects of C. officinalis herbal extract and
clotrimazole on vaginal candidiasis. Results showed that C. officinalis was
effective in healing vaginal discharge, burning, and inflammation and had a
18
longer effect than clotrimazole. An in vitro study on the anti-fungal activity
of the essential oil derived from C. officinalis L. flowers by Gazim et al.
(2008) showed that C. albicans has sensitivity to these essential oils.
In 1998, a study on the antimicrobial effects of C. officinalis in D1 and D3

homeopathic dilutions on C. albicans was conducted using the disc diffusion
and broth dilution method. The results showed that both potencies were
effective in inhibiting C. albicans (De Klerk, 1998).
19
3. METHODOLOGY
3.1 Experimental design
This quantitative experimental research was conducted at the Water and

Health Research Centre (WHRC) with permission granted (Appendix A)
under the supervision of a qualified laboratory technician.
3.2 Candida species
An American Type Culture Collection (ATCC 90028) reference strain of

C. albicans was purchased and stored as per manufacturer instructions at
the WHRC laboratory.
Fourteen isolates of Candida spp. strains from previously analysed samples

were included in the study to determine the effectiveness of the selected
homeopathic tinctures on different Candida spp. strains. The selected
strains were confirmed to be C. albicans (1) C. lusitaniae (6),
C. parapsilosis (5), C. guilliermondii (1), C. inconspicua/lambica (1) and
C. rugosa (1).
3.3 Homeopathic remedies
The homeopathic mother tinctures (Ø) selected to test their effectiveness

against Candida spp. were H. canadensis Ø in 70% ethanol, C. officinalis
Ø in 66% ethanol and a H. canadensis and C. officinalis Ø complex in 68%
ethanol.
3.4 Methods used for analysis
3.4.1 Media preparation
Brain heart infusion (BHI) broth (g/l) and Potato dextrose agar (PDA) (g/l)
were prepared according to the manufacturer’s instructions and autoclaved
for fifteen minutes. The Mueller Hinton broth was prepared by weighing 4.2g
20
of Mueller Hinton powder and dissolved in 200ml distilled water (dHշO).
Mueller Hinton agar was prepared by weighing 7.6g Mueller Hinton powder,
which was dissolved in 200ml of dHշO and poured into sterile petri dishes.
3.4.2 Yeast culture preparation
Fifteen yeast strains were isolated and identified using the VITEK®2
BioM‫ؘ‬erieux (France) machine at the WHRC laboratory. The strains were
stored at -80°C in glycerol and recovered by culturing in BHI broth overnight
at 30-35°C. Growth of the yeast was identified by the turbidity of the broth
and plated onto PDA to confirm the yeast and incubated at 30-35°C
overnight. The plates were checked regularly for yeast growth as it could
take up to 5 days. Once growth was observed, the isolates were gram
stained and plated onto Mueller Hinton agar plates for the experiments and
left overnight for fresh cultures.
3.4.3 Preparation of Hydrastis canadensis and Calendula officinalis

mother tinctures
The Ø’s were purchased from a local homeopathic laboratory, Fusion

Homoeopathics cc, who imports them from a manufacturer in Germany,
Gehrlicher GmbH; the laboratory is able to provide certification and quality
control documentation as proof of the medications’ quality and standard
(Hohl, 2019).
Fusion Homoeopathics cc made up the complex of H. canadensis and

C. officinalis Ø in a 1:1 ratio, which was mixed together in equal parts in a
volume-to-volume manner in a dispensing laboratory, utilising profession
specific and general Good Dispensing Practice guidelines (Deutscher
Apotheker Verlag, 2013).
C. officinalis Ø was made using the HAB3A method with 66% ethanol
concentration and H. canadensis Ø was made using the HAB4A method
21
with 70% ethanol concentration (Appendix B) (Deutscher Apotheker Verlag,
2013).
3.4.4 Kirby Bauer Disk Diffusion method
The Kirby-Bauer Disk Diffusion Susceptibility method is used to measure

the effects of antimicrobial sensitivity against various microorganisms. The
yeast, which was grown overnight was inoculated and plated onto Mueller-
Hinton agar plates (Benkova et al., 2020).
The antimicrobial activity of H. canadensis and C. officinalis Ø was

determined using the Kirby-Bauer disk diffusion method in accordance with
the Clinical Laboratory Standards Institute (Hudzicki, 2009).
The fifteen yeast isolates, including the C. albicans reference strain, were
cultured, and incubated overnight at 30°C. The plates were removed from
the incubator and allowed to reach room temperature (20°C-24°C). A
volume of 1mL saline was aliquoted into 3mL plastic tubes, to which the
yeast isolates were suspended individually to prepare 0.50-0.54 McFarland
standard yeast suspensions. The Densi-check plus (bioM‫ؘ‬erieux) machine
was used to confirm the McFarland standard. The individual yeast
suspension was utilised to swab even fungal lawns onto Mueller-Hinton
plates (Figure 3.1). The plates were allowed to settle for ten minutes at room
temperature before applying the anti-fungal discs.
The Mueller-Hinton Agar (MHA) used in the Kirby-Bauer technique were

used to test the anti-microbial activity of the homeopathic mother tinctures,
with the ethanol samples serving as a control and the anti-fungal as a
positive control.
22
Figure 3. 1 Fungal lawn streaking (Espinel-Ingroff, 2007)
Sterile disks were impregnated with a range of dilutions (10ppm, 20ppm and
25ppm) of H. canadensis Ø, C. officinalis Ø, a combination of
H. canadensis and C. officinalis Ø, and ethanol in 66%, 68% and 70% as
well Fluconazole 25mcg and blank discs were placed individually onto the
surface of the yeast lawn agar plates using sterile forceps.
Inoculated agar plates were inverted and incubated at 35˚C for 24 hours.
After the incubation period, plates were examined for a zone of inhibition
around the impregnated discs. The zones were measured from the edge of
the disc to the outer rim of clear zone using the Automatic colony counter
Scan® 1200 Interscience International (France) to determine if there was
inhibition of growth. The disks used for disk diffusion had a diameter of 6
mm therefore all measurements of <7 mm indicated that no inhibition of
fungal growth had occurred (i.e. resistant); measurements between 8-14
mm showed intermediate inhibition; and those >15 mm showed a sensitivity
of the fungus to the treatment.
The experiments were conducted in triplicate to obtain reliable results.

Appropriate controls, such as the media (blank control), solvent (effect of
solvent [ethanol] alone), negative (no treatment material) and positive (anti-
fungal [Fluconazole 25mcg]) were included to ensure the reliability and
trustworthiness of the methods (Hudzicki, 2009).
23
3.4.5 Microdilution method
The Microdilution method comprises of broth dilution procedures to

determine the Minimum Inhibitory Concentration (MIC), which is a
procedure that determines the lowest concentration of an antimicrobial that
inhibits the growth of an organism and when incubated to inhibit visible
growth. The Broth dilution utilises the 96 well plate, where the microbe is
inoculated into a liquid medium with different concentrations of the
antimicrobial agent (Wiegand et al., 2008).
To determine the MIC of the tinctures, the broth dilution method was used
whereby the compounds, as per Table 3.1, were tested using the serial
dilution method. A 96 well plate was labelled and each well filled with 100μl
Mueller-Hinton broth. The plate was divided into two sections and six wells
per row used for the dilutions of each tincture, as shown in Figure 3.2. Fifty
microliters of each compound to be tested was serially diluted by the
addition of undiluted tincture to the first well, mixed and 50μl removed; this
was then added to the next well and repeated for the next four wells. As per
the Kirby Bauer method, a variety of controls were included. This consisted
of a media control (no yeast), a media and yeast control (no antimicrobials
added), test samples, solvent controls, and antifungal controls (positive)
listed in the previous section.
Table 3. 1 List of compounds added to 96 well plate
A1-A6 Media Only A7-A12 H. canadensis + C.officinalis Ø complex
B1-B6 Yeast Only B7-B12 H. canadensis + C.officinalis Ø complex

C1-C6 H. canadensis Ø C7-C12 H. canadensis + C.officinalis Ø complex
D1-C6 H. canadensis Ø D7-D12 66% ethanol
E1-C6 H. canadensis Ø E7-E12 68% ethanol
F1-F6 C. officinalis Ø F7-F12 70% ethanol
G1-G6 C. officinalis Ø G7-G12 66% Fluconazole
H1-H6 C. officinalis Ø H7-H12 66% blank
24
Figure 3. 2 A 96 well plate template for MIC dilution experiment
A duplicate plate was prepared, and the plates were incubated at 30°C for
18-24 hours. After the incubation period, the plates were measured for
turbidity on the X-Mark Microplate Spectrophotometer (Bio-Rad; USA) at
450nm, whereby the readings of growth and no growth were recorded.
All wells that displayed no growth (clear well) and the consecutive well were
plated onto a Mueller Hinton agar plate and incubated overnight to check if
there was further growth; the results of this were recorded and the plates
were incubated for a further 24 hours to check if the yeast and tincture
reaction was fungistatic or fungicidal.
3.5 Validity and reliability
The Ø’s were acquired from Fusion Homeopathics cc. who imports them
and have certified quality control measures that ensures that their products
are of the premium quality. The study uses the Kirby-Bauer disk diffusion
test and MIC method as recommended by the Clinical Laboratory Standards
Institute (CLSI) (Hudzicki, 2009), and all recommended repeats and
controls were included to ensure reliable results. All experiments were
25
performed under the supervision of a qualified laboratory technician in a
controlled environment to ensure that the results are reliable.
The procedure was performed in triplicate (repeatability) on three

independent days (reproducibility) to ensure reliable results.
H. canadensis Ø, C. officinalis Ø, H. canadensis and C. officinalis complex

Ø along with solvent controls (66%, 68 and 70% ethanol), sterile disc control
(distilled water), and anti-fungal control (Fluconazole 25 mcg) were included
in each test run to ensure that the experimental conditions were correct.
3.6 Data analysis
Results were analysed and interpreted by the researcher with assistance

from a statistician. Frequencies and descriptive testing were done on the
overall sample. The Shapiro-Wilk test was utilised to test for normality and
the Kruskal-Wallis test was performed to measure possible statistically
significant differences among the groups. If statistically significant changes
were noted, the Mann-Whitney comparison tests were performed to check
statistically significant differences between the groups (De Klerk, 2019).
26
4. RESULTS
4.1 Overview
The aim of the study was to investigate the antimicrobial activity of the
homeopathic remedies H. canadensis and C. officinalis Ø complex against
fungi, namely the Candida spp., using the Kirby-Bauer Disk Diffusion
Susceptibility method and Microdilution method.
The Kirby-Bauer Disk Diffusion Susceptibility method, also known as the

Agar Disk Diffusion method, is used to test the sensitivity or resistance of
certain microbes against antimicrobial compounds. This was the method of
choice to test if H. canadensis and C. officinalis Ø complex could inhibit or
kill fungal growth of the C. albicans and Candida spp. strains, respectively.
The disks used for disk diffusion had a diameter of 6 mm therefore all
measurements of <7 mm indicated that no inhibition of fungal growth had
occurred (i.e. resistant); measurements between 8-14 mm showed
intermediate inhibition; and those >15 mm showed a sensitivity of the
fungus to the treatment.
The minimum inhibitory concentration (MIC) of the remedies was defined

as the lowest concentration at which fungal growth was inhibited and was
measured using the Microdilution method.
The Minimum Bactericidal/fungicidal Concentration (MBC/MFC) is the

minimum concentration of a drug that is needed to kill a microbe. It is
determined by sub-culturing broth dilutions that were shown to inhibit growth
of a microbe in the MIC method (Wiegand et al., 2008). Mueller-Hinton agar
plates were streaked with samples from the 96 well plates and incubated
for 48 hours, to determine the presence of viable C. albicans cells after
treatment with the H. canadensis Ø, C. officinalis Ø and the complex of the
two.
27
The H. canadensis Ø was prepared from the rhizome of the plant in 70%
ethanol. The C. officinalis Ø was prepared from the fresh flowers in 66%
ethanol and the complex of the two was prepared in 68% ethanol; these
tinctures were purchased from Fusion Homeopathics cc, a reputable
homeopathic laboratory who imports their tinctures from a manufacturer in
Germany, Gehrlicher GmbH, and can provide certification and quality
control documentation as proof of the medications’ quality and standard
(Hohl, 2019).
Data was analysed and interpreted with the assistance of a statistician (De
Klerk, 2019). Frequencies and descriptive testing were done on the overall
sample. The Shapiro-Wilk Test was used to check for normality and showed
an abnormal distribution of data among the samples given (owing to the
small sample size), therefore non-parametric tests were chosen for further
analysis.
The Kruskal-Wallis Test is a rank-based nonparametric test used to

determine if there are statistically significant differences between two or
more groups of an independent variable (Pallant, 2007). If statistically
significant differences were noted (when the p-value was < 0.05), then the
Mann-Whitney U test was performed.
The Mann-Whitney U test is used to compare differences between two

independent groups when the dependent variable is either ordinal or
continuous, but not normally distributed (Pallant, 2007). This chapter
summarizes and presents the data obtained from the experiments that will
be discussed in further detail in the next chapter.
4.2 Results for the Candida albicans strain
4.2.1 Kirby-Bauer Disk Diffusion method
Paper disks containing H. canadensis Ø, C. officinalis Ø, H. canadensis and

C. officinalis Ø complex, 70% ethanol, 68% ethanol, and 66% ethanol as
28
well as the Fluconazole anti-fungal were allowed to diffuse into the agar
containing the C. albicans and Candida spp. strains, thereby producing a
clear zone around the paper disk. The zone diameters were measured and
recorded accordingly. As shown in Figure 4.1 (A), the paper disks
containing each of the individual tinctures, the combination of tinctures and
the controls were placed evenly apart onto the agar. The H. canadensis Ø
tincture produced a prominent yellow colour, as seen in disk one and disk
three. As shown in Figure 4.1 (B), the paper disks containing (1)
Fluconazole 25mcg and (2) the blank control were placed apart onto the
agar, clearly showing a zone of inhibition around the anti-fungal drug and
no zone of inhibition for the blank control.
A B
2 3
1
1 4 2
6 5
Figure 4. 1 Disk diffusion agar of C. albicans (ATCC 90028) showing

zones of inhibition for A: Remedies (1) H. canadensis (2) C.
officinalis, (3) H. canadensis and C. officinalis complex, (4)
66% Ethanol, (5) 68% Ethanol and (6) 70% Ethanol and B: (1)
Fluconazole 25mcg and (2) blank control.
The experiments were conducted in triplicate, and a summary of the results

are shown in Table 4.1 below. The minimum and maximum zones of
29
inhibition were measured in millimetres (mm) and recorded for each of the
experiments done. Values written in bold text indicate sensitivity to the
treatment.
The H. canadensis and C. officinalis Ø complex inhibited fungal growth of

C. albicans with an average zone of inhibition of 17 mm. However, H.
canadensis Ø on its own had a more notable inhibitory effect with an
average zone of inhibition of 24 mm, while the C. officinalis Ø produced an
average zone of inhibition of 8 mm, indicating only intermediate inhibition.
The 66%, 68% and 70% ethanol solvents used for the preparation of the Ø
showed fungal growth with zones of inhibition of 10 mm. The Fluconazole
25 mcg (anti-fungal positive control) showed the highest inhibitory effect on
C. albicans, with an average zone of inhibition of 29 mm.
Table 4. 1 Summary of the Kirby-Bauer Disk Diffusion susceptibility test

against C. albicans
Sensitivity: > 15 mm; Intermediate Inhibition: 8-14 mm; Resistance: <7 mm

Remedies N Mean Median Minimum Maximum Std. Mean
(mm) (mm) (mm) (mm) deviation Rank
H. 3 24 24 23 25 0.81 17
canadensis Ø
C. officinalis 3 8 8 6 11 2.34 5
Ø
H. 3 17 19 10 20 5.56 12
canadensis &
C. officinalis
Ø complex
66% ethanol 3 10 10 9 11 0.88 7
68% ethanol 3 10 10 10 11 0.36 8
70% ethanol 3 10 10 10 10 0.40 8
Fluconazole 3 29 29 29 29 0.00 20
25mcg
30
The results for the Kruskal Wallis test are shown in Table 4.2. The results
indicate that a statistically significant difference has occurred between the
treatment groups (p = 0.020).
Table 4. 2 Kruskal Wallis test statistics for the Kirby-Bauer Disk Diffusion
test on C. albicans
Kruskal-Wallis H Df Asymp. Sig.

Zone 14.975 6 0.020
The results for the Mann-Whitney test for the Kirby-Bauer Disk Diffusion test
are shown in Table 4.3. This test was used to compare treatments to see
where the statistically significant differences occurred. Statistically
significant results are in bold and are indicated by p values less than 0.050.
Table 4. 3 The Mann- Whitney U test statistics for the Kirby-Bauer Disk
Diffusion on C. albicans
Treatment 1 Treatment 2 p-value

H. canadensis Ø C. officinalis Ø 0.050
H. canadensis Ø H. canadensis & C. officinalis Ø 0.050
complex
H. canadensis Ø 66 % ethanol 0.050
H. canadensis Ø 68% ethanol 0.050
H. canadensis Ø 70% ethanol 0.046
H. canadensis Ø Fluconazole 25mcg 0.037
C. officinalis Ø H. canadensis & C. officinalis Ø 0.127
complex
C. officinalis Ø 66 % ethanol 0.275
C. officinalis Ø 68% ethanol 0.513
C. officinalis Ø 70% ethanol 0.507
C. officinalis Ø Fluconazole 25mcg 0.037
H. canadensis & 66 % ethanol 0.127
C. officinalis Ø complex
31
Table 4.3 continued The Mann-Whiitney U test statistics for the Kirby-
Bauer Disk Diffusion on C. albicans.
Treatment 1 Treatment 2 p-value

H. canadensis & 68% ethanol 0.127
H. canadensis & 70% ethanol 0.268
H. canadensis & Fluconazole 25mcg 0.037
66% ethanol 68% ethanol 0.513
66% ethanol Fluconazole 25mcg 0.037
According to these results, Fluconazole significantly outperformed the H.

canadensis Ø, the C, officinalis Ø, the H. canadensis and C. officinalis Ø
complex, as well as the 66%, 68% and 70% ethanol samples.
The minimum tincture concentration required for inhibition of yeast growth

was tested by the serial dilution method in 96 well plates, as shown in Figure
4.2. The absorbance readings (OD 450nm) were used to determine the
effectiveness of the tinctures against the yeast. Inhibition of growth was
determined by calculating the difference in the pre- and post- incubation
absorbance of wells. A reduction in the corrected absorbance, highlighted
by a negative value or a value less than the yeast absorbance value (0.37
nm), indicated inhibition of yeast growth.
32
Figure 4. 2 96 well plate for MIC dilution of C. albicans (ATCC 90028)
The data for the MIC, as shown in Table 4.4, below confirmed that
H.canadensis and C. officinalis Ø complex and C. officinalis Ø had positive
anti-fungal effects against C. albicans. H.canadensis Ø had a higher
absorbance reading than the yeast but it could be due to the residue in the
tincture that influenced the reading. The p-values <0.050 shows statistically
difference between the treatments, however these had none.
Table 4. 4 Summary of the Microdilution test against C. albicans

Mean Median Maximum Minimum Std. Mean
(nm) (nm) (nm) (nm) deviation Rank
H. canadensis Ø 0.46 0.15 2 0 0.60 19
C. officinalis Ø 0.22 0.23 0 0 0.07 18
H. canadensis & C. 0.06 0.09 0 0 0.24 12
officinalis Ø complex
66% ethanol 0.29 0.31 0 0 0.09 29
68% ethanol 0.24 0.26 0 0 0.09 24
70% ethanol 0.29 0.28 0 0 0.03 30
Fluconazole 25mcg 0.22 0.18 0 0 0.10 20
33
The results for the Kruskal Wallis test are shown in Table 4.5. The results
indicate that a statistically significant difference did not occur between the
treatment groups (p = 0.123), therefore the Mann-Whitney test was not
performed.
Table 4. 5 Kruskal Wallis test statistics for the Microdilution test on

C. albicans
Kruskal-Wallis H Df Asymp. Sig.

Zone 10.041 6 0.123
The first wells of the two Ø’s, which had the highest concentration of the
tinctures, showed growth visually as the wells were cloudy and had residue
in it, however it cannot be established whether the turbidity was due to the
settling of the residue of the tinctures itself or from growth of the yeast. The
wells thereafter showed no growth. The first well had a dry residue
concentration of 0.40 m/m, as per Table 4.6 below, however, the C.
officinalis Ø and the Ø complex did not have any residue settling and did
not seem to affect the absorbance readings.
The exact concentration of the dry residue for the tinctures is not known.
The percentage (m/m) dry residue concentration was given by the supplier
(Appendices C and D). This was to determine a MIC related to the mass
plant extract prepared according to the method HAB4A (H. canadensis Ø),
HAB3A(C. officinalis Ø)(Appendix B), as described in the German
Homeopathic Pharmacopoeia (Deutscher Apotheker Verlag, 2013). The
concentration after dilution was calculated using the following equation:
𝐶𝐶2 =𝐶𝐶1 × 𝑉𝑉1

𝑉𝑉2
Where C1 is the starting concentration given as % m/m dry residue

C2 is the % m/m dry residue concentration after dilution
34
V1 is the volume of C1 added
V2 is the final volume after addition of V1 to the well
Table 4. 6 The percentage of dry residue concentration in each well for
the homeopathic Ø’s
Dry Residue Concentration (%)

Tinctur Well Well 2 Well 3 Well 4 Well 5 Well 6
e 1 4x 8x 16x 32x 64x
2x
H. canadensis 1.2 0.40 0.13 0.04 0.02 0.01 0.00
Ø
C. officinalis Ø 1.7 0.57 0.19 0.06 0.02 0.01 0.00
H. canadensis 1.45 0.48 0.16 0.05 0.02 0.01 0.00
& C. officinalis
Ø Complex
The corrected absorbance readings, as per Table 4.7, showed that the
highest concentration of H. canadensis Ø at the first well had a higher
absorbance reading compared to the H. canadensis and C. officinalis Ø
complex. The absorbance decreased as the H. canadensis Ø was diluted,
however for the C. officinalis Ø, H. canadensis and C. officinalis Ø complex,
ethanol concentrations (66%, 68% and 70%) and Fluconazole, the
absorbance readings increased as the concentrations of the compounds
decreased. The increased absorbance is consistent with the growth of yeast
during the incubation period. The Fluconazole displayed inhibition until the
32x dilution, which demonstrates the directly proportionate relationship
between concentration and growth inhibition. The values in bold are lower
than the yeast values, which show inhibition of the strain.
35
Table 4. 7 Summary of the results for the 2-fold dilution series for
C. albicans
Dilutions in nm 2x 4x 8x 16x 32x 64x

Isolate: C. albicans
Blank -0.01 -0.01 -0.01 0.00 -0.00 -0.00
Yeast only 0.37 0.37 0.36 0.34 0.35 0.36
H. canadensis Ø 1.55 0.81 0.08 0.04 0.11 0.20
C. officinalis Ø 0.09 0.24 0.21 0.23 0.25 0.28
H. Canadensis/ -0.39 0.25 0.05 0.05 0.14 0.29
Calendula officinalis Ø complex
66% Ethanol 0.14 0.24 0.28 0.34 0.36 0.38
68% Ethanol 0.25 0.27 0.51 0.52 0.54 0.67
70% Ethanol 0.28 0.26 0.27 0.30 0.32 0.34
Fluconazole 0.14 0.12 0.14 0.21 0.30 0.38
4.2.3 Minimum Fungicidal Concentration Method (MFC)
The MFC test was performed to check if there was fungal inhibition or fungal
death in the wells that showed no growth. These were visually checked in
the 96 well plates and the first well that showed no growth was plated onto
the Mueller Hinton agar plate, as per Figure 4.3 below, and incubated for
24 hours. The results after 24hrs showed that there was growth on the agar
plate confirming that the C. albicans was inhibited and not killed by the
tinctures.
36
A B
Figure 4. 3 Agar plate of MFC method for C. albicans A: Agar plate reading
showing clear growth after 24 hours B: Grid plate showing wells
that were plated on agar plate
Table 4. 8 List of compounds on grid plate
A1-6 Media only A7-12 H. canadensis & C. officinalis Ø

complex
B1-6 Media & Yeast B7-12 H. canadensis & C. officinalis Ø
complex
C1-6 H. canadensis Ø C7-12 H. canadensis & C. officinalis Ø
complex
D1-6 H. canadensis Ø D7-12 66% EtOH
E1-6 H. canadensis Ø E7-12 68% EtOH
F1-6 C. officinalis Ø F7-12 70% EtOH
G1-6 C. officinalis Ø G7-12 Fluconazole 25mcg
H1-6 C. officinalis Ø H7-12 Blank
4.3 Results for emerging pathogenic Candida spp. strains
The other five Candida spp. strains that were tested included: C. lusitaniae
(n=6) and C. parapsilosis (n=5), C. guilliermondii (n=1),
C. inconspicua/lambica (n=1) and C. rugosa (n=1). The number in brackets
indicates the number of strains tested. These are emerging pathogens
37
which are also are responsible for candidiasis, and with growing resistance
to anti-fungal medications such as Fluconazole (Mixão et al., 2019;
Arendrup, 2013). The strains tested were all obtained from human samples.
Statistical analysis was not done on these strains as the focus of the study
was on C. albicans.
4.3.1 Kirby-Bauer Disk Diffusion method
A summary of the average zones of inhibition for all the Candida species
strains analysed is presented in Table 4.9. Measurements are rounded off
to the nearest decimal.
4.3.1.1 C. lusitaniae
In Figure 4.4, the zone of inhibition is indicated by the clear region

surrounding the paper disk. C. lusitaniae growth is observed by the white
lawn beyond the zone of inhibition.
A
A B
2
1 3 1
6 4 2
5
Figure 4. 4 Disk diffusion agar of C. lusitaniae showing zones of inhibition

for A: Remedies (1) H. canadensis (2) C. officinalis, (3) H.
canadensis and C. officinalis complex , (4) 66% Ethanol, (5)
68% Ethanol and (6) 70% Ethanol and B: (1) Fluconazole
25mcg and (2) blank control.
38
The H. canadensis and C. officinalis complex Ø inhibited fungal growth of
C. lusitaniae with an average zone of inhibition of 19 mm, but H. canadensis
Ø on its own had a more notable inhibitory effect with an average zone of
inhibition of 23 mm, which shows that the microbe is sensitive to the tincture.
The C. officinalis Ø had an average zone of inhibition of only 10 mm. The
66%, 68% and 70% ethanol solvents used for the preparation of the Ø’s
showed fungal growth with zones of inhibition of 9 mm, 10 mm, and 9 mm,
respectively. The Fluconazole 25 mcg showed the highest inhibitory effect
on C. lusitaniae, with an average zone of inhibition of 36 mm.
4.3.1.2 C. parapsilosis
Figure 4.5 highlights the effects of the tinctures on C. parapsilosis, indicated

by the zones of inhibition surrounding the impregnated disks.
A B
B
2
1 3
1
6 4
2
5
Figure 4. 5 Disk diffusion agar of C. parapsilosis showing zones of

inhibition for A: Remedies (1) H. canadensis (2) C. officinalis,
(3) H. canadensis and C. officinalis complex, (4) 66% Ethanol,
(5) 68% Ethanol and (6) 70% Ethanol and B: (1) Fluconazole
25mcg and (2) blank control.
39
The H. canadensis and C. officinalis complex Ø inhibited the fungal growth
of C. parapsilosis with an average zone of inhibition of 35 mm and had the
same effect as H. canadensis Ø on its own. The C. officinalis Ø produced
an average zone of inhibition of only 10 mm. The 66%, 68% and 70%
ethanol solvents used for the preparation of the Ø showed fungal growth
with equal zones of inhibition of 9 mm. The Fluconazole 25 mcg showed the
highest inhibitory effect on C. parapsilosis with an average zone of inhibition
of 47 mm.
4.3.1.3 C. guilliermondii
Figure 4.6 highlights the effects of the tinctures on C. guilliermondii,

indicated by the zones of inhibition surrounding the impregnated disks.
A B
1
2 1
6
3
4 5
Figure 4. 6 Disk diffusion agar of C. guilliermondii showing zones of

(3) H. canadensis and C. officinalis complex, (4) 66%
Ethanol, (5) 68% Ethanol and (6) 70% Ethanol and B: (1)
Fluconazole 25mcg.
40
C. guilliermondii with an average zone of inhibition of 8 mm, but H.
canadensis on its own had a more prominent inhibitory effect with an
average zone of inhibition of 11 mm, which indicates intermediate inhibition.
The C. officinalis Ø had an average zone of inhibition of 8 mm. The 66%,
68% and 70% ethanol solvents used for the preparation of the Ø showed
fungal growth with equal zones of inhibition of 8 mm and 7 mm, respectively.
The Fluconazole 25 mcg showed the highest inhibitory effect on
C. guilliermondii with an average zone of inhibition of 22 mm.
4.3.1.4 C. inconspicua/lambica
Figure 4.7 highlights the effects of the tinctures on C. inconspicua/lambica,

indicated by the zones of inhibition surrounding the impregnated disks.
B
A
2
1
3
6
4 1
5
Figure 4. 7 Disk diffusion agar of C. inconspicua/lambica showing zones of

(3) H. canadensis and C. officinalis complex, (4) 66% Ethanol,
(5) 68% Ethanol and (6) 70% Ethanol and B: (1) Fluconazole
25mcg.
41
C. inconspicua/lambica with an average zone of inhibition of 6 mm, as did
H. canadensis Ø and the C. officinalis Ø, showing that the strain is resistant.
The 66%, 68% and 70% ethanol solvents used for the preparation of the Ø
showed fungal growth with zones of inhibition of 6 mm, respectively. The
Fluconazole 25mcg also showed an inhibitory effect on
C. inconspicua/lambica with an average zone of inhibition of 6 mm. This
whole plate had an average of 6 mm, showing that this strain is resistant
and therefore the Ø’s and the anti-fungal were ineffective.
4.3.1.5 C. rugosa
Figure 4.8 highlights the effects of the tinctures on C. rugosa, indicated by

the zones of inhibition surrounding the impregnated disks.
A B
2 1
1
3 6
4 5
Figure 4. 8 Disk diffusion agar of C. rugosa showing zones of inhibition for

A: Remedies (1) H. canadensis (2) C. officinalis, (3) H.
canadensis and C. officinalis complex, (4) 66% Ethanol, (5)
68% Ethanol and (6) 70% Ethanol and B: (1) Fluconazole
25mcg.
42
The H. canadensis and C. officinalis Ø complex inhibited fungal growth of
C. rugosa with an average zone of inhibition of 17 mm, which was the same
inhibitory effect as H. canadensis with an average zone of inhibition of 17
mm. The C. officinalis Ø had an average zone of inhibition of only 7 mm.
The 66% ,68% and 70% ethanol solvents used for the preparation of the Ø
showed fungal growth with zones of inhibition of 6 mm, respectively. The
Fluconazole 25 mcg showed a prominent effect on C. rugosa with an
average zone of inhibition of 28 mm.
Table 4. 9 Summary of the Kirby-Bauer Disk Diffusion susceptibility test

against C. albicans and other Candida spp. strains.
Average zone of inhibition (mm)*
H. canadensis Ø
C. officinalis Ø
H. canadensis/
66% Ethanol
68% Ethanol
70% Ethanol
Fluconazole
Source
Isolate
C. albicans ATCC 24 8 17 10 10 10 29
90028
C. lusitaniae 23 10 19 9 10 9 36
C. parapsilosis 35 10 35 9 9 9 47
C. guilliermondii Human 11 8 8 8 8 7 22
C. inconspicua/ Human 6 6 6 6 6 6 6
Lambica
C. rugosa Human 17 7 17 6 6 6 28
43
Sensitive: >15 mm; Intermediate: 8-14 mm; Resistance: <7 mm
The results from the Microdilution method to determine the MIC, presented
in Table 4.10, show that H. canadensis Ø, H. canadensis and C. officinalis
Ø complex, as well as C. officinalis Ø exhibited inhibition against Candida
spp. which included C. lusitaniae (6) and C. parapsilosis (5),
C. guilliermondii (1), C. inconspic/lambica (1) and C. rugosa (1). Values
highlighted in bold indicate values less than the yeast value, which show
inhibition of the strain. The yeast value ranged from 0.46 nm in 2x dilution
and until 0.37 nm in the 64x dilution.
Table 4. 10 Summary of the results for the 2-fold Microdilution method for
Candida spp.

Isolate: C. lusitaniae
Blank 0.00 0.00 -0.00 -0.00 -0.00 -0.00
Yeast only 0.48 0.46 0.35 0.33 0.37 0.41
H. canadensis Ø -0.94 0.01 0.08 0.10 0.22 0.32
C. officinalis Ø 0.14 0.28 0.29 0.28 0.31 0.36
H. Canadensis/ -0.40 -0.00 0.08 0.14 0.29 0.41
66% Ethanol 0.27 0.38 0.40 0.41 0.42 0.43
68% Ethanol 0.26 0.36 0.41 0.41 0.42 0.41
70% Ethanol 0.27 0.35 0.38 0.39 0.40 0.40
Fluconazole 0.16 0.18 0.24 0.38 0.40 0.42
Isolate: C. parapsilosis
Blank 0.01 -0.00 0.00 0.07 -0.00 -0.00
Yeast only 0.46 0.46 0.43 0.42 0.43 0.43
H. canadensis Ø -0.68 -0.10 0.00 0.05 0.21 0.24
44
C. officinalis Ø 0.03 0.14 0.17 0.19 0.25 0.30
Table 4.10 continued Summary of the results for the 2-fold Microdilution
method for Candida spp.

H. Canadensis/ -0.85 -0.05 0.04 0.10 0.22 0.30
66% Ethanol 0.35 0.37 0.39 0.41 0.42 0.47
68% Ethanol 0.35 0.35 0.38 0.39 0.42 0.49
70% Ethanol 0.28 0.36 0.34 0.37 0.41 0.45
Fluconazole 0.22 0.24 0.27 0.32 0.36 0.46
Isolate: C. guilliermondii
Blank 0.00 0.00 0.00 -0.01 -0.01 -0.00
Yeast only 0.61 0.66 0.64 0.64 0.64 0.59
H. canadensis Ø -0.83 0.03 0.09 0.13 0.32 0.41
C. officinalis Ø 0.11 0.19 0.14 0.23 0.34 0.67
H. Canadensis/ -0.26 0.10 0.14 0.22 0.50 0.64
66% Ethanol 0.14 0.46 0.47 0.56 0.58 0.78
68% Ethanol 0.15 0.43 0.51 0.52 0.54 0.67
70% Ethanol 0.19 0.34 0.47 0.55 0.55 0.64
Fluconazole 0.63 0.79 0.78 0.67 0.58 0.59
Isolate: C. inconspicua/lambica
Blank 0.00 0.00 0.00 0.00 0.00 0.25
Yeast only 1.29 1.26 1.27 1.26 1.22 1.16
H. canadensis Ø 0.57 1.05 1.42 1.20 1.20 1.10
C. officinalis Ø 1.37 1.09 1.20 1.16 1.22 1.25
H. Canadensis/ 1.04 0.77 1.21 1.24 1.25 1.27
66% Ethanol 0.48 1.02 1.25 1.25 1.25 1.30
45
68% Ethanol 0.86 1.10 1.27 1.25 1.28 1.33
Table 4.10 continued Summary of the results for the 2-fold Microdilution
method for Candida spp.

70% Ethanol 1.02 1.20 1.27 1.27 1.29 1.33
Fluconazole 1.19 1.27 1.28 1.28 1.28 1.31
Isolate: C. rugosa
Blank 0.01 -0.00 -0.00 -0.00 -0.00 -0.00
Yeast only 0.43 0.47 0.36 0.21 0.29 0.37
H. canadensis Ø 0.06 0.39 0.23 0.16 0.15 0.28
C. officinalis Ø 0.08 0.04 0.04 0.09 0.25 0.35
H. Canadensis/ 0.12 -0.03 0.08 0.21 0.21 0.28
66% Ethanol 0.05 0.34 0.42 0.38 0.35 0.31
68% Ethanol 0.10 0.34 0.41 0.44 0.38 0.37
70% Ethanol 0.13 0.34 0.36 0.44 0.35 0.41
Fluconazole 0.18 0.30 0.34 0.40 0.32 0.42
As stated above, a reduction in the corrected absorbance reading

highlighted by a negative value or a value less than the yeast absorbance
value indicated inhibition of yeast growth. The corrected absorbance
readings for the six strains tested of C. lusitaniae, as per Table 4.10, showed
that the 2-fold dilution concentration of H. canadensis Ø had a lower
absorbance reading of -0.94 nm compared to the H. canadensis and
C. officinalis Ø complex with a reading of -0.40 nm. The absorbance reading
increased as the H. canadensis Ø and the Ø complex were diluted, with the
last well having readings of 0.32 and 0.41 nm, respectively. The same was
evident for the C. officinalis Ø, ethanol concentrations (66%, 68% and 70%)
and Fluconazole, where the absorbance readings increased as the
46
concentration of the compounds decreased. The increased absorbance is
consistent with the growth of yeast during the incubation period.
The absorbance readings of the five strains of C. parapsilosis tested

showed that the H. canadensis and C. officinalis Ø complex had lower
absorbance readings of -0.85 nm than the H. canadensis Ø with a reading
of -0.68 nm; the absorbance readings increased as the tinctures were
diluted and the last well had readings of 0.30 nm for the complex and 0.24
nm for H. canadensis. The C. officinalis Ø had an initial absorbance reading
of 0.03 nm and increased with a last well reading of 0.30 nm, the same as
the Ø complex. With regards to the ethanol concentrations (66%, 68% and
70%) and Fluconazole, the absorbance readings increased as the
concentrations of the compounds decreased, however the readings in the
last well at the 64x dilution were all above the yeast value of 0.43 nm
indicating that there was no effect on the compound.
The results for C. guilliermondii, as per the data in Table 4.10, demonstrated
that the H. canadensis Ø, C. officinalis Ø and the Ø complex all had lower
absorbance readings in the first well and steadily increased as the tinctures
were diluted. The first well had readings of -0.83 nm, 0.11 nm, and -0.26
nm, respectively for the three tinctures. The ethanol concentrations of 66%,
68% and 70% also had lower initial absorbance readings and these
increased, however the last well readings of 0.78 nm, 0.67 nm and 0.64 nm
respectively were higher than the last well value of 0.59 nm, which shows
that the ethanol had no effect at this dilution. The strain showed resistance
to the anti-fungal as the values were the same as the yeast value at the last
well, with a reading of 0.59 nm.
The C. inconspicua/lambica strain showed lower absorbance readings for

H. canadensis Ø and the ethanol concentrations of 66% and 68%, with
readings of 0.57 nm, 0.48 nm and 0.86 nm respectively; these increased as
the tincture was diluted, however C. officinalis Ø appeared to have initial
resistance with a higher absorbance reading of 1.37 nm and the readings
47
decreased until the 32x dilution, where it remained the same as the yeast
value, showing that it had no effect. The Ø complex had lower absorbance
readings until the 16x dilution and thereafter it had higher readings than the
yeast, showing that the lower dilution was not effective on this strain. The
70% ethanol had a lower absorbance reading in the first two wells but
increased thereafter and was higher than the yeast absorbance reading.
This indicates that this strain exhibited resistance to the 70% ethanol. The
anti-fungal had a lower absorbance reading than the yeast in the first well
only and as the treatment was diluted it showed higher values than the
yeast, also indicating resistance of this strain to the Fluconazole treatment.
C. rugosa corrected absorbance readings, as per Table 4.10, showed that

the highest concentration of H. canadensis Ø at the first well had a lower
absorbance reading of 0.06 nm compared to the H. canadensis and
C. officinalis Ø complex reading of 0.12 nm. The absorbance increased as
the H. canadensis Ø was diluted. This was also evident in the readings for
the C. officinalis Ø, H. canadensis and C. officinalis Ø complex, the ethanol
concentrations (66%, 68% and 70%) and Fluconazole, where the
absorbance readings increased as the concentrations of the compounds
decreased. The increased absorbance is consistent with the growth of yeast
during the incubation period. The Fluconazole displayed inhibition only at
the 2x dilution which demonstrates resistance of this strain to the anti-
fungal.
4.3.3 Minimum fungicidal concentration (MFC) method
Figure 4.9 below shows Mueller-Hinton agar plates that were streaked with
cells from the wells in Figure 4.2 to confirm the presence of viable Candida
spp. cells after treatment with the H. canadensis Ø, C. officinalis Ø and the
complex of the two (Table 4.11).
These were visually checked in the 96 well plates and the first well that
showed no growth was plated onto a Mueller Hinton agar plate, as per
Figure 4.9, and incubated for 24 hours. These were then checked and
48
thereafter incubated for another 24hrs for a total of 48 hrs. The plate below
clearly shows fungal inhibition and not fungal death due to the regrowth,
and this was evident for all the Candida spp. strains tested.
Figure 4. 9 Agar plate of MFC method of Candida spp. A: Agar plate

reading showing clear growth after 24 hours B: Grid plate
showing wells that were plated on agar plate.
Table 4. 11 List of compounds on grid plate
A1-6 Media only A7-12 H. canadensis & C. officinalis Ø

complex
B1-6 Media & Yeast B7-12 H. canadensis & C. officinalis Ø
complex
C1-6 H. canadensis Ø C7-12 H. canadensis & C. officinalis Ø
complex
D1-6 H. canadensis Ø D7-12 66% EtOH
E1-6 H. canadensis Ø E7-12 68% EtOH
F1-6 C. officinalis Ø F7-12 70% EtOH
G1-6 C. officinalis Ø G7-12 Fluconazole 25mcg
H1-6 C. officinalis Ø H7-12 Blank
49
5. DISCUSSION OF RESULTS
The antimicrobial effects of the homeopathic Ø’s H. canadensis and

C. officinalis complex on C. albicans and other Candida spp., are discussed
below as per the results produced by the Kirby-Bauer Disk Diffusion Method
and Microdilution method.
5.1 Discussion of experimental results for C. albicans
5.1.1 Kirby-Bauer Disk Diffusion Method
The anti-fungal effect of the homeopathic Ø’s were determined by

evaluating which Ø’s inhibited the growth of C. albicans and other strains of
the Candida spp. The disks used for disk diffusion had a diameter of 6 mm,
therefore all measurements of <7 mm indicated that no inhibition of fungal
growth had occurred (i.e., resistant); measurements between 8-14 mm
showed intermediate inhibition, and those >15 mm showed sensitivity of the
fungus to the treatment.
The zone of inhibition for each disk was measured in millimetres (in triplicate
for accuracy). The average measurement was calculated and used as the
measurement of the zone of inhibition, as per Table 4.1.
According to these results, H. canadensis Ø had an average measurement

of 24 mm for C. albicans, which shows that the yeast was sensitive to the
treatment. The three ethanol concentrations produced a zone of inhibition
of growth of only 10 mm each, which shows that the alcohol itself is not
wholly responsible for the antifungal effects of the tinctures. Interestingly
though, C. officinalis Ø produced a zone of inhibition of growth of 8 mm on
C. albicans, which was less than the ethanol concentration of 66% in which
it was prepared in, which produced a zone of 10 mm.
The complex of H. canadensis Ø and C. officinalis Ø had a zone of inhibition

of growth of 17 mm, which was greater than the zone produced by the 68%
ethanol concentration (10 mm) in which it was prepared in; however, still
less than that produced by H. canadensis Ø alone. Both H. canadensis Ø
and the Ø complex displayed >15 mm zones of inhibition towards
C. albicans, which indicates that the yeast was sensitive to the treatment.
The anti-fungal Fluconazole 25mcg produced the largest zone of inhibition
on all three plates with an average of 29 mm, which indicates that the strain
was sensitive to the anti-fungal treatment.
Overall, the results show that H. canadensis Ø and Fluconazole 25mcg had
the highest inhibitory effect on the C. albicans strain, whilst the Ø complex
also showed a notable effect; this result indicates that the C. albicans strain
exhibits a sensitivity to these mother tinctures. C. officinalis Ø on its own
and the ethanol concentrations of 66%, 68% and 70% produced
intermediate effects on the C. albicans strain.
The Kruskal Wallis test revealed that a statistically significant difference

occurred between the treatment groups (p = 0.020). The Mann-Whitney test
was then used to determine where these statistically significant differences
occurred. The results showed that the Fluconazole significantly
outperformed the H. canadensis Ø, the C. officinalis Ø, and the
H. canadensis and C. officinalis Ø complex, as well as the 66%, 68% and
70% ethanol samples. It also showed that the 70% ethanol outperformed
the H. canadensis Ø.
The Microdilution method was used to confirm the results obtained from the
Kirby-Bauer Disk Diffusion Susceptibility test, and to determine the MIC.
Table 4.4 shows a summary of MIC of the H. canadensis Ø, C. officinalis Ø
and the H. canadensis Ø plus C. officinalis Ø complex. The OD readings
showed the differences between the various wells for the tinctures,
Fluconazole, the three ethanol concentrations, as well as the wells that
contained the media and yeast only as a control.
51
H. canadensis Ø showed fungal inhibition at the third well (8x dilution), with
an OD reading of 0.08 nm. The untreated yeast cells in the 8x dilution had
an OD reading of 0.36nm; thus, the inhibitory effect of H. canadensis Ø is
observed by the drastic OD reduction. The inhibition was observed for all
dilutions, thereafter, confirming the effectiveness of H. canadensis Ø
against C. albicans. An observed phenomenon for the H. canadensis Ø
was the increased yeast growth at the higher concentrations of the tincture.
It was expected to have minimal yeast growth at the higher concentrations;
however, the tincture displayed inhibitory effects at lower dilutions instead.
This can possibly be explained according to the homeopathic law of serial
dilutions and minimum dose, whereby the smallest dose given can bring
about cure. Mother tinctures are unprocessed materials that are mixed with
alcohol and are the starting process of all homeopathic preparations; in
homeopathy this is known as the lowest potency but with the highest
concentration of active ingredients. Samuel Hahnemann formulated a
process called potentisation to reduce the dose of the remedy by diluting it.
This process enables the homeopathic potency drug to work by enhancing
its dynamic properties (Chauhan and Gupta, 2012). This phenomenon did
not however occur with the C. officinalis Ø or the complex, and it is not yet
known why this only occurred with the H. canadensis Ø.
The MIC readings for C. officinalis Ø confirmed the inhibition of yeast growth
for the dilution range. The highest level of inhibition was noted at the most
concentrated.
C. officinalis Ø in well one (2x dilution), with an OD value of 0.09 nm. The
analysis showed an average MIC value irrespective of the dilution factor of
0.22 nm, which is below the OD value 0.30 nm for the yeast control.
The combination of H. canadensis Ø and C. officinalis Ø complex

demonstrated the lowest OD value of -0.39nm for all tinctures tested. This
was reiterated with a mean value of 0.06 nm; thus, indicating the
effectiveness of the tincture complex against C. albicans for the
Microdilution method. Interestingly, the OD value increased to 0.25 nm at
52
the second well (4x dilution); thereafter the values decreased to 0.05 nm for
the subsequent dilutions. This pattern may be a result of either human error
during the preparation of the MIC plates or it is as a result of the phenomena
observed for H. canadensis Ø.
The average MIC values for the ethanol controls were 0.29 nm for 66% and
70% ethanol, and 0.24 nm for the 68% ethanol. This data showed that the
ethanol had some level of inhibition against C. albicans. The inhibitory effect
of the ethanol controls on C. albicans were reduced as they were further
diluted.
The Fluconazole had an OD value of 0.14 nm at the highest concentration,

with an increasing OD value as the anti-fungal was diluted. The most diluted
Fluconazole concentration had an OD of 0.38 nm. The value was indicative
of the reduced inhibitory effect of Fluconazole against C. albicans when
diluted 64-fold. The value increased as the dilutions increased; however,
this reading was higher than the yeast reading of 0.36.
5.1.3 Minimum Fungicidal Method (MFC)
This method was used to check whether the tinctures, ethanol and anti-
fungal compounds used in the 96 wells were fungicidal or fungistatic. From
the MFC analysis, it was noted that all the tinctures had a fungistatic
(suppressed growth) effect rather than a fungicidal (killed) effect. The
recovery of the yeast cells, when given nutrients, enabled them to grow and
flourish after the incubation period, which confirmed the fungistatic
mechanism of the tinctures. To determine the fungicidal levels of the
tinctures, further dose experiments need to be conducted.
5.2 Discussion of experimental results for the Candida species
5.2.1 Kirby-Bauer Disk Diffusion Method
The zones of inhibition for the H. canadensis Ø against emerging

pathogenic Candida strains, as shown in Table 4.9, demonstrated that the
53
tincture effectively prevented the growth of C. lusitaniae (23 mm),
C. parapsilosis (35 mm) and C. rugosa (17 mm). The zone of inhibition for
C. guilliermondii was intermediate, with a zone measuring 11 mm.
C. inconspicua/Lambica growth was not inhibited by the tincture, with a
zone of 6 mm; thus, demonstrating resistance.
C. officinalis Ø did not produce as prominent results as H. canadensis Ø,

with an average of 10 mm on both C. lusitaniae and C. parapsilosis, showing
intermediate inhibition of growth. The zones of inhibition for the
C. guilliermondii, C. inconspicua/Lambica and C. rugosa were below 10
mm; thus, indicating the minimal inhibitory effects of the C. officinalis Ø.
The complex of H. canadensis Ø and C. officinalis Ø had a zone of inhibition

of 19 mm for C. lusitaniae, 35 mm for C. parapsilosis, and 17 mm for
C. rugosa, which is indicative of sensitivity of these strains to the tincture
complex. However, C. guilliermondii and C. inconspicua/Lambica displayed
8 mm and 6 mm zones of inhibition respectively; thus, showing
intermediate-resistance effects to the tincture complex. Interestingly a
holistic view reveals that C. inconspicua/Lambica showed resistance to all
treatments, namely the tinctures, ethanol concentrations and Fluconazole
control. This strain has been occurring more frequently in human samples
and is known to be resistant to Fluconazole. As an emerging yeast pathogen
causing invasive infections, it is vital to find alternative or combination
treatments (Guitard et al., 2013).
The C. parapsilosis and C. lusitaniae showed intermediate resistance with

an average of 9 mm for all ethanol concentrations. Fluconazole produced a
large zone of inhibition on C. lusitaniae (36 mm) and C. parapsilosis (47
mm), which indicates the sensitivity of all the Candida strains to this anti-
fungal, with the exception of C. inconspicua/Lambica, which is inherently
resistant to Fluconazole. Even though the anti-fungal showed a greater
zone of inhibition, the results for the H. canadensis Ø, and the H.
54
canadensis and C. officinalis Ø complex are also positive. C. officinalis Ø
only showed minimum growth inhibition on all Candida spp., with an
average of 10 mm, which was less or the same as the ethanol concentration
growths with averages of between 9 and 10 mm.
The Microdilution method was also used to test the effects of the tinctures
on the other Candida spp., and a reduction in the corrected absorbance
reading highlighted by a negative value or a value less than the yeast
absorbance value indicated inhibition of yeast growth.
5.2.2.1 C. lusitaniae
The results of C. lusitaniae show that H. canadensis Ø showed fungal

inhibition at the first well (2x dilution) with an OD reading of -0.94 nm. The
untreated diluted yeast cells had an OD reading of 0.48 nm; thus, the
inhibitory effect of H. canadensis Ø is noted by the extreme OD reduction.
The inhibition was seen for all dilutions, thereafter, confirming the
effectiveness of H. canadensis Ø against C. lusitaniae.
for the dilution range by the yeast cells OD reading of 0.48 nm, compared
to C. officinalis Ø in well one (2x dilution) with an OD value of 0.14 nm.
The combination of H. canadensis Ø and C. officinalis Ø complex

demonstrated the lowest OD value of -0.40 nm of all the tinctures tested.
However, the OD value increased to -0.00nm at the second well (4x
dilution); thereafter the values increased to 0.08nm, until 0.41 nm at the last
well. Even though the values increased with the dilutions, the values were
still below the yeast value, thus confirming inhibition.
The MIC values for the ethanol controls were 0.27 nm for 66% and 70%
ethanol, and 0.26 nm for the 68% ethanol. This data showed that the ethanol
had some level of inhibition against C. lusitaniae. The inhibitory effect of the
55
ethanol controls ended in the second well, and thereafter had no effect on
the strain; this was noted as the values increased in the subsequent wells.
The Fluconazole 25mcg had an OD value of 0.16 nm at the highest

concentration, with an increasing OD value as the anti-fungal was diluted.
The most diluted Fluconazole concentration had an OD of 0.42 nm, while
the yeast had an OD value of 0.41 nm. The value was indicative of the
reduced inhibitory effect of Fluconazole against C. lusitaniae when diluted
64-fold. The value increased as the dilutions increased, and Fluconazole
stopped inhibition at the 16-fold dilution with an OD reading of 0.38 nm. This
reading was higher than the yeast reading of 0.33 nm, which showed no
inhibitory effects.
5.2.2.2 C. parapsilosis
The results of C. parapsilosis demonstrated that H. canadensis Ø showed

fungal inhibition at the first well (2x dilution) with an OD reading of -0.68 nm,
which was the lowest value from all the tinctures tested. The untreated
diluted yeast cells had an OD reading of 0.46 nm; thus, the inhibitory effect
of H. canadensis Ø is observed by the severe OD reduction. The inhibition
was seen for all dilutions, thereafter, confirming the effectiveness of
H. canadensis Ø against C. parapsilosis.
The MIC readings for C. officinalis Ø corroborated the inhibition of yeast

growth for the dilution range by the yeast cells OD reading of 0.46 nm,
compared to the most concentrated C. officinalis Ø in well one (2x dilution),
with an OD value of 0.03 nm.
The combination of H. canadensis Ø and C. officinalis Ø had an OD value

of -0.85 nm. However, the OD value increased to -0.05nm at the second
well (4x dilution), and thereafter the values increased to 0.30 nm at the last
well. Even though the values increased with the dilutions, the values were
still below the yeast values which confirms the effectiveness of the complex
against C. parapsilosis.
56
displayed inhibition against C. parapsilosis until the 32-fold dilution, which
surprisingly was the same as the Fluconazole 25mcg, which had an OD
value of 0.36 nm. The most diluted Fluconazole concentration had an OD
of 0.46 nm, while the yeast had an OD value of 0.43 nm. The value was
indicative of the reduced inhibitory effect of Fluconazole against
C. parapsilosis when diluted 64-fold.
The results of C. parapsilosis demonstrated that H. canadensis Ø showed

fungal inhibition at the first well (2x dilution) with an OD reading of -0.68 nm,
which was the lowest value for all the tinctures tested. The untreated diluted
yeast cells had an OD reading of 0.46 nm, thus the inhibitory effect of
H. canadensis Ø is observed by the severe OD reduction. The inhibition
was seen for all dilutions, thereafter, confirming the effectiveness of
H. canadensis Ø against C. parapsilosis.
The MIC readings for C. officinalis Ø showed the inhibition of yeast growth
for the dilution range, with a yeast cell OD reading of 0.46 nm in comparison
to C. officinalis Ø (2x dilution), with an OD value of 0.03 nm.
The combination of H. canadensis Ø and C. officinalis Ø had an OD value

of -0.85 nm. However, the OD value increased to -0.05nm at the second
well (4x dilution); thereafter the values increased to 0.04nm until 0.30 nm at
the last well. Even though the values increased with the dilutions, they were
still below the yeast values, which confirms the effectiveness of the complex
against C. parapsilosis.
displayed inhibition against C. parapsilosis until the 32-fold dilution, which
surprisingly was the same as the Fluconazole 25mcg which had an OD
value of 0.36 nm. The most diluted Fluconazole concentration had an OD
of 0.46 nm compared to the yeast OD value of 0.43 nm. The value was
57
indicative of the reduced inhibitory effect of Fluconazole against
C. parapsilosis when diluted 64-fold.
5.2.2.3 C. rugosa, C. guilliermondii and C. inconspicua/Lambica
The results of C. rugosa, C. guilliermondii and C. inconspicua/Lambica

demonstrated that H. canadensis Ø showed fungal inhibition at the first well
(2x dilution) with an OD reading of 0.06 nm, -0.83 nm, and 0.57 nm,
respectively. The untreated diluted yeast cells had an OD reading of 0.43
nm, 0.61 nm, and 1.29 nm; therefore, the inhibitory effect of H. canadensis
Ø is observed by the OD reduction. The inhibition was seen for all dilutions,
thereafter, confirming the effectiveness of H. canadensis Ø against
C. rugosa, C. guilliermondii and C. inconspicua/Lambica.
for C. rugosa, C. guilliermondii for all the wells tested, however for
C. inconspicua/Lambica there was no inhibition in the first well with an OD
reading of 1.37 nm, whilst the yeast cells had an OD reading of 1.29 nm.
Inhibition was noted for C. officinalis Ø in well two (4x dilution) with an OD
value of 1.09 nm; this continued until the 64-fold dilution where inhibition
stopped with the increase in dilution.
The complex of H. canadensis Ø and C. officinalis Ø had OD values of 0.12

nm, -0.26 nm, and 1.04 nm, respectively. The OD value for C. rugosa
decreased to -0.03 nm at the second well (4x dilution); thereafter the values
increased to 0.08nm until 0.28 nm at the last well. Even though the values
increased with the dilutions, they were still below the yeast values, which
confirms the effectiveness of the complex against C. rugosa. The results of
C. guilliermondii showed that the values increased after the first well until
the last well where inhibition stopped, with an OD reading of 0.64 nm. C.
inconspicua/Lambica displayed inhibition until the 16-fold dilution, and
thereafter had no effect until the last well at the 64-fold dilution.
58
The values of the control groups are discussed in the paragraphs below:
The MIC values for the ethanol controls of C. rugosa were 0.05 nm for 66%
ethanol, 0.10 nm for 68% ethanol, and 0.13 nm for the 70% ethanol. This
data showed that the ethanol displayed inhibition against C. rugosa until the
8-fold dilution; however, the 70% ethanol inhibition started at the 16-fold
dilution and ended at the 64-fold dilution. The Fluconazole 25mcg had an
OD value of 0.18 nm. The most diluted Fluconazole concentration had an
OD of 0.42 nm, compared to the yeast OD value of 0.37 nm, and reduced
inhibition at the 8x fold dilution, indicating that it was not effective on
C. rugosa with the increased dilutions.
The MIC values for the ethanol control groups of C. guilliermondii were 0.14
nm for 66%, 0.15 nm for 68% ethanol, and 0.19 nm for the 70% ethanol.
This data showed that all the ethanol concentrations displayed inhibition
against C. guilliermondii, until the 32-fold dilution. The Fluconazole 25mcg
had an OD value of 0.18, compared to the yeast OD value of 0.61 nm. The
most diluted Fluconazole concentration had an OD value of 0.59 nm, which
was equal to the yeast OD value. Fluconazole only showed inhibition at the
32-fold dilution, whilst the other wells showed that it was not effective on
C. guilliermondii. This was inconsistent with the other strain readings.
The MIC values for the ethanol controls of C. inconspicua/Lambica were

0.48 nm for 66%, 0.86 nm for 68% ethanol, and 1.02 nm for the 70%
ethanol. This data showed that the 66% concentration had inhibition at the
32-fold dilution, whilst the 68% ethanol showed inhibition at the 4-fold
dilution and16-fold dilution, while the other dilutions had no effect. The 70%
ethanol showed inhibition until the 4-fold dilution, and thereafter had no
effect; this was also inconsistent with other readings. The Fluconazole
25mcg had an OD value of 1.19 nm at the first well (2x dilution). The most
diluted Fluconazole concentration had an OD value of 0.59 nm, which was
less than the yeast OD value of 1.29 nm. Fluconazole only showed inhibition
59
at the 2-fold dilution, whilst the other wells showed that it was not effective
on C. inconspicua/Lambica.
5.2.3 Minimum Fungicidal Concentration (MFC)
This method was used to check whether the tinctures, ethanol

concentrations, and anti-fungal compound used in the 96 wells were
fungicidal or fungistatic against C. lusitaniae, C. parapsilosis, C. rugosa,
C. guilliermondii and C. inconspicua/Lambica. From the MFC analysis, it
was noted that all the tinctures had a fungistatic (suppressed growth) effect
instead of fungicidal (killed) effect. The recovery of the yeast cells, when
given nutrients, enabled them to grow and flourish after the incubation
period, which confirmed the fungistatic mechanism of the tinctures. To
determine the fungicidal levels of the tinctures, further dose experiments
need to be conducted.
5.4 Related research and discussion of results
5.4.1 H. canadensis Ø
H. canadensis Ø is a known anti-fungal, with berberine being its most active

component (Mandal et al. 2020). In this study, H. canadensis Ø was shown
to inhibit the growth of C. albicans and the other Candida spp., exhibiting
fungistatic properties. Overall, H. canadensis Ø was the most effective of
the tinctures against these strains. While Fluconazole was shown to
outperform the tincture, this study provides evidence that H. canadensis Ø
may be an effective alternative anti-fungal in the treatment of
C. albicans and the other Candida spp. This result is supported by a study
carried out by Budree (2004), which showed that H. canadensis Ø inhibited
the growth of C. albicans in vitro using the Kirby-Bauer Disk Diffusion
method. Research on H. canadensis Ø as a homeopathic preparation is
limited, and all other studies relate to the herbal preparation of this medicinal
plant.
60
Wei et al. (2011) that demonstrated how the isolated alkaloid berberine
inhibited the growth of various Candida species, including C. albicans in
vitro using the MIC method. Another study by da Silva et al. (2016)
evaluated the anti-fungal effect of berberine against Fluconazole-resistant
Candida strains and biofilm formation of Candida spp., using the broth
dilution method. The results confirmed that berberine inhibited the growth
of Fluconazole resistant Candida strains and reduced the capability of
biofilm formation. The study by Li et al. (2018) showed the synergism
between Fluconazole and berberine against Fluconazole-resistant
C. albicans in vitro. By exposing the yeast to different concentrations of
Fluconazole and berberine using the time kill curve assay, it was identified
that Fluconazole was more effective when the berberine concentration was
increased.
5.4.2 C. officinalis Ø
C. officinalis Ø is known for its anti-fungal, anti-inflammatory, and anti-

bacterial properties, and has previously been shown to be effective as an
anti-fungal treatment (Faustino et al., 2018). In this study, C. officinalis Ø
was shown to inhibit the growth of C. albicans and the other Candida spp.
with fungistatic effects. However, it was not as effective as the
H. canadensis Ø, or as Fluconazole, as it only showed intermediate effects
towards the various strains. According to the MIC method, however,
C. officinalis Ø inhibited C. albicans from the first well with a 2- fold dilution.
These results show that the tincture performs better in the broth solution
method than the diffusion method, however the reason for this is unknown.
It can be concluded that C. officinalis Ø may potentially be used as an anti-
fungal against most Candida strains, however further testing is required.
Once again, there is very limited research on the homeopathic preparations
of this medicinal plant. De Klerk (1998) performed an in vitro study which
showed that D1 and D3 potencies of C. officinalis were effective in inhibiting
C. albicans, using the disk diffusion method. Other studies relate to the
herbal preparations of this plant. Efstratiou et al. (2012) performed an in
61
vitro disk diffusion study on the antimicrobial activity of the petal extracts of
C. officinalis against gram positive, gram negative and fungal clinical
pathogens; the results showed that the ethanol and methanol extracts had
outstanding anti-fungal activity against C. albicans and C. parapsilosis.
These results support a previous research study done by Verma et al.

(2018), where the anti-fungal effects of floral and leaf extracts of
C. officinalis were tested against various strains of Candida spp. in vitro
which showed that both extracts were effective while the oils of the flower
were effective against Candida spp.
The results in this study were evident for the other Candida spp. strains that
were tested, where it inhibited the yeast from the first well with a 2- fold
dilution; however, the C. inconspicua/Lambica strain showed resistance in
the first well.
The complex of H. canadensis Ø and C. officinalis Ø displayed anti-fungal

effects in both methods that were tested. There are currently no studies
showing the effects of both these tinctures together however the synergistic
effects of herbal medicine as per a review by Ma et al. (2009), shows that if
two herbal ingredients mutually enhance the effect of each other, or the
one increases the effect of the other, then they can be effective as
treatments in combination. In order to understand the standardisation of
homeopathic tinctures and the homeopathic methods, continuous research
should be done, which could be used to advance therapeutic outcomes and
enable more effective treatment protocols.
5.4.3 Ethanol concentrations
Ethanol has the ability to kill off certain microbes; therefore, it is frequently
used as an antiseptic to sterilise equipment and prevent infection, and can
be fungicidal (Elzain et al., 2019). In this study, ethanol was used as a
control, and it revealed that the ethanol concentrations tested on C. albicans
and other Candida spp. had intermediate effects; however, in the MIC, the
62
66% ethanol concentration inhibited the yeast until the 16-fold dilution, the
68% until the 4-fold dilution, whilst the 70% inhibited the yeast in all the
wells, up to the 64-fold dilution; however, the Fluconazole still outperformed
this result. This is supported in a study by Peters et al. (2012) who used the
dose and time method to check which concentration of alcohol was most
effective in the killing of both monomicrobial and polymicrobial biofilms
formed on surfaces. The results showed that four hours was sufficient to kill
C. albicans in 30% ethanol, and this was confirmed by a study done by
Rane et al. (2012).
5.4.4 Fluconazole
Fluconazole is an anti-fungal agent that is used effectively to treat most

fungal infections; however, prolonged use can result in the development of
resistance (Lu et al., 2017). Krishnasamy et al. (2018) showed that
Fluconazole is a commonly used drug that acts as a fungistatic drug rather
than fungicidal drug, which increases its risk for drug resistance. It was also
used chronically to treat infections related to Candida species; however, an
understanding of the molecular mechanisms of the drug is required to avoid
resistance. In this study, Fluconazole outperformed the H. canadensis Ø,
the C. officinalis Ø, the H. canadensis and C. officinalis Ø complex, as well
as the 66%, 68% and 70% ethanol samples in the Kirby-Bauer method. In
the Microdilution method, the H. canadensis and C. officinalis Ø complex
outperformed all of the other treatments tested, but Fluconazole still
inhibited the yeast until the 32-fold dilution. These results are confirmed in
the study by Moosa et al. (2004), which showed that Fluconazole 150mcg
in one dose had fungicidal activity in in vitro settings, to eliminate
C. albicans.
5.5 Limitations of this study
It is essential to understand the limitations of this study in the interpretation

of the results found.
63
Limitations of the study include:
• Studies on the effects of specific doses of H. canadensis Ø,

C. officinalis Ø, and H. canadensis and C. officinalis Ø complex were
not included in the research, which could have provided important
insight in determining which dosage would be more effective in killing
the yeast.
• H. canadensis Ø and C. officinalis Ø are frequently prescribed in
higher potencies in clinical practice; however, this study did not
evaluate the effects of ultra-high dilutions of these remedies.
• This study was carried out in vitro; therefore, the effects of the
therapy in vivo were not ascertained.
• Time-kill studies could be used to test the effects of the
H. canadensis Ø, the C. officinalis Ø, and the H. canadensis and
C. officinalis Ø complex on Candida spp.
64
6. CONCLUSION & RECOMMENDATIONS
6.1 Conclusion
The aim of this study was to test the effectiveness of H. canadensis and
C. officinalis Ø complex as an anti-fungal against C. albicans and other
Candida spp., using the Kirby-Bauer Disk Diffusion and Microdilution
methods.
The results demonstrated that the H. canadensis and C. officinalis Ø

complex inhibited the growth of C. albicans and other Candida spp., which
was confirmed by the Kirby-Bauer Disk Diffusion and Microdilution methods.
The H. canadensis Ø and C. officinalis Ø on their own inhibited the growth
of C. albicans in varying degrees; however, the H. canadensis and the
C. officinalis Ø complex displayed better anti-microbial properties than the
C. officinalis Ø on its own. Fluconazole, however, performed significantly
better than the tinctures.
The minimum fungicidal method was used to check if the treatments were
fungistatic or fungicidal, and overall, the Ø’s were fungistatic. Regardless of
the lower concentration of active ingredients found in homeopathic Ø’s
compared to herbal extract preparations, an anti-fungal effect was
observed.
It was hypothesised that the homeopathic Ø’s of H. canadensis and

C. officinalis, used in combination, will show antimicrobial action against
C. albicans and other Candida spp. This was supported and confirmed by
this study.
In conclusion, the H. canadensis and C. officinalis Ø complex did show

inhibitory effects against C. albicans and other Candida spp., and it was
confirmed to be a fungistatic effect, however the C. inconspicua/Lambica
strain showed resistance. This study has expanded our knowledge of the
antimicrobial effects of H. canadensis Ø and C. officinalis Ø both on their
65
own, and as a complex in vitro. It needs to be ensured that, when using
homeopathic Ø’s, that the correct dose required for it to be fungicidal be
determined. Combined effects of the tinctures with standard anti-fungal
treatment can also be explored in future studies.
6.2 Recommendations
Further research is recommended to confirm the results of this study:
• Studies on the effects of H. canadensis Ø, the C. officinalis Ø, and

the H. canadensis and C. officinalis Ø complex at specific doses
should be done to ascertain at which dose the tinctures are most
effective in killing the yeast.
• H. canadensis and C. officinalis Ø complex in homeopathic potency
can be tested to determine its anti-fungal effects.
• In vivo tests and clinical trials should be done on the H. canadensis
and C. officinalis Ø complex, to check how effective it is.
• Time-kill studies should be done in the lab to establish if the
H. canadensis and C. officinalis Ø complex, and the Ø’s on their own,
are only fungistatic or fungicidal.
• Further research, in investigating the combined effects of
H. canadensis Ø and Fluconazole.
• The use of diluted ethanol as a solvent for the tinctures may be more
suited to obtain the true inhibitory potential of the tinctures in future
studies.
66
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APPENDICES
Appendix A
78
Appendix B
The mother tinctures (ø ) of Calendula officinalis and Hydrastis canadensis

will be made using HAB method 3A and HAB method 4A, respectively
(Deutscher Apotheker Verlag, 2013).
HAB 3A method: Calendula officinalis
• Plants or parts of plants are chopped up

• A sample is used to detect loss on drying
• Instantly add half the amount of 86% alcohol to the chopped plant
elements
• Store in a well- sealed container at a temperature not surpassing
20°C
• Calculate the amount of 86% ethanol (m/m) required for the mass
of the plant
• Leave the mixture to stand for a minimum of 10 days at a
temperature not surpassing 20°C swirling from time to time
• Express and filter
• Use 62% ethanol to adjust to any concentration as required in the
monograph
HAB 4A method : Hydrastis canadensis
• Tinctures are made from dried plants or parts thereof and are
manufactured using the percolation or maceration method.
• 1 part of the drug to 10 parts of 62% ethanol , the mixture is left to
stand for a minimum of 10 days at a temperature not surpassing
20°C
• Filter
79
Appendix C
80
Appendix D
81
Appendix E
82
Appendix F
83
84

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COPYRIGHT AND CITATION CONSIDERATIONS FOR THIS THESIS/ DISSERTATION

How to cite this thesis

A dissertation submitted to the

Supervisor_______ _________ ___7/12/2020__________

Co-supervisor_____ _____________ ___7/12/2020_________

I, Ashica Subramanian, hereby declare that this dissertation is my own

Candida albicans (C. albicans) is the micro-organism responsible for

The aim of this study was to determine the antimicrobial effect of H.

The results demonstrated that the H. canadensis and C. officinalis Ø

This research is dedicated to my three daughters for their continued

I would like to express my gratitude and appreciation to the following people

• My supervisor Dr. Janice Pellow; for your unbelievable work ethic

Table 4. 1 Summary of the Kirby-Bauer Disk Diffusion susceptibility test against

Table 4. 4 Summary of the Microdilution test against C. albicans ...................... 33

Table 4. 8 List of compounds on grid plate ........................................................ 37

Table 4. 9 Summary of the Kirby-Bauer Disk Diffusion susceptibility test against

Table 4. 11 List of compounds on grid plate ...................................................... 49

Figure 2. 2 Candida Biofilm Formation (Gulati and Nobile, 2016) ........................ 6

Figure 2. 3 Homeopathic Potentisation (Homeopathicassociates.com, 2020) ... 13

Figure 2. 4 Hydrastis Canadensis L. Rhizome (Phys.org, 2020). ...................... 14

Figure 2. 5 Calendula officinalis L. plant (Lim, 2014) ......................................... 16

Figure 3. 1 Fungal lawn streaking (Espinel-Ingroff, 2007) ................................. 23

Figure 3. 2 A 96 well plate template for MIC dilution experiment ....................... 25

Figure 4. 1 Disk diffusion agar of C. albicans (ATCC 90028) showing zones of

Figure 4. 4 Disk diffusion agar of C. lusitaniae showing zones of inhibition for A:

Figure 4. 5 Disk diffusion agar of C. parapsilosis showing zones of inhibition for

Figure 4. 6 Disk diffusion agar of C. guilliermondii showing zones of inhibition for

Figure 4. 7 Disk diffusion agar of C. inconspicua/lambica showing zones of

Figure 4. 8 Disk diffusion agar of C. rugosa showing zones of inhibition for A:

1.1 Problem Statement

Candida albicans (C. albicans) is a yeast micro-organism that forms part of

Hydrastis canadensis L. (H. canadensis L.) and Calendula officinalis L.

According to Yang et al. (2014), the synergistic therapeutic effects of herbal

1.2 Aim of the Study

1.3 Objectives of the Study

• The minimum inhibitory concentration (MIC) for the mother tinctures

It was hypothesised that the homeopathic mother tinctures of H. canadensis

1.5 Null Hypothesis

This organism was chosen since it is a common infection amongst women,

2.1 Candida species

Candida albicans (C. albicans), Candida lusitaniae (C. lusitaniae) and

2.1.1 Classification, identification, and morphology of C. albicans

C. albicans is a unicellular oval-shaped yeast that exists on mucosal

At ambient temperature (20–22°C), the yeast reproduces by budding, but

Figure 2. 1 Candida Morphology (Thompson et al., 2011)

The vast majority of persistent Candida infections are associated with

Figure 2. 2 Candida Biofilm Formation (Gulati and Nobile, 2016)

There are several factors that predispose an individual to developing

2.1.3.1 Predisposing conditions

Candida infections may be triggered by underlying conditions that affect

Antibiotics, oral glucocorticoids, and inhaled corticosteroids also put

2.1.3.3 Other risk factors

In women, the use of detergents, douches, and the presence of hormonal

2.1.4.1 Cutaneous candidiasis

2.1.4.2 Oropharyngeal candidiasis

This type of infection is prevalent in HIV patients. Symptoms include a sore

2.1.4.3 Vulvovaginal candidiasis

2.1.4.4 Systemic candidiasis

2.1.5 Conventional treatment of Candida infections

The following anti-fungal medications are commonly used to treat

2.1.5.1 Azole anti-fungals

Other fungal medications such as nystatin and amphotericin B, which are

2.2 Antimicrobial resistance

Drug resistance of Candida infections is on the increase and is a

Supervisor_ _ _7/12/2020__________

Co-supervisor_ _______ _7/12/2020_________