KR102120621B1 - A composition for preventing or treating a Nosema disease comprising an Lespedeza cuneata extract as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing or treating Nosema diseases containing a Lespedeza cuneata extract as an active component, and a composition having antibacterial activity on Nosema apis, causative bacteria of the infectious disease. The composition of the present invention has an excellent antibacterial effect on causative bacteria of Nosema diseases, thereby being able to be usefully used as a therapeutic agent or a functional feed composition for infectious diseases of insects.

Description

A composition for the prevention or treatment of Nozema disease, which includes the non-repair extract as an active ingredient. {A composition for preventing or treating a Nosema disease comprising an Lespedeza cuneata extract as an active ingredient}

The present invention relates to a composition for the prophylaxis or treatment of Nozema disease, including a non-repair extract, having an inhibitory effect on Nosema apis , which is known as a pathogen causing Nozema disease, an infectious disease for bees and the like.

In the ecosystem, bees are indispensable. In particular, bees play a large role in the pollination process of plants. However, the number of bees is steadily decreasing due to the pathogens of bees, and many problems are caused by this.

Include Jose causing representative Jose horsemen to include pathogens that cause infectious diseases in bees do Apis (Nosema apis), or Fannie Bacillus rabae (Paenibacillus larvae), Asda course Blow Apis (Ascosphaera apis) causing chalk disease causing US buzzer bottle .

Nosema apis ( Nosema apis ) is one of the spores infected with honey bee adults, and the disease caused by this, Nosema apis ( Nosema apis ) by the Nosema apis ( Nosema apis ) is infected by the digestive organs and appendages of the bee adult, parasitic and onset It is one of the diseases. The spores of Nosema apis can survive for many years in the external environment and multiply in the body of bees. The influx of pathogens is achieved through the feeding process of bees, and spores germinate in the intestine. After 4-5 days of germination, the cloned spores come out of the cells and germinate again in the surrounding cells.

Although many efforts have been made by researchers to control old disease, control by fumagillin, an antibiotic produced by mold in Aspergillus, and fumigation of ethylene oxide are typically used. However, fumagillin (fumagillin) control technology has been reported not to be heard in some cases, it is also reported that the effect may not be good for the old disease found in Korea. However, it is currently known to be the only substance that can be used to control the disease. Therefore, many researchers have made many attempts to find an active ingredient that inhibits the infection of Nozema , and the researchers used the plant extracts to prevent the infection of Nosema apis , which causes Nozema disease. Excavation of inhibitory substances was performed to confirm the effectiveness of the non-repair extract.

The present invention, as described above, in order to solve the problem of infection and spread of Nojema apis ( Nosema apis ), a causative agent that causes Nozema disease in bees, can effectively suppress and control infection of Nozema apis It aims to provide a substance.

That is, an object of the present invention is to provide a composition for preventing or treating Nozema disease, which includes an unrepaired extract as an active ingredient.

In addition, another object of the present invention is to provide a feed composition comprising Nojema disease inhibitory activity comprising a non-repair extract as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a composition for the prevention or treatment of Nozema disease comprising an unrepaired extract as an active ingredient. The non-repair extract of the present invention is Nosema apis , a pathogen that infects Nosema disease . It is characterized by having antibacterial activity against.

In addition, in order to achieve another object of the present invention, the present invention provides a feed composition comprising an anti-aging agent inhibiting activity comprising an unrepaired extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

Lespedeza cuneata (Chinese bushclover) is a perennial plant of legumes and is commonly used as a raw material for food or feed. It contains a large amount of flavonoids such as quercetin, camperol, and vitexin, and is known to have various effects such as antioxidant, hypoglycemic, and anti-inflammatory properties. Bisuri is a pure Korean word, mainly distributed in Asian countries such as Korea, Japan, and Taiwan, and Australia. It grows from about 50cm to 1m and has a bitter taste. It is more widely called by the name of Yagwanmun.

The "extract" defined in the present invention may be used as an extract obtained by extraction and separation from nature using extraction and separation methods known in the art, and the'extract' defined in the present invention uses an appropriate solvent. It is extracted from the non-repair, for example, includes all of the non-repair crude extract, a polar solvent soluble extract or a non-polar solvent soluble extract.

The non-repair extract of the present invention may use all non-repair leaves, stems, fruits, roots, and non-repair outposts, and water or an organic solvent may be used as the extraction solvent. Without being limited thereto, for example, alcohol having 1 to 4 carbon atoms, acetone (acetone), including purified water, methanol, ethanol, propanol, isopropanol, butanol, and the like ), ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane, or various solvents alone or It can be used in combination, preferably, but can be used a method of extraction with hot water, but is not limited thereto.

As the extraction method, any one of methods such as hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, water vapor distillation, ultrasonic extraction, elution, and compression can be used. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method. There is no limitation in the method for producing the non-repair extract of the present invention, and any known method can be used.

For example, the non-aqueous extract contained in the composition of the present invention may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying of the primary extract extracted by the above hot water extraction or solvent extraction method. . In addition, the primary extract can be obtained by further fractionation using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquidchromatography, etc. It might be.

Therefore, in the present invention, the non-repair extract is a concept including all of the extracts, fractions and refinements, their diluents, concentrates or dried products obtained at each stage of extraction, fractionation or purification.

The non-repair extract may be included in the composition of the present invention in an appropriate concentration for controlling Nozema disease, but is not limited thereto, and may be included in a concentration of 1.0 to 300 μg/mL, more preferably 3.0 to 250 μg/mL, More preferably, it may be a concentration of 6.0 to 200 μg/mL, but is not limited thereto. In one embodiment of the present invention, the antibacterial activity of the non-repair extract was confirmed at a concentration of 6.25 to 200 μg/mL. That is, the non-repair extract of the present invention showed little anti-age control effect at a concentration of 1.0 μg/mL or less, and at a concentration of 300 μg/mL or more, the size of the cell itself in the cell became small, and the number decreased, such as cytotoxicity. It was observed to have.

Nose horse disease is an infectious disease mainly occurring in bees, but it can also occur in insects such as Lepidoptera Trichoplusia ni (silkworm).

In addition, the composition for preventing or treating Nozema disease of the present invention is characterized by having antimicrobial activity against Nosema apis , the causative agent for Nozema disease , and in particular possesses specific antibacterial activity against the strain. , Widespread use is not induced, the incidence of resistance can be minimized, and has excellent bio-stability characteristics.

The composition for preventing or treating Nozema disease comprising the non-repair extract of the present invention may be used alone, or may be used in combination with a known antimicrobial active material or composition.

Although not limited thereto, the composition having antibacterial activity may further include one or more antibacterial or antifungal agents to further maximize antibacterial activity against Nosema apis , and the antibacterial or antifungal agent may include tetracycline, Oxytetracycline, amikacin, gentamicin, tobramycin, streptomycin, netylmycin, kanamycin, ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, romefloxacin, levofloxacin, enoxacin, naphthyridine, Sulfonamide, polymyxin, chloramphenicol, neomycin, paramomycin, colistimetate, bacitracin, vancomycin, rifampin, cycloserine, beta-lactam, cephalosporin, amphotericin, fluconazole, flucytosine Groups containing only, natamycin, myconazole, ketoconazole, corticosteroid, diclofenac, flurbiprofen, ketorolac, suprofen, comoline, rodoxamide, levocarbastin, napazoling, antazoline and phenirami It may be one or more selected from.

The present invention relates to a composition for preventing or treating Nozema disease comprising an unrepaired extract as an active ingredient and a composition having antibacterial activity against Nosema apis , the causative agent of the infectious disease, wherein the composition of the present invention is Nozema disease Since it has excellent antibacterial effect on the causative agent, it can be usefully used as a therapeutic agent or a functional feed composition for infectious diseases of insects.

1 is a Trichoplusia without treatment with Nojema apis and non-repair extracts This is a 400-fold micrograph taken by incubating ni cells for 5 days.
Figure 2 is Trichoplusia infected with Nojema Apis This is a 400-fold micrograph taken by incubating ni cells for 5 days.
Figure 3 is a Trichoplusia treated with Nojema Apis and non-repair extracts This is a 400-fold micrograph taken by incubating ni cells for 5 days.

Hereinafter, the present invention will be described in more detail by examples. It will be apparent to those skilled in the art that these examples are merely for more specifically describing the present invention, and the scope of the present invention is not limited to these examples.

The terms used in the present specification have selected general terms that are currently widely used as possible while considering functions in the present invention, but this may be changed according to the intention or precedent of a person skilled in the art or the appearance of new technologies. In addition, in certain cases, some terms are arbitrarily selected by the applicant, and in this case, their meanings will be described in detail in the description of the applicable invention. Therefore, the terms used in the present invention should be defined based on the meanings of the terms and the contents of the present invention, not simply the names of the terms.

Numeric ranges are inclusive of the numerical values defined in the above range. All maximum numerical limits given throughout this specification include all lower numerical limits as if the lower numerical limits were clearly written. All minimum numerical limits given throughout this specification include all higher numerical limits as higher numerical limits are clearly written. All numerical limits given throughout this specification will include all better numerical ranges within a broader numerical range, as narrower numerical limits are clearly written. The headings provided herein are by way of reference to the specification in various respects or entirely, and should not be construed as limiting the following embodiments.

<Example 1> Preparation of non-repair extract

The non-repair extract of the present invention was used by extracting the non-repair collected from Jeju Island. After crushing the dried unrepaired 1 kg, hot water was extracted with 10 L of 80°C distilled water for 12 hours. The extract was filtered with 8 μm filter paper. After the final filtering, the extracted solution was concentrated with a vacuum rotary evaporator, and then powdered with a freeze dryer, confirming a yield of 6.15%. The powdered sample was dissolved in 100% ethanol and used in the experiment.

<Example 2> Nozema Apis ( Nosema apis )Preparations

Nosema Apis used in this experimental example apis ) was obtained from the Rural Development Administration after 14 days of bee venom disease.

In order to obtain purified Nozema apis, the intestines of bee adults with Nozema disease were extracted and mixed using RIPA buffer. The mixture was mixed using a vortex mixer for 5 minutes so that it could break sufficiently. Thereafter, centrifugation was performed at 400 rpm for 5 minutes and the supernatant was discarded to remove large impurities. The obtained Nozema Apis cells were mixed in 500 μL of distilled water. To remove the bacteria mixed together, 25%, 50%, 75%, and 90% of Perze (Sigma), which was discontinuously accumulated, was placed on a solution of Nozema Apis and centrifuged at 15,000g for 30 minutes. Only the Nozema Apis layer at the bottom was separated and used.

<Example 3> Nozema Apis ( Nosema apis ) Infected cells ( Trichoplusia ni ) Preparations

Trichoplusia , known as an insect that can infect Nozema Apis in addition to bees, as a target for experimenting with antibacterial effects on Nozema Apis Ni cells were used, High five cells were used, Trichoplusia ni cells were grown in Express Five medium (Gibco) containing 2 mL of glutamine, and 6-well plates were used for the raised plates. Trichoplusia raised by the day Nosema was treated with ni cells to be 10 ⁴ Nozema apis/mL, and the extract was treated with a concentration of 50 μg/mL. Observations were made 5 days after treatment to make it easy to confirm the onset of old disease.

<Experimental Example 1> Microscopic observation of Nojema spores

The microscope was first observed at 100 times, and then a portion that could effectively observe the result was further magnified 400 times to obtain a picture.

As a result, as shown in FIG. 1, in the comparison group in which both Nojema apis and non-repair extracts were not treated, Trichoplusia ni cells showed differentiation while maintaining the shape of round cells.

On the other hand, when the Nozema Apis is treated as shown in FIG. 2, the condition of the cells deteriorates, and the cells are abnormally enlarged due to the replication process of the Nozema Apis, and the Nozema Apis spores concentrated in one place can be observed. Could. Also, the number of cells was relatively small.

In order to confirm the antibacterial effect of Nozema Apis of the non-repair extract of the present invention, when Nozema Apis and the non-repair extract were treated together, the cell number and cell size were not different from the normal control. Also, Nozema spores were located outside the cells rather than inside. That is, it can be confirmed from the results of FIG. 3 that the treatment of the non-repair extract is effective in treating Nozema by significantly reducing the number of Nozema spores in the group treated with the non-repair extract compared to the group without treatment with the non-repair extract. there was.

<Experimental Example 2> Cytotoxicity experiment

Based on the results obtained in Experimental Example 4, the following experiment was conducted to find the treatment concentration of the non-aqueous extract that is effective in controlling Nozema Apis without having a cytotoxicity. Based on the initial treatment concentration of 50 μg/mL, the concentration was doubled and the toxicity was observed through the number of cells and cell (spores) size.

As a result, when the concentration of the unrepaired extract is 200 μg/mL or more, Trichoplusia It was observed that the ni cell size decreased, and the number of cells began to stem.

In order to find the minimum concentration effective for controlling Nozema Apis, the minimum treatment concentration was confirmed by sequentially decreasing the concentration to 1/2 in 50 μg/mL in a similar manner. As a result, the size and number reduction effect of Nojema Apis spores began to decrease from 3.125 μg/mL.

Through the above results, it was confirmed that the optimal concentration of the non-repair extract for controlling Nozema Apis is 6.25 to 200 μg/mL.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (8)

A composition for preventing or treating Nozema disease comprising an unrepaired extract as an active ingredient. According to claim 1,
The non-repair extract is purified water, methanol (methanol), ethanol (ethanol), propanol (propanol), isopropanol (isopropanol), butanol (butanol), etc., alcohol having 1 to 4 carbon atoms, acetone (acetone), ether (ether) Extracted to use one or two or more extraction solvents selected from benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane Characterized in that, the composition. According to claim 1,
The composition is Nojema Apis, the causative agent of Nozema disease apis ) A composition for preventing or treating Nozema disease, characterized by having antibacterial activity. According to claim 1,
The composition is a composition for preventing or treating old disease, characterized in that it contains a non-repair extract at a concentration of 1.0 to 300 μg/mL. According to claim 1,
A composition for preventing or treating Nozema disease, further comprising at least one antibacterial or antifungal agent in the composition. The method of claim 5,
The antibacterial or antifungal agent is tetracycline, oxytetracycline, amikacin, gentamicin, tobramycin, streptomycin, netylmycin, kanamycin, ciprofloxacin, norfloxacin, operoxacin, trovafloxacin, romefloxacin, Levofloxacin, enoxacin, naphthyridine, sulfonamide, polymyxin, chloramphenicol, neomycin, paramomycin, colistimetate, bacitracin, vancomycin, rifampin, cycloserine, beta-lactam, cephalosporin, Amphotericin, fluconazole, flucitocin, natamycin, myconazole, ketoconazole, corticosteroid, diclofenac, flurbiprofen, ketorolac, suprofen, comoline, rodoxamide, levocarbastin, napazoling, anta A composition for preventing or treating Nozema disease, characterized in that it is at least one substance from the group consisting of only sleepy and peniramimi. Insect feed composition comprising the unrepaired extract as an active ingredient. The method of claim 7,
The insect feed is characterized in that it comprises an anti-aging agent control activity, insect feed composition.

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