Fitoterapia 78 (2007) 205 210

www.elsevier.com/locate/fitote

Aromatic plant-derived essential oil: An alternative larvicide

for mosquito control
B. Pitasawat a,, D. Champakaew a , W. Choochote a , A. Jitpakdi a , U. Chaithong a ,
D. Kanjanapothi b , E. Rattanachanpichai a , P. Tippawangkosol a ,
D. Riyong a , B. Tuetun a , D. Chaiyasit a
a

Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
b
Chulabhorn Research Institute, Chiang Mai 50200, Thailand
Received 11 November 2005; accepted 8 January 2007
Available online 6 February 2007

Abstract
Five aromatic plants, Carum carvi (caraway), Apium graveolens (celery), Foeniculum vulgare (fennel), Zanthoxylum limonella
(mullilam) and Curcuma zedoaria (zedoary) were selected for investigating larvicidal potential against mosquito vectors. Two
laboratory-reared mosquito species, Anopheles dirus, the major malaria vector in Thailand, and Aedes aegypti, the main vector of
dengue and dengue hemorrhagic fever in urban areas, were used. All of the volatile oils exerted significant larvicidal activity
against the two mosquito species after 24-h exposure. Essential oil from mullilam was the most effective against the larvae of A.
aegypti, while A. dirus larvae showed the highest susceptibility to zedoary oil.
2007 Elsevier B.V. All rights reserved.
Keywords: Larvicidal activity; Aedes aegypti; Anopheles dirus; Carum carvi; Apium graveolens; Foeniculum vulgare; Zanthoxylum limonella;
Curcuma zedoaria

1. Introduction
Interest in the control of Aedes aegypti and Anopheles dirus lies in the fact that they act as vectors of dengue and
dengue hemorrhagic fever, and malaria, respectively, which are serious public health problems in Thailand and many
developing countries. Although some vector-transmitting diseases such as Japanese encephalitis and yellow fever have
been reasonably brought under control by vaccination, no effective vaccine is available for dengue or malaria.
Therefore, the only efficacious approach of minimizing the incidence of these diseases is to eradicate and control
mosquito vectors mainly by application of insecticides to larval habitats, destroying unwanted containers, and
educating the public [1]. During epidemics, these measures are complemented by insecticide space-spraying against
adult mosquitoes. However, aerial toxicants for eradicating A. aegypti are not effective, since this species is highly

Corresponding author. Tel.: +66 53 945343; fax: +66 53 217144, +66 53 945347.
E-mail address: bpitasaw@mail.med.cmu.ac.th (B. Pitasawat).
0367-326X/$ - see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2007.01.003

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B. Pitasawat et al. / Fitoterapia 78 (2007) 205210

domesticated and many adults rest in hidden places indoors [2]. It is acknowledged that attacking breeding places with
larvicides is a potential alternative to diminish mosquito populations.
The most commonly used larvicides are organophosphorus compounds such as temephos, fenthion and
chlorpyrifos, which are highly active against mosquito larvae and other aquatic insects. Organophosphorate temephos
is recommended as the most appropriate larvicide for the control of Aedes and Anopheles, because it has low toxicity
to fish, birds, mammals and humans [3,4]. However, its toxicity to fish and other nontarget organisms as well as the
environment, and the insecticide resistance of arthropods, are being increasingly reported [58]. Even though
susceptibility of various species of mosquito larvae to plant products has been studied extensively, most plant-derived
constituents have shown tendencies to exhibit slower actions and weaker effects compared with synthetic
compounds. The plant-derived natural products, however, still have immense potential for the control of mosquito
vectors, if they are reasonably effective and harmless to beneficial nontarget organisms and the environment.
Determination of the larvicidal activity of AkseBio2, a new botanical natural product derived from a mixture of
various plant extracts, revealed that this product is promising as a larvicide against Culex pipiens and could be useful
in the search of new larvicidal natural compounds [9]. Recently, essential oils derived from plants have received
much interest as potential bioactive agents against mosquito vectors. Many plant oils such as basil, cinnamon,
citronella and thymus are promising as mosquito larvicides [1013]. Investigation of plant oil-derived larvicides used
for mosquito control is, therefore, a vital point of this research. The selection of plants used in this study focused on
those belonging to similar or associated plant families reported to have potential against mosquitoes [10,14,15]. In
addition, herbs used as vegetables and spices in traditional medicine were mainly considered in the search for
effective and safe materials.
2. Experimental
2.1. Plants
Carum carvi L. (Apiaceae), Apium graveolens L. (Apiaceae), Foeniculum vulgare Mill (Apiaceae), Zanthoxylum
limonella Alston (Rutaceae) and Curcuma zedoaria Roscoe (Zingiberaceae) were obtained from commercial suppliers,
Phachinai Industry or E.A.R. Samunpri, in Chiang Mai Province, Thailand. The voucher specimens were deposited in
the Department of Parasitology, Faculty of Medicine, Chiang Mai University, Thailand.
2.2. Extraction and preparation
Essential oils from each plant material were steam-distillated. The obtained oil was dried over anhydrous sodium
sulfate, collected and kept in a light-protected bottle under refrigeration at 4 C for later investigation.
2.3. Test mosquitoes
A. dirus was obtained from the Armed Forces Research Institute of Medical Sciences (AFRIMS). A. aegypti was
collected at various breeding places in Chiang Mai Province, northern Thailand. The free-mating colonies of these
mosquitoes were established in the insectarium of the Department of Parasitology, Faculty of Medicine, Chiang Mai
University, by following the common standard mosquito rearing technique previously described [16]. The mosquito
colony was maintained continuously at 2530 C and 8090% relative humidity under a photoperiod of 14:10 h (light/

Table 1
Physical and organoleptic properties, and yields of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria
Plants

Voucher specimen

Appearance

Color

Odor

Density (g/ml)

% Yield (v/w)

C. carvi
A. graveolens
F. vulgare
Z. limonella
C. zedoaria

PARA-CA-001/1
PARA-AP-001/2
PARA-FO-001/1
PARA-ZA-001/1
PARA-CU-001/2

Liquid
Liquid
Liquid
Liquid
Viscous liquid

Light yellow
Pale yellow
Pale yellow
Light green
Golden yellow

Pepper-like
Orange-like
Pepper-like
Lemon-like
Cineolic-like

0.93
0.89
0.97
0.84
0.90

1.46
1.25
0.77
5.72
1.17

B. Pitasawat et al. / Fitoterapia 78 (2007) 205210

207

dark) for over 20 generations in a laboratory free of exposure to pathogens or insecticides. Freshly molted larvae of A.
dirus and A. aegypti were continuously available for the mosquito larvicidal experiments.
2.4. Determination of larvicidal activity
Mosquito larvicidal assays were carried out according to the standard procedures of the WHO [17], with slight
modifications. Each plant's volatile oil was diluted in dimethylsulphoxide (DMSO) to prepare a serial dilution of test
dosage. Early fourth instar larvae of each mosquito species were selected, transferred in 25 ml of distilled water (25
larvae/beaker), and allowed to acclimatize for 1 h before testing. For experimental treatment, 1 ml of serial dilutions
was added to 224 ml of distilled water in a 500-ml enamel bowl and shaken lightly to ensure a homogeneous test

Table 2
Larvicidal activity of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella, C. zedoaria on A. aegypti
Volatile oil
(ppm)

C. carvi
37.2
46.5
55.8
65.1
74.4
83.7
A. graveolens
26.7
35.6
44.5
53.4
62.3
71.2
F. vulgare
42.7
44.6
46.6
48.5
50.4
52.4
54.3
56.3
Z. limonella
8.4
12.6
16.8
21.0
25.2
29.4
33.6
C. zedoaria
18.0
22.5
27.0
31.5
36.0
40.5
45.0
a

A. aegypti
% Mortality
(Mean SE)

12.67 9.29
22.67 9.02
54.33 8.08
73.00 22.65
87.67 11.59
97.00 3.61
13.67 15.18
41.67 30.83
55.33 31.09
68.00 28.21
78.33 25.66
96.67 4.16
17.67 8.08
25.33 12.34
31.00 14.73
37.33 10.02
48.00 3.00
73.00 6.00
79.00 2.65
88.33 5.86
1.00 1.00
7.33 0.58
21.33 4.04
39.33 8.33
43.33 12.01
67.33 15.31
78.00 5.00
4.33 2.31
12.67 0.58
29.00 7.00
52.33 14.19
63.67 16.50
77.00 16.09
86.67 11.06

Larvicidal activity a
LC50

LC95

LC99

54.62
(52.6256.64)

90.06
(83.09100.76)

119.21
(105.70141.33)

42.07
(39.6244.56)

99.12
(85.00124.85)

160.25
(126.82228.68)

49.32
(48.7049.94)

62.09
(60.0864.88)

70.64
(67.2375.53)

24.61
(23.2925.98)

55.81
(47.5072.01)

88.32
(69.10130.72)

31.87
(30.7133.02)

55.50
(51.0562.30)

75.75
(66.7390.46)

The lethal concentrations with the corresponding 95% confidence intervals are shown in parenthesis.

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B. Pitasawat et al. / Fitoterapia 78 (2007) 205210

solution. Selected mosquito larvae were transferred in distilled water into a bowl of prepared test solution with a final
surface area of 124 cm2 (25 larvae/bowl). Four replicates were run simultaneously yielding a final total of 100 larvae
for each dosage, with at least five dosages providing a range of 0100% mortality. For each test, controls and untreated
sets containing a mixture of 1 ml of diluent DMSO dissolved in 249 ml of distilled water and 250 ml of distilled water,
respectively, were also run for comparison. Symptoms of treated larvae were observed and recorded immediately and at
time-intervals. Mortality and survival were determined after 24 h of exposure, during which time no food was offered
to the larvae. The moribund and dead larvae in the four replicates were combined and expressed as the percentage
mortality for each concentration. Larvae were considered dead or moribund if they were unrousable within a reasonable
period of time, even when gently prodded with a needle, as described in the World Health Organization's technical
report series. They might also show discoloration, unnatural positions, tremors, incoordination, or rigor. Three
independent experiments for each volatile oil against each mosquito species from different rearing batches were carried
out at 2530 C on separate days.

Table 3
Larvicidal activity of volatile oils derived from C. carvi, A. graveolens, F. vulgare, Z. limonella, C. zedoaria on A. dirus
Plant oil
(ppm)

C. carvi
46.5
55.8
65.1
74.4
83.7
93.0
A. graveolens
35.6
44.5
53.4
62.3
71.2
80.1
89.0
F. vulgare
33.9
34.9
35.9
36.9
37.8
38.8
Z. limonella
37.8
42.0
46.2
50.4
54.6
58.8
63.0
C. zedoaria
23.4
25.2
27.0
28.8
30.6
32.4
a

A. dirus
% Mortality
(Mean SE)

4.00 3.61
11.00 10.82
30.00 13.53
57.67 10.21
75.00 5.57
91.33 2.52
10.25 8.34
24.00 8.29
36.00 13.56
50.75 19.47
68.75 4.92
84.25 3.10
92.75 3.30
25.00 14.11
39.00 17.06
63.67 7.02
83.00 5.20
88.00 3.61
93.33 3.06
4.00 1.73
8.33 0.58
13.33 0.58
18.67 4.73
34.00 2.65
59.33 8.62
74.00 9.54
4.33 6.66
7.00 6.00
27.00 11.14
46.33 15.28
54.33 20.50
70.67 14.15

Larvicidal activity a
LC50

LC95

LC99

72.28
(70.2374.53)

104.69
(97.19117.23)

128.87
(115.46152.62)

59.44
(56.8761.98)

111.69
(99.59133.00)

159.06
(133.47208.45)

35.27
(35.0135.50)

38.78
(38.1939.64)

40.90
(39.9542.34)

57.22
(56.1058.51)

76.23
(72.0482.93)

89.52
(82.40101.43)

29.69
(29.0430.37)

40.23
(37.2546.67)

47.70
(42.4060.00)

The lethal concentrations with the corresponding 95% confidence intervals are shown in parenthesis.

B. Pitasawat et al. / Fitoterapia 78 (2007) 205210

209

2.5. Statistical analysis

It was essential to obtain not less than 3 mortality counts of between 10% and 90%. If the mortality in controls was
between 5 and 20%, correction of the observed percentage mortality (%M) was performed by applying the Abbott's
formula [18]:
%M

% test mortality% control mortality

 100
100% control mortality

When the control mortality was over 20%, the tests were discarded and repeated. The average mortality percentage
derived from individual tests was determined. The lethal concentrations of 50%, 95% and 99% (LC50, LC95 and LC99,
respectively) as well as the corresponding 95% confidence intervals (95% CI), used to measure differences between
plant samples, were determined by using a computerized log-probit analysis (Harvard Programming; Hg1, 2).
3. Results and discussion
Steam distillation of C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria yielded from 0.77 to 5.72%
(v/w, dry weight) of volatile oils (Table 1). The highest yield of volatile oil was obtained from Z. limonella, whereas
that of F. vulgare was the lowest. All volatile oils obtained were less dense than water, and their physical and
organoleptic properties presented in Table 1 demonstrated differences in appearance, color and odor.
The larvicidal activities of the essential oils against mosquito larvae under laboratory conditions are given in Tables
2 and 3. All plant oils exerted significant larvicidal potential against A. aegypti (Table 2) and A. dirus (Table 3), after
exposure for 24 h. The lethal dosage of 50% (LC50) on A. aegypti larvae ranged between 24.61 and 54.62 ppm and on
A. dirus between 29.69 and 72.28 ppm. Z. limonella oil was the most effective against A. aegypti with LC50 and LC95
values of 24.61 and 55.81 ppm, respectively. A. dirus larvae showed the highest susceptibility to C. zedoaria oil (LC50
and LC95 values: 29.69 and 40.23 ppm, respectively). After exposure to the essential oils, the treated larvae exhibited
restlessness, sluggishness, tremors and convulsions followed by paralysis at the bottom of the bowl. After that, the
number of toxic affected larvae increased, and some if not all of them subsequently died within 24 h. Some A. aegypti
larvae, treated with fennel oil, paralyzed and sank to bottom of the container, and after a short time of exposure
recovered within 47 h. After 24 h, however, some moribund and dead larvae were found.
Finally, ethnobotanical data and chemical composition of the used plants are reported in Table 4. Chemical
identification by gas chromatography coupled with mass spectrometry (GC/MS) revealed the composition of the
investigated oils. Carvone was a main constituent representing 32.7% of C. carvi oil. A principal component found in
essential oils that were derived from A. graveoles and Z. limonella was limonene (60% and 31%, respectively). F.
vulgare oil was found to contain anethole (85.63%) as a major substance, while a high content of 1,8-cineole (18.5%),
p-cymene (18.42%) and -phellandrene (14.93%) was found in C. zedoaria oil. These compounds have been isolated
Table 4
Ethnobotanical data and chemical composition of C. carvi, A. graveolens, F. vulgare, Z. limonella and C. zedoaria
Plants

English
name

Part
used

C. carvi

Caraway Seed

Phytomedicine and pharmacology

Treatment of digestive disorders; laxative on the

duodenum; antiproliferative; antiulcerogenic; antibacterial
A. graveolens Celery
Seed
Treatment of arthritis, nervousness, hysteria, headaches,
bronchitis, asthma, liver and spleen
disease; hepatoprotecive; sedative;
anticonvulsive; anticarcinogen
F. vulgare
Fennel
Fruit
Treatment of dyspeptic complaints, respiratory
disorder, pediatric colic; promoter of
menstruation; hepatoprotecive
Z. limonella Mullilam Fruit
Treatment of wounds; promoter of digestion;
anthelminthic; gastrointestinal stimulant
C. zedoaria
Zedoary Rhizome Treatment of wounds; promoter of digestion; relief of
flatulence and colic; antiseptic; antimicrobial activity

Chemical composition of
plant oil (GC/MS)

References

Carvone (32.7%), limonene

(17.4%), -terpinene (17.2%)
D-Limonene (60%),
selinene (10%), phthalides (3%)

[2125]
[2631]

trans-Anethole (85.63%), estragole [3237]

(5.27%), D-limonene (3.8%),
p-anisaldehyde (2.68%)
D -Limonene (31%), terpin-4-ol [3840]
(13.94%), sabinene (9.13%)
1,8-Cineole (18.5%), p-cymene
[41,42]
(18.42%), -phellandrene (14.93%)

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B. Pitasawat et al. / Fitoterapia 78 (2007) 205210

from many herbal oils [12,19,20] that showed mosquito larvicidal activity. The efficacy of each of these compounds
and their synergistic mechanisms need to be tested, to see if any have a potential use against mosquitoes.
In conclusion, this study clearly illustrated the efficacy of the investigated herbs, which encourages the development
of alternative active ingredients as effective mosquito larvicides.
Acknowledgements
The authors are thankful to the botanist J.F. Maxwell from CMU Herbarium, Department of Biology, Faculty of
Science, Chiang Mai University. Special thanks also go to the Armed Forces Research Institute of Medical Sciences
(AFRIMS) for providing the test mosquito, Anopheles dirus. Acknowledgment is extended to the Faculty of Medicine
Research Fund, Chiang Mai University for their financial support, and the Faculty of Medicine Endowment Fund for
Research Publication for helping defray the publication cost.
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