KR102080601B1 - Composition for controlling plant diseases including Trevesia palmata, fraction of thereof or a compound isolated therefrom as an active ingredient and method of controlling plant diseases using the same - Google Patents

Abstract

Trevesia palmata (Trevesia palmata) extracts and their fractions or compounds separated therefrom as an active ingredient comprising a composition for controlling plant diseases and a method for controlling plant diseases using the composition, the composition for controlling plant diseases As a natural product, it is harmless to the human body and is biodegradable in nature, so it is effective in controlling plant diseases without causing environmental pollution. Therefore, it can be developed as an eco-friendly biopesticide and can be usefully used for producing high value-added organic products.

Description

Composition for controlling plant diseases including Trevesia palmata extract, fractions or compounds separated therefrom as an active ingredient, and methods for controlling plant diseases using the composition of specifically or a compound isolated therefrom as an active ingredient and method of controlling plant diseases using the same}

The present invention relates to a composition for controlling plant diseases containing Trevesia palmata extract and fractions thereof or compounds separated therefrom as an active ingredient, and a method for controlling plant diseases using the composition.

In crop production, phytopathogens cause plant diseases as one of the harmful factors that inhibit the healthy growth of plants. Plant diseases in large cultivated areas can be a serious social problem because not only will there be significant economic losses due to a significant decrease in crop production, but also a major threat to food security if production disruption occurs.

In modern and modern agriculture, pesticide use has been a major means of controlling plant diseases. However, chemical pesticides sprayed in large quantities for many years have accelerated the emergence of drug-resistant pathogens in the colony of pathogens, increasing the cost of control, and civil society and academia are constantly concerned about the ecosystem disturbances and the killing toxicity of residual pesticides. Comprehensive pest control, using biopesticides as a solution to the problem of resistance and at the same time reducing the use of synthetic pesticides, may be a new alternative to current pesticide-dependent agricultural methods. Biopesticides are developed based on substances derived from biological materials such as plant extracts or using living microorganisms such as fungi, bacteria and viruses in controlling pests. Therefore, biopesticides are natural products derived from nature, have a nature-friendly feature, and have the advantage of being weak or harmless compared to synthetic chemical pesticides.

As a result, policy regulations on the use of synthetic pesticides are strengthening, especially in OECD countries including Korea. The millions of compounds listed in the American Chemical Association's Chemical Information Database (CAS) can be grouped into just a few hundred classes of lead substances. The number of must fall sharply. Therefore, it is necessary to develop a new environmentally friendly plant disease control means to replace synthetic pesticides.

Plants are adapted to the various environments surrounding the plant in order to adapt to alkaloids, terpenoids, flavonoids, glycosides, quinones, coumarins, phenolics, phenolic, It has evolved to biosynthesize various substances such as essential oil and phytoalexin, and it accumulates secondary metabolites that function to inhibit the invasion of phytopathogens and the feeding of pests.

As biopesticides, plant extracts derived from neem, canola, pyrethrin, limonne, tee tree, rotenone and essential oils are used. In addition, Tagetes patula L. essential oil showed strong antibacterial activity against Botrytis cinerea and Penicillium digitatum, and the extract of Mycelialis jajapa L. showed mycelial growth of plant pathogens. It showed inhibitory activity, and the compound from the extract of Diospyros virginiana L. showed antimicrobial effect, and the saponin compound from quinoa (Chenopodium quinoa Willd) was antibacterial against several plant pathogens including gray fungus. A number of studies have been reported to support the high value of plant extracts as plant disease control agents.

Trevesia palmata Roxb.Ex Lindl, belonging to the Araliaceae, is called a snowflake tree or snowflake because of its leaf-like leaves. Evergreen native to India, grows 15 centimeters in diameter and over 8 meters in height. It is distributed in Bangladesh, Colombia, India, Laos, Nepal, Thailand, and Vietnam, and has a tonic effect, which is consumed as a traditional drink, and is known as a valuable ornamental plant that is useful for removing indoor formaldehyde. In some parts of Thailand, the plant is used as a bath for postpartum care. It is known that extracts of the Trebezia palmata plant show efficacy in thrombolytic and arthritis relief, and it has been reported that saponin compounds isolated from these plants have an effect of inhibiting the proliferation of cancer cell lines.

Tommasi, N. D., Autore, G., Bellino, A., Pinto, A., Pizza, C., Sorrentino, R., Venturella, P. (2000) Antiproliferative triterpenoid saponins from Trevesia palmata. J. Nat. Prod. 63, 308-314. Rahman, K. H., Nandi, J. K., Sultana, S., Rahman, S., Hossan, S., Rahmatullah, M. (2014) Phytochemical screening, antihyperglycemic and analgesic activity studies with methanol extract of Trevesia palmata leaves. World J. Pharm. Pharmaceut. Sci. 3, 91-101. Van Khoa, P., Van Nang, B., Hao, N. T. B. (2013) Study on gaseous formaldehyde removal capability of some native plant species in Vietnam. Pharmaceut. Res. 4, 1-7.

One aspect of the present invention is to provide a plant disease control composition containing at least one selected from the group consisting of Trevesia palmata (Trevesia palmata) extract and fractions thereof as an active ingredient.

According to another aspect of the present invention, there is provided a method of treating a plant, a seed thereof, or a habitat thereof, with a composition for controlling plant diseases comprising at least one selected from the group consisting of Trevesia palmata extract and fractions thereof as an active ingredient. It is to provide a plant disease control method comprising a.

Another aspect of the present invention is to provide a compound which is any one selected from the group consisting of hederagenin saponin glycoside compound and asiatic acid glycoside compound.

Another aspect of the present invention provides a composition for controlling plant diseases containing at least one compound selected from the group consisting of a hederagenin saponin glycoside compound and an asiatic acid glycoside compound as an active ingredient. will be.

Another aspect of the present invention is a plant, plant, composition for controlling plant diseases containing at least one compound selected from the group consisting of hederagenin saponin glycoside compound and asiatic acid glycoside compound as an active ingredient, It is to provide a plant disease control method comprising the step of treating the seed or its habitat.

In order to achieve the above object,

According to an aspect of the present invention, there is provided a composition for controlling plant diseases, containing one or more selected from the group consisting of Trevesia palmata extract and fractions thereof as an active ingredient.

In addition, according to another aspect of the present invention, plants, seeds or habitats thereof for controlling plant diseases comprising at least one selected from the group consisting of Trevesia palmata extract and fractions thereof as an active ingredient. There is provided a plant disease control method comprising the step of treating to.

Furthermore, according to another aspect of the present invention, a plant disease control composition containing at least one selected from the group consisting of Trevesia palmata extract and fractions thereof in controlling plant diseases as an active ingredient Use is provided.

Further, according to another aspect of the present invention, there is provided a compound which is any one selected from the group consisting of hederagenin saponin glycoside compound and asiatic acid glycoside compound.

Furthermore, according to another aspect of the present invention, for controlling plant diseases containing at least one compound selected from the group consisting of hederagenin saponin glycoside compound and asiatic acid glycoside compound as an active ingredient A composition is provided.

In addition, according to another aspect of the present invention, for the control of plant diseases containing at least one compound selected from the group consisting of hederagenin saponin glycoside compound and asiatic acid glycoside compound as an active ingredient A plant disease control method is provided that comprises treating a composition to a plant, its seed, or its habitat.

Trevesia palmata (Trevesia palmata) extract, a fraction thereof or a composition for controlling plant diseases containing at least one compound selected from the group consisting of hederagenin saponin glycoside compounds and asiatic acid glycoside compounds as an active ingredient Is a natural product, harmless to the human body, is biodegradable in nature and effective in controlling plant diseases without causing environmental pollution. Therefore, it can be developed as an environmentally friendly biopesticide and can be usefully used in producing high value-added organic products.

1 is a process diagram for separating TP1 and TP2 compounds from butanol fraction of Trevesia palmata extract.
FIG. 2 is a process diagram for separating TP3, TP4 and TP5 compounds from ethyl acetate fraction of Trevesia palmata extract.

Hereinafter, the present invention will be described in detail.

One aspect of the present invention is derived from natural products, harmless to the human body and does not cause environmental pollution, rice blast (Magnaporthe oryzae), tomato ash fungus (Botrytis cinerea), tomato late blight (Phytophthora infestans), wheat rust (Puccinia triticina) It provides a composition for controlling plant diseases exhibiting excellent control effect against plant diseases important in crop production, such as).

The present inventors investigated the control effects of various plant extracts against seven plant diseases in order to develop an eco-friendly plant disease control agent, while Trevesia palmata Roxb.Ex Lindl has excellent control effects on various plant diseases. I found out.

In the present specification, the term "control" is used to mean the prevention and avoidance of diseases and pests, as well as the removal and death. However, plant disease control is the main control mechanism of preventive treatment, so plant diseases of trebezia palmata extract, fractions, hederagenin saponin glycoside compound and asiatic acid glycoside compound Investigation into the control effect was conducted with preventive treatment.

Specifically, it provides a composition for controlling plant diseases containing at least one selected from the group consisting of Trevesia palmata (Trevesia palmata) extract and fractions thereof as an active ingredient.

Herein, the extract may be extracted by using any one or a mixture thereof selected from the group consisting of water, C ₁ to C ₄ lower alcohols, hexane, chloroform, methylene chloride, ethyl acetate, acetone and acetonitrile as a solvent. It may be, but is not limited thereto.

In addition, the fraction of the extract may be fractionated using any one or a mixture thereof selected from the group consisting of hexane, chloroform, ethyl acetate, methylene chloride, methanol, ethanol, butanol and water as a solvent, but is not limited thereto. no.

The plant diseases include Alternaria porri (onion black pattern bacterium), Botrytis cinerea (tomato ash fungus), Coletotrichum coccodes (Capsicum anthrax), Fuzarium Fusarium oxysporum (Tomato rot), Magnaporthe oryzae, Puccinia triticina, Rhizoctonia solani Streptococcus), Blue Maria graminis f. sp. It is caused by, but is not limited to, one selected from phytopathogenic fungal mycelium consisting of Bloomeria graminis f. Sp. Hordei (Barley powdery mildew) and Phytophthora infestans. no.

In addition, the phytopathology is excidoborax avene subsp. Catidore (Acidovorax avenae subsp. Garnensis subsp. Micahganensis (Clavibacter michiganensis subsp. Michiganensis), Pectobacterium carotoborum subsp. Cartoborum (Pectobacterium carotovorum subsp. Carotovorum), Pseudomonas syringe pv. Actinidiae (Pseudomonas syringae pv.actinidiae; kiwi ulcer), Ralstonia solanacearum (foot blight), and xanthomonas arboccola pv. It is caused by any one selected from phytopathogenic bacteria consisting of Xanthomonas arboricola pv. Pruni (Peach bacterial pore pathogen), but is not limited thereto.

The tray chopping Jia arm Mata (Trevesia palmata) extracts or fractions thereof are the following formulas 1 to 5 in any one compound (a hederagenin-3- O Formula 1 represented _{- β - D -glucopyranosyl- (1 →} 3) - α - _L -rhamnopyranosyl- (1 → 2) -α - _L -rhamnopyranosyl- (1 → 2) -α - _L- arabinopyranoside, hederagenin-3- O - β - _D -glucopyranosyl- (1 → 3) -α _L- rhamnopyranosyl- (1 → 2) -α - _L- arabinopyranoside, hederagenin-3- O - β - _D- glucopyranosyl- (1 → 3) -α - _L- rhamnopyranoside, 3- O α - _L- arabinopyranosyl asiatic acid 28- O - β - _D- glucopyranosyl ester, 3- O - α - _L- arabinopyranosyl asiatic acid of formula 5)

[Formula 1]

;

;

[Formula 2]

;

;

[Formula 3]

;

;

[Formula 4]

; 및

; And

[Formula 5]

.

.

In this case, the active ingredient may be included in the composition for controlling plant diseases in a concentration of 100 to 3,000 μg / ml, it may be included in a concentration of 1000 to 3,000 μg / ml. The concentration refers to the concentration contained in the composition when the plant disease is treated to the plant, its seed or its habitat, when the concentration is less than 100 ㎍ / ㎖, the concentration of the active ingredient is too low to control the plant disease If there is a problem that falls, and the concentration is more than 3000 ㎍ / ㎖, the concentration of the active ingredient is too high than necessary, may be uneconomical and adversely affect 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, occurrence, environment, and the like of phytopathogens or phytopathogenic bacteria.

The plant disease control composition may be a Trevesia palmata (Trevesia palmata) extract and a fraction thereof or a simple mixture of the antimicrobial active material isolated from the extract or fraction. Alternatively, the composition for controlling plant diseases is a mixture of the extract, fraction of the extract or an antimicrobial active material isolated therefrom and an inert carrier, the mixture is an emulsion, emulsion, fluidizing agent, wetting powder, granulated wetting powder, It is prepared by adding surfactants and other necessary auxiliaries to the mixture so that they can be formulated into powders, granules and the like. The above mentioned composition for controlling plant diseases can be used as a seed treatment agent by itself or by adding other inert ingredients.

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 oils; Esters such as ethyl acetate and butyl acetate; Nitriles such as acetonitrile and isobutynitrile; 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.

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 leaf powder and walnut husk 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, alkylbenzenes 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.

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 sulfonate forms Aldehyde polycondensates; 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 materials such as aluminum magnesium silicate and alumina sol ( alumina sol), preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxytoluene).

In one embodiment of the present invention, Trevesia palmata (Trevesia palmata) extract and its fractions were found to exhibit a control effect against plant pathogens (see Experimental Examples 1 to 3), from which the extract or fraction is effective It can be seen that the composition for controlling plant diseases containing as a component has a plant disease control effect.

The trebezia palmata extract or fraction may be prepared by a manufacturing method comprising the following steps, but is not limited thereto:

1) extracting by adding an extraction solvent to the Trebezia Palmata;

2) filtering the extract of step 1);

3) concentrating the filtered extract of step 2) under reduced pressure and drying to prepare an extract of trevezia palmata; And

4) extracting the trebezia palmata extract of step 3) additionally extracted with an organic solvent to prepare a trebegia palmata fraction.

In the above method, trebezia palmata of step 1) can be used without limitation, such as grown or commercially available. The trevezia palmata can be used both as a leaf, a stem or a root, 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 alcohols, hexane, chloroform, methylene chloride, ethyl acetate, acetone and acetonitrile, or a mixture thereof, It is not limited to this. The lower alcohol may be methanol, ethanol, propanol, isopropanol or butanol.

Extraction methods may include, but are not limited to, shaking extraction, Soxhlet extraction or reflux extraction. The extraction solvent may be extracted by adding 1 to 10 times to the dried Trevesia palmata amount, and may be extracted by adding 2 to 3 times. The extraction temperature may be 20 ° C to 100 ° C, 20 ° C to 40 ° C, and 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. Further, the number of extraction may be 1 to 5 times, may be repeated extraction 3 to 4 times, may be three times, but is not limited thereto.

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

In the above 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 mixed solution thereof, but is not limited thereto. The fraction may be any one of a solvent fraction obtained by suspending trebezia palmata extract in water and fractionating with hexane, ethyl acetate, butanol or water, and may be an ethyl acetate fraction or a butanol fraction, but is not limited thereto. no. The fraction may be obtained by repeating the fractionation process from 1 to 5 times and 3 times from the trebezia palmata extract, and may be concentrated under reduced pressure after the fraction, but is not limited thereto.

In addition, another aspect of the present invention is to treat plants, seeds or habitats thereof for treating a plant disease control composition containing at least one selected from the group consisting of Trevesia palmata extract and fractions thereof as an active ingredient. It provides a plant disease control method comprising the step of.

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

The control methods of the present invention include treatment of the stems and leaves of the plant, treatment of the place where the plant grows (eg soil), treatment of seeds such as seed sterilization / seed coating and treatment of roots.

Treatment of stems and leaves with the control methods of the present invention may include in particular application on plant surfaces such as, for example, spraying on 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, spraying the liquid treating agent into the soil (irrigation of the liquid treating agent, injection into the soil, dropping of the liquid treating agent). Examples of places to be treated include planting holes, furrows, around replantations, planting furrows, the entire surface of growth, between soil and vegetation, between roots, and under plant stems. , Main furrow, growing soil, nail. Includes a cultivation box, a cultivation tray, and a bed plate. The treatment can be carried out before spraying, during spraying, immediately after spraying, during the simulated cultivation period, before cultivation settlement, at cultivation settlement and at the growth time after cultivation settlement. In the above-mentioned soil treatment, the active ingredient can be applied simultaneously with plants, or a solid fertilizer such as paste fertilizer containing the active ingredient can be applied to the soil. The active ingredient may be mixed in the irrigation liquid, for example injected into irrigation facilities (irrigation tubes, irrigation pipes, sprinklers, etc.), mixed in the overflowing liquid between the furrows, or mixed in a hydroculture medium. Can be. Alternatively, the irrigation liquid and active ingredient may be mixed in advance and used for treatment by suitable irrigation methods, including, for example, the above mentioned irrigation methods and other methods such as sparging and flooding.

The volatilization treatment method according to the control method of the present invention, for example, the volatilization of the sprayed composition by spraying the soil medium for cultivating the plant with the composition for plant disease control of the present invention, and media such as hydroponic medium, mother bed for cultivating the plant It is a method to protect the plant from pests through, in addition to the composition can be placed around the plant to expose the plant to the volatilized gaseous composition.

The seed treatment method of the control method of the present invention is a method of treating seeds so as to be protected from pests with, for example, the plant disease control composition of the present invention, and a specific example thereof is to atomize the suspension of the composition for plant disease control of the present invention. Spray treatment which sprays onto the seed surface; Spraying method of applying the wettable powder, emulsion, fluidizing agent, etc. of the composition for controlling plant diseases of the present invention on the seed surface by itself or by adding a small amount of water; Impregnation treatment in which the seeds are impregnated into a solution of the composition for controlling plant diseases of the present invention for a specific period of time; 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 throughput may vary depending on the type of plant to be treated, the type and frequency of occurrence of pests to be controlled, the form of the formulation, the duration of the treatment, the climatic conditions and the like.

Latex, wetting powders, glidants and the like 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 and the like are usually used for treatment without dilution.

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

In one embodiment of the present invention, Trevesia palmata (Trevesia palmata) extract and its fractions were confirmed to exhibit a control effect against plant pathogens (see Experimental Examples 1 to 3), the extract or fraction as an active ingredient It can be seen that the containing composition for controlling plant diseases can be usefully used for the method of controlling plant diseases.

Furthermore, another aspect of the present invention provides a use of a plant disease control composition containing Trevesia palmata extract and fractions thereof as an active ingredient in plant disease control.

Another aspect of the present invention provides a compound which is any one selected from the group consisting of a hederagenin saponin glycoside compound and an asiatic acid glycoside compound.

Furthermore, another aspect of the present invention is a composition for controlling plant diseases comprising at least one compound selected from the group consisting of hederagenin saponin glycoside compound and asiatic acid glycoside compound as an active ingredient to provide.

The hederagenin saponin glycoside compound is a compound represented by the following Chemical Formulas 1 to 3 (hederagenin-3- O - β - _D -glucopyranosyl- (1 → 3) -α - _L- rhamnopyranosyl- of Formula 1) (1 → 2) - α - L -rhamnopyranosyl- (1 → 2) - α - L -arabinopyranoside, formula (2) of _{hederagenin-3- O - β - D} -glucopyranosyl- (1 → 3) - α - L -rhamnopyranosyl - (1 → 2) - α - L -arabinopyranoside, formula (3) of _{hederagenin-3- O - β - D} -glucopyranosyl- (1 → 3) - α - L -rhamnopyranoside) any one of the compounds selected from the group consisting of Can be.

In addition, the asiatic acid glycoside compound is a compound represented by the following Chemical Formulas 4 and 5 (3- O - α - _L- arabinopyranosyl asiatic acid 28- O - β - _D- glucopyranosyl ester of Chemical Formula 4, Chemical Formula 5 3- O - α - _L- arabinopyranosyl asiatic acid) may be any one compound selected from the group consisting of.

[Formula 1]

;

;

[Formula 2]

;

;

[Formula 3]

;

;

[Formula 4]

; 및

; And

[Formula 5]

.

.

The hederagenin saponin glycoside or asiatic acid glycoside may be isolated from Trevesia palmata extract and fractions thereof.

Solvent fractions may be prepared from trevezia palmata extract, and the fractions may be fractionated by liquid chromatography to separate hederagenin saponin glycosides or asiatic acid glycosides.

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

The plant diseases include Alternaria porri (onion black pattern bacterium), Botrytis cinerea (tomato ash fungus), Coletotrichum coccodes (Capsicum anthrax), Fuzarium Fusarium oxysporum (Tomato rot), Magnaporthe oryzae, Puccinia triticina, Rhizoctonia solani Streptococcus), Blue Maria graminis f. sp. It is caused by, but is not limited to, one selected from phytopathogenic fungal mycelium consisting of Bloomeria graminis f. Sp. Hordei (Barley powdery mildew) and Phytophthora infestans. no.

In addition, the phytopathology is excidoborax avene subsp. Catidore (Acidovorax avenae subsp. Garnensis subsp. Micahganensis (Clavibacter michiganensis subsp. Michiganensis), Pectobacterium carotoborum subsp. Cartoborum (Pectobacterium carotovorum subsp. Carotovorum), Pseudomonas syringe pv. Actinidiae (Pseudomonas syringae pv.actinidiae; kiwi ulcer), Ralstonia solanacearum (foot blight), and xanthomonas arboccola pv. It is caused by any one selected from phytopathogenic bacteria consisting of Xanthomonas arboricola pv. Pruni (Peach bacterial pore pathogen), but is not limited thereto.

The active ingredient may be included in the composition for controlling plant diseases in a concentration of 100 to 3,000 μg / ml, it may be included in a concentration of 1000 to 3,000 μg / ml. The concentration refers to the concentration contained in the composition when the plant disease is treated to the plant, its seed or its habitat, when the concentration is less than 100 ㎍ / ㎖, the concentration of the active ingredient is too low to control the plant disease If there is a problem that falls, and the concentration is more than 3000 ㎍ / ㎖, the concentration of the active ingredient is too high than necessary, may be uneconomical and adversely affect 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, occurrence, environment, and the like of phytopathogens or phytopathogenic bacteria.

Hederagenin saponin glycoside compounds or asiatic acid glycoside compounds may be used in the form of pharmaceutically acceptable salts, and acid addition salts formed by pharmaceutically acceptable free acids are useful as salts. Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid, phosphorous acid, aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Non-toxic organic acids such as dioate, aromatic acids, aliphatic and aromatic sulfonic acids and the like, and organic acids such as acetates, 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 pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, eye Odide, Fluoride, Acetate, Propionate, Decanoate, Caprylate, Acrylate, Formate, Isobutyrate, Caprate, Heptanoate, Propiolate, Oxalate, Malonate, Succinate, Sube Latex, sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobene Sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1 Sulfonates, naphthalene-2-sulfonates, mandelate and the like.

The acid addition salt may be prepared by a conventional method, for example, a heteragenine saponin glycoside compound or an Asiantic glycoside compound is dissolved in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile, and the organic acid or inorganic acid. The precipitate produced by addition may be prepared by filtration and drying, or the solvent and excess acid may be distilled under reduced pressure and dried to crystallize in an organic solvent.

Bases can also be used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are 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 suitable to prepare sodium, potassium or calcium salt as the metal salt. Corresponding salts are also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).

Furthermore, the compound which is any one selected from the group consisting of the hederagenin saponin glycoside compound and the asiatic acid glycoside compound may be prepared therefrom as well as its pharmaceutically acceptable salts. In the form of solvates, optical isomers, hydrates, and the like.

The composition for controlling plant diseases containing at least one compound selected from the group consisting of hederagenin saponin glycoside compounds and asiatic acid glycoside compounds as an active ingredient is a hederagenin saponin glycoside compound or Mix the asiatic glycoside compound with an inert carrier and add surfactants and other necessary auxiliaries to the mixture so that the mixture can be formulated into emulsions, emulsions, glidants, wetting powders, granulated wetting powders, powders, granules, and the like. It is manufactured by. The above mentioned composition for controlling plant diseases can be used as a seed treatment agent by itself or by adding other inert ingredients.

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 oils; Esters such as ethyl acetate and butyl acetate; Nitriles such as acetonitrile and isobutynitrile; 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.

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 leaf powder and walnut husk 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, alkylbenzenes 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.

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 sulfonate forms Aldehyde polycondensates; 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 materials such as aluminum magnesium silicate and alumina sol ( alumina sol), preservatives, colorants and stabilizers such as PAP (acid phosphate isopropyl) and BHT (butylhydroxytoluene).

In one embodiment of the present invention, it was confirmed that the hederagenin saponin glycoside compound or asiatic acid glycoside compound exhibits a control effect against plant pathogens (see Experimental Example 7-9), from the hederagenin saponin glycoside ( It can be seen that the composition for controlling plant diseases containing at least one compound selected from the group consisting of hederagenin saponin glycoside compounds and asiatic acid glycoside compounds as an active ingredient has a plant disease control effect.

Furthermore, another aspect of the present invention is a composition for controlling plant diseases containing at least one compound selected from the group consisting of the hederagenin saponin glycoside compound and the asiatic acid glycoside compound as an active ingredient It provides a plant disease control method comprising the step of treating the plant, its seeds or its habitat.

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

The control methods of the present invention include treatment of the stems and leaves of the plant, treatment of the place where the plant grows (eg soil), treatment of seeds such as seed sterilization / seed coating and treatment of roots.

Treatment of stems and leaves with the control methods of the present invention may include in particular application on plant surfaces such as, for example, spraying on 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, spraying the liquid treating agent into the soil (irrigation of the liquid treating agent, injection into the soil, dropping of the liquid treating agent). Examples of places to be treated include planting holes, furrows, around replantations, planting furrows, the entire surface of growth, between soil and vegetation, between roots, and under plant stems. , Main furrow, growing soil, nail. Includes a cultivation box, a cultivation tray, and a bed plate. The treatment can be carried out before spraying, during spraying, immediately after spraying, during the simulated cultivation period, before cultivation settlement, at cultivation settlement and at the growth time after cultivation settlement. In the above-mentioned soil treatment, the active ingredient can be applied simultaneously with plants, or a solid fertilizer such as paste fertilizer containing the active ingredient can be applied to the soil. The active ingredient may be mixed in the irrigation liquid, for example injected into irrigation facilities (irrigation tubes, irrigation pipes, sprinklers, etc.), mixed in the overflowing liquid between the furrows, or mixed in a hydroculture medium. Can be. Alternatively, the irrigation liquid and active ingredient may be mixed in advance and used for treatment by suitable irrigation methods, including, for example, the above mentioned irrigation methods and other methods such as sparging and flooding.

The volatilization treatment method according to the control method of the present invention, for example, the volatilization of the sprayed composition by spraying the soil medium for cultivating the plant with the composition for plant disease control of the present invention, and media such as hydroponic medium, mother bed for cultivating the plant It is a method to protect the plant from pests through, in addition to the composition can be placed around the plant to expose the plant to the volatilized gaseous composition.

The seed treatment method of the control method of the present invention is a method of treating seeds so as to be protected from pests with, for example, the plant disease control composition of the present invention, and a specific example thereof is to atomize the suspension of the composition for plant disease control of the present invention. Spray treatment which sprays onto the seed surface; Spraying method of applying the wettable powder, emulsion, fluidizing agent, etc. of the composition for controlling plant diseases of the present invention on the seed surface by itself or by adding a small amount of water; Impregnation treatment in which the seeds are impregnated into a solution of the composition for controlling plant diseases of the present invention for a specific period of time; 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 throughput may vary depending on the type of plant to be treated, the type and frequency of occurrence of pests to be controlled, the form of the formulation, the duration of the treatment, the climatic conditions and the like.

Latex, wetting powders, glidants and the like 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 and the like are usually used for treatment without dilution.

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

In one embodiment of the present invention, it was confirmed that the hederagenin saponin glycoside compound or asiatic acid glycoside compound exhibits a control effect against plant diseases (see Experimental Example 7-9), from which the hederagenin saponin glycoside (hederagenin It can be seen that the composition for controlling plant diseases containing at least one compound selected from the group consisting of saponin glycoside compounds and asiatic acid glycoside compounds as an active ingredient can be usefully used for plant disease control methods.

In addition, another aspect of the present invention is an active ingredient comprising at least one compound selected from the group consisting of hederagenin saponin glycoside compound and asiatic acid glycoside compound in controlling plant diseases Provided is a use of the composition for controlling plant diseases.

Hereinafter, it demonstrates in detail by an Example.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1> Trebezia Palmata  Containing extract as an active ingredient Plant disease  Of composition for control Plant disease  Control effect evaluation

Step 1: Trebezia Palmata  Preparation of Extract

In order to prepare a composition for controlling plant diseases, containing trevezia palmata extract as an active ingredient, trebezia palmata extract was prepared. Specifically, the leaves and stem samples of the dried Trevesia palmata are finely chopped, methanol is added, extracted at room temperature for 24 hours, filtered through a filter paper, and the filtrate is concentrated under reduced pressure to obtain 50 g of Trevesia palmata methanol. An extract was obtained.

Step 2: Trebezia Palmata  Containing extract Plant disease  Preparation of Control Composition

For the evaluation of the plant disease control effect of the plant disease control composition comprising the trevezia palmata extract 2 ml of the extract sample of step 1 dissolved in methanol at a concentration of 60 mg / ml was diluted with 38 mL of distilled water, the electrodeposition Tween 20 (Tween 20) was added to a concentration of 0.025% (w / v) to prepare 40 ml of a solution of a plant disease control composition containing Trevezia palmata extract at a concentration of 3,000 μg / ml.

Step 3: Trebezia Palmata  Containing extract Plant disease  Of composition for control plant Bottle Control Activity Survey

Rice Blast Disease (Cacillus: Magnaporthe oryzae), Rice Leaf Blight (Cacillus: Rhizoctonia solani), Tomato Blight Disease (Cacillus: Botrytis cinerea), Tomato Blight (Cacillus Phytophthora infestans) The disease control activities of seven plant diseases, including powdery mildew (causative: Blumeria graminis f. Sp.hordei), wheat red rust (caused: Puccinia triticina), and pepper anthrax (Calletotrichum coccodes), were tested in greenhouse conditions as follows. . The control group used distilled water containing 5% (v / v) methanol and 0.025% (w / v) Tween 20. The experimental results are shown in Table 1.

Specifically, three ports were used for each phytopathogen. After spraying and spraying the control composition solution prepared in step 2 onto the leaf (foliage) and air-dried for 24 hours and then inoculated each phytopathogen. The rice, tomato, barley, pepper and wheat plants used in the experiment were filled with 70% water or horticultural soil in a plastic pot of 4.5 cm in diameter, and then sowing seeds for 1 to 4 weeks in a greenhouse at 25 ± 5 ° C. Cultivated.

Rice blasts are sprayed with 3-4 leaf seedlings and sprayed with a spore suspension (5 × 10 ⁵ spores / ml) of Magnaporthe oryzae (Korea Research Institute of Chemical Technology), which is the causative agent of blasting disease, After the treatment, the incubation was induced for 4 days in a constant temperature and humidity room of 25 ℃, relative humidity 80%.

Rice foliar blight is cultured by culturing Rhizoctonia solani (Korean Research Institute), a causative agent of leaf blight, for 7 days in medium (90 g of wheat bran, 15 g of chaff and 100 ml of distilled water). After inoculation and treatment for 4 days in a 25 ℃ wet chamber, the plant was incubated for 4 days in a constant temperature room at 25 ℃ to induce the onset.

Tomato plague was sprayed with 3 ~ 4 leaf tomato seedlings after spraying the inoculated suspension of jujuja (5 × 10 ⁴ sporangia / ml) of Phytophthora infestans (Gangneung-Wonju National University). The treatment was induced for 2 days in a humidified room at 0 ° C. and incubated in a constant temperature and humidity room at 25 ° C. for 1 day.

Tomato gray mold disease was treated with spore suspension (5 × 10 ⁵ spores / mL) of Botrytis cinerea (Korea Research Institute of Chemical Technology), which is a causative agent of gray mold disease, in tomato seedlings of 3-4 leaves. The incubation was induced by incubation for 3 days in a wet room.

Wheat red rust is suspended in single-leaf seedlings of Puccinia triticina, the causative agent of rust, known as a biotic fungus, in 0.67 g spores / ℓ in a 250 µg / ml Tween 20 solution. After spray treatment and treatment for one day in a 20 ℃ humid room and transferred to a 20 ℃ constant temperature room and incubated for 6 days to induce the onset.

Barley powdery mildew is inoculated with the spores of Blumeria graminis f. Sp. Hordei (Korea Research Institute of Chemical Technology), which is the causative agent of powdery mildew, which is subcultured in host plants. Incubation was induced by incubation for 7 days in a room temperature.

Tomato area blight and tomato late blight were 3 days after inoculation, rice blast was 5 days after inoculation, wheat red rust, barley powdery disease 7 days after inoculation, rice leaf blight blight 8 days after inoculation.

On the other hand, for the control activity test for pepper anthracnose, 70% of gardening topsoil is filled in a plastic pot with a diameter of 5.0㎝, sowing the seeds of the most pepper, growing them in the greenhouse to 3-4 leaves, and then prepared tre Veggie Palmata extract solution was foliar sprayed. After spraying the sample was dried for 24 hours at room temperature and then sprayed inoculated with a spore suspension (4 × 10 ⁵ spores / ㎖) of Collettrichum coccodes (Korea University), the causative agent of pepper anthrax. After 2 days of wet treatment at 25 ° C., the cells were cultivated for 1 day in a constant temperature and humidity room (25 ° C., relative humidity of 80%), and the lesion area ratio (%) was examined after 3 days of inoculation.

Using the area ratio (%) obtained from the above, the control value (%) was calculated according to Equation 1 below.

[Equation 1]

sample density
(μg / ml) Control price (%) RCB RSB TGM TLB WLR BPM PAN Trevezia Palmata
extract 3000 60 0 79 96 83 0 25

(RCB: Rice Blast, RSB: Rice Leaf Blight, TGM: Tomato Blight, TLB: Tomato Blight, WLR: Wheat Red Rust, BPM: Barley Powder, PAN: Red Pepper Anthrax)

As shown in Table 1,

When the extract obtained with methanol from the leaves and stems of Trevezia palma was treated at a concentration of 3,000 μg / ml, it showed control activities against the other four plant diseases except rice leaf blight, pepper anthrax and barley powdery mildew. Confirmed. In particular, it was confirmed that the control against tomato gray mold, tomato late blight, wheat red rust disease showed an excellent control effect of more than 79%. There has been no report on the effects of trevezia palmata extract on plant diseases.

< Example  2> Trebezia Palmata  Of extract Solvent fractions  Of the composition for plant disease control containing as an active ingredient Plant disease  Control effect evaluation

Step 1: Trebezia Palmata  Solvent of extract Fraction  Produce

To prepare a composition for controlling plant diseases containing a solvent fraction of trevezia palmata extract as an active ingredient, trebezia palmata methanol extract was prepared by the same method as in step 1 of Example 1 above. Solvent fractions were prepared using methanol extracts.

Specifically, 50 g of Trevezia palmata leaf and stem methanol extracts were dissolved in 500 ml of distilled water, and extracted twice with the same amount of hexane to obtain a hexane layer and an aqueous layer. The aqueous layer was again extracted twice with the same amount of ethyl acetate and butanol to obtain an ethyl acetate layer, a butanol layer and an aqueous layer. The mixture was concentrated under reduced pressure to give a dried hexane fraction (1.14 g), an ethyl acetate fraction (5.65 g), a butanol fraction (16.84 g) and a water fraction (20.02 g).

Step 2: Trebezia Palmata  Of extract Solvent fractions  Preparation of a composition for plant disease control containing as an active ingredient

2 ml of each sample dissolved in methanol at a concentration of 40 mg / ml was diluted with 38 ml of distilled water for the evaluation of plant disease control effect of the composition for controlling plant diseases containing a solvent fraction of trevezia palmata extract as an active ingredient. In addition, a solution of a composition for controlling plant diseases containing a solvent fraction of Trevezia palmata extract at a concentration of 2,000 μg / ml as an active ingredient was added so that Tween 20 was added to a concentration of 0.025% (w / v). Prepared by ml.

Step 3: Trebezia Palmata  Containing extract Plant disease  Of composition for control Plant disease  Control activity check

Rice Blast Disease (Cacillus: Magnaporthe oryzae), Rice Leaf Blight (Cacillus: Rhizoctonia solani), Tomato Blight Disease (Cacillus: Botrytis cinerea), Tomato Blight (Cacillus Phytophthora infestans) The disease control activity of seven plant diseases including powdery mildew (causal: Blumeria graminis f. Sp.hordei), wheat red rust (caused: Puccinia triticina), and pepper anthrax (Calletotrichum coccodes) was measured in greenhouse conditions. Experiment was carried out in the same manner as 3. The control group used distilled water containing 5% (v / v) methanol and 0.025% (w / v) Tween 20. The experimental results are shown in Table 2.

sample density
(μg / ml) Bottle Control (%) RCB RSB TGM TLB WLR BPM PAN Hexane fraction 2000 30 0 7 7 13 0 58 Ethyl Acetate Fraction 2000 70 5 86 57 3 0 50 Butanol fraction 2000 30 0 75 64 53 0 8 Water fraction 2000 0 0 0 0 0 0 8

(RCB: Rice Blast, RSB: Rice Leaf Blight, TGM: Tomato Blight, TLB: Tomato Blight, WLR: Wheat Red Rust, BPM: Barley Powder, PAN: Red Pepper Anthrax)

As shown in Table 2,

Ethyl acetate fractions showed 70% control against rice blast at 2,000 μg / ml concentration, 86% against tomato gray mold, 57% against tomato late blight and 50% against pepper anthrax. It confirmed that it showed.

At the same concentration, the butanol fraction showed 30% control against rice blast, 75% against tomato gray mold, 64% against tomato late blight and 53% against wheat rust. .

Hexane fraction showed 30% and 58% control value for rice blast and pepper anthrax.

It was confirmed that the water fraction did not exhibit control activity.

From the above results, it was confirmed that the antimicrobial active material of trevezia palmata extract was a non-polar substance, not a water-soluble substance.

Ethyl acetate layer and butanol layer were used separately for purification and purification of the active ingredient based on the highest overall control against several plant diseases.

< Example  3> Trebezia Palmata  Of extract Solvent fractions  As an active ingredient Containing Confirmed the mycelial growth-inhibiting activity of rice blast bacterium in plant disease control composition

In order to understand the basic properties of the antimicrobial active substances contained in trevezia palmata extract, the ethyl acetate fraction and butanol fraction prepared in step 1 of Example 2 were confirmed in vitro antimicrobial activity against rice blast bacteria. . The experimental results are shown in Table 3.

Specifically, the minimum inhibitory concentration (MIC) value was evaluated by a broth micro-dilution method using a 96-well plate. Five mycelial discs of Magnaporthe oryzae were inoculated into 100 ml potato dextrose broth medium in Erlenmeyer flasks and shaken at 150 rpm at 25 ° C for 7 to 10 days. 2 ml of mycelium suspension prepared by grinding with a blade mixer was mixed well with 100 ml potato liquid medium and dispensed into wells by 50 μl.

Finally, 100 μl wells contained Trebezia palmata methanol extract, ethyl acetate fraction and butanol fraction at concentrations of 1, 2, 4, 8, 16, 31, 63, 125, 250, 500 and 1,000 μg / ml, respectively. Dispensed as much as possible, the content of methanol did not exceed 5%. Only 5% methanol was used as a control, and incubated at 25 ° C. for 2-3 days.

sample MIC (μg / ml) Trebezia Palmata Methanol Extract 125 Ethyl Acetate Fraction of Extract 125 Butanol Fraction of Extract 125

As shown in Table 3,

It was confirmed that trevezia palmata methanol extract, ethyl acetate fraction and butanol fraction of the extract completely inhibited mycelial growth of rice blast bacteria at 125 μg / ml.

From the results of Example 3, it was confirmed that the antimicrobial active material contained in the Trebezia palmata extract acted on rice blast bacterium itself under in vitro conditions and showed control activity. The antimicrobial active material contained in the extract was controlled in rice blast bottle. It can be seen that it is an active ingredient showing activity.

< Example  4> Isolation of Antimicrobial Actives 1

Separation was performed using various chromatographic techniques to separate the antimicrobial actives from Trevezia palmata extract.

Specifically, TP1 and TP2 compounds were separated by a process as shown in FIG. 1 to separate the antimicrobial active material from the butanol fraction (16.84 g) obtained in step 1 of Example 2. More specifically, the butanol fraction suspended in 1 L of distilled water was adsorbed onto a Diaion HP-20 (Mitsubishi Chemical) column (6 cm in diameter x 30 cm in length), followed by methanol and distilled water in 0: 100, 20:80, 40: Elution was carried out sequentially with an aqueous methanol solution mixed at a volume ratio of 60, 60:40, 80:20, and 100: 0, eluted once more with acetone, and divided into eight fractions. In order to investigate the chemical characteristics of the fractions, thin layer chromatography (TLC) was used. The samples were adsorbed onto silica gel and then developed with chloroform: methanol: distilled water (14: 6: 1, v / v). TLC pattern analysis was then carried out by color development with p-anisaldehyde reagent. The B7 (2.1 g) fraction was added to a column filled with silica gel (Kiesel gel 60, 70-230 mesh, 500 g; Merck) (7 cm in diameter x 15 cm in length), and chloroform: methanol: distilled water was added 7: The mixed solutions in volume ratios of 3: 0, 14: 6: 1 and 0: 20: 1 were eluted sequentially and divided into 9 fractions. B75 (901.5 mg) fractions were subjected to chromatography on a Sephadex LH-20 (Sigma-Aldrich) column (3 cm in diameter x 90 cm in length). Eluent was divided into two fractions using chloroform: methanol (5: 5, v / v). B751 (143.6 mg) was added to a column filled with silica gel (70-230 mesh, 150 g) (3 cm in diameter x 35 cm in length), followed by chloroform: methanol: distilled water (14: 6: 1, v / v). Eluted and purely isolated Compound TP1 (82.2 mg) from the B7514 fraction out of the five fractions. B752 (639.7 mg) was also chromatographed on the same Sephadex LH-20 column (3 cm in diameter x 90 cm in length) to obtain three fractions. Among them, B7522 (287.0 mg) was added to a column filled with silica gel (70-230 mesh, 150 g) (3 cm in diameter x 35 cm in length), followed by chloroform: methanol: distilled water (14: 6: 1, v / v). Compound TP2 (66.9 mg) and Compound TP1 (101.7 mg) were separated purely by elution.

< Example  5> Isolation of Antimicrobial Actives 2

Separation was performed using various chromatographic techniques to separate the antimicrobial actives from Trevezia palmata extract.

Specifically, TP3, TP4 and TP5 compounds were separated by the process as shown in Figure 2 to separate the antimicrobial active material from the ethyl acetate fraction (5.65 g) obtained in step 1 of Example 2. In more detail, the ethyl acetate fraction dissolved in a chloroform: methanol (9: 1, v / v) solution was added to chloroform: methanol: distilled water (9: 1: 0, 30: 9: 1, 14: 6: 1, 12: A silica gel (70-230 mesh, 500 g) column (diameter 7 cm × 15 cm in length) was chromatographed using 8: 1, 0: 20: 1, v / v / v as a developing solvent. Based on the TLC pattern, a portion of the eluate was adsorbed onto a silica gel thin-film chromatography plate and then developed with chloroform: methanol: distilled water (30: 9: 1, v / v) and then developed with p-anisaldehyde reagent. Divided into dog fractions. The E11 (263.1 mg) fraction was chromatographed on a Sephadex LH-20 column (3 cm in diameter x 90 cm in length) using methanol as the eluting solvent. The fraction was divided into six fractions. Among them, E115 (71.8 mg) was subjected to silica gel (70-230 mesh, 40 g) column (diameter 1 × 25 cm length) chromatography under chloroform: methanol: distilled water (14: 6: 1, v / v / v). 8 fractions were obtained, and compound TP3 (29.4 mg) was isolated purely. In addition, an E10 (435.1 mg) fraction was subjected to Sephadex LH-20 column (3 cm diameter x 90 cm length) chromatography using methanol as eluent and divided into five fractions. Among them, E103 (223.5 mg) was adsorbed onto silica gel (70-230 mesh, 40 g) column (1 cm in diameter x 25 cm in length) and chloroform: methanol: distilled water (14: 6: 1, v / v / v). Elution was divided into 6 fractions. Compound TP4 (28.5 mg) and TP5 (26.2 mg) were separated from the E1032 (38.4 mg) and E1034 (76.4 mg) fractions using preparative high performance liquid chromatography (HPLC). At this time, the conditions of preparative HPLC are as follows. The column was Polaris A C18 (250 mm × 21.2 mm; Agilent), and 80-100% methanol solution was eluted at a rate of 5 ml / min under the concentration gradient to detect the peak detected at 210 nm.

< Example  6> Of antimicrobial active substances  Chemical structure identification

In order to identify the chemical structure of the antimicrobial active material separated in Examples 4 and 5 was carried out the following experiment.

Specifically, nuclear magnetic resonance (NMR) and mass spectroscopy (MS) were used to investigate the chemical structure of the material separated from the Trevesia palmata organic solvent fraction. The sample was dissolved in methanol-d4 (99.5% atom% D, Cambridge Isotope Laboratory) and hydrogen and ¹³ carbon-NMR spectra were obtained on a Bruker AMX-500 instrument. Based on the methanol solvent peak of 3.31 ppm in the hydrogen NMR spectrum and 49.0 ppm in the carbon NMR spectrum, the chemical shift value is expressed as δ ( ppm). ¹ H- ¹ H COSY, HSQC, HMBC and DEPT experiments were used to determine the chemical location of the peaks shown in the spectra. The mass of the compound separated using MALDI-TOF-MS was measured, and molecular weight-based chemical structural characteristics were determined by MS / MS analysis using Bruker Autoflex Speed TOF / TOF equipment. The chemical formula of the isolated compound was completed using high-resolution mass spectroscopy (Synapt G2 HDMS Q-TOF). In addition, the characteristics of the compounds separated by polarimeter (Auto Digital Polarieter, Rudolph Research Analytical) and infrared spectrometer (FT-IR spectroscopy; Identify IR, Smiths) were investigated.

<6-1> Chemical structure of compound TP1

As a result, compound TP1 was detected as a cation of m / z 1081 [M + Na] ⁺ in MALDI-TOF-MS in cation mode, and the chemical formula was determined as C ₅₃ H ₆₆ O ₂₁ . When dissolved in methanol, [α] _D ²¹ showed 8.26 values and was determined in the IR spectrum with a hydroxyl group (3378 cm ^-1 ), a carbonyl group (1692 cm ^-1 ), an alkene (1636 cm ^-1 ), and methyl (1056). cm ^{- 1)} were detected. The NMR analysis results are shown in Table 4 below.

As shown in Table 4 below,

^{In the 13-} carbon NMR spectrum, 53 carbon signals appeared, 30 of which consisted of triterpins and the rest of which constitute 4 sugars. In the hydrogen NMR spectrum, 6 methyls of δ _H 0.70, 0.81, 0.91, 0.94, 0.97, 1.18 ppm (C-24, C-25, C-26, C-27, C-29, C-30), δ _H Methine bound to 5.24 ppm (t, H-12) oxygen and primary alcohol hydrogen signals of δ _H 3.58, 3.33 ppm (m, H-23). Δ _{C in} ¹³ carbon NMR spectrum 13.7, 16.4, 17.7, 26.5, 33.6, 24.0 ppm methyl group, δ _C Two olefins at 123.5, 145.2 ppm, δ _C Methine bound to 82.3 ppm oxygen, δ _C 64.4 ppm primary alcohol and δ _C 182.0 ppm carbonyl carbon signal appeared. Looking at the HMBC spectrum of the triterpin structure, H-9 ( δ _H 1.67-1.57 ppm) is C-25 ( δ _C 16.4 ppm) and C-26 ( δ _C 17.7 ppm); H-11 ( δ _H 1.93-1.85 ppm) is C-13 ( δ _C 145.2 ppm); H-18 ( δ _H 2.85 ppm) is C-28 ( δ _C 182.0 ppm); H-24 ( δ _H 0.70 ppm) is C-3 ( δ _C 82.3 ppm) and C-23 ( δ _C 64.4 ppm); H-27 ( δ _H 1.18 ppm) is C-13 ( δ _C 145.2 ppm); H-29 ( δ _H 0.91 ppm) is C-30 ( δ _C 24.0 ppm); H-30 ( δ _H 0.94 ppm) is adjacent to C-29 ( δ _C 33.6 ppm). These results indicate that the triterpene structure of compound TP1 is α-hederagenin. Analysis of sugars in the HSQC spectrum shows four anomers of δ _H 5.19 (d, J = 1.8 Hz), 4.85 (d, J = 1.7 Hz), 4.52 (d, J = 7.8 Hz), 4.47 (m) ppm anomeric) hydrogen is combined with four anomer carbons of δ _C 101.6, 102.8, 105.7, and 105.2 ppm, respectively. Δ _H 4.47 ( α - _L -Ara) from the HMBC spectrum δ _C 82.3 (C-3 of aglycone); δ _H 101.6 ( α - _L- Rha) and δ _C 76.7 (C-2 of α - _L- Ara); δ _H 105.7 ( β - _D- Glc) and δ _C 82.9 (C-3 of α - _L- Rha); _{δ H 4.85 (α - D -Rha} ) δ _C 70.9 and - shown in the correlation analysis and MALDI-TOF-MSMS analysis between the _{(C-2 of α L -Rha} ) m / z 935 [M + Na Rha] +, Ions of 773 [M + Na Rha Glc] ⁺ , 627 [M + Na Rha Glc Rha] ⁺ determined the binding between hederagenin and sugars, sugars and sugars. Based on these results, the chemical structure of the compound TP1 is represented by hederagenin-3- O - β - _D- glucopyranosyl- (1 → 3) -α - _L- rhamnopyranosyl- (1 → 2) -α - _L- rhamnopyranosyl -(1 → 2) -α - _L -arabinopyranoside was determined, and confirmed that it is a hederagenine saponin glycoside compound.

[Formula 1]

;

;

Position
TP1 δ _C ^a δ _H , mult. ( J ) One 39.6 1.67-1.57, m; 0.98, m 2 26.6 1.93-1.85, m; 1.81-1.68, m 3 82.3 3.63, dd (10.2, 3.6) 4 43.9 5 48.0 1.30-1.27, m 6 18.8 1.49, d (12.3); 1.44-1.32, m 7 33.8 1.67-1.57, m; 1.30-1.27, m 8 40.4 9 49.0 1.67-1.57, m 10 37.6 11 24.5 1.93-1.85, m 12 123.5 5.24, t (3.7) 13 145.2 14 42.9 15 28.8 1.81-1.68, m; 1.08, m 16 24.0 2.01, td (13.4, 3.9); 1.67-1.57, m 17 47.6 18 42.7 2.85, dd (13.9, 4.5) 19 47.2 1.81-1.68, m; 1.13, m 20 31.6 21 34.9 1.44-1.32, m; 1.22, m 22 33.3 1.67-1.57, m; 1.54, d (13.6) 23 64.4 3.58, m; 3.33, m 24 13.7 0.70, s 25 16.4 0.97, s 26 17.7 0.81, s 27 26.5 1.18, s 28 182.0 29 33.6 0.91, s 30 24.0 0.94, s α-L-Ara One 105.2 4.47, m 2 76.7 3.64, m 3 74.1 3.64, m 4 69.9 3.76, m 5 66.0 3.84, m; 3.52, dd (12.4, 1.9) α-L-Rha One 101.6 5.19, d (1.8) 2 70.9 4.25, dd (3.2, 1.8) 3 82.9 3.87, dd (9.5, 3.1) 4 72.4 3.56, m 5 70.0 3.93, dq (9.6, 6.2) 6 17.8 1.27, d (6.0) β-D-Glc One 105.7 4.52, d (7.8) 2 75.4 3.36, m 3 76.6 3.39, m 4 73.7 3.42, d (9.5) 5 76.3 3.48, t (9.0) 6 61.6 3.82, m; 3.67, dd (12.0, 3.7) α-L-Rha (terminal) One' 102.8 4.85, d (1.7) 2' 72.1 3.64, m 3 ' 79.0 3.59, m 4' 72.4 3.84, m 5 ' 70.6 3.99, dq (9.5, 6.2) 6 ' 18.2 1.26, d (6.0)

( ^a Recorded in CD ₃ OD at 500 MHz; δ in ppm and J in Hz)

<6-2> Chemical Structure of Compound TP2

A cation of m / z 935 [M + Na] ⁺ was detected in MALDI-TOF-MS of the isolated compound TP2, and the chemical formula was determined as C ₄₇ H ₇₆ O ₁₇ . MALDI-TOF-MSMS analysis detected 773 [M + Na Rha Glc] ⁺ , 627 [M + Na Rha Glc Rha] ⁺ ions. The NMR analysis results are shown in Table 5 below.

As shown in Table 5, 47 carbon signals appeared in the ¹³ carbon NMR spectrum, 30 of which triterpin constituted the rest, and the other constituted 3 sugars. Since the hydrogen and ¹³ carbon NMR spectra of the compound TP2 were consistent with the data shown in the literature (Tommasi et al., 2000), the compound TP2 was identified as a compound of the formula (2), and it was confirmed that it is a hederagenin saponin glycoside compound.

[Formula 2]

Position
TP2 δ _C ^a δ _H , mult. ( J ) One 39.7 1.70-1.58, m; 0.98, m 2 26.6 1.95-1.85, m; 1.80-1.71, m 3 82.4 3.69, dd (11.8, 5.0) 4 44.0 5 48.1 1.30-1.27, m 6 18.8 1.50, d (13.2); 1.42-1.33, m 7 33.8 1.70-1.58, m; 1.30-1.27, m 8 40.5 9 49.9 1.70-1.58, m 10 37.6 11 24.5 1.92-1.85, m 12 123.6 5.24, t (3.8) 13 145.3 14 43.0 15 28.9 1.80-1.71, m; 1.08, dt (13.6, 3.5) 16 24.0 2.00, td (13.6, 4.1); 1.70-1.58, m 17 47.7 18 42.8 2.85, dd (13.8, 4.5) 19 47.3 1.80-1.71, m; 1.13 ddd (13.7, 4.7, 2.3) 20 31.6 21 34.9 1.42-1.33, m; 1.20, m 22 33.4 1.70-1.58, m; 1.54, dt (13.7, 3.6) 23 64.5 3.62, dd (12.0, 4.6), 3.33 d (8.2) 24 13.7 0.70, s 25 16.4 0.97, s 26 17.8 0.82, s 27 26.5 1.18, s 28 182.0 29 33.6 0.91, s 30 24.1 0.94, s α-L-Ara One 105.0 4.49, dd (5.0, 1.5) 2 76.9 3.65, m 3 74.0 3.65, m 4 69.8 3.76, m 5 65.7 3.84, dd (12.3, 3.9); 3.51, dd (12.3, 2.2) α-L-Rha One 101.7 5.17, d (1.9) 2 71.1 4.24, dd (3.2, 1.9) 3 82.8 3.88, dd (9.5, 3.0) 4 72.3 3.56, t (9.5) 5 70.1 3.92, dq (9.7, 6.2) 6 18.1 1.26, d (6.2) β-D-Glc One 105.8 4.51, d (7.8) 2 75.3 3.31, dd (7.9, 1.32) 3 77.8 3.38, t (8.9) 4 71.3 3.33, d (11.2) 5 77.8 3.57, d (11.3) 6 62.4 3.87, dd (11.9, 2.4); 3.86, dd (12.3, 3.9)

( ^a Recorded in CD ₃ OD at 500 MHz; δ in ppm and J in Hz)

<6-3> Chemical Structure of Compound TP3

The cation detected in MALDI-TOF-MS of compound TP3 was determined by the formula C ₄₁ H ₆₆ O ₁₂ by m / z 773 [M + Na] ⁺ . The NMR analysis results are shown in Table 6 below.

As shown in Table 6, 41 carbon signals appeared in the ¹³ carbon NMR spectrum, 30 of which triterpin constituted the rest, and the other constituted two sugars. Comparing the hydrogen and ¹³ carbon NMR data of compound TP3 with Aliev and Movsumov, 1976, the isolated compound TP3 is represented by hederagenin-3- O - β - _D- glucopyranosyl- (1 → 3)- It was identified as α - _L -rhamnopyranoside, it was confirmed that it is a hederagenin saponin glycoside compound.

[Formula 3]

Position
TP3 δ _C ^a δ _H , mult. ( J ) One 39.7 1.70-1.58, m; 0.98, m 2 26.5 1.95-1.85, m; 1.80-1.71, m 3 82.3 3.62, dd (11.8, 4.7) 4 43.9 5 48.1 1.30-1.27, m 6 18.8 1.50, d (12.3); 1.42-1.33, m 7 33.9 1.70-1.58, m; 1.30-1.27, m 8 40.5 9 49.5 1.70-1.58, m 10 37.6 11 24.5 1.92-1.85, m 12 123.6 5.24, t (3.7) 13 145.5 14 43.0 15 28.9 1.80-1.71, m; 1.08, dt (13.6, 3.5) 16 24.1 1.99, t d (13.6, 3.9); 1.70-1.58, m 17 47.8 18 43.9 2.86, dd (13.9, 4.5) 19 47.4 1.80-1.71, m; 1.13 ddd (13.7, 4.7, 2.3) 20 31.6 21 35.0 1.42-1.33, m; 1.20, m 22 33.4 1.70-1.58, m; 1.53, dt (14.5, 3.7) 23 64.6 3.62, m; 3.33, m 24 13.7 0.70, s 25 16.4 0.98, s 26 17.9 0.83, s 27 26.5 1.18, s 28 182.6 29 33.6 0.90, s 30 24.0 0.94, s α-L-Ara One 104.4 4.56, dd (5.0, 1.5) 2 76.9 3.71, m 3 74.0 3.71, m 4 69.8 3.77, m 5 65.7 3.84, dd (12.0, 5.0); 3.50, dd (11.6, 2.2) α-L-Rha One 101.9 5.16, d (1.7) 2 71.1 3.91, dd (3.5, 1.7) 3 82.8 3.48, d (11.2) 4 72.3 3.37, q (9.5) 5 70.1 3.85, q (6.2) 6 18.1 1.24, d (6.3)

( ^a Recorded in CD ₃ OD at 500 MHz; δ in ppm and J in Hz)

<6-4> Chemical structure of compound TP4

As a result, compound TP4 was detected as a cation of m / z 805 [M + Na] ⁺ in MALDI-TOF-MS in cation mode, and the chemical formula was determined as C ₄₁ H ₆₆ O ₁₄ . NMR analysis results are shown in Table 7 below.

As shown in Table 7,

¹³41 carbon signals appeared in the carbon NMR spectrum, 30 of which consisted of triterpins and the other of which constitute two sugars. In the hydrogen NMR spectrumδ _H 6 methyl of 0.77, 0.86, 0.92, 0.99, 1.08, 1.15 ppm (C-24, C-25, C-26, C-27, C-29, C-30),δ _H Methine bound to 5.28 ppm (t, H-12) oxygen andδ _H 3.60, 3.54 ppm (m, H-23) of primary alcohol hydrogen signal appeared.¹³In the carbon NMR spectrumδ _C 14.4, 17.6, 17.9, 18.0, 21.6, 24.1 ppm methyl groups,δ _C Two olefins of 127.0, 139.3 ppm,δ _C Methine bound to 88.3 ppm oxygen,δ _C 64.4 ppm primary alcohol andδ _C 177.9 ppm of carbonyl carbon signal appeared. Looking at the HMBC spectrum of the triterpin structure, H-9 (δ _H 1.65 ppm) is C-10 (δ _C 38.5 ppm) and C-25 (δ _C 17.9 ppm); H-11 (δ _H 2.00 ppm) is C-13 (δ _C 139.3 ppm); H-18 (δ _H 2.26 ppm) is C-28 (δ _C 177.9 ppm), C-29 (δ _C 17.6 ppm), C-22 (δ _C 25.2 ppm) C-14 (δ _C 43.4 ppm) and C-12 (δ _C 127.0 ppm); H-24 (δ _H 0.77 ppm) is C-3 (δ _C 88.3 ppm) and C-23 (δ _C 64.4 ppm); H-27 (δ _H 1.15 ppm) is C-15 (δ _C 29.2 ppm) and C-13 (δ _C 139.3 ppm); H-29 (δ _H 0.92 ppm) is C-19 (δ _C 40.4 ppm) and C-18 (δ _C 54.2 ppm); H-30 (δ _H 0.99 ppm) is C-21 (δ _C 31.7 ppm) and C-19 (δ _C 40.4 ppm). These results indicate that the triterpene structure of the compound TP4 is asiatic acid, a ursane compound. Analyzing sugars with the HSQC spectrumδ _H 5.37 (d,J = 8.1 Hz), 4.56 (d,J = 4.4 Hz) ppm of two anomeric hydrogens eachδ _C It can be seen that it is combined with two anomer carbons of 95.7 and 106.3 ppm. Appeared in the HMBC spectrumδ _H 4.56 (α-_L-Ara)δ _C 88.3 (C-3);δ _H 5.37 (β-_D-Glc)δ _C Correlation analysis between 177.9 (C-28) was used to determine the binding of asiatic acid to ours. Based on these results, the chemical structure of the isolated compound TP4 is represented by 3-O-α-_L-arabinopyranosyl asiatic acid 28-O-β-_DDetermined by -glucopyranosyl ester, it was confirmed that it is an asiatic acid glycoside compound.

[Formula 4]

Position
TP4 δ _C ^a δ _H , mult. ( J ) One 47.4 2.05, dd (12.6, 4.6); 0.88, m 2 68.0 3.82, dd (12.0, 2.0) 3 88.3 3.48, d (9.5) 4 45.2 5 47.7 1.33, m 6 18.8 1.52, m; 1.40, m 7 33.6 1.68, m; 1.33, m 8 41.0 9 48.9 1.65, m 10 38.5 11 24.5 2.0, m; 0.98, m 12 127.0 5.28, t (3.8) 13 139.3 14 43.4 15 29.2 1.96, m; 1.12, m 16 25.2 2.10, m; 1.78, m 17 49.4 18 54.2 2.26, d (11.3) 19 40.4 1.40, m 20 40.2 21 31.7 1.52, m; 1.33, m 22 37.5 1.78, m; 1.65, m 23 64.0 3.71, m; 3.29, d (11.4) 24 14.4 0.77, s 25 17.9 1.08, s 26 18.0 0.86, s 27 24.1 1.15, s 28 177.9 29 17.6 0.92, d (6.4) 30 21.6 0.99, s α-L-Ara One 106.3 4.31, d (7.6) 2 72.9 3.71, m 3 74.6 3.54, m 4 70.1 3.85, dt (3.5, 1.6) 5 67.8 3.93, dd (12.7, 2.1); 3.65, dd (12.8, 1.3) β-D-Glc One 95.7 5.37, d (8.1) 2 73.9 3.33, m 3 78.2 3.42, m 4 71.1 3.39, m 5 78.5 3.35, d (8.5) 6 62.5 3.80, m; 3.71, m

( ^a Recorded in CD ₃ OD at 500 MHz; δ in ppm and J in Hz)

<6-5> Chemical Structure of Compound TP5

As a result, compound TP5 was detected as a cation of m / z 643 [M + Na] ⁺ in MALDI-TOF-MS in cation mode, and the chemical formula was determined to be C ₃₅ H ₅₆ O ₉ . The NMR analysis results are shown in Table 8 below.

As shown in Table 8 below,

Thirty-five carbon signals appeared in the ^13- carbon NMR spectrum, of which 30 consisted of triterpins and the remainder of one sugar. In the NMR spectrum of Compound TP5, anomer hydrogen δ _H 5.37 (d, J = 8.1 Hz) and carbon δ _C 106.3 of β - _D -glucose linked to carbonyl No. 28 shown in the NMR spectrum of Compound TP4 disappeared. The correlation between δ _H 4.56 ( α - _L -Ara) and δ _C 88.3 (C-3) was confirmed by HMBC spectral analysis. The chemical structure of the separated compound TP5 was represented by 3- O - α of the following Chemical Formula 5. _-L- arabinopyranosyl asiatic acid was determined, and it was confirmed that it is an Asiantic glycoside compound.

[Formula 5]

Position
TP5 δ _C ^a δ _H , mult. ( J ) One 47.4 2.04, m; 0.88, m 2 68.0 3.82, dd (6.6, 4.7) 3 88.3 3.48, dd (9.5, 2.4) 4 45.2 5 47.8 1.33, m 6 18.8 1.52, m; 1.40, m 7 33.7 1.68, m; 1.33, m 8 40.8 9 48.9 1.65, m 10 38.6 11 24.5 2.01, m; 0.98, m 12 126.4 5.26, t (3.6) 13 140.1 14 43.0 15 29.3 1.96, m; 1.12, m 16 25.4 2.10, m; 1.78, m 17 49.9 18 54.5 2.25, d (11.5) 19 40.5 1.40, m 20 40.4 21 31.7 1.52, m; 1.33, m 22 37.6 1.78, m; 1.65, m 23 64.0 3.71, m; 3.29, d (11.4) 24 14.4 0.77, s 25 17.9 1.08, s 26 18.0 0.86, s 27 24.1 1.16, s 28 182.8 29 17.6 0.91, d (6.3) 30 21.7 0.99, s α-L-Ara One 105.0 4.31, d (7.6) 2 72.9 3.72, d (11.5) 3 74.0 3.53, dd (9.6, 3.4) 4 69.8 3.84, dt (3.3, 1.5) 5 65.7 3.93, dd (12.7, 2.1); 3.65, d (12.4)

( ^a Recorded in CD ₃ OD at 500 MHz; δ in ppm and J in Hz)

< Example  7> Hederagenin  Containing saponin glycoside or asiatic acid glycoside compound as an active ingredient Plant disease  Of composition for control Phytopathogenic bacteria  Antimicrobial Activity Evaluation

In order to investigate the antimicrobial activity spectrum of the phytopathogens of the hederagenin saponin glycoside compounds TP1, TP2, and TP3, and the Asiatic acid glycoside compounds TP4 and TP5 isolated in Examples 4 and 5, the following experiment was carried out. Is shown in Table 9.

Specifically, five phytopathogens of the isolated compound Alternaria porri ( Botrytis ), Botrytis cinerea ), Colletotrichum coccodes , Fusarium oxysporum ) Magnaporthe oryzae ) and Phytophthora infestans were evaluated by determining the minimum inhibitory concentration (MIC), the minimum inhibitory concentration, using a broth micro-dilution method using a 96-well plate. It was. Alternaria porri were inoculated in V8 agar medium and incubated at 25 ° C. for 7 days to remove mycelia. Botrytis cinerea was inoculated in potato agar medium and incubated at 20 ° C. for 5 days. Colletotrichum coccodes ) and Magnaporthe oryzae were inoculated in oatmeal agar medium and incubated at 25 ° C. for 7 days and then removed mycelia. Fusarium oxysporum was inoculated in potato agar medium and incubated at 25 ° C. for 7 days to remove mycelia. Phytophthora infestans ) were inoculated in oatmeal agar medium and incubated at 20 ° C for 10 days. All strains were incubated for 2 days in a constant temperature and humidity room with a relative humidity of 80% to induce sporulation. Spores harvested using a potato liquid medium were filtered with a 4-ply gauze to prepare a spore suspension.

Finally, 100 μl wells contained spores of phytopathogens at a concentration of 1 × 10 ⁴ spores / ml. Compounds TP1, TP2, TP3, TP4 and TP5 were 1, 2, 4, 8, 16, 32, and 64, respectively. , 128 and 256 μg / ml was dispensed to include a concentration, the methanol content did not exceed 1%. Only 1% methanol was used as a control, and the experiment was repeated three times for each concentration. After culturing for 72 hours, the OD ₆₀₀ value was measured by UV / Vis spectroscopy, and the concentration at which fungus growth was completely inhibited was determined as the minimum inhibitory concentration. In addition, the concentration of 50% inhibition of mold growth was determined as EC ₅₀ compared to the control OD ₆₀₀ value.

MIC (μg / ml) EC ₅₀ (μg / ml) Plant pathogen TP1 TP2 TP3 TP4 TP5 TP1 TP2 TP3 TP4 TP5 Alternaria porri ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 Botrytis cinerea ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 127 24 44 > 256 35 Colletotrichum coccodes ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 > 256 > 256 ＞ 256 ＞ 256 256 Fusariumoxysporum ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 Magnaporthe oryzae 8 8 4 ＞ 256 32 5 4 2 > 256 5 Phytophthora infestans ＞ 256 ＞ 256 ＞ 256 ＞ 256 ＞ 256 136 205 36 218 128

As shown in Table 9,

All other compounds except TP4 showed activity that completely inhibited the mycelial growth of Magnaporthe oryzae , a causative agent of rice blast, at 4-32 μg / ml, and tomato gray mold at 24-127 μg / ml. It showed antimicrobial activity that inhibits the mycelial growth of Botrytis cinerea by 50%.

All isolated compounds were found in Phytophthora , the causative agent of tomato late blight, at a concentration of 36-218 μg / ml. infestans ) showed 50% inhibition of mycelial growth. In addition, the TP5 compound showed a 50% inhibition of mycelial growth of Colletotrichum coccodes , a causative agent of pepper anthrax, at a concentration of 256 μg / ml. Alternaria of Isolated Compound clear antimicrobial activity against porri ) and Fusarium oxysporum was not found in the concentration range tested.

Example 8 Evaluation of Antibacterial Activity against Phytopathogenic Bacteria of Plant Disease Control Compositions Containing Hederagenin Saponin Glycoside or Asiantic Glycoside Compound

In order to investigate the antibacterial activity spectrum of the heteragenine saponin glycoside compounds TP1, TP2, and TP3, and the asiatic acid glycoside compounds TP4 and TP5 isolated from Examples 4 and 5, against phytopathogenic bacteria, The results are shown in Table 9.

Specifically, the activity of inhibiting the growth of plant bacteriopathogenic bacteria was evaluated using the five compounds TP1, TP2, TP3, TP4 and TP5 isolated from trevezia palmata extract in Examples 4 and 5. Each of the five compounds separated was dissolved in methanol at a concentration of 100 mg / ml and then diluted in methanol by a double dilution method to prepare a stock solution for each concentration. Using a 96-well plate, each isolated compound was finally 1, 2, 4, 8, 16, 32, 64, 128, 256 μg / in 100 ml of soy broth (TSB) medium per well. The stock solution for each concentration was dispensed so as to have a concentration of ml, and the content of methanol did not exceed 1%. 8 plant bacteriopathogenic bacteria excidoborax avene subsp. Catidore (Acidovorax avenae subsp. Garnensis subsp. Micahganensis (Clavibacter michiganensis subsp. Michiganensis), Pectobacterium carotoborum subsp. Cartoborum (Pectobacterium carotovorum subsp. Carotovorum), Pseudomonas syringe pv. Pseudomonas syringae pv.actinidiae (kiwi ulcer), Ralstonia solanacearum (foot blight), Xanthomonas arboricola pv. Antimicrobial activity against Xanthomonas arboricola pv. Pruni (Peach Bacterial Pore Bacteria) was determined by MIC (minimum inhibitory concentration) using a liquid micro-dilution method using a 96-well plate. The minimum inhibitory concentration was calculated and evaluated. After inoculation in TSB medium, Pseudomonas syringe pv. Actinides and Xanthomonas Arvorcola pv. Pruny was shaken at 25 ° C. for 2 days at 30 ° C. at 150 rpm, followed by dilution with TSB medium to give a bacterial concentration of about 1 × 10 ⁷ CFU / ml. Diluted bacterial culture solution was inoculated to finally have about 1 × 10 ⁵ CFU / ml. The antibiotic streptomycin sulfate (Sigma-Aldrich) was used as a control, the addition of only 1% methanol was used as a non-treated group, OD ₆₀₀ value was measured by UV / Vis spectroscopy after 72 hours incubation. The concentration of 50% inhibition of bacterial growth was determined by EC ₅₀ compared to the OD ₆₀₀ value of the control, and the experiment was repeated three times for each concentration.

EC ₅₀ (μg / ml) Phytopathogenic Bacteria SS ^a TP1 TP2 TP3 TP4 TP5 Acidovorax avenae subsp.cattleyae 6 > 256 > 256 182 > 256 > 256 Agrobacterium tumefaciens 53 > 256 > 256 > 256 > 256 > 256 Burkholderiaglumae 7 > 256 > 256 > 256 > 256 213 Clavibacter michiganensis subsp.michiganensis 10 184 218 210 > 256 212 Pectobacterium carotovorum subsp.carotovorum 6 > 256 > 256 > 256 > 256 > 256 Pseudomonas syringae pv. actinidiae 20 > 256 > 256 > 256 > 256 > 256 Xantomonas arboricola pv. pruni 4 > 256 > 256 > 256 234 > 256 Ralstonia solanacearum 3 192 199 > 256 204 > 256

^a streptomycin

As shown in Table 10,

The isolated compounds showed activity inhibiting the growth of phytopathogenic bacteria at concentrations of 182 μg / ml or higher, but the level of antibacterial activity was lower than that of streptomycin used as a positive control. Except for TP4, the isolated compounds were treated with Clavibacter microorganisms at 184-218 μg / ml. It showed 50% inhibition of the growth of microorganisms, and the other compounds except TP3 and TP5 inhibited 50% of the growth of Ralstonia solanacearum at 192-204 μg / ml. Activity was shown. The isolated compounds TP3, TP4 and TP5 were respectively concentrated at the concentration of 182-234 μg / ml. Pruny, Berkholderia Glume, Exidoborax Abene subsp. It showed an activity of inhibiting bacterial growth of the cat tray by 50%. Agrobacterium tumefaciens, Pectobacterium carotoborum subsp. Carotoborum, Pseudomonas Syringe pv. Antibacterial activity against actinidia was not found in the concentration range tested.

< Example  9> Hederagenin  Containing saponin glycoside compound as an active ingredient Plant disease  Of composition for control Plant disease  Control effect evaluation

In order to investigate the in vivo plant disease control activity of the hederagenin saponin glycoside compound isolated from the trebezia palmata extract obtained in Example 4 was carried out as follows. The results are shown in Table 11.

Specifically, the control activities against the rice blast, rice leaf blight, tomato ash mold, tomato blight, wheat red rust, barley flour and red pepper anthracnose of Compound TP1, which were most isolated from trevezia palmata extract, Evaluated. After dissolving the active ingredient in methanol, the final concentration was adjusted to 125 μg / ml, 250 μg / ml and 500 μg / ml by adding 250 μg / ml Tween 20 solution, and the final methanol concentration of all samples was 5%. Fit. In this case, a solution containing 5% methanol and 250 μg / ml Tween 20 was used as a control. Four pots were used for each plant disease, and the active ingredient samples were spray-sprayed on the foliar and air-dried for 24 hours before inoculating each plant pathogen. Control activity against these seven plant diseases was investigated according to the method described in Example 1.

Control price (%) sample Concentration (㎍ / ml) RCB RSB TGM TLB WLR BPM PAN TP1 500 84 10 82 88 70 4 8 TP1 250 69 0 77 50 73 0 8 TP1 125 0 0 39 36 33 0 0

(RCB: Rice Blast, RSB: Rice Leaf Blight, TGM: Tomato Blight, TLB: Tomato Blight, WLR: Wheat Red Rust, BPM: Barley Powder, PAN: Red Pepper Anthrax)

As shown in Table 11, when Compound TP1 was treated at a concentration of 500 μg / ml, more than 80% control of rice blast, tomato gray mold, and tomato blight appeared, and 70% control of wheat rust. Ga appeared. Treatment at 250 μg / ml resulted in 69% for rice blast, 77% for tomato gray mold, 50% for tomato blight, and 73% for wheat rust. Overall, it showed excellent control against rice blast, tomato gray mold, tomato late blight, and wheat rust. .

Claims (17)

Trevesia palmata extract and Botrytis cinerea, tomato lye fungus, which contains at least one selected from the group consisting of fractions thereof, and choletotrichum cocode coccodes; pepper anthrax, Magnaporthe oryzae, Puccinia triticina, and Phytophthora infestans. Phytopathogenic fungal mycelium; And excidoborax avene subsp. Catidore (Acidovorax avenae subsp. Cattleyae; phalaenopsis bacterium brown spot pattern bacterium), Burkholderia glumae, bacterium bacillus bacillus subsp. Clavibacter michiganensis subsp. Michiganensis (Rice ulcer bacterium), Ralstonia solanacearum (foot blight germ) and Xanthomonas arboccola pv. A plant disease control composition caused by any one selected from the group consisting of phytopathogenic bacteria consisting of Xanthomonas arboricola pv. Pruni (Peach bacterial pore pathogen).
The method of claim 1,
The extract is characterized in that the extract using any one or a mixture thereof selected from the group consisting of water, lower alcohols of C ₁ to C ₄ , hexane, chloroform, methylene chloride, ethyl acetate, acetone and acetonitrile as a solvent Composition for controlling plant diseases.
The method of claim 1,
Wherein the fraction is hexane, chloroform, ethyl acetate, methylene chloride, methanol, ethanol, butanol and water any one or a mixed solution selected from the group consisting of water, the composition for controlling plant diseases, characterized in that the fraction is used as a solvent.
The method of claim 1,
The trevesia palmata (Trevesia palmata) extract or a fraction thereof is a composition for controlling plant diseases, characterized in that it comprises one or more compounds represented by any one of the following Formula 1 to:
[Formula 1]

;

[Formula 2]

;

[Formula 3]

;

[Formula 4]

; And

[Formula 5]

.
The method of claim 1,
The active ingredient is a composition for controlling plant diseases, characterized in that contained in the composition for controlling plant diseases in a concentration of 100 to 3,000 μg / ml.
Comprising the step of treating a plant, seed or its habitat with a composition for controlling plant diseases containing at least one selected from the group consisting of the extract of Trevesia palmata (Trevesia palmata) and fractions thereof How to control plant diseases.
Hederagenin saponin glycoside compound represented by Formula 1 below:
[Formula 1]

.
An asiatic acid glycoside compound represented by Formula 5 below:
[Formula 5]

.
Hederagenin saponin glycoside compound represented by the formula (1) containing as an active ingredient,
Botrytis cinerea (tomato ash fungus), Magnaporthe oryzae (P. erythritis), Puccinia triticina (wheat red rust) and Phytophthora infestans (Phytophthora) phytopathogenic fungal mycelium consisting of infestans; And Clavibacter misganogenis subsp. A plant caused by any one selected from the group consisting of Clavibacter michiganensis subsp. Michiganensis (Capsicum ulcer bacterium) and Ralstonia solanacearum (foot blight bacteria); Bottle Control Composition:
[Formula 1]

.
Hederagenin saponin glycoside compound represented by the formula (2) containing as an active ingredient,
Phytopathogenic fungus hyphae consisting of Botrytis cinerea (Tomato gray fungus), Magnaporthe oryzae (Phytophthora bacillus), and Phytophthora infestans; Clavibacter microganensis subsp. A plant caused by any one selected from the group consisting of Clavibacter michiganensis subsp. Michiganensis (Capsicum ulcer bacterium) and Ralstonia solanacearum (foot blight bacteria); Bottle Control Composition:
[Formula 2]

.
Hederagenin saponin glycoside compound represented by the formula (3) containing as an active ingredient,
Phytopathogenic fungal hyphae consisting of Botrytis cinerea (Tomato gray fungus), Magnaporthe oryzae (Phytophthora infestans) and Phytophthora infestans; And excidoborax avene subsp. Catidore (Acidovorax avenae subsp. Cattleyae; phalaenopsis bacterium brown spot pattern bacterium) and Clavibacter microorganisms subsp. A composition for controlling plant diseases caused by any one selected from the group consisting of phytopathogenic bacteria consisting of Clavibacter michiganensis subsp. Michiganensis;
[Formula 3]

;
Containing an asiatic acid glycoside compound represented by the formula (4) as an active ingredient,
Phytopathogenic fungal mycelium of Phytophthora infestans; And Ralstonia solanacearum (foot blight) and Xanthomonas arboricola pv. Plant disease control composition caused by any one selected from the group consisting of phytopathogenic bacteria consisting of Xanthomonas arboricola pv. Pruni (Peach bacterial pore pathogen):
[Formula 4]

.
Containing an asiatic acid glycoside compound represented by the formula (5) as an active ingredient,
Botrytis cinerea (Tomato ash fungus), Colletotrichum coccodes (Capsicum anthrax), Magnaporthe oryzae, and Phytophthora infestans Phytopathogenic fungal hyphae consisting of (Phytophthora infestans; tomato late blight); And Burkholderia glumae (Bacterial rice blight) and Clavibacter microganensis subsp. A composition for controlling plant diseases caused by any one selected from the group consisting of phytopathogenic bacteria consisting of Clavibacter michiganensis subsp. Michiganensis;
[Formula 5]

.
The method according to any one of claims 9 to 11,
The hederagenin saponin glycoside (hederagenin saponin glycoside) compound is a composition for controlling plant diseases, characterized in that separated from Trevesia palmata (Trevesia palmata) extract and fractions thereof.
The method according to claim 12 or 13,
The asiatic acid glycoside compound is a plant disease control composition, characterized in that isolated from Trevesia palmata (Trevesia palmata) extract and fractions thereof.
The method according to any one of claims 9 to 13,
The active ingredient is a composition for controlling plant diseases, characterized in that contained in the composition for controlling plant diseases in a concentration of 100 to 3,000 μg / ml.
A plant disease control method comprising the step of treating the composition of the plant disease control according to any one of claims 9 to 11 to a plant, a seed thereof or a habitat thereof.

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