KR102336810B1 - Composition for Preventing Biofilm Formation Comprising Extract of Elsholtzia ciliata - Google Patents

Abstract

It relates to a composition for inhibiting biofilm formation comprising a perfume oil extract and a composition for preventing and improving diseases related to biofilm formation.

Description

Composition for inhibiting biofilm formation containing a perfume oil extract {Composition for Preventing Biofilm Formation Comprising Extract of Elsholtzia ciliata}

The present invention relates to a composition for inhibiting biofilm formation containing a perfume oil extract as an active ingredient, and a composition for preventing and treating diseases related to biofilm formation.

Biofilm, also called 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).

Streptococcus mutans ( Streptococcus mutans ; S. mutans ) is an important causative agent that attaches to the tooth surface in the oral cavity and causes dental caries (cavities). S. Mutans synthesizes a polysaccharide called glucan using glucose, forms a biofilm with the generated glucan, and attaches it to the teeth, thereby having resistance 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 contamination of medical devices include Enterococcus faecalis. faecalis ) and Staphylococcus aureus , and the like, and may cause osteomyelitis, toxin shock syndrome, food poisoning and skin infections due to contamination of the microorganisms.

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 past. However, when the growth of microorganisms forming a biofilm is inhibited with a material having antibacterial activity, 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, perfume oil 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 biofilm formation of microorganisms containing a perfume oil 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 biofilm formation containing a perfume oil extract as an active ingredient.

As used herein, the term 'biofilm (biofilm)' refers to a membrane-like structure formed by microorganisms, and has structural features composed 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.

Fragrance oil (Elsholtzia ciliata) used as an active ingredient of the composition for inhibiting biofilm formation of the present invention (Thunb.) Hylander) is an aromatic herb in the family Lamiaceae and is used for food, public health and medicinal purposes. It is known to have antiperspirant, diuretic, hydrocephalus, antipyretic and hemostatic effects, but the effect of inhibiting the formation of biofilms by microorganisms is not known.

According to one embodiment of the present invention, the perfume oil extract may be prepared according to a conventional method known in the art. For example, after pulverizing the perfume oil, a solvent commonly used for extraction may be added, and the perfume oil extract may be prepared 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, 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 perfume oil extract includes both the extract by the solvent extraction method described above 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 fragrance oil extract of the present invention. In addition, the perfume oil 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 contain the fragrance oil 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, and the content of the active ingredient according to the method and purpose of use of the composition can be appropriately adjusted.

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

According to an embodiment of the present invention, the microorganism is Streptococcus mutans ( Streptococcus mutans ), Xanthomonas duck material ( Xanthomonas oryzae ) and Yersinia enterocolica ( 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 duck material forms a biofilm in the conduit of rice, it may cause white leaf blight of rice. . In addition, food poisoning, enteritis, etc. may be induced by the biofilm formed by Yersinia enterocolica.

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 in order 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 oral diseases containing 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. 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 ( Streptococcus pyogenes ), Lactobacillus acidophilus ( Lactobacillus acidophilus ), Lactobacillus casei ( Lactobacillus casei ), Actinobacillus viscous sus ( Actinobacillus viscosus ), Aggregatibacter ( Aggregatibacter actinomycetemcomitans ), Porphyromonas gingivalis ( Porphyromonas gingivalis ), Tannerella forsythia , Treponema denticola , Prevotella Intermedia intermedia ) and Fusobacterium nucleatum ( Fusobacterium nucleatum ) It may be selected from the group consisting of.

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. As a typical example of a formulation for parenteral administration, an isotonic aqueous solution or suspension is preferred 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 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 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, weight, individual differences and disease state. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the 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 the disease that causes the most economic loss in rice production and occurs widely around the world. When Xanthomonas oryzae adsorbs 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, so that the rice yield can be maintained normally.

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 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, for example, it may be prepared by adding a solvent and/or a carrier. Suitable inert additives and surface-active substances, for example emulsifying or dispersing agents, may be admixed 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 perfume oil methanol extract, a water extract, or a 50% ethanol extract.

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

According to one embodiment of the present invention, since the perfume oil extract can effectively inhibit the biofilm formation by Yersinia enterocolytica, the composition for inhibiting biofilm formation is used for preventing or treating diseases caused by Yersinia enterocolica. It can be usefully used for

Fragrance oil extract can effectively inhibit the formation of biofilms generated by microorganisms, so 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 a methanol extract of perfume oil on biofilm formation and growth of Streptococcus mutans by Streptococcus mutans: A is a biofilm production amount, B is a cell density measurement result , and the 'dilution ratio' on the X-axis means the dilution ratio of the fragrance oil extract.
Figure 2 is a Yersinia enterocolica (Yersinia extract) enterocolitica ) is a graph showing the results confirming the effect on biofilm formation: A is a methanol extract of perfume oil, B is a 50% ethanol extract of perfume oil, and C is a result of a 95% ethanol extract of perfume oil, and the 'dilution ratio' on the X-axis is a perfume oil extract of the dilution ratio.
3 is a graph showing the results confirming the effect of the perfume oil extract on the growth of Yersinia enterocolitica: A is a methanol extract of perfume oil, B is a 50% ethanol extract of perfume oil, and C is a result of a 95% ethanol extract of perfume oil, X The 'dilution ratio' on the axis means the dilution ratio of the fragrance oil extract.
Figure 4 is a graph showing the results confirming the effect of the perfume oil extract on the biofilm formation of Xanthomonas oryzae : A is a perfume oil methanol extract, B is a perfume oil water extract, and C is a perfume oil 50% ethanol extract result , and the 'dilution ratio' on the X-axis means the dilution ratio of the fragrance oil extract.
5 is a graph showing the results of confirming the effect of the fragrance oil extract on the growth of Xanthomonas duckjae: A is a methanol extract of fragrance oil, B is a fragrance oil water extract, and C is a fragrance oil 50% ethanol extract result, and the X-axis 'dilution ratio'' means the dilution ratio of the fragrance oil extract.

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 fragrance oil 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 water bath at 50° C. for 3 hours. Then, 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% 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 perfume oil extract

2-1. Streptococcus in mutans Inhibitory effect on biofilm formation

Streptococcus mutans GS-5 (Streptococcus mutans GS-5; hereinafter referred to as S. mutans GS-5) strain was purchased from LG Household & Health 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 cultured at 37° C. for 2 days. A single colony was taken from the plate, inoculated into 5 ml of BHI broth, and stationary culture was performed 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 the methanol extract of perfume oil prepared in Example 1 was additionally dispensed. Next, the stationary cultured S. mutans GS-5 was _{inoculated into each well to have an OD 600} of 0.05 and cultured at 37° C. for 24 hours. After 24 hours, by measuring the absorbance (optical density, OD; absorbance) at _{OD 595} , S. mutans The cell density of GS-5 was checked, and the culture medium was removed from the plate, and then 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. Then, 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 inhibited by the perfume oil methanol extract.

2-2. Yersinia in Enterocolica Inhibitory effect on biofilm formation

Yersinia Enterococcus coli urticae (Yersinia enterocolitica ; Hereinafter, Y. enterocolitica ) was smeared on a TYE agar plate (1% tryptone, 0.5% yeast extract, 1.5% agar), and then cultured at 26°C for 2 days. 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 ₄ , 0.2% glucose, 0.02% MgSO ₄ ), the biofilm formation inhibitory effect of the perfume oil extract (1 μl of methanol extract, 5 μl of 50% ethanol and 95% ethanol extract added) was confirmed in the same manner as in 2-1 above, except that it was used.

As a result of the confirmation, as shown in FIG. 2 , it was confirmed that the biofilm formation was inhibited by the perfume oil extract, and in particular, the perfume oil methanol extract significantly inhibited the biofilm formation.

2-3. Inhibitory effect of biofilm formation on Xanthomonas duck material

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.

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 In the _{same manner as in 2-1, except that mM MgCl 2} , 0.18% D-xylose, pH6.5) was used, biomass of the perfume oil extract (1 μl of methanol extract, 5 μl of water extract and 50% ethanol extract was added) The film formation inhibitory effect was confirmed.

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

Those of ordinary skill in the art to which the present invention pertains will 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 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 (9)

A composition for inhibiting biofilm formation containing a perfume oil extract as an active ingredient, the composition comprising:
The biofilm is Streptococcus mutans ( Streptococcus mutans ), Xanthomonas duck material ( Xanthomonas oryzae ) and Yersinia enterocolitica ( Yersinia enterocolitica ) The composition is formed by a microorganism selected from the group consisting of.
According to claim 1, wherein the perfume oil 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.
delete delete delete delete A composition for controlling bacterial leaf blight of rice, containing the composition of claim 1 as an active ingredient.
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