阅读说明：本技术 一种细胞松弛素类化合物的制备方法和应用 (Preparation method and application of cytochalasin compound ) 是由 杨瑞云 覃玉月 莫土香 徐照隆 李俊 于 2019-12-02 设计创作，主要内容包括：本发明提供一种细胞松弛素类化合物的制备方法和应用,属于药物化学技术领域。本发明中细胞松弛素类化合物是指细胞松弛素C和D,是通过广豆根内生真菌炭角菌GDGJ-368接种在大米培养基中发酵,将发酵液通过硅胶柱层析分离得到,所述炭角菌GDGJ-368的保藏号为CCTCC M2019951。本发明为细胞松弛素C和D的制备增添了一种新的制备方法,对于细胞松弛素类化合物的开发利用,具有积极意义。所得的细胞松弛素C和D对大肠杆菌、枯草芽孢杆菌、乙型溶血性链球菌和巨大芽孢杆菌具有抗菌活性,可以应用于制备抗菌药物。(The invention provides a preparation method and application of cytochalasin compounds, and belongs to the technical field of pharmaceutical chemistry. The cytochalasin compounds refer to cytochalasin C and cytochalasin D, are obtained by inoculating sophora subprostratae endophytic fungi xylaria GDGJ-368 into a rice culture medium for fermentation, and separating fermentation liquor through silica gel column chromatography, wherein the preservation number of the xylaria GDGJ-368 is CCTCC M2019951. The invention adds a new preparation method for the preparation of cytochalasin C and cytochalasin D, and has positive significance for the development and utilization of cytochalasin compounds. The obtained cytochalasin C and cytochalasin D have antibacterial activity on Escherichia coli, Bacillus subtilis, beta hemolytic streptococcus and Bacillus megaterium, and can be used for preparing antibacterial drugs.)

1. A method for preparing cytochalasin compounds is characterized in that: the cytochalasin compounds are cytochalasin C and cytochalasin D and are obtained by fermenting sophora subprostratae endophytic fungi xylaria GDGJ-368, and the preservation number of the xylaria GDGJ-368 is CCTCC M2019951.

2. The method for preparing cytochalasin compounds as claimed in claim 1, wherein: the cytochalasin compounds are obtained by inoculating xylaria GDGJ-368 into a rice culture medium for fermentation and separating fermentation liquor through silica gel column chromatography.

3. The method for preparing cytochalasin compounds as claimed in claim 1, further comprising the steps of:

(1) preparing a seed culture medium, inoculating the xylaria GDGJ-368 to a PDA plate culture medium, and culturing to obtain the seed culture medium;

(2) cutting the seed culture medium obtained in the step (1), inoculating the cut seed culture medium into a sterilized rice culture medium, fermenting and culturing to obtain a fermented product, soaking and extracting the fermented product with an organic solvent, and concentrating under reduced pressure to obtain a crude extract;

(3) performing silica gel column chromatography on the crude extract obtained in the step (2), and performing gradient elution by using petroleum ether-ethyl acetate, wherein the gradient range of the petroleum ether-ethyl acetate is as follows: 10:0, 9:1, 7:3, 5:5, 3:7, 0: 10; mixing fractions obtained by gradient elution at ratio of 7:3 and 5:5, concentrating, performing reversed phase C18 column chromatography and Sephadex LH-20 gel column chromatography, and recrystallizing to obtain cytochalasin C and cytochalasin D.

4. The method for preparing cytochalasin compounds as claimed in claim 3, wherein: the PDA plate culture medium is mainly prepared from the following raw materials: 200g/L of potato, 20g/L of glucose and 15-20 g/L of agar powder; the culture condition is constant temperature culture at 28 deg.C for 4-6 days.

5. The method for preparing cytochalasin compounds as claimed in claim 3, wherein: the fermentation condition in the step (2) is that a broad bean-sized thallus is inoculated in each rice culture medium, and the mixture is subjected to static culture for 25 to 50 days at room temperature; the rice culture medium comprises the following components: 50-90g of rice and 150mL of distilled water.

6. The method for preparing cytochalasin compounds as claimed in claim 3, wherein: and (3) the fermentation product in the step (2) is a solid, the organic solvent is ethyl acetate, the extraction times are 1-5 times, and the extracting solutions in each time are combined and then subjected to reduced pressure concentration to obtain a crude extract.

7. The method for preparing cytochalasin compounds as claimed in claim 3, wherein: in the step (3), the specific steps further comprise mixing fractions obtained by gradient elution at a ratio of 7:3 and 5:5, and concentrating to dryness to obtain a solid B;

mixing the solid B with a silica gel column, performing gradient elution by using petroleum ether-ethyl acetate as an eluent according to the volume ratio of 9:1, 7:3, 5:5, 3:7 and 0:10, collecting the part eluted by the petroleum ether-ethyl acetate according to the volume ratio of 7:3, and concentrating to obtain a solid C; collecting the eluted part with the volume ratio of petroleum ether to ethyl acetate of 5:5, and concentrating to obtain a solid D;

and purifying the solid C by using a reverse phase C18 column, eluting by using methanol-water with the volume ratio of 7:3, collecting eluate, and recrystallizing by using methanol to obtain the cytochalasin C.

And purifying the solid D by using a reverse phase C18 column, eluting by using methanol-water with the volume ratio of 7:3, collecting eluate, concentrating, purifying by using a gel chromatography column Sephadex LH-20, and obtaining cytochalasin D by using a dichloromethane-methanol solution with the volume ratio of 1:1 as an eluent.

8. Use of the cytochalasin-like compounds prepared according to any of claims 1-7, characterised in that: refers to the application of the cytochalasin C and the cytochalasin D in the preparation of antibacterial drugs.

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of pharmaceutical chemistry, in particular to a preparation method and application of cytochalasin compounds.

[ background of the invention ]

Cytochalasin, also called phytoalexin, is a hybrid of amino acid/polyketone, and is a natural active compound with unique and novel structure and very good activities of resisting bacteria, inhibiting HIV, viruses, resisting tumors, resisting phytotoxicity and the like. The cytochalasin compounds can be separated from the metabolites of fungi, but the development and application of the compounds are influenced because the content is lower and the yield is lower. Cytochalasin C and cytochalasin D belong to cytochalasin compounds, and no suitable industrial production method for obtaining the compounds is available at present. The fungus GDGJ-368 is a fungus belonging to Xylaria sp, and is isolated from the leaves of Sophora subprostrata (Sophora subprostrata) belonging to Sophora. A large amount of cytochalasin C and D are obtained from metabolites of GDGJ-368 fungi through fungal fermentation, and the cytochalasin C and D have positive significance for development and utilization of cytochalasin compounds.

[ summary of the invention ]

The invention aims to: in view of the problems, the invention provides a preparation method and application of cytochalasin compounds, which are obtained by industrial production for the first time and have positive significance for development and utilization of cytochalasin compounds.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a preparation method of cytochalasin compounds is characterized in that cytochalasin C and cytochalasin D are obtained through fermentation of sophora subprostrata endophytic fungi xylaria GDGJ-368, the xylaria GDGJ-368 is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university, the preservation number is CCTCC M2019951, and the preservation date is 11 months and 19 days in 2019. The structural formulas of cytochalasin C and cytochalasin D are respectively shown in the following formulas 1 and 2:

in the present invention, the cytochalasin-based compound is preferably obtained by inoculating xylaria GDGJ-368 into rice culture medium, fermenting, and separating the fermentation liquid by silica gel column chromatography.

In the present invention, preferably, the method for preparing the cytochalasin compound specifically comprises the following steps:

(1) preparing a seed culture medium, inoculating the xylaria GDGJ-368 to a PDA plate culture medium, and culturing to obtain the seed culture medium;

(2) cutting the seed culture medium obtained in the step (1), inoculating the cut seed culture medium into a sterilized rice culture medium, fermenting and culturing to obtain a fermented product, soaking and extracting the fermented product with an organic solvent, and concentrating under reduced pressure to obtain a crude extract;

(3) performing silica gel column chromatography on the crude extract obtained in the step (2), and performing gradient elution by using petroleum ether-ethyl acetate, wherein the gradient range of the petroleum ether-ethyl acetate is as follows: 10:0, 9:1, 7:3, 5:5, 3:7, 0: 10; mixing fractions obtained by gradient elution at ratio of 7:3 and 5:5, concentrating, performing reversed phase C18 column chromatography and Sephadex LH-20 gel column chromatography, and recrystallizing to obtain cytochalasin C and cytochalasin D.

In the above steps, preferably, the PDA plate medium is mainly made of the following raw materials: 200g/L of potato, 20g/L of glucose and 15-20 g/L of agar powder; the culture condition is constant temperature culture at 28 deg.C for 4-6 days.

Preferably, the fermentation conditions in the step (2) are that a broad bean-sized thallus is inoculated in each rice culture medium, and the mixture is subjected to static culture for 25 to 50 days at room temperature; the rice culture medium comprises the following components: 50-90g of rice and 150mL of distilled water.

Preferably, the fermentation product in step (2) is a solid, the organic solvent is ethyl acetate, the extraction times are 1-5 times, and the extraction solutions in each time are combined and then concentrated under reduced pressure to obtain a crude extract.

Preferably, in the step (3), the specific steps further include combining fractions obtained by gradient elution at 7:3 and 5:5, and concentrating to dryness to obtain a solid B; mixing the solid B with a silica gel column, performing gradient elution by using petroleum ether-ethyl acetate as an eluent according to the volume ratio of 9:1, 7:3, 5:5, 3:7 and 0:10, collecting the part eluted by the petroleum ether-ethyl acetate according to the volume ratio of 7:3, and concentrating to obtain a solid C; collecting the eluted part with the volume ratio of petroleum ether to ethyl acetate of 5:5, and concentrating to obtain a solid D;

and purifying the solid C by using a reverse phase C18 column, eluting by using methanol-water with the volume ratio of 7:3, collecting eluate, and recrystallizing by using methanol to obtain the cytochalasin C.

And purifying the solid D by using a reverse phase C18 column, eluting by using methanol-water with the volume ratio of 7:3, collecting eluate, concentrating, purifying by using a gel chromatography column Sephadex LH-20, and obtaining cytochalasin D by using a dichloromethane-methanol solution with the volume ratio of 1:1 as an eluent.

Experiments prove that the cytochalasin C and the cytochalasin D prepared by the invention have moderate antibacterial activity on escherichia coli, bacillus subtilis, beta hemolytic streptococcus and bacillus megaterium, and can be applied to preparation of antibacterial drugs.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. the cytochalasin C and the cytochalasin D are produced by fermenting the sophora plant endophytic fungi Xylaria sp GDGJ-368(Xylaria sp.) strain for the first time, a new preparation method is added for the cytochalasin C and D preparation, and the method has positive significance for development and utilization of cytochalasin compounds.

2. The content of cytochalasin C in the fermented product obtained under the fermentation condition is more than 8%, the content of cytochalasin D is more than 1%, and the purity of cytochalasin monomer can reach more than 98%. The preparation method is simple and convenient, and the prepared cytochalasin C and D are high in content and suitable for industrial mass production.

3. The cytochalasin C and the cytochalasin D prepared by the invention have antibacterial activity on escherichia coli, bacillus subtilis, beta hemolytic streptococcus and bacillus megaterium, and can be applied to preparation of antibacterial drugs.

[ detailed description ] embodiments

In order that the invention may be more clearly expressed, the invention will now be further described by way of specific examples.

The invention relates to a subprostrate sophora endophytic fungi strain GDGJ-368(Xylaria sp.) which is collected by an inventor from the leaf of subprostrate sophora plant of Jingxi city of Guangxi Baiseo, separated and purified, and identified as the strain by morphology and molecular biology to be a subprostrate sophora; the strain is preserved in China Center for Type Culture Collection (CCTCC) of Wuhan university, Wuhan, China, with the preservation number of CCTCC M2019951 and the preservation date of 2019, 11 months and 19 days.