Antimicrobial and antifungal agents for drug-resistant Staphylococcus aureus or vancomycin-resistant enterococci, derived from lace bugs or their synthetic analogues.

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

Technical Field

[0001] Antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA) or vancomycin-resistant Enterococcus (VRE), antifungal agents, and antibacterial and antifungal fiber products using compounds derived from natural products and their synthetic analogs.

Background Art

[0002] Antibiotics have been defined as "substances produced by microorganisms that inhibit the growth of other microorganisms," but currently, antibacterial agents, antifungal agents, and antiviral agents derived from microorganisms are also broadly regarded as antibiotics. Compounds and synthetic products derived from sources other than microorganisms have been called antibacterial substances and distinguished, but in this study, a new search source for "antibacterial substances" that inhibit the growth of bacteria regardless of their origin was sought and screening was conducted.

[0003] Research on antibiotics has mainly been carried out using soil actinomycetes centered on the genus Streptomyces. Among them, there are quite a few that have been put into practical use as pharmaceuticals and agricultural chemicals, including penicillin, etc., but it is said that it will be difficult to discover antibacterial active substances with new skeletons from the soil in the future. Even in soil where it is said that exploration has been exhausted, there are microorganisms that cannot be cultured with current technology, so it cannot be said that it is impossible to search for new antibacterial substances, but attention is gathering on microorganisms in environments untouched by humans, such as deep-sea microorganisms, as search resources for useful substances. As a background for seeking new compounds, there is always a problem of drug-resistant bacteria (especially methicillin-resistant Staphylococcus aureus (MRSA)) in the research on antibacterial substances. MRSA has become a dominant bacterium in hospitals and other places where there is a selection pressure of conventional antibacterial drugs, and conventional infection control has no effect (Ishii, 2018). In order to inhibit the occurrence of such bacteria, new compounds with different origins and mechanisms of action must be found.

[0004] Insects, which are said to account for more than half of all living organisms, have been identified with various secondary metabolites associated with biological interactions. Microorganisms are also familiar to insects. Some microorganisms live together with insects as symbiotic bacteria, while others can be enemies that infect insects. Antibacterial peptides are well-known as a defense mechanism against bacterial infections in insects. When infected with bacteria, these peptides are transiently synthesized in the body and secreted into the body fluid. Representative examples include apidicin of honeybees, thanatin of stinkbugs, and defensin of stag beetles. It has been found that insect antibacterial peptides disrupt the cell membranes of bacteria. Insect defensin, consisting of 43 amino acid residues isolated from stag beetles, exhibits antibacterial activity against Gram-positive bacteria and also shows activity against MRSA (Furukawa et al., 2004). There are also examples of insects secreting antibacterial substances on their body surfaces to prevent bacterial infections. 1-(2,6-dihydroxyphenyl)dodecan-1-one secreted by nymphs of leafhoppers of the genera Stephanitis and Corythucha was examined for antibacterial activity against 4 species of Gram-positive bacteria including Clavibacter michiganense, the causative agent of tomato canker, and 5 species of Gram-negative bacteria including Agrobacterium tumifaciens, the causative agent of crown gall, and it showed antibacterial activity against Clavibacter michiganense (John W. Neal, JR., et al, 1995). In this paper, structure-activity relationship studies have been carried out, and cyclic compounds and compounds with a phenyl group substituted at the end of the side chain have been examined. These compounds are mainly said to show activity against Gram-positive bacteria. On the other hand, in this paper, similar antifungal activity studies using 1-(2,6-dihydroxyphenyl)dodecan-1-one were carried out against a total of 9 species of fungi, but no antifungal activity was observed against any of the fungi. The nymphs of the Japanese maple leafhopper targeted in this study also secrete 1-(2,6-dihydroxyphenyl)dodecan-1-one.

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] 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. [Non-Patent Document 2] Abstracts of the 2018 Annual Meeting of the Japan Society for Bioscience, Biotechnology, and Agrochemistry (published March 5, 2018), Presentation Number: 3A18p10: Exploration of antimicrobial substances contained in the secretions of the nymphs of the Japanese star anise lace bug. [Overview of the project] [Problems that the invention aims to solve]

[0006] This invention provides antimicrobial agents and antifungal agents that have high antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), or other resistant bacteria, as well as antimicrobial textile products using compounds derived from natural products that are presumed to have low toxicity, and synthetic analogues thereof. [Means for solving the problem]

[0007] Against this backdrop, we conducted a search for antimicrobial substances using insects as a source. In this study, we evaluated the antimicrobial activity against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli, and advanced structure-activity relationship studies focusing on the side chain length and hydroxyl group position of 1-(2,6-dihydroxyphenyl)dodecan-1-one secreted by the nymph of the Japanese star anise lace bug. In particular, we confirmed its effects against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE).

[0008] According to a preferred embodiment of the present invention, the present invention provides an antimicrobial agent and antifungal agent against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE), comprising at least one of the following compounds 1 to 3 as an active ingredient. Alternatively, the present invention provides a textile product using such an antimicrobial and antifungal agent. compound 1 TIFF0007838777000001.tif32135 compound 2 TIFF0007838777000002.tif34135 compound 3 TIFF0007838777000003.tif36135 [Modes for carrying out the invention]

[0009] 1. Secretions of the nymphalid lace bug and its related organisms Stephanitis svensoni, also known as the Japanese star anise lace bug, is an insect belonging to the family Tingidae in the order Hemiptera, and is a pest that feeds on the leaves of Illicium anisatum (Schisandraceae). The nymphs of the Japanese star anise lace bug secrete many interesting compounds from the spines on their backs. In our laboratory, we extracted the nymph secretions with hexane and analyzed them using gas chromatography-mass spectrometry (GC-MS), detecting decanal, dodecanal, 2-undecanone, 3-oxododecanal (houttuynin), 2,6-dihydroxyacetophenone, 1-(2,6-dihydroxyphenyl)dodecan-1-one, 5-hydroxy-2-alkylchromanones, and nonaicosane (Figure I). Among these, 5-hydroxy-2-alkylchromanones are compounds unique to lace bugs and are also found in the secretions of the crested star anise nymphs. 3-Oxododecanal was 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 ant repellent activity (patent application filed in our laboratory) and possesses superior prostaglandin H synthase inhibitory activity compared to aspirin (Jurenka, RA et al, 1989). 1-(2,6-Dihydroxyphenyl)dodecan-1-one has been reported to have remarkable antibacterial activity against the Gram-positive bacterium Clavibacter michiganensis, which causes bacterial leaf blight on maize, and to have growth inhibitory effects on nematodes (John W. Neal, JR., et al, 1995), and possesses prostaglandin H synthase inhibitory activity comparable to aspirin (Jurenka, RA et al, 1989). In this chapter, we focused on 2,6-dihydroxyacetophenone and 1-(2,6-dihydroxyphenyl)dodecan-1-one as aromatic ketones, and investigated their antibacterial activity against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli as part of our efforts to elucidate the biological activity of these compounds.We also verified the antifungal activity of 1-(2,6-Dihydroxyphenyl)dodecan-1-one against four types of fungi.

[0010] <Figure I: GC chromatogram of secretions from nymphs of the Japanese star anise lace bug> TIFF0007838777000004.tif103153

[0011] 1.1 Test Compounds • Essential oil and hexane extract of Japanese star anise leaves • 1-(2,6-dihydroxyphenyl)dodecan-1-one and 2,6-dihydroxyacetophenone and their analogues • Nine compounds from the secretions of the nymphalid lace bug, excluding 3-oxododecanal.

[0012] The structures and abbreviations of the test compounds are shown below (Table I). Hereafter, abbreviations will be used.

[0013] [Table 1]

[0014] The following are abbreviations for compounds similar to 2,6-DHA positional isomers, and these abbreviations will be used hereafter (Table II).

[0015] [Table 2]

[0016] The mass spectra of the secretions of the nymphs of the Japanese star anise lace bug used in the test are shown below (Figures II-1 to II-3).

[0017] <Figure II-1> TIFF0007838777000007.tif149170

[0018] <Figure II-2> TIFF0007838777000008.tif78170

[0019] <Figure II-3> TIFF0007838777000009.tif174170

[0020] 1.2 Synthesis method Synthesis of 2,6-DH4, 2,6-DH6, 2,6-DH8, 2,6-DH10, and 2,6-DH14

[0021] TIFF0007838777000010.tif79155

[0022] <Synthesis of Intermediate A> In CH2Cl2 (5 mL) as the solvent, 0.56 g (5 mmol) of 1,3-cyclohexanedione was reacted with an arbitrary carboxylic acid anhydride (5 mmol) and pyridine (0.44 g, 5.57 mmol) at room temperature for 30 minutes. The solvent was concentrated under reduced pressure, and after liquid-liquid extraction with hexane / diethylether (1:1) and cooled 1N hydrochloric acid, the mixture was dried over anhydrous Na2SO4. After concentrating the solvent under reduced pressure using an evaporator, the crude extract was purified by silica gel column chromatography [hexane / Depositphotos (3:1)].

[0023] A4 (R=3) 1 H-NMR (CDCl3) 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

[0024] A6 (R=5) 1H-NMR (CDCl3) 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

[0025] A8 (R = 7) 1 H-NMR (CDCl₃) data not available GC-MS 17.189 min, 238(M + ), 127, 109, 57, 43

[0026] A10 (R = 9) <​​​​​​​​​​​​​​​​​​​​​Product A (3.4 mmol) was dissolved in 15 mL of CH3CN, and 1 mL of Et3N and 40 μL of acetonecyanohydrine were added. The mixture was reacted at room temperature for 12 hours. The solvent was concentrated under reduced pressure and separated with hexane / SiO2 (1:1), pure water, and dilute hydrochloric acid. After drying over anhydrous Na2SO4, the mixture was concentrated under reduced pressure using an evaporator.

[0029] 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

[0030] <Synthesis of target substance C> Product (2) (1.31 mmol) was dissolved in 5 mL of AcOH, and 21.26 g (0.39 mmol) of Hg(OAc) and 0.33 g of NaOAc were added. 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, and 1N hydrochloric acid was added and stirred for 30 minutes. The mixture was filtered through Celite and washed with hexane / siRNA (3:1). The mixture was washed with water, saturated NaHCO3 aq., and brine. The organic layer was dried over anhydrous Na2SO4, filtered through neutral alumina, and washed with siRNA. After concentration under reduced pressure, the mixture was purified by silica gel column chromatography [hexane / siRNA (5:1)]. The separated aqueous layer was neutralized and appropriately treated as Hg waste.

[0031] C4 (R=3) 1 H-NMR (CDCl3) 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

[0032] C6 (R=5) 1 H-NMR (CDCl3) 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

[0033] C8 (R=7) 1 H-NMR (CDCl3) 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

[0034] C10 (R=9) 1 H-NMR (CDCl3) 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

[0035] C14 (R=13) 1 H-NMR (CDCl3) 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

[0036] <Synthesis of 2,4-DH12> TIFF0007838777000011.tif30155

[0037] (i) Resorcinol (220 mg, 2 mmol) and lauric anhydride (773 mg, 2.02 mmol) were placed in a round-bottom flask, and BF3·OEt2 (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 stirred overnight. After suction filtration, the mixture was thoroughly washed with water. Since the target product is sparingly soluble in hexane, hexane was added and filtration was repeated several times. The target product (300 mg, 51%, Rf 0.23 for Hex:SiO2 (5:1)) was obtained.

[0038] 1 H-NMR (CDCl3) 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

[0039] <Synthesis of 2,5-DH12> TIFF0007838777000012.tif34155

[0040] The reaction was carried out using hydroquinone (220 mg, 2 mmol) as a starting material according to method (i) above, and a monoester (156 mg, 27%) was obtained. The Rf values ​​of the target product and the monoester were almost the same. The reaction proceeded similarly with catechol, and a monoester was also obtained. These monoesters were used as synthetic intermediates 4-HP12 and 2-HP12 for testing. The synthesis method for 2,5-DHP12 was changed to (ii).

[0041] (ii) Hydroquinone (550 mg, 5 mmol) and lauric acid (1.0 g, 5 mmol) were placed in a round-bottom flask, and BF3·OEt2 (1.08 ml, 8.5 mmol, d: 1.12 g / ml) was added dropwise at 80°C. After reacting at that temperature for 1 hour, the temperature was raised to 140°C and the reaction was continued for 2 hours. After cooling to 110°C, 5% Na2CO3aq. (15 ml) was added and the mixture was stirred for 30 minutes. After extraction with ethyl acetate, the mixture was washed with water and brine. After drying over anhydrous Na2SO4, the solvent was removed by distillation. The product was purified by silica gel column (30 g), and the target product had the same Rf value (0.29) as the monoester (Hex: SiO2 (4:1)). The sample consisted of a mixture of pale yellow and black solids. The target substance was a black (dark brown) solid (200 mg) that did not dissolve in 10% SiO2 / Hex, while the pale yellow solid did. Therefore, the separation was due to differences in solubility.

[0042] 1 H-NMR (CDCl3) 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) 13C-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

[0043] <2,4.6-TH12 synthesis> TIFF0007838777000013.tif30155

[0044] The reaction was carried out using phloroglucinol (252 mg, 2.61 mmol) as the starting material according to method (i) above, but the target product was not obtained, and only a monoester was obtained. Next, the reaction was carried out according to method (ii), but the result was the same. Therefore, the synthesis method was changed to (iii).

[0045] (iii) Phloroglucinol (329 mg, 2.61 mmol) and lauric anhydride (1.0 g, 2.61 mmol) were dissolved in anhydrous THF (4 ml), and BF3·OEt2 (0.82 ml, 6.53 mmol) was added at room temperature under a nitrogen atmosphere. After reacting for 48 hours, the mixture was diluted with ethyl acetate and washed three times with 1 M HCl. After washing with brine, the mixture was dried over anhydrous Na2SO4, and the solvent was removed by distillation. The mixture was purified by silica gel column (20 g, 30% HCl / Hex), but the target product had the same Rf value (0.45) as the monoester (Hex: HCl (1:1)). To separate these two components, the ester was hydrolyzed with LiOH.

[0046] The mixture (400 mg, 1.3 mmol) was dissolved in MeOH:H2O (3:1), and LiOH·H2O (109 mg, 2.6 mmol) was added at 0°C. The mixture was reacted overnight at room temperature. 1 M HCl was added to adjust the pH to approximately 2, and the mixture was extracted with ethyl acetate. After washing with brine, the mixture was dried over anhydrous Na2SO4. After removing the solvent by distillation, the mixture was purified using a silica gel column (10 g, 30% siRNA / Hex) to obtain 150 mg (19%) of the target product.

[0047] 1 H-NMR (CDCl3) 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

[0048] <Chemical analysis> 1 H and 13 The 1C nuclear magnetic resonance (NMR) spectrum was obtained using a Bruker Biospin AC-400M instrument. 1 H: 400MHz, 13 The measurement was performed using a 100MHz (C) system with tetramethylsilane (TMS) in deuterated chloroform as the internal standard.

[0049] For GC-MS analysis, a 5975 MSD quadrupole mass filter mass spectrometer (Agilent Technologies) connected to a 6890 N gas chromatograph (Agilent Technologies) was used, and measurements were performed using the electron ionization method (70 eV). An HP-5MS capillary column (inner diameter: 0.25 mm, length: 30 m, film thickness: 0.25 μm, Agilent Technologies) was used, maintained at 60°C for 2 minutes, then increased to 290°C at a rate of 10°C / min, and held at that temperature for 5 minutes.

[0050] 2. Evaluation of antibacterial activity

[0051] 2.1 Preparation of bacteria for preliminary evaluation <Target strains> Staphylococcus aureus (NBRC 12732) Escherichia coli (NBRC 3972)

[0052] <Culture conditions> Staphylococcus aureus: 1 day at 37°C E. coli: 30°C for 1 day (300 rpm during shaking culture).

[0053] The culture medium was prepared to the following composition. • Liquid culture medium Tryptone 10 g Yeast extract 2 g MgSO41 g Distilled water 1 L agar medium Tryptone 10 g Yeast extract 2 g MgSO41 g Distilled water 1 L Agar 15g

[0054] <How to save> By creating a glycerol stock, long-term storage at -80°C is possible. Prepare 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 sterile cryotube, adjusting so that the final glycerol concentration is 20%. Add the glycerol to the cryotube first, and then add the culture medium by decantation after measuring it into a sterile Eppendorf tube. When using the bacteria, thaw them at room temperature, inoculate them onto the agar medium as described above, and then allow to stand at the respective culture temperature (1 day). Although it is possible to store and use the culture medium refrigerated at 4°C for about a week after inoculation, to ensure uniform conditions, we used the culture medium prepared from the glycerol stock the day before the antimicrobial activity test whenever possible.

[0055] 2.2 Preliminary evaluation using the paper disc method

[0056] <Material> Paper disc (Toyo Roshi; 8 mm DIA, Thin) Sterile Petri dish (Atect; SLE, 90mm diameter x 15mm width)

[0057] <Method> The presence or absence of antibacterial activity is determined by the formation of a zone of inhibition. The test medium was prepared by mixing a pre-culture, which had been cultured with shaking for 1 day in the liquid medium described above, with the agar medium described above. (The ratio was 0.5 mL of pre-culture per 25 mL of agar medium / petri dish.) 8 mm paper discs treated with the test compound were placed in the test medium and evaluated after 1 day of incubation. Paper discs treated with methanol, hexane, and ethyl acetate were also used as controls. The paper discs were air-dried on a glass plate for at least 1 hour to completely dry the solvent (Figure III).

[0058] <Figure III> Antimicrobial activity test using the paper disc method JPEG0007838777000014.jpg84139

[0059] <Result> The results of the paper disc method tests are summarized in the following tables. The tables list the compound name, number of tests, and inhibition zone size (mm). In Tables III and IV, the number of trials is indicated in parentheses, and "-" is used if no inhibition zone was formed. Tests were conducted on nymph secretions in which an inhibition zone was formed, using combinations of test compounds, and the results were compared with those of single compounds (Graph I). For the active compounds 1-(2,6-dihydroxyphenyl)dodecan-1-one and 2,6-dihydroxyacetophenone analogs, the mean error was calculated, and graphs of the mean values ​​were created (Graphs II and III).

[0060] Secretions of the nymphalid lace bug and extract of the Japanese star anise leaf [Table 3]

[0061] <Graph I> Inhibition zone size of compounds secreted by nymphs of the Japanese star anise lace bug JPEG0007838777000016.jpg61138

[0062] 2,6-DHP12 and 2,6-DHA and their analogues [Table 4]

[0063] <Graph II> Inhibition circle size of related species (vs. S. aureus) JPEG0007838777000018.jpg68168

[0064] <Graph III> Inhibition circle size of related species (vs. E. coli) JPEG0007838777000019.jpg76149

[0065] 2.3 Examination of the minimum inhibitory concentration for growth

[0066] <Method> The growth of target bacteria was observed in liquid media containing each concentration of the test compound, and the minimum inhibitory concentration of each compound was determined (Figure IV). The test medium consisted of 4.5 mL of liquid medium, 100 μL of pre-culture solution, and 400 μL of DMSO solution of the test compound, totaling 5 mL. Pre-culture was performed overnight in the aforementioned liquid medium, and OD was added before adding to the test medium. 660 nm The OD of the pre-culture was measured. 660 nm A value of around 0.5 is desirable (if it is 0.6 or higher, the OD and bacterial cell count will not be proportional), so it is necessary to consider the appropriate culture time (pre-culture OD in the first experiment). 660 nm The value was 2.498, which is considerably above the standard. The control group received 400 μL of DMSO alone. The inhibitory concentration was determined at 24 hours (OD). 660 nm The determination is made by inoculating the agar plate with bacteria and observing the results. In the initial experiment, the increase in turbidity of the test medium due to the test compound was not taken into consideration, resulting in a pure OD (Oxygen Demand) due to the increase in the number of bacterial cells. 660 nm The OD of each culture medium could not be measured. 660 nm This needs to be measured before starting the culture.

[0067] <Figure IV: Antimicrobial activity test using the MIC method> JPEG0007838777000020.jpg94149

[0068] In the second trial, the OD of each culture medium was measured before the start of the culture. 660 nm The following measurements were taken. 1.5 mL was taken in an Eppendorf tube for pre-culture measurement, and shaking culture was performed in 3.5 mL.

[0069] <Result> Table V below summarizes the initial experimental results when S. aureus cultures containing 2,6-DHA, 2,6-DH6, 2,6-DH8, 2,6-DH10, and 2,6-DH12 at various concentrations were inoculated onto agar plates. The bacteria grew normally in the control group.

[0070] Investigation of minimum inhibitory concentrations of 2,6-DH12 and its analogs (25-800 μg / mL) [Table 5]

[0071] Table VI below summarizes the test results for 2,6-DH6, 2,6-DH8, 2,6-DH10, and 2,6-DH12 at lower concentrations. The numbers in the table represent OD. 660 nm The values ​​in parentheses indicate turbidity before shaking culture. OD after culture of the control. 660 nm The cell count was 1.62 (0.25 before culture), and it grew normally when inoculated onto agar plate.

[0072] Investigation of minimum inhibitory concentrations of 2,6-DH12 and its analogs (1-32 μg / mL) [Table 6]

[0073] <Consideration> This chapter focuses on acetophenone analogs 2,6-dihydroxyacetophenone (2,6-DHA) and 1-(2,6-dihydroxyphenyl)dodecan-1-one (2,6-DH12). As part of our efforts to elucidate the biological activity of these compounds, we investigated their antibacterial activity against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. Qualitative tests were conducted using the paper disc method, and quantitative tests were conducted using the minimum inhibitory concentration (MIC) method.

[0074] The paper disc method is a method for determining the presence or absence of antimicrobial activity by observing the formation of a zone of inhibition by a test compound on an agar plate mixed with the target bacteria. While it allows for a simple evaluation of antimicrobial activity, it is important to note that the size of the zone of inhibition depends on the physical properties of the compound and therefore does not necessarily reflect the strength of the antimicrobial activity. When antimicrobial activity tests were performed using the paper disc method on secretions extracted from the nymphs of the Japanese star anise lace bug, activity against S. aureus was observed (in 2018, there were few occurrences of Japanese star anise lace bugs on Japanese star anise plants such as those on the Kyoto Gakuen University campus, so testing against E. coli could not be performed). To investigate which compounds were the main compounds exhibiting antimicrobial activity, nine compounds from the nymph secretions were tested individually, and zones of inhibition were formed for 2,6-DHA, 2,6-DH12, and 5-HCh7.

[0075] 2,6-DHA showed activity against S. aureus and E. coli at a dose of 300 μg, and 2,6-DH12 showed activity against both bacteria at a dose of 30 μg. 5-HCh7 showed activity only against S. aureus at a dose of 300 μg. 5-HCh7 is a chromanone cyclized between the phenolic hydroxyl group and side chain of an aromatic ketone, and it is presumed that this cyclization reduces its activity. When these three compounds were tested in combination, in the test against S. aureus, 2,6-DHA+2,6-DH12, 2,6-DH12+5-HCh7, and 2,6-DHA+2,6-DH12+5-HCh7 all formed a zone of inhibition at 30 μg, with almost identical zone of inhibition sizes. The average value of the zone of inhibition for 2,6-DHA+5-HCh7 was the largest, but this result showed greater variability compared to the other compounds, and no zone of inhibition was formed at 30 μg. E. In tests against coli, 2,6-DHA + 2,6-DH12 resulted in an inhibition zone size almost equivalent to that of 2,6-DH12 alone. These results suggest that 2,6-DH12 is the primary substance exhibiting antibacterial activity among the secretions of the Japanese star anise lace bug nymph.

[0076] To investigate the origin of these compounds, hexane extraction and steam distillation were performed on the leaves of the Japanese star anise, the host plant of the Japanese star anise lace bug, and the antibacterial activity of both extracts was examined. In the antibacterial activity test using the paper disc method, neither extract formed an inhibition zone. GC-MS analysis was also performed, but no compounds matching those found in nymph secretions were detected, indicating that the components of the Japanese star anise leaves do not contain antibacterial active ingredients derived from nymphs (Figure V).

[0077] <Figure V> GC chromatogram of Japanese star anise leaf extract TIFF0007838777000023.tif52115

[0078] To further our understanding of 2,6-DHA and 2,6-DH12, which exhibited antibacterial activity, we conducted antibacterial activity tests using the paper disc method on their analogues, either synthetically or as standard samples. Compounds with extended side chain carbon lengths were investigated for 2,6-DHA, 2,6-DH4, 2,6-DH6, 2,6-DH8, 2,6-DH10, 2,6-DH12, and 2,6-DH14. At a treatment dose of 30 μg, inhibition zones were formed for both S. aureus and E. coli with 2,6-DH 4, 6, 8, 10, and 12. For 2,6-DHA and 2,6-DH14, inhibition zones were formed at a treatment dose of 300 μg. From this, we hypothesized that the activity is strongest when the side chain carbon length is between 4 and 12.

[0079] Next, the positional isomers of 2,6-DH12 were investigated using 2,5-DH12, 2,4-DH12, and 2,4,6-TH12. Both diol compounds formed an inhibition zone against S. aureus at 300 μg, while only 2,5-DH12 formed an inhibition zone against E. coli at 300 μg. Since no inhibition zone was formed at 30 μg, it can be seen that it is less active than 2,6-DH12. The triol 2,4,6-DH12, in which a hydroxyl group was added, showed activity against S. aureus even at 30 μg, but showed no activity against E. coli. From these results, it is considered that the position of the hydroxyl group is important for antibacterial activity. Furthermore, the number of hydroxyl groups is also important; 2,4,6-TH12, which had a hydroxyl group added at position 4 despite retaining hydroxyl groups at positions 2 and 6 which should show activity, lost its activity against E. coli. Regarding the positional isomers of 2,6-DHA, we investigated 2,4-DHA, 2,5-DHA, and 3,5-DHA, but none of the compounds showed antibacterial activity. We also investigated 2-HA, 3-HA, 4-HA, and 2,6-DHATf, which have one hydroxyl group and two hydroxyl groups protected by trifluoromethanesulfonate, but none of them showed any activity. From these results, it is thought that 2,6-DHA type compounds lose their activity depending on the substitution position of the hydroxyl group, the reduction of hydroxyl groups, and protection.

[0080] For 2,6-DHA, 2,6-DH6, 2,6-DH8, 2,6-DH10, and 2,6-DH12, which showed significant activity, we further investigated their minimum inhibitory concentrations and attempted quantitative evaluation. S. aureus cultured in liquid medium supplemented with each test compound was inoculated onto agar plates, and the growth status was observed after 24 hours. As a result, bacterial growth was observed in the control medium (liquid medium supplemented with DMSO only) and at 25, 50, 100, and 200 μg / mL of 2,6-DHA. From this, the minimum inhibitory concentration of 2,6-DHA at 24 hours is estimated to be 400 μg / mL. For the other compounds, growth was inhibited even at 25 μg / mL, so it was considered necessary to investigate at lower concentrations, and growth inhibition was observed at concentrations of 1, 2, 4, 8, 16, and 32 μg / mL. As in the previous study, when inoculated onto agar plates, growth was observed in 2,6-DH6 at concentrations of 1–32 μg / mL. Growth was observed in 2,6-DH8 at 1–4 μg / mL, 2,6-DH10 at 1 and 2 μg / mL, and 2,6-DH12 at 1–4 μg / mL. The results for 2,6-DH6 contradict the initial results where growth was inhibited at 25 μg / mL, so further trials are needed, but growth was inhibited at least at a concentration of 50 μg / mL. Growth was inhibited in 2,6-DH8 at 8 μg / mL, 2,6-DH10 at 4 μg / mL, and 2,6-DH12 at 8 μg / mL. In summary, these results show that 2,6-DH10 inhibited growth at the lowest concentration of 4 μg / mL, 2,6-DH8 and 2,6-DH12 at similar concentrations of 8 μg / mL, 2,6-DH6 at 50 μg / mL, and 2,6-DHA at 400 μg / mL.

[0081] Regarding absorbance measurement in MIC testing, there is room for improvement in the measurement method. In the initial test, the culture medium was stained by the DMSO solution, making it difficult to measure turbidity after culturing, so measurements were also taken before culturing. This time, the absorbance before culturing was almost the same for all culture media to which samples were added, and no noticeable staining or turbidity was observed when the DMSO solution was added, and it was around 0.25. In this post-culturing measurement, some samples grew even with quite low absorbance after inoculation, so in this test as well, absorbance was not used as the criterion for judgment, and the minimum inhibitory concentration was determined by inoculation onto agar plates. OD 660nm Further consideration of measurement methods is needed for this measurement.

[0082] The mechanisms of action of these compounds have not been elucidated. Antibiotics can be classified according to their mechanisms of action, such as inhibiting bacterial cell wall synthesis (represented by penicillins), damaging bacterial cell membranes (represented by polymyxin B), inhibiting protein synthesis (represented by aminoglycosides and tetracyclines), and inhibiting nucleic acid metabolism (represented by quinones) (Uematsu et al., Simple Pharmacology). S. aureus and E. coli, the subjects of this study, are Gram-positive and Gram-negative bacteria, respectively. Gram-negative bacteria have a cell wall outside the cell membrane, and an outer membrane outside of that. The cell wall of Gram-negative bacteria is about 8 nm thick. In contrast, Gram-positive bacteria do not have an outer membrane, but their cell wall is very thick, about 250 nm thick. To identify the mechanism of action, it is necessary to examine the antibacterial spectrum.

[0083] 2.4 Evaluation of antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) The above-mentioned 2,6-DH8, 2,6-DH10, and 2,6-DH12 were subjected to antibacterial activity evaluation (measurement of minimum inhibitory concentration) against methicillin-resistant Staphylococcus aureus (MRSA) by the Japan Food Research Laboratories (a general incorporated foundation) (Test Report No. 19014456001-001, February 28, 2019, Requestor: Kyoto Gakuen University). As a result, it was confirmed that they possess very high antibacterial activity. The contents of the test report are described below.

[0084] <Specimen> Ethanol solutions of 2,6-DH8, 2,6-DH10, and 2,6-DH12 at a concentration of 8000 μg / mL (submitted to the Japan Food Research Laboratories by the inventors on February 6, 2019). <Exam Overview> Liquid culture media containing the sample at a specified concentration were dispensed into 96-well microplates (hereinafter referred to as "susceptibility testing plates"), and the test bacterial suspension was inoculated. After incubation, the concentration at which bacterial growth was inhibited was defined as the minimum inhibitory concentration.

[0085] <Test Results> Minimum inhibitory concentration (MIC) (μg / mL) 2,6-DH8 2.5 2,6-DH10 1.25 2,6-DH12 1.25

[0086] <Test Conditions> • Test bacterial solution -Test bacterium: Staphylococcus aureus IID 1677 (Methicillin-resistant Staphylococcus aureus: MRSA) -Preculture: Mueller Hinton Agar (Difco), 35℃±1℃, 18~24 hours Bacterial solution preparation: Physiological saline Number of bacteria: Approximately 10 7 mL ·Sensitivity measurement medium: Mueller Hinton Broth (Difco) • Susceptibility testing plate: The concentration of the stock sample solution was set to 8000 μg / mL. Sample dilutions were prepared by gradually diluting the sample by a factor of 2 using 99.5% ethanol. Next, the stock sample solution and each sample dilution solution were added to the susceptibility testing medium in 1 / 99 of the volume, and 100 μL each was dispensed into a 96-well microplate. • Test procedure and determination method: 5 μL of the test bacterial suspension was inoculated into each well of the susceptibility testing plate, and after incubation for a predetermined time, the lowest concentration at which bacterial growth was inhibited was defined as the minimum inhibitory concentration. • Sensitivity test plate culture conditions: 35°C ± 1°C, 18-24 hours

[0087] 2.5 Antimicrobial textile products and their evaluation An antibacterial fiber product was prepared by immersing a surgical mask cloth piece (1 cm x 1 cm, polypropylene nonwoven fabric; AG Clean, Azeas Co., Ltd.) in an ethanol solution (0.1%; mass-volume percentage concentration) in which 2,6-DH12 was dissolved, and then drying it. By precisely measuring the weight of the resulting gauze cloth after drying, it was confirmed that approximately 5.6% of the mask cloth weight was adsorbed. A similar test was conducted on a medical gauze cloth piece (1 cm x 1 cm, cotton; medical gauze, Kawamoto Sangyo Co., Ltd.), and it was confirmed that approximately 3.6% of the gauze cloth weight was adsorbed.

[0088] When surgical mask cloth pieces treated in this manner, as well as original surgical mask cloth pieces that were not treated, were placed in a culture medium containing non-resistant Staphylococcus aureus, following the paper disc method described above, and observed after 24 hours, the growth of Staphylococcus aureus was significantly suppressed around the treated surgical mask cloth pieces. When the same test was conducted with the medical gauze cloth pieces described above, the growth of Staphylococcus aureus was significantly suppressed around the treated gauze cloth pieces.

[0089] 2.5 Evaluation of antimicrobial activity against vancomycin-resistant enterococci (VRE) The above-mentioned 2,6-DH8, 2,6-DH10, and 2,6-DH12 were subjected to antimicrobial activity evaluation (measurement of minimum inhibitory concentration) against vancomycin-resistant enterococci (VRE) by the Japan Food Research Laboratories (a general incorporated foundation) (Test report preliminary report, reception number 19014456, March 22, 2019, requester: Kyoto Gakuen University). As a result, it was confirmed that they possess very high antimicrobial activity.

[0090] <Specimen> Ethanol solutions of 2,6-DH8, 2,6-DH10, and 2,6-DH12 at a concentration of 8000 μg / mL (submitted to the Japan Food Research Laboratories by the inventors). <Exam Overview> Liquid culture media containing the sample at a specified concentration were dispensed into 96-well microplates (hereinafter referred to as "susceptibility testing plates"), and the test bacterial suspension was inoculated. After incubation, the concentration at which bacterial growth was inhibited was defined as the minimum inhibitory concentration.

[0091] <Test Results> Minimum inhibitory concentration (MIC) (μg / mL) 2.6-DH8 5.0 2,6-DH10 2.5 2,6-DH12 2.5

[0092] <Test Conditions> Except for using Enterococcus faecium (vancomycin-resistant enterococcus: VRE) as the test bacterium, the test conditions were the same as those described in "2.4 Evaluation of antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA)" above.

[0093] 2.6 Evaluation of antifungal activity against four types of fungi The antifungal activity (anti-mold properties) of several compounds, including the aforementioned 2,6-DH12, against four types of fungi (molds) was evaluated by the Hygienic Microbiology Research Center, Inc. (Test report: Test request number 2019D-BT-096, March 15, 2019, Test requester: Faculty of Bioenvironmental Sciences, Kyoto Gakuen University). As a result, it was confirmed that the compounds possessed very high antifungal activity. The portion of the test report concerning 2,6-DH12 is described below.

[0094] <Purpose of the test> The antifungal activity of the sample will be investigated based on the shake test method of the Japan Society for International Antimicrobial Agents, with some modifications to the test conditions. <Specimen> 2,6-DH12 <Test mold> Aspergillus brasiliensis NBRC 105649 Penicillium citrinum NBRC 6352 Alternaria alternate NBRC 106339 Fusarium oxysporum NBRC 31631

[0095] <Testing Method> • Preparation of test spore solution The test mold was inoculated onto potato dextrose agar (PDA) and cultured at 25°C for 7-14 days. Spores were then harvested using sterile water with 0.05% Tween 80 added, and the number of spores was reduced to 10 using phosphate-buffered saline. 5 The test spore solution was prepared to achieve a CFU / mL concentration. • Preparation of test samples A test sample was prepared by mixing 10 mg of the sample with 3 mL of dimethyl sulfoxide. • Shaking treatment and measurement of viable cell count 10 mL of the test spore solution was dispensed into centrifuge tubes, and 0.2 mL of the test sample was inoculated into each tube. The tubes were then shaken at 25°C and 150 rpm for 24 hours. After treatment, a 10-fold dilution series of the shaken solution was prepared using physiological saline and inoculated onto Sabouraud dextrose agar (SDLPA) with a neutralizing agent. The inoculated SDLPA was cultured at 22.5 ± 2.5°C for 3 to 5 days, and the number of colonies formed was counted to calculate the number of viable cells.

[0096] <Test Results> • Antifungal test results against Aspergillus brasiliensis (viable cell count measurement results (CFU / mL)) Average values ​​for each test piece 2,6-DH12 1.0×10 2 2.7 × 10 2 1.0 × 10 1 3.0×10 1 Control 7.3 x 10 4 7.7 × 10 4 7.7 × 10 4 6.4 × 10 4

[0097] • Antifungal test results against Penicillium citrinum (viable cell count measurement results (CFU / mL)) Average values ​​for each test piece 2,6-DH12 1.3×10 2 - 3.0×10 1 1.0 × 10 1 Control 2.5 x 10 6 1.6 × 10 6 2.4 × 10 6 3.4 × 10 6

[0098] • Antifungal test results against Alternaria alternata (viable cell count measurement results (CFU / mL)) Average values ​​for each test piece 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

[0099] • Antifungal test results against Fusarium oxysporum (viable cell count measurement results (CFU / mL)) Average values ​​for each test piece 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 × 10 6

[0100] 3. Overall Considerations (Summary) As described above, we investigated the antibacterial activity of the exocrine gland larvae and their analogues. Below, we discuss the structure-activity relationship study.

[0101] Stephanitis svensoni, also known as the Japanese star anise lace bug, is an insect belonging to the family Tingidae in the order Hemiptera, and is a sap-sucking pest of Illicium anisatum. The nymphs of this insect secrete decanal, dodecanal, 2-undecanone, 3-oxododecanal (houttuynin), 2,6-dihydroxyacetophenone, 1-(2,6-dihydroxyphenyl)dodecan-1-one, 5-hydroxy-2-alkylchromanones, and nonaicosane. Among these, 5-hydroxy-2-alkylchromanones are compounds unique to lace bugs and are also found in the secretions of the nymphs of the crested lace bug. 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 ant-repellent activity (patent application filed in our laboratory) and possesses superior prostaglandin H synthase inhibitory activity compared to aspirin (Jurenka, RA et al, 1989). 1-(2,6-dihydroxyphenyl)dodecan-1-one has been reported to have antibacterial activity against the Gram-positive bacterium Clavibacter michiganensis, which causes bacterial leaf blight on maize, and to have growth inhibitory effects on nematodes (John W. Neal, JR., et al, 1995), and possesses prostaglandin H synthase inhibitory activity comparable to aspirin (Jurenka, RA et al, 1989).

[0102] Focusing on acetophenone analogs 2,6-dihydroxyacetophenone (2,6-DHA) and 1-(2,6-dihydroxyphenyl)dodecan-1-one (2,6-DH12), we investigated their antibacterial activity against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli, as part of our efforts to elucidate the biological activity of these compounds. Qualitative testing was performed using the paper disc method, and quantitative testing was performed using the minimum inhibitory concentration (MIC) method. When nine compounds from nymph secretions were tested, inhibition zones were formed for 2,6-DHA, 2,6-DH12, and 5-HCh7. 2,6-DHA showed activity against both S. aureus and E. coli at a dose of 300 μg, while 2,6-DH12 showed activity against both at a dose of 30 μg. 5-HCh7 showed activity only against S. aureus at a dose of 300 μg. 5-HCh7 is a cyclized acetophenone, and it is presumed that this cyclization reduces its activity. When these three compounds were tested in combination, it was determined that 2,6-DHP12 is the main substance exhibiting antibacterial activity in the secretions of the Japanese star anise lace bug nymph. To investigate the origin of these compounds, the antibacterial activity of hexane extract and essential oil from the leaves of the Japanese star anise, which the Japanese star anise lace bug feeds on, was examined. However, neither formed a zone of inhibition, and no peaks consistent with the nymph secretions were detected in GC-MS analysis. Therefore, it is presumed that the Japanese star anise lace bug nymph biosynthesizes these antibacterial components.

[0103] To further our understanding of 2,6-DHA and 2,6-DH12, which exhibited antibacterial activity, we conducted antibacterial activity tests on their analogues. We examined 2,6-DHA, 2,6-DH4, 2,6-DH6, 2,6-DH8, 2,6-DH10, 2,6-DH12, and 2,6-DH14, which have elongated side chains of 2,6-DHA. We hypothesized that activity increases with carbon chain lengths of 4 to 12. Regarding the positional isomers of 2,6-DH12, we investigated 2,5-DH12, 2,4-DH12, and 2,4,6-THP12. Both diol compounds showed weaker activity than 2,6-DH12. The triol 2,4,6-DH12, with an added hydroxyl group, exhibited activity equivalent to 2,6-DH12 against S. aureus, but lost activity against E. coli. We investigated the positional isomers of 2,6-DHA, including 2,4-DHA, 2,5-DHA, and 3,5-DHA, as well as 2-HA, 3-HA, 4-HA (which have one hydroxyl group), and 2,6-DHATf (where the hydroxyl group is protected with trifluoromethanesulfonate). However, no activity was observed in any of the compounds. This suggests that the activity of 2,6-DH12 and 2,6-DHA is significantly weakened by the substitution position of the hydroxyl group, the reduction of the hydroxyl group, or protection.

[0104] In a quantitative evaluation by examining the minimum inhibitory concentration (MIC), in the initial test where the test compound concentrations were set from 25 to 800 μg / mL, bacterial growth was observed at 25, 50, 100, and 200 μg / mL of 2,6-DHA compared to a control medium with only DMSO added. From this, the minimum 24-hour inhibitory concentration of 2,6-DHA was determined to be 400 μg / mL. Since growth was inhibited even at 25 μg / mL for the other compounds, further investigations were conducted at lower concentrations. This test revealed that 2,6-DH10 inhibited growth at the lowest concentration of 4 μg / mL, 2,6-DH8 and 2,6-DH12 inhibited growth at similar concentrations of 8 μg / mL, and 2,6-DH6 inhibited growth at at least 50 μg / mL. It should be kept in mind that the MIC values ​​have a certain range. Because differences in growth status occur depending on the type of bacteria being measured and the drug mechanism, it should be considered that the measured values ​​are subject to fluctuations due to various factors (Tamura et al., "Institutional Problems in MIC Measurement"). The mechanisms of action of these compounds have not yet been elucidated, and further detailed investigation is needed.

[0105] This study confirmed that 2,6-DH8, 2,6-DH10, and 2,6-DH12 possess high antibacterial activity, particularly against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Furthermore, 2,6-DH12 exhibited antifungal activity against the fungi Aspergillus brasiliensis, Penicillium citrinum, and Alternaria alternata. This suggests potential applications in topical skin preparations, cosmetics, and disinfectants, antibacterial agents, antifungal agents, and antifungal agents applied to hands and fingers, all of which can be considered antibacterial and antifungal agents in this invention. Additionally, antibacterial and antifungal textile products can be obtained for use in masks, aprons, and clothing worn by patients and healthcare workers, as well as in bedding materials such as sheets, duvet covers, and pillowcases, or as insoles for footwear and curtains. In particular, its effectiveness and convenience are extremely high because it shows activity against multiple types of resistant bacteria and fungi.

[0106] Insect secretions, which are said to account for half of all living organisms, can be a source for the discovery of bioactive substances useful to humans, including antimicrobial peptides. Furthermore, while the use of previously untapped microbial resources such as marine microorganisms is attracting attention in search of novel antimicrobial substances, microorganisms in the insect habitat are also fully usable as discovery resources. For example, Cordyceps is a well-known fungus that parasitizes insects, and cordycepin has been discovered as an antimicrobial substance in Cordyceps sinensis, which parasitizes the larvae of ghost moths, and Codryceps militaris, which parasitizes the larvae of lepidopteran insects (Y.-J.Ahn, 2000). However, even Cordyceps has only been recognized as one of the drug discovery resources, and currently, microorganisms related to insects are largely untapped (Kobayashi et al., 2014). We hope that the search for useful substances targeting such insect-derived microorganisms will further develop.

[0107] 4.References David J. Maloney, Jing-Zhen Deng, Shelley R. Starck, Zhijie Gao, and Sidney M. Hecht (+)-Myristinin A, a Naturally Occurring DNA Polymerase β Inhibitor and Potent DNA-Damaging Agent (2005) JACS, 127, 4140-4141.

[0108] Yoshinari Enami, Preventing seedling blight by using mites that feed on pathogenic fungi (2003), Tohoku Agricultural Research Center Newsletter 8

[0109] Seiichi Furukawa, Minoru Yamakawa (2004) Insect Antimicrobial Peptides: Biological Defense and Their Applied Chemistry and Biology Vol. 42, No. 1.

[0110] Yoshikazu Ishii (2018) The Importance of Infection Control Based on a One Health Approach. The Chemical Times No. 4 (Issue No. 250) Special Feature: Infection Control - Antimicrobial Resistance (AMR) p2-6

[0111] James E. Oriver, Kenneth R. Wilzer, Rolland M.Waters Synthesis of 1-(2,6-dihydroxyphenyl)-1-alkanones and Benzophenone by aromatization of 2-Acyl-3-hydroxy-2-cyclohexene-1-ones with Mercuric Acetate (1990) Synthesis 1990, 1117-1119.

[0112] Jinbing Liu, Rihui Cao, Qifeng Wu, Chunming Ma, Zihou Wang, Wenlie Peng, Huacan Soug Synthesis and antibacterial evaluation of novel 4-alkyl substituted phenyl β-aldehyde ketone derivatives (2009) European Journal of Medicinal Chemistry 44, 1737-1744.

[0113] 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.

[0114] Kobayashi, H., Takeishi, K., & Adachi, Y. (2014) Search for useful substances focusing on insect-derived microorganisms. Biochemistry, Vol. 86, No. 5, pp. 570-577.

[0115] Kimiko Okabe, Pests of Edible Mushrooms in Japan (2006), "Bulletin of FFPRI," Vol. 5, No. 2 (No. 399), 119-133.

[0116] Russell A. Jurenka, John W. Neal, Jr., Ralph, W. Howard, James E. Oliver, Gray J. Blomquist In Vitro Inhibition Of Prostaglandin H Synthate By Compounds From The Exocrine Secretions Of Lace Bugs (1989) Comp. Biochem. Physiol. 93C(2), 253-2555

[0117] Sydney A. Villaume, Jian Fu, Dr. Ines N'Go, Hui Liang, Huayong Lou, Laurent Kremer, Weidong Pan, Stephane P. Vincent Natural and Synthetic Flavonoids as Potent Mycobacterium tuberculosis UGM Inhibitors (2017) Chemistry -A European Journal, 23(43), 10423-10429.

[0118] Science of Synthesis, 31a, 441-467, 2007; Chem Abstr., 150, 259653w (2009).

[0119] Tamura, S.; Ikedo, M. Problems with MIC measurement (2011) Journal of the Japanese Society of Chemotherapy

[0120] Toshihiko Uematsu, Takahide Nomura, Naohisa Ishikawa, Simple Pharmacology, Revised 3rd Edition, pp. 216-272.

[0121] Young-Joon Ahn, Suck-Joon Park, Sang-Gil Lee, Sang-Cheol Shin, Don-Ha Choi Cordycepin: selective growth inhibitor derived from liquid culture of Cordyceps militaris against Clostridium spp.(2000) J. Agric. Food Chem., 48(7), 2744-2748.

Claims

1. An antimicrobial agent for methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci, comprising at least one of the following compounds 1 to 3 as an active ingredient.

2. An antifungal agent against Aspergillus brasiliensis, Penicillium citrinum, or Alternaria alternata, containing compound 3 listed below as an active ingredient.

3. A textile product for antimicrobial use against methicillin-resistant Staphylococcus aureus or vancomycin-resistant enterococci, which adsorbs or contains the antimicrobial agent of claim 1, as a mask, apron, clothing, bedding material, footwear material, or curtain.

4. Textile products for antifungal use against Aspergillus brasiliensis, Penicillium citrinum, or Alternaria alternata, such as masks, aprons, clothing, bedding materials, footwear materials, or curtains, which adsorb or contain the antifungal agent of claim 2.

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