KR102205138B1 - Composition for Preventing Biofilm Formation Comprising Extract of Morus alba - Google Patents

Abstract

It relates to a composition for inhibiting the formation of a biofilm containing the extract of the bark and a composition for preventing and improving diseases related to the formation of a biofilm.

Description

Composition for Preventing Biofilm Formation Comprising Extract of Morus alba}

The present invention relates to a composition for inhibiting biofilm formation and a composition for the prevention and treatment of diseases related to biofilm formation, containing the extract of Sangbaekpi as an active ingredient.

Biofilm is also called a biofilm, and refers to a state in which microorganisms produce polysaccharides around cells and aggregate with adjacent microorganisms through this to form a film on the surface of a solid or living body. Biofilm-forming microorganisms are 1000 times more resistant to antibiotics than suspended microorganisms, and biofilm formation is bacterial endocarditis, dental caries, chronic sinusitis, food poisoning, and pneumonia. It is known to play an important role in the pathogenesis of various diseases, such as (Non-Patent Document 1).

Streptococcus mutans ( S. mutans ) is an important causative agent that attaches to the tooth surface in the oral cavity and causes dental caries (caries). S. mutans synthesizes a polysaccharide called glucan using glucose, forms a biofilm from the produced glucan, and attaches it to the teeth, thereby becoming resistant to external physical and chemical treatment.

In clinical practice, biofilm contamination on medical devices such as catheters and stents is a problem. Microorganisms that cause medical device contamination include Enterococcus faecalis. faecalis ) and Staphylococcus aureus , and the like, and the contamination of the microorganisms may cause osteomyelitis, toxin shock syndrome, food poisoning, and skin infections.

Therefore, in order to prevent diseases caused by microbial infection, it is necessary to effectively inhibit the formation of a biofilm by microbes. In order to suppress the formation of such a biofilm, an antibacterial activity that inhibits the growth of microorganisms forming a biofilm has been used. However, when the growth of microorganisms forming a biofilm with a material having antibacterial activity is suppressed, only resistant microorganisms remain, and as a result, it may be difficult to remove or inhibit the formation of the biofilm. Therefore, it is necessary to develop a material capable of suppressing the formation of a biofilm while reducing the effect on the growth of microorganisms.

The present inventors completed the present invention by confirming that the natural substance, the extract of Sangbaekpi, has excellent ability to inhibit the formation of biofilm.

1. Korean Patent Registration No. 10-1653662

1.http://www.intron.co.kr/sub/technologies/biofilm.php

An object of the present invention is to provide a composition for inhibiting the formation of a biofilm of microorganisms containing the extract as an active ingredient.

Another object of the present invention is to provide a composition for preventing or treating diseases related to biofilm formation, containing the composition for inhibiting biofilm formation as an active ingredient.

One aspect of the present invention provides a composition for inhibiting the formation of a biofilm containing a Sangbaekpi extract as an active ingredient.

The term “biofilm” as used herein refers to a membrane-like structure formed by microorganisms, and has a structural feature consisting of interconnected microbial cell layers and microbial secretions. Microorganisms secrete enzymes that produce polysaccharides to the outside of cells to form water-soluble or insoluble polysaccharides from sugar substances existing in the external environment, and when such insoluble polysaccharides are continuously accumulated, a thin biofilm in the form of a film is formed. The formed biofilm helps to aggregate between microorganisms and, as a result, may cause diseases such as cystic fibrosis, dental caries, and intractable infections such as sepsis. It can also damage organs or structures and contaminate medical devices such as catheters.

Sangbaek peel used as an active ingredient of the composition for inhibiting biofilm formation of the present invention is a Morus tree of the Morus family. alba L.) or a medicinal herb made from the root bark of a plant of the same genus. Due to its pharmacological action, it is known to have effects such as Jinhae, diuresis, lowering blood pressure, pain relief, and antipyretic, and is also used in seawater and asthma. However, the effect of inhibiting the formation of a biofilm by microorganisms is unknown.

According to an embodiment of the present invention, the Sangbaekpi extract may be prepared according to a conventional method known in the art. For example, a solvent commonly used for extraction may be added after pulverizing the upper bark, and the extract may be prepared by extracting at an appropriate temperature and pressure.

According to an embodiment of the present invention, the solvent is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, alcohol having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, and mixtures thereof. Can be.

According to an embodiment of the present invention, the solvent may be purified water or an alcohol having 1 to 4 carbon atoms.

On the other hand, the Sangbaekpi extract includes not only the extract obtained by the solvent extraction method described above, but also an extract that has undergone a conventional purification process. For example, separation by using an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods may also be included in the Sangbaekpi extract of the present invention. In addition, the extract can be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze drying or spray drying.

The composition for inhibiting biofilm formation of the present invention may contain 0.1 to 20% by weight, or 1 to 10% by weight of the above-mentioned Sangbaekpi extract based on the total weight of the composition, and the content of active ingredients according to the method of use and purpose of the composition Can be adjusted appropriately.

According to one embodiment of the present invention, the biofilm is Streptococcus sp., Xanthomonas sp. sp.) and Yersinia sp. may be formed by microorganisms selected from the group consisting of, but is not limited thereto.

According to an embodiment of the present invention, the microorganism is Streptococcus mutans , Xanthomonas duck material ( Xanthomonas oryzae ) and Yersinia enterocolitica ( Yersinia enterocolitica ) may be selected from the group consisting of, but is not limited thereto.

According to one embodiment of the present invention, dental caries or periodontitis may occur due to the biofilm formed by Streptococcus mutans, and when Xanthomonas ducks form a biofilm in the conduit of rice, it may cause white leaf blight of rice. . In addition, food poisoning and enteritis may be caused by the biofilm formed by Yersinia enterocolitica.

According to one embodiment of the present invention, since the biofilm is formed in a place where the growth or proliferation of microorganisms is possible, such as the surface of a food, the composition for inhibiting formation of the biofilm may be used as a food or cosmetic composition to prevent this. In addition, in order to prevent the formation of biofilm on medical devices such as catheters and stents, the composition for inhibiting biofilm formation according to the present invention may be used as a coating composition.

Another aspect of the present invention provides a composition for preventing or treating oral diseases containing the composition for inhibiting formation of the biofilm as an active ingredient.

According to an embodiment of the present invention, the oral disease may be dental caries or periodontitis, and may be caused by a biofilm formed by oral microorganisms. In addition, the oral microorganisms Streptococcus mutans (Streptococcus mutans), Streptococcus salivarius (Streptococcus salivarius), Streptococcus pick Donnie (Streptococcus gordonii), Streptococcus small debris Augustine (Streptococcus sobrinus), Streptococcus said bus (Streptococcus sanguis ), Streptococcus pyogenes, Lactobacillus acidophilus , Lactobacillus casei , Actinobacillus viscosus , Agregatibacter actinophilus ( Aggregatibacter actinomycetemcomitans ), Porphyromonas gingivalis ( Porphyromonas gingivalis ), Tannerella forsythia , Treponema denticola , Prevotella Intermedia intermedia ) and Fusobacterium nucleatum may be selected from the group consisting of.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. In the above, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause allergic reactions or similar reactions such as gastrointestinal disorders and dizziness when administered to humans.

The pharmaceutical composition according to the present invention may be formulated in a suitable form according to a method known in the art together with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods. As a representative formulation for parenteral administration, an isotonic aqueous solution or suspension is preferable as a formulation for injection. Formulations for injection can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be formulated for injection by dissolving it in saline or buffer. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.

The pharmaceutical composition formulated by the above method may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or intramuscular. In the above,'effective amount' refers to an amount that exhibits a preventive or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention can be appropriately selected according to the route of administration, the subject of administration, age, sex, weight, individual difference, and disease state. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of disease, and in the form of parenteral administration, it may be administered in an effective amount of 1 to 100 mg/kg/day.

Another aspect of the present invention provides a composition for controlling bacterial leaf blight of rice containing the composition for inhibiting formation of biofilm as an active ingredient.

White leaf blight in rice is a disease that causes the most economic loss to rice production and occurs widely worldwide. When Xanthomonas oryzae adsorbs to the ducts of rice to form a biofilm, the passage of the ducts is blocked, and the delivery of nutrients or water is not smooth. Accordingly, the leaves begin to die, and even if the rice dies or survives, rice cannot be normally produced. Therefore, suppressing the formation of biofilm by the xanthomonas duck material can reduce the clogging of the rice conduit, and the rice can grow normally even if it is infected with the xanthomonas duck material, so that the rice yield can be maintained normally.

The composition for controlling white leaf blight of rice can be prepared in the form of a solution, powder and suspension that can be directly sprayed, or in the form of highly concentrated aqueous, oily or other suspensions, dispersions, emulsions, oily dispersions, pastes, dusts, scattering substances or granules, It is not limited thereto. In addition, the composition for control may be used by spraying, spraying, spraying, scattering or pouring.

In addition, the composition for controlling white leaf blight may be formulated in various forms, and, for example, may be prepared by adding a solvent and/or a carrier. Suitable inert additives and surface-active substances such as emulsifiers or dispersants can be mixed into the formulation.

According to an embodiment of the present invention, the composition for controlling white leaf blight of rice may include a composition for inhibiting biofilm formation including a methanol extract or a water extract of Sangbaekpi.

Another aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases caused by Yersinia enterocolitica, containing the composition for inhibiting formation of biofilm as an active ingredient.

According to an embodiment of the present invention, the disease may be selected from the group consisting of acute gastroenteritis, terminal gastroenteritis, sepsis, tuberculous erythema, food poisoning and enteritis, but is not limited thereto.

Yersinia enterocolitica enterocolitica ) is a Gram-negative bacterium belonging to the family Enterobacteriaceae and causes gastroenteritis (food poisoning and enteritis). In humans, various diseases such as acute gastroenteritis, terminal enteritis, sepsis, and tuberculosis erythema occur, and it is known to have resistance to external stimuli or antibiotic treatment by forming a biofilm.

According to an embodiment of the present invention, the composition for inhibiting biofilm formation is used to prevent or treat diseases caused by Yersinia enterocolitica, since the extract of Serrata can effectively inhibit the formation of biofilms by Yersinia enterocolitica. It can be usefully used for purposes.

Since the Sangbaekpi extract can effectively inhibit the formation of a biofilm generated by microorganisms, it can be usefully used as a composition for inhibiting biofilm formation and a composition for preventing or treating diseases related to biofilm formation.

Figure 1 is a graph showing the results of confirming the effect of the methanol extract of cortex bark on the biofilm formation by Streptococcus mutans and the growth of Streptococcus mutans : A is the amount of biofilm production, B is the cell density measurement result And, the'dilution ratio' of the X-axis means the dilution ratio of the methanol extract of Sangbaek skin.
Figure 2 is a graph showing the results confirming the effect on the biofilm formation of Yersinia enterocolitica ( Yersinia enterocolitica ) of the extract of bark: A is a methanol extract of bark, B is a water extract of bark bar, C is a 50% ethanol extract of bark bark And D is the result of the 95% ethanol extract of Sangbaekpi, and the'dilution ratio' of the X-axis indicates the dilution ratio of the extract of Sangbaekpi.
Figure 3 is a graph showing the results of confirming the effect of the extracts of the upper bark on the growth of Yersinia enterocolitica: A is a methanol extract, B is a water extract, C is 50% ethanol extract, and D is 95% Sangbaek It is the result of ethanol extract, and the'dilution ratio' of the X-axis means the dilution ratio of the extract of the extract.
Figure 4 is a graph showing the result of confirming the effect of the extract of the bark bark on the formation of the biofilm of Xanthomonas oryzae : A is the methanol extract of bark, B is the result of the extract of bark bark water,'dilution ratio' of the X-axis Refers to the dilution ratio of the extract of Sangbaekpi.
Figure 5 is a graph showing the result of confirming the effect of the extract of the bark bark on the growth of Xanthomonas duckweed: A is the methanol extract of bark, B is the result of the extract of bark bark, and the'dilution ratio' of the X-axis indicates the dilution ratio of the extract of bark bark. it means.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Preparation of Sangbaekpi extract

100 ml of water, methanol, 50% ethanol and 95% ethanol, respectively, were added to 10 g of upper bark, and extracted for 3 hours in a 50°C constant temperature water bath. Thereafter, the extract was first filtered using a filter paper, and the volume of the filtered first extract was measured.

The water extract was further filtered with a cellulose acetate filter (0.2 µm pore size) and used in the experiment. The methanol extract, 50% and 95% ethanol extract were concentrated to 10% of the total volume of the extract with a rotary evaporator, and then filtered again. Water was added to the filtrate as much as the volume of the first extract, and further filtered through a cellulose acetate filter, and used in the experiment.

Example 2: Inhibitory effect of biofilm formation of the extract of S.

2-1. Streptococcus To mutans Biofilm formation inhibition effect

Streptococcus mutans GS-5 ( Streptococcus mutans GS-5; hereinafter referred to as S. mutans GS-5) strain was distributed from LG H&H Research Institute (Daejeon, Korea) and BHI medium (Brain Heart Infusion medium, Bacto ^TM) , BD Biosciences). S. mutans GS-5 was streaked on a BHI agar plate and then incubated for 2 days at 37°C. A single colony was taken from the plate, inoculated into 5 ml of BHI liquid medium, and stationary cultured at 37°C for 1 day.

100 µl of BHI medium containing 1% sugar was dispensed into a 96-well polyvinyl chloride (PVC) microplate, and 1 µl of methanol extract prepared in Example 1 was additionally dispensed. Next, the stationary cultured S. mutans in each well GS-5 was inoculated so that an OD ₆₀₀ of 0.05 was incubated at 37°C for 24 hours. After 24 hours, the optical density (OD; absorbance) was measured at OD ₅₉₅ to check the cell density of S. mutans GS-5, and after removing the culture medium from the plate, it was gently washed three times with distilled water. 100 µl of a 1% crystal violet solution was dispensed into each well, and the biofilm was stained at room temperature for 15 minutes. After 15 minutes, the mixture was washed three times with distilled water, and 100 µl of 95% ethanol was added to elute the crystal violet and left for 15 minutes. Thereafter, the absorbance was measured at OD ₅₉₅ with a microplate reader to quantify the amount of biofilm formed.

As a result of the confirmation, it was confirmed that the biofilm formation was significantly suppressed by the methanol extract of Sangbaekpi as shown in FIG. 1.

2-2. Yersinia Enterocolitica Biofilm formation inhibition effect

Yersinia enterocolitica enterocolitica ; Hereinafter, Y. enterocolitica ) was plated on a TYE agar plate (1% tryptone, 0.5% yeast extract, 1.5% agar), and then incubated at 26° C. for 2 days. A single cluster was taken from the plate, inoculated into 5 ml of TYE liquid medium, and cultured with shaking at 250 rpm for 1 day at 26°C.

M63 liquid medium (3g/L KH ₂ PO ₄ , 7g/LK ₂ HPO ₄ , 2g/L (NH ₄ ) ₂ SO ₄ , 0.5 mg/L FeSO ₄ , 0.2% glucose, in 96-well polyvinyl chloride microplates, 0.02% MgSO ₄ ) Except for the use of, in the same manner as in 2-1 above, 1 µl of the methanol extract was added, and 5 µl of the water extract, 50% ethanol, and 95% ethanol extract were added respectively) to form a biofilm. The inhibitory effect was confirmed.

As a result, as shown in FIG. 2, it was confirmed that the biofilm formation was suppressed by the extract of Sangbaekpi, and in particular, the methanol extract of Sangbaekpi showed a remarkable effect of inhibiting biofilm formation.

2-3. Inhibitory Effect of Biofilm Formation on Xanthomonas Duck Ash

Xanthomonas oryzae ( Xanthomonas oryzae ; hereinafter, described as X. oryzae ) was spread on YGC agar plates (5% glucose, 0.5% yeast extracts, 1.25% CaCO ₃ , 1.5% agar), and then 2 at 28°C. Incubated for days. Take a single cluster from the plate and inoculate it in 5 ml of 210 liquid medium (0.5% sucrose, 0.8% casein enzymatic hydrolysate, 0.4% yeast extract, 0.3% K ₂ HPO ₄ , 0.03% MgSO ₄ 7H ₂ O) and inoculate at 28°C. It was cultured with shaking at 250 rpm for 2 days.

XOM2 liquid medium (670μM L-methionine, 10 mM sodium L-(+)-glutamate monohydrate, 14.7mM KH ₂ PO ₄ , 40μM MnSO ₄ , 240μ Fe(III)-EDTA, 5 in 96-well polyvinyl chloride microplate) Except for the use of mM MgCl ₂ , 0.18% D-xylose, pH 6.5), the effect of inhibiting biofilm formation of the extract of Siamese bark (1 µl for methanol extract and 5 µl for water extract) was used in the same manner as in 2-1. Was confirmed.

As a result of the confirmation, it was found that the biofilm formation was remarkably suppressed by the methanol extract and water extract of Sangbaekpi, as shown in FIG. 4.

Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (9)

A composition for controlling bacterial leaf blight of rice, which contains the extract of Sangbaek bark as an active ingredient.
The method of claim 1, wherein the extract is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, alcohol having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, and mixtures thereof. A composition that is extracted with a solvent.
The composition of claim 1, wherein the white leaf blight of the rice is caused by a microorganism of the genus Xanthomonas sp.
The composition of claim 3, wherein the microorganism is Xanthomonas oryzae .
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