JP2014148495A - Antibacterial agent, and antibacterial activity enhancing agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an antibacterial agent that contains, as an active ingredient, a novel antibacterial agent, particularly an antibacterial agent effective for drug resistant bacteria.SOLUTION: An antibacterial agent contains dimeric sesquiterpene thioalkaloid.

Description

  The present invention relates to an antibacterial agent effective against drug-resistant bacteria and a pharmaceutical composition containing the antibacterial agent. Further, the present invention relates to an antibacterial activity enhancer for existing antibiotics.

  For bacterial infections, a treatment method is usually employed in which causative bacteria (S. aureus, enterococci, Pseudomonas aeruginosa, pathogenic Escherichia coli, etc.) are bacteriostatic or sterilized by the action of antibacterial agents. However, by using an antibacterial agent, drug-resistant bacteria against the antibacterial agent appear. For example, methicillin-resistant Staphylococcus aureus (MRSA) appears in hospitals where antibacterial drugs are frequently used and is known to be a causative bacterium of mass infection.

  In order to suppress the appearance of drug-resistant bacteria, a treatment method using a combination of a plurality of antibacterial drugs is effective. However, by continuing to employ the same combination of antibacterial agents, the emergence of resistant bacteria against the combination of antibacterial agents is expected. For this reason, it is extremely important to expand the antibacterial drug candidates to be used in combination. In addition, vancomycin is known as a first-line drug for MRSA, but the emergence of vancomycin-resistant bacteria (vancomycin moderately resistant Staphylococcus aureus (VISA)) has also been reported. Therefore, there is a demand for the development of new antibacterial agents, particularly effective against drug-resistant bacteria such as VISA.

  On the other hand, the sesquiterpene dimer thioalkaloid contained in the water lily family plant is a hair growth action (Patent Document 1), a cancer cell invasion inhibitory action (Patent Document 2), a malaria parasite growth inhibitory action (Patent Document 3), etc. However, it has not yet been known that it has antibacterial activity, particularly that it has antibacterial activity against drug-resistant bacteria.

JP 2002-47146 A JP2003-252779A JP 2007-204450 JP

  An object of the present invention is to provide a novel antibacterial agent, particularly an antibacterial agent containing an antibacterial agent effective against drug-resistant bacteria as an active ingredient.

  As a result of diligent research, the present inventors have found that sesquiterpene dimer thioalkaloids contained in the genus Lilyaceae exhibit excellent antibacterial activity against MRSA and VISA, which are drug-resistant bacteria. . In addition, it has surprisingly been found that sesquiterpene dimer thioalkaloids enhance the antimicrobial activity of other existing antimicrobial agents. As a result of further research based on these findings, the present invention was completed.

  That is, the present invention includes the following aspects.

Item 1.
General formula (1):

[Wherein R ¹ represents a hydroxyl group, and R ² represents hydrogen or a hydroxyl group]
A sesquiterpene dimer thioalkaloid represented by the general formula (2):

[Wherein, R ³ and R ⁴ independently represent hydrogen or a hydroxyl group. However, R ³ and R ⁴ do not represent hydrogen]
The antibacterial agent containing the sesquiterpene dimer thioalkaloid represented by these.

Item 2.
The antibacterial agent of Claim 1 which contains the sesquiterpene dimer thioalkaloid represented by General formula (1), and said R2 is a hydroxyl group.

Item 3.
A sesquiterpene dimer thioalkaloid represented by the general formula (1) and / or a sesquiterpene dimer thioalkaloid represented by the general formula (2). Item 3. The antibacterial agent according to item 1 or 2.

Item 4.
The antibacterial agent according to any one of claims 1 to 3, which is for staphylococcus and / or enterococci.

Item 5.
The antibacterial agent according to any one of claims 1 to 4, which is for drug-resistant bacteria.

Item 6.
The antibacterial agent according to claim 5, wherein the drug-resistant bacteria are methicillin-resistant bacteria and / or vancomycin-resistant bacteria.

Item 7.
The antibacterial agent according to any one of claims 1 to 6, further comprising a β-lactam antibacterial agent and / or an aminoglycoside antibacterial agent.

Item 8.
General formula (1):

[Wherein R ¹ represents a hydroxyl group, and R ² represents hydrogen or a hydroxyl group]
A sesquiterpene dimer thioalkaloid represented by the general formula (2):

[Wherein, R ³ and R ⁴ independently represent hydrogen or a hydroxyl group. However, R ³ and R ⁴ do not represent hydrogen]
An antibacterial activity enhancer of β-lactam antibacterial and / or aminoglycoside antibacterial containing a sesquiterpene dimer thioalkaloid represented by the formula:

  ADVANTAGE OF THE INVENTION According to this invention, the antibacterial agent which uses the sesquiterpene dimer thioalkaloid which is a novel antibacterial agent as an active ingredient can be provided. Since the sesquiterpene dimer thioalkaloid has an inhibitory activity against DNA topoisomerase IV universally possessed by bacteria, the antibacterial agent of the present invention can exert antibacterial action against various bacteria.

  In addition, the sesquiterpene dimer thioalkaloid exhibits the same antibacterial activity against MRSA as vancomycin, which is the current first-line drug for MRSA. Furthermore, it exhibits high antibacterial activity against resistant bacteria against vancomycin. Thus, the antibacterial agent of the present invention can exhibit excellent antibacterial activity against drug-resistant bacteria.

  In addition, sesquiterpene dimer thioalkaloids can enhance the antibacterial activity of β-lactam antibacterials and aminoglycoside antibacterials. Therefore, according to the present invention, it is possible to provide an antibacterial agent with enhanced antibacterial activity, which contains these antibacterial agents in combination. Moreover, the appearance of new drug-resistant bacteria can also be suppressed by using antibacterial drugs in combination.

  Furthermore, according to the present invention, an antibacterial activity enhancer for β-lactam antibacterial agents and aminoglycoside antibacterial agents can also be provided. This enhancer can exert antibacterial activity even when these antibacterial drugs are used at a normal concentration or lower (for example, MIC or lower). Arbekacin, which is a kind of aminoglycoside antibacterial agent, causes serious side effects when used at high concentrations. According to the enhancer of the present invention, the use concentration can be lowered while maintaining the antibacterial activity of the antibacterial agent, so that an antibacterial agent such as arbekacin can be used more safely.

A flow chart for extracting a sesquiterpene dimer thioalkaloid from Nuphar japonicum is shown. 1 shows the antibacterial (bacteriostatic) activity of a sesquiterpene dimer thioalkaloid. Fig. 6 shows Topo IV inhibitory activity of sesquiterpene dimer thioalkaloids.

1. Antibacterial Agent The antibacterial agent of the present invention has the general formula (1):

[Wherein R ¹ represents a hydroxyl group, and R ² represents hydrogen or a hydroxyl group]
A sesquiterpene dimer thioalkaloid represented by the general formula (2):

[Wherein R ³ and R ⁴ independently represent hydrogen or a hydroxyl group, but do not represent hydrogen together]
It is an antibacterial agent containing the sesquiterpene dimer thioalkaloid represented by these.

In general formula (1), R ¹ represents a hydroxyl group.

In general formula (1), R ² represents hydrogen or a hydroxyl group. R ² is preferably, for example, a hydroxyl group.

In general formula (2), R ³ and R ⁴ independently represent hydrogen or a hydroxyl group. However, R ³ and R ⁴ do not represent hydrogen.

  As a sesquiterpene dimer thioalkaloid represented by the general formula (1) or (2) contained in the antibacterial agent of the present invention (hereinafter sometimes simply referred to as “sesquiterpene dimer thioalkaloid”), A sesquiterpene dimer thioalkaloid represented by the general formula (1) is preferable.

  Although the sesquiterpene dimer thioalkaloid is a known substance and its production method is also known (Patent Documents 1 to 3), its antibacterial activity is a property newly found by the present inventors. The sesquiterpene dimer thioalkaloid can be extracted from, for example, a plant belonging to the genus Sofione according to a known technique. Therefore, the antibacterial agent of the present invention also includes an antibacterial agent containing a genus plant of the genus Camphorus containing the sesquiterpene dimer thioalkaloid extracted as described above. Examples of the plant belonging to the genus Kouhone include Nuphar japonicum DC. And Nuphar pumilum (TIMM.) DC. In addition to Nuphar luteum, N. japonicum DC. From a fubrotinctum (CASP.) Kitam, N. subintegerrimum Makino), N. pumilum DC. Var. Ozeense (MIKI) Hara, and N. oguraense Miki.

  As long as the antibacterial agent of the present invention is a bacterium having DNA topoisomerase IV, it can be widely used for gram-positive bacteria or gram-negative bacteria regardless of the type of bacteria to be applied. Examples of Gram-positive bacteria to be applied include Staphylococcus (for example, Staphylococcus aureus and Staphylococcus epidermidis), enterococci (for example, Enterococcus), and streptococci (for example, diuretic, quadruplicate, and eight streptococci). Etc., Streptococcus pneumoniae, Haemolytic streptococci), Bacillus spp. (Eg Bacillus anthracis, Bacillus subtilis), Clostridium spp. (Eg tetanus, Clostridium botulinum), Corynebacterium spp. (Eg Diphtheria), Listeria spp., Lactobacillus Examples include genus, Bifidobacterium, Propionibacterium (for example, acne causing acne), and actinomycetes. Examples of Gram-negative bacteria to be applied include Escherichia coli, Salmonella spp., Pseudomonas spp. Among these, the antibacterial agent of the present invention can be preferably used for staphylococci and / or enterococci, and more preferably for staphylococci.

  Since the antibacterial agent of the present invention exhibits excellent antibacterial activity against drug-resistant bacteria, it can be used for drug-resistant bacteria. The drug resistant bacteria to be applied are not particularly limited, and examples include penicillin resistant bacteria, methicillin resistant bacteria, vancomycin resistant bacteria, and the like. Among these, Preferably a methicillin resistant bacterium or a vancomycin resistant bacterium is mentioned. More specifically, methicillin-resistant staphylococci (such as methicillin-resistant Staphylococcus aureus (MRSA)), vancomycin-resistant staphylococci (such as vancomycin medium-resistant Staphylococcus aureus (VISA)), or vancomycin-resistant enterococci ( VRE).

  The antibacterial agent of the present invention may contain only a sesquiterpene dimer thioalkaloid as an active ingredient, or the antibacterial agent of the present invention does not impair the antibacterial activity of the sesquiterpene dimer thioalkaloid, Furthermore, you may contain antibacterial agents other than a sesquiterpene dimer thioalkaloid (it may only be called "other antibacterial agents" in this specification). Examples of other antibacterial agents include β-lactam antibacterial agents (oxacillin, penicillin, ampicillin, methicillin, cefazolin, imipenem silastatin, aztreonam, faropenem, etc.), aminoglycoside antibacterial agents (arbekacin, kanamycin, streptomycin, neomycin, gentamicin ), Tetracycline antibiotics (tetracycline, doxycycline, etc.), lincomycin antibiotics (lincomycin, clindamycin, etc.), chloramphenicol antibiotics (chloramphenicol, etc.), macrolide antibiotics ( Erythromycin, azithromycin, josamycin, etc.), ketolide antibacterials (such as telithromycin), polypeptide antibacterials (such as colistin and bacitracin), glycopeptide antibacterials (such as ba Komycin, teicoplanin), quinolone antibacterials (nalidixic acid, pyromido acid, etc.), new quinolone antibacterials (norfloxacin, lomefloxacin, sparfloxacin, sitafloxacin, etc.), oxazolidinone antibacterials (linezolid, etc.), or sulfa drugs Medicine (ST mixture etc.) etc. are mentioned. Among these, from the viewpoint that the antibacterial activity is enhanced by the sesquiterpene dimer thioalkaloid, preferably β-lactam antibacterial or aminoglycoside antibacterial, more preferably oxalicin, cloxacillin, ampicillin, amoxicillin, sulbenicillin, piperacillin Arbekacin, streptomycin, kanamycin, tobramycin, dibekacin, bekanamycin, amikacin, isepamicin, gentamicin, fradiomycin, ribostamycin, more preferably oxacillin or arbekacin, more preferably arbekacin. Other antibacterial agents may be used alone or in combination of two or more.

  The usage and mode of use of the antibacterial agent of the present invention are not particularly limited as long as it aims at antibacterial against bacteria. For example, it may be used as an antibacterial agent as it is, or various compositions (pharmaceutical composition, cosmetic composition, food composition, medical cleaning composition, dish disinfecting cleaning composition, oral composition, surface You may mix | blend and use for an antibacterial composition etc.).

  The antibacterial agent of the present invention contains an effective amount of the sesquiterpene dimer thioalkaloid and other antibacterial agents desired, or in an amount that can be used in an embodiment in which the concentration is MIC or higher. However, when other antibacterial agents contain β-lactam antibacterials and / or aminoglycoside antibacterial agents, the antibacterial activity of β-lactam antibacterial agents and / or aminoglycoside antibacterial agents depends on sesquiterpene dimer thioalkaloids. As potentiated, these antibacterials exert their antibacterial activity even when used at concentrations below the minimum growth inhibitory concentration (MIC).

  Depending on the use and aspect as described above, the antimicrobial agent of the present invention can be a composition containing an additive. Examples of the additive include a base, a carrier, a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a thickener, a moisturizer, a colorant, a fragrance, Chelating agents, rust inhibitors, metal anticorrosive agents, antifoaming agents, rustproofing agents, extreme pressure additives, metal anticorrosive agents, antifoaming agents, dyes and the like can be mentioned. It is preferable to select and use a pharmaceutically acceptable ingredient and a cosmetically acceptable ingredient among these additives according to the purpose of use. The antibacterial agent of the present invention is converted into an appropriate form (tablet, pill, powder, solution, injection, suspension, emulsion, powder, granule, capsule, etc.) according to the purpose of use. It can be prepared and used.

  The content of the sesquiterpene dimer thioalkaloid in the antibacterial agent of the present invention is not particularly limited as long as the antibacterial action is exhibited. For example, it can be 0.0001% by weight or more, preferably 0.0005 to 1% by weight, more preferably 0.001 to 0.1% by weight.

  The content of other antibacterial agents in the antibacterial agent of the present invention is not particularly limited. For example, 0.0001% by weight or more, preferably 0.0005 to 1% by weight, more preferably 0.001 to 0.1% by weight.

  When the antibacterial agent of the present invention is used for pharmaceutical purposes, or when used in a pharmaceutical composition, the administration method, dosage, etc. are the content of the sesquiterpene dimer thioalkaloid contained, other antibacterial agents Content, dosage form, age / weight of administration subject, and the like.

2. Antibacterial activity enhancer The antibacterial activity enhancer of the present invention has the general formula (1):

[Wherein R ¹ represents a hydroxyl group, and R ² represents hydrogen or a hydroxyl group]
A sesquiterpene dimer thioalkaloid represented by the general formula (2):

[Wherein, R ³ and R ⁴ independently represent hydrogen or a hydroxyl group. However, R ³ and R ⁴ do not represent hydrogen]
A β-lactam antibacterial agent and / or an aminoglycoside antibacterial agent containing an sesquiterpene dimer thioalkaloid represented by the formula:

  A preferred embodiment of the sesquiterpene dimer thioalkaloid is the same as described above.

  The target antibacterial agent that enhances the antibacterial activity is preferably an aminoglycoside antibacterial agent.

  The β-lactam antibacterial agent is not particularly limited. For example, oxacillin, cloxacillin, ampicillin, amoxicillin, sulbenicillin, piperacillin, penicillin, methicillin, cefazolin, imipenem silastatin, aztreonam, faropenem, etc., preferably oxacillin, cloxacillin, ampicillin, amoxicillin, sulpecillin, preferably .

  The aminoglycoside antibacterial agent is not particularly limited. For example, arbekacin, streptomycin, kanamycin, tobramycin, dibekacin, bekanamycin, amikacin, isepamicin, gentamicin, fradiomycin, ribostamycin, neomycin and the like, preferably arbekacin, streptomycin, kanamycin, tobramycin, dibekacin, bekanamycin, amikacin, isepamicin, Examples include gentamicin, fradiomycin, ribostamycin, and more preferably arbekacin.

  The content of the sesquiterpene dimer thioalkaloid in the antibacterial activity enhancer of the present invention is not particularly limited as long as the antibacterial activity of the β-lactam antibacterial agent and / or aminoglycoside antibacterial agent can be enhanced. For example, 0.0001% by weight or more, preferably 0.0005 to 1% by weight, more preferably 0.001 to 0.1% by weight. Further, the antibacterial activity enhancing action is such that the concentration of the sesquiterpene dimer thioalkaloid is MIC or less, for example, 0.00001 to 0.005% by weight, preferably 0.0001 to 0.002% by weight, more preferably 0.8. Even if it is 0002-0.001 weight%, it can exhibit.

  Moreover, the weight part of the sesquiterpene dimer thioalkaloid in the antibacterial activity enhancer of the present invention with respect to 1 part by weight of the β-lactam antibacterial agent present in the affected area to which the antibacterial activity enhancer of the present invention is applied is, for example, 0. 0002-5 parts by weight, preferably 0.001-4 parts by weight, more preferably 0.002-3 parts by weight. The weight part of the sesquiterpene dimer thioalkaloid in the antibacterial activity enhancer of the present invention relative to 1 part by weight of the aminoglycoside antibacterial agent present in the affected area to which the antibacterial activity enhancer of the present invention is applied is, for example, 0.1-20 Part by weight, preferably 0.5 to 15 parts by weight, more preferably 0.8 to 10 parts by weight.

  The antibacterial activity enhancer of the present invention may contain other additives as necessary in the same manner as the above-mentioned “1. Antibacterial agent” to form an appropriate form (tablet, pill, powder, liquid, injection, suspension). Suspending agents, emulsions, powders, granules, capsules, etc.) can be used.

  The usage mode of the antibacterial activity enhancer of the present invention is not particularly limited as long as the antibacterial activity enhancer of the present invention and the β-lactam antibacterial agent and / or aminoglycoside antibacterial agent are coexisting. For example, after applying (for example, applying) the antibacterial activity enhancer of the present invention to an affected part (for example, skin), the antibacterial agent may be applied, or the antibacterial activity enhancer of the present invention and the antibacterial agent may be mixed. It may be applied to the affected area.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1 Measurement of Antibacterial Activity of Kouhone Extract and Identification of Active Ingredient Senkoutsu (Takasago Pharmaceutical Co., Ltd.), a Herbal Medicine that Dried Rhizome Nuphar japonicum The extract was obtained from and fractionated. Furthermore, the antibacterial activity of each fraction was measured. Details of each step (A, B, C) in the flowchart of FIG. 1 are as follows.

Example 1-1: Step A (acquisition of alkaloid fraction)
Centrifugal 4.0 kg was pulverized with a mixer, and about 20 L of 100% methanol per kg was added to the pulverized product, and the mixture was allowed to stand at room temperature for 2 hours for extraction. The obtained extract was filtered under reduced pressure, and the solvent was distilled off from the filtrate to obtain 323 g of a dried product (methanol extract dried product). After 102 g of 100% methanol extract was pulverized with a mixer, the pulverized product was suspended in about 1 L of 1 M hydrochloric acid, and the suspension was transferred to a separatory funnel. About 1 L of chloroform was further added to the separatory funnel, and the mixture was stirred and allowed to stand, and then the chloroform layer was removed (this operation was performed twice using the color of the chloroform layer as a guide). The aqueous layer remaining after the chloroform layer was removed was transferred to a beaker, the pH was adjusted to about 10 by adding an appropriate amount of aqueous ammonia, and then transferred to a separatory funnel. Add approximately the same amount of ethyl acetate as the aqueous layer to the separatory funnel, stir and let stand, and then remove the ethyl acetate layer. Times). Insoluble matter that did not migrate to either the aqueous layer or the ethyl acetate layer was separately collected. The various organic solvent layers and aqueous layers and insolubles thus obtained were each evaporated by an evaporator and dried. The insoluble material was dried using a desiccator. As a result, 10.1 g of dried product (chloroform fraction dried product) from the chloroform layer, 9.0 g of dried product (ethyl acetate fraction dried product) from the ethyl acetate layer, and 113 g of dried product (water fraction dried product) And 64.3 g of dried product (dried product of insoluble fraction) was obtained from the insoluble product.

The minimum inhibitory concentration (MIC) of each dried product against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) was measured by a micro liquid dilution method. Specifically, it was performed as follows. Diluted series in which each dried product was diluted 2-fold with a medium (Mueller Hinton (MH) medium (Difco): meat extract 2.0 g / L, caseinate digestion 17.5 g / L, soluble starch 1.5 g / L) The test bacteria were inoculated into 100 μL of each medium so as to be about 10 ⁴ CFU / well. After inoculation, the mixture was allowed to stand at 37 ° C. for 24 hours, and then the presence or absence of suspension of the medium was evaluated. The minimum concentration with no medium suspension (no growth of bacteria) was defined as the minimum growth inhibitory concentration (MIC). The results are shown in Table 1.

  From Table 1, antibacterial activity was recognized in the ethyl acetate fraction dried product. The ethyl acetate fractions extracted from Senkotsu are sesquiterpene dimer thioalkaloids (6-hydroxythiobinufaridine, 6,6'-dihydroxythiobinufaridine, 6- It is known to contain a large amount of hydroxythionuflutin B and 6′-hydroxythionuflutin B). This suggested that these sesquiterpene dimer thioalkaloids have antibacterial activity.

Example 1-2: Step B (Purification of alkaloid fraction)
The dried ethyl acetate fraction was purified by silica gel column chromatography. Specifically, it was performed as follows. About 400 ml of silica gel swollen with chloroform (70-230 mesh, 60 cm, SIGMA-ALDRICH Co.) was packed in an open column with an inner diameter of 40 mm. 8.17 g of dried ethyl acetate fraction was dissolved in a small amount of mixed solvent 1 (chloroform: ethyl acetate: diethylamine = 20: 1: 1 (v / v)), and the solution was passed through a column packed with silica gel. As a result, the components of the ethyl acetate fraction dried product were retained on the silica gel column. Thereafter, 1200 ml of mixed solvent 1 was passed as an eluent, and then 1200 ml of mixed solvent 2 (methanol: diethylamine = 10: 1 (v / v)) was passed. The liquid eluted from the column was collected in three 800 ml portions, and designated as fr.1, fr.2, and fr.3 in the order of elution. From each fraction, the solvent was distilled off under reduced pressure. As a result, fr. 1 to 5.56 g dry matter (fr. 1 dry matter), fr. 2 to 1.27 g dry matter (fr. 2 dry matter), fr. 3 to 1.30 g dry matter (fr. 3 Dried product) was obtained.

  The minimum inhibitory concentration (MIC) of each dried product against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) was measured in the same manner as in Example 1-1. The results are shown in Table 2. From Table 2, the strongest antibacterial activity was observed in the fr. 1 dried product.

Example 1-3: Step C (Purification of Alkaloid Fraction)
The dried product fr. 1 was further purified by silica gel column chromatography. Specifically, it was performed as follows. About 350 mL of silica gel (70-230 mesh, 60 mm, SIGMA-ALDRICH Co.) swollen with mixed solvent 3 (normal hexane: ethyl acetate: aqueous ammonia = 75: 25: 1 (v / v)) An open column with an inner diameter of 44 mm was packed. By dissolving 0.92 g of fr. 1 dried product in about 2 mL of mixed solvent 3 and passing the solution through a column filled with silica gel, the components of fr. 1 dried product were retained on the silica gel column. . Thereafter, 1000 ml of mixed solvent 3 was passed as an eluent, and then 700 ml of mixed solvent 4 (methanol: aqueous ammonia = 100: 1 (v / v)) was passed. The liquid eluted from the column was sequentially developed by normal phase thin layer chromatography and divided into 10 fractions according to the spot pattern (fr. 1-I to fr. 1-X). From each fraction, the solvent was distilled off under reduced pressure. As a result, fr. 1-I to 34.9 mg dry matter (fr. 1-I dry matter), fr. 1-II to 51.9 mg dry matter (fr. 1-II dry matter), fr. 1-III 91.8 mg dry matter (fr. 1-III dry matter), fr. 1-IV to 36.4 mg dry matter (fr. 1-IV dry matter), fr. 1-V to 50.6 mg dry matter (fr 1-V dry matter), fr. 1-VI to 73.7 mg dry matter (fr. 1-VI dry matter), fr. 1-VII to 14.5 mg dry matter (fr. 1-VII dry matter), fr. 1-VIII to 159.9 mg dry matter (fr. 1-VIII dry matter), fr. 1-IX to 167.5 mg dry matter (fr. 1-IX dry matter), fr. 1-X to 1276.3 mg Of dried product (fr. 1-X dried product) was obtained.

  The minimum growth inhibitory concentration (MIC) of each dried product against methicillin-resistant Staphylococcus aureus (MRSA) was measured in the same manner as in Example 1-1. The results are shown in Table 3. From Table 3, the strongest antibacterial activity was observed in the fr. 1-VIII dried product.

Example 1-4: Identification of active ingredient From the results of thin layer chromatography, fr.1-VIII was considered to be a single compound, and thus its structure was determined by ¹ H-NMR analysis. As a result, chemical shift values shown in Table 4 were obtained. This value almost coincided with that of 6,6′-dihydroxythiobinupharidine, a kind of sesquiterpene dimer thioalkaloid. The specific rotation was also consistent. In Table 4, the chemical shift value of 6,6'-dihydroxythiobinupharidine was extracted from Yoshikawa M. et al., HETEROCYCLES, 1997, Vol. 45, No. 9, pp1815-1824. In the following experiments, fr. 1-VIII dried product was used as 6,6′-dihydroxythiobinufaridine.

  Strong antibacterial activity was observed in fractions containing a large amount of sesquiterpene dimer thioalkaloids that were very similar in structure to each other (Example 1-1), and 6,6 ′, which is actually one of the alkaloids The antibacterial activity was found in -dihydroxythiobinufaridine (Examples 1-3 and 1-4), suggesting strongly that the sesquiterpene dimer thioalkaloids contained in mulberry have antibacterial activity It was done.

Example 2: Analysis of antibacterial activity of sesquiterpene dimer thioalkaloids Various strains of sesquiterpene dimer thioalkaloids (methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and vancomycin moderate Antibacterial activity against resistant Staphylococcus aureus (VISA) was measured. Specifically, the minimum growth inhibitory concentration (MIC) of sesquiterpene dimer thioalkaloid 6,6'-dihydroxythiobinupharidine, and β-lactam antibacterial oxacillin and glycopeptides as comparative controls The MIC of vancomycin, an antibacterial drug, was measured in the same manner as in Example 1-1. The results are shown in Table 5.

  From Table 5, it was shown that 6,6′-dihydroxythiobinufaridine exhibits the same level of antibacterial activity against MRSA, which is a drug-resistant bacterium, as vancomycin, which is a first-line drug for MRSA. In addition, vancomycin showed low antibacterial activity against the resistant bacterium VISA, whereas 6,6'-dihydroxythiobinufaridine also showed strong antibacterial activity against VISA. .

Example 3: Analysis of antibacterial activity enhancing action by sesquiterpene dimer thioalkaloid The antibacterial activity enhancing action of existing antibiotics by sesquiterpene dimer thioalkaloid was examined. Specifically, the minimum growth inhibitory concentration (MIC) of oxacillin, a β-lactam antibacterial agent, vancomycin, a glycopeptide antibacterial agent, and arbekacin, an aminoglycoside antibacterial agent, is a sesquiterpene dimer thioalkaloid. Example 6 shows the case where 6,6′-dihydroxythiobinufaridine alone is added at a concentration (0.5 μg / mL) that does not exhibit antibacterial activity (+) and not added at all (−). Measurement was performed in the same manner as for -1. As a result of this measurement, the MIC of the existing antibiotic is more in the case of 6,6'-dihydroxythiovinufaridine (+) than in the case of 6,6'-dihydroxythiobinufaridine (-). A low value indicates that 6,6′-dihydroxythiobinufaridine enhanced the antibacterial activity of the antibiotic. The results are shown in Table 6.

  From Table 6, 6,6'-dihydroxythiobinufaridine does not exert antibacterial activity enhancing action against vancomycin, a glycopeptide antibacterial agent, but oxacillin, which is a β-lactam antibacterial agent, and aminoglycoside It has been shown that arbekacin, an antibacterial agent, exerts a strong antibacterial activity enhancing effect. In particular, arbekacin exerted an extremely strong antibacterial activity-enhancing effect that lowered the MIC for almost all strains to about 1/4 to 1/8.

Example 4: Analysis of the mechanism of action of a sesquiterpene dimer thioalkaloid 1
In order to clarify whether the antibacterial action of the sesquiterpene dimer thioalkaloid is due to bactericidal action or bacteriostatic action, the survival rate in the presence of the sesquiterpene dimer thioalkaloid was evaluated. Specifically, it was performed as follows. In a medium (dried broth “Nissui” medium: meat extract 5.0 g / L, peptone 15.0 g / L, sodium chloride 5.0 g / L, potassium hydrogen phosphate 5.0 g / L) with sesquiterpene dimer thioalkaloid A certain 6,6′-dihydroxythiobinufaridine was added to various concentrations, and MRSA N315 strain was inoculated there to about 10 ⁹ CFU / mL. After shaking culture at 37 ° C., the number of viable bacteria was measured after 3, 6, 12, and 24 hours. For the number of viable bacteria, after diluting the bacterial solution at each time point with sterile physiological saline at an appropriate magnification, seed it on a dry broth “Nissui” medium, culture at 37 ° C. overnight, and count the number of colonies that appeared. And measured as CFU. The result is shown in figure 2. In FIG. 2, the unit of the vertical axis is log ₁₀ (CFU / mL).

  Fig. 2 shows that even when 6,6'-dihydroxythiovinufaridine was added so that the concentration of MIC (2 μg / ml) was 4 times that of N315 strain (♦ in Fig. 2), the number of viable bacteria was Almost no decrease. This suggests that the antibacterial action of 6,6′-dihydroxythiobinufaridine is due to bacteriostatic action.

Example 5: Analysis of mechanism of action of sesquiterpene dimer thioalkaloid 2
Investigating the effect of sesquiterpene dimer thioalkaloids on the ability of DNA topoisomerase to decatenate (the activity of cleaving and recombining kinetoplast DNA: kDNA) to form circular DNA (minicircular DNA: mcDNA) It was. Specifically, using S. aureus Topoisomerase IV decatenation kit (Inspiralis), it was performed as follows according to the protocol attached to the kit. In the assay solution (50 mM Tris-HCl pH 7.5, 5 mM MgCl ₂ , 5 mM Dithiothreitol, 1.5 mM ATP, 350 mM potassium glutamate, 0.05 mg / ml albumin) included in the kit, 200 ng of kDNA, and Add 1 U S. aureus DNA topoisomerase IV, and adjust the concentration in the solution to various concentrations (0.1 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 10 μM, 15 μM, 20 μM, or 30 μM) '-Dihydroxythiobinufaridine was added to prepare a reaction solution. As a comparative control, a reaction solution containing ciprofloxacin, which is known to inhibit Staphylococcus aureus topoisomerase IV, was also prepared instead of 6,6′-dihydroxythiobinufaridine. These reaction solutions were warmed at 37 ° C. for 30 minutes. The sample after the reaction was electrophoresed on a 1% agarose gel. This agarose gel was stained with 1 μg / ml ethidium bromide solution, and DNA was detected by UV irradiation. Detection was performed with Image Quant LAS4000, and the intensity of the band migrated in the gel was quantified by Image Quant TL. kDNA has a large apparent molecular weight and is hardly electrophoresed. However, when the linked state is eliminated by the action of DNA topoisomerase IV, it becomes mcDNA (minicircle DNA) with a reduced apparent molecular weight and becomes electrophoresed. Therefore, DNA topoisomerase inhibitory activity can be measured by measuring the intensity of the band migrated in the gel. The results are shown in Figure 3.

  From FIG. 3, 6,6′-dihydroxythiobinufaridine inhibited DNA topoisomerase IV in a concentration-dependent manner. Moreover, it was found from the darkness of the band in the gel that 6,6′-dihydroxythiobinufaridine has almost the same inhibitory activity as ciprofloxacin. From the above, it was suggested that the antibacterial activity of the sesquiterpene dimer thioalkaloid is due to the DNA topoisomerase IV inhibitory activity. Since DNA topoisomerase IV is an enzyme that exists universally in bacteria, it is suggested that sesquiterpene dimer thioalkaloids having inhibitory activity of this enzyme exert antibacterial activity against any bacteria. It was. In addition, because of the broad antibacterial spectrum of fluoroquinolone antibacterial agents that have DNA topoisomerase IV inhibitory activity, sesquiterpene dimer thioalkaloids that inhibit this enzyme exert antibacterial activity against any bacteria. It has been suggested.

Claims (8)

General formula (1):

[Wherein R ¹ represents a hydroxyl group, and R ² represents hydrogen or a hydroxyl group]
A sesquiterpene dimer thioalkaloid represented by the general formula (2):

[Wherein, R ³ and R ⁴ independently represent hydrogen or a hydroxyl group. However, R ³ and R ⁴ do not represent hydrogen]
The antibacterial agent containing the sesquiterpene dimer thioalkaloid represented by these. The antibacterial agent of Claim 1 which contains the sesquiterpene dimer thioalkaloid represented by General formula (1), and said R2 is a hydroxyl group. A sesquiterpene dimer thioalkaloid represented by the general formula (1) and / or a sesquiterpene dimer thioalkaloid represented by the general formula (2). Item 3. The antibacterial agent according to item 1 or 2. The antibacterial agent according to any one of claims 1 to 3, which is for staphylococcus and / or enterococci. The antibacterial agent according to any one of claims 1 to 4, which is for drug-resistant bacteria. The antibacterial agent according to claim 5, wherein the drug-resistant bacteria are methicillin-resistant bacteria and / or vancomycin-resistant bacteria. The antibacterial agent according to any one of claims 1 to 6, further comprising a β-lactam antibacterial agent and / or an aminoglycoside antibacterial agent. General formula (1):

[Wherein R ¹ represents a hydroxyl group, and R ² represents hydrogen or a hydroxyl group]
A sesquiterpene dimer thioalkaloid represented by the general formula (2):

[Wherein, R ³ and R ⁴ independently represent hydrogen or a hydroxyl group. However, R ³ and R ⁴ do not represent hydrogen]
An antibacterial activity enhancer of β-lactam antibacterial and / or aminoglycoside antibacterial containing a sesquiterpene dimer thioalkaloid represented by the formula:

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