EFFICIENCY OF CALENDULA sp.

EXTRACT AS MOSQUITO
(Diptera:Culicidae) REPELLENT
UNIVERSITI TEKNOLOGI MARA PERAK

DIPLOMA IN SCIENCE
FACULTY OF APPLIED SCIENCE
UNIVERSITI TEKNOLOGI MARA

JUNE 2015

A PRELIMINARY STUDY OF EFFICIENCY OF CALENDULA sp.

EXTRACT AS MOSQUITO

(Diptera:Culicidae) REPELLENT IN UNIVERSITI TEKNOLOGI MARA PERAK

Mini Project Report Submitted in

Partial Fulfillment of the Requirements for the
Diploma in Science
in the Faculty of Applied Sciences
Universiti Teknologi MARA

JUNE 2015
DECLARATION

We hereby declare that the thesis is based on our original work except for quotations and
citations which have been duly acknowledged. We also declare that it has not been previously or
concurrently submitted for any other report at Universiti Teknologi MARA or other institutions.

_________________________

_________________________

_________________________

_________________________

Date: __________________

Date: __________________

ACKNOWLEDGEMENTS

Upon completion of this project, we would like to express our gratitude to many parties.
Our heartful thanks to UITM Perak Kampus Tapah because give an opportunity to run the
Biology 300 mini project as we can gain new experiences and knowledges. Our grateful
appreciated also to Puan Nadia Nisha Binti Hj Musa as our supervisor because give us an advice
and guidance in this project .Thanks to Puan Nunshaimah Binti Salleh as our co-supervisor
because help us to run the experiment and gives us information.Our gratitude also goes to lab
assistant Puan Naimah Binti because have gave us an opportunity to use FTIR analysis machine
and guide us how to use rotavapour to get an extraction oil.Also thanks to Puan Fatimah Binti
because allows us to use laboratory apparatus along this project.Lastly, thanks to our fellow
friends and family to support in financial site and cooperations.

TABLE OF CONTENTS
Page
DECLARATION

iii

APPROVAL SHEET
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
ABSTRAK

iv
v
vi
vii
viii
x
xi

CHAPTER 1 INTRODUCTION
1.1 Background
1.2 Significance of study
1.3 Objective of study

1
1
2

CHAPTER 2 LITERATURE REVIEW

2.1 Diversity of Kingdom Fungi
2.2 Characteristics of fungi
2.2.1 Body structure
2.2.2 Mode of nutrition
2.3 Life cycle of fungi
2.4 Importance of fungi

3
5
5
6
9
11

CHAPTER 3 MATERIALS AND METHODS

3.1 Collecting macrofungi
3.2 Species identification
3.2.1 Morphological study
3.2.2 Anatomical study

15
15
16
17

CHAPTER 4 RESULTS AND DISCUSSION

4.1 Classification of Microfungi
4.2 Group of Fungi
4.2.1 Gill mushrooms
4.2.1 Boletes
4.2.3 Puffballs and earthballs
4.3 Macrofungi and the environment

20
23
24
29
34
47

CHAPTER 5

50

CONCLUSION

REFERENCES
APPENDICES
CURRICULUM VITAE

51
57
60

LIST OF TABLES

Table
2.1
2.2
2.3
4.1
4.2

Caption
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Ccccc cccccccc ccccccccc cccc
Ddddd dddd dddddd ddddd
Eeeee eeeeeeeeee eeeeeeeeeeee

Page
3
5
10
20
23

LIST OF FIGURES

Figur
e
2.1
2.2
3.1
4.1

Caption
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Bbbbbb bbbbbbb bbbbbbb
Ccccc cccccccc ccccccccc cccc
Ddddd dddd dddddd ddddd

Page
5
6
15
21

4.2
4.3
4.4

Eeeee eeeeeeeeee eeeeeeeeeeee

Fffff ffff ffffffff fffffffff
Gggggggg ggggg gggggg gggggg ggg ggggg

23
25
31

ABSTRACT

A PRELIMINARY STUDY OF EFFICIENCY OF CALENDULA SP. EXTRACT AS

MOSQUITO (DIPTERA: CULICIDAE) REPELLENT IN UNIVERSITI TEKNOLOGI
MARA PERAK

Throughout the year, population of the mosquito in different species was slightly
increases in our country. The problem is when there are several amount of mosquitoes
species that carry a disease to the human and can cause a death. This problem are going to be

solve with our upcoming product in this project. Marigold is the flower that contain Lutein,
an ingredient that are found in many insect repellent product and they have unique aroma
which can repel the insect. This new idea will bring more effective way to repel mosquitoes,
at the same time are friendly for users and environmental. To produce this repellent,
extraction method using rotavapor was used to get the extraction oil of this Marigold flower.
The existence of lutein compound in the Marigold flower was prove by using FTIR analysis
spectrum. The population of mosquitoes was tested for before and after using this repellent
by Bare-leg Catch (mosquito collection method) in the static location. The effectiveness of
lutein was prove when the mosquitoes that have been collected after using this repellent are
decrease.

ABSTRAK
KAJIAN AWAL TENTANG KEBERKESANAN EKTRAK BUNGA CALENDULA
SEBAGAI PENGHALAU NYAMUK DI UNIVERSITI TEKNOLOGI MARA PERAK.

Sepanjang tahun, populasi nyamuk dalam spesies yang berbeza sedikit meningkat di negara
kita. Masalahnya ialah apabila terdapat beberapa jumlah spesies nyamuk yang membawa
penyakit kepada manusia dan boleh menyebabkan kematian. Masalah ini akan terlerai dengan
produk kami dalam projek ini. Marigold adalah bunga yang mengandungi Lutein, iaitu bahan

yang terdapat dalam banyak produk penghalau serangga dan ia mempunyai aroma yang unik
yang boleh menghalau serangga. Idea baru ini akan membawa cara yang lebih berkesan untuk
menghalau nyamuk, pada masa yang sama adalah mesra untuk pengguna dan alam sekitar. Untuk
menghasilkan penghalau ini, kaedah pengekstrakan menggunakan rotavapor telah digunakan
untuk mendapatkan minyak perahan bunga Marigold ini. Kewujudan lutein sebatian dalam
bunga Marigold adalah dibuktikan dengan menggunakan spektrum FTIR analisis. Populasi
nyamuk telah diuji untuk sebelum dan selepas menggunakan penghalau ini dengan
menggunakan Bare-kaki Catch (kaedah koleksi nyamuk) di lokasi yang statik. Keberkesanan
lutein telah dibuktikan apabila bilangan nyamuk yang didapati selepas menggunakan penghalau
ini adalah menurun .

CHAPTER 1

INTRODUCTION

1.1

Background

Over one million worldwide are from mosquito disease every year because of the
diseases that the mosquitoes carried out such as malaria, yellow fever, and others (Burderer et
al,2004).Due to the concern of the health, many repellent were produced to avoid the diseases
which are repel the mosquitoes from the human,plant, and building the structures.

At present, the mosquito formulation usually contain the chemical compound to obtain
the high efficiency of repellent because the excellent mosquito repellent effect.However, these
type of chemicals have a side effect and have potential to be harm to the human(Kwoen et
al,2008).
These are numerous plants and derived products have been investigated and described is
potentially natural sources of mosquitoes repellent due to their eco friendly and biodegradable
nature.Most plant based mosquito repellent currently on the market contain essential oil from one
or more of the following plants;citronella ,grenanium, lemon grass,peppermint neem and
soybean (Prajapathi,2005;Chocohote et al,2007; Gillij et al.,2008).Vasuderan,Kashyap and
Sharma (1997) found that marigold flowers also have potential to become repellent or
mosquitoes.
An active compound marigold flower is use as repellent with an addition of other
chemical. Marigolds have been used in many application such as perfumes,dye,inks,paints and in
the religious ceremonies. From the compound in the flower we can observe that there is lutein
that can be use as a repellent due to the pungent smell from the marigold flowers.
There are several methods to extract the plant based to obtain main component in the
material. There are like steam distillation, hydro distillation, and solvent extraction but this study
focus on extraction using rotavapour method. In this, research the flower petals are extracting to
get the active compound in the marigold using rotavapour. Due to the extraction process which
using rotavapour with using the constant pressure (556amp) between the solute and solid and
also the solute and the solvent also studied.

1.2

Significance of the study

This project was presenting the new problem solving to repel the common insect
that regularly disturb our living home, mosquitoes. Commonly the repellent product that have
been produced was use ingredients base on calendula sp and also only non-organic chemical.
Now, was presenting the innovation of mosquitoes repellent that use flowers as the base
ingredients Calendula sp., or the local name is Marigold flower was find out have a chance to
repel the mosquitoes.

The main extraction methods, namely Rotavapor method, were

investigated using the chosen raw material. In evaluating the extraction methods, some
important factors affecting the extraction process were investigated. These include the
standing or exposure time, solids to solvent ratio and temperature. The effect of these
parameters on the concentration of the extracted lutein, and the rate of extraction were

examined. In this study, it was found that rotavapor method of extractions, acetone yielded
the highest amount of lutein compared to other solvents used in the study. In general, the
amount of lutein extracted was proportional to the standing time and the raw materials to
solvent ratio. In all the operating conditions, maximum achievable lutein concentration was
present, after which a plateau condition was observed. The maximum achievable lutein
concentration was dependent on the operating conditions.

A study on the extraction of lutein from selected locally grown vegetation

was carried out as the first step in developing the most suitable method of producing the
lutein. The active component in Marigold flower can be used as repellent same effective as
DEET and at the same time can lower the cost of mosquitoes repellent. This repellent is nontoxic and very safe for every age group and do not harm pets also. The smell of the oil
relieves from new mosquitoes repellent can calms the body.

We are using marigold in this experiments as repellent because it is cheap

and easy to found. Besides that, we are not using any harmful chemical that give bad effect to
environment.

1.3

Objectives of the study

To determine the efficiency of calendula as repellent to culicidae

CHAPTER 2

LITERATURE REVIEW

2.1 Culicidae

The scientific classification of culicidae start with Kingdom is Animalia, Phylum is Arthropoda,
Class is Insecta, Order is Diptera, Suborder is Nematocera, Infraorder is Culicomorpha,
Superfamily is Culicoidea.

Table 2.1: Life cycle of the mosquito

http://www.wumcd.org/mosquito/lifecycle.gif

2.2 Culicidaes Life Cycle

There are some 3300 species of mosquitoes belonging to 41 genera, all contained
in the family Culicidae. This family is divided into three subfamilies: Toxorhynchitinae,
Anophelinae (anophelines) and Culicinae (culicines).

According to Laurel(2014), the mosquito goes through four separate and distinct stages of its
life cycle which is egg, larva, pupa and adult. Eggs are laid one at a time or attached together to
form "rafts" and float at the surface of water. The larva lives in the water and comes to the
surface to breath . Larvae shed (molt) their skins four time, growing larger after each molt. Most
larvae have siphon tubes for breathing and hang upside down from the water surface. The pupa
stage is resting, non-feeding stage of development, but pupae are mobile, responding to light
changes and moving (tumble) with a flip of their tails toward the bottom or protective areas. This
is the time the mosquito change to adult. The newly emerged adult rests on the surface of the

water for a short time to allow itself to dry and all its body part to harden. The wings have to
spread
out and and dry properly before it can fly.

According to Amrita University(2013), mosquito must have water in which to complete their
life cycle. A female mosquito can produce 500 eggs before she dies. The length of life of adult
mosquito depends on factors like temperature, humidity, sex of the mosquito.

2.2.1

Culicidae characteristics

According to Jessia (2005), a winged termite travelling in swarm will have a small, black,
ant-like head with two with wings that give a slightly violet sheen in the light. Department of
Entomology Texas A&M University (2010), states that winged Formosan termite reproductive or
swarmers are yellowish-brown and 12-15mm (0.5-0.6 inch) in length. According to Dawn
(2001) states that the primary reproductive also called swarmers or alates, vary in body colour
from dark brown to blac

2.2.2

Culicidae lifestyle

According to Leisnham PT, LaDeau SL, Juliano SA (2014). Aedes albopictus would
show a greater increase in the proportion of sites occupied and per site abundance during the
rainy season than Aedes aegypti, due to its high dry season egg mortality and heavy rainy season

competitive superiority. While the proportion of occupied sites increased from early to late rainy
season for Aedes albopictus. They actually decreased for Aedes aegypti, suggesting that rainyseason competition from Aedes albopictus had a particularly severe negative effect on the
distribution of Aedes aegypti. Numerous studies have shown that the seasonal patterns of Aedes
aegypti and Aedes albopictus abundances are linked with local rainfall, and these patterns have
been interpreted as being a result of dry-season egg mortality and rainy-season competitive
ability.

Culex is more sensitive to dry conditions than are Aedes and experience high dryseason mortality resulting in low abundance early in the rainy season, and that abundances would
increase during the rainy season as more water filled container habitats become available as
stated by Leisnham PT, LaDeau SL, Juliano SA (2014). The adult females bite people and
animals throughout the night, indoors and outdoors. During the day they are inactive and are
often found resting in dark corners of rooms, shelters and culverts. Numerous studies have
shown that the seasonal patterns of Aedes aegypti and Aedes albopictus abundances are linked
with local rainfall, and these patterns have been interpreted as being a result of dry-season egg
mortality and rainy-season competitive ability.

For mansonia, this species is very sensitive to low humidities and most males and
unfed females are dead after exposure to sulphuric acid and sodium hydroxide solutions also at a
temperature of 27-29C as stated by B. R. Lacresce (n.d).

Anopheles mosquitos are active between sunset and sunrise. Each species has
specific peak biting hours, and there are also variations in their preference for biting indoors or
outdoors. The anophelines that enter houses to feed often rest indoors for a few hours after
feeding. They may then leave for outdoor sheltered resting sites, among them vegetation, rodent
burrows, cracks and crevices in trees or in the ground, caves and the undersides of bridges.
Alternatively, they may stay indoors for the whole period needed to digest the blood-meal and
produce eggs. Indoor resting is most common in dry or windy areas where safe outdoor resting
sites are scarce.

2.2.3 Mosquitoes Habitat

According to Mosquito World (2015), mosquitoes can live in almost any environment, with
the exception of extreme cold weather. They favor forests, marshes, tall grasses and weeds, and
ground that is wet. Mosquito live in floodwater habitat include drainage ditches that fill during
storm, woodland pools created by melting snow, or spring and early summer rains, floodplains
along the banks of stream and rivers, also irrigated pastures and field.

According to University of Florida (n.d), mosquito habitat divided by two . Floodwater

mosquito and permanent water mosquito. Floodwater mosquito live in moist area that will
eventually dry out and the mosquito eggs also dried and become encased in the cracks and
crevices of the dried mud. This species need water to lay their eggs and the eggs need to dry out
before they can hatch. There are estimate that mosquito that live in floodwater habitat between

0.7 and 1.3 million per acre. While permanent water mosquito cannot withstand drying out.
Water is is necessary for a complete life cycle. This mosquito live in the place that permanently
have water without drying .

According to Orkin (2015), hot, humid environment are most amenable to mosquito growth
and survival. Infestation can occur easily in tropical areas. Mosquito larvae are active in transient
water such as flood water, ditches and woodland pools. Mosquito prefer stagnant water within
which they lay egg such as pond, marshes and swamps.

2.3Natural Plant

Source: hdimgegallery.net

2.3.1 Plant Description

Marigolds are most useful in repelling or warning away insects when planted
along with vegetables and fruits. It has pungent smell that repels insects including
mosquitoes and usually the villagers planted it around their house and farm. According to
Noor Asikin , (2012) this statement is supported by Vasudevan (1997) and also by Sarin
(2004). It also is a potential plant whose essential oil from flowers has been effective
repellent against insects.

According to the RHS Gardening, Calendula derived from the Latin calendae ,
the first day of the month on which interest paid, in reference to the long period of bloom
(2008, as cited by Eileen).Calendula has been used for medical purpose in the treatment of
jaundice and as a substitute for saffron in the treatment of measles and smallpox. Marigold is

beneficial for the eyes ,and old herbal reports.

2.3.2 Active Compound in Marigold Flower

A number of papers are now available on the repellent activities from Marigold
against different type of mosquito species. It has an active ingredient which is lutein. Lutein
is one type of material that can be use in repellent to replace DEET. It obtained from the
Marigold flower petals where it is one of the major constituent of yellow or orange fruits and
vegetables such as mango, papaya, prunes and others.

Lutein is usually useful for preventing cataract and arteriosclerosis, enhances

immunity and also has significant functions for preventing cancer formation where it can
delay cancer development. The functional group of lutein is xanthophyll. This statement is
according to Noor Asikin, (2012),supported by Liu & Fan, (2010). Figure 2.1 shows the
lutein structure with molecular formula is C40H56O2 and molecular weight is 568.87 g/mol. It
is insoluble in water, but soluble in fats and lipophilic solvents.

According to Lim, Serena (2003) there was two main extraction methods, which are
conventional method and rotavapor method, were investigated using the chosen raw material.
Behr Labor Technique (n.d) states that continue the heat solvent vessel until all the solvent has
been evaporated and condensed in the rotavapor extractor. According to The Interactive Lab
Primer (n.d), when a compound of low solubility needs to be extracted from solid mixture at
rotavapor interaction can be carried out. The technique places a specialized piece of glassware in
flask and a condenser. According to Asikin (2012), the content of lutein extracted from Marigold
flower was found to be 2.5354% and the maximum amount of lutein extracted was obtained at
temperature 45C, volume of solvent at 150 ml and extraction time at 30 minutes. The optimum
results demonstrated that temperature was influenced variable on the extraction content of lutein.
Theextractionrateconstant,kofluteindecreasedwith increasing temperature and volume of solvent,
and the k values were (0.0405-0.2712) min-1. According to Hojnik et al. (2008, as cited at
Sakinah,2012) was find that when temperature is increase from 20C to 40C, a small increase in
the final extraction efficiency of lutein can be observed and remains constant with further rise of

temperature to 60C. According to Chromatoqr (2013), the addition of acetone enhance the
extraction yield of lutein in many flowers..

According to Javier(1988), was developed a new reversed-phase HPLC method

for separating in a single step lutein and the different lutein fatty acid esters in colour sources and
in other coloured products. The commonest commercial source of lutein is the flower of the
marigold plant, where it is found esterified with one or two fatty acids and constituents about
90%(w/) of the petals. Lutein and lutein fatty acids ester content in two different parts (petals and
calyces) of flower-head from different types of marigold. (1998, as cited at Roberta).

2.4 FTIR Test

According to Thermo Nicolet corporation (2001), FTIR are stand for

Transform Infrared, the suggested method of infrared spectroscopy that passes through sample.
Nancy(2015) states that FTIR spectrometers usually used in organic synthesis, polymer science,
petro chemical engineering, pharmaceutical industry and food analysis. According to
Magni`(1999), the combination of IR absorption spectroscopy and mass spectrometry allows the
chemical composition and structure of the most relevant plasma-produced neutral species, the
ionic plasma composition and also the chemical composition of the nanometer-sized particles to
be precisely identify. Jonas (2015) states that FTIR can investigate the gas phase during the
chemical vapour deposition of silicon carbide from CH3SiCl3. Andrew (2015) states that FTIR
spectroscopy imaging can determine the concentration in ibuprofen in PEG at which
dimerization of ibuprofen can be avoided. FTIR spectra can be analyze to access the molecular
state and degree of polymer.

According to Yan(2011), stated that Fourier transform infared (FTIR)

spectroscopy was used for obtaining vibrational spectra of the Tagetes erect a petal and
Chrysanthemum petal. The content of lutein esters in the petal was studied by analysis of the
relativeabsorptionintensity.

Source:InstrumentLaboratory

2.5 Rotavapor

According to Department of chemistry Wake Forest University(n.d), the purpose

of rotavapor is to remove low boiling organic chemical, usually solvents from a mixture of a
compound. The solvents are removed by a simple distillation. The rotavapor is design under
vacuum and to heat the sample at the same time. A cold finger is used to condense the vapors to a
liquid, which are trapped in a separate flask. According to University of Toronto Scarborough
(2010), an important concept that this technique applies is that liquid boil when the vapor
pressure is equal to external pressure or atmospheric pressure. The machine utilizes a lower
pressure than atmospheric pressure which allows solvents to boil at lower temperature. Other
than that , The rotation increase the surface area and therefore evaporation proceeds more
rapidly.

Source:InstrumentLaboratory

The extraction efficiency of marigold yellow pigment by different extraction methods

were presented according to Sheng(2001).The stability of this pigment to light and temperature
was also studied. The results showed that the extraction efficiency of marigold pigment was
lvigh to 6,21mg/L by the combined extractant of hexane, acetone, and methanol. Additionally,
marigold

yellow

pigment

was

sensitive

to

light

but

insensitive

to

temperature.

2.6 Bare Leg

According to Renaud(2005), the increased attractiveness was not due to an intrinsic

attractiveness of gametocytes carriers but to the infection status associated with the presence of
gametocytes. The parasites change the infected individuals breath or body odour, as these are
involved in attracting mosquitoes at the distances. While transpiration and body temperature also
attract mosquitoes at these distances, these factors are less likely to be involved in the
manipulation as the infection was asymptomatic in all of the children involved in the study.

According to Minhas S , Sekhon H (20130), human bare-leg catches

(landing catches) of Aedes adults or indoor resting collections of adults are most
commonly used to assess adult Aedes populations. The data collected is used to
calculate the number of Aedes mosquitoes landing / biting on a single human bait per
hour (number per man hour). The survey must assess the mosquito population over a
large area; therefore the collectors should move from house to house, not lingering in
one place. In the same way, indoor resting collections can be made.

2.7DataAnalysis

According to Kent State University(n.d), SPSS is a user friendly

program that facilitates data management and statistical analysis. Creating data on SPSS is enter
the variables and value manually into SPSS to create a new dataset. The variables and values
comes from file or table that have already prepared. Variables are the key part of all data sets. A
variable is name, type, label, or other attributes. According to Institute Digital Research and
Education(2015), creating standardized variables in SPSS is very simple to get the mean,
siginificant value and other simple test statistic.

CHAPTER 3

MATERIALS AND METHOD

3.1 Method
1. Collect 300g of flowers without roots.

Series no:UiTM/P8A2/A/1W823/2015W00010
MODEL:PERKIN ELMER/FRONTIER

2.Do the FTIR test to check the presence of lutein in the petals
2.Click icon on desktop 'spectrum'.

3.Log in
4.Insert basic parameter instrument
5.Then do the background scan
6.Scan the sampel
7.After that, print out the spectrum result.
8.Log out.
9.Separate the petals from the sepals
10.Dry the flower for 3 days under the sunlight
11.Soak the dry flower in the acetone for one week
12.Set the apparatus as shown

ID NO:109162 Rotavapour r-3(Bucchi)

13.Fill 3/4 of water in the heating bath

14.Insert 1/4 of the flower that have been soak in the acetone in the evaporating
flask. Then, connect the conical flask to the vapour duct, then fall off the
metal bracelet and turn the combi-clip clockwise.
15.Turn on the main switch
16.Open the tap
17.Turn on the ON/OFF switch. Then set the up down button to 40-50 degree
Celcius
18.Let off the brake lift. Turn the rotation knob and set the turning speed to (1-10)
19.Turn on the vacuum controller. Press the button menu and set.
20.Turn the selection knob and set the pressure at 556 atm for acetone sample.
21.Press ok and the button start.

22.Wait until all the acetone evaporate then, turn the rotation knob and stop the
evaporating flask.
23.Press the stop button twice to let out the pressure
24.Switch off heating bath and the vacuum controller
25.Press the lift brake in the holder to lift up the evaporating flask
26.Take the sample in the evaporating flask. Clean up the apparatus.
27. Keep the extraction oil in the glass bottle to avoid evaporate and durable
28. By using bare legs method which take 5 days to collect the population of the
mosquitoes with lutein and without lutein for 5 days

Day 1
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm
Day 2
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm
Day 3
1.30pm-7.30pm
17.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm
Day 4
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am
7.30am-1.30pm
Day 5
1.30pm-7.30pm
7.30pm-1.30am
1.30am-7.30am

Name
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha
Farah and Hazwani
Fatin and Nabiha

7.30am-1.30pm

Farah and Hazwani

29. By following shift time which 1.30 p.m until 7.30 p.m and 7.30 p,m to 1.30a,m to 7.30 am
the mosquitoes will collect along the time scheduled.
30. Repeat procedure 29 without lutein for 5 days (2/9/2015-6/9/2015) and with
lutein for 5 days (7/9/2015-11/9/2015)
31. The mosquitoes collected will be kept in bijou bottle.
32.The collected mosquitoes is identify using compound microscope
33.The collected mosquitoes is calculated by using SPSS to get standard
deviation, mean and significance
34.Record all the results in the table

3.2 LIST OF EQUIPMENTS/FACILITIES

1) Rotavapour
2) FTIR
3) Test tube
4) Beaker
5)Measuring cylinder
6)Bijou bottle
7)Spray bottle
8)Compound Microscope

LIST OF CHEMICALS/MEDIA:

1)Lutein(in the petals)

2)500 ml of Acetone

CHAPTER 4
RESULTS AND DISCUSSION

FTIR ANALYSIS SPECTRUM

Ester
lutein

C=C
O-H

N-H
O-H
C-H
C-H
C=O
C=C

3500-3300 (cm-1)
3500-3200 (cm-1)
3100-3000 (cm-1)
3000-2850 (cm-1)
1760-1665 (cm-1)
1680-1640 (cm-1))

Amine
Alcohol
Alkene
Alkane
Ketone,Aldehyde,Ester
Alkene

According to Sivel, (2014) in plant lutein is present either in the form of free lutein in
leafy vegetables or in the form of esters with fatty acids.Marigold flowers contain
0.1%-0.2% dry matter of caretenoids out which 80% are lutein diesters.

DISCUSSION
This project is to produce a repellent for mosquitoes and we choose Lutein as a material
to observe the efficiency. At the end of this project, we can conclude that the Lutein is efficient
to repel the mosquitoes. Lutein also present either in form of free lutein in leafy vegetables or in
the form of esters with fatty acids. Marigolds flowers contain 0.1%-0.2% dry matter of
carotenoids out with 80% are lutein diesters Lutein is found in Marigold or Calendula, that can
be test with the FTIR. From the FTIR analysis spectrum we can see at 1712 cm-1 ester lutein
with functional group of ketone, aldehyde and esters. In addition, at peak 1639 cm-1 which show
functional group of alkene where have the same characteristics at the C=C bond in the chemical
structure of the lutein.
At the beginning, we start our project with calculate the population of the mosquitoes in
the place that we want to observe. We do the observation by the method of bare leg for five
days. By following shift time which is 24 hours and the mosquitoes will collect along the time
scheduled in the bijou bottle. This experiment is repeated for over five days continuously. From
this experiment, we get about 95 species of mosquitoes. After that we differentiate them with
their specific species by using the compound microscope. Those are the species that we can
determined, which are Aedes that have two types , Aedes Aegypti and Aedes Albopictus,
Mansonia, Anopheles and Cullex. We get about 11 species of Aedes aegypti, 9 species of Aedes
Albopictus, 28 species of Mansonia, 15 species of Anopheles and 32 species of Cullex.

We proceed this experiment with repeat the bare legs method by apply the Lutein at our
legs for next five days. The shift time is also same as without Lutein that is 24 hours and again
the mosquitoes will collect along the time schedule in the bijou bottle. We get about 50 species
of mosquitoes. Again we differentiate their specific species by using compound microscope.
The result is about 10 species of Mansonia, 11 species of Anopheles, 29 species of Cullex and
none of the Aedes Aegyti and Aedes Albopictus. So the population of the mosquitoes become
decrease as we apply the Lutein. It can be conclude that Lutein is effective as a repellent of
mosquitoes.
The data we get in the table was continued analyze using simple statistic method ,
SPSS to get the mean , standard division , t value and significant 2 tailed. The mean showed for
pre and post of this experiment was shown as the graph at the result. The mean of mosquitoes
for pre experiment which is without lutein are mostly higher than the mean of the post
experiment with lutein. This shows that the no of mosquitoes are also decrease following the
decreasing of mean. The mean for Aedes species, Aegypti and Albopictus shows the bigger
different that the other species, Mansonia, Anopheles and Culex.
The significant of 2 tailed will show the efficiency of this experiment , which is if
less than the Alpha value= 0.05 is a good result. The significant value for Aedes Aegypti is
0.024, Aedes Albopictus is 0.009, Mansonia is 0.077, Anopheles is 0.428 and Culex is 0.643.
This shows that the Lutein are more effective in repel the Aedes mosquitoes, following by
Mansonia, Anopheles and less effective for Culex. The Aedes mosquito are repel with Lutein
because of the specific smell due to the chemical compound in the Lutein. The no of Aedes
mosquitoes are higher during time from 1.30pm to 1.30 am as shown at the graph.

CONCLUSION
In this project, we can conclude that lutein from Calendula can act as the repellent to Culicidae
by decreasing the population of Culicidae after using lutein especially for Aedes mosquitoes.

REFERENCES

Noor Asikin Ahmad Sfri (2012), Study on Extraction Kinetics and Formation of Natural
Mosquito Repellent Solution from Marigold Flower Extract: Universiti Malaysia Pahang

Eartheasy.com(2001),5 Easy to Grow Mosquito-Repelling Plant at

http://eartheasy.com/blog/2011/04/5-easy-to-grow-mosquito-repelling-plants

Wikepidia (n.d). Marigold at https://en.m.wikepidia.org/wiki/marigold

S.Miroslav, K.Stanislav, F.Miroslav, K.Borivoj & K-vlastiml (2014).Lutein Content In

Marigold Flower at http://www.agriculturejournals.cz/publicfiles/138021.pdf

Dr.P.May, Dr.K.W.Quinn, Flaver Naturextra.Kte GmBh (2014), Supercritical Marigold

FlowerCO2-Extract at
http://www.flavex.com/fileadmin/flavex.de/user_upload/veroeffentlichungen/english/supr
critical_marigold_flower_co2-extract-cst2014.pdf

Flower Expert (2005). Marigold- at

http://theflowerexpert.com/content/growingflowers/flowersandseasons/marigolds

R.James (n.d), Journal of Microbiology and Biology education, Retrieved from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4004744/

Univertsity California chemwiki (n.d), How An FTIR Spectrometers Operates. Retrieved

from
http://chemwiki.ucdavis.edu/Physical_chemistry/spectroscopy/Vibrational_spectroscopy/
How_an_FTIR_spectrometer_Operates

Thermo.Nicolet.Corporation(2001) .Thermo Nicoletto Fourier Transform

Infared.SpectrometryThermo Nicolet .Retrieved from
mmrc.caltech.edu>FTIR>FTIR intro

Jonason.D (2014), Surveys Moths Using Light Traps: Effect Of Weather And Time Of
Year .Retrieved from http://journals.plos.org/plosone/article?
id=10.1371/journal.pone.0092453

A.G.Douglas, (2005), Standard Deviation and Stardard Error . Retrieved from

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1255808/

Lauurence (n.d), The Biology Of Two Species Of Mosquito, Mansonia Africana (THEOBALD)
And Mansonia Uniformis (THEOBALD), Belonging To The Subgenus Mansoniaoides (DIPTERA,

CULICIDAE). Retrieved from http://www.mosquitocatalog.org/files/pdfs/075600-0.pdf

Mosquitos and other biting Diptera .Retrieved from

http://www.who.int/water_sanitation_health/resources/vector007to28.pdf

Minhas S , Sekhon H (2013), Entomological Survey For Dengue Vector:

INTERNATIONAL JOURNAL OF MEDICAL AND APPLIED SCIENCES. Retrieved

from http://earthjournals.org/ijmas_258.pdf

Ralph Harbach (2009), Mosquito Taxonomic Inventory. Retrieved from http://mosquitotaxanomic-inventory.info/itemculexltemgt-classification

Centers for disease control and prevention (n.d). Retrived from

anopheles/www.cdc.gov/malaria/about/biology/mosquitoes

Mosquito Taxonomic Inventory (2008). Retrieved from http://mosquito-taxonomicinventory.info/simpletaxonomy/term/6218

Kyle Harfma (1999), Aedes Albopictus. Retrieved from

http://animaldiversity.org/accounts/aedes-albopictus/

Elsevier B.V. (1991), Journal of Chromatography. Retrieved from

www.sciencedirect.com/science/article/pii/S0021967300942663

Roberta P. (1998), Abstract. Retrieved from

www.sciencedirect.com/science/article/pii/s092666909710005X

Yan.C.Q (2011), Identification of three species of chrysanthemum petal by fourier

transform infared spectroscopy. Retrieved from en,cnki,com.cn/article-en/CJFD TOTALGPSS201103131.htm

Medical Entomology, Westmead Hospital (1996), Aedes Aegyapti. Retrieved from

medent.usyd.edu.au/photos/aedes/.20aegyapti.htm

Mount. L(2014). Mosquito Life cycle. Retrieved from : www.mosquito.org/lifecycle

Amrita.olabs.co.in(2013). Life Cycle of Mosquito. Retrieved from :

amrita.olabs.co.in/?sub=79&brch=15&sim=138&cnt=1

Mosquito World(2015). Your Guide To Effective Mosquito Control. Retrieved

from : www.mosquitoworld.net/about-mosquitoes/habitats/

Florida Medical Entomology Laboratory(n.d), Mosquito habitat. Retrieved from

: mosquito.ifas.ufl.edu/Mosquito_Habitats.htm

Orkin,LLC(2015). Mosquito Habitat. Retrieved from :

www.orkin.com/other/mosquitoes/moaquito-habitats/

APPENDICES

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