WO2016098880A1 - Antibacterial activity potentiator - Google Patents

Antibacterial activity potentiator Download PDF

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Publication number
WO2016098880A1
WO2016098880A1 PCT/JP2015/085487 JP2015085487W WO2016098880A1 WO 2016098880 A1 WO2016098880 A1 WO 2016098880A1 JP 2015085487 W JP2015085487 W JP 2015085487W WO 2016098880 A1 WO2016098880 A1 WO 2016098880A1
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WIPO (PCT)
Prior art keywords
antibacterial
glycopeptide
thioalkaloid
aminoglycoside
antibacterial agent
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PCT/JP2015/085487
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French (fr)
Japanese (ja)
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照夫 黒田
力 波多野
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国立大学法人岡山大学
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Publication of WO2016098880A1 publication Critical patent/WO2016098880A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/7036—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin having at least one amino group directly attached to the carbocyclic ring, e.g. streptomycin, gentamycin, amikacin, validamycin, fortimicins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/62—Nymphaeaceae (Water-lily family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an antibacterial activity enhancer.
  • a treatment method for bacterial infections, a treatment method is usually employed in which causative bacteria (S. aureus, enterococci, Pseudomonas aeruginosa, pathogenic E. coli, etc.) are bacteriostatic or sterilized by the action of antibacterial agents.
  • causative bacteria S. aureus, enterococci, Pseudomonas aeruginosa, pathogenic E. coli, etc.
  • drug-resistant bacteria against the antibacterial agent appear.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Vancomycin is known as a first-line drug for MRSA. Recently, the emergence of vancomycin-resistant bacteria such as vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA) has been reported. Therefore, for example, if the antibacterial activity of vancomycin against vancomycin-resistant bacteria can be enhanced, it will be an effective means to combat this resistant bacteria.
  • VRE vancomycin-resistant enterococci
  • VRSA vancomycin-resistant Staphylococcus aureus
  • the sesquiterpene dimer thioalkaloids contained in the water lily family plant are the hair growth action (Patent Document 1), the cancer cell invasion inhibitory action (Patent Document 2), and the malaria parasite growth inhibitory action (Patent Document 3). It has been reported that it has an action such as the above, and further has an antibacterial action (Patent Document 4). However, it is not yet known that the antibacterial activity of vancomycin against vancomycin-resistant bacteria can be enhanced.
  • JP 2002-47146 A Japanese Patent Laid-Open No. 2003-252779 Japanese Unexamined Patent Publication No. 2007-204450 Japanese Unexamined Patent Publication No. 2014-148495
  • An object of the present invention is to provide an antibacterial activity enhancer of a glycopeptide antibacterial agent against a glycopeptide antibacterial agent (vancomycin or the like) resistant bacterium.
  • the present inventors have found that a sesquiterpene dimer thioalkaloid contained in a water lily family, Sucrose, can enhance the antibacterial activity of glycopeptide antibacterials against glycopeptide antibacterial resistant bacteria. . Furthermore, the present inventors have found that this alkaloid can remarkably enhance the antibacterial activity of aminoglycoside antibacterial agents against the resistant bacteria. As a result of further research based on these findings, the present invention was completed.
  • the present invention includes the following aspects.
  • R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen
  • Item 2. The enhancer according to item 1, comprising a sesquiterpene dimer thioalkaloid represented by the general formula (1) and wherein R 2 is a hydroxyl group.
  • Item 3 A term containing a sesquiterpene dimer thioalkaloid represented by the general formula (1) and / or a sucrose genus plant extract containing the sesquiterpene dimer thioalkaloid represented by the general formula (2) The enhancer according to 1 or 2.
  • Item 4. The enhancer according to any one of Items 1 to 3, wherein the target glycopeptide antibiotic-resistant bacterium is a bacterium having a minimum growth inhibitory concentration of the glycopeptide antibiotic of 16 ⁇ g / mL or more.
  • Item 5. The enhancer according to any one of Items 1 to 4, which is an antibacterial activity enhancer of a glycopeptide antibacterial agent.
  • Item 6. The enhancer according to any one of Items 1 to 5, wherein the target glycopeptide antibacterial-resistant bacterium is a bacterium that is also resistant to an aminoglycoside antibacterial agent.
  • Item 7. The enhancer according to Item 6, wherein the target glycopeptide antibacterial resistant bacterium is a bacterium having an aminoglycoside antibacterial minimum growth inhibitory concentration of 4 ⁇ g / mL or more.
  • Item 8 An antibacterial agent comprising the enhancer according to any one of Items 1 to 7, and a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent.
  • R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen.
  • R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen
  • Item 11 A method for enhancing antibacterial activity of an antibacterial agent against a resistance against a glycopeptide antibacterial agent, comprising using the enhancer according to any one of items 1 to 7 and a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent. .
  • the antibacterial activity of glycopeptide antibacterial agents and / or aminoglycoside antibacterial agents against glycopeptide antibacterial resistant bacteria can be remarkably enhanced.
  • treatment of bacterial infection requires that the minimum growth inhibitory concentration of the antibacterial agent is not more than a certain level. If the enhancer of the present invention is used, the glycopeptide antibacterial agent against the resistant bacteria and / or Alternatively, the minimum inhibitory concentration of aminoglycoside antibacterial agents can be lowered to a concentration effective for treating bacterial infections.
  • sesquiterpene dimer thioalkaloid is known to have antibacterial activity alone (Patent Document 4), it is generally preferred to use at a lower concentration from the viewpoint of further reducing the risk of side effects. Use at lower concentrations is desirable based on historical findings.
  • the antibacterial activity enhancer of the present invention can exert its antibacterial activity enhancing action even if the concentration of the sesquiterpene dimer thioalkaloid is a concentration that cannot exhibit antibacterial activity by itself.
  • the antibacterial activity enhancer of the present invention in combination with a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent, an antibacterial action against target bacteria while using a sesquiterpene dimer thioalkaloid at a lower concentration can be demonstrated.
  • a flow chart for extracting a sesquiterpene dimer thioalkaloid from Nuphar japonicum is shown.
  • Antibacterial activity enhancer The antibacterial activity enhancer of the present invention has the general formula (1):
  • R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen
  • R 1 represents a hydroxyl group.
  • R 2 represents hydrogen or a hydroxyl group.
  • R 2 is preferably, for example, a hydroxyl group.
  • R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen.
  • the sesquiterpene dimer thioalkaloid contained in the antibacterial activity enhancer of the present invention is preferably a sesquiterpene dimer thioalkaloid represented by the general formula (1).
  • the sesquiterpene dimer thioalkaloid is a known substance, and its production method is also known (Patent Documents 1 to 3), but it has a specific antibacterial activity enhancing action (that is, against glycopeptide antibacterial drug-resistant bacteria, The antibacterial activity enhancing action of the glycopeptide antibacterial and / or aminoglycoside antibacterial 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.
  • the antibacterial activity enhancer of the present invention also includes an antibacterial activity enhancer containing an extract of the plant of the genus Camphorus containing the sesquiterpene dimer thioalkaloid extracted as described above.
  • Examples of the plant belonging to the genus Kohone include Nuphar japonicum DC., Nuphar pumilum (TIMM.) DC., Nuphar luteum, Benikohone (N. japonicum DC. Froma fubrotinctum (CASP.) N. subintegerrimum Makino), Okozekone (N. pumilum DC. Var. Ozeense (MIKI) Hara), N. ⁇ ⁇ oguraense Miki and the like.
  • the target antibacterial agent that enhances antibacterial activity is a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent.
  • glycopeptide antibacterial agents are preferable.
  • glycopeptide antibacterial agent examples include vancomycin and teicoplanin, and preferably vancomycin.
  • aminoglycoside antibacterial agent examples include arbekacin, amikacin, streptomycin, kanamycin, tobramycin, dibekacin, bekanamycin, isepamicin, gentamicin, fradiomycin, ribostamycin, neomycin and the like, preferably arbekacin, Amikacin and the like can be mentioned.
  • the target antibacterial drug may be one type or a combination of two or more types.
  • target bacteria of the above target antibacterial drugs are glycopeptide antibacterial drug resistant bacteria. Whether or not a bacterium is resistant to a glycopeptide antibacterial agent can be classified based on an official classification standard, for example, based on a classification standard established by Clinical Laboratory Standards Institute (CLSI) in the United States.
  • CLSI Clinical Laboratory Standards Institute
  • the minimum inhibitory concentration of the glycopeptide antibacterial agent is, for example, 16 ⁇ g / mL or more, preferably 64 ⁇ g / mL or more, more preferably 128 ⁇ g / mL or more, and further Preferably, it can be 256 ⁇ g / mL or more.
  • the antibacterial activity enhancer of the present invention can exert the antibacterial activity enhancing effect more remarkably when the resistance to the glycopeptide antibacterial agent is high as described above.
  • glycopeptide antibacterial agent to which the target bacterium is resistant examples include vancomycin and teicoplanin, and preferably vancomycin.
  • the target bacterium may have resistance to only one of these, or may have resistance to two or more.
  • the target bacterium of the antibacterial activity enhancer of the present invention preferably has resistance to an aminoglycoside antibacterial agent from the viewpoint that the antibacterial activity enhancing action can be exhibited more remarkably. Whether or not it is resistant to an aminoglycoside antibacterial agent can be classified based on a public classification standard, for example, based on a classification standard established by Clinical & Standards Institute (CLSI) in the United States.
  • CLSI Clinical & Standards Institute
  • the minimum growth inhibitory concentration of the aminoglycoside antibacterial agent is, for example, 32 ⁇ g / mL or more, preferably 64 ⁇ g / mL or more, more preferably 128 ⁇ g / mL or more, and still more preferably. It can be 256 ⁇ g / mL or more.
  • the antibacterial activity enhancer of the present invention can exert the antibacterial activity enhancing effect more remarkably against bacteria having high resistance to aminoglycoside antibacterial agents as described above.
  • aminoglycoside antibacterial agent to which the target bacterium is resistant examples include arbekacin, amikacin, streptomycin, kanamycin, tobramycin, dibekacin, bekanamycin, isepamicin, gentamicin, fradiomycin, ribostamycin, neomycin, and preferably arbekacin. Amikacin and the like.
  • the target bacterium may have resistance to only one of these, or may have resistance to two or more.
  • the species of the target bacterium of the antibacterial activity enhancer of the present invention is not particularly limited as long as the wild strain is a species that can be subjected to the antibacterial action of a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent. Bacteria, gram-negative bacteria, etc. can be widely adopted.
  • Gram-positive bacteria include, for example, staphylococci (eg, Staphylococcus aureus, Staphylococcus epidermidis), enterococci (eg, Enterococcus), streptococci (eg, diuretic, quadruple, octococcus, etc.), pneumonia Cocci, hemolytic streptococci), Bacillus (e.g., anthrax), Clostridium (e.g., tetanus, Clostridium botulinum), Corynebacterium (e.g., Diphtheria), Listeria, Lactobacillus, Bifidobacterium Examples include genus Baum, genus Propionibacterium (for example, Acne bacterium causing acne), actinomycetes, and the like.
  • staphylococci eg, Staphylococcus aureus, Staphylococcus epidermidis
  • enterococci eg, Enterococcus
  • Gram-negative bacteria examples include Escherichia coli, Salmonella, Pseudomonas (for example, Pseudomonas aeruginosa), Helicobacter, Influenza, and Neisseria (for example, Neisseria gonorrhoeae and Neisseria meningitidis).
  • Gram-positive bacteria more preferably enterococci and / or staphylococci, and still more preferably enterococci.
  • 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 glycopeptide antibacterial agent and / or aminoglycoside antibacterial agent can be enhanced.
  • it can be 0.000001 to 100% by weight, preferably 0.00001 to 80% by weight.
  • the antibacterial activity enhancer of the present invention is used at a concentration lower than the concentration at which the sesquiterpene dimer thioalkaloid alone can exert the antibacterial action against the target bacteria, for example, the sesquiterpene in the region where the target bacteria exist Even if it is used so that the concentration of the dimeric thioalkaloid is not more than the MIC for the target bacterium, its antibacterial activity enhancing effect can be exhibited.
  • the field of use of the antibacterial activity enhancer of the present invention is not particularly limited as long as it is intended for antibacterial activity against bacteria.
  • it can be used in fields such as the medical field, cosmetic field, food field, cleaning field, oral cavity field, and surface antibacterial field.
  • the use mode of the antibacterial activity enhancer of the present invention is particularly limited as long as the antibacterial activity enhancer of the present invention and the glycopeptide antibacterial agent and / or aminoglycoside antibacterial agent coexist in the region where the target bacteria are present.
  • the antibacterial activity enhancer of the present invention may be applied (for example, applied) to a region (for example, skin) where the target bacterium is present, and then the antibacterial agent may be applied to the region. After applying to the existing region, the antibacterial activity enhancer of the present invention may be applied to the region, or the antibacterial activity enhancer of the present invention and the antibacterial agent may be mixed and then applied to the region. .
  • the antibacterial activity enhancer of the present invention can be a composition containing an additive depending on the intended use and embodiment.
  • 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.
  • the antibacterial activity enhancer of the present invention is prepared in an appropriate dosage form (tablets, pills, powders, solutions, injections, suspensions, emulsions, powders, granules, capsules) according to the purpose of use. Etc.) and can be used.
  • the administration method, dosage, etc. are the content of sesquiterpene dimer thioalkaloid, dosage form, administration It is appropriately selected depending on the age and weight of the subject.
  • the present invention also includes an antibacterial activity enhancer of the present invention and an antibacterial agent containing a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent (hereinafter sometimes referred to as “antibacterial agent of the present invention”). Related.
  • the target bacteria species of the target bacteria, glycopeptide antibacterial drugs and / or aminoglycoside antibacterial drugs, sesquiterpene dimer thioalkaloids and their content, additives, dosage forms, administration methods, dosages, etc. .. Antibacterial activity enhancer ".
  • the content of the glycopeptide antibacterial agent and / or aminoglycoside antibacterial agent in the antibacterial agent of the present invention is not particularly limited. For example, it can be 0.000001 to 70% by weight, preferably 0.00001 to 50% by weight. Even if the antibacterial agent of the present invention is used at a concentration lower than the concentration at which the glycopeptide antibacterial agent and / or the aminoglycoside antibacterial agent alone can exert the antibacterial action against the target bacteria, for example, the region where the target bacteria exist Even if it uses so that the density
  • Example 1 Measurement of antibacterial activity of an extract of kohone and identification of active ingredient Senkotsu (Takasago Pharmaceutical Co., Ltd.), a crude drug obtained by drying the rhizomes of Nuphar japonicum (Japanese name: kohone) according to the flowchart of FIG.
  • the extract was obtained from Lot No. 061207), and the extract was 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.
  • 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.
  • 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)
  • 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 ⁇ L 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.
  • Example 1-2 Step B (Purification of alkaloid fraction)
  • chloroform: ethyl acetate: diethylamine 20: 1: 1 (v / v)
  • 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 fr.fr1 dried product.
  • Example 1-3 Step C (Purification of Alkaloid Fraction)
  • 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 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 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.
  • 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.
  • Example 2 Analysis of antibacterial activity enhancing action by sesquiterpene dimer thioalkaloid The antibacterial activity enhancing action of various antibiotics by sesquiterpene dimer thioalkaloid was examined. Specifically, vancomycin (glycopeptide antibacterial), amikacin (aminoglycoside antibacterial), and arbekacin (aminoglycoside antibacterial) minimal growth against vancomycin sensitive enterococci (VSE) and vancomycin resistant enterococci (VRE) When the inhibitory concentration (MIC) was added to the sesquiterpene dimer thioalkaloid 6,6'-dihydroxythiobinufaridine (DTBN) at a concentration that does not exhibit antibacterial activity alone (1 ⁇ g / mL) ( The measurement was carried out in the same manner as in Example 1-1 for each of (+) and no addition (-).
  • VSE vancomycin sensitive enterococci
  • VRE vancomycin resistant enterococci
  • the FIC index was calculated based on the following formula.
  • a lower FIC index means stronger enhancement activity of the sesquiterpene dimer thioalkaloid. If the FIC index is 0.5 or less, it can be considered that a synergistic effect is exhibited.
  • Table 5 shows the results when Enterococcus faecium was used as the enterococci
  • Table 6 shows the results when Enterococcus faecalis was used as the enterococci.
  • strains with names beginning with ATCC and NCTC are those purchased from ATCC, NCTC, or RIKEN BioResource Center, and other strains are those that have been clinically isolated. is there.

Abstract

Provided is an antibacterial activity potentiator for glycopeptide antibacterial drugs, which is used against glycopeptide antibacterial drug-resistant bacteria such as vancomycin-resistant bacteria. The antibacterial activity potentiator for glycopeptide antibacterial drugs and/or aminoglycoside antibacterial drugs, which is used against glycopeptide antibacterial drug-resistant bacteria, contains a dimeric sesquiterpene thioalkaloid.

Description

抗菌活性増強剤Antibacterial activity enhancer
　本発明は、抗菌活性増強剤に関する。 The present invention relates to an antibacterial activity enhancer.
　細菌感染症に対しては、通常、抗菌薬の作用により原因菌（黄色ブドウ球菌、腸球菌、緑膿菌、病原性大腸菌等）を静菌又は殺菌するという治療方法が採られる。ところが、抗菌薬を用いることによって、その抗菌薬に対する薬剤耐性菌が出現することとなる。例えば、メチシリン耐性黄色ブドウ球菌（MRSA）は、抗菌薬が頻用される病院において出現し、集団感染の起因菌となることが知られている。 For bacterial infections, a treatment method is usually employed in which causative bacteria (S. aureus, enterococci, Pseudomonas aeruginosa, pathogenic E. 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.
　MRSAに対する第1選択薬としてはバンコマイシンが知られているが、近年、バンコマイシン耐性腸球菌（VRE）、バンコマイシン耐性黄色ブドウ球菌（VRSA）等のバンコマイシン耐性菌の出現が報告されている。そこで、例えばバンコマイシン耐性菌に対するバンコマイシンの抗菌活性を増強することができれば、この耐性菌に対抗する有効な手段になると考えられる。 Vancomycin is known as a first-line drug for MRSA. Recently, the emergence of vancomycin-resistant bacteria such as vancomycin-resistant enterococci (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA) has been reported. Therefore, for example, if the antibacterial activity of vancomycin against vancomycin-resistant bacteria can be enhanced, it will be an effective means to combat this resistant bacteria.
　一方、スイレン科コウホネ属植物に含まれるセスキテルペン二量体チオアルカロイドは、発毛作用（特許文献1）、がん細胞浸潤抑制作用（特許文献2）、マラリア原虫増殖抑制作用（特許文献3）等の作用を有すること、さらには抗菌作用（特許文献4）を有することが報告されている。しかしながら、バンコマイシン耐性菌に対するバンコマイシンの抗菌活性を増強できることについては未だ知られていない。 On the other hand, the sesquiterpene dimer thioalkaloids contained in the water lily family plant are the hair growth action (Patent Document 1), the cancer cell invasion inhibitory action (Patent Document 2), and the malaria parasite growth inhibitory action (Patent Document 3). It has been reported that it has an action such as the above, and further has an antibacterial action (Patent Document 4). However, it is not yet known that the antibacterial activity of vancomycin against vancomycin-resistant bacteria can be enhanced.
特開2002－47146号公報JP 2002-47146 A 特開2003－252779号公報Japanese Patent Laid-Open No. 2003-252779 特開2007－204450号公報Japanese Unexamined Patent Publication No. 2007-204450 特開2014－148495号公報Japanese Unexamined Patent Publication No. 2014-148495
　本発明は、グリコペプチド系抗菌薬（バンコマイシン等）耐性菌に対するグリコペプチド系抗菌薬の抗菌活性増強剤を提供することを目的とする。 An object of the present invention is to provide an antibacterial activity enhancer of a glycopeptide antibacterial agent against a glycopeptide antibacterial agent (vancomycin or the like) resistant bacterium.
　本発明者等は鋭意研究した結果、スイレン科コウホネ属植物に含まれるセスキテルペンン二量体チオアルカロイドが、グリコペプチド系抗菌薬耐性菌に対するグリコペプチド系抗菌薬の抗菌活性を増強できることを見出した。さらに、このアルカロイドが当該耐性菌に対するアミノグリコシド系抗菌薬の抗菌活性をも、顕著に増強できることを見出した。これらの知見に基づいてさらに研究を進めた結果、本発明が完成した。 As a result of intensive studies, the present inventors have found that a sesquiterpene dimer thioalkaloid contained in a water lily family, Sucrose, can enhance the antibacterial activity of glycopeptide antibacterials against glycopeptide antibacterial resistant bacteria. . Furthermore, the present inventors have found that this alkaloid can remarkably enhance the antibacterial activity of aminoglycoside antibacterial agents against the resistant bacteria. As a result of further research based on these findings, the present invention was completed.
　即ち、本発明は、下記の態様を包含する。 That is, the present invention includes the following aspects.
　項１．一般式（１）： Item 1. General formula (1):
Figure JPOXMLDOC01-appb-C000007
 ［式中、Ｒ１は水酸基を示し、Ｒ２は水素又は水酸基を示す］
で表されるセスキテルペン二量体チオアルカロイド、及び／又は一般式（２）：
Figure JPOXMLDOC01-appb-C000007
 [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):
Figure JPOXMLDOC01-appb-C000008
 ［式中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない］
で表されるセスキテルペン二量体チオアルカロイドを含有する、
グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性の増強剤。
Figure JPOXMLDOC01-appb-C000008
 [Wherein, R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen]
Containing a sesquiterpene dimer thioalkaloid represented by:
Antibacterial activity enhancer of glycopeptide antibacterial and / or aminoglycoside antibacterial against glycopeptide antibacterial resistant bacteria.
　項２．一般式（１）で表されるセスキテルペン二量体チオアルカロイドを含有し、且つ前記Ｒ２が水酸基である、項１に記載の増強剤。 Item 2. Item 2. The enhancer according to item 1, comprising a sesquiterpene dimer thioalkaloid represented by the general formula (1) and wherein R 2 is a hydroxyl group.
　項３．前記一般式（１）で表されるセスキテルペン二量体チオアルカロイド及び／又は前記一般式（２）で表されるセスキテルペン二量体チオアルカロイドを含有するコウホネ属植物抽出物を含有する、項１又は２に記載の増強剤。 Item 3. A term containing a sesquiterpene dimer thioalkaloid represented by the general formula (1) and / or a sucrose genus plant extract containing the sesquiterpene dimer thioalkaloid represented by the general formula (2) The enhancer according to 1 or 2.
　項４．対象となる前記グリコペプチド系抗菌薬耐性菌が、グリコペプチド系抗菌薬の最小生育阻止濃度が１６μｇ／ｍＬ以上の菌である、項１～３のいずれかに記載の増強剤。 Item 4. Item 4. The enhancer according to any one of Items 1 to 3, wherein the target glycopeptide antibiotic-resistant bacterium is a bacterium having a minimum growth inhibitory concentration of the glycopeptide antibiotic of 16 μg / mL or more.
　項５．グリコペプチド系抗菌薬の抗菌活性増強剤である、項１～４のいずれかに記載の増強剤。 Item 5. Item 5. The enhancer according to any one of Items 1 to 4, which is an antibacterial activity enhancer of a glycopeptide antibacterial agent.
　項６．対象となる前記グリコペプチド系抗菌薬耐性菌が、アミノグリコシド系抗菌薬に対しても耐性を有する菌である、項１～５のいずれかに記載の増強剤。 Item 6. Item 6. The enhancer according to any one of Items 1 to 5, wherein the target glycopeptide antibacterial-resistant bacterium is a bacterium that is also resistant to an aminoglycoside antibacterial agent.
　項７．対象となる前記グリコペプチド系抗菌薬耐性菌が、アミノグリコシド系抗菌薬の最小生育阻止濃度が４μｇ／ｍＬ以上の菌である、項６に記載の増強剤。 Item 7. Item 7. The enhancer according to Item 6, wherein the target glycopeptide antibacterial resistant bacterium is a bacterium having an aminoglycoside antibacterial minimum growth inhibitory concentration of 4 μg / mL or more.
　項８．項１～７のいずれかに記載の増強剤、並びにグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬を含有する抗菌剤。 Item 8. Item 8. An antibacterial agent comprising the enhancer according to any one of Items 1 to 7, and a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent.
　項９．
グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性の増強剤の製造のための、一般式（１）： Item 9.
Formula (1) for the production of an antibacterial activity enhancer of a glycopeptide antibacterial and / or aminoglycoside antibacterial against a glycopeptide antibacterial resistant bacterium:
Figure JPOXMLDOC01-appb-C000009
 ［式中、Ｒ１は水酸基を示し、Ｒ２は水素又は水酸基を示す］
で表されるセスキテルペン二量体チオアルカロイド、及び／又は一般式（２）：
Figure JPOXMLDOC01-appb-C000009
 [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):
Figure JPOXMLDOC01-appb-C000010
 ［式中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない］
で表されるセスキテルペン二量体チオアルカロイドの使用。
Figure JPOXMLDOC01-appb-C000010
 [Wherein, R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen]
Use of a sesquiterpene dimer thioalkaloid represented by:
　項１０．
グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性の増強剤としての使用のための、一般式（１）： Item 10.
General formula (1) for use as a potentiator of antibacterial activity of glycopeptide antibacterials and / or aminoglycoside antibacterials against glycopeptide antibacterial resistant bacteria:
Figure JPOXMLDOC01-appb-C000011
 ［式中、Ｒ１は水酸基を示し、Ｒ２は水素又は水酸基を示す］
で表されるセスキテルペン二量体チオアルカロイド、及び／又は一般式（２）：
Figure JPOXMLDOC01-appb-C000011
 [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):
Figure JPOXMLDOC01-appb-C000012
 ［式中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない］
で表されるセスキテルペン二量体チオアルカロイド。
Figure JPOXMLDOC01-appb-C000012
 [Wherein, R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen]
A sesquiterpene dimer thioalkaloid represented by:
　項１１．
項１～７のいずれかに記載の増強剤、並びにグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬を併用することを含む、該抗菌薬のグリコペプチド系抗菌薬耐性菌に対する抗菌活性の増強方法。 Item 11.
Item 8. A method for enhancing antibacterial activity of an antibacterial agent against a resistance against a glycopeptide antibacterial agent, comprising using the enhancer according to any one of items 1 to 7 and a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent. .
　項１２．
項１～７のいずれかに記載の増強剤、並びにグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬を併用することを含む、グリコペプチド系抗菌薬耐性菌の抗菌方法。 Item 12.
Item 8. An antibacterial method for a bacterium resistant to glycopeptide antibacterials, which comprises using a potentiator according to any one of Items 1 to 7 and a glycopeptide antibacterial and / or aminoglycoside antibacterial.
　本発明の抗菌活性増強剤によれば、グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性を顕著に増強することができる。一般的に、細菌感染症の治療には、抗菌薬の最小生育阻止濃度が一定以下であることが求められるところ、本発明の増強剤を用いれば、上記耐性菌に対するグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の最小生育阻止濃度を、細菌感染症の治療が有効な濃度にまで下げることも可能となる。 According to the antibacterial activity enhancer of the present invention, the antibacterial activity of glycopeptide antibacterial agents and / or aminoglycoside antibacterial agents against glycopeptide antibacterial resistant bacteria can be remarkably enhanced. In general, treatment of bacterial infection requires that the minimum growth inhibitory concentration of the antibacterial agent is not more than a certain level. If the enhancer of the present invention is used, the glycopeptide antibacterial agent against the resistant bacteria and / or Alternatively, the minimum inhibitory concentration of aminoglycoside antibacterial agents can be lowered to a concentration effective for treating bacterial infections.
　また、セスキテルペン二量体チオアルカロイドは単独で抗菌活性を有することが知られているものの（特許文献4）、副作用の危険性をより低減する観点からはより低濃度での使用が好ましいという一般的知見に基づけば、より低濃度での使用が望ましい。本発明の抗菌活性増強剤は、セスキテルペン二量体チオアルカロイドの濃度が単独では抗菌活性を発揮できない濃度であっても、その抗菌活性増強作用を発揮できる。したがって、本発明の抗菌活性増強剤をグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬と併用することによって、セスキテルペン二量体チオアルカロイドをより低濃度で使用しつつも、対象菌に対する抗菌作用を発揮することができる。 In addition, although sesquiterpene dimer thioalkaloid is known to have antibacterial activity alone (Patent Document 4), it is generally preferred to use at a lower concentration from the viewpoint of further reducing the risk of side effects. Use at lower concentrations is desirable based on historical findings. The antibacterial activity enhancer of the present invention can exert its antibacterial activity enhancing action even if the concentration of the sesquiterpene dimer thioalkaloid is a concentration that cannot exhibit antibacterial activity by itself. Therefore, by using the antibacterial activity enhancer of the present invention in combination with a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent, an antibacterial action against target bacteria while using a sesquiterpene dimer thioalkaloid at a lower concentration Can be demonstrated.
Nuphar japonicumからセスキテルペン二量体チオアルカロイドを抽出するフローチャートを示す。A flow chart for extracting a sesquiterpene dimer thioalkaloid from Nuphar japonicum is shown.
　１．抗菌活性増強剤
　本発明の抗菌活性増強剤は、一般式（１）： 1. Antibacterial activity enhancer The antibacterial activity enhancer of the present invention has the general formula (1):
Figure JPOXMLDOC01-appb-C000013
 ［式中、Ｒ１は水酸基を示し、Ｒ２は水素又は水酸基を示す］
で表されるセスキテルペン二量体チオアルカロイド、及び／又は一般式（２）：
Figure JPOXMLDOC01-appb-C000013
 [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):
Figure JPOXMLDOC01-appb-C000014
 ［式中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない］
で表されるセスキテルペン二量体チオアルカロイドを含有する、
グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性増強剤である。
Figure JPOXMLDOC01-appb-C000014
 [Wherein, R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen]
Containing a sesquiterpene dimer thioalkaloid represented by:
It is an antibacterial activity enhancer of glycopeptide antibacterials and / or aminoglycosides antibacterials against glycopeptide antibacterial resistant bacteria.
　一般式（１）中、Ｒ１は水酸基を示す。 In general formula (1), R 1 represents a hydroxyl group.
　一般式（１）中、Ｒ２は水素又は水酸基を示す。Ｒ２は好ましくは例えば水酸基である。 In general formula (1), R 2 represents hydrogen or a hydroxyl group. R 2 is preferably, for example, a hydroxyl group.
　一般式（２）中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない。 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.
　本発明の抗菌活性増強剤に含有されるセスキテルペン二量体チオアルカロイドとして、好ましくは一般式（１）で表されるセスキテルペン二量体チオアルカロイドが挙げられる。 The sesquiterpene dimer thioalkaloid contained in the antibacterial activity enhancer of the present invention is preferably a sesquiterpene dimer thioalkaloid represented by the general formula (1).
　セスキテルペン二量体チオアルカロイドは、公知の物質であり、その製造方法も知られているが（特許文献1～3）、特定の抗菌活性増強作用（すなわち、グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性増強作用）は、本発明者が新たに見出した性質である。セスキテルペン二量体チオアルカロイドは、例えば、コウホネ属植物から公知の手法に従って抽出することができる。したがって、本発明の抗菌活性増強剤には、このようにして抽出された、セスキテルペン二量体チオアルカロイドを含有するコウホネ属植物抽出物を含有する抗菌活性増強剤も包含される。コウホネ属植物としては、例えばコウホネ（Nuphar japonicum DC.）、ネムロコウホネ（Nuphar pumilum (TIMM.) DC.）のほか、Nuphar luteum、ベニコウホネ（N. japonicum DC. froma fubrotinctum (CASP.) Kitam）、ヒメコウホネ（N. subintegerrimum Makino）、オコゼコウホネ（N. pumilum DC. var. ozeense (MIKI) Hara）、オグラコウホネ（N. oguraense Miki）等が挙げられる。 The sesquiterpene dimer thioalkaloid is a known substance, and its production method is also known (Patent Documents 1 to 3), but it has a specific antibacterial activity enhancing action (that is, against glycopeptide antibacterial drug-resistant bacteria, The antibacterial activity enhancing action of the glycopeptide antibacterial and / or aminoglycoside antibacterial 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 activity enhancer of the present invention also includes an antibacterial activity enhancer containing an extract of the plant of the genus Camphorus containing the sesquiterpene dimer thioalkaloid extracted as described above. Examples of the plant belonging to the genus Kohone include Nuphar japonicum DC., Nuphar pumilum (TIMM.) DC., Nuphar luteum, Benikohone (N. japonicum DC. Froma fubrotinctum (CASP.) N. subintegerrimum Makino), Okozekone (N. pumilum DC. Var. Ozeense (MIKI) Hara), N. グ ラ oguraense Miki and the like.
　抗菌活性を増強させる対象抗菌薬は、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬である。これらの中でも、好ましくはグリコペプチド系抗菌薬が挙げられる。 The target antibacterial agent that enhances antibacterial activity is a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent. Among these, glycopeptide antibacterial agents are preferable.
　対象抗菌薬であるグリコペプチド系抗菌薬としては、例えばバンコマイシン、テイコプラニン等が挙げられ、好ましくはバンコマイシンが挙げられる。 Examples of the glycopeptide antibacterial agent that is the target antibacterial agent include vancomycin and teicoplanin, and preferably vancomycin.
　対象抗菌薬であるアミノグリコシド系抗菌薬としては、例えば、アルベカシン、アミカシン、ストレプトマイシン、カナマイシン、トブラマイシン、ジベカシン、ベカナマイシン、イセパマイシン、ゲンタマイシン、フラジオマイシン、リボスタマイシン、ネオマイシン等が挙げられ、好ましくはアルベカシン、アミカシン等が挙げられる。 Examples of the aminoglycoside antibacterial agent that is the target antibacterial agent include arbekacin, amikacin, streptomycin, kanamycin, tobramycin, dibekacin, bekanamycin, isepamicin, gentamicin, fradiomycin, ribostamycin, neomycin and the like, preferably arbekacin, Amikacin and the like can be mentioned.
　対象抗菌薬は、１種でもよいし、２種以上の組み合わせであってもよい。 The target antibacterial drug may be one type or a combination of two or more types.
　上記対象抗菌薬の対象菌（以下、単に「対象菌」と示すこともある）は、グリコペプチド系抗菌薬耐性菌である。グリコペプチド系抗菌薬に対する耐性菌であるか否かは、公的な分類基準に基づいて、例えば米国のClinical and Laboratory Standards Institute（CLSI）によって制定された分類基準に基づいて分類することができる。 The target bacteria of the above target antibacterial drugs (hereinafter sometimes simply referred to as “target bacteria”) are glycopeptide antibacterial drug resistant bacteria. Whether or not a bacterium is resistant to a glycopeptide antibacterial agent can be classified based on an official classification standard, for example, based on a classification standard established by Clinical Laboratory Standards Institute (CLSI) in the United States.
　対象菌のグリコペプチド系抗菌薬に対する耐性の程度としては、グリコペプチド系抗菌薬の最小生育阻止濃度が、例えば１６μｇ／ｍＬ以上、好ましくは６４μｇ／ｍＬ以上、より好ましくは１２８μｇ／ｍＬ以上、よりさらに好ましくは２５６μｇ／ｍＬ以上であることができる。本発明の抗菌活性増強剤は、グリコペプチド系抗菌薬に対する耐性が上記のように高い場合に、抗菌活性増強作用をより顕著に発揮することができる。 As the degree of resistance of the target bacterium to the glycopeptide antibacterial agent, the minimum inhibitory concentration of the glycopeptide antibacterial agent is, for example, 16 μg / mL or more, preferably 64 μg / mL or more, more preferably 128 μg / mL or more, and further Preferably, it can be 256 μg / mL or more. The antibacterial activity enhancer of the present invention can exert the antibacterial activity enhancing effect more remarkably when the resistance to the glycopeptide antibacterial agent is high as described above.
　対象菌が耐性を有するグリコペプチド系抗菌薬としては、例えばバンコマイシン、テイコプラニン等が挙げられ、好ましくはバンコマイシンが挙げられる。対象菌は、これらの１種のみに対して耐性を有していてもよいし、２種以上に対して耐性を有していてもよい。 Examples of the glycopeptide antibacterial agent to which the target bacterium is resistant include vancomycin and teicoplanin, and preferably vancomycin. The target bacterium may have resistance to only one of these, or may have resistance to two or more.
　本発明の抗菌活性増強剤の対象菌は、抗菌活性増強作用をより顕著に発揮することができるという観点からは、アミノグリコシド系抗菌薬に対しても耐性を有することが好ましい。アミノグリコシド系抗菌薬に対する耐性菌であるか否かは、公的な分類基準に基づいて、例えば米国のClinical and Laboratory Standards Institute（CLSI）によって制定された分類基準に基づいて分類することができる。 The target bacterium of the antibacterial activity enhancer of the present invention preferably has resistance to an aminoglycoside antibacterial agent from the viewpoint that the antibacterial activity enhancing action can be exhibited more remarkably. Whether or not it is resistant to an aminoglycoside antibacterial agent can be classified based on a public classification standard, for example, based on a classification standard established by Clinical & Standards Institute (CLSI) in the United States.
　対象菌のアミノグリコシド系抗菌薬に対する耐性の程度としては、アミノグリコシド系抗菌薬の最小生育阻止濃度が、例えば３２μｇ／ｍＬ以上、好ましくは６４μｇ／ｍＬ以上、より好ましくは１２８μｇ／ｍＬ以上、よりさらに好ましくは２５６μｇ／ｍＬ以上であることができる。本発明の抗菌活性増強剤は、アミノグリコシド系抗菌薬に対する耐性が上記のように高い菌に対して、抗菌活性増強作用をより顕著に発揮することができる。 As the degree of resistance of the target bacterium to the aminoglycoside antibacterial agent, the minimum growth inhibitory concentration of the aminoglycoside antibacterial agent is, for example, 32 μg / mL or more, preferably 64 μg / mL or more, more preferably 128 μg / mL or more, and still more preferably. It can be 256 μg / mL or more. The antibacterial activity enhancer of the present invention can exert the antibacterial activity enhancing effect more remarkably against bacteria having high resistance to aminoglycoside antibacterial agents as described above.
　対象菌が耐性を有するアミノグリコシド系抗菌薬としては、例えば、アルベカシン、アミカシン、ストレプトマイシン、カナマイシン、トブラマイシン、ジベカシン、ベカナマイシン、イセパマイシン、ゲンタマイシン、フラジオマイシン、リボスタマイシン、ネオマイシン等が挙げられ、好ましくはアルベカシン、アミカシン等が挙げられる。対象菌は、これらの１種のみに対して耐性を有していてもよいし、２種以上に対して耐性を有していてもよい。 Examples of the aminoglycoside antibacterial agent to which the target bacterium is resistant include arbekacin, amikacin, streptomycin, kanamycin, tobramycin, dibekacin, bekanamycin, isepamicin, gentamicin, fradiomycin, ribostamycin, neomycin, and preferably arbekacin. Amikacin and the like. The target bacterium may have resistance to only one of these, or may have resistance to two or more.
　本発明の抗菌活性増強剤の対象菌の種（species）としては、野生株がグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌作用を受け得る種である限り特に限定されず、グラム陽性菌、グラム陰性菌等を広く採用することができる。グラム陽性菌としては、例えば、ブドウ球菌属菌（例えば黄色ブドウ球菌、表皮ブドウ球菌）、腸球菌（例えばエンテロコッカス属菌）、レンサ球菌属菌（例えば双球菌、４連、８連球菌等、肺炎球菌、溶血連鎖球菌）、バシラス属菌（例えば炭疽菌）、クロストリジウム属菌（例えば破傷風菌、ボツリヌス菌）、コリネバクテリウム属菌（例えばジフテリア菌）、リステリア属菌、ラクトバシラス属菌、ビフィドバクテリウム属菌、プロピオニバクテリウム属菌（例えばニキビの原因となるアクネ菌）、放線菌等が挙げられる。グラム陰性菌としては、例えば、大腸菌、サルモネラ属菌、シュードモナス属菌（例えば緑膿菌）、ヘリコバクター属菌、インフルエンザ菌、ナイセリア属菌（例えば淋菌、髄膜炎菌）が挙げられる。これらの中でも、より確実に本発明の抗菌活性増強作用を発揮できるという観点から、好ましくはグラム陽性菌、より好ましくは腸球菌及び／又はブドウ球菌属菌、よりさらに好ましくは腸球菌が挙げられる。 The species of the target bacterium of the antibacterial activity enhancer of the present invention is not particularly limited as long as the wild strain is a species that can be subjected to the antibacterial action of a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent. Bacteria, gram-negative bacteria, etc. can be widely adopted. Gram-positive bacteria include, for example, staphylococci (eg, Staphylococcus aureus, Staphylococcus epidermidis), enterococci (eg, Enterococcus), streptococci (eg, diuretic, quadruple, octococcus, etc.), pneumonia Cocci, hemolytic streptococci), Bacillus (e.g., anthrax), Clostridium (e.g., tetanus, Clostridium botulinum), Corynebacterium (e.g., Diphtheria), Listeria, Lactobacillus, Bifidobacterium Examples include genus Baum, genus Propionibacterium (for example, Acne bacterium causing acne), actinomycetes, and the like. Examples of Gram-negative bacteria include Escherichia coli, Salmonella, Pseudomonas (for example, Pseudomonas aeruginosa), Helicobacter, Influenza, and Neisseria (for example, Neisseria gonorrhoeae and Neisseria meningitidis). Among these, from the viewpoint that the antibacterial activity enhancing action of the present invention can be exhibited more surely, Gram-positive bacteria, more preferably enterococci and / or staphylococci, and still more preferably enterococci.
　本発明の抗菌活性増強剤中の、セスキテルペン二量体チオアルカロイドの含有量は、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性を増強できる限り特に限定されない。例えば、０．０００００１～１００重量％、好ましくは０．００００１～８０重量％であることができる。本発明の抗菌活性増強剤は、セスキテルペン二量体チオアルカロイドが単独で対象菌に対して抗菌作用を発揮できる濃度よりも低い濃度で使用されても、例えば対象菌が存在する領域におけるセスキテルペン二量体チオアルカロイドの濃度が対象菌に対するＭＩＣ以下となるように使用されても、その抗菌活性増強作用を発揮することができる。 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 glycopeptide antibacterial agent and / or aminoglycoside antibacterial agent can be enhanced. For example, it can be 0.000001 to 100% by weight, preferably 0.00001 to 80% by weight. Even if the antibacterial activity enhancer of the present invention is used at a concentration lower than the concentration at which the sesquiterpene dimer thioalkaloid alone can exert the antibacterial action against the target bacteria, for example, the sesquiterpene in the region where the target bacteria exist Even if it is used so that the concentration of the dimeric thioalkaloid is not more than the MIC for the target bacterium, its antibacterial activity enhancing effect can be exhibited.
　本発明の抗菌活性増強剤の使用分野は、細菌に対する抗菌を目的とするものであれば特に限定されない。例えば、医療分野、化粧分野、食品分野、洗浄分野、口腔分野、表面抗菌分野等の分野において用いることができる。 The field of use of the antibacterial activity enhancer of the present invention is not particularly limited as long as it is intended for antibacterial activity against bacteria. For example, it can be used in fields such as the medical field, cosmetic field, food field, cleaning field, oral cavity field, and surface antibacterial field.
　本発明の抗菌活性増強剤の使用態様は、対象菌が存在する領域において、本発明の抗菌活性増強剤とグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬とが共存状態になる限り、特に限定されない。例えば、本発明の抗菌活性増強剤を対象菌が存在する領域（例えば皮膚）に適用（例えば塗布）した後、該領域に上記抗菌薬を適用してもよいし、上記抗菌薬を対象菌が存在する領域に適用した後、該領域に本発明の抗菌活性増強剤を適用してもよいし、本発明の抗菌活性増強剤と上記抗菌薬を混合してから上記領域に適用してもよい。 The use mode of the antibacterial activity enhancer of the present invention is particularly limited as long as the antibacterial activity enhancer of the present invention and the glycopeptide antibacterial agent and / or aminoglycoside antibacterial agent coexist in the region where the target bacteria are present. Not. For example, the antibacterial activity enhancer of the present invention may be applied (for example, applied) to a region (for example, skin) where the target bacterium is present, and then the antibacterial agent may be applied to the region. After applying to the existing region, the antibacterial activity enhancer of the present invention may be applied to the region, or the antibacterial activity enhancer of the present invention and the antibacterial agent may be mixed and then applied to the region. .
　本発明の抗菌活性増強剤は、使用用途及び態様に応じて、添加剤を含有する組成物であることができる。添加剤としては、例えば基剤、担体、溶剤、分散剤、乳化剤、緩衝剤、安定剤、賦形剤、結合剤、崩壊剤、滑沢剤、増粘剤、保湿剤、着色料、香料、キレート剤、防錆剤、金属防食剤、消泡剤、防錆剤、極圧添加剤、金属防食剤、消泡剤、染料等が挙げられる。使用目的に応じて、これらの添加剤のうち、薬学的に許容される成分、香粧品学的に許容される成分を選択して使用することが好ましい。本発明の抗菌活性増強剤は、使用目的に応じて、慣用の方法により適切な剤形（錠剤、丸剤、散剤、液剤、注射剤、懸濁剤、乳剤、粉末剤、顆粒剤、カプセル剤等）に調製して使用することができる。 The antibacterial activity enhancer of the present invention can be a composition containing an additive depending on the intended use and embodiment. 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 activity enhancer of the present invention is prepared in an appropriate dosage form (tablets, pills, powders, solutions, injections, suspensions, emulsions, powders, granules, capsules) according to the purpose of use. Etc.) and can be used.
　本発明の抗菌活性増強剤を医薬目的で使用する場合、又は医薬組成物に配合して使用する場合、投与方法や投与量等は、セスキテルペン二量体チオアルカロイドの含有割合、剤形、投与対象の年齢・体重などにより適宜選択される。 When the antibacterial activity enhancer of the present invention is used for pharmaceutical purposes or used in combination with a pharmaceutical composition, the administration method, dosage, etc. are the content of sesquiterpene dimer thioalkaloid, dosage form, administration It is appropriately selected depending on the age and weight of the subject.
　２．抗菌剤
　本発明は、本発明の抗菌活性増強剤、並びにグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬を含有する抗菌剤（以下、「本発明の抗菌剤」と示すこともある）にも関する。 2. Antibacterial Agent The present invention also includes an antibacterial activity enhancer of the present invention and an antibacterial agent containing a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent (hereinafter sometimes referred to as “antibacterial agent of the present invention”). Related.
　対象菌、対象菌の種、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬、セスキテルペン二量体チオアルカロイド及びその含有割合、添加剤、剤形、投与方法、投与量等は、上記「１．抗菌活性増強剤」と同様である。 The target bacteria, species of the target bacteria, glycopeptide antibacterial drugs and / or aminoglycoside antibacterial drugs, sesquiterpene dimer thioalkaloids and their content, additives, dosage forms, administration methods, dosages, etc. .. Antibacterial activity enhancer ".
　本発明の抗菌剤中の、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の含有量は、特に限定されない。例えば、０．０００００１～７０重量％、好ましくは０．００００１～５０重量％であることができる。本発明の抗菌剤は、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬が単独で対象菌に対して抗菌作用を発揮できる濃度よりも低い濃度で使用されても、例えば対象菌が存在する領域におけるグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の濃度が対象菌に対するＭＩＣ以下となるように使用されても、その抗菌作用を発揮することができる。 The content of the glycopeptide antibacterial agent and / or aminoglycoside antibacterial agent in the antibacterial agent of the present invention is not particularly limited. For example, it can be 0.000001 to 70% by weight, preferably 0.00001 to 50% by weight. Even if the antibacterial agent of the present invention is used at a concentration lower than the concentration at which the glycopeptide antibacterial agent and / or the aminoglycoside antibacterial agent alone can exert the antibacterial action against the target bacteria, for example, the region where the target bacteria exist Even if it uses so that the density | concentration of the glycopeptide type | system | group antibacterial agent and / or aminoglycoside type | system | group antibacterial agent may become below MIC with respect to an object microbe, the antibacterial effect can be exhibited.
　以下に、実施例に基づいて本発明を詳細に説明するが、本発明はこれらの実施例によって限定されるものではない。 Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
　実施例１：コウホネ抽出物の抗菌活性の測定、及び活性成分の同定
　図１のフローチャートに従って、Nuphar japonicum（和名：コウホネ）の根茎を乾燥させた生薬であるセンコツ（高砂薬業（株）、Lot番号:061207）から抽出液を得て、該抽出液を分画した。さらに各画分の抗菌活性を測定した。図１のフローチャートの各ステップ（Ａ、Ｂ、Ｃ）の詳細については下記のとおりである。 Example 1: Measurement of antibacterial activity of an extract of kohone and identification of active ingredient Senkotsu (Takasago Pharmaceutical Co., Ltd.), a crude drug obtained by drying the rhizomes of Nuphar japonicum (Japanese name: kohone) according to the flowchart of FIG. The extract was obtained from Lot No. 061207), and the extract was 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.
　実施例1－1：ステップＡ（アルカロイド画分の取得）
　センコツ4.0 kgをミキサーで粉砕し、該粉砕物に1 kgにつき約20 Lの100% メタノールを添加し、室温で２時間静置することにより抽出を行った。得られた抽出液を減圧ろ過し、ろ液から溶媒を留去し、323 gの乾燥物（メタノール抽出液乾燥物）を得た。100% メタノール抽出物102 gをミキサーにより粉砕後、該粉砕物を約1 Lの1 M 塩酸に懸濁し、該懸濁液を分液ロートに移した。分液ロートにさらに約1 Lのクロロホルムを加え、撹拌して静置した後、クロロホルム層を除去した（クロロホルム層の着色が薄くなることを目安として、この操作を2回行った）。クロロホルム層が除去された後に残った水層をビーカーに移し、ここにアンモニア水を適量加えることによりpHを約10に調整した後、分液ロートに移した。分液ロートにさらに水層とほぼ同量の酢酸エチルを加え、撹拌して静置した後、酢酸エチル層を除去した（酢酸エチル層の着色が薄くなることを目安として、この操作を計8回行った）。なお、水層と酢酸エチル層のどちらにも移行しない不溶物を別途回収した。得られた各種有機溶媒層及び水層、及び不溶物を、それぞれエバポレーターによって溶媒留去し、乾燥させた。不溶物はデシケーターを用いて乾燥させた。その結果、クロロホルム層から10.1 gの乾燥物（クロロホルム画分乾燥物）、酢酸エチル層から9.0 gの乾燥物（酢酸エチル画分乾燥物）、水層から113 gの乾燥物（水画分乾燥物）、不溶物から64.3 gの乾燥物（不溶画分乾燥物）が得られた。 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.
　各乾燥物のメチシリン耐性黄色ブドウ球菌(MRSA)及びバンコマイシン耐性腸球菌(VRE)に対する最小生育阻止濃度（MIC）を、微量液体希釈法により測定した。具体的には次のように行った。それぞれの乾燥物を培地（Mueller Hinton(MH)培地(Difco):肉抽出エキス2.0 g/L, カゼイン酸消化物 17.5 g/L, 可溶性デンプン1.5 g/L）で2倍ずつ希釈した希釈系列を作成し、各培地100μLに約104 CFU/wellとなるように被検菌を植菌した。植菌後、37℃で24時間静置した後、培地の懸濁の有無を評価した。培地の懸濁が無い（菌の生育が認められない）最小の濃度を最小生育阻止濃度（MIC）とした。結果を表1に示す。 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.
Figure JPOXMLDOC01-appb-T000015
 　表1より、酢酸エチル画分乾燥物に、抗菌活性が認められた。センコツから抽出された酢酸エチル画分は、互いに構造が非常によく類似したセスキテルペン二量体チオアルカロイド（6-ヒドロキシチオビヌファリジン、6，6'-ジヒドロキシチオビヌファリジン、6-ヒドロキシチオヌフルチンB、及び6'-ヒドロキシチオヌフルチンB）を多量に含むことが知られている。このことから、これらのセスキテルペン二量体チオアルカロイドが抗菌活性を有することが示唆された。
Figure JPOXMLDOC01-appb-T000015
 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.
　実施例1－2：ステップＢ（アルカロイド画分の精製）
　酢酸エチル画分乾燥物をシリカゲルカラムクロマトグラフィーにより精製した。具体的には次のように行った。クロロホルムで膨潤させたシリカゲル (70-230 mesh, 60Å, SIGMA-ALDRICH Co.)約400 mlを、内径40 mmのオープンカラムに充填した。酢酸エチル画分乾燥物8.17 gを少量の混合溶媒1（クロロホルム：酢酸エチル：ジエチルアミン＝20：1：1（v/v））に溶解し、該溶解液をシリカゲルが充填されたカラムに通液することにより、酢酸エチル画分乾燥物の成分をシリカゲルカラムに保持させた。その後、溶出液として、混合溶媒1を1200 ml通液し、次いで混合溶媒2（メタノール：ジエチルアミン＝10:1（v/v））を1200 ml通液した。カラムから溶出された液を800 mlずつ3つに分けて回収し、溶出された順にfr. 1、fr. 2、fr. 3とした。各画分から、減圧下で溶媒を留去した。その結果、fr. 1から5.56 gの乾燥物（fr. 1乾燥物）、fr. 2から1.27 gの乾燥物（fr. 2乾燥物）、fr. 3から1.30gの乾燥物（fr. 3乾燥物）が得られた。 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.
　各乾燥物のメチシリン耐性黄色ブドウ球菌(MRSA)及びバンコマイシン耐性腸球菌(VRE)に対する最小生育阻止濃度（MIC）を、実施例1－1と同様に測定した。結果を表2に示す。表2より、fr. 1乾燥物に最も強い抗菌活性が認められた。 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 fr.fr1 dried product.
Figure JPOXMLDOC01-appb-T000016
 　実施例1－3：ステップＣ（アルカロイド画分の精製）
　fr. 1乾燥物をさらにシリカゲルカラムクロマトグラフィーにより精製した。具体的には次のように行った。混合溶媒3（ノルマルヘキサン：酢酸エチル：アンモニア水＝75:25:1（v/v）））で膨潤させたシリカゲル（70-230 mesh, 60 Å,SIGMA-ALDRICH Co.） 約350 mLを、内径44mmのオープンカラムに充填した。fr. 1乾燥物 0.92 gを約2 mLの混合溶媒3に溶解し、該溶解液をシリカゲルが充填されたカラムに通液することにより、fr. 1乾燥物の成分をシリカゲルカラムに保持させた。その後、溶出液として、混合溶媒3を1000 ml通液し、次いで混合溶媒4（メタノール：アンモニア水＝100：1（v/v））を700 ml通液した。カラムから溶出された液を、順次、順相薄層クロマトグラフィーで展開し、スポットのパターンによって10個の画分に分けた（fr. 1-I～fr. 1-X）。各画分から、減圧下で溶媒を留去した。その結果、fr. 1-Iから34.9 mgの乾燥物（fr. 1-I乾燥物）、fr. 1-IIから51.9 mgの乾燥物（fr. 1-II乾燥物）、fr. 1-IIIから91.8 mgの乾燥物（fr. 1-III乾燥物）、fr. 1-IVから36.4 mgの乾燥物（fr. 1-IV乾燥物）、fr. 1-Vから50.6 mgの乾燥物（fr. 1-V乾燥物）、fr. 1-VIから73.7 mgの乾燥物（fr. 1-VI乾燥物）、fr. 1-VIIから14.5 mgの乾燥物（fr. 1-VII乾燥物）、fr. 1-VIIIから159.9 mgの乾燥物（fr. 1-VIII乾燥物）、fr. 1-IXから167.5 mgの乾燥物（fr. 1-IX乾燥物）、fr. 1-Xから1276.3 mgの乾燥物（fr. 1-X乾燥物）が得られた。
Figure JPOXMLDOC01-appb-T000016
 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.
　各乾燥物のメチシリン耐性黄色ブドウ球菌(MRSA)に対する最小生育阻止濃度（MIC）を、実施例1－1と同様に測定した。結果を表3に示す。表3より、fr. 1-VIII乾燥物に最も強い抗菌活性が認められた。 The minimum 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.
Figure JPOXMLDOC01-appb-T000017
 　実施例1－4：活性成分の同定
　薄層クロマトグラフィーの結果から、fr.1-VIIIは単一の化合物であると考えられたため、1H-NMR解析により構造を決定した。その結果、表4に示すケミカルシフト値が得られた。この値は、セスキテルペン二量体チオアルカロイドの一種である6，6'-ジヒドロキシチオビヌファリジン（6,6’-dihydroxythiobinupharidine）のものとほぼ一致した。また比旋光度も一致した。なお、表4中、6,6’-dihydroxythiobinupharidineのケミカルシフト値等は、Yoshikawa M. et al., HETEROCYCLES, 1997, Vol. 45, No. 9, pp1815-1824より抜粋した。以下の実験では、fr. 1-VIII乾燥物を6，6'-ジヒドロキシチオビヌファリジンとして用いた。
Figure JPOXMLDOC01-appb-T000017
 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 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.
Figure JPOXMLDOC01-appb-T000018
 　互いに構造が非常によく類似したセスキテルペン二量体チオアルカロイドを多量に含む画分に強い抗菌活性が認められたこと（実施例1－1）、及び現に該アルカロイドの一種である6，6'-ジヒドロキシチオビヌファリジンに抗菌活性が見出されたこと（実施例1－3、及び1－4）から、コウホネに含まれるセスキテルペン二量体チオアルカロイドは抗菌活性を有することが強く示唆された。
Figure JPOXMLDOC01-appb-T000018
 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), which strongly suggests that the sesquiterpene dimer thioalkaloids contained in mulberry have antibacterial activity It was done.
　実施例２：セスキテルペン二量体チオアルカロイドによる抗菌活性増強作用の解析
　セスキテルペン二量体チオアルカロイドによる、各種抗生物質の抗菌活性増強作用を調べた。具体的には、バンコマイシン（グリコペプチド系抗菌薬）、アミカシン（アミノグリコシド系抗菌薬）、及びアルベカシン（アミノグリコシド系抗菌薬）の、バンコマイシン感受性腸球菌（VSE）及びバンコマイシン耐性腸球菌（VRE）に対する最小生育阻止濃度（MIC）を、セスキテルペン二量体チオアルカロイドである6，6'-ジヒドロキシチオビヌファリジン（DTBN）を単独では抗菌活性を示さない濃度（1 μg/mL）で加えた場合（＋）、及び全く加えない場合（－）それぞれの場合について、実施例1－1と同様に測定した。さらに、測定値に基づいて、下記式に基づいて、FIC indexを算出した。FIC indexがより低い程、セスキテルペン二量体チオアルカロイドの増強活性がより強いことを意味する。FIC indexが0.5以下であれば相乗効果が発揮されているとみなすことができる。 Example 2: Analysis of antibacterial activity enhancing action by sesquiterpene dimer thioalkaloid The antibacterial activity enhancing action of various antibiotics by sesquiterpene dimer thioalkaloid was examined. Specifically, vancomycin (glycopeptide antibacterial), amikacin (aminoglycoside antibacterial), and arbekacin (aminoglycoside antibacterial) minimal growth against vancomycin sensitive enterococci (VSE) and vancomycin resistant enterococci (VRE) When the inhibitory concentration (MIC) was added to the sesquiterpene dimer thioalkaloid 6,6'-dihydroxythiobinufaridine (DTBN) at a concentration that does not exhibit antibacterial activity alone (1 μg / mL) ( The measurement was carried out in the same manner as in Example 1-1 for each of (+) and no addition (-). Furthermore, based on the measured value, the FIC index was calculated based on the following formula. A lower FIC index means stronger enhancement activity of the sesquiterpene dimer thioalkaloid. If the FIC index is 0.5 or less, it can be considered that a synergistic effect is exhibited.
Figure JPOXMLDOC01-appb-M000019
 　腸球菌としてEnterococcus faeciumを用いた場合の結果を表5に、腸球菌としてEnterococcus faecalisを用いた場合の結果を表6に示す。表5及び6に示される株について、ATCC及びNCTCで始まる名称の株はATCC、NCTC、又は理化学研究所バイオリソースセンターより購入した株であり、その他の株は臨床分離されたものを譲り受けた株である。
Figure JPOXMLDOC01-appb-M000019
 Table 5 shows the results when Enterococcus faecium was used as the enterococci, and Table 6 shows the results when Enterococcus faecalis was used as the enterococci. Of the strains shown in Tables 5 and 6, strains with names beginning with ATCC and NCTC are those purchased from ATCC, NCTC, or RIKEN BioResource Center, and other strains are those that have been clinically isolated. is there.
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000020
Figure JPOXMLDOC01-appb-T000021
 　表5及び6に示されるように、6，6'-ジヒドロキシチオビヌファリジンは、バンコマイシン耐性菌に対する各種抗生物質（バンコマイシン、アミカシン、及びアルベカシン）の抗菌活性を顕著に増強した。
Figure JPOXMLDOC01-appb-T000021
 As shown in Tables 5 and 6, 6,6′-dihydroxythiobinufaridine significantly enhanced the antibacterial activity of various antibiotics (vancomycin, amikacin, and arbekacin) against vancomycin-resistant bacteria.

Claims (12)

  1. 一般式（１）：
    Figure JPOXMLDOC01-appb-C000001
     ［式中、Ｒ１は水酸基を示し、Ｒ２は水素又は水酸基を示す］
    で表されるセスキテルペン二量体チオアルカロイド、及び／又は一般式（２）：
    Figure JPOXMLDOC01-appb-C000002
     ［式中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない］
    で表されるセスキテルペン二量体チオアルカロイドを含有する、
    グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性の増強剤。     General formula (1):
    Figure JPOXMLDOC01-appb-C000001
     [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):
    Figure JPOXMLDOC01-appb-C000002
     [Wherein, R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen]
    Containing a sesquiterpene dimer thioalkaloid represented by:
    Antibacterial activity enhancer of glycopeptide antibacterial and / or aminoglycoside antibacterial against glycopeptide antibacterial resistant bacteria.
  2. 一般式（１）で表されるセスキテルペン二量体チオアルカロイドを含有し、且つ前記Ｒ２が水酸基である、請求項１に記載の増強剤。 The enhancer according to claim 1, comprising a sesquiterpene dimer thioalkaloid represented by the general formula (1), and wherein R 2 is a hydroxyl group.
  3. 前記一般式（１）で表されるセスキテルペン二量体チオアルカロイド及び／又は前記一般式（２）で表されるセスキテルペン二量体チオアルカロイドを含有するコウホネ属植物抽出物を含有する、請求項１又は２に記載の増強剤。 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 enhancer according to item 1 or 2.
  4. 対象となる前記グリコペプチド系抗菌薬耐性菌が、グリコペプチド系抗菌薬の最小生育阻止濃度が１６μｇ／ｍＬ以上の菌である、請求項１～３のいずれかに記載の増強剤。 The enhancer according to any one of claims 1 to 3, wherein the target glycopeptide antibiotic-resistant bacterium is a bacterium having a minimum growth inhibitory concentration of the glycopeptide antibiotic of 16 µg / mL or more.
  5. グリコペプチド系抗菌薬の抗菌活性増強剤である、請求項１～４のいずれかに記載の増強剤。 The enhancer according to any one of claims 1 to 4, which is an antibacterial activity enhancer of a glycopeptide antibacterial agent.
  6. 対象となる前記グリコペプチド系抗菌薬耐性菌が、アミノグリコシド系抗菌薬に対しても耐性を有する菌である、請求項１～５のいずれかに記載の増強剤。 The enhancer according to any one of claims 1 to 5, wherein the target glycopeptide antibacterial resistant bacterium is a bacterium having resistance to an aminoglycoside antibacterial agent.
  7. 対象となる前記グリコペプチド系抗菌薬耐性菌が、アミノグリコシド系抗菌薬の最小生育阻止濃度が４μｇ／ｍＬ以上の菌である、請求項６に記載の増強剤。 The enhancer according to claim 6, wherein the target glycopeptide antibacterial resistant bacterium is a bacterium having an aminoglycoside antibacterial minimum growth inhibitory concentration of 4 μg / mL or more.
  8. 請求項１～７のいずれかに記載の増強剤、並びにグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬を含有する抗菌剤。 An antibacterial agent comprising the enhancer according to any one of claims 1 to 7, and a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent.
  9. グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性の増強剤の製造のための、一般式（１）：
    Figure JPOXMLDOC01-appb-C000003
     ［式中、Ｒ１は水酸基を示し、Ｒ２は水素又は水酸基を示す］
    で表されるセスキテルペン二量体チオアルカロイド、及び／又は一般式（２）：
    Figure JPOXMLDOC01-appb-C000004
     ［式中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない］
    で表されるセスキテルペン二量体チオアルカロイドの使用。     Formula (1) for the production of an antibacterial activity enhancer of a glycopeptide antibacterial and / or aminoglycoside antibacterial against a glycopeptide antibacterial resistant bacterium:
    Figure JPOXMLDOC01-appb-C000003
     [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):
    Figure JPOXMLDOC01-appb-C000004
     [Wherein, R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen]
    Use of a sesquiterpene dimer thioalkaloid represented by:
  10. グリコペプチド系抗菌薬耐性菌に対する、グリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬の抗菌活性の増強剤としての使用のための、一般式（１）：
    Figure JPOXMLDOC01-appb-C000005
     ［式中、Ｒ１は水酸基を示し、Ｒ２は水素又は水酸基を示す］
    で表されるセスキテルペン二量体チオアルカロイド、及び／又は一般式（２）：
    Figure JPOXMLDOC01-appb-C000006
     ［式中、Ｒ３及びＲ４は独立して水素又は水酸基を示す。但し、Ｒ３とＲ４は、水素を示すことはない］
    で表されるセスキテルペン二量体チオアルカロイド。     General formula (1) for use as a potentiator of antibacterial activity of glycopeptide antibacterials and / or aminoglycoside antibacterials against glycopeptide antibacterial resistant bacteria:
    Figure JPOXMLDOC01-appb-C000005
     [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):
    Figure JPOXMLDOC01-appb-C000006
     [Wherein, R 3 and R 4 independently represent hydrogen or a hydroxyl group. However, R 3 and R 4 do not represent hydrogen]
    A sesquiterpene dimer thioalkaloid represented by:
  11. 請求項１～７のいずれかに記載の増強剤、並びにグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬を併用することを含む、該抗菌薬のグリコペプチド系抗菌薬耐性菌に対する抗菌活性の増強方法。 A potentiation of the antibacterial activity of the antibacterial agent against a glycopeptide antibacterial drug-resistant bacterium, comprising a combination of the enhancer according to any one of claims 1 to 7 and a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent Method.
  12. 請求項１～７のいずれかに記載の増強剤、並びにグリコペプチド系抗菌薬及び／又はアミノグリコシド系抗菌薬を併用することを含む、グリコペプチド系抗菌薬耐性菌の抗菌方法。 An antibacterial method for a glycopeptide antibacterial drug-resistant bacterium comprising the combined use of the enhancer according to any one of claims 1 to 7, and a glycopeptide antibacterial agent and / or an aminoglycoside antibacterial agent.
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