Mite-Control Activities of Active Constituents Isolated from Pelargonium Graveolens Against House Dust Mites

J. Microbiol. Biotechnol. (2008), 18(10), 1666–1671 Mite-Control Activities of Active Constituents Isolated from Pelargonium graveolens Against House Dust Mites Jeon, Ju-Hyun, Hyung-Wook Kim, Min-Gi Kim, and Hoi-Seon Lee* Bioenviornmental Science Major and Institute of Agricultural Science and Technology, College of Agriculture & Life Science, Chonbuk National University, Chonju 561-756, Korea Received: February 2, 2008 / Accepted: April 2, 2008 The mite-control activities of materials obtained from parts of the world [17, 19, 24]. They are a major source Pelargonium graveolens oil against Dermatophagoides farinae of multiple potent allergens [7, 17, 31], and have been and D. pteronyssinus were examined using an impregnated causally associated with sudden infant death syndrome fabric disk bioassay and were compared with those [13, 18]. Exposure to house dust mite allergens can result shown by commercial benzyl benzoate and N,N-diethyl- in bronchoconstriction and inflammatory reaction of the m-toluamide (DEET). Purification of the biologically airways [4]. Dust mite allergen avoidance measures are active constituents from P. gr av eolen s oil was done by available in daily life. People spend approximately one-third silica gel chromatography and high performance liquid of their lives in bed. Therefore, dust mites in mattresses, chromatography. The structures of the active components bedding, and pillows can contribute greatly to total house were analyzed by EI/MS, 1H-NMR, 13C-NMR, 1H-13C COSY- dust mite exposure [9, 22]. Most houses are co-inhabited NMR, and DEPT-NMR spectra, and were identified as by D. farinae and D. pteronyssinus. However, one species geraniol (C10H18O, MW 154.25, trans-3,7-dimethyl-2,6- usually predominates in each home and makes up more octadien-1-ol) and β-citronellol (C10H20O, MW 156.27, than 70% of the total mite population. These findings 3,7-dimethyl-6-octen-1-ol). Based on the LD50 values, the illustrate the need for cross-antigenicity testing for both most toxic compound was geraniol (0.26 µg/cm2), followed species when a patient undergoes skin testing, and when by β-citronellol (0.28 µg/cm2), benzyl benzoate (10.03 µg/ using immunotherapy [1]. Control of dust mites depends cm2), and DEET (37.12 µg/cm2) against D. farinae. In the on environmental and chemical methods such as fumigation case of D. pteronyssinus, geraniol (0.28 µg/cm2) was the and spraying with acaricidal compounds [2, 8, 17, 19, 21]. most toxic, followed by β-citronellol (0.29 µg/cm2), benzyl However, repeated use of organophosphorus chemicals has benzoate (9.58 µg/cm2), and DEET (18.23 µg/cm2). These resulted in the increase of resistance [17, 28], had undesirable results suggest that D. farinae and D. pteronyssinus may effects on non-target organisms, and fostered environmental be controlled more effectively by the application of geraniol and human health concerns [16, 17]. Therefore, interests and β-citronellol than benzyl benzoate and DEET. have been focused on more selective and new safer Furthermore, geraniol and β-citronellol isolated from P. compounds against house dust mites. graveolens could be useful for managing populations of D. The biological activities of essential oils derived from farinae and D. pteronyssinus. lower (Cryptogamae) and higher plants (Gymnospermae Keywords: Dust mite, Pelargonium graveolens, geraniol, β- and Angiospermae) against several organisms, mainly citronellol bacteria, fungi, and arthropods, have been confirmed in many reports and are mainly due to monoterpenoids and sesquiterpenoids that represent their main components [3, 5, 24]. The essential oil of the rose-scented geranium The most important of the pyroglyphid mites, the American (Pelargonium species, family, Geraniaceae) is widely used dust mite (Dermatophagoides farinae [Hughes]) and the as a floral substitute of the rose scent, and is, therefore, one European dust mite (D. ptertonyssinus [Trouessart]), are of the most valuable natural materials for the perfumery cosmopolitan inhabitants of human dwellings in many and cosmetic industries [27]. Among different essential oils, geranium oil, known as the “women’s oil” because of *Corresponding author Phone: 82-63-270-2544; Fax: 82-63-270-2550; its menstrual and menopausal benefits, has a wide variety E-mail: [email protected] of applications. Besides promoting women’s health, it is MITE-CONTROL ACTIVITIES AND PELARGONIUM GRAVEOLENS 1667 also useful for skin problems like eczema and athlete’s deuteriochloroform with a JNM-LA 400F7 spectrometer (JNM- foot, and for respiratory tract health [15]. It contains ECA600; JEOL Ltd, Japan), at 600 and 150 MHz (TMS as an various compounds, including geraniol, citronellol, nerol, internal standard), respectively, and chemical shifts are given in δ 1 13 linalool, geranyl acetate, and rose oxide [10, 25, 27, 29]. (parts per million). Unambiguous H- and C-NMR chemical shifts 1 1 1 13 However, the acaricidal property of P. graveolens oil has were obtained using DEPT, H- H COSY, and H- H COSY spectra. UV spectra were obtained in chloroform with a Jasco V-550 not yet been investigated. This paper describes a laboratory spectrometer. study to examine the essential oil of the P. graveolens leaves for acaricidal constituents against D. farinae and D. Gas Chromatography-Mass Spectrometry pteronyssinus. Furthermore, the acaricidal activities of The essential oil of P. graveolens leaves was analyzed on a gas active compounds were compared with those of the chromatograph (6890; Agilent, Wilmington, DE, U.S.A.)-mass commonly used benzyl benzoate and DEET. spectrometer (5973IV; Agilent) (GC-MS). The GC column was a 30 m×0.25 mm i.d. DB-5 (0.25-mm film) fused silica capillary column (J&W Scientific, Folsom, CA, U.S.A.). The GC conditions MATERIALS AND METHODS were as follows: injector temperature, 260oC; column temperature, isothermal at 70oC for 3 min, then programmed to rise to 300oC at Chemicals and Plant Materials 10oC/min and be held at this temperature for 5 min; ion source Benzyl benzoate, citral, DEET, farnesol, geranyl acetate, and nerol temperature, 230oC. Helium was used as the carrier gas at a rate of were supplied from Aldrich (Milwaukee, WI, U.S.A.). All other 0.8 ml/min. The effluent of the GC column was introduced directly chemicals were of reagent grade. The leaves of P. graveolens were into the source of the mass spectrometer. Spectra were obtained in purchased from a local market in Chonju. The essential oils tested the EI mode with 70 eV ionization energy. The sector mass analyzer were isolated by steam distillation with cohobation for 8 h as was set to scan from 50 to 600 amu for 2 s. Compounds were described by Cho et al. [6]. Essential oil yield was 0.2% from the identified by comparison with retention times, and the mass spectra steam distillation. obtained with the authentic standards on the GC-MS system were used for analysis. When an authentic sample was not available, the Dust Mites identification was carried out by comparison of experimentally Cultures of D. farinae and D. pteronyssinus were maintained in the obtained mass spectra with those in the mass spectra library (The laboratory for 7 years without exposure to any known acaricide. Wiley Registry of Mass Spectral Data, 6th Ed.). They were reared in plastic containers (15 cm×12 cm×6 cm) containing 30 g of sterilized diet (fry feed No. 1/dried yeast, 1:1 by Dust Mite Bioassay weight) at 25±1oC and 75% relative humidity in continuous An impregnated fabric disk bioassay was used to assess the darkness. The fry feed (Miropa) was purchased from Korea Special acaricidal activity of test samples at 80, 60, 40, 20, 10, 5, 2.0, 1.0, Feed Meal Co. Ltd. (Chonju, South Korea). 0.5, 0.2, 0.1, and 0.05 µg/cm2 [17, 19, 21]. A 20 µl aliquot of test material was applied to disks made of black cotton fabric (0.5 g, Isolation and Identification 5 cm diameter: 700 mesh). Control fabric disks were treated with The oil (24 g) was chromatographed on a silica gel column (Merck only 20 µl of acetone. After the disks were dried in a fume hood 70-230 mesh, 600 g, 10 i.d. × 50 cm) and successively eluted with (19oC) for 30 s, each disk was placed individually in the bottom of a a stepwise gradient of hexane/ethyl acetate (10:0, 9:1, 8:2, 7:3, Petri dish (5 cm diameter×1.2 cm) [17]. Thirty individuals of D. 5:5, and 0:10). The bioactive fraction (8.6 g) was successively farinae (7-10 days old) and D. pteronyssinus (7-10 days old) were rechromatographed on a silica gel column (Merck 70-230 mesh, placed in each Petri dish and covered with a lid. Treated and control 500 g, 5.5 i.d. × 70 cm) using hexane-ethyl acetate (7:3). Column mites were held at 25±1oC and 75% relative humidity in the dark fractions were analyzed by thin layer chromatography (TLC [silica [17, 19, 21]. Mortalities were determined 24 h after treatment under gel 60 F254]; SILC/UV 254, 0.25 mm; Macherey-Nagel, Germany), a binocular microscope (20×). Mites were considered to be dead if and fractions with similar streaking patterns on the TLC plates were appendages did not move when prodded with a pin. All treatments pooled. The active H43 fraction was purified by Prep. HPLC were replicated three times. LD50 values were calculated by probit (Recycling Preparative HPLC; Japan Analytical Industry Co., LTD, analysis [28]. The percentage mortality was determined and Japan) for separation of the biologically active constituent. The first transformed to arcsine square-root values for analysis of variance column was a Jaigel GS Series Column (GS310 50 cm+GS310 (ANOVA).

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