KR20080011958A - Natural insecticide composition containing bisabolangelone and derivative thereof - Google Patents

Abstract

A natural insecticide composition comprising bisabolangelone or a derivative thereof is provided to be able to control effectively acarid, agricultural injurious insects or forest injurious insects and be environment-friendly and harmless to human body. A method for isolating the bisabolangelone from Angelica koreana L. is provided to be able to obtain the bisabolangelone with high yield through a simple recrystallization. A natural insecticide composition comprises 0.001-50 wt.% of bisabolangelone represented by the formula(1) or a derivative thereof, where the derivative is a compound obtained by acetylating or acid-treating the bisabolangelone. The composition is for controlling Dermatophagoides pteronyssinus, Dermatophagoides farinae, Tyrophagus putrescentiae, Tetranychus urticae, Aulacorthum solani or Hyphantria cunea Drury. A method for isolating the bisabolangelone from Angelica koreana L. comprises the steps of: (a) after extracting ground Angelica koreana L. with an organic solvent to obtain a crude extract, drying the crude extract; (b) after adding water and hexane in a ratio of 1:1-3 to the dried extract to fraction a hexane layer, drying the hexane layer; and (c) after dissolving the dried hexane layer in hexane, storing the solution in a refrigerator at a temperature of -5 to -25 deg.C to obtain a precipitated crystal and filtering it.

Description

Natural insecticide composition containing bisabolangelone and derivative thereof}

1 is a diagram illustrating a process of sequential fractionation of active methanol crude extract according to the polarity of an organic solvent according to an embodiment of the present invention.

Figure 2 is a view showing a process of separating the active ingredient by recrystallization and chromatograph the hexane fraction layer of the active methanol crude extract.

The present invention relates to a natural insecticide composition, and more particularly, to a natural insecticide composition including bisabolangelolon and derivatives thereof capable of safely controlling house dust mites, storage mites, spotted mites, aphids, white fire moths and the like.

The dominant species are widespread habitat in the American house dust mite (Dermatophagoides farinae) and European house dust mite (Dermatophagoides pteronyssinus) is the world assumes that cause various allergic won, they are also known association with SIDS. Tyrophagus putrescentiae (Schrank), known as one of the most damaging stock pests, is frequently found in stores with high lipid and protein content, causing allergic disease and contaminants in farmers or workers who handle contaminated stores. It is also known as a carrier of fungi ( Aspergillus spp. , Penicillium spp. , Etc.) that cause abdominal pain in people who consume these stored foods.

In order to control such house dust mites and storage mites, chemical synthesis agents such as pyrimifosmethyl, benzyl benzoate, DEET, pyrethroids, etc. are widely used, but due to the side effects of frequently occurring chemical synthesis agents, Usage is getting less and less. For example, the world's most commercially available benzylbenzoate, due to its allergies, has only been available for limited use after receiving claims from consumers in the United States.

The reduction of agricultural output by agricultural pests such as spotted mites and aphids is somewhat different depending on the production and management techniques of developed and developing countries, but economic loss is about 10-50% per year. Is doing great damage to the trees. Therefore, in order to reduce such damage, various insecticides such as pyrethroid-based, organophosphorus-based and carbamate-based have been used. Although the use of these organic synthetic insecticides has a desired effect in terms of increasing production, their use is limited and reduced due to side effects such as environmental destruction and toxic toxicity. Therefore, there is an urgent need to develop more selective and safe insecticides that can effectively replace these organic synthetic insecticides.

Plants are becoming a popular alternative to chemical pesticides because they are selective against pests, biodegradable with non-toxic compounds, and have little advantage over untargeted organisms and the environment. In addition, pesticides containing some plants or compounds contained therein are classified as minimum risk pesticides under the Festicide Insecticides Insecticides Act (FIFRA), which is regulated by the US federal government, and therefore may be freely used without registration (US EPA). Exemption of Certain Pesticide Substances from Federal Insecticide, Fungicide, and Rodenticide Act Requirements Final Rule, 40 CFR 152.25 (g), May 6, 1996; US Environmental Protection Agency: Washington, DC, 1996.). Therefore, due to the advantages of such insecticides using plants, many studies have been conducted on this, but satisfactory natural insecticides have not been developed yet.

It is therefore an object of the present invention to provide a natural pesticide composition comprising bisabolangelolon and its derivatives which can effectively control mites, agricultural pests or forest pests.

Another object of the present invention is to provide a natural pesticide composition comprising bisabolangelolon and its derivatives which are environmentally friendly and harmless to the human body.

It is still another object of the present invention to provide a method for separating bisabolangelon from active.

In order to achieve the above object, the present invention provides a natural pesticide composition comprising a bisabolangelon represented by the following formula (1) and derivatives thereof.

[Formula 1]

The present invention also extracts the ground actives with an organic solvent to obtain a crude extract, and then drying, adding water and hexane in a ratio of 1: 1 to 3 to the dried active organic solvent crude extract to form a hexane layer. After fractionation, the step of drying and the hexane fractionation layer of the dried active organic solvent crude extract was dissolved in hexane, and then stored in a freezer at -5 ° C to -25 ° C to obtain precipitated crystals, and filtered. It provides a method for separating bisabolangelon from the activity comprising the step.

Hereinafter, the present invention will be described in more detail.

The natural pesticide composition according to the present invention includes bisabolangelon represented by the following formula (1) and derivatives thereof.

The bisabolangelon derivatives include a compound or a monocyclic sesquiterpin compound obtained through the process of acetylation or acid treatment of the nonsabolangelon. Examples of the acid used for the acid treatment of bisabolangelolon include p-toluene sulfonic acid, and the like, wherein 2- (3'-methylbutyl) -3,6-dimethyl-4 -Derivatives such as hydroxybenzofuran (2- (3'-methylbutyl) -3,6-dimethyl-4-hydroxybenzofuran) are produced. The monocyclic sesquiterpin compound (monocyclic sesquiterpenoids) compounds can be exemplified bisabolane (bisabolane), bisabolene (bisabolene), bisabolene (zingiberene), bisabolol (bisabolol), etc. having a structure similar to the bisabolangelon and the basic skeleton have.

The bisabolangelon or derivatives thereof may be used as a pesticide composition together with a conventionally used solvent, and if necessary, neutralizers, carriers, synergists, and the like, which are commonly used for preparing acaricides or insecticide compositions, may be used. It can be added and used. Examples of the solvent include alcohols such as methanol, ethanol, and isopropanol, aliphatic hydrocarbons, aromatic hydrocarbons such as xylene, benzene, and toluene, trichloroethane, and hydrocarbon chlorides such as ethylene dichloride, and ethyl. Ethers such as ether, isopropyl ether, ketones such as acetone, ester solvents such as ethyl formate, ethyl acetate, and mixtures thereof. have. At this time, the content of the bisabolangelon or derivatives thereof may vary depending on the application environment of the neutralizer, formulation, application place, application method, etc., which is used, but preferably 0.001 to 50% by weight based on the pesticide composition. If the content of the bisabolangelon or its derivatives is less than 0.001% by weight, the desired pesticidal effect may not be obtained, and if it exceeds 50% by weight, it is not economically preferable to use more than necessary compounds.

The natural insecticide composition according to the present invention can be prepared and used in the formulation of aerosols, pump sprays, solutions, suspensions, powders, capsules, paints, gels and the like. In particular, the bisabolangelon or derivatives thereof may be microencapsulated in a conventional manner and coated on a cloth such as clothing. An example of such a conventional microencapsulation method is to produce a polymer by reacting monomers dissolved in two solvents, which cannot be mixed with each other, at the interface of the solvent, thereby preparing one of two solvents that cannot be mixed with each other. The dissolved monomers are reacted at the interface of the solvent to form uniform walls on the surface of the core material, thereby diluting by slight changes in the solvent component of the in-situ polymer solution for producing microcapsules. A phase separation method (coacervation method) for preparing a microcapsule wall using a phenomenon of separating the concentrated phase into phases (coacervation); And dispersing the core liquid or core solid in a solvent which dissolves the polymer capable of forming the wall, further dispersing the dispersion in a solvent which cannot be mixed with the solvent, and then slowly evaporating the solvent to the interface of the core material. The solvent evaporation method etc. which deposit a polymer can be illustrated. Here, the wall material (carrier) forming the microcapsules may be used melamine, polyurea, polyurethane, polyamide, urea-formalin resin, melamine-formalin resin, gelatin, albumin, chitosan and the like, melamine or polyurethane It is preferable to use.

The bisabolangelolon and its derivatives according to the present invention can be widely applied to pesticides, acaricides, fragrances, fungicides and other industrial products, quasi-drugs, pharmaceuticals, etc. In particular, European dust mites aka vertical dust mites ( Dermatophagoides pteronyssinus ), American dust mites House dust mites such as Dermatophagoides farinae , storage mites such as Tyrophagus putrescentiae , agricultural pests such as Tetranychus urticae , Aulacorthum solani , and American Triphana moth It is effective for controlling forest pests such as cunea Drury ).

The natural pesticide composition comprising the bisabolangelon and its derivatives according to the present invention preferably acts as a contact poison. This is because the natural insecticide composition acts as a contact poison rather than fumigation, because the insecticidal effect is enhanced. In addition, the natural pesticide composition comprising the bisabolangelon and its derivatives according to the present invention can be used as an inhibitor.

The natural insecticidal composition according to the present invention is an extract of an active ( Ostericum koreanum Kitagawa or Angelica koreana Max.), Angelica silvestris L. , Pimpinella major Huds. Or Dokbok ( Angelica pubescens f. ) Include as an active ingredient. The plants have been reported to contain bisabola gelon (Angelica silvestris L. (Khadzhai YL, Sokolova VE, Farmakol. I. Toksikol. 23 (1960) 37. Khadzhai YL, Sokolova VE, Chem. Abstr. 55 1961) 2920 Novotny L., Samek Z., Sorm F., Tetrahedron Lett. (1966) 3541), Angelica koreana Max. (Hata K., Kozowa M., Baba K., Konoshima M., Chi HJ, Tetrahedron Lett (1970) 4379.), Pimpinella major Huds. (Stahl E., Herting D., Phytochem. (1976) 999.), Angelica pubescens f. Biserrata (Navrot J., Harmatha J., Novotny L., Biochem. Ecol. 12 (1984) 99.)).

The plant body weight is widely used as an herbal medicine, and has been used as an analgesic agent, antipyretic agent, arthritis, rheumatism agent for a long time in East Asia (Namba, T. The Encyclopedia of Wakan-Yaku (Traditional Sino-Japanese Medicines) with Color Pictures; Hoikusha: Osaka, Japan, 1993.), belonging to the benzopyranoid family such as imperatorin (imperatorin), isoimperatorin (isoimperatorin), choreinin (koreanin), oxypeucedanin, prangolarine It is known to include furanocoumarins compounds (Kwon, YS; In, KK; Kim, CM Chemical constituents from the roots of Ostericum koreanum.Kor . J. Pharmacogn. 2000, 31, 284-287.). In addition, the bowels according to the present invention include Chinese bowels ( Notopterygium inisum Ting or Notopterygium forbesii Boiss ) that is used as a tinnitus in Chinese medicine, and is known as the other tinnitus of austerium grossacetium in Korea ( Ostericum grosseserratum ), Angelica koreana Maxim , O. koreanum Kitagawa , Ostericum grossacetium (O. grosseserratum (Maxim.) Kitagawa ), Ostericum prateritum O. praeteritum Kitagawa, sp.nov. ) And Angelica A. miqueliana Maxim , et al. (National Medical Newspaper No. 418. April 23, 2004) )).

Activity extract according to the present invention can be obtained according to a conventional method. For example, the outpost, root, root stem, etc. of the active can be extracted with low alcohols such as butanol, ethanol, methanol, hexane, ether, benzene, chloroform, ethyl acetate or water, preferably with alcohol or water.

1 is a view showing a step of sequentially fractionating the active methanol crude extract according to the polarity of the organic solvent according to an embodiment of the present invention, by sequential fractionation of the active methanol crude extract in an organic solvent, hexane The layer is separated into a chloroform layer, an ethyl acetate layer, a butanol layer and a water layer.

2 is a view illustrating a process of separating the active ingredient by recrystallization and chromatography of the hexane fraction layer of the active methanol crude extract.

Separation method of bisabolangelolon from the actives using the recrystallization method is obtained by extracting the ground actives with an organic solvent to obtain a crude extract, and then drying the water and hexane in the dried active organic solvent crude extract 1: 1 ~ 3 Adding hexane, fractionating the hexane layer, and then drying and dissolving the dried hexane fraction of the crude organic solvent crude extract in hexane, then storing in a freezer at -5 ° C to -25 ° C to precipitate Obtaining the obtained crystals and filtering. Here, when the organic solvent used for extraction is hexane or an aqueous solution of hexane, the process of fractionating the hexane layer by adding water and hexane in a ratio of 1: 1 to 3 may be omitted. When the separation method is used, bisabolangelon having a purity of 95% or more can be obtained simply. In addition, the separation method may further comprise the step of adding only ethyl acetate to the filtered crystals, in order to obtain a high-purity bisabolangelon of 98% or more, and drying only the dissolved solution.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Example 1

end. Sample Preparation

After 2 kg of dried activated oil was evenly ground, the active extract obtained by dipping in 10 liters of methanol for 2 days was filtered twice, and the filtrate was concentrated with a concentrator to obtain 202 g of crude methanol crude extract. Next, 140 g of active methanol crude extract was sequentially partitioned into hexane, chloroform, ethyl acetate, butanol and water, and each was separated into hexane layer (10.6 g), chloroform layer (29.1 g), ethyl acetate layer (2.7 g) and butanol layer (36.7). g) and water layer (55.2 g) were obtained and these fractionation layers were used as bioassay samples.

I. Creatures used for black

The relative humidity of American house dust mite ( Dermatophagoides farinae ), vertical house dust mite ( Dermatophagoide pteronyssinus ), and Tyrophagus putrescentiae for bioassay , temperature 25 ± 5% It was bred in a breeding room maintained at 1 ℃. At this time, artificial feed was put into a 12.5 × 10.5 × 5cm container, and an appropriate amount of saline was poured into the bottom of the container having a volume of 17.5 × 17.5 × 17.5cm. The mites are species bred indoors without exposure to acaricides.

In addition, the spotted mite was distributed from the insect breeding room of the Department of Agricultural Biotechnology, Seoul National University, and the bovine aphid and the American White Bull moth were collected directly from the field and used as an individual for bioassay.

All. Bioassay

A. Bioassay for ticks

The pesticidal activity of the sample against ticks was confirmed by the following fabric-piece contact toxicity. A piece of fiber (diameter 5 cm) treated with a sample prepared in "A of Example 1" (active methanol crude extract, hexane layer, chloroform layer, ethyl acetate layer, butanol layer and water layer) at a content of 50.9 µg / cm 2. After putting into a petri dish, 26 to 38 objects of each of the three ticks were spun onto the petri dish, and the lid was covered. After 24 hours, the state of the tick was confirmed by using a 20 times magnification dissecting microscope, and the ratio of the sample was calculated and shown in Table 1 below. The state of the tick was stimulated each tick with a fine brush or pin, and it was judged that the individual who did not observe the movement of the appendage was dead, and all the tests were repeated three times.

sample Application rate,% (mean ± standard error) Treatment Big legged house dust mite Treatment Vertical Pattern House Dust Mite Treatment Long hair mite Methanol crude extract 94 79 ± 2.3a 98 79 ± 1.8a 101 81 ± 1.4a Hexane layer 106 92 ± 1.1a 107 91 ± 1.2a 103 94 ± 1.3a Chloroform layer 109 19 ± 1.2b 107 18 ± 1.4b 105 11 ± 1.5b Ethyl acetate layer 92 9 ± 2.8b 85 9 ± 3.0b 98 6 ± 2.7b Butanol layer 100 12 ± 3.3b 100 14 ± 2.6b 100 11 ± 2.1b Water layer 96 15 ± 2.8b 99 17 ± 1.3b 93 13 ± 1.6b

From Table 1, it can be seen that the active methanol crude extract and the hexane fraction layer of the active methanol crude extract showed a high rate of fertilization against large house dust mites, vertical house dust mites, and long hair dust mites.

B. Bioassay for Spotted Mite and Pseudomonas Aphids

The insecticidal activity of the samples against spotted mite ( Tetranychus urticae ) and aphid aphid ( Aulacorthum solani ) was confirmed by the following method.

A kidney bean leaf, which can be used as a host of spotted mite and rhizome aphid, was made into sections of 5 cm in diameter, and then placed on cotton wool (5.5 cm in diameter) with water. Next, each of the 20 spotted mite and rhizome aphids were transferred onto the kidney bean leaf slices using a brush, and the insects transferred to the slices were allowed to stand for 1 hour to adapt to the new environment. Next, the sample (active methanol extract and hexane layer) prepared in "A of Example 1" was dissolved in ethanol, and prepared in concentrations of 0.5%, 1%, 2%, 5%, and 10%, respectively. After spraying three times (320 μl) to the spotted mite and moustacus aphid located in the section, after 24 hours, the state of the spotted mite and moustarine aphid was confirmed using a 20x magnification dissecting microscope. All assays were performed in three replicates, and the pesticide rate of the sample was calculated and shown in Table 2 below.

sample density(%) Insecticidal rate,% (mean ± standard error) Spotted mite Vulture aphid Methanol crude extract 10 100 ± 0.0a 100 ± 0.0a 5 92 ± 2.4a 94 ± 1.3a 2 73 ± 1.7b 90 ± 2.1a One 52 ± 2.9c 64 ± 4.3b 0.5 43 ± 3.3c 57 ± 2.3b Hexane layer 10 100 ± 0.0a 100 ± 0.0a 5 96 ± 1.3a 94 ± 1.8a 2 80 ± 2.5b 78 ± 1.5b One 78 ± 1.8b 71 ± 2.8b 0.5 56 ± 3.3c 43 ± 2.1c

From Table 2, the active methanol crude extract shows a high insecticidal rate at a concentration of 2% or more against the spotted mite and the barley aphid, while the hexane fractionation layer of the active methanol crude extract shows a high insecticidal rate even at a concentration of 1% or more. It can be seen.

C. Bioassay for American White Bull Moths

The antifeedant activity of the sample against the American white moth was confirmed by the following leaf immersion method.

After making a hardwood leaf that can be utilized as a host of the American white light moth as a section of 4 cm in diameter by using a cork bore, the prepared section was prepared in the sample of "Example 1" (active methanol crude extract, hexane Layer) The appropriate amount was dissolved in 300 µl of ethanol, and 20 ppm of NBT 40E, a surfactant, was added, and then immersed in a sample solution added with water for a final concentration of 1000 and 2000 ppm for 30 seconds, followed by naturally drying for 4 hours indoors. .

At this time, without using a sample, the one obtained by immersing and drying the sections in a solution containing only the surfactant and ethanol in the same manner was used as a control. Next, put the prepared test piece and one control each in a petri dish of 11 cm in diameter, and then spun five larvae of white fire moth in the center, and after 24 hours to calculate the inhibition rate according to the following formula, Table 3 All assays were performed in three replicates.

[formula]

Feeding inhibition rate% = untreated leaf consumption area-untreated leaf consumption area / untreated leaf consumption area × 100

sample Concentration, ppm Feeding inhibition rate,% (mean ± standard error) Methanol crude extract 2000 97 ± 2.9a 1000 88 ± 3.9b Hexane layer 2000 100 ± 0.0a 1000 98 ± 1.1a

From Table 3, it can be seen that the active methanol extract and the hexane fractionation layer of the active methanol crude extract exhibit a high feeding inhibition rate even at low concentrations for the US white moth.

In this test, the insecticidal rate (fertilization rate) was properly converted for ANOVA, followed by statistical analysis, and the differences between the experimental groups were compared using the Bonferroni multiple comparison method. The lethal concentration value (LD ₅₀ ) was used for probit analysis.

Example 2

end. Isolation of Insecticidal Body by Recrystallization

As shown in Figure 2, the hexane fractionation layer showing the highest activity in Example 1 was further separated and purified by the following method. 10 g of the hexane layer prepared in “A of Example 1” was dissolved in 100 ml of hexane, and then stored in a -20 ° C. freezer. After 48 hours of freezer storage, the white precipitate precipitated in the lower layer was separated by filter paper. Next, the separated precipitate was continuously washed with hexane, and then the hexane solvent remaining in the precipitate was volatilized with nitrogen gas to obtain a pale yellow acicular crystal. The pale yellow crystals were identified as bisabolangelon (purity: 95% or more). Next, in order to obtain a higher purity bisabolangelon, the light yellow crystals were dissolved in 5 ml of ethyl acetate, and only the solution was dried to obtain 0.41 g of a high purity bisabolangelon (H1). Yield: 4.1%, purity: 98% or more).

Next, the acaricide activity of the bisabolangelon (H1) mites separated by the recrystallization method was carried out according to the fiber contact method of Example 1, and the results are shown in Table 4 below.

I. Isolation of Insecticidal Body by Chromatography

As shown in Figure 2, the hexane solution (H2) of the hexane fractionation layer (9.5 g) that was not crystallized in "A of Example 2" was subjected to silica gel column chromatography (Merck 70-230 mesh, 600 g, diameter 5.5 cm, height) 70 cm glass column), the polarity of the solvent was applied sequentially to separate. In this case, the ratio of hexane / ethyl acetate is 100: 0 (2 L), 99: 1 (1 L), 95: 5 (2 L), 90:10 (3 L), 80:20 (2 L), 70:30 (3 L ), 60:40 (1 L), and 0: 100 (1 L) were used sequentially, and finally 500 mL of about 500 ml were obtained by flowing 2 L of methanol. Next, the separated aliquots were developed under hexane: ethyl acetate (70:30) conditions on thin layer chromatography (TLC), and those having similar development distances were collected and finally seven fractionated layers (H21, H22, H23). , H24, H25, H26, H27) were secured.

Next, the acaricide activity against the mite of the hexane dissolution solution (H2) and the fractionation layer (H21, H22, H23, H24, H25, H26, H27) was carried out according to the fiber contact method of Example 1, and the result It is shown in Table 4 below. Fraction layer H26 (2.4 g) showing the best ratio was again separated by silica gel column chromatography by applying the polarity of the solvent sequentially. In this case, chloroform: methanol ratio was 100: 0 (4 L), 500: 1 (1 L), 200: 1 (1 L), 99: 1 (3 L), and 0: 100 (2 L). Approximately 250 ml of 44 aliquots were obtained. The separated aliquots were then developed on thin layer chromatography (TLC) under chloroform: methanol (98: 2) conditions, and those with similar development distances were collected and finally eight fractionated layers (H261, H262, H263, H264, H265, H266, H267, H268).

Next, the acaricide activity against the tick of the fraction layer (H261, H262, H263, H264, H265, H266, H267, H268) was carried out according to the fiber contact method of Example 1, and the results are shown in Table 4 below. It was. Fraction layer H266 (980 mg) showing the best ratio was again separated by silica gel column chromatography by applying the polarity of the solvent sequentially. In this case, chloroform: methanol ratio was 100: 0 (2 L), 500: 1 (1 L), 200: 1 (2 L), 99: 1 (1 L), and 0: 100 (1 L). About 250 ml of 28 aliquots were obtained. The separated aliquots were then developed on thin layer chromatography (TLC) under chloroform: methanol (98: 2) conditions, and those with similar development distances were collected and finally five fractionation layers (H2661, H2662, H2663, H2664, H2665).

Next, the acaricide activity against the tick of the fraction layer (H2661, H2662, H2663, H2664, H2665) was carried out according to the fiber contact method of Example 1, the results are shown in Table 4 below. The fractional layer H2663 (268 mg) showing the best ratio was developed under chloroform: methanol (98: 2) conditions on thin layer chromatography (TLC), and finally two layers (H26631, H26632) were obtained. The acaricide activity of the fractionation layer was carried out, and the results are shown in Table 4 below. In order to further purify H26631 (40.3 mg), which showed strong acaricide activity, high performance liquid chromatography (HPLC) was carried out, and finally an active body, bisabolangelon, was obtained.

Fractional layer Yield (g) Application rate,% (mean ± standard error) Treatment Big legged house dust mite Treatment Vertical Pattern House Dust Mite Treatment Longhair mites H1 0.41 99 97 ± 1.6a 94 99 ± 1.0a 91 100 ± 0.0a H2 9.5 91 93 ± 2.2a 107 92 ± 1.5a 99 98 ± 1.5a H21 1.44 93 1 ± 1.1c 91 5 ± 4.3b 102 10 ± 1.7b H22 1.2 99 9 ± 0.8b 112 11 ± 3.2b 103 8 ± 3.4b H23 0.48 92 7 ± 2.0bc 93 12 ± 1.1b 93 5 ± 1.8b H24 0.24 95 9 ± 1.7b 94 8 ± 2.2b 97 11 ± 2.5b H25 1.38 108 17 ± 2.8b 106 15 ± 5.0b 98 18 ± 3.7b H26 2.48 104 95 ± 0.8a 91 96 ± 2.1a 99 98 ± 1.1a H27 1.77 112 19 ± 4.0b 106 18 ± 3.7b 102 11 ± 2.7b H261 0.02 91 7 ± 1.8b 97 7 ± 1.8b 97 9 ± 1.5b H262 0.18 91 6 ± 1.5b 91 8 ± 4.3b 99 11 ± 3.1b H263 0.67 91 3 ± 1.8b 96 5 ± 2.6b 101 6 ± 1.5b H264 0.31 92 5 ± 2.9b 91 2 ± 1.2b 104 8 ± 1.9b H265 0.07 93 7 ± 2.2b 92 6 ± 1.6b 89 9 ± 1.7b H266 0.98 94 96 ± 1.1a 90 98 ± 1.2a 93 99 ± 0.7a H267 0.17 98 10 ± 3.3b 101 14 ± 1.9b 92 9 ± 1.1b H268 0.14 91 4 ± 2.2b 98 4 ± 1.1b 97 7 ± 1.5b H2661 0.02 94 10 ± 1.7b 114 12 ± 3.5b 96 15 ± 2.5b H2662 0.33 103 13 ± 3.0b 109 14 ± 2.1b 102 11 ± 2.8b H2663 0.27 106 95 ± 0.6a 94 98 ± 1.0a 101 99 ± 1.0a H2664 0.05 95 11 ± 3.3b 98 8 ± 1.8b 104 14 ± 2.3b H2665 0.27 95 4 ± 2.6b 91 6 ± 2.4b 111 8 ± 2.6b H26631 0.04 99 96 ± 0.7a 91 97 ± 1.6a 95 99 ± 1.1a H26632 0.15 96 10 ± 0.6b 97 13 ± 1.1b 92 15 ± 3.2b

From Table 4, it can be seen that the H1 obtained through hexane recrystallization and the hexane dissolution solution H2 of the hexane fractionation layer show high abundance for all three mites. It can also be seen that H26, H266, H2663 and H26631, which are further separated from H2, exhibit strong acaricide activity.

All. Identification of active body

Mass spectrometry (MS) and NMR of H1 obtained by the hexane recrystallization method and H26631 obtained by the chromatographic method confirmed that the active body was bisabolangelone. Spectral values of the compounds are as follows. [R] ²⁰ _D + 195.2 ° (c 0.025, MeOH); UV (EtOH) λ _max (ε) = 247 (33643); IR (KBr, cm ⁻¹ ), Vmax = 3342 (−OH), 1640 (C═O); ¹ H NMR (CDCl ₃ , 400 MHz), δ1.62 (3H, s), 1.72 (3H, s), 1.79 (3H, s), 2.01 (3H, s), 2.65 (1H, d, J) 6.9 Hz), 2.72 (2H, d, J) 6.0 Hz), 3.37 (1H, s), 4.88 (1H, q, J) 4.5 Hz), 5.37 (1H, d, J) 11.4 Hz), 5.99 (1H, m), 6.02 (1 H, m); ¹³ C NMR (CDCl ₃ , 100 MHz), δ 18.2, 24.6, 26.0, 27.3, 34.9, 53.6, 76.1, 78.5, 94.3, 117.7, 127.2, 132.5, 158.1, 160.1, 196.8; EI-MS, relative intensity ( m / z ) = 248 [M] ⁺ (13), 230 (100), 215 (62), 148 (78), 109 (66); HR EI-MS, C ₁₅ H ₂₀ O ₃ found 248.1405 [M < + >], calcd 248.3212.

la. Acaricide Activity of Active Active Body Against Tick

The acaricide activity of bisabolangelolon, the active body of activation, and benzyl benzoate, DEET, and dibutyl phthalate, which is an organic synthetic acaricide, was measured according to the fiber contact method of Example 1. The acaricidal activity was determined based on the half-lethal concentration value (LD ₅₀ ) on the basis of the 24-hour exposure time, and for the large leg dust mite in Table 5, the vertical pattern dust mite in Table 6, and the long hair mite in Table 7 It showed acaricide activity.

Compound, 24 hours Tick Slope (± standard error) LD ₅₀ , μg / cm ² 95% CL ^b RT ^a Bisabolangeloron 274 4.71 ± 0.50 1.92 1.76-2.09 11.23 Benzylbenzoate 295 3.90 ± 0.46 21.56 19.62-23.81 One Diet 282 7.04 ± 0.75 74.20 70.01-78.37 0.29 Dibutyl phthalate 243 6.48 ± 0.77 76.82 72.03-82.00 0.28

^a: benzyl benzoate Relative toxicity = benzyl benzoate of the LD ₅₀ value / LD ₅₀ value of each test compound

^b : Confidence Limit

Compound, 24 hours Tick Slope (± standard error) LD ₅₀ , μg / cm ² 95% CL ^b RT ^a Bisabolangeloron 309 4.76 ± 0.47 1.80 1.65-1.94 9.98 Benzylbenzoate 248 4.50 ± 0.49 17.97 16.30-19.71 One Diet 273 6.17 ± 0.75 75.05 70.44-79.87 0.24 Dibutyl phthalate 296 6.86 ± 0.75 76.95 72.86-81.38 0.23

^a: benzyl benzoate Relative toxicity = benzyl benzoate of the LD ₅₀ value / LD ₅₀ value of each test compound

^b : Confidence Limit

Compound, 24 hours Tick Slope (± standard error) LD ₅₀ , μg / cm ² 95% CL ^b RT ^a Bisabolangeloron 310 4.68 ± 0.40 1.75 1.63-1.91 10.81 Benzylbenzoate 269 4.58 ± 0.51 18.92 16.20-19.70 One Diet 283 6.21 ± 0.69 72.01 71.42-78.75 0.26 Dibutyl phthalate 277 6.90 ± 0.72 75.88 71.92-80.36 0.25

^a: benzyl benzoate Relative toxicity = benzyl benzoate of the LD ₅₀ value / LD ₅₀ value of each test compound

^b : Confidence Limit

From Tables 5, 6 and 7, it can be seen that bisabolangelon has a stronger acaricide activity than organic synthetic acaricides which are commonly used, and thus can be usefully used for controlling ticks. In addition, when applying acaricide with non-sabolangelon as an active ingredient in an indoor environment in which mites inhabit, the half lethal concentration (LD ₅₀ ) is 192mg (large house dust mite) per ㎡ and 180mg (vertical pattern house dust mite) And 175 mg (long hair mite).

hemp. Insecticidal activity of active body against spotted mite and barley aphid

Except for the use of 1% bisabolangelon dissolved in ethanol, the insecticidal activity test was carried out on the spotted mite and the barley aphid in the same manner as in "Example 1, C, B", and the results are shown in Table 8 below. Shown in

insect Division water (100µl / 1time) Insecticidal rate,% (mean ± standard error) Spotted mite 10 99 ± 1.0a 5 94 ± 1.5a 4 76 ± 4.8b 3 54 ± 3.2b 2 24 ± 1.8c Vulture aphid 10 100 ± 0.0a 5 97 ± 1.8a 4 57 ± 2.4b 3 48 ± 3.4b 2 11 ± 2.8c

From Table 8, it can be seen that bisabolangelolon exhibits high insecticidal power when sprayed five or more times on the spotted mite and the barley aphid, it can be usefully used for controlling agricultural pests.

bar. Feeding Inhibitory Activity of Active Roots of the American White Bull Moth

The feeding inhibitory activity test was conducted on the American white fire moth larva in the same manner as in "Example 1, C, C" except that the non-sabolangelon sample liquid having a final concentration of 500, 200, or 100 ppm was used. It is shown in Table 9 below.

Concentration, ppm Feeding inhibition rate,% (mean ± standard error) 500 100 ± 0.0a 200 100 ± 0.0a 100 92 ± 2.8a

From Table 9, it can be seen that bisabolangelolon shows excellent feeding inhibitory activity against the American white fire moth larvae.

Example 3

end. Check mechanism of action

In order to confirm the action of insecticidal (fertilizing) activity of the active extract and bisabolangelolon, after spinning 30 to 40 mites in petri dishes, a hole having a diameter of 3 cm in the center and a fine mesh of 200 mesh Covered Petri Dish lid made of.

Bisabolangelolon, the active body of active, and benzyl benzoate, DEET, and dibutyl phthalate, which are organic synthetic acaricides, were prepared at the concentrations described in Tables 10 to 12 below (fiber 5 cm in diameter). ), Then placed on a wired petri dish lid to prevent ticks from coming into direct contact. Next, a part of the treatment group is sealed to prevent air from passing through (a fully sealed group A), and the other part is exposed to the air as it is (a fully opened group B), and after 24 hours, the state of the tick is checked under a microscope. , Table 10 shows a large house dust mite, Table 11 shows the vertical pattern house dust mite and Table 12 shows the acaricide activity for long hair mites.

Compound, 24 hours exposure Throughput (㎍ / ㎠) Application rate,% Treatment A ^a Treatment B ^b Bisabolangeloron 50.9 109 20 ± 2.4 90 8 ± 0.6 Benzylbenzoate 50.9 105 16 ± 1.2 96 12 ± 1.7 Diet 152.7 107 16 ± 1.4 78 8 ± 2.3 Dibutyl phthalate 152.7 102 10 ± 2.8 90 8 ± 1.4

A ^a : The test piece which closed and closed the lid.

B ^b : Test strips with the lid open allowing air to flow in and out.

Compound, 24 hours exposure Throughput (㎍ / ㎠) Application rate,% Treatment A ^a Treatment B ^b Bisabolangeloron 50.9 108 16 ± 2.4 94 10 ± 2.0 Benzylbenzoate 50.9 110 19 ± 1.3 101 10 ± 0.8 Diet 152.7 104 15 ± 2.4 93 8 ± 2.6 Dibutyl phthalate 152.7 110 12 ± 2.0 100 9 ± 2.2

A ^a : The test piece which closed and closed the lid.

B ^b : Test strips with the lid open allowing air to flow in and out.

Compound, 24 hours exposure Throughput (㎍ / ㎠) Application rate,% Treatment A ^a Treatment B ^b Bisabolangeloron 50.9 102 11 ± 2.9 104 6 ± 2.8 Benzylbenzoate 50.9 106 15 ± 3.1 99 8 ± 1.9 Diet 152.7 105 9 ± 2.1 98 10 ± 3.1 Dibutyl phthalate 152.7 114 8 ± 2.4 105 11 ± 2.6

A ^a : The test piece which closed and closed the lid.

B ^b : Test strips with the lid open allowing air to flow in and out.

From Tables 10, 11 and 12, the killing rate by fumigation is insignificant, and it can be seen that the killing power of bisabolangelon is not by fumigation but by direct contact.

I. Comparison of acaricide activity by fiber

Even if the compound is excellent in activity, the expression of the activity may be limited depending on the environment to be applied. Therefore, the environment in which house dust mites are mainly inhabited is a fiber material, and a variety of fiber materials are treated with bisabolangelon to investigate the change of acaricide activity according to absorption or adhesion to the fiber material.

First, ethanol liquids containing 0.3% bisabolan gelon in various fiber materials (cotton yarn, rayon yarn, nylon yarn, artificial leather, silk yarn) sections (width x length, 3 x 3 cm) were respectively sprayed with sprayers 1, 2, 3 After spraying once and volatilizing for 1 hour, the number of vertical patterned dust mites shown in Table 13 was spun into the center of each fiber section, and rolled carefully as if the roll was rolled, and then the ends of the fiber sections were stapled. Fixed. After the mites were continuously exposed to the compound for 24 hours, the condition of the mites was checked using a 20x magnification dissecting microscope, and the rate of agicide was calculated and shown in Table 13 below. All assays were performed in three replicates.

Type of fiber The number of branches Treatment Application rate,% Cotton yarn 3 63 98 ± 1.1 2 58 96 ± 2.2 One 51 24 ± 1.8 Rayon 3 48 100 ± 0.0 2 77 99 ± 1.0 One 53 44 ± 5.4 Nylon yarn 3 63 100 ± 0.0 2 71 100 ± 0.0 One 60 48 ± 3.4 Leatherette 3 54 96 ± 1.4 2 49 92 ± 2.5 One 51 18 ± 1.8 Silk thread 3 66 94 ± 2.3 2 61 82 ± 2.7 One 57 34 ± 1.9

From Table 13, it can be seen that when spraying 0.3% bisabolangelon two or more irrespective of the type of each fiber yarn, a high acaricide activity of 82% or more. The results suggest that bisabolangelolon can be utilized as a raw material with strong control in a fibrous environment where actual ticks occur.

Example 4

end. Activity of the composition containing active extract

1% by weight of an emulsifier (NBT 40E), 1% by weight of emulsifier (NBT 40E) and 1% by weight of the active extract (ethanol crude extract, crude methanol extract or hexane fraction layer of crude methanol extract) extracted in the same manner as in "A of Example 1" , 10% by weight of ethanol and 87.8% by weight of water were added to prepare a composition containing the active extract. The active extract was diluted to 0.5, 0.1, 0.01 and 0.005% of the composition containing 1.0%, and then placed in a container with a nozzle. Next, 30 to 50 vertical pattern dust mites were spun in the center of a black cloth piece (5 × 5 cm), and the composition containing the active extract was sprayed three times each, and the results were examined after 24 hours. Table 14 shows. All experiments were conducted in three replicates.

extract Active ingredient content and% of fermentation, (mean ± standard error) 0.005 0.01 0.1 0.5 1.0 ethanol 53 ± 4.7 78 ± 5.8 100 ± 0.0 100 ± 0.0 100 ± 0.0 Methanol 70 ± 1.9 93 ± 2.8 100 ± 0.0 100 ± 0.0 100 ± 0.0 Hexane fraction 87 ± 2.4 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0

From Table 14, the reaction of the house dust mite to the composition containing the active organic solvent extracts showed a difference according to the active ingredient content, but in the composition containing more than 0.1% showed a good ratio of 100%. The result is considered to be a difference attributable to the degree of inclusion of bisabolangelolon as the active body.

I. Bisabolangeloron  Activity of the containing composition

In the same manner as in "A of Example 4", a composition containing 1.0, 0.5, 0.1, 0.01, and 0.005% of bisabolangelon was prepared, and acaricide activity assay was performed on vertical pattern dust mites. Is shown in Table 15 below.

Active ingredient content and% of fermentation, (mean ± standard error) 0.005 0.01 0.1 0.5 1.0 89 ± 3.1 100 ± 0.0 100 ± 0.0 100 ± 0.0 100 ± 0.0

From Table 15, it can be seen that the composition (formulation) containing the bisabolangelolon exhibits strong acaricidal activity against the mite dust mites even at 0.005% content.

All. Activity of Fiber Containing Capsules

10 g of active methanol crude extracts, which were ground and ground to an average particle diameter of 20 µm, were put in a metal container, 50 g of 0.1% hydroxypropyl cellulose aqueous solution having an interfacial activity was added, and 20 g of deionized water was added thereto, followed by 1 hour at 40 ° C. It was stirred at 150 rpm. Next, an aqueous solution obtained by mixing 15.7 g of deionized water with 4.3 g of melamine resin was added to the stirring solution, adjusted to pH 4.4 with acetic acid, and reacted at 40 ° C. for 2 hours to contain 10% crude methanol extract. Microencapsulated compositions were prepared. After immersing a previously prepared piece of fiber (horizontal x vertical, 5 x 5 cm) in a microencapsulated composition containing 10% of the crude methanol extract and diluting it to 5, 1 and 0.5%, respectively, for 30 seconds , And dried naturally for 48 hours. Next, after rubbing the dried fiber pieces with both hands about 5 times, bursting the capsule, 20-30 pieces of vertical pattern dust mites were spun onto the fiber pieces, and exposed for 24 hours, and the ratio of the salvage was investigated. 16 is shown.

In addition, except for using bisabolangelon instead of the crude methanol crude extract, after the micro-encapsulated bisabolangelon in the same manner as described above, the ratio of the salvage was investigated, shown in Table 16 below.

% Of application rate, (mean ± standard error) Concentration 0.5 1.0 5.0 10 Methanol extract 14 ± 5.8 76 ± .4 100 ± 0.0 100 ± 0.0 Bisabolangeloron 54 ± 2.7 89 ± 4.6 100 ± 0.0 100 ± 0.0

From Table 16, it can be seen that the fiber immersed in 1.0% or more active organic solvent extract and bisabolangelon-containing microcapsules solution exhibits a strong acaricide activity of 76% or more.

As described above, the natural pesticide composition comprising bisabolangelon and its derivatives according to the present invention can effectively control mites, agricultural pests or forest pests, as well as environmentally friendly and harmless to humans. In addition, the separation method of bisabolangelon from the active according to the present invention can obtain a high yield of nonsabolangelon through a simple recrystallization method.

Claims (10)

A natural pesticide composition comprising bisabolangelon represented by the following formula (1) or a derivative thereof. [Formula 1]

The natural insecticide composition according to claim 1, wherein the bisabolangelon derivative is a compound or a monocyclic sesquiterpin compound obtained through acetylation or acid treatment of bisabolangelon. According to claim 1, wherein the bisabolangelon derivatives are bisabolan, bisabolene, gingiberene, 2- (3'-methylbutyl) -3,6-dimethyl-4-hydroxybenzofuran, bisabolol and mixtures thereof Natural pesticide composition is a compound selected from the group consisting of. The natural insecticide composition according to claim 1, wherein the bisabolangelon or a derivative thereof is prepared in microcapsules. The natural insecticide composition according to claim 1, wherein the natural insecticide composition is for controlling house dust mites, storage mites, spotted mites, aphids or American White Bull moths. The natural pesticide composition of claim 1, wherein the natural pesticide composition acts as a contact poison or an inhibitor. Natural insecticide composition comprising an extract of the plant selected from the group consisting of active, Angelica Silvestris, Pimpinella measure, poisonous activity and mixtures thereof. Extracting the ground actives with an organic solvent to obtain a crude extract, and then drying Adding water and hexane to the dried organic solvent crude extract in a ratio of 1: 1 to 3, fractionating a hexane layer, and drying the mixture;   The hexane fractionation layer of the dried crude organic solvent crude extract was dissolved in hexane, and then stored in a freezer at -5 ° C to -25 ° C to obtain precipitated crystals, followed by filtration from bisabolan. Separation method of gelon. 10. The bisabolan from the activity of claim 8, wherein the solvent used for crude extraction of the activity is selected from the group consisting of methanol, ethanol, butanol, hexane, ether, benzene, chloroform, ethyl acetate, water and mixtures thereof. Separation method of gelon. 9. The method of claim 8, further comprising drying only the dissolved solution after adding ethyl acetate to the filtered crystals.

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