- Email: [email protected]
Acaricidal activity of fennel seed oils and their main components against Tyrophagus putrescentiae, a stored-food mite
ARTICLE IN PRESS
Journal of Stored Products Research 42 (2006) 8–14 www.elsevier.com/locate/jspr
Acaricidal activity of fennel seed oils and their main components against Tyrophagus putrescentiae, a stored-food mite Chi-Hoon Lee, Bo-Kyung Sung, Hoi-Seon Lee Faculty of Applied Biotechnology and Research Center for Industrial Development of Biofood Materials, College of Agriculture & Life Science, Chonbuk National University, Chonju 561-756, South Korea Accepted 23 October 2004
Abstract The acaricidal activities of components derived from Foeniculum vulgare (fennel) seed oils against Tyrophagus putrescentiae adults were examined using direct contact application and compared with those of the compounds benzyl benzoate, dibutyl phthalate and N,N-diethyl-m-toluamide. The biologically active constituent of the F. vulgare seeds was characterized as (+)-carvone by spectroscopic analyses. On the basis of LD50 values, the compound most toxic to T. putrescentiae was naphthalene (4.28 mg/cm2) followed by dihydrocarvone (4.32 mg/cm2), (+)-carvone (4.62 mg/cm2), ()-carvone (5.23 mg/cm2), eugenol (10.62 mg/ cm2), benzyl benzoate (11.24 mg/cm2), thymol (11.42 mg/cm2), dibutyl phthalate (13.11 mg/cm2), N,Ndiethyl-m-toluamide (13.53 mg/cm2), methyl eugenol (39.52 mg/cm2), myrcene (39.88 mg/cm2) and acetyleugenol (72.24 mg/cm2). These results indicate that acaricidal activity of the F. vulgare seed oil could be caused by carvone and naphthalene of which the former is likely to be more important because it is 74.7 times more abundant than naphthalene. Carvone and naphthalene merit further study as potential stored-food mite control agents or as lead compounds. r 2004 Elsevier Ltd. All rights reserved. Keywords: Carvone; Foeniculum vulgare; Natural acaricide; Tyrophagus putrescentiae
Corresponding author. Tel.: +82 63 270 2544; fax: +82 63 270 2550.
E-mail address: [email protected] (H.-S. Lee). 0022-474X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2004.10.004
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1. Introduction Among astigmatid mites, Tyrophagus putrescentiae (Schrank) is a cosmopolitan species commonly encountered infesting a large number of food grains and stored food with a high fat and protein content, such as dried eggs, ham, cheese and different kinds of nuts (Hughes, 1976; Sinha, 1979). It causes serious economic losses (Zdarkova, 1991) as well as reducing nutrient content and germination ability (Krantz, 1955). In addition, the mite is responsible for allergic diseases among farmers and food industry workers handling heavily infested stored products (Hughes, 1976) and causes acute enteritis (Hughes, 1976) and systemic anaphylaxis (Matsumoto et al., 1996) when contaminated food is ingested. The mite also acts as a carrier of bacteria and toxigenic fungi such as Aspergillus spp. in stored grain kept under warm and moist conditions (Franzolin et al., 1999). Use of chemical methods, such as fumigation, spraying with organophosphorus compounds, or treatment with benzyl benzoate, dibutyl phthalate and N,Ndiethyl-m-toluamide, to control T. putrescentiae is prohibited because of human health hazards associated with their consumption. Therefore, the search is on for more selective, natural compounds non-toxic to humans and which do not affect the organoleptic character of the treated product. Research into plant-derived acaricides is now being intensified as it becomes evident that plant-derived acaricides have enormous potential in this regard. Plant extracts or their constituents may provide an alternative to currently used mite-control agents to control stored-food mites (Gulati and Mathur, 1995; Perrucci, 1995; Kim et al., 2004). Since many of them are largely free from adverse effects and have excellent biological activity, they could lead to the development of new classes of possibly safer stored-food mite control agents. In East Asia, the fruits of Foeniculum vulgare Gaertner have long been considered to have medicinal properties attributable to trans-anethole, estragole, d-limonene, (+)-fenchone, a-pinene, g-terpinene and p-cymene (Kim and Ahn, 2001). Little work has been done with respect to managing storedfood mites, although extracts and essential oils of F. vulgare fruits are insecticidal agents (Kim and Ahn, 2001). This paper describes a laboratory study to examine the oil of F. vulgare seeds for storage food mite control constituents against T. putrescentiae adults. The storage food mite control activities of F. vulgare seed oil-derived compounds were compared with those of the commonly used benzyl benzoate, dibutyl phthalate, and N,N-diethyl-m-toluamide.
2. Materials and methods 2.1. Chemicals Asarone, camphene, 2-carene, ()-carvone, dihydrocarvone, eugenol, acetyleugenol, isoeugenol, methyl eugenol, d-limonene, myrcene, naphthalene, terpinolene and thymol were supplied by Sigma (St. Louis, MO, USA). Benzyl benzoate, N,N-diethyl-m-toluamide and dibutyl phthalate were purchased from Aldrich (Milwaukee, WI). All chemicals were of reagent grade. 2.2. Storage mites Cultures of T. putrescentiae had been maintained in the laboratory for 5 yr without exposure to any known acaricide. They were reared in plastic containers (15 12 6 cm) containing 30 g of
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sterilized diet (fry feed No. 1/dried yeast, 1:1 by weight) at 2571 1C and 75% relative humidity (r.h.) in continuous darkness. The fry feed was purchased from Korea Special Feed Meal Co. Ltd., Chonju, Korea. 2.3. Isolation and identification The seeds (6.5 kg) of F. vulgare were purchased from a local market in Chonju and identified by Prof. Sang-Hyun Lee (Forestry Department, Chonbuk National University, Korea). The samples were washed twice with 1 L of distilled water and dried in an oven at 40 1C for 2 days, then finely powdered. The oil (yield 4.8%) of F. vulgare seeds was extracted by steam distillation as previously described (Cho et al., 2002). The oil (15 g) was chromatographed on a silica gel column (Merck 70–230 mesh, 750 g, 6.3 cm i.d. 80 cm) and successively eluted with a stepwise gradient of hexane/ethyl acetate (100:0, 90:10, 80:20, 70:30, 50:50 and 0:100). The bioactive fraction (6.5 g) was successively rechromatographed on a silica gel column, using hexane-ethyl acetate (70:30). Column fractions were analysed by thin layer chromatography (TLC) (silicagel 60 F254) and fractions with similar streaking patterns on the TLC plates were pooled. Preparative high-performance liquid chromatography (prep. HPLC) (Spectra System P2000, Thermo Separation Products) was used for further separation of the constituents. The columns (19 mm i.d. 300 mm, Waters) contained a mPorasil, and were eluted with hexane-ethyl acetate (85:15) at a flow rate of 2.5 mL/min and detected at 280 nm. Finally, the potent active principle (785 mg) was isolated. The structure of the active isolate was determined by instrumental analyses. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded in deuteriochloroform with a JNM-LA 400F7 spectrometer, at 600 and 150 MHz (using tetramethylsilane (TMS) as an internal standard), respectively, and chemical shifts are given in d (ppm). Unambiguous 1H and 13C NMR chemical shifts were obtained using a 1H–1H COSY spectrum as well as a 13C–1H correlation spectrum. UV spectra were obtained in methanol with an Uvikon 922 spectrometer and mass spectra on a JEOL GSX 400 spectrometer. Optical rotation was measured with an Autopol III polarimeter. 2.4. Gas chromatography–mass spectrometry (GC–MS) The oil of F. vulgare seeds was analyzed on a gas chromatograph (HP6890)-mass spectrometer (JMS-600W, JEOL) (GC–MS). The GC column was a 60 m 0.25 mm i.d. DB-WAX (0.25 mm film) fused silica capillary column (J&W Scientific, Folsom, CA, USA). The GC conditions were as follows: injector temperature, 210 1C; column temperature, isothermal at 50 1C for 15 min, then programmed to rise to 200 1C at 2 1C/min and be held at this temperature for 15 min; ion source temperature, 200 1C. Helium was used as the carrier gas at the rate of 0.8 mL/min. The effluent of the GC column was introduced directly into the source of the MS. Spectra were obtained in the EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 50 to 800 amu for 2 s. Compounds were identified by comparison with retention times and the mass spectra obtained with the authentic standards on the GC–MS system used for analysis. When an authentic sample was not available, the identification was carried out by comparison of mass spectra obtained experimentally with those in the mass spectra library (The Wiley Registry of Mass Spectral Data, 6th Ed.).
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2.5. Bioassay An impregnated fabric disk bioassay was used to test acaricidal activity of test materials. Amounts (80, 40, 30, 20, 10, 5, 2.5, 2.0, 1.0, 0.5, 0.25, 0.125 mg/cm2) of each test material dissolved in 100 mL of ethanol were applied to disks of black cotton fabric (0.5 g, 5 cm diameter: 700 mesh). Control fabric disks received 100 mL of ethanol. After drying in a fume hood (18 1C) for 30 s, each piece was placed in the bottom of a Petri dish (5 cm diameter 1.2 cm). Then 30 adult individuals of T. putrescentiae (7–10 days old) were separately placed in each Petri dish which was covered with a lid. Treated and control mites were held at 2571 1C and 75% r.h. in darkness. Mortalities were determined 24 h after treatment under a binocular microscope (20 ). Mites were considered to be dead if appendages did not move when they were prodded with a pin. All treatments were replicated three times. The LD50 values were calculated by probit analysis (SAS, 1990).
3. Results and discussion Acaricidal activity of the oil derived from F. vulgare seeds was tested in various doses against T. putrescentiae adults (Table 1). The LD50 value of the oil was 19.51 mg/cm2 against T. putrescentiae. There was no mortality in the untreated controls. Due to the potent activity of seed oil, isolation of the biologically active component was pursued. Bioassay-guided fractionation of the F. vulgare seed oil afforded an active constituent identified by spectroscopic analyses, including EI-MS, 13CNMR and 1H-NMR, by direct comparison with an authentic reference compound. The biologically active constituent was characterized as the monoterpene (+)-carvone. This compound was identified on the basis of the following evidence. (+)-Carvone, C10H14O: EIMS (70 eV), m/z (% rel int) M+ 150 (21), 135 (14), 122 (9), 108 (67), 94 (10), 82 (100), 67 (7), 54 (56), 39 (23), 27 (4); 1H-NMR (CD3OD, 400 MHz) d 6.77 (1H, s), 4.76–4.78 (1H, d, J ¼ 8 Hz), 2.65 (2H, s), 2.42–2.46 (1H, d, J ¼ 16 Hz), 2.31–2.34 (2H, d, J ¼ 12 Hz), 1.75–1.78 (3H, t, J ¼ 12 Hz), 1.71 (3H, s); 13C-NMR (CD3OD, 100 MHz) d 198.63, 148.26, 145.06, 136.39, 111.60, 44.01, 32.73, 32.66, 21.53, 16.78. The substances identified by GC-MS in the oil of F. vulgare seeds are presented in Table 2. Analysis led to the identification of 14 constituents from the oil of the F. vulgare seeds. The main constituents were apiole (14.73%), asarone (0.4%), camphene (0.6%), 2-carene (0.4%), carvone (44.8%), dihydrocarvone (13.2%), d-limonene (25.5%), 1,4,8-o-menthatriene (0.2%), 1,5,8-pmenthatriene (0.7%), methyl eugenol (0.3%), myrcene (0.3%), naphthalene (0.6%), terpinolene (1.4%) and thymol (1.4%). Together, apiole, carvone, dihydrocarvone and d-limonene made up 98.2% of the oil. Namba (1993) previously reported the main constituents of Foeniculum vulgare fruit oil as t-anethole, p-cymene, estragole, fenchone, d-limonene and terpinene. In this study, the constituents identified from the oil of F. vulgare seeds are different from the constituents of the F. vulgare fruit oil. The acaricidal activity of F. vulgare seed oil-derived compounds against T. putrescentiae adults was examined by direct contact application (Table 1) and compared with that the commonly used benzyl benzoate, N,N-diethyl-m-toluamide and dibutyl phthalate which served as reference materials. Responses varied according to compound and dose. On the basis
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Table 1 Acaricidal activity of constituents derived from Foeniculum vulgare seed oil and synthetic acaricides against Tyrophagus putrescentiaea Compound
LD50 (mg/cm2)
95% confidence limit
Relative toxicityb
Oil Asarone Camphene 2-Carene (+)-Carvone ()-Carvone Dihydrocarvone Eugenol Acetyleugenol Isoeugenol Methyl eugenol d-Limonene Myrcene Naphthalene Terpinolene Thymol Benzyl benzoate N,N-Diethyl-m-toluamide Dibutyl phthalate
19.51 — — — 4.62 5.23 4.32 10.62 72.24 — 39.52 — 39.88 4.28 — 11.42 11.24 13.53 13.11
16.32–22.46 — — — 4.19–5.01 4.88–5.59 3.91–4.75 9.76–11.66 69.67–75.15 — 38.99–40.03 — 39.31–40.39 3.79–4.67 — 10.97–11.94 4.65–17.46 12.43–14.98 11.57–15.06
0.6 — — — 2.4 2.2 2.6 1.1 0.2 — 0.3 — 0.3 2.6 — 1.0 1.0 0.8 0.9
a
Exposed for 24 h. Relative toxicity ¼ LD50 value of benzyl benzoate/LD50 value of each chemical.
b
Table 2 Volatile compounds in Foeniculum vulgare seed oil identified by GC mass spectrometry Peak number
Compound mass spectral dataa
Mass spectral dataa
Retention time (min)
Relative (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Myrcene 1,5,8-p-Menthatriene 1,4,8-o-Menthatriene d-Limonene 2-Carene Dihydrocarvone Terpinolene Camphene Carvone Thymol Methyl eugenol Naphthalene Asarone Apiole
41, 65, 77, 39, 27, 41, 41, 39, 39, 77, 65, 65, 57, 79,
10:82 11:70 13:50 14:34 20:63 30:77 32:10 33:25 33:87 39:25 45:55 52:45 54:79 58:43
0.3 0.7 0.2 25.5 0.4 13.2 1.4 0.6 44.8 1.4 0.3 0.6 0.4 14.73
a
53, 69, 81, 93, 107, 121, 136 77, 91, 105, 119, 134 91, 105, 119, 134 53, 68, 93, 107, 121, 136 39, 53, 79, 93, 105, 121, 136 55, 67, 82, 95, 109, 137, 152 79, 93, 121, 136 41, 79, 93, 121, 136 54, 67, 82, 94, 108, 135, 150 91, 115, 135, 150 77, 91, 107, 147, 163, 178 69, 93, 128 137, 156, 165, 193, 208 87, 105, 137, 151, 176, 209, 222
Major fragmentation ions, base peak (listed first) and other ions in decreasing order of relative abundance.
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of LD50 values, the compound most toxic against T. putrescentiae adults was naphthalene (4.28 mg/cm2) followed by dihydrocarvone (4.32 mg/cm2), (+)-carvone (4.62 mg/cm2), ()-carvone (5.23 mg/cm2), eugenol (10.62 mg/cm2), benzyl benzoate (11.24 mg/cm2), thymol (11.42 mg/cm2), dibutyl phthalate (13.11 mg/cm2), N,N-diethyl-m-toluamide (13.53 mg/cm2), methyl eugenol (39.52 mg/cm2), myrcene (39.88 mg/cm2) and acetyleugenol (72.24 mg/cm2). Acaricidal activities of naphthalene, dihydrocarvone, (+)-carvone and ()-carvone were much more effective than benzyl benzoate, thymol, dibutyl phthalate, N,N-diethyl-m-toluamide, methyl eugenol, myrcene and acetyleugenol. However, no activity was observed for asarone, camphene, 2-carene, isoeugenol, d-limonene and terpinolene. These results indicate that the acaricidal activity of the oil of F. vulgare seeds can mostly be attributed to naphthalene, dihydrocarvone and (+)-carvone. For the acaricidal activity of the seed oil, (+)-carvone is likely to be more important than naphthalene and dihydrocarvone because (+)-carvone is 74.7 and 3.4 times more abundant than naphthalene and dihydrocarvone, respectively. (+)Carvone was about 2.4, 2.9 and 2.8 times more toxic than benzyl benzoate, N,N-diethyl-mtoluamide and dibutyl phthalate, respectively, and naphthalene was about 2.6, 3.2 and 3.1 times more toxic than benzyl benzoate, N,N-diethyl-m-toluamide and dibutyl phthalate, respectively. The acaricidal activity of eugenol and thymol was comparable with that of benzyl benzoate, N,Ndiethyl-m-toluamide and dibutyl phthalate. (+)-Carvone, ()-carvone, dihydrocarvone and naphthalene merit further study as potential stored-food mite control agents or as lead compounds. Plant products are potential sources for stored food-mite control because many of them are selective to pests, with few if any harmful effects on non-target organisms and the environment (Franzolin et al., 1999; Gulati and Mathur, 1995; Perrucci, 1995; Kim et al., 2004). Many plant extracts and phytochemicals are known to possess acaricidal activity against stored food mites (Kim et al., 2004; Sa´nchez-Ramos and Castan˜era, 2001; Macchioni et al., 2002). Naturally occurring acaricidal compounds effective against stored-food mites include fenchone, linalool, linalyl acetate, menthol and menthone from Eucalyptus globulus Labill., Lavandula angustifolia Miller, Lavandula stoechas L. and Menthax piperita L. (Perrucci, 1995), cinnamaldehyde, benzaldehyde, 3-phenylpropionaldehyde, cinnamyl alcohol, salicylaldehyde and 2-hydroxycinnamic acid from Cinnamomum cassia Blume barks (Kim et al., 2004) and terpinene and pulegone from natural monoterpenes (Sa´nchez-Ramos and Castan˜era, 2001). Our study is the first to report acaricidal properties of components derived from F. vulgare seeds against T. putrescentiae adults. In previous studies, the oral LD50 value of carvone and naphthalene for rats was reported as 1.64 g/kg and 0.49 g/kg indicating low acute toxicity to mammals (Sigma Aldrich, 2002; Mallinckrodt Chemicals, 2002). For practical use of F. vulgare seed-derived materials as acaricidal agents, further research should be done on safety issues of this compound for human health, acaricidal mode of action and formulations to improve the acaricidal potency and stability.
Acknowledgments This work was supported by grant No. (R08-2003-000-10009-0) from the Basic Research Program of the Korea Science & Engineering Foundation.
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References Cho, J.C., Kim, J.C., Kim, M.K., Lee, H.S., 2002. Fungicidal activities of 67 herb-derived oils against six phytopathogenic fungi. Agricultural Chemistry and Biotechnology 45, 202–207. Franzolin, M.R., Gambale, W., Cuero, R.G., Correa, B., 1999. Interaction between toxigenic Aspergillus flavus Link and mites (Tyrophagus putrescentiae (Schrank)) on maize grains: effects on fungal growth and aflatoxin production. Journal of Stored Products Research 35, 215–224. Gulati, R., Mathur, S., 1995. Effect of Eucalyptus and Mentha leaves and Curcuma rhizomes on Tyrophagus putrescentiae (Schrank) (Acarina: Acaridae) in wheat. Experimental and Applied Acarology 19, 511–518. Hughes, A.M., 1976. In: Mites of stored food and houses, Technical Bulletin of the UK Ministry of Agriculture, Fisheries and Food, H.M.S.O., London. 400pp. Kim, D.H., Ahn, Y.J., 2001. Contact and fumigant activities of constituents of Foeniculum vulgare fruit against three coleopteran stored-product insects. Pest Management Science 57, 301–306. Kim, H.K., Kim, J.R., Ahn, Y.J., 2004. Acaricidal activity of cinnamaldehyde and its congeners against Tyrophagus putrescentiae (Acari: Acaridae). Journal of Stored Products Research 40, 55–63. Krantz, G.W., 1955. Some mites injurious to farm-stored grain. Journal of Economic Entomology 48, 754–755. Macchioni, F., Cioni, P.L., Flamini, G., Morelli, I., Perrucci, S., Franceschi, A., Macchioni, G., Ceccarini, L., 2002. Acaricidal activity of pine essential oils and their main components against Tyrophagus putrescentiae, a stored food mite. Journal of Agricultural and Food Chemistry 50, 4586–4588. Mallinckrodt Chemicals, 2002. Material Safety Data Sheet, Toxicological information MSDS N0090; Mallinckrodt Baker. Inc. Phillipsburg, NJ. Matsumoto, T., Hisano, T., Hamaguchi, M., Miike, T., 1996. Systemic anaphylaxis after eating storage-mitecontaminated food. International Archives of Allergy and Immunology 109, 197–200. Namba, T., 1993. The Encyclopedia of Wakan-Yaku (Traditional Sino-Japanese Medicines) with Color Pictures. Hoikusha, Osaka, Japan. Perrucci, S., 1995. Acaricidal activity of some essential oils and their constituents against Tyrophagus longior, a mite of stored food. Journal of Food Protection 58, 560–563. Sa´nchez-Ramos, I., Castan˜era, P., 2001. Acaricidal activity of natural monoterpenes on Tyrophagus putrescentiae (Schrank), a mite of stored food. Journal of Stored Products Research 37, 93–101. SAS (Statistical Analysis System) Institute, 1990. SAS/STAT User’s Guide, Version 6. SAS Institute, Cary, NC. Sigma Aldrich, 2002. Material Safety Data Sheet; Toxicological information, Section 11. Sigma-Aldrich Korea Ltd., Yongin, South Korea. Sinha, R.B., 1979. Role of Acarina in stored grain ecosystem. In: Rodriguez, J.G. (Ed.), Recent Advances in Acarology, vol. 1. Academic Press, New York, pp. 263–273. Zdarkova, E., 1991. Stored product acarology. In: Dusbabek, F., Bukva, V. (Eds.), Modern Acarology, Vol. 1. Academia, Prague, pp. 211–218.
Journal of Stored Products Research 42 (2006) 8–14 www.elsevier.com/locate/jspr
Acaricidal activity of fennel seed oils and their main components against Tyrophagus putrescentiae, a stored-food mite Chi-Hoon Lee, Bo-Kyung Sung, Hoi-Seon Lee Faculty of Applied Biotechnology and Research Center for Industrial Development of Biofood Materials, College of Agriculture & Life Science, Chonbuk National University, Chonju 561-756, South Korea Accepted 23 October 2004
Abstract The acaricidal activities of components derived from Foeniculum vulgare (fennel) seed oils against Tyrophagus putrescentiae adults were examined using direct contact application and compared with those of the compounds benzyl benzoate, dibutyl phthalate and N,N-diethyl-m-toluamide. The biologically active constituent of the F. vulgare seeds was characterized as (+)-carvone by spectroscopic analyses. On the basis of LD50 values, the compound most toxic to T. putrescentiae was naphthalene (4.28 mg/cm2) followed by dihydrocarvone (4.32 mg/cm2), (+)-carvone (4.62 mg/cm2), ()-carvone (5.23 mg/cm2), eugenol (10.62 mg/ cm2), benzyl benzoate (11.24 mg/cm2), thymol (11.42 mg/cm2), dibutyl phthalate (13.11 mg/cm2), N,Ndiethyl-m-toluamide (13.53 mg/cm2), methyl eugenol (39.52 mg/cm2), myrcene (39.88 mg/cm2) and acetyleugenol (72.24 mg/cm2). These results indicate that acaricidal activity of the F. vulgare seed oil could be caused by carvone and naphthalene of which the former is likely to be more important because it is 74.7 times more abundant than naphthalene. Carvone and naphthalene merit further study as potential stored-food mite control agents or as lead compounds. r 2004 Elsevier Ltd. All rights reserved. Keywords: Carvone; Foeniculum vulgare; Natural acaricide; Tyrophagus putrescentiae
Corresponding author. Tel.: +82 63 270 2544; fax: +82 63 270 2550.
E-mail address: [email protected] (H.-S. Lee). 0022-474X/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2004.10.004
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1. Introduction Among astigmatid mites, Tyrophagus putrescentiae (Schrank) is a cosmopolitan species commonly encountered infesting a large number of food grains and stored food with a high fat and protein content, such as dried eggs, ham, cheese and different kinds of nuts (Hughes, 1976; Sinha, 1979). It causes serious economic losses (Zdarkova, 1991) as well as reducing nutrient content and germination ability (Krantz, 1955). In addition, the mite is responsible for allergic diseases among farmers and food industry workers handling heavily infested stored products (Hughes, 1976) and causes acute enteritis (Hughes, 1976) and systemic anaphylaxis (Matsumoto et al., 1996) when contaminated food is ingested. The mite also acts as a carrier of bacteria and toxigenic fungi such as Aspergillus spp. in stored grain kept under warm and moist conditions (Franzolin et al., 1999). Use of chemical methods, such as fumigation, spraying with organophosphorus compounds, or treatment with benzyl benzoate, dibutyl phthalate and N,Ndiethyl-m-toluamide, to control T. putrescentiae is prohibited because of human health hazards associated with their consumption. Therefore, the search is on for more selective, natural compounds non-toxic to humans and which do not affect the organoleptic character of the treated product. Research into plant-derived acaricides is now being intensified as it becomes evident that plant-derived acaricides have enormous potential in this regard. Plant extracts or their constituents may provide an alternative to currently used mite-control agents to control stored-food mites (Gulati and Mathur, 1995; Perrucci, 1995; Kim et al., 2004). Since many of them are largely free from adverse effects and have excellent biological activity, they could lead to the development of new classes of possibly safer stored-food mite control agents. In East Asia, the fruits of Foeniculum vulgare Gaertner have long been considered to have medicinal properties attributable to trans-anethole, estragole, d-limonene, (+)-fenchone, a-pinene, g-terpinene and p-cymene (Kim and Ahn, 2001). Little work has been done with respect to managing storedfood mites, although extracts and essential oils of F. vulgare fruits are insecticidal agents (Kim and Ahn, 2001). This paper describes a laboratory study to examine the oil of F. vulgare seeds for storage food mite control constituents against T. putrescentiae adults. The storage food mite control activities of F. vulgare seed oil-derived compounds were compared with those of the commonly used benzyl benzoate, dibutyl phthalate, and N,N-diethyl-m-toluamide.
2. Materials and methods 2.1. Chemicals Asarone, camphene, 2-carene, ()-carvone, dihydrocarvone, eugenol, acetyleugenol, isoeugenol, methyl eugenol, d-limonene, myrcene, naphthalene, terpinolene and thymol were supplied by Sigma (St. Louis, MO, USA). Benzyl benzoate, N,N-diethyl-m-toluamide and dibutyl phthalate were purchased from Aldrich (Milwaukee, WI). All chemicals were of reagent grade. 2.2. Storage mites Cultures of T. putrescentiae had been maintained in the laboratory for 5 yr without exposure to any known acaricide. They were reared in plastic containers (15 12 6 cm) containing 30 g of
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sterilized diet (fry feed No. 1/dried yeast, 1:1 by weight) at 2571 1C and 75% relative humidity (r.h.) in continuous darkness. The fry feed was purchased from Korea Special Feed Meal Co. Ltd., Chonju, Korea. 2.3. Isolation and identification The seeds (6.5 kg) of F. vulgare were purchased from a local market in Chonju and identified by Prof. Sang-Hyun Lee (Forestry Department, Chonbuk National University, Korea). The samples were washed twice with 1 L of distilled water and dried in an oven at 40 1C for 2 days, then finely powdered. The oil (yield 4.8%) of F. vulgare seeds was extracted by steam distillation as previously described (Cho et al., 2002). The oil (15 g) was chromatographed on a silica gel column (Merck 70–230 mesh, 750 g, 6.3 cm i.d. 80 cm) and successively eluted with a stepwise gradient of hexane/ethyl acetate (100:0, 90:10, 80:20, 70:30, 50:50 and 0:100). The bioactive fraction (6.5 g) was successively rechromatographed on a silica gel column, using hexane-ethyl acetate (70:30). Column fractions were analysed by thin layer chromatography (TLC) (silicagel 60 F254) and fractions with similar streaking patterns on the TLC plates were pooled. Preparative high-performance liquid chromatography (prep. HPLC) (Spectra System P2000, Thermo Separation Products) was used for further separation of the constituents. The columns (19 mm i.d. 300 mm, Waters) contained a mPorasil, and were eluted with hexane-ethyl acetate (85:15) at a flow rate of 2.5 mL/min and detected at 280 nm. Finally, the potent active principle (785 mg) was isolated. The structure of the active isolate was determined by instrumental analyses. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded in deuteriochloroform with a JNM-LA 400F7 spectrometer, at 600 and 150 MHz (using tetramethylsilane (TMS) as an internal standard), respectively, and chemical shifts are given in d (ppm). Unambiguous 1H and 13C NMR chemical shifts were obtained using a 1H–1H COSY spectrum as well as a 13C–1H correlation spectrum. UV spectra were obtained in methanol with an Uvikon 922 spectrometer and mass spectra on a JEOL GSX 400 spectrometer. Optical rotation was measured with an Autopol III polarimeter. 2.4. Gas chromatography–mass spectrometry (GC–MS) The oil of F. vulgare seeds was analyzed on a gas chromatograph (HP6890)-mass spectrometer (JMS-600W, JEOL) (GC–MS). The GC column was a 60 m 0.25 mm i.d. DB-WAX (0.25 mm film) fused silica capillary column (J&W Scientific, Folsom, CA, USA). The GC conditions were as follows: injector temperature, 210 1C; column temperature, isothermal at 50 1C for 15 min, then programmed to rise to 200 1C at 2 1C/min and be held at this temperature for 15 min; ion source temperature, 200 1C. Helium was used as the carrier gas at the rate of 0.8 mL/min. The effluent of the GC column was introduced directly into the source of the MS. Spectra were obtained in the EI mode with 70 eV ionization energy. The sector mass analyzer was set to scan from 50 to 800 amu for 2 s. Compounds were identified by comparison with retention times and the mass spectra obtained with the authentic standards on the GC–MS system used for analysis. When an authentic sample was not available, the identification was carried out by comparison of mass spectra obtained experimentally with those in the mass spectra library (The Wiley Registry of Mass Spectral Data, 6th Ed.).
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2.5. Bioassay An impregnated fabric disk bioassay was used to test acaricidal activity of test materials. Amounts (80, 40, 30, 20, 10, 5, 2.5, 2.0, 1.0, 0.5, 0.25, 0.125 mg/cm2) of each test material dissolved in 100 mL of ethanol were applied to disks of black cotton fabric (0.5 g, 5 cm diameter: 700 mesh). Control fabric disks received 100 mL of ethanol. After drying in a fume hood (18 1C) for 30 s, each piece was placed in the bottom of a Petri dish (5 cm diameter 1.2 cm). Then 30 adult individuals of T. putrescentiae (7–10 days old) were separately placed in each Petri dish which was covered with a lid. Treated and control mites were held at 2571 1C and 75% r.h. in darkness. Mortalities were determined 24 h after treatment under a binocular microscope (20 ). Mites were considered to be dead if appendages did not move when they were prodded with a pin. All treatments were replicated three times. The LD50 values were calculated by probit analysis (SAS, 1990).
3. Results and discussion Acaricidal activity of the oil derived from F. vulgare seeds was tested in various doses against T. putrescentiae adults (Table 1). The LD50 value of the oil was 19.51 mg/cm2 against T. putrescentiae. There was no mortality in the untreated controls. Due to the potent activity of seed oil, isolation of the biologically active component was pursued. Bioassay-guided fractionation of the F. vulgare seed oil afforded an active constituent identified by spectroscopic analyses, including EI-MS, 13CNMR and 1H-NMR, by direct comparison with an authentic reference compound. The biologically active constituent was characterized as the monoterpene (+)-carvone. This compound was identified on the basis of the following evidence. (+)-Carvone, C10H14O: EIMS (70 eV), m/z (% rel int) M+ 150 (21), 135 (14), 122 (9), 108 (67), 94 (10), 82 (100), 67 (7), 54 (56), 39 (23), 27 (4); 1H-NMR (CD3OD, 400 MHz) d 6.77 (1H, s), 4.76–4.78 (1H, d, J ¼ 8 Hz), 2.65 (2H, s), 2.42–2.46 (1H, d, J ¼ 16 Hz), 2.31–2.34 (2H, d, J ¼ 12 Hz), 1.75–1.78 (3H, t, J ¼ 12 Hz), 1.71 (3H, s); 13C-NMR (CD3OD, 100 MHz) d 198.63, 148.26, 145.06, 136.39, 111.60, 44.01, 32.73, 32.66, 21.53, 16.78. The substances identified by GC-MS in the oil of F. vulgare seeds are presented in Table 2. Analysis led to the identification of 14 constituents from the oil of the F. vulgare seeds. The main constituents were apiole (14.73%), asarone (0.4%), camphene (0.6%), 2-carene (0.4%), carvone (44.8%), dihydrocarvone (13.2%), d-limonene (25.5%), 1,4,8-o-menthatriene (0.2%), 1,5,8-pmenthatriene (0.7%), methyl eugenol (0.3%), myrcene (0.3%), naphthalene (0.6%), terpinolene (1.4%) and thymol (1.4%). Together, apiole, carvone, dihydrocarvone and d-limonene made up 98.2% of the oil. Namba (1993) previously reported the main constituents of Foeniculum vulgare fruit oil as t-anethole, p-cymene, estragole, fenchone, d-limonene and terpinene. In this study, the constituents identified from the oil of F. vulgare seeds are different from the constituents of the F. vulgare fruit oil. The acaricidal activity of F. vulgare seed oil-derived compounds against T. putrescentiae adults was examined by direct contact application (Table 1) and compared with that the commonly used benzyl benzoate, N,N-diethyl-m-toluamide and dibutyl phthalate which served as reference materials. Responses varied according to compound and dose. On the basis
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Table 1 Acaricidal activity of constituents derived from Foeniculum vulgare seed oil and synthetic acaricides against Tyrophagus putrescentiaea Compound
LD50 (mg/cm2)
95% confidence limit
Relative toxicityb
Oil Asarone Camphene 2-Carene (+)-Carvone ()-Carvone Dihydrocarvone Eugenol Acetyleugenol Isoeugenol Methyl eugenol d-Limonene Myrcene Naphthalene Terpinolene Thymol Benzyl benzoate N,N-Diethyl-m-toluamide Dibutyl phthalate
19.51 — — — 4.62 5.23 4.32 10.62 72.24 — 39.52 — 39.88 4.28 — 11.42 11.24 13.53 13.11
16.32–22.46 — — — 4.19–5.01 4.88–5.59 3.91–4.75 9.76–11.66 69.67–75.15 — 38.99–40.03 — 39.31–40.39 3.79–4.67 — 10.97–11.94 4.65–17.46 12.43–14.98 11.57–15.06
0.6 — — — 2.4 2.2 2.6 1.1 0.2 — 0.3 — 0.3 2.6 — 1.0 1.0 0.8 0.9
a
Exposed for 24 h. Relative toxicity ¼ LD50 value of benzyl benzoate/LD50 value of each chemical.
b
Table 2 Volatile compounds in Foeniculum vulgare seed oil identified by GC mass spectrometry Peak number
Compound mass spectral dataa
Mass spectral dataa
Retention time (min)
Relative (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Myrcene 1,5,8-p-Menthatriene 1,4,8-o-Menthatriene d-Limonene 2-Carene Dihydrocarvone Terpinolene Camphene Carvone Thymol Methyl eugenol Naphthalene Asarone Apiole
41, 65, 77, 39, 27, 41, 41, 39, 39, 77, 65, 65, 57, 79,
10:82 11:70 13:50 14:34 20:63 30:77 32:10 33:25 33:87 39:25 45:55 52:45 54:79 58:43
0.3 0.7 0.2 25.5 0.4 13.2 1.4 0.6 44.8 1.4 0.3 0.6 0.4 14.73
a
53, 69, 81, 93, 107, 121, 136 77, 91, 105, 119, 134 91, 105, 119, 134 53, 68, 93, 107, 121, 136 39, 53, 79, 93, 105, 121, 136 55, 67, 82, 95, 109, 137, 152 79, 93, 121, 136 41, 79, 93, 121, 136 54, 67, 82, 94, 108, 135, 150 91, 115, 135, 150 77, 91, 107, 147, 163, 178 69, 93, 128 137, 156, 165, 193, 208 87, 105, 137, 151, 176, 209, 222
Major fragmentation ions, base peak (listed first) and other ions in decreasing order of relative abundance.
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of LD50 values, the compound most toxic against T. putrescentiae adults was naphthalene (4.28 mg/cm2) followed by dihydrocarvone (4.32 mg/cm2), (+)-carvone (4.62 mg/cm2), ()-carvone (5.23 mg/cm2), eugenol (10.62 mg/cm2), benzyl benzoate (11.24 mg/cm2), thymol (11.42 mg/cm2), dibutyl phthalate (13.11 mg/cm2), N,N-diethyl-m-toluamide (13.53 mg/cm2), methyl eugenol (39.52 mg/cm2), myrcene (39.88 mg/cm2) and acetyleugenol (72.24 mg/cm2). Acaricidal activities of naphthalene, dihydrocarvone, (+)-carvone and ()-carvone were much more effective than benzyl benzoate, thymol, dibutyl phthalate, N,N-diethyl-m-toluamide, methyl eugenol, myrcene and acetyleugenol. However, no activity was observed for asarone, camphene, 2-carene, isoeugenol, d-limonene and terpinolene. These results indicate that the acaricidal activity of the oil of F. vulgare seeds can mostly be attributed to naphthalene, dihydrocarvone and (+)-carvone. For the acaricidal activity of the seed oil, (+)-carvone is likely to be more important than naphthalene and dihydrocarvone because (+)-carvone is 74.7 and 3.4 times more abundant than naphthalene and dihydrocarvone, respectively. (+)Carvone was about 2.4, 2.9 and 2.8 times more toxic than benzyl benzoate, N,N-diethyl-mtoluamide and dibutyl phthalate, respectively, and naphthalene was about 2.6, 3.2 and 3.1 times more toxic than benzyl benzoate, N,N-diethyl-m-toluamide and dibutyl phthalate, respectively. The acaricidal activity of eugenol and thymol was comparable with that of benzyl benzoate, N,Ndiethyl-m-toluamide and dibutyl phthalate. (+)-Carvone, ()-carvone, dihydrocarvone and naphthalene merit further study as potential stored-food mite control agents or as lead compounds. Plant products are potential sources for stored food-mite control because many of them are selective to pests, with few if any harmful effects on non-target organisms and the environment (Franzolin et al., 1999; Gulati and Mathur, 1995; Perrucci, 1995; Kim et al., 2004). Many plant extracts and phytochemicals are known to possess acaricidal activity against stored food mites (Kim et al., 2004; Sa´nchez-Ramos and Castan˜era, 2001; Macchioni et al., 2002). Naturally occurring acaricidal compounds effective against stored-food mites include fenchone, linalool, linalyl acetate, menthol and menthone from Eucalyptus globulus Labill., Lavandula angustifolia Miller, Lavandula stoechas L. and Menthax piperita L. (Perrucci, 1995), cinnamaldehyde, benzaldehyde, 3-phenylpropionaldehyde, cinnamyl alcohol, salicylaldehyde and 2-hydroxycinnamic acid from Cinnamomum cassia Blume barks (Kim et al., 2004) and terpinene and pulegone from natural monoterpenes (Sa´nchez-Ramos and Castan˜era, 2001). Our study is the first to report acaricidal properties of components derived from F. vulgare seeds against T. putrescentiae adults. In previous studies, the oral LD50 value of carvone and naphthalene for rats was reported as 1.64 g/kg and 0.49 g/kg indicating low acute toxicity to mammals (Sigma Aldrich, 2002; Mallinckrodt Chemicals, 2002). For practical use of F. vulgare seed-derived materials as acaricidal agents, further research should be done on safety issues of this compound for human health, acaricidal mode of action and formulations to improve the acaricidal potency and stability.
Acknowledgments This work was supported by grant No. (R08-2003-000-10009-0) from the Basic Research Program of the Korea Science & Engineering Foundation.
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