KR101680791B1

KR101680791B1 - Composition for treatment of honey bee diseases

Info

Publication number: KR101680791B1
Application number: KR1020150073588A
Authority: KR
Inventors: 김기영; 신유경
Original assignee: 경희대학교 산학협력단
Priority date: 2015-05-27
Filing date: 2015-05-27
Publication date: 2016-11-29

Abstract

The present invention relates to a composition for treating infectious diseases of honey bees and, more specifically, to a composition for treating infectious diseases of honey bees which comprises at least one compound derived from a plant extract and is safe to human body.

Description

Technical Field [0001] The present invention relates to a composition for treating an infectious disease of honey bee,

The present invention relates to a composition for treating an infectious disease of bees, and more particularly, to a composition for treating an infectious disease of bees, which comprises not only a single compound derived from a plant extract but also has excellent biocompatibility and specifically acts only on specific causative microorganisms causing diseases on bees, The present invention relates to a composition for treating an infectious disease of a honeybee having a minimized possibility of occurrence.

The domestic bee production market is about 350 billion won, and the multi-function of bees' ecosystem such as pollen of crops is very difficult to quantify. In Korea, the economic value of bees accounted for more than 50% of the total fruit and vegetable production of 12.5 trillion won, and the total ecosystem preservation effect was reported to exceed 50 trillion won.

Despite the economic and ecological importance of honey bees, there is no active research on the honey bees, and there is no antimicrobial or antifungal agent currently on commercial honey bee disease. However, propionic acid has been used as a fumigant to inhibit the growth of fungi that cause disease to bees, and tetracycline or fluoride, which is applied to humans, is used to inhibit the growth of bacteria causing disease to bees do.

There are 15 kinds of bee diseases that have been confirmed in Korea, and there are 15 kinds of diseases such as American beef bottle (AFB), European buzzer bottle (EFB), Chalkbrood, Nosema Disease, Sacbrood, Keratinization virus, keratinization virus, keratinization virus, keratinization virus, keratinization keratinization virus, keratinization keratinization virus, keratinization keratinization virus, keratinization keratinization virus, keratinization keratinization virus, virus, and Kakugo virus. Especially, the number of bees by bee sickness and chalk disease caused by bacteria or fungi is very serious.

In particular, American foulbrood (AFB) is known to be the most virulent among honey bee diseases, and control is very difficult. The causative organism of the American buzzers is Paenibacillus larvae , which is a bacterium and forms a spore. The bee larva is known as the only host of the pathogen, and the vegetative form or spore form of the liver can be observed by a microscope.

The American buzzers are spread from one larva to another, from one to another, from one ape to another ape. It can also be transmitted by contaminated pollen purchased from outside sources, consumption and stunting, and can be transmitted by the infected body of adult adults gathered from the same bunch.

The apoa germinates on the healthy larvae together with the contaminated food, and propagates and rotates the larva. Gradually migrate to other larvae or other populations, or gradually larger areas, by honey or honey from a beehive, such as a worker's bee, or from a beehive hanging from a buzzer bottle to remove this decayed larva. In other words, it is propagated by the use and exchange of polluted sources (flowers), contaminated bee appliances, contaminated consumption, and propagated by the application of the diseased beehive.

On the other hand, European Buzzers disease is a disease caused by melissococcus pluton as a disease which is light on the sickness compared to American buzzers, and causes harm to cones and other hybrid bees, especially weak bee. European buzzer disease is spread by worker bees and docking, and other propagation methods are similar to American buzzer disease. The larvae infected with European buzzer have yellow spots near the head and as the disease progresses, the entire body turns yellow, grayish brown, dark brown and black and decays. European buzzer disease is prone to develop in weak populations, so it is desirable to strengthen the populations' power by feeding rich and high quality honey and pollen. Also, although antibiotic use is a common method of control, resistance may occur, and antibiotics remaining in beekeeping products can affect the human body.

In addition, the bee's chalk disease is a disease that kills bees by transforming the larva into a mummified form and then transforming it into a hard chalk form, first reported by Massen in Germany in 1913, in New Zealand in 1957, (Gillam, Honey bee science, 1, 159-162 (1980)) has been reported to occur in almost all states in California, USA, and in 1971 in Canada. Also, in Japan in 1974, imported beekeepers from both the Akihabara and Canadian provinces, and in 1979, chalky disease was found in Chikugo, one of the worst bee diseases spread worldwide.

The pathogen causing the chalk disease was named Ascosphaera apis in 1955 by Spiltoir et al., And was infected only with bee larvae. The sporangia were resistant to the environment and maintained pathogenic for 15 years (Bailey, Honeybee pathology, 124, Academic press. London, (1981)).

The chalk disease is caused by ingestion of polluted pollen from infected beehives (Mehr et al, Amer. Bee J., 116, 266-268 (1976)), requeening of queen bees in infected beehives (Dejong & Morse, Chalkbrood, New (Herbert et al., J. Apic. Res., 16, 204-208 (1977)), the movement of queen bees, work bees and the like, )). In Korea, it has rapidly increased since the mid 1980s, and is one of the biggest bee diseases now.

The most effective anti-chalk disease control method that has been found so far is incineration of infected beehive, but since the economic loss is too large, studies on anti-cholesterol anti-aging methods have recently been actively pursued.

Considering the domestic situation in which a large number of bees are imported from abroad, it is the best national preventive measure to prevent the infestation of outbreak pathogens through strict quarantine and to suppress domestic inventions. There are no remedies available to control causative organisms that cause disease in Korea, and they depend entirely on imports.

Nonetheless, the well-known antimicrobial or antifungal agents have been developed to inhibit the growth of fungi or bacteria directed against humans or other mammals, and no development of antibiotics specific for bees has been made.

In addition, the use of excessive antibiotics causes the generation of resistant strains, and it is preferable that the use of the antibiotics is minimized because it makes it more difficult to control the spread of the disease in the future. Also, It is urgent to develop a therapeutic composition which acts specifically on diseases of bees.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems of the prior art, and it is an object of the present invention to provide an environmentally friendly composition for treating infectious diseases of bees derived from plant extracts.

It is another object of the present invention to provide a composition for treating infectious diseases which specifically acts only on a specific strain causing bee disease so that side effects and harmfulness to a living body can be minimized.

According to one aspect of the present invention, there is provided a composition for treating an infectious disease of a bee comprising a compound of the following formula (1), a compound of the following formula (2), or a mixture thereof as an active ingredient.

[Chemical Formula 1]

(2)

According to one embodiment, the infectious disease can be a buzzer bottle or a gypsum bottle.

According to one embodiment, it may have antimicrobial activity against causative bacteria of the buzzer bottle or gypsum bottle.

According to one embodiment, the causative bacteria may be Paenibacillus larvae , Melissococcus pluton , or Ascosphaera apis .

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

According to one embodiment, the antimicrobial or antifungal agent is selected from the group consisting of amikacin, gentamicin, tobramycin, streptomycin, nethylamycin, kanamycin, ciprofloxacin, norfloxacin, oproxacin, trovafloxacin, , 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 honeycomb composition for treating infectious diseases of bees contains a single compound derived from a plant extract as an active ingredient, and thus has low toxicity to the body and is excellent in antibacterial or antifungal effect against causative microorganisms, And can be usefully used for treating diseases.

In addition, since the composition for treating infectious diseases of bees exhibits activity only for specific causative microorganisms causing infectious diseases of bees, the total amount of the bees to be used is reduced, so that the incidence of tolerance can be reduced and side effects can be minimized.

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 according to concentration-dependent treatment of anwulignan according to an embodiment of the present invention.
FIG. 2 is a result of evaluating cytotoxicity according to treatment of corosolic acid concentration according to an embodiment of the present invention.
FIG. 3 is a result of evaluating cytotoxicity according to the concentration treatment of myconazole according to the comparative example.

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.

[Chemical Formula 1]

(2)

The therapeutic composition may contain, as an active ingredient, the chemical formula 1 and the chemical formula 2 compound.

The compound of formula (1) is referred to as macelignan or anwulignan and may be 4 - [(2S, 3R) -4- (1,3-benzodioxol- -Dimethylbutyl] -2-methoxyphenol (4 - [(2S, 3R) -4- (1,3-Benzodioxol-5-yl) -2,3-dimethylbutyl] -2-methoxyphenol .

The compound of Formula 1 may be isolated from the Schizandra chinensis extract. The Schizandra chinensis Baillon ( Schizandra chinensis Baillon ) is a medicinal plant which is used as a raw material for medicinal herbs and foodstuffs, such as deciduous wood belonging to the Schizandraceae family. The Schisandra chinensis is Araliaceae and 2 in three paper distributed in Korea, Schisandra hawthorn (Schizandrae) has mainly the Schisandra (S. chinensis) and fruit exhibiting black used as a medicinal is a black Omija tree (S. nigra).

The Schizandra chinensis fruit contains a large amount of lignan compounds such as schizandrin, gomisin-A and gomisin-N as main active ingredients and also contains oil, essential oil, Pigment and so on, it is widely used as a raw material for medicines and food. The above-mentioned Omija has various pharmacological functions including inhibition of hepatotoxicity. Recently, pharmacological effects such as hypoglycemic action, anti-ulcer action, chronic hepatitis treatment effect and central nervous stimulation action have been reported.

According to one embodiment, the Schizosaccharomyces pomaceus 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.

According to one embodiment, the compound of formula 1 contained in the Omiza extract may be isolated by further chromatography, such as silica gel column chromatography, and various known methodologies for separating a single compound may be applied .

On the other hand, the compound of Chemical Formula 2 is referred to as corosolic acid, and is also referred to as Glucosol, 2? -Hydroxyursolic acid. The above corosolic acid can be separated from the plant of Barbaa, a plant of Bacillus subtilis, a leaf of Vipa which is a plant of Rosaceae, and a flesh of a hawthorn tree. The plant is a medicinal plant widely used in the prevention and treatment of diabetes in Southeast Asia and the like, It has been commercialized in the form of tea and its stability has been confirmed.

Plants for isolating corosolic acid may include all known plant species known to produce or contain corosolic acid, callus derived therefrom, plant cells, and plant cell suspension cultures. For example, the plants may be selected from the group consisting of Lagerstroemia speciosa , Eribotrya japonica , Ternstroemia gymnanthera , Crataegus pinnatifida , Tiarella polyphylla , but is not limited thereto, and preferably it may be baranaval. The plant may be used in the whole plant, leaf, flesh, stem, root, flower, preferably leaf part.

According to one embodiment, the barnabas may be concentrated under reduced pressure after hot extraction with a lower alcohol solvent such as methanol or ethanol or a mixed solvent thereof. To more efficiently extract corosolic acid, the barnabas can be milled to 100-200 mesh by a pin mill or a ball mill mill, and can be hydrolyzed three times with 50 to 99% lower alcohol have.

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 hydrophobic or hydrophobic impurities as well as corosolic acid. Therefore, the water-soluble layer may be degreased with a non-polar organic solvent to remove hydrophobic impurities of the barbara, .

In addition, the compound of formula (2) contained in the banaba 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 may be performed by a conventional method well known in the art, and the nonionic resin may be amberlite XAD-2.

The isolated compound of formula (1) and the compound of formula (2) have excellent activity against a specific bacterium or fungus, and thus can be utilized in the treatment of infectious diseases of bees.

According to one embodiment, the infectious disease may be a buzzer bottle or a gypsum bottle, and the composition for treating an infectious disease of a bee may have an antibacterial activity against causative bacteria of a buzzer bottle or a gypsum bottle.

According to one embodiment, the causative bacterium may be Paenibacillus larvae , Melissococcus pluton , or Ascosphaera apis , the composition being specific for the causal organism And may have antimicrobial activity.

That is, the composition for treating an infectious disease of bees has a specific antimicrobial activity against a specific strain, so that it can not be widely used, the incidence of resistance can be minimized, and the biosafety is excellent.

In addition, the composition for treating an infectious disease of bees may further include one or more antimicrobial agents or antifungal agents.

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: Assessment of cytotoxicity

In order to evaluate the cytotoxicity of the compound of formula (1) (anwulignan) and the compound of formula (2) (corosolic acid), it was determined whether each compound inhibited cell growth. In order to confirm the inhibitory effect of the compounds, 10 mg / L of anwulignan and corosolic acid were treated in HepG2 cell line and MTT assay was performed.

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 0.5%. After 24 hours, 100 μl of 5 mg / ml MTT (Sigma Cat. M2128) was added and the activity was measured at 540 nm using an ELISA reader (VersaMax, Molecular Devises, USA). All experiments were repeated three or more times.

FIGS. 1 and 2 are the results of evaluating cytotoxicity according to the treatment of anwulignan and corosolic acid according to an embodiment of the present invention. FIG. 3 is a graph showing cytotoxicity of cells treated with concentrations of myconazole (Miconazole) It is the result of evaluation of toxicity.

As shown in Fig. 1, although anwulignan was treated at a high concentration (10 mg / l), cell growth of 76.6 ± 10.1% was observed compared to the case without anwulignan treatment. That is, anwulignan is low in toxicity to living organisms even when used at high concentration, and it is analyzed that there is almost no toxicity to living organisms when it is used as usual concentration of use.

As shown in FIG. 2, when corosolic acid was treated at a high concentration (10 mg / L), 53.6 ± 8.0% cell growth was observed compared with the case without corosolic acid treatment. The corosolic acid partially inhibited the growth of cells when treated at high concentrations, but the toxicity to the living organisms would be negligible if used at a conventional concentration as an antimicrobial agent.

On the other hand, as shown in FIG. 3, when miconazole, widely used as an antifungal agent, was treated with a high concentration (10 mg / L), cell growth was inhibited by 70% or more and treated with a low concentration (1 mg / It was analyzed that cell growth was inhibited by about 50% and cytotoxicity was high.

That is, the compounds (anwulignan) and (2) (corosolic acid) of the above formula (1) are naturally derived compounds, and even if they are used as antimicrobial agents or antifungal agents, they are low in cytotoxicity and are safe and environmentally friendly because they are low in harmfulness to humans or bees.

Experimental Example 2: Confirmation of antimicrobial activity

The antimicrobial activity of the compound of formula 1 (anwulignan) and the compound of formula 2 (corosolic acid) Paenibacillus larvae , Melissococcus pluton , Ascosphaera apis Respectively.

Ascosphaera To evaluate the antimicrobial activity against apis , we applied the broth micodilution assay proposed by EUCAST. The natural substance was adjusted to a concentration of 10 mg / ml by DMSO (Dimethyl Sulfoxide). For the natural substance, RPMI 1640 (without sodium bicarbonate and with l-glutamine) was adjusted to pH 7.0 using 0.165 M morpholinopropanesulfonic acid, and diluted 2-fold with 2% glucose solution in 96-well -bottomed microtitration plates. At this time, the natural substance used is at most 0.2 mg / l at a maximum of 200 mg / l. The last well was used as a control with only strains. 100 μl of the suspension (0.5 × 10 ⁵ to 2.5 × 10 ⁵ CFU / ml) was added to each well and cultured at 35 ° C for 24 hours or 48 hours. The MIC value was the minimum concentration of the natural product inhibiting the growth of the strain (Cuenca-Estrella M et al., 2002).

On the other hand, bacteria of the MIC value (Paenibacillus larvae, Melissococcus Pluto) was evaluated in some variations the method proposed in EUCAST (Cuenca-Estrella M et al ., 2002, Wiegand et al., 2008). The natural substance was adjusted to a concentration of 10 mg / ml by DMSO (Dimethyl Sulfoxide). Natural materials were diluted 2-fold in succession with appropriate medium for each strain and added to 96-well round-bottomed microtitration plates in 100 μl each. At this time, the natural substance used is at most 0.2 mg / l at a maximum of 200 mg / l. The last well was used as a control with only strains. 100 μl of the suspension (1 × 10 ⁵ to 5 × 10 ⁵ CFU / ml) was added to each well and cultured at 35 ° C for 24 hours or 48 hours. The MIC value was the minimum concentration of the natural product inhibiting the growth of the strain.

Table 1 below compares the antimicrobial activity of anwulignan and corosolic acid and the antimicrobial activity of widely used antimicrobial agents according to the above experiment.

As shown in Table 1, the MIC values of anwulignan were measured, and the results showed that the growth inhibitory effect of Asposphaera apis, Paenibacillus larvae, and Melissococcus pluton was remarkable.

In addition, corosolic acid showed relatively weak antimicrobial activity compared to myconazole , which is widely used, but significantly inhibited the growth of Ascosphaera apis , Paenibacillus larvae, and Melissococcus pluton .

On the other hand, the antimicrobial agents of Comparative Examples 3 to 7, which were used as a control group, exhibited some growth inhibitory activities against the microorganisms, but their effects were weak or no antimicrobial activity was exhibited at all.

That is, the anwulignan and corosolic acid according to the present invention have excellent antimicrobial activity against Ascosphaera apis , Paenibacillus larvae and Melissococcus pluton which cause disease to bees, and their activity is equal or remarkably superior to that of existing antimicrobial agents .

division Ascosphaera apis Paenibacillus larvae Melissococcus pluton MIC-24h
(Mg / l) MIC-48h
(Mg / l) MIC-24h
(Mg / l) MIC-48h
(Mg / l) MIC-24h
(Mg / l) MIC-48h
(Mg / l) Example 1
(anwulignan) 1.56 3.13 3.13 3.13 3.13 3.13 Example 2
(corosolic acid) 12.5 12.5 3.13 200 3.13 3.13 Comparative Example 1
(Miconazole) 1.56 1.56 1.56 3.13 3.13 3.13 Comparative Example 2
(Tetracycline) N.D. N.D. 1.56 1.56 0.78 0.78 Comparative Example 3
(Loganic acid) 100 > 200 > 200 > 200 > 200 > 200 Comparative Example 4
(Tracheloside) 100 > 200 > 200 > 200 > 200 > 200 Comparative Example 5
(Fangchinoline) > 200 > 200 50 > 200 > 200 > 200 Comparative Example 6
(Dehydrocostus lactone 50 50 50 50 > 200 > 200 Comparative Example 7
(Emodin-8-O- [beta] -D-glucopyranoside) > 200 > 200 > 200 > 200 > 200 > 200

N.D: Not determined

Experimental Example 3: Identification of microbial-specific antimicrobial effect

The anwulignan and the corosolic acid of the above formula (1) can be used for the selective antibacterial activity against Paenibacillus larvae , Melissococcus pluton , Ascosphaera apis , Activity.

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.

Table 2 shows the growth inhibitory effect of each of the strains of anwulignan. Table 3 shows the growth inhibitory effects of each strains of corosolic acid.

division Anwulignan MIC-24h (mg / l) MIC-48h (mg / l) Example A. apis 1.56 3.13 P. larvae 3.13 3.13 M. plutonius 3.13 3.13 Comparative Example A. niger > 200 > 200 A. clavatus > 200 > 200 C. albicans > 200 > 200 C. albicans > 200 > 200 C. parapsilosis there is. parapsilosis > 200 > 200 C. tropicalis > 200 > 200 C. tropocalis there is. tropicalis > 200 > 200 C. glabrata > 200 > 200 F. neoformans there is. bacillispora 6.25 6.25 P. guilliermondii 3.13 200 R. oryzae > 200 > 200 S. cerevisiae > 200 > 200 S. cerevisiae > 200 > 200 B. subtilis subsp . spizizenii 1.56 50 E. faecalis > 200 > 200 S. aureus > 200 > 200 S. saprophyticus 6.25 > 200

As shown in Table 2, anwulignan showed antimicrobial activity against Ascosphaera apis , Paenibacillus larvae, and Melissococcus pluton , but there was little or no antimicrobial activity against other strains.

division Corosolic Acid MIC-24h (mg / l) MIC-48h (mg / l) Example A. apis 12.5 12.5 P. larvae 3.13 200 M. plutonius 3.13 3.13 Comparative Example A. niger > 200 > 200 A. clavatus > 200 > 200 C. albicans > 200 > 200 C. albicans > 200 > 200 C. parapsilosis there is. parapsilosis > 200 > 200 C. tropicalis > 200 > 200 C. tropocalis there is. tropicalis > 200 > 200 C. glabrata > 200 > 200 F. neoformans there is. bacillispora 3.13 > 200 P. guilliermondii > 200 > 200 R. oryzae > 200 > 200 S. cerevisiae > 200 > 200 S. cerevisiae > 200 > 200 B. subtilis subsp . spizizenii 6.25 200 E. faecalis > 200 > 200 S. aureus > 200 > 200 S. saprophyticus 12.5 50

As shown in Table 3, corosolic acid showed antimicrobial activity against Ascosphaera apis , Paenibacillus larvae, and Melissococcus pluton , but did not exhibit antibacterial activity against other strains.

That is, since anwulignan and corosolic acid act specifically on some microorganisms and exhibit growth inhibitory effects, they can be effectively used for the treatment of infectious diseases of bees, especially, buzzer diseases and gypsum diseases, It is considered to be small.

Experimental Example 4: Confirmation of antimicrobial effect by combination application

The antimicrobial activity of the compound (anwulignan) and the compound of the formula (2) (corosolic acid) according to the combined application with the existing antimicrobial agent was evaluated.

A broth microdilution assay was used as a chequerboard fashion to determine the antifungal effect of A. apis on the combined use of myconazole and Anwulignan. Myconezol was adjusted to pH 7.0 with 0.165 M morpholinopropanesulfonic acid (RPMI 1640, without sodium bicarbonate and with 1-glutamine) and diluted 2-fold with medium containing 2% glucose (or YPD media). 100 μl of the A. apis suspension (2 × 10 ⁵ to 5 × 10 ⁵ CFU / ml) was added to each well. The dilution of Anwulignan in the direction of 90 ° was 2-fold diluted and added to a 96-well microtiter plate And then cultured at 35 ° C for 24 hours or 48 hours. Myconazole was reduced to a minimum of 0.2 mg / l at a maximum of 200 mg / l, and at least 0.2 mg / l at a maximum of 25 mg / l of anwulignan. The MIC values for each material were confirmed again on each plate.

On the other hand, MYPGP broth was used to investigate the antifungal effects of P. larvae on the use of tetracycline and anwulignan. Tetracycline was reduced to a minimum of 0.1 mg / l at a maximum of 100 mg / l, and at least 0.2 mg / l at a maximum of 25 mg / l of anwulignan. The MIC values for each material were confirmed again on each plate.

To investigate the antifungal effect of M. plutonius on the use of tetracycline and corosolic acid, KBHI broth was used for the susceptibility test under 5% CO ₂ condition. Tetracycline was reduced to a minimum of 0.1 mg / l at a maximum of 100 mg / l and at least 0.2 mg / l at a maximum of 25 mg / l of corosolic acid. The MIC values for each material were confirmed again on 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 4 shows that anwulignan and corosolic acid were added simultaneously with either myconazole or tetracycline, and the growth inhibition was evaluated.

division Target strain FICI-24h (mg / l) FICI-48h (mg / l) Anwulignan + Miconazole A. apis 0.75 + 0.15 0.78 + 0.14 Anwulignan + Tetracycline P. larvae 0.83 + 0.14 0.83 + 0.14 Corosolic acid + Tetracycline M. plutonius 0.29 + 0.07 0.46 + 0.07

As shown in Table 4, the synergistic effects of Anwulignan and Myconazole were not remarkable in combination. However, Anwulignan 0.78 ㎎ / ℓ was used as the only be used Miconazole 0.78 ㎎ / ℓ A. apis . That is, myconezol can be used in combination with Anwulignan to reduce its usage.

In addition, the synergistic effect with the combined use of Anwulignan and tetracycline was not remarkable. However, when 3.13 ㎎ / ℓ of Anwulignan was used, the growth of P. larvae was inhibited even when 0.39 ㎎ / ℓ of tetracycline was used. When 1.58 ㎎ / ℓ of Anwulignan was used, only 0.78 ㎎ / ℓ of tetracycline was used for P. larvae . In other words, tetracycline can be used in combination with Anwulignan to reduce its usage.

On the other hand, Corosolic acid showed a synergistic effect when combined with tetracycline (FICI value = 0.29 ± 0.07, 0.46 ± 0.07). Corosolic acid 1.56 ㎎ / ℓ was used as the tetra-cyclin were used only 0.05 ㎎ / ℓ also completely inhibited the growth of M. plutonius, Corosolic acid 0.39 when using ㎎ / ℓ tetracycline 0.39 ㎎ / ℓ only with M. Fig. and inhibited the growth of plutonius . In other words, corosolic acid can be used in combination with tetracycline, which can significantly reduce usage.

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

A pharmaceutical composition for the treatment of a buzzer or gypsum comprising a compound of the following formula (1), a compound of the following formula (2) or a mixture thereof as an active ingredient.
[Chemical Formula 1]

(2)

delete

The method according to claim 1,
And has antimicrobial activity against causative bacteria of the buzzer bottle or gypsum bottle.

The method of claim 3,
Wherein said causative bacteria are Paenibacillus larvae , Melissococcus pluton , or Ascosphaera apis .

The method according to claim 1,
0.0 > antifungal < / RTI > agent or an antifungal agent.

6. The method of claim 5,
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 > alone.