A kind of ergotane type steroid compound and its preparation method and use

Abstract

The invention discloses an ergotane-type steroid compound, a preparation method and use thereof. The characteristics are that the structural formula of the steroid compound is shown as I. The preparation method comprises the steps of subjecting Aspergillus fusus with a preservation number of CCTCC NO: M2014086 to microbial fermentation to obtain a fermentation product of the ergotane-type steroid compound, then soaking the fermentation product in ethyl acetate to extract a crude extract, and separating and purifying the crude extract through vacuum silica gel column chromatography, medium pressure column chromatography and reverse phase semi-preparative high performance liquid chromatography. The advantage is that the ergotane-type steroid compound has an anti-Staphylococcus aureus effect and can be used to develop drugs for preventing and treating diseases and infections caused by Staphylococcus aureus.

Description

Technical Field

[0001] The present invention relates to an ergotane-type steroid compound, in particular to an ergotane-type steroid compound extracted from marine fungi, and a preparation method and application thereof. Background Art

[0002] Sterols are among the most important small molecules in biology. They are components of the plasma membrane of eukaryotic organisms and play a variety of roles in cell membrane structure and signal transduction. Their derivatives include dozens of physiologically active endogenous substances, such as steroid hormones in vertebrates, ecdysteroids in insects, and brassinosteroids in plants. These substances are involved in regulating cell proliferation, tissue differentiation, and signal transduction. Steroidal drugs, the second largest class of pharmaceuticals, are currently among the best-selling pharmaceutical products on the market and are of great significance for improving quality of life and preventing and treating diseases. Consequently, sterols and their derivatives have attracted continued attention from chemists and biologists.

[0003] The diversity of fungal metabolism often produces natural products with pharmacological activity. The ocean, as the largest ecosystem on Earth, is rich in biological resources. Since the discovery of cephalosporin C with antibacterial activity in marine fungi in 1945, more and more new active natural products have been discovered, indicating that marine fungi have unlimited potential for research and development.

[0004] In the discovery of marine natural products, deduplication can significantly accelerate the discovery of new natural products. Molecular networking, a technique for analyzing MS / MS data based on chemical similarity, has been increasingly used in natural product research since its development, providing guidance and improving the efficiency of discovering new active natural products. The inventors discovered a novel ergotane-type steroid natural product based on the chemical composition of an ethyl acetate extract of a strain of Aspergillus marineus (deposited with the China Center for Type Culture Collection, CCTCC NO: M2014086) fermented with rice. The product was subsequently evaluated for its antibacterial activity. Currently, the chemical structure and antibacterial activity of this compound have not been reported, and therefore, no related drugs are available on the market. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an ergotane-type steroid compound having an inhibitory effect on Staphylococcus aureus, a preparation method thereof and use thereof.

[0006] The technical solution adopted by the present invention to solve the above technical problems is:

[0007] 1. An ergotane-type steroid compound, the structural formula of which is shown in (I);

[0008]

[0009] (I).

[0010] 2. The preparation method of the above-mentioned ergotane-type steroid compound comprises the following steps:

[0011] (1) Fermentation production

[0012] The Aspergillus niger ( Aspergillus ustus) Streak the plate on PDA solid medium and culture it in a 28°C incubator for 7 days. Then, pick a single colony and inoculate it into PDB liquid medium and culture it at 28°C and 180 rpm / min. After three days of culture, inoculate the seed liquid into rice medium at a volume ratio of 10%. Static culture is carried out at 28°C for 30 days to obtain the fermentation product.

[0013] (2) Extraction

[0014] Adding an equal volume of ethyl acetate to the fermented product obtained in step (1) and repeatedly soaking the product three times, then adding an equal volume of a mixture of dichloromethane and methanol to the fermented product and repeatedly soaking the product twice, then distilling the ethyl acetate, dichloromethane and methanol under reduced pressure and concentrating to obtain a crude extract;

[0015] (3) Isolation and preparation of compounds

[0016] The crude extract obtained in step (2) was dissolved in a mixture of equal volumes of dichloromethane and ethyl acetate, and 1.5 times of 200-300 mesh silica gel powder was added to the sample for VLC vacuum column chromatography. Gradient elution was performed using a petroleum ether / ethyl acetate solution with a volume ratio of (100:0) to (0:100) as the eluent. Similar fractions were sequentially combined to obtain a total of 4 components (Fr.A–Fr.D). The collected third component was subjected to reverse medium pressure column chromatography. Gradient elution was performed using a methanol aqueous solution with a volume ratio of 50-100% methanol as the eluent. Similar fractions were sequentially combined to obtain a total of 6 components (Fr.C.1–Fr.C.6). The collected fourth component was evaporated to dryness and then subjected to semi-preparative reverse phase high performance liquid chromatography separation. Acetonitrile / water solution with a volume ratio of 60:40 was used as the eluent to separate and purify the ergotane-type steroid compound, whose structure is shown in (I):

[0017]

[0018] (I).

[0019] The rice culture medium described in step (1) is prepared as follows: 80 g of rice is added to 120 mL of seawater with a salinity of 20%, and then sterilized at 121°C for 15 minutes.

[0020] The volume ratios of the petroleum ether / ethyl acetate solution in the gradient elution in step (3) are 100:0, 95:5, 90:10, 85:15, 80:20, 70:20, 50:50, and 0:100, respectively.

[0021] The gradient eluent in the reverse phase medium pressure column chromatography described in step (3) is methanol and water, with methanol ranging from 50% to 100%, and the elution time is 150 min.

[0022] Step (3) The mass-to-charge ratio was found to be 473.3280 [M + H] by mass spectrometry tracking. + Node.

[0023] The flow rate for the compound separation and preparation by semi-preparative reversed-phase high performance liquid chromatography described in step (3) is 2.0 mL / min.

[0024] 3. Use of the above-mentioned ergotane-type steroid compound in the preparation of a drug for inhibiting Staphylococcus aureus.

[0025] Compared with the prior art, the advantages of the present invention are: an ergotane-type steroid compound and a preparation method and use thereof are obtained by microbial fermentation culture to obtain a fermentation product of sterols, then the fermentation product is soaked and extracted with ethyl acetate, dichloromethane and methanol to obtain a crude extract, and then the crude extract is separated and purified by normal phase silica gel column chromatography, reverse medium pressure column chromatography and reverse phase semi-preparative high performance liquid chromatography to obtain the compound, which has good activity against Staphylococcus aureus and can be used for applications such as drug development for inhibiting related diseases caused by Staphylococcus aureus.

[0026] Aspergillus niger ( Aspergillus ustus ), the bacterium is strain DJ003, with the deposit number CCTCC NO: M2014086, and was deposited in the China Center for Type Culture Collection on March 14, 2014, at Wuhan University, Wuhan, China. DETAILED DESCRIPTION

[0027] The present invention is described in further detail below with reference to the examples.

[0028] Example 1

[0029] The structural formula of an ergotane-type steroid compound is shown in (I):

[0030] (I).

[0031] Example 2

[0032] The method for separating and preparing the ergotane-type steroid compound represented by structural formula (I) in Example 1 specifically comprises the following steps:

[0033] (1) Fermentation production

[0034] The Aspergillus niger ( Aspergillus ustus) A plate of PDA solid medium was streaked and cultured in a 28°C incubator for 7 days. A single colony was then picked and inoculated into PDB liquid medium and cultured at 28°C and 180 rpm / min. After three days of culture, the seed liquid was inoculated into a rice medium at a volume ratio of 10%, and static culture was carried out at 28°C for 30 days to obtain a fermentation product. The rice medium was prepared as follows: 80 g of rice was added to 120 mL of seawater with a salinity of 20%, and then sterilized at 121°C for 15 minutes.

[0035] (2) Extraction

[0036] Adding an equal volume of ethyl acetate to the fermented product obtained in step (1), repeatedly soaking the product three times, then adding an equal volume of a mixture of dichloromethane and methanol to the fermented product, repeatedly soaking the product twice, then distilling the ethyl acetate, dichloromethane and methanol under reduced pressure, and concentrating to obtain a crude extract;

[0037] (3) Isolation and preparation of compounds

[0038] The crude extract was dissolved in methanol and prepared at 0.1 mg / mL for LC-MS analysis. A 2 μL injection volume was used, and eluted with H₂O and CH₃CN (both containing 0.1% formic acid) using the following gradient: 10% CH₃CN / H₂O to 100% CH₃CN for 10 minutes, 100% CH₃CN for 5 minutes, then 100% CH₃CN to 10% CH₃CN / H₂O over 5 seconds, and finally equilibrated with 10% CH₃CN / H₂O for 3 minutes. The flow rate was 0.4 mL / min. Spectra were recorded in positive ion ESI mode over the m / z range of 100–1800, scanning at an energy of 40 eV. The target molecule was isolated and purified using the following purification steps:

[0039] First, the crude extract obtained in step (2) was dissolved in a mixture of equal volumes of dichloromethane and ethyl acetate, and then 1.5 times of 200-300 mesh silica gel powder was added to the sample for VLC vacuum column chromatography. A petroleum ether / ethyl acetate solution with a volume ratio of (100:0)-(0:100) (the volume ratios were 100:0, 95:5, 90:10, 85:15, 80:20, 70:20, 50:50, 0:100) was used as the eluent for gradient elution, and the extracts were combined in sequence. Similar fractions were collected to obtain four components (Fr.A–Fr.D). The third component collected was subjected to reverse medium pressure column chromatography using a methanol-water solution with a volume ratio of 50–100% methanol as the eluent for gradient elution. Similar fractions were sequentially combined to obtain six components (Fr.C.1–Fr.C.6). The fourth component collected was evaporated to dryness and then subjected to semi-preparative reverse phase high performance liquid chromatography using an acetonitrile / water solution with a volume ratio of 60:40 as the eluent at a flow rate of 2 mL / min and a retention time of 23 min to obtain an ergotane-type steroid compound, the structure of which is shown in (I):

[0040] (I).

[0041] Compound I of the present invention is a white powder. High-resolution mass spectrometry (HR-ESI-MS) in positive ion mode gives its quasi-molecular ion peak m / z 473.3280 [M + H] + . Combined 13 C NMR spectroscopy confirmed that its molecular formula is C 30 H 48 O4, the compound 1 H and 13 C NMR spectrum data are shown in Table 1:

[0042] Table 1. 1D and 2D NMR data of compound Ⅰ (CD3Cl3)

[0043]

[0044] Note 1: The signal attribution in this table is based on DEPT, 1 H- 1 H COSY, HSQC, HMBC and NOESY spectrum analysis results; 2: s—singlet, d—doublet, t—triplet, m—multiplet; 3: 1 H was obtained at 600 MHz NMR; 13 C was obtained at 150 MHz NMR.

[0045] Example 3

[0046] Activity and application of the ergotane-type steroid compound described in Example 1

[0047] (1) Experimental samples

[0048] Preparation of the test sample solution: The test sample was the pure Compound I isolated and purified in Example 1. An appropriate amount of sample was accurately weighed and prepared with DMSO to the desired concentration for antibacterial activity testing. The indicator bacteria used in this experiment was Staphylococcus aureus (CMCC(B)26003).

[0049] (2) Experimental methods

[0050] 96-well plate antibacterial test method: In a 96-well plate, MH broth with a final volume of 100 μL / well was used as the basic culture medium, in which the concentration of the compound was diluted to a concentration of 256 to 1 μg / mL by two-fold broth dilution method, and 5×10 6 Add bacterial culture to the plate at a turbidity level of CFU / mL. Incubate the plate at 37°C for 18 hours. Then observe the plate. Add 0.004% MTT to the plate and incubate at 37°C for four hours. Visually observe the color change in each well. The compound concentration corresponding to the last well without color change is determined as the minimum inhibitory concentration (MIC) of the compound. Gentamycin sulfate is also used as a positive control.

[0051] (3) Experimental results

[0052] In the 96-well plate antibacterial test, the MIC of compound I against Staphylococcus aureus was determined to be 64 μg / ml. This indicates that compound I has good antibacterial activity against Staphylococcus aureus.

[0053] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. An ergotane-type steroid compound, characterized in that The structural formula of the ergotane-type steroid compound is shown as (I); （I）。 2. A method for preparing the ergotane-type steroid compound according to claim 1, characterized in that The steps include: (1) Fermentation production The Aspergillus niger ( Aspergillus ustus) Streak the plate on PDA solid medium and culture it in a 28°C incubator for 7 days. Then, pick a single colony and inoculate it into PDB liquid medium and culture it at 28°C and 180 rpm / min. After three days of culture, inoculate the seed liquid into rice medium at a volume ratio of 10%. Static culture is carried out at 28°C for 30 days to obtain the fermentation product. (2) Extraction Adding an equal volume of ethyl acetate to the fermented product obtained in step (1) and repeatedly soaking the product three times, then adding an equal volume of a mixture of dichloromethane and methanol to the fermented product and repeatedly soaking the product twice, then distilling the ethyl acetate, dichloromethane and methanol under reduced pressure and concentrating to obtain a crude extract; (3) Isolation and preparation of compounds The crude extract obtained in step (2) was dissolved in a mixture of equal volumes of dichloromethane and ethyl acetate, and 1.5 times of 200-300 mesh silica gel powder was added to the sample for VLC vacuum column chromatography. Gradient elution was performed using a petroleum ether / ethyl acetate solution with a volume ratio of (100:0)-(0:100) as the eluent. Similar fractions were sequentially combined to collect a total of 4 components; the collected third component was subjected to reverse medium pressure column chromatography. Gradient elution was performed using a methanol aqueous solution with a volume ratio of 50-100% methanol as the eluent. Similar fractions were sequentially combined to collect a total of 6 components; the collected fourth component was evaporated to dryness and then subjected to semi-preparative reverse phase high performance liquid chromatography separation. Acetonitrile / water solution with a volume ratio of 60:40 was used as the eluent to separate and purify to obtain an ergotane-type steroid compound, whose structure is shown in (I): （I）。 3. The method for preparing an ergotane-type steroid compound according to claim 2, wherein The rice culture medium described in step (1) is prepared as follows: 80 g of rice is added to 120 mL of seawater with a salinity of 20%, and then sterilized at 121°C for 15 minutes.

4. The method for preparing an ergotane-type steroid compound according to claim 2, wherein The volume ratios of the petroleum ether / ethyl acetate solution in the gradient elution in step (3) are 100:0, 95:5, 90:10, 85:15, 80:20, 70:20, 50:50, and 0:100, respectively.

5. The method for preparing an ergotane-type steroid compound according to claim 2, wherein The gradient eluent in the reverse phase medium pressure column chromatography described in step (3) is methanol and water, with methanol ranging from 50% to 100%, and the elution time is 150 min.

6. The method for preparing an ergotane-type steroid compound according to claim 2, wherein: The flow rate for the semi-preparative reversed-phase HPLC separation of compounds described in step (3) was 2.0 mL / min.

7. A use of the ergotane-type steroid compound according to any one of claims 1 to 6, characterized in that The ergotane-type steroid compound is used in the preparation of drugs for inhibiting Staphylococcus aureus.

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