KR20190041301A - Composition for Preventing Biofilm Formation Comprising Extract of Mentha arvensis - Google Patents

Abstract

The present invention relates to a composition for inhibiting formation of a biofilm containing a Mentha canadensis extract as an active ingredient, and a composition for preventing and alleviating diseases related to formation of a biofilm. The Mentha canadensis extract is extracted by a solvent selected from the group consisting of purified water, hexane, 1,3-butyleneglycol, alcohol having 1 to 4 carbon atoms, ethylacetate, diethylether, dichloromethane, acetone, and a mixture thereof.

Description

TECHNICAL FIELD The present invention relates to a composition for inhibiting the formation of biofilm,

The present invention relates to a composition for inhibiting the formation of a biofilm containing a peppermint extract as an active ingredient and a composition for preventing and treating diseases associated with the formation of a biofilm.

Biofilm, also called biofilm, is a state in which microorganisms produce polysaccharides around cells and coalesce with adjacent microorganisms to form a film on a solid or living body surface. Biofilm-forming microorganisms are more than 1,000 times more resistant to antibiotics than suspended microorganisms and biofilm formation is associated with bacterial endocarditis, dental caries, chronic sinusitis, food poisoning, And it is known that it plays an important role in the pathogenesis of various diseases (Non-Patent Document 1).

In clinical practice, biofilm contamination of medical devices such as catheters and stents is becoming a problem. Microorganisms that cause contamination of medical instruments include Enterococcus faecalis , and Staphylococcus aureus . Osteitis, toxin shock syndrome, food poisoning, and skin infections can be caused by the contamination of the microorganisms.

Therefore, it is necessary to effectively inhibit microbial biofilm formation in order to prevent disease caused by microbial infection. In order to inhibit the formation of such biofilms, antimicrobial activity that inhibits the growth of microorganisms that form biofilms has been utilized. However, if the growth of microorganisms forming a biofilm is inhibited by a substance having an antimicrobial activity, only microorganisms having resistance are left, and as a result, it may be difficult to suppress the formation or suppression of the biofilm. Therefore, it is necessary to develop a substance capable of inhibiting the formation of biofilm while reducing the influence on microbial growth.

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

1. Korean Patent No. 10-1653662

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

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

It is another object of the present invention to provide a composition for preventing or treating diseases associated with the formation of a biofilm 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 peppermint extract as an active ingredient.

As used herein, the term 'biofilm' refers to a membrane-like structure formed by a microorganism, and has a structural characteristic composed of interconnected microbial cell layers and microbial secretions. The microorganisms secrete the polysaccharide-producing enzyme outside the cell to form a water-soluble or insoluble polysaccharide from the saccharide present in the external environment. When such insoluble polysaccharides are continuously accumulated, a film-like thin biofilm is formed. The formed biofilm can help to aggregate between microorganisms and consequently cause diseases such as cystic fibrosis, dental caries, and refractory infections such as sepsis. It can also cause damage to the organs or structures and can contaminate medical devices such as catheters.

Mentha arvensis var. Piperascens used as an active ingredient of the composition for inhibiting the formation of biofilm according to the present invention is a perennial herb with Lamiaceae. Menthol extracts include isomenthone, isomenthone, pinene, It contains components such as camphene, limonene, zingherone, and shogaol. Menthol is widely used as a fragrance or refreshing agent due to its main component, menthol, but the effect of inhibiting the formation of biofilm by microorganisms is not known.

According to one embodiment of the present invention, the peppermint extract may be prepared according to a conventional method known in the art. For example, a peppermint extract can be prepared by pulverizing a peppermint, then adding a solvent commonly used for the extraction, and extracting at a suitable temperature and pressure.

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

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 peppermint extract includes both the solvent extraction method and the conventional purification method. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatographies (made for separation by size, charge, hydrophobicity or affinity) Fractions obtained through various purification methods can also be included in the peppermint extract of the present invention. In addition, the peppermint extract may be prepared in powder form by an additional process such as vacuum distillation and freeze-drying or spray-drying.

The composition for inhibiting the formation of a biofilm of the present invention may contain the peppermint extract in an amount of 0.1 to 20% by weight or 1 to 10% by weight based on the total weight of the composition. The content of the active ingredient Can be adjusted appropriately.

According to one embodiment of the present invention, the biofilm is selected from the group consisting of Yersinia sp.Yersinia sp.), eromonas sp.Aeromonas sp.) and Xanthomonas sp.Xanthomonas sp.), but the present invention is not limited thereto.

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

According to one embodiment of the present invention, food poisoning, enteritis and the like may occur due to the biofilm formed by Yersinia enterocolitica, and when xanthomonas oryzae forms a biofilm in the conduit of rice, . In addition, biofilms formed by eromonas hydrofilas can cause dropsy disease (Aeromonas infection) in fish and necrotizing fasciitis in humans.

According to one embodiment of the present invention, since the biofilm is formed in a place where microbial growth or proliferation is possible, such as the surface of food, the composition for inhibiting biofilm formation may be used as a food or cosmetic composition in order to prevent it. In addition, a composition for inhibiting biofilm formation according to the present invention may be used as a coating composition in order to prevent the formation of a biofilm on a medical instrument such as a catheter or a stent.

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

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

Yersinia enterocolitica ) is a gram-negative bacterium belonging to the intestinal bacteria and causes gastroenteritis (food poisoning and enteritis). It is known that humans develop various diseases such as acute gastroenteritis, endophthalmitis, sepsis and tuberculous erythema, and are resistant to external stimuli or antibiotic treatment by forming biofilm.

According to one embodiment of the present invention, since the peppermint extract can effectively inhibit biofilm formation by yersinia enterocolitica, the composition for inhibiting biofilm formation can be used for preventing or treating diseases caused by yersinia enterocolitica It can be used as a useful application.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. &Quot; Pharmaceutically acceptable " as used herein refers to a composition that is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, or the like when administered to a human.

The pharmaceutical composition according to the present invention, together with a pharmaceutically acceptable carrier as described above, may be formulated into a suitable form according to a method known in the art. That is, the pharmaceutical composition of the present invention can be prepared in various parenteral or oral administration forms according to known methods. Representative examples of formulations for parenteral administration include isotonic aqueous solutions or suspensions in the form of injection. The injectable formulations 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 formulated for injection by dissolving 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 as described above may be administered in an effective amount through various routes including oral, transdermal, subcutaneous, intravenous, or muscular. The term " effective amount " as used herein refers to an amount that shows 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 depending on the route of administration, subject to be administered, age, sex, weight, individual difference, and disease state. The pharmaceutical composition of the present invention may be administered in an effective amount of 1 to 100 mg / kg / day in the form of parenteral administration, depending on the severity of the disease.

Another aspect of the present invention is a pharmaceutical composition for preventing or treating necrotizing fasciitis caused by eromonas hydrofilas containing the above composition for inhibiting biofilm formation as an active ingredient and a composition for controlling pine cones of fish Lt; / RTI >

Necrotizing fasciitis is a bacterial infectious disease that causes inflammation of the fascia and necrosis of the subcutaneous tissue of the skin. Once necrotizing fasciitis develops, it spreads to the whole body at a rapid rate and can lead to sepsis when the disease progresses.

Drops disease is a bacterial disease that occurs like a pine cone on the scale of a fish. It shows symptoms such as abdominal distension, prominence, hemorrhage, and it is a fatal disease in fish because it is difficult to recover when infected.

Since the peppermint extract can effectively inhibit biofilm formation by eromonas hydrofilas, the composition for inhibiting biofilm formation including peppermint extract may be usefully used for prevention or treatment of necrotizing fasciitis, and for controlling pine cone disease.

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

Blight of blight of rice blight is the most common economic disease in rice production worldwide. When Xanthomonas oryzae is adsorbed on the rice tubing to form a biofilm, the passage of the conduit is blocked and nutrients and water are not smoothly conveyed through the biofilm. As a result, the leaves start to die, and even if rice dies or survives, rice can not be produced normally. Therefore, inhibiting the formation of biofilm by Xanthomonas oryzae can reduce clogging of the rice conduit, and even if infected with Xanthomonas oryzae, the rice can grow normally so that the yield of rice can be maintained normally.

The composition for controlling the blight of blight of blight of rice may be in the form of a directly sprayable solution, a powder and a suspension, or a highly concentrated aqueous, oily or other suspension, dispersion, emulsion, oil dispersion, paste, But is not limited thereto. In addition, the composition for controlling can be used by spraying, spraying, spraying, scattering or swelling.

In addition, the composition for controlling blistering of blighted leaves can be formulated in various forms, for example, by adding a solvent and / or a carrier. Suitable inert additives and surface-active materials, such as emulsifiers or dispersants, may be incorporated into the formulation.

According to one embodiment of the present invention, the composition for controlling blight of blight of blight of rice may include a composition for inhibiting biofilm formation comprising peppermint methanol extract, peppermint water extract, peppermint 50% ethanol extract or peppermint 95% ethanol extract .

Since the peppermint extract can effectively inhibit the formation of the biofilm formed by the microorganism, it can be effectively used as a composition for inhibiting the formation of a biofilm and a composition for preventing or treating a disease associated with the formation of a biofilm.

Figure 1 is a mint extract Yersinia Enterococcus coli urticae (Yersinia enterocolitica ): A is a methanol extract of peppermint, B is a 50% ethanol extract of peppermint, C is a 95% ethanol extract of peppermint, and the 'dilution ratio' of X-axis is peppermint extract Of the dilution ratio.
FIG. 2 is a graph showing the effect of peppermint extract on the growth of Yersinia enterocolitica: A is a methanol extract of peppermint, B is a 50% ethanol extract of peppermint, C is a 95% ethanol extract of peppermint, and X The 'dilution rate' of the shaft means the dilution rate of the peppermint extract.
FIG. 3 is a graph showing the results of confirming the effect of the methanol extract of peppermint on the formation of biofilm by Aeromonas hydrophila and the growth of eromonas hydrofilas : A is the biofilm yield, B is the cell density And the 'dilution ratio' on the X axis means the dilution ratio of the methanol extract of peppermint.
4 is a graph showing the inhibitory effect of the peppermint extract on Xanthomonas oryzae : A is a peppermint methanol extract, B is peppermint water extract, C is peppermint 50% ethanol extract, D is the result of a mint 95% ethanol extract, and the 'dilution ratio' on the X axis is the dilution rate of the peppermint extract.
FIG. 5 is a graph showing the effect of the peppermint extract on the growth of xanthomonas duck: A is a peppermint methanol extract, B is peppermint water extract, C is peppermint 50% ethanol extract and D is peppermint 95% ethanol extract And the 'dilution rate' on the X axis means the dilution rate of the peppermint extract.

Hereinafter, one or more embodiments will be described in more detail by way of examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  1: Production of peppermint extract

100 ml of water, methanol, 50% ethanol and 95% ethanol were added to 10 g of peppermint and extracted for 3 hours in a constant temperature water bath at 50 캜. Then, the extract was firstly 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 used in the experiment. 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. To the filtrate was added water as much as the volume of the primary extract, and further filtered with a cellulose acetate filter and used in the experiment.

Example  2: Inhibitory effect of peppermint extract on biofilm formation

2-1. Yersinia Enterocolitica  Effect of inhibiting biofilm formation in Korea

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 cultured at 26 ° C for 2 days. One single colony was taken from the plate and inoculated into 5 ml of TYE liquid medium and incubated at 26 [deg.] C for one day with shaking at 250 rpm.

To a 96-well polyvinyl chloride (PVC) microplate was added M63 liquid medium (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 ₄ , 0.2% glucose, 0.02% MgSO ₄ ) were dispensed into each well. The peppermint extract (1 占 퐇 of the methanol extract, 5 占 퐇 each of the 50% ethanol extract and the 95% ethanol extract) prepared in Example 1 was further added. Next, the cultured Y. enterocolitica was inoculated in each well to an OD ₆₀₀ of 0.05 and cultured at 26 for 24 hours. After 24 hours, the optical density (OD) was measured at OD ₅₉₅ to confirm the cell density of Y. enterocolitica . After the culture was removed from the plate, the cells were washed gently with distilled water three times. 100 쨉 l 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 three times with distilled water. To elute the crystal violet, 100 mu l of 95% ethanol was added and left for 15 minutes. Then, the absorbance was measured with a microplate reader (OD ₅₉₅ ), and the amount of the biofilm formed was determined.

As a result, as shown in FIG. 1, it was confirmed that the biofilm formation was remarkably inhibited by the peppermint methanol extract, the 50% ethanol extract and the 95% ethanol extract.

2-2. Effect of inhibiting biofilm formation on eromonas hydrofilas

Aeromonas Hydrophila hydrophila ; Hereinafter referred to as A. hydrophila ) was plated on a TSA plate (tryptic soy agar plate (TSB medium supplemented with 1% agar) and then cultured at 30 ° C for 1 day. One single cluster was taken from the plate and inoculated into 5 ml of LB (Luria bertani, Bacto ^TM ) liquid medium and cultured at 30 ° C for 1 day with shaking at 250 rpm.

The inhibitory effect of the methanol extract on the biofilm formation of the peppermint methanol extract was confirmed in the same manner as in 2-1, except that TSB-Y liquid medium (TSB liquid medium containing 0.6% yeast extract) was used in 96-well polyvinyl chloride microplate. Respectively.

As a result, as shown in FIG. 3, it was confirmed that the biofilm formation was significantly inhibited by the methanol extract of peppermint.

2-3. Effect of inhibiting biofilm formation on Xanthomonas duck ash

Xanthomonas oryzae (hereinafter referred to as X. oryzae ) was plated on a YGC agar plate (5% glucose, 0.5% yeast extracts, 1.25% CaCO _{3 ,} 1.5% agar) Lt; / RTI > One plate was 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 cultured with shaking at 250 rpm for 2 days.

To a 96-well polyvinyl chloride microplate was added XOM2 liquid medium (670 μM L-methionine, 10 mM sodium L - (+) - glutamate monohydrate, 14.7 mM KH ₂ PO ₄ , 40 μM MnSO ₄ , 240 μ Fe _{mM MgCl 2, 0.18% D-} xylose,, except that the pH6.5) peppermint extract in the same manner as 2-1 (1㎕ methanol extract, water extract and the 50% ethanol extract and the 95% ethanol extract of each 5 쨉 l) was confirmed to inhibit the formation of biofilm.

As a result, as shown in FIG. 4, it was found that the biofilm formation was remarkably inhibited by the peppermint methanol extract, the peppermint water extract, the peppermint 50% ethanol extract and the peppermint 95% ethanol extract.

It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Accordingly, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (9)

A composition for inhibiting the formation of a biofilm containing an extract of peppermint as an active ingredient.
The method of claim 1, wherein the peppermint extract is selected from the group consisting of purified water, hexane, 1,3-butylene glycol, an alcohol having 1 to 4 carbon atoms, ethyl acetate, diethyl ether, dichloromethane, acetone, Lt; / RTI > solvent.
The method of claim 1, wherein the biofilm is selected from the group consisting of Yersinia sp., Aeromonas sp. sp.) and the genus Xanthomonas sp.). < / RTI >
The method of claim 3, wherein said microorganism is Yersinia Enterococcus coli urticae (Yersinia enterocolitica), Erotic Pseudomonas dihydro pillar (Aeromonas hydrophila and Xanthomonas oryzae. < RTI ID = 0.0 > 11. < / RTI >
A pharmaceutical composition for preventing or treating diseases caused by yersinia enterocolitica, which comprises the composition of claim 1 as an active ingredient.
6. The composition according to claim 5, wherein said disease is selected from the group consisting of acute gastroenteritis, endophthalmitis, sepsis, tuberculous eruption, food poisoning and enteritis.
A pharmaceutical composition for preventing or treating necrotizing fasciitis caused by eromonas hydrofilas containing the composition of claim 1 as an active ingredient.
A composition for controlling the dropsy disease of fish caused by eromonas hydrofilas, comprising the composition of claim 1 as an active ingredient.
A composition for controlling bacterial leaf blight of rice, comprising the composition of claim 1 as an active ingredient.

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