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Isolation and Identification of Mosquito (Aedes aegypti) BitingDeterrent Compounds from the Native American Ethnobotanical Remedy Plant Hierochloë odorata (Sweetgrass) Charles L. Cantrell,*,† A. Maxwell P. Jones,§ and Abbas Ali‡ †

Natural Products Utilization Research Unit, U.S. Department of Agriculture, Agricultural Research Service, University, Mississippi 38677, United States § Department of Plant Agriculture, Gosling Research Institute for Plant Preservation, University of Guelph, Guelph, Ontario N1G 2W1, Canada ‡ National Center for Natural Products Research, The University of Mississippi, University, Mississippi 38677, United States ABSTRACT: Hierochloë odorata (L.) P. Beauv. (Poaceae), commonly known as sweetgrass, has documented use as an insect repellent by the Flatheads of Montana and Blackfoot of Alberta. Both the Flatheads of Montana and Blackfoot of Alberta would use braided plant material in a sachet in clothing or burn them from one end as incense, air/clothing freshener, and insect repellent. This study evaluated the insect-repellent properties of this plant using an in vitro mosquito Aedes aegypti feeding bioassay-directed approach to identify the compound(s) responsible for the observed activities. Evaluation of crude extracts produced from H. odorata revealed that the hydrodistillate had the highest level of mosquito biting deterrence. Fractionation of this extract, followed by re-evaluation for mosquito biting deterrence, produced many active fractions, which were evaluated by spectroscopic techniques and determined to contain phytol, coumarin, and 2-methoxy-4-vinylphenol. Phytol and coumarin were both determined to be responsible for the Ae. aegypti biting deterrency. Scientific evidence reported here validates its traditional use as a biting-insect deterrent. KEYWORDS: Hierochloë odorata, sweetgrass, ethnobotanical remedy, Aedes aegypti, repellent

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INTRODUCTION Utilization of plants and preparations thereof as remedies for the control and management of insect pests has occurred for centuries and is well documented.1−6 Few of these traditional remedies have been investigated systematically to determine the efficacy and understand the chemical constituents present in such preparations specifically responsible for this activity.7−9 The ability to tap into this traditional knowledge base has resulted in the discovery and proliferation of numerous classes of pesticides.10,11 The pyrethroid class of insecticides is an excellent example, with its origin from the Chrysanthemum species. Multiple species of Chrysanthemum flower heads were either crushed and sprinkled over insect-infested plants or burned to repel flying insects.6 Natural pyrethrins or pyrethrum extract isolated from Chrysanthemum species was ultimately used as a starting point for the development of synthetic and semisynthetic pyrethroid insecticides.10 Another example can be seen with aqueous tobacco (Nicotiana spp.) extracts containing alkaloids such as nicotine, which have long been used to control crop insect pests.12 Nicotine preparations such as these have been around for more than 300 years13 and are still in use as biopesticides. Synthetic optimization studies based on the nicotine scaffold have led to the production of some of the most widely used insecticides on the market today such as imidacloprid and acetamiprid. Recent public demand for more “natural” and/or “organic” products has led to a demand for more biopesticides.10 An excellent example is the spinosyns from Dow AgroSciences (Indianapolis, IN, USA), which have had enormous impacts on © 2016 American Chemical Society

both conventional and organic farming. Spinosyns are produced from the fermentation of Saccharopolyspora spinosa with the major constituent being spinosyn A and the next most abundant compound spinosyn D.14 The fermentation product containing both spinosyns A and D is sold commercially under the common name spinosad. Many products containing spinosad are also OMRI (Organic Materials Review Institute) approved, allowing for their use in organic production practices. Hierochloë odorata (L.) P. Beauv. (Poaceae), commonly known as sweetgrass, is an aromatic member of the grass family native to arctic and temperate regions throughout the northern hemisphere.15 This species has traditionally been used for a variety of purposes and is highly revered by several groups of indigenous North Americans.2,16−18 Although the specific uses and beliefs surrounding this plant vary among groups of indigenous people, some of the common uses include basket weaving, medicine, incense and perfume, and a variety of ceremonial applications. It has been used to treat a variety of medical conditions including colds and fevers and to alleviate sharp internal pains.2 However, the medicinal uses of the plant are often overshadowed by its prominence in purification ceremonies, where it is often burned to cleanse the mind, body, and buildings.16 One of the less documented uses of this plant is as an insect repellent. The Flatheads of Montana would store Received: Revised: Accepted: Published: 8352

August 4, 2016 October 14, 2016 October 15, 2016 October 15, 2016 DOI: 10.1021/acs.jafc.6b01668 J. Agric. Food Chem. 2016, 64, 8352−8358

Article

Journal of Agricultural and Food Chemistry

Solvent Extraction. H. odorata leaves (23 g) were air-dried followed by grinding in a Restch PM 400 ball grinder. Ground plant material was extracted at room temperature using 250 mL of hexane providing 324 mg of extractables after evaporation of solvent. Filtered biomass was subsequently extracted using 250 mL of methylene chloride (DCM), providing 354 mg of extractables following evaporation of solvents. This process was repeated using ethanol (95%) as the extraction solvent, providing 1.14 g of extractables. Essential Oil Fractionation. The essential oil (353 mg) was fractionated using an XP-Sil, 100 g, SNAP cartridge (40−63 μm, 60 Å, 40 × 150 mm) running at 40 mL min−1 using a hexane/ethyl acetate step gradient. Three linear steps were used as follows: (step 1) from 100/0 to 80/20 using 1200 mL; (step 2) from 80/20 to 50/50 using 800 mL; and (step 3) from 50/50 to 0/100 using 400 mL. Column eluate was collected into 22 mL portions and, on the basis of TLC similarities, recombined into 12 fractions (A, 1−31, 13 mg; B, 32−24, 53 mg; C, 35−36, 3 mg; D, 37−40, 7 mg; E, 41−43, 8 mg; F, 44−47, 6 mg; G, 48−57, 26 mg; H, 58−64, 22 mg; I, 65−73, 19 mg; J, 74−78, 6 mg; K, 80−110, 17 mg; L, methanol column wash, 102 mg). GC/MS analysis of fraction B indicated the presence of one major compound with a strong National Institute of Standards and Technology (NIST) library match to 6,10,14-trimethyl-2-pentadecanone. Analysis to determine the Kovat index (KI)21,23 of this peak confirmed a match to 6,10,14-trimethyl-2-pentadecanone reported by Karioti et al.24 to be 1845. Final confirmation of fraction B was accomplished by direct comparison of 1H NMR and 13C NMR spectra, GC retention time, and mass spectrum to commercial standard (Alfa Chemistry, Stony Brook, NY, USA), which were all in complete agreement. GC/MS analysis of fraction G indicated the presence of one major compound. Determination of the KI for this major peak to be 1309 indicated a match to 2-methoxy-4-vinylphenol.21 Final confirmation of fraction G was accomplished by direct comparison of 1H NMR and 13 C NMR spectra, GC retention time, and mass spectrum to commercial standard (Sigma-Aldrich), which were all in complete agreement with commercial standard and the literature.25 Fraction H was further purified using a Biotage XP-Sil, 50 g, SNAP cartridge (40−63 μm, 60 Å) using a hexane/acetone step gradient. Three linear steps were used as follows: (step 1) from 100/0 to 90/10 using 996 mL; (step 2) from 90/10 to 80/20 using 495 mL; and (step 3) from 80/20 to 0/100 using 397 mL. Column eluate was collected into 11 mL portions and, on the basis of TLC similarities, recombined into 3 fractions (H1, H2, and H3). The major subfraction (H3) was determined to be phytol on the basis of comparison of 1H NMR and 13 C NMR spectral data with those in the literature.26 Final confirmation was accomplished by comparison of 1H NMR and 13C NMR spectra, GC retention time, and mass spectrum to commercial standard (Sigma-Aldrich), which were all in complete agreement. GC/MS analysis of fraction I indicated the presence of a major compound with a strong NIST library match to coumarin. Determination of the KI for this major peak to be 1432 indicated a match to coumarin. 21 Final confirmation of fraction I was accomplished by direct comparison of 1H NMR spectrum, GC retention time, and mass spectrum to commercial standard (SigmaAldrich), which were all in complete agreement. Mosquito Bioassays. Insects. Ae. aegypti larvae and adults used in these studies were from a laboratory colony maintained at the Mosquito and Fly Research Unit at the Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA. For biting-deterrence bioassays, eggs were hatched and the insects were reared to the adult stage in the laboratory and maintained at 27 ± 2 °C and 60 ± 10% relative humidity (RH) with a photoperiod regimen of 12:12 h (L/D). Adult females (10−20 days old) were used. Mosquito-Biting Bioassays. Experiments were conducted using a six-celled in vitro Klun and Debboun (K&D) module bioassay system20 for quantitative evaluation of biting-deterrent properties of candidate compounds. Briefly, the assay system consists of a six-well reservoir with each of the 3 × 4 cm wells containing 6 mL of blood. As described by Ali et al.,27 a feeding solution consisting of CPDA-1

clothing with braids of sweetgrass or impart the scent by burning the plant under the clothing, which was thought to keep bugs away.2 Both the Flatheads of Montana and the Blackfoot of Alberta would use braided plant material in a sachet in clothing or burn them from one end as incense, air/ clothing freshener, and insect repellent.16 Whereas coumarin is the dominant aromatic compound responsible for the scent of H. odorata and is often credited for any biological activity. Ueyama et al. found that it represented only about 25% of the essential oil, and they went on to identify a total of 169 volatile compounds in an ethanolic extract.19 The objective of the current study was to evaluate the insectrepellent properties of this plant using an in vitro mosquito feeding bioassay-directed approach20 and to identify the compound(s) responsible for any observed activity.

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MATERIALS AND METHODS

Chemicals. Analytical grade solvents used during the experiments were obtained from Sigma-Aldrich (St. Louis, MO, USA). Reference standards and positive controls were obtained as follows: 6,10,14trimethyl-2-pentadecanone (Alfa Chemistry, Stony Brook, NY, USA), 2-methoxy-4-vinylphenol (Sigma-Aldrich), phytol (Sigma-Aldrich), coumarin (Sigma-Aldrich), and N,N-diethyl-m-toluamide (DEET; Sigma-Aldrich). General Experimental Procedures. Column chromatography was performed on a Biotage, Inc. (Charlotte, NC, USA) Isolera pump equipped with an Isolera flash collector and variable-wavelength detector. 1H and13C NMR spectra were acquired on a Bruker (Billerica, MA, USA) Avance 500 MHz in CDCl3. GC/MS/FID Analysis. Fractions and compounds were analyzed on an Agilent 7890A GC system equipped with an autosampler. A DB-5 column (30 m × 0.25 mm fused silica capillary column, film thickness of 0.25 μm) was used operating at the following conditions: injector temperature, 240 °C; column temperature, 60−240 °C at 3 °C/min, held at 240 °C for 5 min; carrier gas, He; injection volume, 1 μL (splitless); MS mass range from m/z 40 to 650; filament delay, 3 min; target TIC, 20,000; prescan ionization, 100 μs; ion trap temperature, 150 °C; manifold temperature, 60 °C; and transfer line temperature, 170 °C. Simultaneous detection with MS and FID was achieved by splitting the column outlet (1:1). The detector temperature for the FID was 300 °C. Kovats indices were calculated using the equation KI (x) = 100[(log RT (x) − log Pz)/(log RT (Pz+1) − log RT (Pz)], where RT (Pz) ≤ RT (x) ≤ RT (Pz+1) and P4 ... P25 are n-paraffins.21 RT is retention time, and x is the compound for which KI is being calculated. Pz and Pz+1 would represent the paraffins with RTs just before and just after x, respectively. Plant Material. H. odorata plants were obtained from Richter’s Herbs (Goodwood, ON, Canada) and grown in greenhouse conditions at the University of Guelph (Guelph, ON, Canada) from January to April 2012. In April, the plants were multiplied through crown division and repotted in 4 L plastic pots filled with Sunshine professional growing media (Sun Gro Horticulture, BC, Canada). The multiplied plants were transferred outside onto black landscaping fabric, watered using drip irrigation, and fertilized with a general purpose fertilizer biweekly. The aerial biomass was harvested in September and placed in trays under forced air at 50 °C until dried, approximately 2 days. This process was repeated in 2013 to provide material for further analysis. A voucher specimen was deposited in the OAC herbarium at the University of Guelph under accession no. 97674. Essential Oil Extraction. Air-dried H. odorata leaves were cut into 1 in. pieces and placed into a 3 L round-bottom flask followed by the addition of 1.2 L of deionized water. Dried leaves were subjected to hydrodistillation for 48 h.22 Hydrodistillation was performed using a Clevenger-type apparatus containing 3 mL of n-pentane. The organic layer from the distillations was combined and dried under a stream of dry nitrogen, resulting in a yield of 353 mg of essential oil. 8353

DOI: 10.1021/acs.jafc.6b01668 J. Agric. Food Chem. 2016, 64, 8352−8358

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Table 1. Evaluation of Hierochloë odorata Extracts and Essential Oil Fractions as Ae. Aegypti Feeding Deterrents Using the K&D Bioassay treatmenta

nb

concentration (μg/cm2)

proportion not biting

standard error

0.84 0.84 0.48 0.44 0.48 0.32

0.075A 0.074A 0.049BC 0.075BC 0.049BC 0.049C

0.88 0.40 0.56 0.80 0.96 0.32

0.05A 0.01BC 0.04B 0.06A 0.04A 0.05C

0.88 0.84 1.00 0.80 0.52 0.28

0.056AB 0.047AB 0.00A 0.00AB 0.08C 0.095D

Experiment 1 DEET (positive control) essential oil (dry) hexane extract methylene chloride extract ethanol extract ethanol (solvent control)

5 5 5 5 5 5

DEET (positive control) essential oil fraction A essential oil fraction B essential oil fractions C−F essential oil fraction G ethanol (solvent control)

5 5 5 5 5 5

DEET (positive control) essential oil fraction H essential oil fraction I essential oil fraction K essential oil fraction L ethanol (solvent control)

5 5 5 5 5 5

4.7 10 10 10 10 n/a Experiment 2 4.7 10 10 10 10 n/a Experiment 3 4.7 10 10 10 10 n/a

a Fractions C−F blended in equal amounts due to limited quantity available for bioassay evaluation. Fraction J not evaluated due to low quantity of sample available. bn, number of replications.

Figure 1. GC-MS total ion chromatogram of H. odorata essential oil. Peaks corresponding to compounds isolated using a bioassay-directed isolation approach are at 20.713 (2-methoxy-4-vinylphenol), 25.777 (coumarin), 41.117 (6,10,14-trimethyl-2-pentadecanone), and 49.643 (phytol). (citrate−phosphate−dextrose−adenine) and ATP was used instead of blood. Green fluorescent tracer dye (www.blacklightword.com) was used to determine feeding by the females. Essential oils, extracts, fractions, and individual compounds were tested in this study. Essential oils, extracts, and fractions were applied at concentrations of 10 μg/cm2, pure compounds at 25 nmol/cm2, and DEET (97%, N,N-diethyl-m-toluamide) (Sigma-Aldrich) at 25 nmol/cm2 as positive control. All treatments were freshly prepared in molecular biology grade 100% ethanol (Fisher Scientific Chemical Co., Fair Lawn, NJ, USA) at the time of bioassay. The temperature of the solution in the reservoirs was maintained at 37 °C by continuously passing warm water through the reservoir using a circulatory bath. Reservoirs were covered with a layer of collagen membrane (Devro, Sandy Run, SC, USA). Test compounds were randomly applied to six 4 × 5 cm areas of organdy cloth and positioned over the membrane-covered CPDA1+ATP solution with a Teflon separator placed between the treated cloth and the six-celled module to prevent contamination of the module. A six-celled K&D module containing five female mosquitoes per cell was positioned over the cloth treatments covering the six

CPDA-1+ATP solution membrane wells, and trap doors were opened to expose the treatments to these females. The number of mosquitoes biting through cloth treatments in each cell was recorded after a 3 min exposure, and mosquitoes were prodded back into the cells to check the actual feeding. Mosquitoes were squashed, and the presence or absence of green fluorescent tracer dye in the gut was used as an indicator of feeding. A replicate consisted of six treatments: four test materials, DEET (a standard biting deterrent), and ethanol-treated organdy as solvent control applied randomly. Five replications each with five females per treatment were conducted for the essential oils, extracts, and fractions. Ten replications were completed for the pure compounds. Statistical Analyses. Proportion not biting (PNB) was calculated using the following formula: PNB = 1 − (total no. of females biting/total no. of females) 8354


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Figure 2. Chemical structures of compounds isolated from H. odorata essential oil. PNB values were analyzed using the ANOVA procedure of SAS, and means were separated using the Ryan−Eino−-Gabrie−-Welsch multiple-range test.

provided PNB values of 0.80 and 0.96, respectively, which were statistically equivalent to DEET. The remaining fractions A and B demonstrated PNB values of 0.40 and 0.56, respectively, neither of which was as effective as DEET. In experiment 3, fractions H, I, and K provided PNB values of 0.84, 1.00, and 0.80, respectively, which were statistically equivalent to that of DEET. The remaining fraction, L, demonstrated a PNB value of 0.52, which was not as effective as DEET. Overall, fractions C−F, G, H, I, and K were all found to be equivalent to DEET; however, fractions G and I seemed to be the most active. Fractions B and L were found to be more active than the solvent control but not as active as DEET. The activity of multiple fractions has also been observed in extracts from other plants and can be the result of inefficient separation of the active constituent or, more often, multiple active compounds.7,9 To determine this and identify the active constituent(s), the most effective fractions were selected for further investigation using chromatographic (CC, GC with retention indices) and spectroscopic techniques (MS, 1H and 13 C NMR). However, the amounts of fractions C−F, K, and L were too low for further purification and characterization and were omitted. Compound Identification. GC/MS analysis of fraction B indicated the presence of one major compound with a strong NIST library match to 6,10,14-trimethyl-2-pentadecanone. Analysis to determine the KI21,22 of this peak confirmed a match to 6,10,14-trimethyl-2-pentadecanone reported by Karioti et al.23 to be 1845 (Figure 2). Final confirmation of fraction B was accomplished by direct comparison of 1H NMR and 13C NMR spectra, GC retention time, and mass spectrum to those of a commercial standard, which were all in complete agreement. Analysis of fraction G by GC/MS indicated the presence of one major compound. Determination of the KI for this major peak to be 1309 indicated a match to 2-methoxy-4-vinylphenol.21 Final confirmation of fraction G was accomplished by direct comparison of 1H NMR and 13C NMR spectra, GC retention time, and mass spectrum to those of a commercial standard, which were all in complete agreement with the commercial standard and the literature.25

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RESULTS AND DISCUSSION Bioassay-Directed Fractionation. Leaves of H. odorata were field grown, harvested, and extracted with hexane, methylene chloride, ethanol and via hydrodistillation. All extracts were evaluated for mosquito biting deterrency against Ae. aegypti at 10 μg/cm2 using an in vitro K&D module bioassay system and compared against DEET at 4.7 μg/cm2 (25 nmol/cm2) and pure ethanol as solvent control. Essential oil produced via hydrodistillation provided a PNB value of 0.84, which was statistically equivalent to that of DEET and more effective than the other extracts (Table 1, experiment 1). Hexane and ethanol extracts demonstrated PNB values of 0.48, whereas the methylene chloride extract gave a PNB value of 0.44. None of the solvent-based extracts were as effective as DEET or the essential oil, nor were they statistically different from the negative control. These results are consistent with other studies in which mosquito-repellent compounds were found in the volatile oils and/or combustion products.7−9,28 The prevalence of mosquito-repellent compounds in volatile extracts comes as no surprise given the importance of olfactory cues in mosquito feeding behavior.29 The lower activity in solvent extracts was likely due to the presence of nonactive compounds, essentially diluting the active components. On the basis of these findings, the essential oil extract was chosen for bioassay-directed fractionation studies. Bioactive essential oil from the hydrodistillation (Figure 1) was fractionated using silica gel column chromatography (CC) and combined into 12 distinct fractions on the basis of their thin layer chromatography (TLC) profile similarity. Fractions were evaluated for mosquito biting deterrency against Ae. aegypti at 10 μg/cm2 using the K&D module bioassay system and compared against DEET at 4.7 μg/cm2 and a solvent control (Table 1, experiments 2 and 3). Because quantities available for some fractions were a limitation in the K&D module bioassay, fractions C−F were evaluated as a mixture in equal amounts, and fraction J was not evaluated, whereas all others were evaluated at 10 μg/cm2. In experiment 2, fractions C−F and G 8355


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6,10,14-Trimethyl-2-pentadecanone has been reported in other volatile oils30,31 and was found to elicit a response when applied to the antennae of Tirathaba mundella,32 suggesting that it may be involved with insect communication. However, when tested as a pure compound in the current study, it was ineffective at deterring mosquito feeding. Although fraction B contained 6,10,14-trimethyl-2-pentadecanone, which was a major constituent in the oil, it was found that this major compound was not highly active. This is one of the reasons that we follow the biological activity throughout the purification process and not always assume that the major constituents are responsible for activity observed in a complex mixture. 2-Methoxy-4-vinylphenol is a common aroma compound found in diverse plant products including fruits such as strawberries33 and tomatoes,34 green tea,35 white wine,36 and some essential oils.37 When tested as a pure compound in this study, it was found to significantly repel mosquitoes, albeit not as effectively as DEET. Previous work has demonstrated it has weak termaticidal activity,37 but to the best of our knowledge this is the first report of its mosquito-deterrent properties. Phytol is common in plants and has previously been identified in essential oils.31,38−40 When tested as a pure compound in previous studies, it was found to have an RC50 (×10−5 mg/cm−2) of 64 compared to 33 for DEET against Anopheles gambiae.39 Similar results were found in the current study, where phytol demonstrated significant repellent activity against Ae. aegypti, but was not as effective as DEET. Coumarin is present in a variety of essential oils19,41−43 and is known to attract some insects while deterring others.43,44 Early studies found that coumarin was an effective repellent against mosquitoes, with a repellency efficiency of >76% when applied to human subjects.45 More recent studies investigated the Ae. aegypti mosquito biting-deterrent constituents present in the fruit oil of Prangos pabularia.46 An analogue of coumarin, suberosin, was isolated from the oil and the authors also included coumarin in the mosquito biting deterrency assays. Both compounds showed biting-deterrent activity, but the activity was lower than that of the positive control, DEET. Similar results were found in the current study using the K&D bioassay, which resulted in significantly fewer mosquitoes feeding than the negative control. However, as with phytol, coumarin was not as effective as DEET. This paper identified three compounds present in sweetgrass with significant mosquito-repellent activity, 2-methoxy-4-vinylphenol, phytol, and coumarin. The presence of these compounds in the volatile oil supports the traditional use as an insect repellent by indigenous North Americans. Altough none of these compounds were as effective as DEET when tested at equal application rates, the crude extract was highly effective when applied at 10 μg/cm2 and could represent a viable natural alternative as pure compound, crude extracts, or using traditional methods. Further research is needed to determine the efficacy of various formulations/approaches, evaluate any potential toxic effects, and assess the duration of the observed activity.

The major constituent present in fraction H was determined to be phytol on the basis of comparison of 1H NMR and 13C NMR spectral data with those in the literature.26 Final confirmation was accomplished by comparison of 1H NMR and 13C NMR spectra, GC retention time, and mass spectrum to those of a commercial standard, which were all in complete agreement. Analysis of fraction I by GC/MS indicated the presence of a major compound with a strong NIST library match to coumarin, known to be the primary constituent present in essential oils of sweetgrass.19 Determination of the KI for this major peak to be 1432 indicated a match to coumarin.21 Final confirmation of fraction I was accomplished by direct comparison of 1H NMR spectrum, GC retention time, and mass spectrum to those of a commercial standard, which were all in complete agreement. Mosquito Biting-Deterrence Evaluation. The four compounds isolated and identified from active fractions were evaluated at 25 nmol/cm2 against female Ae. aegypti mosquitoes in an in vitro K&D module bioassay. Activity of these compounds was compared with DEET and ethanol, solvent control (Figure 3). Compound 6,10,14-trimethyl-2-pentadeca-

Figure 3. Proportion not biting (±SE) values of pure compounds isolated from sweetgrass essential oil against female Ae. aegypti. All compounds were tested at 25 nmol/cm2. Ethanol was the solvent control, and DEET tested at 25 nmol/cm2 was used as positive control.

none was inactive with a PNB of 0.36 and equivalent to solvent control with a PNB value of 0.34. Coumarin, phytol, and 2methoxy-4-vinylphenol were all significantly more active that solvent control with PNB values of 0.64, 0.66, and 0.56. Coumarin and phytol were more active than 2-methoxy-4vinylphenol but not as active as DEET, having a PNB value of 0.78. It is worth mentioning that in Table 1 the crude essential oil activity reported and the activity of the fractions were all evaluated at 10 μg/cm2, whereas in Figure 3 the pure compounds were evaluated at a lower dose for direct comparison with DEET on a molar basis.

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AUTHOR INFORMATION

Corresponding Author

*(C.L.C.) Phone: (662) 915-5898. Fax: (662) 915-1035. Email: [email protected]. Funding

This study was supported in part by a Deployed War-Fighter Protection (DWFP) research program grant funded by the U.S. 8356


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(21) Adams, R. P. Identification of Essential Oil Components By Gas Chromatography/Mass Spectrometry, 4th ed.; Allured: Carol Stream, IL, USA, 2007. (22) Msaada, K.; Hosni, K. Variations in the essential oil composition from different parts of Coriandrum sativum L. cultivated in Tunisia. Ital. J. Biochem. 2007, 56, 47−52. (23) Kováts, E. Gas-chromatographische charakterisierung organischer Verbindungen. Teil 1: Retentionsindices aliphatischer halogenide, alkohole, aldehyde und ketone. Helv. Chim. Acta 1958, 41, 1915− 1932. (24) Karioti, A.; Skaltsa, H.; Demetzos, C.; Perdetzoglou, D.; Economakis, C. D.; Salem, A. B. Effect of nitrogen concentration of the nutrient solution on the volatile constituents of leaves of Salvia f ruticosa Mill. in solution culture. J. Agric. Food Chem. 2003, 51, 6505− 6508. (25) Aldabalde, V.; Risso, M.; Derrudi, M. L.; Geymonat, F.; Seoane, G.; Gamenara, D.; Saenz-Méndez, P. Organocatalyzed decarboxylation of naturally occurring cinnamic acids: potential role in flavoring chemicals production. Open J. Phys. Chem. 2011, 1, 85−93. (26) Kashiwagi, T.; Horibata, Y.; Mekuria, D. B.; Tebayashi, S.; Kim, C. S. Ovipositional deterrent in the sweet pepper, Capsicum annuum, at the mature stage against Liriomyza trifolii (Burgess). Biosci., Biotechnol., Biochem. 2005, 69, 1831−1835. (27) Ali, A.; Cantrell, C. L.; Bernier, U. R.; Duke, S. O.; Schneider, J. C.; Agramonte, N. M.; Khan, I. Aedes aegypti (Diptera: Culicidae) biting deterrence: structure-activity relationship of saturated and unsaturated fatty acids. J. Med. Entomol. 2012, 49, 1370−1378. (28) Wedge, D. E.; Klun, J. A.; Tabanca, N.; Demirci, B.; Ozek, T.; Baser, K. H. C.; Liu, Z.; Zhang, S.; Cantrell, C. L.; Zhang, J. Bioactivity-guided fractionation and GC/MS fingerprinting of Angelica sinensis and Angelica archangelica root components for antifungal and mosquito deterrent activity. J. Agric. Food Chem. 2009, 57, 464−470. (29) Zwiebel, L. J.; Takken, W. Olfactory regulation of mosquito− host interactions. Insect Biochem. Mol. Biol. 2004, 34, 645−652. (30) Sarkhail, P.; Amin, G.; Shafiee, A. Composition of the essential oil of Phlomis persica Boiss and Phlomis chorassanica Bunge from Iran. Flavour Fragrance J. 2004, 19, 538−540. (31) Yayli, N.; Yaşar, A.; Gü leç, C.; Usta, A.; Kolaylı, S.; Coşkunçelebi, K.; Karaoğlu, Ş. Composition and antimicrobial activity of essential oils from Centaurea sessilis and Centaurea armena. Phytochemistry 2005, 66, 1741−1745. (32) Sasaerila, Y.; Gries, R.; Gries, G.; Khaskin, G.; King, S.; Takács, S. Sex pheromone components of male Tirathaba mundella (Lepidoptera: Pyralidae). Chemoecology 2003, 13, 89−93. (33) Pyysalo, T.; Honkanen, E.; Hirvi, T. Volatiles of wild strawberries, Fragaria vesca L., compared to those of cultivated berries, Fragaria × ananassa cv Senga Sengana. J. Agric. Food Chem. 1979, 27, 19−22. (34) Buttery, R. G.; Seifert, R. M.; Guadagni, D. G.; Ling, L. C. Characterization of additional volatile components of tomato. J. Agric. Food Chem. 1971, 19, 524−529. (35) Kumazawa, K.; Masuda, H. Identification of potent odorants in Japanese green tea (Sen-Cha). J. Agric. Food Chem. 1999, 47, 5169− 5172. (36) Guth, H. Quantitation and sensory studies of character impact odorants of different white wine varieties. J. Agric. Food Chem. 1997, 45, 3027−3032. (37) Tellez, M. R.; Khan, I. A.; Kobaisy, M.; Schrader, K. K.; Dayan, F. E.; Osbrink, W. Composition of the essential oil of Lepidium meyenii (Walp.). Phytochemistry 2002, 61, 149−155. (38) Kobaisy, M.; Tellez, M. R.; Webber, C. L.; Dayan, F. E.; Schrader, K. K.; Wedge, D. E. Phytotoxic and fungitoxic activities of the essential oil of kenaf (Hibiscus cannabinus L.) leaves and its composition. J. Agric. Food Chem. 2001, 49, 3768−3771. (39) Odalo, J. O.; Omolo, M. O.; Malebo, H.; Angira, J.; Njeru, P. M.; Ndiege, I. O.; Hassanali, A. Repellency of essential oils of some plants from the Kenyan coast against Anopheles gambiae. Acta Trop. 2005, 95, 210−218.

Department of Defense through the Armed Forces Pest Management Board and USDA-ARS Grant 56-6402-1-612. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Amber Reichley and Solomon Green III for technical assistance. We thank Dr. James J. Becnel, Mosquito and Fly Research Unit, Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, FL, USA, for supplying Ae. aegypti eggs.

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REFERENCES

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