CN109125326A - The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 - Google Patents
The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 Download PDFInfo
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Abstract
Purposes the invention discloses the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 as antibacterial agent.New secondary metabolite LW-4 preparation step are as follows: epigenetic modification adjusting control agent SAHA, paricalcitol, triglycerides are added into fungi culture medium, fermented and cultured, extractive fermentation liquid, mycelium are distinguished with the acetone-water of ethyl acetate, 80%, fermented concentrate is purified through column chromatography, TLC separation, obtains new secondary metabolite LW-4.Have the beneficial effect that the present invention apparently modifies Aspergillus terreus SKL-001 using epigenetic modification adjusting control agent SAHA, the new secondary metabolite LW-4 of isolated one kind.The new secondary metabolite LW-4 of Aspergillus terreus SKL-001 produced by the present invention shows preferable inhibitory activity to Escherichia coli, and minimum inhibitory concentration is 15 μ g/mL, can be developed as the potential purposes for preparing anti-Gram negative bacteria drugs preparation.
Description
Technical field
The present invention relates to drug field, more particularly, to a kind of Aspergillus terreus SKL-001 that can be used as antibacterial agent new secondary generation
Thank to the Preparation method and use of product LW-4.
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 CN 104710396B, 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 purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 in antibacterial agent,
Its new secondary metabolite LW-4 is then extracted through epigenetic modification adjusting control agent SAHA modification Aspergillus terreus SKL-001 fermentation
Purifying obtains.
The present invention in view of the above technology in the problem of mentioning, the technical solution taken are as follows:
New secondary metabolite LW-4 is preparing the purposes in antibacterial agent as effective component, wherein new cometabolism produces
Object LW-4 is Formulas I compound represented, its stereoisomer or pharmaceutically accessible salt, chemical structural formula are as follows:
The preparation method of made new secondary metabolite LW-4 includes the following steps:
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；
Secondary metabolite extraction: with silk by mycelium and separation of fermentative broth；Fermentation liquid is extracted with ethyl acetate, decompression
Concentration, obtains fermentation liquid extract；80% acetone-water, 2- methylalanine and calcium citrate is added in mycelium, impregnates, tissue
Broken instrument is broken, ultrasonic extraction 30min, filters to obtain clear liquid, and ethyl acetate extraction is concentrated under reduced pressure, obtains fermentation mycelium extract；
Merge fermentation liquid extract and fermentation mycelium extract, obtains final fermentation, extraction object；
Isolate and purify: final fermentation, extraction object is eluted with silica gel column chromatography, and elution fraction 3 is eluted with silica gel column chromatography again,
Elution fraction 12 is separated with preparative thin layer chromatography, and the high performance liquid chromatography separation of separation component 2 is purified to get Aspergillus terreus SKL-
001 new secondary metabolite LW-4.
Preferably, fermented incubation time is 30 days in strain fermentation step.
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%；Paricalcitol, triglycerides are added in fungi culture medium, can be with epigenetic modification adjusting control agent
SAHA synergistic effect, changes and dissolves oxygen environment in culture medium, promotes hypha fermentation, meanwhile, promote SAHA and mycelial cell logical
The active site on road combines, and promotes the generation and growth of fungal secondary metabolite, increases the production of mycelium secondary metabolite
Amount and diversity.
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；2- methylalanine and calcium citrate collective effect, pole
The earth infiltration mycelial cell wall, destroys mycelial cell wall by chemical affinities such as hydrogen bond, Van der Waals force, electrostatic attractions
Structure changes cell wall permeability, is broken its molecular structure, broken hole occurs, then the mechanical effect of synergistic supersonic wave, cavitation
Effect and fuel factor promote release, diffusion and the dissolution of substance in mycelial cell, improve the extraction yield of mycelium extract.
Preferably, silica gel column chromatography elutes twice, and eluant, eluent used is successively are as follows: petroleum ether-in purification procedures
Acetone-methanol, petroleum ether-ethyl acetate；The eluant, eluent of high performance liquid chromatography is methanol-water, volume ratio 70:30；It prepares thin
The solvent of layer chromatography is methylene chloride-methanol, volume ratio 50:1.
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；The present invention adds the vertical ossification of pa in the medium
Alcohol, triglycerides generate cooperative gain effect with epigenetic modification adjusting control agent SAHA, promote Aspergillus terreus SKL-001 to generate more
The secondary metabolite of sample；2- methylalanine and calcium citrate is added in mycelium extraction process in the present invention, and collaboration is super
Mechanism, the cavitation of sound wave destroy cell wall, accelerate intracellular organic matter release, improve fermentation material extraction yield；System of the present invention
The new secondary metabolite LW-4 of Aspergillus terreus SKL-001 and its stereoisomer or pharmaceutically accessible salt obtained, has long-acting
Antibacterial anti-inflammatory effect, can be as the purposes of the preparation of preparation prevention and treatment Gram-negative bacteria (such as Escherichia coli).
Specific embodiment
The present invention program is described further below by embodiment:
Embodiment 1:
The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, made new secondary metabolite LW-4 are shown in Formulas I
Compound, its stereoisomer or pharmaceutically accessible salt, chemical structural formula are as follows:
The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, the preparation side of the new secondary metabolite LW-4
Method includes strain fermentation, secondary metabolite extraction and isolates and purifies, specific steps are as follows:
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；Paricalcitol, triglycerides
It is added in fungi culture medium, can acts synergistically with epigenetic modification adjusting control agent SAHA, changes and dissolve oxygen environment in culture medium,
Promote hypha fermentation, meanwhile, promote SAHA in conjunction with the active site of mycelial cell access, promotes fungal secondary metabolite
Generation and growth, increase mycelium secondary metabolite yield and diversity.
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:
The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, made new secondary metabolite LW-4 are shown in Formulas I
Compound, its stereoisomer or pharmaceutically accessible salt, chemical structural formula are as follows:
The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, the preparation side of made new secondary metabolite LW-4
Method includes strain fermentation, secondary metabolite extraction and isolates and purifies, specific steps are as follows:
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；
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:
The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, made new secondary metabolite LW-4 are shown in Formulas I
Compound, its stereoisomer or pharmaceutically accessible salt, chemical structural formula are as follows:
The purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, the preparation side of the new secondary metabolite LW-4
Method includes strain fermentation, secondary metabolite extraction and isolates and purifies, specific steps are as follows:
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；Pa founds bone
Change alcohol, triglycerides are added in fungi culture medium, can increase dissolved oxygen in culture medium, reduce interfacial tension and check thallus
Attachment to bubble promotes hypha fermentation process, and acts synergistically with epigenetic modification adjusting control agent SAHA, promotes SAHA and bacterium
The active site of filament cell pathway combines, and promotes the generation and growth of fungal secondary metabolite, increases mycelium secondary generation
Thank the yield and diversity of product；
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 new secondary metabolite LW-4.
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.
Embodiment 5:
Using Escherichia coli, aerobacteria as pathogenic bacteria model, positive control drug of the Ciprofloxacin as Model of Bacterial is adopted
Preliminary assessment is carried out with bacteriostatic activity of the filter paper doubling dilution to gained compound.It the results are shown in Table 2.
The detection method (doubling dilution solvent diffusion method) of bacteriostatic activity:
The compound of fermentation gained metabolite crude extract and final separation identification is carried out to the screening and survey of bacteriostatic activity
Examination, the screening of bacteriostatic activity use disk diffusion method, and the test of MIC uses doubling dilution.Specific step is as follows:
From in -80 DEG C of refrigerators, by indicator bacteria recovery, 37 DEG C of fermentations for 24 hours, are prepared into seed liquor in liquid bouillon media.
After getting corresponding Solid media for plates ready, seed liquor is spread evenly across spare on plating medium.Take each sample to be tested
(diameter on the filter paper after sterilizing is added dropwise in (concentration of coarse extract is 10mg/mL, final compound concentration 0.1mg/mL) 10 μ L
5mm), it is gently attached at after sample dries on the spare plate for being coated with indicator bacteria, is placed in 28 DEG C of constant temperature in constant incubator
Stationary culture is for 24 hours.Fungistatic effect (i.e. inhibition zone size) is observed after culture.Two times need to be used for active compound
Dilution method continues to test its minimum inhibitory concentration (MIC), until sample concentration when filter paper is not infected just is MIC.
Inhibitory activity of the new secondary metabolite LW-4 of 2 Aspergillus terreus SKL-001 of table to drug-fast bacteria
Test result shows: compound L W-4 shows strong inhibitory activity to Escherichia coli, shows to aerobacteria
Stronger inhibitory activity, the MIC of inhibitory activity is respectively 5 μ g/mL, 19 μ g/mL, therefore can be used as preparing antibacterial agent.
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 (7)
1. the purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001, which is characterized in that the new secondary metabolite LW-4
As effective component, preparing the purposes in antibacterial agent, wherein new secondary metabolite LW-4 be Formulas I compound represented, its
Stereoisomer or pharmaceutically accessible salt, chemical structural formula are as follows:
2. the purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 1, which is characterized in that institute
The preparation method for stating new secondary metabolite LW-4 includes the following steps:
1) strain fermentation: adding epigenetic modification adjusting control agent SAHA, paricalcitol, triglycerides into fungi culture medium,
It stirs evenly, stands, fermented and cultured；
2) secondary metabolite extracts: with silk by mycelium and separation of fermentative broth；Fermentation liquid is extracted with ethyl acetate, and depressurizes dense
Contracting, obtains fermentation liquid extract；80% acetone-water, 2- methylalanine and calcium citrate is added in mycelium, impregnates, and tissue is broken
Broken instrument is broken, ultrasonic extraction 30min, filters to obtain clear liquid, and ethyl acetate extraction is concentrated under reduced pressure, obtains fermentation mycelium extract；It closes
And fermentation liquid extract and fermentation mycelium extract, obtain final fermentation, extraction object；
3) isolate and purify: final fermentation, extraction object is eluted with silica gel column chromatography, and elution fraction 3 is eluted with silica gel column chromatography again, is washed
De- component 12 is separated with preparative thin layer chromatography, and the high performance liquid chromatography separation of separation component 2 is purified to get Aspergillus terreus SKL-
001 new secondary metabolite LW-4.
3. the purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 3, which is characterized in that institute
Stating strain fermentation incubation time is 30 days.
4. the purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 3, which is characterized in that institute
Final concentration of 5-25 μM for stating epigenetic modification adjusting control agent SAHA.
5. the purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 3, which is characterized in that institute
The additive amount for stating paricalcitol is 0.05-0.1%, and triglycerides additive amount is 0.05-0.15%.
6. the purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 3, which is characterized in that institute
The additive amount for stating 2- methylalanine is the 1-3% of culture medium quality, and the additive amount of calcium citrate is the 0.5- of culture medium quality
1.5%.
7. the purposes of the new secondary metabolite LW-4 of Aspergillus terreus SKL-001 according to claim 3, which is characterized in that institute
State silica gel column chromatography, silica gel column chromatography eluant, eluent successively are as follows: petroleum ether-acetone-methanol, petroleum ether-ethyl acetate；Efficient liquid
The eluant, eluent of phase chromatography is methanol-water, volume ratio 70:30；The solvent of preparative thin layer chromatography is methylene chloride-methanol,
Volume ratio is 50:1.
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Cited By (2)
| 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 |
| CN109293662A (en) * | 2018-10-26 | 2019-02-01 | 浙江海洋大学 | A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001 |
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| CN109293662A (en) * | 2018-10-26 | 2019-02-01 | 浙江海洋大学 | A kind of new secondary metabolite LW-4 of Aspergillus terreus SKL-001 |
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