KR20220120955A - Composition for controlling plant diseases comprising Polyalthia longifolia extract or fraction thereof - Google Patents

Abstract

The present invention provides a composition for controlling plant diseases comprising an extract of Polyalthia longifolia or a fraction thereof. The composition for controlling plant diseases comprising an extract of Polyalthia longifolia or a fraction thereof is a natural product, harmless to the human body, and biodegradable in the natural world not to cause environmental pollution. In addition, the composition is effective in controlling plant diseases to be developed as an environmentally friendly biological pesticide and can be usefully used in production of high value-added organic products. The extract of Polyalthia longifolia or the fraction thereof may include a compound represented by any one of formulas 1 to 15.

Description

Composition for controlling plant diseases comprising Polyalthia longifolia extract or fraction thereof

Polyalthia Longifolia longifolia ) It relates to a composition for controlling a plant disease containing an extract and a fraction thereof as an active ingredient, and a method for controlling a plant disease using the composition.

Plant diseases are one of the biological hazard factors that hinder the healthy growth of plants, and affect agricultural production by impairing crop yield and quality. If a large-scale plant disease occurs in a large-scale cultivation area, the crop yield will be greatly reduced, resulting in a large economic loss, and if there is a disruption in the main grain production, it will be a great threat in terms of food security. Serious social problems can arise.

In modern agriculture, chemical pesticides have been used as convenient means for controlling various plant diseases and minimizing losses by effectively managing plant diseases within an economic threshold range. However, as a result of increasing agricultural production by reliance on chemical pesticides for the past several decades, drug-resistant pathogens and pests appeared, concerns about human risk to livestock toxicity, and ecological disturbance due to residual toxicity occurred. There is a need to develop a new plant disease control means to reduce the use of and replace it.

Biopesticides are eco-friendly plant disease control means used to control pathogens, pests and weeds using natural substances derived from nature, and plant extracts and microorganisms are used as main materials.

Plants biosynthesize various secondary metabolites such as alkaloids, terpenoids, flavonoids, glycosides, quinones, coumarins, and phenolic compounds in the body. Some of these substances act as antibiotics or phytoalexin to prevent invasion of pathogens or inhibit the feeding of pests. Rosemary oil (trade name: Milsana), rosemary oil (trade name: Sporan), and tea tree oil (trade name Timorex) are commercially available as bio crop protection agents. A number of studies have been reported.

Polyalthia longifolia (Sonn.) Thwaites belonging to the Annonaceae is a common name of Monoon longifolium (Sonn.) B.Xsu & RMKSaunders, and is a common name for Indian mast tree or Called ulta ashoka tree, it is native to India and is widely distributed in tropical and subtropical regions. In Asia, it was used as a folk remedy to treat skin diseases, fever, high blood pressure, and parasitic diseases, and in India, the bark was used as an antipyretic. Plant extracts are mainly composed of diterpenoids, which exhibit various pharmacological activities such as insect feeding inhibition, opioid receptor detection, cytotoxicity, antimicrobial, anti-inflammatory, lipid lowering, nerve growth, and acetylcholine esterification. known to contain

The present inventors tried to use a polyalthia longifolia plant extract as an environmentally friendly plant disease control means.

Korean Patent Publication 10-2012-039702 (2012.04.25)

One object of the present invention is polyalthia longifolia ( Polyalthia longifolia ) to provide a composition for controlling plant diseases comprising an extract or a fraction thereof.

Another object of the present invention is polyalthia longifolia ( Polyalthia longifolia ) A plant disease control method comprising the step of treating a plant, its seed or its habitat, a composition for controlling plant disease comprising an extract or a fraction thereof will do

The present inventors were investigating the control effect of various plant extracts on various plant diseases in order to develop an eco-friendly plant disease control agent, and it was found that Polyalthia longifolia has an excellent control effect on various plant diseases.

The terminology used in the present application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In the present application, terms such as “comprise” or “have” are intended to designate that a feature, step, structure, or a combination thereof described in the name exists, but one or more other features, steps, structure, or these It should be understood that it does not preclude the possibility of the existence or addition of combinations.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

polyaltia Longifolia ( Polyalthia longifolia ) extract or its fraction containing plant disease Controlling composition, and manufacturing method thereof

In one aspect of the present invention, the present invention provides a composition for controlling plant diseases comprising a polyalthia longifolia extract or a fraction thereof.

In the present specification, the term "control" is intended to be used in the meaning of preventing and avoiding diseases or pests, as well as removing and killing them. However, since the control effect of the preventive treatment is the main control mechanism for plant disease control, the control effect of the Polyalthia longifolia extract or its fractions on plant diseases on plant diseases was tested as a preventive treatment.

In the present invention, the composition is derived from a natural product, is harmless to the human body and does not cause environmental pollution, and has an excellent control effect against plant diseases such as rice blast disease, tomato gray mold disease, tomato late blight, wheat red rust, and red pepper anthrax. and the scope of the plant disease of the present invention is not limited thereto.

In addition, the composition is a Brassica crop black pattern bung ( Alternaria ) brassicicola ), gray mold bacillus ( Botrytis cinerea ), cucumber black star blight ( Cladosporium cucumerinum ), chilli anthrax ( Colletotrichum ) coccodes ), ginseng root rot ( Cylindrocarpon destructans ), tomato wilt disease ( Fusarium ) oxysporum f. sp. lycopersici ), Rice Blast Bacteria ( Magnaporthe oryzae ), potato / tomato late blight ( Phytophthora infestans ) It has a control effect on plant diseases caused by phytopathogenic fungi, such as, preferably, Brassica crops black pattern byeong ( Alternaria brassicicola ), cucumber black star blight ( Cladosporium ) cucumerinum ), pepper anthrax ( Colletotrichum coccodes ), tomato wilt disease ( Fusarium ) oxysporum f. sp. lycopersici ), Rice Blast Bacteria ( Magnaporthe oryzae ), potato / tomato late blight ( Phytophthora infestans ) has an excellent control effect against plant diseases caused by.

In addition, the composition is Phalaenopsis orchid bacterial brown spot disease ( Acidovorax) avenae subsp. cattleyae ), Agrobacterium tumefaciens ), Bacterial Rice Blight Bacterium ( Burkholderia glumae ), Pepper Ulcer Bacteria ( Clavibacter ) michiganensis subsp. michiganensis ), Phalaenopsis soft bacillus ( Dickea ) chrysanthemi ), vegetable blight blight ( Pectobacterium carotovorum subsp. carotovorum ), kiwi ulcer blight ( Pseudomonas syringae pv. actinidiae ), tomato blight blight ( Ralstonia ) solanacearum ), peach bacterial hole fungus ( Xanthomonas arboricola pv. pruni ) has a control effect on plant diseases caused by phytopathogenic bacteria, such as, preferably Phalaenopsis orchid bacterial brown spot blight ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis soft blight It has an excellent control effect on plant diseases caused by the fungus ( Dickea chrysanthemi ).

In the present invention, the extract is water, C ₁ to C ₄ lower alcohols (eg, methanol, ethanol, propanol, isopropanol, butanol, etc.), hexane, chloroform, methylene chloride, ethyl acetate, acetone, acetonitrile And a combination thereof may be extracted using a solvent, preferably C ₁ to C ₄ lower alcohol, more preferably methanol may be used as a solvent. However, in the present invention, the extraction solvent is not limited thereto.

In the present invention, the fraction of the extract may be fractionated using hexane, chloroform, ethyl acetate, methylene chloride, methanol, ethanol, butanol, water, and combinations thereof as a solvent, preferably chloroform, ethyl acetate and It may be fractionated using water as a solvent, and more preferably, the fraction of the present invention may be a chloroform fraction or an ethyl acetate fraction.

In one embodiment of the present invention, the chloroform fraction and ethyl acetate fraction of the polyalthia longifolia extract have excellent control effects, and the antimicrobial active material having a control effect may be a non-polar material rather than a water-soluble material. .

In the present invention, the extract may be a leaf, stem or root extract of Polyalthia longifolia , and the site for extracting the Polyalthia longifolia extract is not limited thereto.

In the present invention, the polyalthia longifolia ( Polyalthia longifolia ) extract or a fraction thereof may include a compound represented by any one of the following Chemical Formulas 1 to 15.

[Formula 1]

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[Formula 2]

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[Formula 3]

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[Formula 4]

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[Formula 5]

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[Formula 6]

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[Formula 7]

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[Formula 8]

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[Formula 9]

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[Formula 10]

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[Formula 11]

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[Formula 12]

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[Formula 13]

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[Formula 14]

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; and

[Formula 15]

.

.

In the present invention,

The compound represented by Formula 1 is polylongifoliaon C;

The compound represented by Formula 2 is polylongifoliaon D;

The compound represented by Formula 3 is polylongifoliaon E;

The compound represented by Formula 4 is polylongifoliaon F;

The compound represented by Formula 5 is polylongifoline A;

The compound represented by Formula 6 is 16 α -hydroxy-cleroda-3,13(14) Z -dien-15,16-olide (16 α -hydroxy-cleroda-3,13(14) Z -dien -15,16-olide);

The compound represented by Formula 7 is 3 α ,16-dihydroxy-cleroda-4(18),13(14) Z -dien-15,16-olide (3α,16-dihydroxy- cleroda -4) (18),13(14) Z -dien-15,16-olide);

The compound represented by Formula 8 is 3 β, 16-dihydroxy-cleroda-4 (18), 13 (14) Z -dien-15,16-olide (3 β 16-dihydroxy-cleroda-4 ( 18),13(14) Z -dien-15,16-olide);

The compound represented by Formula 9 is (4→2)-Aveo-16-hydroxy- cleroda -2,13(14) Z -dien-15,16-olide-3-al ((4→2) abeo -16-hydroxy-cleroda-2,13(14) Z -dien-15,16-olide-3-al);

The compound represented by Chemical Formula 10 is 3,12 E -kolavadien-15-oic acid-16-al (3,12 E -kolavadien-15-oic acid-16-al);

The compound represented by Formula 11 is 16-oxo-cleroda-3,13(14) E -dien-15-oic acid (16-oxo-cleroda-3,13(14) E -dien-15-oic acid) ;

The compound represented by Formula 12 is kolavenic acid;

The compound represented by Formula 13 is labd-13 E -en -8-ol-15-oic acid;

The compound represented by Formula 14 is longimide B; and

The compound represented by Chemical Formula 15 is called longimide B methyl ester.

In the present invention, the compounds represented by Chemical Formulas 1 to 15 may have bactericidal activity, and the “sterilization” may mean physically or chemically destroying all types of microorganisms such as mold, virus, and bacteria. and can also be referred to as sterilization.

In the present invention, the extract or its fraction may be included in the composition for controlling plant diseases at a concentration of 0.1 to 5000 μg/ml. The concentration represents the concentration contained in the composition when treating a plant, its seeds or its habitat in which a plant disease has occurred, and when the concentration is less than 0.1 μg/ml, the concentration of the extract or its fraction is too low to cause a plant disease There is a problem in that the control effect of the is lowered, and when the concentration is more than 5000 μg/ml, the concentration of the extract or its fraction is too high than necessary, which is uneconomical and may cause a negative impact on the environment. However, the amount of the composition for controlling plant diseases is not limited to the above concentration range, and may be appropriately adjusted in consideration of the type of plant pathogen or phytopathogenic bacteria, the degree of occurrence, the environment, and the like.

The composition for controlling plant diseases may be a polyaltia longifolia extract and a fraction thereof or a simple mixture of a bactericidal active compound isolated from the extract or fraction.

The composition of the present invention may be a mixture further containing an inert carrier, and a surfactant in the mixture so that the mixture can be formulated into an emulsion, emulsion, glidant, wettable powder, granulated wettable powder, powder, granule, etc. and other necessary adjuvants. The above-mentioned composition for controlling plant diseases by itself or by adding other inactive ingredients, the present invention can also be used as a seed treatment agent.

In the present invention, examples of the liquid carrier that can be used in the formulation include water; alcohols such as methanol and ethanol; ketones such as acetone and methyl ethyl ketone; aromatic hydrocarbons such as benzene, toluene, xylene, ethylbenzene and methylnaphthalene; aliphatic hydrocarbons such as hexane, cyclohexane, kerosene and light oil; esters such as ethyl acetate and butyl acetate; nitriles such as acetonitrile and isobutyrnitrile; ethers such as diisopropylether and dioxane; acid amides such as N,N-dimethyl formamide and N,N-dimethylacetamide; halogenated hydrocarbons such as dichloromethane, trichloroethane and carbon tetrachloride; dimethyl sulfoxide; and vegetable oils such as soybean oil and cottonseed oil.

Further, examples of solid carriers that can be used in the formulation include fine powders or granules such as minerals such as kaolin clay, attapulgite clay, bentonite, montmorillonite, acid white clay, pyrophyllite, talc, diatomaceous earth and talcite; natural organic substances such as corn leaf stalk powder and walnut hull powder; synthetic organic materials such as urea; salts such as calcium carbonate and ammonium sulfate; synthetic inorganic materials such as synthetic hydrated silicon oxide; As the liquid carrier, aromatic hydrocarbons such as xylene, alkylbenzene and methylnaphthalene; alcohols such as 2-propanol, ethylene glycol, propylene glycol and ethylene glycol monoethyl ether; ketones such as acetone, cyclohexanone and isophorone; vegetable oils such as soybean oil and cottonseed oil; petroleum aliphatic hydrocarbons, esters, dimethylsulfoxide, acetonitrile and water.

Further, examples of surfactants include anionic surfactants such as alkyl sulfate ester salts, alkylaryl sulfonate salts, dialkylsulfosuccinate salts, polyoxyethylene alkylaryl ether phosphate ester salts, lignosulfonate salts and naphthalene sulfo nate formaldehyde polycondensate; and nonionic surfactants such as polyoxyethylene alkyl aryl ethers, polyoxyethylene alkylpolyoxypropylene block copolymers and sorbitan fatty acid esters and cationic surfactants such as alkyltrimethylammonium salts.

Examples of other formulation aids include water-soluble polymers such as polyvinyl alcohol and polyvinylpyrrolidone, polysaccharides such as gum arabic, alginic acid and salts thereof, CMC (carboxymethyl-cellulose), xanthan gum, inorganic substances such as aluminum magnesium silicate and alumina sol ( alumina sol), preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxyltoluene).

In one embodiment of the present invention, it was confirmed through the Examples that the polyaltia longifolia extract and its fractions have a control effect on plant pathogens, and from this, the composition for controlling plant diseases including the extract or fraction is a plant It can be seen that there is a disease control effect.

The polyaltia longifolia extract or fraction may be prepared by a manufacturing method including the following steps, but is not limited thereto.

For example, preparing the extract may include the following steps:

1) extracting by adding an extraction solvent to polyaltia longifolia;

2) filtering the extract of step 1); and

3) Concentrating the filtered extract of step 2) under reduced pressure and drying it to prepare an extract of polyaltia longifolia.

For example, preparing the fraction may include the following steps:

1) extracting by adding an extraction solvent to polyaltia longifolia;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and drying it to prepare an extract of polyaltia longifolia; and

4) preparing a polyaltia longifolia fraction by additionally extracting the polyaltia longifolia extract of step 3) with an organic solvent.

In the above method, the polyaltia longifolia of step 1) may be used without limitation, such as cultivated or commercially available ones. The polyaltia longifolia may have leaves, stems or roots, but is not limited thereto.

The extraction solvent of step 1) may be any one selected from the group consisting of water, C ₁ to C ₄ lower alcohol, hexane, chloroform, methylene chloride, ethyl acetate, acetone and acetonitrile, or a mixed solution thereof, The present invention is not limited thereto. In addition, the lower alcohol may be methanol, ethanol, propanol, isopropanol or butanol.

As the extraction method, shaking extraction, Soxhlet extraction, or reflux extraction may be used, but the present invention is not limited thereto. The extraction solvent can be extracted by adding 1 to 10 times the amount of the dried polyalthia longifolia, and can be extracted by adding 2 to 3 times. The extraction temperature may be in the range of 20°C to 100°C, and may be in the range of 20°C to 40°C, and may be room temperature, but is not limited thereto. In addition, the extraction time may be 10 to 48 hours, may be 15 to 30 hours, may be 24 hours, but is not limited thereto. Furthermore, the number of times of extraction may be 1 to 5 times, and extraction may be repeated 3 to 4 times, and may be 3 times, but is not limited thereto.

In the above method, the vacuum concentration in step 3) may be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying may be vacuum drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

In the method, the organic solvent of step 4) may be any one selected from the group consisting of hexane, chloroform, ethyl acetate, methylene chloride, methanol, ethanol, butanol and water or a mixture thereof, but is not limited thereto. The fraction may be any one of a solvent fraction obtained by suspending the polyaltia longifolia extract in water and fractionating it with chloroform, ethyl acetate, or water, and may be a chloroform fraction or an ethyl acetate fraction, but is not limited thereto. The fraction may be obtained by repeating the fractionation process 1 to 5 times or 3 times from the polyaltia longifolia extract, and may be concentrated under reduced pressure after fractionation, but is not limited thereto.

The compounds represented by Formulas 1 to 15 are isolated from a fraction thereof of the polyaltia longifolia extract, and may have bactericidal activity.

In the present invention, a solvent fraction is prepared from the polyaltia longifolia extract, and the fractions are fractionated by liquid chromatography to separate the compounds represented by the formulas 1 to 15.

The liquid chromatography technique refers to a chromatography technique in which a mobile phase is a liquid, and is performed in a column filled with a stationary phase or a plane to which a stationary phase is attached. The polyalthia longifolia fraction is not limited as long as it is a solvent fraction, but may be a chloroform fraction or an ethyl acetate fraction. As the mobile phase, organic solvents such as water, hexane, methanol, ethanol, acetonitrile, acetone, chloroform, dichloromethane, and ethyl acetate may be used alone or in combination, and as the stationary phase, silica gel, Diaion HP-20 , RP-18 or Sephadex LH-20 may be used, but is not limited thereto.

The compound represented by any one of Chemical Formulas 1 to 15 of the present invention includes not only salts thereof, but also solvates, optical isomers, hydrates, and the like, which can be prepared therefrom by a conventional method.

In the present invention, as the “salt” of the compound represented by any one of Formulas 1 to 15, an acid addition salt formed by a free acid is useful. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, etc., aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. It is obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids, etc., and organic acids such as acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Examples of such salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, fluoride. , acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, Fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, Phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, βhydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.

In addition, the acid addition salt can be prepared by a conventional method, for example, by dissolving the compound represented by any one of Chemical Formulas 1 to 15 in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, etc. It can be prepared by filtering and drying the precipitate formed by adding an inorganic acid, or by distilling the solvent and excess acid under reduced pressure and drying it to crystallize it in an organic solvent.

Also, bases can be used to make metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and evaporating and drying the filtrate. In this case, it is pharmaceutically suitable to prepare a sodium, potassium or calcium salt as the metal salt. The corresponding salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

In the present invention, “isomer” includes “stereoisomer” or “enantiomer”, but stereoisomer may include each diastereomer or a mixture thereof, and optical isomer is Each of the enantiomers as well as mixtures of enantiomers and racemates are included. In the present invention, the compounds represented by Chemical Formulas 7 to 9 include enantiomers in a 1:1 ratio of R form and S form as a chiral carbon center, respectively.

The “hydrate” of the present invention refers to a compound represented by any one of Chemical Formulas 1 to 15 to which water is non-covalently bound by intermolecular forces, and may include a stoichiometric or non-stoichiometric amount of water. Specifically, the hydrate may contain water in a ratio of about 0.25 to about 10 moles based on 1 mole of the active ingredient, and more specifically, about 0.5 moles, about 1.5 moles, about 2 moles, about 2.5 moles, about It may include 3 moles, etc., but is not limited thereto.

The “solvate” of the present invention is one in which the compound represented by any one of Formulas 1 to 15 is bound to a solvent other than water by an intermolecular force, and may include a stoichiometric or non-stoichiometric amount of water. . Specifically, the hydrate may contain water in an amount of about 0.25 to about 10 moles based on 1 mole of the active ingredient, and more specifically, about 0.5 moles, about 1.5 moles, about 2 moles, about 2.5 moles, about It may include 3 moles, about 5 moles, etc., but is not limited thereto.

polyaltia Longifolia ( Polyalthia longifolia ) extract or its fraction containing plant disease using the control composition plant disease control method

In another aspect of the present invention, polyalthia longifolia ( Polyalthia longifolia ) plant disease control composition comprising an extract or a fraction thereof to a plant, its seed or its habitat; Plant disease control method comprising a; provides

The treatment may be indirect spraying of spraying the composition directly on the plant, spraying on the soil in which the plant is growing, or spraying on the medium for culturing the plant.

The control method of the present invention includes treatment of stems and leaves of plants, treatment of places where plants grow (eg soil), treatment of seeds such as seed sterilization/seed coating and treatment of roots.

As treatment of stems and leaves with the control method of the present invention, application on the surface of plants, such as, for example, spraying on stems and leaves, in particular may be included. As the treatment of the soil with the control method of the present invention, for example, spraying onto the soil, mixing with the soil, spraying the liquid treatment agent into the soil (irrigation of the liquid treatment agent, injection into the soil, dripping of the liquid treatment agent) can be included. Examples of places to be treated include planting holes, furrows, around replanting, around planting furrows, the entire surface of the growth site, the area between the soil and plants, the area between the roots, and the area under the stem of the plant. , main furrow, growing soil, nail bed. Includes a seedling box, a seedling tray, and a seedbed. The treatment may be performed before, at the time of, at the time of, immediately after, during the simulated cultivation period, before the cultivation and settlement, at the time of cultivation settlement, and at the growth period after cultivation settlement. In the above-mentioned soil treatment, the active ingredient may be simultaneously applied to the plant, or a solid fertilizer such as a paste fertilizer containing the active ingredient may be applied to the soil. The active ingredient may be mixed in an irrigation liquid, for example injected into an irrigation plant (irrigation tube, irrigation pipe, sprinkler, etc.), mixed in a liquid overflowing between furrows, or mixed into a water culture medium. can Alternatively, the irrigation liquid and active ingredient may be mixed beforehand and used for treatment by suitable irrigation methods, including, for example, the irrigation methods mentioned above and other methods such as spraying and flooding.

As the control method of the present invention, the volatilization treatment method is, for example, the soil in which the plant is cultured with the composition for controlling plant diseases of the present invention, and a hydroponic medium for culturing plants, a medium such as a seed bed, and volatilization of the sprayed composition. It is a method to protect the plant from pests and diseases through the method, and in addition, the composition can be placed around the plant to expose the plant to the composition in the volatilized gaseous state.

The seed treatment method in the control method of the present invention is, for example, a method of treating seeds to be protected from pests with the composition for controlling plant diseases of the present invention, and a specific example thereof is atomizing the suspension of the composition for controlling plant diseases of the present invention. and a spray treatment method of spraying on the seed surface; a spraying treatment method in which the wettable powder, emulsion, glidant, etc. of the composition for controlling plant diseases of the present invention is applied by itself or by adding a small amount of water to the seed surface; Impregnation treatment method of impregnating the seed in the solution of the composition for controlling plant diseases of the present invention for a specific period; film coating treatment and pellet coating treatment.

When a plant or soil for plant growth is treated with the compound according to the present invention, the treatment amount may vary depending on the type of plant to be treated, the type and frequency of occurrence of the pest to be controlled, the formulation form, the treatment period, climatic conditions, and the like.

Emulsions, wettable powders, glidants, etc. are usually diluted with water and then applied for treatment. In this case, the concentration of the active ingredient is usually in the range of 0.0001 to 3% by weight, preferably 0.0005 to 1% by weight. Powders, granules, and the like are usually used for treatment without dilution.

The control method of the present invention can be used in cultivated land or non-arable land such as paddy fields.

Polyalthia Longifolia longifolia ) extract, or a composition for controlling plant diseases containing a fraction thereof, is a natural product that is harmless to the human body and has an excellent effect in controlling plant diseases without causing environmental pollution as it is biodegradable in the natural world. and can be usefully used in the production of high value-added organic products.

1 is a polyalthia longifolia ( Polyalthia longifolia ) is a process diagram for isolating the active compound from the fraction of the extract.
2 is a polyalthia longifolia ( Polyalthia longifolia ) shows the structure of 15 compounds isolated from the extract fraction.
3 is polyalthia longifolia ( Polyalthia longifolia ) shows the three-dimensional structure of Formulas 1 to 5 isolated from the fraction of the extract.

Hereinafter, it will be described in detail by way of Examples.

However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

< Example 1> polyaltia Longifolia ( Polyalthia longifolia ) Preparation of a composition for controlling plant diseases comprising an extract

Step 1: Polyaltia longifolia ( Polyalthia longifolia ) Preparation of extract

Polyalthia Longifolia longifolia ) In order to prepare a composition for controlling plant diseases including the extract, polyaltia longifolia extract was first prepared. Specifically, dry polyalthia longifolia ( Polyalthia Longifolia ) cut into small pieces, add methanol, extract at room temperature for 24 hours, filter paper, and concentrate the filtrate under reduced pressure to obtain 702.9 g of polyalthia longifolia methanol extract. obtained.

Step 2: polyaltia Longifolia containing extract plant disease Preparation of a composition for control

2 ml of the extract sample of step 1 dissolved in methanol to a concentration of 60 mg/ml was diluted with 38 ml of distilled water, and Tween 20, an electrodepositing agent, was added to a concentration of 0.025% (v/v) to a concentration of 3000 μg/ml A composition solution (40 ml) for controlling plant diseases including a methanol extract of polyaltia longifolia was prepared.

< Example 2> polyaltia Longifolia containing extract plant disease Evaluation of plant disease control effect of control composition

Example 2-1: polyaltia Longifolia containing methanol extract plant disease Investigation of plant disease control activity of control composition ( in vivo )

Rice blast disease (causative bacteria: Magnaporthe oryzae ), tomato gray mold disease (causative bacteria: Botrytis ) of the control composition prepared according to Example 1 cinerea ), tomato late blight (causing bacterium: Phytophthora infestans ), wheat red rust (causing bacterium: Puccinia ) triticina ) and chilli anthrax (causative agent: Colletotrichum ) coccodes ) was tested for disease control activity against five plant diseases in greenhouse conditions as follows. At this time, distilled water containing 5% (v/v) methanol and 0.025% (w/v) Tween 20 was used.

Specifically, three ports were used for each plant pathogen. After spraying the control composition solution prepared in step 2 on the leaf surface (foliage), it was air-dried for 24 hours, and then each plant pathogen was inoculated. The rice, tomato, pepper and wheat plants used in the experiment were planted in a plastic pot with a diameter of 4.5 cm with about 80% of the water supply or horticultural top soil, and then sowed seeds and cultivated for 1 to 3 weeks in a greenhouse at a temperature of 25±5℃. did.

Rice blast disease is caused by Magnaporthe oryzae ( Magnaporthe oryzae ), the causative bacterium KACC 46552) of spore suspension (5×10 ⁵ spores/mL) was spray inoculated, treated in a humid room at 25°C for one day, and then grown in a constant temperature and humidity room (temperature 25°C and relative humidity 80%) for 4 days. onset was induced.

Tomato late blight is spray inoculated with a spore suspension (2×10 ⁴ sporangia/mL) of Phytophthora infestans KACC 48738, the causative agent of late blight, on tomato seedlings in the 3-4 leaf stage. The disease was induced by moist treatment for 2 days in a humid room, and cultivation for 1 day in a constant temperature room at 20°C.

Tomato gray mold disease is caused by Botrytis cineria , the causative agent of gray mold disease, on seedlings in the 3-4 leaf stage of tomatoes. cinerea KACC 48736) was treated with a spore suspension (5×10 ⁵ spores/mL), and then cultured for 3 days in a humid room at 20° C. to induce disease.

Wheat red rust is caused by Puccinia triticina, a causative bacterium of rust known as live parasite, on first-leaf seedlings. triticina , Korea Research Institute of Chemical Technology) were suspended in 250 μg/mL Tween 20 solution in an amount of 0.67 g spores/L and sprayed, treated in a humid room at 20°C for one day, and then transferred to a constant temperature room at 20°C. The disease was induced by cultivation for 6 days.

Red pepper anthrax is a spore suspension (4×10 ⁵ spores/mL) of the causative agent of red pepper anthrax, Colletotrichum coccodes KACC 48737, to seedlings in the 3-4 leaf stage of pepper, and spray inoculated, in a humid room at 25°C. After 2 days of moist room treatment, the disease was induced by incubation for 1 day in a constant temperature and humidity room (25℃, 80% relative humidity).

Tomato gray mold and red pepper anthracnose were investigated 3 days after inoculation, tomato late blight 4 days after inoculation, rice blast 5 days after inoculation, and wheat red rust 7 days after inoculation, the lesion area ratio (%) was investigated.

The control value (%) was calculated according to the following Equation 1 using the lesion area ratio (%) obtained from the above, and the results are shown in Table 1 below.

[Equation 1]

Control value (%) = (1-Lesions area ratio of treated group / lesion area ratio of untreated group) X 100

sample density
(μg/ml) Control value (%) RCB TGM TLB WLR PAN Polyaltia longifolia methanol extract 3000 88 50 94 67 78 1000 88 17 94 53 30 500 63 0 91 33 20

(RCB: rice blast disease, TGM: tomato gray mold disease, TLB: tomato late blight, WLR: wheat red rust, PAN: red pepper anthrax)

As shown in Table 1, Polyalthia longifolia ( Polyalthia longifolia ) methanol extract was shown to have an excellent control effect against 5 types of plant diseases. In particular, when treated at a concentration of 3000 μg/mL, an excellent control effect of 78% to 94% was shown, and even when treated at a concentration of 500 μg/mL, it showed an excellent control effect against rice blast and tomato late blight.

Previously, Polyalthia Longifolia ( Polyalthia) longifolia extract, especially Polyalthia longifolia ), the effect of methanol extract on plant disease control has not been reported. Accordingly, the present invention is Polyalthia longifolia ( Polyalthia longifolia ) has technical significance in that it discovered the plant disease control effect of methanol extract.

Example 2-2: polyaltia Longifolia ( Polyalthia longifolia ) containing extracts plant disease Investigation of the bactericidal activity of the control composition ( in vitro )

The phytopathogenic fungi of the composition for control prepared according to Example 1, Brassica crops black-patterned fungi ( Alternaria brassicicola ), cucumber black-starred bacteria ( Cladosporium cucumerinum ), pepper anthracnose ( Colletotrichum ) coccodes ), tomato wilt ( Fusarium oxysporum ) f. sp. lycopersici ), Rice Blast Bacteria ( Magnaporthe oryzae ), Potato/Tomato Blight Bacteria ( Phytophthora ) infestans ) and the phytopathogenic bacteria Phalaenopsis bacterial brown spot bacterium ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis soft blight ( Dickeya chrysanthemi ) by the broth micro-dilution method using a 96-well plate. The minimum inhibitory concentration value was obtained by evaluation.

Brassica crops black-patterned bacteria ( A. brassicicola ) and tomato wilting bacteria ( F. oxysporum f. sp. lycopersici ) were inoculated in PDA (potato dextrose agar) medium and cultured at 25° C. for 10 days. The spores formed were used for the experiment. Potato / tomato late blight ( P. infestans ) was inoculated in PDA (potato dextrose agar) medium and OA (oatmeal agar) medium, cultured at 20 ° C. for 6 days, and then cultured for 4 days under light treatment conditions for 14 hours a day. was used for Red pepper anthrax ( C. coccodes ) and rice blast disease ( M. oryzae ) were inoculated in OA (oatmeal agar) medium and cultured at 25°C for 7 days, then mycelia were removed and a constant temperature and humidity room (25°C, relative humidity 80%) Spore formation was induced by light treatment for 12 hours a day for 2 days.

In the case of phytopathogenic bacteria, after inoculation on TSB medium, Phalaenopsis bacterial brown spot bacillus ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis soft blight ( Dickeya chrysanthemi ) were each cultured with shaking at 30 °C for 2 days (150 r/min) did. For the fungus used in the experiment, spores were harvested using PDB medium, and a spore suspension (1×10 ⁴ pieces/mL) was prepared by filtering with 4-fold gauze, and the bacteria were diluted in TSB medium to obtain a bacterial suspension (1× 10 ⁴ CFU/mL) were prepared.

Finally, 1000 mold spores or bacteria were included in 100 μL well, and the methanol extract of Polyaltia longifolia of the present invention was 1, 2, 4, 8, 16, 32, 63, 125, 250, 500 μg/ It was aliquoted so as to be included at a concentration of mL. At this time, the content of methanol did not exceed 1%. The one in which only 1% methanol was added was used as an untreated group, and the experiment was repeated 3 times for each concentration. After culturing them at 20 ° C or 25 ° C for 2 to 3 days, the minimum concentration at which the growth of mold and bacteria was completely inhibited was determined as the minimum inhibitory concentration (MIC), and the results are shown in Table 2 below.

division plant pathogens Minimum inhibitory concentration
(MIC, μg/mL) phytopathogenic fungi Brassica crops black-spotted fungus ( Alternaria ) brassicicola ) 63 Cucumber black star bacterium ( Cladosporium ) cucumerinum ) 250 Pepper anthrax ( Colletotrichum ) coccodes ) 250 Tomato wilting fungus ( Fusarium ) oxysporum f. sp. lycopersici ) 250 Rice Blast ( Magnaporthe ) oryzae ) 16 Potato/tomato late blight ( Phytophthora ) infestans ) 125 phytopathogenic bacteria Phalaenopsis orchid bacterial brown spot bacterium ( Acidovorax) avenae subsp. cattleyae ) 8 Phalaenopsis Orchid soft fungus ( Dickya chrysanthemi ) 500

As shown in Table 2, Polyalthia longifolia ( Polyalthia longifolia ) extracts obtained with methanol from leaves and stems showed bactericidal activity against 6 types of phytopathogenic fungi. Methanol extract of polyaltia longifolia at concentrations of 16, 63, and 125 μg/mL, respectively, was a rice blast bacillus ( M. oryzae ), a Brassica crop black spot blight ( A. brassicicola ) , and a potato/tomato late blight ( P. infestans ). ) was completely inhibited, and the growth of red pepper anthrax ( C. coccodes ), tomato wilt ( F. oxysporum f. sp. lycopersici ), and cucumber black star blight ( C. cucumerinum ) was completely inhibited at a concentration of 250 μg/mL. showed an inhibitory bactericidal effect.

In addition, Polyalthia longifolia longifolia ) Extracts obtained with methanol from leaves and stems are phytopathogenic bacteria Phalaenopsis Bacterial Brown Spotted Bacteria at a concentration of 8 μg/mL ( A. avenae subsp. cattleyae ), Phalaenopsis soft bacillus at a concentration of 500 μg/mL ( D. chrysanthemi ) showed bactericidal activity to completely inhibit the growth of

< Example 3> polyaltia Longifolia ( Polyalthia longifolia ) Preparation of a composition for controlling plant diseases containing a solvent fraction of a methanol extract as an active ingredient

Step 1: polyaltia Longifolia ( Polyalthia longifolia ) Preparation of solvent fractions of methanol extract

Polyalthia Longifolia longifolia ) In order to prepare a composition for controlling plant diseases containing the solvent fraction of the extract as an active ingredient, the same method as in Step 1 of Example 1 was carried out to prepare a polyalthia longifolia ( Polyalthia ). longifolia ) A methanol extract was prepared, and an organic solvent fraction was prepared using the methanol extract.

Specifically, polyalthia longifolia ( Polyalthia longifolia ) methanol extract was dissolved in 3 L of distilled water and extracted twice with the same amount of chloroform to obtain a chloroform layer and an aqueous layer. The aqueous layer was again extracted twice with the same amount of ethyl acetate to obtain an ethyl acetate layer and an aqueous layer. Thereafter, they were concentrated under reduced pressure to obtain a dried chloroform fraction (50 g), an ethyl acetate fraction (1 g) and a water fraction (25 g).

Step 2: polyaltia Longifolia ( Polyalthia longifolia ) Preparation of a composition for controlling plant diseases containing a solvent fraction of a methanol extract as an active ingredient

Each sample dissolved in methanol to a concentration of 40 mg/mL was diluted with distilled water, and Tween 20 was added to a concentration of 0.025% (w/v) as an electrodepositing agent to obtain a concentration of 1000 μg/mL and 2000 μg/mL of poly Altia Longifolia ( Polyalthia) longifolia ) A solvent fraction (chloroform fraction, ethyl acetate fraction, water fraction) of the methanol extract was prepared by 40 mL each of a composition solution for controlling plant diseases containing as an active ingredient.

< Example 4> polyaltia Longifolia ( Polyalthia longifolia ) Evaluation of plant disease control effect of a composition for controlling plant disease containing the solvent fraction of the extract

Example 4-1: polyaltia Longifolia ( Polyalthia longifolia ) Plant disease control activity of a composition for controlling plant diseases comprising a solvent fraction of methanol extract ( in vivo )

Rice blast of the composition prepared according to Example 3 (causative bacteria: Magnaporthe oryzae ), tomato gray mold disease (causative bacterium: Botrytis ) cinerea ), tomato late blight (causing bacterium: Phytophthora infestans ), wheat red rust (causing bacterium: Puccinia ) triticina ) and chilli anthrax (causative agent: Colletotrichum ) coccodes ) was tested in the same manner as in Example 2-1 in greenhouse conditions for control activity against five plant diseases. In this case, as a control, distilled water containing 5% (v/v) methanol and 0.025% (w/v) Tween 20 was used.

Polyalthia Longifolia longifolia ) The results of analysis of the control effect of the composition containing the solvent fraction of the methanol extract are shown in Table 3.

sample density
(μg/mL) Control value (%) RCB TGM TLB WLR PAN chloroform fraction 2000 75 10 96 60 85 1000 38 0 84 53 63 ethyl acetate fraction 2000 25 0 44 53 55 1000 25 0 19 27 20 water fraction 2000 0 0 0 0 0

(RCB, rice blast (causative bacterium: Magnaporthe ) oryzae ); TGM, tomato gray mold (causative bacterium: Botrytis ) cinerea ); TLB, tomato blight (causative bacterium: Phytophthora ) infestans ); WLR, wheat red rust (causative bacterium: Puccinia ) triticina ); PAN, pepper anthrax (causative bacteria: Colletotrichum coccodes ))

As shown in Table 3, it was found that the chloroform fraction and the ethyl acetate fraction had an excellent control effect on plant diseases. In particular, the chloroform fraction showed an excellent control effect of 96% against tomato blight at a treatment concentration of 2000 μg/mL, and control effects of 75%, 60%, and 85% against rice blast, wheat red rust, and red pepper anthrax, respectively. showed Even at a treatment concentration of 1000 μg/mL, it showed an 84% control value against tomato late blight.

In addition, the ethyl acetate fraction showed a control value of 44% to 55% against tomato late blight, wheat red rust, and pepper anthracnose at a treatment concentration of 2000 μg/mL.

On the other hand, the water fraction at the same treatment concentration did not show control activity, Polyalthia longifolia ( Polyalthia longifolia ) extract was confirmed to be a non-polar substance, not a water-soluble substance.

As described above, the chloroform fraction was used for separation and purification of the control active ingredient based on the fact that the chloroform fraction showed a high level of control activity against tomato late blight, red pepper anthrax, rice blast, and wheat red rust.

Example 4-2: polyaltia Longifolia ( Polyalthia longifolia ) chloroform fraction containing plant disease of the control composition plant disease Check control activity ( in vitro )

Phytopathogenic fungus of the composition comprising the chloroform fraction prepared according to Example 3 brassicicola ), cucumber black star blight ( Cladosporium cucumerinum ), pepper anthrax ( Colletotrichum ) coccodes ), tomato wilt ( Fusarium oxysporum ) f. sp. lycopersici ), rice blast bacillus ( Magnaporthe oryzae ), potato / tomato late blight ( Phytophthora ) infestans ) and phytopathogenic bacteria Phalaenopsis Orchid Bacterial Brown Spotted Bacteria ( Acidovorax avenae subsp. cattleyae ) was evaluated by the broth micro-diLution method using a 96-well plate to obtain the minimum inhibitory concentration value.

Brassica crops black-patterned bacteria ( A. brassicicola ) and tomato wilting bacteria ( F. oxysporum f. sp. lycopersici ) were inoculated in PDA (potato dextrose agar) medium and cultured at 25° C. for 10 days. The spores formed were used for the experiment. Potato / tomato late blight ( P. infestans ) was inoculated in PDA (potato dextrose agar) medium and OA (oatmeal agar) medium, cultured at 20 ° C. for 6 days, and then cultured for 4 days under light treatment conditions for 14 hours a day. was used for Red pepper anthrax ( C. coccodes ) and rice blast disease ( M. oryzae ) were inoculated in OA (oatmeal agar) medium and cultured at 25°C for 7 days, then mycelia were removed and a constant temperature and humidity room (25°C, relative humidity 80%) Spore formation was induced by light treatment for 12 hours a day for 2 days.

In the case of phytopathogenic bacteria, after inoculation in TSB medium, Phalaenopsis bacterial brown spot blight ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis soft blight ( Dickya chrysanthemi ) were cultured with shaking (150 r/min) at 30 °C for 2 days. . For the fungus used in the experiment, spores were harvested using PDB (potato dextrose broth) medium and filtered with 4-fold gauze to prepare a spore suspension, and the bacteria were diluted in TSB (tryptic soy broth) medium to prepare a bacterial suspension. did.

Finally, 1000 mold spores or bacteria were included in 100 μL wells, and methanol extracts were dispensed at concentrations of 1, 2, 4, 8, 16, 31, 63, 125, 250, and 500 μg/mL, respectively. . At this time, the content of methanol did not exceed 1%. The one in which only 1% methanol was added was used as an untreated group, and the experiment was repeated 3 times for each concentration. After culturing them at 20 °C or 25 °C for 2 to 3 days, the lowest concentration at which the growth of mold and bacteria was completely inhibited was determined as the minimum inhibitory concentration (MIC), and the results are shown in Table 4 below. .

division plant pathogens Minimum inhibitory concentration of chloroform fraction
(MIC, μg/mL) phytopathogenic fungi Brassica crops black spot fungus ( Alternaria brassicicola ) 63 Cucumber black star bacterium ( Cladosporium ) cucumerinum ) 250 Pepper anthrax ( Colletotrichum ) coccodes ) 250 Tomato wilting fungus ( Fusarium ) oxysporum f. sp. lycopersici ) 500 Rice Blast ( Magnaporthe ) oryzae ) 31 Potato/tomato late blight ( Phytophthora ) infestans ) 125 phytopathogenic bacteria Phalaenopsis orchid bacterial brown spot bacterium ( Acidovorax) avenae subsp. cattleyae ) 8

As shown in Table 4, Polyalthia longifolia longifolia ), it was confirmed that the chloroform fraction showed bactericidal activity against 5 types of phytopathogenic fungi. Chloroform fractions at concentrations of 31, 63, and 125 µg/mL, respectively, completely inhibit the growth of rice blast bacillus ( M. oryzae ), Brassica crop black blight ( A. brassicicola ), and potato / tomato late blight ( p. infestans ) At a concentration of 250 μg/mL or more, the bactericidal effect of completely inhibiting the growth of cucumber black star blight ( C. showed

In addition, at a concentration of 8 μg/mL, it exhibited a bactericidal activity that completely inhibited the growth of Phalaenopsis orchid bacterial brown spot bacterium ( A. avenae subsp. cattleyae ). In addition, the ethyl acetate fraction completely inhibited the growth of potato/tomato late blight at a concentration of 500 μg/mL.

< Example 5> plant disease Isolation of compounds with control activity

Polyalthia Longifolia longifolia ) silica gel (Kiesel gel 60; 70-230 mesh; Merck) column chromatography, Sephadex LH-20 (Merck) column chromatography, Biotage Isolera One medium-pressure liquid chromatography (mid-pressure) to separate the bactericidal active compound from the extract Separation was performed using a combination of liquid chromatography (MPLC) system and Shimadzu Prominence high pressure liquid chromatography (HPLC) system.

Based on the fact that the chloroform fraction showed a high level of control activity in Example 4, the chloroform fraction was used for separation and purification of the control active ingredient.

Specifically, compounds 1 to 15 , which are bactericidal active substances, were separated from the chloroform fraction (50 g) prepared according to Example 3 by the process shown in FIG. 1 .

1, the chloroform fraction (50 g) was adsorbed on a silica gel column, and then a hexane-methanol-chloroform solution mixed in a volume ratio of 100:0:0 to 0:1:1 was sequentially eluted to 11 fractions ( F1 to F11). After adding the active fraction F10 (3.5 g) to a Sephadex LH-20 column, a chloroform-methanol solution mixed in a 1:1 volume ratio was eluted, and then added to a Biotage SNAP KP-Sil (50 g) column, followed by ethyl in dichloromethane. The active fraction obtained by eluting a solution in which an acetate solution was mixed in a 0-35% volume ratio under a gradient condition was again added to a Biotage SNAP Ultra C18 (30 g) column, and then eluted with a 70%-100% aqueous methanol solution under a concentration gradient condition. Compound 12 (290 mg) was isolated.

Another active fraction F11 (22 g) was adsorbed on a silica gel column, and a solution of chloroform mixed with methanol in a volume ratio of 0-10% was eluted under gradient conditions to obtain four fractions (F11-1, F11-2, F11-3). , F11-4). The active fraction F11-3 (16.5 g) was loaded on a Sephadex LH-20 column, and chloroform-methanol was eluted with a mixed solution (1:1, v/v) to obtain an active fraction F11-33 (9.9 g), which was silica gel. After adsorption to a column, a solution obtained by mixing acetone in 0-35% by volume in dichloromethane was eluted under gradient conditions to obtain three fractions (F11-331, F11-332, F11-333). The active fraction F11-332 (4.5 g) was dissolved in hexane-ethyl acetate (6:1, v/v) solution, and then recrystallized to isolate compound 6 (3.0 g).

Another active fraction F11-333 (0.5 g) was added to a Biotage SNAP Ultra C18 (30 g) column, eluted with 70-100% aqueous methanol solution, and 5 fractions (F11-3331, F11-3332, F11-3333, F11- 3334), and the fraction F11-3333 was isolated as compound 2 (10 mg) through recrystallization. Fraction F11-3331 (100 mg) was loaded on a YMC-Pack ODS-A column (20×250 mm, 5 μm) connected to a Shimadzu Prominence HPLC system, and 60% aqueous acetonitrile solution (containing 0.1% formic acid) was added to 5 mL/ Compound 7 (21 mg), compound 8 (22 mg), and compound 9 (20 mg) were isolated by eluting at a flow rate of min.

The active fraction F11-3332 (30 mg) was loaded on preparative silica gel TLC and developed with hexane-ethyl acetate (2:1, v/v) to isolate compound 5 (8.6 mg). The active fraction F11-3335 (100 mg) was loaded onto a YMC-Pack ODS-A column (20 × 250 mm, 5 μm) connected to a Shimadzu Prominence HPLC system, and 5 mL of 40% aqueous acetonitrile solution (containing 0.1% formic acid) was added. Compound 4 (9.1 mg) and compound 3 (8.2 mg) were separated by eluting at a flow rate of /min.

Another active fraction F11-4 (2.0 g) was added to a Sephadex LH-20 column and eluted with a chloroform-methanol (1:1, v/v) solution to obtain 4 fractions (F11-41, F11-42, F11-43). , F11-44). The active fraction F11-42 (692 mg) was applied to a Biotage SNAP Ultra C18 (30 g) column and eluted with 70-100% aqueous methanol solution to obtain two fractions (F11-411, F11-412). Compound 14 (100 mg) was isolated by recrystallization of fractions F11-411.

After adding the fraction F11-422 (50 mg) to a Biotage SNAP KP-Sil (50 g) column, a solution of 0-35% volume ratio of ethyl acetate in dichloromethane was eluted under a gradient condition to elute Compound 15 (5.3 mg) was isolated.

The active fraction F11-43 (823 mg) was applied to a Biotage SNAP Ultra C18 (30 g) column and eluted with 70-100% aqueous methanol solution to obtain two fractions (F11-431, F11-432). Compound 10 (200 mg) was isolated by recrystallization of fractions F11-431.

Fraction F11-432 (83 mg) was loaded onto a YMC-Pack ODS-A column (20×250 mm, 5 μm) connected to a Shimadzu Prominence HPLC system, and 80% aqueous acetonitrile solution (containing 0.1% formic acid) was added to 5 mL/ Compound 1 (3.1 mg) and compound 13 (15.1 mg) were separated by eluting at a flow rate of min.

Fraction F11-44 (274 mg) was loaded on a Sephadex LH-20 column, and compound 11 (65.0 mg) was isolated by eluting with a mixed solution (1:1, v/v) of chloroform-methanol (FIG. 1).

< Example 6> Identification of the chemical structure of bactericidal active compounds

In order to identify the chemical structure of the bactericidal active material isolated in Example 5, the following experiment was performed.

Specifically, polyalthia longifolia ( Polyalthia longifolia ), nuclear magnetic resonance (NMR) and mass spectrometry (MS) devices were mainly used to elucidate the chemical structure of the material isolated from the chloroform fraction of the extract. After dissolving the sample in CDCl ₃ or CD ₃ OD (Cambridge Isotope Laboratories), ¹ H-NMR, ¹³ C-NMR and 2D-NMR spectra were obtained on a Bruker AMX-400 or AMX-500 instrument. A chemical shift value was expressed as δ ( ppm) based on the peak of tetramethylsilane (TMS) added to the NMR solvent. The chemical positions of the peaks in the spectrum were determined by combining the NMR data shown in the literature and the experimental data of ¹ H- ¹ H COSY, HSQC, HMBC and DEPT. The mass of the isolated compound was measured using ESI-MS, and the chemical formula of the isolated compound was completed using a high-resoLution mass spectroscopy (Synapt G2 HDMS Q-TOF). In addition, the specific optical rotation of the separated compound was measured with a polarimeter (Auto Digital Polarieter, Rudolph Research Analytical) and an infrared analyzer (FT-IR spectroscopy; Identify IR, Smiths).

By combining NMR spectra and data presented in the literature, 5 of the 15 compounds isolated were identified as novel compounds (Compounds 1, 2, 3, 4 and 5) and the remaining 10 as known compounds (Compounds 6 to 15). .

Novel compound 1 is polylongifoliaon C; Novel compound 2 is polylongifoliaon D; Novel compound 3 is polylongifoliaon E; Novel compound 4 is polylongifoliaon F; Novel compound 5 was named polylongifoline A.

In addition, compound 6 is 16α -hydroxy-cleroda-3,13(14) Z -dien-15,16-olide (16α-hydroxy-cleroda-3,13(14) Z -dien-15,16 -olide); Compound 7 is 3α,16-dihydroxy- cleroda -4(18),13(14) Z -dien-15,16-olide (3α,16-dihydroxy-cleroda-4(18),13( 14) Z -dien-15,16-olide); Compound 8 is 3β , 16-dihydroxy-cleroda-4(18),13(14) Z -diene-15,16-olai (3β,16-dihydroxy-cleroda-4(18),13(14) )Z-dien-15,16-olide); Compound 9 is (4→2)-Abeo-16-hydroxy- cleroda -2,13(14) Z -dien-15,16-olide-3-al ((4→2) abeo -16-hydroxy -cleroda-2,13(14) Z -dien-15,16-olide-3-al); Compound 10 is 3,12 E -kolavadien-15-oic acid-16-al (3,12 E -kolavadien-15-oic acid-16-al); Compound 11 is 16-oxo-cleroda-3,13(14) E -dien-15-oic acid (16-oxo-cleroda-3,13(14) E -dien-15-oic acid); Compound 12 is kolavenic acid; Compound 13 is labd-13 E -en -8-ol-15-oic acid; Compound 14 is longimide B; Compound 15 was identified as longimide B methyl ester (FIG. 2).

<6-1> Chemical structure identification of compound 1

Novel compound 1 was isolated as a colorless oil, and was detected as a cation of m/z 369.2043 [M+Na] ⁺ (calculated m/z 369.2042) in HRESIMS analysis, and the chemical formula was determined as C ₂₁ H ₃₀ O ₄ did. By combining the NMR spectrum (Table 5) of the novel compound 1 and the data shown in the literature, it was confirmed that the compound had a clerodane diterpene skeleton.

Finally, the chemical structure of the novel compound 1 is methyl-2,16-dioxo-cleroda-3,12(13)E-diene-15-oate (methyl-2,16-dioxo) represented by the following Chemical Formula 1 -cleroda-3,12(13)E-dien-15-oate) and named the compound polylongifoliaon C. The NMR analysis results of the novel compound 1 are shown in Table 5 below.

[Formula 1]

.

.

Position compound 1 d _c d _H ( J in Hz) One 35.4 2.38 (m)
1.86 (m) 2 199.3 - 3 125.8 5.74(s) 4 172.1 - 5 40.1 - 6 35.3 2.38 (m)
1.86 (m) 7 27.0 1.53 (m) 8 37.5 1.53 (m) 9 40.7 - 10 47.0 1.93 (m) 11 37.3 2.40 (m) 12 158.1 6.67 (t, 7.4) 13 138.5 - 14 29.8 3.31(s) 15 170.4 - 16 193.5 9.40(s) 17 16.3 0.88 (d, 6.1) 18 19.2 1.90 (d, 1.3) 19 18.4 1.13(s) 20 17.5 0.93(s) 3a-OCH3 - - 3b-OCH3 - - 4-OH - - 15-OCH3 52.4 3.67(s) 16-OH - -

(Measured in CDCl ₃ at 400/100 MHz)

As shown in Table 5, in the ¹³ C-NMR spectrum of novel compound 1 , one ketone carbon ( δ _C 199.3), one aldehyde carbon ( δ _C 193.5), one carboxyl carbon ( δ _C 170.4), two quarters nary sp ² carbon ( δ _C 172.1, 138.5), 2 quaternary sp ³ Carbon ( δ _C 40.7, 40.1), 2 methine sp ² carbons ( δ _C 158.1, 125.8), 2 methine sp ³ carbons ( δ _C 47.0, 37.5), 5 methylene sp ³ carbons ( δ _C 37.3, 35.4, 35.3, 29.8, 27.0) and five methyl carbons ( δ _C 52.4, 19.2, 18.4, 17.5, 16.3).

In addition, the positions of hydrogen and carbon were determined through COSY, HMBC and HSQC data analysis, and the three-dimensional structure was completed through NOESY analysis (FIG. 3).

Specifically, in the HMBC spectrum, the hydrogen signal H-1 is with C-2, H-3 is with C-1, C-5, C-18, and H-18 is with C-3, C-4, C-5 It was confirmed that a correlation appeared, and based on the chemical shift values of the carbon signals C-2, C-3, C-4 ( δ _C 199.3, 125.8, 172.1) and the hydrogen signal H-3 ( δ _H 5.74), C- Two positions of oxidation (ketonization) were confirmed. In HMBC analysis, olefin hydrogen H-12 was correlated with C-14 and C-16, and H-16 aldehyde hydrogen was correlated with C-13 and C-14, and NOESY analysis showed correlation between H-12 hydrogen and H-16 aldehyde. A correlation was found between hydrogens. In addition, comparing the NMR data of 3,12E-kolavadien-15-oic acid-16-al shown in the literature, the aldehyde group ( δ _C 193.5; δ _H 9.40) is cis -bonded with H-12 ( δ _H 6.67) olefin hydrogen. confirmed to be in a relationship.

Also, the fact that the hydrogen signal H-14 is a singlet and the hydrogen signal H-12 is a tripLet is that the position of the double bond is at C-12/C-13 instead of C-13/C-14 showed In addition, by comparing the chemical shift values of the C-17, C-19, and C-20 methyl groups ( δ _C 16.3, 18.4, 17.5) of Compound 1 with the literature data, trans -clerodane diterpene) It was revealed that it was a compound of the skeleton, and methyl esterification was confirmed based on the HMBC correlation between methoxy hydrogen and C-15 carbonyl carbon.

<6-2> Novel compound 2 Identification of the chemical structure of

Novel compound 2 was isolated as a colorless oil. In HRESIMS analysis, it was detected as a cation of m/z 385.1991 [M+Na] ⁺ (calculated m/z 385.1991), and the chemical formula was determined as C ₂₁ H ₃₀ O ₅ . By combining the NMR spectrum of the novel compound 2 shown in Table 6 and the data shown in the literature, it was confirmed that the compound had a clerodane diterpene skeleton.

Finally, the chemical structure of the novel compound 2 is methyl-2-oxo-cleroda-3,12(13) E -diene-16-oic acid-15-oate (methyl-2-oxo-) represented by the following Chemical Formula 2 cleroda-3,12(13) E -dien-16-oic acid-15-oate) was determined, and the compound was named polylongifoliaon D. The NMR analysis results of the novel compound 2 are shown in Table 6 below.

[Formula 2]

.

.

Position compound 2 d _c d _H ( J in Hz) One 35.3 2.39 (m)
2.28 (m) 2 199.6 - 3 125.8 5.71(s) 4 172.2 - 5 40.0 - 6 35.3 1.81 (m)1.38 (m) 7 27.0 1.49 (m) 8 37.2 1.50 (m) 9 40.4 - 10 46.7 1.91 (m) 11 37.1 2.21 (m) 12 143.1 7.06 (t, 7.4) 13 126.9 - 14 32.3 3.35 (d, 5.8) 15 170.9 - 16 171.0 - 17 16.2 0.85 (d, 6.0) 18 19.2 1.88 (d, 1.0) 19 18.5 1.10(s) 20 17.4 0.88 (s) 3a-OCH3 - - 3b-OCH3 - - 4-OH - - 15-OCH3 52.4 3.68(s) 16-OH - -

(Measured in CDCl ₃ at 500/125 MHz)

As shown in Table 6, in the ¹³ C-NMR spectrum of novel compound 2 , one ketone carbon ( δ _C 199.6), two carboxyl carbons ( δ _C 171.0, 170.9), two quaternary sp ² carbons ( δ _C 172.2) , 126.9), 2 quaternary sp ³ carbon ( δ _C 40.4, 40.0), 2 methine sp ² carbons ( δ _C 143.1, 125.8), 2 methine sp ³ carbons ( δ _C 46.7, 37.2), 5 methylene sp ³ carbons ( δ _C 37.1, 35.3, 35.3, 32.3, 27.0) and 5 methyl carbons ( δ _C 52.4, 19.2, 18.5, 17.4, 16.2).

In addition, the positions of hydrogen and carbon were determined through COSY, HMBC and HSQC data analysis, and the three-dimensional structure was completed through NOESY analysis (FIG. 3).

The NMR spectrum of Compound 2 was similar to that of Compound 1 , except that one carboxyl group signal appeared instead of one aldehyde group signal. In addition, the fact that the molecular weight difference between Compound 2 and Compound 1 is 16 Da and the correlation between H-12 and H-14 with carboxyl carbon C-16 in the HMBC spectrum is that the aldehyde carbon of Compound 1 is oxidized to a carboxyl group in Compound 2 supported that it was.

<6-3> Novel compound 3 and 4 Identification of the chemical structure of

Novel compounds 3 and 4 were isolated in the form of white wax. In HRESIMS analysis, cations of m/z 387.2148 [M+Na] ⁺ and m/z 387.2146[M+Na] ⁺ (calculated m/z 387.2147) were detected, respectively, and the chemical formula was determined as C ₂₁ H ₃₀ O ₅ . By synthesizing the NMR spectra (Table 7) of the novel compounds 3 and 4 and the data presented in the literature, it was confirmed that the compound had a clerodane diterpene skeleton.

Finally, the chemical structure of the novel compound 3 is (13 S )-methyl-2-oxo-cleroda-3-en-16-oic acid-15-oate ((13 S )-methyl-2- oxo-cleroda-3-en-16-oic acid-15-oate) and named polylongifoliaon E. In addition, the chemical structure of the novel compound 4 is (13 R )-methyl-2-oxo-cleroda-3-ene-16-oic acid-15-oate ((13 R )-methyl-2- oxo-cleroda-3-en-16-oic acid-15-oate) and named polylongifoliaon F. The NMR analysis results of the novel compounds 3 and 4 are shown in Table 7 below.

[Formula 3]

;

;

[Formula 4]

.

.

Position compound 3 compound 4 d _c d _H ( J in Hz) d _c d _H ( J in Hz) One 34.9 2.31 (m) 34.9 2.33 (m) 2 200.9 - 200.9 - 3 125.6 5.71(s) 125.6 5.71(s) 4 173.3 - 173.2 - 5 40.0 - 40.0 - 6 35.6 1.80 (m)1.35 (m) 35.6 1.81 (m)
1.35 (m) 7 26.9 1.49 (m) 26.9 1.49 (m) 8 36.1 1.48 (m) 36.1 1.48 (m) 9 38.8 - 38.8 - 10 45.7 1.81 (m) 45.7 1.81 (m) 11 34.3 1.32 (m) 34.5 1.32 (m) 12 24.5 1.58 (m)1.33 (m) 24.6 1.58 (m)
1.33 (m) 13 41.2 2.81 (m) 41.3 2.80 (m) 14 35.5 2.72 (m)2.43 (m) 35.6 2.73 (m)
2.44 (m) 15 172.5 - 172.4 - 16 178.6 - 178.2 - 17 15.8 0.80 (d, 5.3) 15.8 0.80 (d, 6.2) 18 19.2 1.88 (d, 1.3) 19.1 1.87 (d, 1.3) 19 18.5 1.09(s) 18.5 1.09(s) 20 18.0 0.79 (s) 18.0 0.79 (s) 3a-OCH3 - - - - 3b-OCH3 - - - - 4-OH - - - - 15-OCH3 52.1 3.68(s) 52.0 3.68(s) 16-OH - - - -

(Measured in CDCl ₃ at 400/100 MHz)

As shown in Table 7, since the new compounds 3 and 4 are enantiomers and the NMR data of the two compounds are virtually identical, the chemical structure was first revealed using the NMR data of compound 3 .

In the ¹³ C-NMR spectrum of novel compound 3 , one ketone carbon ( δ _C 200.9), two carboxyl carbons ( δ _C 178.6, 172.5), one quaternary sp ² carbon ( δ _C 173.3), three quaternary sp ³ Carbon ( δ _C 41.2, 40.0, 38.8), 1 methine sp ² carbon ( δ _C 125.6), 2 methine sp ³ carbons ( δ _C 45.7, 36.1), 6 methylene sp ³ carbons ( δ _C 35.6, 35.5, 34.9, 34.3, 26.9, 24.5) and five methyl carbons ( δ _C 52.1, 19.2, 18.5, 18.0, 15.8).

In addition, the positions of hydrogen and carbon were determined through COSY, HMBC and HSQC data analysis, and the three-dimensional structure was confirmed through NOESY analysis (FIG. 3).

The NMR spectrum of Compound 3 was similar to the NMR spectrum of Compound 2 , except that a methylene sp ² signal of C-12/C-13 appeared. In addition, the fact that the molecular weight difference between Compound 3 and Compound 2 was 2 Da supported that methylene sp ² of C-12/C-13 was reduced to methyl. In the ¹³ C NMR spectrum of compound 3 , the chemical shift value of the C-16 carboxyl carbon is slightly cuter than that of compound 4 , which indicates that compounds 3 and 4 are R/S enantiomers centering on the C-13 chiral carbon of each other. (enantiomer) is present.

In NOESY analysis, compound 3 was formed between H-20 methyl hydrogen ( δ _H 0.79) and H-12 hydrogen ( δ _H 1.33), H between H-12 hydrogen ( δ _H 1.33) and H-13 hydrogen ( δ _H 2.81). A correlation was confirmed between -13 hydrogen ( δ _H 2.81) and H-14 hydrogen ( δ _H 2.72), and the chemical structure of compound 3 was determined as ( S )-form enantiomer. On the other hand, in NOESY analysis, compound 4 is between H-12 hydrogen ( δ _H 1.58) and H-13 hydrogen ( δ _H 2.80) between H-13 hydrogen ( δ _H 2.80) and H-14 hydrogen ( δ _H 2.44) It was confirmed that the correlation appeared, and the chemical structure of compound 4 was confirmed to be an enantiomer of the ( R )-form.

<6-4> Novel compound 5 Identification of the chemical structure of

Novel compound 5 was isolated in the form of a white wax. HRESIMS analysis was detected as a cation of m/z 419.2140 [M+Na] ⁺ (calculated m/z 419.2140), respectively, and the chemical formula was determined as C ₂₂ H ₃₆ O ₆ . By synthesizing the NMR spectrum and literature data of the novel compound 5 shown in Table 8, it was confirmed that the compound had a clerodane diterpene skeleton, and finally the chemical structure of the novel compound 5 was converted into a compound represented by Chemical Formula 5 ( 4→2)-abeo-2 S -dimethoxymethyl-4 β ,16 α -dihydroxy-cleroda-13 Z -en-15,16-olide ((4→2) -abeo -2 S - dimethoxymethyl-4 β ,16 α -dihydroxy-cleroda-13 Z -ene-15,16-olide) and named polylongifoline A. The NMR analysis results of the novel compound 5 are shown in Table 8 below.

[Formula 5]

.

.

Position compound 5 d _c d _H ( J in Hz) One 23.1 1.48 (m)
1.22 (m) 2 46.3 2.07 (m) 3 105.4 4.39 (d, 8.6) 4 80.8 - 5 48.6 - 6 29.7 1.56 (m)1.08 (m) 7 27.8 1.40 (m) 8 36.8 1.40 (m) 9 37.4 - 10 45.0 1.99 (m) 11 36.0 1.43 (m) 12 21.3 2.20 (m) 13 170.9 - 14 115.9 5.95(s) 15 171.4 - 16 99.3 6.02(s) 17 15.0 0.75 (overlapped) 18 22.0 0.75 (overlapped) 19 17.2 1.09(s) 20 17.2 0.75 (overlapped) 3a-OCH3 53.8 3.27(s) 3b-OCH3 51.0 3.17(s) 4-OH - 3.86(s) 15-OCH3 - - 16-OH - 7.75 (s)

(Measured in DMSO- d ₆ at 400/100 MHz)

As shown in Table 8, in the ¹³ C-NMR spectrum of the novel compound 5 , one carboxyl carbon ( δ _C 171.4), one quaternary sp ² carbon ( δ _C 170.9), three quaternary sp ³ Carbon ( δ _C 80.8, 48.6, 37.4), 1 methine sp ² carbon ( δ _C 115.9), 5 methine sp ³ carbon ( δ _C 105.4, 99.3, 45.0, 46.3, 36.8), 5 methylene sp ³ carbon ( δ _C 36.0, 29.7, 27.8, 23.1, 21.3) and 6 methyl carbons ( δ _C 53.8, 51.0, 22.0, 17.2, 17.2, 15.0) were found.

In addition, the positions of hydrogen and carbon were determined through COSY, HMBC and HSQC data analysis, and the three-dimensional structure was completed through NOESY analysis (FIG. 3).

Specifically, in the HMBC analysis of compound 5 , the hydrogen signal H-14 was correlated with C-12, C-13, C-15, C-16 and H-16 with C-13, C-14, and C-15. , which is a C-14/H-14 signal ( δ _C 115.9/ δ _H 5.95) and C-16/H-16 signals ( δ _C 99.3/ δ _H 6.02) was found to be the γ -hydroxyl α , β -unsaturated- γ -lactone portion of the clerodane diterpene backbone.

In HMBC analysis, hydrogen signal H-1 is C-2, C-3, C-4, C-9, C-10 and H-3 is C-1, C-2, C-4 and H-18 is C-2, C-4, C-5 and H-19 showed a correlation with C-4, C-5, C-6, and C-10, which is similar to compound polylongifoliaic A with known chemical structure of compound 5 . (4→2) It was shown that it is a clerodane diterpene skeleton with rearranged ring A.

In addition, the HMBC correlation between hydroxy hydrogen 4-OH ( δ _H 3.86) and C-2, C-4, C-5, and C-18 showed that the hydroxy group of compound 5 was located at C-4. . The fact that the chemical shift values of the methyl carbon signals C-17, C-19, and C-20 appear at δ _C 15.0, 17.2, 17.2 means that the main backbone of compound 5 is trans - cleodane diterpine ( trans -clerodane diterpene).

erhauser ffect)는 H-2, H-18, H-19, H-20이 공간적으로 매우 근접해 있음을 나타내며, 이들 수소가 α-배열(α-orientation)을 이루고 있음을 나타내었다. 또한, H-3, 4-OH, H-6, H-10 사이의 핵오버하우저 효과는 이들 수소가 β-배열(β-orientation)을 이루고 있는 것으로 나타났다. 이를 바탕으로 C-2와 C-4의 입체구조를 각각 2(S)-와 4(S)-형태로 결정하였다. The three-dimensional structure of C-2 and C-4 was determined through NOESY analysis, and the nuclear Overhauser effect (nuclear o

erhauser effect) indicates that H-2, H-18, H-19, and H-20 are spatially very close, and that these hydrogens form an α -orientation. In addition, the nuclear Oberhauser effect between H- 3, 4-OH, H-6, and H-10 showed that these hydrogens form a β - orientation. Based on this, the three-dimensional structures of C-2 and C-4 were determined as 2( S )- and 4( S )-forms, respectively.

<6-5> compound 6 inside 15 Identification of the chemical structure of

¹³ C NMR analysis results of compounds 6 to 15 separated by the process of FIG. 1 are shown in Table 9 below.

division ¹³ C NMR data compound 6 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 171.7, 170.6, 144.2, 120.4, 116.7, 99.4, 46.4, 38.6, 38.1, 36.6, 36.2, 34.7, 27.3, 26.7, 21.3, 19.8, 18.2, 18.2, 18.0 , 16.0 compound 7 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 172.0, 170.8 (170.7), 161.8, 117.1 (117.0), 99.9, 99.6 (99.3), 69.8, 48.4, 40.3, 39.4, 37.5, 37.2, 36.8 (36.7) , 34.9, 27.2, 21.3, 20.5, 18.0, 16.0 compound 8 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 171.9, 170.6 (170.6), 157.4 (157.4), 117.2 (117.1), 100.9, 99.4 (99.2), 83.2, 48.5, 40.5, 39.4, 37.1, 36.8 (36.8) ), 34.9 (34.8), 32.4 (32.4), 27.2, 21.4 (21.3), 21.1 (21.1), 20.3 (20.3), 18.0, 15.9 compound 9 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 189.5 (189.4), 173.7 (173.6), 171.6, 170.4 (170.2), 137.1 (137.0), 117.3 (117.0), 99.7 (99.0), 54.1, 51.1, 38.1 (38.0), 37.6 (37.6), 36.7 (36.4), 34.1, 28.3, 26.2 (26.1), 21.9 (21.9), 17.9 (17.9), 17.2 (17.2), 15.1, 9.9 compound 10 ¹³ C NMR (100 MHz in CD ₃ OD): δ _C 195.8, 173.9, 155.4, 144.9, 139.5, 121.6, 49.4, 41.9, 39.5, 38.8, 38.7, 37.7, 30.4, 28.6, 27.7, 20.3, 19.9, 18.3, 18.1, 16.7 compound 11 ¹³ C NMR (100 MHz in CD ₃ OD): δ _C 196.5, 168.4, 155.8, 145.0, 137.5, 121.9, 47.8, 40.3, 39.3, 38.3, 38.2, 37.6, 28.7, 27.8, 20.5, 19.7, 19.1, 18.7, 18.3, 16.4 compound 12 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 171.4, 164.8, 144.6, 120.5, 114.7, 46.6, 38.9, 38.3, 36.9, 36.5, 36.4, 35.1, 27.6, 27.0, 20.1, 19.6, 18.4, 18.1, 16.1 compound 13 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 171.8, 163.6, 114.9, 73.4, 58.9, 56.0, 45.0, 42.4, 42.1, 39.4, 39.2, 33.6, 33.4, 30.7, 23.7, 21.8, 19.5, 18.4, 18.3 , 15.2 compound 14 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 174.7, 173.4, 168.7, 144.7, 144.2, 140.9, 137.8, 126.5, 120.7, 120.6, 116.9, 48.3, 46.6, 41.2, 39.8, 39.2, 38.9, 38.6, 38.3 , 37.9, 36.9, 36.5, 36.4, 35.2, 32.6, 27.8, 27.7, 27.2, 27.2, 24.7, 20.1, 20.1, 19.3, 18.6, 18.5, 18.1, 17.9, 16.8, 16.0 compound 15 ¹³ C NMR (100 MHz in CDCl ₃ ): δ _C 173.0, 170.8, 168.5, 144.6, 144.0, 140.5, 137.4, 126.4, 120.5, 120.3, 116.7, 52.3, 48.1, 46.4, 41.5, 39.7, 39.0, 38.8, 38.4 , 38.3, 37.8, 36.7, 36.3, 36.2, 35.0, 32.4, 27.6, 27.5, 27.1, 26.9, 24.4, 20.0, 20.0, 19.2, 18.4, 18.4, 18.1, 17.8, 16.6, 16.0

Comparing the ¹³ C NMR data in Table 9 with the data shown in the literature, compound 6 is 16 α -hydroxy-cleroda-3,13(14) Z -dien-15,16-olide (16) of Formula 6 α -hydroxy-cleroda-3,13(14) Z -dien-15,16-olide); Compound 7 is 3 α ,16-dihydroxy-cleroda-4(18),13(14) Z -dien-15,16-olide of Formula 7 (3α,16-dihydroxy- cleroda -4(18) ), 13 (14) Z -dien-15,16-olide); Compound 8 is 3 β , 16-dihydroxy-cleroda-4 (18), 13 (14) Z -dien-15,16-olide of Formula 8 (3 β, 16-dihydroxy-cleroda-4 (18) ), 13 (14) Z -dien-15,16-olide); Compound 9 is (4→2)-abeo-16-hydroxy- cleroda -2,13(14) Z -dien-15,16-olide-3-al of Formula 9 ((4→2) -abeo -16-hydroxy-cleroda-2,13(14) Z -dien-15,16-olide-3-al); Compound 10 is 3,12 E - kolavadien -15-oic acid-16-al of Formula 10; Compound 11 is 16-oxo-cleroda- 3,13(14) E - dien-15-oic acid of Formula 11; Compound 12 is kolavenic acid of Formula 12; Compound 13 is labd-13 E -en -8-ol-15-oic acid of Formula 13; Compound 14 is longimide B of Formula 14; Compound 15 was identified as longimide B methyl ester of Chemical Formula 15, and the structures of Chemical Formulas 6 to 15 are as follows.

[Formula 6]

;

;

[Formula 7]

;

;

[Formula 8]

;

;

[Formula 9]

;

;

[Formula 10]

;

;

[Formula 11]

;

;

[Formula 12]

;

;

[Formula 13]

;

;

[Formula 14]

; 및

; and

[Formula 15]

.

.

Each of Chemical Formulas 7 to 9 is an R/S enantiomer based on a chiral carbon, and R form and S form may be present in a ratio of 1:1.

Although the above has been described with reference to the preferred embodiments of the present invention, those skilled in the art can variously modify and change the present invention without departing from the spirit and scope of the present invention as set forth in the claims below. You will understand that you can.

Claims (16)

Polyalthia Longifolia longifolia ) A composition for controlling plant diseases comprising an extract or a fraction thereof. According to claim 1,
The plant disease is any one selected from the group consisting of rice blast disease, tomato gray mold disease, tomato late blight, wheat red rust and red pepper anthrax, a composition for controlling plant diseases. According to claim 1,
The plant disease is a plant disease control composition that is caused by a phytopathogenic fungus or phytopathogenic bacteria. 4. The method of claim 3,
The phytopathogenic fungi are Brassica crops black pattern bung ( Alternaria brassicicola ), cucumber black star blight ( Cladosporium ) cucumerinum ), pepper anthrax ( Colletotrichum coccodes ), tomato wilting fungus ( Fusarium ) oxysporum f. sp. lycopersici ), Rice Blast Bacteria ( Magnaporthe oryzae ) and potato / tomato late blight ( Phytophthora infestans ) A composition for controlling plant diseases that is selected from the group consisting of. 4. The method of claim 3,
The phytopathogenic bacteria is Phalaenopsis Bacterial Brown Spotted Bacteria ( Acidovorax) avenae subsp. cattleyae ) and Phalaenopsis Orchis bacillus ( Dickea ) chrysanthemi ) A composition for controlling plant diseases that is selected from the group consisting of. According to claim 1,
The extract is selected from the group consisting of water, C ₁ to C ₄ lower alcohol, hexane, chloroform, methylene chloride, ethyl acetate, acetone, acetonitrile, and combinations thereof. Composition for controlling plant diseases. According to claim 1,
The fraction of the extract is any one selected from the group consisting of hexane, chloroform, ethyl acetate, methylene chloride, methanol, ethanol, butanol, water, and a combination thereof or a mixture thereof as a solvent. Plant disease that is fractionated Control composition. According to claim 1,
The extract is Polyalthia longifolia longifolia ) A composition for controlling plant diseases which is an extract of leaves, stems or roots. According to claim 1,
The Polyalthia Longifolia longifolia ) extract or a fraction thereof is a composition for controlling plant diseases comprising at least one selected from compounds represented by the following Chemical Formulas 1 to 15:
[Formula 1]

;
[Formula 2]

;
[Formula 3]

;
[Formula 4]

;
[Formula 5]

;
[Formula 6]

;
[Formula 7]

;
[Formula 8]

;
[Formula 9]

;
[Formula 10]

;
[Formula 11]

;
[Formula 12]

;
[Formula 13]

;
[Formula 14]

; and
[Formula 15]

. 10. The method of claim 9,
The compound is a composition for controlling plant diseases that has bactericidal activity. According to claim 1,
The extract or a fraction thereof is contained in the composition for controlling plant diseases at a concentration of 0.1 to 5000 μg / ml, the composition for controlling plant diseases. Polyalthia Longifolia longifolia ) plant disease control composition comprising an extract or a fraction thereof to a plant, its seed or its habitat; plant disease control method comprising a. The method according to claim 12, wherein the control method is carried out by a volatilization treatment method or a seed treatment method. The method according to claim 12, wherein the control method is to treat the composition for controlling plant diseases on the surface of stems and leaves of plants, sterilization and coating of seeds, or in the habitat in which the plants grow. 13. The method of claim 12, wherein the habitat is a planting hole, a furrow, around a replantation, around a planting furrow, the entire surface of a growth site, a part between soil and a plant, a part between roots, a stem of a plant. A control method that is selected from the group consisting of a site, a main furrow, growing soil, a nail bed, a box for seedlings, a tray for seedlings, or a seedbed. The control method according to claim 12, wherein the treatment is performed during the simulated cultivation period of the composition, before cultivation settlement, at the time of cultivation settlement, and during the growth period after cultivation settlement.

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