CN107353274A - Come from the secalonic acid I of penicillium oxalicum and prepare the application of anti-human oesophagus cancer drug - Google Patents

Abstract

The present invention relates to the application of ryeconic acid I derived from Penicillium oxalicum and preparation of anti-human esophagus cancer medicine. The compound has the effect of inhibiting the proliferation of human esophageal cancer cells. Its structural formula is: . Penicillium oxalicum ( Penicillium oxalicum ) IBPT‑6 is cultured by fermentation to obtain a fermented product, and then the compound is isolated and purified from the fermented product. It is confirmed by experiments that the compound has good antitumor activity on human esophageal cancer cell EC9706. It can be used as a drug for inhibiting proliferation of human esophageal cancer cells or an antitumor drug for research on human esophageal cancer.

Description

The ryeconic acid I derived from Penicillium oxalicum and its application in the preparation of anti-human esophageal cancer drugs

technical field

The invention relates to the application of ryeconic acid I derived from Penicillium oxalicum and preparation of anti-human esophageal cancer medicine, and belongs to the field of medicine and biology.

Background technique

Secalonic acids belong to the secondary metabolites of Ergochrome and are xanthone dimers. Since Stoll et al. isolated Secalonic acid A (Secalonic acid A) from fungi in 1952, the series of compounds Secalonic acid (A, B, C, D, E, F, G) have been continuously identified. discovery and research. Secalonic acid compounds have various physiological activities. Taking Secalonic acid D (Secalonic acidD, SAD) as an example, 5 mg/ml of SAD is added to normal saline, within the range of 5-20 mg, that is It can treat early bladder cancer, within the range of 50-100 mg, it is effective for more serious bladder cancer and has no side effects. It has been found through research that some marine fungi can produce ryryronic acid compounds with novel structures and good activities in the secondary metabolism process, which has good medicinal and industrial prospects.

The inventors have learned from research that Penicillium oxalicum ( Penicillium oxalicum ) IBPT-6, (has been preserved in the China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, Wuhan, and the preservation number is: CCTCC NO: M 2013714) the crude extract of the fermentation product has good cell proliferation inhibitory activity, so its active ingredients were studied. The study found that the ryronic acid compound has anti-human esophageal cancer activity, and there is no report on the anti-proliferation activity of the compound on human esophageal cancer cells, so there is no drug related to this in the market.

Contents of the invention

The purpose of the present invention is to provide the ryeconic acid I derived from Penicillium oxalicum and its application in the preparation of anti-human esophageal cancer drugs. The compound has the effect of inhibiting the proliferation of esophageal cancer cells and has anti-human esophageal cancer activity. Its structural formula is:

.

The preparation method of the compound comprises the steps of fermenting and cultivating Penicillium oxalicum ( Penicillium oxalicum ) IBPT-6 to obtain a fermented product, and then separating and purifying the compound from the fermented product. Specific steps are as follows:

1 Fermentation production

The conventional method for cultivating microorganisms is to inoculate Penicillium oxalicum ( Penicillium oxalicum ) IBPT-6 on the PDA solid slant medium and cultivate it in a 28°C incubator for 2 to 3 days, then inoculate it into the culture medium, and then culture it statically at 28°C for 30 days. , to obtain mycelia and fermented liquid; the composition of the cultured liquid: every 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, KH ₂ PO ₄ 0.5 g, MgSO ₄ · 7H ₂ O 0.3 g, NaCl 15.0 g;

2 Obtaining the extract

Separate the mycelium and fermentation broth with gauze. The mycelium was broken by ultrasonic continuously for 3 times with acetone solution (containing 20%~30% water), and the residue was removed by filtration to obtain the crude extract of mycelium containing acetone and water. Concentrate under reduced pressure to remove acetone to obtain an aqueous solution of the crude substance, and then add ethyl acetate at a volume ratio of 1:2 to extract three times to obtain a crude ethyl acetate extract, which is concentrated under reduced pressure until nearly dry to obtain 36.5 g of mycelium extract.

3 Separation and purification of compounds

The mycelia extract was mixed with 100-200 mesh silica gel, and the gradient eluent was petroleum ether: dichloromethane: methanol, and the vacuum silica gel column chromatography was performed. The fractions were combined and separated into fractions AE after simple TLC analysis. Component D (5.9 g) (dichloromethane: methanol v/v = 100:1 eluate) was subjected to pressurized column chromatography on silica gel with dichloromethane: methanol as the gradient eluent, followed by thin-layer chromatography Five subfractions D1-D5 were combined after analysis. Component D2 (1.2 g) was subjected to gel column chromatography (Sephadex LH-20) with chloroform: methanol = 1:2 as the gradient eluent, and four subcomponents D2 were combined after thin-layer chromatography analysis -1~D2-4. Subcomponent D2-3 (212 mg) of D2 was separated by semi-preparative liquid chromatography (ODS-A 1010, 10×250 mm, 5 μm): the separation flow rate was 5 mL/min, and the mobile phase was 55% acetonitrile containing 0.1 % TFA to give the indicated compound (2.7 mg, _tR 13.8 min).

The Penicillium oxalicum ( Penicillium oxalicum ) IBPT-6 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 2013714.

The present invention also protects the application of the compound in the preparation of drugs for inhibiting the proliferation of human esophageal cancer cells, and the application of the compound in the preparation of anti-human esophageal cancer drugs.

Significant advantages of the present invention: as shown in the research, the ryronic acid compound has not been reported and has significant activity of inhibiting the proliferation of human esophageal cancer cells. At present, there is no report on the inhibitory activity of this compound on the proliferation of human esophageal cancer cells. Therefore, it is also available on the market. There are no related drugs yet.

Description of drawings

Figure 1 The main COZY, HMBC and NOE signals of Secalonic acid I.

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 oxalicum ( Penicillium oxalicum ) IBPT-6 (preserved in China Center for Type Culture Collection on December 25, 2013, address: Wuhan University, Wuhan, deposit number Yes: CCTCC NO: M 2013714) Appropriate amount, inoculate on PDA solid slant medium and culture in 28°C incubator for 2 to 3 days.

Take an appropriate amount of Penicillium oxalicum ( Penicillium oxalicum ) IBPT-6 cultured on a slant for 2 to 3 days, and inoculate it into 400 mL 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 ·7H ₂ O 0.3, NaCl 15.0, constant volume] in a 1000mL Erlenmeyer flask, after static culture at 28°C for 30 days, mycelium and fermentation broth were obtained.

2 Obtaining the extract

Separate the mycelium and fermentation broth with gauze. The mycelium was broken by ultrasonic continuously for 3 times with acetone solution (containing 20%~30% water), and the residue was removed by filtration to obtain the crude extract of mycelium containing acetone and water. Concentrate under reduced pressure to remove acetone to obtain an aqueous solution of the crude substance, and then add ethyl acetate at a volume ratio of 1:2 to extract three times to obtain a crude ethyl acetate extract, which is concentrated under reduced pressure until nearly dry to obtain 36.5 g of mycelium extract.

3 Separation and purification of compounds

The mycelia extract was mixed with 100-200 mesh silica gel, and the gradient eluent was petroleum ether: dichloromethane: methanol, and the vacuum silica gel column chromatography was performed. The fractions were combined and separated into fractions AE after simple TLC analysis. Component D (5.9 g) (dichloromethane: methanol v/v = 100:1 eluate) was subjected to pressurized column chromatography on silica gel with dichloromethane: methanol as the gradient eluent, followed by thin-layer chromatography Five subfractions D1-D5 were combined after analysis. Component D2 (1.2 g) was subjected to gel column chromatography (Sephadex LH-20) with chloroform: methanol = 1:2 as the gradient eluent, and four subcomponents D2 were combined after thin-layer chromatography analysis -1~D2-4. Subcomponent D2-3 (212 mg) of D2 was separated by semi-preparative liquid chromatography (ODS-A 1010, 10×250 mm, 5 μm): the separation flow rate was 5 mL/min, and the mobile phase was 55% acetonitrile containing 0.1 % TFA to give the indicated compound (2.7 mg, _tR 13.8 min).

The compound is a yellow oil at room temperature, and the high-resolution electrospray mass spectrometry HRESI-MS gives the molecular ion peak [M+Na] ⁺ (calcd for C ₃₂ H ₂₈ NaO ₁₄ , 659.1377) at m/z : 659.1377, suggesting that the molecular weight is 636, combined with spectral information, the molecular formula is deduced to be C ₃₂ H ₂₈ O ₁₄ . 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.

Table 1 ¹ H and ¹³ C-NMR data of compounds (500 MHz ¹ H and 126 MHz ¹³ C, in DMSO- d ₆ )

Example 2 Test of anti-tumor activity in vitro

1 Experimental samples and experimental methods

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

Cell lines and subculture of cells Tumor cell lines were used, and tumor cells were subcultured in DMEM medium containing 10% FBS at 37°C in an incubator filled with 5% CO ₂ .

Cell Proliferation Inhibitory Activity Test Method

The tumor cells in the logarithmic growth phase were collected by the tetrazolium salt (MTT) method, the cell density was adjusted to 1×10 ⁵ cells per milliliter, and 200 microliters per well were inoculated in a 96-well cell culture plate, and circulated 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, and continue to incubate for 4 hours at 37°C, 2000 rpm Centrifuge for 8 minutes and aspirate the supernatant. Add 100 microliters of DMSO to each well, shake on a micro shaker for 15 minutes, and after the crystals are completely dissolved, use a SPECTRAMAX Plus microplate reader produced by MD to measure the absorbance (OD) value of each well at 570 nm. In the same 96-well plate, set three wells for each concentration of the sample, and set up three wells of blank control and no cell apoptosis wells (if the drug has color, do the corresponding drug concentration without cell apoptosis). The OD value of each well was first calculated without cell apoptosis, and then the average OD value of the three wells was calculated according to IR (%) = (OD _{blank control} - OD _sample ) / OD _{blank control} × 100% to calculate the cell proliferation inhibition rate 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 the proliferation of human esophageal cancer cells

3. Conclusion

The compound has good antitumor activity on human esophageal cancer cells. It can be used as a drug for inhibiting proliferation of esophageal cancer cells or an antitumor drug for research on esophageal cancer.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

Claims (4)

1. compound。

2. the preparation method of compound as claimed in claim 1, it is characterised in that：Fermented and cultured penicillium oxalicum (Penicillium oxalicum) IBPT-6, fermentate is obtained, the compound, institute are then isolated and purified out from fermentate State penicillium oxalicum (Penicillium oxalicum) IBPT-6, it is deposited in Chinese Typical Representative culture on December 25th, 2013 Thing collection, address:Wuhan Wuhan University, deposit number are：CCTCC NO：M 2013714.

3. the compound described in claim 1 is preparing the application in suppressing human esophagus cancer cell hyperproliferation agent.

4. application of the compound in anti-human oesophagus cancer drug is prepared described in claim 1.