JP2020152704A - Antibacterial agents against drug-resistant staphylococcus aureus or vancomycin-resistant enterococcus and antifungal agents comprising stephanitis svensoni-derived polyketide or synthetic analogs thereof - Google Patents

Abstract

To provide antibacterial or antifungal agents comprising a naturally occurring substance or synthetic analogs thereof having high antibacterial activity against methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus and antifungal activity.SOLUTION: Disclosed are an antibacterial agent against methicillin-resistant Staphylococcus aureus or vancomycin-resistant Enterococcus and an antifungal agent, both comprise 2,6-DH8 of the following formula or a homolog thereof as an effective ingredient. Antibacterial/antifungal textile products are also disclosed.SELECTED DRAWING: None

Description

The present invention relates to an antibacterial agent against methicillin-resistant yellow staphylococcus (MRSA) or vancomycin-resistant enterococci (VRE), an antifungal agent, and an antibacterial / antifungal fiber product using a compound derived from a natural product and a synthetic analog thereof.

Antibiotics have been defined as "substances produced by microorganisms that suppress the growth of other microorganisms", but nowadays, antibacterial agents, antifungal agents, and antiviral agents derived from microorganisms are also broadly defined as antibiotics. Has been done. Compounds and synthetic products derived from other than microorganisms have been called antibacterial substances and have been distinguished. In this study, we searched for new search sources for "antibacterial substances" that suppress the growth of bacteria regardless of their origin and screened them.

Research on antibiotics has been carried out mainly on soil actinomycetes centered on the genus Streptmyces. Many of them have been put to practical use as medicines and pesticides such as penicillin, but more soil will be used in the future. It is said that it is difficult to find an antibacterial active substance with a new skeleton. It cannot be said that it is impossible to search for new antibacterial substances because there are microorganisms that cannot be cultivated by the current technology even in the soil that is said to have been thoroughly searched, but as a search resource for useful substances, humans such as deep-sea microorganisms Attention is being focused on microorganisms in the untouched environment. As a background to the search for new compounds, research on antibacterial substances always has the problem of drug-resistant bacteria (particularly methicillin-resistant Staphylococcus aureus (MRSA)). MRSA becomes a dominant bacterium in hospitals where conventional antibacterial drug selection pressure is present, and conventional infection control is ineffective (Ishii, 2018). In order to inhibit the development of such bacteria, it is necessary to find a compound having a new origin or mechanism of action.

In insects, which are said to account for more than half of all organisms, various secondary metabolites have been identified in association with inter-organism interactions. Microorganisms are familiar to insects, and some microorganisms live together as symbiotic bacteria, while others can be foreign enemies that infect insects. A well-known defense mechanism against bacterial infections in insects is antimicrobial peptides. When infected with bacteria, this peptide is transiently synthesized in the body and secreted into body fluids. Typical examples are honeybee apidesin, stink bug tanatin, and beetle defensincin, and insect antibacterial peptides are known to destroy bacterial cell membranes. Insect defensin, which consists of 43 amino acid residues isolated from beetles, exhibits antibacterial activity against Gram-positive bacteria and also activity against MRSA (Furukawa et al., 2004). Insects also secrete antibacterial substances to the surface of the body to prevent bacterial infections. The 1- (2,6-dihydroxyphenyl) dodecan-1-one secreted by Gumbai larvae of the genus Stephanitis and Corythucha are four gram-positive bacteria including the tomato scab bacterium Clavibacter michiganense and the root cancer bacterium Agrobacterium tumifaciens. Antibacterial activity against 5 Gram-negative bacteria including Cavibacter michiganense was investigated and showed antibacterial activity against Clavibacter michiganense (John W. Neal, JR., Et al, 1995). In this paper, a structure-activity relationship study was conducted and examined with cyclized compounds and compounds with a phenyl group substituted at the end of the side chain, but these compounds are mainly active against Gram-positive bacteria. It is said that. On the other hand, in this paper, similar antifungal activity using 1- (2,6-dihydroxyphenyl) dodecane-1-one was examined for a total of 9 fungi, but for all fungi. However, no antifungal activity was observed. The nymphs of Tingidae, which were the subjects of this study, also secrete 1- (2,6-dihydroxyphenyl) dodecane-1-one.

John W. Neal, Jr., James E. Oliver, Raymond H. Fetterer; “In Vitro Antimicrobial and Nematocidal Activity of Acetgenins Identified from Exocrine Secretions of Stephanitis and Corythucha Lace Bugs Numphs (Heteroptera: Tingidae)” (1995) Ann. Entomol . Soc. Am. 88 (4), 496-501. Abstracts of the 2018 Annual Meeting of the Japan Society for Bioscience and Biotechnology (issued on March 5, 2018), Lecture No .: 3A18p10 Search for antibacterial active substances contained in nymph secretions

Antibacterial agents with high antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE) or other resistant strains using naturally occurring compounds that are presumed to be less virulent and their synthetic analogs. Agents, antifungal agents, and antibacterial fiber products using the same are provided.

Against this background, we searched for antibacterial active substances using insects as a search source. In this study, we evaluated the antibacterial activity of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, and side chains of 1- (2,6-dihydroxyphenyl) dodecan-1-one secreted by larvae of Shikimigunbai. We proceeded with structural activity correlation research focusing on the position of length and hydroxyl group. In particular, the effects on methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) were confirmed.

According to a preferred embodiment of the present invention, an antibacterial agent and an antifungal agent against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) containing at least one of the following compounds 1 to 3 as an active ingredient. Is. Alternatively, it is a textile product using such an antibacterial / antifungal agent.
Compound 1

Compound 2

Compound 3

1. 1. Illicium anisatum secretions and their relatives Stephanitis svensoni is an insect of the order Hemiptera Tingidae, an insect that sucks the leaves of Illicium anisatum. Illicium anisatum nymphs secrete many interesting compounds from the thorns on the back. In our laboratory, worm secretions were extracted with hexane and analyzed with a gas chromatograph mass spectrometer (GC-MS). As a result, decanal, dodecanal, 2-undecanone, 3-oxododecanal (houttuynin), 2,6-dihydroxyacetophenone, 1- (2,6-dihydroxyphenyl) dodecan-1-one, 5-hydroxy-2-alkylchromanone, and nonaicosane were detected (Fig. I). Among these, 5-hydroxy-2-alkylchromanones are compounds peculiar to Tingidae, which are also found in the secretions of nymphs of Tingidae. 3-Oxododecanal has been isolated from Houttuynia cordata and has been reported to have antibacterial, antiviral and anti-inflammatory effects (L. Jinbing et al, 2009). 2,6-Dihydroxyacetophenone has a repellent activity against ants (patent pending in our laboratory) and has a prostaglandin H synthase inhibitory activity superior to that of aspirin (Jurenka, RA et al, 1989). It has been reported that 1- (2,6-Dihydroxyphenyl) dodecane-1-one has a remarkable antibacterial activity against the gram-positive bacterium Clavibacter michiganensis and a growth inhibitory effect on nematodes (John W. Neal, JR., Et al, 1995), prostaglandin H synthase inhibitory activity comparable to aspirin (Jurenka, RA et al, 1989). In this chapter, we will focus on 2,6-dihydroxyacetophenone and 1- (2,6-dihydroxyphenyl) dodecane-1-one as aromatic ketones, and as part of the elucidation of the biological activities of these compounds, Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria. The antibacterial activity of the bacterium against Escherichia coli was verified. For 1- (2,6-Dihydroxyphenyl) dodecane-1-one, the antifungal activity of 4 fungi was also verified.

<Fig. I GC chromatogram of Shikimigunbai nymph secretion>

1.1 Test compounds ・ Essential oil and hexane extract of Shikimi leaf ・ 1- (2,6-dihydroxyphenyl) dodecane-1-one and 2,6-dihydroxyacetophenone and their analogs ・ Of the Shikimigunbai larvae secretions, 3- 9 compounds except oxododecanal

The structure of the test compound and its abbreviation are shown below (Table I). Hereinafter described by abbreviation.

<Table I>

The abbreviations of compounds similar to the 2,6-DHA positional isomers are shown below and are described below by abbreviations (Table II).

<Table II>

The mass spectrum of the nymph secretion of Illicium anisatum used in the test is shown below (Figs. II-1 to II-3).

<Fig. II-1>

<Fig. II-2>

<Fig. II-3>

1.2 Synthesis method
Synthesis of 2,6-DH4, 2,6-DH6, 2,6-DH8, 2,6-DH10, 2,6-DH14

<Synthesis of Intermediate A>
Using CH ₂ Cl ₂ (5 mL) as a solvent, 1,3-cyclohexanedione 0.56 g (5 mmol) was reacted with any carboxylic acid anhydride (5 mmol) and pyridine (0.44 g, 5.57 mmol) at room temperature for 30 minutes. It was. The solvent was concentrated under reduced pressure, separated by 1N hydrochloric acid cooled with hexane / diethylether (1: 1), and dried over anhydrous Na ₂ SO ₄ . The solvent was concentrated under reduced pressure on an evaporator and then the crude extract was purified by silica gel column chromatography [hexane / EtOAc (3: 1)].

A4 (R = 3)
^{1 1} H-NMR (CDCl ₃ ) δ5.91 (1H, s), 2.55 (2H, m), 2.46 (4H, m), 2.07 (2H, quin), 1.70 (2H, m) 1.00 (3H, t)
GC-MS 12.892 min 182 (M ⁺ ), 84, 71, 55, 43

A6 (R = 5)
^{1 1} H-NMR (CDCl ₃ ) δ5.91 (1H, s), 2.56 (2H, t), 2.46 (3H, m), 2.07 (2H, quin), 1.70 (2H, t), 1.33 (5H, m) ), 0.91 (3H, t)
GC-MS 15.159 min 210 (M ⁺ ), 99, 84, 71, 55, 43

A8 (R = 7)
^{1 1} H-NMR (CDCl ₃ ) No data
GC-MS 17.189 min, 238 (M ⁺ ), 127, 109, 57, 43

A10 (R = 9)
^{1 1} H-NMR (CDCl ₃ ) δ5.92 (1H, s), 2.55 (2H, m), 2.45 (3H, m), 2.08 (2H, quin), 1.29 (12H, m), 0.89 (3H, t) )
GC-MS 19.323 min 266 (M ⁺ ), 155, 113, 95, 85, 71, 57, 43

A14 (R = 13)
^{1 1} H-NMR (CDCl ₃ ) δ5.92 (1H, s), 2.49 (2H, m), 2.39 (3H, m), 1.67 (2H, quin), 1.28 (20H, m), 0.90 (3H, t) )
GC-MS 30.815 min 322 (M ⁺ ), 211, 113, 97, 85, 71, 57, 43

<Synthesis of Intermediate B>
Product A (3.4 mmol) was dissolved in 15 mL of CH ₃ CN, 1 mL of Et ₃ N and 40 μL of acetone cyanohydrine were added, and the mixture was reacted at room temperature for 12 hours. The solvent was concentrated under reduced pressure and separated with hexane / EtOAc (1: 1) with pure water and dilute hydrochloric acid. After drying over anhydrous Na ₂ SO ₄ , the mixture was concentrated under reduced pressure using an evaporator.

B4 (R = 3) GC-MS 12.410 min 182 (M ⁺ ), 167, 154, 139, 112, 69, 55, 43
B6 (R = 5) GC-MS 14.830 min, 210 (M ⁺ ), 192, 181, 167, 154, 139
B8 (R = 7) GC-MS 17.046 min 238 (M ⁺ ), 167, 154, 139
B10 (R = 9) GC-MS 19.066 min 266 (M ⁺ ), 248, 167, 154, 139
B14 (R = 13) GC-MS 22.605 min 322 (M ⁺ ), 304, 167, 154, 139

<Synthesis of target C>
The product (2) (1.31 mmol) was dissolved in 5 mL of AcOH, Hg (OAc) ₂ 1.26 g (0.39 mmol) and NaOAc 0.33 g were added, and the mixture was stirred at 120-125 ° C. until the precipitate dissolved (2-). 3 hours). After the reaction, the mixture was cooled to room temperature, 1N hydrochloric acid was added, and the mixture was stirred for 30 minutes. The mixture was filtered through Celite and washed with hexane / EtOAc (3: 1). The organic layer was washed with water, saturated acrylamide ₃ aq., Brine, dried over anhydrous Na ₂ SO ₄ , filtered through neutral alumina and washed with EtOAc. After concentration under reduced pressure, the product was purified by silica gel column chromatography [hexane / EtOAc (5: 1)]. The separated aqueous layer was neutralized and then appropriately treated as Hg waste liquid.

C4 (R = 3)
^{1 1} H-NMR (CDCl ₃ ) δ9.57 (2H, br), 7.22 (1H, t), 6.40 (2H, d), 3.11 (2H, t), 1.76 (2H, quin), 1.00 (3H, t) )
GC-MS 15.107 min 180 (M ⁺ ), 165, 147, 137, 81

C6 (R = 5)
^{1 1} H-NMR (CDCl ₃ ) δ9.62 (2H, br), 7.24 (1H, t), 6.41 (2H, d), 3.15 (2H, t), 1.72 (2H, quin), 1.38 (4H, sext) ), 0.93 (3H, t)
GC-MS 17.200 min 208 (M ⁺ ), 190, 165, 152, 137

C8 (R = 7)
^{1 1} H-NMR (CDCl ₃ ) δ10.14 (2H, br), 7.22 (1H, t), 6.39 (2H, d), 3.14 (2H, t), 1.71 (2H, d), 1.34 (10H, sext) ), 0.93 (3H, t)
GC-MS 19.148 min 236 (M ⁺ ), 218, 189, 175, 165, 152, 137

C10 (R = 9)
^{1 1} H-NMR (CDCl ₃ ) δ9.47 (2H, br), 7.24 (1H, t), 6.41 (2H, d), 3.14 (2H, t), 1.73 (2H, quin), 1.34 (12H, sext) ), 0.92 (3H, t)
GC-MS 21.005 min, 264 (M ⁺ ), 246, 189, 165, 151, 137

C14 (R = 13)
^{1 1} H-NMR (CDCl ₃ ) δ9.46 (2H, br), 7.21 (1H, t), 6.38 (2H, d), 3.11 (2H, t), 1.70 (2H, quin), 1.28 (20H, sext) ), 0.88 (3H, t)
GC-MS 24.194 min 320 (M +), 302, 189, 165, 152, 137

<Synthesis of 2,4-DH12>

(i) Resorcinol (220 mg, 2 mmol) and lauric anhydride (773 mg, 2.02 mmol) were placed in a eggplant flask, BF ₃ · OEt ₂ (1.5 ml, 11.9 mmol) was added, and the mixture was reacted at room temperature for 40 hours. 10% NaOAc (7 ml) was added to the reaction mixture, and the mixture was stirred overnight. After suction filtration, it was thoroughly washed with water. Since the target product is poorly soluble in hexane, hexane was added and filtration was repeated several times. The desired product (300 mg, 51%, Rf 0.23 for Hex: EtOAc (5: 1)) was obtained.

^{1 1} H-NMR (CDCl ₃ ) δ 12.85 (1H, s), 7.66 (1H, d), 6.38 (2H, q), 2.89 (2H, t), 1.72 (3H, t), 1.33 (18H, m) , 0.88 (3H, t)
¹³ C-NMR δ 205.34, 165.28, 162.28, 132.38, 113.95, 107.5, 103.60, 38.07, 31.91, 29.61, 29.49, 29.43, 29.39, 29.34, 24.92, 22.69, 14.12
GC-MS 22.820 min 292 (M ⁺ ), 274, 165, 137

<Synthesis of 2,5-DH12>

The reaction was carried out using hydroquinone (220 mg, 2 mmol) as a raw material by the method (i) above, and a monoester (156 mg, 27%) was obtained. The Rf values of the target product and the monoester are almost the same. The reaction proceeded in the same manner with catechol, and a monoester was obtained. These monoesters were tested as synthetic intermediates 4-HP12 and 2-HP12. The synthesis method of 2,5-DHP12 was changed to (ii).

(ii) Place Hydroquinone (550 mg, 5 mmol) and lauric acid (1.0 g, 5 mmol) in an eggplant flask and add 80 BF ₃ · OEt ₂ (1.08 ml, 8.5 mmol, d: 1.12 g / ml). Dropped at ° C. After reacting at that temperature for 1 hour, the temperature was raised to 140 ° C. and the reaction was carried out for 2 hours. After cooling to 110 ° C, 5% Na ₂ CO ₃ aq. (15 ml) was added and the mixture was stirred for 30 minutes. After extraction with ethyl acetate, it was washed with water and brine. After drying over anhydrous Na ₂ SO ₄ , the solvent was distilled off. Purified on a silica gel column (30 g), the desired product had the same Rf value (0.29) as the monoester (Hex: EtOAc (4: 1)). The pale yellow and black solids were mixed, but the target product was a black (blackish brown) solid (200 mg) that was insoluble in 10% EtOAc / Hex, and the pale yellow solid was dissolved and separated due to the difference in solubility.

^{1 1} H-NMR (CDCl ₃ ) δ11.99 (1H, s), 7.28 (1H, s), 7.04 (1H, q), 6.91 (1H, d), 4.73 (1H, s), 2.95 (2H, t) ), 1.75 (1H, t), 1.66 (1H, s), 1.34 (18H, m), 0.90 (3H, t)
¹³ C-NMR δ206.45, 156.75, 147.3, 136.58, 124.65, 114.90, 38.47, 31.91, 29.62, 29.49, 29.43, 29.34, 29.31, 24.46, 22.69, 14.12
GC-MS 22.512 min 292 (M ⁺ ), 274, 189, 165, 152, 137

<Synthesis of 2,4.6-TH12>

The reaction was carried out using phloroglucinol (252 mg, 2.61 mmol) as a raw material by the method (i) above, but the desired product was not obtained and only the monoester was obtained. Next, the method (ii) was performed, but the result was the same. Therefore, the synthesis method was changed to (iii).

(iii) Phloroglucinol (329 mg, 2.61 mmol) and lauric anhydride (1.0 g, 2.61 mmol) were dissolved in anhydrous THF (4 ml), and BF ₃ · OEt ₂ (0.82 ml, 6.53 mmol) was added to room temperature under a nitrogen atmosphere. Added in. After reacting for 48 hours, it was diluted with ethyl acetate and washed 3 times with 1M HCl. After washing with brine, it was dried over anhydrous Na ₂ SO ₄ and the solvent was distilled off. Purification was performed on a silica gel column (20 g, 30% EtOAc / Hex), but the target product had the same Rf value (0.45) as the monoester (Hex: EtOAc (1: 1)). In order to separate these two components, we decided to hydrolyze the ester with LiOH.

The mixture (400 mg, 1.3 mmol) was dissolved in MeOH: H ₂ O (3: 1), LiOH · H ₂ O (109 mg, 2.6 mmol) was added at 0 ° C., and the mixture was reacted overnight at room temperature. The pH was adjusted to about 2 by adding 1M HCl, extracted with ethyl acetate, washed with brine, and dried over anhydrous Na ₂ SO ₄ . After distilling off the solvent, purification was performed on a silica gel column (10 g, 30% EtOAc / Hex) to obtain 150 mg (19%) of the desired product.

^{1 1} H-NMR (CDCl ₃ ) δ 5.82 (2H, s), 3.04 (2H, t), 1.64 (5H, m), 1.30 (20H, t), 0.91 (3H, t)
¹³ C-NMR δ 2.06.10, 164.58, 164.44, 103.98, 94.34, 70.28, 69.96, 48.28, 48.06, 47.85, 47.64, 47.42, 47.21, 47.00, 43.49, 31.71, 29.41, 29.40, 29.34, 29.32, 29.29, 29.13 , 26.16, 26.04, 24.85

<Chemical analysis>
¹ H and ¹³ C nuclear magnetic resonance (NMR) spectra were measured using a Bruker Biospin AC-400M device ( ¹ H: 400 MHz, ¹³ C: 100 MHz) using tetramethylsilane (TMS) in deuterated chloroform as an internal standard.

In the GC-MS analysis, a 5975 MSD quadrupole mass filter type mass spectrometer (Agilent Technologies) connected to a 6890 N gas chromatograph (Agilent Technologies) was used, and the measurement was performed by the electron ionization method (70 eV). The capillary column used HP-5MS (inner diameter: 0.25 mm, length: 30 m, film thickness: 0.25 μm, Agilent Technologies), kept at 60 ° C for 2 minutes, and then raised to 290 ° C at 10 ° C / min. , Held at that temperature for 5 minutes.

2. 2. Antibacterial activity evaluation

2.1 Preparation of bacteria for preliminary evaluation <Target strain>
Staphylococcus aureus (NBRC 12732)
Escherichia coli (NBRC 3972)

<Culture conditions>
Staphylococcus aureus 37 ° C for 1 day Escherichia coli 30 ° C for 1 day (both 300 rpm during shaking culture)

The medium was prepared to have the following composition.
・ Liquid medium
Tryptone 10 g
Yeast extract 2 g
DDL ₄ 1 g
Distilled water 1 L
・ Agar medium
Tryptone 10 g
Yeast extract 2 g
DDL ₄ 1 g
Distilled water 1 L
Agar 15 g

<How to save>
By creating glycerol stock, long-term storage at -80 ° C is possible. Make 40% glycerol in a 100 mL medium bottle (glycerol / pure water = 20 mL / 30 mL) and sterilize in an autoclave at 120 ° C. for 15 minutes. Add 300 μL each of 40% glycerol and culture medium to a sterilized cryotube and adjust the final concentration of glycerol to 20%. Glycerol is placed in the cryotube first, and the culture medium is weighed into a sterile Eppen tube and then decanted. When using the bacteria, thaw them at room temperature, inoculate them on the above agar medium, and then incubate them at each culture temperature (1 day). It can be refrigerated and used at 4 ° C for about 1 week after inoculation, but in order to make the conditions uniform, it was used by raising it from the glycerol stock the day before the antibacterial activity test as much as possible.

2.2 Preliminary evaluation by paper disc method

<Material>
Paper disc (Toyo Roshi; 8 mm DIA, Thin)
Sterilized petri dish (Atect; SLE diameter 90mm x 15mm)

<Method>
The presence or absence of antibacterial activity is determined by the formation of a blocking circle. The test medium was prepared by mixing the preculture, which was shake-cultured in the above liquid medium for one day, with the above agar medium. (The ratio was 25 mL of agar medium / 0.5 mL of preculture for 1 petri dish.) An 8 mm paper disk treated with the test compound was allowed to stand in the test medium and judged 1 day after culture. Paper discs of methanol, hexane, and ethyl acetate were also tested as controls. The paper disc was air-dried on a glass plate for at least 1 hour to allow the solvent to dry completely (Fig. III).

<Fig. III> Antibacterial activity test by paper disc method

<Result>
The test results by the paper disc method are summarized in the table below. The table shows the compound name, the number of tests, and the size of the blocking circle (mm). In Table III and Table IV, the number of trials is shown in parentheses, and when a blocking circle is not formed, it is indicated as-. Among the larvae secretions, those in which a blocking circle was formed were tested by combining test compounds, and a comparative study was conducted with a single case (Graph I). For the active 1- (2,6-dihydroxyphenyl) dodecane-1-one and 2,6-dihydroxyacetophenone analogs, the average error was calculated and graphs of the average values were created (Graphs II and III).

<Table III> Shikimi Gunbai nymph secretion and Shikimi leaf extract

<Graph I> Blocking circle size of compounds secreted by Illicium anisatum nymph

<Table IV> 2,6-DHP12 and 2,6-DHA and their analogs

<Graph II> Analog block size (vs. S. aureus)

<Graph III> Analog block size (vs. E. coli)

2.3 Examination of minimum inhibitory concentration

<Method>
The growth of the target bacteria in the liquid medium supplemented with the test compounds of each concentration was observed, and the minimum inhibitory concentration of each compound was determined (Fig. IV). The test medium was 4.5 mL of liquid medium, 100 μL of preculture solution, and 400 μL of test compound DMSO solution, for a total of 5 mL. Preculture was performed overnight in the liquid medium described above and OD _{660 nm} was measured prior to addition to the test medium. Since it is desirable that the OD _{660 nm} of the preculture is about 0.5 (OD and the number of cells are not proportional to each other when it is 0.6 or more), it is necessary to consider an appropriate culture time (preculture OD ₆₆₀ in the first experiment). _nm is 2.498, which is well above the standard). For control, 400 μL of DMSO alone was added. The inhibition concentration is determined by inoculation / observation on OD _{660 nm} and agar medium after 24 hours. In the first experiment, the increase in turbidity of the test medium due to the test compound was not taken into consideration, so it was not possible to measure pure OD _{660 nm} due to the increase in the number of cells. The OD _{660 nm of} each medium should be measured before the start of culture.

<Fig. IV Antibacterial activity test by MIC method>

In the second test, the OD of each medium was measured at _{660 nm} before the start of culture. 1.5 mL was placed in an Eppen tube for measurement before the start of culture, and shaking culture was performed at 3.5 mL.

<Result>
Inoculate S. aureus culture medium containing 2,6-DHA, 2,6-DH6, 2,6-DH8, 2,6-DH10, and 2,6-DH12 at each concentration in Table V below into an agar medium. The results of the first experiment were summarized. Bacteria grew normally in the control.

<Table V> Examination of minimum inhibitory concentration of 2,6-DH12 and its analogs (25-800 μg / mL)

Table VI below summarizes the test results at lower concentrations of 2,6-DH6, 2,6-DH8, 2,6-DH10, and 2,6-DH12. The numbers in the table are OD _{660 nm} , and the numbers in parentheses are the turbidity before shaking culture. The OD of _{660 nm} after culturing the control was 1.62 (0.25 before culturing), and when inoculated on an agar medium, it grew normally.

<Table VI> Examination of the minimum inhibitory concentration of 2,6-DH12 and its analogs (1-32 μg / mL)

<Discussion>
In this chapter, the biological activities of these compounds, focusing on the acetphenone analogs 2,6-dihydroxyacetophenone (2,6-DHA) and 1- (2,6-dihydroxyphenyl) dodecan-1-one (2,6-DH12). As a part of elucidating the function, we investigated the antibacterial activity of Staphylococcus aureus, a gram-positive bacterium, and Escherichia coli, a gram-negative bacterium. The paper disc method was adopted as the qualitative test, and the minimum inhibitory concentration (MIC method) was adopted as the quantitative test.

The paper disc method is a method for determining the presence or absence of antibacterial activity by forming a blocking circle with a test compound on an agar medium mixed with the target bacteria. Although a simple antibacterial activity evaluation is possible, it should be noted that the size of the blocking circle depends on the physical properties of the compound and does not reflect the strength of the antibacterial activity. When the secretion of Staphylococcus aureus was extracted and the antibacterial activity test was performed by the paper disk method, it showed activity against S. aureus (in 2018, the occurrence of Staphylococcus aureus was low in Shikimi such as on the campus of Kyoto Gakuen University. , E. Not tested on coli). In order to investigate which compound is the main compound showing antibacterial activity, 9 compounds of larvae secretion were individually tested, and 2,6-DHA, 2,6-DH12, and 5-HCh7 blocked circles. Been formed.

2,6-DHA was active against S. aureus and E. coli at 300 μg, and 2,6-DH12 was active against both bacteria at 30 μg. 5-HCh7 showed activity only in S. aureus at a treatment dose of 300 μg. 5-HCh7 is a chromanone cyclized between the phenolic hydroxyl group of the aromatic ketone and the side chain, and it is speculated that such cyclization reduces the activity. When these three compounds were combined and tested, the test for S. aureus showed 2,6-DHA + 2,6-DH12, 2,6-DH12 + 5-HCh7, 2,6-DHA + 2,6. -DH12 + 5-HCh7 formed a blocking circle at 30 μg, resulting in almost the same blocking circle size. We also obtained the result that the average value of the blocking circles of 2,6-DHA + 5-HCh7 was the largest, but this result was more variable than that of other compounds, and no blocking circle was formed at 30 μg. .. E. In tests on coli, 2,6-DHA + 2,6-DH12 had a blocking circle size that was about the same as 2,6-DH12 alone. From these results, it is considered that the main substance showing antibacterial activity among the nymph secretions of Illicium anisatum is 2,6-DH12.

In order to investigate the origin of these compounds, hexane extraction and steam distillation were performed on the leaves of Illicium anisatum, which is the host plant of Illicium anisatum, and the antibacterial activity of both extracts obtained was examined. Neither of them formed a blocking circle in the antibacterial activity test by the paper disc method. GC-MS analysis was also performed, but no compound consistent with larvae secretion was detected, and it was found that the components of the leaves of Shikimi did not contain antibacterial active components derived from larvae (Fig. V).

<Fig. V> GC chromatogram of the extract of Shikimi leaf

In order to further deepen the knowledge about 2,6-DHA and 2,6-DH12 that had antibacterial activity, antibacterial activity tests were conducted by the paper disc method using synthetic products or standard products for these analogs. Compounds with extended side chain carbon chains are 2,6-DHA, 2,6-DH4, 2,6-DH6, 2,6-DH8, 2,6-DH10, 2,6-DH12, 2,6. -Examined DH14. At a treated dose of 30 μg, inhibition circles were formed at 2,6-DH 4, 6, 8, 10, and 12 for both S. aureus and E. coli. At 2,6-DHA and 2,6-DH14, a blocking circle was formed by treatment with 300 μg. From this, it was speculated that the carbon chain of the side chain became more active at 4 to 12.

Next, the positional isomers of 2,6-DH12 were examined using 2,5-DH12, 2,4-DH12, and 2,4,6-TH12. Both diol compounds formed a blocking circle in S. aureus when treated with 300 μg, and only 2,5-DH12 formed a blocking circle against E. coli when treated with 300 μg. It can be seen that the activity is weaker than 2,6-DH12 because 30 μg does not form a blocking circle. Triol 2,4,6-DH12 with added hydroxyl groups showed activity against S. aureus even when treated with 30 μg, but not against E. coli. From these results, it is considered that the position of the hydroxyl group is important in order to have antibacterial activity. Furthermore, the number of hydroxyl groups is also important, and 2,4,6-TH12, which has hydroxyl groups added at the 4-position even though it holds the hydroxyl groups at the 2- and 6-positions, which should show activity, has an activity against E. coli. lost. The positional isomers of 2,6-DHA were examined with 2,4-DHA, 2,5-DHA, and 3,5-DHA, but none of the compounds showed antibacterial activity. We also investigated 2,6-DHATf in which two hydroxyl groups were protected with 2-HA, 3-HA, 4-HA and trifluoromethanesulfonate, which had one hydroxyl group, but none of them showed activity. From these facts, it is considered that the 2,6-DHA type compound loses its activity due to the substitution position of the hydroxyl group, the reduction and protection of the hydroxyl group.

For 2,6-DHA, 2,6-DH6, 2,6-DH8, 2,6-DH10, and 2,6-DH12, which show remarkable activity, the minimum inhibitory concentration was further investigated and a quantitative evaluation was attempted. It was. S. aureus cultured in a liquid medium to which each test compound was added was applied to an agar medium, and the growth condition after 24 hours was observed. As a result, the growth of the bacterium was observed with the control in which only DMSO was added to the liquid medium and 25, 50, 100, 200 μg / mL of 2,6-DHA. From this, it is estimated that the minimum inhibitory concentration of 2,6-DHA for 24 hours is 400 μg / mL. Since the growth of other compounds was inhibited even at 25 μg / mL, it was considered necessary to study at lower concentrations, and growth inhibition at concentrations of 1, 2, 4, 8, 16, and 32 μg / mL was considered. Was observed. When inoculated on an agar medium as in the previous time, growth was confirmed at 1 to 32 μg / mL for 2,6-DH6. Growth was confirmed at 1 to 4 μg / mL for 2,6-DH8, 1 and 2 μg / mL for 2,6-DH10, and 1 to 4 μg / mL for 2,6-DH12. The results of 2,6-DH6 were inconsistent with the initial results of growth inhibition at 25 μg / mL, so further trials are needed, but growth was inhibited at a concentration of at least 50 μg / mL. Growth was inhibited at 8 μg / mL for 2,6-DH8, 4 μg / mL for 2,6-DH10, and 8 μg / mL for 2,6-DH12. Summarizing these results, 2,6-DH10 has the lowest concentration of 4 μg / mL, 2,6-DH8 and 2,6-DH12 have similar concentrations of 8 μg / mL, and 2,6-DH6 has 50 μg / mL. , 2,6-DHA was found to inhibit growth at 400 μg / mL.

Regarding the absorbance measurement in the MIC test, there is room for improvement in the measurement method. In the first test, the medium was colored by the DMSO solution, and it was difficult to measure the turbidity after culturing. Therefore, the measurement was also performed before culturing. This time, the absorbance before culturing was almost the same in the medium to which any sample was added, and no noticeable coloring or turbidity was observed when the DMSO solution was added, which was around 0.25. In the measurement after culturing, there was a sample that grew when inoculated even if the absorbance was considerably low. Therefore, in this test as well, the minimum inhibitory concentration was determined by inoculation on an agar medium without using the absorbance as a criterion. For the measurement of OD _{660 nm} , it is necessary to study the measurement method in the future.

The mechanism of action of these compounds has not been elucidated. Antibacterial agents include inhibition of bacterial cell wall synthesis represented by penicillins, damage to bacterial cell membranes represented by polymyxin B, inhibition of protein synthesis represented by aminoglycosides and tetracyclines, inhibition of nucleic acid metabolism such as kilonones, etc. , Can be classified by mechanism of action (Uematsu et al., Simple pharmacology). The S. aureus and E. coli targeted this time are Gram-positive and Gram-negative bacteria, respectively. Gram-negative bacteria have a cell wall on the outside of the cell membrane and an outer membrane on the outside. The cell wall of Gram-negative bacteria is about 8 nm. On the other hand, Gram-positive bacteria do not have an adventitia, but have a very thick cell wall of about 250 nm. The antibacterial spectrum needs to be examined to identify the mechanism of action.

2.4 Evaluation of antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) For the above 2,6-DH8, 2,6-DH10 and 2,6-DH12, ask the Japan Food Research Laboratories (general foundation). Antibacterial activity evaluation (measurement of minimum inhibitory concentration) against methicillin-resistant Staphylococcus aureus (MRSA) was performed (Test Report No. 19014456001-001, February 28, 2019 Requester: Kyoto Gakuen University). As a result, it was confirmed that it has a very high antibacterial activity. The contents of the test report are described below.

<Sample>
8000 μg / mL ethanol solution of 2,6-DH8, 2,6-DH10 and 2,6-DH12 (submitted by the inventors to the Japan Food Research Laboratories on February 06, 2019).
<Examination outline>
A liquid medium to which a sample was added at an arbitrary concentration was dispensed into a 96-well microplate (hereinafter referred to as a “sensitivity measurement plate”), and then the test bacterial solution was inoculated. After culturing, the concentration at which the growth of the bacterium was inhibited was defined as the minimum inhibitory concentration.

<Test result>
Minimum inhibitory growth concentration (MIC) (μg / mL)
2,6-DH8 2.5
2,6-DH10 1.25
2,6-DH12 1.25

<Test conditions>
・ Test bacterial solution
-Test bacterium: Staphylococcus aureus IID 1677 (methicillin-resistant staphylococcus: MRSA)
- pre-culture: Mueller Hinton Agar (Difco), 35 ℃ ± 1 ℃, 18~24 hours bacterial suspension adjusted solution: Saline number of bacteria: about 10 ⁷ mL
・ Sensitivity measurement medium: Mueller Hinton Broth (Difco)
-Sensitivity measurement plate: The concentration of the sample stock solution was set to 8000 μg / mL. A sample dilution solution was prepared by diluting the sample in steps of 2 times with 99.5% ethanol. Next, the stock solution of the sample and the diluted solution of each sample were added to the susceptibility measurement medium in an amount of 1/99, and 100 μL was dispensed into a 96-well microplate.
-Test operation and determination method: After inoculating 5 μL of the test bacterial solution into each well of the susceptibility measurement plate and culturing for a predetermined time, the minimum concentration at which the growth of the bacteria was inhibited was defined as the minimum inhibitory concentration.
・ Plate culture conditions for sensitivity measurement: 35 ° C ± 1 ° C, 18 to 24 hours

2.5 Antibacterial textile products and their evaluation
Immerse in an ethanol solution (0.1%; mass volume percent concentration) in which 2,6-DH12 is dissolved in a surgical mask cloth piece (1 cm x 1 cm, polypropylene non-woven fabric; AG Clean of Azeas Co., Ltd.) and dry it. As a result, an antibacterial textile product was produced. By precisely measuring the weight of the obtained gauze cloth after drying, it was confirmed that about 5.6% was adsorbed with respect to the weight of the mask cloth piece. As a result of testing a medical gauze cloth piece (1 cm x 1 cm, cotton; Kawamoto Sangyo's medical gauze) by the same method, it was found that about 3.6% of the weight of the gauze cloth piece was adsorbed. I confirmed.

The surgical mask cloth piece after the treatment and the original surgical mask cloth piece without the treatment were allowed to stand in a medium containing non-resistant Staphylococcus aureus according to the above paper disk method, and 24 hours later. As a result of observing the situation, the growth of Staphylococcus aureus was remarkably suppressed around the surgical mask cloth piece after the treatment. When the same test was performed on the above-mentioned medical gauze cloth pieces, the growth of Staphylococcus aureus was remarkably suppressed around the treated gauze cloth pieces.

2.5 Evaluation of antibacterial activity against vancomycin-resistant enterococci (VRE) For the above 2,6-DH8, 2,6-DH10 and 2,6-DH12, we requested the Japan Food Research Laboratories (general foundation) to perform vancomycin. Antibacterial activity evaluation (measurement of minimum inhibitory concentration) against resistant enterococci (VRE) was performed (test report bulletin reception number 19014456 March 22, 2019 Requester: Kyoto Gakuen University). As a result, it was confirmed that it has a very high antibacterial activity.

<Sample>
8000 μg / mL ethanol solution of 2,6-DH8, 2,6-DH10 and 2,6-DH12 (submitted by the inventors to the Japan Food Research Laboratories).
<Examination outline>
A liquid medium to which a sample was added at an arbitrary concentration was dispensed into a 96-well microplate (hereinafter referred to as a “sensitivity measurement plate”), and then the test bacterial solution was inoculated. After culturing, the concentration at which the growth of the bacterium was inhibited was defined as the minimum inhibitory concentration.

<Test result>
Minimum inhibitory growth concentration (MIC) (μg / mL)
2,6-DH8 5.0
2,6-DH10 2.5
2,6-DH12 2.5

<Test conditions>
The test conditions were the same as in "2.4 Evaluation of antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA)" except that the test bacterium was Enterococcus faecium (vancomycin-resistant enterococcus: VRE).

2.6 Evaluation of antifungal activity against 4 types of fungi For multiple types of compounds including the above 2,6-DH12, we requested the Sanitary Microbial Research Center Co., Ltd. to perform antifungal activity against 4 types of fungi (molds). (Antifungal property) was evaluated (Test inspection report: Test request number 2019D-BT-096, March 15, 2019 Test requester: Faculty of Bioenvironment, Kyoto Gakuen University). As a result, it was confirmed that it has a very high antifungal activity. Below, the part related to 2,6-DH12 in the contents of the test report is described below.

<Purpose of test>
The antifungal activity of the sample will be investigated by changing some test conditions based on the antibacterial product technology council shake method.
<Sample>
2,6-DH12
<Test mold>
Aspergillus brasiliensis NBRC 105649
Penicillium citrinum NBRC 6352
Alternaria alternate NBRC 106339
Fusarium oxysporum NBRC 31631

<Test method>
・ Preparation of test spore solution The test mold was inoculated on potato dextrose agar medium (PDA), cultured at 25 ° C for 7 to 14 days, spores were harvested using 0.05% Tween80-added sterile water, and phosphate buffered saline was added. what number of spores was adjusted to 10 ⁵ CFU / mL was tested spore solution used.
-Preparation of test sample A mixture of 10 mg of sample and 3 mL of dimethyl sulfoxide was used as the test sample.
・ Shaking treatment and measurement of viable cell count 10 mL each of the test spore solution was dispensed into a centrifuge tube, and 0.2 mL of the test sample was inoculated therein. Then, it was shaken at 25 ° C. and 150 rpm for 24 hours. After the treatment, a 10-fold dilution series of the shaking solution was prepared using physiological saline and inoculated into a neutralizing agent-added Saburo dextrose agar medium (SDLPA). The inoculated SDLPA was cultured at 22.5 ± 2.5 ° C for 3 to 5 days, the number of colonies formed was counted, and the viable cell count was calculated.

<Test result>
・ Antifungal test results for Aspergillus brasiliensis (live cell count measurement results (CFU / mL))
Average value Each test piece value
2,6-DH12 1.0 × 10 ² 2.7 × 10 ²
1.0 × 10 ¹
3.0 × 10 ¹
Control 7.3 × 10 ⁴ 7.7 × 10 ⁴
7.7 × 10 ⁴
6.4 × 10 ⁴

・ Antifungal test results for Penicillium citrinum (live cell count measurement results (CFU / mL))
Average value Each test piece value
2,6-DH12 1.3 × 10 ² −
3.0 × 10 ¹
1.0 × 10 ¹
Control 2.5 × 10 ⁶ 1.6 × 10 ⁶
2.4 × 10 ⁶
3.4 × 10 ⁶

・ Antifungal test results for Alternaria alternata (live cell count measurement results (CFU / mL))
Average value Each test piece value
2,6-DH12 2.0 × 10 ¹ 2.0 × 10 ¹
−
4.0 × 10 ¹
Control 1.1 × 10 ⁴ 1.7 × 10 ⁴
1.2 × 10 ⁴
4.0 × 10 ⁴

・ Antifungal test results against Fusarium oxysporum (live cell count measurement results (CFU / mL))
Average value Each test piece value
2,6-DH12 4.3 × 10 ⁵ 5.0 × 10 ⁵
7.0 × 10 ⁵
1.0 × 10 ⁵
Control 3.5 × 10 ⁶ 3.0 × 10 ⁶
2.7 × 10 ⁶
4.8 x 10 ⁶

3. 3. Comprehensive consideration (summary)
As described above, the antibacterial activity of the exocrine secretion of Illicium anisatum and its analogs was investigated. The structure-activity relationship study will be considered below.

Stephanitis svensoni is an insect of the family Tingidae of the order Hemiptera, Tingidae, and is a sucking pest of Illicium anisatum. The nymphs of this insect are decanal, dodecanal, 2-undecanone, 3-oxododecanal (houttuynin), 2,6-dihydroxyacetophenone, 1- (2,6-dihydroxyphenyl) dodecan-1-one, 5-hydroxy-2-alkylchromanone, etc. It secretes nonaicosane. Among these, 5-hydroxy-2-alkylchromanones are compounds peculiar to Tingidae, which are also found in the secretions of nymphs of Tingidae. 3-oxododecanal has been isolated from Houttuynia cordata and has been reported to have antibacterial, antiviral and anti-inflammatory effects (L. Jinbing et al, 2009). 2,6-Dihydroxyacetophenone has a repellent activity against ants (patent pending in our laboratory) and has a prostaglandin H synthase inhibitory activity superior to that of aspirin (Jurenka, R.A et al, 1989). It has been reported that 1- (2,6-dihydroxyphenyl) dodecane-1-one has antibacterial activity against the gram-positive bacterium Clavibacter michiganensis and growth inhibitory effect on nematodes (John W. Neal). , JR., Et al, 1995), prostaglandin H synthase inhibitory activity comparable to aspirin (Jurenka, RA et al, 1989).

The biological activity of these compounds, centered on the acetophenone analogs 2,6-dihydroxyacetophenone (2,6-DHA) and 1- (2,6-dihydroxyphenyl) dodecan-1-one (2,6-DH12) As part of the elucidation, the antibacterial activity of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli was investigated. As a method, the paper disc method was adopted as a qualitative test, and the minimum inhibitory concentration (MIC method) was adopted as a quantitative test. When tested on 9 compounds of larvae secretion, blocking circles were formed at 2,6-DHA, 2,6-DH12, and 5-HCh7. 2,6-DHA was active against S. aureus and E. coli at 300 μg, and 2,6-DH12 was active against both bacteria at 30 μg. 5-HCh7 showed activity only in S. aureus at a treatment dose of 300 μg. 5-HCh7 is a cyclized acetophenone, and it is presumed that the activity is reduced by cyclization in this way. When a test was conducted in combination with these three compounds, it is considered that 2,6-DHP12 is the main substance showing antibacterial activity among the secretions of Illicium anisatum. In order to investigate the origin of these compounds, the antibacterial activity of hexane extract and essential oil was examined for the leaves of Shikimi, which is eaten by Shikimi Gunbai, but neither formed a blocking circle, and GC-MS analysis also showed larvae secretions. Since no peak consistent with is detected, it is speculated that the young worms of Illicium anisatum are biosynthesizing these antibacterial active ingredients.

In order to further deepen the knowledge of 2,6-DHA and 2,6-DH12 that had antibacterial activity, antibacterial activity tests were conducted on these analogs. 2,6-DHA, 2,6-DH4, 2,6-DH6, 2,6-DH8, 2,6-DH10, 2,6-DH12 with the carbon chain of the side chain of 2,6-DHA extended , 2,6-DH14 was examined, and it was speculated that the activity became stronger when the carbon chain was 4 to 12. As for the positional isomer of 2,6-DH12, 2,5-DH12, 2,4-DH12, 2,4,6-THP12 were examined, but both diols were more active than 2,6-DH12. Triol 2,4,6-DH12 with a weak hydroxyl group is S. Although it has the same activity as 2,6-DH12 against aureus, it is E. Lost activity against coli. For position isomers of 2,6-DHA, 2,4-DHA, 2,5-DHA, 3,5-DHA, and 2-HA, 3-HA, 4-HA, and trifluoromethanesulfonate, which have one hydroxyl group, form hydroxyl groups. The study was conducted using protected 2,6-DHATf, but no activity was observed in all compounds. From this, it is considered that the activities of 2,6-DH12 and 2,6-DHA are significantly weakened by the substitution position of the hydroxyl group, the reduction and protection of the hydroxyl group.

In the quantitative evaluation by examining the minimum inhibitory concentration, in the initial test in which the test compound concentration was set to 25 to 800 μg / mL, the control in which only DMSO was added to the liquid medium and 25, 50, 100 of 2,6-DHA were used. Bacterial growth was observed at 200 μg / mL. From this, it was determined that the minimum inhibitory concentration of 2,6-DHA for 24 hours was 400 μg / mL. As for other compounds, growth was inhibited even at 25 μg / mL, so lower concentrations were investigated. In this test, 2,6-DH10 grew at the lowest concentration of 4 μg / mL, 2,6-DH8 and 2,6-DH12 grew at the same level of 8 μg / mL, and 2,6-DH6 grew at at least 50 μg / mL. Turned out to block. It must be kept in mind here that the MIC value is a numerical value with a certain range. Since there are differences in the growth state depending on the type of bacteria to be measured and the drug mechanism, it should be considered that the numerical value includes the possibility of fluctuation due to various factors (Tamura et al., MIC measurement system). Problems). The mechanism of action of these compounds has not been elucidated and needs to be investigated in detail in the future.

This study confirmed that 2,6-DH8, 2,6-DH10 and 2,6-DH12 have high antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. It was. In addition, 2,6-DH12 showed antifungal activity against the fungi Aspergillus brasiliensis, Penicillium citrinum, and Alternaria alternata. This has the potential to be applied as an external preparation for skin, cosmetics, and disinfectants, antibacterial agents, antifungal agents, and fungicides applied to hands and fingers, all of which are of the present application. It can be said to be an antibacterial / antifungal agent in the invention. In addition, antibacterial textile products used as masks, aprons, clothes, etc. worn by patients and medical professionals, as bedding materials such as sheets, duvet covers, pillowcases, or as footwear inlays and curtains. And antifungal textile products can be obtained. In particular, since all of them are active against a plurality of types of resistant bacteria and fungi, their usefulness and convenience are very high.

Insect secretions, which are said to account for half of all living organisms, can be a source of search for bioactive substances useful for humans, including antibacterial peptides. In addition, the use of previously untouched microbial resources such as marine microorganisms is attracting attention in search of new antibacterial active substances, but microorganisms in the habitat of insects can also be sufficiently used as search resources. For example, Cordyceps sinensis, which parasitizes ghost moth larvae, and Cordyceps militaris, which parasitizes Lepidoptera larvae, have found cordycepin as an antibacterial active substance, although Cordyceps sinensis is famous as a fungus that parasitizes insects (Y. -J.Ahn, 2000). However, even in Cordyceps sinensis, it is only recognized as one of the drug discovery resources, and at present, microorganisms related to insects are almost unused (Kobayashi et al., 2014). We hope that the search for useful substances targeting these insect-derived microorganisms will further develop.

4. References
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Yoshinari Enami Preventing seedling wilt by using mites that eat pathogenic fungi (2003) News from Tohoku Agricultural Research Center 8

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Claims (5)

An antibacterial agent against methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci, which contains at least one of the following compounds 1 to 3 as an active ingredient.
Compound 1
2,6-DH8
Compound 2
2,6-DH10
Compound 3
2,6-DH12 The antibacterial agent according to claim 1, wherein the total concentration of the compounds 1 to 3 is 2 μg / mL or more. An antifungal agent containing at least one of the following compounds 1 to 3 as an active ingredient.
Compound 1
2,6-DH8
Compound 2
2,6-DH10
Compound 3
2,6-DH12 A mask or other antibacterial textile product that adsorbs or contains the antibacterial agent of claim 1. A mask or other antifungal textile product that adsorbs or contains the antifungal agent of claim 3.