KR101828563B1 - A composition for prevention and treatment of foulbrood - Google Patents

Abstract

The present invention relates to a composition for treating the infectious disease of bees. The present invention provides a composition for prevention and treatment of foulbrood which includes a compound derived from a plant extract, has excellent safety, and prevents foulbrood.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing and treating a disease of a digestive tract,

The present invention relates to a composition for treating infectious diseases of bees, and more particularly, to a composition for treating infectious diseases of bees, which comprises not only a single compound derived from a plant extract but also excellent biocompatibility, And a composition for treating an infectious disease of a bee.

Bees are the most active pollinator in the world and play an important role in bee pollination of various kinds of flowers and plants as well as crops. Although the production of honey, bee wax, royal jelly, pollen, propolis and bee venom, which are directly produced by honey, account for the share of total livestock production (1.2%, Ministry of Agriculture, Forestry and Fisheries, 2010) is not large, but 40% of human-eating vegetable foods are known to be hydrated by bees.

Therefore, it is clear that the decrease in bee populations due to various reasons will have a great impact on the natural ecosystem as well as agricultural production.

In the domestic beekeeping industry, the direct market size of the bee industry is about 400 billion, and the multiple functions of the bee ecosystem such as the pollen of the crops are too large to quantify. The economic value of honeybees in Korea accounted for 6 trillion won, 50% of the total fruit and vegetable production of 12.5 trillion won, and the total ecosystem preservation effect exceeds 50 trillion won.

Bees are farm animals and farm animals. According to the farmhouse, the beehive ranges from several hundreds to several thousand, and in the case of our country, As of 2011, there are about 2 million troops in Korea.

Higher densities are bound to be susceptible to disease. From the viewpoint of infectious pathogens such as fungi, it is an optimal survival condition because there are a lot of individuals that can be infected and are densely populated. Therefore, if the infection is not managed properly and the infection is transmitted to a pathogenic microorganism such as a fungus, it can cause a serious problem of losing the population.

To date, bee-related research on domestic bees has been carried out in the field of pesticide damage survey (Choi, Wai, 1986), Yangbong management and Bongun management (Oh, 1988), management analysis of beekeeping farmers (Kim and Park, 1990) (2009), and the development of bee disease diagnosis technology (Yoon, Byoung-soo, 2013). In addition, research on beekeeping management The bee disease management center has been established at the Agriculture, Forestry and Livestock Quarantine Headquarters, and research on bees has been actively conducted.

However, the Bee Disease Control Center is conducting research on "Diagnosis, prevention and prevention of parasitic diseases and honey bee disease research and development", but does not find any growth inhibitors or anti- .

Among bee infectious diseases that have been confirmed to occur in Korea or in the world, bacterial diseases are known as American Buzzers (AFB) and European Buzzers (EFB).

Buzzers of bees are infectious diseases caused by bacteria, and they are so terrible diseases that they are designated as the first infectious disease of bees in Korea. Two types of buzzer bottles are known: American Buzzers (AFB) and European Buzzers (EFB). There are reports of resistance to pathogens due to the abuse of a number of medicines for the treatment of diseases, and the main causes of deterioration of product quality .

Buzzers are one of the most feared diseases of infectious diseases infecting larvae and are known to be caused by Paenibacillus larvae (AFB). Paenibacillus larva is resistant to bactericides because it can form spores in the form of bacillus (2.5 ~ 5.0 ㎛ × 0.7 ~ 0.8 ㎛) among Gram-positive bacteria, and thus may maintain infectivity for 35 years under certain conditions. It has also been reported that the infection of American buzzers is transmitted by the reuse of contaminated honey or the reuse of contaminated instruments, and the spread of infection among the larvae or ferns may be caused by the exchange or docking of infected bees.

Infections caused by Paenibacillus larva endosperm spores were observed in bees ( Apis mellifera , and it is known that other insects and humans do not cause infection.

The larvae of Paenibacillus larva infected with larvae grow into nutrient cells within 1 day after reaching the midgut through the oral cavity and then proliferate rapidly. The proliferated Paenibacillus larva cells lose nutrients when the larvae die and again form about 500 to 10 million endospores per one larva and infect other larvae.

Other causative agents causing buzzer disease include Melissococcus Plutonious is known, which causes European buzzers. Melissococcus The process and transmission path of plutonious larvae is similar to that of American buzzers, but the disease may be more lightweight than the American buzzers and may be removed by cleaning bees.

The American Boomer Disease (AFB) is officially recognized as the first disease in New Zealand in 1877 and Buzzers disease is one of the most common infectious diseases caused by microorganisms in the world. It was first reported in the central region of Korea in 1950 and has caused serious damage to beekeeping.

American buzzers are mainly found in temperate regions including Korea, and it is widely known that spores of P. larvae , the causative organism, are found in 1.0 to 10% of the population including New Zealand, a developed country of beekeeping. have.

Therefore, the early detection of the US bee sickness is the most important, especially considering the domestic beekeeping situation, which imports a considerable amount of bee populations, it is very important to block the influx of the domestic bee. In addition, Will be.

Also, it is important to think about the causative organism because it is caused by endogenous spores that can survive for several decades, and it is known that spores are present in a large number of the world's vultures.

In case of serious infectious disease in the beehive of the buzzers disease, the rods may be smoked or have a bad smell. Generally, in the case of the infected roe of the buzzers, the roots of the roots are reddish. In the case of bees, there is no force. The abdomen is big. In the case of severe infestation, it is said that the method of immediate incineration is the best method. In case of infectious infection, tetracycline-based antibiotics may be used.

The most effective control method of buzzers known to date is a method of incinerating the infected beehive, but it has a disadvantage of a large economic loss.

Tetracycline-based antibiotics are synthetic compounds that inhibit growth, not drugs that kill bacteria. These antibiotics should be careful about the persistence in honey, so the administration time can be limited to the time of childbearing immediately after wintering. Complete treatment with medicines is difficult, spores can not be removed, and due to the time limit for the use of medicines, prophylactic management of the needles is required rather than treatment for onset.

In addition, when the onset of the buzzer disease is confirmed, the incinerator is incinerated, which causes a large loss. It does not kill the spores of the buzzer, and after a certain period of time, the stability of the antibiotic is reduced (it is known that much of it is destroyed within 1 day of exposure to sunlight). Bacteria can grow again. In addition, tetracycline-based antibiotics kill almost all bacteria, which can kill beneficial bacteria and adversely affect the ecosystem.

Also, the incidence of resistant strains has been reported in many kinds of bacteria due to high frequency of use. Therefore, studies are underway to replace these drugs, but no major results have been reported yet.

In Korea, which mainly imports fishing rod from foreign countries, it is the best national preventive measure to prevent the introduction of pathogens by the quarantine system and to reduce domestic inventions.

As a health food, the presence of antibiotics or products of harmful bacteria in honey bee products is a very serious problem in the competitiveness of honey bee products. Quality products without persistent substances can have a significant impact on the income of farm households because they can take advantage of honey bee products such as cheap honey in other countries

In order to maintain the natural ecosystem as a pollen medium, it is necessary to prevent the decrease of the populations of bees. Therefore, it is urgent to develop a therapeutic agent for a disease such as a pest which is one of the most problematic bees.

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide an environmentally friendly composition for treating a buzzer disease, which is exclusively used for a strain derived from a plant extract and causing a buzzer disease .

According to an aspect of the present invention, there is provided a composition for the prevention and treatment of buzzer disease comprising at least one of compounds represented by the following formulas (1) to (4) as an active ingredient.

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

In one embodiment, the composition may have an antimicrobial activity against causative bacteria of the buzzer.

In one embodiment, the causative organism is Paenibacilus larvae or Melissococcus flutonius, plutonius .

In one embodiment, the composition may further comprise one or more antimicrobial or antifungal agents.

In one embodiment, the antimicrobial or antifungal agent is selected from the group consisting of amikacin, gentamicin, tobramycin, streptomycin, nethylamycin, kanamycin, ciprofloxacin, norfloxacin, oproxacin, trombloxacin, , Enoxysin, naphthyridine, sulphonamide, polymyxin, chloramphenicol, neomycin, paramomomycin, colistimate, baxitracin, vancomycin, tetracycline, rifampin, cycloserine, beta- But are not limited to, spore, amphotericin, fluconazole, flucytosine, natamycin, miconazole, ketoconazole, corticosteroids, diclofenac, flurebiprofen, ketorolac, suiprofen, codolin, rosoxamide, levocabastine, , Anthazoline, and pennylammyone.

According to the present invention, the composition for treating a buzzer disease contains a single compound derived from a plant extract as an active ingredient, so that the toxicity to the body is low and the antimicrobial or antifungal effect against causative bacteria is excellent.

In addition, since the composition for treating an infectious disease of bees exhibits activity only for causative agents of gypsum disease, the total use amount is reduced, thereby reducing the incidence of tolerance and minimizing side effects.

It should be understood that the effects of the present invention are not limited to the above effects and include all effects that can be deduced from the detailed description of the present invention or the configuration of the invention described in the claims.

FIG. 1 shows the results of evaluating cytotoxicity of acetylshikonin according to the concentration of acetylshikonin according to an embodiment of the present invention.
FIG. 2 is a result of evaluating cytotoxicity according to the treatment of 4-Hydroxyderricin according to an embodiment of the present invention.
FIG. 3 is a result of evaluating cytotoxicity according to the concentration treatment of Sophoraflavanone G according to an embodiment of the present invention.
FIG. 4 is a result of evaluating cytotoxicity according to the concentration-dependent treatment of curarinone according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Further, in order to clearly illustrate the present invention in the drawings, portions not related to the description may be omitted.

As used herein, the terminology used herein is intended to encompass all commonly used generic terms that may be considered while considering the functionality of the present invention, but this may vary depending upon the intent or circumstance of the skilled artisan, the emergence of new technology, and the like. Also, in certain cases, there may be a term selected arbitrarily by the applicant, in which case the meaning thereof will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term, not on the name of a simple term, but on the entire contents of the present invention.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

The numerical range includes numerical values defined in the above range. All numerical limitations of all the maximum numerical values given throughout this specification include all lower numerical limitations as the lower numerical limitations are explicitly stated. All the minimum numerical limitations given throughout this specification include all higher numerical limitations as the higher numerical limitations are explicitly stated. All numerical limitations given throughout this specification will include any better numerical range within a broader numerical range, as narrower numerical limitations are explicitly stated. The subject matter provided herein should not be construed as limiting the following embodiments in various aspects or as a reference throughout the specification.

According to an aspect of the present invention, there is provided a composition for the prevention and treatment of buzzer disease comprising at least one of compounds represented by the following formulas (1) to (4) as an active ingredient.

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

The composition may contain at least one of the compounds of the formulas (1) to (4) as an active ingredient.

The compound of formula (I) is referred to as Acetylshikonin and can be prepared by reacting 4 - [(2S, 3R) -4- (1,3-benzodioxol-5-yl) -2,3- -Methoxyphenol [1- (5,8-dihydroxy-1,4-dioxonaphthalen-2-yl) -4-methylpent- 1,4-dioxonaphthalen-2-yl) -4-methylpent-3-enyl] acetate.

The compound of Formula 1 has inflicted (Lithospermum erythrorhizon S. et Z. ) extracts. It belongs to the Borraginaceae family, and it grows in many parts of Korea. It is said to be duck, grasshopper, witch or grasshopper.

The seedlings contain a large amount of naphthoquinone-based compounds such as acetylshikonin, shikonin, alkanan, and isobutylshikonin as main active ingredients, , Essential oils, and pigments, and is widely used as a raw material for medicinal herbs and herbal medicines.

The herb has various pharmacological functions including contraceptive action. Recently, pharmacological effects such as anti-inflammatory activity, antibacterial activity against bacteria, slight anti-cancer activity, and blood circulation promoting action have been reported.

The seedlings may be extracted using water at a low temperature of less than 40 캜 at room temperature or water having a high temperature of 60 캜 or more as a solvent, or may be extracted using alcohol as a solvent. The alcohol may be an aliphatic alcohol having 1 to 6 carbon atoms, and specifically, methanol, ethanol, isopropanol, butanol, hexane and the like may be used, but the present invention is not limited thereto.

The alcohol extract may be dispensed into an organic solvent and water. The organic solvent may be selected from aliphatic hydrocarbons having 1 to 10 carbon atoms, aliphatic halogenated hydrocarbons having 1 to 10 carbon atoms, and esters having 2 to 10 carbon atoms.

The compound of formula (I) contained in the seedling extract can be isolated by additional chromatography, such as silica gel column chromatography, and various known methodologies for separating a single compound can be applied.

On the other hand, the compound of formula (2) is called 4-hydroxyderricin, and it is preferable to use (1) -1- [2-hydroxy-4-methoxy- Phenyl) -3- (4-hydroxyphenyl) prop-2-en-1-yl ((E) -1- [2-hydroxy- ] -3- (4-hydroxyphenyl) prop-2-en-1-one.

The compound of Formula 2 may be isolated from Angelica keiskei extract. The herbaceous plant is a perennial plant belonging to Apiaceae, and is also known as Myron leaf. It grows in the subtropical region and is cultivated throughout Korea.

The chrysanthemum extract contains 4-hydroxyderricin, xanthoangelol, germanium, vitamins and various minerals as major active ingredients and is also used as a raw material for herbal medicine. The herbaceous plant has been reported to have pharmacological effects such as anti-cancer, anti-allergy, anti-inflammatory, anti-aging and liver function improvement.

The plant for isolating 4-Hydroxyderricin is a plant species known to contain 4-Hydroxyderricin, callus derived therefrom, plant cells, and plant cell suspensions A culture solution may be included. The site of use of the plant may be whole plant, leaf, flesh, stem, root, flower, preferably root.

The 4-hydroxyderricin may be subjected to hydrothermal extraction with a lower alcohol solvent such as methanol or ethanol or a mixed solvent thereof, followed by concentration under reduced pressure. In order to more efficiently extract 4-hydroxyderricin, the fresh roots can be pulverized to 100 to 200 mesh by a pin mill or a ball mill, and 50 to 99% Of the lower alcohol three times.

The extracted hot water can be separated into a water insoluble layer and a water soluble layer by filtration to dissolve in distilled water, and the water soluble layer can be degreased with a nonpolar organic solvent such as chloroform, ether or methylene chloride have.

The water-soluble layer may contain a large amount of hydrophilic or hydrophobic impurities as well as 4-hydroxydericin. Therefore, the water-soluble layer may be degreased with a nonpolar organic solvent to remove hydrophobic impurities from the root of the herbarium. Preferably, Degreasing process may be performed three times.

In addition, the compound of formula (2) contained in the fresh root extract can be isolated by chromatographic methods well known in the art. For example, the degreased water-soluble layer may be adsorbed on the nonionic resin for column chromatography after the nonpolar organic solvent is removed. The column chromatography can be carried out by a conventional method well known in the art.

The compound of formula (3) is referred to as Sophoraflavanone G and is a compound of formula (2S) -2- (2,4-dihydroxyphenyl) -5,7-dihydroxy-8- [ Methyl-4-hexen-1-yl] -2,3-dihydro-4H-chromen-4-one ((2S) -2- (2,4- Dihydroxyphenyl) 5,7-dihydroxy-8 - [(2R) -2-isopropenyl-5-methyl-4-hexen-1-yl] -2,3-dihydro-4H-chromen-4-one.

The compound of formula (4) is also referred to as " kurarinone ", and is 2- (2,4-dihydroxyphenyl) -2,3-dihydro- (2,4-Dihydroxyphenyl) -2,3-dihydro-7-methyl-2- (1-methylethenyl) -4-hexenyl] -4- hydroxy-5-methoxy-8- [5-methyl-2- (1-methylethenyl) -4-hexenyl] -4H-1-benzopyran-4-one.

The compounds of Formulas 3 and 4 Sophora (Sophora flavescens Ait root .) extract. The ginseng belongs to the rosemary bean family of the dicotyledonous plant, and it grows widely in the grasses of less than 1000m above sea level. In autumn, the root is picked, washed and dried, and is used as a medicinal product.

The major active ingredients of the ginseng are alkaline and flavonoids including matrine, oxymatrine and sophoraflavanone G and kurarinone, and flavonoid and saponin saponin) and is used as a raw material for herbal medicine. The ginseng has been reported to have pharmacological effects such as anti-cancer action, anti-allergic, antiarrhythmic, antimicrobial action and diuretic as well as bronchial inflammation, parasitic action and paralysis.

Plants for isolating the compounds may include plant species as well as plant cell and plant cell suspension cultures derived therefrom.

In order to effectively extract the sophoraflavanone G and the kurarinone, the ginseng roots can be pulverized into 100-200 mesh by a pin mill or a ball mill pulverizer, It can be extracted by hot water three times with 99% lower alcohol and then concentrated under reduced pressure.

The hot water-extracted extract may be dissolved in distilled water and suspended to be separated into a water-insoluble layer and a water-soluble layer by filtration, and the water-soluble layer may be degreased with a nonpolar organic solvent such as ether or methylene chloride. Since the water-soluble layer may contain various impurities as well as vesicle laplacabanone G or curalinon, the water-soluble layer may be degreased with a non-polar organic solvent to remove impurities from the roots of the ginseng root, Can be performed three times.

In addition, the compounds of formulas (3) and (4) contained in the roots extract of ginseng roots can be isolated by chromatographic methods well known in the art. The compounds of formulas (3) and (4) contained in the goat extract can be isolated by additional chromatography, such as silica gel column chromatography, and various known methodologies for separating a single compound can be applied.

For example, after the nonpolar organic solvent is removed, it can be adsorbed to a nonionic resin for column chromatography. The column chromatography can be carried out by a conventional method well known in the art.

Since the isolated compounds of the formulas (1) to (4) have excellent activity against specific bacteria or fungi, they can be used for prevention and treatment of buzzer disease which is a communicable disease of bees.

Since the composition contains at least one of the compounds of the formulas (1) to (4) as an active ingredient, the composition may have antimicrobial activity against causative bacteria of the buzzer disease, and the causative bacteria may be Paenibacilus larvae or Melisococopteronius ( Melissococcus plutonius ).

That is, since the composition has a specific antimicrobial activity against a specific strain, it can not induce extensive use, can minimize the incidence of resistance, and is excellent in biosafety.

In addition, the composition may further comprise one or more antimicrobial agents or antifungal agents.

Wherein said antimicrobial or antifungal agent is selected from the group consisting of amikacin, gentamycin, tobramycin, streptomycin, nethylamycin, kanamycin, ciprofloxacin, norfloxacin, oproxacin, trovafloxacin, lomeproxacin, levofloxacin, , Sulfonamides, polyomics, chloramphenicol, neomycin, paramomomycin, colistimetate, parkshitracin, vancomycin, tetracycline, rifampin, cycloserine, beta-lactam, cephalosporin, amphotericin, But are not limited to, fluconazole, flucytosine, natamycin, myconazole, ketoconazole, corticosteroids, diclofenac, fluulbifropen, ketorolac, suiprofen, codolin, rosoxamide, levocabastine, ≪ / RTI >

That is, some antimicrobial agents widely used have antimicrobial activity against the causative microorganisms. However, they are resistant to excessive use, and their use is limited due to bio-toxicity.

Accordingly, the antibacterial agent or the antifungal agent can be significantly reduced in the use of the compound of Formula 1 and the compound of Formula 2, and the antibacterial activity can be improved due to the synergistic effect.

The present invention will be further described with reference to the following examples, but it should be apparent that the present invention is not limited by the following examples.

Experimental Example  1: Cytotoxicity test

To evaluate the cytotoxicity of the compound of formula (1) (acetylchiconine), the compound of formula (2) (4-hydroxyderissin), the compound of formula (3) (soporaflabanone G) and the compound of formula (4) In order to determine the effect of each compound on cell growth, we assayed the concentration of acetylcycinine, 4-hydroxyderisin, soporaflabanone G, and cialynolone in HepG2 cell line and MTT assay, respectively.

The cytotoxicity experiments were performed using Hep-G2 cell lines. 2 × 10 ³ cells / 0.1 ml of Hep-G2 cell lines were placed in each well of 96-well tissue culture plates (Falcon), and each substance dissolved in DMSO was added to the culture plates at the indicated concentrations.

Control DMSO was added to each plate so that the maximum concentration did not exceed 0.2%. The activity was measured at 540 nm absorbance using an ELISA reader (VersaMax, Molecular Devises, USA) after the addition of 100 μl (5 mg / ml) MTT (Sigma Cat. M2128) after 24 hours or 48 hours. All experiments were repeated three or more times.

1 to 4 are the results of evaluating cytotoxicity of acetylchiconine, 4-hydroxyderisin, soporaflabanone G and curareonone according to the treatment.

Referring to FIG. 1, when treated with a high concentration (10 mg / L) of acetylchiconine, cell growth of 18.2 ± 5.4% was observed in Hep-G2 cell lines as compared with the case without acetylchiconine treatment. On the other hand, when HaCaT cell line was used at high concentration (10 mg / L), cell growth was observed at 65.7 ± 2.6%.

As compared with the case of myconazole having a cell growth of 14.3 ± 5.2%, the acetylchiconine partially inhibited the growth of cells when treated at a high concentration, but toxicity to the living body is extremely small if it is used at a usual concentration as an antifungal agent Level.

2, when treated with 4-hydroxyderisin at a high concentration (10 mg / L) in Hep-G2 cell lines, cell growth of 89.0 ± 5.3% compared with the case without treatment with 4-hydroxyderisin Respectively. On the other hand, when treated with high concentration (10 mg / L) in the HaCaT cell line, cell growth of 93.1 ± 8.2 was observed.

That is, even if 4-hydroxydericine is used at a high concentration, the toxicity to the living body is very low, and if it is used as a usual concentration, it is analyzed that there is little toxicity to the living body.

Referring to FIG. 3, when hepolablavanone G was treated at a high concentration (10 mg / L), 20.4 ± 17.9% of cell growth was observed in Hep-G2 cell lines, Respectively. On the other hand, when HaCaT cell line was treated with high concentration (10 mg / L), cell growth of 20.6 ± 6.9% was observed. The vesicle Laplacabanone G partially inhibited the growth of cells when treated at a high concentration, but it is analyzed that toxicity to living organisms is extremely low if it is used at a usual concentration as an antimicrobial agent.

Referring to FIG. 4, in the case of treatment with cilarinon at a high concentration (10 mg / L), 51.6 ± 5.3% of cell growth was observed in the Hep-G2 cell lines as compared with the case without curalinon treatment. On the other hand, when HaCaT cell line was used at high concentration (10 mg / L), cell growth of 45.3 ± 2.1% was observed. The curalinon is very toxic to living organisms even if it is used at a high concentration and it is analyzed that there is almost no toxicity to a living body if it is used as a usual use concentration.

That is, the compound of formula (1) (acetylchiconine), the compound of formula (2) (4-hydroxyderissin), the compound of formula (3) (soporaplavanone G) Is used as an antimicrobial agent, its cytotoxicity is lower than that of other synthetic antimicrobial agents. Therefore, it is safe and eco-friendly because it has low harmfulness to humans or bees.

Experimental Example  2: Fanny Bacillus In lava  Antibacterial activity test

( Paenibacilus ) of the compound of formula (1) (acetylchiconine), the compound of formula (2) (4-hydroxyderisin), the compound of formula (3) (soporaplavanone G) larvae and Melissococcus plutonius were evaluated.

To confirm the antimicrobial activity of the compounds of formulas (1) to (4), broth microdilution assay as described in EUCAST (the European Committee on Antimicrobial Susceptibility Testing) was used. Each compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg / ml and used.

Paenibacilus larvae were grown on Luria-Bertani (LB) agar (1% w / v bacto-tryptone, 0.5% w / v bacto- yeast extract, 1% w / v NaCl and 4.5% (MYPGP: 1% of Mueller-Hinton broth, 1.5% of yeast extract, 0.2% of glucose, 0.3% of K ₂ HPO ₄ and 0.1% of sodium pyruvate) .

Melissococcus Plutonius was added to M110 medium (0.25% of peptone), 1% of glucose, 0.2 g of soluble starch, 0.25 g of yeast extract (Oxoid L21), 0.5 g of neopeptone (Difco 0119), 0.2 g of trypticase (BBL211921) 50 mM phosphate buffer (pH 6.7) and 0.025% cysteine hydrochloride). For each compound, the DMSO concentration was maintained at a level lower than 2.5%.

Each compound was continuously diluted 2-fold and then added to a 96-well microtiter plate (100 μl). 100 μl of Paenibacilus larvae and Melissococcus plutonius strain suspension (OD ₆₀₀ = 0.1) was added to each well and cultured at 35 ° C for 24 hours or 48 hours. The concentration of each compound was adjusted from a maximum of 200 mg / l to a minimum of 0.2 mg / l. The MIC values for each substance were confirmed to be the minimum concentrations that do not grow monarchs in each plate.

Table 1 below compares the anti- Paenibacilus larvae activity of the compounds of the formulas (1) to (4) with the antifungal activity of the antifungal agents and the candidate substances widely used.

Referring to Table 1, the compounds of the formulas (1) to (4) showed a remarkable growth inhibitory effect on Paenibacilus larvae by measuring the MIC value.

4- Hydroxyderisin exhibited superior antimicrobial activity compared to the widely used tetracycline, and acetylchoninin, soporaplavanone G and cialynone exhibited relatively insufficient antifungal activity, but the growth of Paenibacilus larvae Respectively.

On the other hand, the compounds of Comparative Examples 3, 5, 6, 8, 10, and 13 to 17, which were used as a control group, exhibited some growth inhibitory activities against the microorganisms, but their effects were weak.

That is, the above acetylchoninin, 4-hydroxyderisin, soporaplavanone G and cialynone showed antimicrobial activity against Paenibacilus larvae causing buzzer disease to bees, and their activities were compared with those of existing antimicrobial agents It is not only equal or superior, but also has a high safety advantage.

division compound P649 (KACC14031) P618 (KACC11540) 24h 48h 24h 48h Example 1 Acetylshikonin 3.125 3.125 12.5 12.5 Example 2 4-Hydroxyderricin <0.78 <0.78 6.25 12.5 Example 3 Sophoraflavanone G 1.56 1.56 3.125 12.5 Example 4 Kurarinone 6.25 6.25 6.25 12.5 Comparative Example 1 Miconazole 1.56 3.125 6.25 12.5 Comparative Example 2 Tetracycline 0.78 0.78 25 50 Comparative Example 3 Alismol 100 100 > 200 > 200 Comparative Example 4 Alpiniumisoflavone > 200 > 200 > 200 > 200 Comparative Example 5 Bisdemethoxy-curcumin 25 > 200 > 200 > 200 Comparative Example 6 (R) - (+) - Dalbergiphenol 100 100 > 200 > 200 Comparative Example 7 Dehydrodieugenol > 200 > 200 > 200 > 200 Comparative Example 8 6,8-Diprenylorobol 25 25 50 > 200 Comparative Example 9 Eupatilin > 200 > 200 > 200 > 200 Comparative Example 10 Falcarindiol 6.25 6.25 50 100 Comparative Example 11 Gagaminine > 200 > 200 > 200 > 200 Comparative Example 12 Galangin > 200 > 200 > 200 > 200 Comparative Example 13 Guaiacin 50 50 50 50 Comparative Example 14 Matairesinoside > 200 > 200 > 200 > 200 Comparative Example 15 Myristargenola > 200 > 200 > 200 > 200 Comparative Example 16 Oleiferin F > 200 > 200 > 200 > 200 Comparative Example 17 Sargachromanol G 6.25 6.25 6.25 50 Comparative Example 18 Timosaponin AIII > 200 > 200 > 200 > 200

On the other hand, the following Table 2 shows the results obtained by measuring the activity of acetylcycinin, 4-hydroxyderisin, plutonius And antimicrobial activity of antimicrobials and candidate substances which are widely used.

Referring to the following Table 2, the compounds of the formulas (1) to (4) showed a remarkable growth inhibitory effect on Melissococcus plutonius by measuring the MIC value. The compounds of the formulas (1) to (4) showed insufficient activity compared to the widely used tetracycline, but the anti- Melissococcus plutonius activity.

On the other hand, the substances of Comparative Examples 8 and 10-13 used as a control group showed some growth inhibitory activities against the microorganisms, but their effects were weak.

That is, the acetylchiconine, 4-hydroxyderisin, the soporaplavanone G and the curalinone according to the present invention are effective against Melissococcus plutonius And exhibits the same or better activity than the existing antimicrobial agents and has a high safety.

division compound 24h 48h Example 1 Acetylshikonin 6.25 6.25 Example 2 4-Hydroxyderricin 3.125 3.125 Example 3 Sophoraflavanone G 3.125 3.125 Example 4 Kurarinone 12.5 12.5 Comparative Example 1 Miconazole 3.125 3.125 Comparative Example 2 Tetracycline 0.78 0.78 Comparative Example 3 Alismol > 200 > 200 Comparative Example 4 Alpiniumisoflavone > 200 > 200 Comparative Example 5 Bisdemethoxy-curcumin > 200 > 200 Comparative Example 6 (R) - (+) - Dalbergiphenol > 200 > 200 Comparative Example 7 Dehydrodieugenol > 200 > 200 Comparative Example 8 6,8-Diprenylorobol 12.5 25 Comparative Example 9 Eupatilin > 200 > 200 Comparative Example 10 Falcarindiol 100 100 Comparative Example 11 Gagaminine > 200 > 200 Comparative Example 12 Galangin > 200 > 200 Comparative Example 13 Guaiacin 50 50 Comparative Example 14 Matairesinoside > 200 > 200 Comparative Example 15 Myristargenola > 200 > 200 Comparative Example 16 Oleiferin F > 200 > 200 Comparative Example 17 Sargachromanol G > 200 > 200 Comparative Example 18 Timosaponin AIII > 200 > 200

Experimental Example  3: Microbial-specific antibacterial effect test

The antimicrobial activity specific to the compound of formula (1) (acetylchiconine), the compound of formula (2) (4-hydroxyderissin), the compound of formula (3) (soporaplavanone G) and the compound of formula (4) Or other species of fungi.

That is, the therapeutic composition comprising the compound as an active ingredient has a specific activity for a specific strain, so that the broad-spectrum use is not induced and the incidence of resistance is minimized. Therefore, the growth inhibitory effect on each strain was measured.

Broth microdilution assay was used. Dilution was repeated twice in order to obtain a maximum concentration of acetylchicine and 4-hydroxydericine of 200 mg / L to at least 0.2 mg / L, and 100 μL of the solution was added to a 96-well microtitre plate . Each strain suspension (1 × 10 ⁵ CFU / ml) was added to each well and cultured at 35 ° C for 24 hours or 48 hours. The MIC values for acetylchoninin and 4-hydroxydericiin were the minimum concentrations at which the strain did not grow in each plate.

The following Tables 3 to 6 show the growth inhibitory effects of acetylcycinin, 4-hydroxyderisin, soporaplavanone G and cirrhinone on the respective strains.

division Acetylciconine MIC-24h (mg / l) MIC-48h (mg / l) Example 1 P. larvae 3.125 3.125 Example 2 M. plutonius 6.25 6.25 Comparative Example 1 A. Baumannii > 200 > 200 Comparative Example 2 B. subtilis subsp . spizizenii 50 > 200 Comparative Example 3 E. Cloacae > 200 > 200 Comparative Example 4 E. coli > 200 > 200 Comparative Example 5 E. faecalis 12.5 12.5 Comparative Example 6 E. faecium 12.5 12.5 Comparative Example 7 L. Monocytogenes 1.56 6.25 Comparative Example 8 M. luteus 3.125 3.125 Comparative Example 9 P. aeruginosa > 200 > 200 Comparative Example 10 S. aureus 25 50 Comparative Example 11 S. saprophyticus 12.5 12.5

As shown in Table 3, acetylcycinin exhibited a high antifungal activity against P. larvae and M. plutonius , but also had antimicrobial activity against L. monocytogenes and M. luteus . In addition, Activity was reduced or no activity was observed at all.

division 4-hydroxyderisin MIC-24h (mg / l) MIC-48h (mg / l) Example 1 P. larvae 6.25 12.5 Example 2 M. plutonius 3.125 6.25 Comparative Example 1 A. niger > 200 > 200 Comparative Example 2 A. Baumannii > 200 > 200 Comparative Example 3 B. subtilis subsp . spizizenii 3.125 6.25 Comparative Example 4 E. Cloacae > 200 > 200 Comparative Example 5 E. coli > 200 > 200 Comparative Example 6 E. faecalis 6.25 12.5 Comparative Example 7 E. faecium 25 25 Comparative Example 8 L. Monocytogenes 0.78 3.125 Comparative Example 9 M. luteus 3.125 3.125 Comparative Example 10 P. aeruginosa > 200 > 200 Comparative Example 11 S. aureus > 200 > 200 Comparative Example 12 S. saprophyticus 6.25 12.5

Referring to Table 4, 4-hydroxydericin was found in P. larvae and M. plutonius , B. subtilis subsp . spizizenii, E. faecalis , L. monocytogenes , M. luteus , and S. saprophyticus , but no antimicrobial activity against other strains was observed.

division Package Laplavanon G MIC-24h (mg / l) MIC-48h (mg / l) Example 1 P. larvae 3.125 6.25 Example 2 M. plutonius 3.125 3.125 Comparative Example 1 A. Baumannii > 200 > 200 Comparative Example 2 B. subtilis subsp . spizizenii 6.25 12.5 Comparative Example 3 E. Cloacae > 200 > 200 Comparative Example 4 E. coli > 200 > 200 Comparative Example 5 E. faecalis 3.125 6.25 Comparative Example 6 E. faecium 12.5 12.5 Comparative Example 7 L. Monocytogenes 0.78 1.56 Comparative Example 8 M. luteus 1.56 1.56 Comparative Example 9 P. aeruginosa > 200 > 200 Comparative Example 10 S. aureus > 200 > 200 Comparative Example 11 S. saprophyticus 12.5 12.5

As shown in Table 5, the antimicrobial activity of bovine Laplacabanone G against P. larvae , M. plutonius , E. faecalis, L. monocytogenes , and M. luteus was shown to be about Or did not appear.

division Curalinone MIC-24h (mg / l) MIC-48h (mg / l) Example 1 P. larvae 6.25 12.5 Example 2 M. plutonius 12.5 12.5 Comparative Example 1 A. Baumannii > 200 > 200 Comparative Example 2 B. subtilis subsp . spizizenii 12.5 25 Comparative Example 3 E. Cloacae > 200 > 200 Comparative Example 4 E. coli > 200 > 200 Comparative Example 5 E. faecalis 25 25 Comparative Example 6 E. faecium 25 25 Comparative Example 7 L. Monocytogenes 6.25 6.25 Comparative Example 8 M. luteus 6.25 6.25 Comparative Example 9 P. aeruginosa > 200 > 200 Comparative Example 10 S. aureus > 200 > 200 Comparative Example 11 S. saprophyticus 12.5 25

As shown in Table 6, curalinon exhibited antibacterial activity against P. larvae , M. plutonius , L. monocytogenes , and M. luteus , but did not exhibit weak or antimicrobial activity against other strains.

That is, since acetylchoninin, 4-hydroxydercin, 4-hydroxyderisin, sulfolaplavanone G and cialynon act specifically on some microorganism species and exhibit the effect of inhibiting the growth, the infectious diseases of bees, It can be used effectively for treatment, and it is considered that the concern of wide use is extremely small.

Experimental Example  4: Antibacterial effect test by combination application

(Complexes with existing antibacterial agents of the compound of formula (1) (acetylchiconine), the compound of formula (2) (4-hydroxyderizine), the compound of formula (3) (soporaplavanone G) The antimicrobial activity was evaluated by application.

Tetracycline and Ascosphaera according to mixed use of each of the above compounds The broth microdilution assay was used as a chequerboard fashion to confirm the antifungal effect on apis .

The concentration of each compound was continuously diluted 2-fold to a minimum of 0.2 mg / L at a maximum of 200 mg / L, and then added to a 96-well microtiter plate in an amount of 100 μL. Each strain suspension (1 × 10 ⁵ CFU / ml) was added to each well and cultured at 35 ° C for 24 hours or 48 hours. The MIC values for each compound were confirmed on each plate at the minimum concentration at which the strain did not grow in each plate.

The synergistic effect of the mixed use was expressed as Fractional inhibitory concentration indexes (FICIs) using the formula FICI = (Ac / Aa) + (Bc / Ba) (Drogari-Apiranthitou et al., 2012).

Ac and Bc are the MIC values of A and B substances, respectively, and Aa and Ba are the MIC values of A and B substances, respectively, when used alone. If the FICI value is 0.5 or less, the synergistic effect is evaluated. If the FICI value is 0.5 to 4, the synergistic effect is not evaluated.

Table 7 shows the inhibition of growth of Paenibacilus larvae after treating acetylchoninin, 4-hydroxyderisin, soporaplavanone G and cialynone with tetracycline simultaneously.

Referring to Table 7, although the synergistic effect of the combined use of acetylchiconine and tetracycline was not significant, the use of acetylcycinine and tetracycline in combination resulted in synergistic effects for each use, thereby reducing the amount of each compound used.

The combined use of 4-hydroxyderisin and tetracycline showed more synergistic effects and 4-hydroxydercin, which has excellent antimicrobial effect, could increase the antimicrobial effect of tetracycline and significantly reduce the amount required for antibacterial.

division FICI Acetylchiconine + tetracycline 0.75 + - 0.12 4-hydroxyderisin + tetracycline 0.625 + - 0.10 Paraffin Laplavanone G + tetracycline 0.83 0.10 Cialynone + tetracycline 0.88 0.10

Table 8 below shows the inhibition of growth of Paenibacilus larvae after treatment with acetylchonin, 4-hydroxyderisin, soporaplavanone G and cialynone simultaneously with Anwulignan.

division FICI Acetylchiconine + angu lignan 0.83 0.12 4-hydroxyderisin + angu lignan 0.88 0.10 Package Lapelavonon G + Anou Lignan 0.625 + - 0.12 Cura Linnon + Angu Lignan 0.75 + - 0.25

Referring to Table 8, a synergistic effect of the combined use of tetracycline and the microparticles of parapraflavanone G and cialin was confirmed. When 0.78 ㎎ / ℓ of bovine Laplavanone G was used, the growth of Paenibacilus larvae was effectively inhibited even when only 0.39 ㎎ / ℓ of Angus lignans was used. In other words, the use of parapolaplavanone G in combination with angular lignan can be significantly reduced.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (5)

A composition for preventing and treating a disease caused by a disease caused by a disease comprising one or more of the following compounds (1) to (4) as an active ingredient.
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

The method according to claim 1,
A composition having an antimicrobial activity against causative bacteria of a buzzer disease. 3. The method of claim 2,
Wherein the causative agent is Paenibacilus larvae or Melissococcus plutonius . The method according to claim 1,
At least one antimicrobial agent or an antifungal agent. 5. The method of claim 4,
Wherein said antimicrobial or antifungal agent is selected from the group consisting of amikacin, gentamycin, tobramycin, streptomycin, nethylamycin, kanamycin, ciprofloxacin, norfloxacin, oproxacin, trovafloxacin, lomeproxacin, levofloxacin, , Sulfonamides, polymyxin, chloramphenicol, neomycin, paramomomycin, colistimetate, bacitracin, vancomycin, tetracycline, rifampin, cycloserine, beta-lactam, cephalosporin, amphotericin, But are not limited to, fluconazole, flucytosine, natamycin, myconazole, ketoconazole, corticosteroids, diclofenac, fluulbifropen, ketorolac, suiprofen, codolin, rosoxamide, levocabastine, Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;

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