The Difference in Effectiveness of Increasing Doses of Cymbopogon

(Lemongrass) Extract When Utilized as a Mosquito Repellent

An Undergraduate Research Submitted to the Faculty of the

Lyceum of the Philippines University - Cavite

International School

In Partial Fulfillment of the Requirements for the

Course Subject Research Capstone

Amante, Jean Magdalene A.

Apelado, Alessandra Lorraine O.

Baral, Kiana Chloe M.

Cortalla, Mithz S.

De los Reyes, Maxine Louisse B.

Deluta, Phatricia S.

Fundal, Rendon T.

Guavis, Jaschryle M.

Longakit, Ericka Mae A.

Pereña, Phoemela Andrea P.

Remolacio, Louis Miguel O.

Santos, Khristine Gel R.

Torres, Elaisse E.
 Abstract

The primary objective of this study was to utilize the active ingredient Cymbopogon

citratus extract as a mosquito repellent. Thus, an experimental study was conducted to

further evaluate its efficacy and duration, utilizing varying doses of the active ingredient,

and examining the possibility of side effects like allergic reactions such as itchiness,

rash, and redness. With that, the researchers examined if higher concentrations would

possess a significant increase in repellency duration and efficacy rate. With a total of six

participants, a random sampling design was conducted to generate two treatment

groups. The 15 mL and the 22.5 mL doses were the main observed variables in the

study. Three randomly selected participants were allocated to respective groups that

would be testing varying doses of Cymbopogon for a total of 8 hours. As a result, the

observed efficacy rate of 15 mL of Cymbopogon extract has no significant difference

when compared to the efficacy of 22.5 mL. However, the study verified that there is a

significant increase in repellency duration, between the two concentrations. Thus,

higher concentrations display longer repellency. With respect to side effects, 66.67% of

the participants have experienced itchiness from the repellent, and no signs of rash or

redness. Therefore, though there is no significant increase in the efficacy rate of 22.5

mL when compared to the 15mL dose of Cymbopogon extract, this study claimed that

utilizing a higher dose of concentration would yield a longer repellency duration

compared to lower doses.

Keywords: Cymbopogon extract, efficacy, mosquito repellent, repellency duration

 Introduction

When infected by viruses, blood-sucking insects like mosquitoes became primary

carriers of fatal mosquito-borne diseases through bites (Centers for Disease Prevention

and Control [CDC], 2016). And as numerous viral diseases like Zika virus, chikungunya,

yellow fever, and dengue are all transmitted by Aedes aegypti, the appearance of

increasing vectors and water stagnation where insects can proliferate poses serious

risks and threats to the country’s public health (European Centre for Disease Prevention

and Control [ECDC], 2016). Given that Aedes aegypti is one of the most predominant

species of mosquito, it is also taken into account that they thrive better indoors and

prefer to propagate in artificial and standing waters. That being said, when usual human

activities that are integrated with water usage are not properly handled, they become

primary drivers of mosquito breeding sites, particularly in urban areas.

Around the globe, dengue has been recognized as the most rampant

vector-borne disease. And particularly in Southeast Asia, the number of dengue cases

over the course of the previous years has increased in spite of the implementation of

preventive measures and disease management (World Health Organization [WHO],

2019). In the Philippines, this disease alone had claimed the lives of 561 people as of

July 13, 2019. Bringing the total number of cases to 130,463 in the year 2019,

individuals infected by this disease suffer from symptoms like headache, joint pain, and

rashes (WHO, 2019). As reported in a severe variant called dengue hemorrhagic fever,

infected individuals experience bleeding, and respiratory difficulties which can be fatal in
rare cases. Hence, as recorded statistics are further analyzed, mosquito-borne diseases

disproportionately affect indigent groups who live in open and unsanitary communities.

Implementing precautionary measures such as formulating repellents and

insecticides is one of the most essential strategies to prevent mosquito-induced

diseases. Given that mosquito repellents are known to be volatile chemicals that induce

mosquito repellency when applied to the skin, regular and frequent application of these

products prevents mosquito contact and bites (Diaz, 2016). Moreover, repellents are

functional if used when human activity coincides with the day-to-day activity patterns of

mosquitoes. However, due to the acclimation of insects, synthetic repellents are now

deemed inefficient (Ojewumi, 2018). Aside from that, the American Mosquito Control

Association (n.d.) presents that most of these repellents contain active ingredients such

as diethyl carbonate, diethyl phthalate, metofluthrin, picaridin, ethyl hexanediol,

essential oils, extracts, and N, N-Diethyl-3-Methylbenzamide (DEET). As most of these

commercial repellents were manufactured using synthetic non-biodegradable chemicals

which are probable to generate environmental risks, high exposure to these chemicals

may also pose hazardous health risks.

Positively, plant-based mosquito repellents have been widely utilized as personal

protection against the host-seeking behavior of mosquitoes. They are obtained through

the knowledge of ethnobotanical studies, which are valuable and reliable sources for the

development and production of emerging natural products. As plant-based repellents

exhibit a pleasant smell and fresh feeling on the skin, their safer nature is advantageous

when compared to synthetic and chemical-based repellents. In addition to numerous

studies that examined the efficiency of plant-based mosquito repellent, the plant that
has the highest repellency effect was the extract of Ligusticum Sinense. Followed by

citronella, pine, peppermint, and Rhizophora, the following plant extracts gave a

protection time that ranges from 9.1 to 11.5 hours. Additionally, plant-based essential

oils are produced by extracting them from plants such as broad-leaved eucalyptus,

lavender, and lemongrass, which also exhibited complete mosquito repellency, and a

protection time that lasts for 8 hours (Asadollahi, et al., 2019).

Cymbopogon citratus, commonly known as Lemongrass, is an aromatic grass

part of the Poaceae or Gramineae group. It is a perennial plant that grows locally in

West India, while lemongrass oil is typically cultured in tropical and subtropical areas

(Toungos, 2019). Although countries like Peru recommended the use of lemongrass for

medicinal purposes, targeting digestive disorders, inflammation, and fever, it is possible

to be used as a treatment for headaches, stomach aches, rheumatic pain, as well as

abdominal pain (Sissons, 2019). Likewise, it is also reliable for short-term medicine

when applied to the skin (Anngraeni et al., 2018). As the plant is also capable of

eradicating germs, the essential oil of Cymbopogon citratus is proven as a substitute for

synthetic fungicides and other sanitation practices. Hence, its high concentration of oil

inhibits the development of fungi in all pathogens (Nyamath and Karthikeyan, 2018).

The essential oil of lemongrass has hence been acknowledged as an organic

supply for insecticides and pesticides. With regards to that, a study conducted by Bhatt

and Kale (2019), utilized fabrics as their control measure to examine the repellency rate

of Cymbopogon oil. When the amount of oil was increased from 50 g/l to 100 g/l, the

repellency rate rose from 40% to 70%. Hence, this study proves that, as the amount of

concentration increases, the rate of repellency response of mosquitoes also increases.

In a study of liquid paraffin solutions of Cymbopogon citratus, concentrations are

associated with the repellency of mosquitoes. While 20% - 25% of high concentrations

are capable of supplying 100% protection that lasts for an hour, lower concentrations

are more likely to exhibit complete repellency in shorter periods (Tilaye et al., 2018).

Similar to a study conducted by Kimutai et al. (2017), increasing the percentage

of lemongrass oil significantly improves the repellency of the mosquito repellent against

sandfly Phlebotomus duboscqi. While further diminishing the frequency of mosquito

bites Cymbopogon citratus was found to exhibit longer repellency duration and insect

deterrence when compared to Tagetes minuta or Southern Cone Marigold. Seeing that

native leaves prove to exhibit potent insecticidal properties at relative concentrations,

these plant-based repellents can be actively used as an alternative to synthetic

repellents (Ojewumi et al., 2018; Kimutai et al., 2017).

Statement of the problem

In conducting this experimental study, the researchers seek to evaluate the

efficacy of varying doses of Cymbopogon (lemongrass) extract when used as the main

ingredient in the production of a mosquito repellent. Particularly, the researchers intend

to acquire all essential information that will help formulate answers to the following

questions:

1. Do varying concentrations exhibit an increasing efficiency rate?

1.1. 4 hours after application

1.2. 8 hours after application

2. Does the duration of repellency increase with the concentration of extract?

 2.1. 15 mL of active ingredient

2.2. 22.5 mL of active ingredient

3. Is there an occurrence of allergic reactions among participants who used the

product?

3.1. Itchiness

3.2. Rash

3.3. Redness

Hypothesis of the Study

Research Question: Is there a significant difference between the efficacy of 15 mL and

22.5 mL concentration of Cymbopogon extract?

The following null hypothesis will be tested in this study:

H₀: There is no significant difference between the efficacy of 15 mL and 22.5 mL

concentration of Cymbopogon extract.

The following alternative hypothesis will be tested in this study:

Hₐ: There is a significant increase in efficacy between 22.5 mL and 15mL concentration

of Cymbopogon extract.

Methodology

The researchers utilized an experimental method in designing the study. This

approach is utilized in a study that involves treatments, clusters, and random allocation
of individuals to respective groups. Therefore, this allows the researchers to alter

independent variables, manage the situation by implementing several control

procedures, and offer each participant an equal chance of being assigned to either

group (Baker 2017; Kabir 2016). Since the primary objective of this study was to

determine the level of effectiveness of the Cymbopogon extract when utilized as a

mosquito repellent, this strategy authorized the researchers to uncover the difference

between the efficacy and duration of varying doses, as well as identify the appearance

of allergic reactions caused by the product. Aside from that, the experimental method is

ideal for this study, as the researchers would have to manipulate the concentrations of

Cymbopogon extract in each formulation. With this design, the researchers were able to

compare the data from the present groups; hence, it assisted in analyzing the results

obtained from the participants, and further evaluating the efficacy of specific plant-based

mosquito repellent.

Participants of the Study and Sampling

A simple random sampling was utilized in this exploration. Regardless of gender

and age, the researchers acquired a total of 6 individuals to participate in the study.

Since the study would compare the efficacy rate of two varying concentrations,

participants were randomly allocated into two groups. One group tested the 15mL

concentration of Cymbopogon extract, while the other one tested the 22.5mL

concentration. In addition to that, due to some restrictions of the ongoing pandemic, this

study was conducted in the local area of the researchers’ household. As contact with
outsiders is limited, the participants of the study only involved the residents of the house

and immediate family members.

Procedure

With the use of simple random sampling, the researchers performed the

experiment with limited participants to examine the repellency of two varying doses of

Cymbopogon repellent. Additionally, the materials required to formulate the product are

lemongrass (Cymbopogon citratus), oil, and alcohol. The chopped lemongrass leaves

were submerged in oil and were boiled to make the extract. The first concentration

consisted of 50 percent extract (15mL) and 50 percent alcohol (15mL), whereas in the

second concentration, 75 percent extract (22.5mL) diluted with 25 percent (7.5mL)

alcohol. While solutions were placed in a spray bottle which has a total volume of 30mL,

the test was primarily subjected to a total of six participants.

Therefore, the researchers created two groups; the first group which consisted of

three randomly picked participants utilized the formula with 15mL lemongrass extract,

while the remaining batch used the product with 22.5mL extract. For both trials, which

started at 7 in the evening, each of the participants applied equal amounts of

plant-based mosquito repellent on their arms and legs. Given that the test was

conducted for eight hours, results were gathered every hour to record the difference in

their effectiveness, and duration of repellency to determine when the reapplication of

the product is needed.

Statistical Analysis
 This study utilized a T-test independent sample to calculate the true difference

between the data of two independent clusters. Since it is an experimental study,

participants were divided into random groups to see if there is a statistical difference

between the efficacy of varying concentrations of Cymbopogon mosquito repellent.

Aside from that, the researchers conducted prescriptive and descriptive analysis for the

discussion of the obtained data. The prescriptive analysis is a method used in making

data-driven decisions. This form of analysis generates prescriptions, which aids in

decision-making in order to make a prediction by utilizing the obtained data from the test

(Šikšnys and Pedersen, 2016). On the other hand, descriptive analysis is a

simplification of data, which helps researchers in organizing the collected information. It

allows the researchers to explain the phenomena by recognizing patterns from the

acquired data to answer who, for what, where, when, and to what extent (Loeb, et al.,

2017). Therefore, these measures enabled the researchers to develop comprehensive

data and causal effects.

Results

Six participants were randomly grouped and were given mosquito repellents with

varying concentrations to justify and test the efficacy rate of the plant-based mosquito

repellent. The first randomly selected group which was composed of 3 participants

tested the mosquito repellent with 15 mL Cymbopogon extract, whereas the remaining

group tested the ones with 22.5 mL Cymbopogon extract. For more reliable results, all

participants were required to apply a uniform number of sprays. Hence, regardless of

the concentration, each participant applied a total of five sprays on each arm, and 10

sprays on each leg.

Table 1. Treatment Test No. 1 Record

Amount of Participant Number of mosquito landing per hour

Cymbopogon

extract

applied Hours

1 2 3 4 5 6 7 8

15 mL
1 0 0 0 1 3 3 4 5

2 0 0 0 0 2 4 3 3

3 0 0 0 0 2 2 5 5

Total 0 0 0 1 7 9 12 13

As presented in table 1, all of the participants who tested the mosquito repellent

with 15 mL lemongrass extract had zero mosquito contact for whole three hours.

However, in the 4th hour, one mosquito landed on the legs of the first participant, while

the rest experienced mosquito landing in their 5th hour

Table 2. Treatment Test No. 2 Record.

Amount of Participant Number of mosquito landing per hour

Cymbopogon
extract Hours

applied

1 2 3 4 5 6 7 8

22.5 mL
1 0 0 0 0 0 2 2 3

2 0 0 0 0 1 2 2 4

3 0 0 0 0 0 1 3 3

Total 0 0 0 0 1 5 7 10

Table 2 illustrates that participants who tested the higher concentration prolonged

the zero mosquito contact for 4 hours. Meanwhile, a mosquito landed on the 2nd

participant’s elbow, a minute before the 5th hour ended. In the 6th hour, a few

mosquitos started landing on the participants.

Table 3. Descriptive statistics and T-test independent sample of the efficacy of variables

Variables M SD

15 mL Cymbopogon 5.25 5.65

extract

22.5 mL Cymbopogon 2.88 3.94

extract

*p.<.05
 The researchers utilized descriptive statistics to summarize the information of the

data, and a t-test independent sample to determine whether the 22.5mL possesses a

greater efficacy rate than 15mL. In each amount of concentration, the total number of

mosquitoes that landed each participant per hour was obtained. As a result, there is no

significant difference between the efficacy of 15 mL (M = 5.25, SD = 5.65) and 22.5 mL

(M = 2.88, SD = 3.94) concentration; t(14) = .98, p = .17. The given result illustrates that

increased concentrations of Cymbopogon extract do not display greater efficacy. In

addition, the calculated Cohen's d is .49, which indicates a moderate effect size.

Table 4. Repellency Duration Record

Treatment Group Participant Protection Hours (zero

mosquito contact)

15 mL 1 3

2 4

3 4

22.5 mL 4 5

5 4

6 5

As observed in the table, the varying Cymbopogon extract concentrations

exhibited differences in the time duration of the repellency. Participants under the 15 mL

treatment group received repellent protection for 3 to 4 hours, whereas those in the 22.5
mL treatment group received protection for 4 to 5 hours before landing attempts

occurred.

Table 5. Allergic Reactions Record

Treatment Did participants experience any allergic reactions?

Group
Participant Itchiness Rash Redness

15 mL 1 Yes No No

2 No No No

3 Yes No No

22.5 mL 4 Yes No No

5 Yes No No

6 No No No

Table 5.1. Percentage of the Allergic reactions associated with the variables

Allergic reactions Yes No

Itchiness 66.67% 33.33%

Rash 0% 100%

Redness 0% 100%
 Moreover, the researchers observed if the participants will encounter any allergic

reactions such as itchiness, rash, and redness when the lemongrass-based repellent

was applied to the skin. To emphasize, none of the respondents experienced mosquito

bites. As a result, four out of six individuals (66.67%) experienced mild itchiness after

applying the repellent, while the two (33.33%) did not. Six out of six (100%) subjects, on

the other hand, did not experience any skin rash or redness.

Discussion

Due to varying weather conditions, the appearance of mosquitoes is natural;

however, their abundance, particularly during rainy seasons, could be detrimental to

dense areas and vulnerable individuals’ health. Hence, in order to prevent acquiring and

spreading mosquito-borne disease, pro-active actions must be implemented to prevent

the spread of the disease. This study aimed to determine if an increased concentration

of Cymbopogon extract will yield a greater efficacy rate .For the participants’ safety, the

researchers decided to identify whether there are any allergic reactions that might occur

once the product comes in contact with the skin. And among the six participants, only

four (66.67%) of them encountered itchiness. While there are zero reports of skin rash

and redness.

As illustrated in the results, increased Cymbopogon extract concentrations did

not exhibit greater efficacy than the ones with lower concentrations. However, it is

emphasized that both doses exhibit enough efficacy to prevent mosquito bites for the

whole eight hours. Furthermore, the repellency duration and concentration are also one

of the matters of concern. In this study, results show that 22.5mL displayed a greater
repellency duration than 15 mL. That being said, various research has supported these

findings. In a study performed by Ojewumi (2017), the experimental result confirmed

that the higher the concentration, the longer the mosquitos will be repelled. As stated in

their findings, 0.5 mL concentration repelled mosquitoes within 1–2 hours of application,

1 mL repelled mosquitoes within 3–5 hours, and 1.5 mL repelled within 6 hours, while

no mosquitoes landed.

That being said, using increasing concentrations gave an indicative result to

consider that the intensity of the extract is relevant to the duration of repellency. In a

similar study, Ettah (2016) recorded that application of 2 mL and 6 mL concentration

elicited 100% mosquito repellency for 2.5 hours and 4.0 hours respectively. All things

considered, the descriptive statistics and t-test independent sample proved that while

concentrations do not exhibit an increase in efficacy rate, they do have a clear

association with the duration of repellency, as the higher concentration exhibited longer

repellency duration.

Conclusion

This study investigated the efficacy of Cymbopogon when utilized as the main

ingredient of mosquito repellent. While using a higher and a lower dose, the research

intended to calculate if higher doses of Cymbopogon would result in greater efficiency

and duration. As concluded in the results, between 15 mL and 22.5 mL concentrations

of Cymbopogon extract, higher concentrations did not display any increase in efficacy

rate. However, it displayed a concentration-dependent duration, as higher

concentrations exhibited longer repellency duration. Therefore, the null hypothesis (Hₒ)
was accepted in terms of examining the difference between the efficacy of varying

concentrations. Although the presented concentrations have no difference in terms of

efficacy, the 22.5 mL possesses anti-mosquito protection for 4 to 5 hours; thus, the

study proved that higher concentrations are associated with longer repellency duration.

As there are no significant differences in efficacy, it is recommended to use higher

doses to prolong the duration and lessen reapplication throughout the day.

Limitation of the Study

Due to some restrictions imposed by the ongoing COVID-19 pandemic, the

product was only tested limitedly in the local area, specifically in the researchers'

households. With limited accessible settings for the experiment, the product was only

tested in an outdoor setting, while the participants of the study were also limited to

volunteer household members. As participants were very limited, the product was not

tested on a larger scale of varied demographics and regions. And in addition to

unpredictable weather conditions, the insufficiency of mosquitoes in the local area

caused difficulties and delays in the research experiment.

Recommendation of the Study

This study aims to motivate individuals to use organic and plant-based repellents

to minimize the possible health and environmental risks that can be acquired from

chemical-based repellents. Given that synthetic non-biodegradable ingredients such as

the chemical DEET contain N, and N Diethyl 3 Methylbenzamide, studies proved that it

may pose risks to health, affecting the central nervous system. (Gershenzon and
Dudareva, 2007, as cited in Maia and Moore, 2011). That being said, exposure to

chemicals can lead to long-term health effects such as skin rashes, eye irritation, and

itching. Using an instrument with botanical derivatives aids reduce the risk of health and

is a much safer alternative than chemical-based repellents. Given that the majority of

plants contain compounds responsible for preventing insects from attacking them,

these compounds have evolved as repellents, which exhibit antibacterial, antiseptic,

anti-inflammatory, and antifungal effects (Pichersky and Gershenzon, 2002, as cited in

Maia and Moore, 2011; Harrewjin et al., 1995, as cited in Maia and Moore, 2011).

Hence, these are used as a personal protection mechanism against host-seeking

mosquitoes for generations. That being said, this study implies that in order to extend

the repellency duration of the product higher concentrations of Cymbopogon extract

must be utilized. Considering that lemongrass mosquito repellents are affordable, can

be made at home, and possess efficiency and enough repellency duration, it is

recommended for people and children to help combat mosquito-borne diseases.

Furthermore, for the studies that will be conducted in the same field, it is suggested to

perform the test in various indoor and outdoor settings to test the product’s actual

performance under various real-life circumstances. With the abundance of active

host-seeking mosquitoes, this method is recommended to further analyze its

effectiveness and duration. Additionally, having more participants in the test will yield

more accurate results, as this will enable us to thoroughly compare the efficiency of

variances of lemongrass extract per hour. With the results showing a positive effect, this

study could have a breakthrough in various industries, aiding cultivators of lemongrass

while assisting public health with its fight against mosquitoes-borne diseases.
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