Guagua National Colleges, Inc.

Sta. Filomena, Guagua, Pampanga

Pre-formulation of Lime Candles as Mosquito Repellent

A Research Title Proposal

Presented to the Faculty of the

Science, Technology, Engineering, and Mathematics Track

Senior High School Department-Regular

Aimee M. Bondoc

Axl Rose S. Songco

Jorenz Mar S. Banal

Kent Janmel O. Panlaqui

Harold C. Castroverde

Alexandra Joy L. Alipio

Darylle Anne B. Cruz

Jeremie Shane M. Tungul

Jann Kyle S. Ronquillo

December 2022
 Table of Contents

Content

Introduction...........................…......................................……............……..............…1

Statement of the Problem…………………………..................…….......…….3

Scope of the Study………………..........................................…………....…. 4

Significance of the Study...........................................................………......….4

Conceptual Framework…….....................................................……….......… 5

Theoretical Framework…….......................................................………...…...6

Hypothesis…………….……............................................................…………7

Objectives of the Study……………...................................................…. ……7

Definition of Terms……….....................................................….......………. 8

Review of Relevant Literature.........................................................……… ………...9

Meta Analysis.........................................................………………………..… ……..14

Method.………………………………………………………………………………22

Participants’ Characteristics .....................................................…….. ………22

Sampling Procedures ......................................………………………………22

Measures and Instruments ...............................................……………………23

Research Design ................................................……………..…….. ………24

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Pre-formulation of Lime Candles as Mosquito Repellent

Introduction

Plants have been used as remedies to treat various diseases all throughout the

world for ages. So far, only a few number of plants have made a substantial impact in

the fight against mosquitoes and other arthropod pests. Instead of a lack of plant

activity, this may be caused by a lack of scientific data. Selection by plants to

produce repellents can be readily explained by their protective role against

phytophagus insects (Maia and Moore, 2012).

Plant essential oils such as citronella have become popular for use as safe

mosquito repellents. Besides citronella oil, numerous other plant extracts have been

available as insect repellents for protection against mosquitoes. Understanding how

alternative natural products function to prevent mosquito human contact is vital in

optimizing tools for personal protection (Tisgratog et al,. 2016)

Citrus Aurantifolia as mentioned in the study of Alessandrello et al,. (2021), is a

plant belonging to the family of Rutaceae and to the genus Citrus. It is a perennial

small evergreen tree which can grow to a height of 3–5 m, with dense ramifications

and whose surface is covered with rigid thorns. The twigs have a quadrangular shape,

and the leaves are elliptical or oval with serrated margins varying in color from

yellow-green to dark green. The flowers are yellowish-white, consisting of 4–5 petals.

The fruit, commonly called lime, has an ellipsoidal shape, with a large diameter

between 3 and 5 cm, initially green in color, but yellow when ripe. The pulp is

greenish yellow and produces an acidic but very perfumed juice. These fruits contain

few white seeds. Lime is a fruit whose use is increasingly widespread in the world,
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both as a fresh fruit for consumption and preparation of juices or drinks. It is also used

in the preparation for jams and candies. The essential oil obtained from the peel is

body lotions, but also in the preparation for detergents or to flavor foods or drinks.

The peel is also widely used, especially in cooking.

As determined by the study of Jain et al,. (2020), Citrus essential oil,

commonly, known as lime oil, has been widely reported in traditional system of

medicine. Industrially, oil is isolated by mainly by hydrodistillation from fruit and

peel of Citrus aurantifolia, family, Rutaceae. Cultivation practice of citrus plants dates

back for over 4000 years and are one of most valuable fruit crops in the world. The

citrus essential oil is globally used in food industry to impart citric flavour and odour

to cuisines.

Lime juice and oil is known to possess multiple biological properties including

anti-cancer, antimicrobial, antioxidant, antiulcer, anti-inflammatory, hypolipidemic,

antityphoid and hepatoprotective properties. Due to potent antibacterial and antifungal

properties, citrus oil is becoming important component of skin care products. The

medicinal importance of plant is due to presence of various secondary metabolites,

alkaloids, carotenoids, coumarins, essential oils, flavonoids, phenolic acids, and

triterpenoids. The citrus oil is rich in aromatic compounds namely, monoterpenes and

their derivatives, aldehydes, ketones, esters, alcohols such as limonene (58.4%), β-

pinene (15.4%), γ-terpinene (8.5%), citral (4.4%) and others. The bitter taste and

aroma of citrus fruit peels is attributed to limonoids. p-caryophyllene constitute 5.7%

of all the sesquiterpenes. On the basis of the available information, we conclude that

citrus oil possess huge potential to be developed into pharmaceutical products.

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According to Adhikari et al,. (2022) Mosquitoes have already developed

resistance toward most of the commercial synthetic repellents. Therefore, searching

for new potential alternatives is the need of the hour.Thus, According to Garg et al,.

(2019), The Oil extracts of traditional plants are safe and non-toxic alternative to

synthetic chemicals against mosquito-borne diseases .

Mosquitoes carry diseases that may be passed on to people or animals through

mosquito bites. Mosquito-borne diseases such as dengue, malaria, filariasis and

Japanese encephalitis are a major global public health issue. According to the World

Health Organization (WHO), more than half of the world’s population are at risk

from diseases spread by mosquitoes. Proper control of mosquitoes lies in personal

protection and public awareness as the most economical method in eradicating

breeding sites and controlling these through environment friendly larvicides. Aedes

albopictus which is an increasingly important disease vector including dengue fever,

has spread with the growth of towns and poor sanitation. Moreover, organic repellent

is one of the most effective ways of controlling outdoor biting of mosquitoes. To

provide an alternative solution for other mosquito repellents, researchers would like to

investigate about the pre-formulation of Lime Candles as Culicidae (Mosquito)

Repellent. Thus, a deeper comprehension and more detailed knowledge of mosquitoes

and the effectiveness of lime-scented candles as a mosquito repellent would be

obtained for this study.

According to the article of Azari et. al. (2019), Mosquitoes (Culicidae) are the

most significant arthropods of medical importance because of the burden of diseases,

such as malaria, encephalitis and filariasis, which are caused by pathogens and

parasites they transmit to humans. In 2007, the most recently published checklist of

Iranian mosquitoes included 64 species representing seven genera. Public databases

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were searched to the end of August 2018 for publications concerning the diseases in

Iran caused by mosquito-borne pathogens. Pertinent information was extracted and

analyzed, and the checklist of Iranian mosquitoes was updated. Six arboviral diseases,

two bacterial diseases, four helminthic diseases and two protozoal diseases occur in

Iran. The agents of these diseases are biologically or mechanically known or assumed

to be transmitted by mosquitoes. The updated checklist of Iranian mosquitoes includes

69 species representing seven or 11 genera depending on the generic classification of

aedines. There is no published information about the role of mosquitoes in the

transmission of the causal agents of avian malaria, avian pox, bovine ephemeral fever,

dengue fever, Rift Valley fever, Sindbis fever, Deraiophoronema evansi infection,

lymphatic filariasis, anthrax and tularemia in Iran. There is just one imported case of

lymphatic filariasis, which is not endemic in the country. It seems arthropods do not

play an important role in the epidemiology of anthrax and ixodid ticks are the main

vectors of the tularemia bacterium. In view of the recent finding of only a few adults

and larvae of Aedes albopictus in southeastern Iran and the absence of Ae. aegypti, it

is not possible to infer the indigenous transmission of the dengue fever virus in Iran.

Considering the importance of mosquito-borne diseases in the country, it is necessary

to improve vector and vector-borne disease surveillance in order to apply the best

integrated vector management interventions as a part of the One Health concept.

The researchers aim to determine the effectiveness of Lime extract against

Aedes Aegypti which can cause severe dengue virus (Rosanty et al,. 2019). If proven

effective, the substance will be used to create a repellant in advance. For the purpose

of supporting the study, this research will provide several questions to specialists and

professionals, particularly biology teachers, nurses, and pharmacists. This study will

be conducted in Guagua National Colleges for the academic year 2022-2023.

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 PLEASE HANAP NG IBANG TRANSITIONAL DEVICE ASIDE

SA ACCORDING TO.

Statement of the Problem

The main objective of this study is to pre-formulate lime candles as mosquito

repellents.

Specifically, the study aims to answer the following specific questions.

1. What is the expert’s opinion on the pre-formulation of lime candles as

mosquito repellent?

2. What are the components of lime that can repel mosquitoes?

3. Is there a significant difference between the use of lime oil extract as mosquito

repellent and other mosquito repellent?

4. What percentages and measures are needed to pre-formulate the essential oil that

was naturally extracted?

5. What is the percentage of repellant protection used with the basic component of

lime extract?

\Scope of the Study

The study focuses on the pre-formulation of lime candles as mosquito

repellents. This study has information on how lime scented candles can be effective in

repelling mosquitoes in the affected area. This study has the knowledge and

information on the pre-formulation of lime candles as mosquito repellent.

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This study focused on the pre-formulation of lime candles as mosquito

repellent. This study has information on how lime candles can be effective in killing

mosquitoes in the affected area.

This study aims to produce a mosquito repellent candle from ethanolic extract

of Citrus aurantiifolia (Lime) against Culicidae (Mosquitoes). The lime essential oil

will be only obtain by the researchers from the fruit itself.

This study requires a medical experts’ insight and opinion on the use

of Lime Candles as Mosquito Repellent in aiming to ensure the safety and developing

of producing the product. Participants in this study includes medical professionals

with specific expertise in biology, such as nurses and pharmacist. The researchers

limited the study only for licensed medical professionals in the school year 2022-2023

of Guagua National Colleges. Each of the respondents were interviewed through face

to face interviews for the researchers to get opinions and recommendations for the

product to be produced perfectly.

Significance of the study

The findings of this study are deemed significant to the following entities.

To the community

This study will be helpful to the residents of the community because lime

citrus candles will be used as mosquito repellent.

To the Economy

This study will be advantageous to the economy because it will offer a more

sensible and affordable alternative. As a mosquito deterrent, homemade organic

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candles are more effective and cost-effective than mosquito coils, as all the

ingredients are easily accessible in nature.

To the Future Researchers

This research study will significantly advance the field. The inputs obtained

from this study can be used as the starting point for future research projects. Future

researchers will have an additional knowledge from the result of this work. This could

lay a solid groundwork for their future research. Lastly, this research can help them

understand how mosquitoes can be repelled by utilizing lime citrus candles.

Conceptual Framework

This study applied the independent variable-dependent variable model to show

the cause and effect relationship between the independent variable and dependent

variable.

Figure 1.

Independent Variable - dependent Variable (IV-DV) Model

INDEPENDENT VARIABLE DEPENDENT VARIABLE

Lime (Citrus aurantiifolia)

Mosquito (Culicidae)

Mosquito Repelent Candle

The independent variable in the model which are Lime (Citrus aurantiifolia)
and Mosquito Repelent Candle will be used against the insect, Culicidae, which is the
 8

dependent variable in this study. This way, the cause and effect relationship between the
variables is seen.

Theoretical Framework

According to the study of Mursiti et. al. (2019), The d-Limonene, an active

ingredients present in the essential oil destroy the wax layer of the insect respiratory

system so that once applied directly, the insects will suffocate. d-Limonene bioassay

results of tests undertaken after treatment of the fabrics, Bioassay results show the

toxic activity of MCT-b-CD finished cotton fabrics treated with different doses of

limonene, and show the effect of exposure time against mosquitoes. The results show

that the repellent, knockdown and killing action against mosquitoes increased with

increasing concentration of limonene in MCT-b-CD finished cotton fabrics within the

range studied (250–1500 mg/m2). Based on the conclusion indicated on their study,

Isolation of 2.6 kg of sweet orange peel produces 24 ml of essential oil.

Characterization of GC-MS essential oils of sweet orange peel containing the main

component compounds were d-Limonene (90.018%), 1,6-Octadien-3-ol, 3,7-

dimethyl- (3,017%), β-Myrcene (2.071%) , Octanal (0.349%), 1-Octanol (0.589%),

1R-α-Pinene (0.688%), 6-Octen-1-ol, 3,7-dimethyl- (0.667%), 3-Cyclohexene-1-

methanol, α, α, 4-trimethyl-, (S) – (1.453%), and Bicyclo [3.1.1] heptane, 6.6-

dimethyl-2-methylene-, (1S) – (1.147%). Bioassay Y test between d-Limonene and

N-hexane compounds showed that d-Limonene is resistant (repellent) and fumigant

(fumigant). Characterization of d-Limonene nanoparticles had an uneven and irregular

surface morphology. Anti-flea activity test by using force-feed method indicates more

than 50% dead blight and showed that orange peel extract (Citrus Sinensis L.) can be

used as a natural insecticide for bed bug controller (Cimex Cimicidae).

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Hypothesis

Null Hypothesis

The oil extract from the Citrus Aurantifolia (Lime), the main

ingredients for the Pre-formulation of Lime Candles, will not be able to

repel mosquitoes.

THERE IS NO SIGNIFICANT DIFFERENCE

Alternative Hypothesis

The oil extract from the Citrus Aurantifolia (Lime), the main

ingredients for the Pre-formulation of Lime Candles, will be able to

repel mosquitoes.

THERE IS A SIGNIFICANT DIFFERENCE

Objective of the Study

The main objective of this study is to pre-formulate lime candles that will be

used to get rid of insects and mosquitoes.

To accomplish the overall objective of the study, the researchers focused on

the following specific objectives:

1. To gather the expert’s opinion on the pre-formulation of lime candles as

mosquito repellent.
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2. To discover the component of lime that will repel mosquitoes.

3. To discover the significant difference between the use of lime oil extract as

mosquito repellent and other mosquito repellent.

4. To determine the percentages and measures needed to pre-formulate the essential

oil that was naturally extracted.

5. To determine the percentage of repellant protection used with the basic component

of lime extract.

Definition of Terms

ARRANGE IT ALPHABETICALLY.

1. Culicidae - Based on the Merriam-Webster dictionary, it is a family of slender

long-legged dipteran flies having the body and appendages partly covered with hairs

or scales and the mouthparts adapted for piercing and sucking, comprising the

mosquitoes, and having active aquatic larvae known as wrigglers. In this paper,

Culicidae are the most significant arthropods of medical importance because of the

burden of diseases, such as malaria, encephalitis and filariasis, which are caused by

pathogens and parasites they transmit to humans as per stated in the article of Azari et.

al. (2019).

2. Dengue - Merriam-Webster dictionary stated that dengue is an acute infectious

disease caused by a flavivirus (species Dengue virus of the genus Flavivirus),

transmitted by aedes mosquitoes Operationally, According to the study of Foster and

Walker (2019), it is the most common well-known human disease caused by

mosquitoes.
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3. Lime -   as per stated in the Merriam-Webster Dictionary, lime is a small globose

yellowish green fruit of a widely cultivated spiny tropical Asian citrus tree (Citrus

aurantifolia) with a usually acid juicy pulp used as a flavoring agent and as a source of

vitamin C. It is operationally defined as a plant belonging to the family of Rutaceae

and to the genus Citrus. It is a perennial small evergreen tree which can grow to a

height of 3–5 m, with dense ramifications and whose surface is covered with rigid

thorns as per stated by Alessandrello et al,. (2021).

4. Repellent - Based on the Merriam-Webster dictionary, repellent means serving or

tending to drive away or ward off, often used in combination, a mosquito-repellent

spray. It is operationally defined as one of the best strategies to avoid mosquito bites,

as per stated in the study of Shooshtari et al,. (2012).

5. Aedes Aegypti- It is the first transmitter of the four viruses that have the most

effect on human health, the viruses causing yellow fever, dengue fever, chikungunya,

and Zika fever. (Souza-Neto, 2019). It is operationally defined as the main carrier of

dengue fever.

6. Hemorrhagic Fever- IAccording in the dictionary ‘Merriam-Webster’,

Hemorrhagic Feverv is any of a diverse group of virus diseases (such as Lassa fever

and Ebola) that are usually transmitted by arthropods or rodents and are characterized

by a sudden onset, fever, aching, bleeding in the internal organs, petechiae, and shock.

It is operationally defined as a virus that is transmitted through the bite of female

Aedes mosquitoes infected with the Dengue virus.

7. Limonene - a widely distributed terpene hydrocarbon C10H16 that occurs in

essential oils (as of oranges or lemons) and has a lemon odor (Merriam-Webster

Dictionary). Operationally, it is defined as an active ingredients present in the

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essential oil of lime that destroy the wax layer of the insect respiratory system so that

once applied directly, the insects will suffocate (Mursiti et. al., 2019)

8. Citrus- in accordance of the dictionary, Merriam-Webster, Citrus are any of a

group of often thorny trees and shrubs (Citrus and related genera) of the rue family

grown in warm regions for their edible fruit (such as the orange or lemon) with firm

usually thick rind and pulpy flesh.

Review of Related Literature

Citrus plants such as lemons, mandarins, oranges, tangerines, grapefruits,

and limes are commonly grown fruits all over the world (Iqbal et, al. 2018).

According to Nagdeve (2021), lime (Citrus aurantifolia) is a small, round, green-color

hybrid citrus fruit. Due to its acidic sour juice and unique aroma, the lime fruit is

widely used in various cuisines to accent flavor. There are several species of citrus

fruits referred to as limes that include key lime, kaffir lime, wild lime, desert lime,

and Persian lime. This zesty, tropical fruit is well-known for its medicinal properties

for ages. Moreover, limes are known for its antioxidant, anti-inflammatory and

antibacterial action, as well as immune-boosting and anticancer properties. And unless

you have gastrointestinal problems such as acid reflux, heartburn, gastritis, or ulcers,

they are quite wonderful options.

As stated in the study of Mangampa et al,. (2017) Lime leaf extract contains

limonoids compounds that serve as larvacide. Tujuan of this research is to make

granule extract lime leaf (Citrus aurantifolia) which has the ability as a mosquito

killer biolarvacide. Their type of research is a laboratory experiment. Their population

in this study were all three instars of Aedes aegypti larvae aged 3-4 days. Their results

showed that there was granule effect of lime leaf extract (Citrus aurantifolia) to the
 13

death of Aedes aegypti mosquito larvae. The granule concentration of lime leaf

extract (Citrus aurantifolia) which is effective in killing Aedes aegypti mosquito

larvae is 15% extract concentration formulated in granule form with percent mortality

of 76.7% larvae.

According to the Journal of medical entomology (2017) The essential oils of

kaffir lime (Citrus hystrix DC.) at four different concentrations (0.5, 1.0, 2.5, and

5.0% v/v) they studied for their repellency, excitation, and knockdown properties

against laboratory strains of Aedes aegypti (L.) and Anopheles minimus Theobald

using an excito-repellency test system. Both contact and noncontact escape responses

to leaf- and peel-derived kaffir lime oils they observed. Comparing unadjusted escape

responses for An. minimus, leaf oil had strong combined irritant and repellent activity

responses at 1–5% concentrations (90.0–96.4% escape) and the strongest spatial

repellent activity at 1% and 2% (85.9% and 87.2% escape, respectively). The peel oil

exhibited good excitation with repellency at concentrations of 2.5% (89.8% escape)

and 5% (96.28% escape), while concentrations 1–5% showed more moderate

repellent activity against An. minimus. For Ae. aegypti, 2.5% leaf oil produced the

greatest response for both contact (56.1% escape) and noncontact (63.3% escape)

trials, while 2.5% produced the strongest response among all concentrations of peel

oil, with 46.5% escape. However, after adjusting the contact trial escape (a measure of

combined excitation and repellency), the estimated escape due to contact alone was a

much weaker response than spatial repellency for both species. Knockdown responses

above 50% they only observed in Ae. aegypti exposed to 5% leaf oil. Kaffir lime oils

were more active against An. minimus than Ae. aegypti mosquitoes. There they

statistically significant differences between leaf (more active) and peel oils at each
 14

concentration against An. minimus in contact and noncontact trials, except at the

highest (5%) concentration.

In accordance with the study of Mursiti et, al. (2019). d-Limonene is used as

insecticide to control ectoparasites of pet animals, but it has activity against many

insects, mites, and microorganisms. Possible attractive effects of limonene to natural

enemies of pests may offer novel applications to use natural compounds for

manipulation of beneficial animals in organic agriculture. The potential of volatile oil

against insects was resistant (repellent), fumigant (fumigant), reduce appetite

(antifeedant), and attract (attractant). The advantage of d-Limonene over synthetic

pesticides in this regard is its biodegradability, which makes it more environmentally

friendly for both fumigation and contact applications. The broad range of

pharmacological properties of d-limonene coupled with low toxicity offers the

possibility of incorporating this compound into various medical and cosmetic

formulations.10 Natural plant products and materials have been used by local people

as insecticides to control insects such as mosquito. Among the most notable plants

having such insecticidal properties are Azadirachta indica (neem) and Citrus sinensis

(sweet orange).

As stated in the study of Shooshtari et al. (2012), Mosquitoes control and

personal protection from mosquito bites is one of the serious ways for preventing of

contagious diseases distribution. In recent years, interest in plant-based products has

been revived because of the development of resistance, cross-resistance and possible

toxicity hazards associated with synthetic insecticides and their rising cost. Various

plant-based products as herbal repellents are safe and biodegradable alternatives to

synthetic chemicals for use against mosquitoes. In the present study, essential oils,

and extracts of six plants "Melissa officinalis, Rosmarinus officinalis, Lavandula

 15

officinalis, Citrus limonum, Eucalyptus globulus and Ocimum basilicum" were

evaluated compared against mosquitoes of Anopheles Stephensi. Finally, repellant

properties of essential oils and extracts as experimental groups and of N, N-diethyl 3 -

methylbenzamide (DEET) as a positive control group were compared. Therefore, to

avoid mosquito bites, one of the best strategies is the use of repellents (Silva and

Ricci, 2019). According to the American Mosquito Control Association (2017),

repellents are substances that help people avoid mosquito bites. They are sold as

aerosols, creams, solids (sticks), pump sprays and liquids. However, repellents do not

kill mosquitoes and other insects, but it will help deter them from biting people.

As stated in the study of Foster and Walker (2019) Culicidae, the mosquito or

insect family, is stated to be comprised of 41 recognized genera incorporating about

approximately 3,500 species. Many of these are vectors of disease pathogens that

have afflicted us humans and domestic animals from the past centuries, with

devastating consequences for tens of the human population. Including here are the

well-known human diseases malaria, filariasis, encephalitis, yellow fever and the

most common which is dengue, but there are many others of medical or veterinary

importance. Complimating matters, many of the human pathogens are shared with

wild animal reservoirs.

According to Wilkerson, et al. (2021) Mosquitoes bite on two, three, or more

different hosts. Thus, some species have co-evolved with pathogens. The effect of

their bites have been one of the most challenging diseases known for both humans and

animals. The Anopheles gambiae are the primary transmitter of malaria parasites to

humans and known as the most dangerous animal in the world. Technically, malaria

has killed many people. Up until now, despite the drugs and mosquito controls,

malaria took the lives of 405,000 every year. There are extensive majority of
 16

mosquitoes that are not involved in pathogen transmission to humans which make a

huge impact on global health.

Based on the study of Takken and Verhulst (2013) Plant sugar and vertebrate

blood are what mosquitoes source o nutrition. When mosquitoes are searching or

looking for blood hosts, some express preferential behavior for some selected species.

In their study, they reviewed the available knowledge on host preference, as this is

expected to affect the life history and transmission of infectious pathogens, host

preference is affected by myriad extrinsic and intrinsic factors. By genetic selection,

inherent factors are determined, that appears to be controlled by adaptive advantages

that result from feeding on certain host species. Although having a genetic basis, host

preference of mosquitoes is characterized by high plasticity mediated by the density

of host species, which by their abundance form a readily accessible source of blood.

Host-selection behavior in mosquitoes is an exception rather than the rule. Those

species that express strong and inherent host-selection behavior belong to the most

important vectors of infectious diseases, which suggests that this behavioral trait may

have evolved in parallel with parasite-host evolution.

Based on the study conducted by Lo et al., (2018) Mosquito-borne diseases are

global problems. The use of topical insect repellents is a key measure recommended

by health authorities to prevent mosquito-borne diseases. Despite more than a hundred

commercial products available in the market, there are relatively few active

ingredients used across these formulations. The most common active components are

diethyltoluamide, picaridin, p-menthane-3,8-diol, ethyl butylacetylaminopropionate

(IR3535) and a range of plant-derived products. Research has shown that each of

these active ingredients vary in the duration of protection provided against biting

mosquitoes. However, the wide spectrum of commercial preparations and various

 17

recommendations from different health authorities make it a challenge to pick an

effective product for end users as well as healthcare personnel. A local market survey

was conducted to assess the mosquito repellent formulae in available products in

Hong Kong, and a review of the current recommendations issued by local and

overseas authorities on choosing the suitable insect repellents is performed with

consideration of recent research and currently available topical repellents in the

market.

While, as reported by Rosanty et al,. (2019) The WHO estimates a 50–100

million dengue infections worldwide every year. Dengue Hemorrhagic Fever (DHF)

is transmitted through the bite of female Aedes mosquitoes infected with the Dengue

virus. Mosquitoes Aedes aegypti is the main vector of the dengue virus. At the time,

there is no cure for dengue so that the control efforts are focused on breaking the

chain of life cycles. One effort to prevent the transmission of the dengue virus is to

avoid vector mosquito bites. Repellent can reduce exposure to the bite of mosquitoes

infected with the dengue virus. The aim of their study was to determine the

effectiveness of the extract of citrus hystrix leaf as a repellent against the Aedes

aegypti mosquitoes. Their research is a laboratory experimental study with a one-shot

case study design. In their study, the extract of citrus hystrix solution was made at a

concentration of 10%, 20%, and 30%, and then the extract solution was used as a

stock to make a 100 gram base lotion that would be used as a repellent. Repellent

effectiveness is seen from the percentage of repellent protection power. The ir

percentage of repellant protection used with the basic ingredients of Citrus hystrix leaf

extract at concentrations of 10%, 20%, and 30%, respectively, at 93.33% 94.67%, and

97.33%. Their extract of citrus hystrix leaf was found to be effective as a repellent

against Aedes aegypti mosquitoes.

 18

As stated in the article of Inkum et al. (2014), Volatile oils from the peels of

sweet orange (Citrus sinensis), lime (Citrus aurantifolia) and lemon (Citrus Limon)

were comparatively evaluated on carpenter ants (Camponotus nearcticus) for repellent

and lethal activity. The volatile oils were extracted by means of steam distillation. The

repellent activity was tested at 2%w∕v concentration with a modified version of World

Health Organization’s (WHO’s) method for mosquito repellent testing. The extracts

had strong repellent activity on the tested ants, C. sinensis recorded the lowest activity

of 82.5% and C. Limon recorded the highest activity of 95.0%.

Based on the study of Kokolakis and Golfinopoulos, (2013). Essential oils (EO)

are key raw materials in the cosmetic, fragrance and food industries. Production of

EO for industry comprises two basic steps: extraction (for example, steam distillation,

hydrodistillation, simultaneous distillation–extraction) and instrumental analysis {for

example, gas chromatography (GC), gas chromatography coupled to mass

spectrometry (GC–MS)} for productivity and quality reasons, respectively. While the

instrumental analysis takes only 15–30 min, extraction lasts at least for several hours.

Extraction is frequently achieved by a prolonged heating with stirring in boiling

water.

According to Adhikari et al,. (2022) Mosquitoes have already developed

resistance toward most of the commercial synthetic repellents. Therefore, searching

for new potential alternatives is the need of the hour. In their study, they evaluated the

repellency of six citrus derived essential oils against Aedes aegypti using the arm in

cage method. The results of their study showed more than 50 percent repellency up to

4 h exposure time at 1 mg/cm2 area for three Citrus with maximum repellency of 81

percent for Citrus aurantifolia. Therefore, two of the major constituents of Citrus

aurantifolia namely citral and limonene, were chosen further for the repellency test.
 19

The results showed higher efficacy of compounds than crude oil. EC50 for

commercial standard repellent DEET was determined for comparison under the same

experimental conditions. To understand the possible mode of action of citral,

limonene and DEET, in silico interactions of these compounds with odorant binding

proteins OBP1 and OBP22 and acetylcholinesterase AChE enzymes were studied.

Their findings revealed positive docking of all of these compounds having affinity

values in the range of − 6.0 to − 6.9 kcal/mol. Overall, their study demonstrated that

limonene and essential oils of Citrus aurantifolia could be the best potential

alternative for synthetic repellents.

As reported by the International Journal of Home Science (2017). In their

study an attempt has been made to approach of developing Mosquito repellency

finishes using natural herbs. The herbal extracts of sweet lime peel was applied on

cotton fabric directly by using pad dry cure method. The Mosquito repellency finishes

of the finished fabric was assessed against mosquito. To enhance the durability of the

finished fabric, wash durability test has been carried out and the results showed good

efficiency of finishes even after 6 domestic washing cotton fabric. This paper

described the textile materials selection, methods of imparting the repellent into the

fabrics, repellency test of treated fabrics. The assessments used in the treated textile

are summarized and conditions of the assessment of repellency relative to this

discussion are presented.

In accordance to Agrawal et al,. (2018) Improving the life and treatment of

diseases of the medicinal plants are helpful, and these are the primary source of the

synthetic and a traditional herbal medicine. The scientists investigates the plants for

their own sake, because of the presence of various life sustaining constituents in

plants. Formulating a mosquito repellent product containing curcumin and embelin

 20

obtained from the curcuma longa and embelia ribes is their objective in their work

The objective of their work was to formulate a mosquito repellent product containing

Curcumin and Embelin obtained from Curcuma longa and Embelia ribes properly.

TLC Technique is the one who confirmed the Isolated Curcumin and Embelin. The

formula used for the cream was checked for distant parameters such as smooth texture

and compatibility with the skin to achieve a pH of 7 that is mild and suitable for the

skin. During that time, phase separation does not occur thermal stability. From their

current study, they concluded that creams containing 1% curcumin and 1.5% embelin

are safe. Effective, gentle on the skin and stable.

As stated in the study of Garg et al,. (2019) The Oil extracts of traditional

plants are safe and non-toxic alternative to synthetic chemicals against mosquito-

borne diseases. Their present investigation aimed to evaluate the mosquito repellent

activity of Eucalyptus oil leaves and Ocimum tenuiflorum oil leaves in plant natural

form of herbal formulation. The plant materials they use was the Eucalyptus oil

leaves, tenuiflorum leaves were collected and subjected to cold maceration for 7 days

using castor oil as solvent. They obtained extracts and formulated into repellent

candle. Their formulation was evaluated for repellent activity and they used standard

method under proper laboratory conditions. The result they got was the candle

formulation gave protection against mosquito bites without any allergic reaction to the

test person. The use of unique formulation methods and natural products, with

mosquito repellent activity may help in reducing the harmful effects of synthetic

mosquito repellents on human health.

In addition, According to Garg, et. al (2019) Using oil extracts of traditional

plants are safe and very non-toxic alternative to synthetic chemicals against different

mosquito-borne diseases. This investigation is aimed to evaluate the mosquito

 21

repellent activity of Ocimum tenuiflorum oil (leaves) and Eucalyptus oil (leaves) in

plant natural form of herbal formulation. The plant materials such as mentioned were

collected and subjected to cold maceration for a week or seven days with the use of

castor oil as the solvent. The obtained extracts were formulated into repellent candles.

The candle formulation gave the protection against mosquito bites without causing

any violent or allergic reactions. The use of unique formulation methods and natural

products, with mosquito repellent activity may help in reducing the harmful effects of

synthetic or chemicalized mosquito repellents.

As explained in the study of Prabhudatta et al,. (2018) The aids prepared from

plant extract are best option for chemically synthesized mosquito repellent. The

ingredients of cow urine and phytochemical compounds of plant extract are

responsible for mosquito repellence. The mosquito coils available in market creates

heavy smoke that can generate respiratory problems especially for patients of Asthma.

Though the ‘Fast card mosquito papers’ are available in market, they contain of TFT

molecules which has adverse effect on respiratory system. By repeated use of

mosquito coils and fast card paper, mosquitoes may acquire resistance against smoke

and TFT molecules. The problems can be reduced by using plant extract paper with

more efficiency for longer time. Along with this, dhoop stick and candles may also be

used. Production of their natural repellents may help common man in earning more

money as it very cheap and eco-friendly.

According to Munawaroh & Astuti, 2010. Soxhlet extraction is a very common

technique for obtaining volatile components from raw materials. It is done by heating

up a solution with a solute of limited solubility in a percolator until it boils, then

condensing and collecting the condensate from a reservoir in which the concentrated

solute can be obtained. According to the research of Gahlot et al. (2018), they
 22

extracted lychee leaves, curry leaves, jamun leaves, and catechu (a by-product of

acacia trees), using maceration and Soxhlet methods, obtained the best yield of oil

using the Soxhlet method. The Soxhlet extraction method is an extraction method that

runs continuously with the aid of heating in which the lime peel sample is in direct

contact with the solvent at a predetermined extraction time and undergoes circulation.

Compared to the maceration method, Soxhlet extraction gives a higher yield.

The use of organic repellents as mosquito repellents are widely use now.

Though there are some existing studies about the use of lime oil extract as a repellent

to mosquitoes, still research about the use of the said extract the given variable are

only few especially, locally. Therefore, the researchers found the need to pre-

formulate lime candles as mosquito repellents.

WHERE IS THE BOTANICAL DESCRIPTION AND TAXONOMY OF

YOUR PLANT?

Meta Analysis

The purpose of this research is to produce essential oil from lime peel so it can be

used as a substitute for synthetic fragrance in soap and to find the best method, type of

solvent, and extraction time in producing it.

Research description

The research begins with the preparation of raw materials. Lime peels were cut into

small pieces, then cleaned to remove dirt and microbes. After that, lime peels were
 23

dried in the oven until the water content of the raw material is 10%. The dry lime peel

was blended and then sieved with a 50 mesh sieve.

For Soxhlet extraction, lime peel powder was weighed as much as 20 grams and

wrapped in filter paper, and put into a thimble. The solvent was put into a 200 ml

flask, then it was heated according to the boiling point of the solvent. The extraction

was carried out for 6, 9, and 12 hours. After the extraction is complete, the extract

obtained is separated between lime peel oil and solvent using a vacuum rotary

evaporator. In this study, several standard analyzes were carried out such as the yield

of oil obtained, the color of oil, oil density using pycnometer, and oil refractive index

with a refractometer based on ISO 3519: 2005 (E). The samples used were analyzed

to determine the presence of limonene in lime peel powder using FTIR (Fourier

Transform Infrared Spectroscopy).

Results and Discussions

Identification of Limonene in Lime Peel Using FT-IR

To prove that the raw material used in this research contains limonene, which is a

compound that affects the distinctive aroma of lime peel, a functional group analysis

of lime peel was carried out using FTIR (Fourier Transform Infrared Spectroscopy).

According to Auta et al. (2018) and Boughendjioua and Samah (2017), the functional

groups that indicate the presence of limonene are C-H (alkane), C-H (aromatic), C-N

(aromatic), C-N (aliphatic), and C = C. The functional groups have different

wavelengths.
 24

From the FTIR spectrum above, there is an absorption band that shows the groups

present in lime peel. The following table shows the functional group data from

limonene, the wave number from the functional group as a result of the analysis, and

the wave range refers to Pavia et al. (2009).

According to the wave numbers obtained from the analysis, it can be concluded that

the sample, namely lime peel, contains limonene. The results of this functional group

test have the same results as the previous studies conducted by Auta et al. (2018) and

Boughendjioua and Samah (2017).

The Effect of Extraction Time on % Yield of Essential Oil From Lime Peel

In this research, the essential oil extraction from lime peel was carried out using two

methods, which was maceration and Soxhlet, using three different solvents, namely

hexane, ethanol, and water. The relation that shows the effect of time on yield with

the maceration method can be seen in Figure 2. (a), and the Soxhlet method can be

seen in Figure 2.(b).

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In the extraction using ethanol as a solvent, there was a decrease in oil yield at 12

hours of extraction time, where the results were not the same as the yields of the other

two solvents, which should have been the highest oil yield at 12 hours of extraction

time. It can occur because the extraction has reached the optimum point at 9 hours so

that the oil yield will decrease. The result of this study is similar to the research

conducted by Amelinda et al. (2018).

Figure 3. (b) shows that in the extraction using the Soxhlet method, the oil yield

increased along with the increase in extraction time. At 6, 9, and 12 hours extraction

times for hexane, the oil yield increased by 5.79%, 5.97%, and 6.15%. While the

extraction using ethanol solvent with extraction times of 6, 9, and 12 hours, the

resulting oil yield was 4.41%, 4.64%, and 4.89%. Extraction, using water as solvent,

cannot be carried out because there is no circulation in the chiffon section of the

Soxhlet tool, causing the thimble to be filled with water so that the water comes out

from the F pipe section, which should function as a steam route in the extraction

process in Soxhlet extraction.

Extraction time is a factor that influences the extraction process. A longer extraction

time generally results in a higher yield. The increase in time will cause an increase in

the contact time between the solvent and the lime peel powder solids, thereby
 26

facilitating the transfer of the mass of essential oils contained in lime peels to the

solvent used.

The results obtained by maceration and Soxhlet methods in this study have the same

results as previous research by Chairunnisa et al. (2019) on the extraction of essential

oils from jujube leaves and research by Giwa et al. (2018) on the extraction of

essential oils from the orange peel, which shows that the longer the extraction time,

the greater the yield obtained.

The Effect of Extraction Method on % Yield of Essential Oil From Lime Peel

One of the main factors determining oil quality in essential oil extraction is the

extraction method. Improper procedures can cause damage to the oil so that natural

and bioactive compounds will be lost. The examples of oil that has been damage can

be seen from discoloration of oil, the odor of oil, and other characteristic, so it should

be avoided. In this study, the essential oil of lime peel was obtained by two methods.

The first method is maceration, using stirring at 150 rpm. The maceration process

with stirring can shorten the extraction time to 6 to 24 hours. The second method was

Soxhlet, this process requires a heating process with temperature that equal to the

boiling point of the type of solvent used. The best lime peel oil yield for each method

can be seen in Figure 4.

 27

Figure 4 shows the highest oil yield using the maceration method and the Soxhlet

method were 5.24% and 6.15%, respectively. The highest yield in maceration and

Soxhlet methods was obtained with an extraction time of 12 hours using hexane as

solvent. From the results obtained, it can be concluded that the Soxhlet method is

more effective than the maceration method. This is similar to the research of Lee et al.

(2017) who extracted essential oils from agarwood leaves using two methods, which

was maceration and Soxhlet. The highest oil yield obtained using the Soxhlet method.

Research from Gahlot et al. (2018) that extracted lychee leaves, curry leaves, jamun

leaves, and catechu (a by-product of acacia trees), using maceration and Soxhlet

methods, obtained the best yield of oil using the Soxhlet method.

The Soxhlet extraction method is an extraction method that runs continuously with the

aid of heating in which the lime peel sample is in direct contact with the solvent at a

predetermined extraction time and undergoes circulation. Compared to the maceration

method, Soxhlet extraction gives a higher yield.

The Effect of Type of Solvent on % Yield of Essential Oil from Lime Peel
 28

Selection of the right solvent is one of the essential factors in extraction. Selectivity,

solubility, cost, and safety must be considered. Solvents with a polarity value close to

the polarity of the solute tend to have better performance and vice versa. In this study,

essential oil from lime peel was obtained using three solvents, namely hexane,

ethanol, and water.

The purpose of using three different types of solvents is to compare the yield of the

extracted oil, so that a more effective solvent can be found. Data of the best lime peel

oil yield obtained for each type of solvent can be seen in Figure 5.

Figure 5 shows that the highest oil yield was obtained using the Soxhlet method for

12 hours using hexane solvent at 6.15%, using ethanol at 4.89%, and using water

solvent at 1.67%. When compared to these three solvents, the hexane solvent is more

effective than ethanol and water.

The results of this study are similar to research by Lee et al. (2017), who extracted

essential oils from agarwood leaves using four different types of solvents, namely

water, ethanol, isopropanol, and hexane, where extraction using hexane as solvents

had the highest yield. The solute will dissolve more easily in the solvent if they have a

polarity that is close enough.

 29

Lime peel oil is a non-polar compound that tends to dissolve in non-polar solvents as

well, where hexane, ethanol, and water have a polarity index of 0.1; 3,9; and 10.2.

The greater the polarity index, the more polar the compound. Therefore, hexane will

more easily dissolve lime peel oil because it is nonpolar so that the yield obtained will

be greater than ethanol and water.

However, the extraction using water with Soxhlet method can’t be done. In Soxhlet

extraction, there are several requirements of the solvent, namely the solvent is more

volatile, the solvent's boiling point is low, and the properties of the compound to be

extracted are similar with the solvent. Between hexane, ethanol, and water, water is a

solvent that has a higher boiling point and is more difficult to evaporate than the other

two solvents. The properties of lime peel oil and water are also different, where water

is a polar compound and hexane is a non-polar compound. So that the extraction

process with Soxhlet method using water as the solvents cannot be done. Water can

be used as a solvent if it is mixed with other nonpolar solvents with a certain ratio, as

was done by Febryanto (2017).

Analysis of the Quality of Essential Oil of Lime Peel

According ISO 3519:2005 (E) The characteristics of the extraction of essential oil

from lime peel were analyzed and compared with the quality standard of lime peel oil

based on ISO 3519: 2005 (E), as shown in Table 2.

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Methods

GENERAL METHODOLOGY

A. Extraction Procedure

FRUIT COLLECTION

SAMPLE AUTHENTICATION

ETHANOLIC EXTRACTION

SOAKING

FILTRATION

FORMULATION

Figure 1: Extraction Procedure

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B. Candle Making Procedure

PREPARATION OF MATERIALS

FILLING (ADDING WAX INTO HEATING TANK)

HEATING (71°C FOR 30 MINS)

COOLING (40°C)

FILLING AND MIXING

CASTING

Figure 2: Candle Making Procedure

C. Quality Testing

MATERIALS TESTS

TOXICOLOGY ASSESSMENT

REPELENCY TESTS

Figure 3: Quality Testing

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Participants’ Characteristics

The participants were selected purposively, since they are the one that suits to

the goal of the study. Experts in medicine are trained when it comes to observing,

experimenting, educating, and ensuring medicines. The researchers have decided to

conduct face to face interviews with experts and professionals, specifically a biology

teacher, nurse, and a pharmacist.

According to Creswell (2013), qualitative interview participants may

use minimum of 3 to maximum of 15. Therefore, the researchers came up with the

decision to have five participants to answer the interview questions.

Sampling Procedures

In conducting the survey, the researchers used purposive sampling. In

purposive sampling, you set out to identify members of the population who are likely

to possess certain characteristics or experiences and to be willing to share them with

you. In this way, you can select the individuals or cases that fit your study, focusing

on a relatively small sample. The review intends to analyze the distinctive citrus lime

candle utilized for mosquito repellents. The researchers then select participants with

good background knowledge. Purposive sampling is a non-probability sampling

technique where participants are chosen, and researchers utilize their knowledge to

pick participants who will aid the study in achieving its objectives. Therefore, the

researchers must consider these unique qualities to assess their study question.

Purposive sampling has been used to select on which participants were to include in

the study because it has allowed the researchers to include participants who were

suitable for the study being performed. It is a non-probability sampling technique

 33

wherein the researchers depend on their own opinion as well as the insight of others

who have prior knowledge of the research.

Measurements and Instruments

Our study utilized the methods of quantitative research. Face to face semi

structured interviews were used for gathering information, insights, and experiences.

A research interview is a beneficial tool that can help researchers accumulate the

information they need wherein they will be able to know the participant’s views,

opinions, insights, and beliefs in a specific manner. The researchers used online and

face-to-face interviews to gain more helpful information that can help make the study

more understandable. The interview is in a form of a semi structured type, which

means the interview questions are open-ended and do allow ideas that are not part of

the questionnaires to be brought up in the interviewee’s answers.

The following are our interview questions which were going to ask our respondents:

1. In what most effective way can you repel mosquitoes?

2. What are the things to consider when pre-formulating mosquito repellents?

3. Do you have any suggestions or additional recommendation regarding the pre-

formulating of lime candles as mosquito repellents?

Research Design

In this study, us researchers have utilized mixed method, mixed method is a

mixture of qualitative and quantitative research design. Qualitative research design is

aimed at discovering how many people think, act or feel in a specific way. In addition,
 34

Quantitative projects or research involve large sample sizes, concentrating on the

quantity of responses, as opposed to gaining the more focused or emotional insight

that is the aim of qualitative research. The standard format in quantitative research

design is for each respondent to be asked the same questions, which ensures that the

entire data sample can be analyzed fairly. The researchers have integrated qualitative

and quantitative research aspects. They used this strategy to provide a more absolute

insight than either quantitative or qualitative analysis alone. This research strategy

was chosen to collect extensive information and data from the medical experts

residing in Pampanga and professionals in the field of the study.