A new use of penicillin A2 from Penicillium citrinum to resist vincristine

Abstract

The present invention relates to a novel use of an alkaloid compound penicillin A2 derived from Penicillium citrinum for resisting vincristine resistance. The structural formula of the penicillin A2 is as follows: Penicillium citrinum )IBPT-5 fermentation product. Experiments have confirmed that the penicillin A2 has good anti-tumor activity against vincristine-resistant cells and can be used as a vincristine-resistant cell proliferation inhibitor or anti-vincristine-resistant drug for anti-tumor research.

Description

Technical Field

[0001] The invention relates to a new use of an alkaloid compound penicillinol A2 derived from Penicillium citrinum for resisting vincristine resistance. Background Art

[0002] In current clinical treatment, many tumors achieve good results after initial drug treatment, but tumor recurrence often occurs in the end. After recurrence, the sensitivity to anti-tumor drugs is significantly reduced, which seriously affects the patient's quality of life and time. Vincristine (VCR) is a diindole compound proposed from Catharanthus roseus in 1962. VCR interferes with tumor cell metabolism by acting on tumor cell microtubules and is widely used in clinical practice (such as acute lymphoblastic leukemia, lymphoma, cervical cancer, breast cancer, etc.). However, in clinical practice, many tumor cells develop drug resistance to VCR soon after contact with it, which eventually leads to chemotherapy failure.

[0003] Alkaloids are a class of nitrogen-containing organic compounds produced by the secondary metabolism of organisms. There are many types of alkaloids in nature, most of which come from plants, so they are also called plant alkaloids. Alkaloids have important physiological effects on humans and animals, including antiasthmatic and antitussive, hypoglycemic, hypolipidemic, antibacterial, antitumor, analgesic, etc., among which antibacterial and antitumor activities are the most prominent. Natural structural alkaloids are an important source of lead compounds discovered in innovative drug research. Currently, there are nearly 100 alkaloid drugs used in clinical practice. Studies have found that some marine fungi can produce alkaloids with novel structures and good activities during secondary metabolism, which have good prospects for medicinal and industrialization.

[0004] The inventors have found that Penicillium citrinum ( Penicillium citrinum The crude extract of the fermentation product of IBPT-5 (which was deposited in China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, Wuhan, with the deposit number: CCTCC NO: M2013713) has good vincristine-resistant cell proliferation inhibition activity, so the active ingredients were studied. The study found that the alkaloid compound has anti-vincristine resistance activity, and there is no report on the inhibitory activity of the compound on resistant cell proliferation, so there is no related drug on the market. Summary of the invention

[0005] The object of the present invention is to provide a new use of the alkaloid compound penicillinol A2 derived from Penicillium citrinum for resisting vincristine resistance.

[0006] The present invention first relates to a strain of Penicillium citrinum ( Penicillium citrinum)IBPT-5, which was deposited in China Center for Type Culture Collection on December 25, 2013. The deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is: CCTCC NO: M 2013713.

[0007] The purpose of the strain is to transform Penicillium citrinum ( Penicillium citrinum )IBPT-5 was fermented and the fermentation broth extract was separated to obtain the compound penicillin A2 with vincristine-resistant cell proliferation inhibitory activity.

[0008] The structural formula of the compound penicillinol A2 is:

[0009] .

[0010] The preparation method of the compound penicillinol A2 comprises the following specific steps:

[0011] (1) Fermentation production

[0012] The conventional method for culturing microorganisms is to take Penicillium citrinum ( Penicillium citrinum ) IBPT-5 was inoculated on a PDA solid slant medium and cultured in a 28°C incubator for 4 days, then inoculated into a culture solution, and after static culture at 28°C for 30 days, mycelium and fermentation solution were obtained; the culture solution consisted of: 20.0 g mannitol, 3.0 g yeast extract, 20.0 g maltose, 10.0 g monosodium glutamate, 10.0 g glucose, 0.5 g KH2PO4, 0.3 g MgSO4, and 30.0 g NaCl per liter of water;

[0013] (2) Obtaining the extract

[0014] The mycelium and the fermentation liquid were separated by gauze, the fermentation liquid was extracted twice with 2 volumes of ethyl acetate, and the extract was distilled to dryness under reduced pressure to obtain an ethyl acetate extract of the fermentation liquid;

[0015] (3) Separation and purification of compounds

[0016] After the ethyl acetate extract was mixed with 100-200 mesh silica gel, it was gradient eluted on a reduced pressure silica gel column with petroleum ether, dichloromethane and methanol as eluents, and the eluate with dichloromethane: methanol v / v=100:1 was taken, and further gradient eluted with dichloromethane and methanol as eluents through pressurized silica gel column chromatography to obtain the eluate with dichloromethane: methanol v / v=50:1, and then reversed-phase silica gel column chromatography with methanol: water v / v=3:2 as solvent was continued to remove impurities, and finally semi-preparative liquid chromatography 1010 type ODS-A, 10×250 mm, 5μm: separation flow rate was 5 mL / min, and the mobile phase was 80% methanol containing 0.1% TFA to obtain tR 8.7 min of the compound.

[0017] The present invention also protects the use of the compound penicillinol A2 in the preparation of vincristine-resistant cell proliferation inhibitory drugs, and the use of the compound penicillinol A2 in the preparation of anti-vincristine-resistant drugs.

[0018] The significant advantages of the present invention are as follows: studies have shown that the alkaloid compound penicillin A2 is relatively rare, and the alkaloid compound penicillin A2 has significant anti-vincristine resistance activity. Currently, there are no reports on the inhibitory activity of the compound penicillin A2 on vincristine-resistant cell proliferation, so there are no related drugs on the market. DETAILED DESCRIPTION

[0019] The compound referred to in the following examples is penicillinol A2, whose chemical structure is:

[0020] .

[0021] Example 1 Fermentation production and separation and purification of the compound

[0022] 1. Fermentation production

[0023] Fermentation culture of production bacteria: According to the conventional method of culturing microorganisms, take Penicillium citrinum ( Penicillium lemon ) IBPT-5 (deposited in China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit number: CCTCC NO: M 2013713) was inoculated in an appropriate amount onto PDA solid slant medium and cultured in a 28°C incubator for 4 days.

[0024] Take the slant culture of Penicillium citrinum for 4 days ( Penicillium citrinum ) IBPT-5 was inoculated into a 1000mL conical flask containing 400mL of culture medium [culture medium composition (g / L): mannitol 20.0, yeast extract 3.0, maltose 20.0, monosodium glutamate 10.0, glucose 10.0, KH2PO4 0.5, MgSO4 0.3, NaCl 30.0 constant volume], and mycelium and fermentation broth were obtained after static culture at 28°C for 30 days.

[0025] 2. Obtaining the extract

[0026] The mycelium and the fermentation liquid were separated with gauze. The fermentation liquid was extracted twice with ethyl acetate at a ratio of 1:2 (v / v), and the extract was distilled to dryness under reduced pressure to obtain 32 g of ethyl acetate extract of the fermentation liquid.

[0027] 3. Separation and purification of compounds

[0028] After the ethyl acetate extract was mixed with 100-200 mesh silica gel, it was gradient eluted with petroleum ether: dichloromethane: methanol as eluent on a reduced pressure silica gel column to obtain 11 components. Component 7 (4.2 g) (eluent of dichloromethane: methanol v / v = 100:1) was further eluted with dichloromethane: methanol as eluent by gradient elution on a pressurized silica gel column, and the obtained subcomponent 7-3 (eluent of dichloromethane: methanol v / v = 50:1) was subjected to reverse phase silica gel column chromatography with methanol-water (v / v3:2) as solvent to remove impurities, and finally semi-preparative liquid chromatography (1010 type ODS-A, 10×250 mm, 5μm): the separation flow rate was 5 mL / min, and the mobile phase was 80% methanol containing 0.1% TFA, to obtain the compound shown (62.4 mg, t R 8.7 min).

[0029] The compound was light yellow oil, and high resolution mass spectrometry (HRESI-MS) was performed on m / z The molecular ion peak [M–H] is given at 296.1865 – , (Calcd for C 16 H 26 NO4, 296.1862), indicating a molecular weight of 297. Combined with the spectrum information, the molecular formula is estimated to be C 16 H 27 NO4. 1 H and 13 C-NMR and other NMR data are shown in Table 1.

[0030] Table 1 Compounds 1 H and 13 C-NMR data (500 MHz, in CDCl3) a)

[0031]

[0032] a) The signal in this table is consistent with the literature "Penicillenols from Penicilliumsp. GQ-7, an Endophytic Fungus Associated with Aegicerascorniculatum, Chem. Pharm. Bull. 56(2) 217-221 (2008)》The data are basically consistent.

[0033] Example 2 In vitro anti-tumor activity test

[0034] 1 Experimental samples and experimental methods

[0035] Preparation of the Test Sample Solution The test sample is the pure compound separated and purified in the above-mentioned Example 1. Accurately weigh an appropriate amount of the sample and prepare a solution of the required concentration with methanol for activity measurement.

[0036] The cell line and cell subculture used the drug-resistant tumor cell line Hela / VCR. The tumor cells were subcultured in DMEM medium containing 10% FBS at 37°C in an incubator filled with 5% carbon dioxide.

[0037] Cell proliferation inhibitory activity test method

[0038] The tumor cells in the logarithmic growth phase were taken by MTT method and the cell density was adjusted to 2×10 per ml. 5 Cells were inoculated into 96-well cell culture plates at 200 μl per well and cultured in an incubator at 37°C with 5% CO2 for 4 hours. 2 μl of sample solution or blank solution was added to each well. After 24 hours of culture, 10 μl of MTT solution (5 mg of MTT per ml of physiological saline solution) was added to each well and cultured for another 4 hours. The cells were centrifuged at 37°C and 2000 rpm for 8 minutes, and the supernatant was removed. 100 μl of DMSO was added to each well and oscillated on a micro-oscillator for 15 minutes until the crystals were completely dissolved. The absorbance (OD) value of each well at 570 nm was measured using the SPECTRAMAX Plus microplate reader produced by MD. Three wells were set for each concentration of the sample in the same 96-well plate, and three blank control wells and cell-free zeroing wells were set (if the drug has color, cell-free zeroing should be performed for the corresponding drug concentration). The OD value of each well was first adjusted to cell-free zero, and then the average OD value of the three wells was taken according to IR (%) = (OD 空白对照 -OD 样品 ) / OD 空白对照 × 100% formula was used to calculate the cell proliferation inhibition rate (IR%) at each concentration.

[0039] 2. Experimental Results

[0040] Cell proliferation inhibitory activity test results

[0041] In the MTT assay, the SPSS 16.0 software was used to process the data and calculate the half inhibitory concentration IC according to the tumor cell proliferation inhibition rate of the compound at different concentrations. 50 The results are shown in Table 2.

[0042] Table 2 Inhibitory activity of compounds on human tumor cell proliferation

[0043]

[0044] 3. Conclusion

[0045] The compound has an obvious proliferation inhibition effect on vincristine-resistant cells and can be used as a vincristine-resistant cell proliferation inhibition drug or an anti-vincristine-resistant drug for anti-tumor research.

Claims

1. Use of penicillin A2 derived from Penicillium citrinum in the preparation of a vincristine-resistant cell proliferation inhibitor, characterized in that: The structural formula of the penicillinol A2 is as follows: ； The vincristine-resistant cells are vincristine-resistant Hela cells.

2. The use of the penicillin A2 derived from Penicillium citrinum in the preparation of a vincristine-resistant cell proliferation inhibitor according to claim 1, characterized in that: The penicillin A2 is obtained from Penicillium citrinum ( Penicillium citrinum ) was isolated and purified from IBPT-5 fermentation product; the Penicillium citrinum ( Penicillium citrinum )IBPT-5 was deposited in China Center for Type Culture Collection on December 25, 2013. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2013713.

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