KR20230169705A - Composition for Inhibiting Persister Cell Comprising Extract of Pine Leaves - Google Patents

Abstract

The present invention relates to a composition for inhibiting persistent cells containing pine needle extract.
In the present invention, persistent bacteria that are not killed even by antibiotic treatment can be effectively inhibited using pine needle extract. Therefore, the composition for inhibiting persistent bacteria of the present invention can be used to prevent and treat infectious diseases caused by bacteria, especially recurrent or chronic infections, and can exhibit excellent effects, and can also be usefully applied to cosmetic compositions for improving dermatitis.

Description

Composition for inhibiting persistent bacteria comprising pine needle extract {Composition for Inhibiting Persister Cell Comprising Extract of Pine Leaves}

The present invention relates to a composition for inhibiting persistent bacteria containing pine needle extract, and more specifically, to a composition capable of inhibiting persistent bacteria that is not killed even by antibiotic treatment using pine needle extract, and pharmaceutical compositions and cosmetic compositions using the same. will be.

Antibiotics are substances that inhibit the growth and proliferation of bacteria. Since the discovery of penicillin, the first antibiotic, various antibiotics have been researched and developed and are widely used to suppress pathogens. For example, Republic of Korea Patent Publication No. 10-2016-0086479 discloses a cyclic depsipeptide-based compound as a compound exhibiting antibacterial activity against Staphylococcus aureus or Salmonella.

However, some bacterial populations exhibit characteristics that do not kill them even with high-concentration antibiotic treatment. These bacteria are called persister cells, and in a 1942 paper by Hobby et al. , Observations on the mechanism of action of penicillin, Exp Biol Med 1942;50:281-5, experimental results showing recurrence of infection despite treatment of Staphylococcus aureus with high concentration of penicillin were reported, and later in the paper Bigger et al. , The bactericidal action of penicillin on Staphylococcus pyogenes , Irish J Med Sci 1944;19:553-68, showed that bacteria were not completely killed when treated with penicillin, and the surviving bacteria were named persistent bacteria.

Persistent bacteria survive with minimal cellular metabolism and do not respond to common antibiotics. Notably, persistent bacteria do not respond to antibiotics regardless of mutation, unlike antibiotic-resistant bacteria that are genetically mutated to become resistant. Specifically, the mechanism of acquiring antibiotic resistance is due to the action of inhibiting the binding of antibiotics to the target through genetic mutation, but the phenomenon in which persistent bacteria do not respond to antibiotics affects the function of the target even though the antibiotic binds to the target. This is due to physiological characteristics that cancel out the effect.

Accordingly, persistent bacteria are not killed even by antibiotic treatment, and there is a problem that the limitations of the treatment effect for infectious diseases cannot be overcome unless the residual problem of such persistent bacteria is solved. In particular, surviving persistent bacteria can proliferate again and cause reinfection, which can pose a serious risk to patients with weakened immune systems. In fact, it has been reported that persistent pathogenic bacteria have a fatal effect on patients with cystic fibrosis, such as HIV infection.

However, in order to suppress persistent bacteria, a strategy different from the conventional antibacterial activity against common or resistant bacteria is required, and it is not easy to discover substances that exhibit a persistent bacterial inhibitory effect.

The purpose of the present invention is to provide a composition capable of inhibiting persister cells.

Another object of the present invention is to provide a pharmaceutical composition that can effectively prevent or treat infectious diseases caused by bacteria by inhibiting persistent bacteria.

Another object of the present invention is to provide a cosmetic composition that can improve dermatitis by inhibiting persistent bacteria.

In order to achieve the above object, the present invention provides a composition for inhibiting persister cells containing pine needle extract.

In the present invention, the persistent bacteria may be bacteria that survive even when treated with antibiotics at 5 times or more than the minimum inhibitory concentration (MIC).

In the present invention, the persistent bacteria may be persistent bacteria of the genus Staphylococcus ( Staphylococcus sp.).

In the present invention, the concentration of the pine needle extract may be 0.001 to 10 g/L.

In the present invention, the composition can be used in combination with antibiotics.

In the present invention, the antibiotics include beta-lactams, quinolones, aminoglycosides, tetracyclines, peptides, and anti-fatigue antibiotics. It may include one or more selected from the group consisting of mupirocin and fusidic acid.

In the present invention, the weight ratio of pine needle extract and antibiotic may be 0.1:1 to 1,000:1.

The present invention also provides a composition for preventing or treating bacterial infectious diseases, comprising the composition for inhibiting persistent bacteria.

In the present invention, the bacterial infectious disease may be an infectious disease caused by Staphylococcus sp.

In the present invention, the bacterial infectious disease may be one or more inflammatory diseases selected from the group consisting of respiratory infection, sinusitis, pneumonia, endocarditis, osteomyelitis, arthritis, cellulitis, gastroenteritis, postoperative wound infection, and dermatitis.

In the present invention, the bacterial infectious disease may be a palindromic or chronic disease.

The present invention also provides a cosmetic composition for improving dermatitis, including a composition for inhibiting persistent bacteria.

In the present invention, the dermatitis may be abscess, folliculitis, boil or acne.

In the present invention, the dermatitis may be recurrent or chronic dermatitis.

In the present invention, persistent bacteria that are not killed even by antibiotic treatment can be effectively inhibited using pine needle extract. Therefore, the composition for inhibiting persistent bacteria of the present invention can be used to prevent and treat infectious diseases caused by bacteria, especially recurrent or chronic infections, and can exhibit excellent effects, and can also be effectively applied to cosmetic compositions for improving dermatitis.

Figure 1 is a graph showing the ratio of persistent bacteria to oxacillin, tobramycin, or ciprofloxacin when pine needle extract was treated at different concentrations in an embodiment of the present invention.
Figure 2 is a graph showing the inhibition rate of persistent bacterial formation for oxacillin, tobramycin, or ciprofloxacin when pine needle extract was treated at different concentrations in an embodiment of the present invention.

Hereinafter, specific implementation forms of the present invention will be described in more detail. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to a composition that can effectively inhibit persister cells.

Persistent bacteria are bacteria that are not affected by treatment with antibiotics, substances that kill bacteria or inhibit their growth, and survive even when exposed to high concentrations or high efficiency antibiotics. For example, the persistent bacteria can be interpreted as bacteria that survive even when treated with antibiotics in an amount 5 times or more, for example, 10 times the minimum inhibitory concentration (MIC). At this time, the minimum inhibitory concentration refers to the minimum concentration at which an antibiotic exhibits an antibacterial effect.

Due to these persistent bacteria, there is a limit to the antibacterial effect of antibiotics, and there is a problem that it is difficult to prevent bacteria from proliferating again after antibiotic treatment. In particular, since a certain percentage of the bacterial population acts as persistent bacteria regardless of genetic mutation, persistent bacteria cannot be suppressed only with general antibacterial activity against bacteria or resistant bacteria. In particular, through previous research, the inventor of the present invention has demonstrated that even if a substance exhibits strong antibacterial activity against bacteria or resistant bacteria, it does not necessarily exhibit inhibitory activity against persistent bacteria, and may actually result in further proliferation of persistent bacteria. It has been confirmed through. According to the survival strategies and experimental results of these persistent bacteria, a new strategy that is completely different from the conventional antibacterial activity against common or resistant bacteria is needed to suppress persistent bacteria.

The present invention is intended to overcome the limitations of the prior art, and was completed by discovering the surprising fact that using pine needle extract can suppress persistent bacteria that are not killed even by antibiotic treatment.

The pine needles used in the present invention refer to the leaves of the pine tree ( Pinus densiflora ). Pine needles contain vitamin A, which is good for blood vessel health, is effective in preventing high blood pressure, and is known to be effective in diseases of the liver, stomach, nervous system, and circulatory system. However, there is no known effect on persistent bacterial inhibition.

According to the present invention, persistent bacteria that are not killed by antibiotic treatment can be very effectively inhibited by using pine needle extract. Therefore, using the present invention, it is possible to solve the problem of recurrence of infection caused by persistent bacteria, which could not be achieved with existing antibiotics alone.

In the present invention, pine needle extract refers to a substance obtained by extracting components of pine needles. The “extract” refers to the extract itself and the extract, such as the extract obtained by extraction treatment of herbal medicine, the diluted or concentrated liquid of the extract, the crude or purified product of the extract, the dried product obtained by drying the extract, the pulverized product of the dried product, or a mixture thereof. It is meant to include extracts of all formulations that can be formed using , and is interpreted to include crude extract and solvent fraction. The raw material to be extracted may be selected from the group consisting of leaves, branches, fruits, seeds, flowers, aerial parts, bark, and roots of plants, and may be the raw material itself or one that has undergone pretreatment such as grinding, drying, or steaming.

In one embodiment of the present invention, pine needle extract can be obtained using a solvent extraction method. Specifically, the extract can be prepared by adding an extraction solvent to pine needles, filtering, concentrating, and freeze-drying.

In the present invention, the extraction solvent is methanol, ethanol, propanol, 1,3-propanediol, butanol, pentanol, hexanol, propylene glycol, dipropylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, butylacetate, It may include one or more selected from the group consisting of chloroform, diethyl ether, dichloromethane, hexane, and water. In an example of the present invention, it was confirmed that when using methanol extract of pine needles, persistent bacteria that are not killed even by antibiotic treatment can be suppressed.

In an exemplary embodiment of the present invention, a pine needle extract can be obtained by adding 1 to 100 mL, preferably 5 to 50 mL, of extraction solvent per gram of pine needles, followed by filtration, concentration and freeze-drying. At this time, the extraction temperature may be room temperature to 100°C, preferably 40 to 60°C, and the extraction time may be 30 minutes to 10 hours, preferably 1 to 5 hours.

In one embodiment of the present invention, supercritical extraction may be used as the extraction method. Supercritical extraction is an extraction method using a supercritical fluid, which is a fluid in a state that exceeds the critical temperature and boundary pressure. Carbon dioxide can be used as the supercritical fluid, and an organic solvent can be used as an auxiliary solvent. Using the supercritical extraction method, only the extracted components can be easily obtained because the fluid vaporizes at normal pressure, and since it uses carbon dioxide, which is inert, tasteless, odorless, and harmless, it can be safely used in the manufacture of food or medicine.

In one embodiment of the present invention, it is also possible to use a compression method of extracting the raw material by applying pressure as an extraction method. For example, the extract can be prepared by pressing pine needles at a pressure of 100 to 2,000 kg/cm ² for 1 minute to 1 hour.

In the present invention, it is also possible to improve extraction efficiency by performing ultrasonic treatment, heat treatment, acid treatment, etc. when preparing the extract.

In the present invention, after obtaining the extract through an extraction process, one or more steps of filtering, separating, concentrating, and drying may be further performed. At this time, each step can be performed by a known method. For example, filtration may be performed by passing the extract through a filter, separation may be performed by chromatographic methods, concentration may be performed through reduced pressure distillation, and drying may be performed using freeze-drying or spray drying. It can be. Additionally, the dried extract can be prepared into powder form through a grinding step and, if necessary, resolvated with an appropriate solvent such as dimethyl sulfoxide (DMSO) or ethanol.

According to the present invention, persistent bacteria can be suppressed by using a composition containing pine needle extract as an active ingredient. At this time, the expression “including as an active ingredient” means containing a sufficient amount to achieve the efficacy or activity of the pine needle extract.

In the present invention, the concentration of the pine needle extract may be 0.001 to 10 g/L, preferably 0.05 to 5 g/L, and more preferably 0.2 to 2 g/L. Within the above range, the effect of suppressing a significant amount of persistent bacteria that are not killed even by antibiotic treatment can be achieved. Relatedly, in an example of the present invention, it was confirmed that the amount of persistent bacteria was significantly reduced as a result of treating pine needle extract at 0.05 to 0.6 g/L, and in particular, it was confirmed that the inhibitory effect was very excellent when the concentration was 0.2 g/L or more.

The persistent bacteria targeted for inhibition in the present invention may be persistent bacteria of the genus Staphylococcus ( Staphylococcus sp.), for example, persistent bacteria of Staphylococcus aureus ( Staphylococcus aureus ). In an example of the present invention, it was confirmed that pine needle extract can significantly inhibit persistent Staphylococcus aureus bacteria even in various antibiotic treatment environments.

In one embodiment of the present invention, the composition for inhibiting persistent bacteria containing the pine needle extract can be used in combination with an antibiotic. For example, the composition for inhibiting persistent bacteria of the present invention may be used after treatment with antibiotics or may be treated together with antibiotics, and the composition may also be used in a form that further contains antibiotics.

In the present invention, the antibiotics include beta-lactams, quinolones, aminoglycosides, tetracyclines, peptides, and radish. One or more types selected from the group consisting of mupirocin and fusidic acid can be used.

Specifically, the beta-lactam antibiotics include oxacillin, methicillin, ampicillin, amoxacillin, amoxicillin, cefadroxil, and cephalothin ( cephalothin, cephalosporin, aztreonam, imipenem, etc., and the quinolone antibiotics include ciprofloxacin, norfloxacin, ofloxacin, There are levofloxacin, moxifloxacin, etc., and the aminoglycoside antibiotics include tobramycin, amikacin, gentamicin, kanamycin, and netilmycin. (netilmicin), sisomicin, butyrosin, neomycin, paromomycin, etc. Tetracycline antibiotics include tetracycline and oxytetracycline. and the like, and the peptide-based antibiotics include vancomycin and bacitracin.

In particular, in the examples of the present invention, it was confirmed that pine needle extract can effectively inhibit persistent bacteria that are not killed by oxacillin, a beta-lactam antibiotic, tobramycin, an aminoglycoside antibiotic, or ciprofloxacin, a quinolone antibiotic. did.

In the present invention, the weight ratio of pine needle extract and antibiotic may be 0.1:1 to 1,000:1, preferably 1:1 to 500:1. Within the above range, persistent bacteria that are not killed even by excessive amounts of antibiotics can be effectively inhibited by the pine needle extract.

In an exemplary embodiment of the present invention, when the antibiotic is a beta-lactam antibiotic such as oxacillin, the weight ratio may be 10:1 to 1,000:1, preferably 20:1 to 500:1, and the antibiotic is Tobra If the antibiotic is an aminoglycoside antibiotic such as mycin, the weight ratio may be 0.5:1 to 1,000:1, preferably 1:1 to 500:1, and if the antibiotic is a quinolone antibiotic such as ciprofloxacin, the weight ratio may be 1: It may be 1 to 1,000:1, preferably 3:1 to 500:1. In this regard, in embodiments of the present invention, the weight ratio of pine needle extract and antibiotic to oxacillin, tobramycin, and ciprofloxacin is 20:1 to 250:1, 0.5:1 to 8:1, and 3:1 to 30, respectively. :1, it was confirmed that a significant amount of persistent bacteria was inhibited. In particular, it was confirmed that persistent bacteria to tobramycin were inhibited by more than 90% when the weight ratio was in the range of 1:1 to 8:1.

While previous studies on substances used in combination with antibiotics were limited to identifying the effect of lowering the MIC of antibiotics, that is, reducing the amount of antibiotics used, the present invention can significantly inhibit persistent bacteria that are not killed even by treatment with high doses of antibiotics. It has technical significance that is distinct from previous studies in that it discovered a material that exists in the past.

Accordingly, the present invention also provides a pharmaceutical composition for preventing or treating bacterial infectious diseases, comprising the composition for inhibiting persistent bacteria.

In the present invention, by using pine needle extract, pathogens that cannot be suppressed by antibiotic treatment can be suppressed, and infectious diseases caused by bacteria can be effectively prevented or treated. In particular, it can exhibit excellent effects on recurrent or chronic infections.

Specifically, the infectious disease to be treated in the present invention may be an infection caused by Staphylococcus sp. Bacteria of the genus Staphylococcus, especially Staphylococcus aureus, cause various inflammatory diseases in the human body, such as respiratory infections, sinusitis, pneumonia, endocarditis, osteomyelitis, arthritis, cellulitis, gastroenteritis, postoperative wound infection, and dermatitis. Using the present invention, These infectious inflammatory diseases can be effectively prevented or treated.

In particular, persistent bacteria proliferate again when antibiotic use is stopped and re-form bacterial colonies, causing recurrence of infection. Such recurrence of infection is especially likely to occur in patients with low immune activity and chronic infectious diseases. Using the present invention, these persistent bacteria can be suppressed, so it can have an excellent effect not only in temporarily preventing or treating infectious diseases, but also in recurrent or chronic diseases.

In addition to the pine needle extract, the pharmaceutical composition may further include an appropriate pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. The pharmaceutically acceptable carriers are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Includes, but is not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition to the above components, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Regarding suitable pharmaceutically acceptable carriers and formulations, they can be preferably formulated according to each ingredient using the method disclosed in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered either orally or parenterally, and parenteral administration includes transdermal administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, etc. Preferably, the pharmaceutical composition of the present invention can be used as an external agent and administered transdermally.

Dosage forms for oral administration include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These dosage forms contain diluents (e.g. lactose, dextrose, water) in addition to the active ingredient. crose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and magnesium or calcium salts thereof and/or polyethylene glycol). Additionally, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid. or disintegrants such as sodium salts thereof or effervescent mixtures and/or absorbents, colorants, flavoring and sweetening agents. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, representative formulations for parenteral administration are injectable formulations, and solvents for the injectable formulation include water, Ringer's solution, isotonic saline solution, or suspension. The sterile fixed oil of the injectable preparation can be used as a solvent or suspending medium, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose. Additionally, the injectable preparation may use fatty acids such as oleic acid.

The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, and activity of the patient's disease. , can be determined based on factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the field of medicine. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a technician with ordinary knowledge.

Specifically, the effective amount of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and is generally administered at 0.001 to 1,000 mg per 1 kg of body weight, preferably 0.01 to 100 mg every day or every other day, or once per day. It can be administered in three to three divided doses. However, since it may increase or decrease depending on the route of administration, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

The present invention can also provide a cosmetic composition for improving dermatitis, including the composition for inhibiting persistent bacteria.

Microorganisms that act as pathogens on the skin, such as Staphylococcus sp., can cause various epidermal infections on the skin, such as abscesses, folliculitis, boils, and acne. However, in the case of general skin antibiotics, persistent bacteria remain even when high doses are used, so the effectiveness of the antibiotic is limited and infections often recur. In particular, in situations where the skin barrier is damaged, such as the skin of atopic dermatitis patients, or when the patient's immunity is weakened, infections occur more easily and worsen symptoms.

In the present invention, persistent bacteria can be suppressed using pine needle extract, so bacterial infections on the skin can be very effectively improved. In addition, it has a special advantage in that, unlike general antibiotics that show temporary effects, it shows excellent effects on chronic dermatitis and recurrent dermatitis.

In the present invention, the formulation of the cosmetic composition can be prepared in any formulation commonly prepared in the art, for example, solution, emulsion, suspension, paste, gel, cream, lotion, powder, soap, cleansing , may be a formulation selected from the group consisting of oil, powder, or spray.

In the present invention, when the formulation of the cosmetic composition is a solution or emulsion, a solvent, solvating agent, or emulsifying agent can be used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan can be used.

In the present invention, when the formulation of the cosmetic composition is a suspension, the carrier component includes 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, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

In the present invention, when the formulation of the cosmetic composition is a paste, gel, cream or lotion, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or Zinc oxide, etc. may be used.

In the present invention, when the formulation of the cosmetic composition is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient, and especially in the case of spray, chlorofluorohydride may be used as a carrier ingredient. May contain propellants such as carbon, propane/butane or dimethyl ether.

In the present invention, when the formulation of the cosmetic composition is cleansing, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linoline derivative, or ethoxylated glycerol fatty acid ester can be used.

The cosmetic formulation of the present invention also includes other ingredients that can have a synergistic effect on the main effect within a range that does not impair the main effect aimed at by the present invention, without difficulty depending on the formulation or purpose of use of the cosmetic. You can select and mix them.

In the present invention, the use and function of the cosmetic composition are not particularly limited and can be appropriately selected depending on the purpose. For example, the cosmetic composition of the present invention includes skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, foundation, essence, nutritional essence. , pack, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, and body cleanser may have one or more uses and functions selected from the group.

In addition to pine needle extract, the cosmetic composition of the present invention may contain ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, moisturizers, solubilizers, vitamins, ultraviolet absorbers, pigments, and fragrances. It may include, and may further include preservatives, disinfectants, etc.

Example

The present invention will be described in more detail through examples below. However, these examples show some experimental methods and configurations to illustratively illustrate the present invention, and the scope of the present invention is not limited to these examples.

Preparation example: Preparation of pine needle extract

30g of pine needles were weighed and placed in a 500mL flask, and 300mL of 95% methanol was added. It was left in a constant temperature water bath at 50°C for 3 hours and stirred once every 30 minutes, a total of 5 times. Next, it was filtered through qualitative filter paper (Whatman Filter paper No. 1) with a diameter of 90 mm, concentrated using a rotary concentrator, and then freeze-dried for 72 hours. The dried extract was dissolved in DMSO to prepare samples to be used in the experiment.

Experimental example: Analysis of persistent bacterial inhibition effect according to pine needle extract treatment

The inhibitory effect of pine needle extract on persistent bacterial formation was confirmed by the following method.

Staphylococcus aureus ATCC 6538 was streaked on TSA (tryptic soy agar) medium and left at 37°C for 24 hours to form colonies. The formed colonies were inoculated into 5 mL of TSB (tryptic soy broth) medium and pre-cultured at 37°C and 250 rpm for 24 hours. The pre-cultured bacteria were inoculated into 5 mL of TSB medium under the condition of OD ₆₀₀ = 0.05, and then cultured at 37°C and 250 rpm for 3 hours and used in the experiment.

In order to confirm the number of viable cells, the S. aureus culture TSB medium was serially diluted with sterile saline solution, then 100 μL of the diluted solution was spread uniformly on a TSA plate and colony forming unit (CFU) was measured.

In order to determine the number of persistent bacteria (persister cells), pine needle extract was used as S. aureus culture was treated with TSB medium at concentrations of 0, 64, 128, 256, and 512 mg/L, and treated with antibiotics at the same time. At this time, oxacillin, tobramycin, or ciprofloxacin was used as the antibiotic, and the concentration was 2.5, 80, and 20 μg/mL, which was 10 times the MIC of each antibiotic. After 24 hours, it was serially diluted with sterile saline solution, and 100 μL of the diluted solution was evenly spread on a TSA plate to measure colony forming unit (CFU).

Using each CFU value obtained from the above experiment, the persister cell ratio and inhibited persister cell formation were calculated according to the formula below.

The percentage of persistent bacteria according to the concentration of pine needle extract in treatment with oxacillin, tobramycin, and ciprofloxacin is shown in Figure 1, and the inhibition rate (%) of persistent bacteria formation is shown in Figure 2.

Referring to the experimental results, even when treated with an excessive amount of antibiotics reaching 10 times the MIC, some bacteria persisted without being killed, but when treated with pine needle extract, the amount of persisting bacteria decreased.

Specifically, when each antibiotic is treated at 10 times the MIC, if the concentration of the pine needle extract is 0.05 g/L or more, the amount of persistent bacteria is significantly reduced, and if it is 0.2 g/L or more, the inhibition rate of persistent bacteria for each antibiotic is more than 80%. Very excellent results were obtained. In particular, when used in combination with tobramycin, it was confirmed that persistent bacteria were inhibited by more than 90% when the concentration of pine needle extract was 0.1 g/L or more.

Through this, it was confirmed that the pine needle extract exhibits excellent inhibitory activity against persistent bacteria that are not killed by the above antibiotics. Among them, it was confirmed that tobramycin exhibited a very excellent inhibitory effect against persistent bacteria.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (14)

A composition for inhibiting persistent cells containing pine needle extract.
According to claim 1,
A composition for inhibiting persistent bacteria, wherein the persistent bacteria survive even when the persistent bacteria are treated with antibiotics at a level of 5 times or more than the minimum inhibitory concentration (MIC).
According to claim 1,
A composition for inhibiting persistent bacteria, wherein the persistent bacteria are persistent bacteria of the genus Staphylococcus ( Staphylococcus sp.).
According to claim 1,
A composition for inhibiting persistent bacteria, wherein the concentration of the pine needle extract is 0.001 to 10 g/L.
According to claim 1,
A composition for inhibiting persistent bacteria, wherein the composition is used in combination with an antibiotic.
According to claim 5,
The above antibiotics include beta-lactams, quinolones, aminoglycosides, tetracyclines, peptides, and mupirocin. A composition for inhibiting persistent bacteria, comprising at least one selected from the group consisting of fusidic acid.
According to claim 5,
A composition for inhibiting persistent bacteria, wherein the weight ratio of pine needle extract and antibiotic is 0.1:1 to 1,000:1.
A composition for preventing or treating bacterial infectious diseases, comprising the composition for inhibiting persistent bacteria according to any one of claims 1 to 7.
According to claim 8,
A composition for preventing or treating a bacterial infectious disease, wherein the bacterial infectious disease is an infectious disease caused by Staphylococcus sp.
According to claim 8,
For the prevention or treatment of a bacterial infectious disease, wherein the bacterial infectious disease is one or more inflammatory diseases selected from the group consisting of respiratory infection, sinusitis, pneumonia, endocarditis, osteomyelitis, arthritis, cellulitis, gastroenteritis, postoperative wound infection, and dermatitis. Composition.
According to claim 8,
A composition for preventing or treating a bacterial infectious disease, wherein the bacterial infectious disease is a palindromic or chronic disease.
A cosmetic composition for improving dermatitis, comprising the composition for inhibiting persistent bacteria according to any one of claims 1 to 7.
According to claim 12,
A cosmetic composition for improving dermatitis, wherein the dermatitis is abscess, folliculitis, boil or acne.
According to claim 12,
A cosmetic composition for improving dermatitis, wherein the dermatitis is recurrent or chronic dermatitis.