KR20190041309A - Composition for Preventing Biofilm Formation Comprising Extract of Prunellae Spica - Google Patents

Abstract

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

[0001] 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 comprising an extract of Chrysanthemum morifolium as an active ingredient and a composition for the prevention and treatment of 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 completed the present invention by confirming that the natural extract and the extract of supernatant are excellent in the 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 which contains an 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 an extract of chrysanthemum 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 prunellae spica used as an active ingredient of the composition for inhibiting the formation of biofilm of the present invention is a perennial plant belonging to the family Lamiaceae ( Prunella vulgaris Linne). It contains triterpenoid, saponin, ursolic acid, hyperoxide, tannin, and caffeic acid. The drug has been found to be effective in the treatment of the liver, It is known to have efficacy in mastitis, breast cancer, diuretic, shinhae, hemostasis, mental anxiety, and acne. However, the effect of inhibiting microbial biofilm formation is not known.

According to one embodiment of the present invention, the herb extract and the herbal extract may be prepared according to a conventional method known in the art. For example, the extract may be prepared by pulverizing the supernatant, adding a solvent conventionally used for extraction, and extracting the supernatant 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 supernatant includes both the extract obtained by the solvent extraction method and the extract obtained through the ordinary 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 extracts of the present invention. In addition, the herb extract can 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 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 can be adjusted appropriately.

According to one embodiment of the present invention, the biofilm is selected from the group consisting of Yersinia sp. And Xanthomonas sp. sp.), but the present invention 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 and the like can be induced by the biofilm formed by Yersinia enterocolitica, and when Xanthomonas duck material forms a biofilm in the duct of rice, .

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 (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, it is possible to effectively inhibit the formation of biofilm by the extract of Eucalypta enterocolitica, so that the composition for inhibiting biofilm formation can be used for preventing or preventing diseases caused by yersinia enterocolitica, And can be usefully used for therapeutic purposes.

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 comprising a methanol extract or a supernatant extract.

And the extract can be effectively used as a composition for inhibiting biofilm formation and a composition for preventing or treating diseases related to biofilm formation since it can effectively inhibit the formation of biofilm formed by microorganisms.

FIG. 1 is a graph showing the results of confirming the effect of the extract on the formation of biofilm of Yersinia enterocolitica : A is a methanol extract, B is a 50% ethanol extract, C is a methanol extract, And a 95% ethanol extract, and the 'dilution rate' on the X axis means the dilution rate of the extract.
FIG. 2 is a graph showing the results of confirming the effect of the extract on the growth of Yersinia enterocolitica: A, methanol extract, B and 50% ethanol extract, C and 95% ethanol extract And the 'dilution rate' on the X axis means the dilution rate of the extract of the primate.
FIG. 3 is a graph showing the inhibitory effect of Xanthomonas oryzae on the biofilm formation of the extract of A. xanthomonas oryzae : A, B, Dilution ratio " means the dilution ratio of the herbal extract.
FIG. 4 is a graph showing the results of confirming the effect of the extract on the growth of Xanthomonas duck: A, H, and B are the results of the supernatant water extract, the X-axis 'dilution ratio' Of the dilution ratio.

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  One: And seconds  Extract preparation

And 100 ml of water, methanol, 50% ethanol and 95% ethanol were added to 10 g / sec, and the mixture was extracted for 3 hours in a constant temperature water bath at 50 ° C. 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: And seconds  Effect of extract on inhibition of 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. 1 [mu] l of the methanol extract prepared in Example 1 was dispensed, and 5 [mu] l of each of the 50% ethanol and 95% ethanol extracts was dispensed. Next, the cultured Y. enterocolitica was inoculated in each well to an OD ₆₀₀ of 0.05 and cultured at 26 ° C 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 extract of the primate.

2-2. Xanthomonas Duck in the ashes  Effect of inhibiting biofilm formation in Korea

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 (using 1 μl of the methanol extract and 5 μl of the water extract) was used in the same manner as in the above 2-1, except that the concentration of the extract of the present invention was changed to 1 mg / ml MgCl ₂ , 0.18% D-xylose, Respectively.

As a result, as shown in FIG. 3, it was found that the biofilm formation was significantly inhibited by the methanol extract and the methanol 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 (7)

Wherein the composition further contains a supernatant extract as an active ingredient.
The method according to claim 1, wherein the herb 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. And Xanthomonas sp. sp.). < / RTI >
The composition according to claim 3, wherein the microorganism is selected from the group consisting of Yersinia enterocolitica and Xanthomonas oryzae .
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 composition for controlling bacterial leaf blight of rice, comprising the composition of claim 1 as an active ingredient.

Images