CN109082445B - Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis - Google Patents

Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis Download PDF

Info

Publication number
CN109082445B
CN109082445B CN201811004688.5A CN201811004688A CN109082445B CN 109082445 B CN109082445 B CN 109082445B CN 201811004688 A CN201811004688 A CN 201811004688A CN 109082445 B CN109082445 B CN 109082445B
Authority
CN
China
Prior art keywords
endophytic fungi
gingko
metabolite
endophytic
ethyl acetate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201811004688.5A
Other languages
Chinese (zh)
Other versions
CN109082445A (en
Inventor
何庆
曾强成
刘帅
周海霞
蒙建州
祁胜文
张洪博
邵奕博
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dezhou University
Original Assignee
Dezhou University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dezhou University filed Critical Dezhou University
Priority to CN201811004688.5A priority Critical patent/CN109082445B/en
Priority to PCT/CN2018/113709 priority patent/WO2019119992A1/en
Priority to US16/469,830 priority patent/US11332707B2/en
Publication of CN109082445A publication Critical patent/CN109082445A/en
Application granted granted Critical
Publication of CN109082445B publication Critical patent/CN109082445B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/062—Ascomycota
    • 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
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/06—Oxygen as only ring hetero atoms containing a six-membered hetero ring, e.g. fluorescein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • 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
    • C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C12P7/26—Ketones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Medical Informatics (AREA)
  • Botany (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Plant Substances (AREA)

Abstract

The invention relates to a metabolite product of gingko endophytic fungi and application thereof in antibiosis, wherein the fungus is gingko endophytic fungi layered Fusarium (Fusarium proliferatum) DZHQ1 with the preservation number of CGMCC No.14983, which is preserved in China general microbiological culture Collection center in 2017, 11 and 28 months, and the metabolite product of the gingko endophytic fungi is obtained by fermentation culture, extraction and separation. The metabolite of the ginkgo endophytic fungi disclosed by the invention shows a relatively obvious inhibition effect in the activity detection of resisting escherichia coli and/or staphylococcus aureus, and has a potential application in preparing a new antibacterial product.

Description

Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis
Technical Field
The disclosure belongs to the field of microbial medicines, and particularly relates to a metabolic product of gingko endophytic fungi and an application of the metabolic product in antibiosis.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Escherichia coli is a typical gram-negative bacillus, and colibacillosis caused by the Escherichia coli is a common disease, veterinary clinical colibacillosis causes serious harm, can cause diseases all the year round, is a common disease and frequently-occurring disease which troubles the development of the breeding industry all the time, and causes serious economic loss to the poultry industry. The drug resistance is easy to generate in the treatment process, the drug resistance spectrum is wide, the drug resistance mechanism is complex, and great difficulty is brought to the prevention and treatment of the disease in poultry industry. Therefore, the search for an effective ingredient against E.coli has been a hot research topic in recent years.
Staphylococcus aureus is one of the most common pathogenic bacteria infected inside and outside hospitals, and clinical isolated bacterial strains obtained by Chinese bacterial drug resistance monitoring networks in dozens of hospitals nationwide for years show that the Staphylococcus aureus accounts for the first 5 clinical pathogenic bacteria. The bacteria can cause severe pneumonia, meningitis, as well as osteoarticular infections, endocarditis and bacteremia. Especially, the bacteria is easily infected by patients with burn wound infection, acute liver failure, blood-borne nephritis and other diseases. In recent years, the emergence of Staphylococcus aureus, which has reduced susceptibility to some drugs, has exposed humans to certain infections that are not treatable with drugs. Therefore, it is important to find an effective ingredient against Staphylococcus aureus.
CN108102928A gingko endophytic fungi and application thereof discloses a gingko endophytic fungi layered Fusarium (Fusarium proliferatum) DZHQ1 with anti-cervical cancer activity separated from gingko bark, the preservation number is CGMCC No.14983, and further discloses a fermentation culture solution of the endophytic fungi and an ethyl acetate extract of the fermentation culture solution, which has therapeutic activity against cervical cancer.
Further developing the research of the gingko endophytic fungi active substance, and having important theoretical significance and potential application value for developing gingko plant medicinal endophytic fungi resources and new microbial medicines in China.
Disclosure of Invention
In view of the background technology, the disclosure further researches and explores CN108102928A 'a strain of gingko endophytic fungi and application thereof' and provides a metabolite product of the gingko endophytic fungi and application thereof in preparation of antibacterial products.
The present disclosure specifically adopts the following technical scheme:
in a first aspect of the disclosure, a metabolite product of a ginkgo endophytic fungus is provided, which is characterized in that: the fungus is gingko endophytic fungi (Fusarium proliferatum) DZHQ1 with the preservation number of CGMCC No.14983, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms and management in 11 months and 28 months in 2017, and the acetic ether extract of the fermentation culture solution is obtained after fermentation culture and acetic ether extraction, and then the metabolic product of the gingko endophytic fungus is obtained after methanol or water dissolution and dissolution.
In a second aspect of the disclosure, there is provided a method for producing a metabolite product of a strain of a ginkgo endophytic fungus, the method comprising: the fungus is gingko endophytic fungi (Fusarium proliferatum) DZHQ1 with the preservation number of CGMCC No.14983, which is preserved in the China general microbiological culture Collection center in 11-28.2017, and the acetic ether extract of the fermentation culture solution is obtained after fermentation culture and acetic ether extraction, and then is dissolved by methanol or water, and the dissolved solution is the metabolite product of the gingko endophytic fungi.
In a third aspect of the disclosure, there is provided the use of the metabolite product of the ginkgo endophytic fungus for the preparation of an antibacterial product.
In a fourth aspect of the present disclosure, an antibacterial composition is provided, which comprises the metabolite product of the ginkgo endophytic fungi as an active ingredient.
In a fifth aspect of the present disclosure, there is provided an antibacterial method comprising the step of inhibiting bacteria using a metabolite product of said endophytic fungus of ginkgo biloba or said antibacterial composition.
One of the technical scheme of the disclosure has the following beneficial effects:
the metabolite of the ginkgo endophytic fungi disclosed by the invention shows a relatively obvious inhibition effect in the detection of escherichia coli and/or staphylococcus aureus resistance, and has a potential application in preparing a new antibacterial product.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the disclosure and, together with the description, serve to explain the disclosure and not to limit the disclosure.
FIG. 1: 17#(17-1 and 17-2) positive ion chromatograms of the samples.
FIG. 2: 18#(18-1 and 18-2) positive ion chromatograms of the samples.
FIG. 3: 17#(17-1 and 17-2) negative ion chromatograms of the samples.
FIG. 4: 18#(18-1 and 18-2) negative ion chromatograms of the samples.
FIG. 5: 17#And 18#The inhibition effect of the methanol solution of the crude extract of the gingko endophytic fungi on escherichia coli.
FIG. 6: 17#And 18#The inhibiting effect of the water solution of the crude extract of the ginkgo endophytic fungi on escherichia coli.
FIG. 7: 17#And 18#The methanol solution of the crude extract of the gingko endophytic fungi has the effect of inhibiting staphylococcus aureus.
FIG. 8: 17#And 18#The inhibiting effect of the water solution of the crude extract of the ginkgo endophytic fungi on staphylococcus aureus is achieved.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
The applicant further researches and explores the previous research result CN108102928A 'a gingko endophytic fungus strain and application thereof' and provides a metabolite product of the gingko endophytic fungus, which is characterized in that: the fungus used is gingko endophytic fungi layer Fusarium (Fusarium proliferatum) DZHQ1 in CN108102928A, the preservation number is CGMCC No.14983, the fungus is preserved in China general microbiological culture Collection center in 11 months and 28 months in 2017, an ethyl acetate extract of a fermentation culture solution is obtained after fermentation culture and ethyl acetate extraction, and then the ethyl acetate extract is dissolved by methanol or water, and the dissolved solution is a metabolite product of the gingko endophytic fungus.
In one or some embodiments of the present disclosure, there is provided a method of fermentation culture and ethyl acetate extraction comprising: digging and taking gingko endophytic fungi cultured in a PDA solid culture medium, inoculating a hypha block into a PDA liquid culture medium, and performing shake culture at 20-28 ℃ at 100-150 r/min for 5-7 d; then adding ethyl acetate (the volume ratio of the ethyl acetate to the fermentation liquor is 1:1-2:1) according to the volume ratio of 1:1-2:1, and continuously culturing for 4-8 d in a shaking table; filtering the fermentation liquor, removing mycelium, and layering to obtain an organic phase containing the secondary metabolite of the gingko endophytic fungi; recovering ethyl acetate to obtain concentrated solution containing secondary metabolite of endophytic fungi of Ginkgo biloba; and finally, drying the concentrated solution to obtain the ethyl acetate extract of the fermentation culture solution.
Wherein, PDA solid culture medium and PDA liquid culture medium are conventional potato culture medium in the prior art, and the formula can be selected from but not limited to: 200g of potatoes, 20g of glucose, 15-20 g of agar and 1000mL of water; or 200g of potatoes, 20g of glucose and 1000mL of water.
In one or some embodiments of the disclosure, the volume ratio of fermentation broth to ethyl acetate is 1: 1-1: 2.
In a specific embodiment of the present disclosure, the active ingredients of the metabolite products of the ginkgo endophytic fungi include, but are not limited to, betaine, scopoletin, harmine, rosmarinic acid, oxypurinol, resveratrol, naringenin, catechin, taxifolin, and xanthohumol.
Further, the active ingredients of the metabolite product of the ginkgo endophytic fungi obtained after the methanol dissolution include, but are not limited to, betaine, scopoletin, harmine, rosmarinic acid, oxypurinol, resveratrol, naringenin, catechin, taxifolin and xanthohumol.
In a specific embodiment of the present disclosure, betaine, scopoletin, harmine, rosmarinic acid, oxypurinol, resveratrol, naringenin, catechin, taxifolin and xanthohumol are present in the HPLC chart at a peak area of 105The above peaks represent species.
In a specific embodiment of the present disclosure, the metabolites of the endophytic fungi of ginkgo biloba are shown in column 6 or column 7 of table 3.
In one or some specific embodiments of the present disclosure, the ratio of ethyl acetate extract to methanol or water is (0.01-0.05) g:1 mL; a further ratio was 0.02g:1 mL.
In an exemplary embodiment of the present disclosure, a method for producing a metabolite product of a strain of a ginkgo endophytic fungus is provided, the method comprising: the fungus is gingko endophytic fungi (Fusarium proliferatum) DZHQ1 with the preservation number of CGMCC No.14983, which is preserved in the general microbiological center of China Committee for culture Collection of microorganisms 28.11.2017, fermented and cultured, ethyl acetate extract of the fermented culture solution is obtained after the ethyl acetate extract is extracted, and then the ethyl acetate extract is dissolved in methanol or water (5-30 min) to obtain a metabolic product of the gingko endophytic fungus after the methanol or water is dissolved.
In a specific embodiment of the present disclosure, post-dissolution filtration membranes; further, the pore size of the filter membrane is 0.22 μm.
In yet another exemplary embodiment of the present disclosure, there is provided a use of the metabolite product of the ginkgo endophytic fungus for the preparation of an antibacterial product.
In a particular embodiment of the present disclosure, the antimicrobial product is an antimicrobial drug or other formulation.
In a specific embodiment of the present disclosure, the metabolite product of the ginkgo endophytic fungi is resistant to escherichia coli and/or staphylococcus aureus.
In a specific embodiment of the present disclosure, the methanol-solubilized metabolite product of the ginkgo endophytic fungi is resistant to escherichia coli.
In a specific embodiment of the present disclosure, the methanol or water-dissolved metabolite product of the endophytic fungus from ginkgo biloba is resistant to escherichia coli and/or staphylococcus aureus.
In an exemplary embodiment of the present disclosure, an antibacterial composition comprising the metabolite product of the ginkgo endophytic fungi as an active ingredient is provided.
In a specific embodiment of the present disclosure, the composition further comprises a pharmaceutically acceptable carrier, wherein the carrier is selected from one or more of diluents, dispersants, stabilizers, disintegrants, and lubricants, such as one or more of starch, sodium carboxymethylcellulose, glycerol, and the like.
In an exemplary embodiment of the present disclosure, there is provided an antibacterial method comprising the step of inhibiting bacteria using a metabolite product of the endophytic fungus of ginkgo biloba or the antibacterial composition, wherein the bacteria is escherichia coli and/or staphylococcus aureus.
In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.
Example 1
A method for preparing ethyl acetate extract of fermentation culture solution can be referred to example 2 of CN108102928A "A strain of Ginkgo Biloba endophytic fungi and its application".
Specifically, the method comprises the following steps: digging and taking ginkgo endophytic fungi cultured in PDA solid culture medium, inoculating the mycelium blocks into a triangular flask filled with 250ml of PDA liquid culture medium, and carrying out shake cultivation at 20 ℃ and 120r/min for 7 d. Ethyl acetate was then added to each flask at a 1:1 ratio by volume and the cultivation was continued in a shaker for 4 d. Filtering the fermentation liquor by 3-4 layers of gauze, then removing mycelium, and layering by using a separating funnel to obtain an organic phase containing the secondary metabolite of the gingko endophytic fungi. Most of ethyl acetate in the organic phase is recovered by using a rotary evaporator to obtain concentrated solution containing the secondary metabolite of the gingko endophytic fungi. And finally, completely drying the concentrated solution by using a vacuum concentration dryer to obtain the ethyl acetate extract of the fermentation culture solution. Wherein the gingko endophytic fungi is gingko endophytic fungi layer Fusarium (Fusarium proliferatum) DZHQ1 with the preservation number of CGMCC No.14983 and the number of 17#Corresponding to the J-1 strain in CN108102928A < one gingko endophytic fungus and application thereof >. Or the gingko endophytic fungus is J-2 strain in CN108102928A 'one strain of gingko endophytic fungus and application thereof', and the number is 18#。
A preparation method of a metabolic product of ginkgo endophytic fungi comprises the following steps of dissolving an ethyl acetate extract of a fermentation culture solution by using methanol, wherein the proportion of the ethyl acetate extract to the methanol in the fermentation culture solution is 0.02g:1mL, and dissolving for 10min to obtain a dissolving solution, namely a metabolite product of the ginkgo endophytic fungi.
Example 2
A preparation method of a metabolite product of ginkgo endophytic fungi comprises the step of dissolving the ethyl acetate extract of the fermentation culture solution in example 1 by using water, wherein the proportion of the ethyl acetate extract of the fermentation culture solution to the water is 0.02 g/mL. Wherein the Ginkgo endophytic fungi is Fusarium delavayi endophytic fungi(Fusarium proliferatum) DZHQ1 with preservation number CGMCC No.14983 and number 17#Corresponding to the J-1 strain in CN108102928A < one gingko endophytic fungus and application thereof >.
Example 3
A preparation method of a metabolic product of ginkgo endophytic fungi comprises the steps of dissolving an ethyl acetate extract of a fermentation culture solution in example 1 by using methanol or water, wherein the proportion of the ethyl acetate extract to the methanol or water of the fermentation culture solution is 0.02g/mL, and filtering the mixture through a 0.22-micrometer filter membrane after dissolving for 10min to obtain a filtrate, namely the metabolic product of the ginkgo endophytic fungi. Wherein the gingko endophytic fungi is gingko endophytic fungi layer Fusarium (Fusarium proliferatum) DZHQ1 with the preservation number of CGMCC No.14983 and the number of 17#Corresponding to the J-1 strain in CN108102928A < one gingko endophytic fungus and application thereof >. Or the gingko endophytic fungus is J-2 strain in CN108102928A 'one strain of gingko endophytic fungus and application thereof', and the number is 18#. To obtain 17#Samples, namely: a metabolite product of the gingko endophytic fungi dissolved by methanol or a metabolite product of the gingko endophytic fungi dissolved by water; 18#Samples, namely: the metabolite product of the gingko endophytic fungi dissolved by the methanol or the metabolite product of the gingko endophytic fungi dissolved by the water.
Example 4
A method for preparing a metabolite product of a ginkgo endophytic fungus, which comprises the following steps: digging out Fusarium proliferatum (DZHQ) 1 cultured in PDA solid culture medium, inoculating mycelia block into triangular flask containing 250ml PDA liquid culture medium, and shake culturing at 25 deg.C and 100r/min for 6 d. Ethyl acetate was then added to each flask at a 2:1 ratio by volume and the culture was continued for 3d on a shaker. Filtering the fermentation liquor by 3-4 layers of gauze, then removing mycelium, and layering by using a separating funnel to obtain an organic phase containing the secondary metabolite of the gingko endophytic fungi. Most of ethyl acetate in the organic phase is recovered by using a rotary evaporator to obtain concentrated solution containing the secondary metabolite of the gingko endophytic fungi. And finally, completely drying the concentrated solution by using a vacuum concentration dryer to obtain an ethyl acetate extract of the fermentation culture solution, dissolving the ethyl acetate extract of the fermentation culture solution by using methanol or water, wherein the ratio of the ethyl acetate extract of the fermentation culture solution to the methanol or water is 0.02g/mL, and dissolving for 10min to obtain a dissolved solution, namely the metabolite product of the endophytic fungi of the ginkgo biloba.
Example 5
A method for preparing a metabolite product of a ginkgo endophytic fungus, which comprises the following steps:
digging three ginkgo endophytic fungi cultured in PDA solid culture medium, inoculating the mycelium blocks into a triangular flask filled with 250ml of PDA liquid culture medium, and performing shake culture at 28 ℃ and 140r/min for 8 d. Ethyl acetate was then added to each flask at a 1.5:1 ratio by volume and the culture was continued on a shaker for 5 d. Filtering the fermentation liquor by 3-4 layers of gauze, then removing mycelium, and layering by using a separating funnel to obtain an organic phase containing the secondary metabolite of the gingko endophytic fungi. Most of ethyl acetate in the organic phase is recovered by using a rotary evaporator to obtain concentrated solution containing the secondary metabolite of the gingko endophytic fungi. And finally, completely drying the concentrated solution by using a vacuum concentration dryer, dissolving the ethyl acetate extract of the fermentation culture solution by using methanol or water, wherein the proportion of the ethyl acetate extract of the fermentation culture solution to the methanol or water is 0.02g/mL, and dissolving for 10min to obtain a dissolved solution, namely the metabolite product of the endophytic fungi in the ginkgo biloba.
Example 6 LC-MS non-Targeted Metabonomics detection
1.1 materials
1.1.1 sample basic information
Samples (ethyl acetate extract of fermentation broth in example 1) in total of 2 groups (17)#,18#) Each set of 2 replicates for a total of 4 samples (17-1, 17-2, 18-1, 18-2).
1.1.2 reagents
Water (water, drochen), reagent: mass Spectrometry acetonitrile (Fisher), formic acid (Sigma-Aldrich)
1.1.3 instruments
Instrument information Table 1
Figure BDA0001783751270000071
1.2 methods
1.2.1 metabolite extraction
To the sample, 1mL of methanol was added to dissolve (sample: methanol 0.02g: 1mL) for 10min, and the sample was introduced through a 0.22 μm filter.
1.2.2 conditions for liquid chromatography-Mass Spectrometry
The analytical instrument for this experiment was Q active plus, Thermo, and the ion source was ESI.
1. Chromatographic conditions
The column was Waters Atlantis T3 (100X 3mm,1.8 mm). The column temperature was 35 ℃. The flow rate was 0.500[ ml/min ].
Mobile phase: equal to "0.1 v/v% HCOOH-H2O"
D.Equate＝"acetonitrile"
The chromatographic conditions are shown in the following table: TABLE 2
Figure BDA0001783751270000072
Figure BDA0001783751270000081
2. Conditions of Mass Spectrometry
Selecting m/z 80-1200 as a scanning range;
resolution ratio: 70,000;
Spectrum data type：Profile；
Capillary voltage：4000V(positive)and 3500 V(negative)；
Capillary Temp:350℃.
2 results and analysis
2.1 Positive ion chromatogram
As shown in fig. 1 and 2.
2.2 anion chromatogram
As shown in fig. 3 and 4.
2.3 substance identification
The original data of LC-MS is imported into MS-DIAL 2.76(MS-DIAL: data independent MS/MSdeconvolution for comprehensive methyl analysis. Nature Methods,12,523-526,2015) software for preprocessing, including peak extraction, de-noising, deconvolution, peak alignment, and three-dimensional data matrix (original data matrix) in CSV format is derived. The extracted peak information was compared with the database, and a full library search was performed on three libraries, MassBank, Respect, GNPS (14951 records total). This three-dimensional matrix comprises the information: sample information, retention time, mass-to-nuclear ratio, and mass spectral response intensity (peak area).
2.4 analysis of results
The results of the evaluation are shown in Table 3 below.
TABLE 3
Figure BDA0001783751270000082
Figure BDA0001783751270000091
Figure BDA0001783751270000101
Figure BDA0001783751270000111
Figure BDA0001783751270000121
Figure BDA0001783751270000131
Figure BDA0001783751270000141
Figure BDA0001783751270000151
Figure BDA0001783751270000161
Figure BDA0001783751270000171
Figure BDA0001783751270000181
Figure BDA0001783751270000191
Figure BDA0001783751270000201
The identified substances in this time are shown in Table 3, wherein the inventors have reported the main active ingredient (the peak area of the main active ingredient is 10) after investigation5Substance represented by the above peak), 17#The method comprises the following steps: betaine, scopoletin, harmine, rosmarinic acid, oxypurinol, resveratrol, naringenin, kaempferol, catechin, taxifolin and xanthohumol; 18#The method comprises the following steps: scopoletin, harmine, rosmarinic acid, oxypurinol, resveratrol, naringenin, kaempferol, catechin, taxifolin and xanthohumol.
Since only the currently known compounds can be detected by liquid quality detection, part of the components with antitumor and antibacterial activities are analyzed by the liquid quality result, and therefore, whether the fermentation liquor contains unknown active compounds or not is still to be further researched.
Example 5 in vitro bacteriostatic study
1. The experimental steps are as follows:
inoculating the bacteria to be detected into a triangular flask filled with 100ml of LB liquid culture medium, and performing shake culture at 37 ℃ at 120r/min for 10 to 12 hours. Preparing 100ml LB solid culture medium, sterilizing, cooling to appropriate temperature, adding bacteria liquid to be detected (adding 100 microliter of bacteria liquid per 100ml solid culture medium), shakingAnd (5) pouring the plate, and standing for solidification. The back of the solidified flat plate is divided into three to four areas, and the samples added in each area are marked. The oxford cup is placed stably in the perfectly central part of each zone, lightly pressed, but not allowed to be inserted into the plate. After setting, 100. mu.L to 200. mu.L of a sample (17 from example 3) was added to each cup#Metabolite of ginkgo endophytic fungi, 18#Metabolite products of ginkgo endophytic fungi). The plate was smoothly placed in a 37 ℃ incubator and the results were observed for 9 to 12 hours.
Note: the method selects a plurality of gingko endophytic fungi for experiments, numbers the gingko endophytic fungi, and selects relatively good experimental results to be presented in the disclosure, wherein 17#、18#Respectively corresponding to J-1 and J-2, 17 in CN 108102928A' A strain of gingko endophytic fungi and application thereof#The strain is gingko endophytic fungi Fusarium proliferatum DZHQ1 with the preservation number of CGMCC No. 14983.
2. The experimental results are as follows:
2.1 inhibitory Effect on Escherichia coli
FIG. 5 shows endophytic fungus 17 from Ginkgo biloba of example 3#、18#The results of experiments on the inhibition of E.coli by methanol solutions of crude extracts and methanol. Therein 17#Represents endophytic fungus 17 of ginkgo biloba#The methanol solution of the crude extract of (4); 18#Representative is Ginkgo endophytic fungus 18#The methanol solution of the crude extract of (4); "A" represents a methanol solution. From the figure 17 can be found#The diameter of the ginkgo endophytic fungi inhibiting zone is 0.9 cm. 18#The inhibitory effect of endophytic fungi and methanol solution is very weak and almost none.
FIG. 6 shows the inhibitory effect of aqueous solutions of crude extracts of Ginkgo endophytic fungi of example 3 on Escherichia coli. Therein 17#B represents endophytic fungus 17 of ginkgo biloba#An aqueous solution of the crude extract of (a); 18#B represents gingko endophytic fungus 18#An aqueous solution of the crude extract of (a); "Water" represents an aqueous solution. As can be seen from the figure, neither of them had bacteriostatic effect。
In summary, the Ginkgo endophytic fungi 17#、18#Middle, 17#The inhibiting effect of endophytic fungi on Escherichia coli is obvious, and 18 is the second#The effect of the endophytic fungi is not very pronounced and is almost 0. In addition, it can be seen from this experiment that the inhibitory effect on E.coli is different for metabolites extracted with different solvents. 17#The methanol solution of the crude extract has obvious inhibition effect on the escherichia coli.
2.2 inhibitory Effect against Staphylococcus aureus
17 from example 3 was used# Sample 18#The samples were subjected to an oxford cup bacteriostatic test. The results are as follows:
FIG. 7 shows the inhibitory effect of methanol solution of crude extract of Ginkgo endophytic fungi of example 3 on Staphylococcus aureus. Among the experimental results, 17#And 18#Has certain inhibiting effect, and the diameters of the inhibition zones are 2cm and 2.9cm respectively.
FIG. 8 is a graph showing the inhibitory effect of an aqueous solution of an endophytic fungus crude extract of Ginkgo biloba of example 3 on Staphylococcus aureus, and it can be seen from the observation that 17, although some inhibitory zones are not clear#The metabolite of the endophytic fungi has certain bacteriostatic effect, 18#The endophytic fungi metabolite has weak and almost no bacteriostatic effect.
Results show that 17#And 18#The gingko endophytic fungi has a certain inhibiting effect on staphylococcus aureus. It can also be seen from this experiment that the inhibitory effect on S.aureus was different for the metabolites extracted with different solvents. 17#The methanol solution and the water solution of the crude extract have obvious inhibiting effect on staphylococcus aureus.
The above embodiments are preferred embodiments of the present disclosure, but the embodiments of the present disclosure are 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 disclosure should be regarded as equivalent replacements within the scope of the present disclosure.

Claims (2)

1. The application of the metabolite of the ginkgo endophytic fungi in preparing products for resisting escherichia coli and/or staphylococcus aureus is characterized in that: the fungus is selected from Fusarium proliferatum (Fusarium proliferatum) of endophytic fungi of Ginkgo bilobaFusariumproliferatum) DZHQ1 with the preservation number of CGMCC No.14983, which is preserved in China general microbiological culture Collection center in 11 months and 28 months in 2017, is subjected to fermentation culture and ethyl acetate extraction to obtain an ethyl acetate extract of a fermentation culture solution, and then is dissolved by methanol to obtain a metabolite product of the ginkgo endophytic fungi;
the specific preparation method of the metabolite product comprises the following steps: digging gingko endophytic fungus cultured in PDA solid culture mediumFusariumproliferatum) DZHQ1 with the preservation number of CGMCC No.14983, inoculating the mycelium blocks into a PDA liquid culture medium, and carrying out shake cultivation at 20-28 ℃ and 100-150 r/min for 5-7 d; then adding ethyl acetate according to the volume ratio of 1:1-2:1, and continuously culturing for 4-8 d in a shaking table; filtering the fermentation liquor, removing mycelium, and layering to obtain an organic phase containing the secondary metabolite of the gingko endophytic fungi; recovering ethyl acetate to obtain concentrated solution containing secondary metabolite of endophytic fungi of Ginkgo biloba; and finally, drying the concentrated solution to obtain an ethyl acetate extract of the fermentation culture solution, then dissolving the ethyl acetate extract by using methanol, and obtaining a metabolic product of the gingko endophytic fungi after the methanol is dissolved.
2. Use according to claim 1, characterized in that: the metabolite product comprises betaine, scopoletin, harmine, rosmarinic acid, oxypurinol, resveratrol, naringenin, catechin, taxifolin and xanthohumol.
CN201811004688.5A 2017-12-21 2018-08-30 Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis Active CN109082445B (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201811004688.5A CN109082445B (en) 2018-08-30 2018-08-30 Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis
PCT/CN2018/113709 WO2019119992A1 (en) 2017-12-21 2018-11-02 Endophytic fungus of ginkgo biloba and metabolite product and application thereof
US16/469,830 US11332707B2 (en) 2017-12-21 2018-11-02 Endophytic fungus from gingko, metabolite product and use thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201811004688.5A CN109082445B (en) 2018-08-30 2018-08-30 Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis

Publications (2)

Publication Number Publication Date
CN109082445A CN109082445A (en) 2018-12-25
CN109082445B true CN109082445B (en) 2020-06-05

Family

ID=64840237

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201811004688.5A Active CN109082445B (en) 2017-12-21 2018-08-30 Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis

Country Status (1)

Country Link
CN (1) CN109082445B (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112159766B (en) * 2020-10-23 2022-06-14 广东省微生物研究所（广东省微生物分析检测中心） Aphyllophora fuscopsis new strain, fermentation method thereof and application thereof in bacteriostasis
WO2022188162A1 (en) * 2021-03-12 2022-09-15 德州学院 Endophytic fungus in ginkgo biloba and anti-bacterial and antitumor application thereof
WO2023150694A1 (en) * 2022-02-04 2023-08-10 Phyto Tech Corp. Natural preservatives and uses thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102676398A (en) * 2012-03-14 2012-09-19 安徽农业大学 Separation and purification method of endophytic fungi from ginkgo biloba
CN102742605A (en) * 2012-06-06 2012-10-24 吉林大学 Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi
CN103194490A (en) * 2013-03-26 2013-07-10 安徽农业大学 Method for preparing phytopathogen-resisting ginkgo biloba L. endophytic-fungus fermentation broth
CN108102928A (en) * 2017-12-21 2018-06-01 德州学院 One plant of gingko endogenous fungus and its application

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102676398A (en) * 2012-03-14 2012-09-19 安徽农业大学 Separation and purification method of endophytic fungi from ginkgo biloba
CN102742605A (en) * 2012-06-06 2012-10-24 吉林大学 Application of gingko endogeny Chaetomium globosum in prevention and control of plant pathogenic fungi
CN103194490A (en) * 2013-03-26 2013-07-10 安徽农业大学 Method for preparing phytopathogen-resisting ginkgo biloba L. endophytic-fungus fermentation broth
CN108102928A (en) * 2017-12-21 2018-06-01 德州学院 One plant of gingko endogenous fungus and its application

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
《2株银杏内生真菌的抑菌活性研究》;王瑞珍;《中国优秀硕士学位论文全文数据库农业科技辑》;20130115;摘要以及第28页 *

Also Published As

Publication number Publication date
CN109082445A (en) 2018-12-25

Similar Documents

Publication Publication Date Title
Silva et al. Studies on antimicrobial activity, in vitro, of Physalis angulata L.(Solanaceae) fraction and physalin B bringing out the importance of assay determination
Abdissa et al. Phytochemical investigation of Aloe pulcherrima roots and evaluation for its antibacterial and antiplasmodial activities
CN109082445B (en) Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis
CN107937453B (en) Preparation method and antibacterial activity application of dichloro-substituted II-type halogenated polyketone compound
CN102643167B (en) Alga endophytic fungus sesquiterpene compound and preparation method and application thereof
Yin et al. Gentiopicrin-producing endophytic fungus isolated from Gentiana macrophylla
US11332707B2 (en) Endophytic fungus from gingko, metabolite product and use thereof
Petit et al. Detection of griseofulvin in a marine strain of Penicillium waksmanii by ion trap mass spectrometry
CN107961233B (en) Application of trichloro-substituted II-type halogenated polyketone compounds
CN108033905A (en) The preparation method and application of compound pencolide
CN115536645B (en) Compound Phomol B, preparation method thereof and application thereof in antibacterial drugs
CN111494364B (en) Application of isopentenyl substituted phenol compound in resisting staphylococcus aureus and methicillin-resistant staphylococcus aureus
CN113499337B (en) Application of kurarinone in preparation of aquatic pathogenic bacteria resistant medicines or feed additives
CN102659547B (en) Ophiobolin sesterterpene compound and preparation and application thereof
CN102190612B (en) Endophytic fungus diterpene alkaloid compound of natural seaweed as well as preparation method and application thereof
CN105476983B (en) A kind of purposes of deep-sea originated from fungus compound Prenylcandidusin C
CN107954964B (en) One kind 3,4,6 3 substitution-α-pyrone derivative and its preparation method and application
CN107746422B (en) Ergosta-7, 22-diene-3-ketaminothiohydrazone, preparation method thereof and application thereof in preparation of antibacterial drugs
Senger et al. Anti-Trichomonas vaginalis activity and chemical analysis of metabolites produced by marine-associated fungi
CN109593074B (en) Separation preparation and application of compound with antibacterial activity in fungus secondary metabolite
Mohanty et al. Development of phytochemical fingerprint of an Indian medicinal plant Chitrak (Plumbago zeylanica L) using high performance thin layer chromatography (HPTLC)
CN107954965B (en) A kind of gamma-pyrone derivative and its preparation method and application
CN109180593A (en) Phenol piperazine alkaloids secondary metabolite and its application
Serben et al. Study of technological parameters of dry extracts for creation of hard gelatin capsules for the treatment of arterial hypertension associated with urolithiasis
CN107739740A (en) A kind of preparation method and application of the Lasiodiplodins compounds in marine fungi source

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant