KR20190041310A - Composition for Preventing Biofilm Formation Comprising Extract of Elsholtzia ciliata - Google Patents

Abstract

The present invention relates to a composition for inhibiting formation of a biofilm containing an Elsholtzia ciliata extract as an active ingredient, and a composition for preventing and alleviating diseases related to formation of a biofilm. The Elsholtzia ciliata 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 an extract of fragrance 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).

Streptococcus mutans ( Streptococcus mutans ; S. mutans ) is an important cause of dental caries (tooth decay) by attaching to the surface of teeth in the oral cavity. S. Mutans are resistant to external physical and chemical treatments by synthesizing glucan, a glucan, by using glucose, and by forming a biofilm with the produced glucan and adhering to the teeth.

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 the natural fragrance extract has excellent ability to inhibit biofilm formation.

1. Korean Patent 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 a microorganism containing an extract of Fragrances 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, which comprises an essence 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.

The perfume oil ( Elsholtzia ciliata) used as an active ingredient of the composition for inhibiting the formation of a biofilm of the present invention (Thunb.) Hylander) is a oriental herb of Lamiaceae and is used for edible, public and medicinal purposes. Sweating, diuretic, species, antipyretic, and hemostatic effects, but the effect of inhibiting the formation of biofilms by microorganisms is unknown.

According to one embodiment of the present invention, the above-described fragrance extract can be produced by a conventional method known in the art. For example, the fragrant oil may be pulverized, and then a solvent commonly used for extraction may be added, followed by extraction at an appropriate 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 above-mentioned fragrant extract includes both the extract obtained by the above-mentioned solvent extraction method and the extract obtained through a usual purification process. 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 fragrance extract of the present invention. In addition, the above-mentioned fragrant extract can be produced in powder form by an additional process such as vacuum distillation, freeze-drying, spray drying and the like.

The composition for inhibiting the formation of a biofilm of the present invention may contain 0.1 to 20% by weight or 1 to 10% by weight of the above-mentioned fragrant extract, based on the total weight of the composition, Can be adjusted appropriately.

According to one embodiment of the present invention, the biofilm is selected from the group consisting of Streptococcus sp., Xanthomonas sp. but are not limited to, microorganisms selected from the group consisting of sp. and Yersinia sp.

According to one embodiment of the present invention, the microorganism is selected from the group consisting of Streptococcus mutans , Xanthomonas oryzae), and Yersinia Enterococcus coli urticae (Yersinia enterocolitica ), but the present invention is not limited thereto.

According to one embodiment of the present invention, dental caries and periodontitis can be caused by biofilm formed by Streptococcus mutans, and when xanthomonas duck material forms a biofilm in the duct of rice, . In addition, food poisoning, enteritis and the like can be induced by the biofilm formed by Yersinia enterocolitica.

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 oral diseases comprising the composition for inhibiting biofilm formation as an active ingredient.

According to one 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. The oral microorganisms may be selected from the group consisting of Streptococcus mutans , Streptococcus salivarius , Streptococcus gordonii , Streptococcus sobrinus , Streptococcus sanguis ), Streptococcus pyogenes, Lactobacillus acidophilus , Lactobacillus casei , Actinobacillus viscosus , Agregatibacter actinomycetemcomitans, (Aggregatibacter actinomycetemcomitans), Fort fatigue Monastir seriously balises (Porphyromonas gingivalis , Tannerella forsythia , Treponema denticola , Prevotella intermedia, intermedia , and Fusobacterium nucleatum . < RTI ID = 0.0 >

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 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 the blight of blight of blight of rice may include a composition for inhibiting biofilm formation, which comprises an extract of spermine methanol, a water extract or a 50% ethanol extract.

Yet another aspect of the present invention provides a pharmaceutical composition for preventing or treating diseases caused by yersinia enterocolitica containing 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). In humans, various diseases such as acute gastroenteritis, endophthalmitis, sepsis and tuberculous erythema are generated, and it is known that they have resistance to external stimuli or antibiotics by forming biofilms.

According to one embodiment of the present invention, the fragrant oil extract can effectively inhibit biofilm formation by yersinia enterocolitica, so that 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.

Since the fragrant oil extract effectively inhibits the formation of the biofilm formed by the microorganism, it can be effectively used as a composition for inhibiting biofilm formation and a composition for preventing or treating diseases related to the formation of biofilm.

FIG. 1 is a graph showing the effect of the methanol extract of the present invention on the biofilm formation by Streptococcus mutans and the growth of Streptococcus mutans : A is the biofilm yield, B is the cell density And the 'dilution ratio' on the X axis means the dilution ratio of the perfume extract.
Figure 2 shows that the perfume extract is a mixture of Yersinia < RTI ID = 0.0 > enterocolitica ): A is a sulphurous methanol extract, B is a 50% ethanol extract, and C is a 95% ethanol extract. The 'dilution ratio' Of the dilution ratio.
FIG. 3 is a graph showing the effect of the fragrance extract on the growth of Yersinia enterocolitica: A is a fragrant methanol extract, B is a 50% ethanol extract of perfume, and C is a 95% ethanol extract of perfume and X The 'dilution ratio' of the shaft means the dilution ratio of the perfume extract.
FIG. 4 is a graph showing the results of confirming the effect of the fragrant extract on the biofilm formation of Xanthomonas oryzae : A is a fragrant methanol extract, B is fragrant water extract, and C is 50% And the 'dilution ratio' on the X axis means the dilution ratio of the perfume extract.
FIG. 5 is a graph showing the results of confirming the effect of the fragrant extract on the growth of xanthomonas duck: A is the result of methanol extract of fennel, B is the extract of fennel, and C is the result of 50% ethanol extract of fennel. &Quot; means the dilution ratio of the perfume 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: Preparation of ointment extract

Water, methanol, 50% ethanol and 100 ml of 95% ethanol were added to 10 g of perfume oil, respectively, and the mixture was extracted in a constant temperature water bath at 50 캜 for 3 hours. 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 extracts, 50% and 95% ethanol extracts were concentrated to 10% of the total volume of extracts by 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 ointment extract on biofilm formation

2-1. Streptococcus Mutans  Effect of inhibiting biofilm formation in Korea

Streptococcus mutans GS-5 (Streptococcus mutans GS- 5; hereinafter abbreviated as S. mutans GS-5) strain is LG Household and Health Care Research Institute (Daejeon, Republic of Korea) received pre-sale BHI medium (Brain Heart Infusion in the medium, Bacto ^TM , BD Biosciences). S. mutans GS-5 was streaked onto BHI agar plates and incubated at 37 [deg.] C for 2 days. One single colony was taken from the plate and inoculated into 5 ml of BHI liquid medium and stationary culture was performed at 37 占 폚 for 1 day.

100 ㎕ of BHI medium containing 1% sugar in 96-well polyvinyl chloride (PVC) microplate was dispensed into each well, and 1 쨉 l of the fragrant methanol extract prepared in Example 1 was further added. Next, S. mutans GS-5, which had been subjected to the above-mentioned incubation, was inoculated into each well at an OD ₆₀₀ of 0.05 and cultured at 37 ° C for 24 hours. After 24 hours, the optical density (OD) was measured at OD ₅₉₅ to determine the presence of S. mutans After confirming the cell density of GS-5, the culture was removed from the plate and gently rinsed three times with distilled water. 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, it was confirmed that the biofilm formation was inhibited by the spermine methanol extract as shown in Fig.

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

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

To the 96-well polyvinyl chloride 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 , (1 μl of methanol extract, 5 μl of 50% ethanol and 95% ethanol extract) was confirmed to inhibit the biofilm formation by the same method as in the above 2-1 except that 0.02% MgSO ₄ was used.

As a result, as shown in FIG. 2, it was confirmed that the biofilm formation was inhibited by the fragrance extract, and the fragrant methanol extract significantly inhibited the biofilm formation.

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, except for using 0.18% D-xylose, pH6.5), and ointment in the same manner as in 2-1 extract (1㎕ methanol extract, water extract, and 50% ethanol extracts of bio 5㎕ added) The film formation inhibiting effect was confirmed.

As a result, as shown in FIG. 4, it was found that the biofilm formation was inhibited by the fragrance extract of all the solvents.

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, which comprises an ointment extract as an active ingredient.
The method of claim 1, wherein the fragrant 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 Streptococcus sp., Xanthomonas sp. sp.) and Yersinia sp.). < / RTI >
The microorganism according to claim 3, wherein the microorganism is Streptococcus mutans , Xanthomonas oryzae oryzae and Yersinia enterocolitica . < RTI ID = 0.0 > 18. < / RTI >
A pharmaceutical composition for preventing or treating oral diseases, comprising the composition of claim 1 as an active ingredient.
6. The composition of claim 5, wherein the oral disease is dental caries or periodontitis.
A composition for controlling bacterial leaf blight of rice, comprising the composition of claim 1 as an active ingredient.
A pharmaceutical composition for preventing or treating diseases caused by yersinia enterocolitica, which comprises the composition of claim 1 as an active ingredient.
9. The composition of claim 8, wherein said disease is selected from the group consisting of acute gastroenteritis, endophthalmitis, sepsis, tuberculous eruption, food poisoning and enteritis.

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