CN109293662A - A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001 - Google Patents
A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001 Download PDFInfo
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Abstract
The invention discloses a kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001, have the following structure:Wherein, R1, R2 are each independently H, OH, C1-C4 alkoxy, C1-C4 alkyl sulphonyl, C2-C4 alkyl acyl, and having one in R1, R2 is H；R3, R4 be each independently H, OH, halogen, amino,
Description
Technical field
The present invention relates to field of biotechnology, more particularly, to a kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001.
Background technique
It is well known that microorganism early has become the important sources of antibiotic, enzyme inhibitor isoreactivity substance.Microorganism due to
Can be with large scale fermentation culture, no raw material limitation will not have become the hot spot in Natural products research field the advantages that welding.
The probability for obtaining noval chemical compound from the microorganism of land in recent years is greatly reduced, and marine-derived microorganism is due to its living environment
Specifically, in addition to it can produce metabolite similar with land, the special noval chemical compound of some structures can be also generated sometimes, shown
Huge exploitation prospect.Aspergillus fungi is all its cometabolism of the important strain of natural products circle research all the time
Product structure novelty skeleton is changeable, and in addition to conventional steroidal, sequiterpene other than the common structures type such as anthraquinone, contains toward contact:
Alkaloids, a variety of skeletons such as peptides, polyketone class, sesterterpene.The compound of these structure novels contains cell toward contact
Poison, antibacterial, the various actives such as antiviral become one of the important sources of marine drug lead compound, cause scholar in the industry
Extensive concern.
The prior art such as Authorization Notice No. is the Chinese invention patent of 104710396 B of CN, and it is next to disclose a kind of lake Liu Shan
Source fungal secondary metabolite derivative and its application as antibacterial agent.Its isolated compound is to methicillin resistance
Golden yellow Portugal coccus (MRSA), drug resistance of vancomycin enterococcus (VRE) have stronger inhibiting effect, to Gram-negative
Bacterium has certain inhibiting effect, and minimum inhibitory concentration MIC is respectively less than 12.5 μM, shows that it can develop as potential antimicrobial
Object, but the lake the Liu Shan source fungal secondary metabolite derivative is unobvious to the inhibitory activity of Gram-negative bacteria.
Summary of the invention
The purpose of the present invention is to provide a kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001, preparation method is
Aspergillus terreus SKL-001 is modified through epigenetic modification adjusting control agent SAHA, generates diversified secondary metabolite, fermentation material separation
Extraction yield is high.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken are as follows:
A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001, be Formulas I compound represented, its stereoisomer or
Pharmaceutically accessible salt, Formulas I have the following structure:
Wherein, R1, R2 are each independently H, OH, C1-C4 alkoxy, C1-C4 alkyl sulphonyl, C2-C4 alkyl acyl,
And having one in R1, R2 is H；R3, R4 be each independently H, OH, halogen, amino,C1-C4 alkyl,
At least one in C2-C4 alkyl acyl, C1-C4 halogenated alkyl, C1-C4 alkyl sulphonyl, C2-C4 alkenyl and R3, R4 be not
H。
A kind of preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, including spore preparation, strain fermentation,
Secondary metabolite is extracted and is isolated and purified, specific steps are as follows:
Spore preparation: the glycerol cryopreservation tube for being placed on the Aspergillus terreus SKL-001 of -80 DEG C of ultra low temperature freezer preservations is placed in cleaning
In station, recovery 10min, the oese after being sterilized with calcination dips the frozen stock solution recovered, on the inclined-plane PDA of fresh configuration
It uniformly crosses in solid medium, then, slant medium is placed in 28 DEG C of constant incubators and cultivates 4 days, cultivates bacterial strain inclined-plane
To maturation, plentiful spore is obtained, it is spare；
Strain fermentation: epigenetic modification adjusting control agent SAHA, paricalcitol, glycerol three are added into fungi culture medium
Ester stirs evenly, and stands, fermented and cultured 30 days, a common fermentation 60L；Paricalcitol, triglycerides are added to fungi culture medium
In, it can act synergistically with epigenetic modification adjusting control agent SAHA, change and dissolve oxygen environment in culture medium, promote hypha fermentation, together
When, promote SAHA in conjunction with the active site of mycelial cell access, promote the generation and growth of fungal secondary metabolite, increases
Add the yield and diversity of mycelium secondary metabolite；
Secondary metabolite extraction: after fermentation, with silk by mycelium and separation of fermentative broth；The bodies such as fermentation liquid addition
Long-pending ethyl acetate stirring extraction 3 times, ethyl acetate is concentrated under reduced pressure, fermentation liquid extract is obtained；The third of 80% is added in mycelium
Ketone-water, 2- methylalanine and calcium citrate impregnate, and are crushed instrument with the ultrasonic tissue of power 800W, with ultrasonic 3s, are spaced 3s
Method then extract 30min for mycelium ultrasonication 45 times, obtain clear liquid with filtered on buchner funnel, be concentrated under reduced pressure, add
Enter isometric ethyl acetate to extract 3 times, obtains fermentation mycelium extract；Merge fermentation liquid extract and fermentation mycelium extracts
Object obtains final fermentation, extraction object；2- methylalanine and calcium citrate collective effect, greatly infiltration mycelial cell wall, lead to
It crosses the chemical affinities such as hydrogen bond, Van der Waals force, electrostatic attraction and destroys mycelial cell wall construction, change cell wall permeability,
It is broken its molecular structure, broken hole occurs, then the mechanical effect of synergistic supersonic wave, cavitation effect and fuel factor, promotes mycelia
The release, diffusion and dissolution of substance in body cell improve the extraction yield of mycelium extract；
It isolates and purifies: final fermentation, extraction object is eluted with silica gel column chromatography, eluant, eluent selects petroleum ether-acetone-
Methanol；Obtained component 3 is eluted with silica gel column chromatography, and eluant, eluent is petroleum ether-ethyl acetate；Obtained component 12 is thin with preparing
Layer chromatography separation, solvent is methylene chloride-methanol, volume ratio 50:1；Obtained component 2, uses high performance liquid chromatography separation
Purifying, eluant, eluent is methanol-water, and volume ratio 70:30 obtains the new secondary metabolite LW-4 of Aspergillus terreus SKL-001.
Preferably, in spore preparation step, frozen stock solution in glycerol cryopreservation tube are as follows: new with 20% glycerol, 3.3% seawater extract
The PDA liquid frozen stock solution of fresh preparation.
Preferably, in strain fermentation step, final concentration of 5-25 μM of epigenetic modification adjusting control agent SAHA.
Preferably, the additive amount of paricalcitol is 0.05-0.1%, triglycerides additive amount in strain fermentation step
For 0.05-0.15%.
Preferably, the additive amount of 2- methylalanine is the 1- of culture medium quality in secondary metabolite extraction step
3%, the additive amount of calcium citrate is the 0.5-1.5% of culture medium quality.
Compared with the prior art, the advantages of the present invention are as follows: the present invention is modified by epigenetic modification adjusting control agent SAHA and is made
Obtain a kind of new compound --- the new secondary metabolite LW-4 of Aspergillus terreus SKL-001；It is vertical that the present invention adds pa in the medium
Ostelin, triglycerides generate cooperative gain effect with epigenetic modification adjusting control agent SAHA, Aspergillus terreus SKL-001 are promoted to produce
Raw diversified secondary metabolite；2- methylalanine and calcium citrate, association is added in mycelium extraction process in the present invention
Mechanism, cavitation with ultrasonic wave destroy cell wall, accelerate intracellular organic matter release, improve fermentation material extraction yield；This hair
The new secondary metabolite LW-4 of Aspergillus terreus SKL-001 made from bright and its stereoisomer or pharmaceutically accessible salt, have
Long-acting bacteriostatic anti-inflammatory effect.
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001, be Formulas I compound represented, its stereoisomer or
Pharmaceutically accessible salt, Formulas I have the following structure:
A kind of preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, including spore preparation, strain fermentation,
Secondary metabolite is extracted and is isolated and purified, specific steps are as follows:
Spore preparation: the glycerol cryopreservation tube of the Aspergillus terreus SKL-001 of -80 DEG C of ultra low temperature freezer preservations is placed on (containing 20%
The PDA liquid frozen stock solution of glycerol, 3.3% seawater extract Fresh) it is placed in clean station, recovery 10min is sterilized with calcination
Oese afterwards dips the frozen stock solution recovered, and uniformly crosses in the inclined-plane the PDA solid medium of fresh configuration, then, tiltedly
Face culture medium is placed in 28 DEG C of constant incubators and cultivates 4 days, and culture bacterial strain inclined-plane to maturation obtains plentiful spore, spare；
Strain fermentation: the addition epigenetic modification adjusting control agent SAHA into fungi culture medium, final concentration of 15 μM,
0.05% paricalcitol, 0.1% triglycerides, stir evenly, and stand, fermented and cultured 30 days, a common fermentation 60L；Pa founds bone
Change alcohol, triglycerides are added in fungi culture medium, can act synergistically with epigenetic modification adjusting control agent SAHA, change culture medium
Interior dissolution oxygen environment promotes hypha fermentation, meanwhile, promote SAHA in conjunction with the active site of mycelial cell access, promotes true
The generation and growth of bacterium secondary metabolite increase the yield and diversity of mycelium secondary metabolite；
Secondary metabolite extraction: after fermentation, with silk by mycelium and separation of fermentative broth；The bodies such as fermentation liquid addition
Long-pending ethyl acetate stirring extraction 3 times, ethyl acetate is concentrated under reduced pressure, fermentation liquid extract is obtained；The third of 80% is added in mycelium
Ketone-water, 2%2- methylalanine, 1% calcium citrate impregnate, and are crushed instrument with the ultrasonic tissue of power 800W, with ultrasonic 3s,
30min is then extracted by mycelium ultrasonication 45 times every the method for 3s, clear liquid is obtained with filtered on buchner funnel, depressurizes dense
Contracting is added isometric ethyl acetate and extracts 3 times, obtains fermentation mycelium extract；Merge fermentation liquid extract and fermented hypha
Body extract obtains final fermentation, extraction object；2- methylalanine and calcium citrate collective effect, greatly infiltration mycelial cell
Wall destroys mycelial cell wall construction by chemical affinities such as hydrogen bond, Van der Waals force, electrostatic attractions, it is logical to change cell wall
Permeability is broken its molecular structure, broken hole occurs, then the mechanical effect of synergistic supersonic wave, cavitation effect and fuel factor, promotes
The release, diffusion and dissolution of substance in mycelial cell improve the extraction yield of mycelium extract；
It isolates and purifies: final fermentation, extraction object is eluted with silica gel column chromatography, eluant, eluent selects petroleum ether-acetone-
Methanol obtains 4 components；Third component is chosen, is eluted with silica gel column chromatography, eluant, eluent is petroleum ether-ethyl acetate；
The 12nd group lease making preparative thin layer chromatography separation is chosen, solvent is methylene chloride-methanol, and volume ratio 50:1 is separated
To 4 component, second component is chosen, is purified through high performance liquid chromatography separation, eluant, eluent is methanol-water, volume ratio 70:
30, obtain the new secondary metabolite LW-4 of compound.
Embodiment 2:
A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001, be Formulas I compound represented, its stereoisomer or
Pharmaceutically accessible salt, which is characterized in that Formulas I has the following structure:
A kind of preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, including spore preparation, strain fermentation,
Secondary metabolite is extracted and is isolated and purified, specific steps are as follows:
Spore preparation: the glycerol cryopreservation tube of the Aspergillus terreus SKL-001 of -80 DEG C of ultra low temperature freezer preservations is placed on (containing 20%
The PDA liquid frozen stock solution of glycerol, 3.3% seawater extract Fresh) it is placed in clean station, recovery 10min is sterilized with calcination
Oese afterwards dips the frozen stock solution recovered, and uniformly crosses in the inclined-plane the PDA solid medium of fresh configuration, then, tiltedly
Face culture medium is placed in 28 DEG C of constant incubators and cultivates 4 days, and culture bacterial strain inclined-plane to maturation obtains plentiful spore, spare；
Strain fermentation: the addition epigenetic modification adjusting control agent SAHA into fungi culture medium, final concentration of 10 μM,
0.08% paricalcitol, 0.1% triglycerides, stir evenly, and stand, fermented and cultured 30 days, a common fermentation 60L；
Secondary metabolite extraction: after fermentation, with silk by mycelium and separation of fermentative broth；The bodies such as fermentation liquid addition
Long-pending ethyl acetate stirring extraction 3 times, ethyl acetate is concentrated under reduced pressure, fermentation liquid extract is obtained；The third of 80% is added in mycelium
Ketone-water, 2%2- methylalanine, 1% calcium citrate impregnate, and are crushed instrument with the ultrasonic tissue of power 800W, with ultrasonic 3s,
30min is then extracted by mycelium ultrasonication 45 times every the method for 3s, clear liquid is obtained with filtered on buchner funnel, depressurizes dense
Contracting is added isometric ethyl acetate and extracts 3 times, obtains fermentation mycelium extract；Merge fermentation liquid extract and fermented hypha
Body extract obtains final fermentation, extraction object；
It isolates and purifies: final fermentation, extraction object is eluted with silica gel column chromatography, eluant, eluent selects petroleum ether-acetone-
Methanol obtains 4 components；Third component is chosen, is eluted with silica gel column chromatography, eluant, eluent is petroleum ether-ethyl acetate；
The 12nd group lease making preparative thin layer chromatography separation is chosen, solvent is methylene chloride-methanol, and volume ratio 50:1 is separated
To 4 component, second component is chosen, is purified through high performance liquid chromatography separation, eluant, eluent is methanol-water, volume ratio 70:
30, obtain the new secondary metabolite LW-4 of compound.
Embodiment 3:
A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001, be Formulas I compound represented, its stereoisomer or
Pharmaceutically accessible salt, which is characterized in that Formulas I has the following structure:
A kind of preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, including spore preparation, strain fermentation,
Secondary metabolite is extracted and is isolated and purified, specific steps are as follows:
Spore preparation: the glycerol cryopreservation tube of the Aspergillus terreus SKL-001 of -80 DEG C of ultra low temperature freezer preservations is placed on (containing 20%
The PDA liquid frozen stock solution of glycerol, 3.3% seawater extract Fresh) it is placed in clean station, recovery 10min is sterilized with calcination
Oese afterwards dips the frozen stock solution recovered, and uniformly crosses in the inclined-plane the PDA solid medium of fresh configuration, then, tiltedly
Face culture medium is placed in 28 DEG C of constant incubators and cultivates 4 days, and culture bacterial strain inclined-plane to maturation obtains plentiful spore, spare；
Strain fermentation: the addition epigenetic modification adjusting control agent SAHA into fungi culture medium, final concentration of 10 μM,
0.1% paricalcitol, 0.15% triglycerides, stir evenly, and stand, fermented and cultured 30 days, a common fermentation 60L；
Secondary metabolite extraction: after fermentation, with silk by mycelium and separation of fermentative broth；The bodies such as fermentation liquid addition
Long-pending ethyl acetate stirring extraction 3 times, ethyl acetate is concentrated under reduced pressure, fermentation liquid extract is obtained；The third of 80% is added in mycelium
Ketone-water, 3%2- methylalanine, 1.5% calcium citrate impregnate, and are crushed instrument with the ultrasonic tissue of power 800W, with ultrasonic 3s,
Then the method for interval 3s extracts 30min for mycelium ultrasonication 45 times, obtain clear liquid with filtered on buchner funnel, depressurize dense
Contracting is added isometric ethyl acetate and extracts 3 times, obtains fermentation mycelium extract；Merge fermentation liquid extract and fermented hypha
Body extract obtains final fermentation, extraction object；
It isolates and purifies: final fermentation, extraction object is eluted with silica gel column chromatography, eluant, eluent selects petroleum ether-acetone-
Methanol obtains 4 components；Third component is chosen, is eluted with silica gel column chromatography, eluant, eluent is petroleum ether-ethyl acetate；
The 12nd group lease making preparative thin layer chromatography separation is chosen, solvent is methylene chloride-methanol, and volume ratio 50:1 is separated
To 4 component, second component is chosen, is purified through high performance liquid chromatography separation, eluant, eluent is methanol-water, volume ratio 70:
30, obtain the new secondary metabolite LW-4 of compound Aspergillus terreus SKL-001.
Comparative example 1:
Paricalcitol, triglycerides, rest part and embodiment 2 complete one are not added in strain fermentation preparation step
It causes.
Comparative example 2:
Epigenetic modification adjusting control agent SAHA is not added in strain fermentation preparation step, rest part and embodiment 2 are complete
Unanimously.
Comparative example 3:
2- methylalanine, calcium citrate are not added in secondary metabolite extraction step, remaining and embodiment 2 complete one
It causes.
Embodiment 4:
Embodiment 2 is set as test group, comparative example 1, comparative example 2 and comparative example 3 are set to control group 1,2 and of control group
Control group 3 compares the weight of final fermentation, extraction object and new secondary metabolite LW-4 obtained, the results are shown in Table 1.
The weight of the final fermentation, extraction object of table 1 and the new secondary metabolite LW-4 of Aspergillus terreus SKL-001
| Group | Final fermentation, extraction object (g) | New secondary metabolite LW-4 (mg) |
| Test group | 18.1 | 11 |
| Control group 1 | 13.1 | 8 |
| Control group 2 | 10.5 | 0 |
| Control group 3 | 18.1 | 9.5 |
As shown in Table 1, test group and the final fermentation, extraction object weight of control group 3 are higher than control group 1, control group 2, explanation
The addition of paricalcitol, triglycerides, epigenetic modification adjusting control agent SAHA can be improved the fermentation journey of Aspergillus terreus SKL-001
Degree makes it generate more secondary metabolites；The weight of the new secondary metabolite LW-4 of test group is higher than control group 1, right
According to group 2, control group 3, illustrate that the addition of 2- methylalanine and calcium citrate can be improved mycelial extraction yield, control group 2
New secondary metabolite LW-4 be 0, it is new to illustrate that the regulation of epigenetic modification adjusting control agent SAHA generates Aspergillus terreus SKL-001
Secondary metabolite LW-4.
Routine operation in operating procedure of the invention is well known to those skilled in the art, herein without repeating.
Technical solution of the present invention is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to restrict the invention, all any modifications made in spirit of the invention,
Supplement or similar fashion substitution etc., should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001 is Formulas I compound represented, its stereoisomer or medicine
The salt that can contact on, which is characterized in that Formulas I has the following structure:
Wherein, R1, R2 are each independently H, OH, C1-C4 alkoxy, C1-C4 alkyl sulphonyl, C2-C4 alkyl acyl, and
Having one in R1, R2 is H；R3, R4 be each independently H, OH, halogen, amino,C1-C4 alkyl, C2-
At least one in C4 alkyl acyl, C1-C4 halogenated alkyl, C1-C4 alkyl sulphonyl, C2-C4 alkenyl and R3, R4 is not H.
2. a kind of preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 as described in claim 1, including spore
Preparation, strain fermentation, secondary metabolite are extracted and are isolated and purified, which is characterized in that the strain fermentation step are as follows: to fungi
Epigenetic modification adjusting control agent SAHA, paricalcitol, triglycerides are added in culture medium, is stirred evenly, and are stood, fermentation training
It supports.
3. the preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 2 a kind of, feature
It is: final concentration of 5-25 μM of the epigenetic modification adjusting control agent SAHA.
4. the preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 2 a kind of, feature
Be: the additive amount of the paricalcitol is 0.05-0.1%, and triglycerides additive amount is 0.05-0.15%.
5. the preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 2 a kind of, feature
It is: the spore preparation step are as follows: take out the glycerol cryopreservation tube of the Aspergillus terreus SKL-001 of -80 DEG C of ultra low temperature freezer preservations
Recovery 10min dips the frozen stock solution recovered with oese, uniformly crosses in the inclined-plane PDA solid medium, 28 DEG C of constant temperature trainings
It supports in case and cultivates to maturation, obtain plentiful spore, it is spare.
6. the preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 5 a kind of, feature
It is: frozen stock solution in the glycerol cryopreservation tube are as follows: with 20% glycerol, the PDA liquid frozen stock solution of 3.3% seawater extract Fresh.
7. the preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 2 a kind of, feature
It is: secondary metabolite extraction step are as follows: after fermentation, with silk by mycelium and separation of fermentative broth；Fermentation liquid is added
Isometric ethyl acetate stirring extraction 3 times, is concentrated under reduced pressure, obtains fermentation liquid extract；The acetone-water of mycelium addition 80%,
2- methylalanine and calcium citrate impregnate, and ultrasonic tissue is crushed instrument and is crushed mycelium, then extract 30min, filter clearly
Liquid is added isometric ethyl acetate and extracts 3 times, is concentrated under reduced pressure, obtains fermentation mycelium extract；Merge fermentation liquid extract with
Fermentation mycelium extract obtains final fermentation, extraction object.
8. the preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 7 a kind of, feature
Be: the additive amount of the 2- methylalanine is the 1-3% of culture medium quality, and the additive amount of calcium citrate is culture medium quality
0.5-1.5%.
9. the preparation method of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 2 a kind of, feature
It is: the purification procedures are as follows: elute final fermentation, extraction object with silica gel column chromatography, eluant, eluent selects petroleum
Ether-acetone-methanol；Obtained component 3 is eluted with silica gel column chromatography, and eluant, eluent is petroleum ether-ethyl acetate；Obtained component 12
It is separated with preparative thin layer chromatography, solvent is methylene chloride-methanol, volume ratio 50:1；Obtained component 2, uses efficient liquid phase
Chromatographic separation and purification, eluant, eluent are methanol-water, and volume ratio 70:30 obtains the new secondary metabolite LW- of Aspergillus terreus SKL-001
4。
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| CN113789267A (en) * | 2021-08-18 | 2021-12-14 | 中国药科大学 | Marine-derived aspergillus terreus M7 with antibacterial effect and separation and application of secondary metabolite thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109053601A (en) * | 2018-09-04 | 2018-12-21 | 浙江海洋大学 | A kind of noval chemical compound LW-1 of the separation and Extraction from Aspergillus terreus secondary metabolite |
| CN113789267A (en) * | 2021-08-18 | 2021-12-14 | 中国药科大学 | Marine-derived aspergillus terreus M7 with antibacterial effect and separation and application of secondary metabolite thereof |
| CN113789267B (en) * | 2021-08-18 | 2023-07-11 | 中国药科大学 | Aspergillus terreus M7 with antibacterial effect from ocean source and separation and application of secondary metabolite thereof |
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