CN104402899A - Citrinin compound penicitrinol L derived from Penicillium citrinum, preparation method and application of citrinin compound penicitrinol L - Google Patents

Abstract

The invention relates to a citrinin compound penicitrinol L derived from Penicillium citrinum and its preparation method and application. The compound has the effect of inhibiting tumor cell proliferation and has antitumor activity. Its structural formula is: . By fermenting and cultivating Penicillium citrinum IBPT-5, obtaining a fermented product, and then separating and purifying the compound from the fermented product. It is confirmed by experiments that the compound has good antitumor activity on the human cervical cancer cell line hela. It can be used as the preparation of cell proliferation inhibitory drugs or anti-tumor drugs for anti-tumor research.

Description

Citrinin compound penicitrinol L derived from Penicillium citrinum and its preparation method and application

technical field

The invention relates to a citrinin compound penicitrinol L derived from Penicillium citrinum and its preparation method and application.

Background technique

Fungi are relatively higher among microorganisms. Fungal natural products are an important source of activity diversity and structural diversity, and many chemical drugs with good therapeutic effects have been developed. For example, antibiotic penicillin (penicillin) series, immunosuppressant cyclosporin A (cyclosporin A), blood lipid-lowering drugs mevastatin (mevastatin), lovastatin (lovastatin) and antifungal drug griseofulvin (griseofulvin), etc. , These all show that fungus is an important treasure house of natural medicine. Citrinin, a well-known secondary metabolite of polyketide fungi, was first isolated from a secondary metabolite of Penicillium citrinum. Studies in recent years have found that citrinin compounds exhibit promising biological activities such as anti-tumor, anti-bacterial, anti-oxidation, anti-fouling, and enzyme inhibition.

The inventors have learned from research that Penicillium citrinum ( Penicillium citrinum ) IBPT-5, (has been preserved in China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, Wuhan, and the preservation number is: CCTCC NO: M 2013713) crude extract of the fermentation product has good cell proliferation inhibitory activity, so its active ingredients were studied. The study found that the alkaloid compound has anti-tumor activity, and there is no report on the anti-proliferation activity of the compound on tumor cells, so there is no drug related to it in the market.

Contents of the invention

The object of the present invention is to provide a kind of citrinin compound penicitrinol L derived from Penicillium citrinum and its preparation method and application. The compound has the effect of inhibiting tumor cell proliferation and has antitumor activity. Its structural formula is:

.

The preparation method of the compound is to culture Penicillium citrinum ( Penicillium citrinum ) IBPT-5 by fermentation, obtain the fermented product, and then separate and purify the compound from the fermented product. The said Penicillium citrinum ( Penicillium citrinum ) IBPT-5 has been It was deposited in the China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, Wuhan, and the deposit number is: CCTCC NO: M 2013713. Specific steps are as follows:

1 Fermentation production

The conventional method for cultivating microorganisms is to inoculate Penicillium citrinum IBPT-5 on the PDA solid slant medium and cultivate it in a 28°C incubator for 4 days, then inoculate it into the culture solution, and cultivate it statically at 28°C for 30 days to obtain Mycelia and fermented liquid; the composition of said cultured liquid: per liter of water contains 20.0 g of mannitol, 3.0 g of yeast extract, 20.0 g of maltose, 10.0 g of monosodium glutamate, 10.0 g of glucose, 0.5 g of KH ₂ PO ₄ , 0.3 g of MgSO ₄ , NaCl 30.0 g;

2 Obtaining the extract

Separate the mycelium and fermentation broth with gauze. The fermentation broth was extracted twice with 2 times the volume of ethyl acetate, and the extract was distilled to dryness under reduced pressure to obtain the ethyl acetate extract of the fermentation broth.

3 Separation and purification of compounds

After the ethyl acetate extract was mixed with 100-200 mesh silica gel, petroleum ether: dichloromethane: methanol was used as the eluent for gradient elution with a decompression silica gel chromatographic column, and the eluate of dichloromethane was collected for Dichloromethane: Methanol is used as the eluent, further eluted by gradient elution of pressurized silica gel column chromatography to obtain dichloromethane: methanol v/v=100:1 eluate, and then passed through semi-preparative liquid chromatography 1010 type ODS -A, 10×250 mm, 5 μm: The separation flow rate was 5 mL/min, the mobile phase was 70% acetonitrile containing 0.1% TFA, and the compound was obtained, and the t _R was 16.7 min.

The invention also protects the use of the compound in the preparation of drugs for inhibiting tumor cell proliferation and the use of the compound in the preparation of antitumor drugs. The tumor cells are Hela cells.

Significant advantages of the present invention: the citrinin compound shown in the research is relatively rare, and the citrinin compound has significant antitumor activity on the human cervical cancer cell line Hela, and can be used as a drug for inhibiting cell proliferation or an antitumor drug in antitumor research. At present, there is no report on the inhibitory activity of the compound on tumor cell proliferation, so there is no drug related to it in the market.

Description of drawings

Figure 1 shows the main COZY, HMBC and NOE signals of the compound penicitrinol L.

detailed description

The chemical structures of the compounds referred to in the following examples:

.

Fermentative production and separation and purification of the compound of embodiment 1

1 Fermentation production

Fermentation culture of production bacteria: According to the conventional method of cultivating microorganisms, take Penicillium citrinum IBPT-5 (preserved in China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, Wuhan, deposit number Yes: CCTCC NO: M 2013713) Appropriate amount, inoculated on PDA solid slant medium and cultivated in 28°C incubator for 4 days.

Take an appropriate amount of Penicillium citrinum ( Penicillium citrinum ) IBPT-5 cultured on the slant for 4 days, and inoculate it into 400mL of culture solution [Culture solution composition (g/L): mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, glucose 10.0, KH ₂ PO ₄ 0.5, MgSO ₄ 0.3, NaCl 30.0 constant volume] in a 1000mL Erlenmeyer flask, after 30 days of static culture at 28°C, mycelium and fermentation broth were obtained.

2 Obtaining the extract

Separate the mycelium and fermentation broth with gauze. The fermented liquid was extracted twice with ethyl acetate 1:2 (v/v), and the extract was distilled to dryness under reduced pressure to obtain 32 g of ethyl acetate extract of the fermented liquid.

3 Separation and purification of compounds

After the extract is mixed with 100-200 mesh silica gel, the eluent is gradient eluted with reduced-pressure silica gel chromatographic column with petroleum ether: dichloromethane: methanol, and 11 components are obtained. Component 3 (1.8 g) (the eluate of dichloromethane) was further eluted with a gradient of pressurized silica gel column chromatography with dichloromethane: methanol as the eluent, and the obtained subcomponent 3-1 (350 mg ) (dichloromethane:methanol v/v=100:1 eluate), and then through semi-preparative liquid chromatography (1010 type ODS-A, 10×250 mm, 5μm): the separation flow rate is 5 mL/min, The mobile phase was 70% acetonitrile with 0.1% TFA to give the compound shown (7.2 mg, _tR 16.7 min).

Compound White powder, high-resolution mass spectrometry HRESI-MS gives the molecular ion peak [M – H] ^– at m/z 361.1671, (calcd. for C ₂₀ H ₂₅ O ₆ , 361.1657), suggesting that the molecular weight is 362, combined with spectral information The molecular formula is speculated to be C ₂₀ H ₂₆ O ₆ . The ¹ H and ¹³ C-NMR data are shown in Table 1, and the main COZY, HMBC and NOE signals are shown in Figure 1.

¹ H and ¹³ C-NMR data of the compounds in Table 1 (500MHz ¹ H and 125MHz ¹³ C , in CDCl ₃ )

The test of embodiment 2 in vitro antitumor activity

1 Experimental samples and experimental methods

Preparation of test sample solution The test sample is the pure product of the compound separated and purified in the above implementation 1. Accurately weigh an appropriate amount of sample, and prepare a solution with the required concentration with methanol for activity measurement.

The subculture of cell lines and cells adopts tumor cell lines, and the tumor cells are subcultured in DMEM medium containing 10% FBS at 37°C in an incubator filled with 5% carbon dioxide.

Cell Proliferation Inhibitory Activity Test Method

Tetrazolium salt (MTT) method Take tumor cells in the logarithmic growth phase, adjust the cell density to 2×10 ⁵ cells per milliliter, inoculate 200 microliters per well in a 96-well cell culture plate, and circulate at 37°C. Incubate for 4 hours in a 5% CO ₂ incubator. Add 2 microliters of sample solution or blank solution to each well, and after 24 hours of incubation, add 10 microliters of MTT solution (5 mg of normal saline solution per milliliter of MTT) to each well, continue to incubate for 4 hours, and centrifuge at 37°C and 2000 rpm 8 minutes, aspirate the supernatant. Add 100 microliters of DMSO to each well, vibrate on a micro shaker for 15 minutes until the crystals are completely dissolved, and measure the absorbance (OD) value of each well at 570 nm using a SPECTRAMAX Plus microplate reader produced by MD Company. In the same 96-well plate, set three wells for each concentration of the sample, and set up three wells for blank control and zero wells without cells (if the drug has color, the corresponding drug concentration should be zeroed without cells). The OD value of each well was adjusted to zero correspondingly without cells, and then the average OD value of the three wells was calculated according to the formula IR (%) = (OD _{blank control} -OD _sample )/OD _{blank control} × 100% to calculate the inhibition rate of cell proliferation at each concentration (IR%).

2. Experimental results

Cell Proliferation Inhibitory Activity Test Results

In the MTT method test, according to the tumor cell proliferation inhibition rate of the compound at different concentrations, SPSS16.0 software was used for data processing and the half inhibitory concentration IC ₅₀ value was calculated. The results are shown in Table 2.

Table 2 Inhibitory activity of compounds on human tumor cell proliferation

3. Conclusion

The compound has a strong proliferation inhibitory effect on tumor cell hela, and can be used as a preparation of hela cell proliferation inhibitor or antitumor agent for antitumor research.

Claims (6)

1. Compound . 2. the preparation method of compound as claimed in claim 1 is characterized in that: ferment and cultivate Penicillium citrinum ( Penicillium citrinum ) IBPT-5, obtain fermented product, then separate and purify this compound from fermented product, described Penicillium citrinum ( Penicillium citrinum ) IBPT-5 was deposited in China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, Wuhan, and the deposit number is: CCTCC NO: M 2013713. 3. the preparation method of compound according to claim 2, is characterized in that: the concrete steps of described preparation method are as follows: (1) Fermentation production The conventional method for cultivating microorganisms is to inoculate Penicillium citrinum IBPT-5 on the PDA solid slant medium and cultivate it in a 28°C incubator for 4 days, then inoculate it into the culture solution, and cultivate it statically at 28°C for 30 days to obtain Mycelia and fermented liquid; the composition of said cultured liquid: per liter of water contains 20.0 g of mannitol, 3.0 g of yeast extract, 20.0 g of maltose, 10.0 g of monosodium glutamate, 10.0 g of glucose, 0.5 g of KH ₂ PO ₄ , 0.3 g of MgSO ₄ , NaCl 30.0 g; (2) Obtaining the extract Use gauze to separate the mycelia from the fermentation broth, extract the fermentation broth twice with 2 times the volume of ethyl acetate, and distill the extract to dryness under reduced pressure to obtain the ethyl acetate extract of the fermentation broth; (3) Separation and purification of compounds After the ethyl acetate extract was mixed with 100-200 mesh silica gel, petroleum ether: dichloromethane: methanol was used as the eluent for gradient elution with a decompression silica gel chromatographic column, and the eluate of dichloromethane was collected for Dichloromethane: Methanol is used as the eluent, further eluted by gradient elution of pressurized silica gel column chromatography to obtain dichloromethane: methanol v/v=100:1 eluate, and then passed through semi-preparative liquid chromatography 1010 type ODS -A, 10×250 mm, 5 μm: The separation flow rate was 5 mL/min, the mobile phase was 70% acetonitrile containing 0.1% TFA, and the compound was obtained, and the t _R was 16.7 min. 4. The compound of claim 1 is used in the preparation of tumor cell proliferation inhibitory drugs. 5. The use of the compound according to claim 1 in the preparation of antitumor drugs. 6. The use according to claim 4, characterized in that: the tumor cells are HeLa cells.