KUMASI TECHNICAL UNIVERSITY

DEPARTMENT OF PHARMACEUTICAL SCIENCES

FINAL YEAR PROJECT

PRODUCTION OF AN INSECT REPELLENT COIL FROM LIME PEELS

NAME: ESHUN ISAAC

INDEX NUMBER: 052041090038

DATE: FEBRUARY 2024

 STATEMENT OF AWARDS
A FINAL YEAR PROJECT SUBMITTED TO THE DEPARTMENT OF
PHARMACEUTICAL SCIENCES, IN PARTIAL FULFILMENT OF THE REQUIREMENT
FOR THE AWARD OF BACHELOR OF TECHNOLOGY [B. TECH] IN
PHARMACEUTICAL SCIENCES.

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 DECLARATION
STUDENT’S DECLARATION
I hereby declare that the information contained in the project report is the result of my own effort
and due citations have been made. I further declare that it contains no material previously
published by another person nor material which has been accepted for the award of any other
degree or diploma in the University, nor elsewhere except where due acknowledgement has been
made.

Name: …………………………………… Signature: …………………………

SUPERVISER(S)’S DECLARATION
I hereby declare that the project report is the results of the project work of the student. I
supervised during the 2022/2023 Academic year.

Name: …………………………………… Signature: …………………………

HEAD OF DEPARTMENT’S DECLARATION

I declare that this project report submitted to me is duly signed by the student and the project
supervisor(s) in accordance with the requirements of Kumasi Technical University.

Name: …………………………………… Signature: …………………………

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 DEDICATION
I dedicate this project to my dearest mom and dad, Mr. and Mrs. Annan for their unflinching
support to my success.

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 ACKNOWLEDGEMENT
I express my profound gratitude to the Almighty God for bringing me this far. I would like to
express my special thanks to my caring lecturer Mr. Seth Obiri Yeboah who have given me this
golden opportunity to do this wonderful project on the topic, “Production of mosquitoes repellent
from Lime peels” which also helped me in doing a lot of research and has been enlightened
about many things. Sir, without your effective supervision, perfect guidance, flawless
experiences, impeccable information and mind-blowing ideas, I would not have completed the
project with such success. I thank you for the encouragement and the ultimate care you give us as
your friends and always ensuring our wellbeing. Also, a special appreciation goes to our teaching
assistant, Mr. Rudock and Mr. Stephen a National Service personnel who assisted us in most of
the activities we carried out, we say a very big thank you for your consistent advice,
encouragement and hope that you give to us each and every blessed day. God bless you and
restore whatever you have lost in helping us come this far. My last appreciation goes to my
group members, Sheridan, Abosi, Belinda, Seth and other colleagues for their unflinching efforts
and helping hands to make this project a better one.

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 TABLE OF CONTENT

Table of Contents
STATEMENT OF AWARDS.........................................................................................................2
DECLARATION.............................................................................................................................3
STUDENT’S DECLARATION..................................................................................................3
SUPERVISER(S)’S DECLARATION........................................................................................3
HEAD OF DEPARTMENT’S DECLARATION........................................................................3
DEDICATION.................................................................................................................................4
ACKNOWLEDGEMENT...............................................................................................................5
TABLE OF CONTENT...................................................................................................................6
LIST OF FIGURES.........................................................................................................................8
LIST OF TABLES...........................................................................................................................9
CHAPTER ONE............................................................................................................................10
INTRODUCTION.........................................................................................................................10
1.1 overview...............................................................................................................................10
1.2. Background of the study.....................................................................................................10
1.3 Statement of problem...........................................................................................................11
1.4 Significance of study............................................................................................................12
1.5 Scope of study......................................................................................................................13
1.6 Aims and objectives.............................................................................................................13
1.6.1 Aim:..................................................................................................................................13
1.6.2 Specific Objectives...........................................................................................................13
1.7 Limitation of study...............................................................................................................13
CHAPTER TWO...........................................................................................................................14
REVIEW OF LITERATURE........................................................................................................14
2.1.0 Overview...........................................................................................................................14
2.2.0 Description of Citrus fruits...............................................................................................14
2.3.0 Chemical description and composition of Limonene.......................................................16
2.4.0 Method of extraction.........................................................................................................16
2.5.0 Physicochemical properties of D-Limonene.....................................................................17
2.6.0 Human exposure...............................................................................................................18

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 2.7.0 Absorption, distribution and metabolism of Limonene....................................................18
2.8.0 Toxicokinetics...................................................................................................................18
2.8.1. Administration.................................................................................................................18
2.8.1. Metabolism.......................................................................................................................19
2.8.2. Elimination.......................................................................................................................19
2.9.0 Effect of Limonene on the respiratory tract......................................................................19
2.9.2 Non-inducibility of pulmonary irritation..........................................................................20
CHAPTER THREE.......................................................................................................................21
METHODOLOGY........................................................................................................................21
3.1. Overview.............................................................................................................................21
3.2. Acquisition of materials and apparatus...............................................................................21
3.3. Materials and apparatus......................................................................................................21
3.4. Extraction of Limonene from lime peels............................................................................22
3.5. Making of Mosquito repellent (coil)...................................................................................22
3.6. Coding and labelling of samples.........................................................................................22
REFERENCES..............................................................................................................................23

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 LIST OF FIGURES
Figure 1: Partly peeled lime fruit Figure 2: Peels of lime fruit Figure 3: sliced lime fruit
peels and amber bottles...............................................................................................................15
Figure 4: Stereoisomers of limonene..........................................................................................17

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 LIST OF TABLES
Table 1: Physicochemical properties of D-Limonene...............................................................18

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 CHAPTER ONE

INTRODUCTION
1.1 overview
This chapter of the study comprises the; background of study, problem of statement, aims and
objectives of study, significance of study, scope of study, limitations of study.

1.2. Background of the study

Herbal medicine remains a cornerstone of healthcare for approximately 75–80% of the global
population, particularly in developing nations, due to its cultural acceptance, compatibility with
the human body, and minimal side effects. Over recent years, there has been a notable rise in its
utilization in developed countries as well [Bilham, Curr. Sci., 1995]. The World Health
Organization (WHO) now defines traditional medicine, including herbal remedies, as therapeutic
practices with centuries-long histories predating modern medicine and still prevalent today
[Gansser, Geology of the Himalayas, 1964]. Traditional remedies encompass a variety of
substances such as medicinal plants, minerals, and organic matter. Herbal drugs specifically refer
to traditional medicines primarily derived from medicinal plant preparations for therapeutic
purposes.

The medicinal benefits of plant materials largely stem from the unique combinations of
secondary compounds they contain. These compounds, which vary between plant species or
groups, contribute to the distinct medicinal actions observed. In contrast, primary products like
carbohydrates, lipids, and proteins are common to all plants and play essential roles in basic
metabolic processes (Wink, 1999). While traditionally viewed as having no direct biochemical
function in cellular maintenance, recent research has illuminated the crucial ecological roles of
secondary compounds in plants (Kaufman, Wink et al., 1999). These compounds serve both
defensive purposes, protecting against herbivores and pathogens, and attract beneficial organisms
like pollinators and symbionts (Kaufman et al., 1999; Wink and Schimmer, 1999).

Over 2,140,000 secondary metabolites are known and are commonly classified according to their
vast diversity in structure, function and biosynthesis. There are seven main classes of secondary
metabolites such as; alkaloids, glycosides, terpenes, phytosterols, fixed oils, volatile oils,
phenolic compounds (tannins, flavonoids, coumarins etc.)
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1.3 Statement of problem
Mosquito-borne diseases afflict approximately 700 million people annually (Fradin et al., 2002).
In response to the dengue epidemic in Pakistan, the usage of mosquito repellents has surged.
Among these, the most renowned is N, N-diethyl-m-toluamide, now referred to as N, N-diethyl-
3-methylbenzamide (DEET) (Mafong et al., 1997). While repellents effectively deter mosquitoes
and decrease the likelihood of bites, they do not offer complete protection against these insects.
However, their use provides individuals with added defense against mosquito-borne illnesses,
thereby reducing community transmission rates. A plethora of mosquito repellent options are
available in the market, including sprays, coils, lotions, and mats (Nasci et al., 2012).
Additionally, some individuals opt for natural remedies such as burning certain herbs or
cultivating plants like horsemint, rosemary, marigolds, and consuming more garlic (Ferreira et
al., 2011). It's worth noting that while some people may be allergic to mosquito repellents, others
are not. Adverse reactions to these repellents can manifest as symptoms like a runny nose,
watery eyes, hoarseness, and in rare cases, seizures (Oransky et al., 1989). Furthermore, there
have been concerns raised about potential severe outcomes such as encephalitis and cancer
associated with mosquito repellents (Roland et al., 1985).

A study conducted on Chinese and Malaysian mosquito coils revealed that the smoke emitted
contains particulate matter (2.5) equivalent to that produced by smoking 75-137 cigarettes, and
burning one mosquito coil releases formaldehyde equivalent to that produced by burning 51
cigarettes (Liu et al., 2003). Another research in Kuala Lumpur indicated that reducing exposure
to mosquito coils among individuals with asthma complaints could potentially decrease the
prevalence of persistent wheezing, chest illnesses, and asthma by up to 29% (Azizi et al., 1991).
Furthermore, a study conducted in Taiwan from 2002 to 2004 found that smokers exposed to
mosquito coils have a lung cancer risk 14 times higher than non-smokers (Chen et al., 2008).
DEET, a common component in many mosquito repellent products, has undergone extensive
toxicity research. While effective, DEET has been associated with seizures, particularly in young
children, as reported in case studies (Lipscomb et al., 1992). Furthermore, ingestion of DEET
can lead to hypotension, seizures, and coma within as little as an hour, with deaths occurring at
serum concentrations of 1 mmol/L, according to reports (Teinenbein et al., 1988).

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A Canadian study highlighted the potential risk of psychosis in adults who applied a product
containing 70% DEET to their skin, as well as the possibility of immediate contact dermatitis
with dermal application (Ellenhorn et al., 1997). Another study found that excessive DEET use
(325 mg/kg daily) beyond the normal human dose resulted in maternal toxicity and low birth
weights of offspring (Schoenig et al., 1994). An analytical investigation conducted in Indonesia
identified bis(chloromethyl) ether (BCME) as a potent lung carcinogen formed during the
combustion of formaldehyde and hydrogen chloride (Krieger et al., 2003). Additionally, Patel et
al. (2012) cautioned against the hazardous effects of chemical mosquito repellents on the skin
and nervous system, citing potential adverse reactions such as skin rashes, swelling, eye
irritation, anaphylactic shock, low blood pressure, and brain swelling in children.
1.4 Significance of study
The significance of this project lies in the development of an affordable and environmentally
friendly mosquito repellent aimed at controlling malaria transmission. By utilizing lime peel
waste, production costs are reduced, rendering the repellent accessible, particularly in rural areas
(Adeniram et al., 2012).

Given the paramount importance of human health in contemporary society, the aim of this
research is to raise awareness about the concealed health risks associated with mosquito repellent
use. It is evident that a significant portion of our community remains uninformed about the
potential hazards posed by these products to our bodies.

Healthcare professionals should be prioritized for training to effectively educate the public on the
proper usage and potential side effects of mosquito repellents. Moreover, awareness programs
utilizing various mediums such as print media, publications, and seminars should be organized.
Companies manufacturing these products should provide comprehensive information, including
their toxicity profiles, to empower consumers to make informed choices.

Ultimately, the goal is to promote sanitation practices, encourage the use of natural alternatives
like mosquito nets or repellents derived from natural sources, and minimize reliance on chemical
repellents wherever possible.

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This study is to ascertain whether Limonene, a dominant secondary metabolite present in the
extract of Lime peels could be a better alternative and a suitable mosquito repellent coil.

1.5 Scope of study

This study was conducted by preparing an extract of lime peels using cold pressing method. With
limonene as the active secondary metabolite which is a terpene; a volatile and essential oil as
well, it was incorporated into a mixture of fine charcoal and starch which acted as our binder and
was shaped into a coil. The lime fruit was purchased from Roman Hill, a marketplace at Kumasi.
The apparatus used were obtained from the Chemistry laboratory of Kumasi Technical
University. Inspections were performed to ascertain how intact the coil is, in that, it does not
break easily, the burning of the coil and the essence of the limonene that comes out of the smoke.

It was finally observed that the coil was intact, the essence from the smoke was appreciable and
the burning was neither too fast nor too slow. This study took place at the department of
Pharmaceutical sciences, the chemistry laboratory to be precise, in Kumasi Technical University.

1.6 Aims and objectives

1.6.1 Aim:
 To produce an insect repellent coil using lime peels.

1.6.2 Specific Objectives

 To prepare an extract of lime peels
 To shape a insect repellant coil
 To produce an insect repellent with less effect on human health

1.7 Limitation of study

 Pure limonene was not qualitatively analyzed.
 The amount of limonene was not quantitatively analyzed.
 Other essential oils were not considered in this study.

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 CHAPTER TWO

REVIEW OF LITERATURE
2.1.0 Overview
There have been numerous scripts or documents by writers, researchers, publishers and reporters
have written and delineated various subjects pertaining to the production of mosquito repellent
coil using lime peels. These published literatures may serve as the means to gain understanding
of the subject matter as well as debates relevant to this particular research. In this chapter of the
study, knowledge gained from articles, books and other literatures are carefully reviewed and
presented in the form of written report. It ultimately seeks to bring clarity and focus to the
research problem stated in the first chapter of this report.

2.2.0 Description of Citrus fruits

The Citrus genus (Rutaceae) encompasses some of the most widely cultivated fruits globally,
including orange, lemon, lime, and mandarin (Kummer et al., 2013). Despite the extensive
cultivation of various species within this genus, Citrus aurantiifolia (Christm) Swingle,
commonly known as lime or key lime, remains relatively underexplored as a source of essential
oil, despite its widespread cultivation (Sharifi-Rad et al., 2017). Noteworthy botanical features of
this species include its fragrant white flowers and ovoid yellow-green fruits. While originally
native to Southern Asia, C. aurantiifolia is now cultivated in several regions of Brazil due to its
nutritional and commercial significance. Recent scientific literature reflects an increasing interest
in the medicinal properties of this species (Al-Aamri et al., 2016).
In Citrus species, essential oil is primarily found in the fruit peel, which contains secretory
cavities (Voo et al., 2012). These fruits thrive in tropical and subtropical regions, particularly in
South East Asia, with countries like India and China being major producers. Citrus cultivation
typically requires deep soils with a pH range of 5.5 to 7.5 to yield high-quality fruits.

Figure 1: Partly peeled lime fruit Figure 2: Peels of lime fruit Figure 3: sliced lime fruit peels and amber
bottles

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Citrus essential oils consist of both volatile and nonvolatile fractions, comprising over 200
compounds. The volatile fraction, which makes up to 99% of the oil, primarily contains
monoterpene and sesquiterpene hydrocarbons, along with their oxygenated derivatives, aliphatic
aldehydes, alcohols, and esters. Conversely, the non-volatile fraction may include hydrocarbons,
sterols, fatty acids, waxes, carotenoids, coumarins, psoralens, and flavonoids (Tranchida et al.,
2012).

Terpenes, the predominant constituents of essential oils, are synthesized by various plant species
and serve diverse functions, including defense mechanisms against herbivores and pathogens, as
well as roles in plant developmental physiology. These compounds have a basic chemical
structure consisting of an isoprene unit and are produced via metabolic pathways in specialized
plant cells (Mewalal et al., 2017; Zulak et al., 2010).

Terpenes display a broad spectrum of biological effects, rendering them valuable in both disease
prevention and treatment. These effects encompass antimicrobial, anti-allergenic, antioxidant,
anti-inflammatory, and immunomodulatory properties (Theis et al., 2003). Furthermore, their
advantageous pharmacokinetic attributes, including lipophilicity and low molecular weight,
contribute significantly to their widespread application in healthcare (Rufino et al., 2015).
Among the most prevalent terpenes in nature is limonene, a primary component of numerous
essential oils derived from Citrus species. Limonene exists as two optical isomers, known as d-
or L-limonene, as well as a racemic mixture (Zulaikha et al., 2015). Exhibiting a colorless liquid
form, limonene possesses a pleasant lemon-like fragrance, leading to its extensive use as a flavor
and fragrance enhancer in various food products, such as fruit juices, candies, chewing gums,
soft drinks, and ice creams. Moreover, limonene is a commonly utilized fragrance in cosmetics
formulations, finding its way into an array of beauty products, including soaps, perfumes,
shampoos, hair conditioners, and shower gels (Hirota et al., 2010). Additionally, limonene serves
as an ingredient in cleaning products and eco-friendly pesticides (Nazaroff et al., 2004). Notably,
it is considered safe for food preservation purposes (Sun et al., 2007) and demonstrates potential
as a green solvent for the extraction of natural products (Chemat et al., 2012).

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2.3.0 Chemical description and composition of Limonene
Often referred to as "D-limonene," the dextrorotatory isomer (+)-limonene (or d-limonene) is a
monocyclic terpene composed of two isoprene units, abundantly synthesized in nature as a
secondary plant metabolite (Ressner, 2011; Mann et al., 2011). This isomer serves as the primary
constituent of the essential oils found in the peel of citrus fruits such as oranges, lemons,
mandarins, grapefruits, and limes. Conversely, the other optical isomer, (-)-limonene or l-
limonene, emits a turpentine scent and is prevalent in plants as a major component of the
volatiles released by oak and pine trees (Schween et al., 1997).

Figure 4: Stereoisomers of limonene

d-Limonene, a colorless oil with limited solubility in water (13.8 mg/L at 25°C) and a pleasant
orange aroma, finds extensive use in the food and cosmetic industries. Primarily sourced from
waste orange peel, which contains approximately 3.8 wt% of d-limonene on a dry weight basis, it
serves various commercial purposes (Pourbafrani et al., 2010).

Initially proposed by Wagner in 1894, the correct molecular formula of d-limonene comprises 76
electrons and exhibits six stable conformational isomers (conformers) (Jansik et al., 2006).

2.4.0 Method of extraction

Essential oils, including those derived from fruit peels such as lime, offer economic potential.
Lime peel waste can be harnessed as a raw material for essential oil production, thereby
increasing both the value of lime peel waste and the income of lime peel farmers (Baser et al.,
2010). Extraction methods for essential oils vary depending on the plant source (Patrascu et al.,
2016).

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Among the extraction methods, maceration stands out as a cost-effective and commonly utilized
technique for extracting active ingredients from plants. This process involves immersing plant
material in a solvent for a specific duration to extract the bioactive compounds (Njila et al.,
2017).

2.5.0 Physicochemical properties of D-Limonene

THE FOLLOWING TABLE PRESENTS SEVERAL PHYSICOCHEMICAL
PROPERTIES OF D-LIMONENE
1-Methyl-4-(1-methylethenyl) cyc1ohexene
Chemical Abstract Name

Cajaputene; carvene; cinene; (+)-dipentene;

d-(+)- limonene; D-(+)-limonene;(+)-
limonene; ®-limonene; ®-(+)-limonene;
Synonyms (+)- para-mentha-1,8-diene;®-(+)-para-
mentha-1,8-diene; 1-methyl-4- isopropenyl
cyc1ohexene-1; Refchole
Description Colourless liquid

Melting point -74.3 °C

Boiling point 175.5-176 °C

Density 0.8411 g/cm3 at 20 °C/4 °C

Solubility insoluble in water; soluble in benzene,

carbon tetrachloride, diethyl, ether, ethanol
and petroleum ether, soluble in glycerine
Optical rotation ~ + 125.6°

Spectroscopy data Infrared, nuclear magne tic resonance and

mass spectral data have been reported

Stability Oxidizes to film in air

Table 1: Physicochemical properties of D-Limonene

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2.6.0 Human exposure
D-Limonene is widely acknowledged as safe for human consumption as a synthetic flavoring
substance, as affirmed by the US Food and Drug Administration (FDA) in 1991. The tolerable
daily intake (TDI) of d-limonene is estimated at 0.27 mg/kg body weight, translating to 16.2 mg
for an adult weighing 60 kg, according to the International Agency for Research on Cancer
(IARC) (IARC, 2018). This dosage of d-limonene is naturally derived from food sources and its
incorporation as a flavoring agent.

2.7.0 Absorption, distribution and metabolism of Limonene

Limonene is typically absorbed in the gastrointestinal tract in both animals and humans, leading
to its dispersion to various body parts such as the liver, lungs, kidneys, and notably, adipose
tissue and the mammary gland (Igimi et al., 1974). Its half-life is estimated to range from 12 to
27 hours.

Inhalation serves as a rapid route for limonene absorption due to its high solubility in blood and
fat. Upon inhalation, limonene quickly enters the bloodstream, aided by a blood-to-air partition
coefficient (Xblood/air) of 42, and its considerable fat solubility (Xoil/air = 5700) further
facilitates absorption (Astrand, 1983). Approximately 70% of the supplied amount is absorbed
by the lungs, attributed to both its solubility properties and the large surface area of the lungs
(Falk, 1993).

Following absorption, limonene swiftly distributes throughout the body and undergoes rapid
metabolism (Anundi et al., 2000). Its blood clearance rate is notably high at 1.1 L kg-1 h-1,
indicating rapid metabolism (Falk, 1993). While about 1% of the total uptake is eliminated
unchanged in the expired air post-exposure, approximately 0.003% is excreted in the urine
(Hagberg, 1993). Notably, limonene exhibits a long half-time in the slow elimination phase,
suggesting accumulation in adipose tissues (Hagberg, 1993).

2.8.0 Toxicokinetics
2.8.1. Administration
According to the IARC limonene monograph (IARC, 2018), two volunteers administered 1.6 g
of 14-carbon d-limonene orally excreted 55% to 83% of the dose in urine within 48 hours. The
primary urinary metabolite observed was 8-hydroxy-p-menth-1-en-9-yl-β-D-

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glucopyranosiduronic acid. Additionally, Falk-Filipsson et al. (Tsuji et al., 1975) exposed eight
male volunteers to d-limonene at atmospheric concentrations of 10, 225, and 450 mg/m3 during
three 2-hour sessions spaced over 2 weeks, while engaged in light physical exercise. Pulmonary
absorption, calculated as the difference between the amount of solvent in inspired and exhaled
air, was notably high, ranging from 63% to 68% for the administered doses, indicating
significant uptake by the lungs.

2.8.1. Metabolism
D-Limonene undergoes rapid and nearly complete metabolism, as evidenced by a blood
clearance rate of 1.1 L/kg h measured up to 21 hours after exposure cessation at 450 mg/m3.
Additionally, limonene or its metabolites are eliminated via respiratory pathways, imparting a
distinct odor to the exhaled air.
2.8.2. Elimination
The authors observed three distinct phases of blood elimination for d-limonene: an initial phase
within 0-15 minutes post-exposure, an intermediate phase characterized by rapid elimination
spanning 16-319 minutes post-exposure, and a terminal phase of slow elimination lasting from
320 to 1300 minutes post-exposure (Holck AR et al., 1991). The blood elimination half-lives
were approximately 2.6 minutes for the initial phase, 32 minutes for the intermediate phase, and
750 minutes for the terminal phase. The extended half-life of the terminal phase suggests
potential accumulation in adipose tissue. Approximately 1% of absorbed d-limonene is excreted
unchanged in the exhaled air post-exposure cessation.

Furthermore, when volunteers were exposed to an atmospheric concentration of 10 mg/m3 of d-

limonene for two hours, it was concluded that percutaneous absorption in humans is relatively
low compared to pulmonary absorption. Thus, d-limonene is readily absorbed by the lungs but
less so by the skin (Karlberg AT et al., 1993).

2.9.0 Effect of Limonene on the respiratory tract

Acute lung injury, particularly when accompanied by inflammation, poses a significant health
concern with high rates of morbidity and mortality, lacking specific treatment options. Chi et al.
(2013) proposed that limonene might mitigate acute lung injury induced by intranasal
lipopolysaccharide (LPS) administration in mice. Their findings demonstrated the efficacy of

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limonene in lung protection, enhancing lung function through its anti-inflammatory properties.
Limonene effectively reduced the inflammatory infiltration of neutrophils and the activity of
myeloperoxidase (MPO). Moreover, it targeted inflammatory cytokines such as TNF-α, IL-1β,
and IL-6, which are pivotal in acute lung injury. Limonene treatment resulted in decreased
activity of these cytokines by attenuating the activation of NF-κB, ERK, JNK, and p38 MAPK
signaling pathways, thereby exhibiting potent anti-inflammatory effects in preventing LPS-
induced acute lung injury.

Bronchial asthma, a chronic inflammatory ailment affecting millions worldwide, poses

substantial challenges. Individuals predisposed to asthma or allergic respiratory conditions often
exhibit heightened sensitivity to inhaled irritants due to airway abnormalities. Hirota et al. (2012)
investigated whether limonene could alleviate allergic symptoms and asthma in a model of
airway inflammation induced by the dust mite Dermatophagoides farinae. Their study revealed
that oral administration of limonene significantly reduced the production of crucial
immunoglobulin markers associated with allergic inflammation and pro-inflammatory cytokines.
Additionally, limonene demonstrated the ability to mitigate bronchoconstriction induced by
acetylcholine administration in mice, indicating its involvement with cholinergic receptors.
These findings underscored the potent therapeutic efficacy of limonene in alleviating allergic
airway conditions, even in the context of asthma.

2.9.2 Non-inducibility of pulmonary irritation

Studies have indicated that limonene, specifically its enantiomers R-(+)-limonene and S-(-)-
limonene, exhibit no significant impact on the respiratory system of humans. Research conducted
on conscious mice exposed to airborne R-(+)- and S-(-)-limonene revealed that both enantiomers
did not elicit pulmonary irritation or general anesthetic effects within certain concentration
ranges (IARC, 2018). Additionally, when applied to the skin in typical amounts found in
fragrances and personal care products, limonene is deemed safe and generally does not affect the
respiratory system of individuals (Dittmar et al., 2019).

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 CHAPTER THREE

METHODOLOGY
3.1. Overview
This chapter of the study describes the step-by-step method carried out to obtain data from the
experiments conducted in this research. The materials and apparatus used are clearly listed and
each method is carefully described in this chapter using simple constructions and language. The
primary purpose of this chapter is to basically make this study reproducible and easily carried out
by individuals and organisations who after reading this report develop interest and wish to carry
out the study again either to affirm the results obtained or develop a new concept from this study.
3.2. Acquisition of materials and apparatus
In this research, the sample utilized was the waste peels of lime fruit. The fruits were purchased
from Roman Hill, a marketplace Kumasi. The fruits were identified as Lime fruits using its
organoleptic properties at the chemistry department of Pharmaceutical Sciences, Kumasi
Technical University by Sir Rudock. The fruits were divided into five (5) groups to obtain
different volumes of the limonene oil and hence obtain different concentrations of the five (5)
repellent.
The extraction was then performed on each of the five (5) different groups of the fruits which
were labeled A to E.
The Charcoal powder was obtained from a charcoal vendor at Asafo, a suburb of Kumasi.
Apart from the sample used, all the other tools and materials were obtained from the Analytical
laboratory of the Pharmaceutical Sciences Department, Kumasi Technical University.
3.3. Materials and apparatus
1. Saucepans
2. Methanol
3. Waterbath
4. Small Basin
5. Grater
6. Conical Flask
7. Citric Acid
8. Maize starch
9. Fine charcoal powder
10. Seive

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3.4. Extraction of Limonene from lime peels.
The fruits were grated with a plastic, the peels were weighed and noted. It was then transferred
into a saucepan. A predetermined volume of Methanol was added to the peels in the ratio of 2:1.
The mixture was then heated in a water bath using the Maceration method to extract the
limonene into the methanol.
The heated mixture was then allowed to cool and then filtered to separate the Marc from the
menstrum.
The Menstrum containing the crude oil was transferred into conical flasks labeled A to E and
stored in a cool and dry place uncovered to allow the Methanol to evaporate out of solution
leaving only the crude oil behind.
The different volumes of oils were transferred into calibrated labeled measuring cylinders for the
production of the repellent.
3.5. Making of Mosquito repellent (coil).
The charcoal powder collected was derived to obtain fine particles of the powder and to remove
any adulterants from the powder. The dry ingredients, charcoal powder, Citric acid, and maize
starch were mixed in a ratio of 2:1:1 in a mortar with a pestle. After thorough mixing, a
measured volume of water was added to the mixture and mixed thoroughly to the desired
consistency of a wet mass. The extracted crude oil was then added with respect to their volumes.
The mass was then molded into a spiral shape using an already-obtained coil.
The molded coils were individually labeled as A to E and was then allowed to dry at room
temperature.
The coils were then distributed to randomly selected people on campus along with printed
questionnaires to use and record their experiences.
3.6. Coding and labelling of samples.
This study is primarily concerned about the level of Limonene used to successfully repel
mosquitoes. In order not to raise any alarming concern and panic in individuals who read this
report and in the general public, the samples were given unique codes. The codes given to the
samples were used extensively throughout this report instead of the respective names or volumes
used. Table 3. below summarizes the volumes of limonene used and their codes given to them.
VOLUME (mls) CODE
5 A
10 B
15 C
20 D
25 E

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 REFERENCES

1. K. Singletary Diet, natural products and cancer chemoprevention J. Nutr. (2000)

2. R. Mewalal et al. Plant-derived terpenes: a feedstock for specialty biofuels Trends Biotechnol.
(2017)

3. A.T. Rufino et al. Evaluation of the anti-inflammatory, anti-catabolic and pro-anabolic effects
of E-caryophyllene, myrcene and limonene in a cell model of osteoarthritis Eur. J. Pharmacol.
(2015)

4. W.W. Nazaroff et al. Cleaning products and air fresheners: exposure to primary and secondary
air pollutants Atmos. Environ. (2004)

5. J. Bai et al. Protective effect of d-limonene against oxidative stress-induced cell damage in
human lens epithelial cells via the p38 pathway Oxid. Med. Cell. Long. (2016)

6. M. Bacanlı et al. The antioxidant and antigenotoxic properties of citrus phenolics limonene
and naringin Food and Chem. Toxicol. (2015)

7. S. Ahmad et al. Hypolipidemic and antioxidant activities of thymoquinone and limonene in

atherogenic suspension fed rats Food Chem. (2013)

8. L. Berliocchi et al. Toxic profile of bergamot essential oil on survival and proliferation of SH-
SY5Y neuroblastoma cells Food and Chem. Toxicol. (2011)

9. R. Russo et al. Implication of limonene and linalyl acetate in cytotoxicity induced by

bergamot essential oil in human neuroblastoma cells Fitoterapia (2013)

10. The stereochemical indicator “D” cannot be applied to limonene as the use of “D”
and “L” notation to describe the absolute stereochemistry is meaningful only for
monosaccharides and amino acids. See: E. C. Ressner, Naming Limonene Correctly.
Chem. Eng. News 2011, 89 (23), 4
11. J. C. Mann, J. B. Hobbs, D. V. Banthorpe, J. B. Harborne, Natural Products: their
Chemistry and Biological Significance, Longman Scientific & Technical, Harlow: UK;
pp. 308–309.
12. J. Schween, R. Dlugi, C. Hewitt and P. Foster, Atmos. Environ.1997, 31, 199-215.
13. M. Pourbafrani, G. Forgacs, I. S. Horvath, C. Niklasson, M. Taherzadeh, Bioresour.
Technol. 2010, 101, 4246-4250.

23
14. B. Jansik, A. Rizzo, L. Frediani, K. Ruud, S. Coriani, J. Chem. Phys. 2006, 125, 1-9.
15. M. A. Śmiałek , M.-J. Hubin-Franskin, J. Delwiche, D. Duflot, N. J. Mason, S.
Vrønning-Hoffmann, G. G. B. de Souza, A. M. Ferreira Rodrigues, F. N. Rodrigues, P.
Limão-Vieira, Phys. Chem. Chem. Phys. 2012, 14, 2056-2064
16. A. F. Thomas, Y. Bèssiere, Nat. Prod. Rep. 1989, 6, 291-309.
17. K. A. D. Swift, Top. Catal. 2004, 27, 143-155.
18. W. A. Duetz, H. Bouwmeester, J. B. van Beilen, B. Witholt, Appl. Microbiol.
Biotechnol. 2003, 61, 269-277.
19. F. Dumeignil, Concept of Biorefinery Comes into Operation: The EuroBioRef
Concept. EuroBioRef Summer School, Castro Marina, Italy, September 18-25, 2011.
20. M. A Teixeira, O. Rodriguez, P. Gomes, V. Mata, A. Rodrigues, Perfume
Engineering: Design, Performance & Classification, Chapter 2, pp. 15-58,
ButterworthHeinemann, Oxford: 2013.

21. Roger S. Nasci, Emily Zielinski-Gutierrez, Robert A. Wirtz, William G. Brogdon Protection
against Mosquitoes, Ticks, & Other Insects & Arthropods Chapter 2 - 2012 Yellow Book -
Travelers' Health – CDC.

22. Marta Ferreira Maia and Sarah J Moore Plant-based insect repellents: a review of their
efficacy, development and testing Malar J. 2011; 10(Suppl 1): S11. Published online 2011,

23. Oransky, S., et al. "Seizures Temporarily Associated with Use of DEET Insect Repellent-
New York and Connecticut." Morbidity and Mortality Weekly Report 1989

24. Lipscomb, J.W., et al. "Seizure following brief exposure to the insect repellent N, N-diethyl-
m-toluamide." Ann.Emerg.Med. 1992

25. Baser K H C and Buchbauer G 2010 Handbook of Essential Oils Science, Technology, and
Applications First Edition (London: Francis Group).

26. Patrascu M and M Radoiu 2016 Rose Essential Oil Extraction from Fresh Petals Using Synergetic
Microwave & Ultrasound Energy: Chemical Composition and Antioxidant Activity Assessment Journal
of Chemistry and Chemical Engineering

24
27. Njila M I N, E Mahdi, D M Lembe, Z Nde, and D Nyonseu 2017 Review on Extraction and Isolation
of Plant Secondary Metabolites 7th International Conference on Agricultural, Chemical, Biological and
Environmental Sciences, Kuala Lumpur (Malaysia) pp 67-72.

28. Karlberg AT, Lindell B. Limonene. In: Beije B,Lundberg P, eds. Criteria documents from the
Nordic Expert Group. Solna, National Institute of Occupational Health, Nordic Council of
Ministers, 1993, 207-247

29. Holck AR, Estep JE, Hemeyer RD. Teratogenicity of dlimonene to Xenopus embryos.
Journal of the American collège of toxicology. 1991; 10:624.

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