MOSQUITO-REPELLING PROPERTIES OF OREGANO LEAVES AND ROOTS

SENIOR HIGH THESIS PROPOSAL PRESENTED TO THE FACULTY

OF THE SENIOR HIGH SCHOOL DEPARTMENT IN

DMMC INSTITUTE OF HEALTH SCIENCES

TANAUAN CITY, BATANGAS

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR SENIOR HIGH SCHOOL

SECTION: 12-CURIE

GALINDEZ, ADRIAN

GONZALES, ARRIANE

MERCADO, BEA FRANCINE A.

2022
 CHAPTER I

THE PROBLEM AND ITS BACKGROUND

This chapter contains the Introduction, Purpose Statement, Statement of the Problem,

Theoretical Framework, Conceptual Framework, Null Hypothesis, Scope and

Delimitations of the study, Significance of the study, and Definition of Terms.

Introduction

Wild Marjoram, Mountain Mint, Origanum, Wintersweet, and Winter Marjoram are

all names for Oregano (Scientific name: Origanum vulgare). This erectly spreading plant

possesses strong aromatic qualities, as well as fleshy leaves and stems. Oregano has heart-

shaped leaves with toothed edges that can grow up to 9 meters in length. Oregano also is

a well-known herbal medication in countries like the Philippines for its powerful anti-

oxidant effects. Oregano has anti-inflammatory, antibacterial, anti-oxidant, anti-fungal,

and anti-viral activities due to the presence of Rosmarinus acid, thymol, and carvacrol.

Flavonoids, triterpenoids, sterols, vitamin C, and vitamin A are all found in oregano.

Recent research has shown that it has antibacterial qualities that can be used to treat

infections of the reproductive system, making it appropriate for women who have recently

given birth (Scott Smith, 2016).

Oregano is a Mediterranean herb that is now grown across the globe. Oregano has

been used medicinally since the ancient Greek and Roman empires, when the leaves were

applied to cure skin ulcers, soothe aching muscles, and act as an antiseptic. Asthma,

cramps, diarrhea, and indigestion have all been treated with oregano in traditional
 2

medicine. An oregano infusion is still used in Greece as a traditional cure for colds and

upset stomachs, as well as to preserve overall health (Singletary, 2010).

Based on the current scientific literature, oregano extracts and individual constituents

consistently have demonstrated antimicrobial actions in vitro toward food-borne

pathogens, although the capacity to counter human infections is not well studied

(Singletary, 2010).

Phytochemicals are plant-based insecticides that kill larvae and adult mosquitos, as

well as repellents that keep mosquito bites at bay. Phytochemicals are extracted from the

entire plant or a specific section of the plant using a variety of solvents, including water,

methanol, acetone, benzene, chloroform, etc., depending on the polarity of the

phytochemical. Phytochemicals are advantageous because of their eco-friendliness, target-

specificity, and lack of resistance development, as well as their lower number of

applications, higher acceptability, and suitable for rural areas. It can be used instead of

synthetic insecticides or in conjunction with other insecticides as part of integrated vector

control strategies Some phytochemicals are poisonous (insecticides) to both adult and

larval mosquitos, while others hinder growth and reproduction or provide an olfactory

stimulation, functioning as a repellent or attractant (Simangan, 2015).

Mosquitoes belong to the Culicidae family of the Diptera order and have a similar

look to other insects. However, anophelines (Anopheles spp. alone) differ from culicides

in a number of crucial ways. Mosquitoes have four stages in their life cycle: eggs, larvae,

pupae, and adults. Under the right circumstances, a large number of mosquitos can hatch

at the same time. Immature stage survival can be fairly high in quickly developing broods,
 3

although estimates for many species indicate that immature stage survival is typically less

than 5%. However, 5% of millions is a significant amount. If they spread illness or

primarily feed on humans, as many species do, they become ideal targets for control actions

regardless of population density (Bohbot et al., 2011).

Mosquitoes leave itchy red rashes on the majority of people in the United States and

Europe. Mosquitoes, on the other hand, can cause significant sickness or death in other

parts of the world. Malaria, a parasitic disease caused by a protozoa parasite, is one of its

side effects. Malaria infects 515 million people each year, and 1 to 3 million people, usually

children, die from yellow fever, which is caused by a virus found in mosquito saliva.

Internal hemorrhaging, unconsciousness, and death can occur in severe situations.

Encephalitis/West Nile causes 200,000 infections and 30,000 fatalities each year;

mosquitos transmit at least six forms of encephalitis disease. Dengue fever, which is caused

by one of four viruses found in mosquito saliva, has a high mortality rate. The epidemic

polyarthritis/Ross River virus, which is endemic to Australia and Papua New Guinea, is

predicted to cause 50 million cases each year, with a fatality rate of 2.5 percent, or an

estimated 125,000 deaths. Although symptoms can last up to six months, only 400 people

are afflicted each year, and the virus is rarely fatal. Elephantiasis occurs when a parasite

lodges in the lymphatic system, causing thickening of the surrounding skin and tissues.

The lower extremities, particularly the genitals, are usually affected. Currently, 120 million

people are afflicted worldwide (Silva, 2018).

Repellents are chemicals that help people avoid being bitten by mosquitos. Mosquito

repellents are essential for anyone working or playing in mosquito-infested areas.

 4

Repellents are designed to be applied directly to the skin. Creams, aerosols, solids (sticks),

pump sprays and liquids are all offered. Spray repellents on outer clothes as well as the

skin are frequently beneficial. Fleas, mosquitoes, ticks, chiggers, and biting flies are all

attracted to it (Chen, 2011).

Purpose Statement

The purpose of this paper is to determine if the Oregano leaves and roots ethanolic

extract are indeed effective as an alternative mosquito repellent. Also, it aims to find out

what benefits it can provide to its future consumer.

Theoretical Framework

Plant-based Repellent Theory

Plant-based repellents have been utilized as a personal protection measure against

host-seeking mosquitoes for generations in traditional practice. Ethnobotanical studies

provide useful information about traditional repellent plants, which can be used to generate

new natural products. Consumers have recently become more interested in commercial

repellents containing plant-based components, which are often seen as "safe" in

comparison to long-established synthetic repellents, but this is not always the case.

Insufficient investigations have followed the standard WHO Pesticide Evaluation Scheme

guidelines for repellent testing up to this point. More standardized research is needed to

better analyze repellent chemicals and develop new products that offer great repellency

while also being safe for consumers. Because plant-based repellents are natural, it is widely

considered that they are safer than DEET. However, some natural repellents are safer than

others, and natural does not always imply safe. DEET has been well tested and has a
 5

favorable safety profile. DEET is used by an estimated 15 million people in the United

Kingdom, 78 million people in the United States, and 200 million people worldwide each

year. DEET does not cause bad effects if it is used appropriately, that is, if it is applied to

the skin at the correct dose (such as that found in a commercial product) and not eaten or

rubbed into the mucous membranes. Since 1946, DEET has been used with only a few

known side effects, most of which were caused by excessive or incorrect repellent use. Its

toxicity has been studied more thoroughly than any other repellent, and it has been found

to be safe for use on children, pregnant women, and breastfeeding mothers. Plant-based

repellents, on the other hand, have not been subjected to the same rigorous testing, with the

majority of them being judged safe merely because they have been used for a long time.

Also, many plant-based repellents contain chemicals that should be avoided (Malar J,

2011).
 6

Conceptual Framework

Leaves Roots

25% Leaves and Roots 25%

50% 50% Leaves, 50% Roots 50%

75% 75% Leaves, 25% Roots 75%

100% 25% Leaves, 75% Roots 100%

Observation and documentation of effects and reactions after the

application of the alternative mosquito repellent made by Oregano
leaves and roots.

Analyzation of gathered feedbacks/data using statistical tools by

categorizing results and reactions.

Recommendation of Oregano leaves and roots ethanolic extract as an

alternative mosquito repellent for commercially available mosquito
repellent.
 7

Statement of the Problem

The study will assess the effectiveness of Oregano leaves and roots ethanolic extract

as an alternative mosquito repellent.

Specifically, this study sought to answer the following questions:

1. What is the level of effectiveness of Oregano leaves ethanolic extract as an alternative

mosquito repellent using the following concentrations?

1.1 100%

1.2 75%

1.3 50%

1.4 25%

2. What is the level of effectiveness of Oregano roots ethanolic extract as an alternative

mosquito repellent using the following concentrations?

2.1.100%

2.2.75%

2.3.50%

2.4.25%

3. What is the level of effectiveness of the combination of Oregano leaves and roots

ethanolic extract as an alternative mosquito repellent using the following concentrations?

3.1 50% Oregano leaves, 50% Oregano roots

3.2 75% Oregano leaves, 25% Oregano roots

3.3 25% Oregano leaves, 75% Oregano roots

 8

4. Is there any significant difference on the level of the effectiveness of Oregano leaves

and roots ethanolic extract as an alternative mosquito repellent compared to positive

control?

5. How may the findings of the study become helpful on repelling mosquito?

Null Hypothesis

H0: The level of effectiveness of Oregano leaves and roots ethanolic extract as an

alternative mosquito repellent is no different from the commercial mosquito repellents sold

in many stores.

Assumption of the Study

According to phDStudent.com, assumptions are things that researchers and peers

who will read the dissertation or thesis accept as true, or at least plausible. In other words,

given the population, statistical test, research design, and other delimitations, any scholar

reading the publication will presume that certain features of the study are true. The

following are the researchers' assumptions for this study:

A1: Oregano contain chemicals with known repellent properties, so it can be the best way

to use in repelling mosquito.

A2: Oregano leaves and roots ethanolic extract is not preferable as an alternative mosquito

repellent because it might cause an allergic reaction to people who have an allergy to plants

in Lamiaceae family.
 9

A3: The data collected from the process of experimentation and direct observation will

dictate the effectiveness of Oregano leaves and roots ethanolic extract as an alternative

mosquito repellent.

Scope and Delimitation of the Study

The goal of this study was to develop an alternative mosquito repellent using an

ethanolic extract of Oregano leaves and roots. The researchers want to create a low-cost

alternative composed of natural substances that isn't damaging to people's health.

Materials for the product's manufacture can be found both in the researchers' home

and in the surroundings. People who use mosquito repellent in their homes are included in

this study. There is no such thing as an age, a status, or a gender.

This study is only for mosquitoes; it is not recommended for other insects including

houseflies, cockroaches, fleas, moths, and others. Furthermore, this study was created

solely to repel mosquitos, not to kill them.

Significance of the Study

The information and findings generated from this study would be helpful and

beneficial to the following:

Children. Because a child's respiratory system is still developing, it is still considered

vulnerable. Chemical exposure should be avoided as much as possible. Furthermore,

children are frequently the victims of dengue fever because they are negligent and lack the

knowledge necessary to protect themselves from mosquito bites. As a result, the outcomes

of this research would be extremely valuable to children.

 10

Future researchers. Researchers interested in studying mosquito repellents made from

natural substances might be interested in this study. This would offer up new possibilities

for future scholars to investigate. The findings, as well as the methodology and design of

this research, would aid in the development of their research problems.

Parents. It is also advantageous to parents since it allows them to leave their children safe

from mosquito bites. They would have a better understanding of how to protect their

children from mosquito-borne illness.

Students. By studying this homemade mosquito repellent, this research will assist students

in gaining information. The findings of this study may provide them with crucial

information about mosquito problems and the development of new products that will assist

in reducing mosquito victims.

Definition of Terms

The terms used in the study have been defined conceptually and operationally for

better understanding.

Carvacrol. A compound of many aromatic plants that are usually used as spices in culinary

and for therapy/prevention purposes in folk medicine. These aromatic plants are including

oregano.

Dengue fever. A mosquito-borne tropical disease caused by the dengue virus. Symptoms

typically begin three to fourteen days after infection. These may include a high fever,

headache, vomiting, muscle and joint pains, and a characteristic skin rash.
 11

Mosquito. A group of small insects that are a type of fly that suck blood from human and

animals.

Oregano. An herb of the mint family, Origanum vulgare, having aromatic leaves. As used

in the present study, it refers to main ingredient that the researchers used to create a

mosquito repellent.

Phytochemicals. A chemical compound produced by plants, generally to help them resist

fungi, bacteria and plant virus infections, and also consumption by insects and other

animals.

Repellent. A substance that deters insects or other pests from approaching or settling.

Thymol. An active ingredient in pesticide products registered for use as animal repellents,

fungicides/fungi stats, medical disinfectants, tuberculocides and virucides.

 CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

This chapter contains literature and studies that are directly and indirectly related to

the present study. Present researchers analyze and give their deep thoughts in code

literature and studies.

Related Literature

Almost everyone has been bitten by a mosquito at some point in their lives. Mosquito

bites can cause skin irritation as a result of an allergic reaction to the mosquito's saliva,

resulting in a red bump and itching. However, transmission of dangerous infections and

viruses such as malaria, dengue fever, Zika, and West Nile virus, which can cause crippling

and potentially fatal effects, may be a more significant consequence of mosquito bites

(Luo, 2019).

Mosquitoes are the most significant single category of insects in terms of public

health, as they serve as vectors for a variety of tropical and subtropical diseases.

Mosquitoes quickly build up their population due to their strong capability for reproduction

and dispersal, as well as their ability to exploit even harsh environmental conditions.

Phytochemicals derived from plants with proven mosquito-control capabilities can be used

instead of synthetic insecticides or in combination with other pesticides in integrated vector

control programs (Pushpalatha, 2015).

Phytochemicals are plant-based insecticides that kill larvae and adult mosquitos, as

well as repellents that keep mosquito bites at bay. Phytochemicals are extracted from the

entire plant or a specific section of the plant using a variety of solvents, including water,
 13

methanol, acetone, benzene, chloroform, etc., depending on the polarity of the

phytochemical. Phytochemicals are advantageous because of their eco-friendliness, target-

specificity, and lack of resistance development, as well as their lower number of

applications, higher acceptability, and suitable for rural areas. It can be used instead of

synthetic insecticides or in conjunction with other insecticides as part of integrated vector

control strategies Some phytochemicals are poisonous (insecticides) to both adult and

larval mosquitos, while others hinder growth and reproduction or provide an olfactory

stimulation, functioning as a repellent or attractant (Simangan, 2015).

The majority of plants have chemicals that protect them from phytophagous (plant-

eating) insects. Repellents, feeding deterrents, poisons, and growth regulators are among

the substances in this category. The majority of them fall into one of five chemical

categories: (1) nitrogen compounds (primarily alkaloids). (2) Terpenoids, (3) Phenolics,

(4) Proteinase Inhibitors, and (5) Growth Regulators are some of the compounds found in

plants. Although these compounds' principal activities are defense against phytophagous

insects, many of them are also efficient against mosquitoes and other biting Diptera,

particularly volatile components generated as a result of herbivory (Malar, 2011).

Plants, extracts, and essential oils have been employed in countries to combat

aggressive mosquitoes that carry malaria, dengue fever, and sleeping sickness, as well as

insects that act as vectors for a variety of other diseases. As a result, mosquitoes are

growing increasingly resistant to insecticides and industrial repellents (Maia & Moore,

2011).
 14

Essential oils in certain plants are known to repel mosquitoes and other insects due

to their odor, but the plants themselves do not keep bugs away just by being present. They

don't naturally produce enough oil into the air to actually "repel" bugs, but they can cover

the odors that mosquitoes are attracted to, making it harder for them to find you (Gabrielle,

2014).

Others claim that oregano essential oil is a mosquito repellent, and some gardeners

grow it in their gardens to repel pests like mosquitoes. Although there is little scientific

evidence to back up this claim, oregano does include two compounds with proven repellent

effects, and because oregano is "generally recognized as safe" by the US Food and Drug

Administration, it doesn't harm to try (Flint, 2018).

There are a lot of volatile oils in this plant. Carvacrol, Thymol, Eugenol, and 1, 8

cineole are the primary volatile oils that have been studied in this plant. Rosmarinus acid

and other antioxidants are also abundant in the plant. Because of its high presence of

phenolic acids and flavonoids, oregano has a significant antioxidant potential. Oregano

contains important components that work together to strengthen the body's natural

resistance. Oregano's volatile oil, in particular, contains powerful phenols like carvacrol

and thymol, which support a healthy microbial habitat in the intestines and throughout the

body. Oregano leaves contain the flavonoid Rosmarinus acid, which appears to assist the

normal chemical cyclooxygenase 2 activity (Cox-2). Cox-2 has been linked to tissue

inflammation. Oregano oil, like other essential oils like Tea Tree oil, appears to have a

special affinity for Candida species as well as a variety of gram positive and gram-negative

bacteria in vitro investigations. Because the majority of Oregano research has been done
 15

in vitro or on animals, there isn't enough evidence to scientifically confirm all of these

activities in humans. Oregano has been proven to have substantial antioxidant capacity in

numerous in vitro models, leading to speculation that it may be beneficial to human health,

however human studies are lacking once again (Brevard, 2017).

38.8% of Thymol, 32.99% of Carvacrol, and 7.9% of P-cymene are abundant in

oregano oils. By contact or fumigation, they are very poisonous to Rhizoperta Dominica.

It's also effective against drosophila. Ocimum gratissum's acaricidal effects are aided by

thymol. Oregano oil is also used topically to repel insects (Heredia et al., 2017).

Oregano includes a number of chemicals, one of which is carvacrol, which is a

natural insect repellant. Plants such as mint and thyme also contain this chemical (Coles,

2013). Man has been exploiting the repellency of plant material for thousands of years,

most simply by hanging bruised plants in buildings, a technique that is still common in

developing nations. Plants have also been utilized for ages as crude fumigants, in which

plants were burned to drive away pesky insects, and later as oil formulations applied to the

skin or garments, which was first documented in ancient Greek, Roman, and Indian

sources. Plant-based repellents are still widely used in this traditional way throughout rural

communities in the tropics because they are the only means of protection from mosquito

bites available for many of the poorest communities, and some of these communities, like

those in Europe and North America, prefer "natural" smelling repellents because plants are

perceived as a safe and trusted means of mosquito bite prevention (Malar, 2011).

A mosquito repellent is a product that is applied to the skin, clothing, or other

surfaces to prevent mosquitos from landing or crawling on them. Mosquitoes can transmit
 16

viruses that can lead to serious illnesses. West Nile virus (WNV) and eastern equine

encephalitis (EEE) are two diseases spread by mosquitos in Massachusetts. Mosquito

repellents can help you avoid getting bitten by a mosquito and minimize your chances of

contracting one of these diseases (Paul, 2014).

The appropriate use of repellents can provide personal protection against tick and

mosquito bites, and the CDC in the United States has advised their usage (2002). N, N-

dimethyl3-methylbenzamide (DEET), as well as permethrin for repellent items applied to

clothing, have been the backbone among repellents licensed for use on human skin for

decades (Demirci et al., 2013; Bernier et al., 2015). Most plants develop secondary

chemicals that repel a wide range of arthropods, including blood feeders that offer no

damage to plants. Origanum vulgare L. has been shown to repel the tick Ambiculus

Americanum (Meng et al., 2016), and Origanum onites L. has been proven to be poisonous

to the tick Rhipicephalus turanicus Pomerantsev.

Oregano comes in a wide variety of species in our surroundings. Some of this is often

used in Middle Eastern and Southern European dishes. The most common and well-known

species of oregano is Origanum vulgare, also known as true oregano or Italian oregano.

This is the familiar herb found on pizzas and in tomato sauces, and it is most abundant on

the Asian continent, particularly in the Philippines. Although some oregano species are

used for décor and cooking, they can also be used in a variety of ways (Jimenez, 2021).

Oregano leaf is probably safe when consumed in food levels and maybe safe when

consumed or applied to the skin in therapeutic amounts. Stomach trouble is one of the
 17

milder side effects. People who are allergic to plants in the Lamiaceae family may

experience an allergic reaction to oregano (Stea et al., 2014).

Related Studies

The following studies have been evaluated and are considered to be relevant and linked to

the current research being discussed by the researchers.

As expressed elaborately in the book Oregano: The Genera Origanum and Lippia.

In most insect repellents, whether chemical or natural, oregano is one of the most

commercially useful components. It can be used because to the presence of carvacrol and

thymol, both of which have antibacterial characteristics and can repel insects. Carvacrol,

the major phenolic constituent of the essential oil of thyme, was screened for

pharmacological effects on the central nervous system and found to have repelling activity

when administered directly to mosquitoes. Thymol is the major component in the essential

oils of oregano and it gives a chemical effect to the insects that makes them repel, whereas

thymol is the major component in the essential oils of oregano and it gives a chemical

effect to the insects that makes them repel.

The study "Repellency of the Origanum Vulgare Essential Oil and Constituents to

the Yellow Fever Mosquito (Aedes aegypti)" was conducted by Carrol, J. F. et al. (2017).

The repellent activity of oregano and essential oil against yellow fever mosquitos was

investigated in laboratory behavioral bioassays (Aedes aegypti). To regulate

Diethyltoluamide, the researchers compared oregano and essential oil (DEET). When

evaluated using human volunteers in a fabric patch assay, oregano and essential oil
 18

demonstrated good repellent action against yellow fever mosquito (Aedes aegypti) with a

minimum effective dosage compared to the reference control DEET.

The prior study and this one is similar in that they both utilized oregano as a mosquito

repellent. However, the researchers focused on a specific mosquito species, the yellow

fever mosquito. Furthermore, the researchers compared oregano's efficiency as a yellow

fever repellant. The current study, on the other hand, aimed to develop a mosquito repellent

composed of oregano leaves and roots that could be utilized against any type of mosquito.

Carpio, E. C. (2014) published a paper titled "Oregano Extract as Mosquito

Repellent." The researcher was interested in seeing if oregano extract could be used as a

mosquito repellant. The oregano extract is reported to be useful as a mosquito repellent. It

is effective not only against dengue fever, but also against all mosquito-borne diseases such

as malaria and filariasis, particularly in high-incidence areas of the country. It's also a

personal care item for preventing mosquito bites, which cause itching, inflammation, and

discomfort in people.

The objectives and research materials employed in the previous study and the current

study are comparable in terms of using oregano extract. Both the previous and current

studies sought to investigate the efficacy of oregano extract as a mosquito repellent.

"Oil of Oregano as Mosquito Repellent," according to a study conducted at the

University of Lowa. Because oregano oil includes carvacrol and thymol, the study

established that it is beneficial for insect control. When applied to clothing, oregano oil
 19

repels anopheles quadrimaculatus, a mosquito species that transmits malaria, for up to 30

minutes.

The previous study and the current study are identical because both studies utilized

oregano as a mosquito repellent. The substances employed by the researchers, however,

were different. Oregano was combined with several ounces of carrier oil, such as coconut

oil, olive oil, or sweet almond oil, in a previous study. The current study, on the other hand,

employed oregano extract as a mosquito repellent and did not use any carrier oil.

SYNTHESIS

Mosquitoes are a type of insect that is universally despised around the world. These

disease-carrying pests spend their existence sucking the blood from anything that moves,

including humans. Plant extracts and essential oils have been used for centuries to combat

aggressive mosquitoes that spread malaria, dengue fever, and sleeping sickness, as well as

insects that act as vectors for a variety of other diseases.

Plants have been used to repel mosquitoes for ages, according to Malar (2011). This

means that there are plants that humans utilize to keep mosquitoes at bay. These mosquito

repellents are vital for reducing the risk of malaria and other mosquito-borne infections in

our environment.

Plants have a number of qualities that can help repel mosquitoes. Oregano is one of

these herbs, with high levels of thymol (38.8%), carvacrol (32.9%), and p-cymene (7.9%).

Thymol contributes to Ocimum gratissimum's acaricidal effects. Oregano oil is often used
 20

as an insect repellent. Oregano is also a natural insect repellant, according to Coles (2013).

Its oil contains a variety of chemicals, including carvacrol, a natural insect repellant.

Similarly, some gardeners use oregano to repel pests like mosquitoes, while others

believe that the essential oil is a mosquito repellent. Oregano, which can be found in

backyards, is extremely beneficial to humans, particularly in repelling or killing

mosquitoes. The researchers developed a mosquito killer or repellent that can help manage

mosquito populations and prevent the spread of diseases transmitted by mosquitos.

 CHAPTER III

RESEARCH METHODOLOGY

This chapter tackled the description of the research design, subject of the study, and

the data gathering as well as the methods and techniques employed in this study.

Research Design

The study will use an experimental design. A problem-solving strategy in which

several attempts are made to reach a solution is known as trial and error. It's a fundamental

mechanism of learning that almost all organisms use to pick up new skills. Trying a

method, seeing if it works, and if it doesn't, trying a different method. This approach is

done until a solution or success is found (Alley Dog, 2020). In this study, the experimental

technique of research will be used because it is the most effective means of establishing a

cause-and-effect relationship within the supplied hypothesis. The goal of the study is to

evaluate and observe the effectiveness of an ethanolic extract of Oregano leaves and roots

as an alternative mosquito repellant.

Sources of Data

The primary source in this study is the information gathered by the researchers

through experimentation and observation. The e-books, published journals, articles, books,

and related studies about Oregano leaves and roots as mosquito repellents from the online

platform were considered secondary sources of data, which were defined by Prachi, J.

(2015) as data that had already been collected and were readily available from other

sources. The leaves and roots of Oregano will be employed in the research. This

necessitates the use of ethanol to extract the content.

 22

Research Locale

The research will be place in the Medical Technology Laboratory at DMMCIHS.

The laboratory in discussion is a location that allows for experimentation, observation, or

practice in a particular field of study. This location was chosen for the study, "Mosquito-

repelling properties of Oregano leaves and roots." The purpose of this study is to see if the

ethanolic extract of Oregano leaves and roots is effective at repelling mosquitoes.

Research Process

Here, the researchers will follow the step-by-step procedure which indicate the

process of the study.

Collection of Oregano Leaves and

Mosquito

Preparation of Materials

Extraction

Observation and Collection of Data

Result
 23

Figure 2. The Research Process of the Study

To conduct this experiment, the researchers will first gather Oregano leaves and roots

from the researchers' property in Trapiche, Tanauan City, Batangas. To capture mosquitos,

the researchers will place a drum filled with 10-20 percent water in a shaded area overnight

and cover it with a mosquito net for 3-4 days. The next step is to prepare the materials.

Bowl, mortar pestle, funnel, casserole pan and burner for boiling procedure, distilled water,

and spray bottle will all be used in the experiment. The researchers also use illustration

board and a plastic cover with dimensions of 10 x 6.5 x 6.5 inches to make an improvised

mosquito glass box. Extraction is the third phase. When using ethanolic extract, it should

evaporate before the extract flows. To establish which concentration was effective, the

extract was diluted in distilled water to 100 percent, 75 percent, 50 percent, and 25 percent.

Following the completion of this experiment, data collection and observation will be used

to test any changes. Finally, the results will show whether the ethanolic extract of Oregano

leaves and roots is effective as an alternative mosquito repellent.

Collection of Oregano Leaves and Mosquito

A total of thirty (30) pieces of oregano leaves and roots will be harvested in a vacant

area near the researcher's home. Only fresh, green leaves and roots were gathered. These

leaves and roots are also clean and dry to avoid other organic substances from being

extracted during the extraction process. If the oregano leaves and roots are damp, the

researchers will be allowed to air dry before being extracted.

The researchers will collect 20 mosquitoes by leaving drum water with a

concentration of 10-20 percent water in a shaded area overnight for 3-4 days, then covering
 24

it with a mosquito net in the morning. An improvised mosquito glass box will also be

constructed by the researchers. Its sides will be made of illustration board, and the five (5)

sides will be covered with a plastic cover that measures 10 x 6.5 x 6.5 inches. The arm will

be inserted on one side, which will be covered with a mosquito net.

Preparation of Materials

The beaker, spray bottle, plastic cover, rubber gloves, illustration board, and mortar

pestle are the materials that will be used in this experiment. The extract of Oregano leaves

and roots will be placed in a beaker and a spray bottle by the researchers. The adapted

insect glass box will be made with a plastic cover and illustration board. Rubber gloves

will be used to protect the hands from any potential dangers while performing the

experiment. To extract the oregano leaves and roots, a mortar and pestle will be employed.

Preparation of Leaves and Roots Ethanolic Extract

Here are the phases in making Oregano leaves and roots ethanolic extract as an

alternative Mosquito repellent.

In the preparation of oregano leaves and roots extract. First, the researchers will

gather some oregano leaves and roots and dry them in the sun. After that, they will grind

the oregano leaves and roots with a mortar pestle and place them in a beaker. Finally, the

researchers will add an exact amount of ethanol to the beaker and wait until the oregano

leaves and roots extract is obtained. Second, the researchers will extract the oregano leaves

and roots with the ethanol, and then test the oregano extract in various quantities, starting

with 100 %, then 75 %, 50 %, and lastly 25 %. The efficiency of the oregano leaves and

roots extract at various levels will be determined after the test. The researchers will
 25

compare it to the positive control after determining it. The weighted mean, frequency test,

and t-test will be used to evaluate the experiment's results.

Methods and Procedure

To do this experiment, the researchers will use the steps provided in the internet and

this will be served as their reference of procedure.

This experiment's guidance will be based on an article published by the World Health

Organization (WHO). Arm-in-Cage tests are a standard method of determining the efficacy

of repellent formulations. This method proved ideal for evaluating topical repellents such

crèmes, moisturizers, and spray formulations, as well as impregnated textiles. There will

be 10-20 mosquitoes in the mosquito box. Prior to the trial, the mosquitoes' wills were

evaluated for host-seeking behavior. This will be accomplished by placing a clean hand in

the cage and counting the number of mosquitos aligned within ten (10) seconds. The

mosquitoes inside the cage will be considered host-seeking if at least five (5) mosquitoes

align on the hand, and the repellency trial will continue. This was done to ensure that the

insects in the mosquito box were looking for food. The hands will be shielded with stocking

socks that the mosquitoes cannot bite before being inserted into the cage housing 10-20

mosquitoes. During the testing, the arm will not be allowed to move. While doing so, the

other researchers will keep track of the time and gather evidence. The bite rate must be at

least 10 landings and/or probing’s every 30-second interval, according to the World Health

Organization (WHO). The control group will consist of the right hand, whereas the

experimental group will consist of the left hand. The covered hand will be put and watched

for the specified concentration periods without the use of mosquito repellant. The
 26

researchers will keep track of the information. The left hand will be sprayed while the

control hand is carefully withdrawn. For around 5 minutes, the hand will be dried. As a

result, the treated hand will be placed in the cage for another 30-second period; however,

testing should not begin until the exposed hand has experienced five (5) mosquito landing

and/or probing rates. Doing such, the researcher will keep track of how many mosquitoes

fall on its arm at various intensities. These events allow the researcher to keep track of the

product's durability. For the data to be valid, the experiment will be repeated four times

(World Health Organization, 2009).

Proper disposal and handling of Specimen

The containers used to store Oregano leaves and roots will be dried after the

experiment and disposed of in a recyclable can, together with the Oregano leaves and roots

that were extracted. The tools and equipment used to collect samples will be sterilized. To

avoid cross-contamination, Oregano leaves and roots extracts will be carefully placed in a

clean and separate container.

Safety Precautions and Procedure

Since Oregano is present in the environment, it's possible that it's been exposed to

dust, which could harm the user's health. To eliminate the accumulated dirt, the researchers

will soak the Oregano leaves and roots in restricted water. The researchers will then ensure

that their bodies and hands are clean before beginning the experiment. Before and after the

experiment, the work surface and table will be cleaned with a 10% bleach or 70% ethanol

solution. The materials used in the experiment will also be sanitized to eliminate any

microorganisms that may have attached to it. When the experiment begins, eating and
 27

drinking are not permitted. Personal protection equipment (PPE) will also be observed in

its proper use.

Interpretation of Positive Control

A positive control, according to Helmenstine (2020), can demonstrate that the

experiment is running as planned and is capable of yielding a positive result. The mosquito

repellent that is commercially available will be employed as a positive control in this

investigation. The researchers will purchase it from a Tanauan City supermarket. This

study will examine the repellency effect of ethanolic extract of Oregano leaves and roots

as an alternative to commercial mosquito repellent.

Statistical Tool

For S.O.P 1, S.O.P 2, and S.O.P 3, a weighted mean and a frequency distribution

table will be used for the level of effectiveness of Oregano leaves and roots ethanolic

extract as an alternative mosquito repellent. The weighted mean is a sort of mean that is

derived by multiplying the weight (or probability) associated with a certain event or

outcome by the associated quantitative outcome, then summing the results. A frequency

distribution is a list, table, or graph that shows the frequency of different outcomes in a

sample. It's used to organize and summarize data.

For S.O.P 4, T-test will be used to determine if there any significant difference on

the level of effectiveness of Oregano leaves and roots ethanolic extract compared to the

positive control. A t-test is an inferential statistic that is used to see if there is a significant

difference in the means of two groups that are related in some way.
 28

Interpretation of Data

The most effective concentration will be determined by testing four different

concentrations on a group of participants. Their effectiveness against mosquitos will be

measured and graded as 0 (zero) – no effect, 1 – slight effect, 2 – moderate effect, and 3 –

heavy effect. After the evaluation, the most effective concentration will be directly

compared to the positive control. The ethanolic extract of Oregano leaves and roots will be

used to determine the efficiency of Oregano leaves and roots as an alternative mosquito

repellent. The interpreted results are based on Grutas, A. et al. (2004).

 29

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