JP7491742B2 - Resistance inhibitor, food and drink composition for resistance inhibition, and method for inhibiting resistance - Google Patents

Description

The present invention relates to a resistance inhibitor that inhibits the resistance of antibiotic-resistant bacteria to antibiotics, and in particular to a resistance inhibitor that inhibits the resistance of bacteria to at least one of β-lactam antibiotics and aminoglycoside antibiotics.

Staphylococcus aureus is a gram-positive cocci that is a normal inhabitant of the skin and nasal cavity of humans and animals. Although it is usually harmless, it is known to cause a variety of diseases when the immune system is weakened. Diseases caused by Staphylococcus aureus range from relatively mild illnesses such as suppuration and food poisoning to severe illnesses such as pneumonia, peritonitis, sepsis, and meningitis.

Antibacterial drugs (antibiotics) such as methicillin have been used to treat Staphylococcus aureus. However, as a result of the worldwide use of antibacterial drugs such as methicillin, methicillin-resistant Staphylococcus aureus (hereinafter also referred to as "MRSA") has emerged, which has acquired resistance to antibacterial drugs. Recent studies have reported that MRSA is a multidrug-resistant bacterium that is resistant not only to β-lactam antibiotics such as methicillin, but also to antibacterial drugs such as aminoglycoside antibiotics and macrolide antibiotics.

When infected with such antibiotic-resistant bacteria, it is difficult to provide effective treatment with currently available antibiotics, and new treatment methods and agents for antibiotic-resistant bacterial infections are being investigated (for example, Patent Document 1).

Japanese Patent Application Laid-Open No. 10-182655

The present invention aims to provide a resistance inhibitor that inhibits the resistance of antibiotic-resistant bacteria to at least one of β-lactam antibiotics and aminoglycoside antibiotics.

The inventors discovered that ginsenoside Rg3 and compound M1 inhibit the resistance of antibiotic-resistant bacteria to β-lactam antibiotics and aminoglycoside antibiotics, making these antibiotics more effective against antibiotic-resistant bacteria, and thus completed the present invention.

［２］前記抗菌薬が、アミノグリコシド系抗菌薬であることを特徴とする［１］に記載の耐性阻害剤。
［３］前記抗菌薬が、ゲンタマイシンとカナマイシンの少なくともいずれか一方であることを特徴とする［２］に記載の耐性阻害剤。
［４］前記抗菌薬耐性菌が、メチシリン耐性黄色ブドウ球菌であることを特徴とする［１］から［３］のいずれか一つに記載の耐性阻害剤。
［５］抗菌薬耐性菌の前記抗菌薬に対する耐性阻害用飲食品組成物であって、下記式（１）で表されるジンセノサイドＲｇ３及び／又は下記式（２）で表される化合物Ｍ１を含有し、前記抗菌薬が、β－ラクタム系抗菌薬とアミノグリコシド系抗菌薬の少なくともいずれか一方である耐性阻害用飲食品組成物。

［６］抗菌薬耐性菌の前記抗菌薬に対する耐性を阻害する耐性阻害方法であって、下記式（１）で表されるジンセノサイドＲｇ３及び／又は下記式（２）で表される化合物Ｍ１を摂取すること含み、前記抗菌薬が、β－ラクタム系抗菌薬とアミノグリコシド系抗菌薬の少なくともいずれか一方である耐性阻害方法。

That is, the gist of the present invention is as follows.
[1] An inhibitor of resistance of an antibiotic-resistant bacterium to an antibiotic, comprising ginsenoside Rg3 represented by the following formula (1) and/or compound M1 represented by the following formula (2), wherein the antibiotic is at least one of a β-lactam antibiotic and an aminoglycoside antibiotic.

[2] The resistance inhibitor described in [1], characterized in that the antibiotic is an aminoglycoside antibiotic.
[3] The resistance inhibitor described in [2], characterized in that the antibiotic is at least one of gentamicin and kanamycin.
[4] The resistance inhibitor according to any one of [1] to [3], characterized in that the antibiotic-resistant bacterium is methicillin-resistant Staphylococcus aureus.
[5] A food and beverage composition for inhibiting the resistance of antibacterial-resistant bacteria to an antibacterial agent, comprising ginsenoside Rg3 represented by the following formula (1) and/or compound M1 represented by the following formula (2), wherein the antibacterial agent is at least one of a β-lactam antibacterial agent and an aminoglycoside antibacterial agent.

[6] A method for inhibiting the resistance of an antibiotic-resistant bacterium to an antibiotic, comprising ingesting ginsenoside Rg3 represented by the following formula (1) and/or compound M1 represented by the following formula (2), wherein the antibiotic is at least one of a β-lactam antibiotic and an aminoglycoside antibiotic.

The present invention provides a resistance inhibitor that inhibits the resistance of antibacterial-resistant bacteria to at least one of β-lactam antibacterial drugs and aminoglycoside antibacterial drugs.

One embodiment of the present invention is described below.

The resistance inhibitor of this embodiment (hereinafter also simply referred to as "inhibitor") contains ginsenoside Rg3 represented by the following formula (1) and/or compound M1 represented by the following formula (2).

Ginsenoside Rg3 is a type of saponin with protopanaxadiol (PPD) as the basic structure, and has a structure in which two glucose molecules are bound to the hydroxyl group at the 3-position. Ginsenoside Rg3 includes two optical isomers, S- and R-, and the inhibitor of this embodiment contains S-form ginsenoside Rg3 (hereinafter also referred to as "ginsenoside Rg3(1)") represented by the above formula (1). Ginsenoside Rg3(1) is also called 12β,20-dihydroxydammar-24-en-3β-yl, 2-O-β-D-glucopyranosyl-β-D-glucopyranoside or protopanaxadiol, 20-O-glucoside. The CAS number of ginsenoside Rg3(1) is 14197-60-5.

Ginsenoside Rg3(1) can be extracted, for example, from red ginseng. Red ginseng is a food produced by heating and drying the root of Panax ginseng (C.A. Meyer) of the Araliaceae family, and is also specified in the 16th Revised Japanese Pharmacopoeia, pages 1488-1489.

The method for extracting ginsenoside Rg3(1) from red ginseng is not particularly limited, and any known method can be used. In addition, the following method may be used to extract ginsenoside Rg3(1) from red ginseng.

First, red ginseng is extracted with water or an alcoholic solvent such as methanol, and the resulting red ginseng extract is purified using an ODS column (OctaDecylSilyl column) to obtain a red ginseng extract with a saponin content of 90% by mass or more. A red ginseng extract with a saponin content of 90% by mass or more can be obtained, for example, by extracting red ginseng with a solvent containing 60% by volume or more of methanol relative to 100% by volume of the solvent. The time for soaking red ginseng in the solvent can be 4 to 48 hours. Among the soaking methods, reflux extraction is preferably used to obtain a red ginseng extract with a saponin content of 90% by mass or more. Reflux extraction is an extraction method in which red ginseng is soaked in a solvent and heated for extraction, while the evaporated solvent and volatile components are cooled and condensed and returned to the solvent in which the red ginseng is soaked. When reflux extraction is performed under pressure, extraction can be performed at low temperatures (e.g., 10 to 40°C) and in a short time (e.g., several tens of minutes to 4 hours). For purification of the ODS column, purified water and 20% by volume of methanol are used as the mobile phase to remove impurities, and for elution of substances adsorbed on the ODS column, 40 to 100% by volume of methanol can be used as the mobile phase.

Next, Rg3 in the red ginseng extract obtained by the purification process is acetylated using pyridine/acetic anhydride (1:1). The reaction time can be, for example, 12 hours, and the reaction temperature can be, for example, 18°C. The acetylated Rg3 is extracted using ethyl acetate, and hydrochloric acid and sodium bicarbonate are added to dryness.

Next, the dried red ginseng extract containing acetylated Rg3 is dissolved in purified water, and then the acetylated Rg3 fraction is separated using an ODS column. The OSD column used to obtain the acetylated Rg3 fraction can use purified water and 40% or less by volume of methanol as the mobile phase, and 40-80% by volume of methanol can be used to elute the adsorbed substance. The acetylated Rg3 fraction can be obtained using an ODS column by increasing the concentration of methanol flowing through the ODS column to 40-80% by volume using a gradient method in which the mixing ratio of purified water and methanol used as the mobile phase is changed every predetermined time. The obtained acetylated Rg3 fraction may be purified using a SiO ₂ column as necessary. Specifically, the obtained acetylated Rg3 fraction can be dried and dissolved in purified water, and then purified using a SiO ₂ column using benzene-acetone (10:1) as the mobile phase.

Next, the separated acetylated Rg3 fraction is subjected to a deacetylation treatment. Deacetylation can be performed using a 1% sodium methoxide methanol solution. The reaction time can be, for example, 8 hours, and the reaction temperature can be, for example, 23°C. The deacetylated Rg3 fraction is dried and dissolved in purified water, and then the S-form of Rg3 (i.e., the fraction of ginsenoside Rg3(1)) is obtained using an ODS column. Here, the S-form of Rg3 can be obtained based on the retention time of a standard product of ginsenoside Rg3(1) (such as commercially available S-form of Rg3). Note that in the ODS column for obtaining the S-form of Rg3, 40 to 100% by volume of methanol can be used as the mobile phase.

In addition, as another method for obtaining ginsenoside Rg3(1), an inorganic acid such as hydrochloric acid or acetic acid or an organic acid such as citric acid or malic acid is added to a red ginseng extract dissolved in water, and the mixture is heated and extracted to remove the sugar groups of the protopanaxadiol (PPD) saponins in the red ginseng extract by hydrolysis, thereby obtaining a protopanaxadiol saponin fraction (hereinafter also referred to as a "PPD saponin fraction") with an increased content of ginsenoside Rg3(1), and the PPD saponin fraction may be used to obtain a ginsenoside Rg3(1) fraction by an ODS column. Note that the method for obtaining a ginsenoside Rg3(1) fraction by an ODS column using a PPD saponin fraction is the same as the method for obtaining a ginsenoside Rg3(1) fraction by an ODS column using the above-mentioned deacetylated Rg3 fraction, so a detailed explanation is omitted. This method makes it possible to efficiently extract the Rg3 fraction.

In addition, in the resistance inhibitor of this embodiment, commercially available ginsenoside Rg3(1) may be used.

Compound M1 (hereinafter also referred to as "compound M1(2)") represented by the above formula (2) is a type of saponin with protopanaxadiol (PPD) as the basic skeleton, and has a structure in which one molecule of glucose is bound to the hydroxyl group at the 20th position. Compound M1(2) is also called (3B,12B)-3,12-dihydroxydammar-24-en-20-yl, β-D-glucopyranoside or 20-O-β-D-glucopyranosyl-20(S)-protopanaxadiol, and is also called compound K. The CAS number of compound M1(2) is 39262-14-1.

Compound M1(2) can be derived from saponins such as ginsenosides Rb1, Rb2, Rc, and Rd contained in red ginseng. The method for deriving compound M1(2) from saponins is not particularly limited, and known methods can be used. For example, a method of treating red ginseng extract with an enzyme (Kyeong-Hwan NOH et al., Bioscience, Biotechnology, and Biochemistry "Ginsenoside Compound K Production from Ginseng Root Extract by a Thermostable β-Glycosidase from Sulfolobus solfataricus", 2009, Vol. 73, No. 2, p. 316-321) or a method of fermenting red ginseng extract with lactic acid bacteria (Jae-Myung Yoo et al., Food Science and Biotechnology "Enhanced production of compound K in fermented ginseng extracts by Lactobacillus brevis", 2019, Volume 28, Issue 3, pages 823-829) can be mentioned. In addition, the following method may be used to derive compound M1 (2) from saponin.

First, water is added to the red ginseng extract extracted with water or an alcohol such as methanol, or the above-mentioned fraction of protopanaxadiol (PPD)-based saponins, and the pH is adjusted to about 4 using hydrochloric acid. The red ginseng extract is the same as the above-mentioned red ginseng extract (red ginseng extract with a saponin content of 90% by mass or more) that can be used to obtain ginsenoside Rg3 (1), so a detailed explanation of the method of obtaining it is omitted. In addition, the fraction of protopanaxadiol (PPD)-based saponins can be obtained by a method that does not use the above-mentioned OSD column that can be used to obtain the Rg3 fraction.

Next, cellulase (e.g., Pectinase G Amano (manufactured by Amano Enzyme Co., Ltd.)) is added to this solution adjusted to approximately pH 4 to carry out an enzyme reaction. The enzyme reaction time can be, for example, 3 days, and the enzyme reaction temperature can be, for example, 50°C.

Next, methanol is added to this solution, and the enzyme is removed by filtration. For example, filter paper can be used for filtration. Next, the filtrate is dried and dissolved in purified water, and then the crude M1 fraction is separated using methanol as the mobile phase in an ODS column. The crude M1 fraction can be obtained based on the retention time of a standard product of compound M1 (2) (such as commercially available compound M1). Next, the crude M1 fraction is dried and dissolved in purified water, and then purified using a chloroform-methanol solution (20:1) as the mobile phase in a SiO ₂ column. Next, the purified crude M1 fraction is dried and dissolved in purified water, and then the M1 fraction can be obtained using methanol as the mobile phase in an ODS column. The M1 fraction can be obtained based on the retention time of a standard product of compound M1 (2).

In addition, in the resistance inhibitor of this embodiment, a commercially available compound M1(2) may be used.

The amount of ginsenoside Rg3 (1) and compound M1 (2) contained in the inhibitor of this embodiment is not particularly limited and can be appropriately set taking into consideration the form (dosage form) and intake amount described below.

The inhibitor of this embodiment may be composed of only ginsenoside Rg3 (1) and/or compound M1 (2), or may contain other components in addition to these components. Examples of other components include pharmacologically acceptable carriers, excipients, integration agents, preservatives, stabilizers, flavorings, pH adjusters, dispersion media, components contained in food and beverages (hereinafter also referred to as "food and beverage components"), components contained in feed, etc.

The form (dosage form) of the inhibitor of this embodiment is not particularly limited, and may be, for example, an external preparation (ointment, patch, etc.) or an injection, but is preferably an internal form such as plain tablets, sugar-coated tablets, granules, powder, liquid, tablet, or capsule (hard capsule, soft capsule). The method of taking the inhibitor of this embodiment can be appropriately set according to the form (dosage form), and is not particularly limited, but can be taken orally, for example. When the inhibitor of this embodiment is used as a food or drink, the inhibitor of this embodiment may contain a food or drink component to be, for example, a beverage (soft drinks such as coffee, juice, and tea drinks, milk beverages, lactic acid bacteria beverages, yogurt beverages, carbonated beverages, etc.), confectionery (chocolate, donuts, pies, cream puffs, gum, jelly, candy, cookies, cakes, puddings, daifuku, mochi, manju, castella, anmitsu, yokan, etc.), or seasonings (dressings, furikake, umami seasonings, soup bases, etc.).

The inhibitor of this embodiment can be a drug, a quasi-drug, or a food or drink. When the inhibitor of this embodiment is a food or drink, it may be a normal food or drink, but it is preferable that it is a health food, a functional food, a nutritional supplement, a supplement, or a food for specified health uses. The method for producing the inhibitor of this embodiment is not particularly limited, and the inhibitor can be formulated by a known method depending on the type of drug, quasi-drug, food or drink, etc.

Examples of recipients who ingest the inhibitor of this embodiment include humans and non-human animals. Examples of non-human animals include higher vertebrates other than humans, particularly non-human mammals, and more specifically, examples include pet animals such as dogs and cats, and livestock such as cows, horses, pigs, and sheep. In addition, the recipient may be a carrier who carries antibacterial resistant bacteria, or a non-carrier who does not carry antibacterial resistant bacteria.

The intake amount of the inhibitor in this embodiment may be any amount that can inhibit the resistance of antibiotic-resistant bacteria to at least one of β-lactam antibiotics and aminoglycoside antibiotics (hereinafter simply referred to as "resistance"), and can be set appropriately depending on symptoms, age, body weight, etc.

The intake amount of ginsenoside Rg3 (1) and/or compound M1 (2) in the inhibitor of this embodiment is not particularly limited, but the oral intake amount (intake amount of ginsenoside Rg3 (1) and/or compound M1 (2)) can be, for example, 0.005 mg or more and 2 mg or less, and further 0.01 mg or more and 0.25 mg or less per 1 kg of the body weight of the person taking the inhibitor. In addition, for example, the transdermal intake amount (intake amount of ginsenoside Rg3 (1) and/or compound M1 (2)) can be 0.01 mg or more and 3 mg or less per transdermal intake of the inhibitor. Note that, when the inhibitor of this embodiment contains both ginsenoside Rg3 (1) and compound M1 (2), the above-mentioned intake amount refers to the total intake amount of these components. The number of intakes is also not particularly limited, and can be, for example, 1 to 4 times per day.

The inhibitor of this embodiment can inhibit the resistance of antibacterial-resistant bacteria to at least one of β-lactam antibacterial drugs and aminoglycoside antibacterial drugs. Here, inhibiting resistance refers to suppressing the resistance of antibacterial-resistant bacteria by taking the inhibitor of this embodiment so that the antibacterial drug acts more easily in a smaller amount than when the inhibitor of this embodiment is not taken. Therefore, if a carrier of antibacterial-resistant bacteria takes the inhibitor of this embodiment, the resistance of the antibacterial-resistant bacteria to the antibacterial drug is suppressed, and the antibacterial drug acts more easily on the antibacterial-resistant bacteria. On the other hand, if a non-carrier of antibacterial-resistant bacteria takes the inhibitor of this embodiment, the resistance of the antibacterial-resistant bacteria to the antibacterial drug can be suppressed when the antibacterial-resistant bacteria are carried. As a result, the antibacterial drug acts more easily on the antibacterial-resistant bacteria.

Antibiotics that are more effective against antibiotic-resistant bacteria are those to which antibiotic-resistant bacteria are resistant, specifically, β-lactam antibiotics and/or aminoglycoside antibiotics.

Examples of β-lactam antibiotics include penicillin antibiotics such as penicillin, carbenicillin, oxacillin, ampicillin, and methicillin, cephem antibiotics such as cefoxitin and cefazolin, carbapenem antibiotics such as doripenem, penem antibiotics such as faropenem, monobactam antibiotics such as aztreonem, and combinations with β-lactamase inhibitors such as tazobactam/piperacillin (TAZ/PIPC).

Examples of aminoglycoside antibiotics include kanamycin, tobramycin, gentamicin, arbekacin, amikacin, streptomycin, dibekacin, bekanamycin, isepamicin, fradiomycin, ribostamycin, and neomycin.

The inhibitor of this embodiment can inhibit resistance to at least one of β-lactam antibacterial drugs and aminoglycoside antibacterial drugs, but from the viewpoint of being more likely to inhibit resistance regardless of the type (strain) of antibacterial drug-resistant bacteria, it is preferable to use an inhibitor of resistance to aminoglycoside antibacterial drugs rather than an inhibitor of resistance to β-lactam antibacterial drugs. Furthermore, among aminoglycoside antibacterial drugs, it is more preferable to use an inhibitor of resistance to gentamicin and/or kanamycin.

The antibiotic-resistant bacteria whose resistance is inhibited by the inhibitor of this embodiment are bacteria that have acquired resistance (resistance) to at least one of β-lactam antibiotics and aminoglycoside antibiotics, and may be bacteria that have acquired resistance to other antibiotics in addition to these antibiotics. A specific example of antibiotic-resistant bacteria is methicillin-resistant Staphylococcus aureus (MRSA). In this specification, methicillin-resistant Staphylococcus aureus (MRSA) refers to both Staphylococcus aureus that is resistant only to methicillin and Staphylococcus aureus that is resistant to multiple types of antibiotics including methicillin.

One aspect of the inhibitor of this embodiment described above includes a food and drink composition for inhibiting the resistance of antibacterial drug-resistant bacteria, which contains ginsenoside Rg3 (1) and/or compound M1 (2) and a food and drink component. The food and drink composition for inhibiting resistance may be a normal food or drink, but may also be a health food, a functional food, a nutritional supplement, a supplement, a food for specified health use, or the like. This food and drink composition for inhibiting resistance can also inhibit the resistance of antibacterial drug-resistant bacteria, like the inhibitor of this embodiment.

The inhibitor of this embodiment can be used as an antibacterial agent for antibacterial-resistant bacteria in combination with the above-mentioned antibacterial agents (β-lactam antibacterial agents and/or aminoglycoside antibacterial agents). The antibacterial agent for resistant bacteria can suppress the resistance to antibacterial agents by the inhibitor, and can kill antibacterial-resistant bacteria whose resistance has been suppressed by the antibacterial agent (or can suppress the growth of antibacterial-resistant bacteria). The antibacterial agent for resistant bacteria can be used, for example, as a pharmaceutical product, and may be a kit formulation in which the inhibitor and the antibacterial agent are separately formulated, or a formulation in which the inhibitor and the antibacterial agent are formulated to be contained in a single agent. The antibacterial agent for resistant bacteria may contain the above-mentioned other components in addition to the inhibitor and the antibacterial agent of this embodiment.

An antibacterial agent for antibacterial-resistant bacteria preferably contains gentamicin and/or kanamycin as the antibacterial agent. The inhibitor contained in the antibacterial agent for antibacterial-resistant bacteria is more likely to inhibit resistance to gentamicin and kanamycin regardless of the type (strain) of antibacterial-resistant bacteria, so the antibacterial agent for resistant bacteria preferably contains gentamicin and/or kanamycin as the antibacterial agent.

Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

[Obtaining ginsenoside Rg3(1)]
First, red ginseng was extracted with a solvent containing 60% or more by volume of methanol to obtain a red ginseng extract. The red ginseng was immersed in the solvent for 4 to 48 hours. Reflux extraction was used to obtain the red ginseng extract. After the red ginseng was extracted with the solvent, the red ginseng extract was purified using an ODS column, and the purified red ginseng extract was dried. In the purification process using the ODS column, purified water and 20% by volume of methanol were used as the mobile phase to remove impurities, and 40 to 100% by volume of methanol was used as the mobile phase to elute the substances adsorbed on the ODS column.

The obtained red ginseng extract was dissolved in water, and an inorganic acid such as hydrochloric acid or acetic acid or an organic acid such as citric acid or malic acid was added, followed by heating and extraction. The sugar groups of the protopanaxadiol (PPD) saponins in the red ginseng extract were removed by hydrolysis, and a PPD saponin fraction with an increased content of ginsenoside Rg3(1) was obtained. The obtained PPD saponin fraction was loaded onto an ODS column to obtain a ginsenoside Rg3(1) fraction. Here, the ginsenoside Rg3(1) fraction was obtained based on the retention time of a standard product of ginsenoside Rg3(1) (such as commercially available S-form Rg3), and 40 to 100% by volume of methanol was used as the mobile phase. The obtained ginsenoside Rg3(1) fraction was dried to obtain ginsenoside Rg3(1).

[Obtaining compound M1(2)]
A PPD-based saponin fraction was obtained in the same manner as when ginsenoside Rg3 (1) was obtained. 500 mL of water was added to about 10 g of the obtained PPD-based saponin fraction, and the pH was adjusted to about 4 using hydrochloric acid. 10 g of cellulase (Pectinase G Amano) was added to this solution, and the enzyme reaction was carried out at 50 ° C. for 3 days. Methanol was added to this solution, and the enzyme was removed by filtration using filter paper, and the filtrate was dried. Next, after dissolving in purified water, the crude M1 fraction was separated using methanol on an ODS column. The methanol concentration (v/v) was changed from 0% to 40% to 80% to 100% to remove impurities in low-concentration methanol, and a crude M1 fraction in high-concentration methanol (40% or more) was obtained. Here, the crude M1 fraction was obtained based on the retention time of a standard product of compound M1 (2) (such as commercially available compound M1 (2)). The obtained crude M1 fraction was dried, dissolved in purified water, and purified using chloroform-methanol = 20:1 (v/v) on a _SiO2 column. The purified crude M1 fraction was then dried. After further dissolving in purified water, the M1 fraction was obtained again using methanol (sequentially changing from 0% to 100% methanol) on an OD column. The M1 fraction was obtained based on the retention time of the standard product of compound M1 (2). The obtained M1 fraction was dried to obtain compound M1 (2).

[Preparation of antibiotic-resistant bacteria]
As antibiotic-resistant bacteria, four types of MRSA (Methicillin Resistant Staphylococcus aureus) were prepared. Specifically, the following MRSA types were prepared: IID1677 (Institute of Medical Science, University of Tokyo), ATCC BAA-1717 (ATCC (American Type Culture Collection)), ATCC33592 (ATCC (American Type Culture Collection)), and ATCC43300 (ATCC (American Type Culture Collection)). It was confirmed in advance that these MRSAs were resistant to the antibiotics used in the test described below.

[Test 1]
The prepared MRSA was cultured in LB medium at 37°C overnight, and a bacterial solution with a turbidity (OD600nm value) of 0.005 (5/ ^3x106 CFU/mL (CFU: Colony Forming Unit, viable cell count)) was prepared in brain heart infusion (BHI) medium. 85μL of the bacterial solution was added to each well of a 96-well microplate to obtain ^1.5x105 CFU/well (CFU: Colony Forming Unit, viable cell count). Next, 10μL of a liquid (hereinafter also referred to as "antibacterial solution") prepared by dissolving an antibacterial agent shown in Table 1 in sterilized water was added to each well (different concentrations of the antibacterial agent were added), and 5μL of the test solution was added to each well.

Three types of test liquids were used: a bacterial liquid containing ginsenoside Rg3 (1) dissolved in 10% aqueous methanol solution, a bacterial liquid containing compound M1 (2) dissolved in 10% aqueous methanol solution, and a bacterial liquid containing 10% aqueous methanol solution as a control. In the wells containing ginsenoside Rg3 (1) and compound M1 (2), the concentrations of ginsenoside Rg3 (1) and compound M1 (2) were fixed at 100 μg/ml. The final methanol concentration in each well was fixed at 0.5%.

After the addition of the antibacterial solution and the test solution, the wells were incubated at 37°C for 24 hours. After incubation, each well was visually inspected to determine whether or not MRSA had grown, and the minimum inhibitory concentration (MIC (μg/mL)) of the antibacterial agent was calculated. In addition, to determine detailed differences, 30 μL of the bacterial solution was mixed with 30 μL of Bac-Titer Gro (Promega) and the amount of bacterial ATP was measured using the chemiluminescence method.

The test results using IID1677 as MRSA are shown in Table 1-1. When no antibacterial agent was added, the addition of ginsenoside Rg3 (1) or compound M1 (2) did not affect the growth (development) of MRSA.
[Table 1-1]

The test results using ATCC BAA-1717 as MRSA are shown in Table 1-2. When no antibacterial agent was added, the addition of ginsenoside Rg3 (1) or compound M1 (2) did not affect the growth (development) of MRSA.
[Table 1-2]

The test results using ATCC33592 as MRSA are shown in Tables 1-3. When no antibacterial agent was added, the addition of ginsenoside Rg3 (1) or compound M1 (2) did not affect the growth (development) of MRSA.
[Table 1-3]

The test results using ATCC 43300 as MRSA are shown in Tables 1-4. When no antibacterial agent was added, the addition of ginsenoside Rg3 (1) or compound M1 (2) did not affect the growth (development) of MRSA.
[Table 1-4]

As shown in Tables 1-1 to 1-4, when β-lactam or aminoglycoside antibiotics were used, the addition of ginsenoside Rg3 (1) or compound M1 (2) reduced the MIC compared to the control without the addition of these ingredients. On the other hand, such a reduction in MIC could not be confirmed when tetracycline or macrolide antibiotics were used.

Here, the MIC refers to the minimum amount of an antibiotic that can inhibit the growth of bacteria. Therefore, a decrease in the MIC means that the antibiotic is more effective. Therefore, from the results of this evaluation, it was understood that the inhibitor of this embodiment was able to inhibit the resistance of MRSA to at least one of β-lactam antibiotics and aminoglycoside antibiotics.

Furthermore, among β-lactam and aminoglycoside antibacterial drugs, kanamycin and gentamicin showed MIC reduction rates of 75% or more, expressed by the following formula (3), regardless of the MRSA used, and the minimum MIC reduction rates were higher than when other antibacterial drugs were used. In other words, it was understood that, among β-lactam and aminoglycoside antibacterial drugs, kanamycin and gentamicin are more likely to inhibit resistance regardless of the type (strain) of antibacterial-resistant bacteria.

[Test 2]
In this evaluation, the concentration of each antibacterial agent added to the wells was fixed at the MIC (MIC when 100 μg/mL of ginsenoside Rg3(1) was added) determined in Evaluation 1. In addition, the concentration of ginsenoside Rg3(1) in the wells (100 μg/ml) used in Evaluation 1 was changed within the range of 25 to 50 μg/ml. Other than these conditions, the presence or absence of MRSA proliferation was confirmed in the same manner as in Test 1. The results were evaluated as ◯ when no proliferation was observed, and × when proliferation was observed.

The test results using IID1677 as MRSA are shown in Table 2-1. When no antibacterial agent was added, the addition of ginsenoside Rg3(1) did not affect the growth (development) of MRSA.
[Table 2-1]

The test results using ATCC BAA-1717 as MRSA are shown in Table 2-2. When no antibacterial agent was added, the addition of ginsenoside Rg3(1) did not affect the growth (development) of MRSA.
[Table 2-2]

The test results using ATCC 33592 as MRSA are shown in Table 2-3. When no antibacterial agent was added, the addition of ginsenoside Rg3(1) did not affect the growth (development) of MRSA.
[Table 2-3]

The test results using ATCC 43300 as MRSA are shown in Table 2-4. When no antibacterial agent was added, the addition of ginsenoside Rg3(1) did not affect the growth (development) of MRSA.
[Table 2-4]

As shown in Tables 2-1 to 2-4, when β-lactam or aminoglycoside antibiotics were used, each antibiotic was effective at the MIC (MIC when Rg3 was added at 100 μg/mL) calculated in Evaluation 1, even when the concentration of ginsenoside Rg3 (1) was changed in the range of 25 to 50 μg/mL.

[Test 3]
In this evaluation, the concentration of each antibacterial agent added to the wells was fixed at the MIC (MIC when 100 μg/mL of compound M1(2) was added) determined in Evaluation 1. In addition, the concentration (100 μg/ml) of compound M1(2) used in Evaluation 1 in the wells was changed within the range of 12.5 to 50 μg/ml. For other conditions, the presence or absence of MRSA proliferation was confirmed in the same manner as in Test 1. The results were evaluated as ◯ when no proliferation was observed, and × when proliferation was observed.

The test results using IID1677 as MRSA are shown in Table 3-1. When no antibacterial agent was added, the addition of compound M1(2) did not affect the growth (development) of MRSA.
[Table 3-1]

The test results using ATCC BAA-1717 as MRSA are shown in Table 3-2. When no antibacterial agent was added, the addition of compound M1(2) did not affect the growth (development) of MRSA.
[Table 3-2]

The test results using ATCC 33592 as MRSA are shown in Table 3-3. When no antibacterial agent was added, the addition of compound M1(2) did not affect the growth (development) of MRSA.
[Table 3-3]

The test results using ATCC 43300 as MRSA are shown in Table 3-4. When no antibacterial agent was added, the addition of compound M1(2) did not affect the growth (development) of MRSA.
[Table 3-4]

As shown in Tables 3-1 to 3-4, when β-lactam or aminoglycoside antibiotics were used, each antibiotic was effective at the MIC determined in Evaluation 1 (MIC when Rg3 was added at 100 μg/mL) even when the concentration of compound M1 (2) was changed in the range of 12.5 to 50 μg/mL.

From the results of tests 1 to 3 described above, it was understood that the inhibitor of this embodiment was able to inhibit the resistance of MRSA to at least one of β-lactam antibiotics and aminoglycoside antibiotics.

Claims (8)

An inhibitor of resistance of an antibacterial-resistant bacterium to an antibacterial agent,
Contains ginsenoside Rg3 represented by the following formula (1):
the antibacterial agent is at least one of a β-lactam antibacterial agent and an aminoglycoside antibacterial agent;
The resistance inhibitor is characterized in that it does not exhibit antibacterial activity against the antibacterial drug-resistant bacteria by itself .
An inhibitor of resistance of an antibacterial-resistant bacterium to an antibacterial agent, comprising:
Contains a compound M1 represented by the following formula (2):
The antibiotic resistance inhibitor is characterized in that the antibiotic is at least one of a β-lactam antibiotic and an aminoglycoside antibiotic.
The resistance inhibitor according to claim 1 or 2 , characterized in that the antibacterial agent is an aminoglycoside antibacterial agent. The resistance inhibitor according to claim 3 , characterized in that the antibacterial agent is at least one of gentamicin and kanamycin. The resistance inhibitor according to any one of claims 1 to 4 , characterized in that the antibacterial drug resistant bacterium is methicillin-resistant Staphylococcus aureus. A food and drink composition for inhibiting the resistance of an antibacterial drug-resistant bacterium to an antibacterial drug,
Contains ginsenoside Rg3 represented by the following formula (1):
the antibacterial agent is at least one of a β-lactam antibacterial agent and an aminoglycoside antibacterial agent;
The food or drink composition for resistance inhibition does not exhibit antibacterial activity against the antibacterial drug-resistant bacteria by itself .
A food and drink composition for inhibiting the resistance of an antibacterial drug-resistant bacterium to an antibacterial drug,
Contains a compound M1 represented by the following formula (2):
The food or drink composition for resistance inhibition, wherein the antibacterial agent is at least one of a β-lactam antibacterial agent and an aminoglycoside antibacterial agent.
A method for inhibiting resistance of an antibacterial-resistant bacterium to an antibacterial agent, comprising:
The method includes ingesting the resistance inhibitor according to claim 1, the resistance inhibitor according to claim 2, the food or drink composition for resistance inhibition according to claim 6, or the food or drink composition for resistance inhibition according to claim 7,
A method for inhibiting resistance (excluding methods for treating humans) in which the antibacterial agent is at least one of a β-lactam antibacterial agent and an aminoglycoside antibacterial agent.