USO08057829B2
(12) United States Patent (10) Patent No.: US 8,057,829 B2 Zhang et al. (45) Date of Patent: Nov. 15, 2011
(54) METHODS FOR REPELLING INSECTS 3.29. A. 658, MaineWOO d SNSEESENTYDROCARBONS 2008.0020078 A1 1/2008 Enan 2008/0057005 A1 3/2008 Lehn 2008, OO69785 A1 3/2008 Jones (75) Inventors: Qinghe Zhang, Spokane Valley, WA 2008.0075796 A1 3/2008 Enan (US); Armenek Margaryan, Spokane 2008. O155886 A1 7, 2008 Okuda Valley, WA (US); Rodney G. 2008/0201796 A1 8/2008 Chappell Schneidmiller, Greenacres, WA (US) 2008/0269.177 A1 10, 2008 Bessette FOREIGN PATENT DOCUMENTS (73) Assignee: Sterling International Inc., Spokane, JP 20O23O8705. A 10, 2002 WA (US) JP 2004051564 * 2, 2004 JP 2004O995.35 * 4, 2004 (*) Notice: Subject to any disclaimer, the term of this WO OO27.197 A1 5.2000 patent is extended or adjusted under 35 OTHER PUBLICATIONS U.S.C. 154(b) by 0 days. Nature's Lather, 2 pages, 2009.* (21) Appl. No.: 12/346,082 Da Silva, T.B.C, et al., “Chemical Constituents and Preliminary Antimalarial Activity of Humiriabalsamifera.” Pharmaceutical Biol (22) Filed: Dec. 30, 2008 ogy 42(2):94-97, Apr. 2004. Flint, H.M. et al., "Caryophyllene: an Attractant for the Green Lacew (65) Prior Publication Data ing.” Environmental Entomology 8(6): 1123-1125, Dec. 1979. Klun, J.A., and M. Debboun, “A New Module for Quantitative Evalu US 2010/O166896 A1 Jul. 1, 2010 ation of Repellent Efficacy Using Human Subjects,” Journal of Medi cal Entomology 37(1): 177-181, 2000. (51) Int. Cl. Kuo, P-M., et al., “Insecticidal Activity of Essential Oil From AOIN 65/00 (2009.01) Chamaecyparis formosensis Matsum.” Holzforschung, 61(5):595 (52) U.S. Cl...... 424/725 North,599. Aug. R.D. 2007. et al., "Agonistic Behavior of the Leaf-Cutting Ant Atta (58) Field of Classification Search ...... None sexdens rubropilosa Elicited by Caryophyllene.” Journal of Insect See application file for complete search history. Behavior 13(1):1-13, 2000. Zhang, A., et al., “A Natural Bridged Tricyclic Sesquiterpene, (56) References Cited Isolongifolenone, is a Repellent Against Blood-Feeding Arthropods.” Poster shown at Entomological Society of America U.S. PATENT DOCUMENTS Annual Meeting, San Diego, Calif., Dec. 9-12, 2007.
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US 8,057,829 B2 1. 2 METHODS FOR REPELLING INSECTS The methods above may further include one or more com USING SESQUITERPENE HYDROCARBONS pounds selected from the group consisting of B-caryophyl AND THEIRDERVATIVES lene, isocaryophyllene, 3-caryophyllene oxide, B-caryophyl lene ketone, C.-humulene, B-patchoulene, C.-patchoulene, BACKGROUND B-elemene, (+)-longipinene, (-)-isolongifolene, (+)-longi folene, and linalool. The methods may further include the use Insects in the order Diptera include many familiar flying of clove oil, patchouli oil, or a mixture thereof. insects such as filth and biting flies and mosquitoes. Many Representative insects that may be repelled include filth species of Diptera are known as vectors for varieties of dis flies, biting flies, blood feeding flies, mosquitoes, and other eases in man, other animals, and plants. For example, diseases 10 flying orbiting insects, mostly from, but not limited to, the Such as dysentery, cholera, and yaws may be transmitted on order Diptera. Filth flies refer to flies that belong primarily to house flies feet and mouthparts. Horse flies and deer flies are known to spread tularemia, loiasis, and trypanosomiasis. the families Muscidae, Calliphoridae, and Sarcophagidae. Black flies are known to spread human onchoceriasis and Representative examples of filth flies include the house fly leucocytozoon infections of poultry. Sand flies are known to 15 (Musca domestica), the black blow fly (Phormia regina), and spread leishmaniasis. Mosquitoes are known to spread bottle flies (Calliphora spp.). Blood feeding or biting flies malaria, encephalitis, yellow fever, and filariasis. Accord refer to flies that include the family Tabanidae (horse flies), ingly, it is important to control flying orbiting insects such as and include flies such as stable flies (Stomoxys calcitrans), flies and mosquitoes, especially in less developed countries, horn flies (Haematobia irritans), and deer flies (Chrysops to minimize and reduce the risk of these insects serving as spp.). Black flies (family Simuliidae) and sand flies (family vectors for diseases in humans. Psychodidae) are also included. Other insects that may be Various chemicals and mixtures have been studied and repelled using the methods disclosed herein include Musca used for insect repelling activities. For example, N,N-diethyl domestica (a housefly), Lucilia sericata (a green bottle fly), m-toluamide (DEET), and dimethyl phthalate are widely Sarcophaga sp. (flesh flies), Muscina Stabulans (a false stable used as insect repellents. DEET is very effective in protecting 25 fly), and Phlebotomus papatasi (a sand fly) and mosquitoes outdoor people from insects. Certain formulations containing including Aedes aegypti, Aedes increpitus, Anopheles DEET have been designed for cosmetic uses, for example, in Stephensi, Anopheles gambiae, and Culex pipiens. Sunscreen products such as lotions. While DEET is an effective repellent, it is not particularly DESCRIPTION OF THE DRAWINGS pleasing in Smell, it stings when applied, and its use causes a 30 number of harmful side effects in humans including injuries The foregoing aspects and many of the attendant advan to eyes, mucous membranes, and sensitive skin. Because tages of this invention will become more readily appreciated DEET is absorbed through the skin, toxic systemic reactions as the same become better understood by reference to the may result even when the compound is only used topically. following detailed description, when taken in conjunction Repeated exposure to DEET has been associated with inci 35 with the accompanying drawings, wherein: dences of seizures, irritability, confusion, insomnia, encepha FIG. 1 shows the antennal responses of a female housefly, lopathy, and coma. sand fly, and three species of mosquitoes to Solid Phase The potential hazards of using a product with DEET as an Microextraction (SPME: CAR/PDMS) samples of clove active ingredient indicate that there is a need for new methods essential oil, recorded by a GC-flame ionization detector for repelling insects. 40 (FID) coupled with an electroantennographic detector (EAD): SUMMARY FIG. 2 shows GC-EAD responses of a female housefly, sand fly, and three species of mosquito antennae to SPME This Summary is provided to introduce a selection of con (CAR/PDMS) samples of patchouli oil, containing cepts in a simplified form, which are further described below 45 C-patchoulene, B-patchoulene, B-elemene, Seychellene, in the Detailed Description. This summary is not intended to C-guaiene, C.-bulnesene, and B-caryophyllene; identify key features of the claimed subject matter, nor is it FIG. 3 shows GC-EAD responses of the female housefly intended to be used as an aid in determining the scope of the antenna to a synthetic mixture of 1-octen-3-ol, (+)-lon claimed Subject matter. gipinene, (-)-isolongifolene, (+)-longifolene, B-caryophyl Methods are disclosed for repelling one or more insects 50 lene, C-humulene, and isolongifolenone; from, but not limited to, the order Diptera and, in particular, FIG. 4 shows the GC-EAD responses of the male housefly for repelling filth flies, biting flies, and mosquitoes. In one antenna to a synthetic mixture of 1-octen-3-ol, B-caryophyl aspect, a method for repelling an insect from an area is pro lene, and isocaryophyllene; vided. The method comprises treating the area with at least FIG. 5 shows the GC-EAD responses of the male false one repellent compound selected from the group consisting of 55 stable fly and the female mosquito Aedes (Ochlerotatus) Seychellene, C-guaiene, and C.-bulnesene, or a mixture increpitus Dyar antennae to a synthetic mixture of linalool, thereof. In another aspect, a method for repelling an insect B-caryophyllene, C.-humulene, and B-caryophyllene oxide; from a human or animal subject is provided. The method FIG. 6 shows the GC-EAD responses of a male house fly, comprises applying to a Subject or to a device worn by the female green bottle fly, female flesh fly, male false stable fly Subject at least one repellent compound selected from the 60 and two species of mosquitoes to a synthetic mixture of group consisting of Seychellene, C-guaiene, and C.-bulnesene, 1-octen-3-ol, linalool B-caryophyllene, C-humulene, or a mixture thereof. In another aspect, a method for inhibit ZE-nepetalactone, EZ-nepetalactone, DEET, and SS220; ing or blocking the biting activity of a mosquito from a human FIG. 7 shows the GC-EAD responses of the female mos or animal Subject is provided. The method comprises apply quito Culex pipiens antenna to a synthetic mixture of 1-octen ing to a subject or to a device worn by the Subject at least one 65 3-ol, B-caryophyllene, and isocaryophyllene; repellent compound selected from a group consisting of Sey FIG. 8 shows the GC-EAD responses of the female mos chellene, C.-guaiene and C.-bulnesene, or a mixture thereof. quito Culex pipiens antenna to a synthetic mixture of 1-octen US 8,057,829 B2 3 4 3-ol, (+)-longipinene, (-)-isolongifolene, (+)-longifolene, and B-Guaiene. Both structures are shown below. B-Guaiene B-caryophyllene, and isolongifolenone; is a known termite attractant from U.S. Pat. No. 6,352,703. FIG.9 shows GC-EAD responses of the sand fly and three However, C.-Guaiene is disclosed herein as an insect repel species of mosquitoes to a synthetic mixture of 1-octen-3-ol, (+)-longipinene, (-)-isolongifolene, (+)-longifolene, B-caryophyllene, C-humulene, ZE-nepetalactone, EZ-nepetalactone, and isolongifolenone; FIG. 10 shows GC-EAD responses of the sand fly, and three species of mosquitoes to a synthetic mixture of 1-octen 3-ol, linalool, isocaryophyllene, B-caryophyllene, C-humu 10 lene, caryophyllene oxide, and DEET: FIG.11 shows a laboratory bioassay of the repellent effects of B-caryophyllene and linalool on the housefly with egg powder used as an attractant; 15 FIG. 12 shows a laboratory bioassay of the repellent effects C-Guaiene B-Guaiene of B-caryophyllene and isocaryophyllene on the housefly , , with egg powder used as an attractant; In one embodiment, the repellent compound is C.-bulne FIG.13 shows a laboratory bioassay of the repellent effects Sene. Bulnesene is a guaiene-type sesquiterpene, which may of B-caryophylene, (+)-longifolene, and (-)-isolongifolene be isolated from patchouli oil. Bulnesene has two isomers, on the housefly with egg powder used as an attractant; and C.-bulnesene and B-bulnesene. Both structures are shown FIG. 14 shows a field bioassay of the repellent effects of below. C-Bulnesene is also known as Ö-guaiene. B-caryophyllene on several species of flies, in which the notation (***) indicates a significant difference between the treatments at PK0.001 by pair-T test. 25 DETAILED DESCRIPTION Methods are disclosed for repelling an insect by using a sesquiterpene hydrocarbon compound or derivative of a ses quiterpene hydrocarbon compound. As used herein, the term 30 “sesquiterpene hydrocarbon refers to any linear or cyclic natural product with C15 skeletons constructed from three (3) five-carbon building-units. As used herein, the term “sesquit C-Bulnesene B-Bulnesene erpene hydrocarbon derivative' refers to any oxidized form of a sesquiterpene hydrocarbon compound, as defined herein. 35 Other sesquiterpene hydrocarbons may be used together Representative oxidized forms include alcohol, oxide, with Seychellene, C.-guaiene, and/or C.-bulnesene and mix ketone, aldehyde, carboxylic acid, lactone, ketal, and ester. tures thereof for repelling insects of the order Diptera. Rep Methods are disclosed for repelling an insect of the order of resentative sesquiterpene hydrocarbons that are useful in Diptera from an area by treating the area with at least one combination with one or more of Seychellene, C.-guaiene, repellent compound selected from the group consisting of 40 C.-bulnesene and mixtures thereof include B-caryophyllene, Seychellene, C-guaiene, and C.-bulnesene, or a mixture isocaryophyllene, C.-humulene, 3-caryophyllene oxide, thereof. These compounds occur naturally in patchouli oil. B-caryophyllene ketone, (+)-longifolene, (-)-isolongifolene, Patchouli oil is derived from several species of plants includ (+)-longipinene, B-patchoulene, C.-patchoulene, and B-el ing Pogostemon cablin, for example. The compounds may be emene. Linalool, although not a sesquiterpene, can be used in isolated in Substantially pure form through well-known sepa 45 combination with other sesquiterpenes disclosed herein. ration techniques, such as distillation. B-caryophyllene is a natural bicyclic sesquiterpene that is a In one embodiment, the repellent compound is Seychel constituent of some essential oils, especially clove oil and the lene. Seychellene is a tricyclic sesquiterpene that may be oil from the stems and flowers of Syzygium aromaticum. isolated from patchouli oil. Alternatively, synthesis of sey Isocaryophyllene is the cis double bond isomer of B-caryo chellene can be performed as described by Fukamiya, N.; 50 phyllene. It is believed that isocaryophyllene is mainly an Kato, M.; and Yoshikoshi, A., Chem. Commun. 1971, 1120. artifact of the isolation process for B-caryophyllene. Seychellene is commonly used in perfume compositions and C.-Humulene is the open ring isomer of B-caryophyllene in flavoring compositions. Seychellene has the following and is a naturally occurring monocyclic sesquiterpene. C.-Hu Structure: mulene is found in the essential oils of Humulus lupulus (hops) from which it derives its name, and in clove oil. The 55 structures of B-caryophyllene, isocaryophyllene, and C.-Hu mulene are shown below.
Seychellene
In one embodiment, the repellent compound is C-guaiene. 65 Guaiene is a hydrocarbon sesquiterpene that may be isolated B-Caryophyllene Iso-caryophyllene from patchouli oil. There are two guaiene isomers, C.-Guaiene US 8,057,829 B2 5 6 -continued Chemically, (+)-longifolene is a tricyclic sesquiterpene. This molecule is chiral, and the enantiomer commonly found CH3 in pines and other higher plants exhibits a positive optical / rotation of about +42.73°. The other enantiomer (optical rota tion of about -42.73) is found in small amounts in some primitive plants like fungi and liverworts. (+)-Longifolene may be obtained by precise distillation of heavy turpentine oil or other essential oils, and is also com Ci-Humulene 10 mercially available. The purity of (+)-longifolene usually is in a range of 55 to 75%. The main impurities in the raw material are sesquiterpene hydrocarbons, such as B-caryophyllene. B-Caryophyllene has a highly strained and reactive endocyclic trans-cyclononene ring, a trans-fused bicyclo (+)-Longifolene, as a tricyclic sesquiterpene in a high 7.2.0 undecane skeleton, and a reactive cyclobutylvinyl energy state, can be rearranged into various derivatives in the moiety; all of which combine to account for its propensity to 15 presence of a Lewis acid under different conditions. Repre undergo facile structural rearrangements. Varieties of rear sentative (+)-longifolene derivatives include various isomers ranged and oxidized forms off-caryophyllene may be used as of (+)-longifolene and various oxidized derivatives of (+)- repellents for insects. Representative B-caryophyllene deriva longifolene and its isomers. tives include oxides and ketones. B-Caryophyllene ketones and isocaryophyllene ketones (-)-Isolongifolene, with a structure shown below, is a well can be obtained by the functionalization of the exocyclic known isomer of (+)-longifolene. U.S. Patent Application methylene group carried out in the presence of the endocyclic Publication No. 2004/242936 describes an isomerization pro double bond, oZonolysis of B-caryophyllene epoxide, or cess to convert (+)-longifolene to (-)-isolongifolene (2.2.7. deoxygenation and conversion of the 2- and 3-alcohol deriva 7-tetramethyltricyclo undec-5-ene) using nano-crystalline tives with high stereospecificity. Representative B-caryophyl 25 Sulfated Zirconia as a Solid Super acid catalyst. Isolongifole lene derived ketones include: none can be made from (-)-isolongifolene as disclosed in U.S. Pat. No. 7,378,557.
30
35 B-Caryophylene ketone Isocaryophyllene ketone (-)-Isolongifolene B-Caryophyllene oxide, shown below, can be obtained by oxidation of the double ring bond in P-caryophyllene. 40 (+)-Longipinene, with the structure shown below, is another sesquiterpene hydrocarbon in the longifolene family.
45
B-Caryophylene oxide 50 (+)-Longifolene, with the structure shown below, is the (+)-Longipinene common chemical name of a naturally-occurring oily liquid hydrocarbon found primarily in the high-boiling fraction of certain pine resins. (+)-Longifolene is the main component of Patchoulene is a sesquiterpene hydrocarbon found in heavy turpentine oil in Chinese Masson's pine. patchouli oil. There are two isomers, C.-Patchoulene and 55 B-Patchoulene, each having the structure as shown below.
60
(+)-Longifolene 65 Ot-Patchoulene B-Patchoulene US 8,057,829 B2 7 8 B-Elemene is a chemical compound that can be extracted folene, (+)-longifolene. The terpene alcohol linalool was also from numerous plants including Curcuma aromatia, and Cur found to be EAD-active. The EAD activities of the above cuma longa linn (all belonging to Ziniberaceae). B-Elemene mentioned compounds are detectable in one or more of the has a structure as shown below. following insects: housefly (Musca domestica L.), green bottle fly (Lucilia sericata), flesh fly (Sarcophaga sp.), and
false stable fly (Muscina stabulans); several mosquitoes such as Culex pipiens, Aedes aegypti, Aedes increpitus, Anopheles gambiae, Anopheles Stephensi, and a sand fly (Phlebotomus papatasi). 10 The GC-EAD antennal response of a female housefly (Musca domestica), sand fly (Phlebotomus papatasi), and mosquitoes including the species Aedes aegypti, Anopheles gambiae, and Anopheles Stephensi were tested with clove B-Elemene essential oil. FIG. 1 is a simultaneous recording of GC flame 15 ionization detection (FID) and electroantennographic detec tion (EAD) of the insectantennae to solid phase microextrac Linalool, or 3,7-dimethyl-3-hydroxy-1,6-octadiene, is a tion (SPME: CAR/PDMS) samples of clove essential oil in a naturally occurring terpene alcohol found in the essential oil 20 ml glass vial for 20 sec. Two EAD-active compounds, of Coriander (Coriandrum sativum), Rosewood (Aniba spp.) B-caryophyllene and C-humulene, and one EAD-inactive (or and Ho oils (Cinnamomum camphora var. linaloolifera and weakly active) compound, eugenol, were identified by GC Cinnamomum camphora var. glavescens). Commercial Syn MS, and confirmed with synthetic standards. As shown in thetic linalool has been prepared by isomerization of geraniol. FIG. 1, two of the three major compounds detected from the The structure of linalool is shown below. SPME sample of the clove essential oil, B-caryophyllene and C-humulene, elicited antennal responses by one or more of 25 the housefly, sand fly, and mosquitoes Aedes aegypti, Anoph eles gambiae, and Anopheles Stephensi, while the third major component, eugenol, was antennally inactive at the dose N tested. No detectable EAD activities from the housefly, sand HO fly, or mosquitoes were found in reaction to any minor com 30 ponents of the clove oil SPME samples. Linalool FIG. 2 shows GC-EAD antennal responses of a female housefly, sand fly, and mosquitoes (Aedes aegypti, Aedes The above-described sesquiterpene hydrocarbons, lina increpitus, and Anopheles gambiae) to SPME (CAR/PDMS) lool, and the derivatives thereof, may be used in substantially samples of patchouli essential oil. Seven major sesquiterpe pure form or may be combined with essential oils, such as 35 nes, C.-patchoulene, B-patchoulene, B-elemene, Seychellene, clove oil and patchouli oil. C-guaiene, C.-bulnesene, and 3-caryophyllene, elicited EAD Clove oil is an essential oil from the clove plant, Syzygium activity by one or more of the housefly, sand fly, and mosquito aromaticum. The main chemical components of clove oil antennae. These EAD-active compounds were identified by include eugenol, eugenol acetate, iso-eugenol, and B-caryo GC-MS. phyllene. Patchouli oil may be extracted from Pogostemon 40 The GC-EAD responses of the female housefly antenna cablin (also known as Pogostemon patchouli) of the Labiatae were tested on a synthetic mixture of 1-octen-3-ol, a known family and is also known as patchouly and puchaput. The attractant used as a positive control, and representative ses chemical components of patchouli oil include 3-patchoulene, quiterpene hydrocarbons and derivatives, (+)-longipinene, C-guaiene, B-caryophyllene, C-patchoulene, Seychellene, (-)-isolongifolene, (+)-longifolene, B-caryophyllene, C.-hu C.-bulnesene, norpatchoulenol, patchouli alcohol, and pogos 45 mulene, and isolongifolenone, with about 100 ng of each tol. compound in the mixture. As shown in FIG. 3, the sesquiter The sesquiterpene hydrocarbons and their derivatives dis pene hydrocarbons, (+)-longipinene, (-)-isolongifolene, (+)- closed herein have been identified as suitable insect repellents longifolene, B-caryophyllene and C-humulene were active on by testing via a gas chromatographic-electroantennographic housefly antennae, while isolongifolenone (a sesquiterpene detection (GC-EAD) method as described in Example 1 50 ketone) was inactive at the dose tested. below. This method can be used to efficiently screen for The GC-EAD responses of the male housefly antenna were potentially behaviorally active compounds, including insect tested on a synthetic mixture of 1-octen-3-ol, and two repre repellents, attractants, deterrents, or attraction-inhibitors sentative sesquiterpenes, B-caryophyllene and isocaryophyl from complex natural Sources or synthetic mixtures. EAD lene, with about 100 ng of each compound in the mixture. As activity shows that the insect has olfactory receptor neurons 55 shown in FIG. 4, both B-caryophyllene and its cis-isomer, for the compound, which is an indicator that the compound isocaryophylene were EAD-active. may be an insect repellentif it comes from a natural repellent The GC-EAD responses of the male false stable fly Mus Source (e.g., essential oils), oran attractant, if coming from an cina stabulans antenna and the female mosquito Aedes attractive natural source. Accordingly, if a compound shows (Ochlerotatus) increpitus Dyarantenna were tested on a syn EAD activity, the compound and any structurally-similar 60 thetic mixture of linalool and two representative sesquiter compound, including any isomer or oxidized derivative, may pene hydrocarbons, B-caryophyllene and C-humulene, and likewise be an insect repellent or attractant. one sesquiterpene oxide, B-caryophylene oxide, with about EAD-active sesquiterpenes from the repellent essential 100 ng of each compound in the mixture. The results are oils, clove oil and patchouli oil, include Seychellene, shown in FIG. 5. The compounds tested appear to be active on C-guaiene, C.-bulnesene, B-caryophyllene, isocaryophyllene, 65 at least one of the insects tested. B-caryophyllene oxide, C-humulene, B-patchoulene, The GC-EAD responses of several species of flies and two C-patchoulene, B-elemene, (+)-longipinene, (-)-isolongi species of mosquito antennae were tested on a synthetic mix US 8,057,829 B2 10 ture of 1-octen-3-ol, linalool, two representative sesquiter the egg powder. In FIGS. 11-13, the bioassay was carried out pene hydrocarbons, 3-caryophyllene and C-humulene, ZE on houseflies using devices known by the designation Trecé nepetalactone, EZ-nepetalactone, and two known insect Pherocon R VI sticky traps. repellents, DEET, and SS220, with about 100 ng of each FIG. 11 shows the laboratory bioassay of the spatial repel compound in the mixture. DEET and SS220 are used as 5 lent effects of B-caryophyllene, linalool, and a mixture of positive controls in the experiment. The compound 1-octen B-caryophyllene and linalool. FIG. 11 shows a graph of the 3-ol is a known attractant for the insects and is also used as a number of house flies per trap plotted againstablank control positive control. The insects tested are species of Musca having no attractant or repellent, a control using only the egg domestica, Lucillia sericata, Sarcophaga sp., Muscina stabu powderattractant, and three combinations of egg powder with 10 two individual repellents, and a combination of repellents. lans, Culex pipiens, and Aedes increpitus. The results are FIG. 11 demonstrates that both B-caryophyllene and linalool shown in FIG. 6. The sesquiterpenes B-caryophyllene and are effective housefly repellents. The blank control showed C-humulene, and linalool appear to be active on at least one of no attraction for flies. Using the egg powder attractant alone the insects tested. resulted in an average of 44 house flies per trap. The egg As shown in FIGS. 5 and 6, antennae of at least one of 15 powder and B-caryophyllene resulted in an average of 3.5 Muscina stabulans, Aedes increpitus, Culex pipiens, Musca house flies per trap. The egg powder and linalool resulted in domestica, Lucilia sericata, and Sarcophaga sp. responded to 5.5 house flies per trap. The egg powder and combination at least one of B-caryophyllene, C-humulene, B-caryophyl B-caryophyllene and linalool resulted in two house flies per lene oxide and linalool. trap. FIG. 11 demonstrates that while both B-caryophyllene The GC-EAD responses of the female mosquito Culex and linalool are house fly repellents, the combination appears pipiens antenna were tested on a synthetic mixture of 1-octen to have a synergistic effect in repelling as evidenced by 3-ol, and two sesquiterpene hydrocarbons, B-caryophyllene attracting the least amount of flies using the combination. and isocaryophyllene, with about 100 ng of each compound FIG. 12 is another bar graph showing results of a bioassay in the mixture. The results are shown in FIG. 7. testing the repellency of B-caryophyllene, isocaryophyllene, The GC-EAD responses of the female mosquito Culex 25 and the combination. The egg powder alone attracted on pipiens antenna were tested on a synthetic mixture of 1-octen average 68 male house flies per trap, and 35 females per trap. 3-ol, and representative sesquiterpene hydrocarbons and Egg powder and B-caryophyllene attracted six male house derivatives including (+)-longipinene, (-)-isolongifolene, flies per trap and 12 female house flies per trap. Egg powder (+)-longifolene, 3-caryophyllene, and isolongifolenone, with and isocaryophyllene attracted 14 male house flies per trap, about 100ng of each compound in the mixture. The results are 30 and 15 female house flies per trap. Egg powder and a combi shown in FIG. 8. nation off-caryophyllene and isocaryophyllene attracted 12 As shown in FIGS. 7 and 8, the sesquiterpene hydrocar male house flies per trap and 6 female house flies per trap. bons, (+)-longipinene, (-)-isolongifolene, (+)-longifolene, FIG. 12 demonstrates that while both tested compounds B-caryophyllene and isocaryophyllene appear to be active on showed repellent activities against houseflies, 3-caryophyl female Culex pipiens antennae. Antennal responses were 35 lene exhibited a stronger repelling activity against the male weak, but repeatable, thus are considered to be true responses. houseflies than the female houseflies. The GC-EAD responses of female antennae of the sand fly, Similarly, a laboratory bioassay of the spatial repellent Phlebotomus papatasi, and three species of mosquitoes, effects off-caryophyllene, (+)-longifolene, and (-)-isolongi Aedes aegypti, Anopheles gambiae, and anopheles Stephensi folene on houseflies was carried out. FIG. 13 is a bar graph were tested on a synthetic mixture of 1-octen-3-ol, six ses 40 plotting the number of house flies per trap attracted for vari quiterpene hydrocarbons including (+)-longipinene, (-)-iso ous combinations of an attractant and repellent compounds. A longifolene, (+)-longifolene, B-caryophyllene, isolongifole blank control attracted two male house flies per trap and one none, and C-humulene, and several known insect repellents female house fly per trap. Egg powder alone attracted 95 male including ZE-nepetalactone, EZ-nepetalactone, with about house flies and 76 female house flies. Egg powder and 100 ng of each compound in the mixture. The results are 45 B-caryophyllene attracted two male house flies and 10 female shown in FIG. 9. house flies per trap. Egg powder and (-)-isolongifolene The GC-EAD responses of female antennae of the sand fly, attracted 30 male house flies and 39 female house flies per Phlebotomus papatasi, and three species mosquitoes, Aedes trap. Egg powder and (+)-longifolene attracted one male aegypti, Anopheles gambiae, and anopheles Stephensi were house fly and 4 female house flies per trap. FIG.13 shows that tested on a synthetic mixture of 1-octen-3-ol, linalool, 50 among three tested compounds, (+)-longifolene is the stron B-caryophyllene oxide, isocaryophyllene, B-caryophyllene, gest repellent against houseflies. In addition, all three com and C-humulene, and DEET, with about 100 ng of each com pounds exhibited stronger repelling activity against male pound in the mixture. The results are shown in FIG. 10. houseflies than against female houseflies. As shown in FIGS. 9 and 10, the compounds B-caryophyl These laboratory bioassays demonstrated that the tested lene, its cis-isomer isocaryophyllene, C.-humulene, (+)-lon 55 sesquiterpene hydrocarbons are effective repellents against gipinene, (-)-isolongifolene, (+)-longifolene, B-caryophyl flies. As shown in FIGS. 11-13, the EAD-active sesquiterpene lene oxide, and linalool appear to be antennally active on one hydrocarbons and derivatives, B-caryophyllene, isocaryo or more of the sand fly (Phlebotomus papatasi) or the mos phyllene (-)-isolongifolene, and (+)-longifolene signifi quitoes, while DEET or isolongifolenone does not appear to cantly reduced the trap catches of houseflies (both sexes) by have elicited antennal activity at the dose tested. 60 50-95%, and in some cases, totally blocked the attraction of Laboratory bioassay tests were carried out to investigate houseflies to the egg powder attractant. the efficacy of some of the EAD-active compounds described A field bioassay of the spatial repellent effects on three above to act as spatial repellents against several insects. The species of flies was carried out with B-caryophyllene, as procedure for the bioassay tests is described in Example 2 described in Example 3 below. The bioassay used the com below. Briefly stated, the bioassay included using egg powder 65 mercially available fly attractant known under the designation (EP) as an attractant for flies, while the compound whose Rescue R and a newly developed reusable prototype trap, insect repellency was being tested was placed in proximity to disclosed in U.S. Pat. No. D561,297, which is incorporated US 8,057,829 B2 11 12 herein by reference. As shown in FIG. 14, B-caryophylene effects not only on the two mosquito species, including Aedes significantly reduced the trap catches of houseflies, bottle Aegypti and Anopheles Stephensi, but also on the sand fly, P flies, and flesh flies by about 50 to about 80%. papatasi. A value of PK0.05 indicates a significant repellency. Laboratory Y-tube behavioral bioassays as described in The blank-blank control tests showed no differences in the Example 4 were conducted to test the spatial repellent effects s number of mosquitoes or sand flies between left and right of clove oil, patchouli oil, B-caryophyllene, isocaryophyl aS. Laboratory K&D module bioassays of the topical repellent lene, (+)-longifolene, C.-guaiene, B-caryophyllene oxide, effects on mosquito species Aedes aegypti and Anopheles B-caryophyllene ketone the combination of B-caryophylene Stephensi were carried out using clove oil, patchouli oil, and isocaryophyllene and (+)-longifolene, on the sand fly, C-guaiene, C.-bulnesene, B-caryophyllene, isocaryophyllene, Phlebotomus papatasi, and mosquitoes, including Aedes 10 (+)-longifolene, the combination off-caryophyllene and iso aegypti and Anopheles Stephensi. The results are shown in caryophyllene and (+)-longifolene, B-caryophyllene oxide, Table 1. and B-caryophyllene ketone, as described in Example 5 TABLE 1. Y-tube Behavioral Bioassays With Potential Spatial Repellent Candidate Chemicals (Mixtures) Against Mosquito and Sand Fly Females Release Number of mosquitoes Rate in each arm Insect Chemical(s) Dispenser (mg/day) Repellent Blank Bottom x2 P SAI* Aedes Blank PE-Bag O 5 7 42 O.333 O.S64 0.17 aegypti Clove Oil PE-Bag 10 4 31 12 20.8 *Spatial Activity Index (SAI) = (Nc-Nt). (Nc + Nt); Nc is the number of females in the blank control arm, Nt is the number of females in the treatment arm. SAI is the measure of the proportion of females in the blankarm over the treatment arm after correcting for proportion of females in the blank arm; SAI varies from -1 to 1, with zero indicating no response, As shown in Table 1, clove essential oil (ca. 10 mg/day below. The bioassays were conducted with a six-cell in vitro release rate), patchouli oil (15 mg/d) and the EAD-active K&D module system, as disclosed in Klun and Debboun, J. sesquiterpenes tested including B-caryophylene and deriva 65 Med. Entomol. 37:177-181 (2000), loaded with an “artificial tives, isocaryophyllene, (+)-longifolene, and C-guaiene, blood” (CPDA+ATP) solution. The results are listed in ranging from 1 to 35 mg/d release, showed spatial repellency Table 2. US 8,057,829 B2 13 14 TABLE 2 K&D Module Behavioral Bioassays With Potential Topical Repellent Candidates Concentration % of biting (SE) (N = 3)* Mosquitoes (Lig/cm) Clove Oil Patchouli Oil C-Guaiene C-Bulnesene Aedes aegypti O (blank) 677b 60 Oc 67 7c 93 7c. 10 7 7a 20 Ob 33 7b 737b 1OO O Oa O Oa O Oa O Oa 500 O Oa O Oa O Oa O Oa 1OOO O Oa O Oa O Oa O Oa 10 (DEET) O Oa 13 13a 7 7a O Oa Anopheles Stephensi O (blank) 67 7c 80.12b 60 Oc 937b 10 20 Ob 477b 40 Ob 87.7b 1OO O Oa O Oa O Oa O Oa 500 O Oa O Oa O Oa O Oa 1OOO O Oa O Oa O Oa O Oa 10 (DEET) O Oa 13 13a 33 7b O Oa % of biting (SE) (N = 3)* B-Caryophyllene + B- Isocaryophyllene + 3 Concentration Caryo- Isocaryo- (+)- (+)-Longifolene Caryophyllene B-Caryophylene Mosquitoes (ug/cm) phyllene phyllene Longifolene (1:1:1) Oxide Ketone Aedes O (blank) 477b 537c 47 7c. 47 13c. aegypti O 4012b 33: 7b S3 7c 33.7bc 100 33 - 24b O Oa 33-13bc 47 13c. 500 O Oa O Oa O Oa 1313ab 1OOO O Oa O Oa O Oa O Oa () (DEET) O Oa O Oa 2012ab 77ab O (blank) 60 Oc 60 Oc 1 277b 20 Obc 5 277b 13 + 7ab O O Oa 77ab 50 O Oa O Oa () (DEET) O Oa 77ab Anopheles O (blank) 60-12c 677b 53-18bc 60 Ob Stephensi O 47-18bc 33 - 13b 537c 6718b 100 4012bc 7: 7a 33 + 7bc 2012a 500 O Oa O Oa O Oa 77 a 1OOO 77 a O Oa O Oa O Oa O (DEET) 13 + 13ab 7 + 7a 277b 77 a O (blank) 60 - 12c 67 7 1 27-13abc 477 cc 5 47 7bc 33 - 7bc O 2012ab 2012ab 50 O Oa O Oa () (DEET) 13 7a 77 a *Data in the same column with same letter, within the same species and chemical, are not significantly different (Ps 0.05) by ANOVA on arcsin sqt Pfollowed by Duncan's multiple-range test, 45 As shown in Table 2, over 50-60% of mosquitoes fed on the Seychellene, C-guaiene, and C.-bulnesene, or a mixture “artificial blood for both mosquito species (Ae. aegypti and thereof. One or more additional compounds selected from An. Stephensi) in the blank control, while DEET (at 10 B-caryophyllene, isocaryophyllene, B-caryophyllene oxide, ug/cm) significantly inhibited/blocked the feeding (biting) B-caryophyllene ketone, C.-humulene, B-patchoulene, activity. The three tested sesquiterpenes showed similar 50 C-patchoulene, B-elemene, (+)-longipinene, (-)-isolongi repellency/feeding deterrent patterns in a clear dose-response folene, (+)-longifolene, and linalool may be used. Further fashion, with lower concentrations (10-100 g/cm or less) more, clove oil or/and patchouli oil may be used together with being inactive, and repellency becoming significant and one or more compounds selected from Seychellene, totally blocking insects at 100-500 ug/cm or higher. B-caryo 55 C-guaiene, and C.-bulnesene, to treat a desirable area for the phyllene oxide and ketone showed even stronger topical purpose of repelling insects from the area. repellency on both species at similar or lower dosages as The dispensing of Such repellent compounds may be those used for DEET. Significant repellency was also found achieved by way of evaporation of the active compounds from for clove and patchouli oils at about 10 mg/d release. a device with either controlled release or passive release. For The sesquiterpene hydrocarbons and derivatives disclosed 60 example, the compounds can be mixed with a Suitable carrier herein may be formulated into suitable devices or dispensers and placed inside a container for controlled release. Repre to provide a spatial repellent. The application of the com sentative control release strategies include absorbing the pounds for spatial deterrence includes, but is not limited to, compounds on a porous Substrate or combining the com the following methods. For use as a spatial repellent, the pounds with a polymeric gel. The release devices can vary in method may include dispensing into an area, where one wants 65 their shapes and/or sizes to fit different settings, such as to provide such inhibiting or deterring effect, an effective incorporating the compounds into ornaments to be incon amount of at least one repellent compound selected from spicuously placed in indoor or outdoor locations, or can be US 8,057,829 B2 15 16 used for dual purposes, such as decorations having insect antenna (Zhang et al., Chemoecology, 10:69-80 (2000)). The repellent properties, and for use in buildings or vehicles. antennal signals were stored and analyzed on a PC equipped In one embodiment, the repellent compounds may be dis with a serial IDAC interface box and the program EAD ver. persed by anaerosol that could be sprayed on the ground or in 2.5 (Syntech, Hilversum, The Netherlands). The compound the air. In another embodiment, the repellent compounds may 5 1-octen-3-ol (known attractant for many flies and mosqui be dispersed by Scattering a powdery Substrate containing the toes) was included in most of the synthetic mixtures, as a repellent compounds. positive control. The results of the experiments are shown in The repelling of an insect of the order Diptera from a FIGS. 1-10 and described above. human or animal Subject can include applying to a subject or to a device worn by the subject at least one repellent com 10 Example 2 pound selected from a group consisting of Seychellene, C-guaiene, and C-bulnesene, or a mixture thereof. Other com The following lab bioassay tests were employed to illus pounds may be used together with Seychellene, C-guaiene, trate the efficacy of the compounds to effectively act as spatial and/or C.-bulnesene to repel an insect from a human or animal repellents for flies. Three sets of tests were conducted in a fly Subject including B-caryophyllene, isocaryophyllene, 15 testing room (23°C., 70% RH, and 12/12 (L/D) light regime B-caryophyllene oxide, B-caryophyllene ketone, C-humu illuminated by fluorescent lights). Two sets of tests were lene, B-patchoulene, C.-patchoulene, B-elemene, (+)-lon carried out using Trécé PheroconR VI traps with sticky gipinene, (-)-isolongifolene, (+)-longifolene, and linalool. inserts, which were set up on poles evenly spaced on a rotat Additionally, Seychellene, C-guaiene, and/or C-bulnesene ing table. The traps were ca. 1 m above a table Surface, and ca. may be used together with clove oil, patchouli oil, or both, on 1 m apart from each other. Two grams of egg powder in a a human or animal Subject for insect repelling purpose. white cap (4 cm in diameter, 0.5 cm in height) used as an The repellent compounds may be applied to a human or attractant was put on the center of the Sticky insert in the animal Subject in varieties of ways for insect repelling pur Sticky trap. The potential repellent candidate chemicals were poses. For example, an effective dose of a repellent com loaded into polyethylene bags, and were hung under the inner pound or a mixture thereof may be applied directly to the skin 25 roof of the traps, 1-1.5 cm above the egg powder attractant. A of the subject. Alternatively, the repellent compounds may be control had the empty white cap and blank polyethylene bag, formulated into creams, lotions, aerosols, or other Suitable while the positive control had only the egg powder and an formulations for topical application. In addition, the repellent empty polyethylene bag. The test table was rotated 1.5 rp.h. compounds may be absorbed on a powdery porous Substrate (moved /s of the rotation during 4 sec, and stayed in place for and applied directly to the subject. 30 5 min. before moving; thus there were 12 movements per hr., The repellent compounds or a mixture of the repellent and resulted in 1.5 rotations) to reduce the positional effect. compounds may be applied to a device worn by a human or About 200-300 houseflies (2-3 days old) were released into animal Subject. In one embodiment, the device may include the testing room for each test. Tests were normally run from an adsorbent Substrate and the repellent compound is 8:00am to 5:00 pm, and the numbers of houseflies caught in adsorbed on the adsorbent substrate. In one embodiment, the 35 each trap were counted after each test. The results of these device may include a diffusion barrier that releases the repel tests are shown in FIGS. 11-13 and described above. lent compound from the device at a predetermined rate. For example, the diffusion barrier may be a polyethylene film. In Example 3 one embodiment, the repellent compound may be placed within a polyethylene film and carried by a human or animal 40 A field trapping experiment was carried out to test the Subject. potential repellency of B-caryophyllene on different filth flies. The experiment uses a commercially available fly attrac EXAMPLES tant known under the designation Rescue R and a newly developed reusable prototype trap disclosed in U.S. Design Example 1 45 Pat. No. D561, 297 (Schneidmiller, 2008), incorporated herein by reference in its entirety. Seven pairs of such traps, The following procedures were employed to test the EAD one with attractant alone and the other with attractant plus the response of an insect antenna to a repellent compound. The B-caryophyllene dispensed through a polyethylene bag GC-EAD technique provides an efficient tool for screening within each pair, were set up in different locations with a behaviorally active repellents. Several synthetic mixtures of 50 suitable level of fly populations. In order to reduce the posi potential insect repellent candidates, mostly sesquiterpene tional effect, the traps within each pair were shifted at least hydrocarbons and derivatives, were analyzed in splitless once before gathering counts. The results of the field trapping mode using a Varian CP-3800 GC equipped with a polar experiments are shown in FIG. 14 and described above. column (Innowax; 1.0 Lum film thickness, 30 mx0.53 mm ID: J & W Scientific), and a 1:1 effluent splitter that allowed 55 Example 4 simultaneous flame ionization detection (FID) and EAD of the separated Volatile compounds. Helium was used as the Clove essential oil, patchouli oil and several EAD-active carrier gas, and the injector temperature was 220° C. The compounds, as well as their partial mixtures with DEET as a column temperature was 50° C. for 2 min, rising to 240° C. at positive control, were tested against a blank as a negative 10°C/min, then held for 10 min. The outlet for the EAD was 60 control in a lab Y-tube olfactometer bioassay on the two held in a humidified air stream flowing at 0.5 liter/min. over mosquito species, Ae. Aegypti and An. Stephensi, and one an antennal preparation. A glass capillary indifferent elec species of sand fly, P. papatasi. Both the mosquitoes and the trode filled with Beadle-Ephrussi Ringer, and grounded via a sand flies used in the study were from colonies maintained at silver wire, was inserted into the severed insects head with the Walter Reed Army Institute of Research, Department of the antennae. A similar recording electrode connected to a 65 Entomology, Silver Spring, Md. This Y-tube bioassay system, high-impedance DC amplifier with automatic baseline drift equipped with CO-enriched air (ca. 1 1/min) as an attractant, compensation was placed in contact with the distal end of one was made from three clear acrylic tubes (4" diameter; 12" US 8,057,829 B2 17 18 length) with symmetrical 120° angles among the three tube nected to a constant temperature water circulator at 38°C. arms; and a screenbutterfly valve on the top part of the bottom The cells were covered with an Edicol collagen membrane releasing tube. These spatial repellency tests were carried out strip. Nylon organdy Strips withink cell patterns were num during 8:00am-12:00 pm (photophase) time period for Ae. bered and randomly treated with five dilutions of the tested aegyptiand 1:00-5:00pm (shifted scotophase) time period for chemical in ethanol (ethanol alone as blank control), plus the species of night-flying mosquitoes and sand fly in a tem DEET (ca. 10 ug/cm) as a positive control. This treated nylon perature and humidity controlled testing room (23-25°C. and organdy Strip was placed on top of the collagen membrane. 20-40% RH). Female mosquitoes or sand flies were stored at The upper section of the K&D module with the six cells each -25-26° C., 50-89% RH, 12:12 hr. (L/D), fed with 10% containing 5 mosquitoes (5-10 dold, starved for 24hr before Sucrose solution before testing. The candidate chemical (indi 10 testing) was placed on top of the nylon strip. The doors to the vidual or mixture) dispenser and a blank control were placed cells were slid open so the mosquitoes could be free to feed on in the upper left or right arm of the Y-tube before releasing the “artificial blood via the collagen membrane if they were 25-40, 6 to 10 day-old female mosquitoes (or 3 to 10 day-old hungry. The experiments were allowed to run for 3 minutes, sand flies) into the bottom tube via a small hole. After a 30-60 and the number of mosquitoes feeding (biting) in each cell sec acclimation period, the butterfly screen valve (door) on 15 was recorded. After each experiment, the nylon and collagen the top of the bottom tube was opened. After 10 min., the strips were removed. The CPDA+ATP solution was removed valve was closed and the numbers of mosquitoes or sand flies and allowed to dry prior to starting the next experiment. The in each arm (including the bottom tube) were counted. In experiments on each chemical and each mosquito species order to minimize the position effect, the same experiment were run three times before the bottom section with circulated was repeated once with the treatment and blank control arms water was cleaned prior to use again. The results are shown in shifted using a new (clean) Y-tube olfactometer and a new Table 2 above. batch of mosquitoes/sand flies. Data from these two replicates While illustrative embodiments have been illustrated and were pooled for X2 goodness of fit test (df-1) between the described, it will be appreciated that various changes can be treatment and blank control. The results of the bioassay are made therein without departing from the spirit and scope of shown in Table 1 above. 25 the invention. Example 5 The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows: 1. A method for repelling phlebotomus papatasi from an Clove essential oil, patchouli oil, and EAD-active potential area, comprising treating the area with an isolated form of repellents, B-caryophyllene, isocaryophyllene, (+)-longi 30 C-guaiene, wherein the phlebotomus papatasi is repelled. folene, and their 1:1:1 mixture, plus C-guaiene, caryophyl 2. The method of claim 1, further comprising treating the lene oxide, and a B-caryophylene ketone, were tested using area with one or more compounds selected from the group the six-cell in vitro K&D module system (Klun and Debboun, consisting of B-caryophyllene, isocaryophyllene, B-caryo J. Med. Entomol. 37:177-181 (2000)) loaded with an “artifi phyllene oxide, B-caryophyllene ketone, C.-humulene, cial blood” (CPDA+ATP) solution on both Ae. aegypti and 35 An. Stephensi in a dose-response fashion. The K&D in vitro B-patchoulene, C-patchoulene, B-elemene, (+)-longipinene, assay system has three components: (1) a PlexiglasTM 29.7 (-)-isolongifolene, (+)-longifolene, Seychellene, C.-bulne sene, and linalool. cm.x7.1 cm K&D module composed of six adjacent cells, 3. The method of claim 1, further comprising treating the each designed to hold mosquitoes and each having a rectan area with clove oil, patchouli oil, or a mixture thereof. gular 3x4 cm floor hole that is opened and closed by a sliding 40 4. The method of claim 1, comprising treating the area with door; (2) a PlexiglasTM six-well 29.7 cmx7.1 cm water water an aerosol carrying the isolated form of alpha-guainene. bath warmed (38° C.) reservoir with six 3x4 cm wells 5. The method of claim 1, comprising treating the area with designed to match the sliding-door openings of the K&D a powdery Substrate carrying the isolated form of alpha module base and to contain 6 mL warmed “artificial blood' guainene. wells covered with a collagen membrane; and (3) a 29.7 45 cmx7.1 cm0.4 cm Teflon R separator having six rectangular openings like the K&D module. The K&D module was con