CHAPTER 1

INTRODUCTION

1.1 Background

During the summers of 2002 and 2003, North America was affected by its

largest-ever outbreaks of arboviral encephalitis. West Nile virus caused 2942 cases of

meningitis or encephalitis in 2002, with 276 deaths, and 2866 cases in 2003, with 246

deaths (Petersen LR, Roehrig JT, Hughes JM, 2004).

Survey of 1,551 inhabitants in six villages of West Irian was undertaken to

determine the presence and distribution of Wuchereria bancrofti and other filariae.

Results confirmed the presence of bancroftian filariasis only in low-lying inland villages

and its absence from coastal villages. Microfilariae were detected in thick blood films

taken during evening hours in 48 of 168 (28.5%) persons in Senggo, and 65 of 190

(34.2%) in Tamnem, a neighboring village situated to the south of the central mountain

range. Males were more frequently infected than females, and infection rates tended to

be highest in those of both sexes 20 years or older (Chester L Harbut, 1999).

All the health problems stated above are mosquito borne disease where the

species involve as the vector is Culex sp. Raining season and floods may increase the

places that are ideal breeding place for the mosquito. As the floodwaters recede, they

will probably leave behind a lot of small puddles and pools of dirty water that will

stagnate as the weather becomes hotter and drier. This is dangerous because dirty,

stagnant water is the perfect breeding ground for Culex mosquitoes, the small brown

species that are the primary vectors of West Nile and St. Louis encephalitis viruses.

(William G Golrey, 2008).

In order to eliminate the vector, many cooperation from all over the world have

introduced many kind of insecticide that are basically organophosphate, DDT,

carbamate, and malathion. The usage of these substances however has its own

downturn such resistance, health and environmental hazard. Malathion for example,

chronic exposure can cause memory impairment (Edwards D, 2006). Besides, these

insecticide do not come cheap causing a wide area mosquito control at time of sudden

outbreak of mosquito borne disease pretty much impossible in the third world.

Bed nets are cheap and completely repel mosquitoes. However only repelling

mosquitoes might no be enough to completely eliminate mosquito borne disease

because the mosquitoes are still free to move around inside the house or building for

any other victim. Therefore,this research’s aim is to make a bednet that is capable in

both repellent and insecticide to reduce mosquito population.

Cinnamon has been known as a safe bioinsecticide. A substance inside

cinnamon – eugenol is what that has been recognized to have insecticidal properties.

This substance is known to have effect on nerve impulse transmission causing paralysis

( Ware, G.W & Whitacre, D.M, 2005 ).

A combination of bed nets and cinnamon’s insecticidal effect will produce a

cheap, safe and nature friendly method in preventing mosquitoes that might be used in

emergency situation such as tsunami, flash flood, or even in hospitals where a mass

protection against mosquitoes is required.

1.2 Problem Statement

Can a cinnamon extract retain its insecticidal properties against Culex sp. when

applied to bednets?

1.3 Objective

1.3.1 General Objective

To measure effectivity of cinnamon extract as insecticide against Culex sp. By

applying the extract on bednets.

1.3.2 Specific Objective

a. To measure concentration of cinnamon extract that should be applied on bednets

to effectively kill mosquitoes

b. To measure the relation between the concentration applied on bed nets and

contact time with mosquitoes to kill them.

1.4 Advantage Of research

a. Can produce a much safer and affordable protection against mosquitoes and

mosquito borne diseases.

b. Can produce a new way in insecticidal method to narrow down the risk of

resistency.

c. Will be a baseline for next research involving evaporation rate of extract to

optimize the level of performance of cinnamon extract.

Chapter 2

Literature Review

2.1 Culex sp

2.1.1 Scientific Classification

Kingdom : Animalia

Phylum : Arthropoda

Class : Insecta

Order : Diptera

Family : Culicidae

Genus : Culex

(Linnaeus, 1758)

2.1.2 Morphology

Culex is a genus of mosquito, and several species act as vectors of important

diseases, such as West Nile virus, filariasis, Japanese encephalitis, St. Louis

encephalitis and avian malaria.

The adult mosquito can measure from 4–10 mm (0.16–0.4 in.), and

morphologically is mainly composed of a head, thorax, and abdomen. It also possesses

wings, which enable it to fly and act as a very important factor in its disease-spreading

abilities (Wikipedia, 2008)

The eggs of Culex is oval shaped very similar to a bullet with blunt end. The

larvae possess a long siphon covered with hairs. Adult Culex are brown in color and

have a pair of wings with long and narrow scales and sharp edge (Juni Prianto et al,

2004).

2.1.3 Life Cycle

Culex mosquitoes lay their eggs on the surface of fresh or stagnant water. The

water may be in buckets, barrels, horse troughs, ornamental ponds, swimming pools,

puddles, creeks, ditches, or marshy areas. Mosquitoes prefer water sheltered from the

wind by grass and weeds.

Culex mosquitoes usually lay their eggs at night. A mosquito may lay a raft of

eggs every third night during its life span. Culex mosquitoes lay their eggs one at a time,

sticking them together to form a raft of from 200-300 eggs. A raft of eggs looks like a

speck of soot floating on the water and is about 1/4 inch long and 1/8 inch wide.

Mosquito larvae, commonly called “wrigglers”, must live in water from 7 to 14

days depending on water temperature. Larvae must come to the surface at frequent

intervals to obtain oxygen through a breathing tube called a siphon. The larva eats algae

and small organisms which live in the water. During growth, the larva molts (sheds its

skin) four times. The stages between molts are called instars. At the 4th instar, the larva

reaches a length of almost 1/2 inch. When the 4th instar larva molts it becomes a pupa.

(Christine, 2004)

Mosquito pupae, commonly called "tumblers", must live in water from 1 to 4 days,

depending upon species and temperature. The pupa is lighter than water and therefore

floats at the surface. It takes in oxygen through two breathing tubes called "trumpets".

When it is disturbed it dives in a jerking, tumbling motion and then floats back to the

surface. The pupa does not eat. The metamorphosis of the mosquito into an adult is

completed within the pupal case. The adult mosquito splits the pupal case and emerges

to the surface of the water where it rests until its body can dry and harden. (Christine,

2004)

Only female mosquitoes bite animals and drink blood. Male mosquitoes do not

bite, but feed on flower nectar. Culex mosquitoes are painful and persistent biters also,

but prefer to attack at dusk and after dark, and readily enter dwellings for blood meals.

Domestic and wild birds are preferred over man, cows, and horses. Culex tarsalis is

known to transmit encephalitis (sleeping sickness) to man and horses. Culex are

generally weak fliers and do not move far from home, although they have been known to

fly up to two miles. Culex usually live only a few weeks during the warm summer

months. Those females which emerge in late summer search for sheltered areas where

they "hibernate" until spring. Warm weather brings her out in search of water on which to

lay her eggs.( WHO, 2008)

2.4 Cinnammon

2.4.1 Nomenclature

Kingdom : Plantae

Divisi : Magnoliophyta

Classs : Magnoliopsida

Order : Laurales

Family : Lauraceae

Genus : Cinnamomum

Species : Cinnamomum burmanni

2.4.2 Description

Cinnamons ( Cinnamomum burmani ) or also known as cassia vera in Indonesia

is widely cultivated by Indonesian people especially in western Sumatera, Jambi, and

northern Sumatera. it is one of several plants whose bark is sold as the spice cinnamon.

The trees, when old enough will produce a thick bark that is aromatic. The main

cultivation from a cinnamon tree are barks from its trunks, branches, leaves and its

roots. (Kardinan 2005)

A cinnamon tree can grow up to 2000 m at an appropriate environment of 500-

1500 m above sea level, and 70-90% of humidity. In Indonesia the tome most

appropriate for cinnamon’s growth is during dry months where the most optimum

temperature is 25 degrees Celsius, ranging from 18 to 27 degrees Celsius. (Kardinan

2005)

2.4.3 Chemical Composition

There are three types of main substance in a cinnamon – cinnamyldehyde,

cinnamylacetate, and eugenol. The latter is believed to have insecticidal effect.

Eugenol (C10H12O2), is an allyl chain-substituted guaiacol. Eugenol is a member

of the phenylpropanoids class of chemical compounds. It is a clear to pale yellow oily

liquid extracted from certain essential oils especially from clove oil, nutmeg, cinnamon,

and bay leaf. It is slightly soluble in water and soluble in organic solvents. It has a

pleasant, spicy, clove-like aroma.

The name comes from a scientific name for the clove, Eugenia aromaticum or

Eugenia caryophyllata. Eugenol is responsible for the aroma of cloves. It is the main

component in the essential oil extracted from cloves, comprising 72-90% of the total.

(Jadhav BK, et al, 2005).

Cinnamon is neurotoxic to mosquitoes, affecting its peripheral nervous system as

well as its central nervous system. Eugenol acts on axon causing a disturbance in

impulse transmission along axon that will prolong excitation phase and cause paralysis

to mosquito body ( Ware dan Whitacre, 2005 ).

Eugenol also acts by blocking Octopamine receptor. This receptor is a

sympatomimetic receptor that control sympathic activity. This will cause

bronchoconstriction and finally kill the mosquito (Enan, 2001).

2.3 Bed Nets

In the poorest parts of the world, where effective window screens are lacking,

insecticide-treated bed nets are arguably the most cost-effective way to prevent malaria

transmission. One bed net costs just $10 to buy and deliver to individuals in need. One

bed net can safely last a family for about four years, thanks to a long-lasting insecticide

woven into the net fabric. ( Anonymous, 2008)  

Studies show that use of insecticide-treated bed nets can reduce transmission as

much as 90% in areas with high coverage rates. Bed nets prevent malaria transmission

by creating a protective barrier against mosquitoes at night, when the vast majority of

transmissions occur. The African malaria mosquitoes generally bite late at night or early

morning, between 10:00 p.m. and 4:00 a.m. A bed net is usually hung above the center

of a bed or sleeping space so that it completely covers the sleeping person. A net

treated with insecticide offers about twice the protection of an untreated net and can

reduce the number of mosquitoes that enter the house and the overall number of

mosquitoes in the area. (Anonymous, 2008)

Chapter 3Conceptual Framework

3.1 Conceptual Framework

Figure 3.1

Figure 3.1 Extraction of cinnamons yields three types of substances; eugenol,

cynnamaldehyde, and cynnamylacetate whereas eugenol is believed to have bioinsecticid

effect. The neurotoxic properties of euganol can affect both central and peripheral nerve

system. It causes an extended period of excitation phase of nerve conductance resulting in

paralysis. It also blocks octopamine receptor which is responsible for sympathetic activity

that result in bronchoconstriction. The application of the extract on bed nets will kill the

mosquitoes getting a direct contact on it.

3.2 research hypothesis

a) Application of cinnamons’ extract on bed nets can kill mosquitoes that get into

contact with it

b) The higher concentration of cinnamon extract, more lethal it will be against Culex

sp.

Chapter 4

Research Method

4.1 Research Design

This research is a true experimental post test only control group design to know

the potential of several concentration of cinnamon extract as insecticide that can kill

mosquitoes when applied to bed nets.

The sample that is being used is adult Culex sp which divided into 6 experimental

groups:

Control Group : In mosquito net without clove extract

Group 1 : In mosquito net applied with clove extract (0.5% concentration)

Group 2 : In mosquito net applied with clove extract (1.0% concentration)

Group 3 : In mosquito net applied with clove extract (1.5% concentration)

Group 4 : In mosquito net applied with clove extract (2.0% concentration)

Group 5 : In mosquito net applied with clove extract (2.5% concentration)

Number of repetition done followed according this formula

6 (n-1) > 16

6n-6 > 16

n > 3.6 ~ 4

Explanation:p = number of groupsn = number of repetition

P (n-1) > 16

According to the formula above, the minimal repetition that should be done is 4

times. In this research repetition is done 5 times.

4.2 Population And Research Sample

In this research, the mosquito from the genus Culex will be used. The sample is

obtained from Parasitology Laboratory of University Brawijaya. 30 mosquitoes will be

used for each repetition of experiment. The mosquitoes will be placed in a box that is

divided into two parts by a net in the middle. Some criteria of the sample will be

considered to minimize errors.

4.2.1 Inclusive Criteria

The inclusive criteria that will be taken into consideration are

i. The mosquitoes are female, because only female mosquitoes are known

to feed on humans blood.

ii. The mosquitoes should not have physical deformation ( i.e. broken legs,

proboscis, wings etc)

4.2.2 exclusive criteria

The exclusive criteria need to be considered are the following

i. The mosquitoes are not too old ( less than two weeks of age) because

mosquitoes that die from old age will cause false positive result.

ii. The mosquitoes are not exposed to any kind of insecticide prior to the

operation

4.3 Time And Location

This experiment will take place in Parasitology Laboratory of Brawijaya University

around February to March 2009.

4.4 Identification Of Variable

4.4.1 Dependent Variable

The number of mosquitoes died

4.4.2 Independent Variable

The amount (concentration) of cinnamon extract that will be applied to bed nets

4.5 Apparatus And Materials

4.5.1 Apparatus

a) Glass box (25 x 25 x 30cm)

b) Bed nets (25 x 25 cm)

4.5.2 Materials

a) Cinnamon extract (from parasitology lab)

b) Culex sp. Mosquitoes

c) Aquadest

d) Sucrose solution

e) Malathion

4.6 Operational definition

a) Cinnamon extract will be obtained from parasitotogy laboratorium

b) Raw cinnamon will be obtained from Pasar Traditional Malang

c) For comparison, negative control that will be used is water+acetone 1%

and for positive control malathion (0.25%) will be used.

d) A box (25 x 25 x30 cm) will be used in this experiment and is divided into

two compartment by a bed net (25 x 25cm) where sucrose and

mosquitoes are placed in each respective compartment.

e) Sucrose will be used as attractant in this experiment.

f) True experiment – post test only control group design is research design

on sample where the control group and experimental group are

considered same before given any action and pre-test is not done.

g) Independent variable is variable that can give difference to dependent

variable if it is changed.

h) Dependent variable is variable that is influence by changes to independent

variable.

4.7 Preparation

4.7.1 Stock Preparation

Cinnamon extract that is kept in the refrigerator is taken out and exposed to room

temperature for 15 minutes until the extract obtain room temperature. Assuming that the

extract is 100%, a 100ml 50% extract will be obtained using dilution method by adding

50 ml extract with 50 ml diluter ( 49.5 ml aquadest with 0.5 ml acetone ).

4.7.2 Experimental solution

From the 50% solution that is obtained from above method, the desired

concentration will be obtained using dilution formula;

M1 x V1 = M2 x V2

Explanation:

M1 = initial concentration from stock (50%)

V1 = volume of stock solution

M2 = desired concentration

V2 = volume of experimental solution

To calculate the volume to which 100 ml cinnamon extract 50% must be diluted

to in order to obtain 25% concentration:

V2 = (M1 x V1) / M2

V2 = (50% x 100) / 25%

V2 = 200 ml

Therefore, 100 ml of cinnamon extract 50% should be diluted to 200 ml in order

to get 25% concentration. Diluting can be done by adding 100ml aquadest to 100ml

cinnamon extract 50%

4.8 Commence of Operation

The experiment will be conducted as the following

a) The test will be done using 5 boxes with bed nets in room temperature

b) Prepare the extract with three different concentrations ( x1, x2, x3 ).

c) Soak the bednets into the extract for 15 minutes

d) Put in sucrose solution into the box and fix the soaked bed net in the middle,

forming a divider that separate the space occupied by the sucrose from the

remaining space

e) Put 30 mosquitoes in the remaining space and securely close the box

f) Repeat by using bednets soaked with malathion as positive control and water +

acetone1% for negative control.

g) Record the number of dead mosquitoes after 6 hours.

h) Repeat this test for four times for every box

i) Measure the potential of cinnamon extract as insecticide when applied to

bednets

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