INTRODUCTION

Background of the Study

Neem the legendary medicinal tree of India, has grown with the human
settlement all over the country and has been an integral part of the Indian way of
life for centuries. The history of the Neem tree is inextricably linked to the history
of the Indian civilization(Neem Foundation, n.d). It is also known as Divine Tree,
Life giving tree, Natures Drugstore(JustNeem, 2011). At four or five years old,
neem can produce flowers and fruit, but only after 10 to 12 years will it produce
economically viable seed quantities (AgroForestyTreeDatabase, n.d).
Every part of the Neem tree has health promoting benefits .They call Neem
tree

as

the

Village

Pharmacy

because

of

its

medicinal

benefits

(Selvester,1999).The leaf is also the part of the neem plant that western medicine
knows the most about. The majority of scientific neem studies was done with
neem leaf or neem leaf extracts (Discover Neem, 2013).
Neem bark has also been utilized for centuries by the Indian and Asian
cultures to treat numerous medical illnesses, prevent pregnancy and also as a
potent pesticide (Jernigan, 2010). Jernigan added also that one of the major uses
of neem bark is as an insect repellent and it offers benefits for humans because
there

is

no

need

to

use

harmful

pesticides

or

chemicals

on

crops...........................................................................
Mosquitoes are very common household insect . The life cycle of this
insect is normally short and breed in stagnant water accumulated in cans, old
tires, flower vases, pots, pails and other containers in the households (GMA
News, 2013) . The female mosquitoes feed on blood while male mosquitoes feed

on plant juices and nectar beacause of having weaker mouth. Also, they are very
common in houses during nightime. They not only disturb sleep but are also
carriers of diseases. Historically, mosquitoes have been identified as the carrier
of malaria and dengue and considered as the most deadliest and dangerous
disease respectively brought about by a mosquito bite (WHO, 2011) . They are
also vectors of filariasis and encephalitis. Lately, the recent outbreaks of dengue
fever which have killed countless of children and adults in many rural and urban
places have been traced to mosquitoes specifically the female of Aedes aegypti.
It is imperative that this insect be eradicated. In addition of this, it is preferred that
this will be controlled; in a less costly manner and environmental friendly.
According to Schumetterer (1981), Azadirachta Indica (Neem) Tree
contains Azadirachtin, a limonoid, that has been reported to have adverse effect
on endocrine system of a bean bettle, Epilachna varivestis, and to cause sterility
in the female insects. Schluter and Schulz (1983) also reported this compound
to cause degradation in larval epidermis preventing the larvae from molting. In
addition, Sridharan (2009) also said that Azadirachtin is mainly responsible for
the insecticidal properties of the neem. Insects are perceptive to smell; they do
not like the smell of neem oil. Azadirachtin disrupts the growth and reproduction
in most of the pest. It is one of the most potent growth regulators. It will repel or
reduce the feeding of many species of pest insects as well as some nematodes.
The problems raised earlier served as reasons why the researchers
embarked to the study to find out the use of Azadirachta Indica (Neem) bark
extract and its effectivity as insecticide against mosquito larvae.This is because
neem tree are abundant in the Philippines and always available as a source of

our raw material and the researchers decided to use the neem bark as the test
plant since majority of the scientific studies in neem were done in neem leaf;
and seeds is hard to find right now.
STATEMENT OF THE PROBLEM
Generally, this study aimed to utilize the neem bark extract as insecticide
against mosquito larvae.
Specifically, it sought to answer the following questions:
1. What are the chemical composition of Azadirachta Indica (Neem) bark
extract?
2. Are neem bark extract derived from Decoction Method and Ethanol
Extraction Method effective as insecticide against mosquito larvae?
3. Is there a significant difference between Azadirachta Indica (Neem)
bark Decoction and Ethanol extraction in terms of their larvicidal
activity?
RESEARCH HYPOTHESES
H01: Azadirachta Indica (Neem) bark extracts derived from Decoction Method
and Ethanol Extraction Method have no effect to mosquito larvae.
H02: There is no significant difference between Azadirachta Indica (Neem) bark
Decoction and Ethanol extraction in terms of their larvicidal activity.
SIGNIFICANCE OF THE STUDY
This study was conducted in order for the researchers to find alternatives
on how to combat illness-carrying mosquitoes by using inexpensive and natural
insecticide from Azadirachta Indica (Neem) bark extract.
The other beneficiaries of the study are the following:

The government, to give them awareness to take necessary actions

in addressing the problems such as conducting seminars in urban
3

and rural areas regarding awareness and prevention of the boost

of health cases brought by mosquitos.

The educational institutions, with the help of teachers, to integrate
to their lessons the use of Azadirachta Indica as insecticide against

mosquito larvae.
The community, especially to areas where illness carried by
mosquitos is rampant, to help them lessen the number of cases
and threat to their health by teaching them the natural and
inexpensive way to fight illness-carrying mosquitos with the use of
Azadirachta Indica.

SCOPE AND LIMITATION

The researchers focused on the use of Azadirachta Indica (Neem) bark
extract as insecticide against mosquito larvae. The Azadirachta Indica (Neem)
bark was collected within the City of Koronadal. The test insects were mosquito
larvae were chosen randomly since the researcher used Complete Randomized
Design and considering their physical appearance after 24-48 hours. The study
was conducted from July 2013 to September 2013 which includes collection and
preparation of materials, gathering of data, experimentation and observation of
variables.
Two methods were used for the extraction of Azadirachta Indica (Neem)
bark. Extraction with distilled water (Decoction Extraction Method) and Extraction
with 95% ethanol solution (Ethanol Extraction Method).
There were five treatments in each of the method of extraction, Treatment
0 (T0), Treatment 1 (T1), Treatment 2 (T2), Treatment 3 (T3) and Treatment 4
4

(T4) consisting of three replicates in each treatment were prepared. T0 is the

negative control which contains 100% distilled water, T1 is the positive control
which contains 100% liquid insecticide, T2 contains 10% Azadirachta Indica
(Neem) bark extract and 90% distilled water, T3 contains 20% Azadirachta Indica
(Neem) bark extract and 80% distilled water, and T4 contains 30% Azadirachta
Indica (Neem) bark extract and 70% distilled water.

Definition of Terms
Azadirachta Indica - the scientific name of neem tree.
Azadirachtin- a complex tetranortri-terpenoid limonoid from the neem tree that is
mainly responsible for the insecticidal properties of the neem.
Bark the outermost layer of the woody part of a neem tree that was used in the
study as insecticide against mosquito larvae.
Extract - is a substance made by extracting a part of a raw material, often by
using a solvent such as ethanol or water.
Insecticide - A chemical substance used to kill insects used as the positive
control in the study.
Lethal Dosage 50 (LD50) - is a measurement used in toxicology studies to
determine the potential impact of toxic substances on different types of
organisms. It is the median lethal dose of a substance, or the amount required to
kill 50% of a given test population. It used to determine the toxicity of the extracts
used in the study.
Limonoids- Phytochemicals, abundant in citrus fruit and other plants of the
families Rutaceae and Meliaceae.
Mosquito Larvae - is the larvae of various kinds of mosquitoes that lay their eggs
on the surface of the water. Also, the test insects used in the study to determine
the effectiveness of the extract as insecticide.

Neem tree is an evergreen tropical tree that belongs to the family Meliacease
(Mahogany). Itis a small to medium-sized treeup to 15m (30 max.) tall, with a
round, large crown up to 10m (20 max.) in diameter; branches spreading; bole
branchless for up to 7.5 m, up to 90 cm in diameter, sometimes fluted at base;
bark moderately thick, with small, scattered tubercles, deeply fissured and flaking
in old trees, dark grey outside and reddish inside, with colourless, sticky foetid
sap.
Mosquito egg- is the first stage in life cycle of a mosquito. Eggs may be laid
individually or connected together to form a raft type of structure
Mosquito- any of various two-winged insects of the family Culicidae, in which the
female of most species is distinguished by a long proboscis for sucking blood.
Mosquito pupa- the comma-shaped stage in the mosquito life cyle. This is the
transition stage between the aquatic stages of the mosquitos life cycle and the
terrestrial adult stage.
Probit Analysis - is commonly used in toxicology to determine the relative toxicity
of chemicals to living organisms. It is used to determine the toxicity level of the
Azadirachta Indica (Neem) bark extracts derived from Decoction Method and
Ethanol Extraction Method.

REVIEW OF RELATED LITERATURE

Azadirachta Indica (Neem Tree)

Neem or Margosa is a botanical cousin of mahogany. It belongs to the
family Meliaceae. The latinized name of Neem - Azadirachta indica - is derived
from the Persian:Azad = Free, dirakht = Tree, i - Hind = of Indian Origin which
literally means: The Free Tree of India.Neem is an attractive broad-leaved,
evergreen tree which can grow up to 30m tall and 2.5m in girth. Its trunk usually
straight is 30-80 cm in diameter. Its spreading branches form a rounded crown of
deep-green leaves and honey-scented flowers as much as 20m across (Neem
Foundation,n.d)
Azadirachta Indica trees may start flowering and fruiting at the age of 4-5
years, but economic quantities of seed are produced only after 10-12 years
(Orwa et al., 2009)
Neem bark is taken from the neem tree which predominately grows in
India and Asia. Neem bark can be used just off the tree or can be freeze dried or
ground into a fine powder for use at a later date. Neem bark has been utilized for
centuries by the Indian and Asian cultures to treat numerous medical illnesses,
prevent pregnancy and also as a potent pesticide (Jernigan, 2010) and Jernigan

also added that one of the major uses of neem bark is as an insect repellent.

Chemical Composition of Azadirachta Indica

According to Chandy (n.d.), the important chemical constituents of
Azadirachta Indica are azardirachtin, 1-maliantriol, salannin, nimbin, nimbidin and
others. The dry flowers on extraction with petroleum-ether yields waxy substances like quercetin, myricetin and b-sitosterol on extraction with alcohol they
produce a pungent essential oil, green amorphores bitter toxic substance,
nonacosane and a sesquiter- pene and a fatty acid fraction. He also added that
the different plant parts contain mostly terpenoids (limonoids). The flowers and
fruits are borne in axillary clusters and when ripe the smooth ellipsoidal drupes
are greenish yellow and comprise a sweet pulp enclosing a seed. The seed
consist of a shell and 1-3 kernels which contain azadirachtin and its homologues
(Mordue, 2000). Mordue added that both the bark and leaves also contain
biologically active molecules but not high levels of azadirachtin which is found
mainly in the seed kernels.
The trunk bark contains nimbrin (0.04%), nimbinin (0.001%), nimbidin
(0.4%), nimbosterol (0.03%), essential oil (0.02%), tannins (6.0%), a bitter
principle margosine and 6-desacetyl nimbinene (Chandy, n.d. & Neem
Foundation,n.d). The trunk bark contains nimbin, nimbinin, nimbosterol, tannins
and a bitter principle margosine (Dabur, 2013).The stem bark contains tannins
(12-16%) and non-tannin (8-11%). The bark contains anti-inflammatory

polysaccharide consisting of glucose, arabinose and fructose at a molar ratio

1:1:1 with molecular weight of 8,400. The bark also yields an antitumor
polysaccharide. Besides polysaccharides, several diterpenoids, viz., nimbinone,
nimbolicin, margocin, nimbidiol, nimbione, etc. have been isolated from stem
bark and root bark (Neem Foundation,n.d)
Neem has rightly been called Sarvaroghari. Modern scientists have
isolated more than 140 compounds from various parts of the Neem tree that
have been evaluated for curative powers. Claimed to be a Village dispensary
the following properties found in Neem, make it one of the best herbal medicines.
Some of its chemical content are Sodium, Potassium, Salts, Chloriphyle,
Calcium, Phosphorus, Iron, Thiamine, Riboflasium, Nicocin, vitamin C, carotene
and oxalic acid (ReadAndDigest.com, n.d.).

Uses of Azadirachta Indica

Neem, identified by WHO/UNEP1989 as an environmentally powerful
natural pesticide, is considered to be one of the most promising trees of the 21st
century for its great potential in pest management, environment protection and
medicine (Nicoletti, 2012).
Azadirachta

indica

(Neem),

an

herb extensively

used

in Ayurveda, Unani and Homoeopathic systems of medicine to treat many health

related problems and ailments (Herbcyclopedia, 2011). Neem is also well known
and used for its medicinal properties from the ancient period (4000 BC); mainly

on the indications of Ayurveda medicine, being very popular, even revered in the
Indian Subcontinent (Gajalakshimi S. & Abbasi S.A., 2004).
In practice, all parts are traditionally used for a variety of indications, but
limiting to the ethnobotanical indications concerning the aim of this paper, we can
recall the use of neem in indigenous medicine as a bitter tonic, antimalarial,
antipyretic, anti-inflammatory, antihelmintic, and for antimicrobial and antiviral
effects (Varie, 1996). It is also known to exert anticancer, antioxidant, woundhealing, and antimicrobial properties is also known to be one of these plants from
which almost every part is used, from the Neem oil extracted from its seeds to
the leaves and branches, to say the least neem benefits extend to various illnes
of health (Herbcyclopedia, 2011). In the article of Pamantong (2008), she stated
that the Neem tree, for the last 4,500 years, healed hundreds of millions in India,
Africa and many other parts of Asia. One of the immediately perceivable impact
of this antifungal, antibacterial and, perhaps, even antiviral king of the
arboretums on the human body is its guaranteed ability to heal or cure many, if
not all, skin diseases or epidermal problems ranging from dandruff, acne,
psoriasis, ringworm, athletes foot, warts, chicken pox, small pox and malaria.
Wong (2012) also stated in her article entitled Neem: What should I know about
it? that Neem is said to help with a number of health problems, including
asthma, constipation, cough, diabetes, gastric ulcers, indigestion, periodontal
disease, and urinary tract infection. Additionally, neem is purported to
reduceinflammation, improve liver health, alleviate pain, preserve eyesight,
stimulate the immune system, and protect against heart disease.
10

In the article of Tandon (n.d.) entitled The Neem Tree, he wrote that neem
leaves are used to treat chickenpox and warts by directly applying to the skin in a
paste form or by bathing in water with neem leaves. In order to increase
immunity of the body, neem leaves are also taken internally in the form of neem
capsules or made into a tea. The tea is traditionally taken internally to reduce
fever caused by malaria. This tea is extremely bitter. It is also used to soak feet
for treating various foot fungi. It has also been reported to work against termites.
In Ayurveda, neem leaves are used in curing neuromuscular pains. Neem leaves
are also used in storage of grains.
Different parts of Neem tree are being used extensively in manufacturing
of soaps, skin creams/lotions, shampoos, toothpastes, beauty aids and toiletries.
In most cases Neem oil/extract is being used for making these cosmetics like
soaps and tooth pastes etc (Johnson, 2010). Neem twigs are used by millions of
Indians as an antiseptic tooth brush. Its oil is used in the preparation of
toothpaste and soap ( Johnson, 2010 & Shiva, 2012).

Life Cycle of Mosquito

The length of the mosquito life cycle varies between species and is
dependent upon environmental conditions such as temperature and moisture
(Orkin, 2013). However, the mosquito goes through four separate and distinct
stages of its life cycle and they are as follows: Egg, Larva, pupa, and adult. Each

11

of these stages can be easily recognized by their special appearance (Alameda

County Mosquito Abatement District, 2013). Their life cycle which can vary in
length depending on temperature and food resources. In the summer time it
takes mosquitoes 3-10 days to totally complete their life cycle from egg to adult
(Renchie, 2007).
The feeding habits of mosquitoes are quite unique in that it is only the
adult females that bite man and other animals. The male mosquitoes feed only
on plant juices. Some female mosquitoes prefer to feed on only one type of
animal or they can feed on a variety of animals. Female mosquitoes feed on
man, domesticated animals, such as cattle, horses, goats, etc; all types of birds
including chickens; all types of wild animals including deer, rabbits; and they also
feed on snakes, lizards, frogs, and toads (Alameda County Mosquito Abatement
District, 2013).
In the life cycle of mosquitoes an egg is the first stage. Depending on the
mosquito species, eggs may be laid individually or connected together to form a
raft type of structure. The Aedes aegypti mosquito lays her eggs individually,
while the Culex pipiens mosquito lays approximately 200 eggs which she unites
to form a raft (MosquitoMagnet.com, 2013). It was also stated in the study of
Anderson and Harrington (n.d.) entiled Mosquito Biology for the Homeowner
that a freshly laid egg is light in color and darkens within a few hours. Mosquito
eggs are oval and about 1/40 th of an inch (0.635mm) long. Eggs are either
deposited singly or as an egg raft depending on the type of mosquito. A standard
egg raft is about 1/4 inch (6.35mm) long and contains 100-200 eggs. Some
12

species of mosquitoes lay their eggs singly and deposit them directly on water or
floating aquatic vegetation. Others will lay their eggs on moist soil that is subject
to periodic flooding, or above the water line in natural and artificial containers.
The number of single eggs laid per batch varies within and between mosquito
species and can range from 60 to 200. Eggs will hatch into larvae within 24 to 48
hours (Orkin, 2013).
The mosquito eggs hatch into larvae or "wigglers," which live at the
surface of the water and breathe through an air tube or siphon (Freudenrich,
2013). Larvae feed mostly on plant and animal debris in the water. This food is
generally low in nutritional value. The nutrition provided by the blood-mealenriched yolk is therefore important for development (Lundquist, M. et.al n.d).
The larvae filter organic material through their mouth parts and grow to about 0.5
to 0.75 inches (1 to 2 cm) long; as they grow, they shed their skin (molt) several
times. Mosquito larvae can swim and dive down from the surface when disturbed
(Freudenrich, 2013). According to Rey (2011), the larvae are enclosed in a hard,
inflexible envelope that is absolutely essential for larval survival, so it cant just be
discarded. In order to grow, mosquito larvae grow a new exoskeleton under the
old one. This new exoskeleton is soft and flexible at first, thus allowing the larvae
to grow. When ready to molt, the larvae then shed the old exoskeleton and the
new one hardens when exposed, to protect the larvaes internal organs. Larger
larvae can be seen floating just above the surface of infested waters.Within
seven to 10 days, larvae enter the pupal stage (Orkin, 2013).

13

According to Rey (2011), the fourth developmental stage of mosquito is

when larvae molt into the comma-shaped stage called the pupa (or tumblers).
This is the transition stage between the aquatic stages of the mosquitos life
cycle and the terrestrial adult stage. Pupa or pupae (plural) live near the surface
of the water, breathing through two horn-like tubes (called siphons) on their back.
(EnchantedLearning.com, 2010). Mosquitoes in this transitional stage are
referred to as tumblers, describing how they propel through the water. In this
stage, the mosquito pupa rests without eating as it prepares to change into an
adult mosquito (MosquitoMagnet.com, 2013). According to American Mosquito
Control Association (2011), the pupal stage is a resting, non-feeding stage of
development, but pupae are mobile, responding to light changes and moving
(tumble) with a flip of their tails towards the bottom or protective areas. In the
study of Freudenrich (2013), the stated that at the end of the pupal stage, the
pupae encase themselves and transform into adult mosquitoes.
Larvae and pupae usually cannot survive without water. If a water source
evaporates before the larvae and pupae within it transform into adult mosquitoes,
those young often will die (Orkin, 2013).
The newly emerged adult rests on the surface of the water for a short time
to allow itself to dry and all its body parts to harden. The wings have to spread
out and dry properly before it can fly (American Mosquito Control Association,
2011). Adult mosquitoes are characterized by having long, slender, needle-like
mouthparts (proboscis), antennae, and legs. Their narrow wings are often

14

covered with minute scales (Anderson and Harrington, n.d.). The adult male
mosqutios emerge first from the pupae (about 2 days earlier than the females)
and form a cloud over the standing water called a nuptial cloud. They wait for
females to emerge and each female takes flight she enters the cloud and mates
with a waiting male. The female only mates once in her life and holds the sperm
for to fertilize all the eggs she will lay in her lifetime (Thompson-Nicola Regional
District, 2011). In the study of Johnsen and Renchie (2007), they cited that
female and male mosquitoes both require carbohydrate sources (nectar, plant
exudates) throughout their life to maintain energy for ying, mating, and seeking
hosts for bloodmeals. Only the female mosquito takes a bloodmeal because she
needs the extra protein to develop eggs. Adult female mosquitoes will then seek
an animal on which to feed. Females are capable of flying for miles (Orkin, 2013).
The process of taking a bloodmeal is how the mosquito is able to vector viruses,
protozoans, and helminthes (worms) to humans and animals. Male mosquitoes
tend to only live a week or two while female mosquitoes can live for up to a
month and produce multiple batches of eggs. Some mosquito species overwinter
as blood fed females and can survive for multiple months (Johnsen & Renchie,
2007).

Habitats of Mosquito
All mosquitoes must have water in which to complete their life cycle. This
water can range in quality from melted snow water to sewage effluent and it can
15

be in any container imaginable. The type of water in which the mosquito larvae is
found can be an aid to the identification of which species it may be. Also, the
adult mosquitoes show a very distinct preference for the types of sources in
which to lay their eggs. They lay their eggs in such places such as tree holes that
periodically hold water, tide water pools in salt marshes, sewage effluent ponds,
irrigated pastures, rain water ponds, etc. (Alameda County Mosquito Abatement
District, 2013).
Aquatic

habitats

are

containers

in

which eggs

develop

into adult mosquitoes. Mosquitoes that transmit dengue lay eggs on the walls of
water-filled containers in the house and patio. The eggs hatch when submerged
in water and can survive for months. Mosquitoes can lay dozens of eggs up to 5
times during their lifetime (Centers for Disease Control and Prevention, 2012).
According to Rutgers (2008), mosquito habitats can be generally grouped
into four types: Running Water, Transient Water, Permanent Water, or Container.
Mosquito species breed in running waters, such as streams. Larvae can be
flushed out when stream volume increases, and to remain in the stream requires
a

large

amount

Anopheles species

of

energy.
are

The

stream

tropical

genus

breeders.

In

Chagasia and

some

addition, Anopheles

quadrimaculatus, Culex territans, and Uranotaenia sapphirina have all been

found in streams, although they prefer other habitats. Stream breeders will find
vegetation along banks with which to anchor themselves or attempt to remain
away from the main flow of the stream by seeking isolated eddies. Transient

16

water sources, such as flooded areas, snowpools, and ditches are used as
breeding grounds for mosquito species whose eggs can withstand desiccation,
such as Aedes and Psorophora. Their life cycles require alternating periods of
wet and dry. Other species, like an opportunistic Culex, might be able to pull off a
single generation during an extended flooded period. Transient water generally
show water quality changes which results in various mosquito species using the
same pool over a period of time. Permanent waters (also known as Semipermanent) are present for extended periods of time and support characteristic
aquatic vegetation. Cattail, rushes and sedges are typical freshwater swamp
vegetation. Genera associated with permanent water are Anopheles, Culex,
Culiseta, Coquillettidia, and Uranotaenia. Eggs of these species are not
desiccant-resistant and must be laid directly on the water. Aedes adults will
oviposit near the edge of the swamp, or within tussocks of vegetation, requiring
later flooding to inundate the eggs for hatching. As with transient waters, there is
a seasonal change in the vegetation, water quality and mosquito species
present. Container water habitat can be found in both natural settings, such as
water held by plants (bromeliads) to artificial settings, such as water found in
tires. The habitat of containers are based on the containers themselves. Treehole
sites generally have tannin-enriched water which is characteristically clear, with
rotting wood at the bottom. Many treehole species now also use artificial sites,
such as tires since they provide insulation against the weather and are more
numerous. Artificial containers are a convenient mode of transporting a species
of mosquito outside of it's natural range.

17

There is a great variety of man-made containers on backyards or patios

that collect rain water or that are filled with water by people where dengue
vectors thrive. Disposing of unused containers, placing useful containers under a
roof or protected with tight covers, and frequently changing the water of animal
drinking pans and flower pots will greatly reduce the risk of dengue infections.
Water storage containers should be kept clean and sealed so mosquitoes cannot
use them as aquatic habitats (Centers for Disease Control and Prevention,
2012).
Culex and Anopheles mosquitoes

are

among

the

most

common

permanent water mosquitoes. These mosquitoes are most active when the
average temperature is above 70 degrees. Their eggs must stay in water in order
to survive and usually will hatch within a couple of days, releasing larvae to begin
the development process (Mosquito World, n.d.).
Many permanent water mosquitoes can also breed in containers that
collect and hold water, such as wading pools, buckets or toys left
outside(Mosquito World,n.d.).

Related Studies
The

neem

tree,

Azadirachta

indica

has

well-known

insecticidal

(Wandscheer et al., 2004, Larvicidal action of ethanolic extracts from fruit

endocarps of Melia azedarach and Azadirachta indica against the dengue
18

mosquito Aedes aegypti) and insect growth regulatory (IGR) constituents

(Sukumar et al., 1991; Batra et al., 1998; Copping & Menn, 2000, Botanical
derivatives in mosquito control: review), and has been used for centuries in India
(Schmutterer, 1990, . Properties and potential of natural pesticides from the
neem tree, Azadirachta indica). Various neem products have been studied
extensively for their phytochemistry and exploitation in pest control programs
(Mulla & Su, 1999, Activity and biological effects of neem products against
arthropods of medical and veterinary importance).
Azadirachta Indica contains limonoids such as azadirachtin, salannin,
deacetylgedunin, gedunin, 17-hydroxyazadiradione, and deacetylnimbin showed
strong antifeedant and growth inhibitor activity against larvae (Nathan et al.,
2005, Efficacy of neem limonoids on Cnaphalocrocis medinalis(Guene)
(Lepidoptera: Pyralidae) the rice leaffolder). Its main chemical composition is a
blend of 3 to 4 related compounds along with over 20 lesser ones, which are
equally as active. The general class of these compounds is triterpenes and within
this category, the most effective are the limonoids, which are abundant in Neem
oil (Sanskrit Documents Org., nd, ). Limonoids are phytochemicals, abundant in
citrus fruit and other plants of the families Rutaceae and Meliaceae. Currently
limonoids are under investigation for a wide variety of therapeutic effects such as
antiviral or Viricide, antifungal, antibacterial, antineoplastic and antimalarial.
Certain limonoids are insecticides such as azadirachtin from the neem tree
(Omnilexica, n.d., Meaning of Limonoid).
At least nine limonoids are effective in inhibiting insect growth, especially
some of the most deadly varieties found in human health and agriculture
19

worldwide. Of these limonoids, azadirachitin has been found to be the main

ingredient for fighting insects and pests, being up to 90% effective in most
instances. It repels and disrupts the life cycle, however does not kill immediately,
but is nonetheless one of the most effective growth and feeding deterrents ever
examined (Sanskrit Documents Org., nd, The Neem Tree).
In the study of Mordue (2000) entitled Azadirachtin from The Neem
Tree Azadirachta indica: Its Action

Against Insects, it was stated that

Azadirachtin is the main component responsible for both antifeedant and toxic
effects in insects. Other limonoid and sulphur-containing compound with
repellent, antiseptic, contraceptive, antipyretic and antiparasitic properties are
found elsewhere in the tree, e.g. leaves, flowers, bark, roots. In the journal by
Scientific Technical and Medical Open Access Journals ( 2013) entitled
Azadirachtin Found in the Bark of Neem Tree Grown in China, Azadirachtin was
found in the bark of introduced neem tree (Azadirachta indica A. Juss) grown in
China by TLC examination. The bioassays made with imported cabbage worm
(Pieris rapae) and Asiatic corn borer (Ostrinia furnacalis) having typical poisoning
reaction to azadirachtin indicated that the extracts from the crushed bark of neem
tree contained a certain amount of azadirachtin. The content of azadirachtin in
the bark of neem tree was tested and calculated to be 0.65 mg / g in dried bark
by HPLC, those findings could be very helpful to directly use the substances from
the neem tree as insecticide and the comprehensive utilization of the products of
the neem tree.

20

Sridharan (2009) states in his study entitled Neem Tree: Melia

Azadirachta and Azadirachta Indica that Azadirachtin is mainly responsible for
the insecticidal properties of the neem. Azadirachtin disrupts the growth and
reproduction in most of the pest. It is one of the most potent growth regulators. It
will repel or reduce the feeding of many species of pest insects as well as some
nematodes. Sridharan also added that azadirachtin is structurally similar to insect
hormones called "ecdysones", which controls metamorphosis in the insects as
they pass from larva to pupa to adult. It affects the corpus cardiacum, an organ
similar to the human pituitary that controls the secretion of hormones.
Metamorphosis requires a careful synchrony of many hormones and other
physiological changes to be successful. Azadirachtin because of its structural
similarity to ecdysone will block the ecdysones action in metamorphosis and the
release of these vital hormones. Insects then will not molt. This of course breaks
their life cycle.
According to Schumetterer(1981, Properties and potential of natural
pesticides from the neem tree, Azadirachta indica), Azadirachtin, a limonoid, has
been reported to have adverse effect on endocrine system of a bean bettle,
Epilachna varivestis, and to cause sterility in the female insects. Schluter and
Schulz (1983) also reported in their study entitled Structural damages caused
by neem in Epilachna verivestis: a summary of histological and ultrastructural
damage this compound to cause degradation in larval epidermis preventing the
larvae from molting. In addition, Sridharan (2009) also cited in his study entitled
Neem Tree: Melia Azadirachta and Azadirachta Indica that Azadirachtin is mainly

21

responsible for the insecticidal properties of the neem. Insects are perceptive to
smell; they do not like the smell of neem oil. Azadirachtin disrupts the growth and
reproduction in most of the pest. It is one of the most potent growth regulators. It
will repel or reduce the feeding of many species of pest insects as well as some
nematodes.

METHODOLOGY

This includes the research design, setting, the participants, sampling

technique, measures, research instrument, research procedure, and statistical
treatment that will be used in the course of this study.

Research Design
This research used an experimental research design. This is an inquiry on
cause-and-effect relationships, and is conducted in a specialized setting, such as
the laboratory, experimental unit or research center. The researcher controlled
and manipulated the independent variable and randomly assigned the subjects to
different conditions or situations (Tan, 2006).
The researcher used the random sampling technique in getting their test
insects. Random sampling technique is a technique where each member of the
population has an equal chance of being selected as subject. The entire process
22

of sampling is done in a single step with each subject selected independently of

the other members of the population (Castillo, 2009).

The research design is divided into two variables: the dependent variable
(x) and the dependent variable (y).
The independent variable (x) corresponds to Azadirachta Indica bark
extract. The distilled water will be the negative control and the liquid insecticide
will be the positive control. The Azadirachta Indica (neem) bark extract derived
from decoction method and Azadirachta Indica (neem) bark extract derived from
ethanolic extraction method are the treatments that will be used; and three (3)
treatments, one (1) as the negative control, one (1) as the positive control and
will be conducted with three (3) replicates each. Each method of extraction will
have the same concentration of distilled water extract.
In treatment 0, it is prepared with zero percent (0%) extract of
Azadirachta Indica bark and one hundred percent (100% ) distilled water as the
negative control and its replicate respectively and for treatment one (1) it is
prepared with zero percent (0%) extract of Azadirachta Indica bark and 100%
liquid insecticide and its replicate respectively; treatment two (2) it is prepared of
ten percent (10%) extract of Azadirachta Indica bark and ninety percent (90%)
distilled water and its replicate respectively; in treatment three (3) twenty percent
(20%) extract of

Azadirachta Indica bark and eighty percent (80%) distilled

water and its replicate respectively; in treatment four (4) thirty percent (30%)
extract of Azadirachta Indica bark and seventy percent (70%) distilled water and
23

its replicate respectively.

The dependent variable (y) represents the percentage

of mortality rate of mosquito wriggler .

24

LEGEND FOR RESEARCH DESIGN

T0- 0% of Azadirachta Indica (Neem) bark extract and 100% distilled water as
the negative control of the sample
R1-first replication of T0
R2- second replication of T0
R3- third replication of T0

T1- 0% of Azadirachta Indica (Neem) bark extract and 100% liquid insecticide as
the positive control of the sample
R1-first replication of T1
R2- second replication of T1
R3- third replication of T1

T2- the addition of 10% of Azadirachta Indica (Neem) bark extract and 90%
distilled water to the sample
R1-first replication of T2
R2- second replication of T2
R3- third replication of T2

25

T3- the addition of 20% of Azadirachta Indica (Neem) bark extract and 80% of
distilled water to the sample.
R1-first replication of T3
R2- second replication of T3
R3- third replication of T3

T4 the addition of 30% of Azadirachta Indica (Neem) bark extract and 70% of
distilled water to the sample.
R1-first replication of T4
R2- second replication of T4
R3- third replication of T4

MATERIALS AND EQUIPMENT

8000 grams of Azadirachta Indica bark
8 liters of distilled water
Liquid insecticde
Mosquito wriggler
1000mL glass beaker (with stopper)
Coarse filter paper
1,600mL 95% Ethanol solution
Rotary Evaporator
Funnel
Erlenmeyer Flask
Cornmill Grinder
30 pcs 500mL calibrated measuring cups
5X 80mm Magnifying glass

26

GENERAL PROCEDURE
A. Collection and Preparation of Plant Extracts
Plant extracts were obtained from the bark parts of the Neem plant
(Azadirachta Indica). All these barks were collected from Barangay Saravia,
Koronadal City.The barks were thoroughly washed under distilled water and airdried for 7 days before grinding using a cornmill grinder and sent to Notre Dame
Marbel University-Chemistry Laboratory for phytochemical analysis and for
decoction and ethanol extraction process.

Bark is separated from its wood

27

A. 1
Decoction Extraction Method

The air-dried Neem bark was

Bark is thoroughly washed
ground using a grinder.
under
distilled
water
before air400gm of the
ground
plant
materials
were divided into 100gm each and were put
drying.

in a 1000mL glass beaker (with stopper) containing 400mL of distilled water and
boiled for 30mins . The boiled extracts were allowed to cool, and then they were
sieved using funnel and coarse filter paper.
A. 2 Ethanolic Extraction Method
The plant extracts were carried out using ethanol by soaking 400gm of the
ground materials in 1, 600mL of 95% ethanol solution for 24 hours, after which
they were filtrated using funnel and coarse filter paper and were concentrated to
dryness under pressure and controlled temperature (40-50 oC)

using rotary

evaporator.
B. CULTURE OF TEST INSECTS
Mosquito larvae will be collected by catching mosquitoes and placing

them

inside a mosquito net with black containers so that the mosquitoes can lay their
eggs.

28

C.PHYTOCHEMICAL ANALYSIS OF THE EXTRACTS

The Phytochemical analysis of the extracts is based on the Phytochemical
Screening by Guevara (2005).

Test for Alkaloids: 4mL of plant extract ethanol extraction method

were

evaporated to dryness using the evaporating dish placed on top of the beaker
which was heated in a hot plate. 5mL of 2M HCL was being added to the residue
and stirred before it was filtered in a testube. Then it was divided into three for
confirmatory tests. The second one was added with Dragendorffs Reagent, and
the other one was added with Mayers Reagent. The third one was added with
3mL and 28% Ammonia until alkaline and added with 10mL Chloroform then
divided into two test tubes. The first chloroform extract was evaporated and
added with 5mL of 2M HCL and then was heated for 2 minutes then filtered and
separated into two portions and tested with Dragendorff and Mayers Reagent.
The other one was added with Aqueous extract and 2M HCL then filtered and
separated into two portions and tested with Dragendorff and Mayers Reagent.
Observation made for the formation of precipitation indicated positive results.

29

Test for Saponins (frothing test): 2mL of plant extracts were diluted with
distilled water to 20ml each was added into test tubes and shaken vigorously.
They were then allowed to stand on testube rack for 1 minute and observation
made for the formation of stable froths which indicated positive results.

30

Test for Flavonoids: 2mL of plant extract derived from

decoction and ethanol extraction method were evaporated to dryness using the
evaporating dish placed on top of the beakers which were heated in a hot plate
then cooled. 9mL of Petroleum ether was added to each of the evaporated plant
extract and sitrred.10mL of 80% ethanol solution was added to each of the
evaporating dish then filtered. Each extract was divided into 3 test tubes for
confirmatory test. The first test tube of each extract will be added with 0.5mL 12M
HCL
and magnesium turnings. The content of the second extract with 12 M HCl were
put in separate beaker with water and place in a hot plate. The third testube
served as the control. The occurrence of a red or orange colouration was
indicative of the presence of flavonoids compounds.

31

Test for Tannins: 10grams of plant material was evaporated to dryness using
evaporating dish. The residue was dissolved in 20 mL of distilled water and 5
drops of NaCl solution then filtered. Then it was divided into 3 parts. The first one
served as control. The second one was added with gelatin-salt reagent. The third
one was added with three drops of 10% of FeCl3 were added to the filtrate. The
appearance of blackish-blue or blackish-green colouration was indicative of
tannins.

32

Test
for

Anthraquinones: 2 evaporating dish were prepared for the test: one for the
extract obtained from Decoction Method and the other one for Ethanol Extraction
Method. 2mL of each extract were placed into each evaporating dish and placed
in a hot plate till dryness. The residue were added with 10mL of distilled water
then filtrated. The filtrate were added with
5ml benzene then divided into two tubes.
One served as control. The other one was
added with 5mL ammonia solution. Red
colouration was indicative of anthraquinones.

33

FLOW CHART
Azadirachta Indica (Neem) Bark Extraction Through Decoction Method

34

100gm of the ground plant material

were put in a 1000mL glass beaker
containing 400mL of distilled water.

Distilled water with ground plant

material were boiled for 30 mins.

The FLOW
boiled CHART
extracts were sieved
using funnel and coarse filter
paper.
Extraction of Azadirachta Indica (Neem) Bark Through Ethanol Extraction
Method

35

400gm of the ground bark was soaked in i,

600mL of 95% ethanol solution for 24 hours.

D. TESTING FOR TOXICITY

The extracts were concentrated to
The soaked plant material were
dryness using rotary evaporator.
sieved using funnel and coarse
Ten (10) larvae were added to each calibrated measuring cup containing
filter paper.
varying concentrations of Azadirachta Indica (Neem) bark extract. Mortality rates
were observed after 24 hours. Mortality rates were compared to determine the
lethal effects and the level of toxicity.
Thirty (30) 500mL calibrated measuring cups will be prepared 15 cups was
used for Decoction Method and 15 cups was used for Ethanol Extraction Method.
The experiment were laid out using Completely Randomized Design (CRD) with
the following treatments: Treatment 0 (T0), Treatment 1 (T1), Treatment 2 (T2),
36

Treatment 3 (T3), and Treatment 4 (T4) consisting of three replicates in each

treatment were prepared. T0 contains

0% Azadirachta Indica (Neem) bark

extract and 100% distilled water as negative control, T1 contains 0% Azadirachta

Indica (Neem) bark extract and 100% liquid insecticide as positive control, T2
contains 10% Azadirachta Indica (Neem) bark extract and 90% distilled water, T3
contains 20% Azadirachta Indica (Neem) bark extract and 80% distilled water,
and T4 contains 30% Azadirachta Indica (Neem) bark extract and 70% distilled
water. Add 10 mosquito wrigglers in each of the glass of different concentration.
Mortality rates of mosquito wrigglers in each glass was observed after 24 hours
with the use of 5x80mm magnifying glass.
Probit Analysis and Lethal Dosage (LD50) was used to determine the
toxicity level of the dosage. 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. As discussed above, the response is always binomial
(e.g. death/no death) and the relationship between the response and the various
concentrations is always sigmoid (having the shape of letter S) (Vincent, 2008).
LD50 is a measurement used in toxicology studies to determine the potential
impact of toxic substances on different types of organisms. It is the median lethal
dose of a substance, or the amount required to kill 50% of a given test
population. It provides an objective measure to compare and rank the toxicity of
substances (Hadley, 2013).
37

FLOW CHART OF THE TESTING FOR TOXICITY

10 larvae were added

to each container
containing varying
concentrations of
Azadirachta Indica
Bark extract.

38

Larvae were
exposed to
extracts for 24
hours.

Mortality rates
were observed
after 24 hours.

E. STATISTICAL TREATMENT
In analyzing the data gathered for this study, the researcher will use OneWay Analysis of Variance (ANOVA), Two-Way Analysis of Variance (ANOVA) , Ttest, Probits Analysis and LD50.
To analyze variance between the effects of Azadirachta Indica (Neem)
Bark Extract obtained from Decoction Method and Ethanol Extraction Method,
One-Way Analysis of Variance (ANOVA) was used. This is a statistical procedure
for testing mean differences among three or more groups by comparing
variability between groups

to variability within groups (Tan, 2006). Two-Way

39

Analysis of Variance was also used. This is a means of comparing multiple levels
of two independent variables. The two-way ANOVA is grounded in the idea that
there are two variables, referred to as factors, affecting the outcome of
the dependent

variable.

To

ANOVA assumes population samples are

equal

in variance,

and

contain

be effective,
normally

two-way

distributed, independent,

sample groups of

equal

size

(BusinessDictionary.com).

The t-test was also used to determine the difference between the means
of two methods of Azadirachta Indica (Neem) bark extraction. This is a parametic
statistical test that shows the difference between the means of two groups of
values. The t distribution is commonly used with samples less than 30 units
(Asperas, 2005).
Probit Analysis and Lethal Dosage (LD50) will be used to determine the
toxicity level of the dosage. 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. As discussed above, the response is always binomial
(e.g. death/no death) and the relationship between the response and the various
concentrations is always sigmoid (having the shape of letter S) (Vincent, 2008).
LD50 is a measurement used in toxicology studies to determine the potential
impact of toxic substances on different types of organisms. It is the median lethal

40

dose of a substance, or the amount required to kill 50% of a given test

population. It provides an objective measure to compare and rank the toxicity of
substances. When comparing LD50 values, a lower value is regarded as more
toxic, as it means a smaller amount of the toxin is required to cause death
(Hadley, 2013).

FLOW CHART OF THE GENERAL PROCEDURE

Collection of Plant Materials and
Culturation of Test Insects

Preparation of Extracts

Phytochemical Screening
41

Test of Insects

Observation and Gathering of

Data

RESULTS AND DISCUSSION OF DATA

Presented in this section are the results of the data gathered on the
Azadirachta Indica (NEEM) bark extract as insecticides against mosquito
larvae.Specifically, it contains the phytochemical screening analysis of the bark
extract, the effectivity of neem bark extract derived from Decoction Method and
Ethanol Extraction Method as insecticide against mosquito larvae, and the
significant difference between Azadirachta Indica (Neem) bark Decoction and
Ethanol extraction in terms of their larvicidal activity.The test involves the
determination of the significant difference between the effects of Azadirachta
42

Indica (Neem) bark extract obtained from Decoction Method and Ethanol
Extraction Method by using ANOVA (Analysis of Variance). T-test is used to show
the difference between the means of two groups of values where the t distribution
is commonly used with samples less than 30 units (Asperas, 2005). Probit
Analysis was used to determine of Toxicity Level of Azadirachta Indica (Neem)
bark extract and Lethal Dosage (LD50) will be used to determine its toxicity level.

Table 1. Phytochemical Screening of Plant Extract

Phytochemical

ALKALOIDS

SAPONINS
FLAVONOIDS
TANNINS
ANTHRAQUINONES

Dragendorffs Test
Mayers Test
Confirmatory Test
Aqueous
Dragendorffs
Mayers
Chloroform Dragendorffs
Mayers
Frothing
Benzopyrene
Leucoanthocyanins
Gelatin-Salt
Ferric Chloride
Ammonia Solution

Decoction

Ethanol

Method

Extraction

Method
+
+

+
+
+

+
+
+
+
+
+
+
+
+
+

43

Legends:
Slight turbidity
Definite turbidity
Heavy precipitation
Not performed

(+)
(++)
(+++)
(-)

Table 1 shows the phytochemical screening results of the plant extract. It

was shown in the table that the plant extract is positive in or contains alkaloids,
saponins, flavonoids, tannins and anthraquinones when it undergone different
confirmatory tests which shown at the middle column of the table.

Table 2. Mortality Rates of Mosquito Larvae Upon Exposure to Azadirachta

Indica (Neem) Bark Extract Obtained Through Decoction Method.
12
10
8
6
4
2
0

Replicate 1
Replicate2
Replicate3

Table 2 shows that among the three treatments (Treatment 2, Treatment 3,

and Treatment 4), Treatment 4 has the highest mortality rate of mosquito upon
exposure to the extract having 100% (10 larvae) in Replicate 1 while Treatment 2
has the lowest mortality rate having 30% (3 larvae) in Replicate1.

Table 3. Mortality Rates of Mosquito Larvae Upon Exposure to

AzadirachtaIndica (Neem) Bark Extract Obtained Through Ethanol
Extraction Method.

44

12
10
8

Replicate 1

Replicate 2

Replicate 3

2
0
Treatment 0 (negative control)

Treatment 2

Treatment 4

Table 3 shows that among the three treatments (Treatment 2, Treatment 3,

and Treatment 4), Treatment 3 and Treatment 4 have the highest mortality rate of
mosquito upon exposure to the extract having 100% (10 larvae) in all replicates
while Treatment 2 has the lowest mortality rate having 20% (2 larvae) in
Replicate 2.
Based from the results on Mortality rates of Mosquito Larvae upon
exposure to Azadirachta Indica (Neem) Bark Extract from the table 2 and 3, it
was proven that both

Azadirachta Indica (Neem) Bark Extract derived from

Decoction Method and Ethanol Extraction Method are both effective as

insecticide against mosquito larvae.

Table 4. Analysis of Variance Between the Effects of Azadirachta Indica

(Neem) Bark Extract Obtained from Decoction Method and Ethanol
Extraction Method.
Summary
Groups
Column 1

Count
15

Sum
81

Average
5.4

Variance
13.4
45

Column 2

Source of
Variation
Between
Groups
Within
Groups
Total

15

100

6.666667

19.38095

SS

df

MS

P-value

F crit

12.03333

12.03333

0.734166

0.39881

4.195972

458.9333

28

16.39048

470.9667

29

Table 4 shows that with the F value of 0.734166, the critical F = 4.195972.
Since the F statistic is smaller than the critical value, we fail to reject the null
hypothesis. Remember from above, the null hypothesis was that all 2 of these
groups' means were equal. So, we fail to reject that there is a significant
difference between Azadirachta Indica (Neem) bark Decoction and Ethanol
extraction in terms of their larvicidal activity. Therefore, it was concluded that
there is no significant difference between Azadirachta Indica (Neem) bark
Decoction and Ethanol extraction in terms of their larvicidal activity since they
both have equal means.

Table 5. Test on the Significant Difference Between Azadirachta Indica

(Neem) Bark Extract obtained from Decoction Method and Ethanol
Extraction Method.

DM
EEM
Difference

N
15
15
15

Mean
5.40
6.67
-1.267

StDev
3.66
4.40
2.282

SE Mean
0.95
1.14
0.589

95% CI for mean difference: (-2.531, -0.003)

T-Test of mean difference = 0 (vs not = 0): T-Value = -2.15 P-Value = 0.050
46

Table 5 shows the difference between the means of two methods of

Azadirachta Indica (Neem) bark extraction. Using the Critical Values for the tDistribution, first we must select the correct critical value from the table to
compare with our calculated value. We do this by computing the degrees of
freedom and the value is 1.697. Since our t-value (t = 2.15) is larger than the
tabled t-value (t = 1.697) this means that there is a small chance that the
population means are the same, and so it is reasonable to conclude that the
means are different. Hence, we reject the null hypothesis that there is no
significant difference between Azadirachta Indica (Neem) bark extract derived
from Decoction Method and Ethanol Extraction Method.

Table 6. Results on Level of Toxicity Using PROBIT ANALYSIS and LETHAL

DOSAGE (LD50) on Decoction Method of Extraction According to FINNEYS
METHOD
Dose
(Stimulus
)

Actual
Percent (%)

Probit Percent

E(R)

Difference

Chi-Square

3.613
4
6.055
2
7.362
5

0.3866

0.0414

-1.0552

0.1839

0.6375

0.0552

0.4

0.3613

10

1.301

0. 5

0.6055

10

1.4771

0.8

0.7362

10

LD50: 14.8458
Table 6 shows toxicity level of Azadirachta Indica (Neem) bark extract
derived from Decoction Method. It was shown that toxicity level of the extract is
at dose 1 having a probit percent of 0.3613 and an LD50 of 14.8458. A lower
47

value is regarded as more toxic, as it means a smaller amount of the toxin is

required to cause death. (Hadley, 2013).

Table 7. Results on Level of PROBIT ANALYSIS and LETHAL DOSAGE

(LD50) RESULTS on Ethanol Extraction Method According to FINNEYS
METHOD
Dose
(Stimulus
)

1
1.301

Actual
Percent (%)

Probit Percent

E(R)

Difference

Chi-Square

0.3
1

10
10

3
9.75

0
-0.2497

0
0.0062

10

9.75

3
9.999
7
10

-0.25

0.0062

0.3
0.975

1.4771
0.975
LD50: 10.8297

Table 7 shows toxicity level of Azadirachta Indica (Neem) bark extract

derived from Decoction Method. It was shown that toxicity level of the extract is
at dose 1 having a probit percent of 0.3 and an LD50 of 10.8297. A lower value is
regarded as more toxic, as it means a smaller amount of the toxin is required to
cause death. (Hadley, 2013).

48

SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

This section presents the summary of study and conclusions made from the
study and the recommendations given by the researchers.
Summary of the Study
The main purpose of the study is to utilize Azadirachta Indica (Neem) bark
extract as insecticide against mosquito larvae and to determine its effectivity.
Two methods were used for the extraction of Azadirachta Indica (Neem)
bark. Extraction with distilled water (Decoction Extraction Method) and Extraction
with 95% ethanol solution (Ethanol Extraction Method).
A Phytochemical Screening, which includes test for Saponins, Tannins,
Alkaloids, Flavonoids and Anthraquinones, was performed to determine the
chemical compounds present in Azadirachta Indica (Neem) bark.
49

There were five treatments in each of the method of extraction, Treatment

0 (T0), Treatment 1 (T1), Treatment 2 (T2), Treatment 3 (T3) and Treatment 4
(T4) consisting of three replicates in each treatment were prepared. T0 is the
negative control which contains 100% distilled water, T1 is the positive control
which contains 100% liquid insecticide, T2 contains 10% Azadirachta Indica
(Neem) bark extract and 90% distilled water, T3 contains 20% Azadirachta Indica
(Neem) bark extract and 80% distilled water, and T4 contains 30% Azadirachta
Indica (Neem) bark extract and 70% distilled water.
Toxicity testing of the extract was determined through monitoring of the
mortality of ten larvae introduced to each calibrated measuring cup containing
different concentration of the extract. Monitoring and observation were made
after 24 hours.
The data gathered from mortality of larvae in toxicity testing was treated
by using Two-Way ANOVA (Analysis of Variance), Probits Analysis, LD50 (Lethal
Dosage 50) and T-Test. The significant difference between the effects of
Azadirachta Indica (Neem) Bark Extract obtained from Decoction Method and
Ethanol Extraction Method was determined by using One-Way and Two-Way
ANOVA (Analysis of Variance). T-test is used to show the difference between the
means of two groups od values where the t distribution is commonly used with
samples less than 30 units (Asperas, 2005). Probits Analysis was used to
determine of Toxicity Level of Azadirachta Indica (Neem) bark extract and Lethal
Dosage (LD50) was used to determine the toxicity level of the dosage.

50

The results from the tests indicated that based on the Phytochemical
Screening Result, Azadirachta Indica (Neem) bark extract contains alkaloids,
saponins, tannins, flavonoids and anthraquinones. ANOVA and T-test results
show that both Azadirachta Indica (Neem) bark extracts derived from Decoction
Method and Ethanol Extraction Method are both effective and has no significant
difference in terms of their larvicidal activity since they both have equal means.
Probit Analysis and LD50 shows that their toxicity level is at the lowest dosage
for it only requires less amount of extract to cause death of the mosquito larvae.

Conclusions
After a thorough analysis of the data gathered, the researchers concluded
that:
1. Azadirachta Indica (Neem) bark extract contains insecticidal property
against mosquito larvae.
2. Azadirachta Indica (Neem) bark extract derived from Decoction
Method and Ethanol Extraction Method are both effective as
insecticide against mosquito larvae.
3. Based on the result from ANOVA Test Result, there is no significant
difference between Azadirachta Indica (Neem) bark extract derived
from Decoction Method and Ethanol Extraction Method in terms of their
larvicidal activity.

Recommendations
The results show that Azadirachta indica (Neem) Bark Extract derived
from Decoction Method and Ethanol Extraction Method are both effective as
51

insecticide against mosquito larvae by mixing 400 grams of plant material to 1,

600ml of water or 95% ethanol solution. The researchers recommend the
following for further study and development of this study:
1. To purify the extract of Azadirachta Indica (Neem) bark to identify its
component that is responsible for the mortality of mosquito larvae with
the use of High-Performance Liquid Chromatography (HPLC) since a
phytochemical screening was conducted.
2. A further study on the comparison of the different parts of Azadirachta
Indica (Neem) in terms of their larvicidal activity.
3. Making a soap or lotion out of Azadirachta Indica (Neem) bark extract.

52

BIBLIOGRAPHY
Alameda County Mosquito Abatement District, (2013). The Life Cycle of
Mosquito. Retrieved July 20, 2013 from
http://www.mosquitoes.org/LifeCycle.html

American Mosquito Control Association (2011). Life Cycle. Retrieved September

4, 2013 from http://www.mosquito.org/life-cycle

Anderson, R and Harrington, L. (n.d.). Mosquito Biology for the Homeowner .

Retrieved September 4, 2013 from
http://entomology.cornell.edu/extension/medent/mosquitofs.cfm
Asperas, C. (2005). Introduction to Basic Nursing Research.
Giuani Prints House, 24 Panghulo Road, Malabon
Business Dictionary ( n.d). Two-way ANOVA. Retrieved September 30, 2013
from http://www.businessdictionary.com/definition/two-way-ANOVA.html
Business Dictionary n.d. Lethal Concentration 50 (LC50). Retrieved August 23,
2013 from http://www.businessdictionary.com/definition/lethalconcentration-50-LC50.html
Castillo, J. (2009).Random Sampling. Retrieved October 1, 2013 from
http://explorable.com/simple-random-sampling
Chandy, K. (n.d). Neem Utilisation Booklet No. 470 Medicinal Plants: MPS-15.
Retrieved August 23, 2013 from
53

https://www.google.com.ph/url?
sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&ved=0CDoQFjAC&ur
l=http%3A%2F%2Fwww.inseda.org%2FAdditional%2520material%2FCD
%2520%2520Agriculture%2520and%2520Environment%2520Education
%2F53-Medicinal%2520Plants%2520(MPS)%2FNeem%2520Utilisation470.doc&ei=Qs4WUuXUOcbtiAf_oDADg&usg=AFQjCNEEwnoY9VIOV9H
tE6uDmQRBlcjijA&sig2=78LRYKRd0dzSpo7MvbkLg&bvm=bv.51156542,d
.aGcdfsd

Dabur.com (2013). Herbal Garden. Retrieved August 23, 2013 from

http://www.dabur.com/Ayurveda-Herbdetails?herbName=Neem

Discover Neem (2013). What Is Neem Leaf Good For?The Uses And Benefits Of
Neem Leaves.Retrieved August 17, 2013 from
http://www.discoverneem.com/neem-leaf.html

ELR GMA NEWS (2013).Stop dengue by cleaning out water containers, other
breeding places of mosquitoes Palace. Retrieved August 17, 2013 from
http://www.gmanetwork.com/news/story/316440/news/regions/stopdengue-by-cleaning-out-water-containers-other-breeding-places-ofmosquitoes-palace

EnchantedLearning.com (2010). Mosquito Life Cycle. Retrieved september 4,

2013 fromhttp://www.enchantedlearning.com/subjects/insects/mosquito/
lifecycle.shtml

Freudenrich, C. (2013). How Mosquitoes Work. Retrieved September 6, 2013

from http://science.howstuffworks.com/zoology/insectsarachnids/mosquito1.htm
54

Gajalakshimi S. & Abbasi S.A. (2004) Neem leaves as a source of fertilizer-cumpesticidevermicompost Biosource Technology, 92, 291-296.
Goddard, M et al., (1995). "Comparison of the up-and-down, conventional LD50,
and fixed-dose acute toxicity procedures".
Guevara, B. (2005). A Guidebook to Plant Screening : Phytochemical and
Biological.University of Santo Tomas Publishing House, Espaa, Manila
Hadley, D. (2013).LD50. Retrieved August 15, 2013 from
http://insects.about.com/od/l/g/LD50.htm About.com Guide
Herbcyclopedia (2011).Neem Benefits (Azadirachta Indica).Retrieve July 20,
2013 from http://www.herbcyclopedia.com/index.php?
option=com_zoo&task=tag&tag=AZADIRACHTA%20INDICA
%20CHEMICAL%20CONSTITUENTS&app_id=5&Itemid=193
Jernigan, K. (2010). Neem Bark Benefits.Retrieved August 16, 2013 from
http://www.livestrong.com/article/78288-neem-barkbenefits/#ixzz2c8b5ilJH
Johnson, L. (2010). The Neem Tree: A Miracle Plant. Retrieved September 16,
2013 from http://ezgrogarden.com/growing-organic/the-neem-tree-amiracle-plant/

Johnsen, M. and Renchie, D. (2007). Mosquito Life Cycle. Retrieved september

10, 2013 from http://texashelp.tamu.edu/001a-hot-topics/pdfs/2008-floodsmosquitoes/the-mosquito-life-cycle.pdf

JustNeem,(n.d).The History Of The Neem Tree. Retrieved August 17, 2013 from
http://www.justneem.com/blog/the-history-of-the-neem-tree/

55

Lundquist, M. et.al (n.d.) Mosquito Reference Manual. Retrieved September 27,

2013 at
http://media.hhmi.org/biointeractive/activities/mosquito/mosquito_referenc
e_manual.pdf

Mordue, J. (2000). Azadirachtin from The Neem Tree Azadirachta indica: Its
Action Against Insects. Retrieved July 25, 2013 from
http://www.scielo.br/scielo.php?
pid=S030180592000000400001&script=sci_arttext

MosquitoMagnet.com 2013. Mosquito Life Cycle. Retrieved september 4, 2013

from http://www.mosquitomagnet.com/advice/mosquito-info/mosquito-funfacts/mosquito-life-cycle
Mulla MS, Su T. (1999). Activity and biological effects of neem products against
arthropods of medical and veterinary importance. Journal of the
AmericanMosquito Control Association 15: 133-152.
Nathan et. al., 2005. Efficacy of neem limonoids on Cnaphalocrocis
medinalis(Guene) (Lepidoptera: Pyralidae) the rice leaffolder. Retrieved
September 2, 2013 from
http://www.sciencedirect.com/science/article/pii/S0261219405000372
Neem Foundation,( n.d). Greening India with Neem.Retrieved August 16, 2013
from http://www.neemfoundation.org/neem-articles/about-neemtree/introduction.html
Nicoletti, M. et al. Neem Tree (Azadirachta indica A. Juss) as Source of
BioinsectidesDepartment of Environmental Biology - University Sapienza
of Rome (2012). Retrieved July 25, 2013 from
http://cdn.intechopen.com/pdfs/25684/InTechNeem_tree_azadirachta_indi
ca_a_juss_as_source_of_bioinsectides.pdf

56

Omnilexica,( n.d). Meaning of Limonoid. Retrieved September 3, 2013 from

http://www.omnilexica.com/?q=limonoid
Orkin, (2013). Mosquito Life Cycle.Retrieved August 17, 2013 from
http://www.orkin.com/other/mosquitoes/mosquito-life-cycle/
Orwa et al. (2009). Azadirachta Indica. Retrieved August 16, 2013 from
http://www.worldagroforestry.org/treedb2/AFTPDFS/Azadirachta_indica.df

Pamantong, E., (2008). Neem the Most Medically Useful Tree of all Time.
Retrieved July, 2, 2013 from
http://showbizandstyle.inquirer.net/lifestyle/lifestyle/view/20080425132742/
Neem-the-most-medically-useful-tree-of-all-time

Readanddigest.com, (n.d). Neem Mother Natures Drugstore Retrieved

September 16, 2013 from http://readanddigest.com/neem-mother-naturesdrugstore/

Renchie, D. (2007). Mosquito Life Cycle. Retrieved September 4, 2013 from

http://www.uaex.edu/Other_Areas/publications/PDF/ag1163.pdf

Rey, J. (2011). The Mosquito. Rerieved September 6, 2013 from

http://edis.ifas.ufl.edu/pdffiles/IN/IN65200.pdf
Rutgers School of Environmental and Biological Sciences, (2008). Larval
Habitats of Mosquitoes. Retrieved September 13, 2013 from
http://www.rci.rutgers.edu/~insects/habitat.htm

57

Sanskrit Documents Organization, (n.d). The Neem Tree. Retrieved September

2, 2013 from sanskritdocuments.org/articles/TheNeemtree.doc

Schluter U, Schulz WD, (1983). Structural damages caused by neem in

Epilachna verivestis: a summary of histological and ultrastructural
damage. 1. Tissues affected in larvae. Proc. 2nd Int. neem conf.
Rottach.Egern, p. 294.
Schmutterer H, Ascher KRS, Rembold H, (1981). Natural pesticides from the
neem tree. Proc. 1st Int. neem Conf. Rottach-Egern, p 294.
Schmutterer H. (1990). Properties and potential of natural pesticides from the
neem tree,Azadirachta indica. Annual Review of Entomology 35: 271-297.
Scientific Technical and Medical Open Access Journals, (2013). Azadirachtin
Found in the Bark of Neem Tree Grown in China. Retrieved July 25, 2013
from http://www.stmopen.net/azadirachtin-found-in-the-bark-of-neem-treegrown-in-china/
Selvester, J. (1999).Neem "The Village Pharmacy". Retrieved August 17, 2013
from http://netowne.com/alt-healing/ayurveda/

Shiva, V. (2012). The Neem Tree - A Case History of Biopiracy. Retrieved July 2,
2013 from http://www.twnside.org.sg/title/pir-ch.htm

Sridharan, L. 2009. Neem Tree: Melia Azadirachta and Azadirachta Indica.

Retrieved September 2, 2013 from
http://www.ncnhdistrict.org/aom/neem.html

Sukumar K, Perich MJ, Boobar LR. (1991). Botanical derivatives in mosquito

58

control: review. Journal American Mosquito Control Association 7: 210237.

Tan,C.B (2006). A Research Guide in Nursing Education.A Text and Workbook.
3rd Edition.Visual Print Enterprises.Makati, Philippines.
Tandon, M. (n.d). The Neem Tree. retrieved: July 18, 2013 from
http://www.organeem.com/neem_tree.html

Varie P.S. (1996). Indian Medicinal Plants. A compendium of 500 species.

Vaidyaratnam. Arya Vaidya Sala Kottakal. Orient Longman.
Wandscheer CB, Duque JE, Da Silva MAN, Fukuyama Y, Wohlke JL, Adelmann
J,Fontana J. D. (2004). Larvicidal action of ethanolic extracts from fruit
endocarps of Melia azedarach and Azadirachta indica against the dengue
mosquito Aedes aegypti. Toxicon 44: 829-835.

World Health Organization (2011).Deadliest Diseases Caused by

Mosquitoes.Retrieved August 17, 2013 from
http://www.who.org.ph/deadliest-diseases-caused-by-mosquitoes.html

59