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CHAPTER I

INTRODUCTION

Background of the Study

Insects are very common vectors of disease. A vector is an organism which spreads

disease from one host to another. Invertebrates spread bacterial, viral and protozoan pathogens

by two main mechanisms. Either via their bite, as in the case of malaria spread by mosquitoes, or

via their faeces, as in the case of Chagas „ disease spread by Triatoma bugs or epidemic typhus

spread by human body lice.

Many insects are responsible for transmitting diseases. Mosquitoes are perhaps the best

known invertebrate vector and transmit a wide range of tropical diseases including malaria,

dengue fever and yellow fever. Another large group of vectors are flies. Sandfly species transmit

the disease leishmaniasis, by acting as vectors for protozoan Leishmania species, and tsetse flies

transmit protozoan trypanosomes (Trypanosoma bruceigambiense and Trypansoma

bruceirhodesiense) which cause African Trypanosomiasis (sleeping sickness). Ticks and lice

form another large group of invertebrate vectors. The bacterium Borrelia burgdorferi, which

causes Lyme disease, is transmitted by ticks and members of the bacterial genus Rickettsia are

transmitted by lice. For example, the human body louse transmits the bacterium Rickettsia

prowazekii which causes epidemic typhus.

Although invertebrate-transmitted diseases remain a major source of illness and death

worldwide, there is one way of controlling it, which is best achieved by avoiding infestation and

using insect repellents. Insect repellents are important public health tools for the prevention of

vector-borne infectious diseases. Actions to reduce vector-borne diseases can result in major


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health gains and relieve an important constraint on development in poor regions. In addition, this

insect repellents help to prevent and control the outbreaks of insect- borne diseases such as

malaria, Lyme disease, dengue fever, and bubonic plague. Insect repellents are an alternative to

the use of insecticides. They may be applied to the skin to protect an individual from the bites of

mosquitoes, mites, ticks and lice.

One of the natural insect repellents is the lemon grass (Cymbopogon citratus), a native

herb from temperate and warm region of South East Asian countries belonging to the family

Poaceae. Lemon grass is native to the Philippines where it is locally known as tanglad . It is a

tufted and perennial grass that grows to as high as 1 meter with leaves of 1 to 1.5 centimeters in

width that grows from a stalk of about 30 to 80 centimeters long with bulbous lower end.

The leaves of lemon grass yield a volatile oil rich (78%-82%) in citral, waxes, flavonoids

(luteolin, isoorientin and orientin), phenolic acids (chlorogenic, caffeic and p-coumaric acids),

triterpenoids such as cymbopogonol and cymbopogone. Lemon grass oil is extracted by steam

distillation. Lemon grass oil has a lemony, sweet smell and is dark yellow to amber and reddish

in color, with a watery viscosity.

Lemongrass is reportedly has a wide variety of therapeutic application. Lemongrass

contains substances that are used to relieve pain, reduce fever, stimulate the uterus and menstrual

flow, and have antioxidant property. Lemongrass is used as herbal medicine for gastro-intestinal

problems, diarrhea, gas, bowel spasms, vomiting, fever, flu, and headaches.

Lemongrass oil mixed with other essential oils such as coconut oil is used as liniment for

back pains, rheumatism, neuralgia, sprains and other body pains. It is useful with respiratory

infections such as sore throats, laryngitis, and fever and helps prevent spreading of infectious

diseases. Lemongrass is also used as an insect repellent.


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Many repellent products pertaining chemical compound like DEET (N,N-Diethyl-meta-

toluamide) used as active chemical ingredient in insect repellent, are now turn to all natural

ingredients as more and more negative data is released. Lemon grass is extremely safe and is

listed on the EPA‟s GRAS list (generally regarded as safe). People all over the world have come

to rely on the potent insect repellent properties of lemon grass and utilize it as a personal and

area spray.

Theoretical and Conceptual Framework

Mosquitoes are mainly considered to be nuisance pests — their bites causing a pruritic

irritation that itches for a day or two and then subsides. Until recently, there were no short- or

long-term sequels from such bites, aside from the rare case of cellulitis or scarring. On a global

basis mosquito-transmitted disease is responsible for a significant amount of morbidity and

mortality. In response, numerous measures have been used to protect people from mosquitoes.

In the recent years and advancement of technology, especially in science and medicine,

the rise of synthetic insect repellent forms have been used to prevent, protect and minimize the

fatalities caused by deadly mosquito strains. Many products have been formulated for such

incidents. These products include sprays, lotions, creams and sticks. Fumigation is also

employed for massive repelling action.

Paradigm of the Study

The study is represented in a paradigm shown in Figure 1. The independent variables are

the Tanglad oil and the commercially available insect repellent. The dependent variable is the


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presence or absence of insects on treated materials. The expected output is the efficacy of the

Tanglad oil as indicated by the absence of insects on the treated material.

Figure 1

Figure 1

Paradigm of the Study

Assumption of the Study

1. The chemical constituents of Tanglad oil can be identified using the different

phytochemical analysis.

2. The insect repellent activity of Tanglad oil can be determined through the observation

of data among actual test done on pests (mosquitoes, ants, cockroaches).

3. The time of efficiency of Tanglad oil in actual contact with the tested animals.

Statement of the Problem

This study aims to determine the effectiveness of Tanglad (Cymbopogon citratus) oil as an

insect repellent. Specifically, it aims to answer the following questions:

1. Will Tanglad oil able to repel insects in a significant manner?2. How long would be the protection time of Tanglad oil against insects?

Input

Tanglad oil onselected

materials

Output

Efficacy of theTanglad oil as

indicated by

the absence of

insects on the

treated

material.

Process

Presence orabsence of

insects ontreated

materials


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3. In which insect is Tanglad oil more effective as an insect repellent?

Statement of Hypothesis

The following hypotheses were formulated based on the stated research problems:

1. Tanglad oil is able to repel insects in a significant manner.2. Tanglad oil will repel insects in a significant amount of time.3. Tanglad oil is equally effective on all insects tested.

Scope and Limitation

The study is focused on the insect repellent activity of Tanglad oil and its effectiveness.

Chemical analysis and the test to determine the insect repelling activity is included in the study.

The Tanglad leaf was purchased at the public market of Baguio City. The extraction of

Tanglad oil was performed at the NSRU, Saint Louis University, Baguio City. To determine the

components of the oil, phytochemical analysis was accomplished at the NSRU, Saint Louis

University. Glassware and chemical reagents were procured from the Saint Louis University

chemistry technician‟s stockroom.

Other than the aforementioned analysis, another observation was made. The performance

of the Tanglad oil as an insect repellent was determined by observing chosen laboratory animals

with written procedures on testing included in this study. The data used in this research was

gathered through observation.


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Significance of the Study

The study was conducted to determine the effectiveness of Tanglad as natural insect

repellent. Extraction of lemon grass was through Steam Distillation Technique. Insect repellents

are important public health tools for the prevention of disease that can be transmitted by insects

(ie. Dengue, Malaria, Lyme disease). Through this study, the knowledge about the use of

Tanglad as insect repellent would be increased and prevention could be done to avoid disease-

carrying insects.


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CHAPTER II

DESIGN AND METHODOLOGY

This chapter presents the different methods and procedures that the researchers will

utilize in the study. It includes the research design, materials, procedures, data gathering tools,

and treatment of data collected from the effectiveness testing.

Research Design

The experimental type of research was used in the study because it aims to acquire

information through experimentation in laboratory conditions. The experimental methods were

utilized to test the hypotheses that were laid down in this research.

This study utilized the oil extracted from Tanglad (Cymbopogon citratus). The oil was

tested to determine the general constituents present.

Mosquitoes, ants and cockroaches are the insects that were used for this study for the

reason that they are the common insects that are present in the environment especially for

mosquitoes because it is the most common vector for a variety of diseases.

In the experimental design presented in the figure, each insect was treated individually

and was done in a span of 20 minutes to ensure accuracy.


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Figure 2

Experimental Design

Mosquitoes Ants Cockroaches

Minutes Negative

Control

Positive

Control

Negative

Control

Positive

Control

Negative

Control

Positive

Control

1st +/- +/- +/- +/- +/- +/-

2n

+/- +/- +/- +/- +/- +/-

3r

+/- +/- +/- +/- +/- +/-

4t

+/- +/- +/- +/- +/- +/-

5t

+/- +/- +/- +/- +/- +/-

6t

+/- +/- +/- +/- +/- +/-

7th

+/- +/- +/- +/- +/- +/-

8th

+/- +/- +/- +/- +/- +/-

9t

+/- +/- +/- +/- +/- +/-

10t

+/- +/- +/- +/- +/- +/-

11t

+/- +/- +/- +/- +/- +/-

12th

+/- +/- +/- +/- +/- +/-

13

t

+/-

+/-

+/-

+/-

+/-

+/-

14t

+/- +/- +/- +/- +/- +/-

15th

+/- +/- +/- +/- +/- +/-

16t

+/- +/- +/- +/- +/- +/-

17t

+/- +/- +/- +/- +/- +/-

18t

+/- +/- +/- +/- +/- +/-

19th

+/- +/- +/- +/- +/- +/-

20th

+/- +/- +/- +/- +/- +/-


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Collection and Preparation of Tanglad .

Tanglad leaves was purchased at the public market of Baguio City. Only green and fresh

leaves were collected, unwanted leaves were removed. Additionally, the leaves were clean to

prevent other organic substances to be extracted in the extraction process. Tanglad leaves will

be cut into small pieces (about ½ inch) prior to extraction.

Extraction of Tanglad Oil

Method: Steam Distillation

Procedure:

1. Assemble the distillation set-up. The 500 mL round bottom flask will be the distilling pot and 250 mL round bottom flask as receiving pot. The source of heat would be the

hot plate.

2. Add the chopped Tanglad leaves into the 500 mL round bottom flask. Add enoughwater so that the distilling pot is about 2/3 full. Turn on the condenser water and seal

the distilling pot.

3. Begin heating the system slowly. Adjust the heat so that the distilling rate is equal to2 drops per second. As the mixture boils and distils, water was lost from the distilling

pot. Add small volumes of water via the separatory funnel. It is important to maintain

the water level to avoid burning of the leaves. Keep the heat a low, steady level.

4. Collect about 300 mL of oil.5. Transfer the oil in a clean, amber bottle.6. Save the oil for further analysis.


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Qualitative Analysis

1. Preliminary Chemical Test for the General Constituents of the Planta. Litmus paper. A blue and red litmus paper were placed in a watch glass. A few

drops of the oil were dropped on these litmus papers. A blue to red change indicates

acidic substance and a red to blue indicates a basic substance

b. Ferric Chloride Test. About 1 mL of the oil was placed in a test tube and a fewdrops of Ferric Chloride TS. A blue green color indicates the presence of tannin,

tannin derivatives, phenol or/and phenolic substances.

c.

Lead Acetate Test. A few mL of the oil was added with a few drops of Lead Acetate

TS. Formation of precipitate indicates the presence of sulfur-containing substances.

d. Fehling’s Test. Equal volumes (2 mL) of Fehling‟s Solution A and B were mixedand 3 mL water was added. The mixture was heated in a water bath. When no brick

red precipitate is formed, 1 mL of the oil was added and heated over a water bath. A

brick red precipitate indicates the presence of a reducing substance. If no precipitate

is formed, acidify the oil with HCl then neutralize with NaOH solution. Perform

again the Fehling‟s Test to this solution, formation of a brick red precipitate indicates

the presence of one or more substances which produced by cleavage upon boiling

with acid reducing substances such as glycosides, glucosides and polysaccharides.

2. Test for the Presence of Alkaloidsa. Mayer’s Test. To about 5 mL of the oil, five drops of Mayer‟s reagent was added. A

cream colored precipitate indicates a positive result.


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b. Wagner’s Test. Another 5 mL of plant extract was added with 5 drops of Wagner‟sreagent. A brown colored precipitate indicates a positive result.

3. Test for the Presence of Saponins.a. Froth Test. In 5 mL of the oil in a test tube 10 mL of distilled water was added,

stopper and shake the test tube vigorously for 30 seconds. Allow to stand for 10

minutes and observe for a “honeycomb froth”. A positive result is obtained when the

honeycomb forth is greater than 2 cm height from the liquid‟s surface and persists

after 10 minutes.

Effectiveness Testing

a. MosquitoesA cage made of wood as a framework with screen on all sides having a dimension

of 50 x 50 x 50 cm was made. On one side it has an opening small enough to enter one

arm, where tested mosquitoes will be removed. Thirty mosquitoes were caught and

transferred to the cage.

For the negative control. A clean dry arm was entered in the cage. The

mosquitoes‟ response was observed if they were repelled or attracted to the arm. Presence

of mosquitoes on the arm was counted every minute and then shaken off. The procedure

was done in a span of 20 minutes. Data collected are recorded.

For the positive control. The same procedure was done but this time the arm was

sprayed with Tanglad oil.


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b. AntsA plant box made of white cardboard having a dimension of 50 x 50 x 8 cm with

its topside open.

For negative control. Biscuit crumbs was use to attract ants. The crumbs are

placed inside the box and the ants are placed in the opposite side. Presence of ants on the

crumbs were counted every minute and then the box was tapped to remove the insects

from the crumbs. This procedure was done in a span of 20 minutes. Data collected were

recorded.

For positive control. The same procedure was done but this time the crumbs was

sprayed with Tanglad oil.

c. CockroachesA plant box made of white cardboard having a dimension of 50 x 50 x 8 cm with

its topside open.

For negative control. Biscuit crumbs was use to attract cockroaches. The crumbs

are placed inside the box and the cockroaches are placed in the opposite side. Presence of

cockroaches on the crumbs were counted every minute and then the box was tapped to

remove the insects from the crumbs. This procedure was done in a span of 20 minutes.

Data collected were recorded.

For positive control. The same procedure was again done but this time the crumbs

was sprayed with Tanglad oil.

NOTE. Cover the topside of the box with a screen when performing with cockroaches to

prevent escape.


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CHAPTER III

DATA, RESULTS AND DISCUSSIONS

This chapter contains the data, results and observations gathered throughout the study.

The presentation is made through tables after which textual presentation of the data follows. The

first three tables present the results of preliminary tests done with the oil. The next two tables,

negative control and positive control, would be the detailed data on the different number of

insects that had contact with the untreated/treated material for every minute in a span of 20

minutes.

Preliminary Tests

1. Chemical Tests for the General Constituents of Tanglad oil

Table 1.1

Chemical Tests for the General Constituents of the Oil

Tests Actual Results

1. Litmus paper Blue to red The blue litmus paper turnedred which indicates that the

sample is acidic

2. Ferric Chloride Test Blue-green The oil contain phenolicsubstances

3.

Lead Acetate Test ( - ) The oil does not have sulfur-

containing component

4. Fehling‟s Test Brick-red precipitate The oil contain an aldehydeso it forms a precipitate


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After the extraction of Tanglad oil, it was tested with blue litmus paper. The blue litmus

paper turned red which indicate the presence of acid and acid salts in the oil.

The oil gives a blue-green solution with Ferric Chloride which indicated the presence of

phenolic substances.

With Lead Acetate, the oil did not produce precipitate. This indicates that the oil does not

have a sulfur-containing component.

For Fehling‟s Test, the oil produced brick -red precipitate. The precipitate is due to the

aldehyde content of the oil.

2. Tests for the Presence of Alkaloids in the Oil

Table 1.2

Tests for the Presence of Alkaloids in the Oil

Tests Actual Results Interpretation

1. Mayer‟s Test ( - ) The oil does not containalkaloids2. Wagner‟s Test ( - ) The oil does not containalkaloids

The oil gave negative results both for Mayer‟s Test and Wagner‟s Test which indicates

the absence of alkaloids.

3. Test for the Presence of Saponin in the Oil

Table 1.3

Test for the Presence of Saponin in the Oil

Test Actual Result Interpretation

1. Froth Test ( - ) The oil does not form bubbles


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therefore it does not contain

saponin

The oil gave a negative result for both tests indicating the absence of saponin

Effectiveness Testing

Table 2.1

Mean Number of Mosquitoes on the Negative and Positive Control

Minutes Negative Control Positive Control

1st 5 -

2n

6 -3

r 8 -

4t

7 -

5t

6 -

6t

9 -

7t

12 -

8t

20 -

9t

20 -

10th

13 -

11th

22 -

12t

24 -

13t 21 -14

t 25 -

15t

27 1

16t

23 -

17t

26 -

18t

22 1

19t

26 -

20t

28 1

General Mean 17.5 0.15

Table 2. shows the number of mosquitoes that had contact with the material and

also contains the mean for the two controls. The number of insects attracted to the test substance

is lesser than that of the negative control. It can be gleaned from the data that the positive


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control (test substance), shows a significant insect repellent activity as compared with the

negative control.

.

Table 2.2

Mean Number of Ants on the Negative and Positive Control

Minutes Negative Control Positive Control

1

st

2 -2n

3 -

3r

5 -

4t

9 -

5t

10 -

6t

12 -

7t

18 -

8t

15 -

9t

17 -

10t

18 -

11th

20 -

12th

22 -13

th 20 1

14t

21 -

15t

25 -

16t

26 1

17t

27 1

18t

30 1

19t

26 2

20t

28 2

General Mean 17.7 0.4

Table 2.2 shows the number of ants that had contact with the material and the mean for

the two controls. It can be gleaned from the data that the positive controls (test substance), show

a significant difference as compared with the negative control since there are lesser ants on the

test substance.


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Table 2.4

Comparison between the Negative and Positive control

Insect Controls Computed t

value

Decision on HO

Mosquitoes Negative andPositive 9.4329 Reject HO

Ants Negative andPositive

9.0683 Reject HO

Cockroaches Negative andPositive

10.1792 Reject HO

The general mean for the negative and test substance, for each insect was subjected to T-

test, and the computed t value was compared to the tabular t value to decide if the null hypothesis

which is that there are no significant difference among the means of the controls. The following

text presents the textual interpretations of the comparisons for each insect:

1. Mosquitoes. The decision is to reject the null hypothesis that there is no significantdifference among the means of the controls, since the computed value of 9.4329 is greater

than the tabular t value of 2.776 at the 0.05 level with 38 as the degree of freedom.


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This can be attributed to the composition of Tanglad oil on the arm that can repel

mosquitoes which is not present on the untreated arm in the negative control (Tyler,

1981).

2. Ants. The decision is to reject the null hypothesis that there is no significant differenceamong the means of the controls, since the computed value of 9.0683 is greater than the

tabular t value of 2.776 at the 0.05 level with 38 as the degree of freedom.

This can be attributed to the composition of Tanglad oil on the biscuit crumbs that can

repel ants which is not present on the untreated biscuit crumbs in the negative control

(Tyler, 1981).

3. Cockroaches. The decision is to reject the null hypothesis that there is no significantdifference among the means of the controls, since the computed value of 10.1792 is

greater than the tabular t value of 2.776 at the 0.05 level with 38 as the degree of

freedom.

This can be attributed to the composition of Tanglad oil on the biscuit crumbs that can

repel cockroaches which is not present on the untreated biscuit crumbs in the negative

control (Tyler, 1981).


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Therefore, the test substance which utilizes Tanglad oil showed a significant result.

Hence, the average number of insects that had contact with the treated material is significantly

different from the average number of insects that had contact with the untreated material. This is

only true at 0.05 level of significance.

Table 2.4

Comparison between Pairs of Insects

Pair No. Insects Computed t Value Decision on HO 1 Mosquitoes and Ants 1.447 Accept HO

2 Mosquitoes andCockroaches

1.677 Accept HO

3 Cockroaches and Ants 0.6726 Accept HO

The general means, for each insect of the positive controls are paired to determine to

which insect Tanglad oil is more effective as an insect repellent. Using the t-test method, the

computed t value was compared to the tabular t value to decide if the null hypothesis which is

there are no significant differences among the different pairs of insects on their response with

Tanglad oil. The following text presents the textual interpretations of the different comparisons:

1. Mosquitoes and Ants. The decision is to accept the null hypothesis that there is nosignificant difference between the means of Mosquitoes and Ants, since the computed t

value of 1.447 is less than the tabular t value of 2.776 at the 0.05 level with 38 as the

degree of freedom.

Both insects do not show significant difference in their response with Tanglad oil. Although,

mosquitoes may be more sensitive than that of the ants.


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2. Mosquitoes and Cockroaches. The decision is to accept the null hypothesis that there isno significant difference between the means of Mosquitoes and Cockroaches, since the

computed t value of 1.677 is less than the tabular t value of 2.776 at the 0.05 level with

38 as the degree of freedom.

Both insects do not show significant difference in their response with Tanglad oil. Although,

mosquitoes may be more sensitive than that of the cockroaches.

3. Cockroaches and Ants. The decision is to accept the null hypothesis that there is nosignificant difference between the means of Cockroaches and Ants, since the computed t

value of 0.6726 is less than the tabular t value of 2.776 at the 0.05 level with 38 as the

degree of freedom.

Both insects do not show significant difference in their response with Tanglad oil.

Although, ants may be more sensitive than that of the cockroaches, this can be attributed

to the high-definition sense of smell of ants that makes them more sensitive (Salisbury,

2012).


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um

ber

of

Ins

ect

s

tha

t

ma

de

con

tact

Figure 3. Number of Insects that had contact with Treated Material in a span of 20 minutes

Number of Minutes

As seen in the graph, as the time progresses the insects are starting to make contact with

the treated material, this implies that the effect of Tanglad oil as an insect repellent diminish with

time. This can be attributed to the property of volatile oils to dissipate into the air ((Tyler, 1981).

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Mosquitoes Ants Cockroaches


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Figure 4. Total number of Insects that made contact with the treated material

Insects

As seen in the graph, mosquitoes were more repelled than ants and cockroaches while

cockroaches were the least repelled by Tanglad oil. This can be attributed to the masked body

odor of the arm that makes the mosquitoes uninterested and their ability to fly away while for

ants they have a high-definition sense of smell that makes them sensitive to odor as compared to

0

2

4

6

8

10

12

14

Mosquitoes

Ants

Cockroaches

Nu

mber

of

tha

t

ma

de

co

nta

ct

with

the

tre

ate

d

ma


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cockroaches which has a lower sense of smell than that of ants and mosquitoes (Gilroy, 2012 and

Salisbury, 2012). Although, this doesn‟t show a significant difference as shown on Table 1.3.

Therefore, by basing the results from the table, tanglad oil is most effective against mosquitoes

and least effective against cockroaches.

CHAPTER IV

SUMMARY, CONCLUSIONS, AND REDOMMENDATIONS

This chapter provides the summary, conclusion, and recommendations of the study based

from the results obtained.

Summary

Insects are the common vectors of many diseases like dengue fever, malaria, etc., to

prevent such diseases the host should be able to repel these vectors. This study presents the

effectiveness of Tanglad (Cymbopogon citratus) oil as an insect repellent.

Specifically, this study aimed to answer the following questions:

4. Will Tanglad (Cymbopogon citratus) oil able to repel insects in a significant manner?5. How long would be the protection time of Tanglad oil against insects?6. In which insect is Tanglad oil more effective as an insect repellent?The experimental type of research was employed in the study. Through observation, data was

gathered.

T-test Method was used as a statistical method to compare the significant difference between

the three insects tested with their response to Tanglad oil. The data gathered were then presented

in a tabular manner.


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Findings

During and after the testing for effectiveness, data were obtained:

1. Tanglad oil is able to repel all the three insects in a significant manner.2. There is no significant difference between the responses of the three insects with the

materials treated with Tanglad oil.

3.

The effect of Tanglad oil decreases in time.

4. When we made a circle of Tanglad oil around an ant it wasn‟t able to get out it juststayed inside the circle.

Conclusions

The study of the insect repellent activity of Tanglad (Cymbopogon citratus) oil revealed

that it is capable of repelling insects. The three insects did not show significant difference as to

their response with Tanglad oil.

Recommendations

For further studies, basing on problems encountered and need for improvement; the

researchers would like to recommend the following:

1. Further studies must formulate Tanglad oil into a topical dosage form like cream,ointment or lotion to provide a longer protection time.


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2. A chemical assay of Tanglad oil will be presented so that we may have a betterunderstanding why such chemical agents are able to repel insects.

3. The oil may be tested also on other insects like flies and termites or on parasiticinsects like Bed bugs. May also be tested on rodents.

4. Further studies may also make use of other natural substances that provides an insectrepellent activity.

5. Further studies regarding the prevention of diseases may be conducted so that theworld becomes a safer place for man to live in.

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