CN113444644B - Application of naphtho-gamma-pyrone compounds in preparation of anti-helicobacter pylori medicines or prevention health-care products - Google Patents
Application of naphtho-gamma-pyrone compounds in preparation of anti-helicobacter pylori medicines or prevention health-care products Download PDFInfo
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- CN113444644B CN113444644B CN202010223692.1A CN202010223692A CN113444644B CN 113444644 B CN113444644 B CN 113444644B CN 202010223692 A CN202010223692 A CN 202010223692A CN 113444644 B CN113444644 B CN 113444644B
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Abstract
The invention discloses application of naphtho gamma-pyrone compounds in preparing anti-helicobacter pylori medicaments or prevention health-care products. The invention provides a marine fungus, which is named as Aspergillus sp.DM94, and has a preservation number: CCTCC NO: m20191003. The method extracts and separates the naphtho-gamma-pyrone compounds from the fermentation culture of the marine fungi by utilizing the polarity difference of the naphtho-gamma-pyrone, has simple and convenient operation, high extraction yield and high product purity, and is suitable for large-scale production. In-vitro antibacterial experiments show that the naphtho-gamma-pyrone compound has better in-vitro antibacterial activity, and the MIC value of the naphtho-gamma-pyrone compound is 4-64 mu G/mL aiming at a helicobacter pylori standard strain G27 and a multi-drug-resistant strain HP159 of levofloxacin, clarithromycin and metronidazole, so that the naphtho-gamma-pyrone compound can be used for preparing antibacterial drugs and preventive health-care foods and has good development prospects.
Description
Technical Field
The invention relates to the field of marine fungus metabolites as new antibacterial agents, and in particular relates to application of naphtho gamma-pyrone compounds in preparation of anti-helicobacter pylori medicines or prevention health-care products.
Background
Helicobacter pylori (Helicobacter pylori) is a gram-negative bacterium that is microaerophilic, often located in the mucous membrane of the human stomach fundus, and infects about half of the population worldwide. Helicobacter pylori infection often causes digestive system diseases such as chronic gastritis, peptic ulcer, and the like. Research shows that helicobacter pylori infection is closely related to the occurrence and development of gastric cancer, and the eradication of helicobacter pylori can reduce the risk of gastric cancer. For the clinical treatment of helicobacter pylori, triple or quadruple therapy recommended by relevant guidelines, including omeprazole, a proton pump inhibitor, amoxicillin and clarithromycin, and the like, has good effects. However, the recent clinical emergence of multidrug resistant strains has greatly reduced antibiotic efficacy, and therefore the development of new anti-helicobacter pylori drugs, especially drugs effective against multidrug resistant strains therein, is of great significance.
Aspergillus niger is often capable of metabolizing structurally diverse secondary metabolites as well as abundant enzymes, and is therefore frequently used for bioconversion/catalysis studies of active natural products. Reports on the use of Aspergillus niger have mainly focused on the transformation studies of medicinal plant components using the enzymes produced by Aspergillus niger. For example, in Hwang et al, Aspergillus niger A-T1 is used to convert flavone in plants into phenolic acid components with antibacterial activity [ patent application, 2019, CN 110093279A 20190806], Zhang et al, Aspergillus niger fermentation is used to increase the yield of luteolin in honeysuckle flower [ patent application, 2017, CN 107022590A 20170808], Ou et al, Aspergillus niger ZJUT712 is used to convert arctiin into aglycone [ patent application, 2009, CN 101358173A 20090204 ].
Naphtho-gamma-pyrones are an important class of aromatic polyketones produced by aspergillus fungi, especially aspergillus niger, including naphtho-gamma-pyrones, bis-naphtho-gamma-pyrones, alpha-pyrones, pyrrolopyrones, and the like. The literature reports that the substances show good xanthine oxidase inhibitory activity, and part of compounds have good antibacterial activity on common pathogenic bacteria such as candida albicans, trichophyton rubrum and the like, and the activity of the compounds is related to ketoconazole. Meanwhile, the literature also reports that the substances have free radical scavenging activity, cytotoxic activity and the like. However, no report has been found so far which reports that naphtho- γ -pyrones derived from Aspergillus niger have anti-helicobacter pylori activity.
Disclosure of Invention
The invention aims to provide application of naphtho-gamma-pyrone compounds in preparing anti-helicobacter pylori medicaments or prevention health-care products.
The naphtho-gamma-pyrones compound is a natural active substance with medicinal value extracted from Aspergillus sp.DM94 (DM 94).
The purpose of the invention is realized by the following technical scheme:
the invention provides an application of a naphtho gamma-pyrone compound or pharmaceutically acceptable salt thereof in preparing anti-helicobacter pylori medicaments or prevention health care products, wherein the structural formula of the naphtho gamma-pyrone compound is shown as formulas (1) to (8):
the invention provides a marine fungus, named Aspergillus fungus DM94(Aspergillus sp.DM94), which is an epiphytic fungus of marine plants, and the preservation information is as follows: the preservation unit is as follows: china Center for Type Culture Collection (CCTCC) with a preservation time of 2019, 12 and 3 months and a preservation address: wuhan university, the preservation number: CCTCC NO: m20191003.
The strain of Aspergillus fungus DM94(Aspergillus sp.dm94) has the following microbiological characteristics:
culturing the bacterial colony in a PDA culture medium at 28 ℃ for 5 days, wherein the diameter of the bacterial colony is 35-40 mm, and culturing the bacterial colony for 10 days is 65-70 mm; the colony is initially white, hypha in the center of the colony is turned into black when the colony is cultured for two to three days, and the whole colony is turned into black when the colony is cultured for six days, has white edges and is cotton-shaped in texture and has no exudate; the reverse side of the colony is light yellow. Conidiophores are generated on aerial hyphae, and conidiophores are spherical. The diameter of the spore stems is 10-12.5 mu m, and the wall thickness is smooth; a top capsule is spherical, the diameter is 30-40 mu m, and the surface of the top capsule is fertile; the spore-forming structure has double layers, wherein the stem base is 10-12 multiplied by 2-3 mu m; 6-8 multiplied by 2-3 mu m of bottle stalk; conidiophore spherical, diameter of 3-4 μm, and rough wall.
The Aspergillus fungus DM94(Aspergillus sp.dm94) has the following molecular biological characteristics: ITS and 18S sequences are shown in SEQ ID NO.1 and 2.
The application of the marine fungus in preparing the helicobacter pylori resistant preparation.
The invention also provides a preparation method of the naphtho gamma-pyrone compound, which comprises the following steps:
(1) activating the Aspergillus fungus DM94(Aspergillus sp.DM94), inoculating into a fermentation culture medium, and performing fermentation culture;
(2) after fermentation culture is finished, adding an organic solvent for extraction, separating to obtain an extract, concentrating the extract to obtain an extract, and separating and purifying the extract to obtain the naphtho gamma-pyrone compounds;
in order to better simulate the marine environment and provide sufficient nutrients for the growth and metabolism of microorganisms, the formula of the fermentation medium in the step (1) is preferably as follows:
the formula is as follows based on 50g of corn kernels: 50g corn kernels, 0.86g yeast extract powder, 2.37g ammonium tartrate and MgSO40.17g，KH2PO40.25g, sea salt (2%) 0.4g, ddH2O20 mL, and natural PH; the corn grains need to be crushed into particles.
Or, the formula is as follows according to the volume of 1L: 1-5 g of starch, 10-20 g of bran, 3-15 g of yeast extract and KH2PO4 1～8g，MgSO4·7H20.1-0.8 g of O, and the balance being seawater;
or, based on 1L of the volume, the formula is as follows: 200-600 g of potatoes, 2-10 g of peptone, 1-5 g of yeast extract, 5-20 g of glucose and the balance of seawater; the initial pH value is 6.0-7.0;
or, the formula is as follows according to the volume of 1L: 10-40 g of cane sugar, 5-20 g of corn flour and NaNO31-4 g, 1-4 g yeast extract and KH2PO4 0.2～0.8g、MgSO4·7H2O 0.2～1g、KCl 0.2～1g、FeSO40.001-0.005 g, and the balance of artificial seawater.
Preferably, the fermentation culture condition in the step (1) is static culture at 20-30 ℃ for 10-40 days. The static culture mode is not to carry out shake flask culture.
Further, the condition of fermentation culture is static culture at 22-26 ℃ for 10-40 days. More preferably, the naphtho-gamma-pyrone compound is statically cultured at 25 ℃ for 20 days, under which the yield of the naphtho-gamma-pyrone compound is the highest.
Preferably, the organic solvent in step (2) is one or two of methanol, ethanol, ethyl acetate and acetone.
Preferably, in the step (2), the extract obtained by leaching the fermentation product is subjected to normal phase silica gel column chromatography and then to reverse phase silica gel column chromatography/high performance liquid chromatography. The method specifically adopts silica gel open column chromatography, ODS column chromatography, preparative HPLC and other methods for separation and purification.
When normal phase silica gel column chromatography is adopted for separation, the eluent can adopt one or more of petroleum ether, n-hexane, ethyl acetate, alcohol and water for gradient elution of the extract.
Preferably, the separation and purification comprises: separating the extract by normal phase silica gel column chromatography, gradient eluting with petroleum ether/ethyl acetate mixture and ethyl acetate at volume ratio of 100:0, 9:1, 8:2, 6:4, and 0:100, and collecting each eluted fraction.
The research of the invention shows that the naphtho-gamma-pyrone compound separated from the marine fungus fermentation culture by the method has better antibacterial activity, so the invention also provides the application of the naphtho-gamma-pyrone compound in preparing anti-helicobacter pylori medicines or prevention health products.
The drug is prepared by taking the naphtho-gamma-pyrone compound or the pharmaceutically acceptable salt thereof as a main active ingredient and adding pharmaceutically acceptable auxiliary materials, and can be prepared into a preparation according to a preparation method recorded in pharmaceutics. The preparation can be injection, infusion solution, powder for injection, granule, tablet, granule, powder, oral liquid, sugar-coated tablet, film-coated tablet, enteric-coated tablet, buccal agent, granule, pill, unguent, pellet, spray, dripping pill, disintegrant, orally disintegrating tablet, pellet, etc.
The health product is prepared by taking the naphtho-gamma-pyrone compound or the pharmaceutically acceptable salt thereof as a main active ingredient and adding acceptable health food auxiliary materials.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention extracts and separates the naphtho-gamma-pyrone compounds from the fermentation culture of marine fungi by utilizing the polarity difference of the naphtho-gamma-pyrone, and the method has simple and convenient operation, high extraction yield and high product purity and is suitable for large-scale production.
(2) In-vitro antibacterial experiments show that the naphtho gamma-pyrone compound provided by the invention has better in-vitro antibacterial activity, and the MIC value of the naphtho gamma-pyrone compound is 4-64 mu G/mL aiming at a helicobacter pylori standard strain (G27) and a multi-drug-resistant strain HP159 of levofloxacin, clarithromycin and metronidazole, so that the naphtho gamma-pyrone compound can be used for preparing antibacterial drugs and preventive health-care foods and has good development prospects.
Drawings
FIG. 1 is a structural formula of naphtho gamma-pyrones of the present invention.
FIG. 2 is HPLC detection spectrum of crude fermentation extract of strain DM 94.
FIG. 3 is a graph of ITS gene sequence cluster analysis of strain DM 94.
FIG. 4 is a diagram of 18S rRNA gene sequence cluster analysis of strain DM 94.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Example 1 isolation of fungus DM94
The strain DM94 is separated from root soil of Bruguiera gymnorrhiza of mangrove, the soil sample is put in a sterile culture dish, air-dried in natural environment, 1g of air-dried soil sample is weighed, 10mL of 50% aged seawater is used for dissolving to obtain 10-1Solution, further diluting to 10-2、10-3Obtaining three gradient sample liquids. Coating 100 μ L of the extract on GPY culture medium, Martin culture medium, CA culture medium, and PDA culture mediumAnd 2 isolation culture media with different dilution concentrations are used in parallel on the SDA culture medium and the fungus No. 2 culture medium, and are cultured for 1-8 weeks at 28 ℃. And observing and picking a single colony, and inoculating the single colony on a No. 2 fungus culture medium to obtain a pure culture strain.
Example 2 molecular characterization of fungal DM94
Extraction of genomic DNA: the DM94 genome DNA template is obtained by a nucleic acid rapid extractor, and the operation is as follows: taking a sterile DNA extraction tube filled with quartz sand, adding 500 μ L of TE, picking an appropriate amount of mycelia, placing into the tube, and extracting for 1min with high speed shaking. Then, 500. mu.L of phenol/chloroform solution was added thereto, and the mixture was centrifuged to obtain genomic DNA.
Amplification of the gene sequence: mu.L of each of 2-fold PCR mastermix 12.5. mu.L, primers ITS1(5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4(5'-TCC TCC GCT TAT TGA TAT GC-3'), (5'-AAC CTG GTT GAT CCT GCC AGT-3' for 18SF and 5'-CGA CGG GCG GTG TGT AC-3' for 18 SR), 2. mu.L of template DNA, and 13.5. mu.L of double distilled water were added. The amplification condition is pre-denaturation at 95 ℃ for 3 min; denaturation at 95 deg.C for 30S, annealing at 59 deg.C for 30S, and extension at 72 deg.C for 30S for 32 cycles; after extension at 72 ℃ for 10 min. The PCR product was detected by 2% agarose gel electrophoresis.
Cutting a target band (about 600bp) for recovery and purification, adding a buffer solution to submerge agar, and carrying out water bath at 42 ℃ until the gel is melted; transferring the glue solution into a collecting tube, and centrifuging to remove waste liquid; washing twice with a washing solution; opening the cover and centrifuging for 1min to volatilize alcohol; the adsorption column is replaced into a new centrifuge tube, 40 mu L of elution buffer solution is added for centrifugation to obtain an amplification product DNA, and the DNA is sent to Huada gene sequencing.
And (3) submitting ITS sequences and 18S sequences to GeneBank for comparison, selecting sequences with higher similarity, carrying out sequence analysis by using Bioedit, and then constructing a phylogenetic tree by using MEGA 7.0. The phylogenetic tree was used to infer that the strain belongs to Aspergillus (FIGS. 3 and 4).
The fungus DM94, designated Aspergillus sp 94(Aspergillus sp.dm94), deposited information: the preservation unit: china Center for Type Culture Collection (CCTCC) with a preservation time of 2019, 12 and 3 months and a preservation address: wuhan university, the preservation number: CCTCC NO: m20191003.
EXAMPLE 3 Mini-preparation of crude fermentation extract of fungus DM94
The strain is cultured in corn solid fermentation medium (corn grain 50 g; ammonium tartrate 2.37 g; yeast extract powder 0.86 g; KH)2PO40.25g；MgSO40.17 g; sea salt 0.4 g; 20mL of distilled water, and natural pH. (Hongkudu, Populus niveus, a high-yielding pseudoshell compound strain Aspergillus oryzae TKYX429 and its application, ZL201310186483.4)) was cultured at 28 ℃ for 4 weeks. Soaking the fermentation product in 80% acetone for 12h, performing ultrasonic extraction for 20min with an ultrasonic extractor, extracting with ethyl acetate for 3-5 times, mixing the extractive solutions, evaporating to dryness, and dissolving with chromatographic methanol. The sample is used for antibacterial activity detection and HPLC detection.
EXAMPLE 4 preliminary examination of antibacterial Activity of fermentation crude extract
The 3 pathogenic bacteria Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 5165 and Candida albicans ATCC 10231 were cultured overnight to prepare 1070.1mL of cfu/mL of bacterial suspension is uniformly coated on a culture medium, after air drying, 20 mu L of crude fermentation extract dissolved in methanol is added on a sterile filter paper sheet (diameter is 6mm), and the sterile filter paper sheet is attached to a flat plate, and pure methanol is used as a blank control. Culturing for 12-24 h at a certain temperature, and observing and measuring the inhibition zone. The detection result shows that the crude fermentation extract of the strain DM94 has the effect of inhibiting staphylococcus aureus and has no effect on other two pathogenic bacteria.
The minimum inhibitory concentration (MIC, 100 mu.L system) of the fungus DM94 fermented extract on helicobacter pylori is detected by a microdilution method. The H.pylori standard strain G27 and the levofloxacin and clarithromycin and metronidazole multidrug-resistant strain (HP159) were provided by the subject group of professor Bighouka of Nanjing medical university. Preparation of MIC plate: 173.6 mu L of culture medium is firstly added into the first hole, and then 6.4 mu L of antibacterial agent is added, and the mixture is diluted to the 7 th hole in a multiple ratio; no drug was added to well 8, and 90. mu.L of the medium was retained as a control with and without drug added. Preparing bacterial liquid: taking helicobacter pylori growing in logarithmic phase on a solid plate, preparing into bacterial suspension by using BHI culture medium, adjusting concentration OD600Is 0.3 (1X 10)8CFU/mL), 10-fold dilution at 1X 107CFU/mL, spare. Inoculating a bacterial liquid:adding 10 μ L into 1-8 th well (the concentration of bacteria liquid in each well is about 1.0 × 10)6CFU/mL), and culturing for 72h to judge the result. The drug concentrations in the 1 st to 7 th wells are 128, 64, 32, 16, 8, 4 and 2 mug/mL respectively. And (5) judging a result: the MIC was taken as the lowest drug concentration that completely inhibited bacterial growth in the wells. The test is only meaningful when the bacteria grow significantly in the 8 th well (i.e., no antibiotic) of the negative control well. When a single jump hole occurs in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If multiple jump holes appear, the result should not be reported, and the test needs to be repeated. Each drug was tested in 3 replicates. The results show that the MIC value of the fungal DM94 fermented extract against the helicobacter pylori standard strain G27 is 128. mu.g/mL.
EXAMPLE 5 HPLC and LC-MS detection of crude fermentation extract
HPLC detection conditions: and (3) chromatographic column: sinochrom ODS-BP, 250X 4.6nm, 5 μm; the mobile phase is mixed with methanol: the water ratio is respectively 10: 90, respectively; 100: 0; 10: and eluting for 90 min to 15min, 15min to 20min and 20min to 25 min. The detection wavelength is 190-400 nm, and the specified wavelength is 254 nm; the amount of the sample was 20. mu.L.
The LC-MS detection conditions are as follows: ThermoFisher column, 150X 2.1mm, 3 μm; the detection wavelength is 254nm, and the sample injection amount is 10 mu L. Mass spectrometry conditions: positive ion mode: ion source ESI, heating temperature 400 ℃, sheath gas flow 30psi, auxiliary gas flow 5psi, nebulizer voltage 4KV, ion transfer tube temperature 325 ℃. Event one: full scanning, wherein the range is 200-2000 m/z; and event two: data Dependent Scan, activation type CID, minimum signal strength 50000, isolation width 2, collision energy 35, charge state 1, activation time 30 ms.
The detection result shows that the DM94 fermented crude extract has a series of absorption (figure 2) in liquid chromatography for 13-18 min, and the maximum absorption peaks are 307nm and 279 nm. Find [ M + H in Mass Spectrometry]+、[M+Na]+、[2M+H]+、[2M+Na]+As a compound, comparison of at least two of the data in the database of Antibase revealed that 28 compounds could be found in the crude fermentation extract, half of which were unknown, indicating new compounds in the product.
EXAMPLE 6 Mass fermentation culture of the fungus DM94
Placing fungus DM94 in GPY solid medium (glucose 20g, peptone 10g, yeast extract 10g, sea salt 15g, agar 20g, ddH)2O1L, pH7.5), in corn solid medium (50g corn kernel, yeast extract powder 0.86g, ammonium tartrate 2.37g, MgSO4 0.17g，KH2PO40.25g, sea salt (2%) 0.4g, ddH2O20 mL, natural pH) was used to ferment a large amount of 1.5 Kg. After 24 days of fermentation at room temperature, the mycelium was soaked overnight with EtOAc for extraction.
EXAMPLE 7 preparation of naphtho-gamma-pyrones
Soaking and extracting a fermentation product of marine fungus DM94 fermented by a corn solid culture medium for 3-5 times by using ethyl acetate (EtOAC). Mixing extractive solutions, and concentrating under reduced pressure to obtain 50g extract. The extract was subjected to silica gel open column chromatography (phi 50X 640mm, 200-300 mesh, 500-700 g), and gradient eluted with petroleum ether-ethyl acetate (100: 0, 98: 2, 95: 5, 9:1, 8:2, 6:4, 0:100) and EtOAC-MeOH (95: 5, 9: 1) to give 11 fractions (Fr.1-Fr.11). Wherein Fr.7[0.2634g, Petroleum/EtOAC (8:2)]By preparative HPLC (YMC-Pack ODS-A, 80% MeOH-H)2O, 3ml/min) to give compound 1(4.0mg) and compound 2(4.8 mg); fr.9[21.8949g, Petroleum/EtOAC (0:100)]Performing ODS open column chromatography (phi 45X 500mm), and gradient eluting with methanol-water (20% -100%) to obtain 21 sub-fractions (Fr.9-1-Fr.9-21). Subfraction Fr.9-11(2.34g, 70% MeOH-H)2O) is subjected to silica gel open column chromatography (phi 25 is multiplied by 130mm, 200 to 300 meshes, 14g) and CH2Cl2Gradient elution with MeOH (100: 0, 100: 1, 100: 2, 100: 4, 0:100) yielded 6 sub-fractions (Fr.9-11-1 to Fr.9-11-6). Fr.9-11-3[ (213.6mg, CH)2Cl2/MeOH(100：1)]By preparative HPLC (YMC-Pack ODS-A, 63% MeOH-H)2O, 3ml/min) to obtain 8 sub-fractions (Fr.9-11-3-1 to Fr.9-11-3-8). Wherein Fr.9-11-3-5(9.6mg, 63% MeOH-H)2O) preparation by two HPLC (YMC-Pack ODS-A, 63% MeOH-H2O, 3ml/min) to obtain compound 6(1.1mg) and Fr.9-11-3-5-1(3.7 mg); fr.9-11-3-5-1(3.7mg) was further prepared by HPLC (YMC-Pack ODS-A, 60% MeOH-H)2O, 3ml/min) to give compound 7(3.2 mg). Fr.9-11-3-7(47.1 mg)，63％MeOH-H2O) preparation by HPLC (YMC-Pack ODS-A, 63% MeOH-H2O, 3ml/min) gave compound 8(10.6 mg). Subfraction Fr.9-12/13(1.18g, 70% MeOH-H)2O) is subjected to ODS medium-low pressure column chromatography (phi 25 is multiplied by 90mm, 12g) and methanol-water (40-100%) gradient elution to obtain 10 sub-fractions (Fr.9-12/13-1-Fr.9-12/13-10), wherein Fr.9-12/13-5(68.2mg, 60% MeOH-H)2O) by preparative HPLC (YMC-Pack ODS-A, 45% ACN-H2O, 3ml/min) to give compound 5(1.5 mg). Sub-fraction Fr.9-14/15(2.51g, 70% MeOH-H)2O) by preparative HPLC (YMC-Pack ODS-A, 50% ACN-H2O, 3ml/min) to give compound 3(12.8mg) and compound 4(14 mg).
Example 8 structural identification of naphtho-gamma-pyrones
Identifying the purity of the prepared compound by TLC and HPLC, identifying the structure of a sample with the purity of more than 98% by mass spectrum, nuclear magnetic resonance and CD spectrum, measuring the nuclear magnetic resonance by a Bruker AV-600NMR spectrometer, and using TMS as an internal standard; the high-resolution mass spectrum is measured by a Waters Synapt G2 mass spectrometer; the CD spectrometer was a JASCO J-810 circular dichroism spectrometer.
From the results of one-dimensional and two-dimensional NMR analyses of the compounds (see tables 1 and 2), it can be seen that the structures of the compounds were determined as shown in fig. 1.
TABLE 1 preparation of naphtho gamma-pyrones 1 to 813C NMR data
Wherein,ain DMSO-d6 bin CDCl3。
TABLE 2 preparation of naphtho-gamma-pyrones 1-81H NMR data
Wherein,ain DMSO-d6 bin CDCl3。
example 9 analysis of anti-helicobacter pylori Activity of naphtho-gamma-pyrones
9.1 helicobacter pylori strain information: standard strain G27; levofloxacin and clarithromycin and metronidazole multiple drug resistant strain (HP159) (provided by professor topic group of chunkka university of south beijing medical science). The bacterial strains are also disclosed in the patent CN201810462768.9, the preparation of zinc linolenate and the application thereof in preparing the anti-helicobacter pylori medicaments.
9.2 culture medium and main reagents, consumables: columbia culture medium, brain heart infusion culture medium, selective antibiotics (vancomycin, polymyxin B and trimethoprim), serum, omeprazole, amoxicillin and clarithromycin; EP tubes, Tip heads, centrifuge tubes, polyethylene 96-well plates, and the like.
9.3 Experimental methods: the minim dilution method is used for detecting the minimum inhibitory concentration (MIC, 100 mu L system) of the drug to helicobacter pylori, and the MIC plate is prepared: 173.6 mu L of culture medium is firstly added into the first hole, and then 6.4 mu L of antibacterial agent is added, and the mixture is diluted to the 7 th hole by times; no drug was added to well 8, and 90. mu.L of the medium was retained as a control for adding bacteria and no drug.
Preparing bacterial liquid: taking helicobacter pylori growing in logarithmic phase on solid plate, preparing into bacterial suspension with BHI culture medium, adjusting concentration OD600 to 0.3(1 × 10)8CFU/mL), diluted 10-fold to 1X 107CFU/mL, spare.
Inoculating a bacterial liquid: adding 10 μ L into 1-8 th well (the concentration of bacteria liquid in each well is about 1.0 × 10)6CFU/mL). Culturing for 72h to judge the result. The drug concentrations in the 1 st to 7 th wells are 64, 32, 16, 8, 4, 2 and 1. mu.g/mL respectively.
9.4 judging the result: the lowest drug concentration that completely inhibited bacterial growth in the wells was taken as the MIC. The test is only meaningful when the bacteria grow significantly in the 8 th well (i.e., no antibiotic) of the negative control well. When a single jump hole occurs in the microdilution method, the highest concentration of drug that inhibits bacterial growth should be recorded. If a plurality of jump holes appear, the result should not be reported, and the test needs to be repeated. Each drug was tested in 3 replicates.
TABLE 3 MIC (μ g/mL) for naphtho γ -pyrone compounds for inhibiting helicobacter pylori
| Compounds | H.pylori G27 | H.pylori HP159 |
| Flavasperone(1) | ＞64 | ＞64 |
| Rubrofusarin B(2) | ＞64 | ＞64 |
| S-Asperpyrone C(3) | 64 | 64 |
| S-Aurasperone A(4) | 8 | 16 |
| S-Fonsecinone A(5) | ＞64 | ＞64 |
| S-Asperpyrone A(6) | 4 | 4 |
| S-Aurasperone B(7) | 8 | 16 |
| S-Aurasperone F(8) | 4 | 4 |
The results are shown in Table 3, which shows that part of naphtho gamma- pyrone compounds 3, 4 and 6-8 derived from Aspergillus fungus DM94 show good anti-helicobacter pylori activity, and MIC values of the naphtho gamma-pyrone compounds for helicobacter pylori common strain G27 and drug-resistant strain HP159 are both 4-64 mu G/mL.
EXAMPLE 10 preparation of dropping pill preparation containing naphtho-gamma-pyrone compounds
0.5g of naphtho gamma-pyrone compound and 10.5g of polyethylene glycol-6000 are uniformly mixed, heated and melted, the mixture is moved into dripping pills for drip irrigation, the medicine is dripped into liquid paraffin with the temperature of 6-8 ℃, oil is removed, and 300 dripping pills are prepared.
EXAMPLE 11 preparation of lyophilized powder for injection containing naphtho-gamma-pyrone compounds
Taking 0.5g of naphtho gamma-pyrone compounds, 4.5g of glucose, 0.9g of sodium thiosulfate and 1000mL of distilled water, uniformly mixing the components, freeze-drying, and subpackaging 400.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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gtccccccgg agagggggac ggcgacccaa cacacaagcc gggcttgagg gcagcaatga 240
cgctcggaca ggcatgcccc ccggaatacc agggggcgca atgtgcgttc aaagactcga 300
tgattcactg aattctgcaa ttcacattag ttatcgcatt tcgctgcgtt cttcatcgat 360
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aggcgccccc ccggcggccg acaagcggcg ggcccgccga agcaacaggg tacaatagac 540
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gagaaacggc taccacatcc aaggaaggca gcaggcgcgc aaattaccca atcccgacac 420
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accttgggtc tggctggccg gtccgcctca ccgcgagtac tggtccggct ggacctttcc 660
ttctggggaa tctcatggcc ttcactggct gtggggggaa ccaggacttt tactgtgaaa 720
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ttctattatg acccgttcgg caccttacga gaaatcaaag tttttgggtt ctggggggag 1080
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Claims (9)
1. A marine fungus characterized by: the Aspergillus sp 94(Aspergillus sp.dm94) is preserved in the China center for type culture collection of Wuhan university in 2019, 12 months and 3 days, with the preservation number: CCTCC NO: m20191003.
2. Use of the marine fungus of claim 1 for the preparation of naphtho γ -pyrones.
3. A preparation method of naphtho gamma-pyrone compounds is characterized by comprising the following steps: the method comprises the following steps:
(1) activating the Aspergillus DM94 of claim 1, inoculating into a fermentation medium, and performing fermentation culture;
(2) after the fermentation culture is finished, adding an organic solvent for leaching, separating to obtain a leaching solution, concentrating the leaching solution to obtain an extract, and separating and purifying the extract to obtain a naphtho gamma-pyrone compound;
the structural formula of the naphtho gamma-pyrone compound is shown in formulas (1) to (8):
4. the production method according to claim 3, characterized in that:
the formula of the fermentation medium in the step (1) is as follows:
the formula is as follows based on 50g of corn kernels: 50g corn kernels, 0.86g yeast extract powder, 2.37g ammonium tartrate and MgSO4 0.17g，KH2PO40.25g, 2% sea salt 0.4g, ddH2O20 mL, pH is natural; the corn grains need to be crushed into particles.
5. The production method according to claim 3, characterized in that:
the formula of the fermentation medium in the step (1) is as follows:
the formula is as follows based on the volume of 1L: 1-5 g of starch, 10-20 g of bran, 3-15 g of yeast extract and KH2PO4 1～8g，MgSO4·7H20.1-0.8 g of O, and the balance being seawater.
6. The production method according to claim 3, characterized in that:
the formula of the fermentation medium in the step (1) is as follows:
the formula is as follows by volume 1L: 200-600 g of potatoes, 2-10 g of peptone, 1-5 g of yeast extract, 5-20 g of glucose and the balance of seawater; the initial pH value is 6.0-7.0.
7. The production method according to claim 3, characterized in that:
the formula of the fermentation medium in the step (1) is as follows:
the formula is as follows by volume 1L: 10-40 g of cane sugar, 5-20 g of corn flour and NaNO31-4 g, 1-4 g yeast extract and KH2PO40.2～0.8g、MgSO4·7H2O 0.2～1g、KCl 0.2～1g、FeSO40.001-0.005 g, and the balance of artificial seawater.
8. The production method according to any one of claims 3 to 7, characterized in that:
the condition of fermentation culture in the step (1) is static culture at 20-30 ℃ for 10-40 days;
the organic solvent in the step (2) is one or two of methanol, ethanol, ethyl acetate and acetone.
9. The method of claim 8, wherein:
the condition of fermentation culture is static culture at 22-26 ℃ for 10-40 days.
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