1

HERBAL-BASED HAND SANITIZER USING EXTRACTS OF THE NEEM

(Azadirachta Indica), HOLY BASIL (Ocimum Sanctum), AND LEMON (Citrus
Limon)

An Investigatory Project
Presented to the Faculty of the Senior High School
CORAZON C. AQUINO HIGH SCHOOL
Gerona, Tarlac

ALEJANDRINO, Kristine R.
BAGASINA, Althea Louise P.
CATAYOC, Dianne Ashly S.
PUERTO, Geoffrey T.
SALMASAN, Camille Ann R.
 March 2022
Chapter 1

INTRODUCTION

Background of the Study

Hands are a primary culprit for the spread of microorganisms (Edmonds-Wilson

et al., 2015). The hands are the parts of the body that have the most contact with other

people and things. Consider how often people unintentionally touch their faces during the

day. When we fail to exercise good hand hygiene, it allows dangerous illnesses to spread

to others. Therefore, hand hygiene is the single most essential, easy, and low-cost method

of avoiding infectious diseases (Varsha et al., 2021).

Several infectious diseases can be spread from one person to another by

contaminated hands. These diseases are caused by pathogenic organisms such as bacteria.

The World Health Organization (2021) reports that pneumonia is the leading infectious

cause of death and that it can be caused by bacteria such as Streptococcus,

Pneumococcus, and Pseudomonas. Furthermore, diarrhea, defined as loose, watery stools

(bowel movements), is also the leading infectious cause of death. According to Shen et

al. (2016), the most common cause of diarrhea was a bacterial infection. With this, hand

hygiene with hand sanitizer or soap washing has been shown to aid in the prevention of

pathogenic bacteria (Rehman et al., 2021). Currently, people are using alcohol-based

hand sanitizers on a large scale to prevent or slow the spread of it.

The majority of hand rubs available for use as sanitizers are alcohol-based. It has

become a widespread preventative measure in healthcare and community settings, which

has increased alcohol demand (Golin et al., 2020). Because the alcohol requirements of

the COVID-19 pandemic make large-scale production incredibly expensive, the WHO
provided two formulas for low-volume alcohol-based hand sanitizer production. The first

formulation comprises 80% (v/v percent) ethanol, 0.12% (v/v percent) hydrogen

peroxide, and 1.45% (v/v percent) glycerol, whereas the second formulation contains

75% (v/v percent) isopropyl alcohol, 0.12% (v/v percent) hydrogen peroxide, and 1.45%

(v/v percent) glycerol (Mahmood et al., 2020). The bulk of hand sanitizers on the market

is made up of various mixtures of isopropyl alcohols, H 202, and ethanol. Toxicity in

individuals and the environment may come from the misuse of these regulations

(Mahmood et al., 2020). The American Association of Poison Control Centers (AAPCC)

recorded 11,324 incidents of alcoholic hand sanitizer exposure in children under the age

of 12 during the first six months of 2020. The aforementioned exposure, according to the

AAPCC, caused nausea and lethargy, disorientation, respiratory arrests, and even death in

infants (AAPCC, 2020). The use of hand sanitizers on a regular basis has also been

linked to an increased risk of several bacterial and viral infections, as well as antibiotic

resistance (Tachikawa, 2020). To mitigate the danger in the alcohol supply chain, an

alcohol-free hand sanitizer with comparable antibacterial effect was required.

Alternative hand sanitizer preparations based on natural and plant resources might

be a feasible answer to the toxicity problem. As an alternative to alcohol-based hand

sanitizers, medicinal herbs with antibacterial and disinfection qualities have been

reported to be effective. According to Alghamdi (2021), herbal-based hand sanitizers are

easy to manufacture and outperform alcoholic hand sanitizers. These herbal mixtures

have been proven to be safe for both human and environmental health (Kalaivani et al.,

2018).
 Furthermore, prior to the development of modern medicine, plants were the

primary treatment for a variety of diseases. With the introduction of new antibiotics,

microorganisms eventually acquire resistance to these medicines. These stimulate the

interest of researchers in plants with antibacterial capabilities. They attempt to capitalize

on the unique capacity of certain naturally occurring substances to exhibit sustained and

extended action against a wide range of pathogenic bacteria (Vaou, 2021).

In this study, the researchers use neem and holy basil leaves, as well as lemon

peel, due to their distinct properties. Neem leaf and its components have been shown to

have immunomodulatory, anti-inflammatory, antihyperglycemic, antiulcer, antimalarial,

antifungal, antibacterial, antiviral, antioxidant, antimutagenic, and anticarcinogenic

properties (Subapriya and Nagini, 2005). Holy basil is a fragrant herb native to the Indian

subcontinent that belongs to the Lamiaceae family. Holy basil leaf has potent

pharmacological actions that include adaptogenic, metabolic, immunomodulatory,

anticancer, anti-inflammatory, antioxidant, hepatoprotective, radioprotective,

antibacterial, antiviral, and antidiabetic effects that have been extensively studied

(Jamshidi and Cohen, 2017). Lemon is a valuable medicinal plant in the Rutaceae family.

Its peel is an excellent source of biologically active compounds known as flavanones,

which have a wide range of biological activity, including antibacterial, antifungal,

antidiabetic, anticancer, and antiviral properties (Dhanavade et al., 2011).

Natural extracts of neem and holy basil leaves, as well as lemon peel, are known

to have antibacterial properties. The goal of this study was to replace chemical products

with eco-friendly products derived from neem, holy basil, and lemon extract against

pathogenic bacteria.
Statement of the Problem

The purpose of this study is to determine the efficacy of neem (Azadirachta

Indica), holy basil (Ocimum sanctum), and lemon (Citrus limon) as herbal-based hand

sanitizer against pathogenic bacteria.

Specifically, it seeks to answer the following questions:

1. Which among the optimum concentration of neem, holy basil, and lemon

extract in the herbal-based hand sanitizer formulation would be the most effective against

pathogenic bacteria?

2. How can the herbal-based hand sanitizer derived from the neem, holy

basil, and lemon extract be described in terms of:

2.1. Texture

2.2. Odor

2.3. Color

2.4. pH

2.5. Viscosity

2.6. Gel Spreadability

3. Based on the findings, what is the level of effectiveness of the herbal-

based hand sanitizer from the neem, holy basil, and lemon extract against pathogenic

bacteria?

Statement of Hypotheses
 1. The treated neem, holy basil, and lemon extract show no potential as an

alternative hand sanitizer.

2. Herbal-based hand sanitizer made of neem, holy basil, and lemon extract

is effective against pathogenic bacteria.

Significance of the Study

Hand hygiene is important since hands may quickly get contaminated. It is

critical, especially in current situations such as pandemic outbreaks, to interrupt the

virus's transmission chain by practicing proper hand sanitization. Herbal-based hand

sanitizer was just as effective against pathogenic bacteria and viruses as conventional

hand sanitizers, but with less toxicity and no negative effects on human tissue.

Furthermore, these herbal plants are easily accessible in gardens and markets. Thus, a

new simple and inexpensive method for providing safe and healthier living through germ-

free hands are discovered.

So, this study will benefit the following:

ENVIRONMENT – The researchers conceived an eco-friendly material for the sake of

the environment. This study cannot contain any chemical that leads the environment to

destroy. The researchers used herbal medicine that can grow well fast in a tropical region

and grow fast and tolerate drought-prone soil Using organic materials will be a

contributor to an improved and safe environment.

COVID-19 PREVENTION - In the global health emergency caused by COVID-19,

multiple experts have mandated the use of hand sanitizers as a safety measure from

COVID-19. The best way to kill germs is by washing hands with soap and water.
However, if there is no access to soap and water, the use of hand sanitizer is

recommended significantly. Hand sanitizers can help to stop the spread of both viruses

and bacteria. Moreover, this research, if proven effective, can help in the prevention of

coronavirus infection.

PEOPLE – The people are the sole beneficiaries of this research. Herbal hand sanitizers

have been shown to be effective against infections and bacteria. The findings were

compared to alcoholic-based hand sanitizer formulations and found to be beneficial to

people, particularly those who are irritated and allergic to alcoholic substances.

Furthermore, this can relieve people's burden of purchasing costly sanitizers and help

them save money because herbal sanitizers can be made at home in a simple and cheap

way.

RESEARCHER – The research will improve the researcher's ability to discover things

on his or her own and apply what he or she has learned for a better future and attributes.

It will also serve as an aid in generating new data and knowledge about herbal medicines.

Scope and Delimitation

This study was conducted in Corazon C. Aquino High School, and only the

extraction process was done at researchers’ houses in the school year 2021 – 2022.

This study was limited only to testing the effectiveness of the neem, holy basil,

and lemon extract if it could be a possible source of hand sanitizer against pathogenic

bacteria.

Definition of Terms
 The following important terms and concepts used in this study are defined for the

benefit of the readers to facilitate their understanding and appreciation of this research

work.

Alcohol-Based Hand Sanitizer. It refers to a hand sanitizer that contains between

60 and 95 percent alcohol, usually in the form of ethanol, isopropanol, or n-propanol

Allergic Reaction. It is used to describe the body’s reaction to a substance.

Aroma. It refers to a distinctive, pervasive, and usually pleasant or savory smell.

Consistency. It refers to the physical nature of a substance.

Extract. It is used to describe a preparation containing the active ingredient of a

substance in concentrated form.

Gel Spreadability. It refers to the ability of a prepared hand gel to distribute

properly when applied to the skin

Herbal-Based Hand Sanitizer. It refers to an alcohol-free hand sanitizer that is

made up of medicinal plants.

Infection. It refers to the invasion of microorganisms such as bacteria and viruses.

Odor. It is used to describe a distinctive smell.

Pathogenic bacteria. Refers to the bacteria that are capable of causing illness and

disease.

pH. It is the measure of acidity or alkalinity of a solution.

Quality. It refers to the totality of features and characteristics of a product that

bear on its ability to satisfy given needs.

Sanitizer. It is a substance used to make something clean and hygienic.

 Viscosity. It refers to the characteristic of some liquids being thick and sticky. It

is a measure of the flow resistance of a fluid.

Review of Related Literature

Neem

The neem is appropriately referred to as the village pharmacy because of its

diverse properties. The neem is a tropical evergreen tree native to the Indian sub-

continent. According to National Research Council in Washington, D.C., neem is one of

the most promising plants, with the potential to benefit everyone on the planet. It has the

potential to be profitable, cost-effective, and minimize the occurrence of pest and parasite

resistance as agricultural productivity, environmental protection, and human and

livestock health care services expand (Brahmachari, 2004).

Neem leaves are compound, imparipinnate, with each comprising 5–15 leaflets.

Neem leaves show therapeutics' role in health management due to the rich source of

various types of ingredients. Leaves contain ingredients such as nimbin, nimbanene, 6-

desacetylnimbinene, nimbandiol, nimbolide, ascorbic acid, n-hexacosanol, and amino

acid, 7-desacetyl-7-benzoylazadiradione, 7-desacetyl-7-benzoylgedunin, 17-

hydroxyazadiradione, and nimbiol. Quercetin and -sitosterol, polyphenolic flavonoids,

were purified from neem fresh leaves and were known to have antibacterial and

antifungal properties and seeds hold valuable constituents including gedunin and

azadirachtin (Alzohairy, 2016).

According to Ghimeray et al. (2009), the neem is useful for the following reasons:
 Anti-bacterial effect. Leaf extracts showed antibacterial activity against all the test

bacteria used (Yerima et al., 2012).

Anti-inflammatory effect. An important property found in Neem extracts is their

ability to work as anti-inflammatory agents (Rupani and Chavez, 2018).

Anti-cancerous effect. Neem and their active compounds play a pivotal role in the

prevention of cancer development and progression (Alzohairy, 2016).

Anti-diabetic effect. An important study suggested that leaves extracts of neem

have significant antidiabetic activity and could be a potential source for the treatment of

diabetes mellitus (Akter et al., 2013).

Antifungal effect. Over 14 common fungus species are sensitive to neem

formulations as fungicides (Mahmoud, 2011).

Wound Healing Effect. Results showed that leave extracts of neem promote

wound healing activity (Osunwoke, 2013).

Holy Basil

Holy Basil is an aromatic shrub and is known as “The Incomparable One,”

“Mother Medicine of Nature” and “The Queen of Herbs,” and is revered as an “elixir of

life” that is without equal for its medicinal properties. It is an adaptogen or herb that

helps with the adaptation. Holy basil’s broad-spectrum antimicrobial activity, which

includes activity against a range of human and animal pathogens, suggests it can be used

as a hand sanitizer, mouthwash, and water purifier as well as in animal rearing, wound

healing, the preservation of foodstuff, and herbal raw materials and traveler’s health

(Cohen, 2014).
 Holy basil is an erect, much-branched sub-shrub 30-60 cm tall, with simple

opposite green or purple leaves. Its leaves have petiole and are ovate, up to 5 cm long.

Holy basil leaf contains a variety of beneficial constituents, including eugenol (a volatile

oil), ursolic acid (a triterpenoid), and rosemarinic acid (a phenylpropanoid). Other active

compounds include caryophyllene and oleanolic acid, carotenoids, vitamin C, calcium,

iron, zinc, and chlorophyll (Yance, 2016).

Studies revealed that holy basil has a lot of benefits that include: Antimicrobial

(including antibacterial, antiviral, antifungal, antiprotozoal, antimalarial, anthelmintic),

mosquito repellent, anti-diarrheal, anti-oxidant, anti-cataract, anti-inflammatory,

chemopreventive, radioprotective, hepato-protective, neuro-protective, cardio-protective,

anti-diabetic, anti-hypercholesterolemia, anti-hypertensive, anti-carcinogenic, analgesic,

anti-pyretic, anti-allergic, immunomodulatory, central nervous system depressant,

memory enhancement, anti-asthmatic, anti-tussive, diaphoretic, anti-thyroid, anti-fertility,

anti-ulcer, anti-emetic, anti-spasmodic, anti-arthritic, adaptogenic, anti-stress, anti-

cataract, anti-leukodermal and anti-coagulant activities (Mahajan, 2013).

Lemon

Lemons are among the most popular citrus fruits in the world (Bjarnadottir,

2019). They are a hybrid of the original citron and lime that grow on lemon trees.

Lemons may be eaten in a variety of ways, but they are highly sour and should not be

eaten alone or as a whole fruit. Instead, they're frequently used as a garnish with meals,

and their juice is frequently utilized to provide a sour flavor. They are an important

component of lemonade.
 Lemons are high in vitamin C and fiber and include a variety of plant

components, minerals, and essential oils. These yellow fruits may potentially offer a

number of health benefits. Lemons may help reduce your chances of heart disease,

cancer, and kidney stones.

Because the adverse effects of herbal medications are typically minor when used

carefully, the use of natural substances in herbal medicines is regarded safer than the use

of contemporary chemical treatments (Berti, 2015).

The lemon fruit is one of the herbal plants that is frequently used as medicine.

Lemon fruit is an herbal plant that contains alkaloid chemicals that have anticancer,

antibacterial, antifungal, antiviral, and antidiabetic properties. Saponin is an alkaloid

molecule that is responsible for antibacterial activity.

Many beneficial substances are found in lemon peel, including flavonoids,

carotenoids, limonoids, tannin, and terpenoids. Lemon peel contains a list of vitamins,

minerals, and fiber like calcium, potassium, and Vitamin C, which gives your body a

nutritional boost. Lemon peels also have some healthy enzymes, which help us to live

healthier lives. So, by throwing away the lemon peel, you are doing a great disservice to

your body. Lemon's bioactive components include antibacterial and antiviral properties

(Ekawati & Darmanto, 2019).

Review of Related Studies

Foreign

Kumar et al. (2018) conducted a study to evaluate the antimicrobial activity of

methanolic leaf extracts of neem and holy basil. The study found that neem was more
effective against E. coli, but holy basil was more effective against S. aureus. However,

when both the neem and holy basil extracts were combined together at equal proportion

at each concentration, they showed better results than either neem or holy basil extract

alone.

The study of Kumar et al. (2018) entitled “Comparative Study on Antimicrobial

Activity of Holy Basil (Ocimum Sanctum) and Neem (Azadirachta Indica) Methanol

Extract,” was cited because it revealed that the combination of holy basil and neem leaf

extract which the present study will be utilizing is effective against microbes, implying

that using it as a hand sanitizer is acceptable. Furthermore, the chemical compositions of

neither plant were unhazardous when combined.

According to the study "Formulation of Herbal Sanitizers and Determining Their

Antimicrobial Activities Against Skin Pathogens" by Patankar and Chandak (2018), three

distinct sanitizers were formulated: lemon, lemon-neem, and neem sanitizer. Lemon and

lemon-neem sanitizers were shown to be effective hand sanitizers. Because neem and

lemon contain bioactive molecules, they can be combined to create a cream for wound

healing and the treatment of various skin ailments. Because of their antibiofilm

properties, neem extract and lemon juice can both be used in toothpaste or mouthwash

liquid to treat dental plaque.

The study of Patankar and Chandak (2018) was cited because it demonstrated that

utilizing neem and lemon as a herbal-based hand sanitizer against pathogenic bacteria is

effective. In addition, the mentioned study combined lemon and neem extract, but the

researchers had no problems with it, demonstrating that there is no harmful response from

their chemical properties.

 The above-mentioned research examined at combining neem and lemon extract,

as well as neem and holy basil extract, and found that it was safe to do so. However, there

have been no research on combining the neem, holy basil, and lemon, which is what the

present study wants to investigate.

Local

Lacuna et al. (2013) conducted research to test the antibacterial activity of

supercritical crude extracts of guava (Psidium Guajava) against four (4) microorganisms.

The guava extract was shown to be antibacterial to two gram-positive bacteria (B. subtilis

and S. aureus), suggesting that the plant may contain active chemical components

capable of killing germs. Furthermore, when compared to the traditional antibiotic

chloramphenicol, guava extract shows a better antibacterial impact against gram-positive

bacteria, which may scientifically confirm its efficiency as an alternative herbal remedy

to cure diseases common in rural regions.

The study of Lacuna et al. (2013) and the present study both dealt with herbal

medicine as a means of combating pathogenic bacteria. However, the cited study focused

on the use of guava. On the other hand, the present study used the extracts of the neem,

holy basil, and lemon as a sanitizer to prevent pathogenic bacteria.

According to the study of Toting et al. (2016) entitled “Formulation of Hand

Sanitizer Gel using the Semi Purified Flavonoids from the outer coverings of the Red

Creole variety of Allium cepa Linn.: A preliminary investigation," the extract from the

onion (Allium cepa) has antibacterial activity against Staphylococcus aureus, Escherichia

coli, Bacillus cereus, Micrococcus luteus, Salmonella typhim. Furthermore, the designed
hand sanitizer gel outperforms the two locally marketed hand sanitizer products in the

Philippines in terms of antibacterial activity.

According to the study of Jacela et al. (2021) entitled “Formulation and

Evaluation of Antibacterial Gel Incorporated with Stachytarpheta Jamaicensis Crude

Ethanolic Leaf Extract Against Staphylococcus Aureus,” which aimed to formulate and

evaluate an antibacterial gel incorporated with kandikandilaan or sentiment (S.

Jamaicensis) leaf extract against Staphylococcus aureus. It was discovered that the

prepared gel exhibits antibacterial activity.

The study of Toting et al. (2016) and Jacela et al. (2021) was prominently cited

since it contributes to the present study's formulation of the method to be used in

evaluating the herbal-based hand sanitizer. Furthermore, the cited studies employed a

herbal plant that showed that it is antibacterial, implying that herbal plants can be used as

a hand sanitizer against pathogenic bacteria.

The previous study helps the current study in evaluating the result product of

herbal-based hand sanitizer. Furthermore, it demonstrates that herbal plants have

antibacterial properties, prompting the researchers to develop a herbal-based hand

sanitizer using extracts of neem, holy basil, and lemon.

 Chapter 2

RESEARCH METHODOLOGY

Experimental Design

Complete Randomized Block

Table 1: The amount of herbal-based hand sanitizer from the extract of the neem, holy

basil, and lemon to be use against pathogenic bacteria.

Treatment Amount R1 R2 R3
Control Group 10ml
(10ml 80% ethanol and
80ml of
distilled water)
Trial 1 10ml 15ml 20ml 25ml
(3ml neem, 3ml holy basil,
and 3ml lemon extract +
10ml 80% ethanol and
80ml of
distilled water)
Trial 2 10ml 15ml 20ml 25ml
(5ml neem, 5ml holy basil,
and 5ml lemon extract +
10ml 80% ethanol and
80ml of
distilled water)
Trial 3 10ml 15ml 20ml 25ml
(7ml neem, 7ml holy basil,
and 7ml lemon extract +
10ml 80% ethanol and
80ml of
 distilled water)
Trial 4 10ml 15ml 20ml 25ml
(9ml neem, 9ml holy basil,
and 9ml lemon extract +
10ml 80% ethanol and
80ml of
distilled water)

Table 1 shows the treatment used as effective antibacterial. Every trial consists of

10ml of herbal-based hand sanitizer. If the amount of treatment is not effective, it will be

replicated three times. Replication 1 will be 15ml, while replication 2 will be 20ml and

replication 3 will be 25ml.

Gathering of Materials

Fresh leaves of neem and holy basil free from diseases were collected from one of

the researchers’ house situated in Danzo, Gerona, Tarlac. While, the peel of lemon was

collected from the Gerona Public Market. All other reagents/chemicals were used as an

analytical grade.

Plant Preparation

The leaves and peels were thoroughly washed 2-3 times with running water and

once with sterile distilled water. The leaves and peels were then air dried in the shade on

sterile blotting paper for a week before being powdered and sewed with a 2mm mesh

size. 

The extraction was carried out according to the method prescribed by Shree

(2020). 1g of shade dried powder from each plant was accurately weighed separately, and
100ml of 80% ethanol was added, sonicated for 60 minutes, and filtered using what's man

filter paper. The solvent extract was stored in an airtight bottle at 50°C until further use.

Preparation of Treatment

The current study is an in vitro study into the antimicrobial efficacy of neem, holy

basil, and lemon extract as herbal-based hand sanitizers against pathogenic bacteria. The

Mueller-Hinton agar well variant of the agar disk diffusion test was used to assess the

antimicrobial efficacy of hand sanitizers. To adjust the turbidity of bacterial suspensions,

the McFarland 0.5 turbidity standard was used as a reference. To test the effectiveness,

10ml of the herbal-based hand sanitizer was added to each of the 5 wells containing the

test organism. As a result, if the treatment dose is ineffective, it will be given three times

more. Replication 1 will be 15 mL in volume, while replications 2 and 3 will be 20 mL

and 25 mL in volume, respectively. This was done for all of the test organisms, and the

plates were incubated in an incubator at 37°C for 24 hours. Following incubation,

antimicrobial efficacy was determined using a digital caliper (mm) by measuring the zone

of inhibition, which indicated the test organism's susceptibility or resistance to the

antibacterial agent.

Collection of Data

The experimental group's responses and reactions to herbal-based hand sanitizer

containing extracts of the neem, holy basil, and lemon were collected for three (3) days of

observation. The data has been collected and is being updated in real-time as responses

are received.
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