CN1542136A - Preparation method of ascosporine B and its application in the preparation of antitumor and antifungal drugs - Google Patents

Abstract

The invention relates to an ascosporine B compound derived from the endophytic fungus of the mangrove plant—Aculetosporum HTF ₃ , a preparation method thereof and its application in the preparation of antitumor and antifungal drugs. The method is the seed culture of endophytic fungi, the fermentation culture of endophytic fungi, the above-mentioned fermentation culture liquid is filtered to remove the fermentation liquid to obtain the thallus, the thallus is dried and soaked repeatedly with ethyl acetate, combined with ethyl acetate, decompressed Concentrate, chromatographically separate, gradient elution, collect the eluate when the ratio of the two is 1/1, and concentrate under reduced pressure to obtain the compound. The ascosporin B compound can be used to prepare antitumor drugs and antifungal drugs. The yield is high, reaching 9 mg/g dry weight of bacteria, and can be used in industrial production.

Description

Preparation method of ascosporine B and its application in the preparation of antitumor and antifungal drugs

(1) Technical field

The invention relates to a preparation method of an ascosporine B compound derived from a mangrove plant endophytic fungus-Aculeella sp.HTF ₃ (Dothiorella sp.HTF ₃ ) and its application in the preparation of antitumor and antifungal drugs .

(2) Background technology

Mangrove is a woody plant community that grows on mud flats in harbor and estuary areas. As a unique sea and land marginal ecosystem, it plays a special role in the natural ecological balance. Mangrove plants are not only important economic plants, but also commonly used medicinal plants in the folk. For example, the bark of the mangrove plant Acanthus ilicifolius can be used to treat hematuria; It can stop bleeding, lax and treat malignant sores. The oil extracted from the seeds can relieve itching and treat ringworm, and it can also treat frostbite and so on.

There are abundant endophytic fungi in mangrove plants, and it has been reported that the medicinal components of mangrove plants are all produced by their endophytic fungi, but there are not many documents (1, Brady, S., Wagenaar, M.W., Singh, M., et al. The cytosporonesisolated from an endophytic fungus. Org. Lett. 2000, 2(25): 4043-4046; 2, Bandaranayake, W.M.. Traditional and medicinal uses of mangroves. Mangroves and Salt Marshes, 1998, 2: 133-148; 3. Bandaranayake, W.M.. Bioactivities, bioactive compounds and chemical constituents of mangrove plants. Wetland Ecology and Management, 2002, 10: 421-452; 4. Lin Y., Wu X., Deng z., et al. The metabolites of the mangrove fungus Verruculina enalia No.2606 from a salt lake in the Bahamas. Phytochemistry, 2002, 59: 469-471) reports have also proved that endophytic fungi of mangrove plants can indeed produce new structures and strong physiological active compound. But at present, the work in this field at home and abroad has just begun, so its research has high theoretical value and practical application value.

(3) Contents of the invention

The object of the present invention is to provide a method for separating and extracting compounds derived from mangrove plant endophytic fungi (cytosporone B) and its application in the preparation of antitumor drugs and antifungal drugs.

The mangrove plant endophytic fungus used in the present invention—Dothiorella sp.HTF 3 (Dothiorella sp.HTF ₃ ) has been preserved in the China Typical Culture Collection Center (CCTCC, within the campus of Wuhan University, China), and the preservation number is CCTCC NO: M203067, and the preservation date is for August 29, 2003.

The structural formula of said ascosporine B (cytosporone B) compound of the present invention is:

  (3,5-Dihydroxy-2-octanonyl)-ethyl phenylacetate

The preparation method of said ascosporine B compound is:

1) Seed culture of endophytic fungi (Dothiorella sp.HTF ₃ ):

The medium is in g. Peel and chop potatoes for 20 g, boil them in water, add glucose 1-3, agar 1.5-2.0 to the filtered filtrate, sea water to 100ml, sterilize, make a test tube slope, pick Take the strains and insert them into the slant, and cultivate them at room temperature for 5-15 days;

2) Fermentation culture of endophytic fungi:

Fermentation medium in g: peel and chop potatoes for 20 g, boil them in water, add glucose 1-3 to the filtered filtrate, adjust the volume of seawater to 100ml, sterilize, and pick the cultured strains on the slant to ferment Culture medium, cultivated at room temperature for 5 to 15 days;

3) obtaining the thallus after filtering the above-mentioned fermentation culture liquid to remove the fermentation liquid;

4) Bacteria are dried, soaked repeatedly with ethyl acetate, combined with ethyl acetate, concentrated under reduced pressure, and the resulting paste is separated by silica gel column chromatography, petroleum ether/ethyl acetate=100/0 to 0/100 Gradient elution, collect the eluate when the ratio of the two is 1/1, and concentrate under reduced pressure to obtain the compound with a content of 9 mg/g dry weight of bacteria.

The experimental data of this compound of gained is:

Colorless oil; EMS: m/z (rel.int.): 323.3[M+H] ⁺ (100), 345.3[M+Na] ⁺ (11.4), 361.2[M+K] ⁺ (4.2); See Table 1 for ¹ H NMR and ¹³ C NMR (400 MHz, CDCl ₃ , ppm).

Said ascosporin B compound of the present invention can be used for preparing antitumor drug, and its antitumor activity detection experiment is as follows:

1) Tumor cell lines

Human B lymphoma Raji cells, human oral dermoid carcinoma KB cells, human osteosarcoma MG-63 cells, human lung cancer A549 cells, human cervical cancer Hela cells, etc.

Table 1. NMR data of ascosporin B position ¹ H-NMR position ¹³ C-NMR 2 3.70)s) 1 171.0 4 6.25(s) 2 41.8 6 6.25(s) 3 136.0 9 4 112.0 10 2.75(t) 5 163.1 11 1.6 6 103.2 12 1.22 7 161.0 13 1.19 8 116.0 14 1.20 9 207 15 1.20 10 43.7 16 0.8 11 25.0 17 4.2(q,7) 12 29.4 18 1.20(t,7) 13 29.2 OH-5 14 31.8 OH-7 15 22.8 16 14.2 17 61.0 18 14.1

2) Material

a MTT (thiazole blue):

Dissolve MTT (Thiazoyl blue) in 0.01mol/L phosphate buffered saline (PBS) to a final concentration of 5mg/ml, filter and sterilize with a 0.22μm microporous membrane, and store in a dark place at 4°C after aliquoting;

b SDS lysate:

100g sodium dodecyl sulfonate (SDS), 10ml 1N HCL, heated to dissolve, distilled water to 1000ml.

c cell culture medium (full culture):

One bag of 10g dry powder RMPI 1640 (Gibco Co.Ltd.) cell culture medium was dissolved in 1L double-distilled water; add 2gNaHCO ₃ and stir well to dissolve, then seal, and place at 4°C overnight to remove impurities by natural precipitation; the next day, add 10%- 15% calf serum that has been inactivated (56°C, 30min) and 1% polyclonal antibody stock solution; after mixing, use a filter membrane with a pore size of 0.22 μm to filter and sterilize.

3) Configuration of compound cytosporone B

Take a certain amount of ascosporin B, dissolve it with methanol and adjust the concentration to 5 mg/ml, filter and sterilize through a filter membrane with a pore size of 0.22 μm, and store it at 4°C for future use.

4) Culture of tumor cells

a Cell activation:

Take a clean beaker, fill it with clean warm water, and adjust the water temperature to 37-40°C; take the cryopreservation tube out of liquid nitrogen and put it into warm water to thaw quickly, and put the frozen cells into a culture bottle pre-filled with cell culture medium. Cultivate under the conditions of 37°C, 5% CO ₂ , and 100% humidity, observe the growth of the cells, replace the culture medium in time, and divide the bottles.

b cell count:

Select the cells in the logarithmic generation phase, digest them with trypsin, transfer them to a centrifuge tube, add whole culture to 10ml, take a drop into the groove on the side of the counting plate, and count under an inverted microscope. Adjust the cell number to 1×10 ⁵ /ml.

c activity detection:

① The 96-well plate was irradiated with ultraviolet light in the ultra-clean workbench for 1 hour;

② Add 80 μl of cell suspension to each well, and incubate for 24 hours at 37°C, 5% CO ₂ , and 100% humidity;

③ Add 20 μl of solution diluted with a series of concentrations in the whole culture gradient, and continue to cultivate for 48 hours;

④ Add 10 μl of MTT solution to each well, shake gently to dissolve the particles, and place at 37°C for 3 hours;

⑤ Take out the culture plate, add 100 μl of 10% SDS solution to each well, and dissolve overnight at 37°C;

⑥ Measure the absorbance of each hole with an enzyme-linked reaction instrument at 570nm (the reference wavelength is 655nm), and calculate the inhibition rate according to the following formula:

Inhibition rate = (OD value of the control group - OD value of the experimental group) / OD value of the control group × 100%

⑦ Take the inhibition rate as the ordinate and the logarithm of the concentration of the tested sample as the abscissa, and find the concentration at which the inhibition rate is 50%, which is IC ₅₀ .

d Test results:

The IC ₅₀ of Cytosporone B on human B lymphoma Raji cells, human oral dermoid carcinoma KB cells, human osteosarcoma MG-63 cells, human lung cancer A549 cells, and human cervical cancer Hela cells are 62.5ng/ml and 4μg/ml, respectively , 1 μg/ml, 4 μg/ml and 1 μg/ml.

Said ascosporin B compound of the present invention can be used for preparing antifungal drug, and its antifungal activity detection experiment is as follows:

1) Fungal indicator strains:

Candida albicans, Aspergillus niger, Trichoderma, Fusarium, Alternaria, white bread mold, Cladosporium

2) Configuration of compound cytosporone B

Take a certain amount of cytosporone B, dissolve it with methanol and adjust the concentration to 1 mg/ml, filter and sterilize through a filter membrane with a pore size of 0.22 μm, and store it at 4°C for later use.

3) Activity detection: (determination of minimum inhibitory concentration MIC)

a Wash the indicator bacteria grown on the PDA (potato-dextrose-agar) slant medium with sterile water to make a bacterial suspension with a spore concentration of ¹⁰⁷ /ml, and store it at 4°C for later use;

b Cytosporone B sample solution was diluted with methanol into a series of concentration gradients according to the two-fold dilution method, and 20 μl was added to the 96-well plate that had been irradiated for 30 minutes under the ultraviolet lamp of the ultra-clean workbench. Dry methanol with blower blower of ultra-clean workbench;

c Dilute the fungal suspension with PD (potato-glucose) medium to 10 ⁴ bacteria/μl, take 100 μl and add it to the above 96-well plate, incubate at 25°C for 48 hours, and observe with the naked eye, the minimum dilution concentration for no fungal formation is for the MIC.

d Experimental results:

The MICs of cytosporone B against Candida albicans, Aspergillus niger, Trichoderma, Fusarium, Alternaria, white bread mold and Cladosporium are: 0.156mg/ml, 0.125mg/ml, 0.0625mg/ml , 0.0625mg/ml, 0.07mg/ml, 0.125mg/ml and 1mg/ml.

In summary, the present invention provides a method for preparing a compound derived from mangrove endophytic fungi, which has a high yield, reaching 9 mg/g dry weight of bacteria, and can be used in industrial production; the compound is effective on a variety of human tumor cells All exhibit good inhibitory effects, and can be used for preparing anti-tumor drugs; the compound also has good inhibitory effects on human pathogenic fungi and plant pathogenic fungi, and can be used for preparing such antifungal drugs.

(4) specific implementation

Example 1

The following examples show the preparation of said ascosporin B compounds.

1) Seed culture of endophytic fungi:

Peel and chop 20g of potatoes, boil them in water for 30min, add 1g of glucose, 1.6g of agar, and 50% seawater to 100ml in the filtered filtrate, sterilize, make a test tube slope, and pick the bacteria into the slope , cultivated at 25°C for 10 days;

2) Fermentation culture of endophytic fungi:

Peel and chop 20g of potatoes, boil in water for 30min, add 1.5g of glucose to the filtered filtrate, dilute to 100ml with 50% seawater, sterilize, pick the cultured strains on the slope into the fermentation medium, and keep at 26°C Under culture for 8 days;

3) obtaining the thallus after filtering the above-mentioned fermentation culture liquid to remove the fermentation liquid;

4) Bacteria are dried, soaked repeatedly with ethyl acetate, combined with ethyl acetate, concentrated under reduced pressure, and the resulting paste is separated by silica gel column chromatography, petroleum ether/ethyl acetate=100/0 to 0/100 Gradient elution, collect the eluate when the ratio of the two is 1/1, and concentrate under reduced pressure to obtain the compound with a content of 9 mg/g dry weight of bacteria.

Example 2

Peel and chop 20g of potatoes, boil them in water for 30min, add 1.5g of glucose, 1.8g of agar, and 50% seawater to 100ml to sterilize the filtered filtrate, make a test tube slope, and pick the bacteria into the slope , cultivated at 30°C for 8 days; then peeled and chopped 20g of potatoes, boiled them in water for 30min, added 2.5g of glucose to the filtered filtrate, adjusted the volume to 100ml with 50% seawater, sterilized, and cultured on the slant The strains were picked into the fermentation medium and cultured at 20°C for 15 days; the above-mentioned fermentation broth was filtered to remove the fermentation broth to obtain bacteria; after drying, the bacteria were repeatedly soaked in ethyl acetate, combined with ethyl acetate, and concentrated under reduced pressure to obtain The paste was separated by silica gel column chromatography, petroleum ether/ethyl acetate=100/0 to 0/100 for gradient elution, and the eluate when the ratio of the two was 1/1 was collected and concentrated under reduced pressure to obtain the compound, the content of which is 9 mg/g dry weight of bacteria.

Example 3

Peel and chop 20g of potatoes, boil them in water for 30min, add 2.0g of glucose, 1.5g of agar, and 50% seawater to 100ml in the filtered filtrate, sterilize it, make it into a test tube slope, and pick the bacteria into it Slope, cultivated at 28°C for 15 days; then peeled and chopped 20g of potatoes, boiled in water for 30min, added 2g of glucose to the filtrate after filtration, adjusted to 100ml with 50% sea water, sterilized, and cultured on the slope The strains were picked into the fermentation medium and cultured at 25°C for 10 days; the above-mentioned fermentation broth was filtered to remove the fermentation broth to obtain bacteria; after drying, the bacteria were repeatedly soaked in ethyl acetate, combined with ethyl acetate, and concentrated under reduced pressure to obtain The paste was separated by silica gel column chromatography, petroleum ether/ethyl acetate=100/0 to 0/100 for gradient elution, and the eluate when the ratio of the two was 1/1 was collected and concentrated under reduced pressure to obtain the compound, the content of which is 9 mg/g dry weight of bacteria.

Example 4

Peel and chop 20g of potatoes, boil them in water for 30min, add 2.5g of glucose, 2.0g of agar, and 50% seawater to 100ml in the filtered filtrate, sterilize, make a test tube slope, pick the strains and insert them Slope, cultured at 20°C for 12 days; then peeled and chopped 20g of potatoes, boiled in water for 30min, added 1g of glucose to the filtrate after filtration, adjusted the volume to 100ml with 50% sea water, sterilized, and cultured on the slope The strains were picked into the fermentation medium and cultured at 30°C for 12 days: the above-mentioned fermentation culture liquid was filtered to remove the fermentation liquid to obtain the bacteria; the bacteria were dried and soaked repeatedly in ethyl acetate, combined with ethyl acetate, concentrated under reduced pressure, and The paste was separated by silica gel column chromatography, petroleum ether/ethyl acetate=100/0 to 0/100 for gradient elution, and the eluate when the ratio of the two was 1/1 was collected and concentrated under reduced pressure to obtain the compound, the content of which is 9 mg/g dry weight of bacteria.

Example 5

Peel and chop 20g of potatoes, boil them in water for 30min, add 3.0g of glucose, 1.7g of agar, and 50% seawater to 100ml in the filtered filtrate, sterilize it, make it into a test tube slope, and pick the strains into it Slope, cultured at 23°C for 5 days; then peeled and chopped 20g of potatoes, boiled in water for 30min, added 3.0g of glucose to the filtrate after filtration, adjusted to 100ml with 50% sea water, sterilized, and cultured on the slope The strains were picked into the fermentation medium and cultured at 28°C for 5 days; the above-mentioned fermentation broth was filtered to remove the fermentation broth to obtain the cells; after drying, the cells were repeatedly soaked in ethyl acetate, combined with ethyl acetate, concentrated under reduced pressure, The obtained paste was separated by silica gel column chromatography, and petroleum ether/ethyl acetate = 100/0 to 0/100 for gradient elution, and the eluate when the ratio of the two was 1/1 was collected and concentrated under reduced pressure to obtain The content of the compound is 9mg/g dry weight of bacteria.

Claims (4)

1. Ascosporin B compound, the preservation number of the used mangrove plant endophytic fungus - Anoculosum HTF3 is CCTCC NO: M203067, which is characterized in that its structural formula is:   (3,5-Dihydroxy-2-octanonyl)-ethyl phenylacetate 2, the preparation method of ascosporine B compound, it is characterized in that its steps are: 1) Seed culture of endophytic fungi: The medium is in g. Peel and chop potatoes for 20 g, boil them in water, add glucose 1-3, agar 1.5-2.0 to the filtered filtrate, sea water to 100ml, sterilize, make a test tube slope, pick Take the strains and insert them into the slant, and cultivate them at room temperature for 5-15 days; 2) Fermentation culture of endophytic fungi: Fermentation medium in g: peel and chop potatoes for 20 g, boil them in water, add glucose 1-3 to the filtered filtrate, adjust the volume of seawater to 100ml, sterilize, and pick the cultured strains on the slant to ferment Culture medium, cultivated at room temperature for 5 to 15 days; 3) obtaining the thallus after filtering the above-mentioned fermentation culture liquid to remove the fermentation liquid; 4) Bacteria are dried, soaked repeatedly with ethyl acetate, combined with ethyl acetate, concentrated under reduced pressure, and the resulting paste is separated by silica gel column chromatography, petroleum ether/ethyl acetate=100/0 to 0/100 Gradient elution, collect the eluate when the ratio of the two is 1/1, and concentrate under reduced pressure to obtain the compound with a content of 9 mg/g dry weight of bacteria. 3. The ascosporin B compound can be used to prepare antitumor drugs. 4. The ascosporin B compound can be used to prepare antifungal drugs.