The Effectiveness of Tomato (Solanum lycopersicum L.

) Leaf

Extract as an Organic Mosquito Repellent

A Topic Proposal

Presented to

Senior High School Unit

Integrated Basic Education Department

San Beda University - Rizal

In partial fulfillment

of the requirements

for Practical Research 2

12 - STEM 6

Lantoria, Kyle Nicole

Geronga, Allana Mae

Mendoza, Jinky Mae

Saito, Austin Christian

S.Y. 2019 - 2020

 CHAPTER I

The Problem and Its Background

This chapter includes the introduction of the topic which is

the effectiveness of tomato leaves as an organic mosquito

repellent, and also its background. It also includes the statement

of the problem and hypothesis that the researchers will tackle and

answer. The study’s significance and specific objectives are also

included. Finally, the scope and delimitations and the definition

of the terms that will be used in the topic are also stated below.

Background of the Study

The Philippines recently declared a national Dengue epidemic

as cases of the mosquito-borne disease continued to rise in the

country. The Department of Health (DOH) reports a total of 146,062

dengue cases from January to July 2019. The epidemic needs to be

addressed immediately so that further fatalities can be avoided.

Proper prevention techniques should be implemented so that

these cases can be reduced. Dengue is a mosquito-borne viral

infection that causes flu-like illness, and occasionally develops

into a potentially lethal complication called severe dengue. In

the wake of the 146,062 dengue related cases recorded since January
up to July 2019, which is 98% higher than the same period last

year, the Philippines declared a national dengue epidemic to enable

the local government units to use their quick response fund and to

further address the situation (Department of Health [DOH], 2019).

As stated by the World Health Organization (WHO, 2019), the

global incidence of dengue has grown dramatically in the recent

decades and about half of the world's population is now at risk.

Although there are no specific treatments for dengue, early

detection, access to proper medical care, and preemptive measures

lower the fatality rates below 1%.

Statement of the Problem

This research attempts to answer the following questions:

1. Which among the ethanolic and aqueous extracts from the

leaf of the tomato will show greater effectivity in repelling

mosquitoes away from the subject?

2. What is the significant difference between the efficacy of

the tomato leaf extract and the commercialized mosquito

repellent that contains DEET?

 3. In a timeframe of 0-3 hours, 3-6 hours , and 6-9 hours,

which among the ethanolic and aqueous tomato leaf extract

will repel more mosquitoes?

Hypothesis

The ethanolic and aqueous extract from the leaf of the tomato

plant has no effect as a repellent against mosquitoes.

Significance of the study

The rationale of this study is to help the readers determine

the efficacy of tomato leaf extract as a natural mosquito

repellent. Furthermore, the results of the study will be of great

benefit to the following:

To the students. This study will help the students to raise

awareness regarding on the prevention of the possible

mosquito related diseases. This study will also help them to

improve their academic competence and upgrade their knowledge

regarding to the said medicinal plant.

To the parents. The findings will also serve as a learning

guide of the parents as to abstain from more serious damage

to their children’s health.

 To the medical schools. The outcome of the study will improve

the school and might be a learning paradigm in line with their

curriculum. This will help them in the advancement of teaching

evaluation approach and clinical leadership.

To the field of medicine. This can benefit the field of

medicine to have new prescription or recommendation in

preventing mosquito breeding grounds.

To the future researchers. The findings of this study can

serve as their reference data in conducting new researches or

proving the validity of their related findings. They might

use this research as a good source of accurate and useful

information for them.

Objectives of the Study

The main objective of this study is to find out the efficacy

of the tomato leaf extract as a mosquito repellent. On the other

hand, the specific objectives of the researchers in conducting

the study are the following:

1. This study aims to determine the time and duration of the

efficacy of the tomato leaf extract as an organic mosquito

repellent.

2. This study also aims to test if the tomato leaf extract

 can be considered as an alternative for any chemical

insecticides and mosquito coils.

3. The researchers intend to prevent dengue outbreak and fight

any possible insect related diseases.

Scope and Delimitations

(HINDI PA PO NASESEND NI KYLE )

Definition of terms

Stated below are the definition of some of the terms that

will be used throughout this research.

Tomatine – is a glycoalkaloid found in tomato leaves that

has fungicidal, antimicrobial, and insecticidal properties

Aqueous - a solvent containing water.

Ethanolic - a solvent containing ethyl alcohol.

Solanum lycopersicum L. – the scientific name of tomato

which that is usually red when matured. It is also the

major source of the antioxidant, lycopene.

Aedes aegypti - is a small, dark mosquito with white lyre

shaped markings and banded legs

DEET - is the active ingredient in many repellent products.

Extraction - selectively dissolves one or more mixture

compounds into a suitable solvent.

Maceration - method used in soaking a substance in a

solvent to soften the substance.

Solvent - a substance that dissolves a solute.

Dengue Virus - is a viral infection transmitted by daytime

biting female Aedes aegypti and Aedes albopictus

mosquitoes.
 CHAPTER II

Review of Related Literature

This chapter includes literature and studies with similar

research topics and methods with this research that would serve as

a support and help to the researchers to accomplish the experiment

without any biases and following the protocols and procedures that

has been conducted by other researchers of the similar topic.

TAXONOMIC CLASSIFICATION OF TOMATO

Kingdom: Plantae
Subkingdom: Tracheobionta
Superdivision: Spermatophyta
Division: Magnoliophyta
Class: Magnoliopsida
Subclass: Asteridae
Order: Solanales
Family: Solanaceae
Genus: Solanum L.
Species: Solanum lycopersicum L.

The tomato is a fruit from the nightshade family native to

South America. Despite botanically being a fruit, it’s generally

eaten and prepared like a vegetable. Tomatoes are the major dietary

source of the antioxidant lycopene, which has been linked to many

health benefits, including reduced risk of heart disease and

cancer. They are also a great source of vitamin C, potassium,

folate, and vitamin K. Usually red when mature, tomatoes can also

come in a variety of colors, including yellow, orange, green, and

purple. What’s more, many subspecies of tomatoes exist with

different shapes and flavor. (Healthline, 2019)

TOMATO LEAVES

According to Luv2Garden, tomato leaves contain a natural

alkaloid [glycoalkaloid] called tomatine sometimes referred to as

lycopersicin which possesses antimicrobial, fungicidal, and

insecticidal properties. Other Solanaceae Alkaloids found in the

tomato plant also have similar properties. Insects particularly

herbivorous insects that chew leaves or suck sap can be combated

with this alkaloid and it is something you can easily make. When

tomato plant leaves are injured or ruptured, they release these

alkaloids which repel and or kills off many harmful insects. These

alkaloids are water soluble and can be easily mixed and diluted

with water to make a potent spray that is toxic to aphids,

whiteflies and other pests but harmless to other plants, pets and

people unless you have an allergy to nightshade family plants.

TOMATINE

Tomatine is a glycoalkaloid, found in the stems and leaves of

tomato plants, and in the fruits at much lower concentrations. It

has fungicidal, antimicrobial, and insecticidal properties.

Chemically pure tomatine is a white crystalline solid at standard

temperature and pressure. Tomatine, as well as the closely related

aglycon (or aglycone) derivative tomatidine have been shown to

have multiple health benefits. It is thought to be similar in

character to saponins and is widely recognized as a strong

immunostimulator. It has been shown to be well tolerated and safe

in mice and does not induce hemolytic activity, tissue damage, or

granuloma formation at the site of inoculation. However, it induces

recruitment of mononuclear cells within 24 hours of immunization.

Heal et al. administered tomatine alongside Plasmodium berghei

circumsporozoite (CS) peptide to BALB/c mice. They found that

splenocytes from the vaccinated mice significantly upregulated

interferon (IFN)-γ compared with controls. Additionally, Morrow et

al. showed the potential of tomatine as an adjuvant in vaccines

protecting against malaria and Francisella tularensis.

(ScienceDirect, 2019)
Ethanolic Extract Method

Ethanol extraction is found easier to penetrate the cellular

membrane to extract the intracellular ingredients from the plant

material (Sindhu, 2014). It is one of the effective solvent

extraction methods that are used to extract fragrant compounds

straight from dry raw materials and wicked oils or concrete due to

organic solvent extraction. The impure substances and some oils

are joined together with ethanol, that is less hydrophobic than

solvents utilized for the purpose of organic extraction, dissolves

most of the oxidized aromatic components, leftover the fats, wax

and other hydrophobic stuffs. The ethanol extractions from dry

substances are known as tinctures, whereas ethanol washes are done

for purifying oils and concretes which are called absolutes

(Prasad, 2018).

Aqueous Extract Method

Water is a universal solvent. It can be used to extract plant

products with antimicrobial activity (Sindhu, 2014). Adding one

liter of hot boiling water and kept the mixture at room temperature

overnight is the aqueous procedure that Nidaullah, Durrani, Ahmad,

Jan & Gul did in their study (2010).

 According to a study by Huzaifa, Bello, Labaran & Olatunde

(2014), the plant should first be sliced, crushed, dried in the

air, and pulverized into powder before extraction. the pulverized

plant will be soaked in distilled water following the ratio, 100

g : 600 mL, for 24 hours. After soaking, filter paper will be used

to separate the residue and the filtrate. The residue will be dried

on a cardboard paper and the filtrate will serve as its extract.

TAXONOMIC CLASSIFICATION OF YELLOW FEVER MOSQUITO

Kingdom: Animalia
Phylum: Arthropoda
Subphylum: Hexapoda
Class: Insecta
Order: Diptera
No Taxon: "Nematocera"
Infraorder: Culicomorpha
Family: Culicidae
Tribe: Aedini
Genus: Aedes
Species: aegypti

According to the Centers for Diseasese Control and Prevention

(CDC), Aedes aegypti is a small, dark mosquito with white lyre

shaped markings and banded legs. They prefer to bite indoors and

primarily bite humans. These mosquitoes can use natural locations

or habitats (for example treeholes and plant axils) and artificial

containers with water to lay their eggs. They lay eggs during the
day in water containing organic material (e.g., decaying leaves,

algae, etc.) in containers with wide openings and prefer dark-

colored containers located in the shade. About three days after

feeding on blood, the mosquito lays her eggs inside a container

just above the water line. Eggs are laid over a period of several

days, are resistant to desiccation and can survive for periods of

six or more months. When rain floods the eggs with water, the

larvae hatch. Generally larvae feed upon small aquatic organisms,

algae and particles of plant and animal material in water-filled

containers. The entire immature or aquatic cycle (i.e., from egg

to adult) can occur in as little as 7-8 days. The life span for

adult mosquitoes is around three weeks. Egg production sites are

within or in close proximity to households. Aedes aegypti do not

remain alive through the winter in the egg stage in colder

climates. Most frequently found in tropical and subtropical areas

of the world, Aedes aegypti historically is considered to be a

primary vector of viral diseases such as the dengue fever,

chikungunya and yellow fever. Aedes aegypti is extremely common in

areas lacking piped water systems, and depend greatly on water

storage containers to lay their eggs. Male and female adults feed

on nectar of plants; however, female mosquitoes need blood in order

to produce eggs, and are active in the daytime. Eggs have the

ability to survive drying for long periods of time, allowing eggs

to be easily spread to new locations. Artificial or natural water

containers (water storage containers, flower pots, discarded

tires, plates under potted plants, cemetery vases, flower pots,

buckets, tin cans, clogged rain gutters, ornamental fountains,

drums, water bowls for pets, birdbaths, etc.) that are within or

close to places where humans live are ideally larval habitats for

this mosquito. This species has also been found in underground

collections of water such as open or unsealed septic tanks, storm

drains, wells, and water meters. Aedes aegypti bites primarily

during the day. This species is most active for approximately two

hours after sunrise and several hours before sunset, but it can

bite at night in well lit areas. This mosquito can bite people

without being noticed because it approaches from behind and bites

on the ankles and elbows. Aedes aegypti prefers biting people but

it also bites dogs and other domestic animals, mostly mammals.

Only females bite to obtain blood in order to lay eggs.

DENGUE VIRUS

As stated by the International Association for Medical

Assistance to Travellers (IAMAT), Dengue is a viral infection

transmitted by daytime biting female Aedes aegypti and Aedes

albopictus mosquitoes. Many people who are infected are

asymptomatic, meaning they don’t have any symptoms, while others

have flu-like symptoms with fever, rash, and muscle, joint, and

bone pain. In its most severe form, the illness can progress to

Dengue Hemorrhagic Fever (DHF) and Dengue Shock Syndrome (DSS),

which can be fatal. There are four closely related Dengue viruses

(DENV-1, DENV-2, DENV-3, DENV-4) which add to the complexity of

the disease. If you are infected with the same serotype (DENV-2,

for example) you become immune to subsequent DENV-2 infections,

but immunity wanes for the other three serotypes over time. As a

result, getting infected by a different serotype (first by DENV-2

and then by DENV-3) increases your risk of developing of DHF. There

is no cure for Dengue. Treatment includes supportive care of

symptoms and a lot of rest.

Use of DEET

According to the United States Environmental Protection

Agency (EPA), DEET (chemical name, N,N-diethyl-meta-toluamide) is

the active ingredient in many repellent products. It is widely

used to repel biting pests such as mosquitoes and ticks. Every

year, an estimated one-third of the U.S. population use DEET to

protect them from mosquito-borne illnesses like West Nile Virus,

the Zika virus or malaria and tick-borne illnesses like Lyme

disease and Rocky Mountain spotted fever. Products containing DEET

currently are available to the public in a variety of liquids,

lotions, sprays, and impregnated materials (e.g., towelettes, roll

on). Formulations registered for direct application to human skin

contain from 5 to 99% DEET. Except for a few veterinary uses, DEET

is registered for use by consumers, and it is not used on food. It

is designed for direct application to people’s skin to repel

insects. Rather than killing them, DEET works by making it hard

for these biting bugs to smell us. After it was developed by the

U.S. Army in 1946, DEET was registered for use by the general

public in 1957. Approximately 120 products containing DEET are

currently registered with EPA by about 30 different companies.

GUIDELINES FOR MOSQUITO REPELLENT TESTING ON HUMAN SKIN

These guidelines are an expanded and updated version of those

recommended by the WHO Pesticide Evaluation Scheme (WHOPES)

Informal Consultation on the evaluation and testing of

insecticides, held at WHO headquarters in Geneva, Switzerland, on

7–11 October 1996 . These were reviewed and recommended by the WHO

Consultation on testing and evaluation of public health

pesticides, held at WHO headquarters on 23–27 February 2009. The

guidelines provide guidance and procedures on laboratory studies,

field trials and evaluation of technical material used in mosquito

repellent products and on the methods used to determine their

application rate(s) and effectiveness. Guidance is also provided

on the single-dose evaluation of formulated repellent products.

With some modification, the guidelines can be used to determine

the repellency of candidate compounds for other flying insects

that blood-feed on humans.

The objective of laboratory studies is to estimate the

effective dose of a repellent and the complete protection time

provided by a repellent after application on the skin.

The specific aims of these tests are:

 to estimate dose–response lines and effective doses (EDs) of

a repellent corresponding to 50% (ED50) and 99.9% (ED99.9)

protection from mosquito landing and/or probing;1

 to estimate the complete protection time (CPT) of a repellent,

which is the time between the application of the repellent

and the first mosquito landing and/or probing.

Testing of repellents on human subjects is the method of choice

as it utilizes the repellent end-user in the testing process and

yields results that are relevant to the actual conditions of use.

Use of laboratory animals or artificial membranes may inadequately

simulate the situation in which repellents for use on human skin

are intended to perform. Tests are carried out on adult human

volunteers who may be selected from among candidates exhibiting

mild or no sensitivity to mosquito bites. Equal numbers of male

and female test volunteers are preferred.

Landing and/or probing behaviour signifies the end-point of the

repellent efficacy test. However, landing is not always associated

with probing, and separate recordings of each behaviour may be

needed. A repellent may provide efficacy by a reduction in biting

activity but not in landing. Alternatively, it may provide efficacy

in terms of landing, but those mosquitoes that land may all bite.

Both scenarios may be important in determining the efficacy of a

repellent.

In preparation for the laboratory studies, the test area of the

volunteer’s skin should be washed with unscented soap and rinsed

with water, then rinsed with a solution of 70% ethanol or isopropyl

alcohol in water and dried with a towel. Given the possibility

that various factors may alter a person's attractiveness to

mosquitoes, and that this may in turn affect the outcome of

repellency assays, test volunteers should avoid the use of

fragrance and repellent products for 12 hours before and during

testing. Volunteers should preferably not be tobacco users, or at

least to have refrained from tobacco use for 12 hours prior to and

during testing.

Standardized mosquito rearing and laboratory testing conditions

are essential to ensure the reliability and reproducibility of

data. Mosquitoes should be reared, maintained and tested (in a

separate space or room) at 27 ± 2 C temperature, ≥80 ± 10% relative

humidity, and a 12:12 (light:dark) photoperiod. Temperate mosquito

species may require modifications to rearing conditions. Stock

populations of adult mosquitoes should have access to sugar

solution but not have been blood- fed. Observations of repellency

should be made using female mosquitoes starved for the preceding

12 hours and, where practical, during times in the diel period

that correspond with biting activity by that species.

Mosquito repellency tests should be conducted with three or more

of the more anthropophilic Aedes (preferably Aedes aegypti), Culex

(preferably Culex quinquefasciatus) and Anopheles (preferably An.

stephensi, An. gambiae or An. albimanus) species. The test species,

strain and age should be reported. Mosquitoes should be contained

during testing using a cage (suggested metal frame for ease of

decontamination, size: 35 –40 cm per side) with a solid bottom and

top, screen or netting on the back, a clear acrylic sheet (for

viewing) on the right and left sides, and a fabric sleeve for
access on the front. Female mosquitoes should be collected from a

stock population cage in which both sexes have been maintained to

allow mating to occur. They should be host-seeking, of uniform

age, preferably 5–7 days post-emergence (use different ages of

mosquitoes when it is more suitable for a particular species and

justify such use in the study report). Active host-seeking females

should be selected to ensure a good response from the test

mosquitoes using an aspirator or an appropriate airflow apparatus.

KAPLAN-MEIER SURVIVAL ANALYSIS

Based on a study conducted by Goel, Khanna, and Kishore

(2014), Kaplan-Meier estimate is one of the best options to be

used to measure the fraction of subjects living for a certain

amount of time after treatment. In clinical trials or community

trials, the effect of an intervention is assessed by measuring the

number of subjects survived or saved after that intervention over

a period of time. The time starting from a defined point to the

occurrence of a given event, for example death is called as

survival time and the analysis of group data as survival analysis.

This can be affected by subjects under study that are uncooperative

and refused to be remained in the study or when some of the subjects

may not experience the event or death before the end of the study,

although they would have experienced or died if observation

continued, or we lose touch with them midway in the study. We label

these situations as censored observations. The Kaplan-Meier

estimate is the simplest way of computing the survival over time

in spite of all these difficulties associated with subjects or

situations. The survival curve can be created assuming various

situations. It involves computing of probabilities of occurrence

of event at a certain point of time and multiplying these

successive probabilities by any earlier computed probabilities to

get the final estimate. This can be calculated for two groups of

subjects and also their statistical difference in the survivals.

This can be used in Ayurveda research when they are comparing two

drugs and looking for survival of subjects.

RELATED STUDIES

A study entitled “Mosquito Repelled by Tomato-Based

Substance; Safer, More Effective than Deet” illustrated how Dr.

Michael Roe from North Carolina State University discovered a

natural compound found in tomatoes can effectively repel insects

that the university patented the substance. The patent shows how

the product may be used against insects. Since the university has

a licensed right to produce the substance as insect repellents,

Insect Biotechnology Inc., a Durham company that has specialties

on developing biochemical insecticides. Dr. Roe and officials from

Insect Biotechnology Inc. are convinced that the substance named

IB-246 has the potential to replace DEET as the active ingredient

in most active insect repellents. DEET (short form N, N-diethyl-

meta-toluamide) is a chemical that is used by many. This was first

developed by the U.S. Department of Agriculture for the army in

1946. The Environmental Protection Agency (EPA) does not stress

any health concern regarding the regular use of DEET products, but

the use of these have been associated with rashes, swelling and

itching, eye irritation, and less frequently, slurred speech,

confusion, and seizure. Products containing large amounts of DEET

are known to be hazardous among children, and the EPA no longer

permits the claims on the products containing DEET that it is safe

for children. In line with this, recent research at Duke University

showed that frequent and prolonged use of DEET among rats caused

brain-cell death and permanent behavioral changes in the animal.

Roe stated that similar to DEET, IB-246 can repel insects

effectively, and on a scale used by the EPA to measure toxicity,

IB-246 is considered slightly safer. Upon testing and further

studying the product, Roe found out that the compound did not only

repel mosquitos, but ticks as well. Subsequent testing showed that

the substance also repels fleas, cockroaches, ants, and biting

flies, as well as insects that are agricultural pests. Since

Toxicology has been done, the product made a big step towards

commercialization.

An online article entitled “Tomato Leaf Extract Repels

Mosquitoes” features a study conducted by Benguet State University

(BSU) Students who tested the efficacy of diluted tomato leaf

extracts in keeping mosquitos away. The extract was diluted to 3

different amounts: 25%, 50%, and 75% of clean water. The three

dilutions were applied to three volunteers' hands and were placed

inside a box containing 30 mosquitos. The study concluded that 25%

of tomato leaf extract with 75% clean water had less mosquito

bites.

On Another research entitled “Application of Tomato Leave

Extract as Pesticide Againstaphis Gossypii Glover (Hemiptera:

Aphididae),” it evaluated the unused parts of tomato plant leaves

as a botanical pesticide. The cotton aphid, Aphid Gossypii Glover

(Hemiptera:Aphididae) is considered to be one of the most serious

pest all over the world. These pests transmit pathogenic viruses

and have a wide host range. Tomato extracts contain phenolic and

flavanoid compounds which were effective upon experiment. In the

study, concentrations used were 250, 500, 750, and 1000 ppm. The

study showed that the higher the concentration level, the higher
the mortality which was 27.5% at 250 ppm and reached 80% at 1000

ppm. The chemical composition of tomato extract was characterized

by GC/MS Analysis that showed the presence of 46 compounds, Pythol

being the major compound containing 16.03%.

On a study entitled “Exploring Wild Tomato Leaf Extracts in

Pesticide Formulations” by Antonious, G. (2016), he argued that

Crude extracts from the leaves of the wild tomato, Lycopersicon

hirsutum f. glabratum accession PI 134417 possess a pestresistance

mechanism in their glandular trichomes (plant hairs) and the

exudates they produce due to the presence of a mixture of methyl

ketones (MKs) that has insecticidal and acaricidal properties.

Type IV and VI glandular trichomes on the leaves of PI 134417 grown

under greenhouse conditions were counted. Major volatile oils from

glandular leaf trichomes were extracted, purified, and quantified

using gas chromatography (GC) and mass spectrometry (GC/MS). The

main objectives of this investigation were to: 1) monitor seasonal

glandular trichomes density on PI 134417 for mass production of

MKs and 2) prepare a simplified formulation of MKs for potential

use of PI 134417 leaf extracts, which could become a valuable

source of natural products, in plant protection against spider

mites and cowpea aphids. Type IV and type VI trichomes were

greatest (P<0.05) in September and October (97 and 238 trichomes

mm-2), respectively. Quantification of MKs in emulsified extracts

of PI 134417 revealed the dominance of 2-tridecanone (the 13-

carbon methyl ketone) compared to other MKs (2-undecanone, 2-

dodecanone, and 2-pentadecanone) on the leaf surface. Crude leaf

extracts of PI 134417 prepared in water caused 33 and 22%

mortality, whereas, water extracts containing 1% Alkamuls (an

organic emulsifier) caused 93 and 82% mortality of spider mites

and cowpea aphis, respectively, 1 d after exposure.

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