KR101409266B1 - Inhibitors of Biofilm and Methods therefor - Google Patents

Abstract

The present invention relates to a biofilm inhibitor and a method of inhibiting a pathogenic microorganism, and more particularly, A biofilm inhibitor and a method of inhibiting comprising at least one substance selected from the group consisting of chrysin, curcumin, daidzein, flavones, genistein and phloretin. . The biofilm inhibitor suppresses biofilm formation by inhibiting surface adhesion of pathogenic microorganisms and is effective for the treatment of inflammatory bowel disease such as colitis.

Description

Inhibitors of Biofilm and Methods therefor < RTI ID = 0.0 >

The present invention relates to biofilm inhibitors and methods of inhibition.

Enterohemorrhagic pathogenic Escherichia coli is a human pathogen that infiltrates into the intestinal tract due to contamination of food or hemorrhagic colitis, that is, blood that can lead to hemolytic uraemia. Recently, it has become known that these pathogens can adhere to and survive on surfaces that are not normally suspended. Thus, biofilm produced by potential pathogens is considered to be an important cause of chronic and recurrent infections, especially due to the ability to form and sustain biofilms on the surfaces of medical devices and in the human body. Recently, the number of studies related to biofilm has increased tremendously, and most of them are aimed at prevention, control, and eradication of fundamental biofilm. In the meantime, very few studies have been conducted on E. coli including pathogenic Escherichia coli , Which is a natural substance that inhibits biofilm. Algae Delisea (5Z) -4-bromo-5- (bromomethylene) -3-butyl-2 (5H) -furanone produced from the pulchra and Dios pyros The compounds such as ursolic acid (3 beta -hydroxy-urs-12-en- 28-oic acid), Group Ⅱ polysaccharide capsule shape that is generated by the non-pathogenic coliform bacteria originating from dendo, the formation of a biofilm significantly Respectively. However, the toxicity of these compounds and their poor water solubility limit their use in the food and pharmaceutical industries.

Therefore, it is urgent to develop a new natural substance which does not inhibit cell growth, is toxic to the human body, is stable, and has excellent biofilm inhibiting effect.

The present invention provides aminoflavone, hydroxyflavone, A biofilm inhibitor and a method of inhibiting comprising at least one substance selected from the group consisting of chrysin, curcumin, daidzein, flavones, genistein and phloretin. .

The present invention relates to a pharmaceutical composition comprising aminoflavone, hydroxyflavone, chrysin, curcumin, daidzein, flavones, genistein and phloretin, Wherein the biofilm inhibitor comprises at least one substance selected from the group consisting of:

The biofilm may be a biofilm of a pathogenic microorganism.

The pathogenic microorganism is Enterobacter pesso transgenic S. serie Escherichia coli (enteropathogenic Escherichia coli), Enterobacter H. Mora S. straight serie Escherichia coli (enterohaemorrhagic Escherichia coli), Pseudomonas air rugi labor (Pseudomonas aeruginosa or Staphylococcus aureus .

The pathogenic microorganism may cause inflammatory diseases.

The substance can regulate the expression of toxin genes, autoinducer-2 importer genes, curli genes or prophage genes.

Such a substance can inhibit the formation of a biofilm by inhibiting the surface adhesion of the pathogenic microorganism.

The surface may be the surface of an organism-derived tissue, organ or medical article.

The present invention relates to a pharmaceutical composition comprising aminoflavone, hydroxyflavone, chrysin, curcumin, daidzein, flavones, genistein and phloretin And treating the surface of the organism or the non-living body with at least one substance selected from the group consisting of the above-mentioned substances.

The amino flavone, hydroxy flavone, One or more substances selected from the group consisting of chrysin, curcumin, daidzein, flavone, genistein and floretine not only inhibit the biofilm of the pathogenic microorganism but also inhibit the biofilm of the pathogenic microorganism, Since it does not show toxicity, it is stable in living body, and it can be applied to various fields such as pharmacy and food science.

FIG. 1A shows aminoflavone, hydroxyflavone, and aminoflavone for E. coli O157: H7 biofilm formation. The effect of chrysin, curcumin, daidzein, flavones, genistein and phloretin on total biofilm formation (OD ₅₇₀ ) It was standardized by (OD ₆₂₀₎ (normalized). 1B shows the effect of inhibiting E. coli O157: H7 biofilm formation with increasing content of floretin.
Fig. 2 is a graph showing the results of the addition of the mutagenic E. coli K-12 BW25113, MG1655, TG1 strain, DH5α and non-pathogenic E. coli ATCC 4157 inhibited the formation of biofilm.
Figure 3 shows the pathogenic E. coli O157: H7 biofilm cells and (3B) symbiotic E. coli (50 μg / ml) when black bars or white bars (3A) K-12 BW25113 The biofilm gene expression was measured by qRT-PCR, and the relative fold expression was compared with the data of white bars (value of 1.0) -fold).
Figure 4 shows the effect of phloretin on pimbrion production in E. coli O157: H7 biofilm cells. Biofilm cells grown in the presence or absence of phloretin (50 [mu] g / ml) will be.
Figure 5 shows the effect of E. coli Cell HT-29 < / RTI > human colonic epithelial cells. FIG. 5A shows the effect of blocking E. coli O157: H7 cells or the symbiotic E. coli K-12 BW25113 cells, and Figures 5B and 5C show the appearance of HT-29 cells in the presence or absence of phloretin (200 μM corresponding to 54.8 μg / ml) Of pathogenic E. coli (Fig. 5B) and the number (Fig. 5C) of O157: H7 cells.
Figure 6 shows the inhibitory effect of phloretin on the adhesion of TNF- [alpha] induced U937 cells to HT-29 human colon epithelial cells, in the presence or absence of phloretin (200 [mu] M corresponding to 54.8 [ (6A) and light intensity (6B) of a U937 cell against HT-29 cells.
FIG. 7 shows the relaxation of TNBS-induced colitis by phloretin in rats, FIG. 7A is a record of mouse body weight from day 1 to 7 days, FIG. 7B is a graph showing the body weight of the colon Fig. 7C shows the wet weight of the distal 5 cm colon region, and Fig. 7D shows the MPO activity of the colon tissue.

The present invention provides biofilm inhibitors and methods of inhibition.

Hereinafter, the present invention will be described in detail.

Antibiotics, which are conventionally used as a therapeutic agent for diseases caused by pathogenic microorganisms, have a problem of tolerance due to drug resistance, and a biofilm inhibitor has been discussed as a solution to this problem. However, biofilm inhibitors have the advantage that they do not cause resistance problems without affecting cell growth, but they also inhibit all the biofilms of symbiotic microorganisms which are beneficial to the host as well as the pathogenic microorganisms. In the present invention, in attempting to develop a therapeutic strategy for controlling only the growth of a biofilm of a pathogenic microorganism without affecting the biofilm of the microorganism which does not cause intracellular toxicity, And the present invention has been completed.

The present invention provides aminoflavone, hydroxyflavone, There is provided a biofilm inhibitor comprising at least one substance selected from the group consisting of chrysin, curcumin, daidzein, flavones, genistein and phloretin .

The microorganisms generally have the property of moving along the fluid and adhering firmly to the surface of the solid medium. The biofilm of the present invention is a layer formed by bonding microorganisms adhered to the surface of a solid medium with extracellular polymeric substance (EPS) secreted by microorganisms. The biofilm is a layer formed of almost all kinds of solids Can be formed on the surface.

The biofilm is commonly found in living, medical and industrial environments that are frequently encountered in the environment. In the case of pathogenic microorganisms which can live in the living body, various kinds of tissues / organs including the host's epithelial cells, bones, teeth, and inner wall of vessels, various artificial implants (catheter, implant) A film can be formed. When biofilm is formed, microorganisms can not only survive in harsh environments but also have a strong resistance to antibiotics and immune cells. Therefore, they are very difficult to remove, cause chronic inflammatory diseases, and cause microbiologically induced corrosion cause. Recent reports indicate that about 65% of all infectious diseases are caused by biofilm formation (Ymele-Leki and Ross, 2007, Applied and Environmental Microbiology 73 (6): 1834-41). Therefore, the inhibition of biofilm by the substance of the present invention may be very important for prevention of corrosion by microorganisms or prevention / treatment of infection by pathogenic microorganisms.

The substance of the present invention may be selected from aminoflavone, hydroxyflavone, Chrysin, curcumin, daidzein, flavones, genistein and phloretin inhibit the biofilm of pathogenic microorganisms as well as inhibit the release of naturally occurring compounds 1 shows that phlorectin is a safe compound which not only exhibits the best biofilm inhibiting effect but also exhibits no toxicity among the above substances, as shown in Fig. 1.

In addition, most conventional biofilm inhibitors showed cytotoxicity leading to drug resistance of microorganisms, but the inhibitor of the present invention did not show cytotoxicity. One example of such cytotoxicity can be confirmed by confirming the growth rate of planktonic cells. As shown in Example 2, the growth rate of floating cells was the same regardless of the presence of floretine in the case of floretine. In the absence of floretine, 1.32 ± 0.01 / h and 50 μg / ml of floretine were present 1.31 0.03 / h, confirming that the materials of the present invention are not toxic.

The biofilm inhibitor of the present invention can selectively inhibit the biofilm of pathogenic microorganisms.

That is, the biofilm inhibitor of the present invention does not affect biofilms of beneficial microorganisms that are symbiotic with the host microorganisms. Specifically, the biofilm inhibitors of the present invention may specifically inhibit the biofilms of pathogenic microorganisms. For example, E. coli Strain It was confirmed that only E. coli O157: H7, which is a pathogenic microorganism, was inhibited without affecting MG1655, TG1, BW25113, DH5α and ATCC 4157.

The type of pathogenic microorganism that is formed inhibits the biofilm in the present invention is not particularly limited, Sirius Bacillus (Bacillus cereus), Agrobacterium Tome Pacifico Enschede (Agrobacterium tumefaciens), Vibrio earn nipi Caicos (Vibrio vulnificus), Vibrio cholera (Vibrio cholerae), Vibrio para Molly Tee hee Caicos (Vibrio parahaemolyticus), Vibrio habe children (Vibrio harveyi), Enterobacter pesso transgenic S. serie Escherichia coli (enteropathogenic Escherichia coli ), enterohaemorrhagic Escherichia coli ), enterotoxigenic Escherichia coli ), enteroinvasive Escherichia coli ), enteroaggregative Escherichia coli , Salmonella enterica enterica), Salmonella Entebbe utility disk (Salmonella enteritidis , Salmonella Typhimurium , Klebsiella pneumoniae , Shigella < RTI ID = 0.0 > flexneri , Pseudomonas aeruginosa , Streptococcus mutans, mutans ), Staphylococcus ( Staphylococcus aureus) aureus , Candidad albicans , Yersinia < RTI ID = 0.0 > enterocolitica , Listeria monocytogenes , and preferably enteropathogenic Escherichia coli, coli ), enterohaemorrhagic Escherichia coli ), Pseudomonas aeruginosa , Staphylococcus ( Staphylococcus aureus) aureus , and more preferably, enterohaemorrhagic Escherichia coli . For example, the pathogenic E. coli may be E. coli O157: H7.

Examples of the diseases caused by the pathogenic microorganisms include, but are not limited to, cystic fibrosis, prostatitis, peridontitis, pneumonia, septicemia, ), Inflammatory diseases, preferably inflammatory diseases, and more preferably, it can be a colitis disease.

The aminoflavone, hydroxyflavone, Inhibition of biofilm by chrysin, curcumin, daidzein, flavones, genistein and phloretin has been associated with toxin genes, autoinducers, -2 autoinducer-2 importer genes, curli genes, or prophage genes.

In one example of the invention, the tin processing Floresta E. coli In the O157: H7 biofilm cell, the toxic gene ( hlyE ), the autoinducer- 2 infector gene ( lsrA and lsrB ), and curly genes ( csgA and csgB ).

In Escherichia coli, the csg gene group is involved in the formation of these collie , type I phyllo and collagen acid from Escherichia coli chromosome. Curl and type I pili are substances essential for biofilm formation, And the colanic acid fills the space between the microorganisms. At this time, the microorganisms attach to the surface of the substance using the collie and the Type I phyllis and form a connection between the coliform bacteria, Colanic acid forms a biofilm by filling the space between them.

The aminoflavone, hydroxyflavone, One or more substances selected from the group consisting of chrysin, curcumin, daidzein, flavones, genistein, and phloretin may inhibit the surface adhesion of pathogenic microorganisms Formation of a biofilm can be suppressed.

The surface may include the surface of an organism or an organism in which the microorganism is capable of forming a biofilm, and may preferably be a tissue, an organ, or a medical article derived from an organism.

For example, various artificial implants applied to biological tissues such as various medical apparatuses, medical equipment, medical facilities / facilities, as well as various living tissues / organs including epithelial cells, bones, teeth, It can be used for implants.

The substance of the present invention is not only excellent in inhibiting the formation of biofilm of pathogenic microorganisms, but also is a compound isolated from nature and does not exhibit toxicity leading to drug resistance to microorganisms. Therefore, it is stable to living organisms and biofilm formation by pathogenic microorganisms Can be widely used in the home, environment, medical, and industrial fields.

At least one substance selected from the group consisting of the amino flavone, hydroxyflavone, chrysin, curcumin, daidzein, flavone, genistein and floretine may be used in an amount effective to prevent or inhibit biofilm formation. The effective amount can be determined according to the conditions including the kind of the pathogenic microorganisms forming the biofilm, the kind and the area of the surface to be treated, the degree of reduction of the desired biofilm formation, the treatment time point and the like, Based on your knowledge of film formation, you will be able to select the appropriate concentration.

The biofilm inhibitor of the present invention can be prepared in various forms in liquid form, spray form or solid form, for specific purposes in the home, industrial, medical, and environmental fields.

In one example of the present invention, aminoflavone, hydroxyflavone, The pharmaceutical composition of the present invention has a biofilm inhibitory activity comprising at least one substance selected from the group consisting of chrysin, curcumin, daidzein, flavones, genistein and phloretin. To provide a food composition.

The food composition of the present invention may contain a sweetening agent, a flavoring agent, a physiologically active ingredient, a mineral, and the like in addition to the above-mentioned materials, and may contain preservatives, emulsifiers, acidifiers, thickeners and the like as necessary.

The food composition may be prepared in the form of various foods, beverages, gums, tea, vitamin complex, health functional foods, and the like. For example, it can be manufactured in the form of gum or tea to inhibit the formation of biofilm in the oral cavity to prevent corrosion of teeth, prevention of tartar formation, prevention and improvement of periodontal disease, and improvement of mouthfeel.

In one example of the present invention, aminoflavone, hydroxyflavone, A pathogenic Escherichia coli biofilm comprising at least one substance selected from the group consisting of chrysin, curcumin, daidzein, flavones, genistein and phloretin. A pharmaceutical composition for preventing or treating an inflammatory disease that exhibits an inhibitory activity can be provided.

The inflammatory disease includes all diseases in which inflammation is induced, preferably inflammatory bowel disease, more preferably colitis and Crohn's disease.

As an example of the present invention, as shown in Example 5, phloretin is converted into E. coli (20 mg / kg / day) was effective in the treatment of inflammation, weight, colon weight and MPO activity. In addition, it was confirmed that ovalbumin 5-ASA (100 mg / kg / day), which was conventionally used in all aspects of the present invention.

The pharmaceutical composition may be prepared by methods known in the art (e.g., Remington's Pharmaceutical Science (current edition; Mack Publishing Company, Easton PA), itself or in combination with a pharmaceutically acceptable carrier, excipient, diluent, Granules, tablets, capsules, injections, and the like. They may also be formulated for oral use (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or oleaginous suspensions) ≪ / RTI >

The active ingredient of the pharmaceutical composition of the present invention, such as amino flavone, hydroxy flavone, Kg / day to 500 mg / kg / day, preferably 0.1 to 50 mg / kg / day, on an adult basis, of at least one substance selected from the group consisting of acyclovir, chrysin, curcumin, daidzein, flavone, And can be increased or decreased depending on the severity of the disease. The scope of the present invention is not limited to the dose.

The present invention relates to a pharmaceutical composition comprising aminoflavone, hydroxyflavone, chrysin, curcumin, daidzein, flavones, genistein and phloretin, And treating the surface of the organism or the non-living body with at least one substance selected from the group consisting of the method for inhibiting biofilm formation.

The method of inhibition of the present invention can be carried out by contacting one or more substances selected from the group consisting of amino flavone, hydroxyflavone, chrysin, curcumin, daidzein, flavone, genistein and floretine with a pathogenic microorganism, It is possible to prevent or inhibit the formation of the biofilm in advance by treating the surface of a tissue, an organ, or a medical article derived from an organism having or proliferating.

The surface may include surfaces of objects used in the home, industry, environment, and medical fields, as well as surfaces derived from living bodies, which may refer to the surfaces of living organisms or inanimate objects from which microorganisms can form biofilms. And may preferably be the surface of an organism-derived tissue, organ or medical article.

For example, it may be the surface of an organism-derived tissue or organ such as a skin or a tooth, and may be used for various medical facilities, equipment, instruments, temporary or permanent artificial implants such as lenses, prosthetic valves, pacemakers, A catheter, or the like, but the scope of the present invention is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Preparation of sample

Aminoflavone, hydroxyflavone, It has been reported that chrysin, curcumin, daidzein, flavones, genistein and phloretin, and vitamin C and vitamin E in dimethyl sulfoxide (DMSO) , And E. coli As a strain, serotype intestinal hemorrhagic E. coli O157: H7 (ATCC 43895, EDL933 strain), a symbiotic E. coli K-12, MG1655, BW25113, TG1, DH5α and non-pathogenic E. coli ATCC 4157 at 37 ° C and 250 rpm in LB medium. The culture was diluted 1: 100 with LB medium and absorbance was measured at 600 nm (OD ₆₀₀ ) using a spectrophotometer (UV-160, Shimadzu, Japan).

< Example  2> Biofilm analysis

Were added to LB medium (300 함유) containing 0, 5, 10, 25, 50, and 100 쨉 g / ml of phloretin and cultured in E. coli Was diluted to an optical density of 0.05 to 600 nm (OD ₆₀₀ ) in a culture of the strain, and then incubated at 37 ° C for 10 hours without shaking. After measuring the cell density (turbidity at 620 nm), rinsing with water three times to remove all the planktonic cells on the plate and adding crystal violet (0.1% (v / v), 300 ㎕) to each well, Was added. After standing at room temperature for 20 minutes, the microplate is emptied and rinsed three times with water. Ethanol (95%, 300 μl) was added to resolve the stained biofilm cells and the absorbance of all the biofilm cells was measured at 570 nm.

As shown in Figure 1, aminoflavone, hydroxyflavone, Chrysin, curcumin, daidzein, flavones, genistein and phloretin have been identified in E. coli O157: H7 inhibited biofilm formation, and vitamin C and E did not affect biofilm formation. Specifically, 25 and 50 μg / ml of phloretin reduced the E. coli O157: H7 biofilm formation by 89% and 93%, respectively (FIG. 1B). The presence of floretine had little effect on the growth rate, showing 1.32 ± 0.01 / h in the absence of floretine and 1.31 ± 0.03 / h in the presence of 50 ㎍ / ml of floretine. In addition, the content of 50 μg / ml of floretine did not show toxicity to E. coli O157: H7 planktonic cells, and decreased biofilm formation.

As shown in Figure 2, phloretin did not inhibit the symbiotic E. coli K-12 biofilm formation. In contrast, in the case of E. coli K-12 BW25113 and non-pathogenic E. coli ATCC 4157, phloretin exhibited no biofilm inhibitory effect on symbiotic E. coli strains MG1655, TG1, BW25113, DH5α and ATCC 4157, The biofilm formation was enhanced in a content-dependent manner.

< Example  3> Transcript  analysis

3-1. gun RNA  detach ( Total RNA isolation )

For transcriptomic analyzes, E. coli O157: H7 and E. coli K-12 BW25113 were inoculated into 250 ml LB medium at 37 ° C and incubated overnight in a 1 L shake flask (1: 100 dilution). To form the biofilm, 10 g of glass wool (Corning Glass Works, Corning, NY) was used and the cells were incubated for 7 hours at 100 rpm with shaking. Only floretin (50 [mu] g / ml) or DMSO was added at the beginning of the culture. As a result of using glass wool to increase the surface area, sufficient RNA can be obtained for DNA microarrays. Before taking the sample, the RNase inhibitor (RNAlater, Ambion, TX, USA) was added and the biofilm cells were immediately cooled with dry ice and 95% ethanol for 30 seconds and then centrifuged at 13,000 g for 2 minutes. The cell fillets were immediately frozen in dry ice and stored at -80 ° C. Total RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA) and treated with 30 units of DNase I purified RNA for 15 min to remove all DNA.

3-2. DNA Microarray  analysis

E. coli GeneChip Genome 2.0 Array (Affymetrix, P / N 900551, Santa Clara, USA) was treated with floretine and then used to study the differentiated gene expression of E. coli O157: H7 cells. DNA microarray analysis was performed with an Affymetrix system.

3-3. Quantitative real-time Reverse transcription Polymerase chain reaction  ( Quantitative real - time  reverse transcription 중합체 chain reaction  ( qRT - PCR ).

CsgA , csgB , stx2A , lsrA , lsrB , hlyE , and mtr in the pathogenic E. coli O157: H7 and the symbiotic E. coli K-12 BW25113 that did not or did not treat the floretine were used to confirm the DNA microarray data QRT-PCR was used to confirm the transcription rate of the cells.

Gene specific primers were used for the gene and the housekeeping control rrsG (Table 1). The level of transcription of the housekeeping gene rrsG was used to normalize the expression data of the gene of interest.

Gene Primer csgA Forward 5'-AGA TGT TGG TCA GGG CTC AG-3 ' Reverse 5'-CGT TGT TAC CAA AGC CAA CC-3 ' csgB Forward 5'-CGT TGT TAC CAA AGC CAA CC-3 Reverse 5'-CCA TAA GCA CCT TGC GAA AT-3 stx2A Forward 5'-GTT CCG GAA TGC AAA TCA GT-3 ' Reverse 5'-CGG CGT CAT CGT ATA CAC AG-3 ' lsrA Forward 5'-AAC ATC CTG TTT GGG CTG GCAA-3 ' Reverse 5'-AAA CAA GCG TTC GGT TTC CGCA-3 ' lsrB Forward 5'-AGC ATC CTG GCT GGG AAA TTG T-3 ' Reverse 5'-AAA TTC TTT CAC CGT GCC GCG T-3 ' hlyE Forward 5'-AAC CGC AGA TGG AGC ATT AG-3 ' Reverse 5'-CAC TGT TTG GGT TGC TTC AA-3 ' mtr Forward 5'-ACC AGC AGC AGA TCC AGA CT-3 ' Reverse 5'-AAG ATC CGA AAA CCA TCG TG-3 ' rrsG Forward 5'-TAT TGC ACA ATG GGC GCA AG-3 ' Reverse 5'-ACT TAA CAA ACC GCC TGC GT-3 '

Studies of the molecular mechanisms of biofilm reduction by phloretin have shown that 70 genes are inhibited, 86 genes are induced, and 156 genes are regulated by phloretin (about 2.5 times or more). In addition, two toxin genes (hemolysin hlyE and Shiga toxin 2 stx2 ), autoinducer-2 importer genes ( lsrACDBF ) and dozens of prophage genes are suppressed (2.5- to 3.8-fold) -related genes, transport-related genes, and stress-related genes (2.5-fold to 4.7-fold). Complete data is available using GEO accession number GSE28144.

qRT-PCR was used to confirm DNA microarray data. A culture independent of the DNA microarray sample was used, and it was confirmed that floretine contained two toxin genes (toxin genes, stx2 And hlyE ), two autoinducer-2 importer genes, lsrA and According to the inhibition of the indole importer gene ( mtr ), the curlin major subunit ( csgA ) and the curlin minor subunit ( csgB ) (Fig. 3A), qRT- PCR showed a different change in expression on E. coli O157: H7 biofilm cells when treated with or without treatment with floretine as compared to DNA microarray data.

As shown in Figures 1 and 2, E. coli K-12 BW25113 enhances biofilm formation, whereas E. coli O157: H7 from after the suppression of E. coli biofilm formation The gene expression of K-12 BW25113 biofilm cells was examined using qRT-PCR. As shown in Fig. 3, E. coli Inhibited genes ( hlyE , lsrA , lsrB , csgA , and csgB ) in O157: H7 biofilm cells were detected in E. coli K-12 BW25113 Highly induced in biofilm cells, whereas stx2 (Shiga toxin 2 gene) was highly expressed in E. coli K-12 strain, mtr Expression of the gene was inhibited in both strains.

< Example  4> Attachment Analysis

4-1. SEM  ( scanning electron microscopy ) Pimbria  analysis

To confirm the production of Pimbria, SEM was used. E. coli O157: H7 cells to form biofilm cells. And grown on a nylon filter (0.5 x 0.5 mm square) at 37 ° C for 24 hours without shaking. Initial turbidity was 0.05 at 600 nm when measuring 300 [mu] l cells. After incubation of E. coli O157: H7 cells in the presence or absence of phloretin (50 μg / ml), 2.5% glutaraldehyde and 2% formaldehyde were added to the pre-fix the cells formaldehyde) and incubated overnight at 4 ° C. Samples were examined at 15 kV Voltage using a SEM S-4100 (Hitachi, Japan), and the range was expanded from × 2,000 to × 25,000.

4-2. Human cell line culture ( Human cell line culture )

HT-29 human colon cancer cell line and U937 human leukemia cell line were purchased from the American Type Culture Collection (Rockville, Mass., USA) and the cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 1% phenylcycline / streptomycin, The cells were grown in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, Calif., USA) supplemented with dimium pyruvate and 2 mM glutamine and the culture medium was changed every other day.

4-3. Monocytes or E. coli  Human Into the colon epithelium  Attachment Analysis

The adhesion of monocyte or E. coli strains to human colon epithelial cells was measured using a co-culture system in which HT-29 colon epithelial cells were co-cultured with U937 monocytic cells or E. coli strains . U937 cells were pre-labeled with BCECF / AM (10 μg / ml) for 1 hour at 37 ° C. The cells were co-cultured in the presence or absence of tumor necrosis factor (TNF) -α for 3 hours at 37 ° C. For bacterial adhesion analysis, E. coli O157: H7 / pCM18 cells and E. coli All of K-12 BW25113 / pCM18 were cultured in LB supplemented with 300 占 퐂 / ml erythromycin for 16 hours at 37 占 폚 to maintain plasmid pCM18 encoding the green light protein. E. coli Cells were harvested by centrifugation at 13,000 rpm for 5 min at 4 ° C, washed with phosphate buffered saline (PBS) and resuspended in PBS. For the floretine experiment, HT-29 cells were pretreated with phloretin (200 μM corresponding to 54.8 μg / ml) and then exposed to TNF-α or E. coli. HT-29 cells were then rinsed twice with PBS to remove unattached cells. U937 monocytic cells or E. coli To measure green glow in HT-29 cells co-cultured with O157: H7 / pCM18, the mixture was dissolved in 0.1% Triton X-100 in 0.1 M Tris, excited at 485 nm and emitted at 520 nm BCECF glare was analyzed using a Fluostar Optima microplate reader (BMG Labtech GmbH, Offenburg, Germany). E. coli into HT-29 cells To measure the attachment of O157: H7 / pCM18, E. coli A negative control with O157: H7 / pCM18 alone was set up and the amount of light produced by this was subtracted from the other experimental groups. In each experiment, E. coli attached to HT-29 cells The number of O157: H7 / pCM18 was analyzed.

Curly (curli) and Pim Calabria, including the Philippines (pili) is E. coli O157: H7 is an important factor in biofilm formation, Floresta Teen E. coli O157: the Curly (curli) gene in cells H7 biofilms and csgA Since the gene expression of csgB was inhibited (Fig. 3A), Pembria production was examined by SEM. As shown in Fig. 4, phloretin significantly reduced the production of Pimbria of E. coli O157: H7.

E. coli O157: H7 for measuring the adhesion to the epithelial cells and E. coli, which coexist as shown in the results of the investigation of the initial step of inducing infection and toxicity, Fig. 5 B More than E. coli K-12 BW25113 cells O157: H7 cells HT-29 showed a more excellent adhesion is attached to the human colon epithelial cells, Floresta tin treatment (200 μM corresponding to 54.8 ㎍ / ml) is E. coli O157: H7 cells to HT-29 epithelial cells, but did not affect the attachment of E. coli K-12 BW25113. Analysis of green light intensity from E. coli K-12 BW25113 cells showed that E. coli O157: H7 attachment to HT-29 epithelial cells was inhibited by floretine.

As shown in Figure 5C, E. coli attached to HT-29 epithelial cells The number of O157: H7 cells was greatly reduced by the treatment with floretine. Thus, phloretin is expressed not only on non-living surfaces (Fig. 1), in E. coli epithelial cells of human colon O157: H7 cells (Fig. 5).

< Example  5> Colitis model

5-1. TNBS - induced rheumatoid arthritis model ( Trinitrobenzene ulfonic  acid ( TNBS ) - induced rat colitis model )

Seven to eight weeks old Sprague Dawley rats purchased from Samtaco bio Korea (Osan, Korea) were fasted for 24 hours, TNBS was injected, and lightly anesthetized with diethyl ether. 0.8 ml of 5% TNBS dissolved in 50% (v / v) ethanol was slowly injected into the rat lumen (8 cm proximal to the anus through the rectum) and placed in a vertical position for 60 seconds. At this time, only 50% (v / v) ethanol was administered to the control group. Control rats were treated similarly, but only 50% (v / v) ethanol was administered instead. To confirm the efficacy of floretine, phloretin (20 mg / kg / day) was administered via the oral route for 5 days after TNBS administration. As a positive control, 5-aminosalicylic acid (5-ASA in 100 mg / kg / day), the active metabolite of sulfasalazine, a drug commonly used for IBD, was used. All rats were killed 7 days after TNBS administration. Macroscopic ulceration of colitis and severity were measured by two independent investigators who did not know whether to treat floretine. Colon tissues approximately 5-7 cm in rectum were cut and used to measure myeloperoxidase (MPO) activity.

5-2. For tissue neutrophil infiltration Myeloper oxidase ( MPO ) activation

MPO activity acts as a marker of leukocyte infiltration into tissues. For the measurement of MPO activity, an MPO assay kit (ASA-001, Cytostore, Canada) was used. The 1 cm long portion of the colon was washed with cold PBS (pH 7.4), weighed and resuspended in ice-cold sample buffer (0.5 ml / 50 mg of tissue) and incubated at 4 ° C with Biospec Products Inc , Switzerland) for 30 seconds. The homogenate was mixed with an additional sample buffer to a final concentration of 50 mg / ml and then centrifuged twice at 10,000 rpm for 5 minutes. Each 20 μl sample solution was mixed with 200 μl of the development reagent (12.5 μl of H ₂ O ₂ containing chromogenic powder in phosphate buffer, 2.5 ml), and the degree of MPO activity of this tissue was immediately measured after mixing at 450 nm absorbance And measured at 1 minute intervals using a spectrophotometer (Molecular Devices, Versa MAX, CA, USA). One MPO activity is defined as the activity required to convert 1 μM H2O2 at 25 ° C for 7 minutes, expressed as Units / g of tissue.

As shown in FIG. 6, TNF-a induces adhesion of U937 pro-mononuclear cells to HT-29 cells, whereas phloretin significantly inhibits adhesion of TNF-a-induced progenitor cells to HT-29 cells Respectively.

In addition, as shown in Figure 7, using the TNBS-induced rat colon model increased body weight in the vehicle-treated control, whereas, in contrast, TNBS inhibited weight gain (Figure 7A). TNBS-treated rats exhibited severe symptoms of colitis such as bloody diarrhea and wasting conditions resulting from large changes in body weight (Figs. 7A and 7B). The weight of the colon tissue (1 cm near the TNBS injected site) was significantly increased by TNBS (FIG. 7C), whereas the length of the distal colon of the cecum was shortened by TNBS (FIG. 7B) Rats treated with phloretin (20 mg / kg / day), such as the drug 5-ASA (100 mg / kg / day), significantly recovered the original body and colon weights.

As shown in FIG. 7D, MPO activity was elevated in TNBS-treated colon, while it decreased again to floretine treatment resulting in an almost vehicle-treated control level (FIG. 7D).

Claims (8)

Which inhibits the formation of a biofilm of a pathogenic microorganism selected from enteropathogenic Escherichia coli or enterohaemorrhagic Escherichia coli , including phloretin, . delete delete The biofilm inhibitor of claim 1, wherein said pathogenic microorganism causes inflammatory disease. 2. The method of claim 1, wherein the phloretin is selected from the group consisting of toxin genes, autoinducer-2 importer genes, curli genes or prophage genes, Lt; RTI ID = 0.0 &gt; expression, &lt; / RTI &gt; The biofilm inhibitor according to claim 1, wherein the phloretin inhibits formation of a biofilm by inhibiting the surface adhesion of the pathogenic microorganism. delete Treating phloretin to the surface of an organism or an organism;
And inhibiting the formation of a biofilm of a pathogenic microorganism selected from enteropathogenic Escherichia coli or enterohaemorrhagic Escherichia coli .

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