CN109971652B - Tibetan pineapple endophytic fungus oncogenes X117 and preparation method and application of fermentation product thereof - Google Patents
Tibetan pineapple endophytic fungus oncogenes X117 and preparation method and application of fermentation product thereof Download PDFInfo
- Publication number
- CN109971652B CN109971652B CN201910229579.1A CN201910229579A CN109971652B CN 109971652 B CN109971652 B CN 109971652B CN 201910229579 A CN201910229579 A CN 201910229579A CN 109971652 B CN109971652 B CN 109971652B
- Authority
- CN
- China
- Prior art keywords
- compound
- culture medium
- strain
- tibetan
- gymnoascoside
- 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
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
- C07D307/58—One oxygen atom, e.g. butenolide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- 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
- C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Tropical Medicine & Parasitology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Botany (AREA)
- Mycology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a Tibetan pineapple endophytic fungus oncogenes X117 and a preparation method and application of a fermentation product thereof. The invention provides endophytic fungi oncogenes X117 of Tibetan pineapple flower for the first time, and the nucleotide sequence of ITS-rRNA gene is shown as SEQ ID NO. 1. The strain can be used for producing a compound Gymnoascol A by fermentation, and is an ideal carrier for developing novel natural medicaments. The invention also provides a method for producing the compound Gymnoascoside A by using the strain through fermentation, and the method is simple, short in period, low in cost, energy-saving and environment-friendly. Meanwhile, the compound Gymnoascolide A is found to have good anti-inflammatory activity for the first time, and has good application prospect and wide development space in the aspect of preparing anti-inflammatory drugs.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a Tibetan pineapple endophytic fungus oncogenes X117 and a preparation method and application of a fermentation product thereof.
Background
Gymnoascoside A [ Ben Clark, et al, J.Nat.prod.2005,68, 1226-1230); tomoo Hosoe, et al, phytochemistry 2005,66,2776-2779]And found to have a moderately high inhibitory K + (60 mM) or norepinephrine pretreated aortic ring Ca 2+ Induced vasodilation effect. 2011 discovers that Gymnoascoside A has activity of inhibiting glycogen synthase kinase-3 (GSK-3 beta) and has potential value of treating type II diabetes mellitus [ DirkSchulz, et al, J.Nat.prod.2011,74,99-101]. As for other bioactive effects of the compound, there is no report at present, including a series of glycosides of derivatives and derivatives thereof of the compound of the type, which are discovered later in succession, but no significant activity of the derivatives is yet found [ Ana Kralj, et al, j.nat.prod.2006,69,995-1000; haruhiro Fujimoto, et al, chem.Pharm.Bull.2006,54 (4) 550-553; daigo Wakana, et al, mycotoxin 2008,58 (1) 1-6]。
Regarding the synthesis of Gymnoascoside A, in 2013 and 2015, U.S. scientists and Chinese scientists have totally synthesized Gymnoascoside A and its derivatives [ Anna N.Parker, et al, tetrahedron Letters 2013,54,5322-5324; lin Mei, et al, tetrahedron 2015,71,3325-3332], however, the step of synthesizing Gymnoascoside A is complicated and high in cost, and is not favorable for realizing large-scale synthesis and application of the compound.
Therefore, it is of great significance to search for the compound Gymnoascoside A from metabolites of natural drugs and further explore and research the pharmacodynamic activity of the compound.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art, provide a Tibetan pineapple endophytic fungus oncogenes X117 capable of producing a compound Gymnoascoside A in large quantity, and explore the process for producing the compound Gymnoascoside A by fermenting the endophytic fungus oncogenes X117 and the application activity of the compound.
The first purpose of the invention is to provide a Tibetan pineapple endophytic fungus oncogenes X117.
The second purpose of the invention is to provide the application of the endophytic fungus oncogenes X117 in the fermentation production of the compound Gymnoascoside A.
The third purpose of the invention is to provide a method for producing a compound Gymnoascoside A by fermentation by using the endophytic fungus oncogenes X117.
The fourth purpose of the invention is to provide the application of the compound Gymnoascoside A in preparing anti-inflammatory drugs.
The above purpose of the invention is realized by the following technical scheme:
the invention provides a Tibetan pineapple endophytic fungus oncogenes X117, which is preserved in Guangdong province microorganism strain preservation center in 2018, 6 and 28 days, and the preservation number is as follows: GDMCC No.60399, deposited at No. 59 building 5 of Mieli Zhonglu No. 100 Guangzhou city.
The nucleotide sequence of the ITS-rRNA gene of the endophytic fungus Onygenes X117 of the Tibetan pineapple is shown as SEQ ID NO. 1.
The endophytic fungus Onygenales X117 is isolated from root of Tibetan medicine Tibetan Artocarpus heterophyllus (Incarvillea younghusbandii Sprague) in autonomous region of Tibet, and identified as fungus of family Pyrococcaceae (Onygenales) by ITS-rRNA. The biological characteristics of the strain are as follows: when the bacterial colony is cultured on a PDA culture medium at a constant temperature of 5-15 ℃, orange villous hyphae are arranged on the surface of the bacterial colony, and orange red spores are generated on the back of the bacterial colony; when the temperature is changed and cultured at 15-20 ℃, the color of the colony is gradually changed from orange to light yellow or white; when the culture temperature exceeds 20 ℃, the strain grows slowly or stops growing and dies gradually.
The endophytic fungus onygenies X117 of the Tibetan pineapple can produce the compound Gymnoaslide A in a large scale, so that the application of the endophytic fungus onygenres X117 in the production of the compound Gymnoaslide A is also within the protection scope of the invention.
The structural formula of the compound Gymnoascoside A is shown as the following formula (I):
in addition, the invention also provides a method for producing the compound Gymnoascoside A, which is to separate and purify a fermentation product of the endophytic fungus Onygenes X117 of the Tibetan pineapple flower to obtain the compound Gymnoascoside A.
Particularly preferably, the method for producing the compound Gymnoaslide A comprises the following steps:
s1, strain culture: inoculating an endophytic fungus Onygenales X117 strain of Tibetan pineapple in a seed culture medium, and culturing;
s2, obtaining fermentation liquor: adding ethyl acetate into the strain obtained in the step S1, and separating to obtain thalli and fermentation liquor;
s3, separating and purifying a compound: and (3) extracting and concentrating the fermentation liquor obtained in the step (S2) to obtain concentrated solution, leaching the thalli obtained in the step (S2) to obtain leaching liquor, mixing the concentrated solution and the leaching liquor, carrying out chromatographic separation, eluting by using an ethyl acetate-petroleum ether system, and recrystallizing to obtain the compound Gymnoascol A.
Preferably, the seed culture medium in step S1 is a potato residue solid culture medium, a potato dextrose broth, a PDA solid culture medium or a rice culture medium.
Preferably, when the seed culture medium in step S1 is a solid culture medium, the culture is a static culture; and in the step S1, when the seed culture medium is a liquid culture medium, the culture is shaking culture.
More preferably, the seed culture medium in step S1 is a potato pulp solid culture medium.
Preferably, the culture of step S1 is a static culture or a shaking culture.
Preferably, the temperature of the static culture is 5-28 ℃.
More preferably, the temperature of the static culture is 10 to 28 ℃.
Preferably, the time of the static culture is 25 to 35 days.
More preferably, the time of the static culture is 30d.
Preferably, the temperature of the shaking culture is 5 to 28 ℃.
Preferably, the shaking culture time is 20 to 30 days.
Preferably, the rotation speed of the shaking culture is 150 to 250rpm.
Preferably, the extractant used in the extraction in step S3 is ethyl acetate and/or chloroform.
Preferably, the volume ratio of the extracting agent to the fermentation liquor is 1-3: 1.
more preferably, the volume ratio of the extractant to the fermentation broth is 2:1.
preferably, the leaching in step S3 is leaching with methanol and/or a methanol-chloroform mixed solution.
Preferably, the volume ratio of methanol to chloroform in the methanol-chloroform mixed solution is 1:1.
preferably, the chromatographic separation in step S3 is performed by silica gel column chromatography.
Preferably, the recrystallization in step S3 is a recrystallization with a petroleum ether-ethyl acetate system.
In addition, the application of the compound Gymnoascolide A in preparing anti-inflammatory drugs also belongs to the protection scope of the invention.
Compared with the prior art, the invention has the following beneficial effects:
(1) The invention provides a endophytic fungus oncogenes X117 of Tibetan pineapple, which can ferment and produce a compound Gymnoascoside A with a unique structure and is an ideal carrier for developing novel natural medicines;
(2) The method for producing the compound Gymnoascoside A by fermenting endophytic fungi Onygenes X117 from Tibet Polo is simple, has short period, is obtained by separating metabolites of natural medicines, has low cost, is energy-saving and environment-friendly, and is suitable for large-scale production of the compound Gymnoascoside A;
(3) The first research of the invention discovers that the compound Gymnoascol A has good anti-inflammatory activity, and has good application prospect and wide development space in the preparation of anti-inflammatory drugs.
Drawings
FIG. 1 is a strain morphology of the endophytic fungus Onygenes X117 of Tibetan pineapple when cultured at 10 ℃ at constant temperature.
FIG. 2 is a strain morphology of the endophytic fungus Onygenes X117 of Tibetan pineapple when cultured at 20 ℃ at constant temperature.
FIG. 3 is a neighbor-joining phylogenetic tree of endophytic fungus Onygene X117 of Tibetan pineapple, constructed using Mega 7.0 software.
FIG. 4 is an EI low resolution mass spectrum of compound Gymnoaslide A prepared in example 2.
FIG. 5 is a 1H-NMR spectrum (CDCl) of compound Gymnoaslide A prepared in example 2 3 ) Drawing.
FIG. 6 is a scheme showing that Compound Gymnoaslide A prepared in example 2 13 C-NMR Spectroscopy (CDCl) 3 ) Drawing.
FIG. 7 shows the H-H COSY spectrum (CDCl) of the compound Gymnoascoside A prepared in example 2 3 ) Figure (a).
FIG. 8 is an HSQC spectrum (CDCl) of the compound Gymnoaslide A prepared in example 2 3 ) Figure (a).
FIG. 9 is an HMBC spectrum (CDCl) of Gymnoaslide A, a compound prepared in example 2 3 ) Figure (a).
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 isolation and characterization of endophytic fungus Onygenes X117 from Tibetan pineapple
1. Isolation of the Strain
Sample preparation: root of Tibetan medicine Tibet Aronia (Incarvillea younghusbandii Sprague) in saline-alkali land beside Bamu measure salt lake in Bango county, tibet autonomous region.
The separation method comprises the following steps: sterilizing the root of fresh Tibetan medicine Tibet Aronia, peeling off outer root bark, inoculating to PDA culture medium, martin culture medium or Chagas culture medium under aseptic condition, and culturing at 20 deg.C below for 5-20 d to obtain single strain oncogenes X117; the obtained strain onynales X117 is preserved at 4 ℃ on an inclined plane by adopting a common PDA culture medium.
2. Identification of strains
The biological characteristics of the strain are that when the strain is cultured on a PDA culture medium at a constant temperature of 5-15 ℃, the surface of a colony is orange short villous hyphae, and the back surface is orange red, so that spores are generated; when the temperature is changed and cultured at 15-20 ℃, the color of the colony gradually changes from orange to light yellow or white (see figure 1); when the culture temperature exceeds 20 ℃, the strain stops growing and gradually dies. In different fermentation processes, a large amount of the compound Gymnoascoside A (see FIG. 2) can be produced.
In addition, a CTAB method is adopted to extract pure cultured DNA of Tibetan medicine endophytic fungi oncogenes X117, a pair of primers ITS1F and ITS4 of an ITS spacer region are adopted to amplify ITS-rRNA gene fragments through a PCR amplification instrument, the reaction system is 50uL, and the reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 40s, annealing at 52 ℃ for 40s, extension at 72 ℃ for 1min, repeating three steps of denaturation, annealing and extension for 30 cycles, and finally extension at 72 ℃ for 10min.
Determining the target fragment to be about 600bp through sephadex electrophoresis detection, and obtaining an ITS-rRNA gene fragment sequence (shown as SEQ ID NO. 1) of the strain through sequencing. Then, carrying out similarity analysis on the sequences by a BLAST online comparison search engine on GenBank to obtain a strain with the maximum similarity of 89%; and a neighbor-joining phylogenetic tree is constructed by adopting Mega 7.0 software, the phylogenetic analysis result is shown in figure 3, and the strain is determined to be the fungus of the family Zymobacteriaceae (oncogenes).
And deposited in Guangdong province microorganism strain collection center (GDMCC) in 2018, 6 months and 28 days, wherein the deposit numbers are GDMCC No:60399.
example 2 fermentation of endophytic fungus Onygenes X117 of Tibetan pineapple and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental method
S1, adopting a potato residue solid culture medium, inoculating a strain of endophytic fungi oncogenes X117 strain in the Tibetan medicinal material into a triangular flask filled with the potato residue solid culture medium, and performing static culture for 30d at the variable temperature of 10-28 ℃;
s2. Adding a methanol-chloroform (v/v 1) mixed solvent to a triangular flask, leaching for 4 times, concentrating the leaching solution by rotary evaporation, and subjecting the obtained extract to chromatography by a common silica gel column chromatography technique, and eluting with an ethyl acetate-petroleum ether system (1: 20, recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The compound Gymnoascoside A obtained by the embodiment has the highest yield which can reach 0.35 g/bottle; the mass spectrum and nuclear magnetic spectrum of the compound Gymnoascoside A obtained in the example are shown in FIGS. 4 to 9.
Example 3 fermentation of endophytic fungus Onygenes X117 of Tibetan pineapple and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental methods
S1, adopting a potato residue solid culture medium, inoculating a strain of endophytic fungi Onygenes X117 strain of the Tibetan medicinal materials into a triangular flask containing the potato residue solid culture medium, and performing standing culture for 25d at the variable temperature of 5-25 ℃;
s2. Adding a methanol-chloroform (v/v 1) mixed solvent to a triangular flask, leaching for 4 times, concentrating the leaching solution by rotary evaporation, and subjecting the obtained extract to chromatography by a common silica gel column chromatography technique, and eluting with an ethyl acetate-petroleum ether system (1: 20, recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoascoside A obtained in this example was 0.05 g/bottle.
Example 4 fermentation of endophytic fungus Onygenes X117 of Tibetan pineapple and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental methods
S1, adopting a potato dextrose liquid culture medium, inoculating a strain of endophytic fungus oncogenes X117 strain of a Tibetan medicinal material into a triangular flask containing the sterilized potato dextrose liquid culture medium, and performing standing culture for 35d at the variable temperature of 5-28 ℃;
s2, adding 200mL of ethyl acetate into each bottle of fermentation liquor, killing the thalli, filtering by using gauze, and separating the thalli and the fermentation liquor;
s3. The fermentation broth is extracted 3 times with ethyl acetate (ethyl acetate: fermentation broth (v/v) 2), rotary-distilled and concentrated, the cells are fan-dried, then leached 4 times with methanol, the leach liquor is combined with the ethyl acetate extract of the fermentation broth after rotary-distilled and concentrated, and subjected to chromatography using a common silica gel column chromatography technique, and eluted with an ethyl acetate-petroleum ether system (1, 20,1, 10,1, 5, 4,1, 3,1: 20, and recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoascoside A obtained in this example was 0.12 g/vial.
Example 5 fermentation of endophytic fungus oncogenes X117 of Tibetan pineapple flower and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental method
S1, adopting a potato dextrose liquid culture medium, inoculating a strain of endophytic fungus oncogenes X117 strain in a Tibetan medicinal material into a triangular flask containing the sterilized potato dextrose liquid culture medium, and culturing for 20 days by a shaking table (with the rotating speed of 150-250 rpm) at the variable temperature of 5-25 ℃;
s2, adding 200mL of ethyl acetate into each bottle of fermentation liquor, killing the thalli, filtering by using gauze, and separating the thalli from the fermentation liquor;
s3, extracting the fermentation liquor for 3 times by using chloroform (chloroform: fermentation liquor (v/v) 2), carrying out rotary evaporation and concentration, drying the thalli by using a fan, leaching for 3 times by using methanol, combining the leaching liquor with an ethyl acetate extracting solution of the fermentation liquor after carrying out rotary evaporation and concentration, carrying out chromatography by using a common silica gel column chromatography technology, and eluting by using an ethyl acetate-petroleum ether system (1: 20, recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoaslide A obtained in this example was 0.15 g/bottle.
Example 6 fermentation of endophytic fungus Onygenes X117 of Tibetan pineapple and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental method
S1, adopting a potato dextrose broth culture medium, inoculating a strain of endophytic fungi X117 strain of a Tibetan medicinal material into a triangular flask containing the disinfected potato dextrose broth culture medium, and performing standing culture for 30d at the variable temperature of 10-28 ℃;
s2, adding 200mL of ethyl acetate into each bottle of fermentation liquor, killing the thalli, filtering by using gauze, and separating the thalli and the fermentation liquor;
s3, extracting the fermentation broth with ethyl acetate (ethyl acetate: fermentation broth (v/v) 3) for 3 times, performing rotary evaporation and concentration, drying the thallus by using a fan, leaching for 4 times with methanol, performing rotary evaporation and concentration on the leaching liquor, mixing the leaching liquor with an ethyl acetate extract of the fermentation broth, performing chromatography by using a common silica gel column chromatography technology, and eluting with an ethyl acetate-petroleum ether system (1: 20, and recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoaslide A obtained in this example was 0.13 g/bottle.
Example 7 fermentation of endophytic fungus Onygenes X117 of Tibetan pineapple and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental methods
S1, adopting a potato dextrose broth culture medium, inoculating a strain of endophytic fungi X117 strain in a Tibetan medicinal material into a triangular flask containing the disinfected potato dextrose broth culture medium, and culturing for 30 days by using a shaker (the rotating speed is 150-250 rpm) at the temperature of 10-28 ℃;
s2, adding 200mL of ethyl acetate into each bottle of fermentation liquor, killing the thalli, filtering by using gauze, and separating the thalli from the fermentation liquor;
s3, extracting the fermentation liquor for 3 times by using ethyl acetate (ethyl acetate: fermentation liquor (v/v) 1), carrying out rotary evaporation and concentration, carrying out fan drying on thalli, leaching for 3 times by using methanol, carrying out rotary evaporation and concentration on a leaching liquor, merging ethyl acetate extracting solutions of the fermentation liquor, carrying out chromatography by using a common silica gel column chromatography technology, and eluting by using an ethyl acetate-petroleum ether system (1: 20, and recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoaslide A obtained in this example was 0.22 g/bottle.
Example 8 fermentation of endophytic fungus oncogenes X117 of Tibetan pineapple flower and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental methods
S1, adopting a PDA solid culture medium, inoculating a strain of endophytic fungi oncogenes X117 strain in the Tibetan medicinal material into a glass plate containing the PDA solid culture medium, and performing static culture for 30d at the variable temperature of 5-25 ℃;
s2, adding 10mL of ethyl acetate into the glass plate overnight, killing the thalli, and stripping the thalli together with a culture medium from the glass plate;
s3, drying the stripped thalli under a fan, leaching for 4 times by using a methanol-chloroform (v/v 1): 20, and recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoaslide A obtained in this example was 0.05 g/bottle.
Example 9 fermentation of endophytic fungus Onygenes X117 of Tibetan pineapple and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental methods
S1, adopting a PDA solid culture medium, inoculating a strain of an endophytic fungi oncogenes X117 strain in a medicinal material in a plate containing the PDA solid culture medium, and performing static culture for 30d at the variable temperature of 10-28 ℃;
s2, adding 10mL of ethyl acetate into the glass plate overnight, killing the thalli, and stripping the thalli together with the culture medium from the glass plate;
s3, drying the stripped thalli under a fan, leaching for 3 times by using a methanol-chloroform (v/v 1:20, recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoascoside A obtained in this example was 0.08 g/vial.
Example 10 fermentation of endophytic fungus Onygenes X117 of Tibetan pineapple and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental method
S1, adopting a rice solid culture medium, inoculating a strain of endophytic fungi Onygenes X117 strain in a Tibetan medicinal material into a triangular flask containing the rice solid culture medium, and performing standing culture for 30d at the variable temperature of 5-25 ℃;
s2. To a flask, a methanol-chloroform (v/v 1) mixed solvent is added, the mixture is extracted 4 times, the extract is concentrated by rotary evaporation, and the obtained extract is subjected to chromatography by a common silica gel column chromatography technique, and eluted with an ethyl acetate-petroleum ether system (1, 20,1, 10,1, 5, 4,1: 20, recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoascoside A obtained in this example was 0.28 g/vial.
Example 11 fermentation of endophytic fungus oncogenes X117 of Tibetan pineapple flower and extraction, separation and purification of Compound Gymnoascoside A
1. Experimental methods
S1, adopting a rice solid culture medium, inoculating a strain of endophytic fungi Onygenes X117 strain in a Tibetan medicinal material into a 1L triangular flask containing the rice solid culture medium, and performing standing culture for 30d at the variable temperature of 10-28 ℃;
s2, adding 1:1, extracting the secondary metabolite, adding a methanol-chloroform (v/v 1): 20, and recrystallizing by using petroleum ether-ethyl acetate to obtain a compound Gymnoascoside A.
2. Results of the experiment
The yield of the compound Gymnoaslide A obtained in this example was 0.2 g/bottle.
EXAMPLE 12 anti-inflammatory Activity assay of the Compound Gymnoaslide A
1. Experimental method
The inhibition rate of the secondary metabolite Gymnoascoside A of endophytic fungus Onygene X117 in Tibetan pineapple on the release of Nitric Oxide (NO) from mouse mononuclear macrophage RAW264.7 induced by LPS is determined by using an LPS-induced mouse mononuclear macrophage RAW264.7 (LPS: 1. Mu.g/mL) model and 420. Mu.M (150. Mu.g/mL) indomethacin as a positive control, and the survival rate of cells is determined by using an MTT method.
2. Results of the experiment
The results are shown in table 1, and it can be seen that compound Gymnoascoside A (GA) has an inhibitory effect on NO release from mouse monocyte macrophage RAW264.7 induced by LPS, and the IC50 value of compound Gymnoascoside A for inhibiting cell release NO is 35.85 +/-3.50 μ M, and the inhibitory effect is significant; in addition, the compound Gymnoascoside A can obviously reduce the survival rate of cells AW264.7, and the compound Gymnoascoside A has good anti-inflammatory activity.
TABLE 1 anti-inflammatory Activity test results for the compound Gymnoaslide A
Note: "- - -" indicates NO inhibition rate.
The above detailed description is of the preferred embodiment for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, that is, it does not mean that the present invention must be implemented by the above embodiment. It will be apparent to those skilled in the art that any modifications to the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific forms, etc., are within the scope and disclosure of the present invention.
Sequence listing
<110> Zhongshan university
<120> Tibetan pineapple endophytic fungus oncogenes X117 and preparation method and application of fermentation product thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 568
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agcttacagc cttccgtagg tgacctgcgg aggatcatta aagtgttagc ctgtgcacct 60
agtgtacagg cgaaactcca cccttgacta ctataccaca tgttgctttg gcgggcctgc 120
cctcgggctg ccggggtttc cctggcccgc gcccgccgaa gaataatgaa ccctttttga 180
attggacgtc tgagttgatt atcaatcatt aaaactttca acaatggatc tcttggttcc 240
ggcatcgatg aagaacgcag cgaaatgcga taagtaatgt gaattgcaga attccgtgaa 300
tcatcgaatc tttgaacgca cattgcgccc cctggtattc cggggggcat gcctgtccga 360
gcgtcattgc aaccttcaag cgcggcttgt gtgttgggcc tcgtcccccg tggacgtgcc 420
cgaaaggcag tggcggcgtc cgtttcggtg cccgagcgta tgggaatcct tataagctca 480
acaggcccgg cggcgctggt cgaaaccata ttttaccggt tgacctcgga tcaggtaggg 540
atacccgctg aacttaagca tatcaaaa 568
Claims (8)
1. Endophytic fungus of Tibet pineapple flowerOnygenalesX117, wherein the strain is deposited at the Guangdong province culture collection center on 28 th 6 th 2018, and the deposit number is: GDMCC No.60399.
2. Endophytic fungus of Tibetan pineapple flower according to claim 1OnygenalesApplication of X117 in production of a compound Gymnoascol A.
3. A method for producing a compound Gymnoascoside A, characterized in that endophytic fungi of the Tibet polo flower of claim 1OnygenalesAnd separating and purifying the fermentation product of X117 to obtain a compound Gymnoascol A.
4. A method according to claim 3, characterized by the steps of:
s1, strain culture: inoculating endophytic fungi of Tibet Aronia in seed culture mediumOnygenalesAn X117 strain is cultured;
s2, obtaining fermentation liquor: adding ethyl acetate into the strain obtained in the step S1, and separating to obtain thalli and fermentation liquor;
s3, separation and purification of a compound: and (3) extracting and concentrating the fermentation liquor obtained in the step (S2) to obtain concentrated solution, leaching the thalli obtained in the step (S2) to obtain leaching liquor, mixing the concentrated solution and the leaching liquor, carrying out chromatographic separation, eluting by using an ethyl acetate-petroleum ether system, and recrystallizing to obtain the compound Gymnoascol A.
5. The method according to claim 4, wherein the seed culture medium of step S1 is a potato pulp solid culture medium, a potato dextrose broth, a PDA solid culture medium or a rice culture medium.
6. The method according to claim 4, wherein when the seed culture medium of step S1 is a solid culture medium, the culturing is static culturing; and in the step S1, when the seed culture medium is a liquid culture medium, the culture is shaking culture.
7. The method according to claim 6, wherein the temperature of the static culture is 5-28 ℃ and the time is 25-35 d; the temperature of the shaking culture is 5-28 ℃, the time is 20-30 d, and the rotating speed is 150-250 rpm.
8. The method according to claim 4, wherein the extractant used in the extraction in step S3 is ethyl acetate and/or chloroform; the volume ratio of the extracting agent to the fermentation liquor is 1-3: 1; and the leaching in the step S3 is leaching by using methanol and/or a methanol-chloroform mixed solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910229579.1A CN109971652B (en) | 2019-03-25 | 2019-03-25 | Tibetan pineapple endophytic fungus oncogenes X117 and preparation method and application of fermentation product thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910229579.1A CN109971652B (en) | 2019-03-25 | 2019-03-25 | Tibetan pineapple endophytic fungus oncogenes X117 and preparation method and application of fermentation product thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109971652A CN109971652A (en) | 2019-07-05 |
| CN109971652B true CN109971652B (en) | 2022-11-22 |
Family
ID=67080464
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910229579.1A Active CN109971652B (en) | 2019-03-25 | 2019-03-25 | Tibetan pineapple endophytic fungus oncogenes X117 and preparation method and application of fermentation product thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109971652B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0539288A (en) * | 1991-08-02 | 1993-02-19 | Kirin Brewery Co Ltd | New physiologically active substance, n-13, its use and production thereof |
| CN101264094A (en) * | 2008-04-16 | 2008-09-17 | 西藏自治区高原生物研究所 | Incarvillea younghusbandii phenylpropanoid glycoside composition and preparation and use thereof |
-
2019
- 2019-03-25 CN CN201910229579.1A patent/CN109971652B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0539288A (en) * | 1991-08-02 | 1993-02-19 | Kirin Brewery Co Ltd | New physiologically active substance, n-13, its use and production thereof |
| CN101264094A (en) * | 2008-04-16 | 2008-09-17 | 西藏自治区高原生物研究所 | Incarvillea younghusbandii phenylpropanoid glycoside composition and preparation and use thereof |
Non-Patent Citations (5)
| Title |
|---|
| 5.8S ribosomal RNA gene and internal transcribed spacer 2, complete sequence * |
| and 28S ribosomal RNA gene, partial sequence.《GenBank ACCESSION：KC894838》.2013,全文. * |
| Chen,B et al..Onygenales sp. HPP-2 internal transcribed spacer 1, partial sequence * |
| Terrusnolides A-D, new butenolides with anti-inflammatory activities from an endophytic Aspergillus from Tripterygium wilfordii;Changxing Qi et al.;《Fitoterapia》;20180827;第130卷;第134-139页 * |
| 基于2(5H)-呋喃酮的C―C成键反应研究进展;王柏文等;《有机化学》;20180517;第38卷(第08期);第1872-1884页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109971652A (en) | 2019-07-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111139188B (en) | Novel skeleton heteroterpene derivative derived from marine fungi and application of novel skeleton heteroterpene derivative in preparation of anti-inflammatory drugs | |
| Kumar et al. | Hyper-production of taxol from Aspergillus fumigatus, an endophytic fungus isolated from Taxus sp. of the Northern Himalayan region | |
| CN108299462B (en) | Mixed source terpene compound and separation method and application thereof | |
| CN113121557B (en) | Marine fungus-derived heterpene compound and application thereof in preparation of anti-inflammatory drugs | |
| CN115197172B (en) | Sesterterpene compound, synthetic gene cluster and synthetic method thereof | |
| CN110117546B (en) | Naphthoquinone compound derived from marine fungi and anti-inflammatory application thereof | |
| CN114606134A (en) | Sponge epiphyte and application thereof in preparation of oxaanthraquinone compounds | |
| CN101126102A (en) | Marine actinomycetes for generating antineoplastic compound Norharmane | |
| CN109971652B (en) | Tibetan pineapple endophytic fungus oncogenes X117 and preparation method and application of fermentation product thereof | |
| CN107099464A (en) | A kind of sclerotium aspergillus and its method for preparing penicillic acid | |
| CN107686817B (en) | Chrysanthemum bud endophytic fungus CYSK-4 and application of Ascomylactam compound produced by same | |
| Kumar et al. | Cultural, morphological and molecular characterization of vinca alkaloids producing endophytic fungus Fusarium solani isolated from Catharanthus roseus | |
| CN110003153B (en) | Benzofuran compound and preparation method and application thereof | |
| CN111407764A (en) | Application of novel skeleton heteroterpene derivative in preparation of medicine for resisting non-alcoholic steatohepatitis and hepatic fibrosis | |
| CN109971651B (en) | Tobacco endophytic fungus and application thereof in preparation of ergosterol 5,8 peroxide | |
| CN107488594B (en) | New penicillium and metabolite antacid A thereof | |
| CN114213428B (en) | Indole alkaloid compound and preparation method and application thereof | |
| CN116103161A (en) | Plant endophytic fungus for producing eupatorium and application thereof | |
| CN111808015B (en) | Phenylalanine-derived cytochalasin as well as preparation method and application thereof | |
| CN113173969B (en) | Marine fungus-derived heteroterpene compound and application thereof in preparation of anti-hepatic fibrosis drugs | |
| CN1534032A (en) | Method of high effect preparing rhoxadunol | |
| CN115011487A (en) | Sponge epiphytic fungus and application thereof in preparation of terpenoid compounds | |
| CN110833560A (en) | Application of 2,4, 6-tri-O-galloyl-D-glucose in decocting of Chinese herbal medicines in preparation of antitumor medicines | |
| CN116199695B (en) | Citrinin derivative, and preparation method and application thereof | |
| CN115044483B (en) | Penicillium fungus W21C371 and application thereof in preparation of curvulin |
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 |