CN108753628A - One plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 - Google Patents
One plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 Download PDFInfo
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Abstract
The invention discloses one plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6, and Guangdong Province's Culture Collection, deposit number were deposited on 03 20th, 2018：GDMCC 60337；Preservation address：5 building, the building of compound the 59th of Xianlie Middle Road, Guangzhou City 100, Guangdong Microbes Inst.Marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 provided by the invention can be generated by fermenting with antibacterium, antimycotic, desinsection or the active material compound A and compound B that inhibit acetylcholine enzyme effect, and the structural formula of compound A and compound B is respectively as shown in formula 1 and formula 2：The bacterial strain is with a wide range of applications in preparing antifungal agent, antibacterial agent, agricultural insecticide or acetylcholinesterase inhibitor.
Description
Technical field
The present invention relates to biotechnologies, more particularly, to one plant of marine fungi pawl aspergillus mutagenic fungi
Aspergillus unguis 6-20-6。
Background technology
Find the cause of disease in clinical treatment at present microorganism drug resistance it is more and more stronger, especially the penicillin of resistance to methoxyl group gold
Staphylococcus aureus (super drug-fast bacteria), pseudomonas aeruginosa (being commonly called as Pseudomonas aeruginosa), vibrio parahaemolytious and Candida albicans
Infection is increasingly severe caused by (being commonly called as Candida albicans), and many conventional antibiotics lose curative effect substantially.
Moreover, the pesticides such as common insecticide in agricultural are made in agricultural production because excessively using various chemical synthetic pesticides
Jeopardize health and the ecological balance at serious food, soil and water pollution, and by food chain, pesticide residue also seriously hinders
The export of farm produce in the China Ai Liao；And biological pesticide has and is not likely to produce drug resistance, safe to non-target organism, environmental-friendly and easy
In the natural degradation the advantages that, have great importance for human health, environmental protection and agricultural sustainable development.
At the same time, it is mainly eserine, galanthamine, huperzine for clinical acetylcholinesterase inhibitor
First, Rivastigmine, Tacrine and donepezil, but these drug prices are expensive, and have centainly other than huperzine
Toxic side effect.
It is, thus, sought for novel anti-bacterial drug, antifungal drug, insecticide or acetylcholinesterase inhibitor.
Invention content
The present invention is existing anti-bacterial drug, antifungal drug, insecticide and the acetyl overcome described in the above-mentioned prior art
The defect of anticholinesterase provides one plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis6-20-6, carries
The marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 of confession can by ferment generate with antibacterium,
Antimycotic, desinsection or the active material for inhibiting acetylcholine enzyme effect.
Another object of the present invention is to provide above-mentioned marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis6-
Applications of the 20-6 in preparing antifungal agent, antibacterial agent, insecticide or acetylcholinesterase inhibitor.
A further purpose of the present invention is to provide a kind of preparation method of depsidone class compound.
In order to solve the above technical problems, the technical solution adopted by the present invention is：
One plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 was protected on 03 20th, 2018
Ensconce Guangdong Province's Culture Collection, deposit number：GDMCC 60337；Preservation address：Xianlie Middle Road, Guangzhou City 100
Number 5 building, the building of compound the 59th, Guangdong Microbes Inst.
The isolated Aspergillus unguis DLEP2008001 of seaweed in Dalian Sea Area, after to its spore use
Atmos low-temperature plasma mutagenesis obtains Aspergillus unguis 6-20-6.Marine fungi pawl aspergillus Aspergillus
Unguis DLEP2008001 were stored in China Committee for Culture Collection of Microorganisms's commonly micro- life on October 29th, 2009
Object center (CGMCC), Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, deposit number are CGMCC No.3372, and strain number is
DLEP2008001。
In addition, the ITS1-5.8S- of the marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6
ITS2rDNA sequences such as SEQ ID NO：Shown in 1.
The ITS1-5.8S-ITS2rDNA sequences of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6
(569bp) is submitted to the GenBank geneseq databases of National Center for Biotechnology Information (NCBI), and accession number is
MH071299。
The marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 have following properties：It is inoculated into
On seawater potato dextrose medium (seawater PDA) tablet, 28 DEG C of cultures, bacterium colony initial stage is green, and the later stage is yellow green, circle
Shape extends around, center projections, and yellowish-brown pigment is produced at the back side；The bacterial strain is quiet in seawater potato sucrose fluid nutrient medium
Anti-acetylcholinesterase, antibacterial, insecticide active substance can be generated by setting fermentation.
Marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 provided by the invention can by fermentation
The structural formula of generation compound A and compound B, compound A and compound B are respectively as shown in formula 1 and formula 2；It is experimentally confirmed,
Compound A and compound B has the effect of antibacterium, antimycotic, desinsection, and in addition compound B also has acetylcholinesterase suppression
System activity.
Therefore, above-mentioned marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 prepare antifungal agent,
Application in antibacterial agent, insecticide or acetylcholinesterase inhibitor, also within protection scope of the present invention.
Preferably, the fungi is Candida albicans.
Preferably, the bacterium is the penicillin of resistance to methoxyl group staphylococcus aureus or pseudomonas aeruginosa.
Preferably, the worm is artemia larva.
The present invention also protects a kind of preparation method of depsidone class compound, the depsidone class compound by
Above-mentioned marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 are prepared by fermentation, extracting and developing；
The depsidone class compound is compound A or compound B；
The structural formula of the compound A and compound B is respectively as shown in formula 1 and formula 2：
Marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 can generate above-mentioned contracting phenol by fermentation
Sour cyclo other compounds, by conventional method in the art can extracting and developing obtain the depsidone class compound.
Preferably, the step of fermentation is as follows：
Pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 are fermented in the medium, collect mycelium and hair
Zymotic fluid.
Preferably, the step of fermentation is as follows：
Pawl aspergillus mutagenic strain Aspergillus unguis 6-20-6 are fermented under fungi culture medium；28 DEG C of standings
Fermentation 15~25 days；Appropriate diatomite is added after cultivation cycle to be filtered, respectively obtains mycelium filter cake and filtrate, i.e.,
Mycelium and zymotic fluid.
The fungi culture medium group becomes：Every liter contains, 400~600mL of murphy juice, 15~25g of sea salt, and sucrose 15~
25g, 400~600mL of pure water or tap water；The PSB culture mediums are through 121 DEG C of sterilizing 20min.
Preferably, the fungi culture medium group becomes：Every liter contains, murphy juice 500mL, sea salt 20g, sucrose 20g, pure water
Or tap water 500mL.
The preparation method of the murphy juice is：Potato decortication is cleaned, and deionized water is added, is heated to boiling 30min, mistake
Filter can boil 500mL murphy juices, freezing per 200g potatoes.
Preferably, the temperature of the fermentation is 28 DEG C, and fermentation time is 20 days.
Preferably, the step of extraction is as follows：
Mycelium and zymotic fluid are obtained after fermentation；Zymotic fluid is after resin adsorption, simultaneously with the elution of the first organic solvent
Concentration；Mycelium is extracted and is concentrated with the second organic solvent；Merge concentrate, obtains crude extract.
Preferably, the resin is macroreticular resin.
Preferably, the macroreticular resin is D101, NKA, XAD-7 or XAD-16.
Preferably, first organic solvent and the second organic solvent are respectively and independently selected from methanol, ethyl alcohol, acetone or acetic acid
One or more of ethyl ester.
Preferably, the step of extraction is as follows：
Mycelium and zymotic fluid are obtained after fermentation；Macroreticular resin is added in zymotic fluid, chromatographic column is transferred to after shaking up,
After draining zymotic fluid and washing, macroreticular resin is eluted with the first organic solvent, is concentrated under reduced pressure；Mycelium filter cake is organic molten with second
Agent soaked overnight, ultrasound are filtered, are concentrated under reduced pressure, in triplicate；Merge enriched product.
It is further preferred that the step of extraction, is as follows：
Mycelium and zymotic fluid are obtained after fermentation；300mL NKA resins are added in per 500mL zymotic fluids, shake up 1h,
After be transferred to chromatographic column, after draining zymotic fluid and washing, resin first with methanol, after wash 3 column volumes respectively with acetone, decompression is dense
Contracting；Mycelium filter cake methanol:Acetone=2:1 soaked overnight, ultrasonic 30min, suction filtration, reduced pressure, in triplicate；Merge dense
Contracting product.
Preferably, the separation includes the following steps：
Crude extract is obtained after extraction；Crude extract is after gel post separation, then by thin-layer chromatography or prepares liquid phase color
Spectrum separation, obtains compound A or compound B.
It is further preferred that the separating step includes：
A. crude extract is obtained after extracting；Crude extract is detached through gel filtration chromatography, and eluent is pure methanol or methanol-
Water collects the first component of mixture that Rf value is 0.29~0.74, solvent is body by being monitored on tlc silica gel plate
Product ratio 10:1 chloroform-methanol, tlc silica gel plate are the Silica gel 60F254 of Merck companies production；
B. the first component of mixture that step a is collected is subjected to gel filtration chromatography again, it is 0.5~0.76 to collect Rf value
Second component of mixture, thin-layer chromatography condition are identical as step a.；
C. the second component of mixture step b collected carries out thin-layer chromatography preparation, and solvent is volume ratio 4:1 oil
Ether-acetone soln, collects the third component of mixture that Rf value is 0.6~0.75, and thin layer chromatography board is the production of Merck companies
Silica gel 60F254；
D. the third component of mixture that step c is collected is subjected to thin-layer chromatography preparation again, solvent is volume ratio 1:3 stone
Oily ether-dichloromethane solution, collects the component that Rf value is 0.65~0.7, and thin layer chromatography board is the production of Merck companies
Silica gel 60F254 obtain the compound A；
E. elution fraction step a collected carries out preparative liquid chromatography separation, and preparative liquid chromatograph is Lisure HP
Plus 50D, chromatographic column used are the SinoChrom ODS-AP that Dalian is produced according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S., 15 μm, 20.0mm × 250mm,
Eluent is volume ratio 3:7~5:5 methanol-water solution；Flow velocity is 5mL/min；Column temperature is room temperature；It is 10 to collect retention time
The component of~20min obtains compound B.
Preferably, the gel of the gel column is sephadex.The sephadex is Sephadex-20.
Preferably, the eluent of the gel column is methanol.
Preferably, it is 4 that the mobile phase of the preparative liquid chromatography, which is volume ratio,:6 methanol-water solution.Volume ratio is 4:6
Methanol-water solution, -60% water of 40% methanol can also be expressed as.
Inventor is found through experiments that compound A and compound B shine on thin layer chromatography board TLC in 254nm ultraviolet lights
It penetrates lower at single black splotch, anisaldehyde colour developing respectively brown and aubergine.
It has also been found that the retention time of compound A and compound B in high performance liquid chromatography is 5.2min and 1.7min, it is high
Effect liquid phase chromatogram instrument is Agilent Infinity II 1260, and column dimensions are 4.6mm × 250mm, and filler is 4 μm of EC.C18,
Mobile phase is 60%MeOH-40%H2O, flow velocity 1mL/min, 30 DEG C, detector DAD, Detection wavelength 210nm of column oven,
254nm, 280nm, 320nm and 365nm.
In addition, compound A and compound B are light yellow solid at normal temperatures and pressures.
It is obtained through antibacterial experiment, the inhibition zone of compound A and compound B to the penicillin of resistance to methoxyl group staphylococcus aureus
Respectively 17.0 ± 0.0 and 17.7 ± 0.6mm, MIC value are respectively 25.6 and 12.8 μM, have and inhibit the penicillin of resistance to methoxyl group gold
The activity of staphylococcus aureus；Compound A and compound B is respectively 11.1 ± 0.2 Hes to the inhibition zone of pseudomonas aeruginosa
16.0 ± 0.0mm, MIC are respectively 51.2 and 102.4 μM, show that above compound has the work for inhibiting P. aeruginosa growth
Property；Compound A and compound B is 8.3 ± 0.6mm to the inhibition zone of vibrio parahaemolytious, and MIC is equal>102.4 μM, show above-mentioned
Compound cannot effectively inhibit vibrio parahaemolytious or activity very weak；Inhibition zones of the compound A and compound B to Candida albicans
Respectively 10.0 ± 0.0 and 15.5 ± 2.2mm, MIC value be respectively>102.4 and 6.4 μM, has and inhibit Candida albicans
Activity；Half lethal dose LCs of the compound A and compound B to artemia larva50Respectively 75.87 and 51.2 μM, show that it has
Insecticidal activity.In addition ICs of the compound B to acetylcholinesterase50It is 56.75 μM, shows that it inhibits to live with acetylcholinesterase
Property.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention obtained one plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6, in 2018
It is deposited in Guangdong Province's Culture Collection, deposit number within 20 days 03 month：GDMCC 60337；Preservation address：Guangzhou is first
5 building, the building of compound the 59th of strong Road 100, Guangdong Microbes Inst.Marine fungi pawl aspergillus mutagenic fungi provided by the invention
Aspergillus unguis 6-20-6 can be generated by fermenting with antibacterium, antimycotic, desinsection or inhibition acetylcholine
The active material of enzyme effect has in preparing antifungal agent, antibacterial agent, agricultural insecticide or acetylcholinesterase inhibitor
It is widely applied foreground.
Description of the drawings
Fig. 1 is the nuclear magnetic resonance spectroscopy of compound A made from embodiment 1.
Fig. 2 is the carbon-13 nmr spectra of compound A made from embodiment 1.
Fig. 3 is the nuclear magnetic resonance spectroscopy of compound B made from embodiment 1.
Fig. 4 is the carbon-13 nmr spectra of compound B made from embodiment 1.
Specific implementation mode
The present invention is further illustrated With reference to embodiment.
The equal cocoa of raw material in embodiment is by being commercially available；
Unless stated otherwise, the present invention uses reagent, method and apparatus for the art conventional reagent, method and are set
It is standby.
Marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 were deposited in extensively on 03 20th, 2018
East saves Culture Collection, deposit number：GDMCC 60337, strain number：6-20-6；Preservation address：In the martyr of Guangzhou
5 building, the building of compound the 59th of road 100, Guangdong Microbes Inst.
Embodiment 1
The present embodiment is the preparation method of marine fungi bromo depsidone class compound.
Step 1. is fermented
Pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 are inoculated in fungi liquid culture medium quiet in 28 DEG C
Fermentation is set, ferments 20 days, appropriate diatomite is added, is filtered, collects mycelium and zymotic fluid respectively.
Wherein, the formula of the fungi liquid culture medium is：Contain following component in every liter：Murphy juice 500mL, sea salt
20g, sucrose 20g, ultra-pure water 500mL.
Wherein, the preparation method of murphy juice is potato decortication, is cleaned, and deionized water is added, is heated to boiling 30min, mistake
Filter can boil 500mL murphy juices, freezing per 200g potatoes.
Step 2. slightly carries
The zymotic fluid that step 1 is obtained shakes up 1h through 300mL NKA resin adsorptions, after be transferred to chromatographic column, drain fermentation
It after liquid and washing, successively uses methanol and acetone that resin is eluted 3 column volumes respectively, is concentrated under reduced pressure, obtains concentrate.It simultaneously will step
The rapid 1 mycelium methanol obtained：Acetone=2:1 soaked overnight, ultrasonic 30min are filtered, and are concentrated under reduced pressure, in triplicate, are obtained dense
Contracting liquid.Gained concentrate is merged, as crude extract；
Step 3. isolates and purifies
A. crude extract step 2 obtained is eluted through gel filtration chromatography with methanol, automatic collector collection eluent, and about 2
~3mL/ is managed, and flow control is dripped in 8~10s mono-；Component characteristics are that solvent is volume ratio 10 on tlc silica gel plate:1
It is unfolded under chloroform-methanol, the component of mixture that Rf value is 0.29~0.74, tlc silica gel plate is Merck companies
The Silica gel 60F254 of production；
B. elution fraction step a collected carries out gel filtration chromatography again, and eluent is pure methanol, flow velocity 0.5mL/min,
The second component of mixture that Rf value is 0.5~0.76 is collected, thin-layer chromatography condition is identical as step a.；
C. elution fraction step b collected carries out thin-layer chromatography preparation, and solvent is petroleum ether：Acetone=4:1；Than moving
Value is 0.6~0.75 component of mixture, and thin layer chromatography board is the Silica gel 60F254 of Merck companies production；
D. elution fraction step c collected carries out thin-layer chromatography preparation, and solvent is petroleum ether：Dichloromethane=1:3；
The component that Rf value is 0.65~0.7 is collected, thin layer chromatography board is the Silicagel 60F254 of Merck companies production, is obtained
Compound A；
E. elution fraction step a collected carries out preparation liquid phase separation, and preparative liquid chromatograph is Lisure HPPlus
50D, chromatographic column used are the SinoChrom ODS-AP that Dalian is produced according to Lyntech Corporation (US) 10177 South 77th East Avenue Tulsa, Oklahoma 74133 U.S., 15 μm, 20.0mm × 250mm, are eluted
Liquid is -60% water of 40% methanol；Flow velocity is 5mL/min；Column temperature is room temperature；The component that retention time is 10~20min is collected, is obtained
To compound B；
Collected target substance also is compliant with following characteristics：
(1) it is in single, uniform black splotch, anisaldehyde colour developing difference that TLC detections, which are collected under the irradiation of 254nm ultraviolet lights,
For brown and aubergine；
(2) compound A and compound B retention times under the conditions of the high-efficient liquid phase analysis of detection are respectively 5.2 Hes
The single chromatographic peak of 1.7min.The high performance liquid chromatograph is Agilent Infinity II 1260, and column dimensions are
4.6mm × 250mm, filler are 4 μm of EC.C18, and mobile phase is 60%MeOH-40%H2O, flow velocity 1mL/min, column oven 30
DEG C, detector DAD, Detection wavelength 210nm, 254nm, 280nm, 320nm and 365nm.
The compound that step 4. is obtained through above-mentioned separation process, is determined as pure compound.
Through Spectrum Analysis, Structural Identification the results show that its be two kinds of antibacterials and desinsection depsidone class natural products, change
The structural formula of conjunction object A and compound B is respectively as shown in formula 1 and formula 2：
Above compound has following physics and chemistry and spectral characteristic：
Compound A nuclear magnetic resonance spectroscopies (1H NMR(500MHz,MeOD)δ6.47(1H,s,H-4),6.40(1H,s,H-
7),5.55(1H,m,H-2'),2.44(3H,s,H1-Me),2.13(3H,s,H9-Me),2.04(3H,s,H-4'),1.82(3H,
Dd, J=6.7,0.5Hz, H-3')), as shown in Figure 1.
Compound A carbon-13 nmr spectras (13C NMR (126MHz, MeOD) δ 165.3 (C=O, C-11), 163.3 (C-3),
161.0(C-4a),153.9(C-8),144.8(C-9a),142.5(C-1),142.4(C-5a),137.1(C-1'),134.2
(C-6),126.5(C-2'),121.5(C-2),116.2(C-11a),113.6(C-9),112.2(C-7),106.7(C-4),
18.5(C1-Me),17.8(C-4'),14.0(C-3'),9.3(C9- Me)), as shown in Figure 2.
Compound B nuclear magnetic resonance spectroscopies (1H NMR (500MHz, MeOD) δ 6.51 (1H, d, J=1.8Hz, H-2), 6.33
(1H, d, J=2.3Hz, H-4), 5.10 (1H, dd, J=6.7,1.2Hz, H-2 '), 2.39 (3H, s, H1-Me),2.16(3H,s,
H9- Me), 2.00 (3H, s, H-4 '), 1.82 (3H, dd, J=6.7,0.8Hz, H-3 ')), as shown in Figure 3.
Compound B carbon-13 nmr spectras (13C NMR(126MHz,MeOD)δ174.41(C7-COOH),165.54(C-4a),
164.03(C-3),163.97(C-11),158.27(C-9a),146.19(C-8),145.96(C-1),141.37(C-5a),
138.97(C-6),134.08(C-1’),122.42(C-2’),116.71(C-2),115.79(C-9),112.92(C-11a),
111.39(C-7),106.57(C-4),21.29(C1-Me),18.83(C-4’),13.86(C-3’),9.22(C9- Me)), such as
Shown in Fig. 4.
Embodiment 2
The present embodiment is the suppression Gram-positive drug-fast bacteria activity experiment of depsidone class compound.
1, staphylococcus aureus is very common germ, and sometimes it can be into causing to infect in human body.It is this
Slight meeting papula and papule on the skin are infected, it is serious, pneumonia or blood infection can be caused.Therefore Staphylococcus aureus
Bacterium is the important target pathogenic microorganism of antibiotics research and development.
2, experimental method：
Antibacterial activity primary dcreening operation is carried out using the filter paper enzyme of international standard, it is strong and weak to weigh antibacterial activity with antibacterial circle diameter
(Clinical and Laboratory Standards Institute.2012.Performance Standards for
Antimicrobial Disk Susceptibility Tests；Approved Standard.Eleventh Edition
M2-A11.)；Secondary screening uses 96 orifice plates, the micro broth dilution method adopted international standards (National Committee for
Clinical Laboratory Standards.2003.Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically,5th ed.Approved
Standard M7-A6.), after cultivating 16~20h, 625nm reads OD values, measures compound to the penicillin of resistance to methoxyl group golden yellow
Bacteriostasis rate under staphylococcic minimal inhibitory concentration (MIC value) and various concentration, culture medium are the Muller-Hinton of standard
Broth bouillon.
Bacteriostasis rate=(ODNot dosing-ODDosing)/(ODNot dosing-ODCulture medium) × 100%
3, experimental result：
The depsidone class compound that above-described embodiment 1 obtains is to the staphylococcus aureus of the penicillin of resistance to methoxyl group
Inhibition zone is respectively 17.0 ± 0.0 and 17.7 ± 0.6mm, and MIC value is respectively 25.6 and 12.8 μM, has and inhibits resistance to methoxy very well
The activity of base penicillin staphylococcus aureus.
Embodiment 3
The present embodiment is the suppression gram prolapse of uterus activity experiment of depsidone class compound.
1, pseudomonas aeruginosa is widely distributed in nature, various water, air, the skin of normal person, respiratory tract and enteron aisle
There is the presence of this bacterium, which often causes postoperative wound infection, can also cause bedsore, abscess, otitis media suppurative etc..
Vibrio parahaemolytious is widely present in marine and marine product, infects bacterium meeting Acute onset, abdominal pain, vomiting, diarrhea
And watery stool.
2, experimental method：
Antibacterial activity primary dcreening operation is carried out using the filter paper enzyme of international standard, it is strong and weak to weigh antibacterial activity with antibacterial circle diameter
(Clinical and Laboratory Standards Institute.2012.Performance Standards for
Antimicrobial Disk Susceptibility Tests；Approved Standard.Eleventh Edition
M2-A11.)；Secondary screening uses 96 orifice plates, the micro broth dilution method adopted international standards (National Committee for
Clinical Laboratory Standards.2003.Methods for dilution antimicrobial
susceptibility tests for bacteria that grow aerobically,5th ed.Approved
Standard M7-A6.), after cultivating 16-20h, 625nm reads OD values, measures compound to pseudomonas aeruginosa and secondary haemolysis
Bacteriostasis rate under the minimal inhibitory concentration (MIC value) and various concentration of vibrios, culture medium are the Muller-Hinton meat soups of standard
Culture medium (culture medium of vibrio parahaemolytious contains 1% sodium chloride).
Bacteriostasis rate=(ODNot dosing-ODDosing)/(ODNot dosing-ODCulture medium) × 100%
3, experimental result：
The depsidone class compound that above-described embodiment 1 obtains is respectively 11.1 to the inhibition zone of pseudomonas aeruginosa
± 0.2 and 16.0 ± 0.0mm, MIC are respectively 51.2 and 102.4 μM, show that above compound has and inhibit pseudomonas aeruginosa
The activity of growth；Inhibition zone to vibrio parahaemolytious is 8.3 ± 0.6mm, and MIC is equal>102.4 μM, show above compound not
It can effectively inhibit vibrio parahaemolytious or activity very weak.
Embodiment 4
The present embodiment is that the fungicidal activities of depsidone class compound are tested.
1, Candida albicans (Candida albicans) are a kind of pathogenic fungus, be typically found in normal person oral cavity, on
Respiratory tract, enteron aisle and vagina, generally quantity is few in normal body, does not cause disease.It, generally can be for opportunistic fungus
Skin, mucous membrane, internal organ and nervous centralis find that the bacterium can cause thrush, angular stomatitis, vaginitis, pneumonia, meningitis, stomach
Scorching, endocarditis, occasional cause septicaemia.Therefore, Candida albicans are the important target cause of diseases of antifungal drug research and development
Microorganism.
2, experimental method：
Antifungal activity primary dcreening operation is carried out using the filter paper enzyme of international standard, it is strong and weak to weigh antibacterial activity with antibacterial circle diameter
(Clinical and Laboratory Standards Institute.2012.Performance Standards for
Antimicrobial Disk Susceptibility Tests；Approved Standard.Eleventh Edition
M2-A11.), secondary screening uses 96 orifice plates, micro sabouraud culture medium dilution method (the National Committee to adopt international standards
for Clinical Laboratory Standards.1997.Reference methed for broth dilution
antifungal susceptibility testing of yeasts:approved standard M27-A,7th
Ed.NCCLS, Wayne, Pa.), after cultivating 16-20h, 625nm reads OD values, measures compound and presses down to the minimum of Candida albicans
Bacteriostasis rate under bacteria concentration (MIC value) and various concentration, culture medium are the sabouraud culture medium of standard.
Bacteriostasis rate=(ODNot dosing-ODDosing)/(ODNot dosing-ODCulture medium) × 100%
3, experimental result：
Inhibition zone to Candida albicans is respectively 10.0 ± 0.0 and 15.5 ± 2.2mm, and MIC value is respectively>102.4
With 6.4 μM, the activity with inhibition Candida albicans.
Embodiment 5
The present embodiment is that the insecticidal activity of depsidone class compound is tested.
1, artemia (Artemia salina) is also known as brine fairy shrimp, belongs to Arthropoda, Crustachia, Anostraca, brine
Fairy shrimp section, genus artemia are a kind of fish food biologies with higher economic value, and a kind of more sensitive for toxin
Important experimental animal, the activity that 2~3 instar larvaes of laboratory cultures can be used for evaluating insecticide is strong and weak.
2, insecticidal activity experimental method：
Artemia is hatched and is collected：At 28 DEG C, the pears through incandescent lamp 1000Lux irradiations are added in the artemia eggs frozen
(the seawater 30g/L of natural coarse sea salt configuration is wherein filled, ovum injected volume is 0.15g/L) in shape glass funnel, it is per minute to be passed through
The air of 420mL is incubated for 24 hours, utilizes the characteristic of artemia phototaxis later, stops ventilation, to the shading of funnel lower half portion, makes dead
The artemia died sinks to bottom, removes dead artemia, after to funnel top half shading, so that artemia is gathered bottom, opening valve
Door collects artemia with clean beaker, and drawing the artemia larva in beaker with the smooth liquid transfer gun head that is open carries out subsequent experimental.
The lethal method of artemia biology：Sample is diluted to series concentration in 96 orifice plates with the continuous sesquialter of absolute methanol, vacuum is dry
200 μ L artemia larvas suspensions (containing 20~30 polypides) are added per hole, and blank control group is arranged for dry removing organic solvent, and 28
After DEG C culture for 24 hours, counted under binocular stereo microscope per larva sum and death toll in hole, the correction calculated per hole is dead
Rate.
Corrected mortality=(the processing hole death rate-control death rate)/control group survival rate × 100%
With corrected mortality-concentration mapping, logarithm Trendline is taken, half lethal dose LC is calculated50。
3, experimental result：
The half lethal dose LC of the depsidone class compound of above-mentioned acquisition50Respectively 75.87 and 51.2 μM, show it
With insecticidal activity.
Embodiment 6
The present embodiment is that the acetylcholine esterase inhibition activity of depsidone class compound is tested.
1, common electric eel acetylcholinesterase is screened using anti-acetylcholinesterasemedicine medicine and carries out external anti-acetylcholine
Esterase active is tested.
2, the experimental method that acetylcholine ester enzyme inhibition rate measures：
Compound concentration is set as：102.4,51.2,25.6,12.8,6.4,3.2,1.6,0.8,0.4,0.2,0.1,
0.05, two multiple holes of each concentration；4 groups, respectively sample sets (+49 μ L PBS+1 μ LDMSO+10 μ L AChE+20 of sample are set
20 μ L ATCh, 37 DEG C of incubation 20min are added in μ L DTNB, 37 DEG C of incubation 10min), sample blank group (+49 μ L PBS+1 of sample
20 μ L ATCh, 37 DEG C of incubation 20min are added in μ L DMSO+10 μ L BSA+20 μ L DTNB, 37 DEG C of incubation 10min), control group
(49 μ L PBS+1 μ LDMSO+10 μ L AChE+20 μ L DTNB, 37 DEG C of incubation 10min, are added 20 μ L ATCh, 37 DEG C of incubations
20min), (20 μ L are added in 49 μ L PBS+1 μ L DMSO+10 μ L BSA+20 μ L DTNB, 37 DEG C of incubation 10min to blank group
ATCh, 37 DEG C incubation 20min), microplate reader 405nm place measurement OD values, suppression of each hole to acetylcholinesterase is calculated according to OD values
Rate processed.
Inhibiting rate=[(ODControl group-ODBlank group)-(ODSample sets-ODSample blank group)]/(ODControl group-ODBlank group) × 100%
With inhibiting rate-concentration mapping, logarithm Trendline is taken, 503nhibiting concentration IC is calculated50。
3, experimental result：
IC of the B compounds of above-mentioned acquisition to acetylcholinesterase50It is 56.75 μM, shows that it is anti-with some strength
Acetylcholine esterase active.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention.For those of ordinary skill in the art, may be used also on the basis of the above description
To make other variations or changes in different ways.There is no necessity and possibility to exhaust all the enbodiments.It is all this
All any modification, equivalent and improvement etc., should be included in the claims in the present invention made by within the spirit and principle of invention
Protection domain within.
Sequence table
<110>Guangdong Ocean University
Shenzhen research institute of Guangdong Ocean University
<120>One plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 569
<212> DNA
<213>Marine fungi pawl aspergillus mutagenic fungi (Aspergillus unguis)
<400> 1
tccgtaggtg aacctgcgga aggatcatta ccgagtgcgg gctgcctccg ggcgcccaac 60
ctcccaccct tgaatactaa acactgttgc ttcggcgggg agccccttcc ggggggcaag 120
ccgccgggga ccactgaact tcatgcctga gagtgatgca gtctgagtct gaattataaa 180
tcagtcaaaa ctttcaacaa tggatctctt ggttccggca tcgatgaaga acgcagcgaa 240
ctgcgataag taatgtgaat tgcagaattc agtgaatcat cgagtctttg aacgcacatt 300
gcgccccctg gcattccggg gggcatgcct gtccgagcgt cattgctgcc cttcaagccc 360
ggcttgtgtg ttgggtcgtc gtcccccccg ggggacgggc ccgaaaggca gcggcggcac 420
cgtgtccggt cctcgagcgt atggggcttt gtcacccgct cgattagggc cggccgggcg 480
ccagccggcg tcatcaatct attttaccag gttgacctcg gatcaggtag ggatacccgc 540
tgaacttaag catatcaata agcggagga 569
Claims (8)
1. one plant of marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6, in preservation on the 20th in 03 month in 2018
In Guangdong Province's Culture Collection, deposit number：GDMCC 60337；Preservation address：Xianlie Middle Road, Guangzhou City 100
5 building, the building of compound the 59th, Guangdong Microbes Inst.
2. marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 according to claim 1, feature
It is, the ITS1-5.8S-ITS2rDNA sequences of the marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6
Row such as SEQ ID NO：Shown in 1.
3. marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 according to claim 1, feature
It is, the marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 have following properties：It is inoculated into seawater
On potato dextrose medium (seawater PDA) tablet, 28 DEG C of cultures, bacterium colony initial stage is green, and the later stage is yellow green, round,
It extends around, center projections, yellowish-brown pigment is produced at the back side；The bacterial strain stands hair in seawater potato sucrose fluid nutrient medium
Ferment can generate anti-acetylcholinesterase, antibacterial, insecticide active substance.
4. any one of claims 1 to 3 marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis 6-20-6 are making
Application in standby antifungal agent, antibacterial agent, insecticide or acetylcholinesterase inhibitor.
5. application according to claim 4, which is characterized in that the fungi is Candida albicans.
6. application according to claim 4, which is characterized in that the bacterium is the penicillin of resistance to methoxyl group Staphylococcus aureus
Bacterium or pseudomonas aeruginosa.
7. application according to claim 4, which is characterized in that the worm is artemia larva.
8. a kind of preparation method of depsidone class compound, which is characterized in that the depsidone class compound is by weighing
Profit requires any one of 1~3 marine fungi pawl aspergillus mutagenic fungi Aspergillus unguis6-20-6 by fermenting, carrying
It takes, detach and be prepared；
The depsidone class compound is compound A or compound B；
The structural formula of the compound A and compound B is respectively as shown in formula 1 and formula 2：
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439705A (en) * | 2018-12-05 | 2019-03-08 | 广州中医药大学(广州中医药研究院) | A kind of microbe preparation method of subergorgic acid |
| CN109943342A (en) * | 2019-04-12 | 2019-06-28 | 徐州工程学院 | A kind of cadmium pollution soil in-situ immobilization agent based on Wei Si Salmonella |
| CN110604731A (en) * | 2019-08-29 | 2019-12-24 | 广东海洋大学深圳研究院 | Application of compound Aspergillus G in preparation of neuroprotective drugs |
| CN113930346A (en) * | 2021-10-25 | 2022-01-14 | 广西中医药大学 | Application of marine-derived aspergillus and fermentation product thereof in mango anthracnose resistance |
-
2018
- 2018-06-20 CN CN201810639846.8A patent/CN108753628A/en not_active Withdrawn
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109439705A (en) * | 2018-12-05 | 2019-03-08 | 广州中医药大学(广州中医药研究院) | A kind of microbe preparation method of subergorgic acid |
| CN109439705B (en) * | 2018-12-05 | 2021-10-01 | 广州中医药大学(广州中医药研究院) | Microbial preparation method of gorgonian acid |
| CN109943342A (en) * | 2019-04-12 | 2019-06-28 | 徐州工程学院 | A kind of cadmium pollution soil in-situ immobilization agent based on Wei Si Salmonella |
| CN110604731A (en) * | 2019-08-29 | 2019-12-24 | 广东海洋大学深圳研究院 | Application of compound Aspergillus G in preparation of neuroprotective drugs |
| CN113930346A (en) * | 2021-10-25 | 2022-01-14 | 广西中医药大学 | Application of marine-derived aspergillus and fermentation product thereof in mango anthracnose resistance |
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