阅读说明：本技术 一种聚酮化合物pyoluteorin及制备方法与用途 (Polyketide pyoluteorin and preparation method and application thereof ) 是由 张卫东 沈云亨 丁婷 李慧梁 徐希科 于 2019-09-27 设计创作，主要内容包括：本发明公开了一种聚酮化合物pyoluteorin,化学结构如式I所示：<Image he="354" wi="454" file="DDA0002218639490000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>所述聚酮化合物pyoluteorin是从湿地土壤来源的黑曲霉中分离制备得到。本发明的聚酮化合物pyoluteorin对人肺腺癌细胞、人结肠癌细胞、人乳腺癌细胞、人三阴性乳腺癌细胞和人三阳性乳腺癌细胞具有较好的细胞毒活性,其中对人三阴性乳腺癌细胞MDA-MB-231的细胞毒活性尤其显著,且对正常乳腺上皮细胞MCF-10A毒性较小,故可用于制备抗肿瘤药物。(The invention discloses a polyketide pyoluteorin, the chemical structure of which is shown as formula I: the polyketide pyoluteorin is obtained by separating and preparing from aspergillus niger derived from wetland soil. The polyketide pyoluteorin has good cytotoxic activity on human lung adenocarcinoma cells, human colon cancer cells, human breast cancer cells, human triple-negative breast cancer cells and human triple-positive breast cancer cells, wherein the polyketide pyoluteorin has good cytotoxic activity on human triple-negative breast cancer cells MDA-MBThe-231 has particularly remarkable cytotoxic activity and has small toxicity to normal mammary epithelial cells MCF-10A, so that the derivative can be used for preparing antitumor drugs.)

1. A polyketide pyoluteorin having the chemical structure shown in formula I:

2. the polyketide pyrolutorin according to claim 1, wherein said polyketide pyrolutorin is isolated from aspergillus niger derived from wetland soil.

3. A process for the preparation of the polyketide pyoluteorin according to claim 1 or 2, comprising the steps of:

filtering Aspergillus niger fermentation liquid to remove mycelium, adding ethyl acetate to carry out liquid-liquid extraction, and concentrating to obtain an ethyl acetate part; the extracted fermentation liquor passes through macroporous resin and is eluted by methanol to obtain a large-polarity part; and combining the ethyl acetate part and the large polar part, separating and purifying to obtain the target compound polyketide pyoluteorin.

4. The method of claim 3, wherein the Aspergillus niger broth is prepared by the steps of:

inoculating the purified aspergillus niger strain into an improved martin solid culture medium, culturing for 7 days at the temperature of 28 ℃, cutting an agar plate containing the strain into small blocks after the culture is finished, inoculating the small blocks into 2L conical flasks, adding 1L of the improved martin liquid culture medium which is sterilized into each conical flask, and fermenting for 28 days at the temperature of 28 ℃ to obtain an aspergillus niger fermentation liquid.

5. The method of claim 4, wherein the Aspergillus niger strain is prepared by a method comprising the steps of:

adding distilled water into fresh wetland soil, diluting and mixing uniformly at a ratio of 1:100, taking 100 mu L of soil diluent, coating the soil diluent on a separation culture medium, culturing for 4 days at the temperature of 28 ℃, picking and purifying after bacterial strains grow out, and obtaining the aspergillus niger strains.

6. The method of claim 5, wherein the isolation medium comprises the following components: improving a martin solid culture medium: 5.0g/L peptone, 1.0g/L dipotassium hydrogen phosphate, 0.5g/L magnesium sulfate, 2.0g/L yeast extract powder, 20.0g/L glucose and 14.0g/L agar; 50mg/L potassium dichromate and 20mg/L nalidixic acid are added into the improved martin solid culture medium as bacteriostatic agents.

7. A process for the preparation of the polyketide pyoluteorin according to claim 3, wherein said isolation and purification comprises the steps of:

subjecting to middle and low pressure reverse phase column chromatography with ODS and Sephadex LH-20 as stationary phase and MeOH-H₂The O10% -100% gave 32 components according to the difference in elution gradient: E1-E1610% -50%; E17-E2850% -100%; E29-E32100%; the component E21 was purified by Sephadex LH-20 with MeOH-H₂Collecting 13 components E21-1-E21-13 by isocratic elution of O70%; wherein the component E21-13 is a target compound, pyoluteorin.

8. Use of a polyketide pyoluteorin according to claim 1 or 2 in the preparation of an anti-tumour medicament.

9. The use of the polyketide pyoluteorin according to claim 8 in the preparation of an anti-tumor medicament, wherein said tumor is breast cancer, triple negative breast cancer, lung adenocarcinoma, colon cancer.

10. The use of a polyketide pyrolutorin in the preparation of an anti-tumor medicament according to claim 8, wherein said anti-tumor medicament is a medicament or pharmaceutical composition comprising said polyketide pyrolutorin as an active ingredient.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a polyketide pyoluteorin separated from Aspergillus niger, a preparation method thereof, and application of the polyketide pyoluteorin in preparation of anti-cancer drugs.

Background

There are more than 180 species of Aspergillus of Mucor, and a large number of secondary metabolites with novel structure and better activity are isolated from this Fungus, and the types of compounds are mostly alkaloids, terpenoids, cytochalasin and lignans (Happi G M, Kouam S F, Talontsi F M, et al. ChemInformim Abstract: A New Dimeric sodium-gamma-pyrone from an Endophylletic fungi, Aspergillus niger AKRNAs specific with the Roots of J.]Chemnform, 2015,70(9): 625-. Aspergillus niger is an attractive species of Aspergillus and produces secondary metabolites with good chemical diversity and biological activity. Naphtho-gamma-pyrones, asperazines and fumonisins isolated from Aspergillus niger have significant antibacterial and antitumor activity (Pan-Pan Z, ShuangS, Jia-Jia L, et al. A. new diphenylolic isocyanate from the marine-derived fungi Aspergillus niger.02[ J-Pan Z, ShuangS, Jia-Jia L]Journal of Asian Natural Products Research,2018: 1-7.). For example, the naphtho-gamma-pyrone compound fonsecinones C isolated from Aspergillus niger 2HL-M-8 has good cytotoxicity and IC for human lung adenocarcinoma cells A549, leukemia cells HL-60 and gastric carcinoma cells MGC-803₅₀Values of 2.8, 0.8 and 2.2. mu.M (Li D H, Han T, Guan L P, et al. New photopheres from wheat-derived fungi niger 2HL-M-8and the human viral anti-proliferative activity, [ J ] respectively]Natural Product Research,2016,30(10): 1116). A novel furan ester derivative isolated from Aspergillus niger BRF074 can significantly inhibit the proliferation of colon cancer cells HCT-116 (Uchoa P K S, Pimenta AT A, Braz-Filho R, et al]Natural Product Research,2017: 1-5.). Previous studies have shown that Aspergillus niger secondary metabolites are presentHas wide application prospect in developing novel antitumor drugs.

In view of the remarkable anti-tumor activity of aspergillus niger secondary metabolites and the less research on soil-derived aspergillus niger compounds in the prior art, the research on the system chemical components of the aspergillus niger derived from wetland soil is urgently needed so as to separate and obtain the compound with better anti-tumor activity.

Disclosure of Invention

The first purpose of the invention is to provide a polyketide pyoluteorin which is a secondary metabolite with remarkable activity and is separated from aspergillus niger of wetland soil sources.

The second purpose of the invention is to provide a preparation method of the polyketide pyoluteorin.

The third purpose of the invention is to provide an application of the polyketide pyoluteorin in preparing an anti-tumor drug.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the invention carries out systematic research on a secondary metabolite of Aspergillus niger from wetland soil, and finds a polyketide pyoluteorin. The in vitro cytotoxic activity screening shows that the compound has better cytotoxic activity on human lung adenocarcinoma cells (A549), human colon cancer cells (HCT116), human breast cancer cells (MCF-7, HCC1954), human triple negative breast cancer cells (MDA-MB-231, MDA-MB-468) and human triple positive breast cancer cells (BT474), wherein the cytotoxic activity on the human triple negative breast cancer cells MDA-MB-231 is particularly obvious, and the compound has lower toxicity on normal breast epithelial cells MCF-10A. The research on the action mechanism of the cell takes MDA-MB-231 as a model shows that pyoluteorin can inhibit the growth of the cell by inducing the G2/M phase cycle block and apoptosis of the cell, and the apoptosis is related to the reduction of Mitochondrial Membrane Potential (MMP) and the increase of the content of Reactive Oxygen Species (ROS) in the cell. Therefore, the polyketide pyoluteorin has great potential to become an anti-tumor drug.

In a first aspect, the present invention provides a polyketide pyoluteorin, the chemical structure of which is shown in formula I:

the polyketide pyoluteorin is prepared by separating from Aspergillus niger (Aspergillus niger) which is a wetland soil source.

The second aspect of the invention provides a preparation method of the polyketide pyoluteorin, which comprises the following steps:

filtering Aspergillus niger fermentation liquid to remove mycelium, adding ethyl acetate to carry out liquid-liquid extraction, and concentrating to obtain an ethyl acetate part; the extracted fermentation liquor passes through macroporous resin and is eluted by methanol to obtain a large-polarity part; and combining the ethyl acetate part and the large polar part, separating and purifying to obtain the target compound polyketide pyoluteorin.

The preparation method of the Aspergillus niger fermentation liquid comprises the following steps:

inoculating the purified aspergillus niger strain into an improved martin solid culture medium, culturing for 7 days at the temperature of 28 ℃, cutting an agar plate containing the strain into small blocks after the culture is finished, inoculating the small blocks into 2L conical flasks, adding 1L of the improved martin liquid culture medium which is sterilized into each conical flask, and fermenting for 28 days at the temperature of 28 ℃ to obtain an aspergillus niger fermentation liquid.

The preparation method of the aspergillus niger strain comprises the following steps:

adding distilled water into fresh wetland soil, diluting and mixing uniformly at a ratio of 1:100, taking 100 mu L of soil diluent, coating the soil diluent on a separation culture medium, culturing for 4 days at the temperature of 28 ℃, picking and purifying after bacterial strains grow out, and obtaining Aspergillus niger bacterial strains.

The composition of the isolation medium is as follows: modified Martin solid medium (5.0g/L peptone, 1.0g/L dipotassium hydrogen phosphate, 0.5g/L magnesium sulfate, 2.0g/L yeast extract powder, 20.0g/L glucose, 14.0g/L agar, etc.), and potassium dichromate (50mg/L) and nalidixic acid (20mg/L) were added to the above modified Martin solid medium as bacteriostatic agents.

The separation and purification comprises the following steps:

subjecting to middle and low pressure reverse phase column chromatography with ODS and Sephadex LH-20 as stationary phase and MeOH-H₂The O10% -100% gave 32 components according to the difference in elution gradient: E1-E1610% -50%; E17-E2850% -100%; E29-E32100%; the component E21 was purified by Sephadex LH-20 with MeOH-H₂Collecting 13 components E21-1-E21-13 by isocratic elution of O70%; wherein the component E21-13 is a target compound, pyoluteorin.

The third aspect of the invention provides an application of the polyketide pyoluteorin in preparation of an anti-tumor drug.

The tumor is breast cancer, triple negative breast cancer, lung adenocarcinoma, colon cancer and the like, especially triple negative breast cancer.

The anti-tumor drug is a drug or a pharmaceutical composition which is composed of the polyketide pyoluteorin as an active ingredient, and comprises a pharmaceutical carrier composition for preparing anti-tumor related drugs; can be administered orally, parenterally, directly in brain, through nasal cavity and brain targeting, by genetic engineering, by receptor mediated transport or by other local routes; the administration dosage form can be pharmaceutically acceptable dosage forms such as tablet (dispersible tablet, buccal tablet, orally disintegrating tablet, sustained release tablet), granule, pill, capsule, emulsion, solution, suspension, injection, instillation, powder injection or aerosol, etc.

The polyketide pyoluteorin has good cytotoxic activity on human lung adenocarcinoma cells (A549), human colon cancer cells (HCT116), human breast cancer cells (MCF-7, HCC1954), human triple negative breast cancer cells (MDA-MB-231, MDA-MB-468) and human triple positive breast cancer cells (BT474), wherein the cytotoxic activity on the human triple negative breast cancer cells MDA-MB-231 is particularly obvious, and the polyketide pyoluteorin has low toxicity on normal breast epithelial cells MCF-10A, so that the polyketide pyoluteorin can be used for preparing antitumor drugs.

Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:

the polyketide pyoluteorin provided by the invention is derived from the microorganism Aspergillus niger, and the mass fermentation and separation of the microorganism are more environment-friendly and resource renewable compared with plant extraction. Chemically, polyketide pyoluteorin has mature synthesis conditions, so that the polyketide has various sources and is rich in resources. In the second aspect, the polyketide pyoluteorin has very obvious cytotoxic activity on human triple negative breast cancer MDA-MB-231 cells, has low toxicity on normal mammary epithelial cells MCF-10A, and is expected to be developed into a medicament for treating triple negative breast cancer.

Drawings

FIG. 1 is a graph showing the inhibition of MDA-MB-231 cells by polyketide pyoluorin at concentrations of 0.032uM, 0.16uM, 0.8uM, 4uM, 20uM, and 100uM for 24, 48, and 72 hours.

FIG. 2 shows that polyketide pyoluteorin is capable of inducing cell G₂Schematic representation of the retarding effect of the/M phase. The experimental data are presented as mean ± sem using two-way analysis of variance (two-way ANOVA), Tukey test, P<0.05 is significantly different (, P)<0.05；**,P<0.01vs.Vehicle)。

FIG. 3 is a schematic representation of MDA-MB-231 apoptosis induced by different doses of polyketide pyoluteorin.

FIG. 4 is a statistical representation of the trend of different doses of the polyketide pyoluteorin in inducing MDA-MB-231 apoptosis. The experimental data are presented as mean ± standard error, with one-way ANOVA, Duncan test, P <0.05 being significantly different (.;, P < 0.05;. P <0.01,. P <0.001vs. vehicle).

FIG. 5, wherein A is a schematic diagram of laser confocal fluorescence image of mitochondrial membrane potential of MDA-MB-231 cells after action of polyketide pyoluorin at different concentrations (a. blank; b.0.1. mu.M pyoluorin; c.0.3. mu.M pyoluorin; d.1. mu.M pyoluorin; e.3. mu.M pyoluorin; f.10. mu.M pyoluorin); b is a schematic diagram of the ratio of JC-1 monomer fluorescence intensity to JC-1 polymer fluorescence intensity and different concentrations of polyketide pyoluteorin.

FIG. 6 is a graph showing the intracellular ROS content of MDA-MB-231 after the action of different concentrations of polyketide pyoluteorin; wherein A is a schematic diagram of DCFH-DA fluorescence intensity detected by a flow cytometer; b is a diagram of the reactive oxygen species ROS content in MDA-MB-231 cells. The experimental data are presented as mean ± standard error, with One-way analysis of variance (One-WayANOVA), Duncan test, P <0.05 was significantly different (.; P < 0.05;. P <0.01,. P <0.001vs. vehicle).

FIG. 7 is a schematic diagram of laser confocal measurement of ROS in MDA-MB-231 cells after action of polyketide pyoluteririn at different concentrations; blank control; b is 0.1. mu.M of pyroluteorin; c is 0.3. mu.M of pyroluteorin; d is 1. mu.M of pyroluteorin; e is 3. mu.M of pyroluteorin; f is 10. mu.M of pyroluteorin.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.