Cymbopogon nardus (Citronella) Leaves Extract: Insecticidal Effect on Pediculus humanus

capitis (Head Lice)

A Thesis Proposal Presented to the Department of Medical Technology, Mountain View

College in Partial Fulfilment of the Requirements
For the Degree

BACHELOR OF SCIENCE IN MEDICAL TECHNOLOGY

ABELLANA, RIXELE JAN V.

CANILLO, WENDELL RHYAN
ISRAEL, CLAIRE A.
POLINTAN, ZARIZ ANNE M.
VILLANUEVA, KAYE ANJELIK F.
ZABALLERO, NOREEN P.

May 2017
Introduction

Head lice (Pediculosis humanus capitis) have been plaguing people for millennia. These

are obligate, blood-feeding human ectoparasites, 1-3mm in length, grayish-whitish color. The

insect has a narrow sucking mouthparts hidden within the head, have short antennae, and three

pairs of clawed legs adapted to grass into the hairs. Head lice move speed of up to 23 cm/min and

they cannot jump or fly (Ko & Elston, 2004; Nutanton et al., 2008; Maunder, 1985). The female

head louse can lay about 150 eggs during a 30- day of her life cycle. The eggs grow and after 1

week, turn into the nymphs. After 7 days, the nymphs mature to adults. Mature female head lice

can then lay eggs after 1.5 days after becoming an adult. The nits can survive for 10 days without

host but the adults can endure for up to 3 days off the host (Ko & Elston, 2004; Dodd, 2001;

Witkowski & Parish, 1997). Head lice feed exclusively on blood and take several feeds each day.

However, they do not excrete liquid urine or feces like other blood feeding insects. Additionally

it has long been recorded that lice are able to survive immersion in water for several hours,

presumably tolerating long periods without oxygen, and specific experimental investigation of

the potential for suffocation by various preparations has not been shown to be currently feasible

(Burgess et. Al. 2009). The criteria for mortality of head lice were strict and were defined as the

complete absence of any vital signs such as gut movement, movement of the limbs, antennae or

movement of the legs with or without stimulation using forceps (Soonwera, M.2015). The vital

state of each louse was classified according to the following categories: vital lice, fully active lice

with normal movements; lice with major vital signs, walking, but unable to walk in a progressive

fashion or no righting reflex when rolled onto the back; lice with minor vital signs, not walking,

but presence of internal (gut) movements, movements of antennae, or leg movements; lice with
no vital signs at all. Definition of ‘mortality’ of head lice: absence of any major or minor vital

signs (Oliveira et. al. 2006).

They are connected to human hosts during all life stages with physical head-to-head contact

as the main route of transmission (Heukelbach 2010). Once accepted as a common and

unremarkable occurrence in society, head lice have attained almost “legendary status” for creating

panic among parents of school-aged children in the United States. Pediculosis is an infestation of

head lice, not an infection. Infestations of head lice mostly affect children between the ages of 3-

12 years. The most common symptom of pediculosis is itching due to sensitization to allergens in

lice saliva. Many times, there are no symptoms. However, Heavy infestations can cause intense

irritation and severe itching. Scratching leads to chafing and secondary bacterial infection. P.

humanus capitis may serve as a vector for some bacterial agents, such as Rickettsia prowazekii

(Robinson et al. 2003). However, inappropriate treatments can pose real and significant health

hazards to the child and his or her household. Effective diagnosis and treatment of head lice

infection are essential for both infested individuals and general population.

Citronella is commonly known for its insect repelling properties especially for mosquitoes.

Currently oil of citronella can be found in over a dozen registered pesticide products. Some of

these products are applied to animal or human skin, ornamental plants, or other outdoor areas. Oil

of citronella can be formulated into sprays, lotions, candles, pellets, and pouches. Mosquito

repellents based on chemicals has a remarkable safety profile, but they are toxic against the skin

and nervous system. Hence, natural mosquito repellents were preferred over chemical mosquito

repellents. Attempts have been made to find out new active ingredients, especially those derived

from natural plants to replace the synthetic insecticides such as pyrethroid (Rani, Wany, Vidyarthi,

& Pandey, 2013).

 Citronella essential oil has more than eighty components, four of the most important ones

are citronellal, eugenol, geraniol and limonene (Rani, Wany, Vidyarthi, & Pandey, 2013). It was

proven to be effective in the control of cattle ticks, citronelal and geraniol as the important active

components (Olivo etal. 2008). It was also proven that citronella has an antifungal activity

(Nakahara et al. 2003). Five commercial plant extracts has been studied (Citronella, Garlic oil,

Neem extract, pine oil and pyrethrum), Citronella was found to be effective in insect repellent for

food packaging, it deters the infestation of muesli, and wheat germ by red flour beetles found in

fruit cartons (Wong et al. 2015). The advanced therapeutic studies enabled the systematic and

controlled use of Citronella oil as an antiparasitic and antibacterial agent (Wany et al. 2013).

Many antipediculosidal agents that are safer and has low toxicity has been developed

recently but none of the available products fulfill the “ideal pediculocide” criteria, which are 100%

effective in killing motile stages and eggs, easy-to-apply formulation, lower toxicological safety,

low cost, having good environmental profile, having a potential to prevent reinfestations and

effectiveness with single treatment (Heukelbach 2010).

Here, in the present study, the researchers will aim to know if Citronella (Cymbopogon

nardus) leaves extract can kill adult head lice faster than commercially prepared pediculosides, if

there is a significant difference in terms of percentage of mortality, time and efficacy of Citronella

extract, Quassinoids (positive control), and tap water (negative control). Furthermore it aims to

add more knowledge to previous studies about head lice infestation and lastly the study aims to

know if Citronella can be a natural alternative remedy for head lice infestation which is economical

and available locally.

METHODOLOGY

This chapter includes the research method, respondents of the study, locale of the study,

sampling design, instrument of the study, data gathering and data processing and analysis.

Research Design

This study used the descriptive research design and focused on measuring the capabilities of

Citronella extract if it is capable of eliminating and eradicating head lice.

Respondent of the Study

The respondents of the study are all female grade 3 to 5 children ages 10-12 years old that

are currently enrolled in a school particularly located in Bukidnon in the school year 2016-2017

who are currently having head lice problem. The researchers have given consent forms to the

elementary principal as well as to the parents of the respondents for them to sign. The signed

consent was returned the next day.

Locale of the Study

This study was conducted in Mountain View College Medical Technology Laboratory, Mt.

Nebo, Valencia city, 8709 Bukidnon.

Instrument of the study

The study used a considerable amount of time of experimentation, observation and lab

testing. The Experimentation have 3 parts: Part I is the extraction of Citronella. Part II is the lice

gathering procedure. Part III was the spraying of the Citronella extract directly on the head lice.
Tap water was used as the negative control and commercial head lice shampoo (Quassinoids) was

used as the positive control.

Part I: Extraction of Citronella

Citronella was collected from Lilingayon, Valencia Bukidnon. It was washed, air-dried,

cut into small pieces and placed in a mechanical mixer and produce 3 ml of extract and filtered

using the filterer attached to the mechanical mixer. 1 ml of Citronella extract, tap water and Lice-

Aliz was separately measured by a 5cc syringe and each was put directly in a sprayer bottle (with

5mm nozzle) which was used for the next part of the process. After extracting, the extract was

immediately transported to the laboratory for testing.

Part II: Lice Gathering procedure

Head lice were gathered using a knit comb and a white paper. Adult head lice were

separated from the nits and nymph by sliding them in the petri dish. The respondents first combed

their hair using a hair comb with large bristles to untangle coiled hair strands. A total of 90 freshly

collected adult head lice were put in 3 petri dishes with hair strands.10 adult head lice were put in

each Petri dishes. Treatment 1 (T1) used Citronella extract, Treatment 2 (T2) used Quassinoid for

the positive control, Treatment 3 (T3) used tap water for the negative control. A plastic sprayer

bottle was used to spray 1 mL of Citronella extract, 1 mL of Lice- Aliz, and 1 mL of tap water in

each petri dish respectively. After gathering, the head lice was immediately transported to the

laboratory for testing.

Part III: Spraying of the Citronella, Tap water and Pediculoside and Observation

Treatment 1(T1) was sprayed directly (with a distance of 3cm) with 1 ml Citronella extract,

Treatment 2 (T2) was sprayed directly (with a distance of 3cm) with 1 ml Pediculoside and
Treatment 3 (T3) was sprayed directly (with a distance of 3cm) with 1 ml tap water. The petri dish

was then put under a microscope and observe the vital signs such as gut movement, movement of

the limbs, antennae or movement of the legs of the head lice. The mortality and the time of

mortality of adult head lice in each treatment was recorded in each set ups. The process was

replicated three times.

Sample Gathering Procedure

The researchers sent a letter to the head teacher of the elementary department of the school

particularly located in Bukidnon for her to sign a week before the conducting process. The

researchers also sent a letter and a waver to the parents for them to sign for an approval in gathering

adult head lice for their school-aged children. Foods, knit combs, and hair combs was given as a

reward for their participation. The collecting of the adult head lice was conducted inside the

classroom. After collecting, head lice was put immediately in the petri dish and immediately

transported back to the laboratory for testing.

Asphyxiation
Results and Discussion:

Head lice that were collected from Students from a certain school in Bukidnon and the

activity levels of lice treated with the negative control which is Tap water, Citronella and the

positive control which is Quassinoids over time are shown in (table 1-1) and the criteria used for

survival of lice were extremely strict: if any even minor signs of life, such as internal

movements(gut movement), or movements of antennae or minimal leg movements were

observed (with or without stimulation by a forceps), the lice were categorized as alive

(Heukelbach et. al 2006) and data gathered was analyzed using One-way ANOVA.

Table 1.1

Trial 1 5 minutes 10 15 20 25 30
minutes minutes minutes minutes minutes
Alive Dead A D A D A D A D A D
10 0 10 0 10 0 10 0 10 0 9 1
Tap water
10 0 6 4 6 4 6 4 6 4 6 4
Citronella
8 2 7 3 6 4 5 5 3 7 0 10
Quassinoids

Trial 2 5 minutes 10 15 20 25 30
minutes minutes minutes minutes minutes
Alive Dead A D A D A D A D A D
10 0 10 0 10 0 10 0 10 0 10 0
Tap water
10 0 10 0 9 1 9 1 9 1 9 1
Citronella
5 5 3 7 0 10 0 10 0 10 0 10
Quassinoids
 Trial 3 5 minutes 10 15 20 25 30
minutes minutes minutes minutes minutes
Alive Dead A D A D A D A D A D
10 0 10 0 10 0 10 0 10 0 10 0
Tap water
10 0 8 2 8 2 8 2 8 2 8 2
Citronella
9 1 0 10 0 10 0 10 0 10 0 10
Quassinoids

The number of alive versus the effectivity of the treatment was (shown in Table 1-2)

analyzed using One-way ANOVA (as shown in figure 2-1) stated 3 Levels, level 1 is the

treatment Tap water, level 2 is the treatment Citronella and level 3 is the treatment Quassinoids.

N is the average of 6 Time and 3 trials (N=18), the Mean is the average number of alive. Level 1

which is the treatment Tap water shows the greater number of alive and Level 3 which is the

Quassinoids shows the lowest number of alive. The Null hypothesis of the Anova is there is no

significant difference in the effectivity in terms of different treatment, and the P value must be

<0.005 to reject the null (P<0.005=reject null). The P value of No. of alive versus treatment is

equal to 0.000 (P=0.000) which states that the Null value should be rejected and there is a

significant difference in the effectivity of the 3 different treatments. In Grouping Informaion

Using Tukey Method as shown in figure 1-2, it shows that there are 3 different groups (A, B and

C) Treatment 1 which is Tap water has a mean of 9.944(M=9.944) belongs to group A,

Treatment 2 which is Citronella has mean value of 8.111 (M=8.111) belongs to group B and

Treatment 3 which is Quassinoids has a mean value of 2.556 (M=2.556) belongs to group C.

Means that do not share a letter are significantly different in terms of effectivity. Treatment 3 is
the most effective because it has the least number of alive, treatment 2 is effective than treatment

1 but is least effective than treatment 3.

Table 1-2 (Trial 1)

Average No. of alive in terms of treatment
12

10

0
5 mins. 10 mins. 15 mins. 20 mins. 25 mins 30 mins.
Level 1 (Tap water) Level 2(Citronella) Level 3(Quassinoids) Column1 Column2

Trial 2

Average No. of alive in terms of treatment

12

10

0
5 mins. 10 mins. 15 mins. 20 mins. 25 mins 30 mins.
Level 1 (Tap water) Level 2(Citronella) Level 3(Quassinoids) Column1 Column2
(Trial 3)

Average No. of alive in terms of treatment

12

10

0
5 mins. 10 mins. 15 mins. 20 mins. 25 mins 30 mins.
Level 1 (Tap water) Level 2(Citronella) Level 3(Quassinoids) Column1 Column2

Figure 2-1
One-way ANOVA: No. of Alive versus Treatment

Source DF SS MS F P
Treatment 2 532.93 266.46 61.45 0.000
Error 51 221.17 4.34
Total 53 754.09

S = 2.082 R-Sq = 70.67% R-Sq (adj) = 69.52%

Level N Mean St Dev

1 18 9.944 0.236
2 18 8.111 1.530
3 18 2.556 3.258

Pooled St Dev = 2.082

Grouping Information Using Tukey Method

Treatment N Mean Grouping

1 18 9.944 A
2 18 8.111 B
3 18 2.556 C

Means that do not share a letter are significantly different.

 The number of alive versus time (as shown on Table 1-3) and analyzed (as shown in figure

2-2). It stated 6 levels which is the time (5 minutes, 10 minutes, 15 minutes, 20 minutes, 25

minutes and 30 minutes). N is the average of the 3 treatments and 3 trials (N=9), the Mean is the

number of alive. The Null hypothesis of the ANOVA is there is no significant difference in the

effectivity in terms of time, and the P value must be <0.005 to reject the null. The P value of the

No. of alive versus time is 0.507 (P=0.507) which states that the null value must be accepted,

meaning there is no significant difference in the effectivity in terms of time. In Grouping

Information Using Tukey Method as shown in figure 1-4, it shows that there is only one

grouping (Group A) for all 6 levels time (5 minutes, 10 minutes, 15 minutes, 20 minutes, 25

minutes and 30 minutes) which means there will be same effect although different time and time

doesn’t matter in terms of effectivity.

Table 1-3
Trial 1
Average No. of alive in terms of time
5 mins. 10 mins. 15 mins. 20 mins. 25 mins. 30 mins.
12

10

0
Treatment 1 (Tap Treatment 2 Treatment
water) (Citronella) 3(Quassinoids)

Trial 2
 Average No. of alive in terms of time
5 mins. 10 mins. 15 mins. 20 mins. 25 mins. 30 mins.
12

10

0
Treatment 1 (Tap Treatment 2 Treatment
water) (Citronella) 3(Quassinoids)

Trial 3

Average No. of alive in terms of time

5 mins. 10 mins. 15 mins. 20 mins. 25 mins. 30 mins.
12

10

0
Treatment 1 (Tap Treatment 2 Treatment
water) (Citronella) 3(Quassinoids)
Figure 2-2
One-way ANOVA: No. of Alive versus Time

Source DF SS MS F P
Time 5 62.8 12.6 0.87 0.507
Error 48 691.3 14.4
Total 53 754.1

S = 3.795 R-Sq = 8.32% R-Aq(adj) = 0.00%

Level N Mean St Dev

5 9 9.111 1.691
10 9 7.111 3.586
15 9 6.556 4.035
20 9 6.444 4.065
25 9 6.222 4.206
30 9 5.778 4.494

Pooled St Dev = 3.795

Grouping Information Using Tukey Method

Time N Mean Grouping

5 9 9.111 A
10 9 7.111 A
15 9 6.556 A
20 9 6.444 A
25 9 6.222 A
30 9 5.778 A

Means that do not share a letter are significantly different.

Conclusion and Recommendation:

Head lice infestation is one of the major public health problems with worldwide

distribution (Shayeghi 2010). Finding effective ways to eradicate head lice is crucial for the

control of head lice infestation in improvised community (Daniel Pilger et al. 2009) .Although

there are antipediculocidal agents available in the market today, still there is no perfect
pediculocide that can 100% eradicate motile stages and eggs of head lice, easy to apply and is

safer and has low toxicity (Heukelbach 2010). Cymbopogon nardus (Citronella) has been

traditionally used as mosquito repellant, and exhibit antibacterial and antifungal activities, after

thorough observation and experimentation, the researchers discovered that Citronella is much

effective than the negative control which is the tap water but is less effective than the positive

control which is Quassinoids, although it didn’t kill adult head lice but the researchers

discovered that in application of Citronella to the head lice, all of them lose their motility and is

paralyzed by the Citronella but the gut movement is still there which shows that it is not 100%

dead ( criteria for mortality includes all movement or vital signs and gut movement) and so

Citronella must be a repellant for lice and not pediculocidal, but it is another objectives that

needs thorough experimentation to conclude its effectivity as a repellant. The researchers

recommends the future researchers, to try applying Citronella directly to the head lice and not

through spraying, and try adjusting the amount of citronella. The researchers also recommend to

measure the effectivity of Citronella in terms of repellency to head lice, Nits and eggs. Time is

also crucial in monitoring mortality of the head lice, for future studies, long time period of

experimentation can also be used. Using Citronella oil and Citronella shampoo is also

recommended instead of leaves extract. Any parts of the plant can also be used.

Scopes and Limitations:

The researchers aim is to know if Citronella leaves extract can be used as

antipediculoside to adult head lice only. The time used in the experimentation was parallel to the

time of mortality on the positive control which is the Quassinoids.

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