THE POTENTIAL OF NEEM EXTRACT,

Azadirachta indica AS NATURAL PESTICIDE ON ANTS,

Ochetellus spp.

NOOR FAIZATUL ASYIKIN BINTI RAZAHA

BACHELOR OF SCIENCE (Hons.) BIOLOGY

FACULTY OF APPLIED SCIENCES
UNIVERSITI TEKNOLOGI MARA

JULY 2017
 THE POTENTIAL OF NEEM EXTRACT,
Azadirachta indica AS NATURAL PESTICIDE ON ANTS,
Ochetellus spp.

NOOR FAIZATUL ASYIKIN BINTI RAZAHA

Final Year Project Report Submitted in

Partial Fulfilment of the Requirements for the
Degree of Bachelor of Science (Hons.) Biology
in the Faculty of Applied Sciences
Universiti Teknologi MARA

JULY 2017
This Final Year Project Report entitled “The Potential of Neem Extract,
Azadirachta indica as Natural Pesticide on Ants, Ochetellus spp.” was submitted
by Noor Faizatul Asyikin binti Razaha, in partial fulfilment of the requirements for
the Degree of Bachelor of Science (Hons.) Biology, in the Faculty of Applied
Sciences, and was approved by

_________________
Hafizah binti Kassim
Supervisor
B. Sc. (Hons.) Biology
Faculty of Applied Sciences
Universiti Teknologi MARA
72000 Kuala Pilah Negeri Sembilan

___________________ _________________________
Lili Syahani binti Rusli Dr. Nor’aishah binti Abu Shah
Project Coordinator Head of Programme
B. Sc. (Hons.) Biology B. Sc. (Hons.) Biology
Faculty of Applied Sciences Faculty of Applied Sciences
Universiti Teknologi MARA Universiti Teknologi MARA
72000 Kuala Pilah 72000 Kuala Pilah
Negeri Sembilan Negeri Sembilan

Date: 18 July 2017

 ACKNOWLEDGEMENTS

First of all, praise to God for His blessings and for giving me strength and
opportunity in completing this project within its expectation and time allocated.
I would like to take this opportunity to express my thanks and gratitude to the
following persons, who have directly and indirectly giving their generous
contribution towards the completion of this project.

First and foremost, I would like to express my special thanks to my supervisor,

Madam Hafizah binti Kassim for her guidance and comments throughout the
period of this project. She also never forgets to give me motivation to complete
this project.

I would also like to give my special thanks to the laboratory assistant of the
Faculty of Applied Sciences for their information about my project. I would also
like to extend my gratitude to my family for their support and motivation. Also,
for my friends that always be there for me and never failed to give me a lot of
encouragement to keep up with this project until this project is completed.

(Noor Faizatul Asyikin binti Razaha)

iii
 TABLE OF CONTENTS

PAGE
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS iv
LIST OF TABLES vi
LIST OF FIGURES vii
LIST OF ABBREVIATIONS viii
ABSTRACT ix
ABSTRAK x

CHAPTER 1: INTRODUCTION
1.1 Background Study 1
1.2 Problem Statements 3
1.3 Significance of the Study 3
1.4 Objectives of the Study 4

CHAPTER 2: LITERATURE REVIEW

2.1 Neem 5
2.1.1 Neem’s leaf 7
2.1.2 Neem’s bark 9
2.1.3 Neem’s flower and fruit 9
2.1.4 Neem’s seed 10
2.2 Active Compound in Neem 10
2.2.1 Azadirachtin 13
2.3 Pests 15
2.4 The Importance of Pest Control 16

CHAPTER 3: METHODOLOGY
3.1 Materials 18
3.1.1 Raw materials 18
3.1.2 Chemicals 18
3.1.3 Apparatus 18
3.2 Methods 19
3.2.1 Preparation of neem seeds and neem leaves 19
3.2.2 Extraction of neem seeds and neem leaves 19
3.2.3 Collection of ant 20
3.2.4 Testing the effectiveness of neem’s insecticide against 20
Ants
3.3 Statistical Analysis 21

iv
CHAPTER 4: RESULTS AND DISCUSSIONS
4.1 The Extraction of Neem Leaves and Neem Seeds 22
4.2 Neem’s Extract Treatment On Ants 23
4.2.1 Behaviour and response 24
4.2.2 Effectiveness of neem solutions 25

CHAPTER 5: CONCLUSIONS AND RECOMMENDATIONS 29

CITED REFERENCES 30
APPENDICES 33
CURRICULUM VITAE 34

v
 LIST OF TABLES

TABLE TITLE PAGE

2.1 Some bioactive compounds from neem 12

2.2 Effects of azadirachtin against insects 14

4.1 Weight of neem crude 22

4.2 Results treatment with insecticide solutions 25

4.3 Effectiveness of different solutions on ants 25

vi
 LIST OF FIGURES

FIGURE TITLE PAGE

2.1 Neem Tree 6

2.2 Neem leaves 8

4.1 Insecticide solutions 23

vii
 LIST OF ABBREVIATIONS

°C Degree Celcius

Cm Centimetre

M Metre

Mm Millimetre

Kg Kilogram

Rs. Indian Rupee

w/v weight/volume

G Gram

Ml Millilitre

viii
 ABSTRACT

THE POTENTIAL OF NEEM EXTRACT, Azadirachta indica

AS NATURAL PESTICIDE ON ANTS, Ochetellus spp.

Pest is known to be a vector to various of diseases and usually is controlled by

using chemical based insecticide. Chemical based insecticide can give harm to
human’s health. Azadirachta indica, also known as neem tree has great potentials
in the fields of pest management as natural bio-pesticide. Therefore, the objectives
of this research are to study the potential of Azadirachta indica used as natural
pesticide against pest such as ant. Also to compare the effectiveness between neem
seeds extract and neem leaves extract as natural pesticide. The crude extract of
neem leaves and neem seeds were prepared for the treatment toward ants along
with positive control which was citronella and distilled water as the negative
control. 360 ants of Ochetellus spp. were captured and trapped in a container. The
behaviour of the ants after exposed to both neem extract and citronella solutions
was positive compared to distilled water. The effectiveness for citronella is 100 %
while neem leaves extract and neem seeds extract were 68.89% and 77.78%
respectively. Hence, both of neem extracts can give an alternative method for
controlling pest such as ants as it contains natural compound such as azadirachtin
which have the ability to act as natural insecticide. Based on statistical analysis,
the result also shows that there is not enough evidence to support the claim of
previous study that the neem seed extract is more effective than the neem leaves
extract. Difference in trees maturity, application technique and perhaps
environmental factors would probably contribute to these inconsistencies.

ix
 ABSTRAK

POTENSI EKSTRAK POKOK SEMAMBU, Azadirachta indica

SEBAGAI RACUN SERANGGA SEMULAJADI KE ATAS SEMUT,
Ochetellus spp.

Serangga perosak juga merupakan pembawa kepada pelbagai jenis penyakit dan
ianya selalu dikawal dengan menggunakan racun serangga yang mengandungi
bahan kimia. Racun serangga yang mengandungi bahan kimia ini boleh membawa
kesan buruk kepada manusia. Azadirachta indica atau lebih dikenali sebagai
pokok semambu mempunyai potensi yang sangat luas di dalam bidang
pengurusan serangga perosak terutamanya sebagai racun serangga semulajadi.
Objektif kajian ini adalah untuk mengkaji potensi Azadirachta indica sebagai
racun serangga semulajadi untuk membasmi serangga perosak seperti semut, dan
juga untuk membandingkan keberkesanan antara ekstrak daripada biji benih
dengan ekstrak daripada daun pokok semambu sebagai racun serangga
semulajadi. Ekstrak daun semambu dan biji benih semambu disediakan untuk
dikaji kesannya keatas semut bersama kawalan positif iaitu citronella dan air
suling sebagai kawalan negatif. 360 ekor semut Ochetellus spp. telah ditangkap
dan diperangkap di dalam bekas. Perlakuan semut setelah didedahkan dengan
kedua ekstrak pokok semambu dan citronella menunjukkan kesan positif
berbanding dengan air suling. Peratus kadar kematian untuk citronella adalah
100% manakala ekstrak daun semambu dan ekstrak biji benih semambu adalah
68.89% dan 77.78%. Oleh itu, kedua jenis ekstrak semambu boleh dijadikan
sebagai kaedah alternatif untuk mengawal serangga perosak seperti semut kerana
ia mengandungi komponen semulajadi seperti azadirachtin dimana ianya boleh
digunakan sebagai racun serangga semulajadi. Keputusan analisa statistik juga
menunjukkan bahawa bukti tidak mencukupi untuk menyokong tuntutan kajian
terdahulu iaitu ekstrak biji benih semambu adalah lebih berkesan daripada ekstrak
daun semambu. Kepelbagaian kematangan di antara berlainan pokok semambu,
teknik-teknik aplikasi dan faktor alam sekitar juga mungkin menjadi antara salah
satu penyebab kepada ketidakselarasan yang berlaku ini.

x
 CHAPTER 1

INTRODUCTION

1.1 Background Study

Azadirachta indica, belong to the Meliaceae family, also known as neem tree.

It has one or two species in the genus of Azadirachta (Hossain et al., 2013).

Neem tree is distributed in diverse locations around the world and is

knowingly native to India, Indian subcontinent (Quelemes et al., 2015) and

Burma (Munoz-Valenzuela et al., 2007). Girish and Shankara Bhat (2008)

stated that neem tree is the most researched tree in the world and said to be

the most propitious tree of 21st century. It has great prospective in the fields

of pest management, environment protection and medicine.

Neem is called “wonder plant” as most of the plant part such as stembark,

rootbark, leaves, flowers, fruits, seeds and seed kernels can be used for good

especially as a natural pesticide (Asogwa et al., 2010). They also have been

used widely in traditional medicine to treat various human illnesses because

they have anti-inflammatory, antiarthritic, antipyretic (used to reduce fevers),

hypoglycemic, spermicidal, antifungal, antibacterial, diuretic (increases

urine flow) and antimalarial properties (Choudhary and Lahti, 2011).

1
Nowadays, natural pesticides from plant are securing popularity in the

community due to rising of the public awareness. It is practically sustainable

replacement and also can reduce environmental pollution compared to

chemical pesticide which can give harm to human’s health resulting from

imprudent usage of their synthetic counterparts. Thus, this natural plant-

derived pesticide can be as an alternative method to control common pest that

live among humans.

Several biologically active chemicals called limonoids such as azadirachtin,

azadirachtol, salannin, nimbin and nimbidin in neem clasp a key ingredient

for diverse activities in controlling pest and microbes. Azadirachtin as the

main element is the most abundant. It dominates a growth disrupting and

larvicidal properties against pest responsible for the toxic effects in insect

(Jadeja et al., 2011). Salako et al., (2008) informed that most of the urban

farmers used neem leaves to make pesticides while in recent studies stated

that neem seeds tend to be more effective due to higher azadirachtin content

in it. Therefore, this study will be run to investigate the potential of extracted

different part of neem plant used as bio-pesticide for controlling pest such as

ant, from Ochetellus spp. and whether it is true that the neem seed extract is

better in term of effectiveness than the leaves extract against the pests.

2
1.2 Problem Statement

Pest has been a huge nuisance to be handled with. Due to close relationship

between pest such as ant and human, the easiest way to get rid of them is by

using broad spectrum of chemical-based pesticide. However, apart from its

effectiveness and convenience, it can pose certain problems like non-

environmental friendly, toxicity, non-biodegradable and may develop

resistance to specific pesticides chemical (Asogwa et al., 2010). As for neem,

both of it seeds and leaves are frequently used to make natural pesticide but

which one is better in term of efficiency to control pest is still a mystery

because there is no concrete proof to measure the statement of neem seeds

works better as bio-pesticide than the leaves.

1.3 Significance of the Study

The knowledge of the effectiveness of Azadirachta indica can give an

alternative method to control pest such as ant as it contains chemical

compound that can work as natural pesticide. This research can give a clearer

view of the efficiency between neem seeds extract and neem leaves extract.

It also gives the information about which part of the neem plant will works

best as natural pesticide. Thus it can also be used as reference to researchers

who intended to further this study in the future.

3
1.4 Objectives of the Study

1. To investigate the potential of Azadirachta indica use as natural

pesticide against pest such as ant.

2. To compare the effectiveness between neem seeds extract and neem

leaves extract as natural pesticide.

4
 CHAPTER 2

LITERATURE REVIEW

2.1 Neem

Neem tree or Azadirachta indica is commonly known as margosa or Indian

Lilac belongs to the family of Meliaceae (Luntz and Nisbet, 2000). Neem is

the most resourceful tree with enormous future. Mostly all of its part such as

leaves, bark, fruits, flowers, seed and oil can be used to make products. This

tree is known to have antifungal, antibacterial, antimalarial, anti-

inflammatory, insecticidal and other biological activities properties

(Choudhary and Lahti, 2011). It has been used in Ayuverdic medicine for

more than 4000 years due to its therapeutic possessions (Girish and Shankara

Bhat, 2008).

There are two species of neem tree, Azadirachta that have been outlined

which are Azadirachta indica A. Juss, the native to Indian sub-continent and

Azadirachta excelsa Kack that confined to Philippines and Indonesia (Girish

and Shankara Bhat, 2008). Neem is a large tree growing about 4.21 m height

in 5 years and attains 8.69 cm diameter in the same year (Singh, 2009). It has

spreading branches forming a broad crown (Figure 2.1). It will grow where

rainfall is little, and thrives in areas that experience extreme heat of up to

5
48°C (Munoz-Valenzuela et al., 2007). The tree has adaptability to a wide

range of climatic and topographic factors. It grows well in hot, dry climates

with an annual rainfall of 450 to 1200 mm. The pH range for neem tree

growth is between pH 4 to pH 10. Neem tree also have the ability to neutralize

acidic soils by a unique property of calcium mining (Girish and Shankara

Bhat, 2008).

Figure 2.1: Neem Tree

6
Nowadays, researches about neem are arising due to its property-rich in

therapeutic possessions and last few decades, especially in controlling pest.

Neem based pesticide are easy to prepare, cheap and highly effectual to be

used especially for economically poor third world country farmers.

Neem bio-pesticide is systemic in nature and provides long term preservation

to plants against pests. Neem was found outstanding in the pest management

affecting rice, cowpea, beans, cabbage and citrus. Many researches has been

done using neem and it is proven that neem can be used mainly as anti-

feedant, moult-inhibiting, ovipositional deterrence and sterilizing potential

which led to the practical application of neem products (Rosen et al., 2014).

The leaves and fruits of neem plant have been found to possess insect

repellent and anti-hormonal properties. It is also known to reduce

ovipositional in a number of different insect species including Corpophilus

hemipterus (dried fruit beetle), Nilaparvata lugens (brown plant hopper) and

Dacus curcurbitac (melon fly) (Watson and Preedy, 2008). According to

Girish and Shankara Bhat (2008), pollinator insects, bees and other useful

organisms are not affected by neem based pesticides.

2.1.1 Neem’s leaf

The shiny dark pinnate compound leaves (Figure 2.2) are up to 30 cm

long. Each leaf has 10-12 serrated leaflets that are 7 cm long by 2.5

cm wide (Munoz-Valenzuela et al., 2007). Substantial amount of

proteins, minerals, carotene and adequate amount of trace minerals

7
contains in neem leaves. They also have significant amount of

digestible crude protein (DCP) and total digestible nutrients (TDN)

so livestock can be fed with neem leaves. Anorexia and skin diseases

are believed can be treated with the helpful of the neem leaves. The

leaf extract has also been announced as non-toxic and non-mutagenic

(Choudhary and Lahti, 2011). Girish and Shankara Bhat (2008)

informed that neem leaves possessed antidermatic, antifungal,

antitumor, antiseptic, antiviral, cosmetics, insecticides, nematicides

and insect repellents properties.

Neem leaf

Figure 2.2: Neem leaves

8
2.1.2 Neem’s bark

Neem bark is cool, bitter, acrid and refrigerant. Tiredness, cough,

fever and worn infestation can be controlled by neem bark. The bark

is reported to heal wounds, annihilate conditions of vomiting, skin

diseases, intemperate thirst and diabetics because they have

antiallergenic, antidermatic, antifungal, antiprotozoal and antitumor

properties (Girish and Shankara Bhat, 2008). The in-vitro studies

show that the bark extracts possess anti-inflammatory activities and

properties to aid the immune system. The chloroform extract of stem

bark is effectively against swelling of the paw due to excess fluidity

in rats (caused by algae), ear inflammation in mice, and inflammatory

stomach conditions in children (Choudhary and Lahti, 2011).

2.1.3 Neem’s flower and fruit

Choudhary and Lahti (2011) also found the slender, branched

clustered flower of Azadirachta indica is white as in inflorescences

with joined sepals. It possessed analgesic property and can be used as

soap and stimulant (Girish and Shankara Bhat, 2008). Normally it

starts fruiting after three to five years, and from its tenth year, it

becomes fully fecund, able to produce up to 50 kg of fruits annually

(Choudhary and Lahti, 2011). Based on Girish and Shankara Bhat

(2008), the neem fruits are yellow, has one seed and fleshy texture.

The fruits are used as laxatives, in treating dry skin, effective in the

treatment of intestinal worms, urinary diseases and piles (Choudhary

9
 and Lahti, 2011). When ripe, the clusters of flowers and fruits are

greenish-yellow, with a sweet pulp, each enclosing a seed.

2.1.4 Neem’s seed

According to Luntz and Nisbet (2000), the seed consists of a shell and

one to three kernels that contain azadirachtin. Phytochemicals

contents in kernels and seeds are also greater than leaves (Salako et

al., 2008). This is why seed and kernel oil are used as antiseptic,

antifungal, and antibacterial agents (Choudhary and Lahti, 2011).

Apparently, fruit colour can indicate maturity and thereby the content

of phytochemicals. For example, yellow fruits have 35% less

azadirachtin than green-yellow fruits whereas triterprenoid content in

fruit changed little from the hard green to yellowish colour with a

highest azadirachtin content in newly ripened fruits and mature seeds

(Gahukar, 2014).

2.2 Active Compound in Neem

Neem has been tested for its nematicidal activity. More than 135 compounds

have been isolated from different parts of neem. These compounds can be

categorized into two major groups which are isoprenoids and others. The

isoprenoids include diterpenoids and triterpenoids containing protomeliacins,

liminoids, azadirone and its derivatives; genudin and its derivatives; vilarin

type of compounds and c-secomeliacins such as nimbin, salannin and

10
azadirachtin (Girish and Shankara Bhat, 2008). These compounds are in

charge for diverse activities such as insect anti-feedant, insect growth

disrupting, insecticidal, nematicidal, fungicidal, and bacterial, etc. The bitter

extracts from neem kernels acquired by alcoholic extraction contain various

bioactive limonoids and are widely used in bio-pesticide formulations (Jadeja

et al., 2011).

The most engrossing compounds from neem are from the kernels and well-

characterized compounds are triterpenoids which all seem to be attained from

the tetracyclic triterpene tirucallol as can be inferred from the stereo chemical

arrangement of the methyl groups at C-10, C-13, C-14 and the side chain at

C-17. There seems to be progression of compounds to higher oxidation states

from nimbin to salannin to azadirachtin as the seeds mature. Besides

azadirachtin and its congeners, there are other compounds isolated from neem

such as meliantriol, salannin and 7-desacetyl-7-benzoylazadirone have the

insect growth-disrupting properties, although not to the event of the

azadirachtin (Govindachari, 1992).

11
 Table 2.1: Some bioactive compounds from neem
(Girish and Shankara Bhat, 2008)

Neem compound Source Biological activity

Anti-inflammatory, Anti-

arthritic, Anti-gastric ulcer,

Nimbidin Seed oil Spermicidal, Antifungal,

Antibacterial, Diuretic

Nimbin Seed oil Spermicidal

Nimbolide Seed oil Antibacterial, Antimalarial

Gedunin Seed oil Antifungal, Antimalarial

Azadirachtin Seed Antimalarial

Mahmoodin Seed oil Antibacterial

Gallic acid, (-) Anti-inflammatory,

epicatechin and catechin Bark Immunomodulatory

Margolone, margolonone

and isomargolonone Bark Antibacterial

Cyclic trisulphide and

cyclic tetrasulphide Leaf Antifungal

12
2.2.1 Azadirachtin

The most notable insecticidal active ingredient of neem is the

tetranortriterpenoid azadirachtin (AZA) (Rosen et al., 2014).

Azadirachtin, a major limonoid from neem seeds, is the main

component responsible for both anti-feedant and toxic effects in

insects. It is the most active and lavish phytochemical in neem and is

obtainable as technical material (up to 96% purity) to which at least

550 insect species are known to be sensitive (Gahukar, 2014).

Azadirachtin is effectively kills the phytophagous insects while

having minimal or no impact on beneficial like pollinators such as

honey bees, bumble bees, and other non-target organisms (Immaraju,

1998).

Anti-feedant and physiological effects of neem were reviewed

especially on behavioural and physiological properties of

azadirachtin. It includes the effects on insect reproduction, direct and

“secondary” anti-feedant. Physiological effects also measure as

growth reduction, increased mortality, abnormal and delayed moults.

These effects are related to disruption of endocrine system controlling

growth and moulting. The moulting effects are due to a disruption in

the synthesis, release of ecdysteroids (moulting hormone) and other

cases of hormones. These effects are categorized in two ways; direct

effects on cells and tissues and indirect effects exerted via the

endocrine system (Luntz and Nisbet, 2000).

13
 Table 2.2: Effects of azadirachtin against insects (Luntz and Nisbet, 2000)

Effects Target Mode of action

Primary anti- Mouthpart & other -Deterrent cell stimulation

feedancy chemoreceptor -Sugar cell inhibition

Secondary anti- Gut -Peristalsis inhibited

feedancy -Enzyme production reduced

Insect growth Cuticle -Blockage of morphogenetic

regulation peptides lead to moulting defects

- Alterations to ecdysteroid

Sterility Reproductive organs leading to reduction in number of

viable eggs and live progeny

-Blockage of cell division post

Dividing cells metaphase in meiosis and mitosis.

Muscles -Loss of muscle tone

Cellular
-Blockage of digestive enzyme
processes
Cell synthetic production in gut

machinery -Inhibition of protein synthesis in

various tissues

14
2.3 Pests

Any living creature which are bothersome, destructive and nuisance to plants,

animals and humans is called as pest. One of the major threats especially to

us human is when they can cause harm to agriculture by feeding on crops and

parasitizing livestock. They also can act as vectors that carry out disease to

other animals or humans like malaria, diarrhoea, dengue and others

(Pimentel, 2002). People have to face this pest problem everyday align with

the rapid growth of human population because they live among us especially

in our house.

According to Braack (2010), the prodigious group of pest is mostly known

as insects. The most diverse species and abundant group of animals in the

world by far range are insects and their relatives (subphylum Hexapoda)

because they may be found in nearly all environments. Hook (2008) stated

that there are roughly about 10 million totals of insects in the environment.

They are important in maintaining the food chain, ecological pollinator, or

pests (Chung et al., 2013). If insects were to dematerialize, the environment

would collapse into chaos (Cranshaw and Redak, 2013), but the existence of

these insects also need to be balance as they could be troublesome to any of

us.

Ants are one of the most diverse species of insect in the world. These species

come in the order hymenoptera which comprises about more than 12,000

described species (Bolton et al., 2006). Hence, ants play important roles in
15
 the ecosystem for example it can be used as an integrative measure of soil

quality, assuming their importance in regulating soil processes which are

crucial to the continued formation of soil and as protection against soil

degradation (Paoletti, 2012). Based on Schowalter (2016), they also can be

huge problems to us as a pest especially because they can lead to food

contamination by leaving their waste behind. In the other hands, the ant bites

also could cause an allergic reaction to some people and it hurt us too. It is

annoying when there is a large colony of ants on our house floor and

surrounding our foods on the table. Pest like ants should be controlled in any

way for a better living.

2.4 The Importance of Pest Control

Pesticide is any substance used to kill pest and is used in agriculture,

medicine industry and for household. There are a few types of pesticides

which are algaecides, bactericide, insecticide, defoliant, fungicides,

herbicides, nematicides, molluscicides and etc. (Marer and Program, 2006).

About one third of world’s agricultural food stuffs get destroyed by more

than 20 000 species of field and storage pests. Pest infestation in India faces

grave loss about Rs. 5000 crores worth of agricultural produce every year.

Synthetic insecticide is used as general method for controlling pest especially

insect. To prevent this loss, large amounts of synthetic pesticides are applied,

out of which only 0.1% reaches the target pests and more than 99%
16
contaminates the ecosystem (Girish and Shankara Bhat, 2008). The problem

with most insecticides is toxicity to non-target organisms where most

insecticides reach water bodies, where they enter food web and via bio

magnification reaching the non-target species, including humans, alarming

toxicity problems. In addition, synthetic pesticide usage has resulted in

development of resistant pests.

A safe yet effectual herbal alternative is therefore advisable from an

ecological point of view, causing virtually no toxicity to the ecosystem

(Watson and Preedy, 2008). Natural pesticides are active principles where it

said to have biologically active ingredients acquired from plants (Asogwa et

al., 2010). There are over 200 pest species belonging to utmost insect orders

of Diptera, Hymenoptera, Coleoptera, Lepidoptera, Orthoptera, Homoptera

and Hemiptera that are vulnerable to the effects of azadirachtin making neem

plant as a broad-range spectrum of bio-pesticide (Immaraju, 1998).

17
 CHAPTER 3

METHODOLOGY

3.1 Materials

The materials used in this study were divided into three parts which were

raw materials, chemicals and apparatus.

3.1.1 Raw materials

The raw materials used were neem leaves and neem seeds. The

sample had been taken from around Seremban area. The ants sample

were collected and ice cream had been bought from the market.

3.1.2 Chemicals

The chemical used was 95% ethanol.

3.1.3 Apparatus

The lists of apparatus were rotary evaporator, beaker, test tube,

conical flask, electric blender, sprayer, electronic balance, stirrer,

measuring cylinder and filter paper.

18
3.2 Methods

Methods used for this study were sample collection of neem seeds and neem

leaves, extraction of neem leaves and neem seeds, and lastly the collection

of ant. The effectiveness of neem extraction from seeds and leaves were

tested on ant.

3.2.1 Preparation of neem seeds and neem leaves

1 kg of Azadirachta indica leaves and fruits were used. Neem leaves

and seeds were washed with water and finally with distilled water to

remove dust. The samples were air-dried for seven days under shade

at room temperature (Hossain et al., 2013). The seeds were peeled to

get the seed kernels. The leaves and seed kernels were grinded into

powder form (Asogwa et al., 2010).

3.2.2 Extraction of neem seeds and neem leaves

As suggested by Obeidat et al., (2012), plant material to solvent

ratio must be 1:10 w/v. In this experiment, fifty grams (50 g) of

both samples were suspended into five hundred millilitres (500 ml)

of 95% ethanol for about 72 hours in a beaker. Ethanol was chosen

to extract terpene compounds, although some flavour top notes

may be disappearing during the evaporation process (Hirasa and

Takemasa, 1998). The extracts were decanted, filtered, and

evaporated using a rotary evaporator at the temperature of 40 C.

Primary purpose of rotary evaporator is to remove solvent by

19
 evaporation process. This apparatus was an environmentally

friendly way to remove volatile solvent from non-volatile solvent

(Keharom et al., 2015). Rotary evaporator apparatus was set up

before proceed with extraction of the filtrate of neem powder and

ethanol. The extraction was performed to obtain neem crude by

using rotary evaporator apparatus.

After the extraction process, the crude of neem leaves and seeds

were obtained. The crude was then transferred into a beaker and

placed it into an oven for three days at 60 oC. This step was

important to get a pure crude. Then, a beaker that filled with crude

was weighed. The weight was recorded to get the weight of the

crude by subtract it with beaker’s weight. The crude extract of both

seeds and leaves were used to test their effectiveness on ant.

3.2.3 Collection of ant

In this step, a thin layer of ice cream was taken and spread inside an

open container. The open container was leaved for a day. Ice cream

were surrounded by colonies of ants inside the open container. Ants

colonies of Ochetellus spp. were trapped inside the container.

3.2.4 Testing the effectiveness of neem’s insecticide against ants

Four types of insecticide set up were distilled water as negative

control, citronella as positive control, seeds extraction solution and

20
 also from the leaves extraction solution. Each new container was

filled with 30 ants. Then, the crude was diluted with ratio of 1 g of

crude sample to 29 ml of distilled water. 30 ml of neem-based

insecticide solution then was sprayed onto sample in each container.

The response was observed and the number of dead ants was recorded

after being leaved for 30 minutes to determine the effectiveness of

neem extract of leaves and seeds. Based on Gudeva et al., (2013), the

effectiveness of neem extraction method can be measured by using Abott

formula.

Effectiveness by Abott (%)

Test mortality (%) − Control mortality (%)

=
100 − Control mortality (%)

3.3 Statistical Analysis

Statistical analysis used was a simple t-test analysis at 95% confidence interval

for comparing two independent means of sample. This analysis is important to

give a clearer view to compare the effectiveness between neem seeds extraction

and the neem leaves extraction. T-test analysis was done by using the SPSS 15.0

version.

21
 CHAPTER 4

RESULTS AND DISCUSSIONS

4.1 The Extraction of Neem Leaves and Neem Seeds

In this experiment, ethanol has been used as solvent. Apparently, the crude

of both neem leaves and seeds samples were difficult to be taken out from

the round bottom flask after the evaporation process of solvent. This happen

because it contains a large amount of gummy sediments. The crude samples

were mostly stick on the glass of round bottom flask thus the crude that had

been obtained only in small amount. The weight of both neem pure crudes

from 1 kg of each seeds and leaves were obtained as shown in Table 4.1.

Table 4.1 Weight of neem crude

Sample Weight (g) of crude

Neem leaves 36.1

Neem seeds 2.1

The crude of both neem leaves and seeds samples contain a large amount of

gummy sediments including azadirachtin. Azadirachtin is a limonoids that

have insecticidal properties as an anti-feedant, repellence, ovipositional

22
 deterrent, moulting inhibition and a growth retardant for a variety of insects

and arthropods (Mala and Muthalagi, 2008).

4.2 Neem’s Extract Treatment on Ants

The crude of seeds and leaves were diluted with ratio of 1 g of crude sample

to 29 ml of distilled water to be made into solution. All insecticide solution

was tested on ants of Ochetellus spp. (Figure 4.1). Their behaviour and

respond for the first five minutes were observed and their mortality rate after

30 minutes were taken.

Figure 4.1 Insecticide solutions

23
4.2.1 Behaviour and response

The first one was treated using distilled water as negative control

which do not give any reaction towards the ants of Ochetellus spp.

Ants which exposed directly towards distilled water gets harder to

walk because their body got wet and getting heavier. Apart from

that, some may walk to the wall of container as they want to escape

from accumulated with distilled water below the container. Ants that

do not exposed to the direct sprayed of distilled water remain

unchanged in their behaviour.

After exposing the ants towards citronella and neem bio-insecticide

solutions, the ants had a positive reaction towards it. The ants were

randomly mobilizing and also uncoordinatedly. After a few minutes,

some of the ants lied on the surface of the container and eventually

die. Citronella has been expected to give positive result as it has been

commercialized in the market. For ants that died by neem bio-

insecticide solutions, this may due to the actions of azadirachtin

towards ant’s cells and tissues after being exposed to the neem bio-

insecticide solution. It is because azadirachtin could causes

inhibition of both cells division and protein synthesis. According to

Luntz and Nisbet (2000), the ant’s muscles will flaccid paralysis,

midgut cells necrosis, regenerating cells of the gut thus making

enzyme production of the midgut decreases. Furthermore,

24
 azadirachtin mostly give stomach poisons to insect. When applied at

higher doses, they exhibit contact toxicity (Gahukar, 2014).

4.2.2 Effectiveness of neem solutions

Results on treatment of ants after 30 minutes with different solutions

were recorded into Table 4.2 and Table 4.3. Negative control

treatment using distilled water showed negative results on the death

of the ants.

Table 4.2 Results treatment with insecticide solutions

Number of ants dead

Solution
Replicate 1 Replicate 2 Replicate 3 Mean

Distilled water 0 0 0 0

Citronella 30 30 30 30
Neem leaves
22 17 23 21
extract
Neem seeds
26 23 21 23
extract

Table 4.3 Effectiveness of different solutions on ants

Solution Effectiveness
Distilled water 0%
Citronella 100%
Neem leaves extract 68.89%
Neem seeds extract 77.78%

25
After treatment, results showed that citronella solution which is

positive control had effectiveness which was 100%. The

effectiveness after treated with neem leaves solution was 68.89%

which was lower than neem seeds solution which was 77.78%. This

result shows that neem seeds solution has more potential as an

insecticide compared to the leaves. Aqueous neem seeds extract was

more toxic compared to neem leaves extract. This result had been

supported by Rejesus and Punzalan (1997), who found that the seed

extract has high toxicity compared to the leaves extract. This may be

due to azadirachtin compound contained in the neem seeds solution

is higher compared to neem leaves solution.

T-test analysis had been done to prove the effectiveness of neem

solution both from seeds and leaves. Levene’s test for equality of

variances result shows that F-statistic of 0.450 with p-value is

0.0539 > 0.05, thus it can be concluded that the variances for the two

groups are equal (equal variances assumed). The result shows that

there is no significant different in mean marks for both classes (p-

value = 0.321 > 0.05). Since there is no significant different in mean,

the null hypothesis is rejected. Therefore, it can be concluded that

there is not enough evidence to support the claim that the neem seed

extract is more effective than the neem leaves extract.

26
Finding from this study was contradicted to that study by Maini et

al., (1993), where it showed that there are significant differences

between neem seeds solution and leaves solution making the seeds

solution is better in terms of effectiveness against pest. Maini et al.,

(1993) had used aqueous extract solution (successive dilutions with

distilled water) to make the experiment against the golden apple

snail while this experiment had used ethanolic extraction to against

ants, from Ochetellus spp. The extraction method that used alcoholic

solution will give a preferable result because these solutions can

extract most of the secondary compound inside a plant than by just

using distilled water.

According to Massaguni and Latip (2012), the factor causing

differences in the findings probably due to azadirachtin content in

each of different neem plant parts. All parts of neem tree contain

azadirachtin, but most abundant in seed kernel. Finding of the study

was expected considering the fact that the content of azadirachtin in

neem varies greatly between locations and other factors may also

contribute to variability. Difference in trees maturity, application

technique and perhaps environmental factors would probably

contribute to these inconsistencies. This study did not analyse on the

active ingredient from prepared extract; however, it is possible that

the azadirachtin content in seeds and leaves could raise the

efficiency of extraction.
27
Azadirachta indica is known to be versatile because it can

effectively harm and kill many types of pest while safe to beneficial

organisms such as earthworms (Salako et al., 2008). The research

done by Yu, 2008 proved that Azadirachta indica can kill broad

range of pest because some naturally occurring compounds in it

shows to be active against different species of insect pests. This is

why it was chosen as a natural bio-pesticide. For example, it can

deteriorate many types of mosquito such as Culex pipens, Culex.

fatigans and Aedes aegypti rather than Citrus sinensis which can

only harm small range of mosquito such as from the group of

Anopheles subpictus (Ghosh et al., 2012).

In contrast with the other study of different natural bio-pesticide,

Sciences, 1971 had proved that neem also can lead to 100%

mortality rate than garlic which only can give up to 69.67% of

mortality rate towards instar larvae. Garlic seems to have property

to repel and prevent insect from feeding especially the suckling pests

but it is not as broad as neem in term of effectiveness against pest.

Furthermore, it shows that garlic is not as versatile as neem where

neem can attack many types of pests to be killed while safe for non-

target organisms. It is said to be the most potent, thus can causing

100% of termite mortality too (Verma et al., 2009).

28
 CHAPTER 5

CONCLUSIONS AND RECOMMENDATIONS

In conclusion, both of neem seeds and leaves extract can be used as natural bio-

pesticide as it has the ability to control pest such as ants. Thus, neem-based

pesticide can work as a good and effective pesticide just like the other commercial

pesticide available in the market.

The effectiveness of neem leaves extract is lower as compared to neem seeds

extract which were 68.89 % and 77.78 % respectively. Neem seeds extract

solution percentage is slightly higher may be due to possibly higher azadirachtin

content in the seeds than in the leaves. From t-test, there is no significant different

in mean marks for both seeds and leaves, thus the null hypothesis is rejected. In a

nutshell, there is not enough evidence to support the claim that neem seeds extract

is more effective to be used than the neem leaves extract.

This research can also be used as references to researcher who intended to further

this study in the future. It is recommended to extend neem extraction research by

using another solvent and method, as both solvent and method may bring huge

influence to the crude’s production.

29
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32
 APPENDICES

Appendix A Weight of Neem’s Crude

Weight (g)
Sample Beaker +
Beaker Crude
crude
Neem leaves 145.7 109.6 36.1

Neem seeds 111.7 109.6 2.1

Appendix B Statistical Analysis

Group Statistics

Std. Std. Error

Name_of_sample N Mean Deviation Mean
Number_of_dead_ants Seed 3 23.3333 2.51661 1.45297
Leaf 3 20.6667 3.21455 1.85592

Independent Samples Test

Levene's Test
for Equality of
Variances t-test for Equality of Means
Sig. 95% Confidence
(2- Mean Std. Error Interval of the
F Sig. t df tailed) Difference Difference Difference

Lower Upper Lower Upper Lower Upper Lower Lower Upper

Number_of_dead_ants Equal
-
variances .450 .539 1.131 4 .321 2.66667 9.21081
2.35702 3.87748
assumed
Equal
variances -
1.131 3.782 .324 2.66667 9.36218
not 2.35702 4.02885
assumed

33
 CURRICULUM VITAE

A. Personal Profile
Full name: Noor Faizatul Asyikin binti Razaha
National IC no: 930305-06-5296
Birth date: 05 March 1993
Citizenship: Malaysian
Place of birth: Pekan, Pahang
Gender: Female
Correspondence address:
BM 31,
Kampung Pulau Jawa,
26600 Pekan,
Pahang Darul Makmur
Telephone No. (HP): 010-5702464
Email address: noorfaizatulasyikin@gmail.com

B. Hobbies and Interests

Socializing with friends and family, watching televisions (documentaries
and news), reading on non-fiction books, play puzzle games and do some
volunteering work.

C. Academic Qualification
2015 - current: Degree in Science (Hons.) Biology UiTM Negeri Sembilan
2011 - 2014: Diploma in science (CGPA 3.14), UiTM Pahang
2006 - 2010: Sijil Pelajaran Malaysia (SPM), Sekolah Seri Puteri, Cyberjaya

D. Work Experience
Cashier at Ikmal Shah café on 2014.

E. Related experiences
Joining sports and management society (Majlis Sukan Pelajar) as treasurer
and manage all the events that the society organized, became one of the
secretariat team for UiTM in Sukan Institut Pengajian Tinggi (SUKIPT)
games on 2016, internship at Zoo Negara of Malaysia and do some
volunteering work at Cheng’s Old Folks Home in Melaka and Kuala Lumpur
Sea Games 2017.

F. Awards
Runner up for best presentation award at Physics Innovation Day.
Top 20 best group in the Technopreneur Wave 1.0 competition.

34