A type II polyketide Actketone, its preparation method and application

Abstract

The present invention discloses a type II polyketide compound actketone, its preparation method and application. The present invention is produced by Actinokineospora diospyrosa NBRC 15665 strain, and the chemical structural formula of the type II polyketide compound actketone is shown in formula (I): #imgabs0# The results of biological activity data show that the type II polyketide compound actketone of the present invention has significant inhibitory effect on breast cancer MCF-7 cells, and its IC 50 value is 26.8 μM.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and is derived from the strain Actinokineospora diospyrosa NBRC15665 of actinomycetes. Specifically, it relates to a type II polyketide compound actketone derived from actinomycetes, its preparation method and application. Background Art

[0002] Natural products are metabolites produced by organisms in nature during the process of evolution to adapt to the environment, compete antagonistically, communicate, resist external invasion, transmit signals, etc. In this sense, natural products themselves are endowed with specific biological activities. Natural products have also always been regarded as an important source for the discovery of new drugs.

[0003] Currently, there is a lack of a type II polyketide compound actketone derived from actinomycetes, its preparation method and application. Summary of the Invention

[0004] The purpose of the present invention is to solve the problems of the prior art, and to provide a type II polyketide compound actketone derived from actinomycetes, its preparation method and application.

[0005] To achieve the above purpose, the present invention adopts the following technical scheme: A type II polyketide compound actketone of the present invention, the chemical structural formula of the type II polyketide compound actketone is shown in formula (I):

[0006]

[0007] The preparation method of the type II polyketide compound actketone of the present invention includes the following steps:

[0008] (1) The actinomycetes Actinokineospora diospyrosa NBRC 15665 strain is cultured by plate fermentation with No. 38.

[0009] (2) The culture medium obtained in step (1) is cut into small pieces, put into medium F, and cultured on a shaker at 30 °C and 120 rpm for 7 days.

[0010] (3) The culture medium obtained in step (2) is extracted with ethyl acetate and concentrated to obtain a crude extract F1.

[0011] (3) The crude extract F1 is subjected to normal-phase silica gel column chromatography. First, it is eluted with petroleum ether and ethyl acetate, and then eluted with dichloromethane and methanol, and a total of 10 fractions are obtained.

[0012] (4) The fourth eluted component was further separated and purified by reverse-phase silica gel, gel column chromatography, and HPLC to obtain the type II polyketide compound actketone.

[0013] Furthermore, in step (1), the culture conditions for plate fermentation in No. 38 plate were culturing at 26 - 30 °C for 7 days.

[0014] Furthermore, in step (4), the HPLC chromatographic conditions were as follows: using a reverse-phase chromatographic column, mobile phase A: water, mobile phase B: acetonitrile, and the gradient elution program was: the mass percentage of mobile phase A was 5%, the mass percentage of mobile phase B was 95%, and the time was 18 min; the HPLC separation and purification process was as follows: semi-HPLC semi-preparative reverse-phase high-performance liquid chromatography: ODS-2 Hypersil column, 5 μm, 250 mm × 10 mm, the mobile phase was: acetonitrile and water with a volume ratio of 4:1, gradient elution at a flow rate of 2 mL / min for 18 min, the pump model was Hitachi pump L-7100, and the ultraviolet lamp model was UV detector L-7400.

[0015] Use of the type II polyketide compound actketone described in the present invention in the preparation of drugs for treating breast cancer.

[0016] Furthermore, use of the type II polyketide compound actketone in the preparation of lead drugs for treating breast cancer diseases.

[0017] Furthermore, the type II polyketide compound actketone can significantly inhibit breast cancer MCF-7 cells.

[0018] Beneficial effects: The present invention for the first time discovered a type II polyketide compound actketone from the fermentation products of the actinomycete Actinokineospora diospyrosa NBRC 15665 strain. Its cytotoxic activity data shows that actketone can significantly inhibit breast cancer MCF-7 cells, and its IC50 value is 26.8 μM, and it can be further used as a lead drug for treating breast cancer diseases. Description of the Drawings

[0019] Figure 1 is 1 1H NMR spectrum.

[0020] Figure 2 position 13 13C NMR spectrum.

[0021] Figure 3 is the DEPT-135 NMR spectrum.

[0022] Figure 4 It is an HSQC NMR spectrum.

[0023] Figure 5 is 1 H- 1 It is an H-H COSY NMR spectrum.

[0024] Figure 6 It is an HMBC NMR spectrum.

[0025] Figure 7 It is a NOESY NMR spectrum. Specific embodiments

[0026] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments.

[0027] Example 1

[0028] A type II polyketide compound actketone of the present invention, and the chemical structural formula of the type II polyketide compound actketone is shown in formula (I):

[0029]

[0030] A preparation method of a type II polyketide compound actketone of the present invention includes the following steps:

[0031] (1) The strain Actinokineospora diospyrosa NBRC 15665 is cultured by plate fermentation with No. 38; the strain Actinokineospora diospyrosa NBRC 15665 is purchased from the NBRC culture collection center in Japan.

[0032] (2) Cut the culture medium obtained in step (1) into small pieces, put it into medium F, and culture it on a shaker at 30 °C and 120 rpm for 7 days;

[0033] (3) Extract the culture medium obtained in step (2) with ethyl acetate and concentrate to obtain a crude extract F1;

[0034] (3) Perform normal-phase silica gel column chromatography on the crude extract F1, first elute with petroleum ether and ethyl acetate, and then elute with dichloromethane and methanol to obtain a total of 10 components;

[0035] (4) Then, the fourth eluted component was separated and purified by reverse silica gel, gel column chromatography, and HPLC to obtain a type II polyketide compound, actketone. The HPLC chromatographic conditions were as follows: a reverse-phase chromatographic column was used. Mobile phase A: water; Mobile phase B: acetonitrile. The isocratic elution program was as follows: the mass percentage of Mobile phase A was 5%, the mass percentage of Mobile phase B was 95%, and the time was 18 min. The HPLC separation and purification process was as follows: semi-HPLC semi-preparative reverse-phase high-performance liquid chromatography: ODS-2 Hypersil column, 5 μm, 250 mm × 10 mm, mobile phase: acetonitrile-water volume ratio = 4:1, gradient elution at a flow rate of 2 mL / min for 18 min; the pump model could be Hitachi pump L-7100, and the UV lamp model could be UV detector L-7400.

[0036] Use of a type II polyketide compound actketone according to the present invention in the preparation of a lead drug for treating breast cancer.

[0037] The type II polyketide compound actketone according to the present invention can significantly inhibit breast cancer MCF-7 cells, and its IC50 value is 26.8 μM.

[0038] Example 2

[0039] The difference between Example 2 and Example 1 is as follows:

[0040] A preparation method of a type II polyketide compound actketone according to the present invention includes the following steps:

[0041] In step (1), the Actinokineospora diospyrosa NBRC 15665 strain was cultured by plate fermentation on a No. 38 plate; the culture conditions for plate fermentation on the No. 38 plate were culturing at 30 °C for 7 days.

[0042] Test Example 1

[0043] Activation of Actinokineospora diospyrosa NBRC 15665 strain.

[0044] The frozen dry powder of the Actinokineospora diospyrosa NBRC 15665 strain was spread on a No. 38 plate medium (peptone: 20.0 g, dipotassium hydrogen phosphate: 1.5 g, magnesium sulfate: 1.5 g, agar: 15.0 g, water 1 L), and cultured in an incubator at 30 °C to obtain the actinomycete.

[0045] Test Example 2

[0046] Fermentation of Actinokineospora diospyrosa NBRC 15665 strain in medium F

[0047] Transfer the strain Actinokineospora diospyrosa NBRC 15665 to plate medium 38#, and culture it in an incubator at 30°C for 7 days. After the mycelium covers the plate, cut it into small squares, transfer them to medium F, and culture them on a shaker at 30°C and 120 rpm for 7 days.

[0048] Test Example 3

[0049] Extraction and isolation of actketone

[0050] Add ethyl acetate to extract the medium F obtained in Test Example 2, and concentrate to obtain extract F1. Perform normal-phase silica gel column chromatography on extract F1. First, elute with petroleum ether and ethyl acetate, and then elute with dichloromethane and methanol to obtain 10 fractions; then, subject the fourth eluted fraction to reverse-phase silica gel, gel column chromatography, and semi-HPLC (chromatographic column: Allsphere ODS-2.5mm column), with an acetonitrile-water system, elute at a flow rate of 2 mL / min, and use an isocratic elution of acetonitrile-water volume ratio = 14:1 for 18 min to obtain the actketone (6.0 mg) described in the present invention. The model of the pump can be Hitachi pump L-7100, and the model of the ultraviolet lamp can be UV detector L-7400.

[0051] Test Example 4

[0052] The structural identification of actketone is shown in the appendix Figures 1-7 acautalides A 1 H and 13 The data attribution of 1H and 13C NMR is shown in Table 1:

[0053] Table 1

[0054]

[0055]

[0056] The structure of actketone is as follows:

[0057]

[0058] Test Example 5

[0059] Cytotoxic activity test

[0060] MCF-7 cells were cultured in MEM complete medium containing 10% fetal bovine serum by volume in a 37 °C incubator containing 5% CO2 by volume, passaged at a ratio of 4:1, and cells in the 3rd to 7th passages were selected for the experiment. Before the toxicity experiment, the cells were pretreated by dissociation with trypsin solution at a volume fraction of 0.25%, and then seeded at a cell density of 10 4 cells / well. After adherent growth for 24 h, the MEM culture medium containing different concentrations of the sample was replaced, and co-incubated for 24 h. Subsequently, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution (5 mg / mL) was added to each well, and after continued culture for 4 h, the original solution was aspirated and 200 μL of DMSO was added, and the absorbance value of each well at 550 nm was read using a 96-well reader. The absorbance value of the blank group was set as 100%, and the cell viability of samples at different concentrations was calculated. Each group was repeated 3 times, and the average value was taken and the error number was calculated.

[0061] The cytotoxicity activity experiment of the present invention shows that actketone has significant inhibitory activity against breast cancer cell cytotoxicity and can be further used as a lead drug for the development of drugs for treating breast cancer diseases. The structure of actketone described in the present invention was determined based on mass spectrometry and nuclear magnetic resonance spectroscopy.

[0062] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A type II polyketide compound actketone, characterized in that: The chemical structural formula of the type II polyketide actketone is shown in formula (I):

2. The preparation method of the type II polyketide compound actketone according to claim 1, characterized in that It includes the following steps: (1) The strain Actinokineospora diospyrosa NBRC 15665 of actinomycetes is cultured by plate fermentation using No. 38 medium. (2) The culture medium obtained in step (1) is cut into small pieces, put into medium F, and cultured in a shaker at 30 °C and 120 rpm for 7 days. (3) The culture medium obtained in step (2) is extracted with ethyl acetate and concentrated to obtain a crude extract F1. (3) The crude extract F1 is subjected to normal-phase silica gel column chromatography. First, it is eluted with petroleum ether and ethyl acetate, and then with dichloromethane and methanol, and a total of 10 fractions are obtained. (4) Then, the fourth eluted fraction is separated and purified by reverse-phase silica gel, gel column chromatography and HPLC to obtain the type II polyketide actketone.

3. The preparation method of the type II polyketide actketone according to claim 2, characterized in that: In step (1), the culture conditions for plate fermentation using No. 38 medium are to culture at 26 - 30 °C for 7 days.

4. The preparation method of the type II polyketide actketone according to claim 2, characterized in that: In step (4), the HPLC chromatographic conditions are as follows: using a reverse-phase chromatographic column, mobile phase A: water, mobile phase B: acetonitrile, and the gradient elution program is: the mass percentage of mobile phase A is 5%, the mass percentage of mobile phase B is 95%, and the time is 18 min; the process of HPLC separation and purification is as follows: semi-HPLC semi-preparative reverse-phase high-performance liquid chromatography: ODS-2 Hypersil column, 5 μm, 250 mm × 10 mm, the mobile phase is: acetonitrile and water with a volume ratio of 4:1, gradient elution at a flow rate of 2 mL / min for 18 min, the model of the pump is Hitachi pump L-7100, and the model of the ultraviolet lamp is UV detector L-7400.

5. Use of the type II polyketide actketone according to any one of claims 1 to 4 in the preparation of a drug for treating breast cancer.

6. The application according to claim 5, wherein: Use of the type II polyketide actketone in the preparation of a lead drug for treating breast cancer diseases.

7. The application according to claim 6, wherein: The type II polyketide actketone can significantly inhibit breast cancer MCF-7 cells.

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