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December, 2004 Journal of Vector Ecology 340

Potential of crude seed extract of celery, Apium graveolens L., against the

mosquito Aedes aegypti (L.) (Diptera: Culicidae)

Wej Choochote, Benjawan Tuetun, Duangta Kanjanapothi1, Eumporn Rattanachanpichai,

Udom Chaithong, Prasong Chaiwong, Atchariya Jitpakdi, Pongsri Tippawangkosol,

Doungrat Riyong, and Benjawan Pitasawat

Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

1Chulabhorn Research Institute, Chiang Mai 50200, Thailand

Received 16 April 2004; Accepted 14 May 2004

ABSTRACT: Crude seed extract of celery, Apium graveolens, was investigated for anti-mosquito potential, including

larvicidal, adulticidal, and repellent activities against Aedes aegypti, the vector of dengue haemorrhagic fever. Theethanol-extracted A. graveolens possessed larvicidal activity against fourth instar larvae of Ae. aegypti with LD

50

and LD95

values of 81.0 and 176.8 mg/L, respectively. The abnormal movement observed in treated larvae indicated

that the toxic effect of A. graveolens extract was probably on the nervous system. In testing for adulticidal activity,

this plant extract exhibited a slightly adulticidal potency with LD50

and LD95

values of 6.6 and 66.4 mg/cm2,

respectively. It showed repellency against Ae. aegypti adult females with ED50

and ED95

values of 2.03 and 28.12

mg/cm2, respectively. It also provided biting protection time of 3 h when applied at a concentration of 25 g%.

Topical application of the ethanol-extracted A. graveolens did not induce dermal irritation. No adverse effects on

the skin or other parts of the body of human volunteers were observed during 3 mo of the study period or in the

following 3 mo, after which time observations ceased. A. graveolens, therefore, can be considered as a probable

source of some biologically active compounds used in the development of mosquito control agents, particularly

repellent products. Journal of Vector Ecology 29 (2): 340-346. 2004.

Keyword Index: Apium graveolens, Aedes aegypti, larvicidal, adulticidal, repellent.

INTRODUCTION

Aedes aegypti (L.) is generally known as a vector

for an arbovirus responsible for dengue fever, which is

endemic to Southeast Asia, the Pacific island area, Africa,

and the Americas. This mosquito is also the vector of

yellow fever in Central and South America and West

Africa. Dengue fever has become an important public

health problem as the number of reported cases continue

to increase, especially with more severe forms of the

disease, dengue haemorrhagic fever and dengue shock

syndrome, or with unusual manifestations such as central

nervous system involvement (Hendarto and Hadinegoro

1992, Pancharoen et al. 2002). About two-fifths of the

world’s population are now at risk of catching dengue

according to the World Health Organization (WHO

2003). At present, the most successful measures to

decrease the incidence of this disease are by personal

protection and control of the vector, Ae. aegypti.

Although there has been much historical research

on the potential of natural plants to protect against

mosquitoes and other insect pests (Granett 1940), the

interest in herbal-based products was subsequently

reduced due to the advent of synthetic chemicals.

However, the interest in anti-mosquito products derived

from natural origin is being revived because the

continued applications of synthetic compounds have

some drawbacks, including the widespread development

of insecticide resistance.

Celery, Apium graveo lens (Umbelliferae),

commonly known in Thailand as “Khuen chaai,” is a

native of Eurasia and is now grown and consumed all

over the world. Leaf stalks and celery seed are used as a

popu lar aromat ic he rb and sp ice (Raf ikal i and

Muraleednaran 2001, Kitajima et al. 2003). Certain

bioactive compounds derived from A. graveolens seeds

have been proven to possess nematocidal activity against

Caenorhabditis elegans and Panagrellus redivivus,

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antifungal activity against Candida albican, C. kruseii

and C. parapsilasis, and mosquitocidal effects against

Ae. aegypti fourth-instar larvae (Rafikali et al. 2000,

Rafikali and Muraleednaran 2001). The literature,

however, offers no data about the adulticidal and repellent

activities of this plant against mosquito vectors. The aimof this work was to investigate the possible anti-mosquito

potential of A. graveolens extract against Ae. aegypti,

the major vector of dengue haemorrhagic fever in

Thailand (Limrat 1997, Saengtharatip 1997), in the

search for an alternative natural product that can be

developed and practically used in the control of recurrent

dengue epidemics.

MATERIALS AND METHODS

Plant extract

Seeds of celery, Apium graveolens, were obtained

from E.A.R. Samunpri, a commercial supplier in ChiangMai province, Thailand. A voucher named PARA-AP-

001 was deposited at the Department of Parasitology,

Faculty of Medicine, Chiang Mai University, Thailand.

Dried and powdered material of this plant (2 kg) was

successively extracted three times by maceration, with

3 L of 95% ethanol at room temperature for 2 d. An

ethanolic extract was suction filtered through a Buchner

funnel and the combined filtrates were concentrated by

a rotary evaporator at 60°C until the solvent completely

evaporated. The residue of ethanol-extracted A.

graveolens was thus obtained, lyophilized, and then kept

at -20°C until testing for larval and adult toxicities and

repellent activity. In preparing test concentrations, the

lyophilized A. graveolens extract was volumetrically

diluted in absolute ethanol and/or Tween 80 at an

appropriate test concentration.

Test mosquitoes

Aedes aegypti larvae, which were derived from

various places with clean stagnant water within Chiang

Mai province, northern Thailand, were colonized and

maintained continuously for several generations since

1997 in a laboratory free of exposure to pathogens,

insecticides, or repellents. The laboratory colony was

maintained at 25-30°C and 80-90% relative humidityunder a photoperiod of 14:10 h (light/dark) in the

insectary of the Department of Parasitology, Faculty of

Medicine, Chiang Mai University, Chiang Mai province.

Under these conditions, the full development from egg

to adult lasted about 3-4 wk. Larvae were fed on finely-

ground dog biscuit. The adult colony was provided with

10% sucrose and 10% multivitamin syrup, and it was

pe riod ical ly blood- fed on rest ra ined ra ts . Two

developmental stages, larvae and adult females, were

continuously available for the experiments.

Human volunteers

Healthy volunteers of both sexes (aged 16-50 y;

weight 43-65 kg), with no history of allergic reactions to

arthropod bites, were recruited from the students andstaff of the Department of Parasitology, Faculty of

Medicine, Chiang Mai University, and they gave written

informed consent before participating in repellent

bioassays. The repellent study was reviewed and

approved by the Research Ethics Committee of the

Faculty of Medicine, Chiang Mai University.

Larvicidal bioassay

The larvicidal bioassay followed the WHO standard

protocols (WHO 1981a) with slight modifications. For

experimental treatment, one ml of A. graveolens extract

dissolved in absolute ethanol was added to 224 ml of

distilled water in a 500-ml enamel bowl, which wasshaken lightly to ensure a homogeneous test solution.

Then 25 early fourth instar larvae of Ae. aegypti in 25

ml of distilled water were transferred to that bowl. Each

experiment was performed in 4 replicates with a final

total of 100 larvae for each concentration. The control

solution was made with 1 ml of ethanol mixed with 249

ml of distilled water, while the untreated solution

contained 250 ml of distilled water only. Symptoms of

treated larvae were observed and recorded immediately

and at timed intervals, and no food was offered to the

larvae. Mortality and survival were registered after 24 h

of the exposure period. The moribund and dead larvae

in four replicates were combined and expressed as a

percentage of larval mortality of each concentration.

Dead larvae were identified when they failed to move

after probing with a needle in the siphon or cervical

region. Moribund larvae were those incapable of rising

to the surface (within a reasonable period of time) or

showing the characteristic diving reaction when the water

was disturbed. They might also show discoloration,

unnatural positions, tremors, uncoordination, or rigor.

All surviving larvae were separately reared and

maintained at 25-30°C and 80-90% relative humidity in

the insectary. Pupation and adult emergence of these

mosquitoes were recorded. The assays were terminated3 d after the last control mosquito emerged. The

experiments were replicated four times.

Adulticidal bioassay

The adulticidal effect of the ethanol-extracted A.

graveolens was assayed following a slightly modified

version of the WHO standard method (WHO 1981b) of

insecticidal deposits on a paper surface. Aliquots of 1.9

ml of 1:2 preparation of appropriate concentrations of

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342 Journal of Vector Ecology December, 2004

ethanol-extracted A. graveolens in Tween 80 or Tween

80 only (for the control group) and ether mixture were

applied to Whatman # 1 filter paper (12x15 cm). The

impregnated paper was hung under a shade overnight,

by which time all the ether had evaporated and the

ethanol-extracted A. graveolenssolution had been spreadevenly. The blood-deprived 5-7-d-old Ae. aegypti

females were then collected and gently transferred to

plastic holding tubes at 25 mosquitoes per tube. The

mosquitoes were allowed to acclimatize in the holding

tube for 1 h before being transferred to a plastic exposure

tube lined with the ethanol-extracted A. graveolens

impregnated paper, and they were exposed for 1 h. At

the end of the exposure period, the mosquitoes were

transferred back to the holding tube, which was kept for

24 h. A pad of cotton wool soaked with 10% sucrose

and 10% multivitamin syrup was placed on the end of

the mesh screen. Four replicates were run for each

concentration, yielding a final total of 100 mosquitoes.Percentage mortality was observed 24 h later and

mortality was determined when the mosquitoes did not

respond to mechanical stimulation. All treatments were

replicated four times at 25-30°C.

Repellent bioassay

Two different treatment methods (dose-response

study and protection time determination) were used to

determine the repellent activity of the ethanol-extracted

A. graveolens against laboratory-reared Ae. aegyptiafter

they were applied to human skin. In the dose-response

test, the procedure for determining effective dosages of

the plant extract against hungry mosquitoes was amodification of the American Society for Testing and

Materials Standard ED 951-83 (ASTM 1983). Tests

were based on the variable dose-fixed time, “free choice

method” described by Buescher et al. (1982) and were

similar to the method described by Coleman et al. (1993,

1994). Because Ae. aegypti is a day biter, the timing of

tests was between 08.00 h and 16.00 h. Evaluations were

carried out in a 10x10x3 m room, at 25-30°C, and relative

humidity of 60-80%. Five circles (29 mm in diameter)

were outlined on the ventral surface of the volunteer’s

forearm using a plastic template and permanent marker.

Applications of 25 ml of the diluent (control) and four

serial dilutions of the ethanol-extracted A. graveolens in

absolute ethanol were applied randomly on the marked

areas. After air drying for 5 min, a plastic cage (4x5x18

cm), divided into 5 compartments, was secured over the

area with rubber bands. Each compartment of the plastic

cage, with a matching cutout on its floor, contained 10

blood-starved 5-7-d-old Ae. aegypti females. The number

of mosquitoes biting on each test site was recorded each

minute for 5 min. Tests were carried out three times on

each repellent-treated area and completed within 25 min

of repellent application. The experiments were conducted

four times on each subject of four human volunteers (2

females, 2 males).

Laboratory repellent tests were also conducted to

determine the repellent protection time using the human- bait technique of the WHO (1996) standard method, with

some modifications. A plastic sleeve was wrapped around

each forearm, with a hole cut in alignment with a 3x10

cm area on the inside part of the forearm, and attached

with double-sided tape. Thus, only a restricted area of

skin was exposed to the mosquitoes, with the hand being

protected with a rubber glove. Approximately 0.1 ml of

25 g % ethanol-extracted A. graveolens dissolved in

absolute ethanol was spread as evenly as possible on the

30 cm2 test area of one forearm of each volunteer. The

other forearm, acting as a control, was treated with

absolute ethanol by the same procedure as that for the

test repellent. After air drying for 1 min, the test armwas put into a 30x30x30 cm3 cage containing 200 unfed

mosquitoes for 3 min. The mosquitoes that landed and

attempted to probe and imbibe any blood were recorded.

If no mosquito bites occurred in the initial 3 min, the

arm was withdrawn from the cage and re-tested every

30 min. The period of repellent protection was then

calculated as the time between the extract application

and the time when at least two mosquitoes bit in the same

3-min exposure or the time when only one mosquito bit

in one exposure period if another bit in the next exposure

period (30 min later). When no confirming bites were

observed in the period after the initial bite, the treated

arm resumed the test until a confirming bite was recorded.

During the experiment, successive introductions of the

control arm were made in the same manner, prior to

inserting the treated arm, in order to confirm the readiness

of the mosquitoes to bite. The same test was repeated on

each subject of 4 human volunteers (2 females, 2 males).

The median complete-protection time was used as a

standard measure of the extract repellency against adult

female Ae. aegypti in the laboratory.

Data management and statistical analysis

It was important to obtain not less than 3 mortality

(repellency) counts of between 10% and 90%. In caseswhere the control mortality (larvicidal and adulticidal

tests) or control non-biting (repellent test) was between

5-20%, the observed percentage mortality (%M) or

repellency (%R) was corrected by Abbott’s formula

(Abbott 1925):

%M (%R) =

% test mortality

(non-biting)

% control mortality

(non-biting)

100 - % control mortality

(non-biting)

-X 100

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Data for the anti-mosquito potential (larvicidal,

adulticidal, and repellent effects) were analyzed by means

of computerized probit analysis (Harvard Programming;

Hg1, 2), yielding a level of effectiveness at 50% and

95% mortality or repellency, and 95% confidence

intervals (95% C.I.).

RESULTS AND DISCUSSION

Ethanolic extract of A. graveolens, with a yield of

1.14% (w/w), was semi-solid, light brownish, and slightly

aromatic. Larvicidal activity of this plant extract against

fourth instar larvae of Ae. aegypti is shown in Table 1.

The susceptibility of Ae. aegypti to serial dilutions of

the ethanol-extracted A. graveolens was dose dependent.

Increasing the plant extract level from 40 to 120 mg/L

increased the larval mortality range from 3 to 100%. High

mortality (> 50% mortality) values were observed at 80

to 120 mg/L. No mortality was observed in control or untreated groups. Mortality of 93-100% was observed

in the highest concentration, 120 ppm of the ethanol-

extracted A. graveolens. At the lowest concentration, 40

mg/L, there was very low mortality (3-5%). However,

in contrast to the control and untreated groups (pupation

rate = 98-100%, emergence rate = 96-98%), many

surviving larvae derived from the concentration of 40

mg/L failed to pupate (6.2-11.3%) and emerge as adults

(11.6-13.2%). The ethanol-extracted A. graveolens

showed promising larvicidal activity with LD50

and LD95

values of 81.0 and 176.8 mg/L, respectively.

Observations carried out through the exposure

period at room temperature revealed that immediately

after exposure to ethanol-extract A. graveolens solution,

all larvae were still active and exhibited a normal

appearance with the siphon pointed up and head hung

down. The process of larval feeding, both collecting-

filtering in the water column and collecting-gathering at

submerged surfaces, were clearly seen. Between 5 and

10 min after treatment, some of the larvae became restless

and frequently sank down and floated up quickly. At 15

min, the restlessness persisted, and tremor and convulsion

at the bottom of the container were observed in

approximately 2-3 larvae. Similar evidence of

restlessness, tremors, and convulsions followed by paralysis was clearly seen at 45 min in approximately 4-

5 larvae. At 1 h, approximately 1-2 moribund and dead

larvae were found. For as long as 4 h after treatment,

approximately one-third of the larvae were paralyzed

and sank to the bottom of the bowl. More and more larvae

exhibited toxic symptoms during 5 to 6 h. Subsequently,

all of them died within 7 h in the 120 mg/L treatment.

The ethanol-extracted A. graveolens did not cause rapid

mortality, suggesting a delayed type of larval killing

property. The symptoms observed in treated larvae were

similar to those caused by nerve poisons, i.e. excitation,

convulsions, paralysis, and death.

The ethanol-extracted A. graveolens showed

adulticidal activity against Ae. aegypti with LD50

and

LD95 values of 6.6 and 66.4 mg/cm2

, respectively. Theextract was found to cause a mosquito knockdown (the

rapidly and normally reversible paralysis) after exposure

to varying concentrations of A. graveolens impregnated

paper. Following exposure to 3.5 to 10.6 mg/cm2 for 5-

15 min, almost all mosquitoes showed signs of paralysis,

i.e. unable to walk and lay at the bottom of the exposure

tube. At the end of a one h exposure period, all

mosquitoes became inactive. Nevertheless, when

transferred from the exposure tube to the holding tube,

approximately one-third of the adults recovered within

1 h. The subsequent record at the end of a 24 h holding

period revealed mortality values ranging from 16 to 76%.

It was also apparent that mortality in adults was dosedependent.

Although the insecticidal potency of the crude seed

extract of A. graveolens against both larva and adult

mosquitoes was lower than that of other plant extracts

against Ae. aegypti and other mosquito species obtained

in comparable laboratory conditions (Pitasawat et al.

1998, Choochote et al. 1999, Mansour et al. 2000, Yang

et al. 2002, 2003), its probable nerve poison to

mosquitoes was certainly not negligible. Rafikali et al.

(2000) isolated 4 compounds from the hexane extract of

celery seed. The first three compounds, b-selinene, 3-n-

bu ty l-4, 5-dihydrophtha lide , and 5-al ly l-2-

methoxyphenol were reported to have a pronounced

larvicidal potential with 100% mortality of fourth instar

Ae. aegypti at 50, 25, and 200 µg/ml, respectively, while

the last one, 1,3-Di [(cis)-9-octadecenoyl]-2-[(cis,cis)-

9-12-octadecadienoyl] glycerol was not biologically

active. Additionally, the knockdown effect on adult

mosquitoes obtained from the present adulticidal

bioassay was very interesting for further repellent study.

In the repellent study, the ethanol-extracted A.

graveolenspossessed significant repellent effect against

Ae. aegypti adults on human volunteers, as shown in

Table 3. The ED50

and ED95

values were 2.03 and 28.12

mg/cm2

, respectively. It also provided a mediancomplete-protection time of 3.0 (2.5-3.0) h against Ae.

aegypti bites when applied at a concentration of 25 g%.

No skin irritation, hot sensations, or rashes were observed

during the 3 mo of the study period or in the following 3

mo, after which time observations ceased, although

dermatitis and phototoxic skin reactions from celery in

some farm or grocery store workers were reported

(Palumbo and Lynn 1953, Seligman et al. 1987).

However, the toxicity of this substance under long-term

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Table 2. Adulticidal activity of the ethanol-extracted A. graveolens against Ae. aegypti.

A. graveolens % Mortality Adulticidal activity

(mg/cm2) (Mean±SE) (95% C.I., mg/cm2)

LD50

LD95

1.8 17.2±1.3 6.6 66.4

3.5 32.0±2.9 (5.6-8.1) (38.0-173.0)

7.0 42.8±4.0

10.6 71.5±3.7

Control 0

Table 3. Initial repellency of the ethanol-extracted A. graveolens against adult female Ae. aegypti.

A. graveolens % Mosquitoes repelled Repellency (mg/cm2)

(mg/cm 2) (Mean±SE) (95% C.I.)

ED50

ED95

1 31.89±1.70 2.03 28.12

2 48.71±3.25 (1.63-2.50) (16.05-75.53)

4 66.38±2.46

6 78.66±3.89

Control 0

Table 4. Repellency (median complete-protection time) of the ethanol-extracted A. graveolensagainst adult female

Ae. aegypti.

Treatment Median complete-protection time (Range, h)

Ethanol-extracted A. graveolens (25 g%) 3.0 (2.5-3.0)

Control (ethanol) 0

Table 1. Larvicidal activity of the ethanol-extracted A. graveolens against fourth instar Ae. aegypti.

A. graveolens % Mortality Larvicidal activity

(mg/L) (Mean±SE) (95% C.I., mg/L)

LD50 LD95

40 3.5±1.0 81.0 176.8

50 13.0±2.2 (71.3-98.2) (129.4-443.7)

60 27.8±1.5

80 44.0±12.8

100 71.2±7.4

120 96.2±2.4

Control 0

Untreated 0

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usage has not yet been investigated and characterized in

humans. Because two h is the minimum protection time

allowed for the sale of mosquito repellents in Thailand,

ethanol-extracted A. graveolens, which provides a

protection time of 2.5-3.0 h, is considered a satisfactory

candidate for improving the practicality and extensionof marketable formulations of repellent. This plant is

cheap and widely cultivated in rural areas, consequently

its commercial exploitation would contribute toward

rural economic development.

In conclusion, A. graveolens offers potential against

Ae. aegypti, particularly in its markedly repellent effect.

Further studies of the active principles involved and their

mode of action, formulated preparations for enhancing

potency and stability, toxicity and effects on non-target

organisms and the environment, and field trials are

needed to recommend A. graveolens as an anti-mosquito

product used to combat and protect from mosquitoes in

a control program.

Acknowledgments

The authors are very grateful to the individuals who

served as subject volunteers and also the staff members

of the Department of Parasitology, Faculty of Medicine,

Chiang Mai University for their cooperation.

Acknowledgment is extended to the Faculty of Medicine

Endowment Fund for its financial support and the Faculty

of Medicine Endowment Fund for Research Publication

for helping to defray the publication cost.

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