Azadirachta Indica (Neem Tree) extract mixed with

Cymbopogon Citratus (Lemongrass) extract as an alternative

acaricide.

A Research Paper
Presented to
Ms. Nova Samonte Sebarios
Science Department
Mactan National Highschool

In the Partial Fulfillment

Of the
Requirements in Research II
Ed Ashley Nicole Duhaylungsod
Khristine Mae Sombelon
Angel Mae Senerpida
(Researchers)

Special Science Class X

S.Y 2022-202
 BIOGRAPHICAL DATA

Researcher: Ed Ashley Nicole Duhaylungsod

Age:16

Address: Angasil Mactan LLC

Date Of Birth: January 11, 2007

Mother: Michell Duhaylungsod

Father: Edwin Tampus

Siblings: Dwin Emmanuel Tampus

Contact no.09517850878

Email Address: ashleynicoleduhaylungsod@gmail.com

Motto: Just keep pushing forward and never give up.

 BIOGRAPHICAL DATA

Researcher: Khristine Mae Sombelon

Age: 16

Address: Gun-ob, Ampallaya Village

Date Of Birth: April 13, 2007

Mother: Josephine Sombelon

Father: Gleen Sombelon

Siblings: Josh Lynn Sombelon

Contact no. 09753203798

Email Address:

Motto: Hope for the best, Prepare for the worst

BIOGRAPHICAL DATA
Researcher: Angel Mae G. Senerpida

Age: 16 years old

Address: Mactan Lapu-Lapu City, Cebu

Date Of Birth: August 23, 2006

Mother: Mariza G. Senerpida

Father: Bobby I. Senerpida

Siblings: King Isaac G. Senerpida

Contact no. 09604372941

Email Address: gelaysenerpida@gmail.com

Mottto: Stop when you’re done, not when you’re tired.

ACKNOWLEDGMENT
We, the researchers, would like to express our gratitude to the following people for making our

study possible:

Nova S. Sebarios, Master Teacher and our SIP adviser, for sharing her ideas, guidance,

encouragement, and continued support throughout the development of the study.

Nym Cosep, Science Teahe and Chemistry Laboratory Custodian, and Alma V. Diola, Science

teacher and the Biology Laboratory Custodian, for letting us use the laboratory tools and

equipment for our experimentation.

Our Class advisers, classmates, and co-students in Mactan National High School for their moral

support.

Our Parents, for their love, encouragement, guidance, inspiration, financial and moral support

and understanding.

And above all, the Almighty GOD,whose blessings and guidance give us courage,determination,

strength, and wisdom to do or best in every educational endeavor that we do.

The Researchers
Ed Ashley Duhaylungsod, Angel Mae Senerpida, and Khristine Mae Sombelon., 2023.

Azadirachta Indica mixed with Cymbopogon Citratus as an alternative acaricide. A Science

Investigatory Project, Mactan National High School, Mactan, Lapu-Lapu City, Cebu.

ABSTRACT

Rhipicephalus sanguineus also called brown dog tick known to infest homes,particularly those

where dogs are kept both indoors and outdoors. This study aimed to investigate the acaricidal

potential of Azadirachta indica and Cymbopogon Citratus crude leaf extract against dog ticks.

Acaricide obtained to observe the mortality rate and exposed to different treatments within 2

days/48hrs .The bioactive compounds in both plant samples are Credelio , Geraniol , Citronellol

and Bravecto. Five treatments were prepared: 100%,75%, 50% and 25% concentrations of crude

leaf extract, Doogies Choice (positive control). Three trials per treatment were conducted. The

mortality rate against dog ticks when exposed to 100% & 75% of crude leaf extract demonstrates

that it can effect the mortality rate of ticks . Therefore, the crude leaf extract of Azadirachta

indica and Cymbopogon citratus has acaricidal potential and can be used as an alternative

treatment for dog ticks.

 BIBLIOGRAPHY

Online Journals

•Prakash Ranjan Dutta1 , Jayanta Kumar Chamuah2 , Akhilesh Kumar3*, Runjun Dowarah4 and

Meraj Haider Khan5 1Comparative Efficacy of Herbal (Lemon Grass Oil and Litsea Grass oil)

and

Synthetic (Deltamithrin, Fipronil, Amitraz) Agents against Rhipicephalus

microplus Infestation in Mithun (Bos frontalis),(2018),221

•panelS.Y. Landau a, F.D. Provenza b, D.R. Gardner c, J.A. Pfister c, E.L. Knoppel c, C.

Peterson c, D. Kababya d, G.R. Needham e, J.J. Villalba bNeem-tree (Azadirachta indica Juss.)

extract as a feed additive against the American dog tick (Dermacentor variabilis) in sheep (Ovis

aries),Volume 165, Issues 3–412 November 2009, Pages 311-317

•Lorena Lopes Ferreira a 1, Jaires Gomes de Oliveira Filho a 1, Gabriel Moura Mascarin b,

Adalberto A. Pérez de León c, Lígia Miranda Ferreira Borges In vitro repellency of DEET and β-

citronellol against the ticks Rhipicephalus sanguineus sensu lato and Amblyomma

sculptumVolume 239 30 May 2017, Pages 42-45

•Wengenmayer, C., Williams, H., Zschiesche, E. et al. The speed of kill of fluralaner

(Bravecto™) against Ixodes ricinus ticks on dogs. Parasites Vectors 7, 525 (2014).
•Murphy, M., Cavalleri, D., Seewald, W. et al. Laboratory evaluation of the speed of kill of

lotilaner (Credelio™) against Ixodes ricinus ticks on dogs. Parasites Vectors 10, 541,2017

Websites

“Azadirachta indica,” description of neem, https://en.wikipedia.org/wiki/Azadirachta_indica

“Azadirachta indica,”classification of neem,

https://sites.google.com/site/neemdatabase1/introduction/classification

“Azadirachtaindica,”geographicaldistribution,https://www.daf.qld.gov.au/__data/assets/
pdf_file/0006/63168/IPA-Neem-Tree-Risk- Assessment.pdf

“Azadirachtaindica,”ecology,
http://worldagroforestry.org/treedb/AFTPDFS/Azadirachta_indica.PDF.

“Azadirachtaindica,”botanicaldistribution,http://www.worldagroforestry.org/treedb/AFTPDFS/
Azadirachta_indica.PDF

“Azadirachta indica,” Leaves https://lra.le.ac.uk/bitstream/2381/30256/1/U533

"Azadirachta Indica"Taxonomy https://en.m.wikipedia.org/wiki/Azadirachta_indica

"Cymbopogon Citratus" description of lemongrass

https://plantvillage.psu.edu/topics/lemon-grass/infos

"Cymbopogon Citratus" classification of lemongrass

https://en.m.wikipedia.org/wiki/Cymbopogon#:~:text=Cymbopogon%2C%20also%20known
%20as%20lemongrass,plants%20in%20the%20grass%20family.

"Cymbopogon citratus"geographical distribution of lemongrass

https://nhb.gov.in/model-project-reports/Horticulture%20Crops/Lemongrass/Lemongrass1.htm

"Cymbopogon Citratus" ecology

https://www.balkep.org/cymbopogon-citratus.html

"Cymbopogon citratus"botanical
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3217679/#:~:text=Cymbopogon%20citratus
%2C%20Stapf%20(Lemon%20grass)%20is%20a%20widely%20used,%2C%20ketones%2C
%20aldehyde%20and%20esters.
Cymbopogon citratus "leaves
https://www.thespruceeats.com/what-is-lemongrass-765171

Taxonomy
https://en.m.wikipedia.org/wiki/Cymbopogon#:~:text=Cymbopogon%2C%20also%20known
%20as%20lemongrass,plants%20in%20the%20grass%20family.

TABLE OF CONTENTS

Title Page
TITLE PAGE i
BIOGRAPHICAL DATA iii
ACKNOWLEDGEMENT vi
ABSTRACT vii
ISEF FORMS viii
RESEARCH PLAN xiv
TABLE OF CONTENTS xx
LIST OF TABLES xxi
LIST OF FIGURES xxii
CHAPTER I. INTRODUCTION
Statement of the Problem 2
Hypotheses 2
Significance of the Study 2
Scope and Limitation 3
Definition of Terms 4
CHAPTER II. REVIEW OF RELATED LITERATURE
The Plant Sample 6
Related Studies on Phytochemical Constituents of Plant Samples 13
The Test Organisms 15
The Positive Control
17
 Conceptual Framework 18
CHAPTER III. METHODOLOGY
Research Design 19
Materials, Tools and Equipment 19
Collection of Plant Materials 20
Preparation of Leaf Extract 20
Preparation of Different Treatments/Concentrations 20
Statistical Analysis 22
Flow Chart 23
CHAPTER IV. RESULTS AND DISCUSSION
Analysis of Acquired Data 26
CHAPTER V. SUMMARY, CONCLUSION AND RECOMMENDATIONS
Summary 28
Conclusion 28
Recommendations 29
BIBLIOGRAPHY 30
APPENDIX
Appendix A. Calculation, Formula and Concentrations of Leaf Extracts and Controls 32
Appendix B. Statistical Analysis of Data 33
Appendix C. Photos 34
PHOTOCOPY OF PROJECT DATA BOOK

LIST OF TABLES
TABLE DESCRIPTION PAGE
1 Zone of Inhibition of the Mortality Rate 33
2 Average Zone of inhibition of mortatily rate 35

LIST OF FIGURES
TABLE DESCRIPTION PAGE
1 Conceptual Framework 29
2 Flow Chart of Methodology 33
3 Average zone of inhibition of the Mortality Rate 36
4 Area within Angasil and Bankal where leaves of 41
Azadirachta indica and Cymbopogon citratus were collected
5 Collection of Rhipicephalus sanguineas 41
6 Preparation of Azadirachta indica and Cymbopogon 42
Citratus crude leaf extract
7 Preparation of Treatments 42
8 Observing the Mortality rate 43
 RESEARCH PLAN
RATIONALE

This preliminary study will investigate the acaricidal potential of the combine crude leaf

extract of two locally available plants, Azadirachta indica and Cymbopogon Citratus, against

Rhipicephalus Sanguineas, Rocky Mountain Spotted fever, Ehrlichiosis (Ehrlichia Canis), and

Lyme disease causes the dogs to be infected or sick. The experiment attempts to provide a

scientific basis in using the crude leaf extract as alternative topical treatment for Rhipicephalus

Sanguineas to those that cannot afford the over-the-counter dog shampoos. The plant samples

will be collected within the vicinity of Angasil Mactan, Lapu-Lapu City and Bangkal Mactan,

Lapu-Lapu city. The Rhipicephalus Sanguineas will be used in the study and will be collected

through swabbing a dog with rashes. The combined crude leaf extract of both plants will be

extracted using a muslin cloth. The experiment will use 5 treatments, four of which consist of the

different concentration of crude leaf extract and Doggies Choice (over-the-counter dog shampoo)

for the positive control. Three trials per treatment will be made. This study will be measured and

observed for 48 hours.

HYPOTHESIS

There is a significant mortality rate observed on the Dog Tick in all treatments using crude

leaf extract of Azadirachta indica and Cymbopogon Citratus which may suggest that the crude

leaf extract has acaricidal potential.

 RESEARCH QUESTIONS

 Can Azadirachta Indica mixed with Cymbopogon Citratus crude leaf extract be used as

alternative acaricide for Rhipicephalus Sanguineas?

 Does Azadirachta Indica mixed with Cymbopogon Citratus crude leaf extract affects the

mortality or condition of the Rhipicephalus Sanguineas?

 What is the maximum inhibitory concentration and minimum concentration of the

Azadirachta Indica and Cymbopogon Citratus crude leaf extract?

ENGINEERING GOALS

The goal of this is to investigate the acaricidal potential of Azadirachta Indica

mixed with Cymbopogon Citratus crude leaf extract against Rhipicephalus Sanguineas.

EXPECTED OUTCOME

•It is expected that Azadirachta Indica mixed with Cymbopogon Citratus crude leaf

extract has acaricidal potential against Rhipicephalus Sanguineas.

•The Azadirachta Indica mixed with Cymbopogon Citratus crude leaf extract affects the

mortality or condition of the Rhipicephalus Sanguineas.

•The Azadirachta Indica mixed with Cymbopogon Citratus crude leaf extract exhibit a

maximum inhibitory concentration and inhibitory concentration against the mortality or

condition of Rhipicephalus Sanguineas..

 PROCEDURES/RESEARCH METHOD

Collection of Plant Materials

Fresh leaves of Azadirachta indica and Cymbopogon Citratus will be collected

within the vicinity of Angasil Mactan, Lapu-Lapu City ang Bangkal Mactan, Lapu-Lapu

City. The collected leaves of Azadirachta indica and Cymbopogon Citratus will be

prepared for extraction.

Preparation of Crude Leaf Extract

The Azadirachta Indica and Cymbopogon Citratus will be separated. 245 grams

of fresh leaves from each sample will be weighed and washed with distilled water. The

plant materials will be pounded using a clean mortar and pestle. The pounded leaves will

then be put in muslin cloth and squeezed to obtain the crude leaf extract. The obtained

extract will be considered as the stock solution. The obtained extract from each plant

sample will be combined (1:1 ratio) and will be considered as the stock solution.

Preparation of Different Treatments/Concentrations

Five treatments or concentrations from the stock solution will be prepared. A

100% concentration (Treatment 1) will be made from the stock solution by measuring 20

ml of the obtained extract. Other concentrations will also be made from the stock solution

to produce 75% (Treatment 2), 50% (Treatment 3), and 25% concentrations (Treatment

4). Positive control (Doggies choice) will be prepared.

Collection and Streaking of Test Organism

Rhipicephalus Sanguineas, also known as the pantropical dog tick, kennel tick, is

a type of tick that may be found all over the world but is more prevalent in hotter areas.

The brown dog tick is distinct from other species of ticks in that it can complete its whole

life cycle indoors. It can be identified by its reddish-brown color, elongated body shape,

and hexagonal basic capitula (flat surface where mouthparts are attached). Adults range

in size from 1.11 to 1.68 mm in width and 2.28 to 3.18 mm in length. The brown dog tick

is known to infest human houses, especially where dogs live both indoors and outdoors.

Rhipicephalus Sanguineas is the most common tick in the world and a well-recognized

vector of many illnesses affecting dogs and occasionally humans.

Statistical Analysis

The data obtained will be subjected to statistical analysis. One-way ANOVA will be used

for the interpretation of the data to ascertain if prominent differences occur in the

measured mortality between the five applied treatments: 100% crude leaf extract, 75%

crude leaf extract, 50% crude leaf extract, 25% crude leaf extract, and positive control

(doggies choice).
 CHAPTER I

INTRODUCTION

Rhipicephalus Sanguineas, also called brown dog tick, is a vector of several pathogens

causing dog diseases including Rocky Mountain Spotted fever, Ehrlichiosis (Ehrlichia Canis),

and Lyme disease. This disease is caused by a bacterial disease spread through the bite of an

infected tick. In general, ticks can be found in and around the ears, around the eyelid, under the

collar and under the front legs of your dog.

The three diseases most commonly implicated in Dogs are Rocky Mountain Spotted Fever,

Ehrlichiosis (Ehrlichia Canis) and Lyme disease. The growth of these ticks can be controlled by

keeping the dogs clean and be treated by continuous use of over-the-counter dog shampoos

containing citronellol, credelio, geraniol , or bravecto.

Most fur parents with infected dogs do not give much attention to its treatment due to

unaffordable over-the-counter dog shampoos and lack of cheaper options for its treatment.

Though there has been know remedies for ticks in dogs, such as Orange and Lemon extract and

tea tree oil, these plants are not readily available in the community and can be very expensive.

Hence, in an attempt to provide an alternative treatment to Rocky Mountain Spotted fever,

Ehrlichiosis (Ehrlichia Canis), and Lyme disease caused by dog ticks, this study was conducted.

This study investigates the acaricidal potential of the crude leaf extract of two known herbal

plants, Azadirachta indica (neem) and Cymbopogon Citratus(lemongrass), on the growth of dog

ticks in dogs causing them to get sick and infected.

 STATEMENT OF THE PROBLEM

The purpose our study is to investigate the effectiveness of Azadirachta Indica (Neem

Tree) mixed with Cymbopogon Citratus (Lemongrass) extract be used as an alternative acaricide

for Rhipicephalus Sanguineas (Dog Tick).

Sub-Problem:

 To determine the effective concentration of Azadirachta Indica and Cymbopogon

Citratus as an alternative acaricide.

 To compare the efficacy of Azadirachta Indica and Cymbopogon Citratus as alternative

acaricide to synthetic products.

 To evaluate the effectiveness of Azadirachta Indica and Cymbopogon Citratus mixed

with lemongrass as an alternative acaricide

HYPOTHESIS

There is no significant result of Azadirachta Indica (neem tree) mixed with Cymbopogon

Citratus (lemongrass) to the mortality of Rhipicephalus Sanguineas (dog tick).

There is significant effect of Azadirachta Indica (neem tree) mixed with Cymbopogon Citratus

(lemongrass) to the mortality of the Rhipicephalus Sanguineas (dog tick).

 SIGNIFICANCE OF THE STUDY

The significance of this study is to lessen the dog ticks that the dog has and to have a

healthy dog, also this study is to prevent dogs to get infected or sick. Having a pet is amazing

and can bring you happiness. This study benefits the people especially for those fur parents

because they’ll be getting a good quality, safer, healthier, and affordable acaricidal treatment for

their dogs. This study gives information to dog lover or those fur parents that this acaricidal

treatment for dog ticks can be a good treatment for them and making the dogs not to get infected

and a healthy life.

SCOPE AND LIMITATIONS

This study is only limited to the leaves of Azadirachta Indica and Cymbopogon

Citratus and not the seeds. This study can only be used by crude leaf extract. This study will only

be dealt with the 3 types of dog tick. The larvae, nymph, and adult tick sizes. The

experimentation process that is used in this study is by placing the ticks inside the petri dish with

a specific amount of extract. The effectiveness of this study will be observed for 2 days. This

study is only limited to the dog ticks itself and not directly to the dogs.

DEFINITION OF TERMS

For better understanding of the study, the following terms were operationally defined and used.
Azadirachta Indica- commonly known as neem, the leaves of this plant is investigated for its

acaricidal potential.

Bioactive Compound- components such as citronellol and credelio, found in the lemongrass and

neem tree leaves used in the study that has direct effect on the Dog ticks.

Citronellol- the active compound found in the lemongrass also known as tanglad known for

stapf as to contain phenolic compounds that are known to have antioxidant property.

Credelio- the active compound found in the neem tree also known for its local name in the

Philippines as margoso known for its antibacterial and antifungal properties.

Crude Extract- the leaf extract used in this study which was obtained by pounding the leaves of

neem tree and lemongrass.

Cymbopogon Citratus- commonly known as lemongrass the leaves of this plant is investigated

for its acaricidal potential.

Distilled Water- used on the solvent for the preparation of different treatments.

Dog Tick- are the pest organism used in this study specially the Rhipicephalus Sanguineas.

Extraction- a method for extracting citronellol, credelio, and geraniol from lemongrass and

neem tree leaves by using a muslin cloth.

Geraniol- the active compound found in the lemongrass also known as tanglad known for stapf

as to contain phenolic compounds that are known to have antioxidant property.

 CHAPTER II

REVIEW OF RELATED LITERATURE

A. Azadirachta Indica (Neem Tree)

Azadirachta Indica commonly known as neem tree & Indian Lilac. It is a tree

in the mahogany family Meliacea. It is one of two species in the genus Azadirachta

Indica. It is native to the northeast of the Indian subcontinent and Indochina but

naturalized and growth around the world in tropical and subtropical areas. Its fruit and

seeds are the source of neem oil.

B. Cymbopogon Citratus (Lemongrass)

Cymbopogon Citratus commonly known as tanglad, Andropogon Citratus. It is

a native aromatic tall sedge family. Lemongrass are barbed, wine grass, silky heads,
 cochin grass, Malabar grass or fever grass is a genus of Asian, African, Australia and

tropical island plants in the grass family.

TAXONOMY

Azadirachta Indica belongs to the family Meliaceae, The Neem Tree is described as

Azadirachta Indica as early as 1830 by De Jussien and in taxonomic

position belongs to order Rutales but recent literature describes that is belongs to order

Sapindales (Biswat et al 2002) as shown below.

KINGDOM: Plantae

DIVISION: Magnoliophata

CLASS: Magnoliopsida

ORDER: Sapindales

FAMILY: Meliaceae

GENUS: Azadirachta

SPECIES: Indica

SCIENTIFIC NAME: Azadirachta Indica

Cymbopogon CItratus (Lemongrass)

Lemongrass belongs to Family Poaceae. The lemongrass is described as Cymbopogon.

Cymbopogon is a genus of about 55 species of grasses, native to warm temperature and tropical

regions of the old world and Oceania.

KINGDOM: Plantae

DIVISION: Magnoliophyta

CLASS: Lilioprida

ORDER: Poales

FAMILY: Poaceae

GENUS: Cymbopogon

SPECIES: Citratus

SCIENTIFIC NAME: Cymbopogon Citratus

Geographical Distribution

Azadirachta Indica is native to the Indian Region and Banladesh in the Indian

subcontinent and to Cambodia, Laos, Myanmar, Thailand, and Vietnam in Indochina. It has been

widely introduced elsewhere in tropical and subtropical regions, from South America to

Indonesia, india is also native of Azadirachta. It is also cultivated in Nepal Pakistan Bangladesh

and Sri-Lanka. Even though neem trees are mostly in Southeastern parts of Asia, they are also

grown in Austrilia, Haiti, West Africa, Puerto Rico, and different parts of America such as

Florida, California, and Oklahma. It can be found from sea level up to an altitude of 1500 m in

places where average annual rainfall ranges from 400 to 1200 mm and where average annual

maximum temperatures may be as high as 40 C It can grow on wide range of soils, from acidic to

alkaline pH, but it does better on shallow, stony, sandy, poor soild, in marginal sloping places or

on rocky crevices. It is a full sunlight species, but it can withstand some shade in its first years.

This plant can ectract nutrients from highly leached sandy soils and can survive extreme pH
conditions, from 3 to 9. In well-drained soils, neem withstanding up to 2500 mm rainfall. It has

some tolerance of salinity and has been used in sugarcane plantation with significant soil salinity.

Cymbopogon Citratus (Lemongrass) is widely cultivated in the states of Kerala,

Karnataka, Tamil, Nadu in the southern region, parts of Uttaranchal in the northern region and

assam in the north-eastern region. At present, East Indian Lemongrass (C. flexuosus) is mainly

cultivated in the western part of India. It can be cultivated throughout Andhra Pradesh either as a

rainfed or irrigated crop in poor and marginal soils having pH 9.6. It is native to Asia (Indochina,

Indonesia, and Malaysia), Africa, and the Americas, but are widely cultivated in temperature and

tropical regions of the world (Jayasinha, 2001). Lemongrass is a tropical plant and as such will

grow best in warm, sunny, and humid conditions of the tropics and subtropics. The plants can be

grown at temperatures ranging from 10 to 33C (50-91.4F) but will grow optimally at

temperatures between 25 and 30C (77-8F) The rhizome is best divided in the spring in areas

where plant can be overwintered outdoors. In colder areas, the bulbous shoot base can be saved

after harvest and stored for use the following Spring. Lemongrass likes it hot, so grow it in an

area full sun and fertile, well-drained soil with a pH of 6.5 to 7.0. Space plants 24 inches apart.

Kick off the growing season by mixing several inches aged compost or other rich organic matter

into your native soil. Lemongrass is a perfect addition to a summer container garden. Growing it

in a pot is easy to move the plant indoors for the winter.

ECOLOGY

A. Azadirachta Indica
 The margosa tree is noted for its drought resistance. Normally it thrives in areas

with sub-arid to sub-humid conditions, with an annual rainfall of 400-1200 mm (1-47 in).

It can grow in regions with an annual rainfall below 400 mm, but in such cases, it

depends largely on ground water levels. Margosa can grow in many different types of

soil, but it thrives best on well drained deep and sandy soils. It is a typical tropical to

subtropical tree and exists at annual mean temperatures of 21–32 °C (70–90 °F). It can

tolerate high to very high temperatures and does not tolerate temperature below 5 °C (41

°F). Margosa is one of a very few shade-giving trees that thrive in drought-prone areas

e.g., the dry coastal, southern districts of India and Pakistan. The trees are not at all

delicate about water quality and thrive on the merest trickle of water, whatever the

quality. In India and tropical countries where the Indian diaspora has reached, it is very

common to see margosa trees used for shade lining streets, around temples, schools, and

other such public buildings or in most people's back yards. In very dry areas the trees are

planted on large tracts of land. The planting of Neem trees helps reduce greenhouse gases

through photosynthesis absorbing large quantities of CO2 and producing oxygen. Besides

neem has remarkable ability to withstand air and water pollution, as well as heat. Neem

also restores and maintains soil fertility.

B. Cymbopogon Citratus
 Lemongrass is a tropical herb that's perennial only in Zones 8 and warmer. In other

regions, grow lemongrass as an annual or in containers that you overwinter indoors (more

on that below). Lemongrass grows quickly and spreads to fill a planting bed or pot.

Expect a plant to reach a size of 3 to 5 feet tall and up to 2 feet wide in a growing season.

Look for lemongrass plants for sale in the herbs section of any garden center. Don’t be

afraid to start with a small pot. Lemongrass is a fast grower — you’ll have a nice clump

by midsummer in most regions. The leaves are blue green in color, turning red in the Fall

and emit a strong lemon fragrance when damaged. Lemongrass produces large compound

flowers on spikes when grown in the tropics, but rarely flowers when grown in more

Northern latitudes. Lemon grass can reach a height of 1.8 m (6 ft) and will grow for

several years, typically its economical lifespan is 4 years. Lemongrass is a tropical plant

and as such will grow best in warm, sunny, and humid conditions of the tropics and

subtropics. The plants can be grown at temperatures ranging from 10 to 33°C (50-91.4°F)

but will grow optimally at temperatures between 25 and 30°C (77–86°F). The grass will

grow in a wide range of soils but grows best in well-draining, fertile loam with a pH

between 5.0 and 8.4. The grass can also be successfully grown in containers. Plants can

be successfully grown in full sun or partial shade. Whether grown in the ground or in

containers, lemongrass is also a good companion for other plants due to its insect

repellent properties. The citronella in lemongrass repels mosquitoes, stable flies, and a

range of other insect pests like whiteflies and aphids.

BOTANICAL CHARACTERISTICS
 A. Azadirachta Indica

Neem trees are attractive broad-leaved evergreens that can grow up to 30 m tall

and 2.5 m in girth. Their spreading branches form rounded crowns as much as 20 m

across. They remain in leaf except during extreme drought when the leaves may fall

off.

Neem can take considerable abuse. For example, it easily withstands pollarding

(repeated lopping at heights above about 1.5 m) and its topped trunk resprouts

vigorously. It also freely coppices (repeated lopping at near-ground level). Regrowth

from both pollarding and coppicing can be exceptionally fast because it is being

served by a root system large enough to feed a full-grown tree.

B. Cymbopogon Citratus

Lemon grass is a tall, perennial sedge throwing up dense fascicles of leaves

from a short rhizome. The culm is stout, erect, up to 1.8 meter high. Leaves are long,

glaucous, green, linear tapering upwards and along the margins; ligule very short;

sheaths terete, those of the barren shoots widened and tightly clasping at the base,

others narrow and separating. It is a short-day plant and produce profuse flowering in

South India. The inflorescence is a long spike about one meter in length. Flowers

borne on decompound spatheate; panicles 30 to over 60 cm long.

LEAVES
 A. Azadirachta Indica

Neem tree are medium to large and elongatend to oblong in shape, averaging

20 to 40cm in length. The vibrant geen leaves are smooth and glossy with sharp,

serrated edge.Neem leaves grow on the branches of neem trees in groups of two, and

each branch produces around eight groupings.

B. Cymbopogon Citratus

Lemongrasses are bluegreen in color, turning red in the fall and emit a strong lemon

fragrance when damaged. Lemongrass produces large compound flowers on spikes when grown

in the tropics, but rarely flowers when grown in more Northern latitudes.

REVIEW OF RELATED STUDIES

According to ( Chaisri, Wasana, Chaisana, Wantida, Pikulkaew, Surachai, Okonogi, Siriporn ,

Surisayathaporn and Witaya ) Citronellol has the natural alternative resources as an alternative

acaricide.

It also has compounds to be used as acaricide (Chaisri, Wasana, Chaisana, Wantida, Pikulkaew,

Surachai, Okonogi, Siriporn , Surisayathaporn and Witaya ) Neem tree were conducted and

used to define the most suitable system for preparing citronellol. Lemongrass is also good in

microemulsions and had stronger acaricide efficacy in citronellol which it's bioactive compound

( Feb 2019 ) done

According to ( Prakash Ranjan Dutta, Jayanta Kumar Chamuah, Akhilesh Kumar, and Runjun

Dowarah ) Geraniol on 12 days of application on Rhipicephalus microplus resulted in very high

efficacy (98.00 %) acaricide and may be recommended for holistic management of nymphal
stages of tick infestation and can be proved as an alternative to synthetic acaricides without

adversely affecting health and environment.

( Meraj Haider Kha ) concludes that lemon grass could be used as a potential substitute to

synthetic acaricides due to natural origin, safe and environment friendly (June, 2018).

According to Rachel‐Ann Suraj, Rhea Rambarran, Krista Ali, Danyelle Harbajan, Roxanne

Charles, and Karla Georges, credelio acaricides may be abused and misused in a desperate

attempt to rid pets of ticks. Neem tree leaf extract (0.25%, 0.5% and 2%) for each trial.

Statistical analysis using the mixed‐effect Poisson regression analysis indicated that there was a

significant difference (p < .05) in the survival of ticks pre‐treatment versus post‐treatment

credelio and all controls when compared to the neem tree. Mortalities for neem leaf extract

ranged from 72.7% to 82% and 38% to 95.3%, respectively, with the greatest

percentagemortalities occurring at the lower concentrations. Neem tree extract can be used as

alternative acaricides( Candice Sant 2020 ).

TEST ORGANISMS

Rhipicephalus Sanguineas, also known as the pantropical dog tick, kennel

tick, is a type of tick that may be found all over the world but is more prevalent in

hotter areas. The brown dog tick is distinct from other species of ticks in that it can

complete its whole life cycle indoors. It can be identified by its reddish-brown color,

elongated body shape, and hexagonal basic capitula (flat surface where mouthparts

are attached). Adults range in size from 1.11 to 1.68 mm in width and 2.28 to 3.18

mm in length. The brown dog tick is known to infest human houses, especially where

dogs live both indoors and outdoors. Rhipicephalus Sanguineas is the most common
 tick in the world and a well-recognized vector of many illnesses affecting dogs and

occasionally humans.

POSITIVE CONTROL

PSIDIUM GUAJAVA FENOBUCARB BPMC GLIRICIDIA SEPIUM (Dog

shampoo) is recommended as a topical treatment for hair loss, itching, scabs, and

infections brought on by allergic diseases, such as flea allergy dermatitis, seasonal

allergies or atopy, food allergies, contact dermatitis from things like soaps and

perfumes, and sarcoptic mange (mites). The drug PSIDIUM GUAJAVA

FENOBUCARB BPMC, commonly known as Gliricidia sepium (Madre de Cacao),

is a potent anti-parasitic with anti-fungal characteristics.Madre de cacao is a popular

mange treatment that is safe for dogs. Once administered, Madre de Cacao functions

as a powerful anti-parasitic, effectively getting rid of fleas that cause mange. Its

negative impacts include the potential for sluggish and messy component extraction.

Doggies Choice, a 3-in-1 shampoo and conditioner, is recommended as an acaricidal

therapy for dog ticks. It aids in scabies defense, tick and flea infection avoidance, and

other skin disease avoidance. Guava extracts, a natural antibacterial that keeps pets

free of bacteria, are included in Doggies' Choice, as well as Maca extracts, a parasite

and fungal repellent.

 CONCEPTUAL FRAMEWORK

This study is focused on the preliminary investigation of the acaricidal

potential of Azadirachta indica (neem) and Cymbopogon Citratus(lemongrass) crude

leaf extract. The standard procedures were followed in the collection of plant

specimen, leaf extraction, preparation of the treatments and in the evaluation of the

acaricidal activity of Azadirachta indica (neem) and Cymbopogon Citratus

(lemongrass) crude leaf extract.

There were four treatments prepared from the stock solution of Azadirachta

indica (neem), and Cymbopogon Citratus (lemongrass) crude leaf extract, namely:

Treatment 1 (100% 20ml pure crude extract), Treatment 2 (75% 15ml pure crude

extract and 5ml distilled water), Treatment 3 (50% 10ml pure crude extract and 10ml

distilled water), and Treatment 4 (25% 5ml pure crude extract and 15ml distilled

water ). One treatment is also prepared for the positive control – Doggies Choice (dog

shampoo) Three trials per treatment were used to limit the errors and to test the

variability between samples. The paradigm, which explains the conceptual framework

in this study, is shown in Figure 1.

INDEPENDENT VARIABLE DEPENDENT VARIABLE

Treatments:

a.T1-100% crude leaf extract Zone of inhibition

b.T2-75% crude leaf extract exhibited by Dog Ticks

c.T2-50% crude leaf extract (Acaricidal Activity)

d.T4-25% crude leaf extract

e.Positive control(Doggies Choice)

Figure 1: Conceptual Paradigm Showing the Independent and Dependent Variables of the
Study

CHAPTER III
METHODOLOGY

RESEARCH DESIGN
This experimental study obtained crude leaf extract of Azadirachta indica and

Cymbopogon Citratus to be investigated for its acaricidal potential against Rhipicephalus

Sanguineas, Rocky Mountain Spotted fever, Ehrlichiosis (Ehrlichia Canis), and Lyme disease.

The plant samples were collected within the vicinity of Angasil Mactan, Lapu-Lapu City and

Bangkal Mactan, Lapu-Lapu City. Dog Ticks used in the study were collected through swabbing

a dog with rashes. Obtained samples were then put in a petri dish.. The experiment is made up of

five treatments, four of which consist of the different concentration of crude leaf extract. Doggies

Choice as the positive control. Three trials per treatment were made. The mortality was recorded

and observed as the acaricidal activity of the crude extract after 48 hours incubation.

Materials, Tools and Equipment

Materials:

Two hundred forty-five grams of each plant sample, Azadirachta indica and

Cymbopogon Citratus, were used for extraction. Over-the counter dog shampoo containing was

used as the positive control.

Tools and Equipments:

 Digital Scale, Tray, Test tube, Beaker (80 ml and 500 ml), Graduated Cylinder (10ml),

Test tube Rack, Test tube Holder, Test tube brush, Forceps, Petri Dish, Mortar and Pestle,

Muslin Cloth, Dropper, Filter Paper, Gloves, and Mesh dish were used in the study.

Collection of Plant Materials

Fresh leaves of Azadirachta indica and Cymbopogon Citratus were collected within the

vicinity of Angasil Mactan, Lapu-Lapu City and Saac Bangkal Mactan, Lapu-Lapu City. The

collected leaves of Azadirachta indica and Cymbopogon Citratus were prepared for extraction.

Preparation of Crude Leaf Extract

The Azadirachta Indica and Cymbopogon Citratus will be separated. 245 grams

of fresh leaves from each sample will be weighed and washed with distilled water. The plant

materials will be pounded using a clean mortar and pestle. The pounded leaves will then be put

in muslin cloth and squeezed to obtain the crude leaf extract. The obtained extract will be

considered as the stock solution. The obtained extract from each plant sample will be combined

(1:1 ratio) and will be considered as the stock solution.

Preparation of Different Treatments/Concentrations

Four treatments or concentrations from the stock solution will be prepared. A

100% concentration (Treatment 1) will be made from the stock solution by measuring 20 ml of

the obtained extract. Other concentrations will also be made from the stock solution to produce

75% (Treatment 2), 50% (Treatment 3), and 25% concentrations (Treatment 4). Positive control

(Doggies Choice) will be prepared.

Statistical Analysis

The data obtained was subjected to statistical analysis. One-way ANOVA was used for

the interpretation of the data to ascertain if prominent differences occur in the measured of

mortality between the five applied treatments: 100% crude leaf extract, 75% crude leaf extract,

50% crude leaf extract, 25% crude leaf extract, positive control (Doggies Choice).

Acaricide Bioassay

The prepared Azadirachta Indica and Cymbopogon Citratus extract will be placed in

a test tube, will be measured in a graduated cylinder, and combined together in a beaker. The

petri dish will be prepared with a filter paper. 6 dog ticks were picked by each size and kept into

the petri dish with the filter paper. Each treatment is tested at 5 applied treatments. 100% crude

leaf extract, 75% crude leaf extract,50% crude leaf extract,25% crude leaf, and positive control

(Doggies Choice). The experiment will be observed by 48 hours.

 FLOW CHART

Preparation
of
Materials and Equipment

Collections
Analysis of
of
Acquired Data
Plant Samples

Preparation
Statistical
of
Analysis
Crude Leaf
Preparation Extract
of
Different
Treatments
 Figure 2. Represents Flow Chart of Methodology

CHAPTER IV
RESULTS & DISCUSSION
The preliminary investigation done on the acaricidal potential of the combined crude leaf

extract of Azadirachta indica and Cymbopogon Citratus against Rhipicephalus Sanguineas.

showed a positive result. The effect of the crude leaf extract on the mortality of the dog tick was

exhibited by the mortality rate formed after the introduction of different treatments.

The mortality rate from three trials of 100% crude leaf extract, 75% crude leaf extract,

50% crude leaf extract, 25% crude leaf extract and controls were reflected in Table 1.

DAY 1 (TABLE 1)
Trial 1 Trial 2 Trial 3 Average

Treatment 1 5 5 5 77.78%
(100%)
Treatment 2 5 4 4 72.2%
(75%)
Treatment 3 4 3 3 55.56%
(50%)
Treatment 4 4 3 2 50%
(25%)
Positive 6 6 6 100%
Control

The data in Table 1 reveal that all treatments display acaricidal activity. The data also show that

the Dog ticks are highly sensitive to the different treatments of the combined crude leaf extract of

Azadirachta indica and Cymbopogon Citratus. This result supports that the crude leaf extract of
the plants under investigation has acaricidal potential against Dog ticks that causes Rocky

Mountain Spotted Fever, Ehrliciosis, and Lyme Disease to dogs and people.

DAY 2 (TABLE 2)
Trial 1 Trial2 Trial3 Average
Treatment 1 6 6 6 100%
(100%)
Treatment 2 6 6 6 100%
(75%)
Treatment 3 6 6 5 94.4%
(50%)
Treatment 4 6 5 5 88.89%
(25%)
Positive Control 0 0 0 0%

Table 2 shows the average zone of inhibition of Dog Ticks after being exposed to crude leaf

extracts at different concentrations. Among the four treatments, the 100% concentration and 75%

of crude leaf extract (pure crude leaf extract) gave the highest mortality rate activity compared to

other concentrations of crude leaf extract. Thus, the 100% concentration is the maximum

inhibitory concentration (MIC) of the crude leaf extract. On the other hand, the 25%

concentration is the minimum inhibitory concentration (MIC) of the crude leaf extract since it

exhibits the least mortality.

The data also describe the negligible difference of the inhibition activity of the positive control

and the pure crude leaf extract against dog ticks. This suggests that the 100% and 75%
concentration of crude leaf extract is as effective as that of Doggies Choice, the over-the-counter

acaricidal shampoo, used as the positive control.

Analysis of the Acquired Data

Established by the data acquired in the study, the crude leaf extract of Azadirachta indica and

Cymbopogon citratus has acaricidal potential against Rhipicephalus sanguineas At 100%

concentration, the acaricidal activity of crude leaf extract is comparable to that of the positive

control . The possible explanation for the acaricidal potential of Azadirachta indica and

Cymbopogon citratus is the presence bioactive compounds in both plant samples. These

substance are Citronellol,Credelio,Bravecto and Geraniol.

In statistical analysis, any insignificant value is high 0.05 level is considered as a

insignificant difference. One-Way ANOVA was used to analyze acaricidal potential of

Azadirachta indica and Cymbopogon citratus against acaricide. The statistical tool reveals a it

might not narrow down which of the pairs of treatments are significantly different.Even though

our data does not suggest the presence of significantly different treatments pairs in one-way

ANOVA,we proceed with the multiple comparison test. . The p-value of 0.2192, suggests

that Azadirachta indica and Cymbopogon citratus crude leaf extract is an effective alternative as

an acaricide treatment for dog ticks especially when used at 100% and 75%concentration.

The average of mortality rate displayed by the different treatments against dog ticks were shown

graphically in Figure 3. The graph showed that the concentration of the leaf extract has direct

effect on its acaricidal activity. Hence, the treatment 1 and 2 concentration of crude leaf extract,

the greater is it mortality rate activity.

 Figure 3.Average Zone of Inhibiti on of
Mortality Rate

100%
90%
80%
70%
60%
percentage

50%
40%
30%
20%
10%
0%
100% 100% 94.40% 88.89% 100%
100% 75% 50% 25% POSITIVE
CONTROL
Treatments

The bioactive compounds considered responsible for the acaricidal activity of Azadirachta indica

and Cymbopogon citratus crude leaf extract are the toxic compounds are

credelio,citronellol,bravecto and geraniol proven to exhibit acaricidal activity against ticks.

 CHAPTER V

Summary,

Conclusion, and Recomendations

SUMMARY

Crude Leaf extract of Azadirachta Indica and Cymbopogon Citratus were investigated for its

acaricidal potential; against Rhipicephalus Sanguineas that causes Rocky Mountain Spotted

Fever, Ehrlichiosis, and Lyme Disease to dogs and people. Azadirachta Indica and Cymbopogon

Citratus leaves were used since it has been known to contain bioactive compounds, citronellol,

credelio, geraniol, and bravecto, which are known as best compounds that can kill dog ticks.

Crude leaf extracts were obtained using mortar and pestle four concentrations were made out of

the obtained stock solution.

CONCLUSION

The exposure of dog ticks to different concentration of crude leaf extract revealed that these

Rhipicephalus sanguineus are highly sensitive to bioactive compound found in the leaf extracts

since the mortality rate was exhibited after 48hrs / 2days of incubation.

The greatest moratlity was observed in treatment 1 (100% concentration ) and positive control

(Doogies Choice).The displayed zone of inhibition in all concentration of crude leaf extracts

confirmed that the leaf extract of Azadirachta Indica and Cymbopogon Citratus has acaricidal

potential.The acaricidal activity exhibited by Azadirachta Indica mixed with Cymbopogon

Citratus crude leaf extract as an alternative acaricide.Though the result of the study showed

positive effect , proper processing is still needed to ensure its appropriateness as an alternative

acaricide treatments.

RECOMMENDATION

We also suggest to those who will conduct to this is to keep the dog ticks active during the

experimental and place it at the center on the filter paper inside the petri dish when putting the

treatments because they’re not could extraneous variable on it.In addition we also suggest in

collecting the dog ticks at its host is to keep them in a spaceous container with small holes for

them to breath,so when you start to experiment them the ticks are still alive in the container.In

last when collecting the dog ticks,we recommend you to have a more that just what you need

because they’re could be a chance that they will die inside the container or they might escape.

APPENDIX A

CALCULATION,FORMULA AND CONCENTRATIONS

OF CRUDE LEAF EXTRACT AND CONTROL

50ml of the stock solution > 100% concentration

( 25ml Azadirachta Indica and 25ml Cymbopogon Citratus )

FORMULA:
C1V1 = C2V2
Treatment 1 ( 100%)
20ml of crude leaf extract
2mL of dropper of 100% extract was used per trial

Treatment 2 ( 75%)
15ml crude leaf extract and 5ml of distilled water
2mL of 75% extract was used per trial

Treatment 3 ( 50%)
10ml of crude leaf extract and 10ml of distilled water
2mL of 50% extract was used per trial

Treatment 4 ( 25%)
5ml of crude leaf extract and 15ml of distilled water
2mL of 25% extract was used per trial

Positive Control
125 ml of Doogies Choice (over-the-counter antiparasitic-antifungal shampoo)
2mL of Doogies Choice Shampoo was used per trial

APPENDIX B

STATISTICAL ANALYSIS OF DATA

ANOVA: Single Factor

Table 1: Zone of Mortality Rate

Treatments: Trial 1 Trial 2 Trial 3 AVERAGE:

1 6 6 6 100%
 2 6 6 6 100%
3 6 6 5 94.45%
4 6 5 5 88.89%

Table 2: : One-way ANOVA Results

Source Sum of Degrees of Mean square F statistic p-value

square SS freedom MS
treatment 0.9167 3 0.3056 1.8333 0.2192
error 1.3333 8 0.1667
total 2.2500 11

APPENDIX C

PHOTOS
 Figure 4. Area within Angasil and Bankal where leaves of
Azadirachta indica and Cymbopogon citratus were collected

Figure 5. Collection of Rhipicephalus sanguineus ( Dog ticks)

Figure 6. Preparation of Azadirachta indica and Cymbopogon citratus Crude Leaf Extract
Figure 7. Preparation of Treatments

Figure 8.Observing of Mortality Rate