CN1809368B - Compositions and methods for controlling insects - Google Patents

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

Compositions and methods for controlling insects

Cross References to Related Applications

This application claims priority to US Provisional Application Serial No. 60/465,320, filed April 24, 2003, and US Provisional Application Serial No. 60/532,503, filed December 24, 2003, which are hereby incorporated by reference in their entirety.

field of invention

The present invention relates to compositions, methods, cell lines and reporters related to the control of insects.

Background of the invention

Animals have chemosensory and mechanosensory systems, which can recognize a variety of environmental stimuli and produce behavioral responses. Behavioral studies have been performed to understand the genetics of these systems. The olfactory system plays an important role in the survival and life of some species, including insects.

Drosophila is a model organism for the study of sensory systems due to its ability to perform mutational analysis by molecular techniques, conduct behavioral and electrophysiological analysis, and its olfactory system is comparable to its mammalian counterpart.

The insecticidal activity of various chemicals or mixtures has been studied for several years with the aim of obtaining a compound that is selective for invertebrates such as insects but has little or no activity against vertebrates such as mammals, fish, poultry and other species. Products that are completely non-toxic and additionally do not remain in the environment nor damage the environment.

Most known and commercial products with sufficient insecticidal activity have toxic or deleterious effects on mammals, fish, poultry or other species that are not the target of the product. For example organophosphorus compounds and carbamates inhibit the activity of acetylcholinesterase in insects as well as in all kinds of animals. Dimeform and the related formamidines are known to act on octopamine receptors in insects, but have been withdrawn from the market due to their cardiotoxic potential in vertebrates, carcinogenicity in animals and variable action on different insects . Other compounds that are less toxic to mammals and other non-target species are often difficult to identify.

Summary of the invention

The present invention includes compositions for controlling insects and methods of using these compositions. The present invention includes insect control compositions comprising one or more botanical essential oils, and methods of using these compositions. The botanical essential oils have a synergistic effect when used in combination. The composition may include a fixed oil, which is a fixed, fragrance-free vegetable oil. Additionally, these compositions may consist of generally recognized as safe (GRAS) compounds.

The present invention includes compositions comprising one or more botanical essential oils and an insect control agent, and methods of using these compositions. Examples of insect control agents include DEET and D-pyrethrin. The plant essential oil and the insect control agent have a synergistic effect when used in combination. For example, an insect control effect of 29% DEET can be obtained with 5% DEET in the compositions of the present invention.

The present invention includes methods of screening compositions for insect control activity. The present invention includes cell lines stably transfected with tyramine receptor (TyrR), Or83b olfactory receptor (Or83b), or Or43a olfactory receptor, which can be used to screen compositions for insect control activity.

The present invention includes a method of generating a report identifying one or more compositions having insect control activity. The term "report" means a record or description that exists in a printed file, database, computer system or other medium.

For simplicity, the term "insect" will be used throughout this application; however, it should be understood that the term "insect" refers not only to insects, but also to arachnids, larvae, and similar invertebrates. Also for the purposes of this application, the term "insect control" means having a repellent effect, an insecticidal effect or both. By "repellent effect" is meant an effect wherein more insects are excluded from hosts and areas treated with the composition compared to control hosts or areas not treated with the composition. In some embodiments, a repellent effect refers to an effect wherein at least about 75% of the insects are excluded from the host and area treated with the composition. In some embodiments, a repellent effect refers to an effect wherein at least about 90% of the insects are repelled from the host and area treated with the composition. "Insecticidal effect" means an effect wherein treatment with the composition results in the death of at least about 1% of the insects. In this regard, compositions of LC1 to LC100 (lethal concentration) or LD1 to LD100 (lethal dose) can cause insecticidal effects.

In some embodiments, the pesticidal effect is one in which treatment with the composition results in the death of at least about 5% of exposed insects. In some embodiments, the pesticidal effect is one in which treatment with the composition results in the death of at least about 10% of exposed insects. In some embodiments, the insecticidal effect is one in which treatment with the composition results in the death of at least about 25% of the insects. In some embodiments the insecticidal effect is one in which treatment with the composition results in the death of at least about 50% of the exposed insects. In some embodiments the insecticidal effect is one in which treatment with the composition results in the death of at least about 75% of exposed insects. In some embodiments the insecticidal effect is one in which treatment with the composition results in the death of at least about 90% of exposed insects. In some embodiments of the invention, treatment with such concentrations or doses can knock down insects within seconds to minutes.

The compositions of the present invention may be used to control insects, either directly on the host, or in an area where the host is located, eg, indoors in a living space, outdoors in a yard or garden. For the purposes of this application, a host is defined as a plant, human or other animal.

Brief description of the drawings

Figure 1 shows the receptor-specific binding of Schneider cells transfected with tyramide receptors;

Figure 2 shows the ³ H-tyramine saturation binding curves of membranes prepared with Schneider cells expressing tyramine receptors after incubation with different concentrations of ³ H-tyramine in the presence or absence of unlabeled tyramine ;

Figure 3 shows the ³ H-tyramine inhibition binding curves of membranes prepared with Schneider cells expressing tyramine receptors after incubation with ³ H-tyramine in the presence or absence of different concentrations of unlabeled tyramine ;

Figure 4 shows membranes prepared with Schneider cells expressing tyramine receptors in the presence or absence of different concentrations of unlabeled ligands: tyramine (TA), octopamine (OA), dopamine (DA), and serum The ³ H-tyramine inhibition binding curve under the condition of amine (SE);

Figure 5 shows the membranes prepared from Schneider cells expressing tyramine receptors after incubation with ³ H-tyramine in the presence or absence of different concentrations of clove oil (LFO) and black seed oil (BSO). ³ H-tyramine inhibition binding curve;

Figure 6 shows that membranes prepared from Schneider cells expressing tyramine receptors react with ³ H-tyramine in the presence or absence of LFO or BSO or with different concentrations of unlabeled tyramine (TA). After incubation, ³ H-tyramine ( ³ H-TA) inhibits binding to the membrane;

Figure 7 shows the tyramine-dependent changes in cAMP levels in Schneider cells expressing tyramine receptors in the presence or absence of forskolin and tyramine;

Figure 8 shows the tyramine-dependent changes in cAMP levels in Schneider cells expressing tyramine receptors treated with clove oil and black seed oil in the presence or absence of forskolin and tyramine;

Figure 9 shows the tyramine-dependent changes in cAMP levels in Schneider cells expressing tyramine receptors treated with forskolin in the presence or absence of tyramine, clove oil and black seed oil;

Figure 10 shows the saturation binding curves of ³ H-tyramine to membranes prepared from Schneider cells expressing Or83b receptors;

Figure 11 shows the saturation binding curves of ³ H-tyramine to membranes prepared from Schneider cells expressing Or43a receptors;

Figure 12 shows the forskolin-dependent changes in cAMP levels in Schneider cells expressing Or83b receptors;

Figure 13 shows the ionomycin-dependent changes in intracellular Ca2 ⁺ levels in Schneider cells expressing Or83b receptors;

Figure 14 shows the change of intracellular Ca2 ⁺ level dependent on ionomycin in Schneider cells expressing Or43a receptor;

Figure 15 shows the tyramine-dependent changes in intracellular ^Ca levels in control Schneider cells, Schneider cells expressing Or83b receptors, and Schneider cells expressing Or43a receptors;

Figure 16 shows that various plant essential oils, including LFO, ^piperonal , diethyl phthalate, and α-terpineol with Or83b and Interaction between Or43a receptors;

Figure 17 shows that ^various plant essential oils, including BSO, quinine, sabinene, α-thujone, α-pinene, d - Limonene, and the interaction between p-thymelin and the Or43a receptor;

Figure 18 shows various plant essential oils, including BSO, ^quinine , sabinene, α-thujone, α-pinene, d - Limonene, and the interaction between p-thymelin and the Or83b receptor;

Figure 19 shows various plant essential oils, including geraniol, ^linalyl anthranilate, phenylacetaldehyde, linalool, α- Interactions between terpineol, t-anethole, terpinene 900, lindenol, and eugenol with Or83b and Or43a receptors;

Figure 20 shows the relationship between various plant essential oils, including thyme oil, carvacrol, and thymol, and Or83b and Or43a receptors in Schneider cells expressing olfactory receptors incubated with ³ H-tyramine. interaction;

Figure 21 shows the relationship between various plant essential oils, including piperonal, piperonyl alcohol, piperonyl acetate, and piperonylamine, and Or83b and Or43a receptors in Schneider cells expressing olfactory receptors after incubation with ³ H-tyramine interaction between

Figure 22 shows the effect of ionomycin, tyramine, and agarwood anthranilate on intracellular Ca ^levels in Schneider cells expressing the Or43a receptor;

Figure 23 shows the effect of linalool, perillyl alcohol, t-anethole, geraniol, phenylacetaldehyde and eugenol on intracellular ^Ca levels in Schneider cells expressing the Or43a receptor;

Figure 24 shows the effect of piperonyl alcohol, piperonyl acetate, and piperonylamine on ^{intracellular} Ca levels in Schneider cells expressing Or43a receptors;

Figure 25 shows the effect of α-terpineol, lindenol, and terpinene ⁹⁰⁰ on intracellular Ca levels in Schneider cells expressing Or43a receptors;

Figure 26 shows the effect of thyme oil, thymol, and carvacrol on intracellular ^Ca levels in Schneider cells expressing the Or43a receptor;

Figure 27 shows the effect of LFO on intracellular Ca ^levels in Schneider cells expressing Or43a receptors or Or83b receptors;

Figure 28 shows BSO, α-pinene, p-thymelin, d-limonene, sabinene, quinine, l-carvone, d-carvone in Schneider cells expressing Or43a receptor or Or83b receptor Effect of ketone, and α-thujone on intracellular Ca ²⁺ level;

Figure 29 shows the tyramine-dependent changes in cAMP levels in Schneider cells expressing Or83b receptors in the presence or absence of tyramine, LFO and BSO;

Figure 30 shows the tyramine-dependent changes in cAMP levels in Or83b receptor-expressing Schneider cells treated with LFO and BSO in the presence or absence of tyramine and forskolin;

Figures 31A and 31B show the nucleotide and peptide sequences of tyramine receptors;

Figures 32A and 32B show the nucleotide and peptide sequences of the Or43a olfactory receptor; and

Figures 33A and 33B show the nucleotide and peptide sequences of the Or83b olfactory receptor

Detailed description of the invention

The present invention relates to compositions, methods, cell lines and reporters related to the control of insects. The insect control is associated with one or more receptors, including the tyramine receptor (TyrR), the Or83b olfactory receptor (Or83b), and the Or43a olfactory receptor (Or43a).

The present invention includes methods of screening compositions for insect control activity. The invention includes Drosophila Schneider cell lines stably transfected with TyrR, Or43a, or Or83b, which can be used to screen compositions for insect control activity. The nucleic acid and peptide sequences of TyrR are shown in Figure 31A and Figure 31B. The nucleic acid and peptide sequences of Or43a are shown in Figure 32A and Figure 32B. The nucleic acid sequence and peptide sequence of Or83b are shown in Figure 33A and Figure 33B.

Possible insect control activity can be identified by detecting the affinity of the detection composition to each receptor in a cell line expressing TyrR, Or83b, and/or Or43a. Potential insect control activity can also be identified by detecting intracellular cAMP and/or Ca2 ⁺ in cell lines expressing TyrR, Or83b, and/or Or43a after treatment with the detection composition. There are similarities between the TyrR receptors of different insect species, the Or83b receptor and the Or43a receptor. Likewise, Drosophila Schneider cell lines expressing these receptors can be used to screen compositions for insect control activity against different insect species.

The present invention includes compositions for controlling insects and methods of using these compositions. The present invention includes insect control compositions comprising one or more botanical essential oils, and methods of using these compositions. Plant essential oils have a synergistic effect when used in combination. Compositions of the present invention may include any of the following oils, or mixtures thereof:

t-anthole Lime Oil Pepperbase

Black Seed Oil (BSO) d-Limonene Piperonyl Acetate

Camphene Anthranilate Agarwood Ester Piperonol

Carvacrol Linalool Piperonamine

d-carvone lindenol benzoquinone

l-Carvone Methyl Citrate Sabinene

1,8-cineole Methyl dihydrojasmonate α-Terpinene

p-Thymelin Myrcene Terpinene 900

Diethyl phthalate                                

Eugenol Phenylacetaldehyde γ-Terpineol

Geraniol α-pinene 2-tert-butyl-p-benzoquinone

Isopropyl Citrate β-Pinene α-Thujone

Lemongrass Oil Piperonal Thyme Oil

Clove Oil (LFO) Thymol

Compositions of the present invention may also include any of the following oils, or mixtures thereof:

Allyl Sulfur β-Elemene Menthyl Salicylate

Allyl Trisulfide γ-Elemene Myrtleenal

Allyl Disulfide Elmol Dimethyl Neral Acetate

Anethole Artemisinin Nerolidol

Artesinyl acetate 2-Ethyl-2-hexen-1-ol Nonanone

Benzyl Acetate Eugenol Acetate 1-Octanol

Benzyl alcohol α-farnesene E ocimenone

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

Bisabolene Oxide Fenzene Ocimene

α-Bisabolol Furandiene Eucalyptol-1, Octyl Acetate

Bisabolol Oxide 3-Diene Peppermint Oil

Oxidized bisabolol β eucalyptus-1,4-diene α-phellandrene

Bornyl acetate

β-Bourbongeranene 5)-Dien-6-one Piperonal

Alpha-Gininol Furan Sesquiterpenes Prenal

Camphene Geraniol Pulegone

α-Bornene Geraniyl Acetate Sabinene

α-Bornene Acetaldehyde Grandgerene D Hibinoyl Acetate

Camphor germanne B α-santalene

Eugenolene Oxide α-Guerene

Matriculene α-Humulene Sativen

Cinnamaldehyde α-Ionone δ-Ostene

cis verbenol β-ionone β-sesquphelandrene

Citral A Isoborneol Spartanol

Citral B

Citronellal Isomenthone α-Terpinene

Citronellol Isopulegone 4-Terpineol

Citronellol Acetate Jasmone α-Terpinolene

Citronellyl Formate

α-Cubacene Limonene α-Thujene

Wild Peppermint Oil Thymol Methyl Ether

α-Picolin Alcohol

Cryptonone

Curcumone Methallyl Trisulfide Transverbeenol

d-Carvone Menthol Verbenoenone

l-Carvone 2-Methoxyfurandiene Yomogi Alcohol

Artemisininone Menthone Gingerene

Diallyl Trisulfide Menthyl Acetate Dihydrotagentone

Dihydropyrocururone Methyl Cinnamate

In those compositions containing more than one oil, each oil comprises from about 1% to about 99% by weight of the composition mixture. For example, one composition of the present invention includes about 1% thymol and about 99% geraniol. Optionally, the composition may also contain a fixed oil, which is a fixed, fragrance-free vegetable oil. For example, the composition may include one or more of the following fixed oils:

Castor Oil Mineral Oil Safflower Oil

corn oil olive oil sesame oil

Cumin Oil Peanut Oil Soybean Oil

For example, one composition of the present invention includes about 1% thymol, about 50% geraniol and about 49% mineral oil. Additionally, these compositions may consist of generally recognized as safe (GRAS) compounds such as: thyme oil, geraniol, lemongrass oil, clove oil, black seed oil, lime oil, eugenol, castor oil, mineral oil, and safflower oil.

The present invention encompasses compositions comprising one or more botanical essential oils and an insect control agent, eg, DEET, and D-alletlirin, and methods of using these compositions. The plant essential oil and the insect control agent have a synergistic effect when used in combination. For example, an insect control effect of 29% DEET can be obtained with 5% DEET in the compositions of the present invention.

The composition of the present invention may contain two or more plant essential oil compounds and/or derivatives thereof, natural and/or synthetic, including racemic mixtures, enantiomers, diastereoisomers thereof , hydrates, salts, solvates and metabolites in admixture with a suitable carrier and optionally a suitable surfactant.

Suitable carriers may include any carrier known in the art suitable for plant essential oils, as long as the carrier has no adverse effect on the composition of the present invention. The term "carrier" as used herein means an inert or liquid substance, which may be inorganic or organic, synthetic or natural, which is mixed with the active compound or which is made suitable for use in a container or carton or other the form of an object, or a form suitable for storage, transport and/or handling. In general, any substance commonly used in the manufacture of repellents, insecticides, herbicides or fungicides is suitable. The compositions of the present invention may be used alone, or in combination with solid and/or liquid dispersion carriers and/or other known suitable active agents such as other repellents, insecticides, or acaricides, nematicides, fungicides Agents, bactericides, rodenticides, herbicides, fertilizers, growth regulators, and if necessary, can also be formulated into specific formulations for specific applications, such as solutions, emulsions, suspensions, powders, ointments, and granules to spare. The composition of the present invention can be mixed with traditional inert insecticide diluents or diluents used in traditional insect control agents, such as traditional dispersion vehicles such as gases, solutions, emulsions, suspensions, concentrated emulsions, spray powders, ointments , soluble powders, release agents, granules, foams, pastes, tablets, aerosols, natural and synthetic substances filled with active compounds, microcapsules, coating compositions for use on seeds, and preparations for combustion, such as fumigating boxes, Fumigation pots and pans, and ULV cold and warm mist formulations.

The compositions of the present invention may further comprise surface active agents. Examples of surface active agents, i.e. conventional carrier aids, which can be used in the present invention include emulsifying agents such as nonionic and/or anionic emulsifying agents (e.g. polyethylene oxide esters of fatty acids, polyethylene oxide ethers of fatty alcohols , alkyl sulfate, alkyl sulfonate, aryl sulfonate, albumin hydrolyzate, etc., especially alkyl aryl polyglycol ether, magnesium stearate, sodium oleate, etc.); and/or Dispersing agents such as lignin, sulfite waste liquor, methyl cellulose, etc.

The compositions of the present invention are useful for controlling insects, either directly in the treatment of a host, or in the area in which the host is located. For example. The host can be treated directly with a cream or spray, which can be applied topically or topically, eg to human skin. The composition, when applied to a host, such as a human, can be formulated into a variety of personal products or cosmetics for use on the skin or hair. For example, the following substances may be used: perfumes, colorants, pigments, dyes, colognes, skin creams, body lotions, deodorants, talcum powder, bath oils, soaps, shampoos, conditioners, and styling agents.

For animal, human or non-human hosts, direct therapy can also be achieved by oral delivery of the composition formulation. For example, the composition can be enclosed in a liquid capsule for swallowing.

An area may be treated with a composition of the invention, for example, by a spray, such as an aerosol or pump spray, or a burning agent, such as a candle or incense containing the composition. Of course, different treatments may be used without departing from the spirit and scope of the invention. For example, the composition may be contained in household products such as air fresheners (including "heated" air fresheners, in which the insect repellent is released by heating, such as electric heating, or combustion); hard surface cleaners; or laundry products (eg laundry detergents, conditioners containing compositions).

The invention is further illustrated by the following specific but non-limiting examples. Although values, results and/or data are included in the examples, the following examples are prophetic. Examples 1 to 5 relate to the preparation of cell lines capable of expressing the tyramine receptor (TyrR) and the screening of compositions using the cell lines. Examples 6 to 11 relate to the preparation of cell lines expressing the Or83b receptor, the preparation of cell lines expressing the Or43a receptor, and the screening of compositions using these cell lines. Examples 12 to 34 relate to determining the repellent and/or insecticidal effects of the compositions.

Example 1

Preparation of Schneider cell line stably transfected with tyramine receptor (TyrR)

A. PCR amplification and subcloning of Drosophila melanogaster tyramine receptors

Tyramine receptors were amplified from the head cDNA phage library GH of Drosophila melanogaster obtained from the Berkeley Drosophila Genome Project (Baumann, A., 1999, Drosophila melanogaster mRNA for octopamine receptor, splice variant 1B NCBI direct submission, AccessionAJ007617) . The nucleic acid and peptide sequences of TyrR are shown in Figures 31A and 31B. Phage DNA was purified from liquid culture lysates of this library. (Baxter, et al., 1999, Insect Biochem Mol Biol 29, 461-467). Used to amplify the open reading frame of the Drosophila tyramine receptor (TyrR) (Han, et al., 1998, J Neurosci 18, 3650-3658; von Nickisch-Rosenegk, et al., 1996. Insect BiochemMol Biol 26, 817-827) were the 5' oligo: 5' gccgaattc gc cacc ATGCCATCGGCAGATCAGATCCTG 3' and the 3' oligo: 5' taatctagaTCAATTCAGGCCCAGAAGTCGCTTG 3'. Capital letters indicate matches to tyramine receptors. The underlined nucleotides indicate the added Kozak sequence (Grosmaitre, X., Jacquin-Joly, E., 2001 Mamestra brassicae putative octopamine receptor (OAR) mRNA, complete cds. NCBI direct submission, Accession AF43878). The 5' oligonucleotide also includes an EcoR I site and the 3' oligonucleotide includes an Xba I site. Perform PCR with Vent polymerase (New England Biolabs) under the following conditions: about 95° C. for about 5 minutes for about 1 cycle; about 95° C. for about 30 seconds; about 70° C. for about 90 seconds for about 40 cycles; Approximately 70°C for approximately 1 cycle for approximately 10 minutes.

The PCR product was digested with EcoR I and Xba I, subcloned into pCDNA 3 (Invitrogen), and both strands were sequenced by automated DNA sequencing (Vanderbilt Cancer Center). The protein translated from this open reading frame correctly matches the published tyramine receptor sequence (Saudou, et al., The EMBO Journal vol 9 nol, 6-617). For expression in Drosophila Schneider cells, this TyrR ORF was cleaved from cDNA3 and inserted into pAC5.1/V5-His(B)[pAc5(B)] via Eco RI and Xba I restriction sites.

For transfection, Drosophila Schneider cells were stably transfected with pAc5(B)-TyrR ORF by the calcium phosphate-DNA co-precipitation method as described in the Invitrogen Drosophila Expression System (DES) manual. The co-precipitation method is the same for transient or stable transfection, except that an antibiotic-resistant plasmid is used for stable transfection. From these, at least ten clones of stably transfected cells were selected and propagated individually. Whole cell binding/uptake ^with3H -tyramine was used to select stable clones expressing the receptor. In this assay, cells were washed and collected in insect saline (170 mM NaCl, 6 mM KCl, 2 mM NaHCO ₃ , 17 mM glucose, 6 mM NaH ₂ PO ₄ , 2 mM CaCl ₂ , and 4 mM MgCl ₂ ). About 3 million cells in about 1 mL of insect saline were co-incubated with 4 nM ³ H-tyramide for about 5 minutes at 23°C. Centrifuge the cells for approximately 30 seconds and aspirate the binding solution. The cell pellet was washed with approximately 500 μL of insect saline, and the cells were resuspended and transferred to scintillation fluid. 50 [mu]M unlabeled tyramide was included in the reaction to determine non-specific binding. Binding was quantitatively counted for radioactivity using a liquid scintillation beta-counter (Beckman, Model LS1801).

B. Selection of the clone with the highest level of functionally active tyramine receptor protein

Tyramine receptor binding/uptake was performed to determine which of the transfected clones had the highest level of functionally active tyramine receptor protein. Tyramine receptor experiments were performed with about 10 vegetative lines and about 2 pAc(B) as controls. ³ H-Tyramine (approximately 4 nM/reaction) was used as tracer with or without 50 [mu]M unlabeled tyramine as specific competitor. In this assay, cells are cultured on plates and harvested in approximately 3 ml of medium for cell counting, adjusting the number of cells to approximately 3 x ¹⁰⁶ cells/ml. At the same time approximately two pAcB clones were used as controls. Approximately 1 ml of cell suspension was used in each reaction. In specific binding, about 3 clones expressed high levels of active tyramine receptor protein. Clones with the highest specific tyramine receptor binding were selected for further study. Selected clones were propagated and stored in liquid nitrogen. Aliquots of selected clones were cultured for whole-cell binding and plasma membranes were prepared for kinetic and screening studies. The control pAcB did not have any specific binding to tyramine receptors.

C. Schneider Cells Transfected with Tyramine Receptors for Screening Efficacy of Compositions Interacting with Tyramine Receptors

Cells transfected with tyramine receptors (approximately 1 x ¹⁰⁶ cells/ml) were cultured in each well of a multiwell plate. Approximately 24 hours after seeding the cells, the medium was removed and replaced with approximately 1 ml of insect saline (approximately 23°C). Various concentrations of ³ H-tyramide (approximately 0.1-10 nM) were added with or without 10 μM unlabeled tyramide and incubated at room temperature (RT). After approximately 20 minutes of incubation, the saline was quickly aspirated to terminate the reaction and washed at least once with approximately 2 ml of insect saline (approximately 23° C.). Cells were lysed in approximately 300 ml of 0.3 M NaOH for approximately 20 minutes at RT. The lysed cells were transferred to about 4 ml of Liquid Scintillation Solution (LSS), vortexed vigorously for about 30 seconds, and then counted radioactivity with a liquid scintillation β-counter (Beckman, Model LS1801) (LSC).

Referring to Figure 1, receptor specific binding data are expressed as fmol of specific binding per 1 x ¹⁰⁶ cells and were determined as a function ^of3H -tyramine concentration. The difference between the value in the absence of 10 μM unlabeled tyramine and the value in the presence of 10 μM unlabeled tyramine was the specific binding value. As shown in Figure 1, there is maximum specific binding at approximately 5 nM ³ H-tyramide. Untransfected cells were unresponsive even at concentrations of tyramide as high as approximately 100 μM.

In order to study the kinetics of tyramine receptors in cells stably transfected with pAcB-TyrR, crude extracts of membrane fractions were prepared from transfected cells and used to calculate the equilibrium dissociation constant (K _d ), the maximum binding capacity ( B _max ), equilibrium inhibitory dissociation constant (K _i ) and EC ₅₀ (effective concentration for 50% inhibition of binding). A preliminary study was performed to determine the optimal concentration of membrane protein with receptor binding activity. In this study, different concentrations of protein (approximately 10-50 μg/reaction) were incubated in approximately 1 ml of binding buffer (50 mM Tris, pH 7.4, 5 mM MgCl ₂ and 2 mM ascorbic acid). Reactions were initiated with the addition of approximately 5 nM ³ H-tyramine, with or without approximately 10 μM unlabeled tyramine. After approximately 1 hour of incubation at room temperature, the reaction was terminated by filtration through a GF/C filter (VWR) previously soaked in approximately 0.3% polyethyleneimine (PEI). Filters were washed once with approximately 4 ml of ice-cold Tris buffer and allowed to air dry prior to measurement of residual radioactivity by LSC. Binding data were analyzed by curve fitting (GraphPad software, Prism). The data demonstrate no difference between approximately 10, 20, 30 and 50 μg protein/reaction in specific binding to the tyramine receptor. Therefore, use approximately 10 μg protein/reaction.

To determine the _Bmax and _Kd values of WyrR-expressing membrane tyramine receptors (TyrR), saturation binding assays were performed. Briefly, approximately 10 μg of protein were incubated with various concentrations (approximately 0.2-20 nM) ^of3H -tyramine. Binding data were analyzed by curve fitting (GraphPad software, Prism) to determine the _Kd for binding of tyramine to its receptor.

To determine the affinity of several ligands for TyrR, several compounds were tested for their ability to inhibit binding of approximately 2 nM ³ H-tyramine at increasing concentrations. For saturation and inhibition assays, total and non-specific binding were determined in the absence and presence of approximately 10 [mu]M unlabeled tyramide, respectively. Receptor binding reactions were incubated for 1 hour at room temperature (RT) under light-limited conditions. Reactions were terminated by filtration through a GF/C filter (VWR) and air dried prior to measurement of residual radioactivity by LSC. Binding data were analyzed by curve fitting (GraphPad software, Prism).

Referring to FIG. 2 , there is shown the saturation binding curve of ³ H-tyramine ( ³ H-TA) to membranes prepared from Schneider cells expressing tyramine receptors, ³ H-tyramine to pAcB-TyrR stably transfected Tyramine receptors in cells have high affinity, the measured K _d is about 1.257nM, and the measured B _max is about 0.679pmol/mg protein.

Referring to Figure 3, which shows the relationship between ³ H-tyramine ( ³ H-TA) and membranes prepared from Schneider cells expressing tyramine receptors in the presence or absence of different concentrations of unlabeled tyramine (TA). The EC ₅₀ and K _i of tyramine for inhibiting binding of tyramine receptors in Schneider cells expressing tyramine receptors are approximately 0.331 μM and 0.127 μM, respectively.

To determine the pharmacological profile of the tyramine receptor (TyrR), a number of potential neurotransmitters were tested for their ability to replace membrane-bound ³ H-tyramine ( ³ H-TA) expressing the tyramine receptor. Referring to Figure 4, which shows the relationship between ³ H-tyramine and membranes prepared from Schneider cells expressing tyramine receptors in the presence or absence of different concentrations of unlabeled ligands (including tyramine (TA), octopamine (OA ), dopamine (DA), and seroamine (SE)) inhibited binding. Tyramine exhibits the highest affinity for Drosophila TyR ( _K1 is about 0.127 μM, _EC50 is about 0.305 μM). Octopamine, dopamine and seroamine were not as effective as tyramine at displacing ³ H-tyramine binding.

Referring to Table A, the K _i and EC ₅₀ of the ligands are listed, which are ranked in order of capacity: tyramine > octopamine > dopamine > serotonin, which indicates that it is likely that stably transfected Schneider cells express functionally active tyramine Amine receptors.

Ligand K _i (μM) _EC50 (μM) Tyramine (TA) Octopamine (OA) Dopamine (DA) Serumamine (SE) 0.1272.8685.7478.945 0.3057.45614.94023.260

Likewise, Schneider cells expressing tyramine receptors are useful as models for research and screening of compositions capable of interacting with tyramine receptors.

Example 2

Effects of Treatment of Cells Expressing Tyramine Receptors and Compositions on Intracellular [cAMP]

Cells were cultured on plates and the medium was changed the day before treatment. When the cells reached approximately 95% confluency, the medium was aspirated and washed with 5 ml insect saline (170 mM NaCl, 6.0 mM KCl, 2.0 mM NaHCO ₃ , 17.0 mM glucose, 6.0 mM NaH ₂ PO ₄ , 2.0 mM CaCl ₂ , 4.0 mM MgCl ₂ ; pH 7.0) to wash the cells once. Add approximately 20ml of insect saline, and gently scrape to collect the cells. An aliquot of cells was counted with a hemocytometer, and the cells were centrifuged at 1000 RPM for approximately 5 minutes. Resuspend the cells at 3 x ¹⁰⁶ cells/ml. IBMX was added to a concentration of about 200 µM. Then about 1ml of the cell suspension was aliquoted for processing. Forskolin (cAMP inducing reagent), tyramine or different candidate combinations are added and the cells are incubated at about 27°C for about 10 minutes.

Treated cells were centrifuged at approximately 13000 g for approximately 10 seconds. The solution was aspirated and about 1 ml of 70% ethanol at about -20°C was added. Spin down the cell pellet and store the sample at -20°C overnight. After extraction with ethanol, the cell debris was pelleted by centrifugation at about 13000g for about 5 minutes. Transfer the supernatant to a test tube and freeze dry with a spin dryer. The resulting extract was resuspended in approximately 100 μl TE for cAMP detection.

cAMP detection is based on the competitive binding of endogenous cAMP and ³ H-cAMP to cAMP-binding proteins. The assay was performed using ^the3H -cAMP Biotrak system (Amersham Biosciences) according to the manufacturer's instructions. Briefly, about 50 μl of cell extract was incubated with about 50 μl of ³ H-cAMP and about 100 μl of cAMP binding protein on ice for about 2-4 hours. Activated charcoal (approximately 100 μl) was then added and the solution was centrifuged at approximately 4°C for approximately 3 minutes. About 200 μl of the reaction mixture was taken and the level of ³ H-cAMP was determined by scintillation counting. The level of endogenous cAMP in the cells was calculated using a standard curve of approximately 0 to 16 pmol of cold cAMP per reaction.

Example 3

Effects of Treatment of Cells Expressing Tyramine Receptors and Compositions on Intracellular [Ca ²⁺ ]

Intracellular calcium ion concentrations ([Ca ²⁺ ]i) were determined using the fluorescent indicator furan-2 acetoxymethyl (AM) ester (Enan, et al., Biochem. Pharmacol vol 51, 447-454). In this study, cells expressing tyramine were cultured under standard conditions. Cell suspension was prepared with detection buffer (140 mM NaCl, 10 mM HEPES, 10 mM glucose, 5 mM KCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ ), and the cell number was adjusted to approximately 2×10 ⁶ cells per ml. Briefly, approximately 1.0 ml of the suspension (approximately 2×10 ⁶ cells) was incubated with 5 μM furan 2/AM at approximately 28° C. for approximately 30 minutes. After incubation, cells were pelleted by centrifugation at 3700 rpm for approximately 10 seconds at room temperature, and then resuspended in approximately 1.5 ml of assay buffer. Changes in [Ca ²⁺ ]i were analyzed spectrofluorometrically in the presence or absence of the tested compounds. Excitation wavelengths are approximately 340 nm (resulting from Ca ²⁺ -bound furan-2) and approximately 380 nm (corresponding to Ca ²⁺ -free furan-2). Monitor the fluorescence intensity at an emission wavelength of approximately 510 nm. No artificial fluorescence absorption was observed with any of the compounds. The ratio of approximately 340/380nm was calculated and plotted as a function of time.

Example 4

Clove Oil and Black Seed Oil for Tyramine Receptor Binding in Cells Expressing Tyramine Quantifiers

Active Effects

To determine whether specific oils, i.e., male clove oil (LFO) and black seed oil (BSO), could interact with tyramine receptors and modulate the functional expression of tyramine receptors, samples from stably transfected and untreated Membranes prepared from transfected Schneider cells were analyzed for ³ H-tyramide incorporation.

For binding ^to3H -tyramine at the receptor site, the binding protocol described above was used. Dose response experiments with LFO and BSO (approximately 1-100 μg/ml) were performed to determine their effect on the inhibitory binding of3H ^- tyramine to membranes prepared from Schneider cells expressing tyramine receptors. Referring to Figure 5, which shows the inhibitory binding of ³ H-tyramine with membranes prepared using Schneider cells expressing tyramine receptors in the presence or absence of various concentrations of LFO and BSO, demonstrating the Treatment resulted in the inhibition ^of3H -tyramine and its receptors in a dose-dependent manner. The EC ₅₀ values of LFO and BSO were close to 10 μg/ml and 20 μg/ml, respectively.

Turning now to Figure 6, which shows ³ H-tyramine with membranes prepared from Schneider cells expressing tyramine receptors in the presence or absence of LFO or BSO or together with approximately 1 and 10 μM unlabeled tyramine Inhibition of binding under , LFO (approximately 25 μg/ml) alone can inhibit the binding of ³ H-tyramine to its receptor. This effect was inconclusive for approximately 10 [mu]M (approximately 1.74 [mu]g/ml) of unlabeled tyramine. In addition, LFO enhanced the ability of unlabeled tyramine to inhibit ³ H-tyramine binding only when the unlabeled tyramine was used at a concentration of about 1 μM. On the other hand, BSO (approximately 25 [mu]g/ml) was less capable of inhibiting ³ H-tyramine binding than LFO. But regardless of the concentration of unlabeled tyramine, BSO can significantly increase the ability of unlabeled tyramine to inhibit ³ H-tyramine binding. These two oils had no effect on ³ H-tyramine binding in untransfected Schneider cells.

Likewise, LFO and BSO interact differently with tyramine receptors. Without wishing to be bound by any theory or mechanism, it is likely that LFO and tyramine compete for the same binding site, whereas BSO acts at a different receptor site than the endogenous ligand (tyramine). Certain other oils, including those specifically listed in this application, can also interact with tyramine receptors.

Example 5

Effects of clove oil and black seed oil on intracellular [cAMP] in cells expressing tyramine receptors

Effect

To study detection chemical-dependent coupling of tyramine receptors, pAcB-TyrR was stably expressed in Schneider cells. Transfected and untransfected cells were treated with tyramide (approximately 10 μM), LFO (approximately 25 μg/ml) and BSO (approximately 25 μg/ml) in the presence or absence of forskolin (FK) (approximately 10 μM). cAMP production was measured using ^the3H -cAMP detection kit (Amersham) as described above.

To ensure that the cAMP cascade is functionally active in this cell model, forslcolin, a cAMP inducer, was used as a standard reagent. As shown in Figures 7 to 9, which show the presence or absence of tyramine (about 10 μM) and forskolin (about 10 μM) after treatment with LFO (about 25 μg/ml) and BSO (about 25 μg/ml) Tyramine-dependent changes in cAMP levels in Schneider cells expressing tyramine receptors, increased only in transfected cells after treatment with forskolin compared to baseline levels of cAMP in cells treated with solvent (ethanol) alone up about 19 times.

On the other hand, tyramine slightly decreased cAMP production. However, tyramine significantly antagonized forskolin-stimulated cAMP levels in tyramine-expressing cells, suggesting that tyramine receptors couple to Gαi _/o in the presence of tyramine, as shown in Figure 7. Only in the transfected cells was the cAMP level reduced by approximately 34% and 25% after treatment with LFO and BSO, respectively (Fig. 8). While tyramine enhanced the effect of LFO on cAMP production in tyramine receptor transfected cells, there was no change in cAMP levels when BSO and tyramine were treated together except for the effect of BSO itself, as shown in Figure 8. The LFO- and BSO-induced decrease in cAMP levels in Schneider cells expressing tyramine receptors was reduced in the presence of forskolin, as shown in FIG. 9 .

Treatment with other specified botanical essential oils, including those specifically listed in this application, can result in changes in intracellular cAMP levels in cells expressing tyramine receptors.

Example 6

Preparation of Schneider cells stably transfected with olfactory receptors (Or83b and Or43a)

A. RT-PCR amplification and subcloning of olfactory receptors Or83b and Or43a in Drosophila melanogaster

Total RNA was extracted from the head and antennae of wild-type Drosophila melanogaster using Trizol reagent (Invitrogen). This was homogenized together with Trizol in a motorized Teflon pestle and glass homogenizer. RNA was then extracted according to the manufacturer's instructions, including removal of proteoglycans and polysaccharides by precipitation. Total RNA was reverse transcribed with MuLV reverse transcriptase (Applied Biosystems) using oligo-dT as a primer. PCR amplification of open reading frames was performed using the following oligonucleotides: Or83b sense orientation: 5′ taa gcggcc gc ATGACAACCTCGATGCAGCCGAG 3′; Or83b antisense orientation 5′ataccgcggCTTGAGCTGCACCAGCACCATAAAG 3′; Or43a sense orientation 5′taa gcggccgc ATGACAATCGAGGATATCGGCCTGG 3 '; and Or43a antisense orientation 5' ata ccgcgg TTTGCCGGTGACGCCACGCAGCATGG 3'. Capital letters indicate an exact match to the Or83b and Or43a receptor sequences. Oligonucleotides in sense orientation include Not I sites, and oligonucleotides in antisense orientation include SacII sites. Two restriction sites are indicated by underlined nucleotides. The oligonucleotide in the antisense orientation does not include a stop codon, so the V5 phenotype of the pAC 5.1 plasmid is linked to the translated protein. For PCR amplification of Or83b, Vent polymerase (New England Biolabs) was used and the reaction conditions were as follows: about 95°C for about 5 minutes for about 1 cycle; about 95°C for about 30 seconds; about 70°C for about 90 seconds About 40 cycles; about 70°C, about 10 minutes for about 1 cycle. For PCR amplification of Or43a, use the Failsafe PCR Premix Selection Kit (Epicentre Technologies), and the reaction conditions are as follows: about 95°C, about 5 minutes for about 1 cycle; about 95°C, about 30 seconds; about 60°C, about 30 seconds and about 70°C, about 90 seconds for about 40 cycles; about 70°C for about 10 minutes for about 1 cycle. Failsafe Master Mix Buffer F yields the correct size product. The PCR product was digested with Sac II and Not I, gel purified and ligated into pAC 5.1/V5 His B (Invitrogen). Inserts on both strands were sequenced using an automated fluorescent sequencer (Vanderbilt Cancer Center). The proteins encoded by the OR83b ORF and Or43a ORF are identical to the sequence information found on PubMed and in the genome sequence on the website. The nucleotide and peptide sequences of Or43a are shown in Figures 32A and 32B. The nucleotide and peptide sequences of Or83b are shown in Figures 33A and 33B.

Transfection was performed using the calcium phosphate-DNA co-precipitation method, and Drosophila Schneider cells were stably transfected with pAc5(B)-Or83b ORF or pAc5(B)-Or43a ORF as described in the Invitrogen Drosophila Expression System (DES) manual above. At least about ten clones of Or83b or Or43a stably transfected cells were picked and cultured separately. Stable clones were analyzed by RT-PCR to check whether they expressed the corresponding mRNA. RNA was extracted from cells using Trizol according to the manufacturer's instructions. Total RNA was reverse transcribed with MuLV reverse transcriptase. PCR was performed with Vent polymerase and the following primers: Or83b sense orientation primer and Or83b antisense orientation primer; Or43a sense orientation primer and Or43a antisense orientation primer. PCR products were analyzed by agarose gel electrophoresis and compared to control Schneider cell RNA used for RT-PCR. A clone highly expressing Or83b-mRNA or Or43a-mRNA was used for further studies to study protein expression (Western blot), and signaling (cAMP production and [Ca2+]) after treatment with tyramide and specific plant essential oils.

RT-PCR was used to determine which clones expressed the Or83b and Or43a genes. Agarose gel analysis indicated that for Or83b, about 4 out of about 10 clones produced a product of the correct size of about 1.46 kb. For Or43a, approximately 2 clones produced approximately 1.1 kb of the correct size product. These products were not obtained when PCR was performed on control Schneider cells. Clones expressing the mRNA were selected for additional studies on the receptor.

B. The ability of Schneider cell lines transfected with Or83b receptors or Or43a receptors to screen compositions that interact with Or83b and Or43a receptors

In order to investigate whether the Or83b receptor and the Or43a receptor contain specific binding sites for tyramine, the membranes expressing the Or83b receptor or the Or43a receptor were prepared from cells expressing the two receptors as described above, and used for binding with ³ H - Tyramine competitively binds. Binding assays were performed exactly as described above for TyrR-expressing cells. As shown in Figure 10, which shows the saturation inhibition curves of ³ H-tyramine and membranes prepared from Schneider cells expressing the Or83b receptor in the presence or absence of approximately 20 μM unlabeled tyramine, and Figure 11 The same experimental results are shown in cells expressing Or43a receptors, ³ H-tyramine specifically binds to Or83b and Or43a receptors. As shown in Table B, the Kd for binding of tyramine to the Or83b receptor is approximately 96.90 nM and the _Bmax is approximately 4.908 pmol/mg protein. For Or43a, the corresponding values are a Kd of about 13.530 nM and a _Bmax of about 1.122 pmol/mg protein.

Receptor type K _i (nM) B _max (pmol/mg protein) TyrROr83bOr43a 1.25796.90013.530 0.6794.9081.122

Form B

Example 7

  cAMP Production in Cells Expressing Olfactory Receptors

To ensure that the cAMP cascade was functionally active in this cell model, forskolin, a cAMP inducer, was used as a standard reagent. Cyclic AMP levels were determined using the cAMP assay described in Example 2 above. As shown in Figure 12, which shows a forskolin-dependent change in cAMP levels in cells expressing the Or83b receptor, there was an approximately 13-fold increase over baseline cAMP levels in cells treated with approximately 10 μM forskolin at room temperature for approximately 10 minutes. Similar results were obtained for cells expressing the Or43a receptor. Likewise, cells expressing olfactory receptors have functionally active cAMP cascades.

Example 8

Intracellular fluctuations of Ca2+ in cells expressing olfactory receptors

Intracellular Ca2 ⁺ levels were determined using the method described in Example 3 above. Calcium fluctuations following treatment with ionomycin (a Ca ²⁺ -inducing agent) and tyramide in cells expressing Or83b or Or43a receptors. In particular, referring to Figures 13 and 14, when approximately 2 μM ionomycin was added to cells expressing Or83b or Or43a, the ratio of fluorescence excited at 340 nm and 380 nm correlated with intracellular calcium levels, and a significant increase in intracellular calcium was observed. In cells expressing Or83b and Or43a, calcium was increased approximately 3.8-fold and 7-fold after treatment with ionomycin, respectively. Referring to FIG. 15 , about 10 μM of tyramine also induced about 1.18-fold and 3.5-fold increases in intracellular calcium in cells expressing Or83b and Or43a, respectively.

Overall, the pharmacological analysis data demonstrate that these cell models transfected with the Or83b receptor gene or the Or43a receptor gene can express functional protein receptors.

Example 9

Binding activity of different plant essential oils to olfactory receptors and receptor downstream signaling

The role of signal pathways

Cells expressing an olfactory receptor were used to study the interactions between plant essential oils and these receptors and the signaling cascades downstream of each receptor.

For binding activity, membranes prepared from each cell model were used to study the interaction between plant essential oils and receptor binding sites. Referring to Figure 16, the following oils interacted with olfactory receptors: clove oil (LFO), diethyl phthalate, alpha-terpineol, and piperonal.

Likewise, referring to Figures 17 and 18, the following oils interact with olfactory receptors: black seed oil (BSO), α-pinene, benzoquinone, p-thymelin, sabinene, α-thujone and d- Limonene.

Similarly, referring to Figures 19 to 21, the following oils are also capable of interacting with olfactory receptors: geraniol, linalyl anthranilate, phenylacetaldehyde, linalool, alpha-terpineol, t-anethole, Terpinene 900, lindenol, eugenol, thyme oil, carvacrol, thymol, piperonal, piperonyl alcohol, piperonyl acetate, and piperonylamine.

Certain other oils, including those specifically identified in this application, can also interact with olfactory receptors.

Example 10

Effects of different plant essential oils on intracellular Ca2+ in cells expressing Or43a receptors

The role of volatility

To determine the effect of different botanical essential oils on intracellular calcium fluctuations, intact cells from each cell model were assayed, as described above. Changes in intracellular Ca2 ⁺ levels were calculated from the difference in the 340/380 fluorescence ratio before and approximately 150 seconds after treatment. As shown in Figure 22, treatment with ionomycin and tyramide induced Ca2 ⁺ fluctuations in control cells but only marginally increased intracellular Ca2 ⁺ levels in cells expressing the Or43a receptor.

Referring to Figures 22 to 28, the following oils are capable of causing calcium fluctuations in cells expressing the Or43a receptor: linalyl anthranilate, linalool, perillyl alcohol, t-anethole, geraniol, phenylacetaldehyde, Eugenol, piperonol, piperonyl acetate, piperonylamine, α-terpineol, lindenol, terpinene 900, thyme oil, thymol, carvacrol, LFO, BSO, α-pinene, p-thymol , d-limonene, sabinene, quinine, l-carvone, d-carvone, and alpha-thujone. Finally, as shown in Figure 24, treatment with piperonal reduced intracellular Ca2 ⁺ levels in cells expressing the Or43a receptor.

Treatment with certain other plant essential oils, including those specified in this application, can also induce changes in intracellular Ca2 ⁺ levels in cells expressing the Or43a receptor.

In addition, treatment with certain other plant essential oils, including those specified in this application, can induce changes in intracellular Ca2 ⁺ levels in cells expressing the Or83b receptor.

Example 11

Effects of different plant essential oils on cAMP production in cells expressing olfactory receptors

Function

To determine the effect of different plant essential oils on intracellular cAMP production, as well as the tyramine-dependent changes in cAMP in cells expressing an olfactory receptor, cells from each cell model were treated with LFO (approximately 50 μg/ml) and BSO (approximately 50 μg/ml) was treated in the presence or absence of tyramine (approximately 20 μM) and forskolin (approximately 10 μM), and intracellular cAMP was determined using the assay described in Example 2.

As shown in Figures 29 and 30, treatment with the following oils resulted in increased cAMP levels in cells expressing the Or43a receptor: tyramine; LFO; BSO; LFO and tyramine; BSO and tyramine; forskolin; tyramide and forskolin ; LFO and forskolin; LFO, forskolin and tyramide; BSO; and BSO, tyramide and forskolin.

Still referring to Figures 29 and 30, cAMP production in cells expressing the Or83b receptor increased by about 34%, 32% and 64%, respectively, after treatment with about 20 μM tyramine, about 50 μg LFO/ml and about 50 μg BSO/ml. Treatment with tyramine and LFO together had an antagonistic effect on cAMP production (approximately 24%) compared to the effect of each alone. On the other hand, treatment with BSO and tyramine together had a synergistic effect (approximately 300% increase) on cAMP production.

In the presence of forskolin (approximately 10 [mu]M), cAMP production was increased approximately 3000-fold. When tyramine or LFO was given to cells pretreated with forskolin, cAMP production was only increased by about 10-13%, in addition to the effect of forskolin itself. Addition of BSO to cells pretreated with forskolin increased cAMP levels by only about 22% in these cells, in addition to forskolin-induced cAMP production.

Also, treatment with certain other plant essential oils, including those specifically listed in this application, resulted in changes in the amount of intracellular cAMP in cells expressing Or43a or Or83b receptors.

Example 12

Toxicity of the composition to Drosophila melanogaster

Two acetone solutions (approximately 1% and 10%) of the composition were prepared. Add the acetone solution of the detected concentration to the glass bottle (about 5ml), and mark it about 3cm from the bottom of the bottle. Rotate the glass bottle so that a film of the detection solution remains on the inner surface of the glass bottle except for the area between the mark and the bottle neck. All vials were air-dried for 10 seconds to ensure complete evaporation of the acetone, and then the flies were placed in the treated vials. After the acetone had completely evaporated, about 10 adult mixed-sex flies were added to each bottle, and the bottles were stopped with cotton plugs. Death was observed after approximately 24 hours.

Example 13

Male clove oil (LFO) and black seed oil (BSO) for wild-type Drosophila and tyramine receptors

Toxicity of mutant Drosophila

The toxicity of LFO and BSO can be determined using wild-type Drosophila melanogaster and tyramine receptor mutant flies as models. The toxicity of these oils was studied by the method described in Example 12 above. Referring to Tables C and D below, both chemicals were toxic to wild type Drosophila. LFO is about 300 times more toxic to Drosophila than BSO. The LC ₅₀ of LFO is about 25-30 ng/mm ² , the corresponding value of BSO is about 94 μg/cm ² . On the other hand, LFO was approximately 1000-fold less toxic than BSO to tyramine receptor mutant flies. Toxicity of two chemicals to Drosophila can be mediated through tyramine receptors. Mutations in tyramine receptors significantly reduced the toxicity of LFO to flies, while the same mutant flies became more susceptible to BSO.

Form C

Form D

Example 14

The effect of the composition on repelling field ants

Adult insects were arbitrarily selected for testing the repelling effect of the composition, and the insects were not individually marked. Approximately 5 insects were used for each replication. Each treatment was replicated approximately 3 times. Untreated control assays were performed under the same conditions but using only solvent (acetone) for equal populations/replicates. Filter paper (approximately 80 cm ² ) was treated with the composition (approximately 100 mg in 300 ml acetone). After about 3 minutes of air drying, the filter paper was placed on the plate to repel insects. Place the insects on the dish, one insect at a time at the far end of the dish. The time spent on the untreated surface of each filter paper or dish was recorded up to approximately 300 seconds with one or more stopwatches. Calculate the repellency rate (RR) according to the following formula:

RR = [(time spent on control surface - time spent on treated surface)/total time tested]. The composition is considered repellent if RR > 0, that is to say more insects are repelled on the treated surface than the control surface; if RR < 0 the composition is considered not repellent .

Example 15

The repelling effect of clove oil (LFO) and black seed oil (BSO) on field ants

The repelling effects of LFO (about 1.4 mg/cm ² ) and BSO (about 1.4 mg/cm ² ) on field ants were investigated by the method described in Example 14 above. As shown in Tables E and F, BSO proved to be more repellent than LFO for field ants. The repellency of LFO and BSO to field ants was 90% and 100%, respectively. In addition, LFO and BSO can also cause 100% mortality of field ants within 24 hours.

Form E

Form F

The residual effect of BSO on repelling ants was studied using BSO-treated discs. Use five ants per day according to the above expelling method. The time course of BSO toxicity was also determined. In toxicity experiments, ants were exposed to the same treated surface for approximately 10 seconds, then transferred to fresh containers, and mortality data were recorded approximately 24 hours after exposure. Use five ants per day. As shown in Table G, BSO can repel ants for up to 4 days. Time after surface treatment, days Repellency% Day 1 100 day 2 100 3rd day 100 day 4 100

Form G

Example 16

d-limonene, α-pinene, and p-thymotin used alone or in combination for repelling field ants

removal effect

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

Referring to Table 16H, d-limonene, α-pinene, and p-thymotin each have repellency individually. Whereas mixing the oils into Composition A, a composition comprising approximately one-third each of d-limonene, alpha-pinene and p-thymelin, had a synergistic effect with a greatly increased percentage repellency.

Table H

Also, referring to Table I, d-limonene and α-pinene each have repellency alone. However, mixing these oils into Composition B, a composition containing half of d-limonene and half of alpha-pinene, had a synergistic effect, with a greatly increased percentage repellency.

Table I

Example 17

Linalool, d-limonene, alpha-pinene, p-thymelin and thyme oil alone or in combination

                                                                                     

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

Referring to Table J, while d-limonene, α-pinene, p-thymelin and thyme oil each have repellent properties individually, Composition C, a composition comprising approximately 25% of each oil, had repellent properties of Exceeds the effect of either component oil alone.

Table J

Likewise, as shown in Table K, while linalool, alpha-pinene, p-thymelin and thyme oil are each individually repellent, composition D, a composition comprising approximately 25% of each oil , the repellency exceeds the effect of any one component oil used alone.

Form K

Similarly, as shown in Table L, while linalool, α-pinene, and p-thymetin are each individually repellent, Composition E, a composition comprising approximately one-third of each oil , the repellency exceeds the effect of any one component oil used alone.

Table L

Example 18

α-pinene, thyme oil, α-thujone, sabinene alone or in combination for field ants

                              

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

While α-pinene, thyme oil, α-thujone and sabinene are each individually repellent, as shown in Table M, Composition F, a composition comprising approximately 25% of each oil, Enhanced repellency.

Table M

Example 19

d-limonene, p-thymelin, thymol, carvacrol and geraniol alone or in combination

              The effect of repelling field ants

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Table N, while d-limonene, p-thymelin, thymol and carvacrol are each individually repellent, Composition G, a composition comprising about 25% of each oil, had Sexuality exceeds the effect of any one component oil used alone.

Table N

Similarly, as shown in Table O, while d-limonene, p-thymolin and thymol are each individually repellent, Composition H, a composition comprising approximately one-third of each oil, The repellency exceeds the effect of any one component oil used alone.

Table O

Similarly, as shown in Table P, while d-limonene, p-thymol, thymol and geraniol are each individually repellent, Composition I, a composition comprising approximately 25% of each oil , the repellency exceeds the effect of any one component oil used alone.

Table P

Example 20

Agarwood anthranilate, alpha-pinene, d-limonene, p-thymelin, and geranium

The repelling effect of alcohol alone or in combination on field ants

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Table Q, while geraniol, d-limonene, p-thymelin and linalyl anthranilate were each individually repellent, composition J, a composition containing about 40% geraniol, had about 30 The combination of % d-limonene, about 10% p-thymelin, about 10% alpha-pinene, and about 10% agarwood anthranilate repelled more than either of the component oils alone.

Table O

Example 21

d-limonene, thymol, alpha-terpineol, piperonyl acetate, piperonylamine, and piperonal

  The effect of repelling field ants alone or in combination

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Table R, while d-limonene, thymol, α-terpineol, piperonyl acetate, piperonylamine and piperonal were each individually repellent, composition K, a composition containing approximately 20% d- A combination of limonene, about 30% thymol, about 20% α-terpineol, about 10% piperonyl acetate, about 10% piperonylamine and about 10% piperonal, repells more than any of the component oils effect when used alone.

Table R

Example 22

Geraniol, d-limonene, eugenol, lindenol, and phenylacetaldehyde alone or in combination

                                                          

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Table S, while geraniol, d-limonene, eugenol, lindenol, and phenylacetaldehyde were each individually repellent, composition L, one containing about 50% geraniol, about 20% d - Compositions of limonene, about 10% eugenol, about 10% lindenol, and about 10% phenylacetaldehyde, repellant more than either component oil alone.

Form S

Example 23

Effects of geraniol, lemongrass oil, eugenol and mineral oil alone or in combination on repelling field ants

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Table T, while geraniol, lemongrass oil, and eugenol are each repellent, composition M, a composition containing about 50% geraniol, about 40% lemongrass oil, and about 10% clove The repellency of the combination of phenols exceeds the effect of any one component oil used alone. Geraniol, lemongrass oil, and eugenol are all generally recognized as safe (GRAS compounds) by the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA) and have received an exemption from EPA's pesticide registration.

Table T

Likewise, as shown in Table U, while geraniol and lemongrass oil were each repellent, composition N, a composition containing about 70% geraniol and about 30% lemongrass oil, was more repellent than The effect of using any one of the component oils alone.

Table U

Furthermore, as shown in Table V, the addition of mineral oil to form Composition O, a composition comprising about 60% geraniol, about 30% lemongrass oil, and about 10% mineral oil, did not affect the geraniol and lemongrass oils. Synergy of herbal oils. Mineral oil alone is not repellent, but it stabilizes the composition, limiting the evaporation of the active ingredient. Mineral oil is a GRAS compound along with geraniol and lemongrass oil.

Table V

Example 24

Geraniol, thymol, lemongrass oil and mineral oil alone or in combination for field ants

                            

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Table W, while geraniol, thymol and lemongrass oil are each repellent, composition P, one containing about 50% geraniol, about 20% thymol, about 20% lemon Combinations of grass oil, and approximately 10% mineral oil, repelled more than either component oil alone. Geraniol, thymol, lemongrass oil, eugenol, and mineral oil are all generally recognized as safe (GRAS compounds) by the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA) and have received EPA approval for insecticidal agent registration exemption.

Table W

Example 25

Black Seed Oil (BSO), Clove Oil (LFO), Geraniol, Thymol, Lemongrass Oil

The effect of repelling carpenter ants with mineral oil alone or in combination

The filter paper was treated with the tested oil to test the repelling effect of different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Table X, geraniol, thymol and thyme oil each had repellent properties. As shown in Table Y, compositions Q to V, containing different combinations of BSO, LFO, geraniol, thymol, thyme oil, mineral oil, safflower oil and castor oil, all showed enhanced repellency.

Table X

Table Y

Example 26

 The effect of the commercial repellant 29% DEET on carpenter ants

In order to compare the repelling effect of different compounds composed of different plant essential oils, an insect control agent was tested, commercial repellant 29% DEET, trade name REPEL_ (Wisconsin Pharmacal Company, Inc, Jackson, WY), for wood Ant repellency by treating filter paper with 29% DEET. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. As shown in Table Z, the percent repellency of 29% DEET at day 0 was approximately 98.4%. The percentage repellence of LFO, BSO and the composition of the invention was comparable and sometimes higher than that of 29% DEET.

Table Z

Example 27

The commercial repellent DEET, alone or with geraniol, thymol, and lemongrass oil

or in combination with geraniol, D-limonene, eugenol, LINDENOL, and phenylacetaldehyde

          Effective against carpenter ants

The filter paper was treated with the tested oil to test the repelling effect of the commercial repellant DEET and different plant essential oils. After standing at room temperature for 5 minutes, put the filter paper in the plate and put the ants in at one time. Repellency was determined as described in Example 14 above. Each oil is tested individually. In addition, various oils were mixed into compositions and tested.

As shown in Tables AA and BB, treatment with DEET at a concentration of about 5-10% showed no signs of repellency. However, as shown in Table AA, when combined with composition W, a composition containing about 25% geraniol, 10% thymol, 10% lemongrass oil and mineral oil (45% to 55%, depending on the final concentration of DEET Concentration) when used in combination, the repellency is close to 100. Likewise, as shown in Table BB, when combined with composition X, a composition containing about 25% geraniol, 10% d-limonene, 5% eugenol, 5% lindenol, 5% phenylacetaldehyde and mineral oil (45% % to 55%, depending on the final concentration of DEET), when the composition was used in combination, the repellency was close to 97% to 98%. Also, as shown in Figures AA and BB, the repellency of the various oils increased when they were used in combination with DEET.

Form AA

Table BB

Example 28

Insecticidal effect of the composition on head lice

Live adult head lice Pediculus humanus were collected from girls and boys aged 4 to 11 years living in the Karmos region of Alexandria, Egypt. The insects were collected with a fine-toothed lice comb and clustered together. Collected lice were placed in trays and used for this study within approximately 30 minutes of collection.

Various concentrations of the composition for testing were prepared in water. In order to compare the insecticidal efficacy of these compositions with that of commercially available pediculicidal agents, ivermectin was dissolved in water. About 1 ml of each concentration of the composition was applied on the disc, about 1 ml of ivermectin in water, and about 1 ml of water on the control disc. Place approximately 10 adult head lice in each tray.

Continue to observe the treatment and control discs and observe the LT ₁₀₀ . LT refers to the time required to kill a given percentage of insects; thus LT ₁₀₀ refers to the time required to kill 100% of the lice. Head lice are considered dead if they do not respond to hard objects.

Example 29

Group including Geraniol, D-Limonene, Benzyl Alcohol, P-Thymelin, and Clove Oil

Insecticidal effect of the compound on head lice

Composition Y, a combination comprising about 20% p-thymelin, about 40% clove oil (LFO), about 30% benzyl alcohol, and about 10% mineral oil, was studied using the method described above in Example 28. material, insecticidal effect. The LT ₁₀₀ of this composition was compared to a commercially available pediculicidal agent, ivermectin. As shown in Table CC, the lice treated with composition Y died faster than the lice treated with ivermectin. deal with LT100(min) Composition Y 3 Ivermectin 5

table CC

Example 30

The repelling effect of the composition on mosquitoes

A. Oral delivery

The repellent effect of orally delivered compositions was tested in hairless or shaved mice and guinea pigs. Orally administer a test oil (e.g., clove oil (LFO) or black seed oil (BSO)) or a test composition (e.g., a composition containing geraniol, d-limonene, eugenol, and lindenol) to approximately 10 rodents . A control substance, such as mineral oil, is administered orally to approximately 10 rodents. After approximately 30 minutes, place each rodent in a closed container. Approximately 20 mosquitoes were placed into each container. Observe each container for approximately 1 hour. The time each insect remained on the rodent and the number of wounds left by the insect on the rodent's skin were recorded. The insects spend less time on the rodents that received the test composition than on the rodents that received the control substance. The rodents that received the test composition had fewer wounds than the rodents that received the control substance.

B. Local Delivery

The effect of topically delivered compositions was tested in hairless or shaved mice and guinea pigs. A test oil (e.g., clove oil (LFO) or black seed oil (BSO)) or a test composition (e.g., a combination containing geraniol, d-limonene, eugenol, and lindenol) was administered topically to the skin of approximately 10 rodents. things). A control substance, such as mineral oil, is topically administered to the skin of approximately 10 rodents. After approximately 30 minutes, place each rodent in a closed container. Approximately 20 mosquitoes were placed into each container. Observe each container for approximately 1 hour. The time each insect remained on the rodent and the number of wounds left by the insect on the rodent's skin were recorded. The insects spend less time on the rodents that received the test composition than on the rodents that received the control substance. The rodents that received the test composition had fewer wounds than the rodents that received the control substance.

Example 31

The repelling effect of the composition on mosquitoes

About three cages, each containing about 100 tropical house mosquitoes (culex quinquefasciatus ) about 7 to 10 days old. Starves mosquitoes for about 12 hours. Each cage had four containers, each filled with cotton soaked in sugar water.

Three of the four containers were arbitrarily treated with 1000 ppm (approximately 1 mg/l) of the test composition and the other container served as an untreated control. The containers were placed in four opposite corners of each cage, and mosquito landings were counted at time intervals of approximately 0, 1, 2, 4, and 6 hours after the test composition was added to each container. Containers were removed from cages between exposure time intervals. Each exposure interval lasted approximately 5 minutes.

This method was used to test the repellency of the compositions described in Table DD. combination Composition (expressed as % by weight) EE 10% DEET, 45% LFO, 45% Cumin Oil AAA 50% Geraniol, 40% Thyme Oil, 10% Lemongrass Oil BB 50% LFO, 50% Cumin Oil

Form DD

LFO, cumin oil, geraniol, thyme oil, and lemongrass oil are all generally recognized as safe (GRAS compounds) by the Environmental Protection Agency (EPA) and the Food and Drug Administration (FDA), and have received EPA approval for insecticidal agent registration exemption.

Mosquito landings were counted at time intervals of approximately 0, 1, 2, 4, and 6 hours after addition of the test composition to each container, as shown in Table DD. The number of drops is shown in Table EE. Percent repellency was calculated using this data and the data in Table FF. Compositions EE, AA and BB exhibited almost 100% repellency in each exposure time interval. Even after 6 hours, the composition was 100% repellent against mosquitoes.

Table EE

Table FF

Example 32

Examination of the repelling effect and insecticidal effect of the composition containing plant essential oils on red ants

Test method

The insecticidal effect of different compositions containing plant essential oils on red ants was tested by the following method. Cardboard was treated with approximately 20 [mu]l of each detection composition, and the treated cardboard was placed one sheet per vial. Untreated cardboard was placed in a control bottle. The cardboard was then treated with approximately 20 [mu]l of 100% DEET and placed in a vial to compare the insecticidal efficacy of the composition with DEET, a known commercial insect control agent. About three red ants were placed in each vial, and the mouth of the vial was sealed with cotton to prevent the insects from escaping. Insects were exposed to the composition for approximately one hour or less and mortality was recorded.

The repelling effect of different compositions containing plant essential oils on red ants was tested by the following method. Cardboards were treated with approximately 200 [mu]l of each detection composition and placed in trays. Place untreated cardboard in the control pan. The cardboard was then treated with approximately 200 [mu]l of 100% DEET and placed in a tray to compare the repellency of the composition and DEET. Red ants were placed in each dish. The behavior of the insects and the number of stays of the insects on the treated cardboard were monitored for approximately 5 minutes. The number of times a red ant stayed was recorded.

The detection of residual insecticidal effect and repelling effect is to treat the cardboard with the test composition, and keep the treated cardboard under laboratory conditions for a predetermined period of time (for example, 0 minutes, 6 hours, 1 day, 3 days, 5 days, 7 days). days), red ants were exposed to the treated cardboard using the method described above.

Example 33

Repelling effect and insecticidal effect of composition containing plant essential oil on red ants

The insecticidal effect and repelling effect of the composition were tested by the method described in Example 32, as shown in Table GG. Untreated discs were neither toxic to nor repellent red ants. combination Composition (expressed as % by weight) Z 20% d-limonene, 10% lindenol, 10% eugenol, 10% phenylacetaldehyde, 50% geraniol AAA 50% Geraniol, 40% Thyme Oil, 10% Lemongrass Oil BB 50% LFO, 50% Cumin Oil CC 20% d-limonene, 20% thyme oil, 20% geraniol, 20% alpha-pinene, 20% p-thymelin DD 10% DEET, 18% d-limonene, 18% thyme oil, 18% geraniol, 18% alpha-pinene, 18% p-thymelin EE 10% DEET, 45% LFO, 45% Cumin Oil FF 44% LFO, 44% Cumin Oil, 10% Geraniol, 2% Thyme Oil

Table GG

Each composition resulted in 100% mortality when exposed to the cardboard for approximately 0 minutes, 6 hours, 1 day, 3 days, 5 days, or 7 days after treatment of the cardboard with the compositions, compared with DEET quite.

As shown in Table HH, red ants were repelled by the compositions used to treat cardboard. Furthermore, the compositions are superior to DEET in terms of residual capacity, they retain repellency for at least one week after application to cardboard, whereas DEET loses repellency after 1 day. Table HH shows the number of times red ants stayed on the treated cardboard. The time listed in the table, 0 minutes, 6 hours, 1 day, 3 days, 5 days, or 7 days, refers to the time elapsed between treating the cardboard with the composition and exposing the red ants to the cardboard treated. Approximate time.

Table HH

Example 34

Repelling effect and insecticidal effect of composition containing plant essential oil on red ants

The insecticidal effect and repelling effect of the composition were tested by the method described in Example 32, as shown in Table JJ. Treatment with each composition had repellent and insecticidal effects. combination Composition (expressed as % by weight) GG 10% d-limonene, 30% thyme oil, 35% geraniol, 10% alpha-pinene, 10% p-thymelin, 5% phenylacetaldehyde HH 15% d-limonene, 50% geraniol, 15% alpha-pinene, 15% p-thymelin, 5% phenylacetaldehyde JJ 50% d-limonene, 50% p-thymelin KK 33.3% d-limonene, 33.3% p-thymelin, 33.3% alpha-pinene LL 50% d-limonene, 50% thyme oil MM 50% Thyme Oil, 50% Alpha-Pinene NN 33.3% thyme oil, 33.3% alpha-pinene, 33.3% p-thymelin OO 50%-pinene, 50% p-thymelin PP 25% Linalool, 25% Alpha-Pinene, 25% p-Thymelin, 25% Thyme Oil QQ 33.3% linalool, 33.3% alpha-pinene, 33.3% p-thymol RR 33.3% d-limonene, 33.3% p-thymol, 33.3% thymol SS 25% d-limonene, 25% p-thymol, 25% thymol, 25% geraniol

Table JJ

It will be apparent to those skilled in the art that various modifications and changes can be made in the present invention without departing from the scope and spirit of the invention. The description and examples are illustrative only, and do not limit the scope and spirit of the invention. References and publications cited herein are hereby incorporated by reference.

It is to be understood that, unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, etc. used in the specification, examples and claims can be varied in all instances by the introduction of the term "about". Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification, examples, and claims are approximations that may vary depending upon the determined desired properties of the invention.

Claims (55)

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.

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