CN1809368B

CN1809368B - Compositions and methods for controlling insects

Info

Publication number: CN1809368B
Application number: CN2004800173698A
Authority: CN
Inventors: 伊塞姆·伊那恩
Original assignee: Tyratech Inc
Current assignee: Tieretche
Priority date: 2003-04-24
Filing date: 2004-04-26
Publication date: 2011-02-09
Anticipated expiration: 2024-04-26
Also published as: ZA200508646B; CN1809368A

Abstract

The present invention comprises compositions, methods and cell lines related to controlling insects. An embodiment of a composition comprises a plant essential oil and targets at least one receptor of insects chosen from tyramine receptor, Or83b olfactory receptor, and Or43a olfactory receptor, resulting in a change in the intracellular levels of cAMP, Ca2+, or both in the insects.

Description

Composition and the method for control insect

The cross reference of related application

The application requires the priority of U.S. Provisional Application sequence number of submitting on April 24th, 2,003 60/465,320 and the U.S. Provisional Application sequence number of submitting on December 24th, 2,003 60/532,503, and the full text that is incorporated herein them as a reference.

Invention field

The present invention relates to the composition relevant, method, cell-line and report with controlling insect.

Background of invention

Animal has chemoreception and mechanoreception system, can discern multiple environmental stimulus, produces behavior reaction.Carried out behaviouristics research to understand the genetics of these systems.Olfactory system is at some species, comprises in the existence of insect and the life having important effect.

Fruit bat is a kind of model organism of research sensory system, and this is because it can carry out mutation analysis by molecular engineering, carry out behaviouristics analysis and electrophysiology analysis, and its olfactory system is suitable with mammiferous corresponding system.

Over several years after deliberation the insecticidal activity of various chemicalses or mixed agent, its objective is obtain a kind of to invertebrate for example insect have selectivity, but vertebrate such as mammal, fish, poultry and other species are had little or no toxicity, and in environment, do not retain the product that does not also destroy environment in addition.

Major part has the known of enough insecticidal activities and all has toxicity or illeffects with business-like product for the mammal, fish, poultry or other species that are not the target of this product.But organic phosphorus compound and carbamate activity of acetylcholine esterase inhibition all in the animal of insect and all kinds for example.Galecron and the relevant known octopamine receptor that acts on insect of carbonamidine, but because they make vertebrate that cardiotoxic possibility be arranged, animal is had carcinogenicity and different to different insect effects, thereby withdrawn from market.Other compound that mammal and other non-target earnest kind are had less toxicity often is difficult to differentiate.

Summary of the invention

The present invention includes the composition of control insect and use these method for compositions.The present invention includes the composition of control insect, it comprises one or more essence oil from plant, and uses these method for compositions.Described essence oil from plant has synergy when being used in combination.Described composition can comprise fixed oil, and it is a kind of nonvolatile plant oil that does not have fragrance.In addition, these compositions can be by it is generally acknowledged that safe (GRAS) compound forms.

The present invention includes and contain one or more essence oil from plant and insect control combination of agents thing, and use these method for compositions.The example of insect control reagent comprises DEET and D-allethrin.Described essence oil from plant and described insect control agent combination have synergy when using.For example, the insect of 29%DEET control effect DEET with 5% in composition of the present invention just can obtain.

The present invention includes screening and have the active method for compositions of insect control.The present invention includes with tyrasamine acceptor (TyrR), Or83b olfactory receptor (Or83b), or the cell-line of Or43a olfactory receptor stable transfection, it can be used for screening the composition with insect control activity.

The present invention includes to produce and differentiate that one or more have the method for reporting of the active composition of insect control.Term " report " refers to be present in typescripts, database, record or explanation in computer system or other medium.

For the sake of simplicity, the application will use term " insect " in the whole text; But, be to be understood that term " insect " not only refers to insect, also refer to spider guiding principle animal, larva and similar invertebrate.And be the application's purpose, term " insect control " is meant to have dispel effect, insecticidal effect or have two effects simultaneously." dispel effect " is meant such effect, wherein compares with contrast host or zone that described compositions-treated of no use is crossed, has more insect to be excluded outside the host and zone that cross with described compositions-treated.In some embodiments, dispel effect is meant such effect, wherein has at least about 75% insect to be excluded outside the host and zone that cross with described compositions-treated.In some embodiments, dispel effect is meant such effect, wherein has at least about 90% insect to be excluded outside the host and zone that cross with described compositions-treated." insecticidal effect " is meant such effect, wherein causes about at least 1% insect death with compositions-treated.In this, the composition of LC1 to LC100 (lethal concentration) or LD1 to LD100 (lethal dose) can cause insecticidal effect.

In some embodiments, described insecticidal effect is such effect, wherein causes the insect death of about at least 5% exposure with compositions-treated.In some embodiments, described insecticidal effect is such effect, wherein causes the insect death of about at least 10% exposure with compositions-treated.In some embodiments, described insecticidal effect is such effect, wherein causes about at least 25% insect death with compositions-treated.Described in some embodiments insecticidal effect is such effect, wherein causes the insect death of about at least 50% exposure with compositions-treated.Described in some embodiments insecticidal effect is such effect, wherein causes the insect death of about at least 75% exposure with compositions-treated.Described in some embodiments insecticidal effect is such effect, wherein causes the insect death of about at least 90% exposure with compositions-treated.In some embodiments of the present invention, handle and insect can be knocked down between by several minutes in the several seconds with this concentration or dosage.

Composition of the present invention can be used for controlling insect, perhaps directly handles the host, the zone of perhaps handling the host and being positioned at, for example indoor living space, outdoor courtyard or garden.Be the application's purpose, the host is defined as plant, people or other animal.

The accompanying drawing summary

Fig. 1 has shown the receptor-specific combination with the Schneider cell of tyrasamine acceptor transfection;

Fig. 2 shown film with the Schneider cell preparation that can express the tyrasamine acceptor under the condition that has or do not exist unlabelled tyrasamine with variable concentrations ³After the H-tyrasamine is cultivated ³The saturated binding curve of H-tyrasamine;

Fig. 3 shown film with the Schneider cell preparation that can express the tyrasamine acceptor under the condition of the unlabelled tyrasamine that has or do not exist variable concentrations with ³After the H-tyrasamine is cultivated ³The H-tyrasamine suppresses binding curve;

Fig. 4 has shown with the film of the Schneider cell preparation that can express the tyrasamine acceptor at the unlabelled part that has or do not exist variable concentrations: tyrasamine (TA), and octopamine (OA), dopamine (DA), and under the condition of serotonin (SE) ³The H-tyrasamine suppresses binding curve;

Fig. 5 shown film with the Schneider cell preparation that can express the tyrasamine acceptor under the condition of flos caryophylli oil (LFO) that has or do not exist variable concentrations and black seed oil (BSO) and ³After the H-tyrasamine is cultivated ³The H-tyrasamine suppresses binding curve;

Fig. 6 shown with the film of the Schneider cell preparation that can express the tyrasamine acceptor under the condition that has or do not exist LFO or BSO or with the condition of the unlabelled tyrasamine (TA) of variable concentrations under with ³After the H-tyrasamine is cultivated, ³The H-tyrasamine ( ³H-TA) combine with the inhibition of described film;

Fig. 7 has shown that cAMP level in the Schneider cell of expressing the tyrasamine acceptor under the condition that has or do not exist forskolin and tyrasamine depends on the variation of tyrasamine;

Fig. 8 has shown the variation that depends on tyrasamine under the condition that has or do not exist forskolin and tyrasamine with cAMP level in the Schneider cell that can express the tyrasamine acceptor of flos caryophylli oil and black seed oil processing;

Fig. 9 has shown in existence or has not had tyrasamine that the cAMP level depends on the variation of tyrasamine in the Schneider cell of handling with forskolin under the condition of flos caryophylli oil and black seed oil that can express the tyrasamine acceptor;

Figure 10 has shown ³The H-tyrasamine and the saturated binding curve of film with the Schneider cell preparation that can express the Or83b acceptor;

Figure 11 has shown ³The H-tyrasamine and the saturated binding curve of film with the Schneider cell preparation that can express the Or43a acceptor;

Figure 12 has shown that in expressing the Schneider cell of Or83b acceptor cAMP level depends on the variation of forskolin;

Figure 13 has shown in expressing the Schneider cell of Or83b acceptor Ca in the born of the same parents ²⁺Level depends on the variation of ionomycin;

Figure 14 has shown in expressing the Schneider cell of Or43a acceptor Ca in the born of the same parents ²⁺Level depends on the variation of ionomycin;

Figure 15 shown at contrast Schneider cell, can express the Schneider cell of Or83b acceptor and can express in the Schneider cell of Or43a acceptor Ca in the born of the same parents ²⁺Level depends on the variation of tyrasamine;

Figure 16 shown the Schneider cell that can express olfactory receptor with ³Wherein various essence oil from plant after the H-tyrasamine is cultivated comprise LFO, piperonal, ethyl phthalate, and the interaction between alpha-terpineol and Or83b and the Or43a acceptor;

Figure 17 shown the Schneider cell that can express olfactory receptor with ³Wherein various essence oil from plant after the H-tyrasamine is cultivated comprise BSO, quinine, sabinene, α-absinthol, α-sobrerone, d-citrene, and the interaction between p-cymene and the Or43a acceptor;

Figure 18 shown the Schneider cell that can express olfactory receptor with ³Wherein various essence oil from plant after the H-tyrasamine is cultivated comprise BSO, quinine, sabinene, α-absinthol, α-sobrerone, d-citrene, and the interaction between p-cymene and the Or83b acceptor;

Figure 19 shown the Schneider cell that can express olfactory receptor with ³Wherein various essence oil from plant after the H-tyrasamine is cultivated comprise geraniol, ortho-aminobenzoic acid agalloch eaglewood ester, phenylacetaldehyde, linalool, alpha-terpineol, t-anethole, terpinene 900, bodhi alcohol, and the interaction between eugenol and Or83b and the Or43a acceptor;

Figure 20 shown the Schneider cell that can express olfactory receptor with ³Wherein various essence oil from plant after the H-tyrasamine is cultivated comprise thyme linaloe oil, carvacrol, and the interaction between thymol and Or83b and the Or43a acceptor;

Figure 21 shown the Schneider cell that can express olfactory receptor with ³Wherein various essence oil from plant after the H-tyrasamine is cultivated comprise piperonal, piperitol, acetic acid pepper ester, and the interaction between pepper amine and Or83b and the Or43a acceptor;

Figure 22 has shown ionomycin in expressing the Schneider cell of Or43a acceptor, and tyrasamine and ortho-aminobenzoic acid agalloch eaglewood ester are for Ca in the born of the same parents ²⁺The effect of level;

Figure 23 has shown linalool in expressing the Schneider cell of Or43a acceptor, perillyl alcohol, and the t-anethole, geraniol, phenylacetaldehyde and eugenol are for Ca in the born of the same parents ²⁺The effect of level;

Figure 24 has shown piperitol in expressing the Schneider cell of Or43a acceptor, and acetic acid pepper ester and pepper amine are for Ca in the born of the same parents ²⁺The effect of level;

Figure 25 has shown alpha-terpineol in expressing the Schneider cell of Or43a acceptor, and bodhi alcohol and terpinene 900 are for Ca in the born of the same parents ²⁺The effect of level;

Figure 26 has shown thyme linaloe oil in expressing the Schneider cell of Or43a acceptor, and thymol and carvacrol are for Ca in the born of the same parents ²⁺The effect of level;

Figure 27 has shown Ca in LFO is for born of the same parents in the Schneider cell that can express Or43a acceptor or Or83b acceptor ²⁺The effect of level;

Figure 28 has shown BSO in the Schneider cell that can express Or43a acceptor or Or83b acceptor, α-sobrerone, and the p-cymene, the d-citrene, sabinene, quinine, the l-carvol, the d-carvol, and α-absinthol is for Ca in the born of the same parents ²⁺The effect of level;

Figure 29 shown and existed or do not have tyrasamine, under the condition of LFO and BSO in expressing the Schneider cell of Or83b acceptor the cAMP level depend on the variation of tyrasamine;

Figure 30 shown under the condition that has or do not exist tyrasamine and forskolin in the Schneider cell that can express the Or83b acceptor of LFO and BSO processing the cAMP level depend on the variation of tyrasamine;

Figure 31 A and 31B have shown the nucleotide sequence and the peptide sequence of tyrasamine acceptor;

Figure 32 A and 32B have shown the nucleotide sequence and the peptide sequence of Or43a olfactory receptor; And

Figure 33 A and 33B have shown the nucleotide sequence and the peptide sequence of Or83b olfactory receptor

Detailed Description Of The Invention

The present invention relates to the composition relevant with controlling insect, method, clone and report. The control of described insect and one or more are receptor related, comprise tyrasamine acceptor (TyrR), Or83b olfactory receptor (Or83b) and Or43a olfactory receptor (Or43a).

The present invention includes the method that screening has the active composition of insect control. The present invention includes by TyrR, Or43a, or the fruit bat Schneider clone of Or83b stable transfection, it can be used for screening the composition with insect control activity. The nucleotide sequence of TyrR and peptide sequence are shown in Figure 31 A and Figure 31 B. The nucleotide sequence of Or43a and peptide sequence are shown in Figure 32 A and Figure 32 B. The nucleotide sequence of Or83b and peptide sequence are shown in Figure 33 A and Figure 33 B.

Can be by detect expressing TyrR, Or83b, and/or detection composition is differentiated the insect control activity that may exist with the affinity of each acceptor in the clone of Or43a. Also can be by detecting the expression TyrR after processing with detection composition, Or83b, and/or cAMP and/or Ca in the born of the same parents in the clone of Or43a²⁺Differentiate that the insect control that may exist is active. The TyrR acceptor of different species of insect has similitude between Or83b acceptor and the Or43a acceptor. Similarly, the fruit bat Schneider clone of expressing these acceptors can be used for screening different species of insect is had the active composition of insect control.

The present invention includes the composition of control insect and the method for using these compositions. The present invention includes the composition of control insect, it comprises one or more essence oil from plant, and the method for using these compositions. When being used in combination, essence oil from plant has synergy. Composition of the present invention can comprise any in the following oil or its mixture:

T-anthole limette oil piperonyl

Black seed oil (BSO) (R)-4-isopropenyl-1-methyl-1-cyclohexene acetic acid pepper ester

Amphene anthranilate agalloch eaglewood ester piperitol

Carvacrol linalool pepper amine

D-carvol lindenol benzoquinones

L-carvol methylcitrate sabinene

1,8-cineole MDJ α-terpinene

P-cymene myrcene terpinene 900

Ethyl phthalate perillyl alcohol alpha-terpineol

Eugenol phenylacetaldehyde γ-terpineol

Geraniol α-sobrerone 2-the tert-butyl group-p-benzoquinones

Isopropyl citrate β-sobrerone α-absinthol

Lemongrass oil piperonal thyme linaloe oil

Flos caryophylli oil (LFO) thymol

Composition of the present invention can also comprise any in the following oil or its mixture:

Allyl sulfide beta-elemene Menthyl salicylate

Pi-allyl trithio γ-elemene myrte

Allyl disulfide Elmol acetate neral dimethyl ester

Anethole chavicol methyl ether nerolidol

Acetate artemisol ester 2-ethyl-2-hexen-1-ol nonanone

Benzyl acetate NSC 1242 1-octanol

Phenmethylol α-farnesene E ocimenone

Bergaptene (Z, E)-β-farnesene Z ocimenone

Oxidation bisabolene fenchone ocimene

α-bisabolol furanodiene furans folium eucalypti-1, octyl acetate

Oxidation bisabolol 3-diene peppermint oil

Oxidation bisabolol β furans folium eucalypti-1,4-diene α-phellandrene

Big Mang ox 1,10 (1 β-phellandrene of bornyl acetate furans

The other geranium wilfordii alkene 5 of beta waves)-diene-6-ketone piperonal

α-cadinol furans sesquiterpene Prenal

Amphene geraniol pulegone

α-borneene acetate spiceleaf alcohol ester sabinene

α-borneene acetaldehyde germacrene D sabinyl acetate

Camphor germacrene B α-santalene

Trans-Caryophyllene oxide α-gurjunene santalol

Chamazulene α-humulene Sativen

Cinnamic acid α-Zi Luolantong δ-selinene

Along verbenol alpha, beta-lonone β-sesquphelandrene

Citral A isoborneol spathulenol

The different furans germacrene of citral B tagetone

Citronellal isomenthone α-terpinene

Citronellol isopulegone 4-terpineol

Citronellyl acetate jasmone α-terpinolene

Citronellyl formate flos caryophylli oil α-terpinyl acetate

α-copaene citrene α-thujene

Wild mint oil linalool thymol methyl ether

α-anti-cloves the alkene of costus root alcohol linalyl acetate

Cryptone lindestrene pinocarveol

The anti-verbenol of curzerenone methyl allyltrisulfide

D-carvol menthol verbenone

L-carvol 2-methoxyl group furanodiene Yomogi alcohol

Davanone menthones zingiberene

Diallyl trithioether acetate ester in Meng Dihydrotagentone

Dihydro pyrocurcuzerenone methyl cinnamate

In those contained composition more than a kind of oil, every kind of oil constituted about 1% to about 99% of described composition mixture by weight.For example, a kind of composition of the present invention comprises about 1% thymol and about 99% geraniol.Randomly, described composition can also contain fixed oil, and it is the nonvolatile plant oil that does not have fragrance.For example, described composition can comprise one or more following fixed oils:

Castor oil mineral oil safflower oil

Corn oil olive oil sesame oil

Cymin oil peanut oil soybean oil

For example, a kind of composition of the present invention comprises about 1% thymol, about 50% geraniol and about 49% mineral oil.In addition, these compositions can be by it is generally acknowledged that safe (GRAS) compound forms, for example: thyme linaloe oil, geraniol, lemongrass oil, flos caryophylli oil, black seed oil, limette oil, eugenol, castor oil, mineral oil, and safflower oil.

The composition that the present invention includes contains one or more essence oil from plant and insect control reagent, for example, and DEET, and D-alletlirin, and use these method for compositions.Described essence oil from plant and described insect control agent combination have synergy when using.For example, the insect of 29%DEET control effect DEET with 5% in composition of the present invention just can obtain.

Composition of the present invention can contain two or more plant essence oil compositions and/or its derivative, natural and/or synthetic, comprise its racemic mixture, enantiomter, diastereoisomer, hydrate, salt, solvate and metabolite, and they are mixed in appropriate carriers and optional suitable surfactant.

Appropriate carriers can comprise any carrier that is applicable to essence oil from plant well known in the art, as long as this carrier does not have harmful effect to composition of the present invention.Terminology used here " carrier " is meant inertia or liquid substance, it can be inorganic matter or organic matter, synthetic or natural, it mixes mutually with reactive compound or it is made into to be applicable to the form of container or carton box or other object to be processed, perhaps is suitable for the form that stores, transports and/or operate.Generally speaking, be generally used for preparing expellent, insecticide, any material of weed killer herbicide or fungicide all is suitable.Composition of the present invention can use separately, perhaps with solid and/or liquid dispersion agent carrier and/or for example other expellent, insecticide or miticide, nematocide, fungicide, bactericide, rat-bane, weed killer herbicide, fertilizer, the mixed use of growth regulator of other known suitable active agent, if desired, also can make the particular formulations of application-specific, for example make solution, emulsion, suspension, powder, paste and particle with standby.Composition of the present invention can be controlled the mixed use of the thinner that uses in the reagent with traditional inertia insecticide thinner or traditional insect, for example traditional dispersible carrier such as gas, solution, emulsion, suspension, emulsifiable concentrates, spray powder, paste, soluble powder, interleaving agent, particle, foam, paste, tablet, aerosol, be full of reactive compound natural and synthetic, microcapsules, be used in the coating composition on the seed, and burning preparation, for example stifling box, fumigation tank and stifling dish, and cold mist of ULV and warm fog preparation.

Composition of the present invention further can comprise surface-active agents.Can be used for surface-active agents of the present invention, the example that is traditional carrier auxiliary agent comprises emulsification reagent, nonionic and/or anion emulsification reagent (the PEO ester of fatty acid for example, the PEO ether of fatty alcohol, alkyl sulfate for example, alkylsulfonate, arylsulphonate, albumin hydrolysate etc., particularly alkylaryl polyglycol ether, dolomol, enuatrol etc.); And/or disperse for example lignin of reagent, sulfite waste liquor, methylcellulose etc.

Composition of the present invention can be used for controlling insect, can be directly used in to handle the host, perhaps is used to handle the zone at host place.For example.Can directly handle the host with creme or spraying, can external application or local for example people's the skin that is used for.Composition is applied to the host, for example the people time, can make multiple individual product or cosmetics to be used for skin or hair.For example, can use following substances: perfume, colouring agent, pigment, dyestuff, Gu Longshui, face cream, skin lotion, deodorant, talcum powder, bath oil, soap, shampoo, hair conditioner and setting agent.

For animal, people or inhuman host also can directly treat by oral delivering compositions preparation.For example, composition can be wrapped in the iquid capsule and swallow.

Can be with certain zone of compositions-treated of the present invention, for example, by spray, for example aerosol or pump sprays, perhaps incendiary agent, for example candle or contain the stacte of described composition.Certainly, different processing methods can be used and the spirit and scope of the present invention can be do not deviated from.For example, composition can be contained in the household products, for example air freshener (comprise " heating " air freshener, wherein insect repellent by the heating, for example electrical heating, or the burning and discharge); Hard surface cleaner; Or laundry articles for use (laundry detergents that for example contains composition, conditioning agent).

The present invention further illustrates by following specific but non-restrictive example.Though comprise numerical value in an embodiment, result and/or data, following embodiment is Deuteronomic.Embodiment 1 to 5 relates to the preparation of the cell-line that can express tyrasamine acceptor (TyrR) and with the screening of this cell-line to composition.Embodiment 6 to 11 relates to the preparation of the cell-line that can express the Or83b acceptor, can express the preparation of the cell-line of Or43a acceptor, and with the screening of these cell-lines to composition.EXAMPLE l 2 to 34 relates to dispel effect and/or the insecticidal effect of determining composition.

Embodiment 1

Preparation with the Schneider cell-line of tyrasamine acceptor (TyrR) stable transfection

A. the pcr amplification of Drosophila melanogaster tyrasamine acceptor and subclone

From deriving from the Berkeley Drosophila Genome Project (Baumann, A., 1999, Drosophila melanogaster mRNA for octopamine receptor, splice variant 1B NCBI direct submission, amplification tyrasamine acceptor among the cDNA phage library GH of Drosophila melanogaster AccessionAJ007617).The nucleotide sequence of TyrR and peptide sequence are shown in Figure 31 A and 31B.Purifying phage DNA from the liquid culture lysate in this library.(Baxter，et?al.，1999，Insect?Biochem?Mol?Biol?29，461-467)。Open reading frame (Han, et al., 1998, J Neurosci 18, the 3650-3658 of fruit bat tyrasamine acceptor (TyrR) are used to increase; Von Nickisch-Rosenegk, et al., 1996.Insect BiochemMol Biol 26, oligonucleotides 817-827) they are 5 ' oligonucleotides: 5 ' gccgaattc Gc CaccATGCCATCGGCAGATCAGATCCTG 3 ' and 3 ' oligonucleotides: 5 ' taatctagaTCAATTCAGGCCCAGAAGTCGCTTG 3 '.Capitalization is represented to mate with the tyrasamine acceptor.The nucleotide of underscore is represented the Kozak sequence (Grosmaitre that adds, X., Jacquin-Joly, E., 2001 Mamestra brassicae putative octopamine receptor (OAR) mRNA, complete cds.NCBI direct submission, Accession AF43878).5 ' oligonucleotides also comprises an EcoR I site, and 3 ' oligonucleotides comprises an Xba I site.(New England Biolabs) carries out PCR under the following conditions with the Vent polymerase: about 95 ℃, carried out about 1 circulation in about 5 minutes; About 95 ℃, about 30 seconds; About 70 ℃, carried out about 40 circulations in about 90 seconds; About 70 ℃, carried out about 1 circulation in about 10 minutes.

The PCR product is digested with EcoR I and Xba I, and subclone checks order to two chains by automated DNA order-checking (Vanderbilt Cancer Center) in pCDNA 3 (Invitrogen).Thus the protein of open reading frame translation can correctly mate disclosed tyrasamine receptor sequence (Saudou, et al., The EMBOJournal vol 9 nol, 6-617).In order in fruit bat Schneider cell, to express, this TyrR ORF is cut out from CDNA3, be inserted among the pAC5.1/V5-His (B) [pAc5 (B)] by Eco RI and Xba I restriction site.

For transfection, with fruit bat Schneider cell with pAc5 (B)-TyrR ORF by the described calcium phosphate-DNA coprecipitation method of operation manual stable transfection as the fruit bat expression system (DES) of Invitrogen.Coprecipitation method all is the same for temporary transient or stable transfection, will use the plasmid of antibiotic resistance during except stable transfection.Therefrom select the clone of at least ten stable transfected cells and breeding separately.With ³The H-tyrasamine carries out full cell combination/absorption to select the stable clone of expressing described acceptor.In this detects, with cell washing and be collected in insect salt solution (170mM NaCl, 6mMKCl, 2mM NaHCO ₃, 17mM glucose, 6mM NaH ₂PO ₄, 2mM CaCl ₂And 4mM MgCl ₂) in.With about 300 ten thousand cells and the 4nM in about 1mL insect salt solution ³The H-tyrasamine was cultivated about 5 minutes altogether at 23 ℃.With about 30 seconds of cell centrifugation, inhale and remove binding soln.Cell precipitation is with about 500 μ L insect salt water washings, resuspended and go in the scintillation solution with cell.Comprise the unlabelled tyrasamine of 50 μ M in the reaction to determine non-specific binding.With liquid scintillation beta-counter (Beckman, Model LS1801) the radioactivity quantitative counting is carried out in combination.

B. selection has the clone of the functional activity tyrasamine receptor protein of highest level

Carry out tyrasamine receptors bind/absorption so which has the functional activity tyrasamine receptor protein of highest level among the clone who determines commentaries on classics seven.Nourishing and generating with about 10 is to carry out the experiment of tyrasamine acceptor, with about 2 pAc (B) in contrast.With ³H-tyrasamine (approximately 4nM/ reaction) as tracer, wherein contains or does not contain the unlabelled tyrasamine of 50 μ M as the specificity competitor.In this detects, cultured cell on plate, and be collected in about 3ml medium and carry out cell counting, adjust cell number to about 3 * 10 ⁶Individual cell/ml.Use about two pAcB clone simultaneously in contrast.Use about 1ml cell suspension in each reaction.In the specificity combination, the high-caliber active tyrasamine receptor protein of about 3 clonal expressions is arranged.Choosing the clone with the highest specificity tyrasamine receptors bind further studies.Make the clonal expansion of choosing and be stored in the liquid nitrogen.The clone's that cultivation is chosen aliquot is carrying out full cell combination, and the preparation plasma membrane is to carry out dynamics and screening study.Contrast pAcB combines without any specificity with the tyrasamine acceptor.

C. with the Schneider cell of tyrasamine acceptor transfection be used to screen can and the effect of the composition of tyrasamine acceptor interaction

Cultivate in each hole on porous plate with tyrasamine acceptor (about 1 * 10 ⁶The cells transfected of individual cell/ml).Behind the inoculating cell about 24 hours, remove medium, replace about 1ml insect salt solution (about 23 ℃).The adding variable concentrations ³H-tyrasamine (approximately 0.1-10nM) wherein contains or does not contain the unlabelled tyrasamine of 10 μ M, and cultivates in room temperature (RT).After cultivating about 20 minutes, inhale fast and desalt water, at least once with about 2ml insect salt solution (about 23 ℃) washing with cessation reaction.Under RT with cytolysis in the NaOH of about 300ml 0.3M about 20 minutes.With the dissolving cell go to about 4ml liquid scintillation solution (Liquid Scintillation Solution) (LSS) in, about 30 seconds of violent rotation, use liquid scintillation beta-counter (Beckman, Model LS1801) (LSC) to carry out radiocounting then.

With reference to Fig. 1, the receptor-specific binding data is expressed as per 1 * 10 ⁶The fmol amount of individual cell-specific combination, and be determined as ³The function of H-tyrasamine concentration.Lack the value under the condition of the unlabelled tyrasamine of 10 μ M and exist the difference between the value under the condition of the unlabelled tyrasamine of 10 μ M to be the specificity associated value.As shown in Figure 1, at about 5nM ³Has maximum specificity combination under the H-tyrasamine.Even the cell of untransfected is also not reaction when tyrasamine concentration is up to about 100 μ M.

In order to study dynamics with tyrasamine acceptor in the cell of pAcB-TyrR stable transfection, prepare the crude extract of membrane component from cells transfected, be used for calculated equilibrium dissociation constant (K _d), maximum binding capacity (B _Max), balance suppresses dissociation constant (K _i) and EC ₅₀(to being combined with the valid density of 50% inhibition).Carry out preliminary research to determine to have the best film protein concentration of receptor-binding activity.In this research, the albumen of variable concentrations (approximately 10-50 μ g/ reaction) is at about 1ml binding buffer liquid (50mM Tris, pH7.4,5mM MgCl ₂With the 2mM ascorbic acid) the middle cultivation.Add about 5nM ³The H-tyrasamine wherein contains or does not contain the unlabelled tyrasamine of about 10 μ M with initial action.After room temperature is cultivated about 1 hour, filter with cessation reaction by GF/C filter (VWR), filter is immersed in about 0.3% the polymine (PEI) in advance.With the ice-cooled Tris buffer solution washing filter of about 4ml once, before measuring residual radioactivity that it is air-dry with LSC.(GraphPad software Prism) is analyzed binding data with the curve match.Digital proof does not have difference between about 10,20,30 and 50 μ g albumen/reactions in the specificity combination of tyrasamine acceptor.Therefore, use about 10 μ g albumen/reactions.

In order to measure the B of tyrasamine acceptor (TyrR) on the film of expressing WyrR _MaxAnd K _dValue is carried out saturated in conjunction with test.Briefly, with about 10 μ g albumen and variable concentrations (approximately 0.2-20nM) ³The H-tyrasamine is cultivated together.(GraphPad software Prism) is analyzed binding data, determines the K of tyrasamine and its receptors bind with the curve match _d

In order to determine the affinity of several parts to TyrR, detected several compounds when concentration is cumulative to about 2nM ³The ability of the inhibition of H-tyrasamine combination.Detect for saturated and inhibition, under the condition that does not have and exist the unlabelled tyrasamine of about 10 μ M, determine total combination and non-specific binding respectively.The receptors bind reaction is to cultivate 1 hour in room temperature (RT) under the limit optical condition.Filter with cessation reaction by GF/C filter (VWR), before measuring residual radioactivity that it is air-dry with LSC.(GraphPad software Prism) is analyzed binding data with the curve match.

With reference to Fig. 2, wherein shown ³The H-tyrasamine ( ³H-TA) with saturated binding curve from the film of the Schneider cell preparation of expressing the tyrasamine acceptor, ³The H-tyrasamine is to having high affinity with the tyrasamine acceptor in the cell of pAcB-TyrR stable transfection, the K of mensuration _dApproximately be 1.257nM, the B of mensuration _MaxApproximately be 0.679pmol/mg albumen.

With reference to Fig. 3, wherein shown ³The H-tyrasamine ( ³H-TA) combine with the inhibition of film under the condition of the unlabelled tyrasamine (TA) that has or do not exist variable concentrations from the Schneider cell preparation of expressing the tyrasamine acceptor, tyrasamine is to the EC of the tyrasamine acceptor in the Schneider cell of expressing the tyrasamine acceptor ₅₀And K _iBe respectively about 0.331 μ M and 0.127 μ M.

In order to determine the pharmacology pattern of tyrasamine acceptor (TyrR), detected many possible neurotransmitters and replaced expressing the film combination of tyrasamine acceptor ³The H-tyrasamine ( ³H-TA) ability.With reference to Fig. 4, wherein shown ³H-tyrasamine and the inhibition combination of film under the condition of the unlabelled part (comprising tyrasamine (TA), octopamine (OA), dopamine (DA), and serotonin (SE)) that has or do not exist variable concentrations from the Schneider cell preparation of expressing the tyrasamine acceptor.Tyrasamine shows the highest affinity (K to fruit bat TyR _iBe about 0.127 μ M, EC ₅₀Be about 0.305 μ M).Octopamine, dopamine and serotonin are replacing ³The H-tyrasamine in conjunction with last not as tyrasamine effective.

With reference to table A, listed the K of part _iAnd EC ₅₀, it arranges in order by ability and is tyrasamine＞octopamine＞dopamine＞serotonin, and this shows that the Schneider cellular expression of stable transfection probably has the tyrasamine acceptor of functional activity.

Part	?K _i(μM)	?EC ₅₀(μM)
			Tyrasamine (TA) octopamine (OA) dopamine (DA) serotonin (SE)	?0.127 2.868 5.747 8.945	?0.305 7.456 14.940 23.260

Similarly, the Schneider cell of expression tyrasamine acceptor can be effectively as the model of studying and screen the composition of energy and tyrasamine acceptor interaction.

Embodiment 2

The processing of the cell of expression tyrasamine acceptor and composition are for the effect of [cAMP] in the born of the same parents

Cell is cultivated on plate, changes medium the previous day handling.When cell reaches approximate 95% when converging, inhale and remove medium, with the about 27 ℃ insect salt solution of 5ml (170mM NaCl, 6.0mM KCl, 2.0mM NaHCO ₃, 17.0mM glucose, 6.0mM NaH ₂PO ₄, 2.0mM CaCl ₂, 4.0mM MgCl ₂PH7.0) washed cell once.Add about 20ml insect salt solution, gently scrape collecting cell.Aliquot with the hemacytometer pair cell is counted, then with centrifugal about 5 minutes of cell 1000RPM.With cell resuspended be 3 * 10 ⁶Individual cell/ml.It is about 200 μ M that adding IBMX makes it concentration.Telling about 1ml cell suspension then handles.Add Forskolin (cAMP induces reagent), tyrasamine or different candidate set compounds are cultivated cell about 10 minutes at about 27 ℃.

With the cell handled centrifugal about 10 seconds of about 13000g.Solution is removed in suction, adds approximately-20 ℃ 70% ethanol of about 1ml.Cell precipitation is smashed in rotation, sample is placed-20 ℃ spend the night.After the ethanol extracting, centrifugal about 5 minutes of about 13000g with the sedimentation cell fragment.Supernatant is gone in the test tube, with the freeze drying of Rotary drying instrument.The extract that obtains is resuspended among about 100 μ l TE, is used for cAMP and detects.

CAMP detect be based on endogenous cAMP and ³H-cAMP is to the protein-bonded competitive combination of cAMP.With ³H-cAMP Biotrak system (Amersham Biosciences) carries out this detection, according to the description operation of manufacturer.Briefly, with about 50 μ l cell extracts and about 50 μ l ³H-cAMP and about 100 μ l cAMP cultivated about 2-4 hour in conjunction with the albumen ice bath.Add active carbon (about 100 μ l) then, with solution about 4 ℃ centrifugal about 3 minutes.Get about 200 μ l reactant mixtures, measure with scinticounting ³The level of H-cAMP.Calculate the level of endogenous cAMP in the cell with the calibration curve of the cold cAMP of about every reaction 0 to 16pmol.

Embodiment 3

The processing of the cell of expression tyrasamine acceptor and composition are for [Ca in the born of the same parents ²⁺] effect

Acetoxy-methyl (AM) ester with fluorescence indicator furans-2 is measured intracellular free calcium level ([Ca ²⁺] i) (Enan, et al., Biochem.Pharmacol vol 51,447-454).In this research, under standard conditions, cultivate the cell that to express tyrasamine.Week is detected buffer solution (140mM NaCl, 10mM HEPES, 10mM glucose, 5mM KCl, 1mM CaCl ₂, 1mM MgCl ₂) the preparation cell suspension, adjust cell number to every ml about 2 * 10 ⁶Individual cell.Brief, with about 1.0ml suspension (about 2 * 10 ⁶Individual cell) cultivated about 30 minutes at about 28 ℃ with 5 μ M furans 2/AM.After the cultivation, room temperature 3700rpm was resuspended in about 1.5ml and detects in the buffer solution with sedimentation cell in centrifugal about 10 seconds then.Under the condition of the chemical combination medicine that has or do not exist detection with sepectrophotofluorometer analysis [Ca ²⁺] variation of i.Excitation wavelength is that about 340nm is (by in conjunction with Ca ²⁺Furans-2 produce) and about 380nm (corresponding to not containing Ca ²⁺Furans-2).Monitor the fluorescence intensity of the emission wavelength of about 510nm.All do not observe any artificial fluorescent absorption when using any compound.Calculate the ratio of about 340/380nm, and be depicted as the function of time.

Embodiment 4

Flos caryophylli oil and black seed oil are for the tyrasamine receptors bind in the cell of expressing tyrasamine amount body

Active effect

In order to determine specific oil, that is, flos caryophylli oil (LFO) and black seed oil (BSO) whether can with tyrasamine acceptor interaction and the functional expression that whether can regulate the tyrasamine acceptor, use from the film analysis of the Schneider cell preparation of stable transfection and untransfected ³The combination of H-tyrasamine.

For on the receptor site with ³The combination of H-tyrasamine is used above-mentioned in conjunction with experimental program.The dose response experiments of having carried out LFO and BSO (approximately 1-100 μ g/ml) with determine they for ³The effect that the H-tyrasamine combines with inhibition from the film of the Schneider cell preparation that can express the tyrasamine acceptor.With reference to Fig. 5, wherein shown ³The H-tyrasamine has proved with LFO and BSO processing to cause in dose-dependent mode with the inhibition combination of film under the condition of LFO that has or do not exist variable concentrations and BSO with the Schneider cell preparation that can express the tyrasamine acceptor ³The inhibition of H-tyrasamine and its acceptor.The EC of LFO and BSO ₅₀Value is respectively near 10 μ g/ml and 20 μ g/ml.

See Fig. 6 now, wherein shown ³The H-tyrasamine is with existing or do not exist LFO or BSO's or combine with inhibition under the condition of the about 1 and 10 unlabelled tyrasamines of μ M with the film of the Schneider cell preparation that can express the tyrasamine acceptor, and LFO (about 25 μ g/ml) can suppress separately ³The H-tyrasamine combines with its acceptor.This effect is also uncertain for the effect of the unlabelled tyrasamine of about 10 μ M (about 1.74 μ g/ml).In addition, when to have only concentration when the unlabelled tyrasamine that uses be about 1 μ M, LFO has just strengthened unlabelled tyrasamine to be suppressed ³The ability of H-tyrasamine combination.On the other hand, BSO (about 25 μ g/ml) suppresses ³The energy force rate LFO of H-tyrasamine combination is poor.But no matter how much concentration of unlabelled tyrasamine is, BSO can both significantly increase unlabelled tyrasamine to be suppressed ³The ability of H-tyrasamine combination.This two kinds of oil are in the Schneider cell of untransfected ³The combination of H-tyrasamine is without any effect.

Similarly, LFO and BSO are different with the interaction of tyrasamine acceptor.Do not wish to be subjected to the restriction of any theory and mechanism, LFO and tyrasamine are competed at same binding site probably, and BSO acts on and the different acceptor site of endogenous ligands (tyrasamine).Other specific oil, comprise those clear and definite oil of listing in this application also can with the tyrasamine acceptor interaction.

Embodiment 5

Flos caryophylli oil and black seed oil are for [cAMP] in the born of the same parents in the cell that can express the tyrasamine acceptor

Effect

Depend on the coupling that detects chemical substance in order to study the tyrasamine acceptor, make pAcB-TyrR stably express in the Schneider cell.With tyrasamine (about 10 μ M), LFO (about 25 μ g/ml) and BSO (about 25 μ g/ml) handle the cell of transfection and untransfected under the condition that has or do not exist forskolin (FK) (about 10 μ M).Use as mentioned above ³H-cAMP detection kit (Amersham) is measured the generation of cAMP.

Have functional activity in order to ensure the cAMP cascade in this cell model, use forslcolin, a kind of cAMP derivant is as standard reagent.Shown in Fig. 7 to 9, shown that wherein cAMP level in the Schneider cell of the expression tyrasamine acceptor after handling under the condition that has or do not exist tyrasamine (about 10 μ M) and forskolin (about 10 μ M) with LFO (about 25 μ g/ml) and BSO (about 25 μ g/ml) depends on the variation of tyrasamine, only in cells transfected, compared with the baseline values of cAMP in the cell of only using solvent (ethanol) processing and increased about 19 times handling later on the cAMP level with forskolin.

On the other hand, tyrasamine makes the generation of cAMP that slight reduction be arranged.But the remarkable antagonism of tyrasamine is expressed the cAMP level that forskolin stimulates in the cell of tyrasamine, and this shows that the tyrasamine acceptor exists under the condition of tyrasamine and G _{α i/o}Coupling, as shown in Figure 7.Only in cells transfected, after handling, find that the cAMP level has reduced about 34% and 25% (Fig. 8) respectively with LFO and BSO.And tyrasamine has strengthened the effect that LFO produces for the cAMP in the tyrasamine acceptor transfectional cell, when handling together with BSO and tyrasamine except the effect of BSO self the cAMP level without any variation, as shown in Figure 8.The phenomenon that the cAMP level that LFO and BSO cause in the Schneider cell of expressing the tyrasamine acceptor reduces exists under the condition of forskolin and has reduced, as shown in Figure 9.

With other specific essence oil from plant, comprise that those clear and definite oil processings of listing in this application can cause expressing the change of cAMP level in the born of the same parents in the cell of tyrasamine acceptor.

Embodiment 6

The preparation Schneider cell of olfactory receptor (Or83b and Or43a) stable transfection

A. the RT-PCR of Drosophila melanogaster olfactory receptor Or83b and Or43a increases and subclone

With Trizol reagent (Invitrogen) from the wild type Drosophila melanogaster the head and feeler the extraction total RNA.With it together with Trizol homogenate in the Teflon pestle of motor driven and glass homogenizer.Specification according to manufacturer extracts RNA then, comprises by precipitation and removes proteoglycan and polysaccharide.With MuLV reverse transcriptase (Applied Biosystems) total RNA is carried out reverse transcription as primer with oligo-dT.Use following oligonucleotides to carry out the pcr amplification of open reading frame: Or83b sense orientation: 5 ' taa Gcggcc GcATGACAACCTCGATGCAGCCGAG 3 '; Or83b antisense orientation 5 ' ataccgcggCTTGAGCTGCACCAGCACCATAAAG 3 '; Or43a sense orientation 5 ' taa GcggccgcATGACAATCGAGGATATCGGCCTGG 3 '; And Or43a antisense orientation 5 ' ata CcgcggTTTGCCGGTGACGCCACGCAGCATGG 3 '.Capitalization is represented to mate fully with Or83b and Or43a receptor sequence.The oligonucleotides of sense orientation comprises Not I site, and the oligonucleotides of antisense orientation comprises the SacII site.Two restriction sites are represented by the nucleotide of underscore.The oligonucleotides of antisense orientation does not comprise terminator, so the V5 phenotype of pAC 5.1 plasmids is joined with translation albumen.For the pcr amplification of Or83b, use Vent polymerase (New England Biolabs), reaction condition is as follows: about 95 ℃, carried out about 1 circulation in about 5 minutes; About 95 ℃, about 30 seconds; About 70 ℃, carried out about 40 circulations in about 90 seconds; About 70 ℃, carried out about 1 circulation in about 10 minutes.For the pcr amplification of Or43a, use Failsafe PCR Premix Selection kit (Epicentre Technologies), reaction condition is as follows: about 95 ℃, carried out about 1 circulation in about 5 minutes; About 95 ℃, about 30 seconds; About 60 ℃, about 30 seconds and about 70 ℃ carried out about 40 circulations in about 90 seconds; About 70 ℃, carried out about 1 circulation in about 10 minutes.Failsafe premix buffer solution F produces the product of correct size.The PCR product is digested with Sac II and Not I, and gel-purified also is connected on the pAC 5.1/V5 His B (Invitrogen).(Vanderbilt Cancer Center) checks order to the insert on two chains with automatic fluorescence sequenator.Or83b open reading frame and Or43a open reading frame coding albumen and PubMed on and the genome sequence on the website in the sequence information that finds be identical.The nucleotide sequence of Or43a and peptide sequence are shown in Figure 32 A and 32B.The nucleotide sequence of Or83b and peptide sequence are shown in Figure 33 A and 33B.

Carry out transfection with the calcium phosphate-DNA coprecipitation method, as described in above-mentioned InvitrogenDrosophila Expression System (DES) operation manual, with pAc5 (B)-Or83b ORF or pAc5 (B)-Or43a ORF stable transfection fruit bat Schneider cell.Picking is the clone of about ten Or83b or Or43a stable transfected cells at least, cultivates respectively.With the RT-PCR method stable clone is analyzed to detect them whether express corresponding mRNA.From cell, extract RNA with Trizol, according to the specification operation of manufacturer.With the MuLV reverse transcriptase total RNA is carried out reverse transcription.Carry out PCR:Or83b sense orientation primer and Or83b antisense orientation primer with Vent polymerase and following primer; Or43a sense orientation primer and Or43a antisense orientation primer.Analyze the PCR product with agarose gel electrophoresis, and compare with the contrast Schneider cell RNA that is used for RT-PCR.The clone who expresses Or83b-mRNA or Or43a-mRNA with a height is cooked further research, with protein expression (Western blot) and the signal conduction (cAMP product and [Ca2+]) of research after with tyrasamine and specific plant essence oil processing.

Determine which clonal expression Or83b and Or43a gene with RT-PCR.Agarose gel analysis shows for Or83b have about 4 clones to produce the big or small correct product of about 1.46kb in about 10 clones.For Or43a, about 2 clones produce the big or small correct product of about 1.1kb.When being carried out PCR, contrast Schneider cell do not obtain these products.Select the clone that can express described mRNA to carry out other research of relevant acceptor.

B. with the Schneider cell-line of Or83b acceptor or the transfection of Or43a acceptor be used to screen can with the ability of the composition of Or83b and Or43a acceptor interaction

In order to study the specific binding site whether Or83b acceptor and Or43a acceptor contain tyrasamine, can express the film of Or83b acceptor or Or43a acceptor as mentioned above from the cell preparation that can express two kinds of acceptors, be used for ³The competitive combination of H-tyrasamine.Just the same in conjunction with the method that detects with the cell of expressing TyrR, as mentioned above.As shown in figure 10, wherein shown ³H-tyrasamine and the saturated inhibition curve of film under the condition that has or do not exist the unlabelled tyrasamine of about 20 μ M from the Schneider cell preparation that can express the Or83b acceptor, and shown the same experimental result of cell of expressing the Or43a acceptor among Figure 11, ³The H-tyrasamine combines with Or83b and Or43a receptor-specific.As shown in Table B, the Kd of tyrasamine and Or83b receptors bind approximately is 96.90nM, B _MaxApproximately be 4.908pmol/mg albumen.For Or43a, corresponding value is that Kd is about 13.530nM, B _MaxBe approximately 1.122pmol/mg albumen.

Acceptor type	?K _i(nM)	?B _max(pmol/mg albumen)
			?TyrR Or83b Or43a	?1.257 96.900 13.530	?0.679 4.908 1.122

Table B

Embodiment 7

The generation of cAMP in the cell of expression olfactory receptor

Have functional activity in order to ensure the cAMP cascade in this cell model, use forskolin, a kind of cAMP derivant is as standard reagent.With the cAMP detection assay ring AMP level described in the foregoing description 2.As shown in figure 12, shown that wherein cAMP level in the cell of expressing the Or83b acceptor depends on the variation of forskolin, with the forskolin of about 10 μ M in room temperature treatment increased about 13 times than baseline cAMP level in about 10 minutes cell.The cell of expressing the Or43a acceptor has also obtained similar result.Similarly, the cell of expression olfactory receptor has the cAMP cascade of functional activity.

Embodiment 8

Fluctuation in the born of the same parents of Ca2+ in the cell of expression olfactory receptor

Measure Ca in the born of the same parents with the method described in the foregoing description 3 ²⁺Level.In the cell of expressing Or83b or Or43a acceptor, use ionomycin (a kind of Ca ²⁺Induce reagent) and tyrasamine is handled later on, and generation calcium fluctuates.Particularly, with reference to Figure 13 and 14, when adding about 2 μ M ionomycin in the cell of expressing Or83b or Or43a, 340nm and 380nm excited fluorescent are than interrelated with the cellular calcium level, and observing cellular calcium increases significantly.In the cell of expressing Or83b and Or43a, after handling with ionomycin, calcium has increased about 3.8 times and 7 times respectively.With reference to Figure 15, the tyrasamine of about 10 μ M also can induce cellular calcium to increase about 1.18 times and 3.5 times respectively in the cell of expressing Or83b and Or43a.

Generally, pharmacology is analyzed these protein receptors that can express function with Or83b acceptor gene or Or43a acceptor gene cells transfected model of digital proof.

Embodiment 9

Different essence oil from plant is for the letter in conjunction with activity and acceptor downstream of olfactory receptor

The effect of number pathway

With the cell research essence oil from plant of expressing a kind of olfactory receptor and the signal transduction cascade in the interaction between these acceptors and every kind of acceptor downstream.

For in conjunction with active, will be used to study the interaction between essence oil from plant and the receptor binding site from the film of every kind of cell model preparation.With reference to Figure 16, following oil and olfactory receptor interact: flos caryophylli oil (LFO), ethyl phthalate, alpha-terpineol, and piperonal.

Similarly, with reference to Figure 17 and 18, following oil and olfactory receptor interact: black seed oil (BSO), α-sobrerone, benzoquinones, p-cymene, sabinene, α-absinthol and d-citrene.

Similarly, with reference to Figure 19 to 21, following oil also can interact with olfactory receptor: geraniol, ortho-aminobenzoic acid agalloch eaglewood ester, phenylacetaldehyde, linalool, alpha-terpineol, t-anethole, terpinene 900, lindenol, eugenol, thyme linaloe oil, carvacrol, thymol, piperonal, piperitol, acetic acid pepper ester, and pepper amine.

Other specific oil comprises what those spelt out in this application, also can interact with olfactory receptor.

Embodiment 10

In the born of the same parents of different essence oil from plant for the Ca2+ in the cell of expressing the Or43a acceptor

The effect of fluctuation

In order to determine of the effect of different essence oil from plant, detect with the intact cell of every kind of cell model, as mentioned above for the cellular calcium fluctuation.According to calculating Ca in the born of the same parents with the difference of handling 340/380 afterwards about 150 seconds fluorescence ratio before handling ²⁺The variation of level.As shown in figure 22, can induce Ca in the control cells with ionomycin and tyrasamine processing ²⁺Fluctuation, but in the cell of expressing the Or43a acceptor, can only increase Ca in the born of the same parents very minutely ²⁺Level.

With reference to Figure 22 to 28, following oil can cause the calcium fluctuation in the cell of expressing the Or43a acceptor: ortho-aminobenzoic acid agalloch eaglewood ester, linalool, perillyl alcohol, t-anethole, geraniol, phenylacetaldehyde, eugenol, piperitol, acetic acid pepper ester, pepper amine, alpha-terpineol, lindenol, terpinene 900, thyme linaloe oil, thymol, carvacrol, LFO, BSO, α-sobrerone, p-cymene, d-citrene, sabinene, quinine, l-carvol, d-carvol, and α-absinthol.At last, as shown in figure 24, handle and in the cell of expression Or43a acceptor, to reduce Ca in the born of the same parents with piperonal ²⁺Level.

With other specific essence oil from plant, comprise that those essential oils that spell out in this application processing also can cause Ca in the born of the same parents in the cell of expressing the Or43a acceptor ²⁺The variation of level.

In addition, with other specific essence oil from plant, comprise that those essential oils that spell out in this application processing can cause Ca in the born of the same parents in the cell of expressing the Or83b acceptor ²⁺The variation of level.

Embodiment 11

Different essence oil from plant is for the generation of cAMP in the cell of expressing olfactory receptor

Effect

For the effect of determining that different essence oil from plant produces for cAMP in the born of the same parents, and cAMP depends on the variation of tyrasamine in expressing a kind of cell of olfactory receptor, the cell of every kind of cell model is handled under the condition that has or do not exist tyrasamine (about 20 μ M) and forskolin (about 10 μ M) with LFO (about 50 μ g/ml) and BSO (about 50 μ g/ml), determined cAMP in the born of the same parents with the detection method described in the embodiment 2 then.

Shown in Figure 29 and 30, in the cell of expressing the Or43a acceptor, can cause the cAMP level to increase with following oil processing: tyrasamine; LFO; BSO; LFO and tyrasamine; BSO and tyrasamine; Forskolin; Tyrasamine and forskolin; LFO and forskolin; LFO, forskolin and tyrasamine; BSO; And BSO, tyrasamine and forskolin.

Still with reference to Figure 29 and 30, with about 20 μ M tyrasamines, the generation of expressing cAMP in the cell of Or83b acceptor after about 50 μ g LFO/ml and about 50 μ g BSO/ml handle has increased about 34%, 32% and 64% respectively.Handle together with the generation that compares cAMP with each effect of handling separately with tyrasamine and LFO and to have antagonism (about 24%).On the other hand, the generation of handling together cAMP with BSO and tyrasamine has synergy (approximately increasing by 300%).

Under the condition that has forskolin (about 10 μ M), the generation of cAMP has increased about 3000 times.When giving tyrasamine or LFO with the pretreated cell of forskolin, except the effect of forskolin self, the generation of cAMP has only increased about 10-13%.To adding BSO with the pretreated cell of forskolin, the cAMP that induces except forskolin in these cells produced, the level of cAMP had only increased about 22%.

Also have, handle with some other plant essential oil, as comprise the plants essential oil of clearly listing in this application, the quantitative changeization of cAMP in the cell in the cell of feasible expression Or43a or Or83b acceptor.

Embodiment 12

Composition is for the toxicity of Drosophila melanogaster

Two kinds of acetone solns (about 1% and 10%) of preparation composition.The acetone soln of adding detectable concentration in vial (approximately 5ml) makes marks counting about 3cm from the bottle bottom surface.The rotary glass bottle all leaves the detection solution film on the inner surface of the vial the feasible zone between mark and bottleneck.Air-dry 10 seconds of all vials to guarantee that acetone evaporates fully, are placed fruit bat the vial of processing then.After treating that acetone has evaporated fully, in each bottle, add about 10 other fruit bats of adult miscibility, clog bottle with tampon.Observe death after about 24 hours.

Embodiment 13

Flos caryophylli oil (LFO) and black seed oil (BSO) are for wild type fruit bat and tyrasamine acceptor

The toxicity of sudden change fruit bat

Can determine the toxicity of LFO and BSO with wild type Drosophila melanogaster and tyrasamine receptor mutation fruit bat as model.Study these oily toxicity with the method described in the foregoing description 12.With reference to following table C and D, two kinds of chemical substances are all toxic for the wild type fruit bat.LFO is about 300 times of BSO to the toxicity of fruit bat.The LC of LFO ₅₀Approximately be 25-30ng/mm ², the analog value of BSO is about 94 μ g/cm ²On the other hand, LFO lacks about 1000 times for the toxicity of tyrasamine receptor mutation fruit bat than BSO.Two kinds of chemical substances can be regulated by the tyrasamine acceptor for the toxicity of fruit bat.The sudden change of tyrasamine acceptor has significantly reduced the toxicity of LFO for fruit bat, and same mutant fruit bat is for the BSO more susceptible that becomes.

Table C

Table D

Embodiment 14

Composition is for the dispel effect of field ant

Select adult insects to be used for the dispel effect of detection composition arbitrarily, insect is not carried out separate marking.Use about 5 insects when carrying out repetition at every turn.The each processing approximately repeated 3 times.Untreated control test is carried out under the same conditions, just to the colony of equivalent/repeat only to use solvent (acetone).Handle filter paper (about 80cm with described composition (about 100mg is in 300ml acetone) ²).After air-dry about 3 minutes, filter paper is placed on the dish, insect is driven away.Insect is placed on the dish, once places an insect at the far-end of plate.Be recorded in the time used on the untreatment surface of each filter paper or plate with one or more stopwatches, until about 300 seconds.Be calculated as follows expeling rate (RR):

Used time on used time-treatment surface on the RR=[(contrast surface)/total time of detecting].If RR＞0 then is considered as described composition and has dispel effect, that is to say in comparison on the treatment surface has more insect to be purged according on the surface; If RR＜0 then described composition is regarded as there is not dispel effect.

Embodiment 15

Flos caryophylli oil (LFO) and black seed oil (BSO) are for the dispel effect of field ant

With (the about 1.4mg/cm of the method research LFO described in the foregoing description 14 ²) and BSO (about 1.4mg/cm ²) for the dispel effect of field ant.As show shown in E and the F, prove that BSO is stronger than LFO expeling property for the field ant.LFO and BSO are respectively 90% and 100% for the expeling of field ant.In addition, LFO and BSO also can cause the field ant that 100% lethality is arranged in 24 hours.

Table E

Table F

The dish research BSO that use is handled with BSO drives away the residual effect of ant.According to above-mentioned expelling method during every day with five ants.Determine the time-histories of the toxicity of BSO simultaneously.In toxicity test, ant was exposed to same treatment surface about 10 seconds, transfer to then in the fresh container, expose the dead data of about 24 hour records in back.Every day is with five ants.As show shown in the G, BSO can drive away ant and reach 4 days.

Time after the surface treatment, day	Expeling property %
		The 1st day	100
The 2nd day	100
		The 3rd day	100
The 4th day	100

Table G

Embodiment 16

The d-citrene, α-sobrerone and p-cymene are used alone or in combination driving for the field ant

Remove effect

With inspection oil processing filter paper to detect the dispel effect of different essence oil from plant.Room temperature was placed after 5 minutes, and filter paper is placed dish, disposablely put into ant.As definite expeling as described in the above-mentioned embodiment 14.Every kind of oil detects separately.In addition, also various oil are mixed into composition and detect.

With reference to table 16H, the d-citrene, α-sobrerone and p-cymene all have expeling separately for every kind.And oil is mixed into composition A, and a kind of d-citrene that comprises, α-sobrerone and p-cymene be each composition of 1/3rd approximately, has synergy, and driving away percentage increases greatly.

Table H

Similarly, with reference to table I, d-citrene and α-sobrerone all have expeling separately for every kind.And these oil are mixed into composition B, and a kind of composition that contains d-citrene and α-sobrerone each half has synergy, and driving away percentage increases greatly.

Table I

Embodiment 17

Linalool, the d-citrene, α-sobrerone, p-cymene and thyme linaloe oil are alone or in combination

Use is for the dispel effect of field ant

With reference to table J, though the d-citrene, α-sobrerone, p-cymene and thyme linaloe oil all have expeling separately for every kind, composition C, a kind of each composition of about 25% of every kind of oil that comprises, expeling surpass the effect that any component oil uses separately.

Table J

Similarly, shown in table K, though linalool, α-sobrerone, p-cymene and thyme linaloe oil all have expeling, composition D separately for every kind, a kind of each composition of about 25% of every kind of oil that comprises, expeling surpass the effect that any component oil uses separately.

Table K

Similarly, shown in table L, though linalool, α-sobrerone and p-cymene all have expeling separately for every kind, composition E, a kind of each composition of about 1/3rd of every kind of oil that comprises, expeling surpass the effect that any component oil uses separately.

Table L

Embodiment 18

α-sobrerone, thyme linaloe oil, α-absinthol, sabinene are used alone or in combination for the field ant

Dispel effect

Though α-sobrerone, thyme linaloe oil, α-absinthol and sabinene all have expeling separately for every kind, shown in table M, composition F, a kind of comprising each composition of about 25% of every kind of oil, the enhancing of expeling property.

Table M

Embodiment 19

The d-citrene, the p-cymene, thymol, carvacrol and geraniol make alone or in combination

Use dispel effect for the field ant

As show shown in the N, though the d-citrene, the p-cymene, thymol and carvacrol all have expeling separately for every kind, composition G, a kind of each composition of about 25% of every kind of oil that comprises, expeling surpass the effect that any component oil uses separately.

Table N

Similarly, shown in table O, though the d-citrene, p-cymene and thymol all have expeling separately for every kind, composition H, a kind of each composition of about 1/3rd of every kind of oil that comprises, expeling surpass the effect that any component oil uses separately.

Table O

Similarly, shown in table P, though the d-citrene, the p-cymene, thymol and geraniol all have expeling separately for every kind, composition I, a kind of each composition of about 25% of every kind of oil that comprises, expeling surpass the effect that any component oil uses separately.

Table P

Embodiment 20

Ortho-aminobenzoic acid agalloch eaglewood ester, α-sobrerone, d-citrene, p-cymene, and spiceleaf

Alcohol is used alone or in combination the dispel effect for the field ant

As show shown in the Q, though geraniol, the d-citrene, p-cymene and ortho-aminobenzoic acid agalloch eaglewood ester all have expeling separately for every kind, and composition J a kind ofly contains about 40% geraniol, about 30%d-citrene, about 10%p-cymene, the composition of about 10% α-sobrerone and about 10% ortho-aminobenzoic acid agalloch eaglewood ester, expeling surpass the effect that any component oil uses separately.

Table O

Embodiment 21

The d-citrene, thymol, alpha-terpineol, acetic acid pepper ester, pepper amine, and piperonal

Be used alone or in combination dispel effect for the field ant

As show shown in the R, though the d-citrene, thymol, alpha-terpineol, acetic acid pepper ester, pepper amine and piperonal all have expeling, composition K separately for every kind, a kind ofly contain about 20%d-citrene, about 30% thymol, about 20% alpha-terpineol, about 10% acetic acid pepper ester, the about composition of l0% pepper amine and about 10% piperonal, expeling surpass the effect that any component oil uses separately.

Table R

Embodiment 22

Geraniol, the d-citrene, eugenol, lindenol and phenylacetaldehyde are used alone or in combination

Dispel effect for the field ant

As show shown in the S, though geraniol, the d-citrene, eugenol, lindenol and phenylacetaldehyde all have expeling, composition L separately for every kind, a kind ofly contain about 50% geraniol, about 20%d-citrene, about 10% eugenol, approximately 10%lindenol, with the composition of about 10% phenylacetaldehyde, expeling surpass the effect that any component oil uses separately.

Table S

Embodiment 23

Geraniol, lemongrass oil, eugenol and mineral oil are used alone or in combination the dispel effect for the field ant

As show shown in the T, though geraniol, lemongrass oil and eugenol all have expeling for every kind, composition M, a kind of about 50% geraniol, about 40% lemongrass oil of containing, with the composition of about 10% eugenol, expeling surpass the effect that any component oil uses separately.Geraniol, lemongrass oil and eugenol all are that environmental protection institution (EPA) and Food and Drug Administration (FDA) it is generally acknowledged safe (GRAS compound), and have obtained the insecticide registration exemption of EPA.

Table T

Similarly, shown in table U, though geraniol and lemongrass oil all have expeling for every kind, composition N, a kind of composition that contains about 70% geraniol and about 30% lemongrass oil, expeling surpass the effect that any component oil uses separately.

Table U

In addition, as shown in Table V, add mineral oil and form composition O, a kind ofly comprise about 60% geraniol, the composition of about 30% lemongrass oil and about 10% mineral oil can not influence the synergy of geraniol and lemongrass oil.Mineral oil is expeling property not separately, but can make composition stable, has limited the volatilization of active component.Mineral oil is the same with geraniol and lemongrass oil all to be the GRAS compound.

Table V

Embodiment 24

Geraniol, thymol, lemongrass oil and mineral oil are used alone or in combination for the field ant

Dispel effect

As show shown in the W, though geraniol, thymol and lemongrass oil all have expeling for every kind, composition P, a kind of about 50% geraniol, about 20% thymol, about 20% lemongrass oil of containing, with the composition of about 10% mineral oil, expeling surpass the effect that any component oil uses separately.Geraniol, thymol, lemongrass oil, eugenol and mineral oil all are that environmental protection institution (EPA) and Food and Drug Administration (FDA) it is generally acknowledged safe (GRAS compound), and have obtained the insecticide registration exemption of EPA.

Table W

Embodiment 25

Black seed oil (BSO), flos caryophylli oil (LFO), geraniol, thymol, lemongrass oil

Be used alone or in combination dispel effect with mineral oil for carpented ant

As shown in Table X, geraniol, thymol and thyme linaloe oil all have expeling for every kind.As show shown in the Y, composition Q contains BSO to V, LFO, and geraniol, thymol, thyme linaloe oil, mineral oil, the various combination of safflower oil and castor oil, expeling property all strengthens to some extent.

Table X

Table Y

Embodiment 26

Commercial expellent 29%DEET is for the dispel effect of carpented ant

In order to compare with the dispel effect of the different compounds of forming by different essence oil from plant, detected insect control reagent, commercial expellent 29%DEET, commodity are called REPEL_ (Wisconsin Pharmacal Company, Inc, Jackson, WY), for the expeling of carpented ant, method is to handle filter paper with 29%DEET.Room temperature was placed after 5 minutes, and filter paper is placed dish, disposablely put into ant.As definite expeling as described in the above-mentioned embodiment 14.As show shown in the Z, 29%DEET is about 98.4% at the 0th day expeling percentage.LFO, the expeling percentage of BSO and composition of the present invention is suitable, sometimes than the expeling rate height of 29%DEET.

Table Z

Embodiment 27

Commercial expellent DEET, separately or and geraniol, thymol, and lemongrass oil

Or geraniol, the D-citrene, eugenol, LINDENOL and phenylacetaldehyde are used in combination

Dispel effect for carpented ant

With inspection oil processing filter paper to detect the dispel effect of commercial expellent DEET and different essence oil from plant.Room temperature was placed after 5 minutes, and filter paper is placed dish, disposablely put into ant.As definite expeling as described in the above-mentioned embodiment 14.Every kind of oil detects separately.In addition, also various oil are mixed into composition and detect.

Shown in Table A A and BB, be that the DEET of about 5-10% handles and not show the sign of expeling with concentration.But, shown in Table A A, when with composition W, a kind ofly contain about 25% geraniol, 10% thymol, when the combination of compositions of 10% lemongrass oil and mineral oil (45% to 55%, depend on the ultimate density of DEET) was used, expeling property was near 100.Similarly, as show shown in the BB, when with composition X, a kind of about 25% geraniol, 10%d-citrene, 5% eugenol of containing, 5%lindenol, when the combination of compositions of 5% phenylacetaldehyde and mineral oil (45% to 55%, depend on the ultimate density of DEET) was used, expeling property was near 97% to 98%.And, shown in figure AA and BB, expeling property increase when various oil and DEET are used in combination.

Table A A

Table BB

Embodiment 28

Composition is for the insecticidal effect of head louse

Collect adult head louse Pediculus humanus alive on one's body from the young girl and the boy of age 4 to 11 years old who lives in Karmos area, Egyptian Alexandria.Spy lice comb with fine and closely woven tooth is collected described insect, flocks together.The louse of collecting is placed dish, after collection, be used for this research in about 30 minutes.

The composition of the variable concentrations that preparation is used to detect in water.For the insecticidal effect of these compositions is compared with commercial obtainable effect of killing lice reagent, that ivermectin is water-soluble.Use the composition of every kind of concentration of about 1ml on dish, approximately the ivermectin aqueous solution of 1ml is used about 1ml water on the contrast dish.In each dish, put into about 10 adult head louses.

Continue to observe and handle and the contrast dish, and observe LT ₁₀₀LT is meant the needed time of the insect that kills given percentage; So LT ₁₀₀Be meant 100% the needed time of louse of killing.If head louse does not react for hard thing then is considered as its death.

Embodiment 29

Comprise geraniol, D-citrene, phenmethylol, the group of P-cymene and flos caryophylli oil

Compound is for the insecticidal effect of head louse

With the compound Y of method seminar described in the foregoing description 28, a kind ofly comprise about 20%p-cymene, about 40% flos caryophylli oil (LFO), the composition of about 30% phenmethylol and about 10% mineral oil, insecticidal effect.LT with said composition ₁₀₀With the obtainable lice reagent that kills of commerce, ivermectin is compared.As show shown in the CC, the louse that usefulness composition Y handles is faster than the louse death of handling with ivermectin.

Handle	LT100 (minute)
		Composition Y	3
Ivermectin	5

Table CC

Embodiment 30

Composition is for the dispel effect of mosquito

A. oral delivery

With there not being the dispel effect that mouse hair or that scraped hair and cavy detect the composition of oral delivery.Detect oil (for example flos caryophylli oil (LFO) or black seed oil (BSO)) or detection composition (for example containing geraniol, d-citrene, the composition of eugenol and lindenol) for about 10 rodent orally gives.Give the about contrast of 10 rodent orally gives material, for example mineral oil.After about 30 minutes, every rodent is placed in the container of sealing.In each container, put into about 20 mosquitoes.Observed each container about 1 hour.Write down the number of the wound that every insect stays time that rodent stops and described insect on one's body on rodentine skin.It is few that described insect has accepted to contrast the time that the rodent of material on stops at the time ratio that the rodent of having accepted detection composition stops on one's body.The rodent wound on one's body of accepting detection composition lacks than the rodent of accepting the contrast material.

B. local delivery

With there not being the effect that mouse hair or that scraped hair and cavy detect the composition of local delivery.Detect oil (for example flos caryophylli oil (LFO) or black seed oil (BSO)) or detection composition (for example containing geraniol, d-citrene, the composition of eugenol and lindenol) for about 10 rodentine local skins.Contrast material for about 10 rodentine local skins, for example mineral oil.After about 30 minutes, every rodent is placed in the container of sealing.In each container, put into about 20 mosquitoes.Observed each container about 1 hour.Write down the number of the wound that every insect stays time that rodent stops and described insect on one's body on rodentine skin.It is few that described insect has accepted to contrast the time that the rodent of material on stops at the time ratio that the rodent of having accepted detection composition stops on one's body.The rodent wound on one's body of accepting detection composition lacks than the rodent of accepting the contrast material.

Embodiment 31

Composition is for the dispel effect of mosquito

About three cages, each is equipped with the tropical tame mosquito (culex quinquefasciatus) in about 100 about 7 to 10 day ages.Mosquito was starved about 12 hours.Each cage has four containers, and each container all has been full of the cotton that is soaked with syrup.

Three in four containers detection composition with 1000ppm (approximately 1mg/l) are handled arbitrarily, and another container is as untreated contrast.Container is placed on four relative angles of each cage, in time interval about 0,1,2,4 and 6 hours after each container adding detection composition, falling of mosquito counted.Between the time interval that exposes, container is shifted out from cage.Each time interval that exposes continues about 5 minutes.

Detect the dispel effect of the composition described in the table DD with the method.

Composition	Composition (being expressed as the % of weight)
		EE	10%DEET, 45%LFO, 45% cymin oil
AA
		50% geraniol, 40% thyme linaloe oil, 10% lemongrass oil
BB
		50%LFO, 50% cymin oil

Table DD

LFO, cymin oil, geraniol, thyme linaloe oil and lemongrass oil are that environmental protection institution (EPA) and Food and Drug Administration (FDA) it is generally acknowledged safe (GRAS compound), and have obtained the insecticide registration exemption of EPA.

In time interval about 0,1,2,4 and 6 hours after each container adding detection composition, falling of mosquito being counted, shown in table DD.The number that falls is shown in table EE.Drive away percentage with the data computation among these data and the table FF.In the time interval of each exposure, composition EE, AA and BB show almost 100% expeling.Even after 6 hours, described composition also has 100% expeling to mosquito.

Table EE

Table FF

Embodiment 32

Contain of the inspection of the composition of essence oil from plant for dispel effect and the insecticidal effect of Formica rufa

The survey method

The different components that contains essence oil from plant detects by following method for the insecticidal effect of Formica rufa.Every kind of detection composition with about 20 μ l is handled cardboard, puts one in each bottle of the cardboard after the processing.Untreated cardboard is placed in the control bottle.Handle cardboard with the 100%DEET of about 20 μ l again and be placed in the bottle with comparative composition and DEET a kind of insecticidal effect of known commodity insect control reagent.In each bottle, put into about three Formica rufas, seal bottleneck with cotton and prevent the insect escape.Insect is exposed to about hour of described composition or shorter time and writes down lethality.

The different components that contains essence oil from plant detects by following method for the dispel effect of Formica rufa.Handle cardboard and cardboard is placed in the dish with every kind of detection composition of about 200 μ l.Untreated cardboard is placed in the contrast dish.Handle cardboard with the 100%DEET of about 200 μ l again and be placed in the dish dispel effect with comparative composition and DEET.In each dish, put into Formica rufa.The number of times that behavior and the insect of insect stays on the cardboard of handling was monitored about 5 minutes.Write down the number of times that Formica rufa stays.

The detection of remaining insecticidal effect and dispel effect is to handle cardboard with detection composition, and a period of time that the cardboard of handling is kept being scheduled under laboratory condition (for example 0 minute, 6 hours, 1 day, 3 days, 5 days, 7 days), the cardboard that Formica rufa is exposed to handled with said method.

Embodiment 33

Contain dispel effect and the insecticidal effect of the composition of essence oil from plant for Formica rufa

With the insecticidal effect and the dispel effect of the method detection composition described in the embodiment 32, shown in table GG.Untreated dish did not both have toxicity not drive away Formica rufa to Formica rufa yet.

Composition	Composition (being expressed as the % of weight)
		Z	The 20%d-citrene, 10%lindenol, 10% eugenol, 10% phenylacetaldehyde, 50% geraniol
AA
		50% geraniol, 40% thyme linaloe oil, 10% lemongrass oil
BB
		50%LFO, 50% cymin oil
CC			The 20%d-citrene, 20% thyme linaloe oil, 20% geraniol, 20% α-sobrerone, 20%p-cymene
	DD
		10%DEET, 18%d-citrene, 18% thyme linaloe oil, 18% geraniol, 18% α-sobrerone, 18%p-cymene

EE

	10%DEET, 45%LFO, 45% cymin oil
		FF	44%LFO, 44% cymin oil, 10% geraniol, 2% thyme linaloe oil

Table GG

After crossing cardboard with described compositions-treated, when being exposed to cardboard about 0 minute, 6 hours, 1 day, 3 days, 5 days, or 7 days the time, each composition can both cause 100% lethality, and is suitable with DEET.

As show shown in the HH, Formica rufa is used to handle the composition of cardboard and drives away.In addition, aspect remaining ability, described composition is better than DEET, and they have kept at least one week being applied to cardboard rear-guard removing solid capacity, and DEET has just lost the expeling ability after 1 day.Table HH has shown the number of times that Formica rufa stays on the cardboard of handling.The time of listing in the table, 0 minute, 6 hours, 1 day, 3 days, 5 days, or 7 days, be meant with the described cardboard of described compositions-treated and Formica rufa be exposed to the time proximity of the process between the cardboard of handling.

Table HH

Embodiment 34

Contain dispel effect and the insecticidal effect of the composition of essence oil from plant to Formica rufa

With the insecticidal effect and the dispel effect of the method detection composition described in the embodiment 32, shown in table JJ.All have dispel effect and insecticidal effect with every kind of compositions-treated.

Composition	Composition (being expressed as the % of weight)
		GG	The 10%d-citrene, 30% thyme linaloe oil, 35% geraniol, 10% α-sobrerone, 10%p-cymene, 5% phenylacetaldehyde
HH	The 15%d-citrene, 50% geraniol, 15% α-sobrerone, 15%p-cymene, 5% phenylacetaldehyde
		JJ	The 50%d-citrene, the 50%p-cymene
KK	The 33.3%d-citrene, 33.3%p-cymene, 33.3% α-sobrerone
		LL	The 50%d-citrene, 50% thyme linaloe oil
MM
		50% thyme linaloe oil, 50% α-sobrerone
NN			33.3% thyme linaloe oil, 33.3% α-sobrerone, 33.3%p-cymene
	OO	The 50%-sobrerone, the 50%p-cymene
PP			25% linalool, 25% α-sobrerone, 25%p-cymene, 25% thyme linaloe oil
	QQ	33.3% linalool, 33.3% α-sobrerone, 33.3%p-cymene
RR			The 33.3%d-citrene, 33.3%p-cymene, 33.3% thymol
	SS	The 25%d-citrene, 25%p-cymene, 25% thymol, 25% geraniol

Table JJ

It will be appreciated by those skilled in the art that, can modifications and variations of the present invention are and can not deviate from scope and spirit of the present invention.Specification and embodiment only are examples, and are not to be to limit the scope of the invention and spirit.List of references and publication that this paper index is used all are incorporated herein by reference at this.

Should be appreciated that unless otherwise noted, specification, employed all expression compositions in embodiment and the claim, characteristic such as reaction condition, the numeral of the quantity that waits can change by introducing term " approximately " in all examples.Correspondingly, unless otherwise noted, specification, the digital parameters that occurs in embodiment and the claim all is an approximative value, can be according to the present invention determined desirable characteristics and changing.

Claims

1. composition of controlling invertebrate comprises: at least two kinds of oil, the fixed described invertebrate of wherein said composition target be selected from the tyrasamine acceptor, at least one acceptor of Or83b olfactory receptor and Or43a olfactory receptor can make cAMP in the born of the same parents of described insect, Ca2+, or the level of the two changes the collaborative invertebrate control action of described change generation, and wherein said at least two kinds of oil comprise two kinds or more kinds of oil, are selected from: t-anthole, black seed oil, amphene, carvacrol, d-carvol, the l-carvol, 1, the 8-cineole, the p-cymene, ethyl phthalate, eugenol, geraniol, Isopropyl citrate, lemongrass oil, flos caryophylli oil, limette oil, d-citrene, ortho-aminobenzoic acid agalloch eaglewood ester, linalool, lindenol, methylcitrate, MDJ, myrcene, perillyl alcohol, phenylacetaldehyde, α-sobrerone, β-sobrerone, piperonal, piperonyl, acetic acid pepper ester, piperitol, pepper amine, quinine, Chinese juniper is peaceful, α-terpinene, terpinene 900, alpha-terpineol, γ-terpineol, the 2-tert-butyl group-p-benzoquinones, α-absinthol, thyme linaloe oil, and thymol.

2. composition as claimed in claim 1 wherein uses described composition to handle and can obtain dispel effect.

3. composition as claimed in claim 1 wherein uses described composition to handle and can obtain insecticidal effect.

4. composition as claimed in claim 2 further comprises at least a fixed oil, and wherein said at least a fixed oil is selected from: castor oil, corn oil, cymin oil, mineral oil, olive oil, peanut oil, safflower oil, sesame oil, and soybean oil.

5. composition as claimed in claim 1, wherein selected grease separation is from flos caryophylli oil, geraniol and thyme linaloe oil.

6. composition as claimed in claim 2, wherein said grease separation is from flos caryophylli oil, geraniol, and thyme linaloe oil.

7. composition as claimed in claim 4, wherein selected grease separation is from flos caryophylli oil, geraniol, thyme linaloe oil, and cymin oil.

8. composition as claimed in claim 7, wherein selected grease separation be from about by weight 44% flos caryophylli oil, about by weight 10% geraniol, about by weight 2% thyme linaloe oil and about by weight 44% cymin oil.

9. composition as claimed in claim 1, wherein selected grease separation is from d-citrene, thyme linaloe oil, geraniol, α-sobrerone and p-cymene.

10. composition as claimed in claim 2, wherein selected grease separation is from d-citrene, thyme linaloe oil, geraniol, α-sobrerone and p-cymene.

11. composition as claimed in claim 9, wherein said grease separation is from about by weight 20% d-citrene, about by weight 20% thyme linaloe oil, about by weight 20% geraniol, about by weight 20% α-sobrerone and about by weight 20% p-cymene.

12. composition as claimed in claim 9 further comprises phenylacetaldehyde.

13. composition as claimed in claim 10 further comprises phenylacetaldehyde.

14. composition as claimed in claim 12, wherein selected grease separation is from about by weight 10% d-citrene, about by weight 30% thyme linaloe oil, about by weight 35% geraniol, α-sobrerone of about by weight 10%, 10%p-cymene and about by weight 5% phenylacetaldehyde.

15. composition as claimed in claim 1, wherein selected grease separation are certainly: geraniol, thyme linaloe oil, and lemongrass oil.

16. composition as claimed in claim 2, wherein selected grease separation are certainly: geraniol, thyme linaloe oil, and lemongrass oil.

17. composition as claimed in claim 15, wherein selected grease separation are certainly: about by weight 50% geraniol, about by weight 40% thyme linaloe oil, about by weight 10% lemongrass oil.

18. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene, lindenol, eugenol, phenylacetaldehyde, and geraniol.

19. composition as claimed in claim 2, wherein selected grease separation are certainly: d-citrene, lindenol, eugenol, phenylacetaldehyde, and geraniol.

20. composition as claimed in claim 19, wherein selected grease separation is from about by weight 20% d-citrene, about by weight 10% lind enol, about by weight 10% eugenol, about by weight 10% phenylacetaldehyde and about by weight 50% geraniol.

21. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene, geraniol, α-sobrerone, p-cymene, and phenylacetaldehyde.

22. composition as claimed in claim 2, wherein selected grease separation are certainly: d-citrene, geraniol, α-sobrerone, p-cymene, and phenylacetaldehyde.

23. composition as claimed in claim 21, wherein selected grease separation is from about by weight 15% d-citrene, about by weight 50% geraniol, α-sobrerone of about by weight 15%, about by weight 15% p-cymene and about by weight 5% phenylacetaldehyde.

24. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene and p-cymene.

25. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene, p-cymene, and α-sobrerone.

26. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene and α-sobrerone.

27. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene and thyme linaloe oil.

28. composition as claimed in claim 1, wherein selected grease separation are certainly: α-sobrerone and thyme linaloe oil.

29. composition as claimed in claim 1, wherein selected grease separation are certainly: p-cymene, α-sobrerone and thyme linaloe oil.

30. composition as claimed in claim 1, wherein selected grease separation are certainly: p-cymene and α-sobrerone.

31. composition as claimed in claim 1, wherein selected grease separation are certainly: linalool, p-cymene, thyme linaloe oil, and α-sobrerone.

32. composition as claimed in claim 1, wherein selected grease separation are certainly: linalool, p-cymene, and α-sobrerone.

33. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene, p-cymene, and thymol.

34. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene, p-cymene, thymol and geraniol.

35. composition as claimed in claim 1, wherein selected grease separation are certainly: α-absinthol, α-sobrerone, sabinene, β-sobrerone, p-cymene and citrene.

36. composition as claimed in claim 2, wherein selected grease separation are certainly: α-absinthol, α-sobrerone, sabinene, β-sobrerone, p-cymene and citrene.

37. composition as claimed in claim 1, wherein selected grease separation are certainly: ethyl phthalate, alpha-terpineol, piperonal, linalool, γ-terpineol, MDJ, methylcitrate and Isopropyl citrate.

38. composition as claimed in claim 2, wherein selected grease separation are certainly: ethyl phthalate, alpha-terpineol, piperonal, linalool, γ-terpineol, MDJ, methylcitrate and Isopropyl citrate.

39. composition as claimed in claim 1, wherein selected grease separation are certainly: d-citrene and p-cymene, α-sobrerone, thyme linaloe oil, linalool, thymol, and geraniol.

40. composition as claimed in claim 2, wherein selected grease separation are certainly: d-citrene and p-cymene, α-sobrerone, thyme linaloe oil, linalool, thymol, and geraniol.

41. composition as claimed in claim 1, wherein said composition comprise at least two kinds of oil that are selected from following substances: d-citrene, p-cymene, α-sobrerone, thyme linaloe oil, thymol, geraniol, lemongrass oil, black seed oil, flos caryophylli oil, mineral oil, and phenylacetaldehyde.

42. composition as claimed in claim 2, wherein said composition comprise at least two kinds of oil that are selected from following substances: d-citrene, p-cymene, α-sobrerone, thyme linaloe oil, thymol, geraniol, lemongrass oil, black seed oil, flos caryophylli oil, mineral oil, and phenylacetaldehyde.

43. a composition of controlling invertebrate comprises at least two kinds of oil that are selected from following substances: black seed oil, amphene, the d-carvol, 1-carvol, 1, the 8-cineole, Isopropyl citrate, lemongrass oil, flos caryophylli oil, limette oil, ortho-aminobenzoic acid agalloch eaglewood ester, methylcitrate, MDJ, myrcene, perillyl alcohol, phenylacetaldehyde, α-sobrerone, β-sobrerone, piperonyl, pepper amine, quinine, Chinese juniper is peaceful, α-terpinene, terpinene 900, γ-terpineol, the 2-tert-butyl group-p-benzoquinones, and α-absinthol.

44. composition as claimed in claim 43 further comprises at least a fixed oil, wherein said at least a fixed oil is selected from: castor oil, corn oil, cymin oil, mineral oil, olive oil, peanut oil, safflower oil, sesame oil, and soybean oil.

45. composition as claimed in claim 44, wherein selected grease separation are certainly: flos caryophylli oil and cymin oil.

46. a composition of controlling insect comprises:

At least a essence oil from plant; With

A kind of insect control reagent, the ultimate density of wherein said insect control reagent have the active required concentration of insect control when using separately less than described insect control reagent, and wherein said at least a essence oil from plant is selected from following oil: t-anthole, black seed oil, amphene, carvacrol, d-carvol, l-carvol, 1,8-cineole, p-cymene, ethyl phthalate, eugenol, geraniol, Isopropyl citrate, lemongrass oil, flos caryophylli oil (LFO), limette oil, d-citrene, ortho-aminobenzoic acid agalloch eaglewood ester, linalool, l indenol, methylcitrate, MDJ, myrcene, perillyl alcohol, phenylacetaldehyde, a-sobrerone, β-sobrerone, piperonal, piperonyl, acetic acid pepper ester, piperitol, pepper amine, quinine, Chinese juniper is peaceful, α-terpinene, terpinene 900, alpha-terpineol, γ-terpineol, the 2-tert-butyl group-p benzoquinones, α-absinthol, thyme linaloe oil, and thymol.

47. composition as claimed in claim 46, wherein said insect control reagent is DEET.

48. composition as claimed in claim 46, wherein said insect control reagent is DEET.

49. composition as claimed in claim 48, wherein the ultimate density of DEET is low at least to 10%.

50. composition as claimed in claim 49, wherein the ultimate density of DEET is low at least to 5%.

51. composition as claimed in claim 47 further comprises fixed oil, wherein said fixed oil is selected from: castor oil, corn oil, cymin oil, mineral oil, olive oil, peanut oil, safflower oil, sesame oil, and soybean oil.

52. composition as claimed in claim 51, wherein selected grease separation are certainly: flos caryophylli oil and cymin oil.

53. composition as claimed in claim 52, wherein selected grease separation is from: about by weight 45% flos caryophylli oil and about 45% cymin oil by weight.

54. composition as claimed in claim 47, wherein selected grease separation are certainly: d-citrene, thyme linaloe oil, geraniol, α-sobrerone and p-cymene.

55. composition as claimed in claim 54, wherein selected grease separation is from about by weight 18% d-citrene, about by weight 18% thyme linaloe oil, about by weight 18% geraniol, about by weight 18% α-sobrerone and about by weight 18% p-cymene.