Executive Summary

Brief statement of the problem: Synthetic antiseptic soap is more expensive making it

unaffordable for consumers seeking effective skincare solution and the microbial resistance of

chemicals substances used in soap, along with the potential use of harmful chemical, posses

significant risk to public health and the environment. This issue not only impacts the

effectiveness of soap against resistance microorganisms but also raises concern about the

negative consequences of harmful chemicals on human health and the ecosystem. Addressing

these challenges requires innovative approaches to develop soap formulations that are both

effective against microbial resistance and free from harmful chemical, ensuring the wellbeing of

consumers and the environment while maintaining cost effectiveness.

Brief background information: skin infection, such as bacterial and fungal dermatoses, pose

significant health challenges. Conventional treatments often involve synthetic antimicrobial

agents, which may have adverse effects and contribute to many have adverse effects and

contribute to antibiotic resistance. Herbal remedies, on the other hand, offer a natural alternative

with potential therapeutic benefits. Curcuma longa, commonly known as turmeric, has

demonstrated antimicrobial, anti-inflammatory, and wound-healing properties. This study aims

to to harness these properties by formulating an herbal soap for topical use.

Research Questions:

i. Can herbal soap be formulated using C. longa?

ii. What are physicochemical properties of Curcuma long as a herbal soap?

iii. What is the in vitro and in vivo antimicrobial effect of the herbal soap containing C longa?
 iv. Does the herbal soap cause any adverse reaction or allergies on animal subject during a

prolong use of two weeks?

Specific objective:

i. To formulate herbal soap containing Curcuma longa extract.

ii. To evaluate the physicochemical properties of the soap formulation.

iii. To assess the in vitro and in vivo antimicrobial effect of Curcuma longa rhizorm against

staphylococcus aureus and Trichophyton rubrum before and after the herbal soap

formulation.

iv. To observe any adverse reaction or allergies in animal subject during a prolong use of two

week.

Theoretical framework: The research will be based on the formulation and evaluation of herbal

soap against bacterial and fungi skin infections using C. longa as the main active ingredients.

The study will involve selecting an appropriate animal model (guinne pig), isolating S. aureus

and T. rubrum from infected individuals, obtaining ethical approval, and designing controlled

experiments. Animal will receive topical applications of the formulated soap while from

microbiological perspective, bacterial and fungal susceptibility will be exam at different

concentration of the formulated soap. Data will be collected on the microbial load reduction,

safety and skin reaction. considering the urgent need for alternative treatments due antimicrobial

resistance thus statiscally analysis of this research will determine the soaps antimicrobial efficacy

and potential as an alternative treatment. This theoretical frame work integrates concepts from

herbal medicine, microbiology and health behavior theories, bridging traditional wisdom with

scientific rigor
Methodology: A quantitative experimental research designed shall be used for the study. Data

shall be collected through the measurement of the zone of inhibition and observation. The data

shall be analysed using Microsoft excel 2019, chat and tables.

Introduction

Majority of the commercial soap today sold in the market are highly incorporated with harmful

chemical agent such as aluminum, barium, bis phenols, plastics formaldehyde, triclosan, sodium

lauryl sulfate, dioxane, fragrance, cocamidopropyl betaine, butylated hydroxy anisole and

butylated hydroxytoluene, triethanolamine and methyllisothiazoline and

methylchloroisothiaoline which are absorbed into the body via the internal lungs from

vaporization of the chemicals as well as skin absorption with negative side effects on the health

of individuals (Aiello et al ., 2007). Synthetic antiseptic soap is expensive and unaffordable

especially in developing countries, herbal soap or herbal cosmetics provide an affordable and

cheap with comparative health and safety benefits (Joshi and Pawal, 2015; Sharma et al., 2008).

The aim of this work is to conduct an experimental study on the in vitro and in vivo effect of

Curcuma longa, against S. aureus and Trichophyton rubrum and the herbal formulation of

medicated soap against skin infection cause by fungi and bacterial. This seek in providing an

alternative means in treating skin infections cause by S. aureus and T. rubrum using which is

more affordable and safer for managing bacterial and fungal skin condition if proving effect.

Background of the study

The skin is the largest organ in the body that act as the first line of defense against pathogenic

agents with underlying tissue (Dryden, 2009). Skin and soft tissue infections are very common

within the population due to constant exposure of the skin to pathogenic agents (Dryden, 2009).
Studies have shown that skin infection cause by bacterial, viral, and fungal ranges from 42 to

65% of the overall skin illness (Enfeksiyonlari., et al 2015)). The most bacterial skin infections

is cause by Staphylococcus aureus which is commonly found on the skin particularly in moist

areas such as the anterior nares (nose), axilla and groin and mucous membrane and causes skin

infections such as cellulitis, folliculitis, boils and abscesses (Debra, louisa et al., 2023).

Trichophyton rubrum is another dermatophytic fungus in the phylum Ascomycota that causes

athletes-foot, fungal infection of the nail, jock itch and ring worm (Zaugg et al., 2009)

Since ancient civilization soap has been one of most important and the oldest chemical product

of removing dirt and eliminating pathogenic agents on the skin. Soap is a chemical product made

by the chemical process of saponification of an alkali salt such as wood ash or strong alkali

solution with a long chain fatty acid (Grace X. F, Sowmya K et al, 2015). The most common

used fat or oils for production of soap through saponification reactions are animal tallow,

coconut oil, palm oil, kernel oil and linseed oil. (Kubmarawa, 2000). Similarly, potassium and

sodium hydroxides are widely used as the caustic alkaline for the purpose. (Eromosele, 1997).

Herbal soap preparation is a medicine or drugs containing phytochemical from different

botanical sources and has antibacterial and antifungal agents which involve the uses of plants

parts such as like leaves, stem, roots and fruits for the treatment of injuries or disease or to

achieve good health (Kareru, et al., 2010). This preparation possess antimicrobial property are

administered topically and available to apply in various forms like creams, lotion, gel, soap,

solvent extract or ointment the variety of creams and soap properties have been used to treat

various skin disorder (Bandyopadhyay et al., 2004). Herbal skin care formulation can be made

from collection of various herbal plant part such as leaves, roots, bark, fruits seed and flowers
which are topically applied as antimicrobial and anti-inflammatory agents in treating skin disease

such as eczemas, ringworm and pruritus (Sharma, et al., 2014).

Antibacterial agents and antifungal agents play crucial roles in combating infection caused by

bacterial and fungi, these agents are used to target specific pathogens and prevent their growth or

eliminate them (Andrea et al., 2019). The incorporation of antibacterial and antifungal agent into

soap products for the treatment of skin infection has been a common practice. However, several

challenges have emerged over time such as the development of microbial resistance by bacterial

and fungi to these agents. This resistance reduces the effectiveness of treatment (Deborah A et

al., 2017, Paloma et al., 2023). The distinctive characteristics of soap production for both

industrial and domestic uses depend specifically on the type of oil which give the product

specific criterial like aroma, clarity, color, low moisture content, absence of fat and rancid odor

(Okoye et al., 1999, Manji et al., 2013). The quality soap is achieved by reacting different

concentration of oil or fat with lye and the quality of the soap depends on the hardness, the

ability of the soap to penetrate and remove dirt, conditional lathering potential and the antiseptic

nature of the soap (Manji et al., 2013). Majority of the commercial soap today sold in the market

are highly incorporated with harmful chemical agent such as aluminum, barium, bis phenols,

plastics formaldehyde, triclosan, sodium lauryl sulfate, dioxane, fragrance, cocamidopropyl

betaine, butylated hydroxy anisole and butylated hydroxytoluene, triethanolamine and

methyllisothiazoline and methylchloroisothiaoline which are absorbed into the body via the

internal lungs from vaporization of the chemicals as well as skin absorption with negative side

effects on the health of individuals (Aiello et al ., 2007). Synthetic antiseptic soap is expensive

and unaffordable especially in developing countries, herbal soap or herbal cosmetics provide an
affordable and cheap with comparative health and safety benefits (Joshi and Pawal, 2015;

Sharma et al., 2008).

Herbal cosmetic has many potential health benefits such as antioxidant, anticancer and

antimicrobial properties that manages various skin and hair conditions. The presence of

phytochemicals such as vitamins, proteins, terpenoids and other bioactive ingredients rejuvenate,

freshen and protect the hair and skin from various conditions such as psoriasis, eczema, skin

dryness, skin cancers, sun burn, skin dryness, boil, candidiasis, athletes’ foot, chicken pox solar

keratosis, dermatitis, impetigo and others (Fathima et al., 20011, Kapoor, 205; Joshi and Pawal,

2005). Neem, turmeric and tulsi are all-natural plant ingredients in herbal soap, and this

combination has antibacterial, antifungal, and anti-inflammatory properties (Sankholkar et al.,

2009).

The aim of this work is to conduct an experimental study on the herbal analysis and evaluation of

Curcuma longa, Ocimum tenuiflorum and Azadirachta indica against S. aureus, Trichophyton

rubrum and the herbal formulation of medicated soap against skin infection cause by fungi and

bacterial.

1.1 Problem statement

Synthetic antiseptic soap is more expensive making it unaffordable for consumers seeking

effective skincare solution and the microbial resistance of chemicals substances used in soap,

along with the potential use of harmful chemical, posses significant risk to public health and the

environment. This issue not only impacts the effectiveness of soap against resistance

microorganisms but also raises concern about the negative consequences of harmful chemicals

on human health and the ecosystem. Addressing these challenges requires innovative approaches
 to develop soap formulations that are both effective against microbial resistance and free from

harmful chemical, ensuring the wellbeing of consumers and the environment while maintaining

cost effectiveness.

1.2 RATIONAL

The high cost of medicinal soap used for specific skin conditions, cosmetic or therapeutic

purposes has act as barrier for low income individuals from accessing essential skincare products

and the scarcity of affordable alternatives of medicinal soap for specific skin conditions has

worsen skin conditions and an increase in health care expenses. challenges have emerged over

time such as the development of microbial resistance by bacterial and fungi to these agent, this

resistance reduces the effectiveness of treatment and many commercial medical soap contain a

high amount of synthetic chemical substance and additives such parabens, sulfate and artificial

fragrance causes harm to human health and the aquatic ecosystems thus use of herbal soap as an

alternative could be a potential benefit in the treatment of skin infection since it is relatively

cheap, has little or no adverse effect and ecofriendly and more effective against skin infections.

1.3 Hypothesis

I. Extract from Curcuma longa, will produce antifungal and antibacterial effects that meets

both the Encyclopedia of Industrial Chemical standards and KEBs standards respectively.

II. The antibacterial and antifungal soap produced from Curcuma longa, extract will cure

diseases such as ring worm, boils, wounds, pityriasis and even pimples.

1.4 Research question

v. Can herbal soap be formulated using C. longa?

 vi. What are physicochemical properties of Curcuma long as a herbal soap?

vii. What is the in vitro and in vivo antimicrobial effect of the herbal soap containing C longa?

viii. Does the herbal soap cause any adverse reaction or allergies on animal subject during a

prolong use of two weeks

1.5 General objective

To formulate herbal soap and evaluate the effect of Curcuma longa against S. aureus,

Trichophyton rubrum..

1.6 Specific objectives

v. To formulate herbal soap containing Curcuma longa extract

vi. To evaluate the physicochemical properties of the soap formulation.

vii. To assess the in vitro and in vivo antimicrobial effect of Curcuma longa rhizorm against

staphylococcus aureus and Trichophyton rubrum before and after the herbal soap

formulation.

viii. To observe any adverse reaction or allergies in animal subject during a prolong use of two

week.

1.7 Significant of the study

 This research contributes to the development of natural and effective alternatives to synthetic

antimicrobial soaps.

 The findings may lead to the production of safe and eco – friendly herbal soap for health skin

which are more affordable and easily assessable in nature.

 This finding will greatly impact the public health sector by decreasing the cost of health care

and reliance on chemical - based products which will potential prevent skin infections.

1.8 Limitation of the study

 This research work focuses on in vitro experiment and it may not fully represent real world

conditions.

 The herbal soaps effectiveness may differs based on the skin type of individuals and usage

patterns

 The study did not explore long term effects or chronic use of the soap.

1.9 Definition of terms

Dirty t dispersion:

Wetting time:

Foaming stability:

Moisture content

Ph:

Optimal inhibitory concentration:

Phytochemicals:

Bactericidal:

CHAPTER TWO

LITERATURE REVIEW
2.1 A review of S. aureus, R. rubrum and T. versicolor

Staphylococcus aureus and Trichophyton rubrum are common pathogens that can cause skin

infections, each presenting unique challenges. Staphylococcus aureus, a bacterium, is known for

its resistance to antibiotics, making treatment difficult can lead to conditions like cellulitis and

impetigo, causing symptoms such as redness, swelling, and pus-filled lesions (Venanzio vella et

al., 2021). On the other hand, Trichophyton rubrum, a fungus, causes superficial skin infections

like athlete's foot and ringworm. These infections are characterized by itchy, red, and scaly skin

(Smith et al., 2020). Thus Understanding their pathogenesis, epidemiology, method of

transmission and classification of these pathogens will provide us with an inside view of these

pathogens.

2.1.1 Overview of Staphylococcus aureus.

Staphylococcus aureus is a member of Bacillota gram - positive facultative anaerobe bacteria

which is spherical in shape and is one of the normal body flora often found on the skin, nares and

mucous membrane of healthy individuals (Masalha et al., 2001, Wertheim et al., 2005).

2.3.1 Botanical classification of Staphylococcus aureus

Domain: Bacteria

Kingdom: Eubacteria

Phylum: Firmicutes

Class: Bacilli

Order: Bacillales
Family: Staphylococcaceae

Genus: Staphylococcus

Species: S. aureus

2.2.1 Morphological description of S. aureus

S. aureus are spherical shape gram – positive bacterial which appear in cluster resembling a

bunch of grapes under an electronic light microscope after gram staining (Licitra G, 2013). The

cell wall contains a thick peptidoglycan layer and lack motility, flagella and spores (Brooks et al,

2007). Staphylococcus aureus is a facultative anaerobe that grows at an optimum temperature of

37ºC and an optimum pH of 7.5, on a nutrient agar it appears as a large white or yellow colony

(Liu et al., 2005).

2.3.1 Virulence factor, pathogenesis and mode of transmission of S. aureus

S. aureus produces various virulence factors such as alpha cytotoxin hemolysin also known Hla

or alpha toxin that bind to metalloprotease ADAM10 which initiate the breakdown of the

epidermis cells of the skin (86) . Staphylococcus aureus skin infection can be transmitted from

one person to another by coming with a contaminated skin individual and touching contaminated

object (Felix, 2022). Staphylococcus aureus causes serious skin infection ranging from mild to

server such folliculitis is the inflammation of the follicles leading to pustules or red bumps

(Laureano et al., 2014), impetigo which is highly contagious superficial infection impacting the

keratin cells of the epidermis (56) more common in warm and humid climates (64) and it is

characterized by honey-colored crusts and small blister (56), cellulitis is another skin infection
cause by staphylococcus aureus affecting deeper layers of the skin causing redness, warmth and

swelling (39), boils a painful pus-filled lump caused by infected hair follicles (55)

2.1.2 Overview of Trichophyton rubrum

Trichophyton rubrum is a dermatophyte fungus in the phylum Ascomycota which is exclusively

clonal (Graser et al., 199) anthropophilic saprotroph that colonizes the upper layers of death skin

and it is the most common cause of tinea pedis (athletes ffoot), tinea corporis (ringworm), and

onychomycosis (nail infection) worldwide (Zaugg et al., 2009)

2.2.2 Botanical classification of Trichophyton rubrum

Kingdom: Fungi

Division: Ascomycota

Class: Eurotiomycetes

Order: Onygenalles

Family: Arthrodermataceae

Genus: Trichophyton

Species: T. rubrum

2.3.2 Morphological description of Trichophyton rubrum

Trichophyton rubrum is an anthropophilic saprotroph which are white and cottony like on their

surfaces although some colony may appear red, yellowish or brownish underside (Kane, Julius,

1997). Trichophyton rubrum colonizes the upper layers of death skin and it is the most common
cause of athletes-foot, fungal infection of the nail, jock itch and ring worm (Zaugg et al., 2009).

It colonizes the skin by secretion more than 20 specific proteases that including exopeptidases

and exopeptidases which allow for the digestion of human keratin, collagen and elastine (kwong-

Chung, Bennett et al., 1992).

4.1.1 Virulence factor, pathogenesis and mode of transmission of Trichophyton rubrum

Trichophyton rubrum, a dermatophytic fungus, is a common cause of skin infections worldwide.

Its virulence factors include enzymes (such as keratinase, protease, lipase, and others) that aid in

tissue invasion. Additionally, adhesion m echanisms allow T. rubrum to adhere to keratinized

tissues, which is essential for colonization (Martínez-Herrera et al., 2023). The pathogenesis

involves the invasion of the outermost skin layer (stratum corneum), triggering an inflammatory

response. Interestingly, T. rubrum evades the host immune system and can persist chronically

(Majid Kadhim, 2019). Transmission occurs via direct contact (skin-to-skin) and indirectly

through contaminated objects (fomites). Proper diagnosis, antifungal treatment, and hygiene

practices are crucial for managing T. rubrum infections effectively (Ann packeu et al., 2021)

2.1.3 Overview of Curcuma longa

Curcuma longa is this highly branched perennial herbaceous plant belonging to the family of

Zingiberaceae and can grow up to 1m tall, with short stem. Curcuma longa is widely distributed

throughout tropical and subtropical regions of the words and is it is widely cultivated in Asiatic

countries, mainly in India and China (Eigner et al., 1999). Curcuma longa has a yellowish to

orange cylindrical aromatic rhizomes (Kew science, Kew gardens, 2018). The leaves are

alternate and are arranged into two rows. They are divided into leaf shealth, petiole, and leaf

blade. (Kew science, Kew gardens, 2018). From the leaf sheath, a false stem is formed. The
petiole is 50 to 115cm long. The simplest leaf blades are usually 76 to 115cm long and rarely up

to 230cm. they have a width of 38 to 45cm and are oblong to elliptical, narrowing at the tip (Kew

science, Kew gardens, 2018).

2.3.3 Botanical classification of Curcuma longa

Kingdom: Plantae

Class: Liliopsida

Order: Zingiberales

Family: Zingiberaceae

Genus: Curcuma

Species: C. longa

2.3.3 physicochemical composition of Curcuma longa

The phytochemical analysis ethanolic extract of turmeric shows important preliminary

phytochemical constituent such as tannin, saponins, phenolic compound, phytosterols,

terpenoids, alkaloids and flavonoids which has very important pharmacological activity such as

antimicrobial, antimutagenic, anti-inflammatory and antioxidant (Rajesh, Rao et al., 2013).

Polyphenolic compounds are the main chemical constituent in the turmeric rhizome and there are

curcumin, demethoxycurcumin, and bisdemethoxycurcumin, collectively known as curcuminoids

(3-6%). In the In the 19th century turmeric rhizomes were identified as Curcumin and there were

primarily used as coloring agents. 1-hydroxy-1, 7-bis (4-hydroxy-3- methoxyphenyl) - (6E) - 6-

heptene-3,5-dione is another phenolic molecule found in turmeric rhizome. The pale yellow to
orange_yellow volatile oil (4-6%) obtained from turmeric consist of a number of mono and

sesquiterpenes (Moghadamtousi et al., 2014)

FIGURE 2: chemical structure of Curcuma longa

2.5.3 pharmacological properties of Curcuma longa

Inflammatory Disorders: According to Sandur et al. (2007), Inflammatory markers which

induces inflammatory cytokines in the presence to stimuli are C-reactive protein (CRP),

complements, and fibrinogen. curcumin, demethoxycurcumin, and bisdemethoxycurcumin are

the active compounds in C. longa that inhibit TNF-induced NF-κB activation. The methoxy

groups on the phenyl ring were discovered to be responsible for their actions. C. longa extract

was examined to improve serum inflammatory markers and mental health and mood disturbance

in healthy participants who are overweight (Uchio et al., 2021). Researchers discovered that
curcumin has anti-inflammatory properties by inhibiting the pro-inflammatory transcription

factor (NF-κB) in 1995. They also discovered the molecular mechanism that underlies this

inhibition (Singh and Aggarwal, 1995)

Antioxidant Properties C. longa and its curcumin constituent have significant antioxidant

activity, equivalent to both vitamin C and vitamin E, in both water- and fat-soluble extracts.

Curcumin can help the body rid itself of hydroxyl radicals, singlet oxygen, superoxide radicals,

nitrogen dioxide, and NO. Curcumin pretreatment was proven to reduce ischemia-induced

mutations in the heart (Dikshit et al., 1995).

Reduces dark spots and hyperpigmentation: Turmeric contains an active compound called

curcumin which helps lighten dark spots and reduce hyperpigmentation by limiting the

overstimulation of melanin production, turmeric soap gradually fades dark sports and even out

skin tone. Studies have shown that constant used of turmeric soap over four weeks can results to

14% decrease in hyperpigmentation (Elizabeth, 2022).

Anticancer Activity: Annapurna et al. (2011) evaluated the ability of C. longa prophylactically

and therapeutically, i.e., pre-induction treatment and post-induction treatment via oral and topical

application to modulate the N-methyl-N-nitrosourea-induced mammary cancer in rats for 24

weeks. Prophylactic topical application given at 200 mg/kg of C. longa has significantly reduced

the mean tumor volume compared with therapeutic topical application. This was the first report

to show the anticancer activity of C. longa with topical application in a breast cancer model. In

an in vivo research involving the topical application of curcumin in CD-1 mice and dietary

administration of 1% C. longa, 0.05% of its ethanol extract significantly reduced tumor

incidence, tumor burden, and tumor volume in dimethyl benz[a]anthracene (DMBA)- initiated

and 12,O-tetradecanoylphorbal-13-acetate (TPA)- promoted skin tumors (Huang et al., 1988).

2.5.3 Mechanism of action of Curcuma longa

Curcumin has been reported for its antibiofilm activity through the inhibition of bacterial quorum

sensing systems and removal of already formed biofilms (Loo et al., 2016, Shukla et al., 2020).

Previous studies have shown a broad spectrum of antimicrobial properties of curcumin by

various pharmacological point of action (Shariifi S et al., 2020). It has been shown that curcumin

can inhibit bacterial DNA replication and alter gene expression. Moreover, it damages bacterial

cell membrane and reduces the motility of microorganism (Tyagi et al., 2015). The in vitro

studies have shown that curcumin inhibits the polymerization of FtsZ protofilaments and disturbs

the GTPase activity in cytoskeleton of B. subtilis, E. coli (Kaur et al., 2010) and S. aureus (Teow

et al., 2016). Other investigations have shown that curcumin stimulated apoptosis – like response

in E. coli (Yun et al., 2016). In turn, Alawan et al., (2017) observed its anti – adhesive effects

against C. albican biofilm formation at sub inhibitory concentration of MIC/2.

Alkaloids are secondary metabolites that are widely used in cosmetics in the production of

cream, tonics, lotions, face and hair mask to solve skin problems such as inflammations,

discoloration of the skin, antiaging and reduce the formation of cellulitis (Anna et al., 2021).

Alkaloid have a wide range of pharmacological activity including antibacterial activity (Alibis et

al., 2021). Most alkaloid exert their effect via efflux pump inhibitor which is consider as the

most potential effect of the antibacterial activity (Khameneh et al., 2019). Alkaloid has numerous

mechanism of actions such as disruption of bacterial cells membrane, inhibit the activities of

bacterial enzymes (Yan Y et al., 2021)

Tannins are phenolic compound found in plants which are known widely for their antioxidant

properties on the skin (Daliah Spiegel, 2007). Apart from their antioxidant properties on the skin

tannins, tannins have other pharmacological function such as anti – inflammatory properties that

can help minimize skin redness and inflammation, they help to remove excessive oil from pores

without drying out the skin. (Thayers, 2022).

2.2 Physicochemical properties of soap

2.2.1 Moisture content

Moisture content refers to the amount of water present in a soap sample. It is a parameter used in

assessing the shelf life, stability and texture of a product. Higher moisture content in soap would

leas to reaction of excess water with unsaponified fat to give fatty acid and glycerol in a process

called hydrolysis of soap on storage (Sarfaraz et al., 2019). According to the international

standard and Encyclopedia of industrial chemical analysis (2007), soap product should have a

moisture content ranging from 10% to 15%.

2.2.2 Analysis of PH

PH represents the acidity or alkalinity of a solution. For soap, PH influences their compatibility

with skin and cleaning efficacy (Sari and Suryari, 2018). Higher PH value in soap are due to the

incomplete hydrolysis resulting from saponification process. It can be overcome by adding

excess fat or oil to reduce the harshness of the soap (Warra et al., 2011). The higher the PH

value above the recommended range of …….. proposed by intentional standard indicate the soap
is corrosive to the skin. According to study conducted by Dash et al., 2014 showed that the PH of

turmeric is slightly acidic which ranges from 5.8 to 6.9.

2.2.3 Analysis of Total Fatty Matter

Total fatty matter is one of the most important characteristics describing the quality of soap. It

represent the total amount of fatty matter, mostly fatty acids, that can be separated from a soap

sample after splitting with a mineral acid usually hydrochloride acid (Betsy et al., 2013) or it

simple represent the percentage of fatty in the soap (Ajala et al., 2016). TFM is a measure of

identifying the amount of fatty matter present in soap. Bureau of Indian Standards (BIS) has

categorized bath or toilet soaps as ‘normal’, ‘baby, transparent, and antibacterial soaps. The last

three are called specialty soaps targeted to specific users. A toilet soap is a cosmetic by law and

it must fulfill the requirements of the relevant Indian standard. BIS categorized toilet soaps in to

three grades based on the total fatty matter present in them. If TFM is above 76%, grade I, which

is having good quality. TFM above 60%, belongs to grade II and TFM above 50% belongs to

grade III. According to International Standards (ISO), good quality soaps must have TFM above

76% (Lewkowltsch, 1922; Bureau of Indian standards, 2011 ). The lower TFM value indicate

unreacted NaOH in the mixture (Awang et al., 2001). However, dry skin needs soap which

contain higher percentage of TFM content (80%), which make skin smooth by rehydrating and

additionally the high oil content within the soap acts as a lubricant throughout the day (Mak-

Mensah and Firempong, 2011)

2.2.4 Free Caustic Alkali

The amount of alkali the amount of alkali free to counter and avert the soap from becoming oily

(Ashrafy et al., 2016) and it determine the abrasiveness of any soap (Ali and Geetha, 2001). High

amount of free caustic soda in soap product will lead to skin irritation, skin dryness and scaling

which can cause the skin to become susceptible fungal attack. This is because excess alkali will

saponify the fat and oil that is normally present on the skin as a protective coat to form soluble

soap and therefore become washed away and thereby rendering the skin dry (Ngambes et al.,

2014) thus the recommended free caustic alkali should be less than 0.1% according to East

African standard 318 (2002).

2.2.5 Total Alkali content

Total alkali contents is the amount of total alkali present in a soap product which include alkali

component such as sodium / potassium hydroxides, oxides, carbonates or bicarbonate (Vivian et

al., 2014). According to the BIS, good quality soap must have less than 5% of total alkali content

while according to ISO soap must have a total alkali content below 2% (Betsy et al., 2013)

2.3 Physicochemical properties of Curcuma longa

The analysis of the phytochemical composition of the aqueous and ethanol extracts of tumeric

(Curcuma longa L) obtained from High Level market Makurdi, Benue State, Nigeria showed the

presence of Alkaloids, Steroids, Saponins, Flavonoids, Phenols, Tannins, Carbohydrate and

Protein (Ahola et al., 2023).

2.4 Determination of antimicrobial activity

CHAPTER THREE

RESEARCH METHODOLOGY

3.1 RESEARCH DESIGN

The research follows an experimental design where the aim is to formulate and evaluate an

antimicrobial soap using Curcuma longa, Ocimum tenuiflorum against S. aureus, Trichophyton

rubrum. The study involves both qualitative and quantitative assessments.

3.2 Study area

 The study will be conducted at the department of chemistry at the university of Buea,

Cameroon.

3.3 Study population

My study population includes:

 Healthy volunteers: These individuals participated in the evaluation of herbal soaps safety

and efficacy and include only six participant and

 S. aureus and Trichophyton rubrum.

3.4 Research Materials and Equipment

Mortar and Pestle, Conical flask, Beaker, Measuring cylinders, Water bath, Filter papers, Petri

dishes, Auto clave, Capillary tube, Inoculation wire loop, Funnels, Syringes, Culture plates

mannitol salt, potato glucose agar and sabouraud destrose agar), weighing balance, foil paper,

conical flask, Oven, Refrigerator, Condenser. Chemical reagent used includes Fehling solutions

(A and B), Methanol, distilled water, Ferric chloride 3.5% (3.5 mL of FeCl 3 in 96.5 mL of

solvents), Dilute tetraoxosulphate (IV) acid (H2SO4), Sodium hydroxide (NaOH), Zinc chips,

Meyer’s reagent, Dragendoff’s reagent, Ammonia, Chloroform (CHCl 3), Nutrient agar, Ethyl

acetate, Acetone, Aqueous hydrochloric acid (1.0% HCl), Ethanol, Benzene and methanol

3.5 Sample collection and Preparation

S. aureus sample was collected by nasal swabbing the noise of a laboratory personal for three

seconds, Trichophyton rubrum sample were collected from 6 infected school children at Ndongo

primary schools in Molyko Buea with the consent of the school authorities. The site of infection

where the sample was collected was disinfected using methylated spirit. Different sterile swab

sticks was used for each child. Each sample was labeled and according to each place of

collection, the samples were labelled and packaged. The collected samples were immediately

wrapped tightly to avoid air contamination, and were brought to the chemistry laboratory in UB.

Curcuma longa where purchase from Limbe botanical garden.

3.5.1 Preparation of Plant Extract

The turmeric rhizomes were carefully peeled with a knife, rinsed with potable water, sliced and

dried in the oven at 50 °C for 24 hours. The dried samples were milled using a Blender into

powder and allowed to pass through a sieve with a nominal mesh size of 0.2 mm in diameter.

Twenty grams (20.0 g) of the powdered tumeric was dissolved in 100 mL sterile distilled water

and allowed to soak for 24 hours; after which the mixture was filtered through whatman’s filter

paper number 1 and kept for the analysis

3.5.2 Preparation of Sabouraud’s Dextrose Agar

Sabouraud’s Dextrose Agar (SDA) medium was used for the isolation of the organisms. 65g of

the medium powder is to be dissolved in 1litre of distilled water. Therefore, 24g per 369.2ml of

distilled water was prepared for 20 petri-dishes. The conical flask was covered with wad of

cotton wool, wrapped with aluminum foil. The conical flask was shaken gently to allow proper
dissolution of the medium. The medium was then autoclaved at 121oC for 15minutes and

allowed to cool to 50oC. Chloramphenicol was added as antibacterial agents. Aseptically, 2ml of

sterile distilled water was added to the chloramphenicol powder. It was allowed to dissolve by

mixing it gently. Then, 10ml of distilled water was added and mix together. This was distributed

in 2ml amounts into appropriate sterile containers. Each 2ml volume was sufficient for 500ml of

Sabouraud’s agar medium. The final concentration is 0.4mg/ml. The chloramphenicol solution

was added to the medium and the plates were poured (Cheesbrough,2010).

3.5.3 Preparation of Mannitol salt Agar

10g of mannitol salt agar was weighed using an electronic balance and dissolved separately and

dissolved in 369.2ml and 90ml of distilled water respectively in a conical flask and heated using

a water bath to completely dissolve. Mannitol agar was then sterilizes in an autoclave at a

pressure of 121 degrees for 15mins to ensure that the media is free from microorganisms. The

container or conical flask was removed from the autoclave and allow cool at temperature of 50

degree and the sterilize medium was then immediately poured into 8 petri dishes of 100mm and

bubbles where remove by flaming. Following solidification of the agar , place the petri dishes in

a plastic sleeve and store at 4 degree until needed.

3.6 Microbial Analysis

The swab sticks were innorculated in to their various petri dish the plates were then incubated at

room temperature (25oC) for 48 days. After incubation, the agar plates were observed for fungal

and staphylococcus aureus growth. On sabouraud dextrose agar Trichophyton rubrum appeared
as cream white colored with a reverse side of the colony exhibiting a wine - red pigment while

staphylococcus aureus had a small round yellowish appearance in Mannitol salt agar.

3.6.1 Microscopic examination

Sample were collected from the successifly culture S. aureus, Trichophyton rubrum on the

culture media fixed on a slide and various test were perform. For staphylococcus aureus gram

staining was done which appeared purple under the microscope while the identification of the

Trichophyton rubrum isolates was done by its staining procedure. To study the morphology of

the isolates, thin layer of the Trichophyton rubrum mycelia was spread on clean glass slides and

teased by adding a few drop of lactophenol cotton blue stain on each slide. The slides were

covered with cover-slips and visualized under microscope at X40 magnification for

Trichophyton rubrum appeared as septate hyphae with a spaghetti like structure and had an

appearance of a spaghetti and meatball structure respectively.

3.7 Soap herbal Preparation

The herbal soap was produced using cold process soap making. The cold process method is one

of the most common ways to produce soap even at home. This process gets its name from the

general low temperature used to mill the type of the soap. The low temperature or sufficient

temperature is to ensure the liquefaction of the fat used. This soap making process requires exact

measurements of alkali and fat amounts and computing their ratio, using saponification charts to

ensure that the finished product mild and skin friendly. (Donkor,1997).

3.7.1 Ingredient of soap base formulation & its role/uses

Various Ingredients used in the preparation of soap base listed below:

Table 1: ingredients used in soap formulation and their roles

S.No. Ingredient Role/ use

1. Sodium hydroxide Lye

2. Coconut oil Anti-ageing,soothe skin

3. Olive oil Moisturizer, emollient

4. Distilled water Aqueous vehicle

5. Vit C&E, anti-inflammatory, antioxidant, antibacterial antifungal

Turmeric extract
and anti-acne

6. Glycerine Moisturizing agent& solvent

3.7.2 Determination of Lye and the Amount of Water

The amount of lye in the recipe depends on the mass of the fat or oil (Childers, 2000)

Mass of lye= Mass of fat or oil x Sodium factor or

 (Amount of Fat) × (Saponification Value of the Fat) = (Amount of Lye)

 (Amount of Lye) ÷ 0.3 = (Total Weight of Lye Water Solution)

 (Total Weight of Lye Water Solution) – (Amount of Lye) = (Amount of Water)

Table 2: Olive and Coconut oil for soap preparation

Table 1: First trial

OIL AMOUNT SAP AMOUNT OF AMOUNT OF LYE AMOUNT OF

VALUE LYE USED WATER WATER

SOLUTION SOLUTION

Olive 20 ml 0.135 2.7ml 9 ml 6.3 ml

Coconut 50 ml 0.183 9.18 ml 30.5 ml 21.32ml

Table 2: Final experiment

Oil Amount Sap value Amount of lye Used Amount of Lye amount of

Water Solution water solution

Castor 18.4 g 0.135 2.5g 8.3 g 5.8g

Coconut 45.15 g 0.183 8.3 g 27.5g 19g

3.7.3 Preparation of soap bas

For preparing the soap base, first 20ml of the olive oil and 50ml of the coconut oil was mixed in

a 500 ml beaker. Put the mixed coconut and olive oil in the water bath and stir-boil it until a
strong consistency is forms at a temperature between 40 to 45 °C. Then take 10.8 g sodium

hydroxide dissolve in 26.3 ml distilled water in another beaker and mixed properly. After

preparing, this solution was added slowly in the mixture olive and coconut oil and it was

constantly stirred. The mixture was boil at 40–45 °C until base consistency is attained and then

this mixture was used as a soap base. This is shown on the table below.

Table4: Preparation of soap bas

S.No. Ingredients Quantity

Coconut oil 50ml

1.

2. Sodium hydroxide 13.20 g

3. Distilled water 24ml

3.7.4 Formulation of Herbal Soap

Take 50 ml of soap base in a beaker and put on water bath at 45 . Then add the all ingredient

(turmeric extract, tulsi extract glycerine and neem extract) with continuous stirring. Boil the

mixture on the water bath at 45 and soap mixture is prepared. Prepared soap mixture is filled in

moulds and the mould is put in the refrigerator for 15 min. After solidification cut the soap

mould using cutter or blade and then obtained herbal soap. As shown on the table below

S.No Material/Ingredients Quantity

1. Soap base 50g

 2. Turmeric extract 10ml

6. Glycerine 5ml

3.8 Preparation of turmeric soap extract for sensitivity test

The soap extract (5g) was transferred into a small bottle and 10ml of distelide water was added

to it, the bottle was closed and shaken vigorously to ensure mixing. Double serial dilution was

done with this to obtain 50mg/ml, 25mg/ml and 12.5mg/ml of the soap concentration

respectively. 0.1mL of each of the 2 days culture of the test organisms was aseptically

transferred onto the Petri dishes containing 20 mL of the Mannitol salt agar and Sabourad

dextrose agar. The inoculums were spread around the plate using a sterile cotton swab to give

uniform distribution of the test organisms in the agar and allowed to set. Wells were aseptically

bored into the plates containing each of the organisms using a cork borer of 5 mm in an

inoculating chamber. The bottom of the Wells was sealed with 50.0 mg, 25.0 mg and 12.5 mg of

different concentrations of the extracts and the plates were incubated at 37 for 18-24 hours.

The diameter of the zones of bacteria and fungi inhibitions around each Well was measured

using a meter rule. The experiment was performed in three replicates.

3.8.1 Determination of minimum inhibitory concentration (MIC)

Minimum inhibitory concentrations (MIC) were determined by agar dilution assay using

Nutrient Agar respectively. The concentration of the extract test ranged from 50.0 mg/mL, 25.0

mg/mL and 12.5.0 mg/mL. In this case, agar diffusion techniques were performed in agar plate

containing various concentrations of the extracts. The plate were inoculated with 100 µL of the

test organisms containing 105 cfu/mL Log phase bacteria and incubated at 37 for 18-24 hrs,
the lowest concentration of the extract that inhibited the growth of the bacteria was determined

as the MIC

3.8.2 Determination of minimum bactericidal concentration (MBC)

At the concentration in which the organism isolate shows positive test small quantity was picked

into a small culture media of S. aureus and T. rubrum aseptically using aflame ware loop and

then incubated on Mannitol and Sabourad dextrose agar at 370 c for 24 hours in the incubator

this was done to subculture the isolate. (The MBC was determined according to the National

Committee for clinical standard 1999).

3.9. Physicochemical analysis of Herbal Soap formulation

In order to verify the efficacy and quality of the final formulations, the following

physicochemical characteristics were tested such as colour, aroma, pH, clarity, dirt dispersion,

foam height, foam retention, skin irritation, and saponification value, etc.The herbal soap

formulation was tested using the standard approaches.

3.14.1 Determination of Color

The herbal soap formulation was visualize using a white background so that the colour could be

determined and so that the clarity of formulations could be seen.

3.14.2 Determination Odor/Aroma

The odor was evaluated by heating the sample on a hot plate and it was inhaled by five different

people, including both males and females.

3.14.3 Determination of the shape

organoleptic properties of the herbal soap formulation such as shape and clarity, was done by

sensory and visual examination.

3.14.4 PH determination of herbal soap

10g of the powdered soap were weighed and dissolved in 100ml of distilled water in the

volumetric flask in order to prepare 10% soap solution. The pH of the 10% soap solution was

determined with a pH meter. 2g of the prepared soap was also dissolved in 10ml of distilled

water and stirred till the sample dissolved. The pH was determined with a pH meter. three

different readings that were obtained from each of the samples after they were each examined

three times.

3.14.5 Determination of Dirt dispersion of the formulated soap

10g of the powdered soap were weighed and dissolved in 100ml of distilled water in the

volumetric flask in order to prepare 10% soap solution and 10ml of the soap solution was

transfer into measuring cylinder. Two drops of ink were added in to the sample solution. The

measuring cylinder was then shaken ten times while being covered by a hand. The ink is present

concentrate in the foam is considered to be of low quality, investigate that.The remaining dirt

particles are then found in the water section. The amount of ink found in the foam was noticed.

3.14.5 Determination of Wetting time

a piece of cotton fabric was cut it into a disc shape with a diameter of 2mm, and was weighed t

wet. 10g of the powdered soap were weighed and dissolved in 100ml of distilled water in a

beaker. The disc made of fabric was allowed to float freely on top of the 10% herbal soap

formulation solution. The amount of time that it took for the fabric disc to go from floating to

sinking was carefully recorded and referred to as the wetting time. A higher wetting efficiency is
associated with a shorter time to sink. The experiment was repeated three times and the average

was calculated.

3.14.6 Evaluation of the foam forming ability

The Cylinder Shake Method was used to determine the Foaming ability of the herbal soap

formulation. 50 ml of a 10% of the herbal soap formulation solution was put in a 100 ml

measuring cylinder and shaken vigorously 10 times. After shaking for 1 minute, the height of the

foam that had formed was measured and recorded the total volume of foam. This was done three

times to confirm the results.

3.14.7 Evaluation of the Foam stability

The Cylinder Shake Method was utilised to determine the Foaming ability. First, in a 100 ml

measuring cylinder, we put 50 ml of a 1% sample solution. The cylindrical container was

covered up with the use of the hand and shaken vigorously 10 times. The volume of the foam

after ten minutes was calculated.

3.14.8 Evaluating the Moisture content

10g of the material were heated in a hot air oven at 100 to 105 degrees Celsius for an hour. After

that deducted the true weight of the tarred china dish from the total weight of the sample and

dish. The weight of the material was recorded, and the method for calculating the percentage of

the moisture content that can be found in it is shown below formula.

Moisture content = (Difference in weight/initial weight) x100

3.16 Skin Irritation test

 PROJECT MANAGEMENT

TIMELINE

February April April May June June

Conception of

topic

Development

of proposal

Proposal

defence
Correction of

proposal

Data

collection

Data analyses

and

completion of

research work

Defence

BUDGET

Item Estimated Cost in South African

Rand

Laboratory fees 10, 000

Transportation (field study) 2000

Sample collection 3000

Ethical approval fee 5000

Petri dish 4800

Culture media 8000

4 Guinea pig 10,000

Purchasing of chemical 20,000

Services (photocopying, printing, and binding) 10,000

Miscellaneous 10,000

Total 82, 800 CFA