KR102099534B1 - Composition for repressing or removing Staphylococcus spp. biofilm comprising the extract of Orostachys japonicas - Google Patents

Abstract

The present invention relates to a composition for inhibiting or removing Staphylococcus aureus bio-films containing Wasong extract and various applications thereof. Wasson extract of the present invention contains a quercetin (quercetin) or camperol (kaempferol) component, inhibits the cell surface adhesion of Staphylococcus aureus and PBP2a latex (latex) agglutination reaction, mecillin resistance biomarker mecA gene and coding therewith Since it inhibits the PBP2A (penicillin binding protein 2A) protein, it has an effect of inhibiting staphylococci and staphylococci having methicillin resistance, and thus can be usefully used in the pharmaceutical and health functional food fields.

Description

Composition for inhibiting or removing staphylococcus aureus biofilm containing Wasong extract {Composition for repressing or removing Staphylococcus spp. biofilm comprising the extract of Orostachys japonicas}

The present invention relates to a composition for inhibiting or removing a staphylococcal biofilm comprising a Wasong extract and various applications thereof.

In its natural state, most bacteria use polysaccharides or proteins to form biofilms, a community of sticking microbes that attach to the surface. The biofilm is a form in which an extracellular matrix (mucosal surface) made of a polymer matrix (polysaccharide and polypeptide is the main component) surrounds the bacterial colony. That is, the biofilm is a complex composed of a bacterial colony, which is a solid biological surface, and an outer membrane, which is a non-biological surface. Biofilms are common in nature, such as the slime found in rocks and ponds. Biofilm can be said to be a small city made of bacteria, in which bacteria communicate with each other and defend against the outside world. Due to this, the biofilm exhibits resistance to various environmental stresses including antibiotics, and contributes to bacterial resistance to chronic infection.

Biofilms are often found in relation to bacterial infectious diseases in addition to the general natural environment. It may form in human organs, may appear in the form of plaques on teeth, and may form on industrial equipment or medical implants. Because of this, biofilm has been the subject of interest of scholars studying periodontal disease, pneumonia accompanying cystic fibrosis, and earache in the middle ear. In a 2002 report by the National Institutes of Health, it was estimated that up to 80% of the bacterial population was spreading pathogens through the formation of such biofilms. Separately, antibiotics that have shown efficacy against planktonic bacteria are also said to have a greater tendency to lose their efficacy when bacteria form biofilms (Trends Microbiol 9: 34-39, 2001).

It is said that when bacteria form a biofilm, antibodies, etc., cannot penetrate the outer membrane existing in the biofilm, thereby incapacitating the host immune system and increasing the resistance of bacteria to antibiotics by about 1,000 times. Antimicrob Agents Chemother 47: 3407-3414, 2003). The cause of the increase in resistance to antibiotics due to the formation of such a biofilm is not yet clearly identified, but the formation of the biofilm reduces the dependence on the surrounding environment and the exchange action, and the outer membrane composed of mucous polysaccharides contains antibiotics for individual bacteria. This is because when a biofilm is formed, non-resistant bacteria present in the biofilm also acquire resistance factor-related genes through horizontal gene transfer from the surrounding resistance bacteria, thereby preventing resistance bacteriosis. It can be explained because it proceeds. Therefore, when the biofilm is formed, it can be considered that it has become an antibiotic-resistant state, and the action of antibiotics widely used in the treatment of infectious diseases becomes difficult, resulting in a problem that the therapeutic effect of the antibiotic is weakened. In other words, the formation of a biofilm means that it enters a state of chronic bacterial infection. In this case, as described above, the susceptibility of bacteria to antibiotics is lowered, so even using antibiotics has little effect. To overcome this, simply over-prescribing antibiotics increases the antibiotic resistance of bacteria only. In particular, infections caused by bacteria forming biofilms are more serious because they are often caused by multi-drug resistance bacteria that are resistant to various antibiotics.

Among the bacteria that form biofilms, Staphylococcus aureus is a Gram positive pathogenic bacterium, causing purulent, abscess formation, various purulent infections, and sepsis. . Staphylococcus aureus is a very dangerous pathogen, the world's highest level with an average of 73% resistance to the antibiotic methicillin investigated in Korea. This means that 73% of Staphylococcus aureus, which does not die even with antibiotics, is a very serious pathogen. When a biofilm is formed by Staphylococcus aureus, chronic infection is caused, and treatment of a disease caused by Staphylococcus aureus with a biofilm is particularly difficult compared to treatment of a disease caused by a biofilm formed by other bacteria. This is because, even if drugs are used for the treatment of diseases, there is difficulty in drug delivery due to the biofilm, and even if the drugs for diarrhea are delivered, treatment based on existing antibiotics is not effective due to the nature of Staphylococcus aureus, which has many resistant strains.

Regarding the inhibition of bacterial biofilm formation, Streptomyces albus extract inhibits the biofilm formation of Vibrio harvey (Applied Microbiology and Biotechnology 76: 1137-1144, You et al. 2007) ), indole-3-acetaldehyde produced from Rhodococcus BFI 332 is known to inhibit the formation of a biofilm of intestinal bleeding E. coli O157:H7 (Korean Patent Laid-Open No. 10-2013-0128141). In addition, with regard to the biofilm inhibition of Staphylococcus aureus, a method of using lysostaphin is known (Antimicrob Agents Chemother 47: 3407-3414, 2003), but Staphylococcus aureus is not sensitive to lysostaphin. There are quite a few lysostaphinresistant strains (J Clin Microbiol 11: 724-727, 1980; Antimicrob Agents Chemother 47: 3407-3414, 2003), and Staphylococcus aureus, which has acquired resistance to lysostaphin, is occurring. There is a problem in

As described above, if the microbial biofilm is not effectively suppressed and removed, it may cause serious problems in industry and medicine. Therefore, in order to prevent the incapacitation of antibiotic treatment due to the formation of a biofilm, a new material or an existing antibiotic capable of suppressing the formation of the biofilm and removing the biofilm even if the biofilm is formed can be applied to the biofilm. There is a need to develop a material that can decompose the existing outer membrane.

It is an object of the present invention to provide a composition for inhibiting or removing a staphylococcus spp. biofilm comprising an extract of Orostachys japonicas as an active ingredient.

In addition, it is an object of the present invention to provide a composition for inhibiting antibiotic resistance against staphylococcus aureus comprising a Wasong extract as an active ingredient.

In addition, it is an object of the present invention to provide a pharmaceutical composition for preventing and treating staphylococcal infection, comprising a Wasong extract as an active ingredient.

In addition, it is an object of the present invention to provide an antibacterial composition for inhibiting staphylococcus aureus comprising a Wasong extract as an active ingredient.

In addition, it is an object of the present invention to provide a health functional food composition for inhibiting staphylococcus aureus comprising a Wasong extract as an active ingredient.

It is also an object of the present invention to provide a feed composition for inhibiting staphylococcus aureus comprising a Wasong extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for inhibiting or removing a staphylococcus spp. biofilm containing an extract of Orostachys japonicas as an active ingredient.

In addition, the present invention provides a composition for inhibiting antibiotic resistance against Staphylococcus aureus, comprising the Wasong extract as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of staphylococcal infection, comprising the Wasong extract as an active ingredient.

In addition, the present invention provides an antimicrobial composition for inhibiting staphylococcus aureus comprising a Wasong extract as an active ingredient.

In addition, the present invention provides a health functional food composition for inhibiting staphylococcus aureus comprising the Wasong extract as an active ingredient.

In addition, the present invention provides a feed composition for inhibiting staphylococcus aureus containing the Wasong extract as an active ingredient.

Wasong extract of the present invention contains quercetin or camperol component, inhibits cell surface adhesion of staphylococcus aureus and PBP2a latex agglutination reaction, and mecA gene, which is a methicillin resistance biomarker, is encoded therein. Since it inhibits the PBP2A (penicillin binding protein 2A) protein, it has the effect of inhibiting staphylococcus aureus and staphylococcus having resistance to methicillin, so it can be usefully used in pharmaceutical and health functional food fields.

1 is a diagram showing an extraction solvent and an extraction sequence used in the Wasong extraction of the present invention.
Figure 2 shows an HPLC chromatogram, (1) of (A) is quercetin alone, (2) is a Wasong extract alone, (3) is a result of analysis of quercetin + Wasong extract, ( B) is a diagram showing the results of analysis of (1) camphorol (Kaempferol) alone, (2) Wasong extract alone, and (3) Camphorol + Wasong extract.
Figure 3 is a diagram showing the result of confirming the MRSA biofilm formation (%) in the microtiter plate containing the Wasong extract of each concentration.
Figure 4 is a diagram showing the results of confirming the cell adhesion (%) in the microtiter plate containing the Wasong extract at each concentration.
5 is a diagram showing the results of confirming by confocal laser scanning microscopy (CLSM) by staining MRSA biofilm with SYTO 9 after treating each concentration of Wasong extract.
6 is a diagram showing the results of confirming the degree of aggregation of PBP2a of MRSA treated with Wasong extract.
7 is a diagram showing the results of confirming the mecA gene expression level of MRSA after treatment with Wasong extract at each concentration.

The present invention provides a composition for inhibiting or removing a staphylococcus spp. biofilm containing an extract of Wasong (Orostachys japonicas ) as an active ingredient.

In the present invention, the term "biofilm" is a complex composed of a bacterial colony, which is a solid biological surface, and an outer membrane, which is a non-biological surface. In addition, it exhibits resistance to various environmental stresses, including antibiotics, and contributes to bacterial resistance to chronic infection.

The Wasong extract is extracted with one or more primary extraction solvents selected from the group consisting of distilled water, methanol and ethanol, and is not limited thereto, preferably in order to correctly extract methanol or major components of Wasong.

The extraction solvent is at least one secondary extraction solvent selected from the group consisting of ethylacetate (EtOAc), n-hexane, dichloromethane (DCM), and n-butanol (BuOH), but preferably It is not limited to ethyl acetate, as long as it is to correctly extract the main components of Wasong.

The Wasong extract is one or more selected from the group consisting of camperol, quercetin, catechin, myricetin, luteolin, and apigenin, but preferably Camphorol or quercetin, but not limited thereto.

* The Wasong extract may inhibit the expression of a PBP2A (penicillin binding protein 2A) protein or MecA gene, thereby inhibiting the production of a biofilm of staphylococcus aureus.

The Staphylococcus aureus is a Gram-positive bacteria belonging to the genus Staphylococcus aureus , and can be classified as Staphylococcus aureus, Staphylococcus epidermidis , and Staphylococcus saprophyticus. For example, S. aureus , S. simiae , S. auricularis , S. carnosus , S. condiment ( S. condimenti ), S. assiliensis , S. piscifermentans , S. simulans , S. capitis , S. S. caprae , S. epidermidis , S. saccharolyticus , S. devriesei , S. haemolyticus (S. haemolyticus), S. hoe Nice (S. hominis), S. Croix mojen's (S. chromogenes), S. Felice (S. felis), S. Delfini (S. delphini), S. high Nukus (S. hyicus ), S. intermedius , S. lutrae , S. microti , S. muscae, S. pseudoin the midius It may be ( S. pseudointermedius ), S. rostri , or S. gallinarum , but is preferably Staphylococcus aureus and is not limited thereto.

In addition, the staphylococcus of the present invention may include methicillin-resistant staphylococcus aureus , and methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus epidermidis , MRSE) or methicillin-resistant Staphylococcus saprophyticus , but preferably methicillin-resistant Staphylococcus aureus, but is not limited thereto.

In addition, the present invention provides a composition for inhibiting antibiotic resistance against Staphylococcus aureus, comprising the Wasong extract as an active ingredient.

The antibiotics are methicillin-based, penicillin-based, cephalosporin-based, monobactam-based, aminoglycoside-based, macrolide-based, tetracycline-based, and tetracycline-based antibiotics. )-Based, glycopeptide-based, lincomycin-based, and quinolone-based one or more selected from the group consisting of, but is preferably methicillin-based, but is not limited thereto.

In addition, the present invention provides a pharmaceutical composition for the prevention and treatment of staphylococcal infection, comprising the Wasong extract as an active ingredient.

These infections include caries, periodontitis, otitis media, musculoskeletal infection, necrotizing fasciitis, bile duct infection, osteomyelitis, bacterial prostatitis, mastitis, dermatitis, sepsis, purulent disease, food poisoning, impetigo, bacteremia, endocarditis, enteritis, cystic fibrosis pneumonia , Meloidosis, nosocomial infection, ICU pneumonia, urinary catheter cystitis, peritoneal dialysis (CAPD) peritonitis and biliary stent blockage. It may be abnormal, but is not limited thereto.

Wasong extract according to the present invention can be used to be obtained by extraction and separation from nature using an extraction and separation method known in the art, "extract" as defined in the present invention is extracted from Wasong using an appropriate solvent , For example, a crude extract of Wasong, a polar solvent-soluble extract or a non-polar solvent-soluble extract. Any suitable solvent for extracting the extract from Wasong may be used as long as it is a pharmaceutically acceptable organic solvent, and water or an organic solvent may be used, but is not limited thereto. Can be used. As the extraction method, any one of methods such as hot water extraction, cold precipitation extraction, reflux cooling extraction, solvent extraction, steam distillation method, ultrasonic extraction method, elution method, and compression method may be used. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method.

There is no limitation on the method for preparing the Wasong extract of the present invention, and any known method may be used. For example, the Wasong extract included in the composition of the present invention can be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying of the primary extract extracted by the hot water extraction or solvent extraction method described above. . In addition, the primary extract was further purified using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. You can also get it. Therefore, in the present invention, the Wasong extract is a concept including all extracts, fractions, and purified products obtained in each step of extraction, fractionation, or purification, and their dilutions, concentrates, or dried products.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the Wasong extract. Any of the adjuvants known in the art may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase its immunity.

The pharmaceutical composition according to the present invention may be prepared in a form in which an active ingredient is incorporated in a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention are not limited thereto, but lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The pharmaceutical compositions of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions according to a conventional method. .

In the case of formulation, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient in the active ingredient, such as starch, calcium carbonate, sucrose, lactose, gelatin. It can be prepared by mixing and the like. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used diluents. I can. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like may be used. As a base for suppositories, witepsol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the individual's age, weight, sex, and physical condition. It is apparent that the concentration of the Wasong extract contained in the pharmaceutical composition can be selected in various ways depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg/ml. When the concentration is less than 0.01 μg/ml, pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg/ml, toxicity to the human body may occur.

In addition, the present invention provides an antimicrobial composition for inhibiting staphylococcus aureus comprising a Wasong extract as an active ingredient.

Since the antimicrobial composition includes the above-described pharmaceutical composition, descriptions of the contents overlapping with the above-described composition of the present invention are omitted in order to avoid excessive complexity of the present specification due to the description of the overlapped contents.

In addition, the present invention provides a health functional food composition for inhibiting staphylococcus aureus comprising the Wasong extract as an active ingredient.

The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as an additional component, as in a conventional food composition, in addition to containing the Wasong extract as an active ingredient.

Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The above-described flavoring agent can be advantageously used as a natural flavoring agent (taumatin), stevia extract (eg, rebaudioside A, glycyrrhizin, etc.), and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. There is this.

In addition, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, in addition to the active ingredient Wasong extract. , Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juice and fruit juice beverages and vegetable beverages.

The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of inhibiting staphylococcus. In the present invention, the term'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functions for the human body pursuant to the Health Functional Food Act No.6727, and with respect to the structure and function of the human body. It means ingestion for the purpose of obtaining useful effects for health use such as controlling nutrients or physiological effects. The health functional food of the present invention may contain ordinary food additives, and whether it is suitable as a food additive is determined according to the general rules and general test methods of food additives approved by the Food and Drug Administration, unless otherwise specified. It is judged according to the standards and standards. Examples of the items listed in the'Food Additives Code' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as reduced pigment, licorice extract, crystalline cellulose, high color pigment, and guar gum; Mixed preparations, such as a sodium L-glutamate preparation, an alkali additive for noodles, a preservative preparation, and a tar color preparation, etc. are mentioned. For example, in a health functional food in the form of a tablet, a mixture of Wasong extract, an active ingredient of the present invention, mixed with an excipient, a binder, a disintegrant, and other additives, is granulated by a conventional method, and then a lubricant is added thereto and compression molding. Alternatively, the mixture may be directly compression molded. In addition, the health functional food in the form of a tablet may contain a mating agent or the like as needed. Among the health functional foods in the form of capsules, hard capsules can be prepared by filling a conventional hard capsule with a mixture of Wasong extract, which is the active ingredient of the present invention, with additives such as excipients, and soft capsules include Wasong extract as additives such as excipients. The mixture mixed with can be prepared by filling in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary. The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention, Wasong extract, excipients, binders, disintegrants, etc., by conventionally known methods. Alternatively, the surface may be coated with a material such as starch or talc. Health functional foods in the form of granules can be prepared in granular form by a mixture of excipients, binders, disintegrants, etc. of Wasong extract, which are the active ingredients of the present invention, in granular form, and if necessary, flavoring agents, flavoring agents, etc. It may contain.

In addition, the present invention provides a feed composition for inhibiting staphylococcus aureus containing the Wasong extract as an active ingredient.

The feed composition of the present invention may further include ingredients added to conventional feed. Examples of ingredients added to such feed may include grain powder, meat powder, and beans. In the above, the grain powder may be one or more selected from rice flour, wheat flour, barley flour, and corn flour. In the above, the meat powder may be a meat powder obtained by powdering at least one selected from chicken, beef, pork, and ostrich meat. Beans in the above may be used one or more selected from soybeans, kidney beans, peas, and black beans.

In the feed composition of the present invention, in addition to the ingredients added to the conventional feed mentioned above, in order to increase the nutritional properties of the feed, in addition to grain powder, meat powder, and beans, any one or more selected from nutrients and minerals may be added, and feed quality It may contain at least one selected from an anti-fungal agent, an antioxidant, an anticoagulant, an emulsifier, and a binder to prevent the deterioration of the.

The composition may be coated on a medical device, a medical material, or a medical implant. For example, the coating composition may coat tissues or organs derived from organisms such as skin and teeth, and various medical facilities, equipment, instruments, lenses, artificial valves, pacemakers, surgical It is possible to coat medical items such as pins, insertion conduit, and catheter, but the scope of the present invention is not limited thereto.

The present invention will be described in more detail through the following examples. However, the following examples are only for embodiing the contents of the present invention, and the present invention is not limited thereto.

Example 1. Experimental Preparation and Experimental Method

1-1. Wasong( Orostachys japonicas ) Extract preparation

Wasong provided by Jirisan Wasong (Sancheong, Korea) was dried, cut and made into powder. The powdered Wasong (200g) was extracted by boiling twice for 3 hours with 95% methanol (MeOH) as a primary solvent. The extract was concentrated by rotary evaporation at 50, the concentrate was suspended in water, and then fractionated using each of the following solvents as a secondary solvent: n-hexane, dichloromethane (DCM), ethyl Acetate (EtOAc), n-butanol (BuOH), and water (H ₂ 0). Each of the solution fractions was concentrated by rotary evaporation at 40, and stored at -20 degrees before use. The EtOAc fraction was analyzed by thin layer chromatography (TLC) to confirm whether a constituent of Wasong such as camperol or quercetin was included.

1-2. Bacterial strain and culture conditions

MRSA (Methicillin resistant Staphylococcus aureus ) was obtained by isolation from clinical specimens at Seoul National University Hospital, and the bacteria were cultured in a medium nutrient agar (Difco Laboratories) containing Bacto beef extract (3mg/mL) and Bacto peptone (5mg/mL). Or, digest of casein (17mg/mL), papaic digest of soybean (3mg/mL), dextrose (2.5mg/mL), sodium chloride (5mg/mL) and dipotassium phosphate ( Tryptic soy broth (Becton, Dickinson and Company) containing 2.5mg/mL) was cultured at 35°C.

1-3. Confocal laser scanning microscopy (CLSM)

MRSA biofilm was confirmed on a glass-bottom confocal dish (SPL Life sciences). After exposing 0.5mg/mL and 1.0mg/mL Wasong extract for 24 hours, the biofilm was LIVE/DEAD. BacLight Bacterial Viability Kit (Invitrogen) was used to stain according to the manufacturer's manual: Briefly, the bacterial suspension was mixed with 1 μl SYTO 9 mixture and propidium iodide (1:1), and 15 Thereafter, each 5 μl sample suspension was pipetted onto a slide glass, covered with a coverslip, and then confocal laser scanning micro scope (LSM 510 META, Carl Zeiss, Germany) It was confirmed as.

1-4. Biofilm inhibition assay

The biofilm inhibition assay is a conventional method [Materaci et al. 2012, Agents Chemother., 56(12): 6366-71]. Briefly, 4 mg/mL, 2 mg/mL, 1 mg/mL, and 0.5 mg/mL of Wasong extract were prepared in a 96-well microtiter plate. For the final addition, an aliquot of the MRSA suspension was added to each well so as to be ^{1×10 5} CFU/mL in a final 200 μl volume of each well. The plate was incubated at 35 degrees for 24 hours. After incubation, each well is washed with a physiological buffered saline (PBS) buffer, and a crystal violet staining method for quantifying biofilm production [Durham-Colleran et al. (2010), Microb. Ecol., 59(3):457-465], and the experiment was performed three times.

1-5. Microtiter attachment test

The microtiter adhesion assay is a conventional method [Overhage J. et al. (2008), Infect. Immun., 76(9): 4176-4182]. Specifically, 4mg/mL, 2mg/mL, 1mg/mL, and 0.5mg/mL Wasong extract were prepared in a 96-well microtiter plate. For the final addition, an aliquot of the MRSA suspension was added to each well so as to be ^{1×10 7} CFU/mL in a final 200 μl volume of each well. The plate was incubated at 35 degrees for 1 hour. Then, after washing with PBS, the percentage of cell adhesion was determined by the crystal violet staining method [Durham-Colleran et al. (2010), Microb. Ecol., 59(3):457-465], and the experiment was performed three times.

1-6. HPLC analysis of Camperol, Quercetin and Wasong extract

Camperol, quercetin, and Wasong extract were analyzed by high performance liquid chromatography (HPLC), and the experiment was conducted by a conventional method [Federico F. et al. (1991), J. Sci. Food. Agric., 56(1): 49-56]. An Agilent 1100 series liquid chromatography system (Palo Alto, CA, USA), which includes a vacuum degassing device, a quaternary gradient pump, an autosampler and a diode array detector (DAD), was used and is controlled by Agilent ChemStation software. A hydrosphere C18 column (250 mm x 4.6 mm id, 5 um, YMC Co., Ltd., Kyoto, Japan) was used at a column temperature of 25. The applied-mobile phase gradient is methanol/water/formic acid (50:47:3 v), the flow rate is 1.0 mL/min, and the UV detection is 340 nm. 5 or 10 µl of methanol was injected into camphorol, quercetin, or Wasong extract, respectively.

1-7. PBP2a latex agglomeration test for MRSA biofilm

Conventional method [Carolina S. et al. (2015), Molecules, 20(3): 4473-4482], and MRSA was incubated at 37 degrees for 24 hours in a 50 mm diameter Petri dish of 10 mL TSB containing Wasong extract at a concentration of 1 mg/mL. After incubation, the culture appendages were carefully removed, 0.5 mL PBS was added, and the biofilm layer was scraped using a sterile 5 µl inoculation loop to fill the inner diameter (approximately 1.5 x 10 ⁹ CFU/mL given). The obtained bacterial biofilm was measured for PBP2a according to the manual of the MRSA screening kit (Denka Seiken, Tokyo, Japan). The quasi-quantitative measurement of PBP2a production in biofilms is a conventional method [Zhao WH et al. (2001), Agents Chemother. 45(6): 1737-1742], the cohesive strength was observed as-and ++, and the non-reactive control latex in which PBP2a was not produced was indicated as -.

1-8. Comparison of RNA extraction, cDNA synthesis and qRT-PCR

MRSA including 2mg/mL, 1mg/mL and 0.5mg/mL Wasong extract was incubated until the late exponential phase (24 hours). Total RNA and DNA were extracted using ExiProgenTM Fully Automated Nucleic Acid Extraction System and ExiPrep™ Viral DNA/RNA Kit (Bioneer Co., Korea). For the extracted total sample, DNA was removed using RiboclearTM plus kit (GeneAll biotechnology Co., Korea), and RNA was left. RNA concentration was measured at 260 and 280nm absorbance, and 300ng RNA was synthesized cDNA using M-MLV Reverse Transcriptase kit (Bioneer Co., Korea). Expression of the target gene was measured by qRT-PCR using the synthesized cDNA. qRT-PCR was performed with ExicyclerTM 96 Real-Time Quantitative PCR System and AccuPower® Dualstar™ qPCR PreMix kit (Bioneer Co., Korea). The qRT-PCR performance conditions were 95 to 5 minutes, initial denaturation from 95 to 5 seconds, 95 to 5 seconds 45 times, and 60 to 5 seconds. The results were analyzed with ExicyclerTM 96 3.0 software (Bioneer Co., Korea). The threshold cycle method (2 ^-ΔΔCT ) was performed according to the conventional method [Livak KJ et al. (2001) Methods, 25(4): 402-408], the change in gene expression of each sample was measured compared to the control (same condition without Wasong extract), and qRT-PCR was repeated three times. The mecA and 16s rRNA gene sequences are shown in Table 1 below.

Experimental Example 1. Inhibitory effect according to type of extraction solvent of Wasong

As shown in Example 1-1, Wasong powder (200 g) was repeatedly extracted with distilled water and 100 distilled water twice. In addition, the Wasong powder was treated and extracted at 60 degrees using 95% ethanol (EtOH) as a primary extraction solvent. In addition, 95% methanol (MeOH) was treated with 50 to extract the Wasong powder. Each extract was placed cold at room temperature and filtered using a filter paper (Whatman filter paper No. 1, Whatman, UK). Then, it was concentrated using a rotary evaporator (Rotary evaporator N-1000, EYELA, Japan). In order to obtain the powder of each concentrate, it was freeze-dried using a Freeze Dryer (Clean-Vac 8B, Hanil Research & Development, Korea) and stored at -20 degrees. Thereafter, using a disk diffusion method, the inhibitory effect of Staphylococcus aureus , Pseudomonas aeruginosa and Escherichia coli of the Wasong extract extracted with each of the extraction solvents was confirmed. The results are shown in Table 2 below.

As shown in Table 2, it was confirmed that the Wasong extract using methanol as the extraction solvent had an antibacterial effect, and it was confirmed that Staphylococcus aureus was particularly more effectively inhibited than the Wasong extract extracted with other extraction solvents. I did.

Accordingly, according to the results of Table 2, methanol was used as the primary extraction solvent during Wasong extraction, and as shown in FIG. 1, n-hexane and dichloromethane were used as secondary extraction solvents in the fraction extracted with methanol. (dichloromethane, DCM), ethylacetate (EtOAc), n-butanol (BuOH), or water (H ₂ O) was additionally used to obtain a Wasong extract and treated with each of the above bacteria. As a result, it was confirmed that the Wasong extract fractionated with ethyl acetate as a secondary extraction solvent showed the highest inhibitory effect, and the Wasong extract was used in the following experiment.

Experimental Example 2. Analysis confirmation of Wasong extract

Wasong is known to contain flavonoid components such as quercetin or camperol, and it was confirmed by HPLC analysis whether the Wasong extract of the present invention contained the above substances.

Specifically, it was carried out according to the method of Examples 1-6, and analysis of quercetin or campferol alone; Wasong extract alone analysis; Quercetin+ Wasong extract; Or camperol (kaempferol) + Wasong extract was compared. The results are shown in Fig. 2 and Table 3.

As shown in FIG. 2, as a result of HPLC analysis, it was confirmed that the Wasong extract of the present invention showed the same peak as that of quercetin or camperol, and the Wasong extract of the present invention was confirmed to contain quercetin or camphorol. I did.

In addition, as shown in Table 3, specifically, it was confirmed that quercetin contained 3.75% and camphorrol was 0.94%.

Experimental Example 3. Confirmation of biofilm formation inhibitory effect of methicillin resistant Staphylococcus aureus (MRSA)

Bacterial biofilms are known to protect colonies and cells. In order to confirm the inhibitory effect of MRSA on biofilm formation according to Wasong extract treatment, the method of Example 1-4 was performed. Wasong extract was treated with 4mg/mL, 2mg/mL, 1mg/mL and 0.5mg/mL. The results are shown in FIG. 3.

As shown in Figure 3, when 0.5, 1, 2, 4 mg/mL treatment of Wasong extract, it was confirmed that the biofilm inhibitory effect was shown to 91.91% ± 14.93, 67.66% ± 7.67, 50.45% ± 9.11 and 38.91% ± 4.77. I did. In addition, a significant inhibitory effect appeared even at a treatment amount of 1 mg/mL or more, and it was confirmed that the Wasong extract of the present invention had an excellent MRSA inhibitory effect.

Experimental Example 4. Confirmation of inhibition of cell surface adhesion of MRSA

When a bacterial biofilm is produced, the bacteria adhere to the cell surface to form a colony, and when the cells interact with the cell, the bacteria form a multilayered structure that forms a biofilm. Therefore, in order to inhibit MRSA, cell adhesion must be inhibited. Therefore, according to the method of Example 1-5, 0.5, 1, 2, 4 mg/mL of Wasong extract was treated to the well, and then MRSA was added to confirm its effect of inhibiting cell adhesion. The results are shown in FIG. 4.

As shown in FIG. 4, when 0.5, 1, 2, 4 mg/mL of Wasong extract of the present invention was treated, 81.93% ± 3.45, 44.14% ± 6.15, 43.48% ± 6.19 and 45.80% ± 5.42 were adhered to the surface of MRSA cells. It was confirmed to suppress. In addition, it was confirmed that a significant inhibitory effect exists at a treatment amount of 1 mg/mL or more.

Experimental Example 5. SYTO 9 staining and confirmation of the inhibitory effect of MRSA through confocal laser scanning microscopy (CLSM)

In order to confirm the inhibitory effect of MRSA according to the treatment of Wasong extract of the present invention, MRSA + DMSO (dimethyl sulfoxide) group, MRSA + Wasong extract (0.5mg/mL) group, and MRSA + Wasong extract ( 1.0mg/mL) was stained with SYTO 9 to check the degree of fluorescence, and the inhibition of MRSA was confirmed by CLSM. The results are shown in FIG. 5.

As shown in FIG. 5, when the Wasong extract was treated with 1.0 mg/mL, it was confirmed that the SYTO 9 fluorescence expression was low, and the MRSA inhibitory effect was exhibited.

Experimental Example 6. PBP2a latex (latex) agglutination inhibition effect on MRSA biofilm confirmed

Biofilm formation of MRSA is mediated by PBP2a, and PBP2a is a modified protein that avoids the antibacterial action of beta-lactam antibiotics, which is associated with multiple toxicity factors.

Therefore, in order to inhibit the MRSA biofilm, the effect of inhibiting PBP2a latex aggregation reaction was confirmed, and according to the method of Example 1-7, Wasong extract was treated with 0.5, 1.0 and 2.0 mg/mL, and PBP2a latex (latex) aggregation The MRSA control was expressed as ++, and the MSSA in which the PBP2a aggregation reaction did not appear was indicated as-and the effect of Wasong extract treatment was confirmed. The results are shown in Table 4 and FIG. 6.

As shown in Table 4 and FIG. 6, it was confirmed that the Wasong extract was effectively inhibited by inhibiting PBP2a latex aggregation when treated with 0.5, 1.0, and 2.0 mg/mL, thereby effectively inhibiting MRSA.

Experimental Example 7. mecA gene expression inhibition effect

The mecA gene is a biomarker of resistance to methicillin and other beta-lactam antibiotics and encodes the PBP2a protein. Therefore, in order to confirm the inhibitory effect of methicillin-resistant Staphylococcus aureus MRSA according to the mecA gene inhibitory effect, 0.5 and 1.0 mg/mL Wasong extract were treated with MRSA, and qRT-PCR by the method of Example 1-8. This was confirmed by analysis. The results are shown in FIG. 7.

As shown in FIG. 7, it was confirmed that the expression of the mecA gene was significantly inhibited when 0.5 and 1.0 mg/mL of Wasong extract were treated, and it was confirmed that MRSA was effectively suppressed.

Therefore, as shown in Figure 8, the Wasong extract extracted with methanol as the primary extraction solvent of the present invention inhibits the activity of Staphylococcus aureus , Pseudomonas aeruginosa , and Escherichia coli to prevent antibacterial It was confirmed that the effect was present. In addition, the Wasong extract extracted with ethyl acetate as a secondary extraction solvent again in the methanol extract contains quercetin or campferol, and inhibits adhesion of MRSA to the cell surface and aggregation of PBP2a latex. Thus, it was confirmed that the biofilm formation of MRSA was suppressed. In addition, it was confirmed that the expression of the mecA gene, which is a methicillin resistance biomarker, was inhibited, and consequently, MRSA activity was effectively inhibited.

<110> inje university industry-academic cooperation foundation <120> Composition for repressing or removing Staphylococcus spp. biofilm comprising the extract of Orostachys japonicas <130> PN1906-299 <150> KR 1020170132693 <151> 2017-10-12 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for 16s r RNA of Staphylococcus aureus <400> 1 atacgttccc gggtcttgta 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for 16s r RNA of Staphylococcus aureus <400> 2 caccccaatc atttgtccca 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for mecA gene for PBP 2a <400> 3 gcacttgtaa gcacaccttc a 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for mecA gene for PBP 2a <400> 4 ctggaacttg ttgagcagag g 21

Claims (9)

Methicillin-resistant Staphylococcus aureus, MRSA, comprising as an active ingredient an extract of Orostachys japonicas extracted with methanol as a primary extraction solvent and ethylacetate (EtOAc) as a secondary extraction solvent. ) Biofilm (biofilm) composition for inhibiting or removing. According to claim 1,
The wasson extract is characterized in that it includes at least one selected from the group consisting of camperol (Kaempferol), quercetin, catechin, myricetin, luteolin and apigenin. A composition for inhibiting or removing methicillin-resistant Staphylococcus aureus biofilm. According to claim 1,
The wasson extract is characterized in that to inhibit the expression of PBP2A (penicillin binding protein 2A) protein or MecA gene, methicillin-resistant Staphylococcus aureus biofilm inhibition or removal composition. According to claim 1,
The wasson extract is characterized in that to inhibit the production of a biofilm (biofilm) of Staphylococcus aureus, methicillin-resistant Staphylococcus aureus biofilm inhibition or removal composition. A pharmaceutical composition for the prevention and treatment of methicillin-resistant Staphylococcus aureus infection, comprising as a primary extraction solvent methanol and a washi extract extracted with ethyl acetate as a secondary extraction solvent as an active ingredient. The method of claim 5,
The infectious diseases include tooth decay, periodontitis, otitis media, musculoskeletal infection, necrotizing fasciitis, bile duct infection, osteomyelitis, bacterial prostatitis, mastitis, dermatitis, sepsis, purulent disease, food poisoning, impetigo, bacteremia, endocarditis, enteritis, cystic fibrosis 1 selected from the group consisting of pneumonia, meloidosis, nosocomial infection, ICU pneumonia, urinary catheter cystitis, peritoneal dialysis (CAPD) peritonitis and biliary stent blockage A pharmaceutical composition for preventing and treating methicillin-resistant Staphylococcus aureus infection, characterized in that it is more than a species. An antimicrobial composition for inhibiting methicillin-resistant Staphylococcus aureus, comprising as a primary extraction solvent methanol and a second extract solvent, ethyl acetate extracted with ethyl acetate as an active ingredient. A health functional food composition for preventing and improving methicillin-resistant Staphylococcus aureus, comprising as a primary extraction solvent methanol and a washi extract extracted with ethyl acetate as a secondary extraction solvent as an active ingredient. Methicillin-resistant Staphylococcus aureus prevention and improvement feed composition comprising as a primary extraction solvent methanol and a secondary extraction solvent ethyl acetate extracted with ethyl acetate as an active ingredient.

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