Mosquito Repellency Potential of Sinturis (Citrus nobilis) Rinds

Essential Oil Against Aedes aegyptiA Research Study by

Awingan, M.A., Balicanta, C.J.J, Bascuna, R.S., Brillante, E.M.

Daguit, S.E.J., Eala, M.A., Gan, P.R., Jazmines, J.C., Limbo, A.R.

Magnaye, E.J., Mag-usara, R.C., Mata, M.K. and Rodolfo, R.C.

Background of the Study• Dengue Fever – among most prevalent diseases-caused by vector Aedes aegypti-can be prevented by mosquito repellents- Some commercial products contain harmful chemicals- Citrus known to have mosquito-repellant potential

Aedes aegypti mosquito during a blood meal(aedes.caltech.edu)

Background of the StudyCitrus nobilis- widely cultivated and commercially sold in the Philippines- contains citric acid, vitamins A, B and C, hesperidin, methyllanthranillic acid, methyl ester, and monoterpenes such as l-limonene, γ-terpene and ß-phellandrene

Citrus nobilis fruits

Statement of the Problem1. Occurrence of dengue fever in the country 2. Current preventive measures have been

found to have harmful side effects

Objectives of the Study• This study aimed to determine the repellent

activity of sinturis (C. nobilis) essential oil against adult Aedes aegypti.

- determined repellency potential through mean mosquito

landings and probings across different C.nobilis extract concentrations

Significance of the Study• Possible alternative to current preventive

measures for this mosquito-borne viral infection

• Potential usage of C. nobilis essential oils as an alternative natural mosquito repellent with lower risk of side effects

Scope and Limitations• Protection potential of the crude extracts calculated only in

terms of the mean number of landings/probings of adult A. aegypti mosquitoes on human skin

• The study did not determine/identify:

- the length of protection time

- active components in extract responsible for mosquito repellency

- any possible side effects

Review of Related Literature• Dengue

- mosquito-borne (A. aegypti/ A. albopiticus) viral disease - may develop into:

odengue hemorrhagic fever (DHF)- results in internal bleeding, low platelet counts, and blood plasma leakage

odengue shock syndrome (DSS), causes lethal low blood pressure levels- may be prevented by insect repellents

Review of Related Literature• Most common chemicals used as insect repellents: DEET,

Permethrin, and Picaridin

• N,N-Diethyl-meta-toluamide (DEET) - Affects the special antennal sensilla of mosquito, causing inherent aversion to chemical (“true repellent”)

• Permethrin - neurotoxin, also a cure for some skin diseases, but toxic to bees, beneficial insects and aquatic flora and fauna. Likely to be carcinogenic when taken orally

• Picaridin – overstimulates mosquito’s sensory organs, potentially lethal with excess and improper application

Review of Related Literature•Natural plant extracts for repellent use

- centella, peppermint oil, guava, morning glory vine•Sinturis (Citrus nobilis)

- culinary and medical uses- larvicidal and repellent properties

(Akram, et al., 2010) against A. albopictus

Review of Related Literature• WHO Mosquito Repellency Testing Guidelines

- estimate effective doses (ED) corresponding to 50%-99.9% protection

- estimate complete protection time (CPT)

- elimination of bias:

- washout with unscented soap and water, then 70% ethanol

- participants to avoid fragrance products and tobacco smoking

Data Sampling• WHO standard sampling design: crossover method

• 6 human volunteers, 3 males and 3 females, aged 16-18 years old, no history of allergic reaction to insect/arthropod bites, with informed consent

•Left and right arm control; applied increasing concentrations of extract to left arm

• Statistical Analyses: Friedman’s Test and multiple paired t-tests between controls and treatment concentrations

Materials• Sinturis (C. nobilis) fruit

- Fifteen kilograms of fruit obtained from Batangas

• A. aegypti mosquitoes - Pathogen-free female mosquitos (aged 3-7 days) from

Research Institute for Tropical Medicine and DOST (total of 600)

- starved for 12 hours to ensure biting activity

Materials• Arm-cages (40 cm x 40 cm x 40 cm)

- contained 75-100 mosquitoes each

• Materials for design control:- ethanol, latex gloves (non-penetrable), unscented

soap, dropper, vacuum cleaner (for mosquito disposal)

MethodologyProcurement of materials

Verification of Plant Material as Sinturis (C. nobilis)

Extraction of essential oil from Sinturis (64 mL, 6.7% yield)

MethodologyRepellency test against female A. aegypti mosquitos via arm-

cage method

Application of increasing concentrations of essential oil to

male volunteer

Application of increasing concentrations of essential oil to

female volunteer

Observation of the number of landings/probings

Observation of the number of landings/probings

Computation of protection exhibited by repellent

(5%, 10%, 15%, 20%)

Methodology

Extract treatments in varying concentrations (5%, 10%, 15%,

20%, 25%)

Extraction of Sinturis rind essential oil through water

distillation

Preparation of Sinturis (C. Nobilis) fruits

Methodology

Mosquitoes probing a volunteer arm inside the arm-cage

Application of extract treatment on arm

Results

TreatmentNumber of Mosquito Landings/Probings

R1 R2 R3 R4 R5 R6 Mean P%

T0a : Left Arm Control 13 16 17 10 25 2918.3333

3a

T1: 5% C. nobilis

essential oil7 5 13 7 14 15

10.16667b

41.35%

T2: 10% C. nobilis

essential oil1 1 1 4 4 7

3.000000c

82.69%

T3: 15% C. nobilis

essential oil0 0 0 0 1 1

0.333333d

98.08%

T4: 20% C. nobilis

essential oil0 0 0 0 0 0

0.000000d

100.00%

T0b : Right Arm

Control11 15 17 10 24 21

16.33333a

* Treatments with common letter of notation mean that they are not significantly different

Table 1: Individual and mean Aedes aegypti landings and probings and calculated protection percent for each treatment*

Results• Mean number of A. aegypti mosquito landings and/or probings ranged from 0 to 18.333• Left arm control T0a exhibited the highest mean mosquito landings/probings (18.333), followed by the right arm control T0b (16.333)

Results•Twenty percent C. nobilis essential oil (T4) exhibited the lowest mean mosquito landings, with a mean of 0.000• This was followed by 15% essential oil (T3), with a mean of 0.333, 10% essential oil (T2), with a mean of 3.000, and 5% essential oil (T1), with a mean of 10.167.

Discussion and AnalysisPaired t-test Results  p-value

LC v RC 0.083600LC v 5% 0.00320LC v 10% 0.00072LC v 15% 0.00064LC v 20% 0.00080

5% v 10%

0.00200

5% v 15%

0.00083

5% v 20%

0.00109

10% v 15%

0.01256

10% v 20%

0.01505

15% v 20%

0.08734

Table 2: P-values from the multiple paired t-tests conducted

• No significant difference between left and right arm controls

• No significant difference between 15% and 20% treatments

• Optimum concentration may lie between 15%-20%

Discussion and Analysis

0% 5% 10% 15% 20% 25%0

5

10

15

20 18.3333333333333

10.1666666666667

3 0.333333333333333 0 0

Concentration vs Mean Mosquito Land-ings

ConcentrationMean

Mosq

uit

o L

an

din

gs

Figure 1. Concentration of C. nobilis essential oil vs. mean number of mosquito landings/probings

• Based on the statistical analysis, the decreasing trend can be attributed largely to the effect of the C. nobilis essential oil on repellency.• Increased protection as the concentration of the essential oil is increased.

Discussion and AnalysisSkin area/biting site

% insects biting (SE) nControl SS220 Picaridin DEET

24cm2   60 12cm2, treated skin

35.0 0.0 1.6 0

12 cm2, untreated skin

40.0 23.0 (5.4)

35 (4.5) 25 (5.6)

Total 75.0 (5.0)

12 cm2 60 6 cm2, treated skin

41.7 0.0 0.0 1.6

6 cm2, untreated skin

35.0 20.0 (5.2)

41.7 (6.4)

27.0 (5.7)

 

Total 76.7 (5.5)

 

Table 3. Percentage of Ae. aegypti biting on adjacent treated and untreated half-cell 12- and 24-cm2 skin surfaces with (n) number of mosquitoes tested against each treatment (test 2)a b

aTreatments of SS220, Picaridin, and Deet were each at a dose of 48 nmol of compound/cm2 skin. bLifted from Klun, Khrimian and Debboun (2006) (Table 2)

Results from the essential oil concentration of 20% are comparable to the results of the study by Klun, Khrimian and Debboun (2006), which made use of DEET with ethanol and picaridin with ethanol as mosquito repellents.

Conclusion Given the comparable repellent capacities between DEET, Picaridin, and C. nobilis, there is sufficient evidence to suggest that essential oil from Citrus nobilis rinds could be a component in mosquito repellent products.

Hence, C. nobilis could be considered as a potential alternative to DEET and Picaridin.

Recommendation

Time Table

Budget

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