THE PROCESSING AND FORMULATION OF ANTIBACTERIAL AND

ANTIFUNGAL HERBAL OINTMENT FOR SKIN DISORDERS USING ALOE

VERA AND GARLIC
 CHAPTER ONE

Introduction

1.1 Background of the Study

Skin disorders are a prevalent global concern, affecting individuals of all ages and
demographics [WHO, 2020]. These conditions encompass a wide range of ailments,
including bacterial and fungal infections, eczema, psoriasis, and acne. Conventional
treatments for skin disorders often rely on synthetic medications such as antibiotics,
antifungals, and corticosteroids [Adityasuta et al., 2014]. However, these medications can be
associated with side effects such as allergic reactions, antibiotic resistance, and skin thinning
[Grice et al., 2008]. This has spurred a growing interest in exploring alternative therapeutic
options, particularly those derived from natural sources like plants.

Plants have been a cornerstone of traditional medicine for centuries, offering a rich source of
bioactive compounds with potential health benefits [Heinrich et al., 2010]. Two readily
available and well-studied plants with promising antimicrobial properties are aloe vera (Aloe
barbadensis Miller) and garlic (Allium sativum L.).

1.1.1 Aloe vera

Aloe vera is a succulent plant with a long history of use in traditional medicine for wound
healing, skin conditions, and various ailments [Reynolds, 2002]. The gel extracted from its
leaves contains a complex mixture of bioactive compounds, including polysaccharides,
vitamins, minerals, and enzymes [Surjushe et al., 2008]. Studies have shown that aloe vera
gel possesses antibacterial and antifungal properties attributed to various compounds,
including aloin, mannose-6-phosphate, and saponins [Choi & Chung, 2005; Hamidi et al.,
2013]. Its anti-inflammatory and wound healing properties further contribute to its potential
effectiveness in treating skin disorders [Davis et al., 2004].

1.1.2 Garlic

Garlic is a bulbous plant belonging to the Liliaceae family and has been used for centuries for
its medicinal properties [Fleming, 2001]. Garlic contains a variety of sulfur-containing
compounds, including allicin, which is considered its primary bioactive component
[Cavallaro et al., 2006]. Allicin exhibits a broad spectrum of antimicrobial activity against
bacteria, fungi, and viruses [Sills et al., 1998]. Studies have demonstrated the effectiveness of
garlic extracts against various skin pathogens, including Staphylococcus aureus, Escherichia
coli, and Candida albicans [Banerjee et al., 1996; Adetumbi et al., 2010].

1.1.3 Rationale for the Study

The emergence of antibiotic-resistant bacteria and the potential side effects of conventional
medications necessitate the exploration of alternative therapeutic options for skin disorders.
Both aloe vera and garlic exhibit promising antibacterial and antifungal properties, suggesting
their potential application in topical treatments. However, limited research directly compares
the efficacy of these two plant extracts in treating skin infections.

This project aims to address this gap in knowledge by processing and formulating an
antibacterial and antifungal herbal ointment using aloe vera and garlic extracts. We will then
compare the efficacy of these ointments against common skin pathogens.

1.2 Statement of the Problem

The increasing incidence of antibiotic-resistant bacteria and the potential side effects of
conventional medications pose challenges in the management of skin disorders. This study
seeks to investigate the potential of formulating antibacterial and antifungal ointments using
readily available plant extracts (aloe vera and garlic) as an alternative therapeutic option.

1.3 Aims and Objectives of the Study

1.3.1 Aims

The main aim of this project is to develop and compare the efficacy of topical herbal
ointments formulated with aloe vera and garlic extracts for treating skin disorders caused by
bacterial and fungal pathogens.
1.3.2 Objectives
 To extract and prepare antibacterial and antifungal formulations from aloe vera gel
and garlic cloves.
 To formulate ointments incorporating the prepared aloe vera and garlic extracts.

 To evaluate the in vitro antibacterial and antifungal activity of the formulated

ointments against common skin pathogens.

 To compare the efficacy of the aloe vera and garlic-based ointments in terms of their
antimicrobial activity.

 To assess the physical properties and stability of the formulated ointments.

1.4 Significance of the Study

The findings of this study have the potential to contribute to the development of safe and
effective topical treatments for skin disorders using natural resources. Here's how:

 Reduced Dependence on Conventional Medications: This study could provide

evidence for the effectiveness of herbal ointments, thereby offering an alternative to
conventional medications and potentially reducing dependence on antibiotics.

 Combating Antibiotic Resistance: The development of effective herbal treatments

could help combat the growing problem of antibiotic resistance by providing a non-
antibiotic approach to treating bacterial skin infections.

 Natural and Safe Option: The use of natural plant extracts like aloe vera and garlic
offers a potentially safer option with fewer side effects compared to some
conventional medications.

 Improved Patient Care: By providing additional therapeutic options, this study could
contribute to improved patient care and management of skin disorders.
  Reduced Healthcare Costs: The use of herbal ointments, if proven effective, could
potentially lead to reduced healthcare costs associated with conventional medications
and their side effects.

1.5 Scope of the Study

This study will focus on the development and evaluation of topical ointments formulated with
aloe vera gel and garlic extracts. The in vitro antibacterial and antifungal activity of these
ointments will be assessed against selected common skin pathogens. The study will not
investigate the in vivo efficacy or safety of the ointments in humans.

1.6 Research Questions

This study aims to answer the following research questions:

1. Can antibacterial and antifungal formulations be effectively prepared from aloe vera gel and
garlic cloves?
2. Is it feasible to formulate stable and physically acceptable ointments incorporating the
prepared aloe vera and garlic extracts?
3. Do the formulated ointments exhibit antibacterial activity against common skin pathogens?
4. Do the formulated ointments exhibit antifungal activity against common skin pathogens?

1.7 Research Hypotheses

Research Question 1: Can antibacterial and antifungal formulations be effectively prepared

from aloe vera gel and garlic cloves?

 Null Hypothesis (H0): Antibacterial and antifungal formulations cannot be effectively

prepared from aloe vera gel and garlic cloves using the chosen extraction methods.
 Alternative Hypothesis (H1): Antibacterial and antifungal formulations can be
effectively prepared from aloe vera gel and garlic cloves using the chosen extraction
methods.
Research Question 2: Is it feasible to formulate stable and physically acceptable ointments
incorporating the prepared aloe vera and garlic extracts?

 Null Hypothesis (H0): It is not feasible to formulate stable and physically acceptable
ointments incorporating the prepared aloe vera and garlic extracts.

 Alternative Hypothesis (H1): It is feasible to formulate stable and physically

acceptable ointments incorporating the prepared aloe vera and garlic extracts.

Research Question 3: Do the formulated ointments exhibit antibacterial activity against

common skin pathogens?

 Null Hypothesis (H0): The formulated ointments do not exhibit antibacterial activity
against common skin pathogens.

 Alternative Hypothesis (H1): The formulated ointments exhibit antibacterial activity

against common skin pathogens.

Research Question 4: Do the formulated ointments exhibit antifungal activity against

common skin pathogens?

 Null Hypothesis (H0): The formulated ointments do not exhibit antifungal activity
against common skin pathogens.

 Alternative Hypothesis (H1): The formulated ointments exhibit antifungal activity

against common skin pathogens.
1.8 Definition of Terms

 Antibacterial: Refers to the ability of a substance to kill or inhibit the growth of

bacteria.

 Antifungal: Refers to the ability of a substance to kill or inhibit the growth of fungi.

 Skin disorders: A broad term encompassing various conditions affecting the skin,
including infections, inflammatory conditions, and acne.

 Topical: Refers to the application of a medication to the surface of the skin.

 Ointment: A semisolid dosage form for topical application containing a medication

dispersed in a greasy base.

 In vitro: Refers to experiments conducted in a controlled laboratory environment,

typically using isolated cells or tissues.

 Pathogen: An organism that causes disease.

 Efficacy: The capacity to produce a desired effect.

 CHAPTER TWO

2.0 Literature Review

2.1 Introduction

Chapter one introduced the background, rationale, and objectives of this project investigating
the potential of aloe vera and garlic extracts in formulating topical ointments for skin
disorders. This chapter delves into the existing body of knowledge relevant to the study. We
will explore the scientific literature on:

 The antimicrobial properties of aloe vera and garlic.

 The use of herbal ointments for treating skin disorders.
 The mechanisms of action of aloe vera and garlic against bacteria and fungi.
 A comparison of the antimicrobial efficacy of aloe vera and garlic.

2.2 Conceptual Framework

The conceptual framework for this study is based on the premise that aloe vera and garlic
possess inherent antimicrobial properties that can be harnessed to develop topical ointments
for treating skin disorders caused by bacterial and fungal pathogens.

2.2.1 Independent Variables

Type of Extract

Aloe vera gel extract: The framework identifies "aloe vera gel extract" as an independent
variable. However, further exploration can reveal different extraction methods and their
potential impact on the final product:
  Solvent extraction: Techniques like ethanol or methanol extraction can isolate specific
bioactive compounds from aloe vera gel. The choice of solvent can influence the yield
and properties of the extract, potentially affecting its antimicrobial activity.

 Hydrothermal extraction: This method utilizes hot water to extract bioactive

compounds. It offers a potentially gentler approach compared to organic solvents and
might preserve a broader spectrum of beneficial components in the extract.

 Freeze-drying: This method involves freezing and then removing water from aloe
vera gel under vacuum. The resulting lyophilized powder can be further processed
into an extract for ointment formulation.

Garlic extract: Similar to aloe vera, garlic extract can be obtained through various methods:

 Alcoholic extraction: Similar to aloe vera, ethanol or other alcohols can be used to
extract bioactive compounds from garlic cloves. This method offers high yield but
might also extract less desirable components.

 Aqueous extraction: Water-based extraction can be a gentler approach, potentially

preserving more water-soluble bioactive compounds like allicin. However, the yield
and stability of the extract might be lower compared to alcoholic extraction.

 Solvent-free extraction methods: Techniques like supercritical fluid extraction or

microwave-assisted extraction offer potentially cleaner and more environmentally
friendly methods for extracting bioactive compounds from garlic.

Concentration of Extract in the Ointment: The concentration of the extract in the

formulated ointment is another crucial independent variable. This section can be
expanded to explore:
o Minimum inhibitory concentration (MIC): Determining the MIC of the aloe
vera and garlic extracts against target pathogens (Staphylococcus aureus and
 Candida albicans) can guide the selection of effective concentration ranges for
the ointments.

o Dose-dependent effects: Studies suggest a dose-dependent effect for both aloe

vera and garlic extracts, meaning higher concentrations exhibit greater
antimicrobial activity. This framework can explore the intended range of
concentrations to be tested in the ointments.

o Cytotoxicity considerations: While the focus is on antimicrobial activity, it's

important to consider potential cytotoxic effects of the extracts on healthy
human cells. The framework can incorporate concentration ranges that balance
efficacy against potential cytotoxicity.

2.2.2 Dependent Variables

Antibacterial Activity against Common Skin Pathogens: This section can be

expanded to specify the specific methods used to evaluate antibacterial activity. Here
are some potential approaches:
o Disc diffusion assay: This commonly used method involves placing discs
containing the formulated ointments on agar plates inoculated with the target
bacteria (e.g., Staphylococcus aureus). The presence of a clear zone of
inhibition around the disc indicates antibacterial activity.

o Broth microdilution assay: This method involves testing different

concentrations of the ointments in a liquid broth medium containing the target
bacteria. The MIC can be determined as the lowest concentration that inhibits
bacterial growth.

o Time-kill assay: This assay measures the rate at which the ointments kill
bacteria over time. This can provide valuable insights into the potency of the
antibacterial activity.
 Antifungal Activity against Common Skin Pathogens: Similar to antibacterial
activity, the framework can be expanded to detail the methods used to evaluate
antifungal activity:
o Agar dilution method: This method involves incorporating different
concentrations of the ointments into solidified agar media before inoculation
with the target fungus (e.g., Candida albicans). The MIC can be determined as
the lowest concentration that inhibits fungal growth.

o Fluorescent microscopy: This technique can be used to visualize the effects of

the ointments on fungal cells. Staining with specific dyes can reveal damage to
cell membranes or disruption of fungal morphology.

Physical Properties and Stability of the Formulated Ointments: This section can
be further elaborated to encompass key physical properties relevant to the ointment's
functionality and user experience:
o Viscosity: It refers to the resistance of the ointment to flow. An appropriate
viscosity ensures ease of application and prevents the ointment from being too
runny or too stiff.

o Spreadability: This property determines how easily the ointment can be spread
on the skin. A good spreadability allows for even application and patient
comfort.

o pH: The pH of the ointment should ideally be close to neutral (pH 7) to

minimize potential irritation to the skin.

o Stability: Evaluating the stability of the formulated ointments over time is

crucial. This can involve monitoring changes in appearance, viscosity, pH, and
antimicrobial activity over a defined storage period.

2.2.3 Control Variables

The framework identifies several control variables that will be kept constant throughout the
study to ensure reliable comparisons between the aloe vera and garlic ointments. Here, we
can delve deeper into the rationale behind these controls:

 Extraction methods for aloe vera gel and garlic: Standardizing the extraction methods
for both plant materials ensures consistency in the types and concentrations of
bioactive compounds present in the final extracts. This allows for a more accurate
comparison of the inherent antimicrobial properties of aloe vera and garlic.

 Ointment base composition: The chosen ointment base plays a crucial role in the
physical properties and stability of the final product. Selecting a common base for
both aloe vera and garlic ointments ensures that any observed differences in activity
are primarily due to the type of extract, not variations in the base material.

 Incubation conditions for evaluating antimicrobial activity: Maintaining consistent

incubation conditions (temperature, time) during the assays for both bacterial and
fungal activity is essential for reliable comparisons. This ensures that any observed
differences in activity are not due to variations in the testing environment.

2.2.5 Conclusion on the Conceptual Framework

By expanding the conceptual framework as detailed above, we gain a more comprehensive

understanding of the key variables and considerations involved in the study. This refined
framework will guide the research process, ensuring a systematic and controlled approach to
developing and evaluating the aloe vera and garlic-based topical ointments.

2.3 Theoretical Frameworks

The theoretical frameworks will explore plant secondary metabolites and antimicrobial
defense, mode of action of antimicrobials, and wound healing and anti-inflammatory
properties. We will delve deeper into these frameworks, specifically focusing on the
comparison between aloe vera and garlic in their potential benefits for treating skin disorders
caused by Staphylococcus aureus and Candida albicans.

2.3.1 Plant Secondary Metabolites and Antimicrobial Defense

Both aloe vera and garlic produce a diverse array of secondary metabolites known to
contribute to their antimicrobial activity. Understanding the specific types and mechanisms of
these compounds is crucial for comparing their effectiveness against S. aureus and C.
albicans.

2.3.1.1 Aloe Vera

 Polysaccharides: These complex sugars, particularly glucomannans present in aloe

vera gel, have been shown to exhibit antibacterial and antifungal activity [Choi &
Chung, 2005]. Their proposed mechanisms include:

 Interference with cell wall synthesis: These polysaccharides can hinder the formation
of bacterial and fungal cell walls, leading to cell death. This mechanism might be
particularly effective against S. aureus, which relies on a peptidoglycan cell wall.

 Modulation of immune response: Certain polysaccharides in aloe vera may stimulate

the immune system to enhance the body's defense against microbial infections. This
could offer a broader benefit beyond direct antimicrobial action.

2.3.1.2 Garlic

 Allicin: The primary bioactive compound responsible for garlic's antimicrobial

activity is allicin, formed from alliinase upon garlic tissue damage. Allicin exhibits a
broad spectrum of activity against both bacteria and fungi, including S. aureus and C.
albicans. Here's how it disrupts these pathogens:
  Disruption of cell membranes: Allicin disrupts the cell membranes of bacteria and
fungi, leading to leakage of cellular contents and cell death. This mechanism can be
highly effective against both S. aureus and C. albicans.

 Inhibition of enzyme activity: Allicin can inhibit essential enzymes involved in

bacterial and fungal metabolism, thereby hindering their growth and survival. This
might be particularly effective against S. aureus by inhibiting enzymes crucial for cell
wall synthesis.

2.3.1.3 Comparison

 Both aloe vera and garlic possess secondary metabolites with antimicrobial activity.
However, their mechanisms of action differ.

 Polysaccharides in aloe vera primarily target cell wall synthesis, while allicin in garlic
disrupts cell membranes and inhibits essential enzymes in a broader manner. This
suggests that garlic might have a wider spectrum of activity and potentially be more
effective against various pathogens.

 Aloe vera might offer additional benefits through immune modulation, which could
be complementary to its direct antimicrobial effects.

2.3.2 Mode of Action of Antimicrobials

2.3.2.1 Aloe Vera

The proposed mechanisms of action for aloe vera's antimicrobial activity extend beyond
disrupting cell wall synthesis. Here's a detailed breakdown:

 Interference with cell wall synthesis: As mentioned earlier, polysaccharides in aloe

vera gel may interfere with the formation of bacterial and fungal cell walls. This could
be particularly effective against S. aureus, which has a peptidoglycan cell wall.
  Disruption of cell membranes: Certain compounds in aloe vera, such as
anthraquinones, might disrupt the cell membranes of microbes, causing leakage of
cellular contents and compromising their viability. While less prominent than
polysaccharides, this mechanism could contribute to the overall antimicrobial activity.

 Inhibition of enzyme activity: Although less studied compared to other mechanisms,

enzymes like lysozyme in aloe vera can potentially inhibit essential enzymes involved
in bacterial growth and metabolism. This could further weaken the bacteria and
enhance the effectiveness of other antimicrobial mechanisms.
 Immunomodulation: Aloe vera may stimulate the immune system to enhance the
body's defense mechanisms against microbial infections. This broader effect could
provide additional benefits in managing skin disorders.

2.3.2.2 Garlic

Garlic's primary antimicrobial activity stems from allicin, which acts through several potent
mechanisms:

 Disruption of cell membranes: Allicin disrupts the phospholipid bilayer of bacterial

and fungal cell membranes, leading to leakage of cellular contents and cell death. This
broad-spectrum mechanism is effective against both S. aureus and C. albicans.

 Inhibition of enzyme activity: Allicin can inactivate essential enzymes involved in

various microbial processes, including those crucial for energy production, protein
synthesis, and DNA replication. This disrupts the normal functioning of the microbes
and hinders their growth and survival.

 Disruption of DNA synthesis: Studies suggest that allicin might interfere with DNA
synthesis in bacteria and fungi, further impeding their ability to replicate and grow.
This mechanism could contribute to the long-term effectiveness of garlic against these
pathogens.
2.3.2.3 Comparison

 Aloe vera and garlic exhibit distinct modes of action. Aloe vera's mechanisms are
more diverse, potentially targeting cell wall synthesis, cell membranes, enzyme
activity, and the immune system. Garlic, on the other hand, relies primarily on
disrupting cell membranes and inhibiting essential enzymes, with a possible
additional effect on DNA synthesis.

 The broader range of mechanisms employed by aloe vera might offer some
advantages in terms of hindering the development of resistance by microbes.
However, the potency of allicin in disrupting cell membranes appears to be highly
effective against both S. aureus and C. albicans.

2.3.3 Wound Healing and Anti-inflammatory Properties

Beyond direct antimicrobial activity, both aloe vera and garlic possess properties that can be
beneficial in treating skin disorders. Here's a detailed comparison:

2.3.3.1 Aloe Vera

 Wound healing: Aloe vera gel has been shown to promote wound healing by
stimulating several processes:
o Fibroblast proliferation: Aloe vera can stimulate the production of fibroblasts,
the cells responsible for building new collagen fibers. This promotes wound
closure and tissue regeneration.

o Angiogenesis: Aloe vera may also promote the formation of new blood
vessels, which improves the delivery of oxygen and nutrients to the wound
site, facilitating healing.

o Collagen synthesis: Aloe vera can increase the production of collagen, a

crucial protein component of the skin. This strengthens the newly formed
tissue and promotes scar reduction.
  Anti-inflammatory properties: Aloe vera possesses anti-inflammatory compounds that
can help reduce inflammation associated with various skin conditions, including those
caused by S. aureus and C. albicans. This can alleviate pain, discomfort, and redness,
promoting faster healing and patient comfort.

2.3.3.2 Garlic

 Wound healing: While less studied compared to aloe vera, some evidence suggests
that garlic extracts might also promote wound healing. Mechanisms may include:

o Stimulation of collagen synthesis: Garlic extracts might stimulate the

production of collagen, similar to aloe vera. This can strengthen the healing
tissue.

o Improved blood circulation: Certain compounds in garlic may improve blood

circulation, potentially enhancing the delivery of nutrients and oxygen to the
wound site.

 Anti-inflammatory properties: Garlic exhibits anti-inflammatory activity due to the

presence of compounds like allicin and allyl methyl sulfide. This can help reduce
inflammation associated with skin disorders, providing symptomatic relief and
promoting healing.

2.3.3.3 Comparison

 Both aloe vera and garlic possess anti-inflammatory properties, which can be
beneficial in managing skin disorders.
  Aloe vera appears to have a more established role in promoting wound healing
through various mechanisms. However, further research is needed to fully understand
the wound healing potential of garlic extracts.

 The combination of aloe vera's wound healing properties and garlic's potent
antimicrobial activity could be a promising avenue for future research in developing
topical treatments for skin disorders.

2.3.3.4 Conclusion on Theoretical Frameworks

By delving deeper into the theoretical frameworks, we gain a comprehensive understanding

of the potential mechanisms by which aloe vera and garlic can exert beneficial effects in
treating skin disorders caused by S. aureus and C. albicans. While both plants possess
antimicrobial and anti-inflammatory properties, their specific mechanisms of action and
additional benefits like wound healing differ. This knowledge base lays the foundation for the
research design and guides the development of aloe vera and garlic-based topical ointments
for further evaluation in the study.

2.4 Empirical Review

Chapter 2 provided a foundational review of existing empirical research on the use of aloe
vera and garlic for treating skin disorders caused by Staphylococcus aureus and Candida
albicans. Here, we delve deeper into this existing literature, conducting a more extensive and
comparative analysis of in vitro and in vivo studies to gain a comprehensive understanding of
the current evidence base.

2.4.1 In vitro Studies on the Antimicrobial Activity of Aloe vera and Garlic
2.4.1.1 Staphylococcus aureus

Aloe vera: A substantial body of in vitro research has investigated the antibacterial activity
of aloe vera gel against S. aureus. Here's a breakdown of key findings and considerations:
  Antibacterial activity: Studies by Surjushe et al. (2008) and Oulad et al. (2009)
demonstrated the in vitro antibacterial activity of aloe vera gel extracts against S.
aureus. The minimum inhibitory concentration (MIC) varied depending on the
extraction method and aloe vera species, ranging from 0.625% to 50%.

 Mechanisms of action: Studies by Eshuis et al. (2004) and Hamoda et al. (2010)
suggest that polysaccharides and anthraquinones in aloe vera gel might be responsible
for its antibacterial activity against S. aureus. These compounds may interfere with
cell wall synthesis and protein synthesis in S. aureus, respectively.

 Limitations: While these studies provide evidence for in vitro activity, further
research is needed to explore the optimal extraction methods, concentrations, and
formulations for topical applications against S. aureus infections.

Garlic: Similar to aloe vera, numerous in vitro studies have investigated the antibacterial
activity of garlic extracts against S. aureus:

 Antibacterial activity: Studies by Adetumbi et al. (2010) and Banerjee et al. (1996)
demonstrated the broad-spectrum antibacterial activity of garlic extracts, including
against S. aureus. The MICs were generally lower compared to aloe vera, suggesting
potentially higher potency.

 Mechanisms of action: The primary mechanism of action attributed to garlic's

antibacterial activity is the action of allicin. Allicin disrupts the cell membrane of S.
aureus, leading to leakage of cellular contents and cell death. It might also inhibit
essential enzymes involved in bacterial growth and metabolism.

 Limitations: Similar to aloe vera, further research is needed to translate these in vitro
findings into effective topical formulations for treating S. aureus infections on the
skin.
2.4.1.2 Candida albicans

Aloe vera: Existing research suggests potential antifungal activity of aloe vera against
Candida albicans:

 Antifungal activity: Studies by Hamidi et al. (2013) demonstrated that ethanol extracts
of aloe vera exhibited the most potent antifungal activity against C. albicans, with a
MIC of 31.25 µg/ml. Eshuis et al. (2004) and Choi & Chung (2005) suggested that
mannose-6-phosphate and polysaccharides in aloe vera gel might contribute to its
antifungal activity.

 Mechanisms of action: The proposed mechanisms include disrupting the fungal cell
wall or interfering with fungal adhesion to host cells. However, further research is
needed to elucidate the exact mechanisms by which aloe vera exerts its antifungal
activity.

 Limitations: More in vitro studies are necessary to explore the antifungal activity of
different aloe vera extracts and their effectiveness against various strains of C.
albicans.

Garlic: Similar to S. aureus, garlic extracts have also been shown to possess antifungal
activity against Candida albicans:

 Antifungal activity: Studies by Hajheydari et al. (2014) and Szejtli & Pocsi (2000)
demonstrated the in vitro antifungal activity of garlic extracts against C. albicans. The
mechanisms are likely similar to its antibacterial activity, with allicin disrupting the
fungal cell membrane and potentially inhibiting essential enzymes involved in fungal
growth.

 Limitations: While these studies provide evidence for antifungal activity, further
research is needed to optimize extraction methods, formulations, and concentrations
for topical applications against Candida albicans infections.
2.4.1.3 Comparison

 Both aloe vera and garlic extracts exhibit in vitro antimicrobial activity against
Staphylococcus aureus and Candida albicans. However, the available data suggests
that garlic extracts might possess a broader spectrum of activity and potentially higher
potency compared to aloe vera, particularly against S. aureus.

 The specific mechanisms of action differ, with aloe vera primarily targeting cell wall
synthesis and fungal adhesion, while garlic disrupts cell membranes and inhibits
essential enzymes in microbes.

2.4.2 In vivo Studies on the Efficacy of Aloe vera and Garlic for Treating Skin Disorders

In contrast to the abundance of in vitro studies, research on the in vivo efficacy of aloe vera
and garlic for treating skin disorders remains limited. Here, we critically analyze the existing
studies and identify key areas for further investigation.

2.4.2.1 Staphylococcus aureus Infections

 Aloe vera: Limited in vivo research has been conducted on the efficacy of aloe vera
for treating skin disorders caused by S. aureus. Some studies have investigated its
wound healing properties in humans, but not specifically in the context of S. aureus
infections.

o Wound healing studies: A review by Ernst (2000) analyzed studies on the

topical use of aloe vera for treating burns and chronic wounds. The review
concluded that aloe vera might offer some benefits in wound healing, but more
research is needed to confirm its efficacy against S. aureus-infected wounds.

 Garlic: Similar to aloe vera, limited in vivo research exists on the efficacy of garlic
for treating S. aureus skin infections. Studies have primarily focused on its use as a
dietary supplement or preventative measure.
2.4.2.2 Candida albicans Infections
 Aloe vera: No conclusive in vivo studies have been identified that directly investigate
the efficacy of aloe vera for treating Candida albicans infections on the skin.

 Garlic: Similar to S. aureus infections, in vivo research on the effectiveness of garlic

for treating Candida albicans skin infections are scarce. Existing research focuses
more on its use for systemic candidiasis.

2.4.2.2 Comparison and Limitations

 In vivo research on the efficacy of aloe vera and garlic for treating skin disorders
caused by S. aureus and C. albicans is scarce. This highlights the need for well-
designed clinical trials to bridge the gap between in vitro findings and real-world
applications.

 The limited in vivo research on aloe vera primarily focuses on its wound healing
properties, not specifically on its antimicrobial activity against S. aureus.

 Studies on garlic have not directly evaluated its effectiveness against topical S. aureus
or C. albicans infections.

2.4.3 Safety Considerations

 Aloe vera: Topical application of aloe vera gel is generally considered safe for most
individuals. However, allergic reactions can occur in some cases.

 Garlic: Topical application of garlic can cause skin irritation in some individuals. The
concentration and duration of application are crucial factors in avoiding irritation.
2.4.4 Conclusion on the Empirical Review

This expanded review of the empirical literature highlights the limitations of current research
on the in vivo efficacy of aloe vera and garlic for treating skin disorders. While in vitro
studies suggest promising antimicrobial activity, translating these findings into effective
topical treatments requires further in vivo research. The review also emphasizes the need for
safety considerations when using these plant-based products. This knowledge gap
underscores the importance of the present study, which aims to develop and evaluate aloe
vera and garlic-based topical ointments for their efficacy and safety in treating skin disorders
caused by S. aureus and C. albicans.

2.5 Summary of the Literature Review

By delving deeper into the conceptual framework, theoretical frameworks and empirical
review, we have gained a comprehensive understanding of the potential benefits and
limitations of aloe vera and garlic in treating skin disorders. Here's a concise summary of the
key takeaways:

 The conceptual framework: The key elements involved in developing and evaluating
topical ointments formulated with aloe vera and garlic extracts for treating skin
disorders caused by Staphylococcus aureus and Candida albicans was explained.

 Theoretical Frameworks: Both aloe vera and garlic possess antimicrobial properties
that can potentially combat S. aureus and C. albicans. Their mechanisms of action
differ, with aloe vera targeting cell wall synthesis, cell membranes, and immune
modulation, while garlic primarily disrupts cell membranes and inhibits essential
enzymes. Additionally, aloe vera offers potential benefits in wound healing.

 Empirical Review: In vitro studies provide evidence for the antimicrobial activity of
both aloe vera and garlic extracts against S. aureus and C. albicans. However, in vivo
research on their efficacy for treating skin disorders is scarce. The limited in vivo
studies on aloe vera focus on wound healing, not specifically on S. aureus infections.
 CHAPTER THREE

3.0 Methodology

This chapter meticulously details the methodology employed to develop topical ointments
formulated with aloe vera and garlic extracts obtained through solvent extraction using 50%
ethanol. The primary focus lies in investigating the efficacy of 50% ethanol for extracting
bioactive compounds with potential antimicrobial activity against Staphylococcus aureus (S.
aureus) and Candida albicans (C. albicans).

3.1 Materials

High-quality materials specifically chosen for solvent extraction with 50% ethanol will be
utilized:

Plant Materials
 Aloe vera: Fresh aloe vera leaves will be sourced from a local organic farm known
for sustainable practices. Leaves will be selected based on maturity, with firm
texture and minimal blemishes, ensuring optimal extract quality (El Moussaoui et
al., 2023).

 Garlic: Fresh garlic cloves will be purchased from a reputable grocery store,
ensuring cloves are firm, plump, and free of mold or sprouting, to maximize the
presence of bioactive compounds (Mitra et al., 2022).

Extraction Solvent
 Food-grade Ethanol (50% v/v): This concentration is chosen to balance
efficient extraction of bioactive compounds from the plant materials with
minimizing solvent usage, promoting environmental sustainability (El
Moussaoui et al., 2023).

Other Materials
 Distilled Water: Used for rinsing aloe vera leaves and preparing media for
antimicrobial assays.

 Analytical Balance: Ensures precise weighing of plant materials and extracts

for accurate yield determination.
  Blender (Optional): Facilitates efficient processing of aloe vera gel into a
uniform consistency for better extraction (El Moussaoui et al., 2023).

 Rotary Evaporator: Employed for gentle and controlled solvent removal

during extract concentration.

 Sterile Mortars and Pestles: Used for crushing garlic cloves to maximize
surface area and facilitate efficient extraction of bioactive compounds (Mitra
et al., 2022).

 Autoclave: Ensures sterilization of media and equipment for aseptic

procedures, minimizing contamination risks.

 Laminar Flow Hood: Provides a sterile environment for handling extracts and
preparing ointments, maintaining product safety.

 Filter Paper (Whatman No. 1): Facilitates filtration of the extracts after the
extraction process.

 Glassware (flasks, beakers):Used for extraction procedures and solvent

removal.

 Ointment Base Components (described in Section 3.3).

 Media and Reagents for Antimicrobial Assays (described in Sections 3.4 and
3.5).

3.2.1 Extraction of Bioactive Compounds from Aloe vera using 50% Ethanol

This section details the protocol for extracting bioactive compounds from aloe vera gel using
50% ethanol:

Preparation
1. Washing and Peeling: Fresh aloe vera leaves will be thoroughly washed under
running tap water to remove dirt, debris, and potential surface contaminants.
They will be patted dry with clean, lint-free paper towels to minimize water
carryover (El Moussaoui et al., 2023).
 2. Gel Separation: Using a sharp knife, the outer rind of the aloe vera leaves will
be carefully peeled to expose the inner gel, the primary source of bioactive
compounds.

3. Gel Processing: The aloe vera gel will be processed depending on the desired
consistency:
o For a smooth texture, the gel can be blended using a sanitized blender for a
uniform consistency, facilitating efficient extraction (El Moussaoui et al.,
2023).
o If a less processed form is preferred, the gel can be diced into small pieces to
increase surface area for extraction.

4. Weighing: A predetermined amount of aloe vera gel (e.g., 50 grams) will be

weighed accurately using the analytical balance to ensure consistent extraction
parameters.

Extraction
1. Solvent Selection and Ratio: The weighed aloe vera gel will be transferred to a
clean, sterile flask. Fifty percent (v/v) ethanol will be added to the gel in a
solvent-to-gel ratio of 2:1 (v/w). This ratio balances efficient extraction with
minimizing solvent usage (El Moussaoui et al., 2023).

2. Extraction Time and Agitation: The flask will be securely sealed, and the
mixture will be gently agitated using a magnetic stirrer for a defined extraction
time (e.g., 3 hours). This agitation facilitates optimal contact between the
solvent and the plant material, promoting efficient extraction of bioactive
compounds (El Moussaoui et al., 2023).

3. Filtration: After the designated extraction time, the mixture will be filtered
using Whatman No. 1 filter paper placed in a funnel. The filtrate collected in a
clean flask will contain the aloe vera extract in 50% ethanol solution. The
remaining aloe vera gel residue can be discarded responsibly following proper
biowaste disposal practices.

Concentration
1. Rotary Evaporation: The ethanol extracts needs to be concentrated to remove
the solvent and obtain a more potent extract. The rotary evaporator will be
employed for this purpose, following the manufacturer's instructions.
 2. Temperature Control: A reduced pressure and a water bath temperature around
40°C will be maintained during the evaporation process. This gentle approach
minimizes thermal degradation of the extracted bioactive compounds from
aloe vera, preserving their efficacy (El Moussaoui et al., 2023).

3. Concentrated Extract: The concentrated aloe vera extract obtained using 50%
ethanol will remain in the flask after the ethanol evaporates. The weight of the
concentrated extract will be recorded to determine the extraction yield,
providing insights into the efficiency of the process.

Standardization and Storage

1. Documentation: All parameters of the extraction process, including the ethanol
concentration (50%), solvent-to-gel ratio (2:1), extraction time (3 hours), and
temperature (40°C) used, will be meticulously documented in a designated
laboratory notebook. This ensures consistent and repeatable extractions in the
future for comparative analysis and quality control.

2. Storage: The concentrated aloe vera extract obtained using 50% ethanol will
be stored in a separate, airtight, light-protected amber glass vial. This
minimizes exposure to light and air, which can degrade the bioactive
compounds (El Moussaoui et al., 2023). The vial will be stored at a cool
temperature (e.g., 4°C in a refrigerator) to further ensure the stability and
potency of the extract for downstream applications.

Evaluation of Extract Quality

1. Antimicrobial Activity Assay: The antimicrobial activity of the obtained aloe
vera extract (using 50% ethanol) will be evaluated against S. aureus using a
disc diffusion assay (or another suitable method) as described in Section 3.4.
The diameter of the zone of inhibition surrounding the discs containing the
extract will be measured. A larger zone of inhibition indicates greater
antibacterial activity of the extract (National Center for Complementary and
Integrative Health, 2023).

2. Total Phenolic Content (TPC) Assay (Optional): An optional assay for total
phenolic content (TPC) can be performed to indirectly assess the concentration
 of bioactive compounds in the extract. Phenolic compounds are known to
contribute to the antimicrobial properties of aloe vera (El Moussaoui et al.,
2023). This assay will provide a quantitative comparison of the phenolic
content in the extract obtained using 50% ethanol.

By evaluating both antimicrobial activity and TPC, we can gain valuable insights into the
effectiveness of the 50% ethanol extraction process for aloe vera.

3.2.2 Extraction of Bioactive Compounds from Garlic using 50% Ethanol

This section details the protocol for extracting bioactive compounds from garlic cloves using
50% ethanol:

Preparation
1. Peeling and Weighing: Fresh garlic cloves will be peeled to remove the outer
skin. A predetermined amount of cloves (e.g., 20 grams) will be weighed
accurately using the analytical balance to ensure consistent extraction
parameters (Mitra et al., 2022).

2. Crushing: The garlic cloves will be crushed or finely ground using sterile
mortars and pestles to increase the surface area and facilitate efficient
extraction of bioactive compounds from the plant material (Mitra et al., 2022).

Extraction
1. Solvent Selection and Ratio: Similar to aloe vera, the crushed garlic material
will be transferred to a clean, sterile flask. Fifty percent (v/v) ethanol will be
added to the crushed garlic in a solvent-to-garlic ratio of 10:1 (v/w). This
higher solvent-to-material ratio accounts for the denser structure of garlic
cloves compared to aloe vera gel (Mitra et al., 2022).

2. Extraction Time and Agitation: The flask will be securely sealed, and the
mixture will be gently agitated using a magnetic stirrer for a defined extraction
time (e.g., 48 hours). The extended extraction time reflects the tougher cell
walls of garlic cloves compared to aloe vera gel, allowing for more efficient
extraction of bioactive compounds (Mitra et al., 2022).
 3. Filtration: After the designated extraction time, the mixture will be filtered
using Whatman No. 1 filter paper placed in a funnel. The filtrate collected in a
clean flask will contain the garlic extract in the 50% ethanol solution. The
remaining garlic residue can be discarded responsibly following proper
biowaste disposal practices.

Concentration

The concentration procedure for the garlic extract will follow the same steps as outlined for
aloe vera extracts in Section 3.2.1 (3) using the rotary evaporator at a low temperature
(around 40°C) to minimize degradation of the heat-sensitive bioactive compounds present in
garlic (Cavallaro et al., 2010).

Standardization and Storage

Similar to aloe vera extracts, the concentrated garlic extract obtained using 50% ethanol will
be standardized through meticulous documentation of the extraction process and stored in a
separate, airtight, light-protected amber glass vial at a cool temperature (e.g., 4°C in a
refrigerator) to further ensure the stability and potency of the extract.

Evaluation of Extract Quality

1. Antifungal Activity Assay: The antifungal activity of the obtained garlic
extract (using 50% ethanol) will be evaluated against C. albicans using an agar
dilution method (or another suitable method) as described in Section 3.5. The
minimum inhibitory concentration (MIC) of the extract for inhibiting fungal
growth will be determined. A lower MIC value indicates greater antifungal
activity (National Center for Complementary and Integrative Health, 2023).

2. Allicin Content Assay (Optional): An optional assay can be performed to

quantify the allicin content in the garlic extract. Allicin is a well-known
antifungal compound found in garlic (Cavallaro et al., 2010). By measuring
the allicin content, we can gain a more direct understanding of the potential
antifungal activity of the extract obtained using 50% ethanol.

3.2.3 Selection of Optimal Ethanol Concentration

This section describes an optional step to compare the effectiveness of 50% ethanol with
another ethanol concentration 70%for extracting bioactive compounds from aloe vera and
garlic:
 1. Repeat Extraction with Different Ethanol Concentration: The entire extraction
procedures outlined in Sections 3.2.1 and 3.2.2 will be repeated using 70%
(v/v) ethanol instead of 50% for both aloe vera and garlic.

2. Evaluation of Extracts: The concentrated extracts obtained using 70% ethanol

will be evaluated for quality using the same methods employed for 50%
ethanol extracts (Section 3.2.1 & 3.2.2):
 Antimicrobial activity assays (disc diffusion for S. aureus, MIC for C.
albicans).
 Optional assays (TPC for aloe vera, allicin content for garlic).

3. Comparison and Selection: The results obtained from the extracts prepared
with 50% and 70% ethanol will be compared. The concentration (either 50%
or 70%) that yields extracts with higher antimicrobial activity and/or higher
content of relevant bioactive compounds (TPC or allicin) will be selected for
further ointment formulation (Section 3.3).

3.3 Ointment Base Components

This section details the components chosen for formulating the topical ointments
incorporating the aloe vera and garlic extracts obtained using 50% ethanol:

 Petrolatum: This will serve as the primary base for the ointment, providing a
thick, occlusive layer for effective topical application (Gupta et al., 2019).

 White Beeswax: This natural wax will add structure and stability to the
ointment, while also offering a degree of emolliency for a smoother
application (Gupta et al., 2019).

 Cetiol® V (cetearyl alcohol): This emulsifying agent will facilitate the

incorporation of the aqueous aloe vera/garlic extracts into the oil-based
petrolatum, ensuring a stable and uniform ointment (Gupta et al., 2019).

The specific ratios of these components will be optimized to achieve a desirable ointment
consistency (e.g., spreadability, viscosity) suitable for topical application. This optimization
process may involve pilot studies with varying ratios to identify the formulation with the
most favorable characteristics.
Additional Considerations
 Sterilization: All ointment base components will be sterilized using appropriate
methods (e.g., filtration for heat-sensitive components) to ensure a sterile final
product.

 Preservatives (Optional): Depending on the storage conditions and desired shelf

life of the final ointment, the addition of a safe and approved preservative may be
necessary. The selection and concentration of the preservative will be determined
based on relevant regulations and compatibility with other ointment components.

3.4 Antimicrobial Activity Assay against S. aureus

This section details the protocol for evaluating the antimicrobial activity of the aloe vera and
garlic extracts obtained using 50% ethanol against Staphylococcus aureus (S. aureus):

Materials
 Mueller-Hinton Agar (MHA) plates
 Overnight culture of S. aureus (bacterial strain)
 Sterile swabs
 Antibiotic disc (positive control, e.g., oxacillin)
 Extract-impregnated discs (prepared as described below)

Preparation of Extract-Impregnated Discs

1. Sterile filter paper discs (6 mm diameter) will be obtained.

2. A predetermined volume of each concentrated extract (aloe vera and garlic) obtained
using 50% ethanol will be pipetted onto separate sterile discs. The volume will be
chosen to ensure a measurable zone of inhibition upon application.

3. The extract-impregnated discs will be allowed to dry completely under aseptic

conditions in a laminar flow hood to ensure solvent evaporation.

Disc Diffusion Assay

1. MHA plates will be streaked uniformly with the overnight culture of S. aureus using a
sterile swab.
 2. The prepared extract-impregnated discs, along with a positive control antibiotic disc
(e.g., oxacillin), will be placed onto the inoculated MHA plates using sterile forceps,
ensuring even distribution.

3. The inoculated MHA plates will be incubated at 37°C for 24 hours to allow bacterial
growth.

Evaluation
1. After incubation, the diameter of the clear zone of inhibition surrounding each disc
will be measured using a ruler. A larger zone of inhibition indicates greater
antibacterial activity of the extract or antibiotic control.
2. The experiment will be performed in duplicate or triplicate for each extract to ensure
reproducibility of the results.

Interpretation

The diameter of the zones of inhibition will be compared between the aloe vera and garlic
extracts, as well as the positive control antibiotic. This comparison will provide insights into
the relative antibacterial activity of the extracts obtained using 50% ethanol.

3.5 Antimicrobial Activity Assay against C. albicans

This section details the protocol for evaluating the antifungal activity of the garlic extract
obtained using 50% ethanol against Candida albicans (C. albicans):

Materials
 RPMI 1640 medium with L-glutamine and phenol red
 Yeast extract
 D-(+)-Glucose
 Sterile 96-well microplates
 Overnight culture of C. albicans (fungal strain)
 Microplate reader capable of measuring absorbance at 540 nm

Preparation of Broth Microdilution Assay

1. A serial dilution of the concentrated garlic extract obtained using 50% ethanol will be
prepared in RPMI 1640 medium to create a range of concentrations for testing.
 2. The RPMI 1640 medium will be supplemented with yeast extract and D-(+)-glucose
to support fungal growth.

3. An overnight culture of C. albicans will be diluted to a standardized inoculum density.

Microdilution Assay
1. Aliquots of the diluted extract solutions will be dispensed into a sterile 96-well micro
plate.

2. The standardized inoculum of C. albicans will be added to each well containing the
extract dilutions.

3. A growth control well containing only RPMI medium with supplements and inoculum
will be included for comparison.

4. A sterility control well containing only RPMI medium with supplements but without
inoculum will also be included.

5. The 96-well microplate will be incubated at 30°C for 48 hours to allow fungal growth.

Evaluation
1. After incubation, the microplate reader will be used to measure the absorbance of
each well at 540 nm. A decrease in absorbance compared to the growth control well
indicates fungal growth inhibition by the extract.

2. The minimum inhibitory concentration (MIC) of the extract will be determined as the
lowest concentration that inhibits visible fungal growth (indicated by minimal change
in absorbance compared to the growth control).

Interpretation

The MIC value obtained for the garlic extract will be indicative of its antifungal activity
against C. albicans. A lower MIC value signifies greater antifungal potency of the extract.
3.6 Statistical Analysis

All experiments, including antimicrobial activity assays (disc diffusion and microdilution),
will be performed in duplicate or triplicate to account for potential variability. The data
collected (zone of inhibition diameters and MIC values) will be subjected to statistical
analysis using appropriate software. Descriptive statistics (mean, standard deviation) will be
calculated to summarize the data for each extract. Depending on the normality of the data
distribution, parametric (e.g., one-way ANOVA) or non-parametric (e.g., Kruskal-Wallis test)
tests will be employed to compare the antimicrobial activity of aloe vera and garlic extracts,
as well as to assess any significant differences between the extracts obtained using 50% and
70% ethanol (if the optional section 3.2.3 is conducted).

Statistical significance will be set at a level of p < 0.05. Reporting the results of the statistical
analysis will enhance the robustness of the study and provide a more objective interpretation
of the observed antimicrobial activity of the extracts.

3.7 Safety Considerations

Throughout the methodology, safety will be a paramount concern:

 Personal Protective Equipment (PPE): During extraction procedures involving

ethanol, gloves, safety glasses, and appropriate attire will be worn to minimize
exposure to flammable solvents.

 Aseptic Techniques: Sterile techniques will be strictly followed during extract

preparation, ointment formulation, and antimicrobial assays to prevent
contamination.

 Waste Disposal: All waste materials, including used solvents, filter paper, and
biowaste from plant materials, will be disposed of responsibly according to local
regulations and safety protocols.

 Ointment Base Sterilization: Depending on the chosen components, appropriate

sterilization methods will be employed for the ointment base to ensure a safe final
product.

 Preservative Selection (if applicable): If preservatives are incorporated into the

ointment formulation, only safe and approved preservatives will be chosen,
adhering to relevant regulations.
By adhering to these safety considerations, potential hazards will be minimized throughout
the research process.

3.8 Conclusion

This chapter has meticulously detailed the methodology employed for extracting bioactive
compounds from aloe vera and garlic using 50% ethanol. The extraction procedures,
evaluation of extract quality through antimicrobial assays, and ointment base formulation
have all been outlined. Statistical analysis will ensure robust interpretation of the results.
Safety considerations have been addressed to ensure a responsible research approach. The
findings from this methodology will contribute to the development of topical ointments with
potential antimicrobial activity for further investigation.