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Oct 25, 2015 - Seon-Mi Seo,. †. Chan-Sik Jung,. §. Jaesoon Kang,. #. Hyo-Rim Lee,. Γ. Sung-Woong Kim,. §. Jinho Hyun,. Δ,Π and Il-Kwon Park*,Γ...
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Larvicidal and Acetylcholine Esterase Inhibitory Activity of Apiaceae Plant Essential Oils and Their Constituents against Aedes albopictus, and Formulation Development Sun-Mi Seo, Chan Sik Jung, Jaesoon Kang, Hyo Rim Lee, Sung Woong Kim, Jinho Hyun, and Il-Kwon Park J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b03586 • Publication Date (Web): 25 Oct 2015 Downloaded from http://pubs.acs.org on November 3, 2015

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Journal of Agricultural and Food Chemistry

Journal of Agricultural and Food Chemistry

Larvicidal and Acetylcholine Esterase Inhibitory Activity of Apiaceae Plant Essential Oils and Their Constituents against Aedes albopictus, and Formulation Development

Seon-Mi Seo1, Chan-Sik Jung2, Jaesoon Kang3, Hyo-Rim Lee4, Sung-Woong Kim2, Jinho Hyun5,6 and Il-Kwon Park4,6* 1

Lifetree Biotech Co., Ltd. Maesonggosaek-ro, Kwonsun-gu, Suwon, Gyeonggido, 441-813, Republic of Korea; 2Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-712, Republic of Korea; 3Gyeongnam Department of Environmental

Toxicology and Chemistry, Korea Institute of Toxicology, Jin-Ju, Gyeongnam, Republic of Korea; 4Department of Forest Sciences; 5Department of Biosystems and Biomaterials Science & Engineering; 6Research Institute of Agriculture and Life Science, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea

*

To whom correspondence should be addressed (Telephone +82-2-880-4751; Fax

+82-2-873-3560; E-mail [email protected]).

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1

ABSTRACT

2

We evaluated the larvicidal activity of 12 Apiaceae plant essential oils and their components

3

against the Asian tiger mosquito, Aedes albopictus, and the inhibition of acetylcholine

4

esterase with their components. Of the 12 plant essential oils tested, ajowan (Trachyspermum

5

ammi), caraway seed (Carum carvi), carrot seed (Daucus carota), celery (Apium graveolens),

6

cumin (Cuminum cyminum), dill (Anethum graveolens), and parsley (Petroselinum sativum)

7

resulted in >90% larval mortality when used at 0.1 mg/mL. Of the compounds identified,

8

α-phellandrene,

9

cuminaldehyde, neral, (S)-+-carvone, trans-anethole, thymol, carvacrol, myristicin, apiol, and

10

carotol resulted in >80% larval mortality when used at 0.1 mg/mL. Two days after treatment,

11

24.69%, 3.64%, and 12.43% of the original amounts of the celery, cumin, and parsley oils,

12

respectively, remained in the water. Less than 50% of the original amounts of α-phellandrene,

13

1,8-cineole, terpinen-4-ol, cuminaldehyde, and trans-antheole were detected in the water at 2

14

days after treatment. Carvacrol, α-pinene, and β-pinene inhibited the activity of Ae.

15

albopictus acetylcholinesterase with IC50 values of 0.057, 0.062, and 0.190 mg/mL,

16

respectively. A spherical microemulsion of parsley essential oil-loaded PVA (polyvinyl

17

alcohol) was prepared and the larvicidal activity of this formulation was shown to be similar

18

to that of parsley oil.

α-terpinene,

p-cymene,

(-)-limonene,

(+)-limonene,

γ-terpinene,

19 20

Keywords: Apiaceae plant essential oils, larvicidal activity, Asian tiger mosquito,

21

acetylchholine esterase inhibition, residue in water

22 23 24 25

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Journal of Agricultural and Food Chemistry

INTRODUCTION

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The Asian tiger mosquito, Aedes albopictus Skuse, has become increasingly important in

28

public health throughout the world because of its quick spread from its native geographical

29

origin, East Asia. Ae. albopictus also plays an important role in outbreaks of dengue virus

30

(DENV) and chikungunya virus (CHIKV).1 To control the adults and larvae of Ae. albopictus,

31

various synthetic pesticides including insect growth regulators (methoprene, novaluron, and

32

pyriproxifen), organophosphates (temephos), and pyrethroids are widely used around the

33

world.2,3 Although these pesticides are effective, the continued use of synthetic pesticides for

34

several years has resulted in unintended side effects, including environmental and human

35

health concerns and undesirable effects on natural enemies and nontargeted organisms.4,5

36

Recently, Ae. albopictus resistance to larvicides, mainly temephos, has been reported in Asia,

37

Central and South America, and Europe.2,3

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Plant essential oils and their constituents could be good sources for mosquito-controlling

39

agents. Essential oils can be easily extracted with steam distillation, and contain several

40

volatile compounds including alcohols, aldehydes, ketones, esters, aromatic phenols, lactones,

41

monoterpenes, and sesquiterpenes.6 Moreover, plant essential oils are considered safe for

42

humans and are widely used as fragrances and flavoring agents in foods and beverages.7,8 Seo

43

et al.9 recently reported that temephos is about 0.77 million-fold more toxic than ajowan

44

essential oil against water flea, Daphnia magna.

45

Many plant essential oils have been reported to exhibit larvicidal activity against

46

mosquitoes. Leaf and twig essential oils from Clausena excavate showed larvicidal activity

47

against Aedes aegypti and Ae. Albopictus.10 Moreover, the essential oil of Tagetes patula was

48

toxic to the larvae of three mosquito species.11 Similarly, essential oils extracted from Amyris

49

balsamifera, Daucus carota, and Pogostemon cablin demonstrated strong larvicidal activity

50

against Culex pipiens pallens.12 Furthermore, plant essential oils and their constituents have

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been reported to evaporate easily in water; thus, their residues are of little concern.9,12,13

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In this study, we evaluated the larvicidal activity of 12 Apiaceae plant essential oils and we

53

identified the individual compounds from the active oils against the Asian tiger mosquito. In

54

addition, we determined their residue levels in water, and verified feasibility of formulation for

55

field application. Because many phytochemicals have been known to inhibit acetylcholine

56

esterase,14 we quantitated their abilities to inhibit acetylcholine esterase to investigate their mode

57

of action.

58 59

MATERIALS AND METHODS

60

Chemicals. Myrcene (95%), (+)-limonene (97%), dihydrocarvone (98%), carveol (97%),

61

cuminaldehyde (98%), (S)-(+)-carvone (96%), (R)-(-)-carvone (>98%), (-)-α-pinene (99%),

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(-)-β-pinene (99%), 1,8-cineole (99%), and trans-anethole (99%) were purchased from

63

Sigma-Aldrich (Milwaukee, WI, USA). α-Terpinene (85%), p-cymene (95%), γ-terpinene

64

(97%), linalool oxide (97%), menthol (99%), terpinen-4-ol (97%), and thymol (>99%) were

65

purchased from Fluka (Buchs, Switzerland). (+)-α-Pinene (>95%), camphene (80%),

66

β-caryophyllene (>90%), β-pinene (94%), α–phellandrene (65%), (-)-limonene (95%), bornyl

67

acetate (70%) and carvacrol (95%) were purchased from Tokyo Kasei (Tokyo, Japan).

68

Myristicin (99%), elemicin (99%), allytetramethoxybenzene (99%) and apiol (99%) were

69

isolated from parsley oil. Carotol (99%) were isolated from carrotseed oil. Silicagel was

70

purchased from Merck (0.006-0.2mm). Neral (98%) was synthesized in the laboratory. Values

71

in parentheses indicate the purities of the compounds.

72

Plant Essential Oils. The plant essential oils used in this experiment are listed in Table

73

1. Essential oils were purchased from Jinarome (NY, USA, www.jinarome.com) and Oshadhi

74

Ltd. (Cambridge, UK).

75

Insects. Ae. albopictus cultures were maintained in the laboratory, without exposure to

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any insecticides. Ae. albopictus adults were maintained on a 10% sugar solution. A live

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mouse in a steel cage was supplied for blood under a Korea National Institute of Health

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Institutional Animal Care and Use Committee (KCDC-020-11-2A) protocol approved for this

79

study. Larvae were reared in plastic pans (24 × 35 × 5 cm) containing sterilized food and

80

water. Colonies were reared at 26 ± 1 oC, with a relative humidity of 60 ± 5%, under a 16:8 h

81

light:dark cycle.

82

Gas Chromatography. Gas chromatography (GC) analysis of ajowan, dill, celery,

83

caraway, and cumin oils was performed using an Agilent 7890N system (Santa Clara, CA,

84

USA) equipped with a flame ionization detector (FID). The retention times of the compounds

85

were compared with those of authenticated compounds using DB-1MS and HP-INNOWAX

86

columns (30 m × 0.25 mm i.d., film thickness: 0.25 µm, J&W Scientific). The oven

87

temperature was programmed to be isothermal at 40 oC for 1 min, raised to 250 oC at a rate of

88

6 oC/min, and held at this temperature for 4 min. Helium was used as the carrier gas at a rate

89

of 1.5 mL/min. The configurations of limonene, α-pinene, and β-pinene were determined using

90

a chiral Beta DEX 120 column (30 m × 0.25 mm i.d., film thickness: 0.25 µm, Supelco,

91

Bellefonte, PA, USA). The oven temperature was maintained at 100 oC for 20 min, and the

92

flow rate of the carrier gas was 1.0 mL/min. A Beta DEX 225 column (30 m × 0.25 mm i.d.,

93

film thickness: 0.25 µm, Supelco, Bellefonte, PA, USA) was used for the separation of

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carvone. The oven temperature was programmed to be isothermal at 130 oC for 10 min and

95

then raised to 200 oC at a rate of 10 oC/min. The carrier gas had a flow rate of 1.0 mL/min.

96

The retention indices were obtained in relation to a homologous series of n-alkanes (C7-C20),

97

under the same operating conditions as for the GC-FID analysis. The components were

98

further identified by enhancing the integrated area by co-injection with the essential oil and

99

authentic samples. The oil components were then quantified by adding 2 internal standards

100

undecane (purity: 99%; Wako) and pentadecane (purity: 99%; Wako), without the use of any

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correction factors.

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Gas Chromatography-Mass Spectrometry. A gas chromatograph (Agilent 7890A) and

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a mass spectrometer (Agilent 5975C MSD) were used for gas chromatography-mass

104

spectrometry (GC-MS) analysis using a DB-5MS column (30 m × 0.25 mm i.d., film

105

thickness: 0.25 µm, J&W Scientific). The oven temperature was the same as that used for the

106

GC-FID analysis. The flow rate of the carrier gas (helium) was 1.0 mL/min. The GC column

107

effluent was introduced directly into the MS source via a transfer line at 250 oC. Ionization

108

was obtained by electron impact (70 eV, source temperature: 230 oC), and the scan range was

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41-400 amu. Most of the essential oil components were identified by comparing the mass

110

spectra of each peak with those of authenticated samples obtained from the NIST MS library.

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Isolation of Myristicin, Apiol, Allytetramethoxybenzene, Elemicin, and Carotol.

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The bioassay-guided isolation procedure of active compounds from parsley and carrot seed

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oils is shown in Figure 1. Small amounts of parsley (40 g) and carrot seed (1 g) oils were

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subjected to SiO2 gel column chromatography (hexane/diethyl ether 100/0 → 0/100).

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Myristicin (5.5 g), apiol (140 mg), allytetramethoxybenzene (275 mg) and elemicin (190 mg)

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were isolated from parsley oil, whereas carotol (0.8g) was isolated from carrot seed oil. The

117

isolation

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allytetramethoxybenzene (purity 99%) and elemicin (purity 99%) is shown in Figure 1. Pure

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myristicin, apiol, allytetramethoxybenzene, and elemicin were isolated from the

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hexane/diethyl ether (100/1), hexane/diethyl ether (100/2), hexane/diethyl ether (100/3), and

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hexane/diethyl ether (100/4) fractions, respectively. Carotol (purity 99%) was isolated from

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the hexane/diethyl ether (90/10) fraction and used for bioassays. NMR spectra were obtained

123

on a Varian UI 500 NMR spectrometer (500 MHz for 1H spectra and 125 MHz for

124

spectra) at the Korean Basic Science Institute using TMS as an internal standard.

125

procedure

used

for

myristicin

(purity

99%),

apiol

(purity

99%),

13

C

Larvicidal Activity Test. A larvicidal activity test was performed as previously

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described.15 Briefly, Apiaceae plant essential oils and their constituents were serially diluted

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from an initial 0.01% (weight/volume) stock solution prepared in acetone. One milliliter of

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each oil or compound was suspended in 200 mL of water in 270 mL paper cups. Ten early

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third instar Ae. albopictus larvae were transferred individually into the cup using a glass

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pipette. A separate set of cups that received 1 mL of acetone only served as the controls.

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Treated and control larvae were maintained at the same conditions used for colony

132

maintenance, and larval mortality was investigated 48 h after treatment. Larvae were not

133

provided with food during bioassay. All treatments were replicated 4 times.

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Residues in Water. To quantitate the residues of essential oils and their constituents in

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water, the essential oils and their components were dissolved in acetone and added to a glass

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beaker (250 mL) filled with distilled water (200 mL). The initial concentration of all essential

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oils and their constituents was 0.1 mg/mL. One milliliter of a test solution was extracted at

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days 2 and 7 for quantitation of the remaining residues of Apiaceae plant oils and their

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constituents. The sampled solutions were stirred and mixed with a small amount of NaCl, and

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the solution was extracted with 2 mL of hexane containing undecane as an internal standard.

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The concentrations of undecane were adjusted to 0.01 mg/mL and 0.001 mg/mL for test

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solutions extracted on days 2 and 7, respectively. The extracted solution was directly

143

analyzed by GC-FID, with the same oven temperature as that used for the GC-FID analysis.

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Oil residues were quantified by comparing the total areas of all constituents with that of the

145

IS.

146

Acetylcholinesterase Inhibition. Fifty mosquito larvae were collected randomly and

147

used to generate crude protein extracts. Mosquito larvae were ground in 0.1 M Tris-HCl

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supplemented with 0.02 M NaCl, 0.5% Triton X-100 (pH 7.8), and a protease inhibitor

149

cocktail (Sigma-Aldrich, St. Louis, MO, USA) using a glass tissue grinder (Wheaton

150

Industries Inc., Millville, NJ, USA) on ice. The resultant extract was spun by centrifugation at

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17,000 ×g for 15 min at 4 °C to eliminate insect tissue debris and the supernatant containing

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soluble protein was collected. The protein content of each extract was quantified by the

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Bradford protein assay. The ability of each extract to inhibit AChE activity was evaluated

154

using the modified Ellman method.16 Briefly, each test chemical was completely dissolved in

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acetone (Sigma-Aldrich) to a concentration of 100 mg/mL and the protein was diluted to 0.2

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µg/µL in 0.1 M Tris-HCl referred as above. Reaction mixtures consisted of 1 µL of test

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compound and 79 µL of protein and were placed in 96-well microplates. Reactions were

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incubated for 10 minutes at room temperature; control reactions received acetone only. 10 µl

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of

160

5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) were added to the reaction mixtures. AChE

161

activity was evaluated by measuring the absorbance at 405 nm at 30 sec intervals for 20

162

minutes at RT using an iMark microplate absorbance reader (Bio-Rad, Hercules, CA, USA).

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The values were used to calculate the initial velocity (Vo) for each reaction. All experiments

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were performed at least in triplicate. The inhibition rate of each chemical was calculated

165

according to the following formula:

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10

mM

acetylthiocholine

iodide

(ASChI)

and

10

µL

of

4

mM

Inhibition activity (%) = 100 – [(Vo of chemical treatment/Vo of control treatment) ×

167

100]

168 169

To determine the IC50 values of α-pinene, β-pinene, and carvacrol, the following

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concentrations were used: 1, 0.5, 0.2, 0.1, and 0.05 mg/mL. Three replicates were performed

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for each treatment at each concentration. The rates of AChE inhibition were calculated

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according to the formula described above, and the IC50 values were determined by probit

173

analysis.17

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Microemulsion of Parsley Oil with Polyvinyl Alcohol. For field application of plant

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essential oils as control agents for Ae. albopictus larvae, it is necessary to develop the

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appropriate formulations. A microemulsion of parsley essential oil in water was prepared by

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adding 100 µL of essential oil to 900 µL of 1 wt% PVA (polyvinyl alcohol, 15000 Da,

178

Sigma-Aldrich) solution in deionized water. Next, the mixture was homogenized at 65 W for

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1 min in a VCX130 apparatus (Sonic & Materials, USA). The parsley essential oil

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microemulsion was stored in a refrigerator until further use. The microemulsion was

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observed with a dark-field microscope (Nikon ECLIPSE LV 100, Nikon Instruments Inc,

182

USA). The larvicidal activity of the parsley essential oil microemulsion was investigated as

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described above. Parsley oil and polyvinyl alcohol were used for as positive and negative

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controls, respectively.

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Statistical Analysis. The percentages of mortality of Ae. albopictus mosquito larvae

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were transformed to arcsine square-root values prior to analysis of variance (ANOVA).

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Treatment mean values were compared and separated using Scheffe’s test.17 Mean (±SE)

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values of untransformed data have been reported.

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RESULTS AND DISCUSSION

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Larvicidal Activities of Plant Essential Oils. The larvicidal activities of the various

192

plant essential oils against Ae. albopictus are listed in Table 1. Among the plant essential oils

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tested, dill, celery, caraway seed, cumin, carrot seed, parsley, and ajowan essential oils

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showed strong larvicidal activity against Ae. albopictus (≥90% mortality) at a concentration

195

of 0.1 mg/mL. The larvicidal activities of ajowan, parsley, and celery essential oils were 80%,

196

60%, and 60%, respectively, at a concentration of 0.05 mg/mL. All other oils yielded less

197

than 50% mortality. Plant essential oils belonging to the Apiaceae family have been shown to

198

have insecticidal activity against German cockroaches (Blattella germanica)18 and rice

199

weevils (Sitophilus oryzae)19. Dill, caraway, and cumin oils have also demonstrated strong

200

fumigant toxicities against German cockroaches and rice weevils. However, celery, carrot

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seed, parsley, and ajowan oils have not shown to have strong fumigant toxicities against

202

German cockroaches or rice weevils. In this study, dill, celery, caraway, cumin, carrot seed,

203

parsley, and ajowan oils exhibited strong insecticidal activity against Ae. albopictus. We used

204

the direct contact method to investigate the larvicidal activities of Apiaceae plant essential

205

oils against Ae. albopictus. Thus, the insecticidal activities of celery, carrot seed, parsley, and

206

ajowan might be attributed mainly to the direct contact of the oils with the larval cuticle.

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Chemical Analysis of the Active Oils. The chemical compositions of the ajowan, cumin,

208

dill, celery, and caraway oils are given in Table 2. We previously performed a detailed

209

chemical analysis of ajowan, cumin, dill, and caraway oils,18 thus, in the present study we

210

analyzed optical isomer of α-pinene, β-pinene, carvone, and limonene. The α-pinene and

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carvone isomers in dill oil were identified as (+)-α-pinene and (S)-(+)-carvone, whereas

212

limonene consisted of both (+)-limonene (20.21%) and (–)-limonene (0.27%). Only

213

(+)-α-pinene, (+)-β-pinene, and (+)-limonene were identified in cumin oil. (S)-(+)-carvone

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(48.7%) was the most abundant compound in caraway oil, followed by (+)-limonene (24.2%),

215

cis-carveol (0.4%), and trans-carveol (0.3%). Two optical isomers of limonene were

216

identified in ajowan oil, (+)-limonene and (–)-limonene at 0.36% and 0.08%, respectively.

217

The chemical compositions of celery, carrot seed, and parsley essential oils have previously

218

been reported by other groups. Kiralan et al.20 analyzed the chemical compositions of parsley

219

and celery essential oils. The most abundant compound of parsley oil was α-pinene (22.89%),

220

followed

221

allyltetramethoxybenzene (13.56%), myristicin (7.45%), and elemicin (2.11%). Limonene

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(76.63%) and α-selinene (11.12%) were the main compounds of celery oil, with all other

223

compounds present at less than 2%. In our analysis, the most abundant compound in parsley

224

oil was 1,3,8-p-menthatriene (18.5%), followed by α-pinene (17%), myristicin (14.2%),

225

β-pinene (11.08%), 1,8-cineole (10.09%), β-myrcene (4.01%), allyltetramethoxybenzene

by

β-pinene

(19.16%),

elemicin

(13.56%),

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(11.27%),

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(0.36%), elemicin (0.5%), and apiol (0.36%). Limonene (69.66%) was identified as the most

227

abundant compound in celery oil, followed by β-pinene (1.09%), β-myrcene (0.84%), and

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β-caryophyllene (0.69%). Carotol (86.29%) was the only component identified in carrot seed

229

oil. The chemical composition of the essential oil of a single species can vary according to

230

cultivation region, date of harvest, storage time, extraction method, edaphic factors, and

231

climatic factors.21

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Isolation of Myristicin, Apiol, Allytetramethoxybenzene, Elemicin, and Carotol. To

233

isolate the active compounds that are not commercially available from parsley and carrot seed

234

oils, we used open column chromatography coupled with bioassay-guided isolation (Figure 1).

235

Four compounds were isolated from the active fraction of parsley seed oil, whereas one

236

compound was isolated from the active fraction of carrot seed oil. Each isolated compound

237

was identified by 1H- and

238

13

239

consistent with previous reports.12,22-25

13

C-NMR, DEPT, 1H-1H COSY, and HMQC. The 1H- and

C-NMR data of carotol, allytetramethoxybenzene, apiol, elemicin, and myristicin were

240

Larvicidal Activities of Essential Oil Constituents. The larvicidal activities of the

241

various essential oil constituents are shown in Table 3. Of the compounds tested,

242

α-phellandrene, α-terpinene, p-cymene, (-)-limonene, (+)-limonene, γ-terpinene, thymol,

243

carvacrol, myristicin, apiol, and carotol resulted in ≥95% mortality of Ae. albopictus larvae at

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a concentration of 0.1 mg/mL. Cuminaldehyde, neral, and (S)-(+)-carvone yielded 82.5%

245

mortality. At 0.05 mg/mL, myristicin, carotol, and carvacrol yielded 92.5%, 90%, and 80%

246

mortality, respectively. γ-Terpinene and (+)-limonene resulted in 77.5% and 70% mortality,

247

respectively. All other compounds yielded