阅读说明：本技术 一种海洋真菌土曲霉丁内酯类化合物丁内酯-i的制备方法 (Preparation method of butyrolactone-I of marine fungus aspergillus terreus butyrolactone compound ) 是由 张翼 聂影影 宋采 千忠吉 张永平 雷晓凌 李亚娟 洪鹏志 杨静明 杨文聪 冯妍 于 2018-02-26 设计创作，主要内容包括：本发明涉及一种海洋真菌土曲霉丁内酯类化合物丁内酯-I的制备方法。如式(I)所示的海洋真菌土曲霉代谢产物丁内酯类化合物丁内酯-I在制备抗外周及神经炎症和抗神经退行性疾病药物的应用。<Image he="579" wi="700" file="DDA0002220357140000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明发现丁内酯-I除可清除DPPH自由基外,还可较好地清除ABTS自由基和OH自由基；具有较好的抗氧化、抗炎；以及具有较好的神经保护的活性；因此,在制备制备抗外周及神经炎症药物和抗神经退行性疾病药物方面具有良好的应用前景。同时,本发明提供的丁内酯-I的制备方法,可成功制备得到该丁内酯-I,方法简单,易于实现大规模生产；且还可通过诱导剂的添加来优化培养条件,从而大幅提高其产量。(The invention relates to a preparation method of a marine fungus aspergillus terreus butyrolactone compound butyrolactone-I. The application of butyrolactone compounds of marine fungus aspergillus terreus metabolite butyrolactone-I shown in formula (I) in preparing medicines for resisting peripheral and neuroinflammation and neurodegenerative diseases. The invention finds that butyrolactone-I can remove DPPH free radicals and can also well remove ABTS free radicals and OH free radicals; has good antioxidation and anti-inflammatory effects; and has better neuroprotective activity; therefore, the method has good application prospect in the preparation of the anti-peripheral and anti-neuroinflammation drugs and the anti-neurodegenerative disease drugs. Meanwhile, the invention providesThe preparation method of the lactone-I can successfully prepare the butyrolactone-I, is simple and is easy to realize large-scale production; and the culture condition can be optimized by adding an inducer, so that the yield of the strain is greatly improved.)

1. A preparation method of a marine fungus aspergillus terreus butyrolactone compound butyrolactone-I shown in formula (I) is characterized by comprising the following preparation steps:

s1, fermentation: inoculating marine fungus aspergillus terreus C23-3 into a fungus liquid culture medium, standing and fermenting, and collecting mycelium and fermentation liquor;

s2, crude extraction: extracting the fermentation liquor obtained in the step S1 with ethyl acetate, concentrating, ultrasonically extracting mycelia with an organic solvent, concentrating, and mixing the obtained concentrates to obtain a crude extract;

s3, separation and purification: performing gradient chromatography on the crude extract obtained in the step S2 through a reduced pressure silica gel column, detecting through silica gel thin layer chromatography, and continuously performing gel column chromatography, reverse phase silica gel column chromatography and reverse phase preparative liquid chromatography on the elution component containing the target substance for separation and purification to obtain the butyrolactone-I;

wherein the marine fungus Aspergillus terreus C23-3 is preserved in Guangdong center for microbial strains in 2018, 1 month and 22 days, and the preservation number is GDMCC No. 60316;

2. the process for producing butyrolactone-I according to claim 1, wherein the conditions of the gradient chromatography on silica gel under reduced pressure in step S3 are: sequentially carrying out gradient elution by using petroleum ether-ethyl acetate and chloroform-methanol, and collecting gradient elution components with the volume ratio of the eluent of 100: 0-0: 100.

3. The preparation method of butyrolactone-I according to claim 1, wherein the reverse phase silica gel column is ODS reverse phase silica gel column chromatography, and the mobile phase is methanol-water in a volume ratio of 2:3 to 3: 2.

4. The process for the preparation of butyrolactone-I according to claim 1, wherein the conditions of the purification by reverse phase preparative liquid chromatography are: the mobile phase is methanol-water with the volume ratio of 1: 3-3: 2, 10-15 g of reverse phase silica gel is filled in a silica gel column, the flow rate is 4-8 mL/min, a main peak with the retention time of 5-20 min is collected, and the obtained collection is butyrolactone-I.

5. The process for preparing butyrolactone-I according to claim 1, wherein the fungal liquid medium of step S1 further contains an inducer ZnCl ₂An inducer ZnCl in the culture medium ₂The content of (b) is 0.0001-1 mM/mL.

Technical Field

The invention relates to the technical field of microorganisms, in particular to a preparation method of a marine fungus aspergillus terreus butyrolactone compound butyrolactone-I. The invention discloses a preparation method and application of a butyrolactone compound I, belonging to the patent division with application number of 2018101601117 and application date of 2018.02.26, and named as 'preparation method and application of a marine fungus aspergillus terreus butyrolactone compound'.

Background

β -amyloid (A β), phosphorylated tubulin (Tau), accumulation of unbound iron, metabolism of Dopamine (DA) and the like can stimulate the generation of Reactive Oxygen Species (ROS), cause oxidative stress, damage neurons and cause cognitive or motor dysfunction, the continuous activation of oxidative stress states can further promote the aggregation and deposition of A β and phosphorylated Tau, mitochondrial damage and the like, and finally cause the occurrence of neurodegenerative diseases and accelerate the pathological process thereof.

The existing medicines for treating the clinical Alzheimer disease are mainly AChE inhibitors, including physostigmine, galantamine, huperzine A, rivastigmine, amsacrazidine tacrine, piperidine donepezil and the like, and the medicines for treating the Parkinson disease are mainly levodopa, amantadine, selegiline and the like, are high in price, and have certain toxic and side effects except huperzine A. Therefore, the screening of the natural antioxidant-anti-inflammatory-neuroprotective agent with low toxicity, good activity and high yield has important practical significance for the prevention and treatment of neurodegenerative diseases.

Disclosure of Invention

The invention aims to overcome the defects and defects of large toxic and side effects and high price of the existing Alzheimer disease treatment drugs, and provides application of a marine fungus Aspergillus terreus butyrolactone compound butyrolactone-I shown in formula (I) in preparation of a peripheral and neuroinflammation resistant drug. The inventor of the invention finds that butyrolactone-I can clear away after multiple researchesBesides DPPH free radical, the following functions are also provided: (1) ABTS free radicals and OH free radicals can be well eliminated; (2) can inhibit NO and active oxygen in cells, thereby having good antioxidant and anti-inflammatory effects; (3) for MPP ⁺Induced nerve cell damage has reversal effect and neuroprotective activity; therefore, butyrolactone-I has good application prospects in eliminating ABTS free radicals and OH free radicals, preparing anti-peripheral and neuroinflammation medicines and treating neurodegenerative diseases.

The invention also aims to provide application of the marine fungus aspergillus terreus metabolite butyrolactone compound butyrolactone-I shown in the formula (I) in clearing ABTS free radicals and OH free radicals.

Another purpose of the invention is to provide a marine fungus aspergillus terreus metabolite butyrolactone compound butyrolactone-I as MPP shown in formula (I) ⁺The induced nerve cell damage reversal agent is applied to the preparation of the medicament for treating the neurodegenerative diseases.

Another object of the present invention is to provide a process for producing butyrolactone-I as described above.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of butyrolactone compounds butyrolactone-I, which is a metabolic product of aspergillus terreus of marine fungi shown in formula (I), in preparing medicaments for resisting peripheral and neuroinflammation,

generally, butyrolactone compounds have an effect of scavenging DPPH radicals, but the present inventors found that butyrolactone compounds butyrolactone-I have an effect of scavenging ABTS radicals and OH radicals in addition to this effect; in the concentration range of 20-100 mu M, the compound can inhibit the generation of NO and active oxygen in RAW264.7 and BV-2 cells, which is related to the fact that the compound can obviously inhibit the expression of iNOS and COX-2 proteins in the cells, so that the compound has good anti-inflammatory and anti-oxidation effects and has wide application prospects in the aspect of preparing anti-peripheral and neuroinflammation medicines.

Preferably, the use of butyrolactone-I as described above for the inhibition of NO and reactive oxygen species production in peripheral and neuronal cells.

The application of butyrolactone compounds butyrolactone-I, which is a metabolic product of aspergillus terreus, shown in formula (I) in the aspect of eliminating ABTS free radicals and OH free radicals can be widely used for preparing antioxidants.

The marine fungus aspergillus terreus metabolite butyrolactone compound butyrolactone-I shown in the formula (I) is used as MPP ⁺The induced nerve cell damage reversal agent is applied to the preparation of the medicament for treating the neurodegenerative diseases.

The inventors of the present invention have found that butyrolactone-I is responsible for MPP ⁺The induced nerve cell damage has a reversing effect, so the nerve cell damage has a neuroprotective effect and has a potential better therapeutic effect on neurodegenerative diseases.

Preferably, the butyrolactone-I is applied to the preparation of a medicament for treating Alzheimer's disease or Parkinson's disease.

The invention provides a method for preparing butyrolactone-I from a metabolite of marine fungus aspergillus terreus.

A preparation method of a marine fungus aspergillus terreus butyrolactone compound butyrolactone-I shown in formula (I) comprises the following preparation steps:

s1, fermentation: inoculating marine fungus aspergillus terreus C23-3 into a fungus liquid culture medium, standing and fermenting, and collecting mycelium and fermentation liquor;

s2, crude extraction: extracting the fermentation liquor obtained in the step S1 with ethyl acetate, concentrating, ultrasonically extracting mycelia with an organic solvent, concentrating, and mixing the obtained concentrates to obtain a crude extract;

s3, separation and purification: performing gradient chromatography on the crude extract obtained in the step S2 through a reduced pressure silica gel column, detecting through silica gel thin layer chromatography, and continuously performing gel column chromatography, reverse phase silica gel column chromatography and reverse phase preparative liquid chromatography on the elution component containing the target substance for separation and purification to obtain the butyrolactone-I;

wherein the marine fungus aspergillus terreus C23-3 is stored in the Guangdong province microbial strain preservation center in 2018, 1 month and 22 days, the preservation number is GDMCC No.60316, and the preservation address is No. 59 floor 5 of Michelia Tokyo 100, Guangdong province, Guangzhou.

According to the invention, when the metabolic product of marine fungus aspergillus terreus C23-3 is analyzed, the metabolic product contains butyrolactone-I, and the butyrolactone-I in the collected mycelium liquid and fermentation liquid is subjected to crude extraction and purification to obtain the high-purity butyrolactone compound.

The fungal liquid culture medium used in the present invention is a conventional culture medium, and preferably, the formulation of the fungal liquid culture medium in step S1 is: 400-600 mL of fresh potato juice, 15-25 g of sea salt, 15-25 g of sucrose, 3-8 g of peptone, 400-600 mL of distilled water and 6-8 of pH value.

Preferably, the fermentation condition in the step S1 is standing fermentation for 18-25 days.

Preferably, the number of times of ethyl acetate extraction in step S2 is 2-4 times.

Organic solvents commonly used in the art can be used for the mycelium extraction in the step S2 of the present invention, and preferably, the organic solvent in the step S2 is one or more of chloroform, acetone, ethyl acetate, ethanol and methanol.

Preferably, the number of times of the organic solvent ultrasonic extraction in the step S2 is 2-4.

Preferably, the conditions of the reduced pressure silica gel column gradient chromatography in step S3 are: sequentially carrying out gradient elution by using petroleum ether-ethyl acetate and chloroform-methanol, and collecting gradient elution components with the volume ratio of the eluent of 100: 0-0: 100.

Preferably, the conditions for silica gel thin layer chromatography are: developing agent on the silica gel thin layer chromatography plate is petroleum ether-ethyl acetate solution with the volume ratio of 2:1, and R is collected _fA mixture component having a value of 0.2 to 0.8.

Preferably, the mobile phase of the gel column chromatography is methanol, and the flow rate is 0.5-1 mL/min.

Preferably, the reverse phase silica gel column is ODS reverse phase silica gel column chromatography, and the mobile phase is methanol-water with a volume ratio of 2: 3-3: 2.

Preferably, the conditions for the purification by reverse phase preparative liquid chromatography are: the mobile phase is methanol-water with the volume ratio of 1: 3-3: 2, 10-15 g of reverse phase silica gel is filled in a silica gel column, the flow rate is 4-8 mL/min, a main peak with the retention time of 5-20 min is collected, and the obtained collection is butyrolactone-I.

In order to improve the yield of butyrolactone-I in the metabolic product of marine fungus aspergillus terreus C23-3, the invention finds a chemical inducer ZnCl through a plurality of tests ₂。

Preferably, the fungal liquid medium of step S1 further contains an inducer ZnCl ₂The inducer ZnCl ₂The content of (b) is 0.0001-1 mM/mL.

The inducer ZnCl ₂The application of the method for improving the yield of butyrolactone compounds-I of the marine fungus Aspergillus terreus metabolite is also within the protection scope of the invention.

Compared with the prior art, the invention has the following beneficial effects:

the invention finds that butyrolactone-I can remove DPPH free radicals, can well remove ABTS free radicals and OH free radicals, has good antioxidant, anti-inflammatory and neuroprotective activities, and has good application prospect in the aspect of preparing medicines for resisting neurodegenerative diseases.

Meanwhile, the preparation method of butyrolactone-I provided by the invention can successfully prepare the butyrolactone-I, is simple and is easy to realize large-scale production; and the culture condition can be optimized by adding an inducer, so that the yield of the strain is greatly improved.

Drawings

FIG. 1 shows the nuclear magnetic resonance hydrogen spectrum of butyrolactone-I, a butyrolactone compound provided by the present invention.

FIG. 2 is a nuclear magnetic resonance carbon spectrum of butyrolactone-I, a butyrolactone compound provided by the present invention.

FIG. 3 shows the cytotoxicity of butyrolactone I of butyrolactone compounds provided by the present invention and the inhibition of LPS-induced intracellular NO of BV-2(A) and RAW264.7 (B).

FIG. 4 shows the effect of butyrolactone I, a butyrolactone compound provided by the present invention, on the inhibition of LPS-induced intracellular ROS in BV-2(A) and RAW264.7 (B).

FIG. 5 shows the effect of butyrolactone-I, a butyrolactone compound, provided by the present invention, on the expression of RAW264.7 intracellular iNOS (A) and COX-2(B) proteins.

FIG. 6 shows butyrolactone-I vs MPP of butyrolactone compounds provided by the present invention ⁺Induced protection of SH-SY5Y cells.

FIG. 7 is a HPLC comparative analysis spectrum of ethyl acetate extract before and after chemical induction of marine fungus Aspergillus terreus C23-3.

Detailed Description

The present invention will be further described in detail with reference to the following specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.