KR20210130693A - Composition for Preventing Biofilm Formation Comprising Extract of Prunellae Spica - Google Patents

Abstract

The present invention relates to a composition for inhibiting biofilm formation and a composition for preventing and alleviating diseases related to biofilm formation which contain a Prunella vulgaris extract. The Prunella vulgaris extract can effectively inhibit the formation of biofilms produced by microorganisms, the Prunella vulgaris extract can be usefully used as the composition for inhibiting biofilm formation, and the composition for preventing or treating diseases related to biofilm formation.

Description

A composition for inhibiting biofilm formation containing a seaweed extract {Composition for Preventing Biofilm Formation Comprising Extract of Prunellae Spica}

The present invention relates to a composition for inhibiting biofilm formation and a composition for preventing and treating diseases related to biofilm formation, which contain a hagogi extract as an active ingredient.

A biofilm, also called a biofilm, refers to a state in which microorganisms produce polysaccharides around cells and aggregate with adjacent microorganisms as a medium to form a film on a solid or living body surface. Microorganisms that form biofilms are more than 1000 times more resistant to antibiotics than microorganisms in a suspended state, and biofilm formation is associated with 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).

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

Therefore, in order to prevent diseases caused by microbial infection, it is necessary to effectively inhibit the formation of biofilms of microorganisms. In order to suppress the formation of such a biofilm, an antibacterial activity that inhibits the growth of microorganisms forming a biofilm was used in the prior art. However, if the material having antibacterial activity inhibits the growth of microorganisms forming the biofilm, 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 inhibiting the formation of a biofilm while reducing the effect on the growth of microorganisms.

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

1. Republic of Korea Patent No. 10-1653662

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

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

As used herein, the term 'biofilm (biofilm)' refers to a membrane-shaped structure formed by microorganisms, and has structural features 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 present in the external environment, and when these insoluble polysaccharides are continuously accumulated, a thin biofilm in the form of a film is formed. The formed biofilm helps aggregation between microorganisms, and as a result, can cause diseases such as cystic fibrosis, dental caries, and intractable infections such as sepsis. It can also cause damage to organs or structures and contaminate medical devices such as catheters.

Hagocho (prunellae spica) used as an active ingredient of the composition for inhibiting biofilm formation of the present invention refers to an above-ground part of a perennial plant (Prunella vulgaris Linne) belonging to the Lamiaceae family. It contains triterpenoid, saponin, ursolic acid, hyperoxide, tannin and caffeic acid. It is known to be effective for mastitis, breast cancer, diuresis, antitussives, hemostasis, mental anxiety, and militia. However, the effect of inhibiting biofilm formation by microorganisms is not known.

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

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

According to one 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 hagocho extract includes both the extract by the above-described solvent extraction method and the extract that has undergone a conventional purification process. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity, or affinity), etc. Fractions obtained through various purification methods may also be included in the extract of the present invention. In addition, the hagocho extract may 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 include 0.1 to 20% by weight, or 1 to 10% by weight of the plant extract based on the total weight of the composition. The content can be appropriately adjusted.

According to an embodiment of the present invention, the biofilm may be formed by a microorganism selected from the group consisting of Yersinia sp. and Xanthomonas sp., but is not limited thereto.

According to one embodiment of the present invention, the microorganism may be selected from the group consisting of Yersinia enterocolitica and Xanthomonas oryzae, but is not limited thereto.

According to one embodiment of the present invention, food poisoning, enteritis, etc. can be induced by the biofilm formed by Yersinia enterocolica, and when Xanthomonas duckjae forms a biofilm in the conduit of rice, white leaf blight of rice can be prevented. can provoke

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 on the surface of food, the composition for inhibiting biofilm formation can be used as a food or cosmetic composition to prevent this. In addition, in order to prevent biofilm formation 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 pharmaceutical composition for preventing or treating diseases caused by Yersinia enterocolica, containing the composition for inhibiting biofilm formation as an active ingredient.

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

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

According to one embodiment of the present invention, since the extract of Hagogonia can effectively inhibit the biofilm formation by Yersinia enterocolica, the composition for inhibiting biofilm formation can be used to prevent or prevent diseases caused by Yersinia enterocolica. It can be usefully used for therapeutic purposes.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier. As used herein, 'pharmaceutically acceptable' refers to a composition that is physiologically acceptable and does not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions 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 forms for parenteral or oral administration according to known methods. A typical example of a formulation for parenteral administration is an isotonic aqueous solution or suspension as an injection formulation. Formulations for injection may be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component may be dissolved in saline or buffer to be formulated for injection. In addition, formulations for oral administration include, but are not limited to, powders, granules, tablets, pills and capsules.

The pharmaceutical composition formulated in the above manner may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. As used herein, the term 'effective amount' refers to an amount exhibiting a prophylactic or therapeutic effect when administered to a patient. The dosage of the pharmaceutical composition according to the present invention may be appropriately selected according to the route of administration, administration, age, sex, body weight, individual differences and disease state. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the degree of disease, and may be administered in an effective amount of 1 to 100 mg/kg/day in the form of parenteral administration.

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

Rice blight is a disease that causes the greatest economic loss in rice production and is widespread worldwide. When Xanthomonas oryzae is adsorbed to the conduit of rice to form a biofilm, the passage of the conduit is blocked, thereby preventing the smooth delivery of nutrients or water. As a result, the leaves begin to die, and even if the rice dies or survives, rice cannot be produced normally. Therefore, suppressing the formation of a biofilm by Xanthomonas duckweed can reduce the clogging of rice ducts, and even if infected with Xanthomonas duckweed, rice can grow normally, thereby maintaining normal rice yield.

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

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

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

Since the extract of seaweed can effectively inhibit the formation of biofilms 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.

1 is a graph showing the results of confirming the effect of an extract of Hagogi on the biofilm formation of Yersinia enterocolitica : A is a methanol extract of Hagogi, B is a 50% ethanol extract of Hagogi, C is This is the result of the 95% ethanol extract of hagogi root, and the 'dilution ratio' on the X-axis means the dilution ratio of the hago root extract.
Figure 2 is a graph showing the results of confirming the effect of the extract of hagogi on the growth of Yersinia enterocolitica: A is a methanol extract of hagogi, B is a 50% ethanol extract of hagogi, and C is a 95% ethanol extract of hagfish. As a result, the 'dilution ratio' on the X-axis means the dilution ratio of the H.
Figure 3 is a graph showing the results of confirming the biofilm formation inhibitory effect of the Hagogi extract against Xanthomonas oryzae : A is the methanol extract of Hagogi and B is the result of the water extract, and the X-axis ' The dilution ratio 'means the dilution ratio of the hago root extract.
Figure 4 is a graph showing the results of confirming the effect of the extract of Hagonia on the growth of Xanthomonas duckjae: A is a methanol extract of Hagogi and B is a result of a water extract of Hagogi, and the 'dilution ratio' of the X-axis is the extract of of the dilution ratio.

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

Example 1: Preparation of hagocho extract

100 ml of each of water, methanol, 50% ethanol, and 95% ethanol was added to 10 g of seaweed, and the mixture was extracted in a water bath at 50° C. for 3 hours. Thereafter, the extract was first filtered using filter paper, and the volume of the filtered primary extract was measured.

The water extract was further filtered with a cellulose acetate filter (0.2 μm pore size) and then used for the experiment. The methanol extract, 50% ethanol extract 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 as much as the volume of the primary extract to the filtrate, and it was further filtered through a cellulose acetate filter and used in the experiment.

Example 2: Inhibitory effect on biofilm formation of Hagogi extract

2-1. Inhibitory effect of biofilm formation on Yersinia enterocolica

Yersinia enterocolitica (hereinafter referred to as Y. enterocolitica ) was streaked on a TYE agar plate (1% tryptone, 0.5% yeast extract, 1.5% agar), and then at 26°C for 2 days. incubated during A single colony was taken from the plate, inoculated into 5 ml of TYE broth, and cultured with shaking at 26° C. for 1 day at 250 rpm.

_{M63 broth (3 g/L KH 2} PO ₄ , 7 g/LK ₂ HPO ₄ , 2 g/L (NH ₄ ) ₂ SO ₄ , 0.5 mg/L FeSO in 96-well polyvinyl chloride (PVC) microplates ₄ , 0.2% glucose, 0.02% MgSO ₄ ) was dispensed at 100 μl each. Here, 1 μl of the methanol extract prepared in Example 1 was dispensed, and 5 μl of each of 50% ethanol and 95% ethanol extract was dispensed. Next, the cultured Y. enterocolitica was inoculated into each well to have an OD ₆₀₀ of 0.05 and cultured at 26° C. for 24 hours. After 24 hours, the absorbance (optical density, OD; absorbance) was measured at _{OD 595} to confirm the cell density of Y. enterocolitica , and after removing the culture medium from the plate, it was gently washed 3 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, it was washed 3 times with distilled water, and 100 μl of 95% ethanol was added to elute the crystal violet and left for 15 minutes. Thereafter, the amount of the biofilm formed was quantified by measuring the absorbance _{at OD 595 with a microplate reader.}

As a result of the confirmation, as shown in FIG. 1 , it was confirmed that the biofilm formation was remarkably inhibited by the extract of Hagogi.

2-2. Inhibitory effect of biofilm formation on Xanthomonas duck material

Xanthomonas oryzae (hereinafter referred to as X. oryzae ) was smeared on YGC agar plates (5% glucose, 0.5% yeast extracts, 1.25% CaCO _3, 1.5% agar), followed by 2 at 28°C. incubated for one day. A single colony is taken from the plate and inoculated into 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 inoculated at 28°C. Shaking culture was performed at 250 rpm for 2 days.

In a 96-well polyvinyl chloride microplate, XOM2 broth (670 μM L-methionine, 10 mM sodium L-(+)-glutamate monohydrate, 14.7 mM KH ₂ PO ₄ , 40 μM MnSO ₄ , 240 μ Fe(III)-EDTA, 5 mM MgCl ₂ , 0.18% D-xylose, pH6.5), except that the biofilm formation inhibitory effect of the extract (1 μl of methanol extract, 5 μl of water extract) was used in the same manner as in 2-1 above. was confirmed.

As a result of the confirmation, as shown in FIG. 3 , it was found that the biofilm formation was significantly inhibited by the methanol extract and the water extract of Hagogi plant.

Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated 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 (4)

As a composition for inhibiting the formation of a biofilm containing a hago leaf extract as an active ingredient,
The biofilm is Xanthomonas genus (Xanthomonas sp.) The composition is formed by microorganisms. According to claim 1, wherein the extract is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, alcohols 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 microorganism is Xanthomonas oryzae . A composition for controlling bacterial leaf blight of rice, containing the composition of claim 1 as an active ingredient.

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