In Vitro With New Template 2

DR. CARLOS S.
LANTING COLLEGE
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“In Vitro Acaricidal Activity of Pandan(Pandanus amaryllifolius) Leaf Extract Against Brown Dog
Tick(Rhipicephalis sanguineus) Nymphs”Agustin;Macarilao
CHAPTER I
THE PROBLEM AND IT’S BACKGROUND
In this section, the researcher gives a brief background and also information about the
study. The proponent will also present in this chapter the statement of the problem significance
of the study, and as well as the scope and limitation.
Background of the Study
Figure 1. Rhipicephalus sanguineus
The brown dog tick has the scientific name of Rhipicephalus sanguineus. These ticks
can be found on dogs living in both urban and rural areas, being highly adapted to live within
human dwellings and being active throughout the year not only in tropical and subtropical
regions, but also in some temperate areas. Depending on the factors such as climate and host
availability, Rhipicephalus sanguineus can complete up to four generations per year. This
observation suggests that the risk of human parasitism by Rhipicephalus sanguineus could
increase in areas experiencing warmer and longer summer like the Philippines (Parola et al.
2005, Vitale et al., 2006).
Rhipicephalus sanguineus has also been reported to vector the protozoan parasite
Hepatozoon canis, which causes canine hepatozoonosis in dogs (Baneth et al. 2001., Nordgren
& Craig., 1984) as well as Babesia canis also called canine babesiosis in dogs (Dantas-Torres,
2008).
DR. CARLOS S. LANTING COLLEGE
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Ticks are commonly controlled using chemical insecticide which has been considered as
the best method of controlling ticks. Insecticide can be toxic to animals and humans and are
harmful to the environment if they are not properly used. Moreover, resistance to insecticides
may also develop and it can be inherited by the next generations (George, 2000). In most areas
of the world, ticks have become resistant to various ectoparasites like arsenic and
organophosphates (Wharton, 1983). Thus, natural biodegradable insecticide of plant origin had
been an alternative.
Pandanus amaryllifolius was first decribed by Scottish botanist William Roxburgh in 1831
and revised by American botanist Benjamin C. Stone in 1978. Its leaves are used in food
preparation in Asian countries as flavoring agent. It was primarily used for giving a particular
fragrance to rice preparation. Later on it was used in bread preparation to give a typical nutty
flavor. The aroma compound is 2-acetyl-1-pyrroline derived from amino acid- phenylalanine.
Along with the aromatic properties, the leaves have compounds with antiviral and
antioxidant properties. This species also used in some traditional medicine such as remedy for
toothache and for decreasing glucose concentration or hypoglycemic effect, the rots extract are
used to cure thyroid problems. It also used for preparing lotion along with ash and vinegar to
treat measles, in the treatment of leprosy, sore throat and as diuretic in the Philippines.
Powdered Pandanus amaryllifolius leaves may be used against Callosobruchus chinensis,
an insect pest infestation of mung-bean seeds (Setyowati & Siemonsma, 1999). In addition,
Pandanus amaryllifolius is used as an environmentally friendly pest management tool to keep
out cockroach (Warke et al., 2012).
Statement of the Problem

This study will determine the acaricidal activity of Pandanus amaryllifolius
(Pandan) against Brown Dog Tick (Rhipicephalus sanguineus) Nymphs.
Specifically, it will answer the following questions:
1. What is the mortality rate of Rhipicephalus sangiuneus nymphs treated with different
concentrations of pandan ethanolic leaf extract at specific time interval?
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2. Which concentration of pandan ethanolic leaf extract is most effective in killing Rhipicephalus
sanguineus nymphs?
3. What is the lethal concentration (LC) of pandan leaf extracts in LC 50% and LC 90% mortality?
Significance of the Study

This study can be used to analyze the extent of the acaricidal activity of Pandanus
amaryllifolius in vitro. If the acaricidal activity of Pandan leaves against Rhipicephalus
sanguineus was conducted and successfully affect ticks, therefore the following persons will
benefit this study:
To pet owners specifically dog owners: The infected dog with Rhipicephalus sanguineus will
be successfully eradicate the ticks from them that causes rushes and other skin illnesses in their
pet with little or no expense at all. It also helps to avoid
diseases that people can get in ticks such as ehrlichiosis, babesiosis and Lyme disease.
To the community and environment: This help our community to realize that plants in the
environment are not only herbal plant, food flavorings or a display in our backyard but also
decrease the population of pests. It also helps the environment to develop of how plants in our
environment can alter into useful products.
To the future researchers: The result of this study will serve as their guide in conducting the
experiment related to this.
The result of the study can help in developing low cost, environment and effective vector
control strategies.
Scope and Limitations
The focus of this study is to determine the acaricidal activity of pandan leaf extract
against Rhipicephalus sanguineus (Brown Dog) tick only and not on the tick that was attached
to the skin of the dog. The data will be used in the research gathered through observation.
Pharmacological mode of action of the extract nor its pathologic effects on the tick was not
examined.
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CHAPTER 2
REVIEW OF RELATED LITERATURE AND STUDIES
This chapter is a combination of all the facts of the researcher’s gathered through blogs,
books, journals, and as well as magazines and online references that are related in this study
which will be very helpful to the proponents and could contribute knowledge regarding the
research.
Rhipicephalus sanguineus
Rhipicephalus sanguineus, commonly called kennel tick, or pan-tropical dog tick, is a
species of tick which is found worldwide, but more commonly in warmer climates (Gray, et al.,
2013). It is characterized by its small, red-brown in color which was in uniform color and the
reason why it also called red dog tick on other parts of the world. Its mouthparts are easily seen
viewed from above and the body is flattened associated with tear drop shape.
The brown dog tick (Rhipicephalus sanguineus) is the most widespread tick in the world
and a well-recognized vector of many pathogens affecting dogs and occasionally humans. This
tick can be found on dogs living in both urban and rural areas, being highly adapted to live
within human dwellings and being active throughout the year not only in tropical and subtropical
regions, but also in some temperate areas. Depending on factors such as climate and host
availability, Rhipicephalus sanguineus can complete up to four generations per year. Recent
studies have demonstrated that ticks exposed to high temperatures attach and feed on humans
and rabbits more rapidly. This observation suggests that the risk of human parasitism by Rh.
sanguineus could increase in areas experiencing warmer and/or longer summers, consequently
increasing the risk of transmission of zoonotic agents such as Rickettsia conorii and Rickettsia
rickettsia (Dantas-Torres, F., 2010 ).
The brown dog tick has been implicated in the transmission of several Ehrlichia species.
The bacterial agent Ehrilichia canis, responsible for canine monocytic ehrlichiosis is
transmitted by R. sanguineus . Other Ehrlichia agents associated with R. sanguineus include: E.
ewingii, responsible for canine granulocytic ehrlichiosis in dogs but can also cause illness in
humans; and E. chaffeensis, also known as human monocytic ehrlichiosis (HME), a type of white
blood cell which is due to its predilection for monocytes (Murphy et al. 1998).
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The brown dog tick has also been reported to transmit the bacterium Rickettsia ricksettsii,
causing Rocky Mountain Spotted Fever (RMSF) in humans and domestic canines in the United
States (Dantas-Torres., 2007) and Mexico.
In regions of southern Europe, Africa and Asia, the brown dog tick is the main vector
for Rickettsia conorii, which is the bacteria responsible for causing Mediterranean spotted fever
in humans as well as Rickettsia massiliae, recently implicated as a cause of human
disease (Parola et al. 2005, Vitale et al. 2006). R. massiliae was also recently found in R.
sanguineus from Arizona (Eremeeva et al. 2006). R. sanguineus has also been reported to vector
the protozoan parasite Hepatozoon canis, which causes canine hepatozoonosis in dogs (Baneth et
al. 2001) as well as Babesia canis also called canine babesiosis in dogs (Dantas-Torres 2008).
Bartonella henselae, the bacteria which causes cat scratch disease in humans, is typically
associated with fleas, but was recently detected in R. sanguineus ticks in California (Wikswo et
al. 2007). It is currently unknown whether it is a competent vector for B. hensaelae. Brown dog
ticks have also been experimentally infected with Bartonella vinsonii (Billeter et al 2012). R.
sanguineus is also considered a possible vector for Anaplasma plattys and Babesia gibsoni to
domestic dogs (CAPC, 2012).
Description
The adult stage of Rhipicephalus sanguineus or the brown dog tick is red-brown in
coloration, lacking any distinctive markings or body patterns with an elongated body shape and
hexagonally shaped mouth parts (Lord 2011; Keirans and Litwak 1989). Adult females are
between 1/8 to 1/4 inches in length.
Life Cycle
Rhipicephalus sanguineus is unique among tick species as it can complete its
entire life cycle indoors, therefore infestations in homes or kennels can occur rapidly (Lord.,
2011). This tick requires three blood meals to complete development from each life stage to the
next. Dogs are the preferred primary host across all life stages (larva, nymph and adult) in North
America. The brown dog tick attains sexual maturity and mates only on the host. A female may
begin feeding without the presence of a male but will not become fully engorged unless she
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mates with a male since the ingestion of a blood meal is a stimulus for production of
spermatocytes (sperm cells) in males and oocytes (egg cells) in females. (Dantas-Torres, 2010).
Figure 2. Brown Dog Tick Life Cycle

An adult female typically feeds on a host for approximately one week and after mating,
the engorged female detaches from the host, drops off and finds a safe area for egg development
(typically cracks or crevices in or near a home, garage or dog kennel). A female may begin
oviposition or egg-laying anywhere from as soon as four days to several weeks after detaching
from the host (Lord 2011; Dantas-Torres 2010). The oviposition or egg-laying period can last for
up to several weeks (Koch 1982). These ticks are very prolific– an individual fully blood-fed
gravid female is capable of laying an average of 1500-4000 eggs (Dantas-Torres 2010; Koch
1982). The number of eggs deposited depends on a number of factors including the weight of the
female, the amount of blood ingested and the length of the oviposition period (Koch 1982). The
ambient temperature also influences the length of time each stage feeds and the time required for
development and moulting where feeding and development are generally faster at higher
temperatures but survivability is greater at cooler temperatures and higher relative humidity
(Lord 2011).
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Larvae
After hatching, the larvae wait months while waiting for a host. The tiny light brown
larvae also called the "seed tick" have 6 legs and move to a lower area to attach to a dog at the
first opportunity. Should a host not be readily available, they can survive for 8 months without a
blood meal and water. After larvae attach to a host they engorge themselves for 3-9 days,
changing from a flattened to a globular shape, and light brown to bluish-gray color. They leave
the host, seek a sheltered area, and will undergo moulting for 6-23 days to become an 8-legged
reddish-brown nymph (Yates III, 1992).
Nymph
Body of dog tick nymphs showed maximum width behind scutum and narrowing
noticeably anteriorly. The scutum has a pair of elongated eyes at its greatest width while the
posterior margin is well convex, posto-lateral margins are straight and antolateral margins are
straight and slightly convergent to broadly round scapulae (Pegram et al., 2014)
Moulting occurs about one week after the blood meal, and nymphs emerge to climb
vegetation or vertical surfaces to again wait for a host. The feeding of the nymphs will last about
four days, after which they again to drop off and molt into the adult stage (Kaiser-Wilhelm-
Allee., 2016) Nymphs can survive for extended periods without food or water.
Hosts
R. sanguineus will feed on a variety of hosts but domestic dogs are the preferred host in the
United States. They can attach anywhere on a dog, but particularly prefer the head, ears,
between the toes, back, inguinal region and axilla (Dantas-Torres 2010).
When seeking a host, the brown dog tick is typically utilizes a hunting strategy or actively
pursues hosts but has also been known to adopt an ambush strategy or “questing behavior”
(Dantas-Torres 2010).
Chemical Control
Control of the brown dog tick requires extreme persistence by the pet owner. If the
infestation is throughout the residence the dog, home, and yard will need to be chemically treated
simultaneously. Incomplete treatments will prolong the infestation and result in an over use of
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pesticides. Insecticides to control R.sanguineus should have a reasonable residual efficacy.

Most organophosphates and carbamates qualify, as opposed to the pyrethrins. However,
pyrethrins are safe as a shampoo bath for the pet. The primary objective when applying a
pesticide to control this pest is to concentrate on those areas which are mostly visited by the dog.
Outdoor applications may include the crawl space beneath homes, kennels, and favorite napping
areas in the yard and/or garage. If fabrics need to be sprayed, read the pesticide label first for
possible staining precautions. If in doubt, spray a small and inconspicuous area, allow the
pesticide to dry, and observe for staining. If upholstered furniture requires treatment, remove the
cushions and spray all crevices and seams. Attention should also be directed to carpet edges,
baseboards, doorways, and floor/wall cracks and crevices. Treatment of outdoor and indoor areas
may have to be repeated depending upon the severity of the infestation and pesticide label
instructions. Do not treat if ticks are not observed on the pet or premises. Treatment of the pet
can be done by the owner or a veterinarian. A wide variety of commercial products are available
for baths or dips. An internal parasite, Hunterellus hookeri Howard, of the nymphal stage also is
an aid in the control of this pest (Mallis., 1982).
Pour on
Pour on applications are easy to apply and are long acting. One of the most effective
products is the topically applied fipronil, Frontline. It spreads over the skin and is deposited in
sebaceous material. It is effective for up to one month against ticks.
Sprays and washes
Dogs treated with dips and washes must be thoroughly wet to the skin, with particular
attention being given to inside the ears and between the toes. Generally these products are only
effective for a limited time. Because the brown dog tick is a three host tick, regular chemical
treatments at seven to 14 day intervals are required to break its life cycle. Washes generally
contain organophosphates, synthetic pyrethroids or formamidines. Take care in applying these
and follow safety instructions. In the cattle tick dips only Barricade ‘S’ is registered for use on
dogs for the control of cattle ticks. One of its active ingredients is chlorfenvinphos. Do not use at
intervals of less than three weeks.
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Collars
These are another effective method of applying chemical for prolonged periods.
However, exposure to water will affect the duration of protection. Collars generally contain
organophosphates, synthetic pyrethroids or formamidines.
With all chemical treatments, it is very important to read and follow the manufacturers’
recommendations. Remember that some of these chemicals also affect human health. There are a
limited number of treatments registered for use on animals under three months. In the Top End
weather conditions are favorable for tick survival for most of the year, except for short periods in
the driest times and the wettest times. As long as dogs are monitored for tick burden, year round
treatment would not be warranted. Commence control as soon as ticks are seen on the dog. Treat
all dogs at the same time.
Other Control Measures
Constant reinfestation will occur if the dog’s environment is contaminated with tick larvae
and nymphs. Treat the dog’s resting areas with an acaricidal spray. These are often
organophosphate chemicals such as maldison or diazinon. Bedding, crevices in the kennel and
any other items in the vicinity will harbour ticks so all of these must also be treated or removed.
If ticks have invaded your house, a commercial pest control operator should be consulted. Ticks
will shelter in cracks and crevices associated with brickwork, skirting boards and mouldings and
the use of powders/dusts with a residual effect are recommended. Products should be used only
according to directions. Dogs should be regularly checked for ticks. Pull off any ticks and
destroy them. Ticks in the ear removed using blunt-ended tweezers.
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Botanical products
Pandan
Figure 3. Pandanus amaryllifolius

Pandan (Pandanus amaryllifolius) is a tropical plant of the family Pandanaceae in the
screw pine genus. Pandan leaf is often known as screw pine, because they resemble the Ananas
plant (pineapple) with the spiral arrangement of long, narrow and strap-shaped green leaves
(Wongpornchai, 2006). This sprawling perennial plant is native to the Southeast Asia and has
been introduced on other tropical regions. It produces spirally arranges leaves about 1 to 1 ½
inches wide and up to 18 inches in length. This shrubby perennial plant has fan shaped clusters
of long and narrow leaves (Palmer, 2012)
Pandanus amaryllifolius is well-known as the only Pandanus with scented leaves (Setyowati
& Siemonsma, 1999; Wakte et al., 2009), the sweet smell of freshly harvested leaves similar to
the fragrance of expensive Basmati in India, Jasmine in Thai, and Kaorimari rice in Japan.
(Wakte et al., 2009). It also contains compounds that repel some species of cockroaches and are
a traditional insect repellent.
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Uses of Pandan
Pandan leaves also contain a number of essential oils and chemicals that cockroaches
find unpleasant. Terpenes and sesquiterpene hydrocarbons make up between 6 and 42 percent of
the oils in the leaves, according to the National University of the Singapore researchers. Pandan
leaves contain substances repellant to vermins such as cockroaches and mosquitos.
Terpenes are a large and diverse class of organic compounds produced by a variety of
plants, particularly conifers, though also by some insects such as termites or swallowtail
butterflies, which emit terpenes from their osmeterium. Terpenes and terpenoids are the primary
constituents of the essential oils of many types of plants and flowers.
Pandan leaves also contain volatile oil, an essential oil found in plants, which helps to
repel vermins. (Warke et al, 2012)
Homemade bio pesticide produced from Pandan (Pandanus amaryllifolius Roxb.)
against American cockroach (Periplaneta amaricana L.) was conducted in Cavite State
University- Science High School. In all of the treatments produced, only the one hundred pandan
extract show effectiveness though it was slow. Homemade bio-pesticide is effective in
controlling German cockroaches, with approximately 43 percent as the highest rate of
effectiveness based on the results of the study (Bayaset et al., 2014).
Another genus of Pandan (Pandanus odoratissimus L.) was used against common household
cockroaches (Periplaneta americana). The study was conducted in the Cebu Doctor’s University
and the results of the experiments shows one hundred percent Pandanus odoratissimus L.
(pandan) leaf extract was demonstrated to have an insecticidal effect on cockroaches
(Castañeda.,et al., 2013).
Pandan can also use as a mosquito killer spray and show 60 (sixty) percent mortality rate
in 100 (one hundred) percent concentration of leaf extract. (Zabal., 2013)
Kantutai (Lantana camara, Linn)
The Lantana camara, Linn leaf extract show insecticidal property against Brown Dog
Tick (Rhipicephalus sanguineus) and has the mortality rate of 66 (sixty-six) and 44 (forty-four)
percent using 500 (five hundred) ppm and 1000 (one thousand) ppm at long minutes of
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postexposure. It was also tested by oral administration in mice to know the median lethal dose
and it was concluded that it has no harmful effect on animals. (Protomartir, 2011)
Camphor Tree
The camphor tree (Cinnamonium camphora) crude leaf extract was tested against Brown
Dog Tick (Rhipicephalus sanguineus) and it show of decrease in locomotory activity and
flattened of scutum of the tick. It show higher mortality rate as the concentration increases
(Rivera et al.,2012).
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CHAPTER 3
STUDY FRAMEWORK
In this chapter it contains the paradigm of the study. It discusses about how the study
is done from the beginning until the end and also, it contains thee treatments used against Brown
Dog Tick Nymphs.
INPUT PROCESS OUTPUT

Pandan (Pandanus
amaryllifolius)
as ethanolic leaf extract: 1. Brown
1. Collection of
Dog Tick
Test Organisms
2. Collection of treated with
T1: 100% Ethanolic Plant Material different
Plant extract 3. Preparation concentration
T2: 75% Ethanol, of Plant Extracts s of Pandan
75mL Extract leaf ethanolic
4. Treatment
with 25 mL
distilled water and Control extract
T3: 50% Ethanol, Groups 2. Lethal
50mL Extract Preparation concentration
with 50 mL 5. Nymphal s (LC) of
distilled water Bioassay
T4: 25% Ethanol, pandan leaf
6. Application ethanolic
25mL Extract
with 75 mL in vitro Extract in LC
distilled water 7. Observation
50% and LC
T5: Negative control 8. Statistical
(Distilled water) 90%
analysis
mortality.
Figure 4. Conceptual Framework of In vitro Acaricidal Activity of Pandan Leaf

Extract against Brown Dog Tick
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Conceptual Framework of the Study

In analyzing the acaricidal activity of Pandan (Pandanus amaryllifolius) against Brown
Dog Tick (Rhipicephalus sanguineus) Nymphs, the study was anchored on the I-P-O model of
the Systems Theory as the research paradigm as shown in Figure 4.
The input variables focused on the treatments from the plant extracts that will be
used in the study and the process of testing it in vitro. The input variables are assessed in
accordance to the process involve in the study. As the end result, the expected output of the
researcher is whether or not the Pandan (Pandanus amaryllifolius) leaf ethanolic extract is
effective treating Brown Dog Tick (Rhipicephalus sanguineus).
Definition of Term
Acaricidal activity is the manner of killing certain pest.
In vitro means doing the research in a test tube or petridish, or anywhere that doesn’t involve
testing in human.
Nymph is the stage after the larval stage of the nymph and it is also before the adult stage
Pandanus amaryllifolius were used as flavoring agent and insect repellant among cockroaches.
It have marginal and midrib prickles on the leaves.
Rhipicephalus sanguineus or Brown Dog Ticks are the pest that cause diseases among dogs and
can affect human.
Leaf Extract is the product of from Pandan that will be used to test against Rhipicephalus
sanguineus.
Assumptions
The effectiveness of ethanolic leaf extract against Brown Dog Tick will be effective for
high concentrations and prolong exposure of ethanolic leaf. We are expecting that the many
drops of ehtanolic leaf extract and few minutes that brown dog tick exposed will bring any effect
to the tick. It can be cause to tick hard to more anymore and as well as it will bring mortality.
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CHAPTER 4
METHODOLOGY
The test parasite to use is the Brown Dog Tick Nymphs (Rhipicephalus sanguineus).
The materials are the following: Pandan leaves (Pandanus amaryllifolius), knives, scissors,
blender, cheesecloth, beakers, filter papers, syringe, and rotary evaporator. Methods to be used are
summarized using schematic diagram. (Appendix Figure 2.)
Research Design
This research used experimental design, specifically Randomized Complete Block Design
(RCDB) with five treatment groups of ten replications each using 10 nymphs per replication.
The treatments are the following:
T1: 100% Ethanolic Pandan leaf extract
T2: 75% ( 75mL extract with 25 mL distilled water)
T3: 50% (50mL extract with 50 mL distilled water)
T4: 25% ( 25mL extract with 75 mL distilled water)
T5 : Distilled water
Preparation of the Mixture
Four kilo of Fresh leaves of Pandan (Pandanus amaryllifolius) will be collected at
Mabuhay Homes 2000, Paliparan 2, Dasmariñas City, Cavite. Fresh Leaves samples will be
submitted at the Botanical Herbarium, University of the Philippines Los Baños, Laguna for the
identification and authentication of the plant material. Other parts of the plant will not be included
in the study.
Freshly collected Pandan leaves will be air-dried for 1 month. After air-drying the leaves
will be grinded using blender. The grinded leaves will be placed in a jar with the solvent ethanol.
The grinded leaves will be soaked for 3 days. The ethanolic extract obtained will be filtered using
cheesecloth and will be submitted to College of Allied Health Professionals in Dr. Carlos S.
Lanting College, Sangandaan, Quezon City to conduct the study.
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Gathering and Rearing of Test Insects

Free engorged and mated adult female tick will be reared in the Research Laboratory,
Department of Medical Laboratory Science, College of Allied Health Professionals, Dr. Carlos S.
Lanting College, Quezon City for 4 (four) days to 4 (four) weeks, until eggs are laid. The eggs
will hatch to become larvae and will be feed once with blood meal and water. In larval stage, it
will engorge themselves for 3-9 days so it will be feed with blood meals until it change from a
flattened to globular shape, and light brown to bluish-gray color. The developing larvae will be
observed until it undergoes moulting for 6-23 days to become an 8 (eight) legged reddish-brown
nymph. The nymphs will then be acclimatized for 24 hours. The nymph stage of brown dog tick
will be used as test parasites.
Data Gathering Procedure
Ten Rhipicephalus sanguineus nymphs will be placed in a petri dish using a medicine
dropper for each treatment. Ten replicates will be allocated for the solution. Each petri dish will
be labelled accordingly with its treatment and replicate each number for easier counting and
recording.
Nymphal Bioassay
One ml of the solution will be applied in each replicate using a 3 cc syringe. Dog tick
nymphs mortality will be recorded after incubation for 15, 30, 60, 120, and 180 minutes without
any nutritional supplements. Moribund nymphs are counted and added to dead nymphs for
calculating percentage mortality. Dead nymphs are those that cannot be induced to move when
they are probed with a needle in the siphon or cervical region. Moribund nymphs are those
incapable of rising to or not showing the characteristic diving reaction when the water is
distributed, as described in the World Health Organization's (WHO) technical report (2005). The
experiment will be performed at 28 variables 1°C. Determining killed nymphs percentage of
mortality will be computed using the formula:
Mortality rate= Number of tick deaths _ x 100
Number of treated Brown Dog Tick
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Statistical Treatment
The number of killed Rhipicephalus sa nguineus nymphs at each observation period will
be counted and results will be tabulated. The mortality rate of Rhipicephalus sanguineus nymphs
from each treatment group will be computed. Treatment group will be compared using Analysis
of variance (ANOVA). Mortality data from all replicates will be pooled for toxicity analysis. The
data will then be subjected to Log- Probit Regression analysis to determine the median lethal
concentration (LC50) and (LC90) to kill 90% of the treated nymphs.
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CHAPTER 5
RESULTS AND DISCUSSION OF DATA GATHERED
In this chapter, it discusses the results gathered from the experimental research done. It contains
he tabular data of the results such as the raw data of the dead ticks, mortality rate, and as well as
the tabular data regarding lethal concentration.
Length of Time
Treatments Replication 1 Replication 2 Replication 3
Time(mins.) 15 30 60 120 180 15 30 60 120 180 15 30 60 120 180
T1- 100% 10 10 10 10 10 10 10 10 10 10 9 10 10 10 10
T2- 75% 10 10 10 10 10 10 10 10 10 10 9 10 10 10 10
T3- 50% 10 10 10 10 10 10 10 10 10 10 8 10 10 10 10
T4- 25% 7 8 10 10 10 10 10 10 10 10 7 10 10 10 10
T5- water 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Table 1. Raw Data of Result of Acaricidal Activity of Pandanus amaryllifolius leaf extract
against Brown Dog Tick
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Mortality Rate of Rhipicephalus sanguineus
MORTALITY (%)
TREATMENTS
15 30 60 120 180
T1- 100% Ethanolic Extract 97.7a 100.0a 100.00a 100.00a 100.0a
T2- 75% Ethanolic Extract 97.78a 100.0a 100.00a 100.00a 100.00a
T3-50% Ethanolic Extract 85.89a 100.0a 100.0a 100.0a 100.0a
T4- 25% Ethanolic Extract 73.33c 85.89a 100.0a 100.0a 100.0a
T5- Distilled Water 0 0 0 0 0
Table 2. Percent mortality of R.sanguineus after treatment with different concentrations of

P.amaryllifolius aqueous and ethanolic extract
Table 2 shows the percent mortality of R. sanguineus after treatment with different
concentrations of ethanolic leaf extract. The negative control exhibited by 0 percent mortality in
180 minutes exposure time validating the study. A two way ANOVA was conducted that
examined the interaction of substrates and concentrations for all time.
In 15 minutes, the 25% ethanolic leaf extract caused 73.33c percent rate of mortality
which is the lowest while the 75 percent and 100 percent ethanolic leaf extract caused the highest
mortality rate for the exposure time.
As a result of the experiment, the mortality rate of R. sanguineus varied depending on
the concentration of the prepared leaf extract. The 100 percent ethanolic yielded the highest
mortality for within 15 minutes exposure period it already exhibited 97.7a percent mortality rate,
while the 25 percent ethanolic extract exhibited 73.33c percent mortality only, the lowest rate
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record among all the extracts. But in the 60 to 180 minutes exposure time, the mortality rate of
75 and 100 percent ethanolic extract both exhibits 100 percent mortality rate.
In conclusion, the leaf extract of Pandanus amaryllifolius can be used for killing
Rhipicephalus sanguineus. This is due to the active substances that can be found in the
P.amaryllifolius leaf extract such as the having 42.15% of phytol, 16.81% of squalene, and
6.17% of pentadecanal.
The result of the experiment is in line with the finding of other studies evaluating the
acaricidal activity of plant extracts with the same phytochemical analysis. A study entitled In
vitro Insecticidal Activity of Pandanus odoratissimus L. against common household cockroaches
(Periplaneta americana) supports the finding of this study that Pandan leaf extract can be used
as acaricide. Cockroches and Ticks belong to the same phylum of parasite which is
Arthropoda.(Dantas-Torres, 2008).
Toxicity of Pandanus amaryllifolius Leaf Extract
The mortality data obtained were subjected to probit analysis in order to estimate the
LC50. The LC50 values estimated for Pandan leaf extract at 15 minutes with 25 percent
concentration 28.970 and the LC 90 is 63.943. Probit Analysis is commonly used in toxicology
to determine the relative toxicity of chemicals to living organisms. This is done by testing the
response of an organism under various concentrations of each of the chemicals in question and
then comparing the concentrations at which one encounters a response. The LC50 for liquids or
LD50 for solid ate the most widely used outcomes of the modern dose-response experiments.
The LC50/LD50 represent the concentration (LC50) or dose (LD50) at which 50% of the
population responds. (Vincent., 2008). The study Acaricidal
Activity of tropical extracts against citrus mites and their effect on predator and citrus
mite that evaluated the acaricidal activity of 30 tropical plants extracts against citrus mite and
their effect on predator and citrus plants. The citrus rust mite Phyllocoptruta oleivora and the
citrus red mite Panonichus citri were used as mite test, while Harmonia axyridis was a predator
test. Plant materials were extracted with ethanol using infusion methods. Bioassays were
conducted by leaf-residual feeding method. The results showed that some new resources of
botanical acaricide screened from 30 plant species. Not all ethanol extracts screened showed very
21
strong or strong acaricidal activity. Study utilizing different plants and evaluating their acaricidal
activity reported the dose response data of the identified 95 percent ethanolic extracts, the LC 90
and LC50 values of D.metel fruit extract were determined as 7.13 (95 percent confidence interval
of 6.17-8.23) and 2.7 percent (95 percent CI 2.4-3.04), respectively. The LC90 and LC50 values
of A.mexicana extract were calculated as 11.30 percent (95 percent CI 5.56-6.49), respectively,
against the reference susceptible IVRI-I line of R. (B.) microplus (Ghosh et al., 2014).
Comparing to the said studies of the P.amaryllifolius leaf extract is less toxic and safer to use
having an LC50 of 28.970
PROBABILITY 95% CONFIDENCE LIMIT FOR 95% CONFIDENCE LIMIT FOR
CONCENT LOG
Estimate LOWER UPPER ESTIMATE LOWER UPPER
BOUND BOUND BOUND BOUN

D
PROBIT .010 6.883 .000 15.417 .838 -3.967 1.188
.020 8.145 .000 26.894 .911 -3.423 1.288
.030 9.064 .001 17.905 .957 -3.078 1.253
.040 9.823 .002 18.708 .992 -2.818 1.272
.050 10.486 .002 19.389 1.021 -2.607 1.288
.060 11.086 .004 19.898 1.045 -2.427 1.301
.070 11.641 .005 20.531 1.066 -2.270 1.312
.080 12.161 .007 21.030 1.085 -2.129 1.323
.090 12.654 .010 21.495 1.102 -2.000 1.332
.100 13.125 .013 21.933 1.118 -1.882 1.341
.150 15.271 .040 23.856 1.184 -1.394 1.378
.200 17.224 .099 25.253 1.236 -1.006 1.407
.250 19.097 .212 27.068 1.281 -.674 1.432
.300 20.953 .421 28.564 1.321 -.375 1.456
.350 22.833 .795 30.061 1.359 -.100 1.478

22
.400 24.773 1.449 31.611 1.394 .161 1.500
.450 26.806 2.583 33.277 1.428 .412 1.522
.500 28.970 4.542 35.167 1.460 .657 1.546
.550 31.308 7.915 37.506 1.496 .898 1.574
.600 33.878 13.609 40.944 1.530 1.134 1.612
.650 36.756 22.259 47.988 1.565 1.348 1.681
.700 40.054 31.061 68.282 1.603 1.492 1.834
.750 43.946 36.561 121.601 1.643 1.563 2.085
.800 48.726 40.441 250.654 1.688 1.607 2.399
.850 54.958 44.153 600.015 1.740 1.645 2.778
.900 63.943 48.586 1826.321 1.806 1.687 3.262
.910 66.325 49.664 2392.433 1.822 1.696 3.379
.920 69.014 50.846 3208.996 1.839 1.706 3.506
.930 72.096 52.162 4433.343 1.858 1.717 3.647
.940 75.701 53.655 6362.551 1.879 1.730 3.804
.950 80.033 55.392 9609.898 1.903 1.743 3.983
.960 85.441 57.484 15605.482 1.932 1.760 4.193
.970 92.592 60.140 28334.033 1.967 1.779 4.152
.980 103.034 63.829 62642.741 2.013 1.805 4.797
.990 121.933 70.048 218931.126 2.086 1.845 5.340
Table 4. Lethal Concentration (LC50) of Pandanus amaryllifolius Leaf Extract

23
CHAPTER 6
CONCLUSIONS, IMPLICATIONS AND RECOMMENDATIONS
In this chapter, it discuss about the summary of all the results gathered from the
experimental research , what we concluded as researchers and what we recommend for the future
researchers who will use our study as reference.
SUMMARY
The study In Vitro Acaricidal Activity of Pandan (Pandanus Amaryllifolius against Brown
Dog Tick Nymphs (Rhipicephalus sanguineus) was conducted at the Department of Medical
Laboratory Science of College of Allied Health Professionals from July to September 2017. The
general objective was to determine the acaricidal activity of Pandan (Pandanus amaryllifolius)
leaf extract against Brown Dog Tick nymphs (Rhipicephalus sanguineus). Specific objectives
were: to determine the mortality rate of R. sanguineus treated with different concentrations of
ethanolic leaf extract prepared from P. amaryllifolius, negative control which is the distilled water
at different time intervals; To determine what concentrations of the prepared extracts was most
effective in killing ticks; and lastly to determine the toxicity of the prepared extracts by
comparing the toxicity of other plant extracts that used animals in their previous study.
For the preparation of plant material, pandan (Pandanus amaryllifolius) plants were
collected from Dasmarinas, Cavite. It was air dried, chopped and powdered by blending, then
soaked with ethanol extract. The filtered extracts were gathered then placed in a jar. For the tick’s
preparation, Brown Dog Ticks Nymphs (Rhipicephalus sanguineus) were collected from heavily
infested dogs with no previous acaricidal treatment from selected owners at Mabuhay Homes
2000, Paliparan 2 Dasmarinas City Cavite, using twizzer then transferred to a clean specimen
container.
The study used randomized design, 5 treatments each with 3 replicates. Treatments are
made from 25%, 50%, 75% , 100% plant extracts mixed with ethanol and water, and with
distilled water as the negative control. Ticks were acclimatized from collecting jar to the petri
dishes in the laboratory, and then it was sprayed with treatments and was observed through
24
time. The dead ticks were counted at 15, 30, 60, 120, 180 minutes post exposure time. After
gathering of results percents mortality was computed, tabulated and analysed using Two-Way
Analysis of Variance (ANOVA).
Result showed that the highest mortality rate can be seen at the highest concentration
with longest exposure. 25% ethanolic extract was the least effective and from 50% , 75% and
100% ethanolic extracts were the most effective.
To know the toxicity of the extract, probit analysis was made in the 25 % ethanolic
solution at 15 mins exposure time because the study was after the solution that kills 50% of the
tick population. The result of the lethal dose for Pandanus amaryllifolius 25% leaf extract was
higher compared to other studies. This was the dose recommended that can kill 50% of a test
population.
CONCLUSION
Conclusions were made by answering the objectives of the study as follows:
1. Determine the mortality rate of R. sanguineus treated with different

concentration prepared P. amaryllifolius, negative control- distilled water and ethanolic leaf
extracts at a different time intervals. The researchers concluded that the mortality rate
Rhipicephalus sanguineus varied with the leaf extracts concentration with different time
intervals., based on the fact that the highest mortality rate where observed in the highest
concentrations with the longest exposure of time supported by the result.
2. Determine which concentration of the prepared extracts is most effective in
killing ticks. The results shows that Pandan (Pandanus amaryllifolius) leaf extracts were effective
against the brown dog tick nymphs (R. sanguineus), howerever based on the comparison of the
data gathered, the 100% concentration was the most effective having the highest means for all the
exposure time, while 25% was the least effective having the lowest means or the average number
of ticks that it can kill for all exposure time. However, the ethanolic solution is found to be more
effective than other solution supporting by the comparison of the results based on the shortest
25
time of exposure wherein percents of other solution used shows the lowest mortality results
compared ethanolic extracts.
3. Determine the toxicity of the prepared extracts. The probit analysis for toxicity
that was made in the 25 percent ethanolic solution at 15 minutes exposure time showed that 25
percent ethanolic solution can kill up to 50 percent of tick nymphs population at a little higher
concentration compared to other studies with probit analysis, which means it is less toxic.
RECOMMENDATIONS
Based on the findings of the study, the following was recommended:
1) The study of the activity of the Pandan (Pandanus amaryllifolius) against brown dog
tick nymphs (Rhipicephalus sanguineus) opens a new door of opportunity to further focus on
developing the effectiveness of the plant by trying different solutions and concentrations.
2) Researchers are highly recommending to use lower amount of concentration of the
solution to see the toxicity if it will be lower.
3) It is also suggested to explore some possibilities of combining other acaricidal plant and
see the best results
4) Conduct experiment with the same variables and try to make other acaricidal product
aside from spray (shampoo or powder).
5) Conducing experiment it is recommended to investigate the activity of the Pandan (P.
amaryllifolius) to other species of ticks and other arachnids or insects.

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DR. CARLOS S.

Background of the Study

Figure 1. Rhipicephalus sanguineus

Statement of the Problem

Significance of the Study

Figure 2. Brown Dog Tick Life Cycle

pesticides. Insecticides to control R.sanguineus should have a reasonable residual efficacy.

Figure 3. Pandanus amaryllifolius

INPUT PROCESS OUTPUT

Figure 4. Conceptual Framework of In vitro Acaricidal Activity of Pandan Leaf

Conceptual Framework of the Study

Gathering and Rearing of Test Insects

Treatments Replication 1 Replication 2 Replication 3

Time(mins.) 15 30 60 120 180 15 30 60 120 180 15 30 60 120 180

Mortality Rate of Rhipicephalus sanguineus

T1- 100% Ethanolic Extract 97.7a 100.0a 100.00a 100.00a 100.0a

T2- 75% Ethanolic Extract 97.78a 100.0a 100.00a 100.00a 100.00a

T3-50% Ethanolic Extract 85.89a 100.0a 100.0a 100.0a 100.0a

T4- 25% Ethanolic Extract 73.33c 85.89a 100.0a 100.0a 100.0a

T5- Distilled Water 0 0 0 0 0

Table 2. Percent mortality of R.sanguineus after treatment with different concentrations of

Estimate LOWER UPPER ESTIMATE LOWER UPPER

BOUND BOUND BOUND BOUN

PROBIT .010 6.883 .000 15.417 .838 -3.967 1.188

.020 8.145 .000 26.894 .911 -3.423 1.288

.030 9.064 .001 17.905 .957 -3.078 1.253

.040 9.823 .002 18.708 .992 -2.818 1.272

.050 10.486 .002 19.389 1.021 -2.607 1.288

.060 11.086 .004 19.898 1.045 -2.427 1.301

.070 11.641 .005 20.531 1.066 -2.270 1.312

.080 12.161 .007 21.030 1.085 -2.129 1.323

.090 12.654 .010 21.495 1.102 -2.000 1.332

.100 13.125 .013 21.933 1.118 -1.882 1.341

.150 15.271 .040 23.856 1.184 -1.394 1.378

.200 17.224 .099 25.253 1.236 -1.006 1.407

.250 19.097 .212 27.068 1.281 -.674 1.432

.300 20.953 .421 28.564 1.321 -.375 1.456

.350 22.833 .795 30.061 1.359 -.100 1.478

.400 24.773 1.449 31.611 1.394 .161 1.500

.450 26.806 2.583 33.277 1.428 .412 1.522

.500 28.970 4.542 35.167 1.460 .657 1.546

.550 31.308 7.915 37.506 1.496 .898 1.574

.600 33.878 13.609 40.944 1.530 1.134 1.612

.650 36.756 22.259 47.988 1.565 1.348 1.681

.700 40.054 31.061 68.282 1.603 1.492 1.834

.750 43.946 36.561 121.601 1.643 1.563 2.085

.800 48.726 40.441 250.654 1.688 1.607 2.399

.850 54.958 44.153 600.015 1.740 1.645 2.778

.900 63.943 48.586 1826.321 1.806 1.687 3.262

.910 66.325 49.664 2392.433 1.822 1.696 3.379

.920 69.014 50.846 3208.996 1.839 1.706 3.506

.930 72.096 52.162 4433.343 1.858 1.717 3.647

.940 75.701 53.655 6362.551 1.879 1.730 3.804

.950 80.033 55.392 9609.898 1.903 1.743 3.983

.960 85.441 57.484 15605.482 1.932 1.760 4.193

.970 92.592 60.140 28334.033 1.967 1.779 4.152

.980 103.034 63.829 62642.741 2.013 1.805 4.797

.990 121.933 70.048 218931.126 2.086 1.845 5.340

Table 4. Lethal Concentration (LC50) of Pandanus amaryllifolius Leaf Extract

Conclusions were made by answering the objectives of the study as follows:

1. Determine the mortality rate of R. sanguineus treated with different

Based on the findings of the study, the following was recommended:

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