CN105399708A - Anti-liver fibrosis Penicilfuranone A compound as well as pharmaceutical composition and application thereof - Google Patents

Abstract

A pharmaceutical composition consisting of penicilfuranone A (Penicilfuranone? A) represented by structural formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient and at least one pharmaceutically acceptable carrier, its preparation method, and Its application in the preparation of drugs for preventing or treating liver fibrosis. Penicillium furanone A is a furan polyketone compound with a novel chemical structure, which has significant anti-hepatic fibrosis activity in vitro. It has a novel chemical structure and strong activity, and has many advantages as an anti-hepatic fibrosis drug.

Description

Anti-hepatic fibrosis penicillin furanone A compound and its pharmaceutical composition and application

Technical field:

The invention belongs to the technical field of medicines, in particular, it relates to Penicilfuranone A, a furanone polyketide active compound with anti-hepatic fibrosis activity, a preparation method thereof, a pharmaceutical composition using the compound as an active ingredient, and its preparation for treating liver fibrosis. application in medicine for diseases.

Background technique:

Furanone polyketides are a class of secondary metabolites produced by fungi, which have been reported in the secondary metabolites of fungi Aspergillusrugulosus, Cephalosporiumgregatum, and Penicilliumdaleae. So far, there are less than 20 furan polyketide compounds reported in the literature, and their molecular weights are all about 300. Previous studies have shown that this type of compound has antibacterial effects, causes plant withering, and inhibits mammalian Y family DNA enzymes. The compound of the present invention has a novel structure with a molecular weight of 486, which is completely different from the previously reported compounds. So far, no furanone polyketide compound with novel structure of the present invention has been reported in the prior art, and there is no literature report on the research on the anti-hepatic fibrosis activity of this furanone polyketide compound.

Invention content:

The object of the present invention is to provide furanopolyketone compound A isolated from endophytic fungus Penicilliumdaleae, its preparation method, and its application in medicine, especially in anti-hepatic fibrosis medicine.

In order to realize the above-mentioned purpose of the present invention, the present invention provides following technical scheme:

Penicillofuranone A compound represented by the following structural formula (I) or a pharmaceutically acceptable salt thereof,

A pharmaceutical composition, which comprises the penicillofuranone A compound or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier and/or diluent.

According to the penicillin furanone A compound or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is hydrochloride, hydrobromide, nitrate, sulfate, phosphate, tartrate , citrate, formate, acetate, oxalate.

The present invention also provides a preparation method of penicillium furanone A compound or a pharmaceutically acceptable salt thereof. The fermentation product of penicillium furanone APenicilliumdaleae is taken, extracted with an organic solvent, concentrated to obtain an extract, extracted with ethyl acetate, and silica gel Chromatographic column, preparative high performance liquid chromatography and other methods to obtain the compound of structural formula (I).

The more specific preparation method is as follows: take Penicilliumdaleae mycelium and inoculate PDA in sterilized potato glucose culture solution, shake it in a shaker at 170 rpm at room temperature for five days, make seed solution, and transfer the seed solution to In sterilized rice culture medium, culture aseptically at 28°C for 40 days, extract the mycelium after 40 days of culture in rice culture medium with ethyl acetate, extract four times in total to obtain ethyl acetate extract, use silica gel column Chromatography, high-performance liquid chromatography-mass spectrometry, preparative high-performance liquid chromatography to process the ethyl acetate extract, and finally obtain penicillium furanone A, and its preparative liquid chromatography conditions are: 12mL/min, UVλ _max 230nm, MeCN-H ₂ O45:55, retention time 15.0min.

In the above-mentioned preparation method, the penicillium furanone A compound can be further salted with an appropriate acid, and the appropriate acid is selected from hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, tartaric acid, citric acid, formic acid, acetic acid, oxalic acid or Other suitable organic or inorganic acids.

The present invention also provides the application of the penicillofuranone A compound or the pharmaceutical composition in the preparation of anti-hepatic fibrosis medicine.

The pharmaceutical composition of the present invention can be in the form of a preparation suitable for oral or injection administration.

The formulations for oral administration described herein are tablets, sustained-release tablets, controlled-release tablets, lozenges, hard or soft capsules, dropping pills, pellets, aqueous or oil suspensions, emulsions, dispersible powders or Granules, oral solutions, syrups or elixirs, and the preparations for injection are in the form of sterile aqueous or oily solutions, sterile powders, liposomes, emulsions, microemulsions, nanoemulsions or microcapsules.

The compound penicillin furanone A of the present invention has a novel structure and a molecular weight of 486, which is completely different from the structure of the previously reported compounds, and has obvious anti-hepatic fibrosis activity, and its mechanism is related to blocking TGF-β1 to stimulate hepatic stellate cells The overactivation of Penicilfuranone A is related to the ability to form extracellular matrix. At a concentration lower than 32 μM, Penicilfuranone A has no obvious hepatotoxicity.

Description of drawings:

Fig. 1 is the structural representation of PenicilfuranoneA of the present invention;

Fig. 2 is the X-single crystal diffraction structure schematic diagram of PenicilfuranoneA of the present invention;

Fig. 3 is the effect of Penicilfuranone A of the present invention on the growth activity of normal human liver cells (MTT method; treatment 48h);

Fig. 4 is that Penicilfuranone A of the present invention inhibits the expression of proteins related to the formation of hepatic stellate cell fibrosis under the induction of TGF-β1 (westernblot method; A is the rat T6 hepatic stellate cell strain, and B is the human LX-2 hepatic stellate cell strain) .

detailed description:

The substantive content of the present invention will be further described below with reference to the accompanying drawings, but the present invention is not limited thereto.

Example 1:

Preparation of compound PenicilfuranoneA:

Separation process: Take a small amount of Penicilliumdaleae mycelium and inoculate it in 1000ml sterilized potato dextrose culture solution (PDA), and shake it in a shaker at 170 rpm for five days at room temperature to make a seed solution. The seed solution was transferred to 2.0 kg of sterilized rice medium, and cultured aseptically for 35 days at 25°C. The mycelium after 35 days of cultivation in rice culture medium was extracted with 5 L of ethyl acetate, extracted five times in total, 5 L each time, to obtain 18 g of ethyl acetate extract. Using silica gel column chromatography, high performance liquid chromatography mass spectrometry, preparative high performance liquid chromatography to process 18g of ethyl acetate extract, finally get PenicilfuranoneA12mg, its preparative liquid chromatography conditions are: 12mL/min, UVλ _max 230nm, MeCN- H ₂ O 45:55, retention time 15.0min.

Example 2:

PenicilfuranoneA structure analysis:

Compound PenicilfuranoneA, golden needle-like crystals, [α] ²² D–6 (c0.2, MeOH). The ESI-MS spectrum gives the quasi-molecular ion peak as m/z509[M+Na] ⁺ , combined with high-resolution positive ion HR-ESI-MS (m/z[M+Na] ⁺ 509.1792, the calculated value is C ₂₆ H ₃₀ O ₉ Na, 509.1782) and the information provided by ¹³ CNMR and DEPT spectra, it is determined that its molecular formula is C ₂₆ H ₃₀ O ₉ , and its degree of unsaturation is 12. The UV spectrum has absorption at 196 (4.5), 222 (4.6), 359 (4.0), 495 (2.6) nm, indicating that there are large conjugated groups in the molecule. Analysis of ¹ H and ¹³ CNMR spectra (Table 1), combined with HSQC, HMBC, ¹ H- ¹ HCOSY, and ROESY spectra showed that the structure of the compound PenicilfuranoneA was obtained. Finally, the above analysis was further confirmed by X-single crystal diffraction analysis, and its stereo configuration was confirmed (Fig. 2). The X-single crystal diffraction data of PenicilfuranoneA have been uploaded to the Cambridge University single crystal database www.ccdc.cam.ac.uk , and its accession number is: CCDC1042018.

Table 1. NMR spectral data of PenicilfuranoneA (δinppm, JinHz)

^a Tested on 600MHz instrument, the solvent is DMSO-d ₆ . ^b Tested on 600MHz instrument, the solvent is acetone-d ₆ .

Example 3:

Toxicity test of PenicilfuranoneA on normal human hepatocytes:

Normal human liver cells (L-02 cell line) were purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; RPMI1640 medium was produced by Hyclone; fetal bovine serum was produced by Gibco; dimethylsulfoxide (DMSO) and thiazole Blue (MTT) is the product of sigma company; automatic microplate reader (U.S., Thermo company); constant temperature _CO incubator (U.S., Thermo company); normal human hepatocyte L-02 cell line uses RPMI1640 medium (containing 10% Inactivated fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin) were routinely subcultured in an incubator at 37° C., saturated air humidity, and containing 5% CO ₂ . Take the L-02 cell line in the exponential growth phase, centrifuge at 1000×g for 5 minutes, discard the supernatant, mix well with the corresponding medium to make a cell suspension, count and dilute to a concentration of ³ ×104 cells/mL The cell suspension was inoculated in a 96-well plate, 200 μL per well, placed in a cell culture incubator for 24 hours and administered, and the cell control group and 11 concentrations of PenicilfuranoneA sample groups (0.125, 0.25, 0.5, 1, 2, 4 . 5mg/mL stock solution), continue to incubate in the cell culture incubator for 4h, centrifuge and discard the supernatant, add 100uL DMSO to each well, shake slightly to dissolve the purple crystals completely, and measure the absorbance of each well at 570nm with a microplate reader ( OD value), calculate the average OD value of 3 duplicate wells, and calculate the cell survival rate: cell survival rate (%)=(mean OD value of cells in the administration group-mean background OD)/(mean OD value of cells in the control group-mean background OD )×100%.

Hepatic cell injury is often considered to be an inducing factor for liver fibrosis. Therefore, drugs can prevent the process of liver fibrosis by protecting liver cells, at least by themselves, they should not be toxic to liver cells. The results of MTT test found that after being treated with various concentrations of PenicilfuranoneA for 48 hours, at a very high concentration (32μM), human normal liver cells did not appear to significantly inhibit the activity, which means that at a concentration lower than 32μM, PenicilfuranoneA did not See obvious liver cytotoxicity, the results are shown in Figure 3.

Example 4:

The effect of Penicilfuranone A on the activation of hepatic stellate cells and the expression of extracellular matrix components stimulated by TGF-β1 was detected by western blot

The human hepatic stellate cell LX-2 cell line was quoted from the China Center for Type Culture Collection; the rat hepatic stellate cell T6 cell line was cited from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; cultured in DMEM medium and RPMI1640 respectively Substratum (containing 10% inactivated fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin) was routinely subcultured in an incubator containing 5% CO ₂ at 37°C. Hepatic stellate cell lines LX-2 and T6 in the logarithmic growth phase were seeded in 6-well culture plates with ⁵ ×104 cells/well. After the cells were cultured overnight, they were cultured synchronously with corresponding serum-free medium. After 24 hours, the medium was changed in groups, and recombinant human TGF-β1 (Peprotech, USA) with a final concentration of 10 ng/ml and different concentrations of Penicilfuranone A (1, 2 and 4 μM) were added respectively, and a solvent control group (0.5% DMSO) was respectively set up. and SB431542 (TGF-β1 type I receptor kinase inhibitor, American Cayman Company) positive control group with a concentration of 10 μM. Cells were collected after 48 hours of drug action, lysed with protein lysate, centrifuged at 12000×g at 4°C for 30 minutes at low temperature, and the supernatant was collected. The protein concentration of the supernatant was determined by BCA method. Add an equal volume of 2×SDS loading buffer (125mmol/LTris-HCl, 20% glycerol, 0.01% bromophenol blue, 4% SDS, 200mmol/LDTT), adjust the loading protein concentration of each group to be consistent, and heat in a boiling water bath 5min, SDS-polyacrylamide gel electrophoresis, transfer membrane, TBST containing 5% skimmed milk powder, after blocking for 1h, add α-smooth muscle actin (α-SMA), vimentin (vimentin), type I collagen (typeIcollagen) respectively ), connective tissue growth factor (CTGF) and internal control β-actin antibody, incubated overnight at 4°C. Wash 3 times with TBST, add horseradish enzyme-labeled goat anti-rabbit IgG secondary antibody, react with the membrane for 1 h at 37°C, wash the membrane 3 times with TBST, perform chemiluminescence according to the instructions of the ECL kit, and analyze the expression of related proteins.

After activation, hepatic stellate cells can produce a large amount of extracellular matrix components, so they play a key role in the occurrence and development of liver fibrosis. TGF-β1 is the strongest factor known to induce the activation of hepatic stellate cells. It not only induces the activation of hepatic stellate cells, but also up-regulates the expression levels of cell markers α-SMA and vimentin, and also increases the typeIcollagen-based cells in the liver. Synthesis of extracellular matrix. Western blot results showed that after Penicilfuranone A treated hepatic stellate cells for 48 hours, it could significantly reduce the expression of TGF-β1-induced cell activation marker proteins α-SMA and vimentin, and at the same time significantly reduce the ability of hepatic stellate cells to synthesize typeIcollagen, which down-regulates vimentin The expression ability is even stronger than the positive control drug SB431542. In addition, CTGF, as the most important regulatory protein downstream of TGF-β1, can cooperate with TGF-β1 to promote the massive production of extracellular matrix in liver tissue. The results of Western blot also found that the expression level of CTGF protein in hepatic stellate cells was significantly reduced after the treatment of PenicilfuranoneA dose groups compared with the TGF-β1 induction group. The above results suggest that Penicilfuranone A has an obvious anti-hepatic fibrosis effect, and its mechanism is related to blocking the excessive activation of hepatic stellate cells and the formation of extracellular matrix stimulated by TGF-β1. The results are shown in Figure 4.

Example 5:

Penicilfuranone A was prepared according to Example 1, and the excipient was added according to the ratio of its weight to the excipient of 1:1, and then granulated and compressed into tablets.

Embodiment 6:

Penicilfuranone A was prepared according to Example 1, and excipients were added at a weight ratio of 1:2 to the excipients, granulated and compressed into tablets.

Embodiment 7:

Prepare Penicilfuranone A according to Example 1, and make capsules according to the conventional capsule preparation method.

Embodiment 8:

Obtain Penicilfuranone A according to embodiment 1, then make tablet according to the following method:

Embodiment 9:

Capsules: Penicilfuranone A, 100mg

Appropriate amount of starch

Magnesium Stearate Appropriate amount

Preparation method: mix Penicilfuranone A with auxiliary agents, sieve, mix evenly in a suitable container, and put the obtained mixture into hard gelatin capsules.

Example 10:

Preparation method: Add one ingredient at a time to an appropriate volume of double-distilled water with stirring until completely decomposed, and then add another ingredient. After making up to 2 ml with water, the solution is filtered on a sterile filter, filled into bottles and divided according to the appropriate doses.

Example 11:

Dropping pills: Penicilfuranone A1g

Polyethylene glycol 60009g

Preparation method: Preparation of PenicilfuranoneA and Polyethylene Glycol 6000 melt solution: Weigh PenicilfuranoneA according to the above-mentioned prescription amount, add appropriate amount of absolute ethanol, after slightly heating to dissolve, add the prescription amount into the Polyethylene Glycol melt solution (60 ℃ water bath insulation ), stir and mix evenly until the ethanol is completely evaporated, and keep it in a water bath at 60°C for 30 minutes. Under the condition of 85 ℃, control the dropping speed, drip into the condensate drop by drop, wait for the condensation to be complete, pour off the condensate, collect the dropping pills, drain and remove the condensate on the pills with filter paper, place them in a silica gel desiccator or Let it dry naturally.

Claims (9)

1. the mould furanone A compound shown in following structural formula (I) or its pharmacy acceptable salt,

2. pharmaceutical composition, it comprises mould furanone A compound according to claim 1 or its pharmacy acceptable salt and the pharmaceutically acceptable carrier of at least one and/or thinner.

3. mould furanone A compound as claimed in claim 1 or its pharmacy acceptable salt, wherein said pharmacy acceptable salt is hydrochloride, hydrobromate, nitrate, vitriol, phosphoric acid salt, tartrate, Citrate trianion, formate, acetate, oxalate.

4. the preparation method of mould furanone A compound according to claim 1 or its pharmacy acceptable salt, get mould furanone APenicilliumdaleae fermented product, with organic solvent extraction, concentrate to obtain medicinal extract, be extracted with ethyl acetate respectively, silica gel column chromatography, preparative high performance liquid chromatography method obtains mould furanone A compound.

5. the preparation method of mould furanone A compound as claimed in claim 4 or its pharmacy acceptable salt, it is characterized in that getting Penicilliumdaleae mycelium is inoculated in PDA in sterilizing potato dextrose broth, shake five days in shaking table with 170 rpms under room temperature, make seed liquor, seed liquor is transferred in the rice medium of sterilizing, sterile culture 40 days under 28 DEG C of conditions, the mycelium after 40 days is cultivated with ethyl acetate extraction and application rice medium, extract four times altogether, obtain ethyl acetate extract, utilize silica gel column chromatography, high performance liquid chromatography mass spectrometry, preparative high performance liquid chromatography process ethyl acetate extract, finally obtain mould furanone A, its preparative liquid chromatography condition is: 12mL/min, UV λ _max230nm, MeCN-H ₂o45:55, retention time 15.0min.

6. the mould furanone A compound as described in claim 4 or 5 or the preparation method of its pharmacy acceptable salt, it is characterized in that mould furanone A compound further with suitable sour salify, suitable acid be selected from hydrochloric acid, Hydrogen bromide, nitric acid, sulfuric acid, phosphoric acid, tartrate, citric acid, formic acid, acetic acid, oxalic acid or other be applicable to organic acid or mineral acid.

7. mould furanone A compound according to claim 1 or the application of pharmaceutical composition according to claim 2 in the medicine preparing anti-hepatic fibrosis.

8. pharmaceutical composition according to claim 2, is characterized in that its dosage form as suitable for oral administration or drug administration by injection.

9. pharmaceutical composition according to claim 8, the dosage form of wherein said oral administration is tablet, slow releasing tablet, controlled release tablet, lozenge, hard or soft capsule, dripping pill, micropill, water-based or oil suspension, emulsion, dispersible powder or granule, oral liquid, syrup or elixir, and the dosage form of described drug administration by injection is water-based or oily solution, sterilized powder, liposome, emulsion, microemulsion, nano-emulsion or the micro-capsule of sterilizing.