KR20170027523A - solubilized formulation with nematicidal activity and method for manufacturing thereof - Google Patents
solubilized formulation with nematicidal activity and method for manufacturing thereof Download PDFInfo
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- KR20170027523A KR20170027523A KR1020150124275A KR20150124275A KR20170027523A KR 20170027523 A KR20170027523 A KR 20170027523A KR 1020150124275 A KR1020150124275 A KR 1020150124275A KR 20150124275 A KR20150124275 A KR 20150124275A KR 20170027523 A KR20170027523 A KR 20170027523A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/08—Oxygen or sulfur directly attached to an aromatic ring system
- A01N31/16—Oxygen or sulfur directly attached to an aromatic ring system with two or more oxygen or sulfur atoms directly attached to the same aromatic ring system
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- A01N63/04—
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S424/00—Drug, bio-affecting and body treating compositions
- Y10S424/10—Insect repellent
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S43/00—Fishing, trapping, and vermin destroying
- Y10S43/90—Liquid insecticide sprayer
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- Y10S514/919—
Abstract
The present invention relates to novel ( Bursaphelenchus), which causes a great deal of damage to coniferous trees including horticultural crops, edible crops, and pine trees, and the like, and more particularly, xylophilus & lt ; / RTI & gt; Solubilizing agents and methods for their preparation
Description
The present invention relates to novel ( Bursaphelenchus), which causes a great deal of damage to coniferous trees including horticultural crops, edible crops, and pine trees, and the like, and more particularly, xylophilus & lt ; / RTI & gt; A solubilizing agent and a process for producing the same.
At present, there are pine tree bruises ( Bursaphelenchus xylophilus ) caused by the pine tree and pine trees are seriously damaged. This plant disease, also known as "pine tree aids", is not treated once it is infected and almost all pine trees are killed. In Korea, pine trees are mainly damaged in the southern region, and pine trees are mainly damaged in the central region.
Pine reeves ( Bursaphelenchus xylophilus is a taxonomic animal that belongs to the family Metazoa, Eumetazoa, Bilateria, Pseudocoelomata, Nematoda, and Nemata. . The length is not less than 1 mm, but the breeding power is amazing, so the pair will increase to 200,000 in 20 days. Pine re-infestation can not penetrate into trees or move between trees by themselves, but it must be done through mediators. Monochamus alternatus or M. saltuarius , which is an insect species , mainly produces diseased pine trees or pine trees and lays eggs. When the eggs of the parasites born in the trees infected with the re-infestation become pupa in the spring, When they are gathered around, and when the fowls emerge during the middle of May to the end of July, the rewinds attached to the body move together. The foliage that escapes from the woody part mainly feeds on newly grown branches (1-2 year old branches) of a healthy tree, and infects pine tree rehabilitate through the wound. In the case of diseased pine trees, the whole leaf turns brown and usually dies within one year.
The cause of pine wilt disease, which first occurred in Japan in 1905, has not been elucidated for a long time. In Japan, most of the pine native forests have disappeared, except for landscape plants with high historical value. In Korea, the first case occurred in 1988 in Geumjeongsan, Busan. In the early days, it was only eradicated in Busan area due to the eradication of airplanes, early detection and elimination of infected trees, and active injection of trees around the infected tree. However, due to damage to beneficial insects such as bees by air spraying of insecticides, protests by environmental groups due to environmental pollution, and movement of victims by the people, there was no effective control, and in the end, A re-infectious disease was caused.
It is known that pine reeves do not only affect Pinus , but also some species of Abies , Picea and Larix . In Korea, Gyeongsang Province, Jeolla Province and Gangneung regions were predominantly in pine trees, while in the western part of Gangwon Province and in Gyeonggi Province, they were predominantly in pine trees. These differences in the tree-damaged species in the southern and central regions are known to be due to differences in the mediators that mediate pine tree re-growth. In the southern region, the densities of the dung beetles are high. In the central region, the densities of the northern dung beetles are high, which is presumed to be due to the fact that the preferred plants differ from each other.
Methods for controlling this plant disease include: 1) prophylactic injection of nematicide into a healthy tree, 2) aerial spraying of synthetic pesticides or antagonistic microorganisms for controlling the pest, 3) Such as disruption of the infected tree, combustion and fumigation. However, the injection method and infectious tree control method are not cost effective and can not be effectively applied due to the difficulty of controlling pine forest in high mountainous area. Synthetic insecticides are the most effective method for aerial spraying of medicines from May to July when medicines are mainly active, but they are not applicable to urban and nearby areas because of the side effects of synthetic insecticides. Although there are methods to control environmentally friendly mediators by the application of antagonistic microorganisms, it is not effective yet because it is less effective than synthetic pesticides. Although the effect of organic synthetic pesticides is excellent, there are problems such as reduction of benefit benefit due to continuous use and abuse, development of resistance and toxicity to make up. Accordingly, it is necessary to develop effective environmental control methods.
DISCLOSURE OF THE INVENTION The present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a method for controlling pesticides, such as pine nutrients, which damage pine cones including horticultural crops, edible crops, Have Solubilizing agent and a process for producing the same.
It is also an object of the present invention to provide a water-soluble or oil-soluble solubilizing agent of an insoluble compound having pesticidal activity against pine tree worms.
In order to solve the above-mentioned problems, the present invention relates to a method for the treatment of bursaphenlenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; Solubilizer is provided.
[Chemical Formula 1]
In Formula 1, R 1 and R 2 are each independently -H, -OH or -OCH 3 .
According to a preferred embodiment of the present invention, the compound represented by Formula 1 of the solubilization agent of the present invention may be a compound represented by Formula 1-1.
[Formula 1-1]
According to another preferred embodiment of the present invention, the solubilization agent of the present invention may be a water-soluble agent or an oil-soluble agent.
According to another preferred embodiment of the present invention, the compound represented by the above formula (1) of the solubilizing agent of the present invention is selected from the group consisting of Sparassis latifolia ) culture medium.
According to another preferred embodiment of the present invention, the culture broth of the solubilizing agent of the present invention may be cultured for 50 to 70 days at a pH of 4.0 to 7.0 and at a temperature of 15 to 20 ° C.
According to another preferred embodiment of the present invention, the medium of the solubilizing agent of the present invention is selected from potato starch, dextrose, yeast extract, peptone and distilled water water). < / RTI >
According to another preferred embodiment of the present invention, the compound represented by Formula 1 of the solubilizing agent of the present invention is obtained by dividing the culture of P. japonicus strain with an extraction solvent containing ethyl acetate (CH 3 COOC 2 H 5 ) May be derived from one ethyl acetate fraction.
According to another preferred embodiment of the present invention, the concentration of the compound represented by Formula 1 in the solubilizing agent of the present invention may be 120 ppm or more.
According to another preferred embodiment of the present invention, the solubilizing agent of the present invention can exhibit 80% to 100% live nematode rate after 72 hours of treatment based on the following formula (1).
On the other hand, the present invention relates to (1) a sparassis latifolia ) is fractionated with an extraction solvent containing ethyl acetate (CH 3 COOC 2 H 5 ) and concentrated to prepare crude extract; (2) separating the crude extract to prepare a compound represented by Formula 1; Having a nematocide activity on Bursaphelenchus xylophilus (Bursaphelenchus xylophilus), comprising: and (3) a compound represented by the above formula (1) Preparation step of preparing a solubilizing agent comprising as an active ingredient And a method for producing a solubilizing agent.
According to a preferred embodiment of the present invention, the compound represented by Formula 1 in the method for preparing a solubilizing agent of the present invention may be a compound represented by Formula 1-1.
[Formula 1-1]
According to another preferred embodiment of the present invention, the step (2) of the method for producing a solubilizing agent of the present invention comprises separating the crude extract through column chromatography or recrystallization, ≪ / RTI >
According to another preferred embodiment of the present invention, the step (3) of the method for preparing a solubilizing agent of the present invention comprises mixing the compound represented by Formula 1, a salt forming agent and a C 1 -C 4 alcohol solvent, And then dried to prepare a water-soluble solubilizing agent.
According to another preferred embodiment of the present invention, the salt formers of the solubilizing agent of the present invention are selected from the group consisting of sodium hydroxide (NaOH), sodium hydrogencarbonate (NaHCO 3 ), sodium chloride (NaCl), sodium carbonate (Na 2 CO 2 ) And sodium peroxide (Na 2 O 2 ).
According to another preferred embodiment of the present invention, the step (3) of the method for producing a solubilizing agent of the present invention comprises dissolving and dispersing a compound solution containing the compound represented by the formula (1), an oil, a fatty acid and a surfactant in a solvent And an oil-soluble solubilizing agent can be prepared using a spray dryer.
[Equation 1]
The solubilizing agent having the nematicidal activity of the present invention and the method for producing the same can have a nematocidal activity against the pine tree rehabilitating pine tree which causes great damage to coniferous trees including various horticultural crops, edible crops and pine trees.
It is also possible to provide water-soluble or oil-soluble solubilizers of insoluble compounds having pesticidal activity against pine tree worms.
Hereinafter, the present invention will be described in more detail.
As described above, pine and pine trees are seriously damaged by the pine wilt disease caused by Bursaphelenchus xylophilus in Korea. This plant disease, also known as "pine tree aids", is not treated once it is infected and almost all pine trees are killed. In Korea, pine trees are mainly damaged in the southern region, and pine trees are mainly damaged in the central region.
Methods for controlling this plant disease include: 1) prophylactic injection of nematicide into a healthy tree, 2) aerial spraying of synthetic pesticides or antagonistic microorganisms for controlling the pest, 3) Such as disruption of the infected tree, combustion and fumigation. However, the injection method and infectious tree control method are not cost effective and can not be effectively applied due to the difficulty of controlling pine forest in high mountainous area. Synthetic insecticides are the most effective method for aerial spraying of medicines from May to July when medicines are mainly active, but they are not applicable to urban and nearby areas because of the side effects of synthetic insecticides. Although there are methods to control environmentally friendly mediators by the application of antagonistic microorganisms, it is not effective yet because it is less effective than synthetic pesticides. Although the effect of organic synthetic pesticides is excellent, there are problems such as reduction of benefit benefit due to continuous use and abuse, development of resistance and toxicity to make up.
Accordingly, it is necessary to develop a new and effective environmentally friendly control method.
The solubilizing agent having the nematicidal activity of the present invention and the method for producing the same can have a nematocidal activity against the pine tree rehabilitating pine tree which causes great damage to coniferous trees including various horticultural crops, edible crops and pine trees.
It is also possible to provide water-soluble or oil-soluble solubilizers of insoluble compounds having pesticidal activity against pine tree worms.
The solubilizing agents of the present invention comprises a compound represented by the following general formula (1) as an active ingredient, and has a nematocide activity on Bursaphelenchus xylophilus (Bursaphelenchus xylophilus).
[Chemical Formula 1]
In Formula 1, R 1 and R 2 are each independently -H, -OH or -OCH 3 .
The compound represented by the above formula (1) is generally a poorly soluble substance, and it is advantageous for the insecticidal activity only when the poorly soluble substance is dissolved. Therefore, the effect of the present invention, that is, Bursaphelenchus xylophilus , the solubilizing agent of the present invention is advantageous in that it is solubilized by solubilizing the compound represented by the above formula (1), and the solubilizing agent of the present invention contains the solubilized compound represented by the formula (1) as an active ingredient can do.
In addition, the compound represented by Formula 1 may be a compound represented by Formula 1-1.
[Formula 1-1]
In addition, the solubilizing agent may be a water-soluble agent or an oil-soluble agent.
When the solubilizing agent is a water-soluble agent, it may be in a salt form composed of the compound represented by the formula (1), preferably the compound represented by the formula (1-1) -solubility) and can be used as a solubilizing agent having nematicidal activity against the pine tree.
When the solubilizing agent is an oil-soluble agent, a solution containing the compound represented by the formula (1), preferably the compound represented by the formula (1-1) and an oil, a fatty acid and a surfactant is dissolved in a solvent And / or dispersed in water, and may be a solid content prepared using a spray dryer, and the solid content can be used as a solubilizing agent having an aldehyde activity against pine tree worms by oil-solubility.
On the other hand, the compound represented by the above formula (1) Zinnia mushrooms (Sparassis latifolia ) culture medium.
The blossoms mushroom is edible mushrooms belong to the Democratic name mushroom neck (Aphyllophorales), zinnia and mushrooms (Sparassidaceae), zinnia mushrooms in (Sparassis). In Japanese, it is called Hanabiratake, and in English it is called cauliflower mushroom because its fruiting body is 10 ~ 25㎝ in size and has a pale yellow or white as a whole (Shin HJ et al., J. Life Sci. 17, 1290-1293, 2007). The mushroom is mainly distributed in Korea, Japan, China, North America, Europe and Korea. It grows in stems and stumps near the root of conifer in May to September, and is found mainly in July (Oh DS et al , Korean J. Mycol. 37, 33-40, 2009).
The mushrooms are classified into seven species according to the DNA test results.
Sparassis spathulata Is broadly sprouted in the eastern United States. It grows vertically as a regular, flat fan-shaped gab with a tee-shaped pattern from the ground-buried and branching bases, There is no clamp connection between the cells.
Sparassis radicata Is characterized by the presence of a cramped connection between mycelial cells and cells. It is characterized by the fact that it grows in the western part of the United States, and the fan-shaped petals (gut) irregularly curl a lot.
Sparassis crispa Is a species that grows mainly in Europe and the eastern United States and has a cusp-shaped connection between mycelial cells and cells. It is characterized by a fan-shaped petal that grows from repeatedly divided short branches.
Sparassis brevipes , Which has a scientific name, is mainly grown in northern Europe and has features that do not have a crotch connection part of mycelial cells. Sparassis miniensis Which has a scientific name, is predominantly in the northwestern region of Spain and is characterized by the absence of the crotch connecting portion of mycelial cells.
Sparassis cystidiosa Blossom mushroom with the scientific name of which is mainly characterized by the angle of the connecting portion of mycelial cells dotgo in Thailand, Sparassis latifolia Which has a scientific name, is mainly grown in Korea, Japan, China, Russia and Far East Asia region, and has a characteristic feature of the connecting part of mycelial cells.
In the present invention, all of the seven kinds of mushrooms described above can be used, and preferably, Sparassis latifolia May be used. More preferably, a Japanese mushroom having a scientific name of Sparassis latifolia which is inhabited in Korea can be used.
The above-mentioned mushroom ( Sparassis latifolia ) is a culture medium obtained by isolating and identifying P. japonicus mushroom strain, wherein the mushroom strain is cultivated at a pH of 4.0 to 7.0, preferably at pH 4.0 to 5.0, at 15 to 30 ° C, preferably at 20 to 25 ° C For 50 to 70 days, preferably 55 to 65 days, but not always limited thereto.
In other words, Sparassis latifolia ) culture medium can be used without limitation as long as it is a culture condition capable of maintaining a high level of live nematode activity against the pine reovirus ( Bursaphelenchus xylophilus) .
On the other hand, when the culture is carried out at a temperature lower than 15 캜, the growth of the strain is very slow, which may cause problems of long-term culture. When the culture is carried out at a temperature exceeding 30 캜, A problem of death may occur.
In addition, when the culture is cultured at a pH lower than 4.0, there is a problem that the growth is slow and the cultivation time is prolonged. When the culture is performed at pH exceeding 7.0, the growth is drastically slowed, Lt; / RTI >
The culture medium for cultivating the mushroom strain may be selected from a variety of media available in the art, such as potato starch, dextrose, yeast extract, peptone and distilled water Distilled water may be used. More preferably, a PDB (potato dextrose broth) medium containing potato starch, dextrose, and distilled water is used. Can be used.
The above-mentioned mushroom ( Sparassis latifolia ) culture broth will be described in more detail. Sparassis latifolia ) strain is cultured in a potato dextrose agar (PDA) medium containing potato starch, dextrose, distilled water and agar at 15 ° C to 30 ° C, Cultured in a medium at 20 ° C to 25 ° C for 20 days to 40 days, preferably for 25 days to 35 days, and then cultured at a pH (including potato starch), dextrose and distilled water The cells may be cultured in a medium of PDB (potato dextrose broth) of 4.0 to 7.0, sterilized at 100 ° C to 150 ° C for 10 to 30 minutes, and cultured at 18 to 30 ° C for 50 to 70 days.
Furthermore, the compound represented by the formula (1) of the present invention may be derived from an ethylacetate fraction obtained by fractionating the culture of P. japonicus with an extraction solvent containing ethyl acetate (CH 3 COOC 2 H 5 ) Wherein the compound represented by the above formula (1) derived from the fractions is more effective than the compound represented by the above-mentioned formula (1) derived from the culture of the mushroom strain of Bombyx mori, against Bursaphelenchus xylophilus ) may be more excellent.
Meanwhile, the solubilizing agent of the present invention may have a concentration of the compound represented by the formula (1) of 120 ppm or more, and 80 to 100% of the carnivorous rate after 72 hours of treatment based on the following formula (1).
[Equation 1]
Further, the present invention relates to a method for producing a pine bug ( Bursaphelenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; A method for producing a solubilizing agent is as follows.
First, in step (1) of the method for preparing a solubilizing agent, the culture medium of Sparassis latifolia strain is fractionated with an extraction solvent containing ethyl acetate (CH 3 COOC 2 H 5 ), and concentrated to obtain crude extract .
Specifically, in step (1), the culture of P. japonicus strain cultured for 40 days to 80 days is filtered through a filter paper, and the filtered filtrate is transferred to a separatory funnel and ethyl acetate (CH 3 COOC 2 H 5 ) And the mixture can be fractionated by stirring. The crude extract can be prepared by fractionating the ethyl acetate layer with a vacuum evaporator.
Next, the crude extract is separated in step (2) of the method for preparing a solubilizing agent to prepare a compound represented by the following formula (1).
[Chemical Formula 1]
In Formula 1, R 1 and R 2 are each independently -H, -OH or -OCH 3 .
The compound represented by Formula 1 may be a compound represented by Formula 1-1.
[Formula 1-1]
Specifically, the step (2) can be carried out by separating the crude extract through column chromatography to prepare the compound represented by the formula (1). More specifically, the crude extract is dissolved in acetonitrile ) Solvent, followed by separation using preparative column chromatography.
The acetonitrile solvent means acetonitrile and may be 100% acetonitrile or mixed with other solvent.
In this case, water, alcohol, ethyl acetate, chloroform, hexane and the like can be preferably used as other solvent. When water is used as a solvent for the acetonitrile solvent, 60 to 90% by weight, preferably 65 to 85% % Aqueous solution of acetonitrile may be used.
Meanwhile, in step (2), the crude extract can be separated by recrystallization to prepare the compound represented by the formula (1). Specifically, the crude extract is dissolved in an alcohol solvent, At 0 캜 to 8 캜 to induce crystal nuclei, and then to crystallize and recrystallize the compound to produce the compound represented by the above formula (1). The recrystallized compound of Formula 1 may further comprise a filter.
The alcohol solvent may be any alcohol selected from the group consisting of C 1 -C 4 lower alcohols, preferably methanol. The alcohol solvent of the present invention means alcohol, and may be 100% alcohol or mixed with other solvent. In this case, water, ethyl acetate, chloroform, hexane and the like can be preferably used as other solvents.
Next, a solubilizing agent containing, as an active ingredient, the compound represented by the formula (1) prepared in the step (2) in the step (3) of the method for producing a solubilizing agent is prepared.
Specifically, in step (3), a water-soluble solubilizing agent may be prepared by mixing the compound represented by Formula 1, a salt forming agent, and a C 1 -C 4 alcohol solvent, followed by drying.
The water-soluble solubilizing agent may be in the form of a salt composed of the compound represented by the formula (1), and the salt forming agent may be selected from the group consisting of sodium hydroxide (NaOH), sodium hydrogencarbonate (NaHCO 3 ) Sodium carbonate (Na 2 CO 2 ) and sodium peroxide (Na 2 O 2 ), preferably sodium hydroxide (NaOH). The C 1 -C 4 alcohol solvent may be any one selected from the group consisting of C 1 -C 4 lower alcohols, but ethanol may be preferably used. The alcohol solvent of the present invention means alcohol, and may be 100% alcohol or mixed with other solvent. In this case, water, ethyl acetate, chloroform, hexane and the like can be preferably used as other solvents.
More specifically, in step (3), 1 to 10 mg of the compound represented by the formula (1), 50 to 150 mM of sodium hydroxide (NaOH) and 0.1 to 10 ml of an ethanol solvent are mixed at 50 ° C to 90 ° C, And then dried to prepare a water-soluble solubilizing agent in the form of a compound / sodium salt represented by the general formula (1).
In step (3), the compound of formula (1), the oil, the fatty acid and the surfactant may be dissolved and dispersed in a solvent and may be dissolved in an oil-soluble solvent using a spray drier. A subject can be produced.
At this time, the oil-soluble solubilizing agent may be in a solid-state form.
The oil may also be a phospholipid such as caprylic / capric triglyceride, alpha-bisabolol, tocopheryl acetate, liposome, phosphatidylcholine, etc. phospholipid, di-c12-13 alkyl malate, coco-captylate / caprate, cetyl octanoate and hydrogenated castor oil. (hydrogenated castor oil).
The fatty acid may also be selected from the group consisting of oleic acid, stearyl alcohol, myristic acid, linoleic acid or lauric acid, capric acid, caprylic acid, caprylic acid, caproic acid, and the like.
Surfactants are hydrophilic or lipophilic substances in the presence of polar or non-polar groups present in the molecule, or they exist in a state where they can maintain equilibrium between these two extremes. They are hydrophilic-lipophilic (HLB) balance) and can be used as a key index in the screening of various surfactants. The surfactant is used as an emulsifier, a surface adsorbent, a penetrating agent and a dispersing agent by lowering the interfacial tension between two interfaces such as water-oil. Particularly when the critical micelle concentration is higher than the critical micelle concentration, solubility of the poorly soluble drug It can be widely used for increasing the utilization rate.
The surfactant may be selected from the group consisting of sodium lauryl sulfate and its derivatives, poloxamer and its derivatives, intermediate chain triglyceride (MCT), labrasol, transcutol, polyoxyethylene sorbitan monolaurate (hereinafter referred to as " Tween 20 "), polyoxyethylene sorbitol, polyoxyethylene sorbitol, (Tween 40), polyoxyethylene sorbitan monostearate (hereinafter Tween 60), and polyoxyethylene sorbitan monooleate (hereinafter Tween 80)), sorbitan esters (e.g., (Hereinafter referred to as Span 20), sorbitan monopalmitate (hereinafter referred to as Span 40), sorbitan monostearate (hereinafter referred to as Span 60), sorbitan monooleate (hereinafter referred to as Span 80), sorbitan monolaurate Non-cancer triturate (hereinafter referred to as Span 25 ) Sorbitan trioleate (hereinafter Span 85) sorbitan tristearate (hereinafter referred to as Span 65)], cremophor, PEG-60 hydrogenated castor oil, PEG- 40 hydrogenated castor oil, sodium lauryl glutamate, and disodium cocoamphodiacetate, preferably at least one of an anionic interface Tween 20, Tween 40, Tween 60, Tween 80, sorbitan esters Span 20, Span 25, Span 40, Span 60, Span 65, Span (surfactant), sodium lauryl sulfate (SDS) 80, and Span 85, and more preferably, at least one of sodium lauryl sulfate and Tween 80. [
Further, the solvent may include at least one of water, acetone, and ethanol.
Also, the spray dryer may be used by spraying at a preheating time of 10 minutes to 50 minutes, a spray drying speed of 3 to 7 ml / min, and an injection temperature of 70 to 150 ° C.
Further, the above step (3) may be carried out by mixing 0.2 to 0.3 g of the product "Tween 80" (manufactured by Uniqema) and "Tween 20" (manufactured by Uniqema) having a volume ratio of 0.8 to 1.2 g, polyethylene glycol (PEG) having a weight average molecular weight of 600 and polyethylene glycol (PEG) having a weight average molecular weight of 400, 0.2 to 0.3 g, trade name " Arlacel 165 " (Atlas Powder (Manufactured by Sigma-aldaich), trade name "Kolliphor SR" (manufactured by Sigma-aldaich) or trade name "Kolliphor 25" (manufactured by Sigma-aldaich) were dissolved in 16 to 24 ml of an ethanol solvent, ) Was added to prepare a mixed solution.
To the prepared mixed solution, 0.8-1.2 g of sodium lauryl sulfate is added and dissolved in 8 to 12 mL of water to prepare an oil-soluble solubilizing agent.
The present invention will now be described more specifically with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, and should be construed to facilitate understanding of the present invention.
[ Example ]
Preparation Example 1: Isolation and Identification of Staphylococcus aureus
From the Forest Research Institute of Chemical Laboratory Microbiological and mushrooms neck Material morphological classification and blossoms mushrooms (Sparassis through ITS analysis latifolia ) strain KFRI 747 strain (Strain) was used.
Preparation Example 2 : Pine Isolation and identification
Nematodes were isolated from the wood of pine woodworm infected with Berkman funnel method. Pine woodworms were isolated purely from the isolated nematodes using the morphology and genetic characteristics of pine woodworms.
The purely isolated pine needles were cultivated by subculture in the medium after pre - culture of the gray mold disease ( Botrytis cineria ) without sclerotia.
Example One
(1) Sparassis mushroom ( Sparassis) latifolia ) was cultivated in a culture room at 23 占 폚 for 30 days using a potato dextrose agar (PDA) medium containing potato starch, dextrose, distilled water and agar The cells were inoculated into a PDB (potato dextrose broth) medium containing Potato Starch, dextrose and distilled water and cultured at 23 ° C for 60 days to obtain Sparassis latifolia ) Culture broth.
(2) Mushrooms produced by sparassis latifolia ) was filtered with a filter paper (Watman, C5), and the filtrate was fractionated with ethyl acetate (CH 3 COOC 2 H 5 ) using a separating funnel. The ethyl acetate layer was evaporated using a evaporator ) To obtain 3 g of crude extract.
(3) The crude extract thus prepared was dissolved in an 80% by weight aqueous solution of acetonitrile, and Compound 1 was isolated and purified by preparative column chromatography (Flash chromatography system, Japan).
In this case, ODS-A (40 mm × 200 mm, YMC-GEL, S-75 μm, Japan) was used as a separating filler resin, and an EYELA ceramic pump (VSP-3050) and a UV moniter Respectively.
(4) Separation and Purification Compound 1 1 mg, 100 mM sodium hydroxide (NaOH) and 1 ml of an ethanol solvent were mixed at 70 캜 and completely dried using a drying device to obtain a water- soluble solubilizing agent.
Example 2
(1) Sparassis mushroom ( Sparassis) latifolia ) was cultivated in a culture room at 23 占 폚 for 30 days using a potato dextrose agar (PDA) medium containing potato starch, dextrose, distilled water and agar The cells were inoculated into a PDB (potato dextrose broth) medium containing Potato Starch, dextrose and distilled water and cultured at 23 ° C for 60 days to obtain Sparassis latifolia ) Culture broth.
(2) Mushrooms produced by sparassis latifolia ) was filtered with a filter paper (Watman, C5), and the filtrate was fractionated with ethyl acetate (CH 3 COOC 2 H 5 ) using a separating funnel. The ethyl acetate layer was evaporated using a evaporator ) To obtain 3 g of crude extract.
(3) The crude extract thus prepared was dissolved in an 80% by weight aqueous solution of acetonitrile, and Compound 1 was isolated and purified by preparative column chromatography (Flash chromatography system, Japan).
In this case, ODS-A (40 mm × 200 mm, YMC-GEL, S-75 μm, Japan) was used as a separating filler resin and an EYELA ceramic pump (VSP-3050) and a UV moniter .
(4) Separation and Purification Compound 1.0 g, Tween 80 / Tween 20 (0.25 g), PEG 600 / PEG 400 (1: 1, v / v) 0.25 g, sodium lauryl sulfate 1.0 g, Arlacel 165 and 0.25 g of Kollidon 25 were dissolved and dispersed in 10 ml of water, and the mixture was preheated for 30 minutes using a spray dryer (Buchi B-290), at a spray drying rate of 5 ml / min, at 110 占 폚 To prepare an oil-soluble solubilizing agent in the form of a solid powder.
Experimental Example 1: Structural analysis of compound 1
Compound 1 isolated in Example 1 and Example 2 was analyzed by thin layer chromatography (TLC) and i-class UHPLC (Water). The separation solvent was 50 wt% acetonide Was analyzed by gradient elution in aqueous 80% by weight acetonitrile solution for 30 minutes.
The molecular weight of Compound 1 was determined in a negative mode using an LC / MS spectrometer (API-2000, AB) and electrospray ionization mass spectrometry (ESI-MS) Using nuclear magnetic resonance (NMR), it was confirmed that the crystal structure was as follows.
Compound 1:
1) Properties: Crystal (Crystal)
2) Molecular weight: 196 g / mol
3) Molecular formula: C 10 H 12 O 4
4) 1 H NMR (600 ㎒ , CD 3 OD): δ6.3 (1H, d, J = 2.4 Hz, H-5), δ6.2 (1H, m, H-3), δ3.9 (3H , s, -OMe),? 3.7 (3H, s, -OMe),? 2.5 (3H, s,
5) 13 C NMR (150 MHz, CD 3 OD):? 24.4 (C-7),? 55.2 (-OMe x 2),? (C-5),? 143.1 (C-6),? 163.8 (C-4),? 165.6 (C-2),? 171.7
6) ESI-MS m / z 195 [MH] -
Experimental Example 2
The pine reeves of Preparation Example 2 were suspended in sterile distilled water and then transferred to a sterilized container in 0.5 mL of suspension containing about 100 of the pine reeves.
Next, a chemically water-soluble substance obtained by solubilizing the solubilizer prepared in Example 1 was added to the above-mentioned container, and a carnivorous activity test was carried out.
The solubilized material was added at 40 ppm, 80 ppm, 100 ppm, 120 ppm, 140 ppm, 160 ppm, 180 ppm, 200 ppm, 220 ppm, 240 ppm and 260 ppm, respectively, of the solubilizer prepared in Example 1.
The live nematode activity assay was carried out by observing the container with a microscope and confirming the number of dead pine reindeer.
The percentage of live nematodes (%) was measured by the following formula (1), and the results are shown in Table 1.
[Equation 1]
Experimental Example 3
The pine reeves of Preparation Example 2 were suspended in sterile distilled water and then transferred to a sterilized container in 0.5 mL of suspension containing about 100 of the pine reeves.
A substance obtained by chemically solubilizing the solubilizing agent prepared in Example 2 was added to the above-mentioned container to carry out a test for activity of nematode.
The solubilized material was added with 40 ppm, 80 ppm, 100 ppm, 120 ppm, 140 ppm, 160 ppm, 180 ppm, 200 ppm, 220 ppm, 240 ppm and 260 ppm, respectively, of the solubilizing agent prepared in Example 1.
The nematode activity assay was carried out by observing the vessel with a microscope and confirming the number of dead pine reindeer. After 72 hours, it was measured using tweezers.
The percentage of live nematodes (%) was measured by the following formula (1), and the results are shown in Table 1.
[Equation 1]
ppm
ppm
ppm
ppm
ppm
ppm
ppm
ppm
ppm
ppm
ppm
As shown in Table 1, when the amount of the solubilizer prepared in Examples 1 and 2 was 100 ppm or more, it was confirmed that the activity of the nematode was confirmed.
In addition, it was confirmed that more than 120 ppm of the solubilizing agent prepared in Examples 1 and 2 showed 90% or more of the live nematode rate after 72 hours from the nematode activity test.
Claims (15)
[Chemical Formula 1]
In Formula 1, R 1 and R 2 are each independently -H, -OH or -OCH 3 .
Wherein the compound represented by the formula (1) is a compound represented by the following formula (1-1): Bursaphelenchus xylophilus ) . & lt ; / RTI & gt ;
[Formula 1-1]
Characterized in that the solubilizing agent is a water-soluble agent or an oil-soluble agent. Bursaphelenchus xylophilus ) . & lt ; / RTI & gt ;
The compound represented by the above formula (1) Zinnia mushrooms (Sparassis The present invention relates to a pine tree bush ( Bursaphelenchus) xylophilus ) . & lt ; / RTI & gt ;
Wherein the culture broth is cultivated in a medium of pH 4.0 to 7.0 and 15 to 20 ° C for 50 to 70 days.Bursaphelenchus xylophilus)≪ / RTI > Solubilizing agent.
Wherein the medium comprises at least one of potato starch, dextrose, yeast extract, peptone, and distilled water.Bursaphelenchus xylophilus )≪ / RTI > Solubilizing agent.
The compound represented by Formula 1 is derived from an ethyl acetate fraction obtained by fractionating a culture of P. japonicus strain with an extraction solvent containing ethyl acetate (CH 3 COOC 2 H 5 ). Bursaphelenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; Solubilizing agent.
Wherein the concentration of the compound represented by the formula (1) is 120 ppm or more. Bursaphelenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; Solubilizing agent.
Characterized in that the solubilizer exhibits 80-100% live nematode after 72 hours of treatment according to the following formula (1): Bursaphelenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; Solubilizing agents;
[Equation 1]
(2) separating the crude extract to prepare a compound represented by Formula 1 below; And
(3) preparing a solubilizing agent comprising a compound represented by the following formula (1) as an active ingredient;
( Bursaphelenchus < RTI ID = 0.0 > RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; A method for producing a solubilizing agent;
[Chemical Formula 1]
In Formula 1, R 1 and R 2 are each independently -H, -OH or -OCH 3 .
Wherein the compound represented by the formula (1) is a compound represented by the following formula (1-1): Bursaphelenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; A method for producing a solubilizing agent.
[Formula 1-1]
Wherein the step (2) comprises separating the crude extract through column chromatography or recrystallization to produce a compound represented by the formula (1), wherein the Bursaphelenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; A method for producing a solubilizing agent.
Wherein the step (3) comprises mixing a compound represented by the formula (1), a salt forming agent and a C 1 -C 4 alcohol solvent, followed by drying to prepare a water-soluble solubilizing agent Bursaphelenchus RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; A method for producing a solubilizing agent.
The salt-forming agent is sodium hydroxide (NaOH), sodium hydroxide (NaOH), sodium bicarbonate (NaHCO 3), sodium chloride (NaCl), sodium carbonate (Na 2 CO 2), and sodium peroxide (Na 2 O 2) at least one of ( Bursaphelenchus < RTI ID = 0.0 > RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; A method for producing a solubilizing agent.
In step (3), the compound solution containing the compound represented by Formula 1, oil, fatty acid, and surfactant is dissolved and dispersed in a solvent, and an oil-soluble solubilizing agent is added thereto using a spray drier ( Bursaphelenchus < RTI ID = 0.0 > RTI ID = 0.0 & gt; xylophilus & lt ; / RTI & gt; A method for producing a solubilizing agent.
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| KR101827442B1 (en) * | 2017-11-22 | 2018-02-08 | 비앤에스코퍼레이션 주식회사 | Nematocidal Composition Comprising Alkyl Amine and Fatty Acid |
| CN114632338A (en) * | 2022-03-28 | 2022-06-17 | 河北惠尔信新材料有限公司 | Titanium dioxide washing mother liquor concentration treatment system |
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| KR20090057503A (en) | 2007-12-03 | 2009-06-08 | 한국화학연구원 | Composition for control of pine wilt disease using nematocidal agents and crude extracts from the fruit rind of bombay mace and its controling process |
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| KR20090057503A (en) | 2007-12-03 | 2009-06-08 | 한국화학연구원 | Composition for control of pine wilt disease using nematocidal agents and crude extracts from the fruit rind of bombay mace and its controling process |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR101827442B1 (en) * | 2017-11-22 | 2018-02-08 | 비앤에스코퍼레이션 주식회사 | Nematocidal Composition Comprising Alkyl Amine and Fatty Acid |
| CN114632338A (en) * | 2022-03-28 | 2022-06-17 | 河北惠尔信新材料有限公司 | Titanium dioxide washing mother liquor concentration treatment system |
| CN114632338B (en) * | 2022-03-28 | 2024-01-19 | 河北惠尔信新材料股份有限公司 | Titanium white water washing mother liquor concentration processing system |
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