ACKNOWLEDGEMENT

“There are no secrets to success. It is the result of preparation, hard work and learning

from failure.”

The researchers would like to thank and give gratitude to the people who helped,

assisted, and encourage the researchers on the journey.

The researchers would like to acknowledge the individuals and the group of people who

made this research possible.

To Mr. Rolando M. Bulatao who gave the researchers the knowledge and thoughts to

accomplish the study.

To Ms. Mariel Regina Karla D. Corpuz who gave the primary idea for the researchers

the idea about the primary knowledge to the study.

To the peers and friends who supported him throughout the whole journey of the

research.

To God almighty for the strength, knowledge, and wisdom

Finally, the researchers would like to dedicate the success of the research to their family

for the support that the researchers have received.

-The researchers

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 TABLE OF CONTENTS

TITLE PAGE i

ACKNOWLEDGEMENT ii

TABLE OF CONTENTS iii

INTRODUCTION 1

Background of the study 1

Statement of the Problem 3
Hypotheses of the Study 3
Significance of the Study 3
Scope and Limitations 4
Review of Related Literature 4
Review of Related Studies 5
Definition of Terms 6
Research Paradigm 7
METHODOLOGY 8

Collection of Camachile Leaves 8

Extraction of Camachile Leaves 8
Preparation of Test Mosquito Larvae 8
Larvicidal Assay 8
Statistical Analysis 10

FLOW DIAGRAM OF EXPERIMENTAL PROCEDURE 11

BIBLIOGRAPHY 12

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INTRODUCTION

Background of the Study

Dengue fever is the widely cause of death in the Philippines. The risk of transmission

is highest during rainy season, which typically occurs on May to November. Department of

Health’s Disease Surveillance Report shows elevated dengue fever activity nationwide, with

51,622 dengue fever cases recorded Jan. 1- June 18 2022 (Crisis24, 2022). This was higher

compare to last year with 32,610 cases reported in 2021. Central Luzon is the most affected

(6,641 cases), followed by Central Visayas (6,361 cases) and Zamboanga Peninsula (4,767

cases). This report represents the most complete information available as of July 5 (Crisis24,

2022). Health authorities reported roughly 226,497 cases of dengue fever in the Philippines in

2022, 78,208 cases in 2021 and 90,135 in 2020. (Crisis24, 2022).

Dengue Malaria, and other mosquito-borne diseases are very harmful to humans. Some

patients die and some survives. But with today’s technology, the outbreak can be controlled,

lessened, or even stopped with the use of larvicidal products and insect repellents. However,

most of the mosquito repellents and larvicidal products are quite expensive and have a negative

impact on our environment.

The Philippines is known for having various species of wild plants. One of the wild

plants that can be found in the forests is the Camachile (Pithecellobium dulce) tree. The

Camachile is commonly used in cookery. The pods of the Camachile are used as an oil for

cooking. Moreover, based on the Philippine Medicinal Plants, it contains tannins. Tannins are

a class of astringent, polyphenolic biomolecules that can be used as an insecticide and larvicide.

Tannins have a larvicidal and repellent properties, tannins are used is mostly larvicidal products

(Silvateam, 2004). Animals that have been subjected to continuous administration

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of purified tannins showed damage in their liver and kidney tissue. However, humans revealed

that tannin-rich food is found to be safe for humans. Overconsumption may lead to negative

side effects. Tannins are toxic to insects due to oxidizing in insects with high pH guts, forming

semiquinone radicals and quinones, as well as other reactive oxygen species Tannins, in

particular, are endogenous inhibitors of the growth of numerous species of pests. They act as

an antibiotic or as an anti-nutritional deterrent against insects and aphids (SilvaTeam, 2023).

In addition, Camachile extracts containing tannins can be used as a natural larvicides.

Utilizing the Camachile extract as a natural larvicide could help not just the community

but also the government, in fighting and controlling the mosquito-borne diseases such as

dengue and malaria. The extract can be applied in stagnant water where the larvae of dengue

carrier mosquito. With that, the number of vectors can be controlled and also the victims of the

diseases can be lessened. Prevention through the use of natural larvicides from camachile

extract are far better than using costly larvicidal products. With the use of natural larvicide,

there will be no negative effect of it in the environment, so this will be safer and cost efficient.

Moreover, this natural larvicide can be a big help for the government and can show a lot of

differences when compared to the traditional toxic chemical approach to mosquito control,

that’s why this study is conducted.

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Statement of the Problem

This study generally aims to evaluate the Larvicidal activity of Camachile

(Pithecellobium dulce) leaf extract to yellow fever mosquito larvae.

Specifically, it is intended to answer the following:

1. Did the larvicide control the mosquito outbreaks?

2. Which treatment is the most effective in killing mosquito larvae?

3. Which is the most effective drying techniques for the leaves

Hypothesis of the Study

1. Yes, the larvicide will control the mosquito outbreaks

2. Treatment 4 (20% (6.0 mL extract) + 24 mL distilled water is the most effective.

3. The more effective drying method is oven drying in drying the leaves.

Significance of the Study

In the Philippines, dengue and malaria are regarded as one of the major health problems.

Over the past years, there’s been no change in the number of people who get sick. The battle

between mosquitoes and humans is constantly raging. Many people are bitten each year and

not a few of them die as a result. The government have tried to control the number of vectors

but failed miserably. Over the years many people have studied and done some experiments on

how to control or reduce the number of people who get sick. But still, some people pass away

because of how expensive the products are to cure or prevent the disease.

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 With the help of today’s advanced technology, the number of vectors as well as the

number of people who got ill due to the mosquito bites could be lessened because this study

aims to kill the larvae of the mosquito. Not so many natural larvicides are made to this date so,

producing one will provide a huge help to the community.

Scope and Limitations

This study will be focused mainly on evaluating the Larvicidal activity of

Camachile (Pithecellobium dulce) leaf extract to Yellow Fever Mosquito larvae. The camachile

leaves will be processed to produce the extract. In the drying process of the camachile leaves,

the drying method used is oven-drying. The researcher sets a limitation in the amount of tannin

extract that will be the most effective

This will be conducted at Philippine Rice Research Institute Laboratory, Barangay

Maligaya Science city of Munoz Nueva Ecija. The study will be conducted on August-

December 2023.

Review of Related Literature

Tannin, also called tannic acid, any of a group of phenolic compounds in woody

flowering plants that are important deterrents to herbivores and have a number of industrial

applications. As secondary metabolites, tannins are sequestered in vacuoles within the plant

cell, which protects the other cell components. They occur normally in the roots, wood, bark,

leaves, and fruit of many plants, particularly in the bark of oak (Quercus) species and in sumac

(Rhus) and myrobalan (Terminalia chebula). They also occur in galls, pathological growths

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resulting from insect attacks. Tannins may be classified chemically into two main groups,

hydrolyzable and condensed. Hydrolyzable tannins (decomposable in water, and react to form

other substances) yield various water-soluble products, such as gallic acid and protocatechuic

acid and sugars. Gallotannin, or common tannic acid, is the best known of the hydrolysable

tannins. It is produced by extraction with water or organic solvents from the galls of certain

trees, notably the Aleppo oak (Quercus infectoria) and Chinese nutgall (Rhus chinensis). Tara,

the pod from Caesalpinia spinosa, a plant indigenous to Peru, contains a gallotannin similar to

that from galls and has become an important source for refined tannin and gallic acid. The

European chestnut tree (principally Castanea sativa) and the American chestnut oak (Q.

Montana) yield hydrolysable tannins important in leather manufacture. Condensed tannins, the

larger group, form insoluble precipitates called tanner’s reds, or phlobaphenes. Among the

important condensed tannins are the extracts from the wood or bark of quebracho (Schinopsis),

mangrove (various genera and species), and wattle (Acacia).

Review of Related Studies

There are some studies which particulate in the decreasing number of mosquito larvae

of different larvicides. One of these studies is called “Elimination of mosquito larvae

through utilization of mosquito dunks out of tubli (Derris elliptica) extract” which is

accomplished by Aguirre (2015). The main objective for this study is to utilize the tubli extract

as mosquito dunks. Evaluation of larvicidal assays of different kinds of treatments was done to

identify the most effective for killing mosquito larvae. The treatment that has the highest

mortality rate was observed in treatment 3 (5%) and in treatment 4 (15%). The treatment 3

contained 5% aqueous extract while the treatment 4 has the combination of 15%

aqueous extract (100%) while the lowest was noted in the treatment 1 which is the control

group, the distilled water with only a mean of 1. One sample t-test revealed that T2 (positive

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control) T3 (5% aqueous extract), and T4 (15% aqueous extract) is not significantly different

to the T1 (negative control). While the treatment 1, the distilled water was significantly

different from treatment 2, treatment 3, and treatment 4. Therefore, the treatment 3 and 4 was

the most sufficient in eliminating larvae. This also indicates the effectiveness of the tubli extract

in eliminating mosquito larvae even in a small dilution or concentration. Furthermore, results

showed that there’s a 10% mortality rate in T1 (negative control), that is due to the

environmental factors (Aguirre, 2015).

Tirwani et al (2019) conducted a study entitled “Larvicidal Activity of Ketapang

Leaf Fraction (Terminalia catappa L) on Aedes aegypti Instar III Phytochemical screening

revealed a water-ethanol fraction of ketapang leaf contained tannin, saponin, and flavonoid

compounds. The water-ethanol fraction with 1800 ppm concentration possessed significant

larvicidal effect comparable to temephos. Probit test revealed an LC50 value of 1563.082

μg/ml.

Definition of Terms

Assay- a procedure for the examination or determination of quality or strength of a substance.

Dengue- is a mosquito-borne disease that occurs in tropical and subtropical areas of the world.

Extract- a substance produced by extracting a part of a raw material, often by using a solvent

such as water.

Larvicide- an insecticide that specifically targets the larval life of an insect especially

mosquitoes

Mortality rate- a measure of the number of deaths in a particular population scaled to the size

of the population, per unit of time.

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Tannins- also called tannic acid, any of a group of phenolic compounds in woody flowering

plants that are important deterrents to herbivores and have a number of industrial applications.

Vectors- a living organism that transmits an infectious agent from an infected animal to a

human or another animal.

Research Paradigm

The experimental variable in this study is the camachile tree. This will be developed

into larvicides. The controlled factors are the grinding process, and the time of oven drying.

the diagram below shows the factors that will be used in this study.

INDEPENDENT DEPENDENT VARIABLE

VARIABLES
Larvicidal activity of
Camachile Leaves Camachile Leaves

Extraneous Variables
• Grinding process
• Time of oven drying
• Type of solvent

Figure 1. The Research Paradigm of the Study

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METHODOLOGY

Collection of Camachile leaves

Fresh leaves of camachile leaves will be collected from Munoz, Nueva Ecija. The

leaves will be washed thoroughly 2-3 times with distilled water.

Extraction of Camachile leaves

The leaves of camachile were oven-dried and grinded in a mixer. 30 grams of grinded

leaves will be crushed in a 100 mL of 80% ethanol and placed in a water bath with 50-60°C

for 3 minutes and will be filtered through Whatman No. 1 filter paper. The filtrate will be

centrifuged at 2,500 rpm for 15 minutes and will be stored in sterile bottles at 4°C. (Aguirre,

2015)

Preparation of Test Mosquito Larvae

Black colored containers with water will be prepared. It will be placed in dark places

for a week. The containers will be checked for the presence of the mosquito larvae. The larvae

in the containers will be collected using a spoon and transferred into another container and

kept. (Aguirre, 2015)

Larvicidal Assays

The bioassays will be performed and carried out in four treatments in triplicate using

15 randomly chosen larvae for each replicate assay.

The treatment will be as follows:

T1 - Soaked with boiling water

T2 - Soaked with 80% ethano

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Table 1. Boiling water

Treatment Percentage of extract Component

T1 0 30 mL boiling water

T2 5 28.5 mL boiling water

T3 15 25.5 mL boiling water

T4 20 24 mL boiling water

Table 2. 80% Ethanol

Treatment Percentage of extract Component

T1 0 30 mL distilled water

T2 5 28.5 mL 80% ethanol

T3 15 25.5 mL 80% ethanol

T4 20 24 mL 80% ethanol

The collected larvae will be submerged in each conducted treatment. The mortality rate

will be measured and percentage mortality was computed using Abbott’s formula as shown

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below after 24 and 48 hours. The larvae will be considered dead if they are unrousable within

a short period of time even when gently prodded.

n in T after treatment
Mortality Rate (%)= (1 – ) 100
n in Co after treatment

Where: n = Insect population

T = treated

Co = control

Statistical Analysis

One sample LSD was used to determine the significant difference of the means to

determine significance among treatments using Statistical package for Social Sciences (SPSS

software.

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BIBLIOGRAPHY
Aguirre, Catherine May. 2015. “Elimination of Mosquito Larvae through Utilization of
Mosquito Dunks out of Tubli (Derris Elliptica) Extract,” December, 32.

“Mosquito-Borne Diseases - American Mosquito Control Association.” 2013. Mosquito.org.

2013.https://www.mosquito.org/page/diseases?&hhsearchterms=%22mosquito+and+
diseases%22.

“Philippines: Increased Number of Dengue Fever Cases Reported Nationwide through June.”
2022. Philippines: Increased Number of Dengue Fever Cases Reported Nationwide
through June | Crisis24. June 5, 2022.
https://crisis24.garda.com/alerts/2022/07/philippines-increased-number-of-dengue-
fever-cases-reported-nationwide-through-june.

Redo, Thaswin, Triwani Triwani, Chairil Anwar, and Salni Salni. 2019. “Larvicidal Activity
of Ketapang Leaf Fraction (Terminalia Catappa L) on Aedes Aegypti Instar III.”
Open Access Macedonian Journal of Medical Sciences 7 (21): 3526–29.
https://doi.org/10.3889/oamjms.2019.760.

Mukherjee, Pulok K. 2019. “Chapter 7 - Bioactive Phytocomponents and Their Analysis.”

Edited by Pulok K. Mukherjee. ScienceDirect. Elsevier. January 1, 2019.
https://www.sciencedirect.com/science/article/abs/pii/B9780128133743000077.

Izawa, Kunisuke, Yusuke Amino, Masanori Kohmura, Yoichi Ueda, and Motonaka Kuroda.
2010. “4.16 - Human–Environment Interactions – Taste.” Edited by Hung-Wen (Ben)
Liu and Lew Mander. ScienceDirect. Oxford: Elsevier. January 1, 2010.
https://www.sciencedirect.com/science/article/abs/pii/B9780080453828001088.

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FLOW DIAGRAM OF EXPERIMENTAL PROCEDURE

Collection of leaves samples

Sorting and washing

Oven-drying

Grinding

Crushed in 75 mL 80% Ethanol solution

Water bathing

Filtering

Centrifuging

Storing in sterile bottles

Experimenting Larvicidal Activity

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