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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”, “Nature’s 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

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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

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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
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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
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(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.

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) in diameter. with small. in which the female of most species is distinguished by a long proboscis for sucking blood. with colourless. bark moderately thick. deeply fissured and flaking in old trees. Itis a small to medium-sized treeup to 15m (30 max. sticky foetid sap. Eggs may be laid individually or connected together to form a “raft” type of structure Mosquito. dark grey outside and reddish inside. with a round.is the first stage in life cycle of a mosquito.Neem tree – is an evergreen tropical tree that belongs to the family Meliacease (Mahogany). bole branchless for up to 7. It is used to determine the toxicity level of the Azadirachta Indica (Neem) bark extracts derived from Decoction Method and Ethanol Extraction Method.) tall.the comma-shaped stage in the mosquito life cyle.5 m. This is the transition stage between the aquatic stages of the mosquito’s life cycle and the terrestrial adult stage.is commonly used in toxicology to determine the relative toxicity of chemicals to living organisms. 6 .any of various two-winged insects of the family Culicidae. Probit Analysis . scattered tubercles. Mosquito egg. large crown up to 10m (20 max. sometimes fluted at base. Mosquito pupa. up to 90 cm in diameter. branches spreading.

is derived from the Persian:Azad = Free. 2009) Neem bark is taken from the neem tree which predominately grows in India and Asia. dirakht = Tree.n.Neem is an attractive broad-leaved. 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. 2010) and Jernigan 7 . Neem bark has been utilized for centuries by the Indian and Asian cultures to treat numerous medical illnesses. i .d) Azadirachta Indica trees may start flowering and fruiting at the age of 4-5 years.. evergreen tree which can grow up to 30m tall and 2.Azadirachta indica .Hind = of Indian Origin which literally means: ‘The Free Tree of India’.REVIEW OF RELATED LITERATURE Azadirachta Indica (Neem Tree) Neem or Margosa is a botanical cousin of mahogany.5m in girth. Its spreading branches form a rounded crown of deep-green leaves and honey-scented flowers as much as 20m across (Neem Foundation. Its trunk usually straight is 30-80 cm in diameter. prevent pregnancy and also as a potent pesticide (Jernigan. but economic quantities of seed are produced only after 10-12 years (Orwa et al. It belongs to the family Meliaceae. The latinized name of Neem .

salannin. nimbidin (0. The dry flowers on extraction with petroleum-ether yields waxy substances like quercetin. 1-maliantriol. tannins (6. nimbosterol (0. The bark contains anti-inflammatory 8 . nimbinin. The seed consist of a shell and 1-3 kernels which contain azadirachtin and its homologues (Mordue. nimbinin (0.04%). Chemical Composition of Azadirachta Indica According to Chandy (n. the important chemical constituents of Azadirachta Indica are azardirachtin. nimbidin and others. green amorphores bitter toxic substance.The stem bark contains tannins (12-16%) and non-tannin (8-11%). tannins and a bitter principle margosine (Dabur. essential oil (0.001%).0%).). 2013).02%).pene and a fatty acid fraction. The trunk bark contains nimbrin (0.d).03%). The trunk bark contains nimbin. nimbosterol. a bitter principle margosine and 6-desacetyl nimbinene (Chandy.also added that one of the major uses of neem bark is as an insect repellent. nimbin. 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.d. myricetin and b-sitosterol on extraction with alcohol they produce a pungent essential oil. 2000). He also added that the different plant parts contain mostly terpenoids (limonoids).4%). n.d. nonacosane and a sesquiter. & Neem Foundation.n. 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.

Uses of Azadirachta Indica Neem. Azadirachta indica (Neem). Iron. Modern scientists have isolated more than 140 compounds from various parts of the Neem tree that have been evaluated for curative powers. carotene and oxalic acid (ReadAndDigest. etc.). make it one of the best herbal medicines. Potassium. Unani and Homoeopathic systems of medicine to treat many health related problems and ailments (Herbcyclopedia. nimbidiol.400. Nicocin. 2011).polysaccharide consisting of glucose. several diterpenoids. 2012). Riboflasium. viz. an herb extensively used in Ayurveda. have been isolated from stem bark and root bark (Neem Foundation. The bark also yields an antitumor polysaccharide. Calcium.d) Neem has rightly been called Sarvaroghari. Phosphorus. vitamin C. identified by WHO/UNEP1989 as an environmentally powerful natural pesticide.com. margocin. nimbione. Claimed to be a ‘Village dispensary’ the following properties found in Neem.n. nimbinone. n.d. Chloriphyle. is considered to be one of the most promising trees of the 21st century for its great potential in pest management. Neem is also well known and used for its medicinal properties from the ancient period (4000 BC). Besides polysaccharides. mainly 9 . environment protection and medicine (Nicoletti. Salts. Some of its chemical content are Sodium.. nimbolicin. arabinose and fructose at a molar ratio 1:1:1 with molecular weight of 8. Thiamine.

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

Neem leaves are also used in storage of grains. Larva. toothpastes. Its oil is used in the preparation of toothpaste and soap ( Johnson. neem leaves are used in curing neuromuscular pains. In order to increase immunity of the body. Neem twigs are used by millions of Indians as an antiseptic tooth brush. beauty aids and toiletries. 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. skin creams/lotions.d. neem leaves are also taken internally in the form of neem capsules or made into a tea. It has also been reported to work against termites. the mosquito goes through four separate and distinct stages of its life cycle and they are as follows: Egg. 2010). 2010 & Shiva. This tea is extremely bitter. shampoos. 2012).In the article of Tandon (n. In most cases Neem oil/extract is being used for making these cosmetics like soaps and tooth pastes etc (Johnson. pupa. However. Each 11 . It is also used to soak feet for treating various foot fungi. In Ayurveda.) entitled The Neem Tree. 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). and adult. The tea is traditionally taken internally to reduce fever caused by malaria. Different parts of Neem tree are being used extensively in manufacturing of soaps.

com. all types of wild animals including deer. Some female mosquitoes prefer to feed on only one type of animal or they can feed on a variety of animals. In the summer time it takes mosquitoes 3-10 days to totally complete their life cycle from egg to adult (Renchie. The male mosquitoes feed only on plant juices.d.635mm) long. and they also feed on snakes. domesticated animals. 2013). It was also stated in the study of Anderson and Harrington (n. 2007). The feeding habits of mosquitoes are quite unique in that it is only the adult females that bite man and other animals. such as cattle. eggs may be laid individually or connected together to form a “raft” type of structure. rabbits. Some 12 .) entiled “Mosquito Biology for the Homeowner “ that a freshly laid egg is light in color and darkens within a few hours. all types of birds including chickens. 2013). 2013). horses. Their life cycle which can vary in length depending on temperature and food resources. lizards. A standard egg raft is about 1/4 inch (6. In the life cycle of mosquitoes an egg is the first stage. Eggs are either deposited singly or as an egg raft depending on the type of mosquito. Depending on the mosquito species. and toads (Alameda County Mosquito Abatement District. Mosquito eggs are oval and about 1/40 th of an inch (0. while the Culex pipiens mosquito lays approximately 200 eggs which she unites to form a raft (MosquitoMagnet. The Aedes aegypti mosquito lays her eggs individually. frogs. goats.35mm) long and contains 100-200 eggs.of these stages can be easily recognized by their special appearance (Alameda County Mosquito Abatement District. Female mosquitoes feed on man. etc.

so it can’t just be discarded. 2013).al n. The number of single eggs laid per batch varies within and between mosquito species and can range from 60 to 200. 2013). Mosquito larvae can swim and dive down from the surface when disturbed (Freudenrich.75 inches (1 to 2 cm) long.species of mosquitoes lay their eggs singly and deposit them directly on water or floating aquatic vegetation. Larvae feed mostly on plant and animal debris in the water. thus allowing the larvae to grow. The nutrition provided by the blood-mealenriched yolk is therefore important for development (Lundquist. as they grow.Within seven to 10 days. This new exoskeleton is soft and flexible at first. According to Rey (2011). 2013). the larvae then shed the old exoskeleton and the new one hardens when exposed. larvae enter the pupal stage (Orkin. Larger larvae can be seen floating just above the surface of infested waters. The mosquito eggs hatch into larvae or "wigglers. inflexible envelope that is absolutely essential for larval survival. Eggs will hatch into larvae within 24 to 48 hours (Orkin. Others will lay their eggs on moist soil that is subject to periodic flooding. When ready to molt.d)." which live at the surface of the water and breathe through an air tube or siphon (Freudenrich. or above the water line in natural and artificial containers. In order to grow. M. This food is generally low in nutritional value. they shed their skin (molt) several times. 13 . to protect the larvae’s internal organs. The larvae filter organic material through their mouth parts and grow to about 0.5 to 0. 2013). the larvae are enclosed in a hard. et. mosquito larvae grow a new exoskeleton under the old one.

In the study of Freudenrich (2013). Mosquitoes in this transitional stage are referred to as “tumblers. the pupal stage is a resting. 2011). the fourth developmental stage of mosquito is when larvae molt into the comma-shaped stage called the pupa (or tumblers). 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. In this stage. 2013). but pupae are mobile.com. the stated that at the end of the pupal stage. breathing through two horn-like tubes (called siphons) on their back. non-feeding stage of development. 2013). This is the transition stage between the aquatic stages of the mosquito’s life cycle and the terrestrial adult stage.” describing how they propel through the water. Their narrow wings are often 14 . the mosquito pupa rests without eating as it prepares to change into an adult mosquito (MosquitoMagnet. and legs. (EnchantedLearning. 2010). Pupa or pupae (plural) live near the surface of the water. Adult mosquitoes are characterized by having long.com. responding to light changes and moving (tumble) with a flip of their tails towards the bottom or protective areas. antennae. needle-like mouthparts (proboscis). If a water source evaporates before the larvae and pupae within it transform into adult mosquitoes. According to American Mosquito Control Association (2011). the pupae encase themselves and transform into adult mosquitoes. Larvae and pupae usually cannot survive without water. slender.According to Rey (2011). those young often will die (Orkin. The wings have to spread out and dry properly before it can fly (American Mosquito Control Association.

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”. protozoans. They wait for females to emerge and each female takes flight she enters the cloud and mates with a waiting male. Some mosquito species overwinter as blood fed females and can survive for multiple months (Johnsen & Renchie. 2007). This water can range in quality from melted snow water to sewage effluent and it can 15 . n.). Habitats of Mosquito All mosquitoes must have water in which to complete their life cycle. 2011).d. 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. mating. plant exudates) throughout their life to maintain energy for flying. and helminthes (worms) to humans and animals. they cited that female and male mosquitoes both require carbohydrate sources (nectar. and seeking hosts for bloodmeals. The process of taking a bloodmeal is how the mosquito is able to vector viruses.covered with minute scales (Anderson and Harrington. 2013). 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. In the study of Johnsen and Renchie (2007). Females are capable of flying for miles (Orkin. 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.

such as streams. 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.be in any container imaginable. or Container. They lay their eggs in such places such as tree holes that periodically hold water. Larvae can be flushed out when stream volume increases. irrigated pastures. 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. Mosquito species breed in running waters. and Uranotaenia sapphirina have all been found in streams. 2013). Mosquitoes can lay dozens of eggs up to 5 times during their lifetime (Centers for Disease Control and Prevention. In Chagasia and some addition. are The stream tropical genus breeders. the adult mosquitoes show a very distinct preference for the types of sources in which to lay their eggs. and to remain in the stream requires a large amount Anopheles species of energy. Transient 16 . Culex territans. (Alameda County Mosquito Abatement District. Anopheles quadrimaculatus. Permanent Water. sewage effluent ponds. According to Rutgers (2008). Aquatic habitats are containers in which eggs develop into adult mosquitoes. 2012). Also. although they prefer other habitats. etc. tide water pools in salt marshes. The type of water in which the mosquito larvae is found can be an aid to the identification of which species it may be. rain water ponds. mosquito habitats can be generally grouped into four types: Running Water. Transient Water.

might be able to pull off a single generation during an extended flooded period. Aedes adults will oviposit near the edge of the swamp. The habitat of containers are based on the containers themselves. water quality and mosquito species present. rushes and sedges are typical freshwater swamp vegetation. Cattail. Many treehole species now also use artificial sites. or within tussocks of vegetation. Genera associated with permanent water are Anopheles. such as tires since they provide insulation against the weather and are more numerous. such as Aedes and Psorophora. such as water found in tires. there is a seasonal change in the vegetation. Container water habitat can be found in both natural settings. Artificial containers are a convenient mode of transporting a species of mosquito outside of it's natural range. and Uranotaenia. Culiseta. snowpools. Treehole sites generally have tannin-enriched water which is characteristically clear. and ditches are used as breeding grounds for mosquito species whose eggs can withstand desiccation. Transient water generally show water quality changes which results in various mosquito species using the same pool over a period of time. with rotting wood at the bottom. As with transient waters. Culex.water sources. Permanent waters (also known as Semipermanent) are present for extended periods of time and support characteristic aquatic vegetation. 17 . Eggs of these species are not desiccant-resistant and must be laid directly on the water. like an opportunistic Culex. Their life cycles require alternating periods of wet and dry. Other species. such as water held by plants (bromeliads) to artificial settings. such as flooded areas. requiring later flooding to inundate the eggs for hatching. Coquillettidia.

buckets or toys left outside(Mosquito World.). Related Studies The neem tree.d. Water storage containers should be kept clean and sealed so mosquitoes cannot use them as aquatic habitats (Centers for Disease Control and Prevention. 2012).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. Their eggs must stay in water in order to survive and usually will hatch within a couple of days. Many permanent water mosquitoes can also breed in containers that collect and hold water.d. Culex and Anopheles mosquitoes are among the most common permanent water mosquitoes. and frequently changing the water of animal drinking pans and flower pots will greatly reduce the risk of dengue infections. n.n. Disposing of unused containers.).. such as wading pools. 2004. placing useful containers under a roof or protected with tight covers. Azadirachta indica has well-known insecticidal (Wandscheer et al. Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue 18 . These mosquitoes are most active when the average temperature is above 70 degrees. releasing larvae to begin the development process (Mosquito World.

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

contraceptive.g. azadirachitin has been found to be the main ingredient for fighting insects and pests. 20 . 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. it was stated that Azadirachtin is the main component responsible for both antifeedant and toxic effects in insects. Azadirachtin was found in the bark of introduced neem tree (Azadirachta indica A. 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. flowers. In the journal by Scientific Technical and Medical Open Access Journals ( 2013) entitled Azadirachtin Found in the Bark of Neem Tree Grown in China. being up to 90% effective in most instances. Juss) grown in China by TLC examination.. It repels and disrupts the life cycle. The content of azadirachtin in the bark of neem tree was tested and calculated to be 0.worldwide. e. bark. Other limonoid and sulphur-containing compound with repellent. In the study of Mordue (2000) entitled “Azadirachtin from The Neem Tree Azadirachta indica: Its Action Against Insects”.65 mg / g in dried bark by HPLC. roots. The Neem Tree). nd. antiseptic. antipyretic and antiparasitic properties are found elsewhere in the tree. but is nonetheless one of the most effective growth and feeding deterrents ever examined (Sanskrit Documents Org. leaves. Of these limonoids. however does not kill immediately.

In addition. It affects the corpus cardiacum. Epilachna varivestis. Azadirachta indica). has been reported to have adverse effect on endocrine system of a bean bettle. Sridharan also added that azadirachtin is structurally similar to insect hormones called "ecdysones". Insects then will not molt.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. 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. Azadirachtin. which controls metamorphosis in the insects as they pass from larva to pupa to adult. Properties and potential of natural pesticides from the neem tree. a limonoid. It is one of the most potent growth regulators. According to Schumetterer(1981. Azadirachtin because of its structural similarity to ecdysone will block the ecdysone’s action in metamorphosis and the release of these vital hormones. Azadirachtin disrupts the growth and reproduction in most of the pest. This of course breaks their life cycle. an organ similar to the human pituitary that controls the secretion of hormones. Sridharan (2009) also cited in his study entitled Neem Tree: Melia Azadirachta and Azadirachta Indica that Azadirachtin is mainly 21 . Metamorphosis requires a careful synchrony of many hormones and other physiological changes to be successful. and to cause sterility in the female insects. It will repel or reduce the feeding of many species of pest insects as well as some nematodes.

The researcher controlled and manipulated the independent variable and randomly assigned the subjects to different conditions or situations (Tan. Research Design This research used an experimental research design. It is one of the most potent growth regulators. The researcher used the random sampling technique in getting their test insects. It will repel or reduce the feeding of many species of pest insects as well as some nematodes. and statistical treatment that will be used in the course of this study. Azadirachtin disrupts the growth and reproduction in most of the pest. setting. research instrument. This is an inquiry on cause-and-effect relationships. experimental unit or research center. and is conducted in a specialized setting. Random sampling technique is a technique where each member of the population has an equal chance of being selected as subject.responsible for the insecticidal properties of the neem. sampling technique. they do not like the smell of neem oil. The entire process 22 . such as the laboratory. Insects are perceptive to smell. 2006). METHODOLOGY This includes the research design. the participants. measures. research procedure.

of sampling is done in a single step with each subject selected independently of the other members of the population (Castillo. Each method of extraction will have the same concentration of distilled water extract. one (1) as the negative control. 2009). in treatment four (4) thirty percent (30%) extract of Azadirachta Indica bark and seventy percent (70%) distilled water and 23 . In treatment 0. 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. The research design is divided into two variables: the dependent variable (x) and the dependent variable (y). one (1) as the positive control and will be conducted with three (3) replicates each. The independent variable (x) corresponds to Azadirachta Indica bark extract. 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. 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. in treatment three (3) twenty percent (20%) extract of Azadirachta Indica bark and eighty percent (80%) distilled water and its replicate respectively. and three (3) treatments.

its replicate respectively. 24 . The dependent variable (y) represents the percentage of mortality rate of mosquito wriggler .

third replication of T2 25 .second replication of T2 R3.second replication of T0 R3.0% 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.LEGEND FOR RESEARCH DESIGN T0.third replication of T0 T1.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 R2.0% of Azadirachta Indica (Neem) bark extract and 100% distilled water as the negative control of the sample R1-first replication of T0 R2.

T3.second replication of T3 R3. R1-first replication of T3 R2.the addition of 20% of Azadirachta Indica (Neem) bark extract and 80% of distilled water to the sample.600mL 95% Ethanol solution Rotary Evaporator Funnel Erlenmeyer Flask Cornmill Grinder 30 pcs 500mL calibrated measuring cups 5X 80mm Magnifying glass 26 .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. R1-first replication of T4 R2.second replication of T4 R3.third replication of T3 T4 – the addition of 30% of Azadirachta Indica (Neem) bark extract and 70% of distilled water to the sample.

Koronadal City. All these barks were collected from Barangay Saravia. Collection and Preparation of Plant Extracts Plant extracts were obtained from the bark parts of the Neem plant (Azadirachta Indica). Bark is separated from its wood 27 .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.GENERAL PROCEDURE A.

600mL of 95% ethanol solution for 24 hours. 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. A. in a 1000mL glass beaker (with stopper) containing 400mL of distilled water and boiled for 30mins . and then they were sieved using funnel and coarse filter paper. 28 . 1 Decoction Extraction Method The air-dried Neem bark was Bark is thoroughly washed ground using a grinder.A. The boiled extracts were allowed to cool. 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. under distilled water before air400gm of the ground plant materials were divided into 100gm each and were put drying. B. 2 Ethanolic Extraction Method The plant extracts were carried out using ethanol by soaking 400gm of the ground materials in 1.

and the other one was added with Mayer’s Reagent. The third one was added with 3mL and 28% Ammonia until alkaline and added with 10mL Chloroform then divided into two test tubes. Then it was divided into three for confirmatory tests. 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. The second one was added with Dragendorff’s Reagent. The other one was added with Aqueous extract and 2M HCL then filtered and separated into two portions and tested with Dragendorff and Mayer’s Reagent.C. 29 . 5mL of 2M HCL was being added to the residue and stirred before it was filtered in a testube. Observation made for the formation of precipitation indicated positive results.PHYTOCHEMICAL ANALYSIS OF THE EXTRACTS The Phytochemical analysis of the extracts is based on the Phytochemical Screening by Guevara (2005). 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 Mayer’s Reagent.

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.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. 30 .

5mL 12M HCL and magnesium turnings. The third testube served as the control. 31 .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. The content of the second extract with 12 M HCl were put in separate beaker with water and place in a hot plate. Each extract was divided into 3 test tubes for confirmatory test. The first test tube of each extract will be added with 0. The occurrence of a red or orange colouration was indicative of the presence of flavonoids compounds. 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.

The residue was dissolved in 20 mL of distilled water and 5 drops of NaCl solution then filtered.Test for Tannins: 10grams of plant material was evaporated to dryness using evaporating dish. The first one served as control. 32 . The third one was added with three drops of 10% of FeCl3 were added to the filtrate. Then it was divided into 3 parts. The appearance of blackish-blue or blackish-green colouration was indicative of tannins. The second one was added with gelatin-salt reagent.

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. Red colouration was indicative of anthraquinones. The filtrate were added with 5ml benzene then divided into two tubes. One served as control. 33 . The residue were added with 10mL of distilled water then filtrated. 2mL of each extract were placed into each evaporating dish and placed in a hot plate till dryness. The other one was added with 5mL ammonia solution.

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 .

Treatment 2 (T2). The experiment were laid out using Completely Randomized Design (CRD) with the following treatments: Treatment 0 (T0). 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. D. TESTING FOR TOXICITY The extracts were concentrated to The soaked plant material were dryness using rotary evaporator. Treatment 1 (T1). Mortality rates were compared to determine the lethal effects and the level of toxicity. 600mL of 95% ethanol solution for 24 hours.400gm of the ground bark was soaked in i. varying concentrations of Azadirachta Indica (Neem) bark extract. Mortality rates were observed after 24 hours. sieved using funnel and coarse Ten (10) larvae were added to each calibrated measuring cup containing filter paper. 36 .

It is the median lethal dose of a substance. 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. Probit Analysis is commonly used in toxicology to determine the relative toxicity of chemicals to living organisms. 2008). and Treatment 4 (T4) consisting of three replicates in each treatment were prepared. 37 . or the amount required to kill 50% of a given test population. Mortality rates of mosquito wrigglers in each glass was observed after 24 hours with the use of 5x80mm magnifying glass. LD50 is a measurement used in toxicology studies to determine the potential impact of toxic substances on different types of organisms.g. T1 contains 0% Azadirachta Indica (Neem) bark extract and 100% liquid insecticide as positive control. and T4 contains 30% Azadirachta Indica (Neem) bark extract and 70% distilled water. Probit Analysis and Lethal Dosage (LD50) was used to determine the toxicity level of the dosage. the response is always binomial (e. 2013). T2 contains 10% Azadirachta Indica (Neem) bark extract and 90% distilled water. As discussed above. death/no death) and the relationship between the response and the various concentrations is always sigmoid (having the shape of letter S) (Vincent. Add 10 mosquito wrigglers in each of the glass of different concentration. T0 contains 0% Azadirachta Indica (Neem) bark extract and 100% distilled water as negative control. It provides an objective measure to compare and rank the toxicity of substances (Hadley. T3 contains 20% Azadirachta Indica (Neem) bark extract and 80% distilled water.Treatment 3 (T3).

38 .FLOW CHART OF THE TESTING FOR TOXICITY 10 larvae were added to each container containing varying concentrations of Azadirachta Indica Bark extract.

Two-Way Analysis of Variance (ANOVA) . 2006). One-Way Analysis of Variance (ANOVA) was used. Ttest. Two-Way 39 . This is a statistical procedure for testing mean differences among three or more groups by comparing variability between groups to variability within groups (Tan.Larvae were exposed to extracts for 24 hours. Probits Analysis and LD50. STATISTICAL TREATMENT In analyzing the data gathered for this study. E. Mortality rates were observed after 24 hours. the researcher will use OneWay Analysis of Variance (ANOVA). To analyze variance between the effects of Azadirachta Indica (Neem) Bark Extract obtained from Decoction Method and Ethanol Extraction Method.

referred to as factors. LD50 is a measurement used in toxicology studies to determine the potential impact of toxic substances on different types of organisms. As discussed above.Analysis of Variance was also used. affecting the outcome of the dependent variable. 2008). The t distribution is commonly used with samples less than 30 units (Asperas. and contain be effective. the response is always binomial (e. Probit Analysis and Lethal Dosage (LD50) will be used to determine the toxicity level of the dosage. 2005).com). To ANOVA assumes population samples are equal in variance. The t-test was also used to determine the difference between the means of two methods of Azadirachta Indica (Neem) bark extraction. death/no death) and the relationship between the response and the various concentrations is always sigmoid (having the shape of letter S) (Vincent.g. sample groups of equal size (BusinessDictionary. Probit Analysis is commonly used in toxicology to determine the relative toxicity of chemicals to living organisms. independent. This is a parametic statistical test that shows the difference between the means of two groups of values. 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. normally a two-way distributed. It is the median lethal 40 . The two-way ANOVA is grounded in the idea that there are two variables. This is a means of comparing multiple levels of two independent variables.

or the amount required to kill 50% of a given test population. FLOW CHART OF THE GENERAL PROCEDURE Collection of Plant Materials and Culturation of Test Insects Preparation of Extracts Phytochemical Screening 41 . It provides an objective measure to compare and rank the toxicity of substances. a lower value is regarded as more toxic.dose of a substance. When comparing LD50 values. as it means a smaller amount of the toxin is required to cause death (Hadley. 2013).

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

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.Indica (Neem) bark extract obtained from Decoction Method and Ethanol Extraction Method by using ANOVA (Analysis of Variance). 2005). Table 1. Phytochemical Screening of Plant Extract Phytochemical ALKALOIDS SAPONINS FLAVONOIDS TANNINS ANTHRAQUINONES Dragendorff’s Test Mayer’s Test Confirmatory Test Aqueous Dragendorff’s Mayer’s Chloroform Dragendorff’s Mayer’s Frothing Benzopyrene Leucoanthocyanins Gelatin-Salt Ferric Chloride Ammonia Solution Decoction Ethanol Method Extraction - Method + + + + + + + + + + + + + + + 43 . 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.

44 . Table 2. 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. 12 10 8 6 4 2 0 Replicate 1 Replicate2 Replicate3 Table 2 shows that among the three treatments (Treatment 2. tannins and anthraquinones when it undergone different confirmatory tests which shown at the middle column of the table.Legends: Slight turbidity Definite turbidity Heavy precipitation Not performed (+) (++) (+++) (-) Table 1 shows the phytochemical screening results of the plant extract. Treatment 3. Mortality Rates of Mosquito Larvae Upon Exposure to Azadirachta Indica (Neem) Bark Extract Obtained Through Decoction Method. Table 3. and Treatment 4). saponins. Mortality Rates of Mosquito Larvae Upon Exposure to AzadirachtaIndica (Neem) Bark Extract Obtained Through Ethanol Extraction Method. flavonoids. It was shown in the table that the plant extract is positive in or contains alkaloids.

4 Variance 13. and Treatment 4). Analysis of Variance Between the Effects of Azadirachta Indica (Neem) Bark Extract Obtained from Decoction Method and Ethanol Extraction Method. Based from the results on Mortality rates of Mosquito Larvae upon exposure to Azadirachta Indica (Neem) Bark Extract from the table 2 and 3.4 45 . Treatment 3.12 10 8 Replicate 1 6 Replicate 2 4 Replicate 3 2 0 Treatment 0 (negative control) Treatment 2 Treatment 4 Table 3 shows that among the three treatments (Treatment 2. Summary Groups Column 1 Count 15 Sum 81 Average 5. 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. 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.

050 46 . DM EEM Difference N 15 15 15 Mean 5.195972 458.531. 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. Since the F statistic is smaller than the critical value. Remember from above.589 95% CI for mean difference: (-2.Column 2 Source of Variation Between Groups Within Groups Total 15 100 6.40 6.03333 0. Therefore.666667 19.14 0. -0.282 SE Mean 0.195972. So.734166 0.39048 470.734166.267 StDev 3. Test on the Significant Difference Between Azadirachta Indica (Neem) Bark Extract obtained from Decoction Method and Ethanol Extraction Method. the null hypothesis was that all 2 of these groups' means were equal.39881 4.003) T-Test of mean difference = 0 (vs not = 0): T-Value = -2.9667 29 Table 4 shows that with the F value of 0.03333 1 12.40 2. Table 5.66 4.15 P-Value = 0.95 1.9333 28 16. the critical F = 4. we fail to reject the null hypothesis.67 -1.38095 SS df MS F P-value F crit 12. 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.

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. first we must select the correct critical value from the table to compare with our calculated value.3866 0. Hence. Since our t-value (t = 2. A lower 47 .362 5 0. It was shown that toxicity level of the extract is at dose 1 having a probit percent of 0.15) is larger than the tabled t-value (t = 1.4771 0.697) this means that there is a small chance that the population means are the same.6375 0.8458 Table 6 shows toxicity level of Azadirachta Indica (Neem) bark extract derived from Decoction Method.8 0. 5 0.6055 10 5 1.0552 0.3613 10 4 1. Using the Critical Values for the tDistribution.613 4 6.0414 -1.055 2 7.Table 5 shows the difference between the means of two methods of Azadirachta Indica (Neem) bark extraction.7362 10 8 LD50: 14. and so it is reasonable to conclude that the means are different.3613 and an LD50 of 14.697. We do this by computing the degrees of freedom and the value is 1.0552 1 0. Results on Level of Toxicity Using PROBIT ANALYSIS and LETHAL DOSAGE (LD50) on Decoction Method of Extraction According to FINNEY’S METHOD Dose (Stimulus ) Actual Percent (%) Probit Percent N R E(R) Difference Chi-Square 3.8458. Table 6.1839 0.301 0.4 0.

975 1.999 7 10 -0. 2013).0062 1 10 9. A lower value is regarded as more toxic. (Hadley.8297 Table 7 shows toxicity level of Azadirachta Indica (Neem) bark extract derived from Decoction Method.75 0 -0. 2013). Table 7.0062 0.value is regarded as more toxic. as it means a smaller amount of the toxin is required to cause death.75 3 9. 48 .4771 0. It was shown that toxicity level of the extract is at dose 1 having a probit percent of 0. (Hadley.975 LD50: 10. Results on Level of PROBIT ANALYSIS and LETHAL DOSAGE (LD50) RESULTS on Ethanol Extraction Method According to FINNEY’S METHOD Dose (Stimulus ) 1 1.25 0.3 1 10 10 3 9.2497 0 0.3 and an LD50 of 10.8297.3 0. as it means a smaller amount of the toxin is required to cause death.301 Actual Percent (%) Probit Percent N R E(R) Difference Chi-Square 0.

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. Flavonoids and Anthraquinones. Extraction with distilled water (Decoction Extraction Method) and Extraction with 95% ethanol solution (Ethanol Extraction Method). was performed to determine the chemical compounds present in Azadirachta Indica (Neem) bark.SUMMARY. A Phytochemical Screening. which includes test for Saponins. Tannins. CONCLUSIONS AND RECOMMENDATIONS This section presents the summary of study and conclusions made from the study and the recommendations given by the researchers. Two methods were used for the extraction of Azadirachta Indica (Neem) bark. 49 . Alkaloids.

Monitoring and observation were made after 24 hours. Treatment 2 (T2). 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. 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. The data gathered from mortality of larvae in toxicity testing was treated by using Two-Way ANOVA (Analysis of Variance). 2005). 50 . Treatment 3 (T3) and Treatment 4 (T4) consisting of three replicates in each treatment were prepared. 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. 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. LD50 (Lethal Dosage 50) and T-Test. T1 is the positive control which contains 100% liquid insecticide. Probit’s Analysis.There were five treatments in each of the method of extraction. T0 is the negative control which contains 100% distilled water. 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). Treatment 0 (T0). Treatment 1 (T1). T2 contains 10% Azadirachta Indica (Neem) bark extract and 90% distilled water.

the researchers concluded that: 1. 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. tannins.The results from the tests indicated that based on the Phytochemical Screening Result. flavonoids and anthraquinones. Azadirachta Indica (Neem) bark extract derived from Decoction Method and Ethanol Extraction Method are both effective as insecticide against mosquito larvae. 2. Azadirachta Indica (Neem) bark extract contains insecticidal property against mosquito larvae. 3. 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. Azadirachta Indica (Neem) bark extract contains alkaloids. 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. saponins. Based on the result from ANOVA Test Result. Conclusions After a thorough analysis of the data gathered. Recommendations The results show that Azadirachta indica (Neem) Bark Extract derived from Decoction Method and Ethanol Extraction Method are both effective as 51 .

2. The researchers recommend the following for further study and development of this study: 1. Making a soap or lotion out of Azadirachta Indica (Neem) bark extract. 3. 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. A further study on the comparison of the different parts of Azadirachta Indica (Neem) in terms of their larvicidal activity.insecticide against mosquito larvae by mixing 400 grams of plant material to 1. 52 . 600ml of water or 95% ethanol solution.

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