WO2012173452A1 - Antimicrobial agent made of ginsenoside compound k or derivatives thereof - Google Patents

Abstract

The present invention relates to an antimicrobial agent made of ginsenoside compound K or derivatives thereof. The present invention further relates to a pharmaceutical composition, to food additives, or to animal feed additives, containing the antimicrobial agent, for preventing or treating diseases caused by a variety of bacteria such as Staphylococcus strains including Staphylococcus aureus and antibiotic-resistant Staphylococcus aureus. The present invention further relates to a composition for antimicrobial quasi-drugs or to cosmetics, containing the antimicrobial agent.

Description

Antibacterial agent with ginsenoside compound shock or derivatives thereof

The present invention relates to an antimicrobial agent of ginsenoside compound K or derivatives thereof. In addition, the present invention is a pharmaceutical composition, food additive or feed additive for the prevention or treatment of diseases caused by various bacteria, including Staphylococcus aureus, including Staphylococcus aureus and antibiotic resistant Staphylococcus aureus containing the antimicrobial agent ; And it relates to antimicrobial composition or cosmetic for antimicrobial containing the antimicrobial agent.

Since penicillin was developed in the early 1940s, many antimicrobial agents have been developed and used for the treatment of infectious diseases, but the isolation of antimicrobial resistant bacteria is increasing in clinical samples. In particular, methicillin-resistant Staphylococcus aureus has become a clinical problem.

Antibacterial resistance of bacteria is largely classified into two types. Endogenous resistance is inherently resistant to bacterial species, such as E. coli resistance to oxacillin or vancomycin and E. coli resistance to cephalosporin, and acquired resistance is originally susceptible. The bacteria acquired resistance and are caused by acquiring mutations or new DNA. Mutation is a natural phenomenon that occurs regardless of antimicrobial exposure, and rifampin resistance is a representative example. Methods of obtaining new DNA include conjugation, transduction, and transformation. Conjugation is the transmission of certain plasmids with resistance genes to other bacteria, the most common mechanism of resistance transmission. Of recent interest in the transfer of resistance by conjugation is transposon. There may be a number of resistance genes, which are also called "jumping genes" because they can transfer from one plasmid or chromosome to another plasmid or chromosome. Transduction is a mechanism for obtaining DNA derived from other bacteria and becoming resistant, and is known in pneumococci, gonococci, meningococci, H. influenzae, and the like. Bacteria tolerate antimicrobial agents include impermeability (a decrease in the amount of antimicrobial agents that enter and accumulate in bacterial cells), changes in target components, and inactivation of antimicrobial agents by enzymes. Changes in target protein are resistant when the penicillin binding protein (PBP) produced by bacteria is low in affinity for β-lactam antimicrobials. An example of inactivation of the antimicrobial agent is an example of β-lactamase production, and the effluent is reported in tetracycline as the antimicrobial agent is released out of bacteria.

Resistance of various pathogens is a problem, but the most important ones are Methicillin Resistance Staphylococcus aureus (MRSA), Enterobacteriaceae and Glucose Nonfermented Gram-negative Bacteria, Penicillin Resistance S. pneumoniae Pneumoniae, glycopeptide, high concentrations of aminoglycoside resistant enterococcus and β-lactamase producing H. influenzae. More than two-thirds of MRAS isolated from general hospitals have been found to be due to PBP2a production by the mecA gene. S. pneumoniae was susceptible to penicillin in the past, but now resistant bacteria are increasing, enterococcus faecium is increasing in ampicillin resistant strains, and in 1998, vancomycin resistance in some hospitals. Enterococci are increasing. In the 1990s, enterococci resistant to high concentrations of aminoglycosides appeared and resistance strains to high concentrations of aminoglycosides were required for strains isolated from patients with endocarditis, bacteremia, meningitis and osteomyelitis. Enterobacteriaceae were resistant to TEM-1 or SHV-1 β-lactamase production, and Escherichia coli or K. pneumoniae caused by extended-spectrum β-lactamase production recently became common.

We have found that Staphylococcus aureus, in particular, against methicillin resistant Staphylococcus aureus As a result of intensive research to develop an antimicrobial agent that exhibits antimicrobial activity, Compound K or a derivative thereof, which is a kind of saponin produced by the modification of natural saponin by bacteria, has been shown to be effective against Staphylococcus aureus, in particular its antibiotic resistant bacteria. It was confirmed that it represents. In addition, it was confirmed that the compounds exhibit antimicrobial activity against Staphylococcus aureus strains other than Staphylococcus aureus and various bacteria, and completed the present invention.

It is a main object of the present invention to provide an antimicrobial agent of ginsenoside compound K (C-K) or derivatives thereof.

It is another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of diseases caused by various bacteria, including Staphylococcus aureus, including Staphylococcus aureus and antibiotic resistant Staphylococcus aureus containing the antimicrobial agent as an active ingredient. will be.

Still another object of the present invention is to provide an antimicrobial composition for antimicrobial containing the antimicrobial agent as an active ingredient.

Still another object of the present invention is to provide a food additive for preventing or improving diseases caused by various bacteria, including Staphylococcus aureus, including Staphylococcus aureus and antibiotic resistant Staphylococcus aureus containing the antimicrobial agent as an active ingredient. will be.

Another object of the present invention to provide a feed additive for the prevention or improvement of diseases caused by various bacteria, including Staphylococcus aureus, including Staphylococcus aureus and antibiotic resistant Staphylococcus aureus containing the antimicrobial agent as an active ingredient. will be.

Another object of the present invention to provide a cosmetic containing the antimicrobial agent as an active ingredient.

Since the antimicrobial agent of the present invention exhibits specific antimicrobial activity against various bacteria including Staphylococcus aureus, including Staphylococcus aureus resistant to Staphylococcus aureus, in particular, antibiotics, it is effective for infection of various bacteria, particularly antibiotic resistant bacteria. It may be widely used for the prevention or treatment of diseases caused by.

1 is a schematic view showing a method for producing Compound K. FIG.

Figure 2 is a photograph showing the concentration-dependent growth inhibition of Staphylococcus aureus according to the concentration of compound K. A) Staphylococcus aureus subsp . aureus KCTC13197, B) MRSA N315, C) MRSA MW2, D) MRSA COL. The numbers on each culture dish indicate the amount of compound K used (μg).

Figure 3 is a diagram showing the specific growth rate change of Staphylococcus aureus against the concentration of compound K.

Figure 4 is a photograph of the experimental results for determining the minimum inhibitory concentration of compound K.Staphylococcus aureusA) DSM20231, B) MRSA N315, C) MRSA MW2, D) MRSA COL.

Numbers on each tube indicate the concentration of compound K (μg / ml). Each tube is the result of 24 hours incubation and SC is a sterile control.

5 is a death curve for MRSA MW2 by compound K. FIG.

6 is a molecular structure of a halogenated compound K derivative. A) Compound K, B) Halogenated sugar derivatives, C) Halogenated dammarane derivatives.

7 is a graph showing specific growth rate changes of various Staphylococcus aureus strains for the concentration of Compound K.

FIG. 8 shows Bacillis subtilis, Acinetobacter calcoaceticus, Purulent Streptococcus (Streptococcus pyogenes), Enterococcus pykalis for the concentration of Compound K. FIG. (Enterococcus faecalis) and a diagram showing the specific growth rate changes of mycobacteria (Mycobacterium smegmatis).

9 is a diagram showing the antimicrobial activity of ginsenoside compounds other than Compound K against Staphylococcus aureus.

Until now, compound K has been shown to have anti-inflammatory activity (Park EK, et al., Biol. Pharm. Bull., 28: 652-656, 2005), liver protection (Lee HU, et al., Liver Int., 25: 1069-1073, 2005), treatment of liver fibrosis (Park EJ, et al., Planta Med., 72: 1250-1253, 2006), inhibition of tumor growth (Jung SH, et al., Int. J. Cancer, 118-490-497, 2006), antioxidant activity (Bae EA, et al., Arch. Pharm. Res., 27: 1136-1140, 2004), antiallergic action (Choo MK, et al., Planta Med , 69: 518-522, 2003), neurodegenerative disease prevention (Tohda C., et al., Neuropsychopharmacology, 29: 860-868, 2004), skin protective action (Shin YW, et al., J. Pharmacol Sci., 99: 83-88, 2005) and the like, but the antimicrobial activity has not been reported yet.

The present invention confirmed that Compound K alone showed antimicrobial activity against antibiotic resistant bacteria such as Staphylococcus aureus, especially methicillin resistant Staphylococcus aureus without the aid of other antibiotics (Examples 2, 3 and 4). Furthermore, staphylococcus strains other than the Staphylococcus aureus, for example, byproductive Staphylococcus aureus (Staphylococcus saprophyticus), Staphylococcus carnosus, Staphylococcus coch Staphylococcus cohnii, Staphylococcus xylosus (Example 6), as well as Bacillis subtilis, Acinetobacter calcoaceticus, Streptococcus (Streptococcus) It was confirmed that the bacterium (Example 7) also exhibited antibacterial activity such as Streptococcus pyogenes, Enterococcus faecalis, and Bacillus mycobacterium smegmatis. The present invention is based on this.

Accordingly, one embodiment of the present invention provides an antimicrobial agent of ginsenoside compound K of formula 1 or a derivative thereof.

Formula 1

"Compound K" does not exist in ginseng itself, but saponins such as ginsenosides Rb1, Rb2, Rc, and Rd present in ginseng or red ginseng are caused by the action of intestinal microorganisms such as bifidus or soil microorganisms. Is a saponin converted into an absorbable form at.

Meanwhile, examples of compound K derivatives exhibiting antimicrobial activity include halogenated compounds K such as halogenated sugar derivatives and halogenated dammarane derivatives, and preferred examples thereof include compounds of the following Chemical Formulas 2 and 3.

As another example of the compound K derivative exhibiting the antimicrobial activity, the compound K may include a compound K fatty acid derivative having a fatty acid bound thereto. The fatty acid capable of forming the compound K fatty acid derivative showing the antimicrobial activity may be a saturated or unsaturated fatty acid having 10 to 20 carbon atoms, but may be included without limitation as long as the conjugate with the compound K may exhibit antimicrobial activity.

According to a specific embodiment of the present invention, the compound K halide is actinomycetes exhibiting halogenase activityAmycolatopsis mediterraneiTo Compound K fatty acid derivative combined with fatty acid was produced by mixing the ground bovine liver with Compound K and reacting with alcohol.

Formula 2

Formula 3

In Formula 2, three X's are each independently OH, Cl, Br, or I, except that X is all OH;

In Formula 3, two Y's are each independently H, Cl, Br, or I, except that Y is all H.

Staphylococcus aureus in the present invention includes antibiotic resistant Staphylococcus aureus.

According to another embodiment, the present invention provides Staphylococcus aureus strains, including Staphylococcus aureus, including antibiotic resistant Staphylococcus aureus, including Staphylococcus aureus, and antibiotic-resistant Staphylococcus aureus, as an active ingredient of Compound K or derivatives thereof. Bacillus subtilis, including Staphylococcus saprophyticus, Staphylococcus carnosus, Staphylococcus cohnii, Staphylococcus xylosus (Bacillis subtilis), Acinetobacter calcoaceticus, Purulent Streptococcus (Streptococcus pyogenes), Enterococcus faecalis, and Staphylococcus aureus Matisse; Mycobacterium smegmatis) and other bacteria (hereinafter referred to as 'various bacteria including staphylococcus strains') It provides a pharmaceutical composition for the prevention or treatment of diseases caused by harm. The compound may include a pharmaceutically acceptable salt thereof. Acid addition salts formed with pharmaceutically acceptable free acids may be useful. Organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, sulfurous acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, metalsulfonic acid may be used as the organic acid. , Acetic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, citric acid, aspartic acid, and the like can be used.

The addition salt according to the present invention is dissolved in a conventional method, i.e., a compound of Formula 1 or a derivative thereof in a water-miscible organic solvent such as acetone, methanol, ethanol, or acetonitrile and added with an equivalent or excess of an organic acid or an inorganic acid. It can be prepared by adding an aqueous acid solution followed by precipitation or crystallization, or by evaporating the solvent or excess acid followed by suction filtration of the dried or precipitated salt.

The present invention may include all of the compounds of Formula 1 / derivatives thereof and pharmaceutically acceptable salts thereof, as well as possible solvates, hydrates and stereoisomers that may be prepared therefrom, within the scope of the invention.

"Diseases caused by various bacteria, including Staphylococcus aureus strains" are dermatitis, various skin infections, food poisoning, pneumonia, meningitis, osteomyelitis, endocarditis, TSS (Toxic shock) caused by infection of a variety of bacteria, including staphylococcus strains syndrome), bacteremia, sepsis and the like.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease caused by various bacteria including Staphylococcus aureus by administration of the composition, and "treatment" refers to staphylococcus by administration of the composition. Means any action that improves or benefits the symptoms of diseases caused by various bacteria, including cocci strain.

In addition, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The carrier includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl Pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

The term "pharmaceutically acceptable" of the present invention refers to compositions and molecules that are physiologically acceptable and typically do not cause unexpected reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by other generally known pharmacopoeia for use in mammals and in particular humans.

The pharmaceutical composition may be formulated together with the carrier in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, or the like, external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be used. When formulated as such, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, in the compound K or derivatives thereof. , Sucrose or lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The content of Compound K or a derivative thereof included in the pharmaceutical composition of the present invention is not particularly limited, but may be included in 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, based on the total weight of the final composition. Include as.

In another embodiment of the invention, the invention provides a method of administering an antimicrobial agent of Compound K or a derivative thereof to a subject having a disease caused by various bacteria, including Staphylococcus strains, at a therapeutically effective dosage. Staphylococcus strains, including Staphylococcus aureus and antibiotic resistant Staphylococcus aureus, including, for example, Staphylococcus saprophyticus, Staphylococcus carnosus , Staphylococcus cohnii, Staphylococcus xylosus, as well as Bacillis subtilis, Acinetobacter calcoaceticus, Purulent Streptococcus (Staphylococcus cohnii) Streptococcus pyogenes, Enterococcus faecalis and Provided are methods for treating diseases caused by bacteria such as Staphylococcus aureus (Mycobacterium smegmatis). At this time, the composition may be used alone or in combination with other pharmaceutical compositions.

The term "therapeutically effective dosage" or "therapeutically effective amount" or "effective amount" as used herein means an amount of Compound K or derivative thereof that is sufficient to result in a therapeutic response. A therapeutic response can be any response that a user (ie, investigator) will recognize as an effective response to treatment, such as by evaluating symptoms and surrogate clinical markers. Thus, the therapeutic response will generally be alleviation of one or more symptoms of the disease or disorder.

The term "individual" in the present invention means a living organism, and means a mammal, although not particularly limited thereto.

The term "mammal" of the present invention refers to "high mammals" that feed their offspring with milk secreted by the mammary gland, including, for example, mice, rats, rabbits, dogs, cats, and especially humans. Mammalian "refers to any organism of the class.

Compound K or a derivative thereof constituting the antimicrobial agent of the present invention is a saponin in a form that can be absorbed into the body by enterobacteriaceae, so there are no problems such as toxicity and side effects.

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the compound K or derivative thereof of the present invention is preferably administered at 0.01 to 100 mg / kg (body weight) per day, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times.

The pharmaceutical composition may be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

As another aspect, the present invention provides an antimicrobial quasi-drug composition comprising an antimicrobial agent of compound K or a derivative thereof as an active ingredient.

A "quasi drug" is a fiber, rubber product or the like used for the treatment, alleviation, treatment or prevention of human or animal diseases, with weak or no direct action on the human body, and which is not an instrument or machine; A similar article, one of the preparations used for disinfection, insecticide and similar uses for the prevention of infectious agents, of an article used for the purpose of diagnosing, treating, reducing, treating or preventing diseases of humans or animals. Means any article other than a machine, machine or apparatus, which is not a machine or apparatus, and which is used for the purpose of pharmacologically affecting the structure and function of a person or animal.

When the antimicrobial agent of the present invention is used as an quasi-drug additive, the antimicrobial agent may be added as it is, or used together with other quasi-drugs or quasi-drug components, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the intended use.

The quasi-drug composition of the present invention is not limited thereto, but may preferably be a disinfectant cleaner, a shower foam, a gagreen, a wet tissue, a detergent soap, a hand wash, a humidifier filler, a mask, an ointment, or a filter filler.

As still another embodiment of the present invention, the present invention provides Staphylococcus aureus strains, including Staphylococcus aureus, including antibiotic-resistant Staphylococcus aureus, and Staphylococcus aureus, which comprise an antibacterial agent of Compound K or a derivative thereof as an active ingredient. Bacillus, including (Staphylococcus saprophyticus), Staphylococcus carnosus, Staphylococcus carnosus, Staphylococcus cohnii, Staphylococcus xylosus Bacillis subtilis, Acinetobacter calcoaceticus, Purulent Streptococcus (Streptococcus pyogenes), Enterococcus faecalis, and Bacillus sp. Food additives for the prevention or amelioration of diseases caused by bacteria such as therium smegmatis (Mycobacterium smegmatis) to provide.

The food additive can be used is not particularly limited, and the content of the compound K or derivatives thereof included in the food additive is also not particularly limited, these are considering the ease of infection, the degree of infection of various bacteria, including staphylococcus strains It can be easily determined by those skilled in the art as needed.

Examples of the food to which the antimicrobial agent of the present invention may be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionary, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, Drinks, alcoholic beverages and vitamin complexes, and the like, may include all of the health functional foods in the conventional sense, may include foods used as feed for animals.

In addition, when the antimicrobial agent of the present invention is added to and used in a beverage, various sweeteners, flavoring agents or natural carbohydrates, etc. may be added as additional components, as in general beverages. The natural carbohydrate can be glucose, monosaccharides such as fructose, maltose, disaccharides such as sucrose, polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, erythritol. The ratio of the natural carbohydrate is not limited thereto, but may be preferably about 0.01 to 0.04 g, more preferably 0.02 to 0.03 g per 100 ml of the food containing the antimicrobial agent of the present invention. The sweetener may be a natural sweetener such as taumartin, stevia extract and a synthetic sweetener such as saccharin, aspartame.

In addition to the above, the food containing the antimicrobial agent of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, Glycerol, alcohols, carbonation agents used in carbonated drinks, and the like. Others may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks.

As another embodiment, the present invention is Staphylococcus aureus strains, including Staphylococcus aureus and antibiotic-resistant Staphylococcus aureus comprising an antimicrobial agent of the compound K or derivatives thereof as an active ingredient, for example, byproductive Staphylococcus aureus (star Bacillus subtilis, including Staphylococcus saprophyticus, Staphylococcus carnosus, Staphylococcus cohnii, Staphylococcus xylosus Bacillis subtilis, Acinetobacter calcoaceticus, Purulent Streptococcus (Streptococcus pyogenes), Enterococcus faecalis, and Staphylococcus aureus Provides a feed additive for the prevention or improvement of diseases caused by bacteria such as Megmatis (Mycobacterium smegmatis) do.

Like the food additive, the feed additive can be used is not particularly limited, and the content of the compound K or derivatives thereof included in the feed additive is also not particularly limited, these are infections of various bacteria, including staphylococcus strains In view of the ease and degree of infection, those skilled in the art can easily determine as necessary.

As another aspect, the present invention provides a cosmetic comprising an antimicrobial agent of the compound K or a derivative thereof. The compound is as described above, and may include a pharmaceutically acceptable salt thereof.

Cosmetics of the present invention can be prepared in the form of general emulsion formulations and solubilized formulations. The emulsified formulations include nutrient cosmetics, creams, essences, etc., and the solubilized formulations include soft cosmetics. Suitable formulations include, but are not limited to, solutions, gels, solid or pasty anhydrous products, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionics (liposomes) obtained by dispersing an oil phase in an aqueous phase, for example In the form of a vesicle dispersant, may be in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. It may also be in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetics may additionally contain fatty substances, organic solvents, solubilizers, thickening and gelling agents, emollients, antioxidants, suspending agents, stabilizers, blowing agents, fragrances, surfactants, water, ionic or nonionic emulsifiers, fillers, metal ions Commonly used adjuvants such as containment agents, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetic compositions. It may contain.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are merely to illustrate the invention, the present invention is not limited by the following examples. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.

Example 1 Preparation and Separation of Compound K

Compound K was prepared by the method of biotransformation from a ginsenoside mixture of diol-based saponins (protopanaxadiol, PPD) (FIG. 1). 1 is a schematic view showing a method for producing Compound K. FIG.

Specifically, ginsenosides Rb1 and Rb2 belonging to PPD were subjected to 50 mM Tris HCl buffer (pH7.5) using BlpA, a ß-glucosidase derived from Rhodanobacter ginsenosidimutans Gsoil3054. It reacted at 37 degreeC for 24 hours, and was converted into Rd.

In addition, ginsenoside Rc included in the PPD mixture was prepared using 50% Tris HCl buffer (pH7.5) environment using Araf3054, which is an arabinoofuranosidase derived from the Rhodanobacter ginsenocidimutans Gsoil3054. To Rd by reacting at 37 ° C. for 24 hours.

The converted ginsenoside Rd was converted to Compound K via F2 using BgpA, a ß-glucosidase from Terrabacter Gsoil3082.

Compound K is then extracted with the same volume of n-butanol, reduced pressure to evaporate the solvent, dissolved in methanol again, adhered to ODS resin (ZEOprep 60 C ₁₈ 40-63 μm, ZEOCHEM) and the concentration of methanol in the mobile phase Separation was increased by 100% and purity was confirmed by HPLC.

Example 2 Concentration-dependent Growth Inhibition of Staphylococcus Aureus by Compound K

Mueller Hinton Broth (MHB)Staphylococcus aureussubsp. aureus KCTC 13197), MRSA MW2, MRSA N315, MRSA COL were incubated, and the cell culture solution was evenly spread on a flat plate MHB medium with a sterile swab.

Ginsenoside compound K dissolved in a dimethyl sulfoxide (DMSO) solution in the plate MHB solid medium was added dropwise to disc-shaped filter paper, respectively 0, 2, 4, 6, 8 or 10 ug, Incubated overnight under conditions (Figure 2). Figure 2 is a photograph showing the concentration-dependent growth inhibition of Staphylococcus aureus according to the concentration of compound K. As shown in Figure 2, Compound K was confirmed to exhibit a concentration-dependent antimicrobial activity against Staphylococcus aureus or its antibiotic-resistant bacteria. In addition, by applying a compound K in a broader concentration range for the same strain was confirmed the antimicrobial activity by compound K. Dispense 150 μl of MHB into each well using sterile 96-well microplates (Falcon 3072), add up to 128 mg / l of compound K, and in turn so that the minimum concentration of compound K is 2 mg / l. Serial dilution was performed 1/2. After diluting 1/10 of the seed culture incubated at 37 ° C. for 24 hours and inoculating each with 2% (v / v) of the volume of the medium, the tape was gas permeable (Breathe-Easy; Diversified Biotech, USA), and absorbance at 590 nm at 10 minute intervals with agitation using a Bioplate Microstation at 25 ° C. for 48 hours to obtain a growth curve and analyzed by SigmaPlot program. 3 is a graph showing the concentration dependent growth inhibition of various Staphylococcus aureus as a function of the concentration of Compound K as a function of specific growth rate for CK concentration. As shown in Figure 3, Compound K was confirmed to exhibit a concentration-dependent antimicrobial activity against Staphylococcus aureus. In Table 1 are shown by calculating the EC ₅₀ value. As shown in Table 1, the compound K of the present invention was confirmed to exhibit EC ₅₀ growth inhibition activity of less than 16 ㎍ / ㎖ for Staphylococcus aureus.

On the other hand, Staphylococcus aureus (Staphylococcus aureus subsp. Aureus) disk diffusion test, the same manner described above by using the TSB (Tryptic Soy Broth) instead of MHB to identify the compound K growth inhibitory activity of the standard strain (Type strain) The experiment was conducted.

Table 1

Example 3 Determination of Minimum Inhibitor Concentration of Staphylococcus Aureus by Compound K

Four kinds of Staphylococcus aureus subsp. Aureus KCTC DSM20231, MRSA MW2, MRSA N315, and MRSA COL were cultured in Mueller Hinton Broth (MHB), and the cell culture solution was diluted to 1/100 (v / v). 10 μl of the diluent was aliquoted and compound K dissolved in DMSO was inoculated into MHB liquid medium at 1, 2, 4, 8, 16, or 32 μg / ml (w / v) at 37 ° C. for 1 day. Incubated at 16 and 24 hours to observe the turbidity visually (Fig. 4). Figure 4 is a photograph showing the minimum inhibitory concentration of Staphylococcus aureus or its antibiotic-resistant bacteria by Compound K. As shown in Figure 4, the minimum inhibitory concentration of Compound K against MRSA Staphylococcus aureus was found to be 16 ㎍ / ㎖.

Example 4 Determination of Death Curves for Compound K of MRSA MW2

MRSA MW2 was incubated in Mueller Hinton Broth (MHB), the cell culture solution was diluted to 1/100 (v / v), and 10 μl of the diluent was aliquoted and 0.5X MIC (minimum inhibitory) was dissolved in compound K dissolved in DMSO. concentration, 1X MIC, 2X MIC, 4X MIC, added to MHB liquid medium, incubated at 37 ℃ constant temperature, and aliquoted at a predetermined time.^-One To 10^-9Until After dilution, plated on MHB solid medium to count the colony produced after 1 day (Fig. 5). Figure 5 is a photograph showing the killing curve (killing-curve) of the MRSA MW2 strain by compound K.

Example 5 Preparation of Derivatives of Compound K

In order to produce compound K fatty acid derivative using acyltransferase system, 100 g of fresh bovine liver was ground by adding 200 ml of Tris buffer (Tris 50 mM, pH7.0) to compound K (0.1 mg / ml) and After mixing and reacting at 37 ° C. for 24 hours, it was extracted with butanol to produce Compound-K in which fatty acids were bound.

In addition, actinomycetes which exhibit halogenase activity to produce ginsenoside halogen derivativesAmycolatopsis mediterraneiTo To produce halogenated derivatives of Compound K (FIG. 6).

Example 6: Antimicrobial Activity of Staphylococcus aureus Strains of Compound K

In the method described in Example 2, Staphylococcus bacteria Staphylococcus aureus subspecies Aureus KCTC1916 and KCTC1927, byproducts of the Staphylococcus bacterium in Mueller Hinton Broth (MHB) (Staphylococcus saprophyticus) subspecies Saprophyticus KCTC3345, Staphylococcus carnosus subspecies Tarnosus KCTC3580, Staphylococcus cohnii subspecies KCTC3574, Staphylococcus xylosus KCTC3342 was incubated, and the cell culture was evenly spread on plated MHB solid medium using a sterile swab.

Ginsenoside compound K dissolved in DMSO solution was added to the disk-type filter paper, respectively, in a flat MHB solid medium, and 5 or 10 µg was added dropwise and reacted overnight at 37 ° C in constant temperature (FIG. 7). Figure 7 is a photograph showing the concentration-dependent growth inhibition of various staphylococcus bacteria according to the concentration of compound K. As shown in Figure 7, Compound K was confirmed to exhibit a concentration-dependent antimicrobial activity against Staphylococcus aureus or various staphylococcus bacteria.

Example 7: Antibacterial activity against strains other than Staphylococcus aureus strains of Compound K

Bacillis subtilis, Acinetobacter calcoaceticus in Nutrient Broth (NB) and Bovine heart infusion broth (BHI) in the same manner as in Example 2 in Streptococcus Strains such as Piogenes (Streptococcus pyogenes), Enterococcus faecalis, and Bacillus sp. (Mycobacterium smegmatis) were precultured. In order to confirm the antimicrobial activity of Compound K by applying Compound K in a wide concentration range, 150 μl of NB or BHI was dispensed into each well using sterilized 96-well microplates (Falcon 3072). After the addition of 128 mg / l of compound K, serial serial dilution was carried out in sequence so that the minimum concentration of compound K was 2 mg / l. After diluting 1/10 of the seed culture incubated at 37 ° C. for 24 hours and inoculating each with 2% (v / v) of the volume of the medium, the tape was gas permeable (Breathe-Easy; Diversified Biotech, USA), and absorbance at 590 nm at 10 minute intervals with agitation using a Bioteck Microstation at 25 ° C. for 48 hours to obtain a growth curve and analyzed by SigmaPlot program (FIG. 8).

8 is a graph showing the concentration dependent growth inhibition of various strains other than Staphylococcus as a function of the concentration of Compound K as a function of specific growth rate for CK concentration. As shown in Figure 8, Compound K was confirmed to exhibit a concentration-dependent antimicrobial activity against other strains than Staphylococcus aureus bacteria. Table 2 shows the calculated EC ₅₀ value. As shown in Table 2, Compound K of the present invention was confirmed to exhibit an EC ₅₀ inhibitory activity of less than 16 ㎍ / ㎖ for strains other than Staphylococcus aureus.

TABLE 2

Comparative Example 1 Antibacterial Activity of Staphylococcus Aureus by Different Kinds of Ginsenoside Compounds

The antibacterial activity of the compounds was confirmed by culturing Staphylococcus aureus and treating various kinds of ginsenoside compounds in a similar manner to Example 2. Specifically, Staphylococcus aureus standard strains were cultured in Tryptic Soy Broth (TSB), and the cell culture solution was evenly plated on a flat TSB solid medium with a sterile swab.

Ginsenosides F1, F2, Rh2 and PPTol dissolved in dimethyl sulfoxide solution in the plate TSB solid medium were respectively added 0, 1, 3, 5 or 10 ㎍ to disk-type filter paper, and at 37 ° C constant temperature. Reaction overnight (FIG. 9). 9 is a photograph showing the concentration-dependent growth inhibition of Staphylococcus aureus according to the concentration of the ginsenosides. As shown in Figure 9, other ginsenoside compounds other than Compound K did not show antimicrobial activity against Staphylococcus aureus or its antibiotic-resistant bacteria.

Claims (14)

1. An antibacterial agent comprising ginsenoside compound K of formula 1 or a derivative thereof.

   [Formula 1]

   
2. The method of claim 1,

   An antibacterial agent characterized in that the derivative of Compound K is a fatty acid derivative or halide of Compound K.
3. The method of claim 2,

   The compound K fatty acid derivative is an antimicrobial agent characterized in that a fatty acid selected from the group consisting of saturated or unsaturated fatty acids having 10 to 20 carbon atoms is combined.
4. The method of claim 2,

   The halide of compound K is an antimicrobial agent characterized by the following formula (2) or (3).

   [Formula 2]

   

   [Formula 3]

   

   In Formula 2, three X's are each independently OH, Cl, Br, or I, except that X is all OH;

   In Formula 3, two Y's are each independently H, Cl, Br, or I, except that Y is all H.
5. The method of claim 1,

   Staphylococcus, Staphylococcus, Bacillis subtilis, Acinetobacter calcoaceticus, Streptococcus pyogenes, Enterococcus pyogenes An antimicrobial agent that exhibits antimicrobial activity against bacteria selected from the group consisting of Enterococcus faecalis and Enterococcus faecalis (Mycobacterium smegmatis).
6. The method of claim 5,

   The staphylococcus strain is Staphylococcus aureus, Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus saprophyticus, Staphylococcus carnosus , Staphylococcus cohnii, Staphylococcus xylosus, and methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium selected from the group consisting of Antibacterial.
7. Staphylococcus (Staphylococcus) strain, Bacillis subtilis, Acinetobacter calcoaceticus containing the antimicrobial agent of any one of Claims 1-4 as an active ingredient. (Acinetobacter calcoaceticus), purulent streptococcus (Streptococcus pyogenes), Enterococcus faecalis and mycobacteria (Mycobacterium smegmatis). Pharmaceutical compositions for the prevention or treatment of diseases caused by bacteria.
8. The method of claim 7, wherein

   The disease includes Staphylococcus strains, Bacillis subtilis, Acinetobacter calcoaceticus, Streptococcus pyogenes, Streptococcus pyogenes. ), Enterococcus faecalis and Bacillus (mycobacterium smegmatis) is a composition caused by the infection of the bacteria selected from the group consisting of.
9. The method of claim 7, wherein

   The disease is a dermatitis, skin infection, food poisoning, pneumonia, meningitis, osteomyelitis, endocarditis, TSS (Toxic shock syndrome), bacteremia, or sepsis.
10. The method of claim 7, wherein

    The composition further comprises a pharmaceutically acceptable carrier.
11. The quasi-drug composition for antimicrobial containing the antimicrobial agent of any one of Claims 1-4 as an active ingredient.
12. Staphylococcus (Staphylococcus) strain, Bacillis subtilis, Acinetobacter calcoaceticus containing the antimicrobial agent of any one of Claims 1-4 as an active ingredient. (Acinetobacter calcoaceticus), purulent streptococcus (Streptococcus pyogenes), Enterococcus faecalis and mycobacteria (Mycobacterium smegmatis). Food additives for the prevention or improvement of diseases caused by bacteria.
13. Staphylococcus (Staphylococcus) strain, Bacillis subtilis, Acinetobacter calcoaceticus containing the antimicrobial agent of any one of Claims 1-4 as an active ingredient. (Acinetobacter calcoaceticus), purulent streptococcus (Streptococcus pyogenes), Enterococcus faecalis and mycobacteria (Mycobacterium smegmatis). Feed additives for the prevention or improvement of diseases caused by bacteria.
14. Cosmetics containing the antimicrobial agent of any one of Claims 1-4 as an active ingredient.

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