Insecticidal and Acetylcholine Esterase Inhibition Activity of ...

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Insecticidal and Acetylcholine Esterase Inhibition Activity of Asteraceae Plant Essential Oils and Their Constituents against Adults of the German Cockroach (Blattella germanica) Hwa-Jeong Yeom,†,‡ Chan-Sik Jung,†,‡ Jaesoon Kang,§ Junheon Kim,∥ Jae-Hyeon Lee,⊥ Dong-Soo Kim,# Hyun-Seok Kim,¶,□ Pil-Sun Park,¶,□ Kyu-Suk Kang,¶,□ and Il-Kwon Park*,¶,□ †

Division of Forest Insect Pests and Diseases, Korea Forest Research Institute, Seoul 130-712, Republic of Korea Gyeongnam Department of Environmental Toxicology and Chemistry, Korea Institute of Toxicology, Jin-Ju, Gyeongnam Republic of Korea ∥ Institute of Agriculture and Life Science/BK21+, Gyeongsang National University, Jinju, Gyeongnam 660-701, Republic of Korea ⊥ Department of Seed & Seedling Management, Korea Forest Seed and Variety Center, Chungju, Chungcheongbuk-do 380-941, Republic of Korea # Southern Forest Research Center of the Korea Forest Research Institute, Jinju, Gyeongnam 660-300, Republic of Korea ¶ Department of Forest Science and □Research Institute of Agriculture and Life Science, College of Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Republic of Korea §

ABSTRACT: The fumigant and contact toxicities of 16 Asteraceae plant essential oils and their constituents against adult male and female Blattella germanica were examined. In a fumigant toxicity test, tarragon oil exhibited 100% and 90% fumigant toxicity against adult male German cockroaches at 5 and 2.5 mg/filter paper, respectively. Fumigant toxicities of Artemisia arborescens and santolina oils against adult male German cockroaches were 100% at 20 mg/filter paper, but were reduced to 60% and 22.5% at 10 mg/filter paper, respectively. In contact toxicity tests, tarragon and santolina oils showed potent insecticidal activity against adult male German cockroaches. Components of active oils were analyzed using gas chromatography, gas chromatography−mass spectrometry, or nuclear magnetic resonance spectrometer. Among the identified compounds from active essential oils, estragole demonstrated potent fumigant and contact toxicity against adult German cockroaches. β-Phellandrene exhibited inhibition of male and female German cockroach acetylcholinesterase activity with IC50 values of 0.30 and 0.28 mg/mL, respectively. KEYWORDS: Asteraceae plant essential oils, fumigant toxicity, contact toxicity, German cockroach, acetylcholinesterase inhibition

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INTRODUCTION The German cockroach, Blattella germanica (L.) is the most common cockroach found in human residences. Cockroaches are considered a significant insect pest with regard to hygiene, because not only are they a source of allergy1 but they also spread several intestinal diseases such as cholera, diarrhea, and dysentery.2 To control German cockroaches, several contact or residual insecticides such as organophosphorus, carbamate, pyrethroids, and hydramethylnon have been used,3−5 but these pesticides have many side effects, including environmental and human health problems, and are susceptible to resistance.4−6 Because of multiple side effects caused by synthetic pesticides, the development of environmentally friendly German cockroach control agents is essential.7−9 Plant essential oils extracted by steam distillation are good resources for developing German cockroach control agents because they are known to have many bioactivities including insecticidal and repellent activity against German cockroaches.8−13 Another advantage of plant essential oils is their high volatility. The main constituents of essential oils are monoor sesquiterpenoids, which are highly volatile.14,15 High volatility reduces concerns of residue problems. Furthermore, constituents of some plant essential oils are known to act synergistically with respect to insecticidal activity,8,9 which could retard the © 2015 American Chemical Society

development of resistance if the oil constituents have different modes of action. In this study, we investigated the fumigant and contact toxicities of Asteraceae plant essential oils and their constituents against male and female adult German cockroaches to find alternatives to current insecticides. We also measured the acetylcholinesterase (AChE) inhibition activities of constituents from active Asteraceae plant essential oils to better understand their mode of action.

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MATERIALS AND METHODS

Plant Essential Oils and Chemicals. Essential oils of Artemisia af ra and davana were purchased from Jinarome (Anyang, Gyeonggi Province, Korea). Artemisia arborescens, chamomile blue, chamomile roman, chamomile wild, chrysantheme abs., costus root, elecampane roots, erigeron, Eriocephalus punctulatus, Helichrysum bracteiferum, helichrysum (Immortella), santolina, tagetes extra-s, and tarragon were purchased from Oshadhi (Weinstrasse, Bühl/Baden, Germany). These plant essential oils are listed in Table 1. Camphene (80%), estragole (98%), (+)-limonene (97%), myrcene (95%), and cis-ocimene

Received: Revised: Accepted: Published: 2241

December 8, 2014 January 31, 2015 February 9, 2015 February 9, 2015 DOI: 10.1021/jf505927n J. Agric. Food Chem. 2015, 63, 2241−2248

Article

Journal of Agricultural and Food Chemistry Table 1. List of Asteraceae Plant Essential Oils Tested common name of essential oil

scientific name

source

part

country of origin

Artemisia af ra Artemisia arborescens chamomile blue chamomile roman chamomile wild chrysantheme abs. costus root davana elecampane roots erigeron Eriocephalus punctulatus Helichrysum bracteiferum helichrysum (Immortella) santolina tagetes extra-s tarragon

Artemisia af ra Artemisia arborescens Chamomilla matricaria Anthemis nobilis Ormensis multicaulis Chrysanthemum morifolium Saussurea lappa Artemisia pallens Inula racemosa Coniza canadensis Eriocephalus punctulatus Helichrysum bracteatum Helichrysum angustifolium Santolina chamaecyparissus Tagetes minuta Artemisia dracunculus

Jinarome Oshadhi Oshadhi Oshadhi Oshadhi Oshadhi Oshadhi Jinarome Oshadhi Oshadhi Oshadhi Oshadhi Oshadhi Oshadhi Oshadhi Oshadhi

flowering plant flowering plant blossoms blossoms blossoms leaves roots leaves roots flowering plant flowering plant blossoms blossoms plant blossoms plant

South Africa Morocco Nepal France Morocco India India India India Canada South Africa South Africa Croatia Spain Egypt France

Isolation and Identification of β-Thujone and Artemisia Ketone. To isolate β-thujone, 1 mL of A. arborescens oil was chromatographed on a SiO2 column (Wakogel 200; Wako Pure Chemicals, Osaka, Japan) eluting with a diethyl ether−hexane gradient. β-Thujone and camphor coeluted with 10:90 (ether:hexane) solution. This mixture was rechromatographed on a 10% AgNO3-impregnated SiO2 column, and β-thujone eluted with 10% ether−hexane solution in 98.9% purity (395 mg). Artemisia ketone was isolated from santolina oil. Santolina oil (1 mL) was chromatographed on a SiO2 column eluting with a diethyl ether−hexane gradient. Artemisia ketone (purity 80%) was isolated with 10:90 (ether: hexane) solution. To obtain pure artemisia ketone, the 10% ether fraction was rechromatographed on a SiO2 column eluting with 1:99 ether:hexane. The purity of isolated artemisia ketone (245 mg) was 98.6%. Isolated β-thujone and artemisia ketone were subjected to NMR analysis and were used for bioassays. Synthesis of β-Phellandrene. β-Phellandrene (purity 98.2%) was synthesized in the laboratory and was subjected to bioassay. The synthesis procedure and NMR data have been well documented in our previous study.16 Fumigant Toxicity Test. The fumigant toxicity of Asteraceae plant essential oils and their constituents was determined using a glass cylinder (diameter, 9.5 cm; height, 19 cm) with a wire sieve installed 9.5 cm above the bottom. Asteraceae plant essential oils or their components were applied to a paper disk (8 mm, Advantec). The treated paper disk was transferred to the bottom lid of the glass cylinder. The lid of the glass cylinder was sealed with Para-film to prevent the leakage of the oil or constituents (Pechiney Plastic Packaging Company, Chicago, IL, USA). Ten adult male or female German cockroaches were placed on the sieve, which prevented direct contact of the cockroaches with the test oils and constituents. Test cockroaches were maintained at 25 ± 1 °C and 60% RH. Mortality was determined 48 h after treatment. All treatments were replicated 4 times. Contact Toxicity Test. The appropriate dose of Asteraceae plant essential oil or individual constituents was dissolved in acetone. Adult male and female German cockroaches were anesthetized using CO2, and the oils or constituents in acetone were topically applied to the abdomen of adult male and female German cockroaches with a microapplicator (Burkard, U.K.). Control insects received only acetone (1 μL). Treated adults (10 adults/replication) were transferred to Petri dishes (diameter, 9.5 cm; height, 2 cm), which were maintained at 25 ± 1 °C and 60% RH. Mortality was determined 24 h after treatment. Each assay was replicated 5 times. Acetylcholinesterase Inhibition. Crude protein was obtained from two adult male and female cockroaches. Adult cockroaches were soaked in 0.1 M Tris-HCl mixed with 0.02 M NaCl, 0.5% Triton X-100 (pH 7.8), and a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and then they were ground using a glass tissue grinder (Wheaton Industries Inc., Millville, NJ, USA) in ice. The ground

(90%) were purchased from Sigma-Aldrich (Milwaukee, WI, USA). Camphor (>97%), terpinen-4-ol (99%), γ-terpinene (97%), and pcymene (95%) was purchased from Fluka (Buchs, Switzerland). βCaryophyllene (>90%), (+)-α-pinene (95%), and β-pinene (94%) were obtained from Tokyo Kasei (Tokyo, Japan). Acetone (99.8%) was purchased from Merck. Dichlorvos (purity 98.6%) was purchased from Chem Service (West Chester, PA, USA), and deltamethrin (purity 96.8%) was supplied from Dongbu Farm Hannong (Seoul, Republic of Korea). Insects. German cockroaches, B. germanica, were reared in plastic boxes (54.3 × 18.8 × 36.3 cm, L × W × H) without exposure to any insecticide. The cockroaches were supplied with water from a glass flask fitted with a cotton stopper and dried mouse food (Purina feed). The cockroaches were maintained at 25 ± 1 °C and 60% RH (relative humidity) under a 16:8 h light:dark cycle. Gas Chromatography. Gas chromatography (GC) analysis of Artemisia arborescens, santolina, and tarragon oils was carried out using an Agilent 7890A (Agilent Technologies, Santa Clara, CA, USA), equipped with DB-1MS or HP-INNOWAX columns (30 m × 0.25 mm internal diameter [i.d.]; film thickness, 0.25 μm; J&W Scientific, Folsom, CA, USA) and a flame ionization detector (FID). Retention times of essential oil components were compared with those of authentic compounds. The oven temperature of the gas chromatograph was programmed as isothermal at 40 °C for 1 min, raised at a rate of 6 °C/ min to 250 °C, and maintained at 250 °C for 4 min. Helium was used as the carrier gas at a flow rate of 1.5 mL/min. Retention indices were obtained in relation to a homologous series of n-alkanes (C7−C20; DB1MS), (C8−C22; HP-INNOWAX) under the same gas chromatography operating conditions. Further identification of oil constituents was confirmed by increasing the integrated area by coinjection with oil and standard samples. Gas Chromatography−Mass Spectrometry. Constituents of Asteraceae plant essential oils were also analyzed using a tandem gas chromatograph (Agilent 7890A) mass spectrometer (Agilent 5975C MSD) (GC-MS) (Agilent Technologies). A DB-5MS column (30 m × 0.25 mm i.d.; film thickness, 0.25 μm; J&W Scientific) was used, and the column oven temperature program was the same as used for GC-FID analysis. Helium was used as the carrier gas at a flow rate of 1.0 mL/min. The effluent of the column was introduced directly into the source of the MS via a transfer line (250 °C). Ionization was attained using electron impact (70 eV; source temperature, 230 °C). The scan range was 41− 400 amu. Most components of the plant essential oils were tentatively confirmed by comparing the mass spectra of each peak with those of standard samples in the NIST MS library. NMR spectra were obtained on a Varian UI 500 NMR spectrometer (500 MHz for 1H spectra and 125 MHz for 13C spectra) at Korean Basic Science Institute using TMS as an internal standard. 2242

DOI: 10.1021/jf505927n J. Agric. Food Chem. 2015, 63, 2241−2248

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Journal of Agricultural and Food Chemistry Table 2. Fumigant Toxicity of 16 Asteraceae Plant Essential Oils against Adult of German Cockroaches mortality (%, mean ± SE, N = 40a) male plant essential oils Artemisia af ra Artemisia arborescens chamomile blue chamomile roman chamomile wild chrysantheme abs. costus root davana elecampane roots erigeron Eriocephalus punctulatus Helichrysum bracteiferum helichrysum (Immortella) santolina tagetes extra-s tarragon control

20b

10

female 5

2.5

1.25

20

10

5

2.5

70 ± 4.1 bcc 100 a

2.5 ± 2.5 c 60 ± 12.9 b

−d 15.0 ± 6.5 b

− −

− −

17.5 ± 7.5 cd 25 ± 6.5 bc

− −

− −

− −

2.5 ± 2.5 ef 85.0 ± 5.0 ab

− 22.5 ± 8.5 c

− −

− −

− −

0d 25.0 ± 6.5 bc

− −

− −

− −

57.5 ± 2.5 c 7.5 ± 4.8 def

10 ± 4.1 c −

− −

− −

− −

0d 0d

− −

− −

− −

0f 27.5 ± 7.5 de 60 ± 4.1 bc

− − 0c

− − −

− − −

− − −

0d 35 ± 2.9 bc 42.5 ± 2.5 b

− − 0b

− − −

− − −

0f 30 ± 4.1 d

2.5 ± 2.5 c

− −

− −

− −

0d 0d

− −

− −

− −

5 ± 5.0 def

−

−

−

−

0d

−

−

−

0f

−

−

−

−

0d

−

−

−

100 a 0f 100 a 0f F16,51 = 145.810 p < 0.0001

22.5 ± 9.6 c − 100 a 0c F8,27 = 36.054 p < 0.0001

− − 100 a 0b F2,9 = 209.400 p < 0.0001

− − 90 0

− − 15.0 ± 6.5 0

37.5 ± 4.8 bc 0d 100 a 0d F16,51 = 67.796 p < 0.0001

2.5 ± 2.5 b − 100 a 0b F3,12 = 1574.333 p < 0.0001

− − 50 ± 14.1 0

− − 10 ± 8.2 0

a Number of insects tested. bmg/filter paper. cMeans within a column followed by the same letters are not significantly different (Scheffé’s test). dNot tested.

Table 3. Contact Toxicity of 16 Asteraceae Plant Essential Oils against Adult of German Cockroaches mortality (%, mean ± SE, N = 50a) male

female

plant essential oils

2b

1

0.5

2

Artemisia af ra Artemisia arborescens chamomile blue chamomile roman chamomile wild chrysantheme abs. costus root davana elecampane roots erigeron Eriocephalus punctulatus Helichrysum bracteiferum helichrysum (Immortella) santolina tagetes extra-s tarragon control

70 ± 9.5 bcdec 82 ± 5.8 abc 74 ± 8.0 abcd 12.5 ± 3.7 hi 96 ± 2.4 ab 42 ± 4.9 efg 46 ± 6.8 defg 58 ± 7.3 cdefg 66 ± 6.8 cdef 50 ± 6.3 defg 32 ± 3.7 gh 98 ± 2.0 ab 98 ± 2.0 ab 100 a 40 ± 10.5 fgh 100 a 0i F16,68 = 66.609 p < 0.0001

16 ± 2.4 cd 42 ± 10.2 bc 16 ± 5.1 cd − 54 ± 6.8 b 14 ± 4.0 cd 24 ± 7.5 bcd 18 ± 2.0 cd 6 ± 2.4 d 50 ± 5.5 b − 30 ± 8.4 bcd 50 ± 3.2 b 86 ± 9.3 a 0d 100 a 0d F14,60 = 51.721 p < 0.0001

−d 6 ± 4.0 b − − 16 ± 4.0 ab − − − − 12 ± 5.8 b − − 20 ± 6.3 ab 12 ± 8.0 b − 44 ± 11.2 a 0b F7,32 = 5.891 p < 0.0001

30 ± 10.5 cdef 26 ± 4.0 cdef 16 ± 4.0 def 14 ± 5.1 ef 48 ± 9.7 bcd 24 ± 10.8 cdef 20 ± 6.3 def 38 ± 8.0 bcde 30 ± 5.5 cdef 16 ± 2.4 def 20 ± 8.4 def 30 ± 8.4 cdef 54 ± 7.5 bc 70 ± 4.5 ab 6 ± 2.4 ef 100 a 0f F16,68 = 31.488 p < 0.0001

1

18 ± 3.7 abc

6 ± 4.0 bc 32 ± 2.0 a 14 ± 2.4 b 0c F4,20 = 18.750 p < 0.0001

a Number of insects tested. bmg/adult. cMeans within a column followed by the same letters are not significantly different (Scheffé’s test). dNot tested.

cockroach extract was centrifuged at 17000g for 15 min at 4 °C to eliminate insect tissue debris. The concentration of crude protein was measured by the Bradford protein assay using BSA as the standard protein. The AChE inhibition activity was evaluated using the modified

Ellman method.17 Test chemicals were dissolved in acetone to give a concentration of 100 mg/mL. Mixtures of 1 μL of test compound and 79 μL of crude cockroach protein were placed in 96-well microplates, which were incubated for 10 min at rt (room temperature). The control 2243


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Journal of Agricultural and Food Chemistry Table 4. Chemical Analysis of Artemisia arborescens, Santolina, and Tarragon Oils retention index

a

no.

compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

α-pinene camphene sabinene β-pinene β-myrcene p-cymene β-phellandrene limonene cis-ocimene artemisia ketone γ-terpinene β-thujone camphor terpinen-4-ol estragole β-caryophyllene sum

DB-1 929 941 964 969 982 1011 1019 1020 1027 1043 1049 1095 1117 1160 1174 1415

Innowax

composition rate (%) Artemisia arborescens

santolina

tarragon

identification

2.33 2.51 4.21 − 7.22 2.09 − − − − 2.28 43.72 22.93 3.03 − 2.56 92.88

− − 8.99 8.84 11.46 − 25.67 − − 31.20 − − − − − − 86.16

1.46 − − − − − − 4.87 3.02 − − − − − 88.7 − 98.10

GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS, NMR GC, GC-MS GC, GC-MS, NMR GC, GC-MS GC, GC-MS GC, GC-MS GC, GC-MS

1020 1064 1122 1107 1165 1273 1209 1200 1238 1350 1247 1444 1519 1611 1676 1600

a

Not detected.

Figure 1. Chemical structure (A), 1H NMR (B), and 13C NMR (C) chemical shift values of β-thujone, and chemical structure of artemisia ketone (D). received acetone only. 10 μL of acetylthiocholine iodide (ASChI, 10 mM) and 10 μL of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, 4 mM) were added to the mixtures of test compounds and crude protein. AChE activity was evaluated by estimating the initial velocity (V0) for 20 min at 30 s intervals at 405 nm and rt using an iMark microplate absorbance reader (Bio-Rad, Hercules, CA, USA). The primary AChE inhibition assay was replicated at least 3 times. The percentage inhibition of each chemical was calculated according to the following formula:

exhibited 100% fumigant toxicity against male German cockroaches at 20 mg/filter paper. The fumigant toxicity of tarragon was 100% at 10 and 5 mg/filter paper, but declined to 90% at 2.5 mg/filter paper. Fumigant toxicities of Artemisia arborescens, and santolina were 60% and 22.5% at 10 mg/filter paper, respectively. Chamomile roman showed 85% mortality at 20 mg/filter paper, but declined to 22.5% at 10 mg/filter paper. Other oils showed moderate or weak fumigant toxicity against male German cockroaches. In a test with female German cockroaches, only tarragon showed potent fumigant toxicity at 20 mg/filter paper. Fumigant toxicities of tarragon against female German cockroaches were 100% and 50% at concentrations of 10 and 5 mg/filter paper, respectively. Other oils exhibited moderate or weak toxicity against female German cockroaches at 20 mg/filter paper. In contact toxicity tests, santolina and tarragon essential oils showed 100% insecticidal activity against adult male German cockroaches at 2 mg/♂ (Table 3). Insecticidal activities of tarragon and santolina were 100% and 86% at 1 mg/♂, but were reduced to 44% and 12% at 0.5 mg/♂, respectively. Helichrysum bracteiferum, helichrysum (Immortella), and chamomile wild exhibited 98%, 98%, and 96% mortality against male German cockroaches at 2 mg/♂, but declined to 30%, 50%, and 54% at 1 mg/♂, respectively. Contact toxicities of other oils against male German cockroaches were less than 85% at 2 mg/♂. In tests with female cockroaches, tarragon and santolina exhibited 100% and 70% mortality at 2 mg/♀,

inhibn act. (%) = 100 − [(V0 of chemical treatment /V0 of control treatment) × 100] To determine the IC50 value of β-phellandrene, the following concentrations of β-phellandrene were used: 1, 0.5, 0.2, 0.1, and 0.05 mg/mL. All treatments were replicated 3 times at each concentration. The AChE inhibition activity was evaluated according to the formula described above, and the IC50 was determined by probit analysis.18 Statistical Analysis. Percentage mortality and primary AChE inhibition rates were transformed to arcsine square-root values for analysis of variance. Treatment mean values were compared and analyzed using Scheffé’s test.18 Mean (±SE) values of untransformed data are reported.

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RESULTS AND DISCUSSION Fumigant and Contact Toxicities of Plant Essential Oils. Fumigant and contact toxicities of 16 Asteraceae plant essential oils varied according to oil type and dose (Table 2). Artemisia arborescens, santolina, and tarragon essential oils 2244


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− − − − 2.5 ± 5.0 b − − − 100 a 0b F2,9 = 1561.0 p < 0.0001

respectively, while the other oils exhibited less than 55% mortality at 2 mg/♀. Many plant essential oils have been reported to exhibit fumigant or contact toxicity against German cockroaches.8−10,12,13 Yeom et al.8 reported that plant essential oils belonging to Apiaceae exhibited potent fumigant and contact toxicity against German cockroaches. Alzogaray et al.19 and Yeom et al.9 also investigated the insecticidal activities of plant essential oils belonging to Myrtaceae. However, fumigant and contact toxicities of the plant essential oils used in this study against German cockroaches have not been reported in any other study. Chemical Constituents of Plant Essential Oils. The chemical compositions of Artemisia arborescens, santolina, and tarragon essential oils are shown in Table 4. The most abundant components of Artemisia arborescens were β-thujone (43.72%) followed by camphor (22.93%), β-myrcene (7.22%), sabinene (4.21%), terpinen-4-ol (3.03%), β-caryophyllene (2.56%), camphene (2.51%), α-pinene (2.33%), γ-terpinene (2.28), and p-cymene (2.09%). Artemisia ketone (31.20%), β-phellandrene (25.67%), β-myrcene (11.46%), sabinene (8.99%), and β-pinene (8.84%) were detected as the main constituents in santolina oil. Estragole (88.75%) was identified as the most abundant compound in tarragon oil followed by limonene (4.87%), cisocimene (3.02%), and α-pinene (1.46%). Chemical analyses of Artemisia arborescens, santolina, and tarragon essential oils have been reported previously in other studies.20−22 While the main components of these oils were similar to those previous reports, there were some differences in minor components and composition quantities. Beyrouthy et al.20 reported that βthujone (68.5%), chamazulene (12.3%), terpinen-4-ol (1.5%), myrcene (1.1%), linalool and cis-thuyanol-4-ol (1%), α-thujone (0.9%), and sabinene (0.8%) were detected as the main components of Artemisia arborescens. Chamazulene, linalool, cis-thuyanol-4-ol, and α-thujone were not detected in our study. The difference of some oil components and composition amount could be attributed to the origin of Artemisia arborescens. Beyrouthy et al.20 and we used Artemisia arborescens essential oils from Lebanon and Moroco, respectively. Artemisia ketone, camphor, and β-phellandrene were analyzed as the main compounds in santolina oil by Demirci et al.,21 while camphor was not detected in our study. Arabhosseini et al.22 analyzed the constituents of French tarragon oil and found estragole, methyl eugenol, ocimene, and sabinene as the main constituents. In our study, the amount of estragole was higher, and methyl eugenol and sabinene were not identified. Galambosi and Peura23 suggested that constituents of plant essential oils differ widely according to production conditions such as harvesting date and storage time, as well as climatic or edaphic factors. Isolation and Identification of β-Thujone and Artemisia Ketone. Based on 1H- and 13C NMR, DEPT, 1H−1H COSY, and HMQC, each signal of β-thujone was assigned as shown in Figure 1. β-Thujone: 13C (ppm) 12.49 (C10, −CH3), 14.71 (C6, −CH2), 19.72 (C9, −CH3), 19.79 (C8, −CH3), 24.63 (C5, −CH), 27.42 (C1, -C), 32.30, (C7, −CH), 41.73 (C2, −CH2), 45.41 (C4,-CH), 218.51 (C3, -CO); 1H (ppm) −0.04 (6a, 1H, dd, J = 5.5, 4), 0.59 (6b, 1H, tq, J = 7, 2), 1.00 (8, 3H, d, J = 7), 1.03 (10, 3H, d, J = 7), 1.44 (5, 7, 2H, m), 2.12 (2a, 1H, d, J = 18.5), 2.54 (2b, 1H, d, J = 18.5, 2), 2.71 (4, 1H, m). Chemical shift values of the proton at C6 of α-thujone and β-thujone were reported as 0.12 and −0.05 ppm, respectively.24 1H NMR analysis of the isolated thujone showed only a chemical shift value at −0.05 ppm. Thus, the isolated thujone was determined

a

Number of insects tested. bmg/filter paper. cMeans within a column followed by the same letters are not significantly different (Scheffé’s test). dNot tested.

− − − − 40 ± 29.4 b − − − 100 a 0c F2,9 = 35.077 p < 0.0001 − − − − 80 ± 16.3 b − − − 100 a 0c F2,9 = 126.0 p < 0.0001 − − − − 100 a − 10 ± 4.1 bc 17.5 ± 4.8 b 100 a 0c F4,15 = 317.526 p < 0.0001 15 ± 2.9 bc 10 ± 7.1 bc 10 ± 11.5 bc 0c 100 a 12.5 ± 4.8 bc 47.5 ± 15.5 b 100 a 100 a 0c F9,30 = 54.071 p < 0.0001 − − − − 67.5 ± 5.0 b − − − 100 a 0c F2,9 = 1249.0 p < 0.0001 − − − − 100 − − − 100 0 − − − − − 100 a 22.5 ± 22.2 b − 12.5 ± 4.8 b 100 a 0b F4,15 = 82.436 p < 0.0001 artemisia ketone camphene camphor β-caryophyllene estragole cis-ocimene β-phellandrene β-thujone dichlorvos control

compounds

20b

c

27.5 ± 4.8 b 7.5 ± 2.5 bc 22.5 ± 7.5 bc 0c 100 a 82.5 ± 4.8 a 100 a 100 a 100 a 0c F9,30 = 148.745 p < 0.0001

d

− − − − 100 a 35.0 ± 2.9 b 20.0 ± 4.1 b 90.0 ± 5.8 a 100 a 0b F5,18 = 43.145 p < 0.0001

5

female

10 10

male

5

2.5

1.25

20

mortality (%, mean ± SE, N = 40a)

Table 5. Fumigant Toxicity of Constituents from Artemisia arborescens, Santolina, and Tarragon Oils against Adult of German Cockroaches

2.5

1.25

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Table 6. Contact Toxicity of Constituents from Santolina and Tarragon Essential Oils against Adult of German Cockroaches mortality (%, mean ± SE, N = 50a) male b

female

compounds

1

0.5

0.25

1

artemisia ketone camphene camphor β-caryophyllene estragole cis-ocimene β-phellandrene deltamethrin control

74 ± 11.7 abc 4 ± 2.4 d 16 ± 4.0 cd 46 ± 6.8 bc 100 a 42 ± 7.3 bc 56 ± 2.4 b 100 a 0d F8,36 = 48.784 p < 0.0001

30.0 ± 3.1 b −d − 22 ± 3.7 b 100 a 22 ± 7.3 b 10 ± 4.5 bc 100 a 0c F6,28 = 124.354 p < 0.0001

− − − − 34 ± 7.5 b − − 100 a 0c F2,12 = 138.5 p < 0.0001

40 ± 4.5 b 0e 0e 0e 20 ± 3.2 cd 6 ± 2.4 de 26. ± 4.0 bc 100 a 0e F8,36 = 186.404 p < 0.0001

a Number of insects tested. bmg/adult. cMeans within a column followed by the same letters are not significantly different (Scheffé’s test). dNot tested.

Figure 2. Blatella germanica acetylcholinesterase inhibition rates of constituents identified in essential oils from Artemisia arborescens, santolina, and tarragon oils at 1 mg/mL. Mean values corresponding to each treatment with different letters are significantly different from one another (male: F7,16 = 109.235, p < 0.0001, female, F7,16 = 230.853, p < 0.0001, Scheffé’s test).

Table 7. IC50 Values of β-Phellandrene against Adult German Cockroach Acetylcholinesterase Activity male

a

female a

compounds

IC50 (mg/mL)

slope

95% cl

β-phellandrene

0.30

2.19 ± 0.16

0.26−0.34

χ

IC50 (mg/mL)

slope

95% cl

χ2

2.95

0.28

1.82 ± 0.14

0.24−0.34

0.29

2

Confidence limit.

to be β-thujone. NMR data of artemisia ketone was assigned in our previous study.25

Fumigant and Contact Toxicities of Individual Constituents. The fumigant and contact toxicities of individual 2246


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al.30 also found that weight differences between male and female German cockroaches affected the insecticide susceptibility to synthetic insecticides such as organophosphates, carbamates, and pyrethroids. Primary AChE Inhibition Assay and IC50 Determination. AChE inhibition by artemisia ketone, camphene, camphor, βcaryophyllene, estragole, cis-ocimene, and β-phellandrene was determined, since inhibition activities of the other constituents were reported in our previous study.8,9 The primary inhibition activities of various isolated constituents against German cockroach acetylcholinesterase are summarized in Figure 2. βPhellandrene gave the highest inhibition (81.28%), followed by cis-ocimene (44.10%) and estragole (25.04%) (Figure 2). Inhibition activities of the other compounds were less than 20%. In the primary inhibition assay, only β-phellandrene showed >50% inhibition. IC50 values of β-phellandrene against male and female acetylcholinesterase were 0.30 and 0.28 mg/mL, respectively (Table 7). Insect acetylcholinesterase inhibition activity of many phytochemicals has been investigated in several studies.16,25,31−38 German cockroach AChE inhibition activity of phytochemicals from plant essential oils has also been studied by Yeom et al.8,9 They reported that insecticidal activity of carvacrol and dihydrocarvone correlated with their ability to inhibit German cockroach acetylcholinesterase. However, there was no relationship between AChE inhibition and most other active compounds. In our study, only β-phellandrene showed potent AChE inhibition activity, while most of the other active compounds revealed moderate or weak inhibition activity. Seo et al.25 reported that β-phellandrene exhibited potent AChE inhibition activity against Japanese termites. The results of our study and other research indicate that insecticidal activity of βphellandrene correlates with its ability to inhibit AChE. Although the mode of action of phytochemicals from plant essential oils has been reported by some research groups to include AChE, a definitive mechanism has yet to be completely established.8,9,16,25,31,39,40 Our results indicate that Asteraceae plant essential oils and their components can be used as fumigants or spray-type control agents against German cockroaches. However, further studies including safety of the oils and their constituents to humans and nontarget organisms, formulations, and their modes of action are necessary for practical use of Asteraceae plant essential oils and their constituents as novel cockroach-control agents.

constituents from Artemisia arborescens, santolina, and tarragon essential oils are shown in Tables 5 and 6. Fumigant or contact toxicities of α-pinene, β-pinene, β-myrcene, p-cymene, limonene, γ-terpinene, and terpinen-4-ol were not tested in this study, because their toxicities to German cockroaches have been reported in our previous studies.8,9 In a fumigant toxicity test, estragole was the most toxic component to adult males. Fumigant toxicities of estragole were 100% and 67.5% at 2.5 and 1.25 mg/filter paper, respectively. β-Thujone, β-phellandrene, and cis-ocimene showed 100%, 100%, and 82.5% fumigant toxicity against adult males at 20 mg/filter paper, but their activities were reduced to 90%, 20%, and 35% at 10 mg/filter paper, respectively. Other compounds showed weak activity. In tests with adult females, estragole exhibited 100% fumigant toxicity at 20 and 10 mg/filter paper, but its toxicity was reduced to 80% and 40% at 5 and 2.5 mg/filter paper, respectively. The fumigant toxicity of β-thujone was 100% at 20 mg/filter paper, but was reduced to 17.5% at 10 mg/filter paper. Other compounds showed moderate or weak toxicity at 20 mg/filter paper. In a contact toxicity test, only estragole exhibited potent activity against adult males. The contact toxicity of estragole was 100% at 1 and 0.5 mg/♂, but was reduced to 34% at 0.25 mg/♂. Artemisia ketone produced 74% mortality at 1 mg/♂, but the mortality decreased to 30% at 0.5 mg/♂. Other compounds exhibited moderate or weak contact toxicity. In a contact toxicity test with female German cockroaches, all test compounds showed moderate or weak toxicity at 1 mg/♀. However, fumigant or contact toxicity of all test compounds was weaker than that of conventional pesticides such as dichlorvos and deltamethrin (Tables 5 and 6). Insecticidal activities of phytochemicals from plant essential oils against German cockroaches have been reported in several studies.8−10,13 Yeom et al.8,9 reported fumigant and contact toxicities of constituents from Apiaceae and Myrtaceae plant essential oils. They found that carvone, 1,8cineole, trans-dihydrocarvone, cumminaldehyde, trans-anethole, p-cymene, γ-terpinene, carvacrol, thymol, carveol, terpinen-4-ol, terpinolene, and α-terpinene demonstrated potent fumigant or contact toxicities against male and female German cockroaches. Philips et al.10 and Philips and Appel13 also tested fumigant and contract toxicities of phytochemicals from plant essential oils including carvacrol, 1,8-cineole, trans-cinnamaldehyde, citronellic acid, eugenol, geraniol, limonene, linalool, menthone, αpinene, β-pinene, and thymol. In our study, estragole showed the most potent fumigant toxicity to male and female German cockroaches and contact toxicity to male German cockroaches. Estragole is the main constituent of basil oil, and its insecticidal activity against fruit flies (Ceratitis capitata, Bactrocera dorsalis, Bactrocera cucurbitae), maize weevil (Sitophilus zeamais), azuki bean weevil (Callosobruchus chinensis), rice weevil (Sitophilus oryzae), and cigarette beetle (Lasioderma serricorne) have been reported in several studies.26−28 However, this is the first report on its fumigant and contact toxicity against adult German cockroaches. Many studies have identified β-thujone and βphellandrene as components of plant essential oils.20,21 However, there are few reports on their insecticidal activity because they are not commercially available.29 In this study, adult male German cockroaches were more susceptible than adult female German cockroaches to plant essential oils and their constituents. The weight of female German cockroaches (average weight of 20 females was 104.9 mg) was about twice that of the male German cockroaches (54.2 mg). This weight difference may be the main reason for the different susceptibility to test oils and their constituents. Lee et

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AUTHOR INFORMATION

Corresponding Author

*(Tel: +82-2-880-4751. Fax: +82-2-873-3560. E-mail: parkik1@ snu.ac.kr. Author Contributions ‡

H.-J.Y. and C.-S.J. contributed equally to this work.

Funding

This work was supported by a Research Resettlement Fund for the new faculty of Seoul National University (Project No.: 50020140209) and a grant from Korea Forest Service (Project No.: S111414L080110) to I.-K.P. Notes

The authors declare no competing financial interest.

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