KR102634924B1 - Composition for controlling plant diseases comprising Polyalthia longifolia extract or fraction thereof - Google Patents

Abstract

The present invention is Polyalthia longifolia longifolia ) extract or a fraction thereof.
Polyalthia longifolia of the present invention longifolia ) extract and its fractions, the composition for controlling plant diseases is a natural product that is harmless to the human body, biodegrades in the natural world, and is effective in controlling plant diseases without causing environmental pollution, so it can be developed as an environmentally friendly biological pesticide. It can be useful in producing high value-added organic products.

Description

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

Polyalthia longifolia longifolia ) extract and fractions thereof as active ingredients, and a method for controlling plant diseases using the composition.

Plant diseases are one of the biological hazards that impede the healthy growth of plants and affect agricultural production by reducing crop yield and quality. If a plant disease occurs in a large-scale cultivation area, crop production will decrease significantly, causing great economic loss, and if production of staple grains is disrupted, it will pose a major threat to food security, such as the Great Famine in Ireland or the Great Famine in Bengal in India. Serious social problems may arise.

In modern agriculture, chemical pesticides have been used as a convenient means to control various plant diseases and minimize losses by effectively managing plant diseases within the economic threshold range. However, as a result of increased agricultural production relying on chemical pesticides over the past several decades, drug-resistant pathogens and pests have emerged, concerns have arisen about human risk due to toxicity to livestock, and ecosystem disturbance due to residual toxicity has arisen, leading to the emergence of chemical pesticides. There is a need to reduce the use of and develop new plant disease control methods to replace them.

Biocrop protection agent (biopesticide) is an eco-friendly plant disease control method 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, which are then stored in the body. Some of these substances act as antibiotics or phytoalexins, preventing the invasion of pathogens or inhibiting the feeding of pests. Knotweed extract (brand name Milsana), rosemary oil (brand name Sporan), and tea tree oil (brand name Timorex) are commercially available as biocrop protection agents, and various other plant extracts are highly valuable as plant disease control agents. A number of research results have been reported.

Polyalthia longifolia (Sonn.) Thwaites, belonging to the Annonaceae family, is a common name (synonym) for Monoon longifolium (Sonn.) B. It is called the false Ashoka tree, and is native to India and 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 a fever reducer. The main ingredient of the plant extract is diterpenoid, which exhibits various pharmacological activities such as insect feeding inhibition, opioid receptor detection, cytotoxicity, anti-microbial, anti-inflammatory, lipid lowering, nerve growth, and acetylcholine esterification. It is known to contain

The present inventors attempted to use Polyalthia longifolia plant extract as an environmentally friendly means of controlling plant diseases.

Korean Patent Publication 10-2012-039702 (2012.04.25)

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

Another object of the present invention is to provide a plant disease control method comprising treating plants, their seeds, or their habitat with a composition for controlling plant diseases containing a Polyalthia longifolia extract or a fraction thereof. It is done.

While investigating the control effect of various plant extracts on various plant diseases in order to develop an eco-friendly plant disease control agent, the present inventor discovered that Polyalthia longifolia has an excellent control effect on various plant diseases.

The terms used in this application are only used to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, terms such as “comprise” or “have” are intended to designate the presence of the features, steps, structures, or combinations thereof described above in the name, but are intended to indicate the presence of one or more other features, steps, structures, or these. It should be understood that the existence or addition of combinations is not excluded in advance.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless explicitly defined in the present application, should not be interpreted in an ideal or excessively formal sense. No.

polyartia longifolia ( Polyalthia longifolia ) extract or its fraction containing plant disease Composition for pest control and method for producing the same

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

In this specification, the term “control” is used to mean not only prevention and avoidance of diseases or pests, but also removal and death. However, since the main control mechanism for plant disease control is the control effect through preventive treatment, the control effect of Polyalthia longifolia extract or its fractions on plant diseases was investigated using preventive treatment.

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

In addition, the composition is a black spot pathogen of Brassica plants ( Alternaria) . brassicicola ), gray mold fungus ( Botrytis cinerea ), cucumber black star fungus ( Cladosporium cucumerinum ), pepper anthracnose fungus ( Colletotrichum coccodes ), ginseng root rot fungus ( Cylindrocarpon destructans ), tomato wilt fungus ( Fusarium oxysporum f. sp. lycopersici ), rice blast fungus ( Magnaporthe oryzae ) and potato/tomato late blight fungus ( Phytophthora infestans ). It is effective in controlling plant diseases caused by phytopathogenic fungi, and is preferably the brassica plant black spot fungus ( Alternaria brassicicola ) and cucumber black spot fungus ( Cladosporium ). cucumerinum ), pepper anthracnose ( Colletotrichum coccodes ), tomato wilt fungus ( Fusarium oxysporum f. sp. lycopersici ), rice blast fungus ( Magnaporthe oryzae ) and potato/tomato blight fungus ( Phytophthora infestans ).

In addition, the composition is Phalaenopsis orchid bacterial brown spot disease ( Acidovorax avenae subsp. cattleyae ), fruit tree root nodule fungus ( Agrobacterium) tumefaciens ), bacterial rice blight ( Burkholderia glumae ), pepper canker ( Clavibacter) michiganensis subsp. michiganensis ), Phalaenopsis orchid soft rot fungus ( Dickeya ) chrysanthemi ), vegetable soft rot ( Pectobacterium carotovorum subsp. carotovorum ), kiwi canker ( Pseudomonas syringae pv. actinidiae ), tomato green rot ( Ralstonia ) It is effective in controlling plant diseases caused by plant pathogenic bacteria such as solanacearum ) and peach bacterial borer ( It has an excellent control effect against plant diseases caused by fungi ( Dickeya chrysanthemi ).

In the present invention, the extract is water, C ₁ to C ₄ lower alcohol (e.g., methanol, ethanol, propanol, isopropanol, butanol, etc.), hexane, chloroform, methylene chloride, ethyl acetate, acetone, acetonitrile. and a combination thereof can be used as a solvent, preferably a C ₁ to C ₄ lower alcohol, more preferably methanol. However, the extraction solvent in the present invention is not limited to this.

In the present invention, the fractions of the extract can be fractionated using hexane, chloroform, ethyl acetate, methylene chloride, methanol, ethanol, butanol, water, and combinations thereof as solvents, 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 Polyalthia longifolia extract have excellent pest control effects, and the antibacterial active substance having a pest control effect may be a non-polar substance rather than a water-soluble substance. .

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

In the present invention, the Polyalthia longifolia longifolia ) extract or fractions thereof may include a compound represented by any one of the following 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]

.

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 -diene-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 -diene-15,16-olide (3 α ,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 -diene-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 -diene-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 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. ;

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 Formula 15 is named longimide B methyl ester.

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

In the present invention, the extract or fraction thereof may be included in a composition for controlling plant diseases at a concentration of 0.1 to 5000 μg/ml. The concentration refers to the concentration contained in the composition when treating a plant, its seeds, or its habitat in which a plant disease has occurred. If the concentration is less than 0.1 μg/ml, the concentration of the extract or fraction thereof is too low and causes plant disease. There is a problem in that the control effect is low, and if the concentration exceeds 5000 μg/ml, the concentration of the extract or fraction thereof is too high than necessary, making it uneconomical and may have 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 can be appropriately adjusted considering the type, occurrence level, environment, etc. of plant pathogens or plant pathogenic bacteria.

The composition for controlling plant diseases may be a simple mixture of Polyaltea longifolia extract and fractions thereof, or 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 may be added to the mixture so that the mixture can be formulated into an emulsion, emulsion, fluidizing agent, wettable powder, granulated wettable powder, powder, granules, etc. and adding other necessary auxiliaries. The composition for controlling plant diseases mentioned above can also be used as a seed treatment agent according to the present invention by itself or by adding other inert ingredients.

In the present invention, examples of liquid carriers 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.

Additionally, 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 materials such as corn stalk powder and walnut shell powder; synthetic organic substances such as urea; salts such as calcium carbonate and ammonium sulfate; Includes synthetic inorganic materials such as synthetic hydrated silicon oxide; As liquid carriers, 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; Includes petroleum aliphatic hydrocarbons, esters, dimethylsulfoxide, acetonitrile and water.

Additionally, 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 sulfosulfons. 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 auxiliaries include water-soluble polymers such as polyvinyl alcohol and polyvinylpyrrolidone, polysaccharides such as gum arabic, alginic acid and its salts, CMC (carboxymethyl-cellulose), xanthan gum, inorganic substances such as aluminum magnesium silicate and alumina sols ( alumina sol), preservatives, colorants and stabilizers such as PAP (isopropyl acid phosphate) and BHT (butylhydroxylitholuene).

In one embodiment of the present invention, it was confirmed through 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 containing the extract or fraction was used to control plant diseases. It can be seen that it has a disease control effect.

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

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

1) Extracting Polyaltia longifolia by adding an extraction solvent;

2) filtering the extract of step 1); and

3) Preparing an extract of Polyaltea longifolia by concentrating the filtered extract of step 2) under reduced pressure and drying it.

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

1) Extracting Polyaltia longifolia by adding an extraction solvent;

2) filtering the extract of step 1);

3) preparing an extract of Polyaltea longifolia by concentrating the filtered extract of step 2) under reduced pressure and then drying it; and

4) Preparing a Polyaltea longifolia fraction by additionally extracting the Polyaltea longifolia extract from step 3) with an organic solvent.

In the above method, Polyaltea longifolia in step 1) can be used without limitation, whether cultivated or commercially available. The Polyaltea longifolia can be used in all its leaves, stems, or roots, but is not limited thereto.

The extraction solvent in 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. It is not limited to this. Additionally, the lower alcohol may be methanol, ethanol, propanol, isopropanol, or butanol.

Extraction methods include shaking extraction, Soxhlet extraction, or reflux extraction, but are not limited thereto. The extraction solvent can be extracted by adding 1 to 10 times the amount of dried Polyaltea longifolia, and can be extracted by adding 2 to 3 times the amount. The extraction temperature may be 20°C to 100°C, 20°C to 40°C, or room temperature, but is not limited thereto. Additionally, the extraction time may be 10 to 48 hours, 15 to 30 hours, or 24 hours, but is not limited thereto. Furthermore, the number of extractions may be 1 to 5 times, extraction may be repeated 3 to 4 times, and may be repeated 3 times, but is not limited thereto.

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

In the above method, the organic solvent in 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 mixed solution thereof, but is not limited thereto. The fraction may be any one of the solvent fractions obtained by suspending the Polyaltia longifolia extract in water and then 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 from the Polyaltia longifolia extract by repeating the fractionation process 1 to 5 times, 3 times, 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 fractions of Polyaltea longifolia extract and may have bactericidal activity.

In the present invention, a solvent fraction is prepared from the Polyaltia longifolia extract, and the fraction is fractionated using liquid chromatography to isolate the compounds represented by Formulas 1 to 15.

The liquid chromatography technique refers to a chromatography technique in which the mobile phase is a liquid, and is performed in a column filled with a stationary phase or a plane to which the stationary phase is attached. The Polyaltia 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 can be used alone or in combination, and as the stationary phase, silica gel and Diaion HP-20 can be used. , RP-18 or Sephadex LH-20 can be used, but are not limited to these.

The compound represented by any one of Formulas 1 to 15 of the present invention includes not only its salt, but also all forms such as solvates, optical isomers, and hydrates that can be prepared therefrom by conventional methods.

In the present invention, an acid addition salt formed from a free acid is useful as a “salt” of a compound represented by any one of Formulas 1 to 15. 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 dioate, aromatic acids, aliphatic and aromatic sulfonic acids, and organic acids such as acetate, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, and fumaric acid. These salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide, and 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, methyl benzoate, dinitro benzoate, hydroxybenzoate, methoxybenzoate, Phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, Includes tartrate, methanesulfonate, propane sulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, etc.

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

Additionally, metal salts can be made using bases. 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 insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically appropriate to prepare sodium, potassium, or calcium salts as metal salts. Additionally, the corresponding salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (e.g., silver nitrate).

In the present invention, “isomers” include “stereoisomers” or “enantiomers,” and stereoisomers may include diastereomers individually or mixtures thereof, and optical isomers may include diastereomers, respectively, or mixtures thereof. It includes not only individual enantiomers, but also mixtures and racemates of enantiomers. In the present invention, the compounds represented by Formulas 7 to 9 each have a chiral carbon center and include enantiomers of R form and S form in a 1:1 ratio.

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

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

polyartia longifolia ( Polyalthia longifolia ) extract or its fraction containing plant disease Using a pest control composition plant disease Control method

In another aspect of the present invention, a plant disease control method comprising the step of treating a plant, its seeds, or its habitat with a composition for controlling plant diseases containing a Polyalthia longifolia extract or a fraction thereof. provides.

The treatment may be indirect spraying, in which the composition is sprayed directly on the plant, sprayed on the soil in which the plant grows, or sprayed on a medium for cultivating the plant.

The control method of the present invention includes treatment of the stems and leaves of the plant, treatment of the area where the plant grows (e.g. soil), treatment of the seeds such as seed sterilization/seed coating, and treatment of the roots.

Treatment of stems and leaves with the control method of the invention may in particular include application on the plant surface, for example by spraying the stems and leaves. Treatment of the soil with the control method of the present invention may include, for example, spraying on the soil, mixing with the soil, application of the liquid treatment agent into the soil (irrigation of the liquid treatment agent, injection into the soil, dripping of the liquid treatment agent). Examples of places to be treated include planting holes, furrows, around planting holes, around planting furrows, the entire surface of the growth area, the area between the soil and the plant, the area between roots, and the area below the stem of the plant. , main furrow, growth soil, nail bed. Includes seedling growing boxes, seedling growing trays, and seedbeds. Treatment can be carried out before spraying, at the time of spraying, immediately after spraying, during a simulated cultivation period, before cultivation establishment, at cultivation establishment, and during the growing season after cultivation establishment. In the above-mentioned soil treatment, the active ingredient may be applied simultaneously to the plants, or a solid fertilizer such as a paste fertilizer containing the active ingredient may be applied to the soil. The active ingredient can be mixed in the irrigation liquid, for example injected into irrigation equipment (irrigation tubes, irrigation pipes, sprinklers, etc.), mixed in the liquid that floods between the furrows, or mixed in the water culture medium. You can. Alternatively, the irrigation liquid and active ingredients can be premixed and used for treatment by suitable irrigation methods, including, for example, the irrigation methods mentioned above and other methods such as spraying and flooding.

The volatilization method of the control method of the present invention is, for example, spraying the composition for controlling plant diseases of the present invention on media such as soil for cultivating plants, hydroponic media for culturing plants, and seedbeds to volatilize the sprayed composition. This is a method of protecting the plant from pests and diseases through this method. In addition, the plant can be exposed to the volatilized gaseous composition by placing the composition around the plant.

The seed treatment method using the control method of the present invention is, for example, a method of treating seeds to be protected from pests and diseases with the composition for controlling plant diseases of the present invention, and a specific example thereof is atomizing a 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, fluidizing agent, etc. of the composition for controlling plant diseases of the present invention is applied on the seed surface by itself or by adding a small amount of water; An impregnation treatment method in which seeds are impregnated in a solution of the composition for controlling plant diseases of the present invention for a specific period of time; Includes film coating treatment method and pellet coating treatment method.

When plants or soil for plant growth are treated with a 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 pests to be controlled, formulation type, treatment period, climatic conditions, etc.

Emulsions, wettable powders, fluidizing agents, etc. are usually diluted with water and then sprayed 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, etc. are usually used for processing without dilution.

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

Polyalthia longifolia longifolia ) extract, or a fraction thereof, is a natural product that is harmless to the human body, biodegrades in the natural world, and has an excellent effect in controlling plant diseases without causing environmental pollution, so it could be developed as an environmentally friendly biological pesticide. and can be useful in producing high value-added organic products.

Figure 1 shows Polyalthia longifolia ( Polyalthia longifolia ) This is a process diagram for separating active compounds from fractions of extract.
Figure 2 shows Polyalthia longifolia ( Polyalthia longifolia ) extract fractions.
Figure 3 shows Polyalthia longifolia ( Polyalthia longifolia ) extract, showing the three-dimensional structures of formulas 1 to 5 isolated from the fractions.

Hereinafter, it will be described in detail through examples.

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

< Example 1> polyartia longifolia ( Polyalthia longifolia ) Preparation of a composition for controlling plant diseases containing extracts

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

Polyalthia longifolia In order to prepare a composition for controlling plant diseases containing an extract of Polyaltea longifolia ), an extract of Polyaltea longifolia was first prepared. Specifically, dried Polyalthia longifolia ( Polyalthia longifolia ) leaf and stem sample (5.8 kg) was cut into small pieces, added with methanol, extracted at room temperature for 24 hours, filtered through filter paper, and the obtained filtrate was concentrated under reduced pressure to obtain 702.9 g of Polyalthia longifolia methanol extract. Obtained.

Step 2: polyartia longifolia containing extracts plant disease Preparation of composition for pest control

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

< Example 2> polyartia longifolia containing extracts plant disease Evaluation of plant disease control effect of control composition

Example 2-1: polyartia longifolia Contains methanol extract plant disease Investigation of plant disease control activity of control compositions ( in vivo )

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

Specifically, three pots were used for each plant pathogen. The control composition solution prepared in step 2 was sprayed on the foliage, air-dried for 24 hours, and then inoculated with each plant pathogen. The rice, tomato, pepper, and wheat plants used in the experiment were filled with 80% of water supply or horticultural topsoil in a plastic pot with a diameter of 4.5 cm, then sown seeds and grown in a greenhouse at a temperature of 25 ± 5°C for 1 to 3 weeks. did.

Rice blast is caused by Magnaporthe oryzae , the causative agent of rice blast in seedlings at the 2-3 leaf stage. KACC 46552) spore suspension ( ⁵ The outbreak was induced.

Tomato late blight is caused by spraying 3-4 leaf stage tomato seedlings with a zoospore suspension (2×10 ⁴ sporangia/mL) of Phytophthora infestans KACC 48738, the causative agent of late blight, at 20°C. Disease was induced by treatment in a wet room for 2 days and cultivation in a constant temperature room at 20°C for 1 day.

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

Wheat red rust is caused by Puccinia triticina, the causative agent of rust, known as an active parasitic bacterium on first-leaf seedlings. triticina (Korea Research Institute of Chemical Technology) spores were suspended in a 250 μg/mL Tween 20 solution at an amount of 0.67 g spores/L and inoculated by spray. After being treated in a wet room at 20°C for one day, they were transferred to a constant temperature room at 20°C. Disease was induced by cultivation for 6 days.

Pepper anthracnose is caused by spraying and inoculating seedlings at the 3-4 leaf stage of pepper with a spore suspension (4×10 ⁵ spores/mL) of Colletotrichum coccodes KACC 48737, the causative agent of pepper anthracnose, in a wet room at 25°C. After treatment in a humid room for 2 days, disease was induced by culturing in a constant temperature and humidity room (25°C, 80% relative humidity) for 1 day.

The disease spot area ratio (%) was examined 3 days after inoculation for tomato gray mold and pepper anthracnose, 4 days after inoculation for tomato late blight, 5 days after inoculation for rice blast, and 7 days after inoculation for wheat red rust.

Using the disease spot area ratio (%) obtained above, the control value (%) was calculated according to Equation 1 below, and the results are shown in Table 1 below.

[Equation 1]

Control value (%) = (1-disease area ratio of treatment group/disease area rate of untreated group)

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

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

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

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

Example 2-2: polyartia longifolia ( Polyalthia longifolia ) containing extracts plant disease Investigation of bactericidal activity of control compositions ( in vitro )

The phytopathogenic fungi of the control composition prepared according to Example 1 include Brassica family crop black spot fungus ( Alternaria brassicicola ), cucumber black spot fungus ( Cladosporium cucumerinum ), and pepper anthracnose fungus ( Colletotrichum coccodes ), tomato wilt fungus ( Fusarium oxysporum) f. sp. lycopersici ), rice blast fungus ( Magnaporthe oryzae ), potato/tomato blight fungus ( Phytophthora infestans ) and the plant pathogenic bacteria Phalaenopsis brown spot ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis orchid soft rot ( Dickeya chrysanthemi ) were tested using the broth micro-dilution method using a 96-well plate. Through evaluation, the minimum inhibitory concentration value was obtained.

Brassica family crop black spot fungus ( A. brassicicola ) and tomato wilt fungus ( F. oxysporum f. sp. lycopersici ) were inoculated on PDA (potato dextrose agar) medium and incubated at 25°C for 10 days, and the formed spores were used in the experiment. Potato/tomato late blight bacteria ( P. infestans ) were inoculated into 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 conditions of 14 hours a day, and the formed spores were tested. It was used in . Pepper anthracnose ( C. coccodes ) and rice blast fungus ( M. oryzae ) were inoculated into OA (oatmeal agar) medium and cultured at 25°C for 7 days, then mycelia were removed and placed in 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 plant pathogenic bacteria, after inoculation on TSB medium, Phalaenopsis orchid bacterial brown spot fungus ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis orchid soft rot fungus ( Dickeya chrysanthemi ) were each cultured with shaking (150 r/min) for 2 days at 30°C. did. For the fungi used in the experiment, spores were harvested using PDB medium, filtered through 4-ply gauze to prepare a spore suspension (1 × 10 ⁴ /mL), and bacteria were diluted in TSB medium to produce a bacterial suspension (1 × 10 ⁴ CFU/mL) was prepared.

Ultimately, 100 μL wells contained 1000 fungal spores or bacteria, and the Polyaltea longifolia methanol extract of the present invention was added at 1, 2, 4, 8, 16, 32, 63, 125, 250, and 500 μg/, respectively. It was dispensed to contain a concentration of mL. At this time, the methanol content did not exceed 1%. One with only 1% methanol added was used as an untreated group, and the experiment was repeated three 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 2 below.

division plant pathogens Minimum inhibitory concentration
(MIC, μg/mL) plant pathogenic fungi Black spot disease fungus ( Alternaria) on cabbage crops brassicicola ) 63 Cucumber black star spot fungus ( Cladosporium cucumerinum ) 250 Capsicum anthrax ( Colletotrichum) coccodes ) 250 Tomato wilt fungus ( Fusarium) oxysporum f. sp. lycopersici ) 250 Rice blast fungus ( Magnaporthe oryzae ) 16 Potato/tomato blight fungus ( Phytophthora infestans ) 125 plant pathogenic bacteria Phalaenopsis orchid bacterial brown spot disease ( Acidovorax avenae subsp. cattleyae ) 8 Phalaenopsis orchid soft rot fungus ( Dickeya chrysanthemi ) 500

As shown in Table 2, Polyalthia longifolia longifolia ) leaves and stems with methanol showed bactericidal activity against six types of plant pathogenic fungi. Polyaltea longifolia methanol extract was used at concentrations of 16, 63, and 125 μg/mL, respectively, against rice blast fungus ( M. oryzae ), brassica plant black spot fungus ( A. brassicicola ) , and potato/tomato late blight fungus ( P. infestans ) completely inhibited the growth of pepper anthracnose ( C. coccodes ), tomato wilt fungus ( F. oxysporum f. sp. lycopersici ), and cucumber black spot fungus ( C. cucumerinum ) at a concentration of 250 μg/mL. It showed an inhibitory bactericidal effect.

Additionally, Polyalthia longifolia longifolia ) leaves and stems with methanol. At a concentration of 8 μg/mL, the extract obtained was Phalaenopsis orchid brown spot fungus ( A. avenae subsp. cattleyae ), a plant pathogenic bacterium, and Phalaenopsis soft rot ( D. chrysanthemi ) at a concentration of 500 μg/mL. It showed bactericidal activity that completely inhibited the growth of.

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

Step 1: polyartia 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 step 1 of Example 1 was performed to prepare Polyalthia longifolia ( Polyalthia) . longifolia ) methanol extract was prepared, and an organic solvent fraction was prepared using the methanol extract.

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

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

Each sample dissolved in methanol at a concentration of 40 mg/mL was diluted with distilled water, and the electrodeposition agent Tween 20 was added to a concentration of 0.025% (w/v) to prepare polystyrene at a concentration of 1000 μg/mL and 2000 μg/mL. Polyalthia longifolia longifolia ), 40 mL each of a composition solution for controlling plant diseases containing the solvent fractions (chloroform fraction, ethyl acetate fraction, and water fraction) of the methanol extract as active ingredients was prepared.

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

Example 4-1: polyartia longifolia ( Polyalthia longifolia ) Plant disease control activity of a composition for plant disease control containing the solvent fraction of methanol extract ( in vivo )

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

Polyalthia longifolia longifolia ) The results of the 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 price (%) 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 disease (causing bacteria: Magnaporthe oryzae ); TGM, tomato gray mold disease (causing bacteria: Botrytis cinerea ); TLB, tomato late blight (causing agent: Phytophthora infestans ); WLR, wheat red rust (causing agent: Puccinia triticina ); PAN, pepper anthracnose (causing bacteria: Colletotrichum coccodes ))

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

Meanwhile, the water fraction did not show control activity at the same treatment concentration, showing that Polyalthia longifolia longifolia ) extract, it was confirmed that the antibacterial active substance was a non-polar substance, not a water-soluble substance.

Based on the fact that the chloroform fraction showed a high level of control activity against tomato late blight, pepper anthracnose, rice blast, and wheat red rust, the chloroform fraction was used for isolation and purification of the active control ingredient.

Example 4-2: polyartia longifolia ( Polyalthia longifolia ) Chloroform fraction containing plant disease Control composition plant disease Confirm control activity ( in vitro )

The composition containing the chloroform fraction prepared according to Example 3 was used against the plant pathogenic fungus, Brassica family crop black spot fungus ( Alternaria) brassicicola ), cucumber black star fungus ( Cladosporium cucumerinum ), pepper anthracnose fungus ( Colletotrichum coccodes ), tomato wilt fungus ( Fusarium oxysporum) f. sp. lycopersici ), rice blast fungus ( Magnaporthe oryzae ), potato/tomato late blight fungus ( Phytophthora infestans ) and the plant pathogenic bacterium Phalaenopsis orchid ( Acidovorax). avenae subsp. cattleyae ) was evaluated by the broth micro-dilution method using a 96-well plate, and the minimum inhibitory concentration value was obtained.

Brassica family crop black spot fungus ( A. brassicicola ) and tomato wilt fungus ( F. oxysporum f. sp. lycopersici ) were inoculated on PDA (potato dextrose agar) medium and incubated at 25°C for 10 days, and the formed spores were used in the experiment. Potato/tomato late blight bacteria ( P. infestans ) were inoculated into 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 conditions of 14 hours a day, and the formed spores were tested. It was used in . Pepper anthracnose ( C. coccodes ) and rice blast fungus ( M. oryzae ) were inoculated into OA (oatmeal agar) medium and cultured at 25°C for 7 days, then mycelia were removed and placed in 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 brown spot fungus ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis orchid soft rot fungus ( Dickeya chrysanthemi ) were cultured with shaking (150 r/min) for 2 days at 30°C. . For the fungi used in the experiment, spores were harvested using PDB (potato dextrose broth) medium and filtered through 4-layer gauze to prepare a spore suspension, and the bacteria were diluted in TSB (tryptic soy broth) medium to prepare a bacterial suspension. did.

Finally, 100 μL wells were allowed to contain 1000 fungal spores or bacteria, and methanol extract was dispensed at concentrations of 1, 2, 4, 8, 16, 31, 63, 125, 250, and 500 μg/mL, respectively. . At this time, the methanol content did not exceed 1%. One with only 1% methanol added was used as an untreated group, and the experiment was repeated three 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 molds 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) plant pathogenic fungi Brassica family crops black spot disease ( Alternaria brassicicola ) 63 Cucumber black star spot fungus ( Cladosporium cucumerinum ) 250 Capsicum anthrax ( Colletotrichum) coccodes ) 250 Tomato wilt fungus ( Fusarium) oxysporum f. sp. lycopersici ) 500 Rice blast fungus ( Magnaporthe oryzae ) 31 Potato/tomato blight fungus ( Phytophthora infestans ) 125 plant pathogenic bacteria Phalaenopsis orchid bacterial brown spot disease ( Acidovorax avenae subsp. cattleyae ) 8

As shown in Table 4, Polyalthia longifolia longifolia ) was confirmed to have bactericidal activity against five types of plant pathogenic fungi. The chloroform fraction completely inhibited the growth of rice blast fungus ( M. oryzae ), brassica plant black spot fungus ( A. brassicicola ), and potato/tomato late blight fungus ( p. infestans ) at concentrations of 31, 63, and 125 μg/mL, respectively. At a concentration of 250 μg/mL or more, it has a bactericidal effect that completely inhibits the growth of cucumber black spot fungus ( C. cucumerinum ), pepper anthracnose ( C. coccodes ), and tomato wilt fungus ( F. oxysporum f. sp. lycopersici ). indicated.

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

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

Polyalthia longifolia longifolia ) extract, silica gel (Kiesel gel 60; 70-230 mesh; Merck) column chromatography, Sephadex LH-20 (Merck) column chromatography, and Biotage Isolera One medium-pressure liquid chromatography (mid-pressure) were used to separate the bactericidal active compounds from the extract. Separation was performed using a combination of a liquid chromatography (MPLC) system and a 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 isolated from the chloroform fraction (50 g) prepared according to Example 3 through the process shown in FIG. 1.

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

Another active fraction F11 (22 g) was adsorbed on a silica gel column and eluted with a solution of 0-10% volume ratio of methanol in chloroform under concentration gradient conditions to produce four fractions (F11-1, F11-2, F11-3). , F11-4). Active fraction F11-3 (16.5 g) was loaded on a Sephadex LH-20 column and eluted with a chloroform-methanol mixed solution (1:1, v/v) to obtain active fraction F11-33 (9.9 g), which was separated into silica gel. After adsorption to the column, 3 fractions (F11-331, F11-332, and F11-333) were obtained by eluting with a solution of dichloromethane mixed with acetone at a 0-35% volume ratio under concentration gradient conditions. 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 and eluted with 70-100% methanol aqueous solution to produce 5 fractions (F11-3331, F11-3332, F11-3333, F11- 3334) was obtained, and fraction F11-3333 was separated into 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 5 mL/mL of 60% aqueous acetonitrile solution (containing 0.1% formic acid). Compound 7 (21 mg), compound 8 (22 mg), and compound 9 (20 mg) were separated by elution at a flow rate of min.

Active fraction F11-3332 (30 mg) was loaded on preparative silica gel TLC and developed with hexane-ethyl acetate (2:1, v/v) to separate 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 elution 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 chloroform-methanol (1:1, v/v) solution to produce four fractions (F11-41, F11-42, F11-43). , F11-44). Active fraction F11-42 (692 mg) was added to a Biotage SNAP Ultra C18 (30 g) column and eluted with 70-100% methanol aqueous solution to obtain two fractions (F11-411, F11-412). Fraction F11-411 was recrystallized to isolate compound 14 (100 mg).

Fraction F11-422 (50 mg) was added to a Biotage SNAP KP-Sil (50 g) column, and then eluted with a 0-35% volume ratio of ethyl acetate solution in dichloromethane under concentration gradient conditions to obtain compound 15 (5.3). mg) was isolated.

The active fraction F11-43 (823 mg) was added 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). Fraction F11-431 was recrystallized to isolate compound 10 (200 mg).

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 at 5 mL/mL. Compound 1 (3.1 mg) and Compound 13 (15.1 mg) were separated by elution 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 separated by eluting with a chloroform-methanol mixed solution (1:1, v/v) (Figure 1).

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

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

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

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

New compound 1 is polylongifoliaon C; New compound 2 is polylongifoliaon D; New compound 3 is polylongifoliaon E; New compound 4 is polylongifoliaon F; New 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 -diene-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-dien-15,16-olide); Compound 9 is (4→2) -abeo -16-hydroxy-cleroda-2,13(14) Z -diene-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; 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 (Figure 2).

<6-1> Identification of chemical structure of compound 1

New 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 its chemical formula was determined to be C ₂₁ H ₃₀ O ₄ did. By combining the NMR spectrum of New Compound 1 (Table 5) and data presented in the literature, it was confirmed that it was a compound with the clerodane diterpene skeleton.

Finally, the chemical structure of new compound 1 is methyl-2,16-dioxo-cleroda-3,12(13)E-diene-15-oate (methyl-2,16-dioxo) represented by the following formula 1: -cleroda-3,12(13)E-dien-15-oate), and the compound was named polylongifoliaon C. The NMR analysis results of new 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 new compound 1 , there was one ketone carbon ( δ _C 199.3), one aldehyde carbon ( δ _C 193.5), one carboxyl carbon ( δ _C 170.4), and two quarter carbons. Quaternary 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) appeared.

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 (Figure 3).

Specifically, in the HMBC spectrum, the hydrogen signal H-1 is connected to C-2, H-3 is connected to C-1, C-5, and C-18, and H-18 is connected to C-3, C-4, and C-5. It was confirmed that a correlation appears, and based on the chemical shift values of carbon signals C-2, C-3, and C-4 ( δ _C 199.3, 125.8, 172.1) and hydrogen signal H-3 ( δ _H 5.74), C- Oxidation (ketogenesis) at 2 positions was 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 in NOESY analysis, H-12 hydrogen and H-16 aldehyde were correlated. A correlation appeared between hydrogen. 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 the H-12 ( δ _H 6.67) olefin hydrogen. It was confirmed that they were in a relationship.

In addition, the hydrogen signal H-14 is a singlelet (singLet) and the hydrogen signal H-12 is a triplet (tripLet) because the position of the double bond is at C-12/C-13 instead of C-13/C-14. indicated. In addition, by comparing the chemical shift values of C-17, C-19, and C-20 methyl groups ( δ _C 16.3, 18.4, 17.5) of Compound 1 with literature data, trans -clerodane diterpene ( trans -clerodane diterpene) It was found to be a skeletal compound, and methyl esterification was confirmed based on the HMBC correlation between methoxy hydrogen and C-15 carbonyl carbon.

<6-2> New compounds 2 Identification of the chemical structure of

New 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 its chemical formula was determined to be C ₂₁ H ₃₀ O ₅ . By combining the NMR spectrum of new compound 2 shown in Table 6 and data presented in the literature, it was confirmed that it was a compound of the clerodane diterpene skeleton.

Finally, the chemical structure of the new compound 2 is methyl-2-oxo-cleroda-3,12(13) E -diene-16-oxoic acid-15-oate (methyl-2-oxo- cleroda-3,12(13) E -dien-16-oic acid-15-oate), and the compound was named polylongifoliaon D. The NMR analysis results of new 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, ^{the 13} C-NMR spectrum of new compound 2 shows one ketone carbon ( δ _C 199.6), two carboxylic carbons ( δ _C 171.0, 170.9), and 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 five methyl carbons ( δ _C 52.4, 19.2, 18.5, 17.4, 16.2) appeared.

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 (Figure 3).

The NMR spectrum of Compound 2 was similar to the NMR spectrum of Compound 1 , except that one carboxyl group signal appeared instead of one aldehyde signal. In addition, the difference in molecular weight 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 indicates that the aldehyde carbon of Compound 1 is oxidized to a carboxyl group in Compound 2 . It was supported that it was done.

<6-3> New compounds 3 and 4 Identification of the chemical structure of

New compounds 3 and 4 were isolated in the form of white wax. In HRESIMS analysis, cations were detected at m/z 387.2148 [M+Na] ⁺ and m/z 387.2146[M+Na] ⁺ (calculated m/z 387.2147), respectively, and the chemical formula was determined to be C ₂₁ H ₃₀ O ₅ . By combining the NMR spectra of new compounds 3 and 4 (Table 7) and data presented in the literature, it was confirmed that they were compounds with the clerodane diterpene skeleton.

Finally, the chemical structure of the new compound 3 is (13 S )-methyl-2-oxo-cleroda-3-en-16-oxoic 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 new compound 4 is (13 R )-methyl-2-oxo-cleroda-3-en-16-oxoic 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 new 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, new compounds 3 and 4 are enantiomers of each other and the NMR data of the two compounds are virtually identical, so the chemical structure was first revealed using the NMR data of compound 3 .

¹³ C-NMR spectrum of new compound 3 shows 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), and three quaternary sp carbons. 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) appeared.

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

The NMR spectrum of compound 3 appeared similar to the NMR spectrum of compound 2 , except that the 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 lower than that of compound 4 , which means that compounds 3 and 4 are R/S enantiomers centered on the chiral carbon of C-13. It indicates that it exists as an enantiomer.

In NOESY analysis, compound 3 has H between H-20 methyl hydrogen ( δ _H 0.79) and H-12 hydrogen ( δ _H 1.33) and H between H-12 hydrogen ( δ _H 1.33) and H-13 hydrogen ( δ _H 2.81). It was confirmed that there was a correlation between -13 hydrogen ( δ _H 2.81) and H-14 hydrogen ( δ _H 2.72), and the chemical structure of compound 3 was determined to be a ( S )-type enantiomer. Meanwhile, in NOESY analysis, compound 4 was found between H-12 hydrogen ( δ _H 1.58) and H-13 hydrogen ( δ _H 2.80) and between H-13 hydrogen ( δ _H 2.80) and H-14 hydrogen ( δ _H 2.44). It was confirmed that a correlation appeared, and the chemical structure of Compound 4 was confirmed to be a ( R )-type enantiomer.

<6-4> New compounds 5 Identification of the chemical structure of

New compound 5 was isolated in the form of white wax. In HRESIMS analysis, each was detected as a cation of m/z 419.2140 [M+Na] ⁺ (calculated m/z 419.2140), and its chemical formula was determined to be C ₂₂ H ₃₆ O ₆ . By combining the NMR spectrum and literature data of new compound 5 shown in Table 8, it was confirmed that it was a compound of the clerodane diterpene skeleton, and finally, the chemical structure of new compound 5 was determined as a compound represented by 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 - It was determined as dimethoxymethyl-4 β ,16 α -dihydroxy-cleroda-13 Z -ene-15,16-olide) and named polylongifoline A. The NMR analysis results of new 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 (overlapping) 18 22.0 0.75 (overlapping) 19 17.2 1.09 (s) 20 17.2 0.75 (overlapping) 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 new compound 5 , one carboxyl carbon ( δ _C 171.4), one quaternary sp ² carbon ( δ _C 170.9), and 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 carbons ( δ _C 105.4, 99.3, 45.0, 46.3, 36.8), 5 methylene sp ³ carbons ( δ _C 36.0, 29.7, 27.8, 23.1, 21.3), and six methyl carbons ( δ _C 53.8, 51.0, 22.0, 17.2, 17.2, 15.0) appeared.

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 (Figure 3).

Specifically, in the HMBC analysis of compound 5 , hydrogen signals H-14 were correlated with C-12, C-13, C-15, and C-16, and H-16 was correlated with C-13, C-14, and C-15. This represents the C-14/H-14 signal ( δ _C 115.9/ δ _H 5.95) and C-16/H-16 signal ( δ _{C 99.3} / δH 6.02) was found to be the γ -hydroxyl α , β -unsaturated- γ -lactone portion of the clerodane diterpene skeleton.

In HMBC analysis, the hydrogen signal H-1 is connected to C-2, C-3, C-4, C-9, and C-10, H-3 is connected to 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, similar to the compound polylongifoliaic A, whose chemical structure of compound 5 is known. (4→2) It was shown to be 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 shifts of the methyl carbon signals C-17, C-19, and C-20 appear at δ _C 15.0, 17.2, and 17.2 indicates that the main skeleton of compound 5 is trans -chloradane diterpine (like compound 1 ) trans -clerodane diterpene).

Through NOESY analysis, the three-dimensional structures of C-2 and C-4 were determined, and the nuclear Overhauser effect (nuclear o erhauser ffect) 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 Overhauser 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

The ¹³ C NMR analysis results of compounds 6 to 15 separated through the process in 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, 1 6.0

By comparing the ¹³ C NMR data in Table 9 and the data shown in the literature, compound 6 was 16 α -hydroxy-cleroda-3,13 (14) Z -diene-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 -diene-15,16-olide (3 α ,16-dihydroxy-cleroda-4(18) of Formula 7 ),13(14) Z -dien-15,16-olide); Compound 8 is 3 β, 16-dihydroxy-cleroda-4(18),13(14) Z -diene-15,16-olide (3 β, 16-dihydroxy-cleroda-4(18) of formula 8 ),13(14) Z -dien-15,16-olide); Compound 9 is (4→2) -abeo -16-hydroxy-cleroda-2,13(14) Z -diene-15,16-olide-3-al((4→2) -abeo of the formula 9 -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 the 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 Formula 15, and the structures of 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]

; and

[Formula 15]

.

Each of Formulas 7 to 9 is an R/S enantiomer centered on a chiral carbon, and the R form and S form may exist in a ratio of 1:1.

Although the present invention has been described above with reference to preferred embodiments, those skilled in the art may make various modifications and changes to the present invention without departing from the spirit and scope of the present invention as set forth in the following patent claims. You will understand that it is possible.

Claims (16)

A composition for controlling plant diseases containing Polyalthia longifolia extract or fractions thereof,
The plant disease is caused by phytopathogenic fungi or phytopathogenic bacteria,
The phytopathogenic fungus is selected from the group consisting of cucumber black star fungus ( Cladosporium cucumerinum ) and potato/tomato late blight fungus ( Phytophthora infestans ),
A composition for controlling plant diseases, wherein the plant pathogenic bacteria are selected from the group consisting of Acidovorax avenae subsp. cattleyae and Dickeya chrysanthemi . According to paragraph 1,
A composition for controlling plant diseases, wherein the plant disease is tomato late blight. delete delete delete According to paragraph 1,
The extract is extracted using a solvent selected from the group consisting of water, C ₁ to C ₄ lower alcohols, hexane, chloroform, methylene chloride, ethyl acetate, acetone, acetonitrile, and combinations thereof. Composition for. According to paragraph 1,
The fraction of the extract is a plant disease that is fractionated using any one selected from the group consisting of hexane, chloroform, ethyl acetate, methylene chloride, methanol, ethanol, butanol, water, and combinations thereof, or a mixed solution thereof as a solvent. Composition for pest control. According to paragraph 1,
The extract is Polyalthia longifolia A composition for controlling plant diseases, which is an extract of leaves, stems or roots of (longifolia ). According to paragraph 1,
A composition for controlling plant diseases, wherein the Polyalthia longifolia extract or fraction thereof contains at least one selected from the compounds represented by the following 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]
. According to clause 9,
A composition for controlling plant diseases, wherein the compound has bactericidal activity. According to paragraph 1,
The extract or fraction thereof is included in the composition for controlling plant diseases at a concentration of 0.1 to 5000 μg/ml. It includes the step of treating a plant, its seeds, or its habitat with a composition for controlling plant diseases containing a Polyalthia longifolia extract or a fraction thereof,
The plant disease is caused by phytopathogenic fungi or phytopathogenic bacteria,
The phytopathogenic fungus is selected from the group consisting of cucumber black star fungus ( Cladosporium cucumerinum ) and potato/tomato late blight fungus ( Phytophthora infestans ),
A method for controlling plant diseases, wherein the plant pathogenic bacteria are selected from the group consisting of Phalaenopsis orchid bacterial brown spot fungus ( Acidovorax avenae subsp. cattleyae ) and Phalaenopsis soft rot fungus ( Dickeya chrysanthemi ). The method of claim 12, wherein the control method is carried out by a volatilization method or a seed treatment method. The control method according to claim 12, wherein the composition for controlling plant diseases is treated on the surface of the stem and leaves of the plant, sterilized and coated seeds, or treated in the habitat where the plant grows. The method of claim 12, wherein the habitat is a planting hole, a furrow, around a planting hole, around a planting furrow, the entire surface of the growth area, the area between the soil and the plant, the area between roots, and under the stem of the plant. A control method selected from the group consisting of a site, main furrow, growing soil, bed, seedling box, seedling tray or seedbed. The control method according to claim 12, wherein the treatment is performed using the composition during a simulated cultivation period, before cultivation establishment, during cultivation establishment, and during the growth period after cultivation establishment.