CN111533756A - Spiroketal compound derived from plant pathogenic fungi, preparation method and application - Google Patents
Spiroketal compound derived from plant pathogenic fungi, preparation method and application Download PDFInfo
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- CN111533756A CN111533756A CN202010350603.XA CN202010350603A CN111533756A CN 111533756 A CN111533756 A CN 111533756A CN 202010350603 A CN202010350603 A CN 202010350603A CN 111533756 A CN111533756 A CN 111533756A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
Abstract
The invention discloses spiroketal compounds derived from plant pathogenic fungi, which have the structural formula shown as follows:
the spiroketal compound is obtained by fermenting, culturing and separating epicoccum nigrum. The compound epicpirocins A-H provided by the invention belongs to a novel spiroketal compound, has mature extraction method,the method has the advantages that the process is simple, the yield of the obtained product is high, the structure of the product is correct through nuclear magnetic resonance and mass spectrum detection, the compounds epicipirorins A, B, D, G and H have certain inhibitory activity to staphylococcus aureus and methicillin-resistant staphylococcus aureus, and the compounds epicpirocins A-D have certain inhibitory action to candida albicans.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to spiroketal compounds derived from plant pathogenic fungi, and a preparation method and application thereof.
Background
The genus Epicoccum (Epicoccum) belongs to the phylum Ascomycota, Ascomycetes, Xyleriales, Haematolaceae, widely distributed around the world, is a plant pathogenic fungus and also belongs to an endophytic fungus, often parasitizes on the surface of putrefying plants, and has been shown by studies to be colonized on leaves growing in water at low temperature of 0 ℃, and thus is also considered to be a facultative marine fungus. Nigrum can produce a variety of active natural products, for example, the antimicrobially active compounds epicorazines a and B; the antineoplastic active compounds orevaccae and acetosellin; beta-glucose aldolase inhibiting active compound ent-epicoccinG, epicoccins M, epicoccins T and anticancer active compound taxol. At present, very few compounds with a spiro [ isobenzofuran-1, 3' -isochroman ] skeleton are reported, only few chemical syntheses are reported, and fewer natural products containing the skeleton are important natural products with cytotoxic, antibacterial, nematicidal and antiviral activities. The spiroketal compound with the framework type is selected for deep research, and has good prospect for finding out a compound with a new structure and new activity.
Disclosure of Invention
The first purpose of the invention is to provide a spiroketal compound derived from plant pathogenic fungi.
The second purpose of the invention is to provide a preparation method of the spiroketal compounds derived from plant pathogenic fungi.
The third purpose of the invention is to provide the application of the spiroketal compounds derived from plant pathogenic fungi.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides spiroketal compounds derived from plant pathogenic fungi, and the structural formula is shown as follows:
the spiroketal compounds are obtained by fermenting, culturing and separating Epicoccum nigrum 09116(Epicoccum nigrum).
The second aspect of the present invention provides a preparation method of the spiroketal compounds derived from plant pathogenic fungi, which comprises the following steps:
sterilizing a solid fermentation culture medium, inoculating 5% of seed liquid by volume to the solid fermentation culture medium, standing and culturing at 28 ℃, harvesting a solid fermentation product after 40 days, wherein the solid fermentation product is all substances in a container, and separating and purifying the solid fermentation product to obtain the spiroketal compound derived from the plant pathogenic fungi.
The solid fermentation medium is rice: water (W: V) ═ 2: 3.
The preparation method of the seed liquid comprises the following steps: respectively filling seed culture medium into multiple glass bottles, sterilizing at 121 deg.C for 20min, inoculating with Pleurotus nigrococcus strain, and rotary culturing at 28 deg.C on rotary table (rotation speed of 220rpm) for 7d to obtain seed solution.
The seed culture medium consists of the following components: potato extract, glucose, agar and water; the concentrations of the components in the seed culture medium are respectively (g/L): potato extract 200g/L, glucose 20g/L and agar 20 g/L.
The culture method of the epicoccum nigrum-containing plate strain comprises the following steps:
sterilizing the plate culture medium at 121 ℃ for 20min, preparing a plate, culturing at 37 ℃ for 3d until the surface moisture is slightly dry and no foreign bacteria grow, inoculating the epicoccum nigrum 09116 strain spores to the plate culture medium, culturing at 28 ℃ for 10d, wherein the appearance is meat red, aerial hyphae are plump, and the plate strain can be collected and used when no bacteria are infected, thus obtaining the plate strain.
The plate culture medium consists of the following components: potato extract, glucose, agar and water; the concentrations of the components in the plate culture medium are respectively (g/L): potato extract 200g/L, glucose 20g/L and agar 20 g/L.
The strain preservation method comprises the following steps: storing in 25% glycerol freezing tube at-80 deg.C.
The method for separating Epicoccum nigrum (Epicoccum nigrum)09116 is as follows:
cleaning the plant sample at the leaf part of the fresh Chilean san Diego grass by using sterile water, sucking surface water by using absorbent paper, and cutting the plant sample into small pieces for surface disinfection treatment: rinsing with 75% alcohol for 3min, rinsing with sterile water for 4-5 times, rinsing with 5% sodium hypochlorite solution for 3min, rinsing with sterile water for 4-5 times, sucking water with sterile filter paper, and shearing the surface-sterilized material into 0.5cm pieces2And (3) putting the small blocks into a flat plate containing a strain isolation culture medium, culturing at a constant temperature of 28 ℃ for 3-15d until a small amount of hyphae are generated at the edge of the tissue block of the experimental group, and transferring the small blocks into another culture flat plate for culturing by adopting a hypha tip picking method in time.
The method for obtaining the spiroketal compounds derived from plant pathogenic fungi by separating and purifying the solid fermentation product comprises the following steps:
extracting the solid fermented product with ethyl acetate for three times, filtering the extractive solution to remove solid fermented product, collecting supernatant, concentrating the supernatant, evaporating to dryness, weighing to obtain crude extract, separating by silica gel vacuum liquid chromatography (85 × 200mm column), and mixing with dichloromethane and methanol at a ratio of 1: 0,100: 1,50: 1,20: 1,10: 1,4: 1,1: 1,0: 1 gradient elution of crude extract to give 10 fractions G1-G10, further eluting the G5 fraction with methanol as mobile phase and Sephadex LH-20 column (4 × 108mm) to give 23 subfractions (G5N1-G5N23), the subfraction G5N7 using ODS-MPLC for separation and gradient elution with 10% -100% aqueous acetonitrile for 60 minutes to give 17 fractions G5N7M1-G5N7M17, G5N7M15 fraction purified by semi-preparative RP-HPLC using YMC-Ph (10 × 250mm) at a flow rate of 4.0mL/min using gradient eluent: 0min, 20% methanol water solution; 20min, 50% methanol in water to obtain epicipirosin A and epicipirosin B; the G5N7M17 fractions were purified by semi-preparative RP-HPLC using Cosmosil π -nap (10X 250mm) with a flow rate of 4.0mL/min, eluting with 30% methanol water, to give the compounds epicipirosin C and epicipirosin D; fractions of G5N7M10 were purified by semi-preparative RP-HPLC using Cosmosil π -nap (10X 250mm), flow rate 4.0mL/min, eluting with 22% acetonitrile in water, to give the compounds epicosphipricin E and epicosphiricin F; fractions of G5N7M12 were purified by semi-preparative RP-HPLC using Cosmosil π -nap (10X 250mm) at a flow rate of 4.0mL/min, eluting with 35% aqueous methanol to give the compounds epicosphipricin G and epicosphipricin H.
The third aspect of the invention provides an application of the spiroketal compound derived from plant pathogenic fungi in preparing antibacterial drugs.
The antibacterial agent has the activity of resisting staphylococcus aureus, methicillin-resistant staphylococcus aureus and candida albicans, and the pathogenic strains are as follows: staphylococcus aureus strain ATCC6538, methicillin-resistant staphylococcus aureus, a clinical strain isolated by hospital 309 of the people's liberation force of china, and candida albicans strain SC 5314.
The fourth aspect of the present invention provides a pharmaceutical composition, which comprises the spiroketal compound derived from plant pathogenic fungi, its cis-trans isomer, solvate or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.
The fifth aspect of the invention provides an application of the pharmaceutical composition in preparing antibacterial drugs.
Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:
the compound epicpirocins A-H provided by the invention belongs to a new spiroketal compound, the extraction method is mature, the process is simple and convenient, the obtained product has high yield, the structure is correct through nuclear magnetic resonance and mass spectrum detection, the compounds epicpirocins A, B, D, G and H have certain inhibitory activity to staphylococcus aureus and methicillin-resistant staphylococcus aureus, and the compounds epicpirocins A-D have certain inhibitory action to candida albicans.
Drawings
FIG. 1 is a UV spectrum of epicopologicins A-H, a compound of the present invention.
FIG. 2 is a HR-ESI-MS spectrum of epicipirorin A, a compound of the present invention.
FIG. 3 is a HR-ESI-MS spectrum of epicipirorin B, a compound of the present invention.
FIG. 4 is a HR-ESI-MS spectrum of epicipirorin C, a compound of the invention.
FIG. 5 is a HR-ESI-MS spectrum of epicpirocin D, a compound of the present invention.
FIG. 6 is a HR-ESI-MS spectrum of epicipirorin E, a compound of the invention.
FIG. 7 is a HR-ESI-MS spectrum of epicipirocin F, a compound of the invention.
FIG. 8 is a HR-ESI-MS spectrum of epicipirorin G, a compound of the present invention.
FIG. 9 is a HR-ESI-MS spectrum of epicopolicin H, a compound of the present invention.
FIG. 10 shows the dissolution of epicpirocin A of the compound of the present invention in DMSO-d6In (1)1H-NMR spectrum.
FIG. 11 shows the dissolution of epicipirosin B compound of the present invention in DMSO-d6In (1)1H-NMR spectrum.
FIG. 12 shows the dissolution of epicpirocin C in DMSO-d of the compound of the present invention6In (1)1H-NMR spectrum.
FIG. 13 shows the dissolution of epicpirocin D of the compound of the present invention in DMSO-D6In (1)1H-NMR spectrum.
FIG. 14 shows the solubility of epicipirosin E and epicipirosin F, the compounds of the invention, in DMSO-d6In (1)1H-NMR spectrum.
FIG. 15 shows the solubility of epicpirocin G and epicpirocin H compounds of the present invention in DMSO-d6In (1)1H-NMR spectrum.
FIG. 16 shows the dissolution of epicpirocin A of the compound of the present invention in DMSO-d6In (1)13C-NMR spectrum.
FIG. 17 shows the dissolution of epicipirosin B compound of the present invention in DMSO-d6In (1)13C-NMR spectrum.
FIG. 18 shows the dissolution of epicpirocin C of the compound of the present invention in DMSO-d6In (1)13C-NMR spectrum.
FIG. 19 shows the dissolution of epicpirocin D of the compound of the present invention in DMSO-D6In (1)13C-NMR spectrum.
FIG. 20 shows the dissolution of epicipirosin E and epicipirosin F, the compounds of the present invention, in DMSO-d6In (1)13C-NMR spectrum.
FIG. 21 shows the solubility of epicpirocin G and epicpirocin H in DMSO-d of the compounds of the invention6In (1)13C-NMR spectrum.
FIG. 22 shows the dissolution of epicpirocin A of the present invention in DMSO-d6DEPT NMR Spectrum in (1).
FIG. 23 shows the dissolution of epicipirosin B compound of the present invention in DMSO-d6DEPT NMR Spectrum in (1).
FIG. 24 shows the dissolution of epicpirocin C of the compound of the present invention in DMSO-d6DEPT NMR Spectrum in (1).
FIG. 25 shows the dissolution of epicpirocin D of the compound of the present invention in DMSO-D6DEPT NMR Spectrum in (1).
FIG. 26 shows the dissolution of epicipirosin E and epicipirosin F, the compounds of the present invention, in DMSO-d6Dept nmr spectrum in (1).
FIG. 27 shows the solubility of epicipirosin G and epicipirosin H in DMSO-d, compounds of the invention6Dept nmr spectrum in (1).
FIG. 28 shows the dissolution of epicpirocin A of the present invention in DMSO-d6In (1)1H-1H COSY spectra.
FIG. 29 shows the dissolution of epicipirosin B compound of the present invention in DMSO-d6In (1)1H-1H COSY spectra.
FIG. 30 shows the dissolution of epicpirocin C of the compound of the present invention in DMSO-d6In (1)1H-1H COSY spectra.
FIG. 31 shows the dissolution of epicpirocin D of the compound of the present invention in DMSO-D6In (1)1H-1H COSY spectra.
FIG. 32 shows the solubility of epicipirosin E and epicipirosin F, compounds of the invention, in DMSO-d6In (1)1H-1HCOSY spectra.
FIG. 33 shows the solubility of epicpirocin G and epicpirocin H compounds of the present invention in DMSO-d6In (1)1H-1HCOSY spectra.
FIG. 34 shows the dissolution of epicpirocin A of the compound of the present invention in DMSO-d6HSQC spectrum in (1).
FIG. 35 shows the dissolution of epicpirocin B of the present invention in DMSO-d6HSQC spectrum in (1).
FIG. 36 shows the dissolution of epicpirocin C of the compound of the present invention in DMSO-d6HSQC spectrum in (1).
FIG. 37 shows the dissolution of epicpirocin D of the present invention in DMSO-D6HSQC spectrum in (1).
FIG. 38 shows the dissolution of epicpirocin E and epicpirocin F, compounds of the invention, in DMSO-d6HSQC spectrum in (1).
FIG. 39 shows the dissolution of epicpirocin G and epicpirocin H in DMSO-d of the compounds of the present invention6HSQC spectrum in (1).
FIG. 40 shows the dissolution of epicpirocin A of the present invention in DMSO-d6HMBC spectrum in (1).
FIG. 41 shows the dissolution of epicipirosin B as a compound of the present invention in DMSO-d6HMBC spectrum in (1).
FIG. 42 shows the dissolution of epicpirocin C of the compound of the present invention in DMSO-d6HMBC spectrum in (1).
FIG. 43 shows the dissolution of epicpirocin D of the present invention in DMSO-D6HMBC spectrum in (1).
FIG. 44 shows the solubility of epicipirosin E and epicipirosin F, compounds of the invention, in DMSO-d6HMBC spectrum in (1).
FIG. 45 is a compound of the present inventionEpicospirocin G and Epicospirocin H are dissolved in DMSO-d6HMBC spectrum in (1).
FIG. 46 shows the dissolution of epicpirocin A of the compound of the present invention in DMSO-d6NOESY spectrum of (1).
FIG. 47 shows the dissolution of epicipiroricin B as a compound of the present invention in DMSO-d6NOESY spectrum of (1).
FIG. 48 shows the dissolution of epicpirocin C in DMSO-d of the compound of the present invention6NOESY spectrum of (1).
FIG. 49 shows the dissolution of epicpirocin D of the present invention in DMSO-D6NOESY spectrum of (1).
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Preparation of spiroketal compounds
Potato extract was purchased from BD corporation, USA under catalog number 2022-01-31; glucose was purchased from Shanghai Tantake technologies, Inc. under product catalog number G61055A; agar was purchased from glass instruments, east China, Qingdao.
The preparation method of spiroketal compounds derived from plant pathogenic fungi comprises the following steps:
preparation of spiroketal compounds by fermentation
1. Seed culture
(1) Sterilizing a plate culture medium at 121 ℃ for 20min, preparing a plate, culturing at 37 ℃ for 3d until the surface moisture is slightly dry and no foreign bacteria grow, inoculating Epicoccum nigrum 09116 strain spores to the plate culture medium, culturing at 28 ℃ for 10d, wherein the appearance is flesh red, aerial hypha is full, and the plate strain can be collected and used when no bacteria are infected, thus obtaining the plate strain.
The plate culture medium consists of the following components: potato extract, glucose, agar and water; the concentrations of the components in the plate culture medium are respectively (g/L): potato extract 200g/L, glucose 20g/L and agar 20 g/L.
The pH value of the plate culture medium is natural.
The method for separating Epicoccum nigrum (Epicoccum nigrum)09116 is as follows:
cleaning the plant sample at the leaf part of the fresh Chilean san Diego grass by using sterile water, sucking surface water by using absorbent paper, and cutting the plant sample into small pieces for surface disinfection treatment: rinsing with 75% alcohol for 3min, rinsing with sterile water for 4-5 times, rinsing with 5% sodium hypochlorite solution for 3min, rinsing with sterile water for 4-5 times, sucking water with sterile filter paper, and shearing the surface-sterilized material into 0.5cm pieces2And (3) putting the small blocks into a flat plate containing a strain isolation culture medium, culturing at a constant temperature of 28 ℃ for 3-15d until a small amount of hyphae are generated at the edge of the tissue block of the experimental group, and transferring the small blocks into another culture flat plate for culturing by adopting a hypha tip picking method in time.
The preparation method of the strain isolation medium comprises the following steps:
potato extract 200g/L, glucose 20g/L and agar 20g/L, sterilizing at high temperature, and adding mixed solution of penicillin 100mg/L and streptomycin 200 mg/L20 mL when preparing plate.
The method for preserving the Epicoccum nigrum (Epicoccum nigrum)09116 comprises the following steps: storing in 25% glycerol freezing tube at-80 deg.C.
(2) 40mL of seed medium was placed in each of 250mL glass bottles, sterilized at 121 ℃ for 20min, and inoculated with a plate-shaped piece (using the plate-shaped strain obtained in step (1)). Performing rotary culture (rotation speed of 220rpm) on a rotary shaking table at 28 deg.C for 7d to obtain seed liquid.
The seed culture medium consists of the following components: potato extract, glucose, agar and water; the concentrations of the components in the seed culture medium are respectively (g/L): potato extract 200g/L, glucose 20g/L and agar 20 g/L;
the pH value of the seed culture medium is natural.
2. Fermentation culture
Preparing a solid fermentation medium (the components of the solid fermentation medium: rice: water (W: V): 2:3, pH value is natural), subpackaging 160g of rice and 240mL of water in a 1000mL triangular flask, sterilizing (sterilizing at 121 ℃ for 20min), inoculating the seed solution obtained in the step 1 to the solid fermentation medium according to the inoculation amount of 5% (volume percentage), standing and culturing at 28 ℃, and harvesting a solid fermentation product after 40d, wherein the solid fermentation product is all substances in a container.
Secondly, separating and purifying spiroketal compounds and identifying
1. Separation and purification of spiroketal compounds
Extracting the solid fermented product with ethyl acetate for three times, filtering the extractive solution to remove solid fermented product, collecting supernatant, concentrating the supernatant, evaporating to dryness, weighing to obtain crude extract, separating by silica gel vacuum liquid chromatography (85 × 200mm column), and mixing with dichloromethane and methanol at a ratio of 1: 0,100: 1,50: 1,20: 1,10: 1,4: 1,1: 1,0: 1 gradient elution is carried out on the crude extract, and 10 components G1-G10 are obtained in total. The G5 fraction was further eluted with Sephadex LH-20 column (4X 108mm) using methanol as mobile phase to give 23 sub-fractions (G5N1-G5N 23). Subfractions G5N7 were separated using ODS-MPLC and eluted with a gradient of 10% to 100% acetonitrile in water for 60 min to give 17 fractions G5N7M1-G5N7M 17. The G5N7M15 fraction was purified by semi-preparative RP-HPLC using YMC-Ph (10X 250mm) at a flow rate of 4.0mL/min using a gradient eluent: 0min, 20% methanol water solution; 20min, 50% aqueous methanol to give epicipirosin A and epicipirosin B compounds. Fractions of G5N7M17 were purified by semi-preparative RP-HPLC using Cosmosil π -nap (10X 250mm) at a flow rate of 4.0mL/min, eluting with 30% methanol water, to give compounds epicosphipricin C and epicosphiricin D. Fractions of G5N7M10 were purified by semi-preparative RP-HPLC using Cosmosil π -nap (10X 250mm) at a flow rate of 4.0mL/min, eluting with 22% acetonitrile in water, to give the compounds epicosphipricin E and epicosphiricin F. Fractions of G5N7M12 were purified by semi-preparative RP-HPLC using Cosmosil π -nap (10X 250mm) at a flow rate of 4.0mL/min, eluting with 35% aqueous methanol to give the compounds epicosphipricin G and epicosphipricin H.
2. Identifying the spiroketal compounds epicpirocins A-H.
The compound epicopolipirins A-H obtained above was identified:
(1) appearance: all are amorphous pale yellow powders.
(2) Solubility: is easily dissolved in methanol and hardly dissolved in water.
(3) Ultraviolet spectrum: the ultraviolet spectrum of the methanol solution of the compound epicopologicins A-H has a maximum absorption peak at 304.0nm, the ultraviolet spectrum is shown in figure 1, and figure 1 is the ultraviolet spectrum of the compound epicopologicins A-H of the invention. The ultraviolet spectrum testing instrument is a Mariner System 5304 instrument.
(4) Mass spectrum: FIG. 2 is a HR-ESI-MS spectrum of epicipirorin A, a compound of the present invention, [ M-H ]]-The peak is m/z 405.08176, suggesting that the most probable molecular formula is C19H18O10. FIG. 3 is a HR-ESI-MS spectrum of epicosphirocinB of the compound of the present invention, showing [ M-H]-The peak is m/z 405.08167, suggesting that the most probable molecular formula is C19H18O10. FIG. 4 is a HR-ESI-MS spectrum of epicipirorin C, a compound of the present invention, [ M-H ]]-The peak is m/z405.08163, suggesting that the most probable molecular formula is C19H18O10. FIG. 5 is a HR-ESI-MS spectrum of epicpirocin D, a compound of the present invention, [ M-H ]]-The peak is m/z 405.08173, suggesting that the most probable molecular formula is C19H18O10. FIG. 6 is a HR-ESI-MS spectrum of epicipirorin E, a compound of the present invention, [ M-H ]]-The peak is m/z 391.06622, suggesting that the most probable molecular formula is C18H16O10. FIG. 7 is a HR-ESI-MS spectrum of epicipirorin F, a compound of the invention, [ M-H ]]-The peak is m/z 391.06616, suggesting that the most probable molecular formula is C18H16O10. FIG. 8 is a HR-ESI-MS spectrum of epicipirorin G, a compound of the present invention, [ M-H ]]-The peak is m/z 391.06622, suggesting that the most probable molecular formula is C18H16O10. FIG. 9 is a drawing of the compound epicospi of the inventionHR-ESI-MS spectrum of rocin H, showing [ M-H ] thereof]-The peak is m/z 391.06622, suggesting that the most probable molecular formula is C18H16O10. HR-ESI-MS spectrum test adopts a ThermalFisher Orbitrap Q active mass spectrometer and methanol as a solvent.
(5) Nuclear magnetic resonance spectroscopy: FIG. 10 shows the dissolution of epicpirocin A of the compound of the present invention in DMSO-d6In (1)1H-NMR spectrum, FIG. 11 shows that epiciprocin B of the present invention is dissolved in DMSO-d6In (1)1H-NMR spectrum, FIG. 12 shows that epiciprocin C of the compound of the present invention is dissolved in DMSO-d6In (1)1H-NMR spectrum, FIG. 13 shows that epicpirocinD of the present invention is dissolved in DMSO-d6In (1)1H-NMR spectrum, FIG. 14 shows that the compounds epicpirocin E and epicpirocin F of the present invention are dissolved in DMSO-d6In (1)1H-NMR spectrum, FIG. 15 shows that the compounds epicpirocin G and epicpirocin H of the present invention are dissolved in DMSO-d6In (1)1H-NMR spectrum. FIG. 16 shows the dissolution of epicpirocin A of the compound of the present invention in DMSO-d6In (1)13C-NMR spectrum, FIG. 17 shows that epiciprocin B of the present invention is dissolved in DMSO-d6In (1)13C-NMR spectrum, FIG. 18 shows that epiciprocin C of the compound of the present invention is dissolved in DMSO-d6In (1)13C-NMR spectrum, FIG. 19 shows that epiciprocin D of the present compound is dissolved in DMSO-D6In (1)13C-NMR spectrum, FIG. 20 shows that the compounds epicipirosin E and epicipirosin F of the present invention are dissolved in DMSO-d6In (1)13C-NMR spectrum, FIG. 21 shows that the compounds epicpirocin G and epicpirocin H of the present invention are dissolved in DMSO-d6In (1)13C-NMR spectrum. DEPT NMR spectra of 8 additional compounds (shown in FIGS. 22-27, FIG. 22 shows that epiciprocin A of the present invention is dissolved in DMSO-d6DEPT NMR Spectroscopy in (1), FIG. 23 shows that the compound epicpirocin B of the present invention is dissolved in DMSO-d6DEPT NMR spectra in (1), FIG. 24 shows that epicpirocin C of the present compound is dissolved in DMSO-d6DEPT NMR Spectroscopy in (1), FIG. 25 shows that epicpirocin D of the present invention is dissolved in DMSO-D6DEPT NMR spectra in (1), FIG. 26 shows that the compounds epicpirocin E and epicpirocin F of the present invention are dissolved in DMSO-d6DEPT NMR Spectroscopy inFIG. 27 shows the solubility of epicpirocin G and epicpirocin H compounds of the present invention in DMSO-d6DEPT NMR spectrum of (1),1H-1h COSY spectrum (shown in FIGS. 28-33, FIG. 28 shows that the compound epicpirocin A of the present invention is dissolved in DMSO-d6In (1)1H-1H COSY spectrum, FIG. 29 shows that the compound epicpirocin B of the present invention is dissolved in DMSO-d6In (1)1H-1H COSY spectrum, FIG. 30 shows that the compound epicpirocin C of the present invention is dissolved in DMSO-d6In (1)1H-1HCOSY spectra, FIG. 31 shows that the compound of the present invention, epicpirocin D, is dissolved in DMSO-D6In (1)1H-1H COSY spectra, FIG. 32 shows that the compounds epicpirocin E and epicpirocin F of the present invention are dissolved in DMSO-d6In (1)1H-1H COSY spectra, FIG. 33 shows that the compounds epicpirocin G and epicpirocin H of the present invention are dissolved in DMSO-d6In (1)1H-1H COSY spectrum), HSQC spectrum (as shown in FIGS. 34-39, FIG. 34 shows that the compound epicipirosin A of the present invention is dissolved in DMSO-d6FIG. 35 shows the HSQC spectrum of the compound epicpirocin B of the present invention dissolved in DMSO-d6FIG. 36 shows the HSQC spectrum of the compound epicpirocin C of the present invention dissolved in DMSO-d6FIG. 37 shows the solubility of epicpirocin D of the present invention in DMSO-D6FIG. 38 shows the solubility of epicpirocin E and epicpirocin F, the compounds of the present invention, in DMSO-d6FIG. 39 shows the solubility of epicpirocin G and epicpirocin H in DMSO-d6HSQC spectrum of (1)) and HMBC spectrum (as shown in FIGS. 40-45, FIG. 40 shows that the compound epicpirocin A of the present invention is dissolved in DMSO-d6FIG. 41 shows the HMBC spectrum of the compound epicpirocin B of the present invention dissolved in DMSO-d6FIG. 42 shows the HMBC spectrum of the compound epicpirocin C of the present invention dissolved in DMSO-d6FIG. 43 shows the HMBC pattern of the compound epicpirocin D of the present invention dissolved in DMSO-D6FIG. 44 shows the solubility of epicpirocin E and epicpirocin F of the compounds of the present invention in DMSO-d6FIG. 45 shows the solubility of epicpirocin G and epicpirocin H in DMSO-d6HMBC spectrum) of 8 compounds were investigatedAnd to1H and13the C signals were assigned as shown in tables 1 and 2. The relative configuration of the compounds epicpirocins A-D was then determined from the respective NOSEY spectra (as shown in FIGS. 46-49, FIG. 46 shows the solubility of epicpirocin A in DMSO-D6In (1), FIG. 47 shows the dissolution of epicpirocin B in DMSO-d as a compound of the present invention6In (1), FIG. 48 shows the dissolution of epicpirocin C of the compound of the present invention in DMSO-d6In (1), FIG. 49 shows the dissolution of epicpirocin D of the compound of the present invention in DMSO-D6NOESY spectrum of (iii) and finally the structure as follows:
TABLE 1 preparation of epicopolipirins A-D compounds1H and13assignment of peaks in C-NMR spectra
TABLE 2 preparation of epicopolipirins E-H compound1H and13assignment of peaks in C-NMR spectra
NMR measurements of the Compounds epicopolicolinic A-H Using Bruker 600MHz (1H 600MHz；13C150 MHz). The solvent of the compound epicpirocins A-H is DMSO-d6(solvent Peak correction)H2.50/C39.52)。
Spiroketal compound antibacterial activity test
(1) Antibacterial assay for Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Streptococcus mutans and Streptococcus sanguis
The strains tested included: staphylococcus aureus strain ATCC6538, methicillin-resistant Staphylococcus aureus strain, Streptococcus mutans strain ATCC UA159, Streptococcus sanguinis strain ATCC 10556, which were isolated in Hospital 309 of the people's Release force of China. The inhibition of the growth of the selected bacteria by the compound is determined by a continuous dilution method, and the minimum inhibitory concentration of the compound against different bacterial strains is obtained. The Minimum Inhibitory Concentration (MIC) is the minimum concentration of drug required to inhibit bacterial growth. Vancomycin was selected as a positive control in this experiment.
The test bacteria were cultured in Mueller-Hinton Broth (MHB) medium to log phase, and the cultured bacteria were diluted to 10 with the medium4cfu/mL concentration was seeded in 96-well cell culture plates containing 78 μ L of bacterial suspension per well, 2 μ L added to 2-fold serial dilutions of each compound. The sample and control were dissolved in dimethyl sulfoxide (DMSO) at a final concentration of no more than 0.05%. A series of sample concentrations of 4000 to 31.3. mu.g/mL were obtained by serial dilution, and incubated aerobically at 37 ℃ for 16 hours. The absorbance before and after the culture is measured by a microplate reader at 600nm, the Minimum Inhibitory Concentration (MIC) value is calculated according to the change of the absorbance, and all the experiments are carried out in parallel for 3 times.
The results of the experiment are shown in table 3:
TABLE 3
In-vitro antibacterial activity studies show that the compounds epicosphirocins A, B, D, G and H have certain inhibitory activity on staphylococcus aureus and methicillin-resistant staphylococcus aureus (MIC is 64 mu G/mL).
(2) Antifungal assay
The strains tested included: candida albicans strain SC 5314. The inhibition of the growth of the selected bacteria by the compound is determined by a continuous dilution method, and the minimum inhibitory concentration of the compound against different bacterial strains is obtained. The Minimum Inhibitory Concentration (MIC) is the minimum concentration of drug required to inhibit bacterial growth. In the experiment, amphotericin B is selected as a positive control drug, and only DMSO treatment is set as a negative control.
Single colonies of Candida albicans strain SC 5314 were suspended in RPMI 1640 at a concentration of 1 × 104cfu/mL were seeded in 96-well cell culture plates containing 78. mu.L of fungal suspension per well, 2. mu.L of each compound added in 2-fold serial dilutions. The sample and control were dissolved in dimethyl sulfoxide (DMSO) at a final concentration of no more than 0.05%. A series of sample concentrations of 4000 to 31.3. mu.g/mL were obtained by serial dilution and the plates were incubated at 35 ℃ for 16 hours. The absorbance before and after the culture is measured by a microplate reader at 600nm, the Minimum Inhibitory Concentration (MIC) value is calculated according to the change of the absorbance, and all the experiments are carried out in parallel for 3 times.
The results of the experiment are shown in table 4:
TABLE 4
The compounds epicpirocins A-D have certain inhibition effect on Candida albicans (MIC value is 64mg/L) by taking DMSO and amphotericin B as controls.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A spiroketal compound derived from plant pathogenic fungi is characterized in that the structural formula is as follows:
2. the spiroketal compound derived from plant pathogenic fungi as claimed in claim 1, wherein the spiroketal compound is isolated from epicoccum nigrum 09116 by fermentation culture.
3. A method for preparing spiroketal compounds derived from plant pathogenic fungi according to claim 1 or 2, which comprises the following steps:
sterilizing a solid fermentation culture medium, inoculating 5% of seed liquid by volume to the solid fermentation culture medium, standing and culturing at 28 ℃, harvesting a solid fermentation product after 40 days, wherein the solid fermentation product is all substances in a container, and separating and purifying the solid fermentation product to obtain the spiroketal compound derived from the plant pathogenic fungi.
4. The method according to claim 3, wherein the solid fermentation medium is rice: 2:3 of water;
the preparation method of the seed liquid comprises the following steps: respectively filling seed culture medium into multiple glass bottles, sterilizing at 121 deg.C for 20min, inoculating with Pleurotus nigrococcus strain, and rotary culturing at 28 deg.C for 7d to obtain seed solution.
5. The method according to claim 4, wherein the seed culture medium comprises the following components: potato extract, glucose, agar and water; the concentrations of the components in the seed culture medium are respectively as follows: potato extract 200g/L, glucose 20g/L and agar 20 g/L;
the culture method of the epicoccum nigrum-containing plate strain comprises the following steps:
sterilizing the plate culture medium at 121 ℃ for 20min, preparing a plate, culturing at 37 ℃ for 3d until the surface moisture is slightly dry and no foreign bacteria grow, inoculating the epicoccum nigrum 09116 strain spores to the plate culture medium, culturing at 28 ℃ for 10d, wherein the appearance is meat red, aerial hyphae are plump, and the plate strain can be collected and used when no bacteria are infected, thus obtaining the plate strain.
6. The method of claim 5, wherein the plating medium comprises the following components: potato extract, glucose, agar and water; the concentrations of the components in the plate culture medium are respectively as follows: potato extract 200g/L, glucose 20g/L and agar 20 g/L.
7. The method according to claim 3, wherein the step of separating and purifying the solid fermentation product to obtain the spiroketal compounds from the plant pathogenic fungi comprises the following steps:
extracting the obtained solid fermentation product for three times by using ethyl acetate, filtering an extracting solution to remove the solid fermentation product, collecting a supernatant, concentrating and evaporating the supernatant to dryness, weighing to obtain a crude extract, separating by using silica gel vacuum liquid chromatography, and using dichloromethane to methanol according to the following ratio of 1: 0,100: 1,50: 1,20: 1,10: 1,4: 1,1: 1,0: 1 gradient elution of the crude extract to give 10 fractions G1-G10 in total, further eluting the G5 fraction with methanol as mobile phase and Sephadex LH-20 column to give 23 subfractions G5N1-G5N23, the subfraction G5N7 with ODS-MPLC for 60 minutes, gradient elution with 10% -100% aqueous acetonitrile to give 17 fractions G5N7M1-G5N7M17, the G5N7M15 fraction purified by semi-preparative RP-HPLC using YMC-Ph at a flow rate of 4.0mL/min using gradient eluent: 0min, 20% methanol water solution; 20min, 50% methanol in water to obtain epicipirosin A and epicipirosin B; the G5N7M17 fraction was purified by semi-preparative RP-HPLC using Cosmosil π -nap at a flow rate of 4.0mL/min, eluting with 30% methanol water, to give the compounds epicipiroricin C and epicipiroricin D; fractions of G5N7M10 were purified by semi-preparative RP-HPLC using Cosmosil π -nap at a flow rate of 4.0mL/min, eluting with 22% acetonitrile in water, to give compounds epicosphipricin E and epicosphiricin F; fractions of G5N7M12 were purified by semi-preparative RP-HPLC using Cosmosil π -nap with a flow rate of 4.0mL/min, eluting with 35% aqueous methanol to give the compounds epicosphipricin G and epicosphiricin H.
8. Use of the spiroketal compound derived from plant pathogenic fungi according to claim 1 or 2 in the preparation of antibacterial drugs.
9. A pharmaceutical composition comprising the spiroketal compound derived from a plant pathogenic fungus, its cis-trans isomer, solvate or pharmaceutically acceptable salt thereof according to claim 1 or 2 and a pharmaceutically acceptable carrier.
10. Use of a pharmaceutical composition according to claim 9 for the preparation of an antibacterial medicament.
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Citations (2)
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| EP1870405A1 (en) * | 2006-06-22 | 2007-12-26 | Bioprojet | Carbonylated (Aza)cyclohexanes as dopamine D3 receptor ligands |
| CN107951878A (en) * | 2016-10-14 | 2018-04-24 | 复旦大学 | Purposes of the Spiroketals polyacetylene compound in anti-outer pump drug-resistant staphylococcus aureus hypersitization medicine is prepared |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1870405A1 (en) * | 2006-06-22 | 2007-12-26 | Bioprojet | Carbonylated (Aza)cyclohexanes as dopamine D3 receptor ligands |
| CN107951878A (en) * | 2016-10-14 | 2018-04-24 | 复旦大学 | Purposes of the Spiroketals polyacetylene compound in anti-outer pump drug-resistant staphylococcus aureus hypersitization medicine is prepared |
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| NGUYEN DINH LUYEN ET AL.: "Aspermicrones A-C, novel dibenzospiroketals from the seaweed-derived endophytic fungus Aspergillus micronesiensis", 《THE JOURNAL OF ANTIBIOTICS》 * |
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