KR102670270B1 - Composition for inhibiting biofilm formation and antibacterial against Gram-positive bacteria and fungi comprising indole derivatives - Google Patents

Abstract

The present invention relates to a composition for inhibiting biofilm formation and antibacterial against one or more species selected from the group consisting of Gram-positive bacteria and fungi containing an indole derivative as an active ingredient. More specifically, indole-3-carbinol is used to treat skin inflammation and It showed the effect of suppressing biofilm formation without cell death of trouble-causing Gram-positive acne bacteria (C. ances), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans), and 3,3'- Diindolylmethane (DIM) exhibits strong antibacterial and antibiofilm activity against the above-mentioned bacteria, and as it has been confirmed that it improves the antibacterial activity of antibiotics through concurrent use with antibiotics, the indole-3-carbinol or 3,3 A composition containing '-diindolylmethane as an active ingredient may be provided as an antibacterial agent, antibiotic adjuvant, or antibiofilm composition, and may be provided as a cosmetic composition for improving skin inflammatory diseases.

Description

Composition for inhibiting biofilm formation and antibacterial against Gram-positive bacteria and fungi comprising indole derivatives}

The present invention contains an indole derivative, 3,3'-diindolylmethane (DIM) or indole-3-carbinol, as an active ingredient, and is effective against Gram-positive bacteria and fungi. This is a technology related to biofilm formation inhibition and antibacterial compositions.

In areas infected with bacteria, mucous-like bacterial colonies formed by a large number of bacteria exist in the form of mucus. This mucous-like bacterial complex is called a biofilm, biofilm, or biofilm. Biofilms form on various solid surfaces, either biological or non-biological surfaces. Bacterial biofilm is a complex surrounded by a polymer matrix outer membrane composed of polysaccharides, polypeptides, and various biomaterials. Inside the biofilm, bacteria communicate with each other to defend against the external environment. Through this, biofilms enable bacteria to survive even under various environmental stresses, including antibiotics.

Bacterial biofilms are very frequently found in the general natural environment as well as in the medical field related to bacterial infectious diseases. It can form on human skin and organs, appear in the form of tartar on teeth, and can also form on industrial equipment, medical equipment, and body implants. Due to these problems, biofilms cause cavities, periodontal disease, pneumonia accompanying cystic fibrosis, earache in the middle ear, skin infections accompanying burns, and skin infections such as athlete's foot. It has been a subject of interest for scholars studying diseases, etc. The U.S. National Institutes of Health estimated in a 2002 report that up to 80% of bacterial groups cause infection by pathogenic bacteria through the formation of biofilms.

Antibiotics that are effective against planktonic bacteria independently of biofilm formation tend to lose their effectiveness when bacteria form biofilm. When bacteria form a biofilm, antibodies etc. cannot penetrate the thick biofilm, so the bacteria's resistance to antibiotics can increase by about 1,000 times. The cause of this increase in antibiotic resistance due to biofilm formation is not yet clearly known, but can be explained in the following three ways. The first is “Changes in the physiological life cycle of microorganisms.” When a biofilm is formed, bacterial metabolism and proliferation tend to decrease. At this time, the bacteria inside the biofilm have a significantly reduced exchange rate with the surrounding environment for metabolism and proliferation, resulting in lowered susceptibility to antibiotics. The second is “physical blocking of the outer membrane composed of polysaccharides and proteins.” The electrical properties of the polysaccharide and protein complex that forms the mucoid outer membrane of bacteria have a tendency to bind to antibiotics, thereby physically preventing the spread of antibiotics. In other words, the antibiotic is not delivered well to each bacteria, resulting in a decrease in its efficacy. The third is a speculation related to the general mechanism of antibiotic resistance acquisition, which is “production of antibiotic inhibitory factors.” The most well-known substances as antibiotic decomposition and inhibitory factors that inhibit the efficacy of antibiotics include beta-lactamases produced by Pseudomonas bacteria. When a biofilm is formed, bacteria that were not antibiotic-resistant inside the biofilm tend to acquire resistance factor genes through horizontal gene transfer from surrounding antibiotic-resistant bacteria and become resistant. In other words, when a biofilm is formed at the site of infection, the appearance of antibiotic-resistant bacteria becomes frequent.

For these reasons, when a biofilm is formed, resistance to antibiotics widely used to treat infectious diseases develops, weakening the treatment effect of antibiotics, and entering a state of chronic bacterial infection. In this case, as described above, the sensitivity of bacteria to antibiotics decreases, making it difficult to eradicate bacterial infections even when using a normal amount of antibiotics, leading to overprescription of antibiotics. As a result, the antibiotic resistance of bacteria only increases and the emergence of antibiotic-resistant superbugs occurs. This will lead to dire consequences such as mass production. Therefore, treating bacterial infections with biofilm formation simply with antibiotics has limitations and cannot be an effective treatment. In particular, infections caused by bacteria forming biofilms are more serious because they are often caused by multi-drug resistant bacteria that are resistant to various antibiotics.

Therefore, in order to solve the above problem, the effect of antibiotics and antifungal agents can be maximized by suppressing biofilm formation by suppressing biofilm or outer membrane production present in the biofilm, changing the surface properties of bacteria, or inducing changes in the growth pattern of bacteria. The development of a treatment is necessary.

Republic of Korea Patent Publication No. 10-2014-0044053 (published on April 14, 2014)

The present invention provides 3,3'-diindolylmethane (DIM) to prevent or improve diseases caused by Acne acne, Staphylococcus aureus and Candida albicans or their biofilms, which are Gram-positive bacteria that cause dermatitis diseases and skin troubles. ) and indole-3-carbinol to provide an antibacterial and biofilm formation-inhibiting composition containing as an active ingredient.

The present invention relates to a gram-positive bacteria containing one or two or more selected from 3,3'-diindolylmethane (DIM) and indole-3-carbinol as an active ingredient. And it provides a composition for inhibiting biofilm formation against one or more species selected from the group consisting of fungi.

The present invention provides an antibacterial composition for at least one selected from the group consisting of Gram-positive bacteria and fungi containing 3,3'-diindolylmethane (DIM) as an active ingredient.

In addition, the present invention contains one or two or more selected from 3,3'-diindolylmethane (DIM) and indole-3-carbinol as active ingredients, Provides a cosmetic composition for preventing or improving dermatitis disease or skin trouble caused by one or more types selected from the group consisting of gram-positive bacteria and fungi.

According to the present invention, indole-3-carbinol kills Gram-positive acne bacteria (C. ances), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans), which cause skin inflammation and trouble, without cell death. It showed an effect of suppressing biofilm formation, and 3,3'-diindolylmethane (DIM) showed strong antibiofilm activity against the above bacteria, and was confirmed to improve the antibacterial activity of antibiotics through concurrent use with antibiotics. Accordingly, the composition containing indole-3-carbinol or 3,3'-diindolylmethane as an active ingredient may be provided as an antibacterial agent, antibiotic adjuvant, or antibiofilm composition, and may be provided as a cosmetic composition for improving skin inflammatory diseases. You can.

Figure 1 shows the results of confirming the effect of various indole derivatives on the cell growth of C. acnes, showing the minimal inhibition concentration (MIC), which is the minimum growth inhibition concentration of each indole derivative for C. acnes.
Figure 2 shows the results of confirming the anti-biofilm activity of indole derivatives against Acne acnes, Figure 2A shows the results of screening biofilm formation of Acne acnes in the presence of 0.1 mM indole derivatives, Figures 2B, 2C and 2D show indole, 2C and 2D respectively. The results show the growth of acne bacteria in the presence of 3,3'-diindolylmethane and indole-3-carbinol by concentration and time, and Figures 2E, 2F and 2G show indole and 3,3'-diindolylmethane, respectively. and indole-3-carbinol, the degree of biofilm formation and inhibition of acne bacteria was tested at each concentration, and the error bars represent the standard deviation. (*, P < 0.05 vs. untreated control)
Figure 3 shows the results confirming the inhibition of biofilm formation of acne bacteria by antibiotics in the presence or absence of 3,3'-diindolylmethane, and Figures 3A, 3B and 3C show benzoyl peroxide, a drug commercially available as an antibiotic for inhibiting and killing acne bacteria. This is the result of confirming the degree of biofilm inhibition after treatment with benzoyl peroxide, gentamycin, and ciprofloxacin. Figures 3D, 3E, and 3F show that the biofilm formation inhibition ability of benzoyl peroxide, gentamicin, and ciprofloxacin is 40-70%. This is the result of measuring the effect of 3,3'-diindolylmethane on inhibiting biofilm formation at different concentrations, and the error bar represents the standard deviation. (*, P < 0.05 vs. untreated control)
Figure 4 shows the results of confirming the antibiofilm activity of 3,3'-diindolylmethane against Candida albicans (C. albicans) and Staphylococcus aureus (S. aureus) under anaerobic and aerobic conditions. Figures 4A and 4B are These are the results of confirming the degree of biofilm formation under aerobic conditions for Candida albicans and Staphylococcus aureus, respectively. Figures 4C and 4D are the results measured after culturing Candida albicans and Staphylococcus aureus under anaerobic conditions, respectively, and the error bars represent the standard deviation. It shows. (*, P < 0.05 vs. untreated control)
Figure 5 shows the results confirming the anti-biofilm effect of 3,3'-diindolylmethane on Acne bacteria, and Figure 5A shows the inhibition of biofilm of Acne bacteria by 3,3'-diindolylmethane (0, 0.02 or 0.05 mM). was observed as 2D and 3D images, Figure 5B is the result confirmed by confocal laser scanning microscopy (CLSM), and Figure 5C is the image observed by confocal laser scanning microscopy of the plastic plate through the COMSTAT program. As a result of analyzing the amount (biomass), thickness (thickness), and biofilm formation surface area (coverage) of the biofilm formed on the floor surface, the scale size in Figure 5B is 100 μm. (*, P < 0.05 vs. untreated control) Figure 5D is a Scanning Electrone Microscope (SEM) image formed with or without 3,3'-diindolylmethane (0, 0.02, or 0.05 mM), with yellow and white rulers. Sizes represent 3 μm and 750 nm, respectively, and None represents the untreated control.
Figure 6 shows the results confirming the effect of 3,3'-diindolylmethane on multibacterial biofilm. Figure 6A shows acne bacteria and Staphylococcus aureus, Figure 6B shows acne bacteria and Candida albicans, and Figure 6C shows acne under anaerobic conditions. This is the result of confirming the biofilm formation inhibitory activity of 3,3'-diindolylmethane on multibacterial biofilms of bacteria, Candida albicans, and Staphylococcus aureus, and Figure 6D shows acne bacteria cultured in the presence of 3,3'-diindolylmethane. , This is the result of exploring three types of biofilm containing Candida albicans and Staphylococcus aureus in 2D and 3D. Figure 6E is an image observed with a confocal laser scanning microscope, and Figure 6F is the bottom surface of a plastic plate through the COMSTAT program. As a result of analyzing the amount (biomass), thickness (thickness), and biofilm formation surface area (coverage) of the biofilm formed, the scale size indicates 100 μm. (*, P < 0.05 vs. untreated control group) Figure 6G shows the results of scanning electron microscopy (SEM) confirmation of the degree of biofilm formed by three types of bacteria according to the concentration of 3,3'-diindolylmethane. Yellow and white ruler sizes represent 10 and 3 μm, respectively, and None represents the untreated control.
Figure 7 shows the results of confirming the effect of indole, 3,3'-diindolylmethane and indole-3-carbinol on the production of extracellular polymeric substances (EPS) of Acne acne. Figures 7A, 7B and 7C are This is the result of measuring extracellular membrane matrix (EPS) production in the presence of indole, 3,3'-diindolylmethane, and indole-3-carbinol, respectively. (*, P < 0.05 vs. untreated control)
Figure 8 shows the results confirming the inhibitory effect of 3,3'-diindolylmethane on hyphal filamentation and aggregation of Candida albicans, Figure 8A shows the results confirming that 3,3'-diindolylmethane inhibits filamentous hyphal growth in PDB medium, and Figure 8B shows As a result of confirming that fungal cell aggregation was inhibited in RPMI-1640 medium containing 10% bovine serum, the scale size indicates 100 μm in Figures 8A and 8B, and None indicates the untreated control group.
Figure 9 shows the results of confirming the level of gene expression of acne bacteria treated with 3,3'-diindolylmethane. Acne bacteria were treated with 3,3'-diindolylmethane and the total RNA of acne bacteria was extracted and analyzed in real-time. This is the result of analysis using reverse transcription PCR (qRT-PCR). (* = P < 0.05 vs. untreated control)
Figure 10 shows the effect and mechanism of action of indole-3-carbinol and 3,3'-diindolylmethane derived from cruciferous plants on single and polymicrobial biofilms of Acne acne, Candida albicans, and Staphylococcus aureus. It shows the research results of the invention.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention believe that the indole derivatives, 3,3'-diindolylmethane (DIM) or indole-3-carbinol, are Gram-positive bacteria that cause dermatitis diseases and skin troubles, such as Acne acne, Staphylococcus aureus, Candida albicans or As it was confirmed that it has the effect of suppressing biofilm formation, the present invention was completed to prevent or improve diseases caused by Gram-positive bacteria and fungi.

The present invention relates to a gram-positive bacteria containing one or two or more selected from 3,3'-diindolylmethane (DIM) and indole-3-carbinol as an active ingredient. And it is possible to provide a composition for inhibiting biofilm formation against one or more species selected from the group consisting of fungi.

The Gram-positive bacteria may be selected from the group consisting of Cutibacterium acnes (C. acnes) KCCM 41747 (ATCC 6919) and Staphylococcus strains. More specifically, the Staphylococcus aureus is It may be methicillin-susceptible and resistant Staphylococcus aureus, but is not limited thereto.

The fungus may be Candida albicans, and more specifically, the Candida albicans may be a fluconazole-resistant Candida albicans DAY185 fungus, but is not limited thereto.

The indole-3-carbinol may inhibit biofilm formation without cell death.

The 3,3'-diindolylmethane is a multi-species biofilm in which Cutibacterium acnes (C. acnes), Candida albicans (C. albicans), and Staphylococcus aureus (S. aureus) form a biofilm together. This may inhibit its formation.

The present invention can provide an antibacterial composition for at least one selected from the group consisting of Gram-positive bacteria and fungi containing 3,3'-diindolylmethane (DIM) as an active ingredient.

The 3,3'-diindolylmethane is composed of Gram-positive bacteria Cutibacterium acnes (C. acnes), methicillin-susceptible and resistant Staphylococcus strains, and fluconazole-resistant Candida albicans. It can help kill one or more bacteria selected from the group.

In addition, an adjuvant for enhancing the antibacterial effect of an antibiotic or antibacterial agent against one or more types selected from the group consisting of Gram-positive bacteria and fungi containing 3,3'-diindolylmethane (DIM) as an active ingredient. It can be provided as .

The adjuvant may be treated in combination with an antibiotic or antibacterial agent to improve the antibacterial effect of the antibiotic or antibacterial agent, and the antibiotic may be selected from the group consisting of Gentamycin and Ciprofloxacin, but is not limited thereto. no.

In addition, the present invention contains one or two or more selected from 3,3'-diindolylmethane (DIM) and indole-3-carbinol as an active ingredient. It is possible to provide a cosmetic composition for preventing or improving dermatitis disease or skin trouble caused by one or more types selected from the group consisting of gram-positive bacteria and fungi.

The dermatitis disease or skin trouble may be caused by Cutibacterium acnes, Staphylococcus strains, Candida albicans, or their biofilm.

The dermatitis diseases include comedonal acne, papular acne, pustular acne, crystalline acne, cystic acne, acne vulgaris, acne fulminans, coagulative acne, keloid acne, psoriasis, atopic dermatitis, seborrheic dermatitis, It may be selected from the group consisting of contact dermatitis, nummular dermatitis, staphylococcal scalded skin syndrome, folliculitis, impetigo, abscess, cellulitis, toxic epidermal necrolysis, epidermolysis syndrome, candidiasis, candidal nail fungus, and general dermatitis. there is.

The cosmetic composition contains, in addition to the active ingredients 3,3'-diindolylmethane (DIM) and indole-3-carbinol, stabilizers, solubilizers, vitamins, pigments and It may contain conventional auxiliaries such as flavorings, and carriers.

The cosmetic composition can be prepared in any formulation commonly prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, oil, powder foundation, emulsion foundation, It may be formulated as a wax foundation or spray, but is not limited thereto. More specifically, it can be manufactured in the form of sun cream, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, pack, spray, or powder.

When the formulation is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivatives, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide can be used as the carrier ingredient. .

If the formulation is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as the carrier ingredient. In particular, in the case of a spray, chlorofluorohydrocarbon, propane/butane may be used as the carrier ingredient. Alternatively, it may include a propellant such as dimethyl ether.

When the formulation is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -Butyl glycol oil, fatty esters of glycerol, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystalline cellulose. , aluminum metahydroxide, bentonite, agar or tracant, etc. can be used.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples. Examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Strains, compounds and culture conditions

Acne bacteria include Cutibacterium acnes strain KCCM 41747 (ATCC 6919) Gram-positive bacteria (isolated from human facial acne), and Staphylococcus aureus include methicillin-susceptible Staphylococcus aureus and Candida albicans. The fluconazole-resistant Candida albicans strain DAY 185 was used. Solid media with agar added to Reinforced Clostridium Media (RCM) and liquid media without agar were used as media for culturing acne strains, and the experiment was performed at 37°C under anaerobic conditions. For anaerobic conditions, BD GasPak™ EZ Gas Generating Anaerobic Pouch Systems (Fisher Scientific, Pittsburgh, USA) pouches were used. Staphylococcus aureus strains were cultured in LB (Lysogeny broth) medium at 37°C, and Candida albicans strains were cultured in PDB (potato dextrose broth) medium at 37°C.

A total of 20 indoles are 7-azaindole, 7-benzyloxyindole, 3,3-diindolylmethane (DIM), 7-formylindole, 7-hydroxyindole, indole, indole-3-acetamide, indole-3-acetic acid, indole-3-acetonitrile , indole-3-butyric acid, indole-3-carbinol, indole-3-carboxyaldehyde, indole-7-carboxylic acid, indole-3-propionic acid, isatin, 7-methoxyindole, 7-methylindole, methyl indole-7-carboxylate , 7-nitroindole and 2-oxindole were purchased from Sigma Aldrich (St. Louis, MO, USA), Wako Chemicals Inc. (Richmond, VA, USA), or Combi-Blocks, Inc. (San Diego, CA, USA). used.

Dimethyl sulfoxide (DMSO) was used as a solvent to dissolve all indole derivatives, and DMSO (0.1% v/v) was used as a negative control. DMSO below 0.1% showed no effect on bacterial growth or biofilm formation.

2. Measurement of planktonic cell growth and confirmation of minimum growth inhibitory concentration (MIC)

To confirm the effect of indole on the planktonic growth of acne bacteria, cell culture medium cultured for 6 days in a 96-well plate was diluted 1:50 in fresh RCM medium and re-inoculated. Indole derivatives were treated. The plate was cultured at 37°C under anaerobic conditions, and anaerobic conditions were maintained using an anaerobic pouch. After 96 hours of incubation, cell turbidity was checked at 600 nm using a spectrophotometer (Optizen 2120UV spectrophotometer).

3. Biofilm formation inhibition assay

Crystal violet analysis was performed to quantify biofilm formation. Briefly, acne bacteria cultured for 96 hours were diluted 1:50 with sterilized RCM medium and cultured for 6 days under anaerobic conditions at 37°C in the presence or absence of indole.

To quantify biofilm, the plate was washed three times with distilled water, planktonic cells were discarded, and only the biofilm cells attached to the well were stained with 0.1% crystal violet for 20 minutes. Afterwards, the plate was washed three times with sterile distilled water, crystal violet was extracted using 95% ethanol, and the absorbance was measured at 570 nm using a spectrophotometer (Multiskan EX microplate reader (Thermo Fisher Scientific, Waltham, MA). . Results are expressed as the average value of at least 12 replicate wells.

The percentage of inhibition indicates relative biofilm formation (100 x biofilm formation with chemical/biofilm formation of untreated control). In addition, the antibacterial and antibiofilm efficacy of benxoyl peroxide, gentamycin, and ciprofloxacin, which are well-known acne treatment antibiotics, and 3,3-diindolylmethane were compared.

4. Analysis of multi-species biofilm formation of Acne acne, Staphylococcus aureus and Candida albicans

To generate multispecies biofilms of Acne acnes, Staphylococcus aureus, and Candida albicans, Acne acnes (59 ± 4 × 10 ⁷ CFU), Staphylococcus aureus cells (59 ± 4 × 10 ⁷ CFU), and Candida albicans cells (23 ± 3 × 10 ⁵ CFU) were simultaneously inoculated into mixed RCM/LB/PDB medium (1:1:1) and cultured. Biofilm formation was analyzed under anaerobic conditions at 37°C for 6 days using anaerobic pouches. To form two types of biofilm, Acne acne and Staphylococcus aureus or Acne acne and Candida albicans were inoculated into RCM/LB (1:1) or RCM/PDB medium (1:1) in a 96-well plate and incubated under anaerobic conditions. Cultured for 6 days at 37°C.

5. Observation by live imaging microscopy, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM)

To quantify the biofilm of P. acnes or the mixed biofilm of P. acnes/Staphylococcus aureus/Candida albicans in the presence or absence of 3,3-diindolylmethane, the samples were cultured at 37°C for 6 days under anaerobic conditions using anaerobic pouches. Afterwards, planktonic cells were removed by washing three times with PBS, and real-time biofilm was confirmed at various magnifications using an optical microscope iRiS ^TM Digital Cell Imaging System. Biofilm images were reconstructed into 2D and 3D pictures using ImageJ.

Acne biofilm or mixed Acne acne/Staphylococcus aureus or Acne acne/Staphylococcus aureus/Candida albicans biofilm was cultured in 96-well polystyrene plates without agitation under anaerobic conditions for 6 days by adding 3,3-diindolylmethane at different concentrations. did. Afterwards, the planktonic cells were washed three times with water, discarded, and stained with carboxyfluorescein diacetate succinimidyl ester. Biofilm cells on the plate were visualized with a 488 nm Ar laser (emission wavelength 500–550 nm) using a confocal laser scanning microscope (Nikon Eclipse Ti, Tokyo, Japan). COMSTAT software was used to determine biofilm volume (μm ³ /μm ² ), average biofilm thickness (μM) and substrate coverage (%). For each experimental sample, two independent cultures of bacteria were used and at least 10 random spots were analyzed.

Multispecies biofilms on nylon membranes were observed using scanning electron microscopy (SEM). Briefly, single or mixed species were treated in the presence or absence of 3,3-diindolylmethane in 96-well plates, and a nylon membrane (0.4 × 0.4 mm square) was added to each well.

Biofilms were grown on nylon membranes by culturing together for 6 days at 37°C under anaerobic conditions without agitation. Afterwards, the biofilm cells attached to the nylon membrane were fixed with 2.5% glutaraldehyde and 2% formaldehyde for 20 h, successively fixed with osmium tetroxide, and incubated in an ethanol series (50, 70, 80, 90, 95, and 100%) for 30 minutes each, and isoamyl acetate for 20 hours.

After critical point drying, the cells on the filter were coated with palladium/gold, and the cells were observed and imaged using a voltage of 15 kV under an S-4800 scanning electron microscope (Hitachi, Tokyo, Japan).

6. Extracellular matrix production (extracellular polymeric substance, EPS) analysis

For EPS quantification, 50 ml of control and indole-treated Acne bacteria were cultured at 37°C for 6 days. After incubation, the culture was centrifuged at 8,228 × g for 10 minutes and the supernatant was separated.

The precipitated cells were washed with sterilized PBS to remove the culture supernatant. 50 ml of isotonic buffer (10mM Tris/HCl, pH 8.0, 10mM EDTA, 2.5% NaCl) was added to the cell pellet, and the mixed bacteria were left at 4°C overnight. Afterwards, the cell suspension was mixed for 5 minutes and centrifuged at 8,228 × g for 10 minutes. The supernatant containing EPS bound to the cells was separated from the cells. Cold ethanol was added to the supernatant at a ratio of 1:3 and left at -20°C overnight. The precipitated EPS was collected by centrifugation at 18,514 × g for 30 minutes at 4°C, and the precipitate was washed with 70% ethanol, dried, and weighed. The percentage inhibition of EPS production by indole was calculated by expressing the treated EPS weight as a percentage of the control weight.

7. Hyphal formation and cell aggregation assay for Candida albicans

To observe filamentous growth and hyphal formation, a real-time video microscopy system was used. Briefly, Candida albicans cells were diluted 1:50 in 1 ml of PDB medium containing various concentrations of indole, indole-3-carbinol, or 3,3-diindolylmethane, re-inoculated, and incubated at 37°C for 24 hours without agitation. It was cultured for some time. Cells were then visualized using the iRiS ^™ Digital Cell Imaging System according to the manufacturer's instructions. At least four cultures were used. For cell aggregation analysis, Candida albicans was cultured overnight in 1 ml of mycelium promotion medium (RPMI-1640, GE Healthcare Bio-Sciences, Pittsburgh, PA) supplemented with 10% bovine serum. One culture was diluted 1:50.

For cell aggregation assessment, Candida albicans cells were seeded in 1 ml of hyphal promotion medium (RPMI-1640, GE Healthcare Bio-Sciences, Pittsburgh, PA) supplemented with 10% bovine serum (GE Healthcare Bio-Sciences) in 1.7 ml tubes. After culturing for 20 hours, it was diluted 1:50, inoculated, and cultured at 37°C for 24 hours without stirring. After incubation, cells were visualized using iRiS ^™ Digital Cell Imaging at 4× and 10× magnification, and at least five independent experiments were performed.

8. Quantitative real-time reverse transcription PCR (qRT-PCR) for gene expression studies

For gene expression analysis, 15 ml of P. acnes with an initial turbidity of 0.05 at OD600 (23 ± 3 × 10 ⁵ CFU) was inoculated into RCM liquid medium in a 15 ml tube and cultured for 3 days at 37°C without agitation under anaerobic conditions. After incubation, cells were cultured in the presence or absence of 3,3-diindolylmethane (0.1mM) for an additional 24 h at 37°C without agitation. To prevent RNA degradation, RNAlater (RNase inhibitor, Ambion, TX, USA) was used before harvesting cells. Total RNA was extracted and washed using the Qiagen RNeasy mini Kit (Valencia, CA, USA).

qRT-PCR was used to determine the expression of biofilm- and virulence-related genes, namely PPA1761, PPA1796, PPA2105, PPA380, hly, PPA0149, btuR, cbiL, roxP, tly and PPA0349. Expression of 16s rRNA was not affected by 3,3-diindolylmethane. It was performed using SYBR Green master mix (Applied Biosystems, Foster City, CA, USA) and the ABI StepOne Real-Time PCR System (Applied Biosystems). At least two independent cultures and four PCR reactions were used.

9. Statistical analysis

The number of replicates for each experiment is provided for each method, and results were expressed as mean ± standard deviation. One-way analysis of variance followed by Dunnett's test using SPSS version was performed for statistical analysis. A P value of <0.05 was considered significant. In the figure, * marks indicate significant changes between treated and untreated samples.

<Example 1> Confirmation of antibacterial and antibiofilm activity of indole against acne bacteria

To evaluate the antibiofilm properties against acne bacteria, 20 indoles at a concentration of 0.1 mM were screened in a 96-well plate as shown in Figure 1.

As a result, among the indole derivatives tested as shown in Figure 2A, 7-azaindole, 7-benzyloxyindole, 3,3'-diindolylmethane (DIM), and indole-3-carbinol at a concentration of 0.1 mM significantly inhibited biofilm formation by Acne bacteria. whereas indole and other indoles were less effective.

The antibacterial concentration of 20 indole analogs against Acne acne was investigated using minimum growth inhibitory concentration (MIC) analysis. As a result, as shown in Figure 1, the MICs of most indole analogs were >5 mM, while the MICs of indole-3-carbinol, 3,3'-diindolylmethane, 7-methylindole, and 7-nitroindole were each >5 after 6 days of culture. mM, 0.1mM, 1.0mM and 2mM.

Additionally, referring to the planktonic growth curve in Figure 2, indole up to 0.1 mM does not inhibit cell growth, 3,3'-diindolylmethane at 0.1 mM completely inhibits cell growth, and indole-3 at 0.1 mM -Carbinol was confirmed to partially inhibit cell growth.

From the above results, it was confirmed that biofilm formation of acne bacteria was partially suppressed due to the antibacterial activity of 3,3'-diindolylmethane and indole-3-carbinol.

<Example 2> Confirmation of the acne biofilm inhibition effect of indole-3-carbinol, 3,3’-diindolylmethane and antibiotics

From the previously confirmed biofilm experiment results, it was confirmed that indole, indole-3-carbinol, and 3,3'-diindolylmethane inhibit the biofilm formation of acne bacteria in a concentration-dependent manner.

More specifically, as shown in Figure 2E, 1 mM (117 μg/ml) of indole significantly inhibited the formation of P. acnes biofilm, and this result is the first report that indole has antibacterial film activity against P. acnes. However, as shown in Figure 2F, 0.1mM (32 μg/ml) of 3,3'-diindolylmethane almost inhibited biofilm formation, while Indole-3-carbinol inhibited the formation of P. acnes biofilm in a concentration-dependent manner as shown in Figure 2G. , lower than that of 3'-diindolylmethane.

Meanwhile, the antibiofilm activity of 3,3'-diindolylmethane was compared with three antibiotics used as common acne treatments: benzoyl peroxide, gentamycin, and ciprofloxacin. Referring to Figure 3, biofilm formation of acne bacteria was suppressed in a concentration-dependent manner. Benzoyl peroxide, commonly used in the treatment of acne, showed a lower inhibitory effect on biofilm formation of acne bacteria than 3,3'-diindolylmethane, while gentamycin and the broad-spectrum antibiotic ciprofloxacin were more effective in inhibiting cell growth and biofilm formation of acne bacteria. For example, as shown in Figures 3A to 3C, 200 μg/ml of benzoyl peroxide inhibited biofilm formation by 60% with an MIC of 400 μg/ml. 20 μg/ml of gentamycin inhibited biofilm formation by 98% at an MIC of 100 μg/ml, and 1 μg/ml of ciprofloxacin inhibited biofilm formation by 94% at an MIC of 5 μg/ml.

<Example 3> Confirmation of the effect of the combination of 3,3'-diindolylmethane and antibiotics on the anti-biofilm effect against acne bacteria

The effect of the combination of 3,3'-diindolylmethane and antibiotics on the antibiofilm effect against acne bacteria was confirmed. As a result, antibiofilm activity was improved when 3,3'-diindolylmethane and antibiotics were used together. More specifically, as shown in Figure 3E, 5 μg/ml of Gentamycin reduced biofilm formation by 53%, while Gentamycin (0.5 μg/ml) and 3,3'-diindolylmethane (0.05 mM) reduced biofilm formation by 93%. . Additionally, as shown in Figure 3F, 0.5 μg/ml of ciprofloxacin reduced biofilm formation by 37%, while treatment with ciprofloxacin (0.5 μg/ml) and 3,3’-diindolylmethane (0.05 mM) reduced biofilm formation by 84%. However, as shown in Figure 3D, 3,3'-diindolylmethane did not improve the efficacy of benzoyl peroxide.

<Example 4> Confirmation of anti-biofilm activity of 3,3’-diindolylmethane against biofilm formation of Candida albicans and Staphylococcus aureus

Acne acne often forms biofilms on the skin with other microorganisms, such as Staphylococcus aureus or Candida albicans, and these biofilms tend to be more resistant to antibacterial agents. Therefore, the inhibitory effect of 3,3'-diindolylmethane on Staphylococcus aureus or Candida albicans biofilms was investigated under aerobic and anaerobic conditions.

As a result, as shown in Figures 4A and 4B, biofilm formation by Candida albicans and Staphylococcus aureus was inhibited in a dose-dependent manner by 3,3'-diindolylmethane after culturing for 1 day under aerobic conditions. More specifically, 0.1mM of 3,3'-diindolylmethane inhibited Candida albicans biofilm formation by 94% and Staphylococcus aureus biofilm formation by 71% under aerobic conditions. Additionally, 3,3'-diindolylmethane significantly inhibited biofilm formation by Candida albicans and Staphylococcus aureus after culturing for 6 days under anaerobic conditions.

As the minimum growth inhibitory concentration (MIC) of 3,3'-diindolylmethane for Candida albicans and Staphylococcus aureus was confirmed to be >1mM, the antibacterial activity of 3,3'-diindolylmethane partially affects the antibiofilm activity. It could be suggested that he was crazy.

<Example 5> Confirmation of biofilm inhibition of acne bacteria by 3,3’-diindolylmethane through microscopic observation

Biofilm inhibition was analyzed using a live cell imaging system and CSLM and SEM. Referring to Figure 5A, the 3-D color mesh plot confirmed that 3,3'-diindolylmethane inhibits biofilm formation by Acne bacteria at 0.02 or 0.05 mM.

Additionally, as shown in Figures 5B and 5C, CLSM and COMSTAT analyzes better revealed biofilm changes. Additionally, untreated Acne acnes formed high-density biofilms (thickness greater than 40 μm and surface coverage of nearly 100%), while Acne acnes treated with 0.02 or 0.05 mM 3,3'-diindolylmethane significantly reduced biofilm density and thickness. decreased. More specifically, biofilm biomass, thickness, and surface coverage were reduced >88, 87, and 95%, respectively, by 0.05 mM 3,3'-diindolylmethane compared to the untreated control.

In addition, through 5D SEM analysis, it was confirmed that 3,3'-diindolylmethane reduces the production of EPS in biofilms and reduces the cell density and biofilm formation of acne bacteria. Interestingly, 3,3’-diindolylmethane treatment increased cell length, indicating inhibition of cell division. More specifically, the cell size of P. acnes increased by 240% (3.0 ± 0.3 μm) and 252% (3.2 μm) in the presence of 0.02 or 0.05 mM 3,3′-diindolylmethane, respectively, compared to the untreated control (1.25 ± 0.4 μm). ± 0.2 μm) increased.

<Example 6> Antibiofilm activity of 3,3'-diindolylmethane against multiple biofilms of Acne acne, Candida albicans, and Staphylococcus aureus

As 3,3'-diindolylmethane effectively inhibits biofilm formation by Acne acne, Candida albicans, and Staphylococcus aureus, the anti-biofilm effect of 3,3'-diindolylmethane on multibacterial biofilm containing 2-3 species was confirmed. did.

Acne and Staphylococcus aureus in a 1:1 mix of RCM and LB, Acne acne and Candida albicans in a 1:1 mix of RCM and PDB, and Staphylococcus aureus in a 1:1:1 mix of RCM, LB, and PDB. Cocci and Candida albicans were used, and two mixtures of the two species and one mixture of the three species were inoculated into the medium, and the effect of 3,3'-diindolylmethane on the biofilm formation was confirmed.

As a result, as shown in Figures 6A to 6C, 3,3'-diindolylmethane inhibited biofilm formation in a concentration-dependent manner in all cases. In particular, as shown in Figure 6C, 0.05 mM 3,3'-diindolylmethane inhibited biofilm formation by 69% after culturing for 6 days under anaerobic conditions. The above results are the first report on three types of biofilms: Acne acne, Candida albicans, and Staphylococcus aureus.

In addition, multi-species biofilms were observed using a live cell imaging system, CLSM, and SEM. As a result, 3,3'-diindolylmethane was found to inhibit three types of biofilm in all three methods. More specifically, as shown in Figures 6D to 6F, the untreated group formed a biofilm with a thickness of approximately 19 μm with almost 100% surface coverage, but 3,3'-diindolylmethane at a concentration of 0.02 or 0.05 mM decreased biofilm density and biofilm density in a concentration-dependent manner. thickness was reduced. On the other hand, as shown in Figure 6F, the three types of biofilms were less dense and thinner than the single P. acnes biofilm. Additionally, as shown in Figure 5B, SEM visualized the presence of Acne acne, Candida albicans, and Staphylococcus aureus in the mixed biofilm. On the other hand, in the untreated control group as shown in Figure 6G, Candida albicans formed pseudohyphae and yeast cells that were much larger than the round cells of Staphylococcus aureus or the rod-shaped cells of Acne acne. Meanwhile, treatment with 0.02 mM 3,3'-diindolylmethane prevented adhesion of most Candida albicans cells, and few Staphylococcus aureus cells were observed. In particular, 0.05 mM DIM significantly reduced the production of EPS in the three biofilms, and only a few Staphylococcus aureus cells remained, while no Candida albicans cells and Acne acne cells remained.

<Example 7> Confirmation of the effect of 3,3’-diindolylmethane on reducing extracellular matrix (EPS) production in acne bacteria biofilm

Extracellular matrix (EPS) production is a key feature of biofilm formation and helps protect cells from external environmental challenges. Extracellular matrix production by P. acnes was measured in the presence of indole, 3,3’-diindolylmethane, and indole-3-carbinol.

Referring to Figures 7A to 7C, indole at a concentration of up to 2 mM had no effect on EPS production, but 3,3'-diindolylmethane and indole-3-carbinol reduced extracellular matrix production in Acne acnes in a dose-dependent manner. , 0.05mM of 3,3'-diindolylmethane reduced the extracellular matrix production of Acne bacteria by up to 90%.

From the above results, it was confirmed that the antibiofilm activity of 3,3'-diindolylmethane was partially caused by inhibiting extracellular matrix production.

<Example 8> Confirmation of inhibition of mycelial growth and cell aggregation of Candida albicans with 3,3'-diindolylmethane and indole-3-carbinol

As heterozygous conversion of yeast cells into hyphal cells and cell aggregation are prerequisites for Candida albicans biofilm development, microscopy and cell aggregation assays were performed to study the effect of indole on Candida albicans morphology.

As a result, large cell aggregates entangled by hyphae and hyphae were observed in untreated Candida albicans after 24 hours. In more detail, as shown in Figure 8A, treatment with 0.1 mM 3,3'-diindolylmethane or indole-3-carbinol inhibited hyphae formation, and most yeast and pseudohyphal cells were observed. Additionally, as shown in Figure 8B, indole at concentrations up to 0.2 mM had no effect on this heterotypic transition, but 0.05 mM 3,3'-diindolylmethane clearly inhibited hyphal formation and cell aggregation.

From the above results, it was confirmed that 3,3'-diindolylmethane strongly inhibits 3,3'-diindolylmethane biofilm formation by inhibiting hyphae formation and cell aggregation.

<Example 9> Changes in gene expression caused by 3,3’-diindolylmethane in acne bacteria

qRT-PCR was performed to confirm the effect of 3,3'-diindolylmethane on the expression of 11 biofilm and virulence-related genes in Acne acne. Overall, 3,3'-diindolylmethane regulated the expression of several lipase genes, hyaluronate lyase genes, and toxicity-related genes, while no changes in the expression of housekeeping genes (16s rRNA) were confirmed, as shown in Figure 9. In particular, the expression of lipase genes (PPA1796 and PPA2105), hyaluronate lyase (hly), and precorrin-2 C(20)-methyltransferase (cbiL) genes was decreased, while the expression of hemolysin (tly) was decreased by 0.1 mM of 3,3'- It was increased 3-fold by diindolylmethane.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (9)

Contains 0.05mM to 0.2mM of 3,3'-diindolylmethane (DIM) as an active ingredient, and contains Cutibacterium acnes (C. acnes) and Staphylococcus strains. ) and a composition for inhibiting biofilm formation of fluconazole-resistant Candida albicans. The composition for inhibiting biofilm formation according to claim 1, wherein the composition further comprises indole-3-carbinol as an active ingredient. The composition for inhibiting biofilm formation according to claim 1, wherein the Staphylococcus aureus is methicillin-susceptible and resistant Staphylococcus aureus. delete The composition for inhibiting biofilm formation according to claim 2, wherein the indole-3-carbinol inhibits biofilm formation without cell death. The method according to claim 1, wherein the 3,3'-diindolylmethane is a multi-biofilm in which Cutibacterium acnes (C. acnes), Candida albicans (C. albicans), and Staphylococcus aureus (S. aureus) form a biofilm together. A composition for inhibiting biofilm formation, characterized in that it inhibits species biofilm formation. Contains 0.05mM to 0.2mM of 3,3'-diindolylmethane (DIM) as an active ingredient, and protects against Cutibacterium acnes (C. acnes) and Staphylococcus strains. ) and a cosmetic composition for preventing or improving dermatitis disease or skin trouble caused by biofilm of fluconazole-resistant Candida albicans. The cosmetic composition of claim 7, wherein the composition further comprises indole-3-carbinol as an active ingredient. The method of claim 7, wherein the dermatitis disease includes comedonal acne, papular acne, pustular acne, crystalline acne, cystic acne, acne vulgaris, acne fulminans, agglutinative acne, keloid acne, psoriasis, and seborrheic dermatitis. , nummular dermatitis, staphylococcal scalded skin syndrome, folliculitis, impetigo, abscess, cellulitis, toxic epidermal necrolysis, epidermal exfoliation syndrome, candidiasis, candidal nail fungus, and general dermatitis. Composition.