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

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.

Many repellent products pertaining chemical compound like DEET (N,N-Diethyl-metatoluamide) 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 EPAs 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 peststheir 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

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. Input Tanglad oil on selected materials Process Presence or absence of insects on treated materials Output 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?

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 technicians 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.

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 diseasecarrying insects.

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.

Figure 2 Experimental Design Mosquitoes Minutes 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 14th 15th 16th 17th 18th 19th 20th Negative Control +/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/Positive Control +/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/Ants Negative Control +/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/Positive Control +/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/Cockroaches Negative Control +/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/Positive Control +/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/+/-

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 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 enough water 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 to 2 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.

Qualitative Analysis 1. Preliminary Chemical Test for the General Constituents of the Plant a. 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 few drops 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. Fehlings Test. Equal volumes (2 mL) of Fehlings Solution A and B were mixed and 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 Fehlings 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 Alkaloids a. Mayers Test. To about 5 mL of the oil, five drops of Mayers reagent was added. A cream colored precipitate indicates a positive result.

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b. Wagners Test. Another 5 mL of plant extract was added with 5 drops of Wagners reagent. 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 liquids surface and persists after 10 minutes.

Effectiveness Testing a. Mosquitoes A 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. Ants A 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. Cockroaches A 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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Data Gathering Tools Observation Data will be collected by monitoring materials treated with the formulated insect repellent made from Tanglad oil and the control, materials that are not treated. Treated materials will be exposed to an environment that has been a habitat for insects such as mosquitoes and flies. Data will be based on the presence or absence of insects and its density on both the control and the treated materials.

Treatment of Data T-test was used as a statistical tool by the researchers in the study to aid in testing hypothesis and arriving at a conclusion for the different problems. Two kinds of hypotheses were used namely the Null hypotheses (Ho), which stated that there was no significant difference between the means of the tests, and the alternative hypotheses (Hl), which stated that there was significant difference between the two or among the means compared. If the computed value is lesser than the tabular value, the null hypothesis is accepted. If the computed value is greater than the tabular value, the alternative hypothesis is accepted. To compare the effect of Tanglad oil as an insect repellent against mosquitoes, ants and cockroaches, the T-test for the insects means was used. The t-test was used in testing the significance of difference between means when the samples are small n < 30. The use of the Ttest involves the use of the degree of freedom of the distribution (df). The level of significance used is at 0.05.

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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 1. Litmus paper Actual Results Blue to red The blue litmus paper turned red which indicates that the sample is acidic 2. Ferric Chloride Test Blue-green The oil contain phenolic substances 3. Lead Acetate Test (-) The oil does not have sulfurcontaining component 4. Fehlings Test Brick-red precipitate The oil contain an aldehyde so 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 Fehlings 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 1. Mayers Test 2. Wagners Test Actual Results (-) (-) Interpretation The oil does not contain alkaloids The oil does not contain alkaloids

The oil gave negative results both for Mayers Test and Wagners 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 1. Froth Test Actual Result (-) Interpretation 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 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 14th 15th 16th 17th 18th 19th 20th General Mean Negative Control 5 6 8 7 6 9 12 20 20 13 22 24 21 25 27 23 26 22 26 28 17.5 Positive Control 1 1 1 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 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 14th 15th 16th 17th 18th 19th 20th General Mean Negative Control 2 3 5 9 10 12 18 15 17 18 20 22 20 21 25 26 27 30 26 28 17.7 Positive Control 1 1 1 1 2 2 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 2.3 Mean Number of Cockroaches on the Negative and Positive Control Minutes 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th 14th 15th 16th 17th 18th 19th 20th General Mean Negative Control 3 6 11 10 11 13 11 14 20 23 20 21 23 22 23 25 26 28 25 29 18.2 Positive Control 1 3 1 1 2 4 0.6

Table 2.3 shows the number of cockroaches 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 because there are lesser number of cockroaches found on the test substance
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Table 2.4 Comparison between the Negative and Positive control Insect Mosquitoes Ants Cockroaches Controls Negative and Positive Negative and Positive Negative and Positive Computed t value 9.4329 9.0683 10.1792 Decision on HO Reject HO Reject HO Reject HO

The general mean for the negative and test substance, for each insect was subjected to Ttest, 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 significant difference 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 difference among 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 significant difference 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 Mosquitoes and Ants 1 Mosquitoes and 2 Cockroaches Cockroaches and Ants 3 Computed t Value 1.447 1.677 0.6726 Decision on HO Accept HO Accept HO 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 no significant 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 is no 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 no significant 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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Figure N3. Number of Insects that had contact with Treated Material in a span of 20 minutes um ber of Ins ect s tha
1.5 4.5 4 3.5 3 2.5 2

t
1

ma
0.5

de con tact wit Number of Minutes

As seen in the graph, as the time progresses the insects are starting to make contact with h the treated material, this implies that the effect of Tanglad oil as an insect repellent diminish with the time. This can be attributed to the property of volatile oils to dissipate into the air ((Tyler, 1981). tre
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ate d

Figure 4. Total number of Insects that made contact with the treated material Nu mb er of tha t ma de co nta ct wit h the tre ate d ma ter ial

14 12 10 8 6 4 2 0 Mosquitoes Ants Cockroaches

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
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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 doesnt 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 wasnt able to get out it just stayed 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 better understanding 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 parasitic insects like Bed bugs. May also be tested on rodents. 4. Further studies may also make use of other natural substances that provides an insect repellent activity. 5. Further studies regarding the prevention of diseases may be conducted so that the world becomes a safer place for man to live in.

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