Comparative Analysis of Lemongrass Oil (Cymbopogon citratus),

Oregano Oil (Origanum vulgare), Lemon Juice (Citrus limon),

and Green Cross Alcohol as Hand Sanitizer
Against Staphylococcus
Aureus

A Research Proposal by the Faculty of NDSSM

In Partial Fullfillment of the requirements of Research 10

By:
Aratuc, Potre Aleissa
Casa, Jasper
Sinoy, Glendel Marie G.
Non, Mary Casandra R.

October 30 2022
 APPROVAL SHEET

The Research entitled “Comparative Analysis of Lemongrass Oil (Cymbopogon

citratus), Oregano Oil (Origanum vulgare), Lemon Juice (Citrus limon), and
Green Cross Alcohol as Hand Sanitizer Against Staphylococcus Aureus”
prepared and submitted by Mary Casandra Non, Potre Aleissa Aratuc, Glendel
Marie Sinoy, and Jasper Casa, in Partial Fulfillment of the requirements for the
Research 10 Subject, has been examined and is recommended for acceptance
and approval.

ABBEGAIL ANDRINO
Adviser

Panel of Examiners
Approved by the Committee on Oral Examination

Accepted and approved in partial fulfillment of the requirements for the Research
10 subject

Date
 Acknowledgment

This Research Study would not have been possible without the assistance and
encouragement of various people. The researchers would like to offer their
sincere appreciation for the learning opportunities and capabilities provided by
the following:

To Ms. Abbegail Andrino, the researcher’s adviser, for her advisory, ideas,
expertise, patience, time, drive, and her generously contributed tremendous
knowledge. Her support and teachings are much valued and appropriately
acknowledged. Without her providing management for our research study, this
study would not be possible.

To the members of the panel for contributing their valuable time, expertise,
and energy for reviewing this study. For their constructive feedback and support
in helping the study enhance, including their ideas that helped the researchers
broaden the scope of the research study.

To the researcher’s family and friends who have always been constructive
and who patiently sustained them financially, morally, and spiritually, those who
helped them finish the study and lifted their spirits. The researchers were
immensely driven and inspired to conduct this research study.

To God, thank you for the strength, blessings, lifting the researcher’s
spirits, wisdom, and love that he gave. For the guidance and unconditional
affection he consistently provided the researchers, both for the study and in their
day-to-day operations. This study would not be possible without him.
 TABLE OF CONTENTS

Page
TITLE PAGE……........................……………………………………………….........i
APPROVAL SHEET……………………………………………………………..........ii
ACKNOWLEDGEMENT………………………………………………………...........iii
ABSTRACT……………………………………………………………………............iv
TABLE OF CONTENTS…………………………………………………...................v
LIST OF FIGURES……………………………………………..........................…...vii
I.THE PROBLEM
Introduction...…………………………………………………....................................6
Statement of the Problem…………...................……………............................….. 7
Scope and Delimitation………………...……………………….................................8
Significance of the Study…………………………..……………...............................8
II. REVIEW OF RELATED LITERATURE AND STUDIES
Related Literature
Antibacterial Activity of Essential Oils………………………………........................9
Alcohol as Antibacterial Hand
Sanitizer…………………………............................10
Oregano oil as Antibacterial Hand
Sanitizer…………….......................................12
Lemongrass oil as Antibacterial Hand Sanitizer……..........
…………....................15
Lemon Juice as Antibacterial Hand Sanitizer……………………………...............16
Related Studies
Foreign…………………...……………………………………..................................17
Local...………………...………………………………………...................................20
Conceptual Framework…………………………………………..............................22
Hypothesis……………………………………………………...................................22
Definition of Terms……………...…………………………………...........................23
III. Methodology
Research Design……………………………………………….................................24
Locale of the
Study………………………………………………..............................26
Subjects of the
Study…………………………………………..................................26
Research Instrument…………………………….....…………….............................26
Data Gathering Procedures…………………………................…..........................26
Data Analysis……………………………………………………...............................28
Statistical Treatment............................................................................................29

List of Figures
Figure 1: Conceptual
Framework…………………………………….......................22
Figure 2: Research Design........
……………………………………….....................25
Figure 3: Statistical
Treatment..............................................................................29

Abstract

A comparative analysis of Lemongrass oil (Cymbopogon citratus), Oregano Oil

(Origanum vulgare), Lemon Juice (Citrus Limon), and Green Cross alcohol as
Hand Sanitizer against gram-positive Staphylococcus Aureus was conducted.
The hand sanitizers were made using 30 mL of distilled water, 0.5 g of carbopol,
2 drops triethanolamine, 0.2 grams propil paraben, 1 milliliter of glycerine
dissolved in 10 milliliters of 70% alcohol. The homogenized mixture was added
with the essential oils of Lemongrass and Oregano. The lemon juice was treated
using the same process. Antibacterial activity of Lemongrass oil (Cymbopogon
citratus), Oregano Oil (Origanum vulgare), Lemon Juice (Citrus Limon), and
Green Cross alcohol were determined using agar-well diffusion method. The
samples of each plant were tested to Gram-positive (Staphylococcus aureus).
Against the Gram-positive bacterium, all three of the plant extracts and green
cross alcohol exhibited antibacterial activity against the Gram-positive bacterium.

Chapter I

THE PROBLEM

Introduction
Hand hygiene is a term used to cover both hand washing using soap and water,
and cleaning hands with waterless or alcohol-based hand sanitizers (Minnesota
Dept. Of Health,2020). In order to prevent the spread of illnesses, hand hygiene
aims to sufficiently lower bacterial populations on the skin. To offer superior
cleaning services, there are various efficient antimicrobial cleaning agents
accessible. Despite some drawbacks linked to transient residual effects, alcohol-
based cleaning solutions are particularly effective against gram negative and
gram positive bacteria. However, some of these cleaning products could be
pricey and hard to get. Instead of costly commercial antibacterial remedies, a
number of plants with antimicrobial properties can be used.

According to ER Ekawati et.al (2019), Lemon fruit is an herbal plant that has the
main content of alkaloid compounds which have the function as anticancer,
antibacterial, antifungal, antiviral and antidiabetic. Furthermore, Alkaloid
compounds that are responsible for antibacterial substances are saponin. Lemon
(Citrus limon) juice contains many bioactive compounds such as flavonoids,
carotenoids, limonoid, tannin, and terpenoids. Moreover, Essential oils
fractionally distilled from plants have drawn increased attention because of its
multiple pharmacological properties like antibacterial, antifungal, and antiviral
activities (Wińska et al., 2019). To elaborate further, the lemongrass plant, which
thrives in tropical and subtropical regions of the world, is the source of
lemongrass essential oil. In addition to this, Lemongrass is used as a natural
remedy to heal wounds and help prevent infection. According to Naika et.al
(2010), lemongrass essential oil was effective against a variety of drug-resistant
bacteria. Along with this, Oregano oil is another product made from the oregano
plant, Origanum vulgare. It contains a higher concentration of the helpful
compounds that occur naturally in the plan (Johnson, 2019). The main
components of Oregano Oil are carvacrol and thymol, which have strong
biological activities, including anti-inflammatory, antibacterial and anti-oxidation
(Govaris et al., 2010). In contrast to herbal plants, Alcohol or hand sanitizers are
alternative options for removing bacteria. Green cross alcohol is made from
isopropyl and ethyl. Isopropanol is used in soaps and lotions as an antiseptic
(NCI Thesaurus, n.d). Isopropyl Alcohol is an isomer of propyl
alcohol with antibacterial properties. Although the precise mechanism of
Isopropanol's disinfectant action is not known, It may kill cells by denaturing cell
proteins and DNA, interfering with cellular metabolism, and dissolving cell lipo-
protein membranes. Whereas, Ethanol is a primary alcohol that is ethane in
which one of the hydrogens is substituted by a hydroxy group. It has a role as an
antiseptic drug, a polar solvent, a neurotoxin, a central nervous system
depressant, a teratogenic agent, a NMDA receptor antagonist, a protein kinase C
agonist, a disinfectant, a human metabolite, a Saccharomyces cerevisiae
metabolite, an Escherichia coli metabolite and a mouse metabolite (Chemical
Entities of Biological Interest, 2020). According to Green Cross Incorporated,
Green Cross Alcohol provides everyday, accessible disinfecting with its range of
isopropyl- and ethyl-based rubbing alcohols. 99.9% effective against bacteria and
viruses. It also has moisturizing variants to prevent skin dryness. Improving
hygiene habits and protecting the health of generations.

Staphylococcus Aureus is a gram-positive bacteria that causes a wide variety of

clinical diseases. This bacteria frequently causes infections, both in settings
where they are contracted in the community and in hospitals. Due to the rise of
multi-drug resistant forms like MRSA (Methicillin-Resistant Staphylococcus
aureus), treatment remains challenging. S. aureus does not normally cause
infection on healthy skin, however, if it is allowed to enter the internal tissues or
bloodstream, it may cause a variety of potentially serious infections (Taylor et
al.,2022). The study's objective is to evaluate and compare lemon (Citrus limon)
juice, oregano (Origanum vulgare) oil,lemongrass (Cymbopogon citratus) oil, ang
store bought Green Cross Alcohol as antibacterial hand sanitizer against
Staphylococcus aureus.
Statement of the Problem

This study aims to compare the antibacterial efficacy of lemon grass oil and
oregano oil and lemon juice as hand sanitizer. The researchers were guided by
the following questions:

1. Which among the natural substances affect the antimicrobial efficacy of

Lemongrass Oil (Cymbopogon citratus), Oregano Oil (Origanum vulgare), Lemon
Juice (Citrus limon) as a Hand Sanitizer against Staphylococcus Aureus?
2. Is there a significant difference in the antibacterial potency between the
natural extracts and the commercially bought hand sanitizer against
staphylococcus aureus?
3. How can antimicrobial activities of diluted plant extracts and alcohol be used to
assess how they are efficient against Staphylococcus aureus?
4. Which of the test solutions showed a zone of inhibition towards the presence
of Staphylococcus Aureus?

Scope and Delimitation

This study will only make use of Lemon (Citrus Limon) Juice and two essential
household oils, Lemongrass (Cymbopogon citratus), Oregano (Origanum
vulgare), and Green cross alcohol. Moreover, Staphylococcus aureus will also be
used as a bacteria in the study. Additionally, the Notre Dame Siena School Of
Marbel Science Laboratory will serve as the site for the experiment for the
researchers.

Significance of the Study

The goal of this study is to compare the potency of lemongrass oil, oregano oil,
lemon juice, and store bought Green cross alcohol as hand sanitizer against
Staphylococcus Aureus. The research study will be highly beneficial to the
following:
The health workers, this study will provide them a natural daily hand sanitizer that
is safe and inexpensive. Also, this will help health workers avoid illnesses that
they can acquire from hospitals and health centers.

The students, in this study, will provide them knowledge about the properties of
lemon juice, lemongrass oil, oregano oil, and Green cross alcohol. Also, it will
provide students with a safe and easy-to-use hand sanitizer that will protect them
from bacteria and viruses.

The personnel, this study will provide them with an inexpensive and reliable day-
to-day hand sanitizer. This will also protect them from harmful illnesses.

The future researchers, this study will help them even more if they decide to
pursue this as a research topic in the future. The data presented here will assist
future researchers in improving their research and also serve as a foundation for
their research.

Chapter II

REVIEW OF RELATED LITERATURE AND STUDIES

Related Literature

Antibacterial Activity of Essential Oils

According to Chouhan et al. in an article published in 2017, Essential oils have
been reported to have significant antiseptic, antibacterial, antiviral, antioxidant,
anti-parasitic, antifungal, and insecticidal activities. As a result, essential oils can
be used to treat infectious diseases. Plant extracts and essential oils have been
screened on a global scale as potential sources of novel antimicrobial
compounds, agents promoting food preservation, and alternatives to treat
infectious diseases. Essential oils, also known as volatile oils, are aromatic oily
liquids that are extracted from plant sources (leaves, buds, fruits, flowers, herbs,
twigs, bark, wood, roots and seeds). The most popular way to acquire essential
oils for commercial use is by a process known as steam or hydro-distillation,
which was initially created by Arabs in the medieval times.

Nikolc et al. (2013), evaluated the chemical composition and biological activity of
essential oils from five Lamiaceae species, including Mentha piperita, Lavandula
angustifolia, Mentha pulegium, Salvia lavandulifolia, and Satureja montana. It
was also revealed that the fully produced essential oil from dried oregano leaves
(Origanum vulgare) has antimicrobial properties. The biological characteristics of
essential oils are often dictated by their primary constituents, which include two
categories with separate biosynthetic origins. The two primary classes are
terpenes and terpenoids, whereas the other group is made up of low-molecular-
weight elements from the aromatic and aliphatic families.

Alcohol

Alcohol is among the most effective hand sanitizers due to their rapid
antimicrobial efficacy, convenience and good tolerance on the skin (Tushar et
al.,2021). According to study, Alcohol is crucial for inhibiting the transmission of
infectious bacteria, but given that it contains chemicals that are toxic and
combustible, accidents might arise. Improper alcohol usage can also lessen the
alcohol's ability to destroy germs.

Alcohol contains either isopropyl alcohol or ethyl alcohol, which are major
components that are prevalent in alcohol in varying amounts. Additionally,
alcohol aids in destroying the bacterial proteins' structural integrity. According to
research, ethanol possesses antibacterial properties that are effective against
bacteria like E. Staphylococcus aureus and E. coli. Alcohol-based hand sanitizer
is effective against bacteria and viruses including Escherichia coli, Enterococcus
faecalis, Acinetobacter baumannii, Candida albicans, methicillin-resistant
Staphylococcus aureus, Pseudomonas aeruginosa, SARS-CoV, Ebola virus,
Middle East respiratory syndrome coronavirus (MERS-CoV) and Zika virus
(Tushar et al.,2021).
Alcohol as Antibacterial Hand Sanitizer

Alcohol as an Antibacterial Hand Sanitizer, is the most prevalent hand sanitizer

(Ochwoto, M., Muita, L., Talaam, K. et al., 2017). According to Antimicrobial
Resistance and Infection Control reports, in order to improve the efficacy of
formulations, they also could incorporate additional active substances like
quaternary ammonium compounds, povidone-iodine, triclosan, or chlorhexidine.
Alcohols work by denaturing proteins, and their optimum concentration range is
between 60 and 80 percent. However, while proteins are difficult to denature in
the absence of water, concentrations of alcohol higher than 80% are less
effective. Alcohols exhibit effective in vitro germicidal action against a variety of
fungal strains as well as Gram-positive and Gram-negative vegetative bacteria

Alcohol as an antibacterial hand sanitizer can reliably and quickly reduce bacteria
throughout a wide germicidal spectrum. On the other hand, alcohol as an
antibacterial hand sanitizer makes use of chemicals with antiseptic properties to
produce antimicrobial effects. These chemicals have different modes of working
and function according to their chemical functional groups. Furthermore, it was
also mentioned in this study that the researchers will make use of the efficacy of
Alcohol as an Antibacterial Hand Sanitizer. In addition, the alcohol hand sanitizer
serves as an alternative when the juice or oil extracts are not readily available or
easily accessible.

Sanitizers containing an alcohol concentration between 60–85% are suggested

to kill 99.99% of microorganisms on hands. The sanitizers which are effective on
bacteria are effective in virus and vice versa (Gizachew, M., Desalegn, M,
Anduale,, H. et al., 2021). Hand sanitizer can play a key role in preventing the
transmission of pathogenic microorganisms but may also lead to harmful impacts
on the human body if not used appropriately. The ingredients used in hand
sanitizer preparations may cause serious health problems if not used carefully
(Tushar, S., Prakash, K., Shyamal D., 2021).

Green Cross Alcohol

Green Cross Alcohol, according to study, provides the breakthrough protection of

these advantages: kills germs on contact, forms a protective barrier surrounding
support to help resist against bacteria for up to five hours, inhibits the
transmission of bacteria through hands, and is non-drying. With its selection of
rubbing alcohols made from isopropyl and ethyl, it provides extensive, accessible
disinfection. Green Cross Alcohol furthermore comes in restorative variations to
reduce infections brought by bacteria. According to Green Cross, it has been
proven that alcohol improves domestic hygienic practices.

A variety of personal hygiene and sanitation products from Green Cross have
shielded generations of Filipinos against disease. Green Cross offers goods that
assist in daily health protection for millions of people. Green Cross has expanded
their product range beyond its original offering of rubbing alcohol to include hand
sanitizers, insect repellents, and bath soaps. With its Triple Acting System,
Green Cross Alcohol instantaneously eliminates 99.9% of disease-causing
microorganisms, offers hours of antibacterial protection, and nourishes skin with
its moisture-lock composition. (Green Cross, Incorporated, 2014)

Oregano oil as Antibacterial Hnad Sanitizer

Oregano has been a valuable source of natural products for human health
maintenance for a long time, particularly in the last decade, when natural
therapies have been the subject of more extensive research (Force et
al.,2000).Oregano's volatile oil has traditionally been used to treat respiratory
problems, indigestion, dental caries, rheumatoid arthritis, and problems with the
urinary tract.Oregano has been used as an antibacterial, antifungal, anticoccidial,
and antispazmolytic medicine (Ertas et al.,2005), a powerful antioxidant
(Lamaison et al.,1991), antagonist (Okazaki et al.,2002) as well as an anti-
inflammatory (Kelm et al.,2000)agent. Additionally, it is antiseptic and stimulates
digestion (Cabuk et al.,
2003).Oregano's most significant constituents are limonene, gamma-cariofilene,
rho-cymenene, canfor, and linalol.Carvacol, thymol, and alpha-pinene (Arcila-
Lozano et al.,2004).According to Tian & Lai (2006), the essential oil of oregano
primarily contains thymol and carvacrol, which are responsible for the oil's
antioxidative, antimicrobial, and antifungal properties (Proestos et al.,
2006).2005).

A number of studies on the antibacterial effects of spices, essential oils, and their
derivatives have been reported. The antibacterial properties of spices and their
essential oils have been known for a long time.Antimicrobial compounds could be
obtained from plant essential oils.The antimicrobial properties of plant
compounds against a wide variety of microorganisms, including foodborne
pathogens, have been the subject of numerous studies.Essential oils are derived
naturally from plant materials.Due to their antibacterial, antifungal, antioxidant,
and anti-carcinogenic properties, they are utilized as natural additives in
numerous foods.Due to the fact that herbal remedies have been used to treat a
variety of infectious diseases throughout human history, screening natural
medicinal plants is becoming increasingly popular.

Natural antimicrobial compounds are essential not only for food preservation but
also for the management of microbial-related human and plant diseases.Studies
on how essential oils and spice extracts and their constituents can stop the
growth of pathogen microorganisms have recently received a lot of attention.It
has also been known for some time that the derivatives of some crude drugs and
spices contain antifungal substances.The antimicrobial properties of essential
oils or their main components have been the subject of a few studies.Oregano
(Origanum vulgare) and clove (Eugenia caryophyllata) essential oils have been
found to be among the most effective compounds against E. coli strains in recent
research.

Staphylococcus Aureus

Staphylococci are common members of the microbiome of the human skin.

Opportunistic pathogens such as Staphylococcus aureus and Staphylococcus
epidermidis are responsible for significant disease in a number of species.The
various functions of the human skin are discussed, including its function as an
antimicrobial barrier and the staphylococcal mechanisms used to colonize and
combat the various stresses in this niche.A variety of adhesins, surface proteins,
and secreted enzymes are used to successfully combat the acid mantle's
antimicrobial peptides, enzymes, and lipid matrix components during
colonization.These bacteria are able to withstand desiccation, osmotic and acid
stresses, and other environmental conditions thanks to additional mechanisms.

The natural habitat of Staphylococcus Aureus in humans is the skin and

nasopharynx. It has the potential to trigger a wide range of infections that affect
the skin and soft tissues, endovascular locations, and internal organs.
Staphylococcus Aureus remains a significant pathogen, causing high mortality
and morbidity in both the community and hospitals.The organism can spread
through the bloodstream from a superficial location to internal organs, where it
can establish a metastatic infection focus.Surgical wounds and implanted
medical devices are the most common sources of infection for hospital patients.
In the latter scenario, the bacteria may either colonize the implanted device and
cause local damage or spread.Additionally, food contaminated with enterotoxins
can result in food poisoning.The economically important ruminant mastitis is also
caused by Staphylococcus Aureus.

According to Cartron, Hurd, et al. in an article published in 2014, Staphylococcus

aureus is a very adaptable bacterium that thrives in the human nose as a
commensal.This organism is an opportunistic pathogen that causes numerous
infections and deaths worldwide, despite the fact that it is carried unharmed by
30% of humans.The alarming spread of antibiotic resistance exacerbates the
issue;Methicillin-resistant S. aureus (MRSA) is a particular strain that is prevalent
in hospitals and is beginning to spread throughout the community.One important
aspect of S. aureus' ability to spread from host to host is its ability to live in the
nares and on the skin. Sebum's skin fatty acids, in particular, have been shown
to be effective staphylocidal agents. In fact, a Toll-like receptor-mediated
pathway in mice results in increased fatty acid production and protection against
S. aureus skin infections, both of which are important components of our innate
defenses.Cis-6-hexadecanoic acid (C6H) is the human skin fatty acid that is most
effective against staphylococci. C6H topical treatment reduces S. aureus levels
in atopic dermatitis patients, who also have a higher rate of S. aureus
colonization and decreased C6H levels.In addition, we have demonstrated that
both cutaneous and systemic S. aureus disease models can be treated with
purified C6H. S. aureus is also killed by fatty acids in abscesses. Human sebum
and C6H inhibit the production of virulence determinants and the induction of
antibiotic resistance by S. aureus and other important pathogens at sublethal
concentrations, in addition to being bactericidal. As a result, there are a number
of ways that fatty acids can weaken potentially harmful bacteria.

Staphylococcus Aureus can withstand skin fatty acids thanks to a number of

mechanisms. Turner, Rauter, Foster, et al., (2014), discovered that nasal
colonization necessitates the production of the major S. aureus surface protein,
IsdA, in response to the absence of iron in the human host. Through its C-
terminal domain, IsdA helps skin resist fatty acids by making the cells more
hydrophilic.S. aureus cannot survive on human skin unless this domain is
present.As a result, S. aureus' capacity to colonize a host is largely dependent on
how it interacts with fatty acids found in human skin.Skin fatty acids' bactericidal
mechanism against S. aureus is still unknown, despite its significance.These
substances probably cause membrane disruption because they are
surfactants.Many central metabolic processes are also inhibited by fatty acids,
but this may happen indirectly through the uncoupling of ATP synthesis.
Additionally, toxic lipid hydroperoxides may be produced by the accumulation
and incorporation of linoleic acid. Turner, Rauter, Foster, et al. wanted to
understand how the major staphylocidal skin fatty acid C6H affects S. aureus in
this study.Through C6H's ability to disrupt essential membrane functions, this
revealed multiple killing mechanisms.

Lemongrass oil as antibacterial and sanitizer

Cymbopogon citratus or also known as Lemon grass are used in traditional

medicine as remedies for the treatment of various diseases (MN Boukhatem,
2014). According to Dr. Gayathri Ramesh Et.al, Cymbopogon citratus (DC.)
stapf, commonly known as lemongrass and other Cymbopogon species is a tall,
coarse grass with a strong lemon taste. It is a perennial herb widely cultivated in
the tropics and sub-tropics, designates two different species, East Indian
Cymbopogon flexuosus (DC.) stapf and West Indian, Cymbopogon citratus (DC.)
stapf. The use of lemongrass was found in folk remedy for coughs, consumption,
elephantiasis, malaria, ophthalmia, pneumonia and vascular disorders.
Researchers have found that lemongrass holds antidepressant, antioxidant,
antiseptic, astringent, bactericidal, fungicidal, nervine and sedative properties.
Further, many workers had reported about the antibacterial activity of lemongrass
oil against a diverse range of organisms comprising gram positive and gram
negative organisms, yeast and fungi. The chemical composition of the oil
analysed by gas chromatography (GC)/mass spectrometry (MS) identified fifteen
components where neral (39.0%), geranial (33.3%), limonene (5.8%) and geranyl
acetate (4.2) were the most abundant constituents.

In tropical nations, particularly in Southeast Asia, the herb Cymbopogon citratus,

Stapf (Lemon grass), is commonly utilized. Aromatherapy employs the plant's
essential oil. Terpenes, alcohols, ketones, aldehydes, and esters make up the
majority of the substances found in Cymbopogon citratus. Essential oils that
contain Citral, Citral, Nerol Geraniol, Citronellal, Terpinolene, Geranyl acetate,
Myrcene, and Terpinol Methylheptenone are a few of the documented
phytoconstituents. The plant is also said to contain phenolic substances such
luteolin, isoorientin 2'-O-rhamnoside, quercetin, kaempferol, and apigenin as well
as flavonoids. Numerous pharmacological activity, including anti-amoebic,
antibacterial, antidiarrheal, antifilarial, antifungal, and anti-inflammatory
characteristics, have been linked to Cymbopogon citratus, according to studies.
Antimalarial, antimutagenic, antimycobacterial, antioxidant, hypoglycemic, and
neurobehavioral properties, among others, have also been researched (G Shah,
2011).

In a study by Naik et al.,(2010), lemongrass oil was found effective against many
test organisms except P. aeruginosa. Gram positive organisms (S. aureus, B.
cereus and B. subtilis) were found more susceptible than gram negative
organisms (E. coli, K. pneumoniae, P. aeruginosa). The antibacterial activity was
found progressively increasing with the increase in concentration of oil. The
maximum effect was found at 30% concentration and minimum effect was
observed at 5% concentration of oil. Results with methanol extract of lemon
grass plant prevented bacterial growth of
Staphylococcus aureus, Bacillus cereus and Escherichia coli which with
increasing concentration, their antibacterial effect also increased. This showed
that most plant extracts have inhibition effects on Gram positive bacteria and little
effect on Gram negative bacteria. This inhibition effect can be related to its active
compounds that include: steroids and terpenoids, alkaloids, citral, geraniol,
flavonoids, eugenol, cytronolal, geranyl acetate, beta cariofiln, tannins, phenolic
compounds, saponin and farnsul. In another study by Soares et al., C. citratus
essential oil exhibited high antifungal and antibacterial properties. Although
inhibition was higher in Gram positive bacteria and effect in Gram negative
bacteria was also shown. At the tested concentrations of the oils an extensive
Candida spp. inhibition was noted. This effect of inhibition was particularly
pronounced in Non Candida Albicans (NCA) species. The activity of Lemon
grass oil over Gram-positive multidrug-resistant bacterial strains and over C.
albicans and NCA species indicated a potential role in preventing spreading of
hospital infections, namely by its use as an antiseptic for topical and for hand
cleaning.

Lemon Juice

Rutaceae's lemon plant is a significant therapeutic herb. Lemons are grown for
their various parts, including their leaves, stems, roots, and flowers, primarily for
their alkaloids, which have anticancer properties and the ability to fight clinically
significant bacterial strains when extracted in crude form. Because citrus
flavonoids are present, they have biological properties such as antibacterial,
antifungal, antidiabetic, anticancer, and antiviral properties. They work as direct
antioxidants and free radical scavengers, and they can control enzymatic
processes and stop cell division. When pathogens, such as bacteria, invade
plants, they appear to play a protective role.
both viruses and fungi.
A study by Maruti et al.,(2011), reveals that lemon peel is an effective
antibacterial agent in addition to being an astringent. Since certain skin flora,
such as Pseudomonas and Micrococcus, can grow in the presence of sebum,
especially when it is secreted in excess (in some people), and cause purulent
skin infections as well as serve as a predisposing factor for other types of skin
infections like acne, the authors claim that the simple use of lemon juice can
prevent such types of infections and could help in maintaining a healthy and clear
complexion wholesome skin. By using several types of lemon extracts, Nada et
al.,2013 evaluated pure cultures of 6 types of gram positive bacteria, 8 types of
gram negative bacteria, and 1 yeast isolates by different types of citrus peel juice
and delicious lemon extracts. More than other forms of extracts, lemon juice has
antibacterial properties. Lemon juice exhibited suppression when used against
Gram-positive bacteria like S. Enterococcus fecalis, S. Agalactiae, and Aureus.
S. S. Pyogenes and pneumonitis. Lemon juice prevents the growth of gram-
negative bacteria, specifically E. Enterobacter aerogenes,K. Pneumoniae, M.
mutans, S. typhi, and Proteus species. Catarrhalis. We were surprised to find
that the extract of lemon juice was efficient against Candida albicans, as there
was no action against Candida in the current trial. This focuses on the need for
more research on the antifungal effectiveness of lemon juice. Both gram positive
and gram negative bacteria were found to have decreased, they discovered.
They came to the conclusion that lemon extracts had an important role as an
antimicrobial effect.

Foreign Studies

According to Mohd, I., Bashir, A., Javid, A (2010) Lemongrass oil was
investigated for activity against Staphylococcus aureus, using Agar Diffusion
Method and Broth Dilution Method. Minimum inhibitory concentration (MIC) and
minimum bactericidal concentration (MBC) were determined by the Broth Dilution
Method. Lemongrass was found effective against all the test organisms except P.
aeruginosa. Gram positive organisms were found more sensitive to lemon grass
oil as compared to gram negative organisms. The test organisms were found
inhibited by Lemon grass oil at lower concentrations in the Broth Dilution Method
as compared to the Agar Diffusion Method.
Cymbopogon Citratus against Staphylococcus, commonly known as lemongrass
and other Cymbopogon species is a tall, coarse grass with a strong lemon taste.
Lemongrass is a perennial herb widely cultivated in the tropics and sub-tropics,
designating two different species, East Indian Cymbopogon. Cymbopogon
citratus has been cultivated over many years for medicinal purposes in different
countries throughout the world. The use of lemongrass was found in folk remedy
for coughs, consumption, elephantiasis, malaria, ophthalmia, pneumonia and
vascular disorders.

Researchers have found that lemongrass holds antidepressant, antioxidant,

antiseptic, astringent, bactericidal, fungicidal, nervine and sedative properties.
Further, many workers had reported about the antibacterial activity of lemongrass
oil against a diverse range of organisms comprising gram positive and gram
negative organisms, yeast and fungi. The lemongrass oil was found to be
effective against Staphylococcus aureus (S. aureus). The development of
bacterial resistance to presently available antibiotics has necessitated the search
for new antibacterial agents. Hence the present study was carried out to find out
the antibacterial activity of lemongrass oil against the selected pathogenic
bacteria (Mohd, I., Bashir, A., et al., 2010).

According to a study by Cui et al. (2019), Oregano essential oil is an effective

natural antibacterial agent and has been found to be effective in inhibiting MRSA.
In their experiment, the effects of oregano essential oil on respiratory
metabolism, energy metabolism and genetic material of MRSA were studied with
the metabolism as the starting point. Furthermore, the main component of
oregano essential oil, carvacrol, can be chimeric with DNA, and oregano
essential oil inhibits the relative expression of the pvl gene. The results of the
study are that oregano essential oil affects the permeability of the cell membrane
and causes irreversible damage to the cell membrane. This result is consistent
with the results of Cui et al. (2018). Secondly, the study found that oregano
essential oil can inhibit the respiratory metabolism of MRSA by affecting the
metabolites and key enzymes of the TCA cycle.
According to a study by Hashemi et al. (2017) about the antibacterial effect of
fermented and unfermented juice of Lemon, Fermented juice showed an
acceptable antibacterial activity against E. coli O157: H7 and S. Typhimurium.
Results also showed that the fermented juice had higher antibacterial properties
against S. Typhimurium in comparison to E. coli O157: H7 (P < 0.05). These
findings are in agreement with results of Pereira et al. (2011), who claimed that L.
casei could control the growth of other bacteria as well as juice spoilage during
fermentation of cashew apple juice. The interaction of lactic acid on the external
membrane of Gram-negative pathogenic bacteria could increase
permeabilization and consequently allow other antibacterial compounds to enter
into the bacteria (Alakomi et al., 2000). However, the analytical methods for
detecting pathogens inside probiotics or products with probiotics are not fully
validated and accurate internationally. Contrary to the herbal plants, In clinical
practice, poisoning with ethylene glycol, methanol, and isopropyl alcohol is
common. These alcohol-related intoxications can present with high anion gap
metabolic acidosis and increased osmolality. Toxicity and clinical symptoms are
due to the accumulation of their metabolites, causing increased anion gap, rather
than the parent compounds that are associated with an increase of serum
osmolality. Clinical manifestations result from abnormalities of neurologic,
cardiopulmonary, and renal function. Laboratory abnormalities when present are
helpful for diagnosis but may be absent depending on the time of ingestion and
time of presentation. Fomepizole and ethanol are potent inhibitors of alcohol
dehydrogenase and reduce generation of toxic metabolites. Hemodialysis is an
effective way of detoxification because it can remove unmetabolized alcohol in
addition to the organic anions. High index of suspicion and early diagnosis can
prevent the significant morbidity and mortality associated with these intoxications
(Jammalamadaka et al.,2010).

In a study by Ramesh et al.(2016) about Evaluation of Antibacterial Efficacy of

Lemongrass oil, eucalyptus oil, and lemon juice, The results are, Lemon juice
and lemon grass oil had almost same effects on the microorganisms as
compared to eucalyptus oil. But the oils were ineffective against spores and
Candida. Lemon juice can be used as an efficient hand sanitizer but since the
sample size was very small, further research and development of these oils in
consumer products is warranted.

The plant leaves extracts were used for antibacterial activity on Staphylococcus
aureus microorganisms by the Kirby Bauer agar disc diffusion method. This study
was carried out on lemongrass plant leaf extracts in different concentrations of all
solvents. The leaf extracts from different solvents were tested for their
scavenging activity against the stable free radical DPPH in quantization using a
spectrophotometric assay Balachandar Balakrishnan, et al.)

Antibacterial activities of the essential oil of oregano (Origanum vulgare L.)

against Staphylococcus aureus were determined. The minimum inhibitory
concentration (MIC) for oregano oil against various bacteria varied: while
M.luteus (16 µg/ml) and B.cereus (32 µg/ml) were susceptible to oregano oil, the
others exhibited partly resistance. Except MRSA S. aureus (250 µg/ml), ampicillin
had more effect on K. pneumoniae (128 µg/ml) and C. albicans (128 µg/ml) than
oregano oil, but very low concentrations of the essential oil were sufficient to
prevent microbial growth. Of the bacteria tested, Micrococcus luteus and Bacillus
cereus proved to be most susceptible to oregano oil. Oregano oil possessed
strong antimicrobial activity compared with the antibiotic. Gram-positive bacteria
were more sensitive to the antimicrobial agent in spice than Gram-negative ones.
(Journal of food, agriculture & environment vol. 8, 2010).

In accordance with the study, the antibacterial activities of spices and their
essential oils have been known for a long time, and a number of researches on
the antibacterial effect of spices, essential oils and their derivatives have been
reported. Plant essential oils are the potentially useful source of antimicrobial
compounds. Numerous studies have been published on the antimicrobial
activities of plant compounds against many different types of microorganisms,
including foodborne pathogens. Essential oils are natural products extracted from
vegetal materials. They are used as natural additives in many foods due to their
antibacterial, antifungal, antioxidant and anti-carcinogenic properties. Screening
of natural medicinal plants is now being popular because many infectious
diseases are known to have been treated with herbal remedies throughout
history.

Local Studies

Morilla et al. (2014), stated that In the Philippines, the plants that are mostly
used among indigenous people were recorded to be food plants and plants used
for various other purposes mainly construction and crafts. Since most indigenous
people used to live considerably isolated from the mainstream society, they
heavily relied on natural products for food and construction. The plants used as
medicine contain a wide range of substances that can be used to treat chronic as
well as infectious diseases. They are rich in secondary metabolites and essential
oils of therapeutic importance. The role/importance of medicinal plants/traditional
medicine cannot be underestimated in the Philippine setting because many
medicinal plants are being utilized ever since as topical/external ointments,
liniments, poultices that is for wounds, skin diseases, muscle pains, massage
and aromatherapies (Penecilla et al.,2011).

Lemongrass (Cymbopogon citratus) is a plant that usually grows in tropical or

subtropical places like Asia, South America, and Africa. In the Philippines, it is
commonly known as tanglad. It has long, thin leaves that's used as a scent,
flavoring for food, and as a medicinal plant in many cultures (Medenilla,2020). In
a study of Onawunmi et al. (2008) , Lemongrass oil obtained by the steam
distillation of the fresh aerial parts of Cymbopogon citratus (DC.) Stapf was
investigated for antibacterial activity. The results obtained suggest that the oil
possesses rapid bactericidal activity against Gram-negative and Gram-positive
bacteria. According to Herbanext Laboratories, Origanum amboinicus (Philippine
Oregano) contains rosmarinic acid, ursolic acid, thymol, and carvacrol that have
anti-inflammatory, antibacterial, antioxidant, antifungal and antiviral properties.
Oregano also consists of flavonoids, triterpenoids, vitamins, and minerals. Based
on an article by Philippine Herbal Medicine, In other countries, the plant is
primarily used as a culinary ingredient. However, in countries like the Philippines,
Oregano is a known herbal medicine for its strong anti-oxidant properties.
Oregano contains a rosmarinic acid compound, thymol, and carvacrol that are
responsible for its anti-inflammatory, anti-bacterial, anti-oxidant, antifungal and
antiviral properties. Flavonoids, triterpenoids, sterols, vitamin C, and vitamin A
are also present in oregano. Recent research have shown that it has antibacterial
qualities that can be used to treat infections of the reproductive tracts, making it
the perfect medication to give to new mothers. In the Philippines, oregano leaves
are traditionally boiled in the home as a treatment for burns, rheumatism,
myalgia, and otalgia in addition to treating throat infections, burns, colic,
dyspepsia, and nasal congestion.

In addition to the traditional home remedy of boiling oregano leaves to treat

burns, headaches, colic, dyspepsia, and throat infections, oregano is also used
to treat rheumatism, myalgia, and otalgia in the Philippines. In 2009, the Bureau
of Agricultural Research of the Department of Agriculture funded a project to
make soap and tea from oregano. A professionally manufactured syrup has
already been on the market recently, while oregano wine, oregano juice, and
oregano vinegar production are all in the works. These assertions of tradition
appear to be supported by several earlier investigations that have shown this
plant to have a variety of medicinal characteristics, including antibacterial,
antiepileptic, and antioxidant capabilities. Oregano's antibacterial properties have
been proven effective, among others, against Escherichia coli and
Staphylococcus aureus. In 2009, the Bureau of Agricultural Research of the
Department of Agriculture funded a project to make tea from oregano as
well as soap. Production of oregano wine, oregano juice, and oregano
vinegar are also in the works. Recently, a commercially prepared syrup
has already been available in the market. Several previous studies appear
to support these claims of tradition by demonstrating various therapeutic
properties of this plant, such as antimicrobial, antiepileptic, and antioxidant.
The antimicrobial effect of oregano has been demonstrated against
Staphylococcus aureus and Escherichia coli, among others (Manlubatan et
al.,2022).

Conceptual Framework
The Conceptual Framework of the study is shown below.

Independent Variable Dependent Variable

Figure 1. Conceptual Framework

Hypothesis

H1: There is no significant difference between the test solutions of natural

substances that affect the antimicrobial efficacy of Lemongrass Oil (Cymbopogon
citratus), Oregano Oil (Origanum vulgare), Lemon Juice (Citrus limon) as a Hand
Sanitizer against Staphylococcus Aureus.
H2: There is no significant difference in the antibacterial potency between the
natural extracts and the commercially bought hand sanitizer against
Staphylococcus Aureus.
H3: There is no significant difference in the test solutions of antimicrobial
activities of diluted plant extracts and alcohol that assesses their efficiency
against Staphylococcus Aureus.
H4: There is no significant difference between the test solutions that showed a
zone of inhibition towards the presence of Staphylococcus Aureus.

Definition of terms:
The researcher defined these terms conceptually and operationally.

Alcohol- Conceptually, alcohol is an organic compound containing one or more

hydroxyl groups bound to a saturated carbon atom (Biology Online, 2022).
Operationally, Alcohol can reduce the growth of bacteria as well as viruses, and
fungi. It can also serve as a disinfectant.
Hand Sanitizer- Conceptually, Hand sanitizer is an agent applied to the hands
for the purpose of removing common pathogens (Rogers,2015). Operationally,
hand sanitizer can be used as a disinfectant against microorganisms.
Juice- Conceptually, Juice is the extractable fluid contents of cells or tissues
(Merriam-Webster, 2022). Operationally, the juice of the lemon can be used as
an antibacterial component against staphylococcus aureus.
Lemon (Citrus limon)- Conceptually, Lemon is an acid fruit that is botanically a
many-seeded pale yellow oblong berry produced by a small thorny citrus tree
and that has a rind from which an aromatic oil is extracted (Merriam-Webster,
2022). Operationally, Lemon will be used as an antibacterial component against
staphylococcus aureus.
Lemongrass (Cymbopogon citratus)- Conceptually, a lemongrass
(Cymbopogon citratus) is a robust habit native to southern India and Ceylon that
is grown in tropical regions for its lemon-scented foliage used as a seasoning
and that is the source of an aromatic essential oil (Merriam-Webster, 2022).
Operationally, Lemongrass and its components will be used as an alternative
hand sanitizer against staphylococcus aureus.
Oil- Conceptually, Oil is defined as any of numerous unctuous combustible
substances that are liquid or can be liquefied easily on warming, are soluble in
ether but not in water, and leave a greasy stain on paper or cloth (Merriam-
Webster,2022). Operationally, As an alternative to hand sanitizer against
staphylococcus aureus, two essential oils will be employed.
Oregano (Origanum vulgare)- Conceptually, Oregano is a Mediterranean
variety of wild marjoram ( Origanum vulgare), with pungent leaves (Collins
Dictionary, 2016). Operationally, Oregano will be used as a component to
evaluate the antibacterial effectiveness of Oregano Essential Oil as hand
sanitizer against staphylococcus aureus.
Staphylococcus Aureus- Conceptually, Staphylococcus aureus is a gram-
positive bacteria that causes a wide variety of clinical diseases. Infections caused
by this pathogen are common both in community-acquired and hospital-acquired
settings (Taylor et al., 2022). Operationally, Staphylococcus aureus will be used
as a bacterium for comparing the efficacy of lemon juice, two essential oils, and
green cross alcohol as hand sanitizer.
Zone of Inhibition- Conceptually, This is an area of media where bacteria are
unable to grow, due to presence of a drug that impedes their growth. (Biology
LibreTexts, 2021).
Operationally, Zone of Inhibition will help assess the potency of the extracts as
hand sanitizer and green cross alcohol against staphylococcus aureus.

Chapter III

METHODOLOGY
 This Chapter presents the method of research used in conducting this
study. It includes research design, subjects of the study, research instrument
gathering procedure, and statistical used in the data analysis.

Research Design

In this study, the comparative design will be used. A comparative study is

a kind of method that analyzes phenomena and then puts them together to
find the points of differentiation and similarity (MokhtarianPour, 2016).
Moreover, Comparative research method can be defined as a research
methodology in which aspects of social science or life are examined across
different cultures. It is a form of qualitative approach or analysis in which
different types of methods like case study analysis are used by the
researchers to elucidate the similarities and differences between the entities
or countries. Comparative research method combines theory or theoretical
concepts with data collection (Given, 2008). According to Richardson (2018),
In comparative research, data are collected primarily by observation, and the
goal is to determine similarities and differences that are related to the
particular situation or environment of the two groups.

The researchers will utilize the research design to compare the

effectiveness of Lemongrass Oil (Cymbopogon citratus), Oregano Oil
(Origanum vulgare), Lemon Juice (Citrus limon), and Green Cross Alcohol
against Staphylococcus Aureus.

Hand sanitizer Formulation at 80°C, 30 mL of distilled water will be added

to approximately 0.5 g of carbopol, and the mixture will be agitated at 200 rpm
for five minutes. The researchers will then make a mixture of 2 drops
triethanolamine, 0.2 grams propil paraben, and 1 milliliter of glycerine
dissolved in 10 milliliters of 70% alcohol. The mixture of alcohol and carbopol
will be homogenized. The homogenized mixture will be added with the
 essential oils of Lemongrass and Oregano. The lemon juice will be treated
using the same process.

Figure 2. Research Design

Locale of the Study

The experiment will take place at the Notre Dame-Siena School of Marbel,
located at Alunan Ave 9506 in Koronadal City, South Cotabato. The
experimentation will be conducted in the school laboratory.

Subjects of the Study

The bacterium that will be used in the study is Staphylococcus aureus. Purposive
sampling will be used by the researchers in this study. Purposive sampling, also
known as judgmental, selective, or subjective sampling, is a form of non-
probability sampling in which researchers rely on their own judgment when
choosing members of the population to participate in their surveys (Jordan,2021).
In this study, the researchers chose Staphylococcus Aureus as bacterium in
determining the antibacterial efficacy of Lemongrass Oil, Oregano Oil, Lemon
juice, and Green cross alcohol as hand sanitizer since S. aureus is found in the
environment and is also found in normal human flora, located on the skin and
mucous membranes of most healthy individuals (Taylor et al.,2022).

Research Instrument

In this study, the researchers will be using different tools and apparatus such as
petri dish, nutrient agar (MERCK), nutrient broth powder, sterile cork borer,
micropipette, incubator, test tubes, vernier caliper, erlenmeyer flask, filter paper
and the bacteria, Staphylococcus Aureus in conducting the experiment. The
researchers will also be using a checklist to record all the data during the
experiment. More so, the researchers will also be using devices such as video
recorders and cameras in order to accurately record the process.

Data Gathering Procedure

The researchers will conduct their study using the following approach:

Hand Sanitizer Formulation:

At 80°C, 30 mL of distilled water will be added to approximately 0.5 g of carbopol,
and the mixture will be agitated at 200 rpm for five minutes. The researchers will
then make a mixture of 2 drops triethanolamine, 0.2 grams propil paraben, and 1
milliliter of glycerine dissolved in 10 milliliters of 70% alcohol. The mixture of
alcohol and carbopol will be homogenized. The homogenized mixture will be
added with the essential oils of Lemongrass and Oregano. The lemon juice will
be treated using the same process.
Testing of Antibacterial Efficacy:
The antibacterial activity of Lemongrass oil, Oregano Oil, lemon juice, and Green
cross alcohol against the Staphylococcus Aureus bacterium culture are
determined using the agar well diffusion method.
Preparation of sample dilutions:
In 1 ml of DMSO, extracts (15 mg) will be fully dissolved. Additionally, standard
antibiotic solutions (2 mg/ml of ampicillin) will be obtained. The positive and
negative controls will be pure DMSO solutions and normal antibiotic solutions.
Preparation of media (nutrient broth and nutrient agar):
The researchers will evaluate the alcohol and plant extracts for antibacterial
activity using Gram-positive Staphylococcus aureus bacteria. The nutritional
broth medium will be made with 50 ml of filtered water and 0.65 g of nourishing
broth for the bacterial inocula growth. The remedy will be autoclaved after the pH
is increased to 7.0. Furthermore, 100 ml of water and 2.0 g of nutritional agar will
be combined to generate the nutritional agar medium. The pH will be raised to
7.0, and distilled water will be used to create the solution. The solution will go into
an autoclave.
Preparation of Inocula:
The inoculum will be made by centrifuging plate of Staphylococcus Aureus from
a 24-hour-old culture in nutrient broth (SIGMA) then combining the resulting
mixture with physiological normal saline solution to reach the McFarland turbidity
standard [106 colony forming unit (CFU) ml-1].
Preparation of seeded agar plates:
The seeding of nutrient agar will then be done using this inoculum. To make
nutritional agar, the researchers will mix 2.0 g of nutrient agar (MERCK) with 100
ml of distilled water, then set the pH to 7.0 and autoclave the agar.
Pouring of test solutions ; incubation and measurement of zone of
inhibitions:
75 ml of nutritional agar will be poured into petri dishes and left to harden. A
sterile cork borer will be used to create wells on each plate under aseptic
circumstances, and 100 ml of test solution will then be pipetted into each well
using a micropipette. At 37 °C, these plates will be incubated. The diameter of
the clear zones of inhibition will be measured using a ruler following a 24-hour
incubation period. Antibacterial activity of dilutions of each plant extract and
alcohol will be determined against Staphylococcus Aureus.

Data Analysis

The Three-way Analysis of Variance (ANOVA) will be used to compare the

significant similarities and differences between Lemongrass Oil (Cymbopogon
citratus), Oregano Oil (Origanum vulgare), Lemon Juice (Citrus limon), and
Green Cross Alcohol as Hand Sanitizer Against Staphylococcus Aureus.

Three-way Analysis of Variance (ANOVA) determines what effect, if any, three

factors had on an outcome. Three-way ANOVAs are useful for gaining an
understanding of complex interactions where more than one variable may
influence the result. A three-way ANOVA is also known as three-factor ANOVA.
The researchers will be able to determine whether the variability of the outcomes
is due to chance or to the factors in the analysis (Will Kenton, 2021).

Statistical Treatment

The One way Analysis of Variance (ANOVA) will be used to compare the
significant similarities and differences between Lemongrass Oil (Cymbopogon
citratus), Oregano Oil (Origanum vulgare), Lemon Juice (Citrus limon), and
Green Cross Alcohol as Hand Sanitizer Against Staphylococcus Aureus.

The one-way analysis of variance (ANOVA) is used to determine whether there

are any statistically significant differences between the means of three or more
independent (unrelated) groups. (Laerd Statistics, n.d.)

The researchers will use this formula of one-way Analysis of Variance (ANOVA):
.

Figure 3: Statistical Treatment

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