CN110627699B - Polyketide pyoluteorin and preparation method and application thereof - Google Patents

Abstract

The invention discloses a polyketone compound pyoluteorin, the chemical structure is shown in formula I:

The polyketide pyoluteorin is isolated and prepared from Aspergillus niger derived from wetland soil. The polyketide compound pyoluteorin of the present invention 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 human triple-negative The cytotoxic activity of breast cancer cell MDA-MB-231 is particularly significant, and it is less toxic to normal breast epithelial cell MCF-10A, so it can be used for the preparation of anti-tumor drugs.

Description

A kind of polyketone compound pyoluteorin and preparation method and use

technical field

The invention belongs to the technical field of medicine, in particular to a polyketide compound pyoluteorin isolated from Aspergillus niger, a preparation method thereof, and the application of the compound in the preparation of anticancer drugs.

Background technique

There are more than 180 species of Aspergillus fungus in the family Mucoraceae worldwide. A large number of secondary metabolites with novel structure and good activity have been isolated from this genus of fungi. The types of compounds are mostly alkaloids, terpenoids, cellular Relaxin and lignans (Happi GM, Kouam SF, Talontsi FM, et al.ChemInform Abstract: A NewDimeric Naphtho-γ-pyrone from an Endophytic Fungus Aspergillus niger AKRNAssociated with the Roots of Entandrophragma congoense Collected in Cameroon[J]. Cheminform, 2015, 70(9):625-630.). Aspergillus niger is one of the more attractive species of Aspergillus fungi, and its secondary metabolites have good chemical diversity and biological activity. Naphtho-γ-pyrones, asperazines and fumonisins isolated from Aspergillus niger have significant antibacterial and antitumor activities (Pan-Pan Z, ShuangS, Jia-Jia L, et al. A new diphenolic metabolite isolated from the marine-derived fungus Aspergillus niger. 02 [J]. Journal of Asian Natural Products Research, 2018: 1-7.). For example, the naphtho-γ-pyrone compound fonsecinones C isolated from Aspergillus niger 2HL-M-8 has good cytotoxicity against human lung adenocarcinoma cell A549, leukemia cell HL-60 and gastric cancer cell MGC-803 , the IC ₅₀ values were 2.8, 0.8 and 2.2 μM, respectively (Li DH, Han T, Guan LP, et al. Newnaphthopyrones from marine-derived fungus Aspergillus niger 2HL-M-8 and their in vitro antiproliferative activity.[J].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 PKS, Pimenta AT A, Braz-Filho R, et al. Newcytotoxic furan from the marine sediment-derived fungi, Aspergillus niger [J]. Natural Product Research, 2017: 1-5.). Previous studies have shown that Aspergillus niger secondary metabolites have great application prospects in the development of new anti-tumor drugs.

In view of the significant antitumor activity of the secondary metabolites of Aspergillus niger and the lack of research on soil-derived Aspergillus niger compounds in the prior art, it is urgent to carry out systematic chemical composition research on Aspergillus niger derived from wetland soils, in order to isolate and obtain better compounds. Compounds with antitumor activity.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a polyketide compound pyoluteorin, a secondary metabolite with significant activity, isolated from Aspergillus niger derived from wetland soil.

The second object of the present invention is to provide a preparation method of the polyketide compound pyoluteorin.

The third object of the present invention is to provide an application of the polyketide compound pyoluteorin in the preparation of antitumor drugs.

In order to achieve the above object, the technical scheme adopted in the present invention is as follows:

The invention systematically studies the secondary metabolites of a strain of Aspergillus niger from wetland soil, and discovers a polyketide, pyoluteorin. The in vitro cytotoxic activity screening found that the compound was effective against human lung adenocarcinoma cells (A549), human colon cancer cells (HCT116), human breast cancer cells (MCF-7, HCC1954), and human triple-negative breast cancer cells (MDA-MB). -231, MDA-MB-468) and human triple-positive breast cancer cells (BT474) have good cytotoxic activity, among which the cytotoxic activity of human triple-negative breast cancer cells MDA-MB-231 is particularly significant, and the normal Mammary epithelial cells MCF-10A were less toxic. Using MDA-MB-231 cells as a model to study the mechanism of action, the study shows that pyoluteorin can inhibit cell growth by inducing cell G2/M phase cycle arrest and apoptosis, and apoptosis and mitochondrial membrane potential (MMP) decrease and the increase of intracellular reactive oxygen species ROS content. Therefore, the polyketide compound pyoluteorin in the present invention has great potential to become an antitumor drug.

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

The polyketide pyoluteorin is isolated and prepared from Aspergillus niger derived from wetland soil.

A second aspect of the present invention provides a preparation method of the polyketide pyoluteorin, comprising the following steps:

The Aspergillus niger fermentation broth was filtered to remove mycelium, ethyl acetate was added to carry out liquid-liquid extraction, and ethyl acetate fractions were obtained after concentration; the extracted fermentation broth was passed through macroporous resin, and eluted with methanol to obtain macropolar fractions ; Combine the ethyl acetate part and the large polar part, separate and purify to obtain the target compound polyketide pyoluteorin.

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

The purified Aspergillus niger strain was inoculated into the modified Martin solid medium, and cultured at a temperature of 28°C for 7 days. After the cultivation, the agar plate containing the strain was cut into small pieces and inoculated into a 2L conical flask. , each conical flask was added with sterilized 1L modified Martin liquid medium, and fermented for 28 days at a temperature of 28°C to obtain Aspergillus niger fermentation broth.

The preparation method of described Aspergillus niger strain comprises the following steps:

Add fresh wetland soil into distilled water to dilute and mix evenly at a ratio of 1:100, take 100 μL of soil dilution and spread it on the separation medium, cultivate it for 4 days at a temperature of 28 °C, and pick and purify after the strain grows. A strain of Aspergillus niger was obtained.

The components of the separation medium are 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.), potassium dichromate (50mg/L) and nalidixic acid (20mg/L) were added to the above-mentioned modified Martin solid medium as bacteriostatic agents.

The separation and purification include the following steps:

Through medium and low pressure reversed-phase column chromatography, with ODS and Sephadex LH-20 as stationary phase, and MeOH-H ₂ O 10%-100% according to different elution gradients, 32 components were obtained: E1-E16 10%-50% ; E17-E28 50%-100%; E29-E32 100%; Component E21 was collected by Sephadex LH-20 with MeOH-H ₂ O 70% isocratic elution to obtain 13 components E21-1-E21-13; , component E21-13 is the target compound pyoluteorin.

The third aspect of the present invention provides an application of the polyketide compound pyoluteorin in the preparation of antitumor drugs.

The tumor is breast cancer, triple negative breast cancer, lung adenocarcinoma, colon cancer, etc., especially triple negative breast cancer.

The anti-tumor drug is a drug or a pharmaceutical composition composed of the polyketide compound pyoluteorin of the present invention as an active ingredient, including a combination with a pharmaceutical carrier, for preparing an anti-tumor-related drug; Intracerebral direct administration, nasal brain targeted administration, genetic engineering, receptor-mediated transport administration or other local route administration; Sustained-release tablets), granules, pills, capsules, emulsions, solutions, suspensions, injections, drips, powder injections or aerosols and other pharmaceutically acceptable dosage forms.

The polyketide compound pyoluteorin of the present invention is effective against 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) have good cytotoxic activity, among which the cytotoxic activity of human triple-negative breast cancer cells MDA-MB-231 is particularly significant, and the normal breast epithelial cells. MCF-10A is less toxic, so it can be used to prepare antitumor drugs.

Owing to adopting the above-mentioned technical scheme, the present invention has the following advantages and beneficial effects:

The polyketide pyoluteorin provided by the invention comes from the microorganism Aspergillus niger, and the mass fermentation and separation of the microorganism is more environmentally friendly and resource renewable than plant extraction. From a chemical point of view, the polyketide compound pyoluteorin has relatively mature synthesis conditions, so the source of the compound is diverse and abundant. Second, the polyketide compound pyoluteorin has very significant cytotoxic activity against human triple-negative breast cancer MDA-MB-231 cells, and is less toxic to normal breast epithelial cells MCF-10A, and is expected to be developed as a treatment for triple-negative breast cancer medicine.

Description of drawings

Figure 1 is a graph showing the inhibitory effect of polyketide pyoluteorin on the growth of MDA-MB-231 cells, the concentration of 0.032uM, 0.16uM, 0.8uM, 4uM, 20uM, 100uM polyketide pyoluteorin after 24, 48, 72h on MDA - Inhibition rate of MB-231 cells.

Figure 2 is a schematic diagram showing that the polyketide pyoluteorin can induce cell G ₂ /M arrest. The experimental data are expressed as mean ± standard error, using two-way ANOVA, Tukey test, P<0.05 is significant difference (*,P<0.05;**,P<0.01vs.Vehicle) .

Figure 3 is a schematic diagram of the induction of apoptosis of MDA-MB-231 cells by different doses of the polyketide compound pyoluteorin.

Figure 4 is a schematic diagram showing the trend of apoptosis of MDA-MB-231 cells induced by different doses of the polyketide compound pyoluteorin. The experimental data are expressed as mean ± standard error, using one-way ANOVA, Duncan test, P<0.05 is considered significant difference (*, P<0.05; **, P<0.01, *** , P<0.001 vs. Vehicle).

In Figure 5, A is the schematic diagram of the confocal fluorescence image of mitochondrial membrane potential of MDA-MB-231 cells after the action of different concentrations of polyketide compound pyoluteorin (a. blank control; b. 0.1 μM pyoluteorin; c. 0.3 μM pyoluteorin; d. 1 μM pyoluteorin; e. 3 μM pyoluteorin; f. 10 μM pyoluteorin); 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.

Figure 6 is a schematic diagram of the content of reactive oxygen species ROS in MDA-MB-231 cells after the action of different concentrations of the polyketide compound pyoluteorin; A is a schematic diagram of the fluorescence intensity of DCFH-DA detected by flow cytometry; B is a schematic diagram of the intracellular MDA-MB-231 cells Schematic diagram of reactive oxygen species ROS content. The experimental data are expressed as the mean ± standard error, using one-way analysis of variance (One-Way ANOVA), Duncan test, P<0.05 is significant difference (*, P<0.05; **, P<0.01, ***, P<0.001 vs. Vehicle).

Figure 7 is a schematic diagram of the laser confocal detection images of reactive oxygen species ROS in MDA-MB-231 cells after the action of different concentrations of polyketide compound pyoluteorin; A is blank control; B is 0.1 μM pyoluteorin; C is 0.3 μM pyoluteorin; D is 1 μM pyoluteorin ; E is 3 μM pyoluteorin; F is 10 μM pyoluteorin.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below with reference to the preferred embodiments. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

Example 1: Preparation of polyketide pyoluteorin

1. Sample source

From the wetland soil of Shanghai Gongqing National Forest Park.

2. Bacteria fermentation, extraction separation and purification methods

The recovered fresh wetland soil was added to distilled water at a ratio of 1:100 to dilute and mix evenly. Take 100 μL of soil dilution and spread it on the separation medium (separation medium: modified Martin solid medium (5.0g/L peptone, 1.0g/L). L dipotassium hydrogen phosphate, 0.5g/L magnesium sulfate, 2.0g/L yeast extract powder, 20.0g/L glucose, 14.0g/L agar, etc.), add potassium dichromate (50mg/L) to the above modified Martin solid medium /L) and nalidixic acid (20mg/L) as a bacteriostatic agent.), cultured for 4 days at a temperature of 28°C, picked and purified after the strains grew to obtain Aspergillus niger strains .

The above-purified Aspergillus niger strain was inoculated into the 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.), cultured for 7 days at a temperature of 28°C. After the cultivation, the agar plates containing the strains were cut into small pieces and inoculated into 2L conical flasks (2L×32, each conical flask was added with sterilized 1L modified Martin liquid 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, etc.)), fermented at 28°C for 28 days to obtain Aspergillus niger fermentation broth. After the fermentation, the fermentation broth was filtered to remove the mycelium, liquid-liquid extraction was performed 5 times with ethyl acetate (30 L), and the ethyl acetate fraction (12 g) was obtained after concentration. The extracted fermentation broth was passed through a macroporous resin (Japan 30 DIAION HP-20) and eluted with 100% methanol to obtain a large polar part (2g). Merge the above two parts (12g of ethyl acetate part+2g of large polar part), through medium and low pressure reversed-phase column chromatography, and use ODS and Sephadex LH-20 as stationary phase, and use different ratios of methanol and water as eluent, 32 fractions were obtained according to different elution gradients with MeOH-H ₂ O 10%-100%, E1-E16 10%-50%; E17-E28 50%-100%; E29-E32 100%; E1 (532 mg ), E2(2.5g), E3(120mg), E4(110mg), E5(83mg), E6(149mg), E7(96mg), E8(181mg), E9(98mg), E10(132mg), E11( 320mg), E12 (423mg), E13 (89mg), E14 (310mg), E15 (736mg), E16 (203mg) 10%-50%; E17 (76mg), E18 (134mg), E19 (234mg), E20 ( 260mg), E21 (478mg), E22 (135mg), E23 (229mg), E24 (112mg), E25 (180mg), E26 (419mg), E27 (256mg), E28 (146mg) 50%-100%; E29 ( 980 mg), E30 (760 mg), E31 (156 mg), E32 (69 mg) 100%.

Component E21 was collected by isocratic elution with MeOH-H ₂ O 70% by Sephadex LH-20 to obtain 13 components E21-1-E21-13; among them, component E21-13 (7.5mg) was the target compound polyketone Compound pyoluteorin.

3. Structure determination of the polyketide pyoluteorin

Using ¹ H-NMR, ¹³ C-NMR, and ESIMS to obtain the nuclear magnetic and molecular weight information of the compound, and comparing the carbon spectrum and hydrogen spectrum data with the known compounds, it is determined that the compound is shown in formula I:

Polyketide pyoluteorin: brown powder; ESIMS (negative) m/z 269.7 [MH] ⁻ ; ¹ H-NMR (500 MHz, CD ₃ OD) δ: 7.08 (1 H, t, J=8.3 Hz, H-10), 6.56 (1H, s, H-4), 6.35 (2H, d, J=8.3Hz, H-9, H-11); ¹³ C-NMR (125MHz, CD ₃ OD) δ: 185.9 (C=O) ,158.3(C×2,C-8,C-12),132.9(CH-10),132.5(C-5),122.0(C-2),119.4(CH-4),115.4(C-7) ,112.1(C-3),108.2(C×2,CH-9,CH-11).

Example 2: Cytotoxic activity test

1. Experimental materials

Human glioma cells (U87), human lung adenocarcinoma cells (A549), human colon cancer cells (HCT116), human breast cancer cells (MCF-7, HCC1954), human chronic myelogenous leukemia cells (K562), human Triple-negative breast cancer cells (MDA-MB-231, MDA-MB-468), human triple-positive breast cancer cells (BT474), and human normal breast epithelial cells (MCF-10A) were purchased from ATCC.

The polyketide pyoluteorin was prepared from Example 1.

2. Cytotoxic activity screening and mechanism of action research methods

(1) Detection of cytotoxic activity by CTG method

Different tumor cells growing in log phase were collected, and the cells were counted and seeded in a 96-well culture plate, with 2000 cells/well, and 90 μL of DMEM medium (4.5 g/L D-glucose, L-glutamic acid, 110 mg/well) was added to each well. L sodium pyruvate, etc., containing serum), cultured at 37°C under 5% CO ₂ (MDA-MB-231 cell culture does not require CO ₂ ), after the cells adhered, 10 μL of the polyketide compound pyoluteorin to be tested was added at different concentrations Continue to cultivate for 72h. After the culture was terminated, 100 μL of CTG solution was added to each well and allowed to stand at room temperature for 10 min. After standing, the chemiluminescence value of each well was measured on a microplate reader.

(2) Time-dose-dependent detection of cytostatic rate

CTG method: The drug was diluted with DMEM medium to the experimental design concentration (0.032, 0.16, 0.8, 4, 20, 100 μM). According to the experimental groups, the cells were counted and then seeded in a 96-well culture plate, with 2000 cells/100uL/well, and 90uL of LDMEM medium (containing serum) was added to each well to culture overnight. After overnight, 10uL (0.032, 0.16, 0.8, 4, 20, 100μM) of the polyketide compound to be tested was added with different concentrations as the experimental group, and 10uL of DMEM medium was added to the blank control group, and three duplicate wells were set up at 37°C. After 24h, 48h and 72h in the incubator, 100uL of CTG was added to each well and incubated at room temperature for 10min in the dark. After incubation, the chemiluminescence value of each well was measured by a microplate reader. The inhibition rate and IC ₅₀ were calculated according to the following formula: inhibition rate (%)=(chemiluminescence value of blank control group−chemiluminescence value of experimental group)/chemiluminescence value value of blank control group×100%. Taking the concentration of the added compound as the abscissa and the inhibition rate (%) as the ordinate, the IC ₅₀ was calculated by the direct graphic method.

(3) Cell cycle detection

The cell cycle was detected using a cell cycle detection kit.

The polyketide compound to be tested, pyoluteorin, was diluted with DMEM medium to the experimental design concentration (0.1, 0.3, 1, 3, 10 μM). The cultured MDA-MB-231 cells were trypsinized and then seeded in a 6-well plate, 2 mL of cell suspension was added to each well, and cultured in a 37°C incubator. For the experimental group, the polyketide compound pyoluteorin that has been prepared with different concentrations to be tested was added. For the blank control group, an equal volume of DMEM medium was added, and the cells were placed in a centrifuge tube after being placed at 37°C for 24 hours. , the cells in each group were centrifuged at 1500 rpm for 5 min, 1 mL of 70% methanol was added, and incubated at 4°C for 15 min. After incubation, the cells in each group were centrifuged at 1500 rpm for 5 min, and 500 uL of PI solution (50 ug/mL PI, 100 ug/mL RNaseA, 0.05% Triton X-100) was added, and incubated at 37°C for 40 min. After incubation, the cells in each group were centrifuged at 1500 rpm for 5 min, added with 1 mL of PBS, centrifuged at 1500 rpm for 5 min, resuspended in 500 uL of PBS, and the DNA content of each cell cycle was detected by flow cytometry.

(4) Detection of apoptosis

Apoptosis was detected by Annexin V-FITC/PI double staining.

The polyketide compound to be tested, pyoluteorin, was diluted with DMEM medium to the experimental design concentration (0.1, 0.3, 1, 3, 10 μM). The cultured MDA-MB-231 cells were trypsinized and then seeded in a 6-well plate, 2 mL of cell suspension was added to each well, and placed in a 37°C incubator for 24 hours. For the experimental group, the polyketide compound pyoluteorin that has been prepared at different concentrations to be tested was added, and for the blank control group, an equal volume of DMEM medium was added, and it was placed in a 37°C incubator for 24 hours. After incubation, the cells of each group were collected and centrifuged at 1000 rpm for 5 min. Adjust the cell density to 1 x 10 ⁶ /mL with 1 x Annexin V BindingSolution. Take 100uL of cell suspension, add 5uL of AnnexinV-FITC conjugate and 5μL of PI solution in sequence, and store at room temperature for 15min in the dark. After the end, 400uL of 1×AnnexinVBinding Solution was added and detected by flow cytometer within 1h.

(5) Detection of mitochondrial membrane potential

Mitochondrial membrane potential was detected by mitochondrial membrane potential detection kit (JC-1).

The polyketide compound to be tested, pyoluteorin, was diluted with DMEM medium to the experimental design concentration (0.1, 0.3, 1, 3, 10 μM). The cultured MDA-MB-231 cells were trypsinized and then seeded in a 6-well plate, 2 mL of cell suspension was added to each well, and placed in a 37°C incubator for 24 hours. For the experimental group, the polyketide compound pyoluteorin that has been prepared at different concentrations to be tested was added, and for the blank control group, an equal volume of DMEM medium was added, and it was placed in a 37°C incubator for 24 hours. Prepare JC-1 staining working solution according to the kit instructions. After the incubation, the cells of each group were collected and placed in a centrifuge tube at 1000 rpm for 5 min to remove the cell culture medium. Rinse once with PBS, resuspend in 1 mL of cell culture medium, add 1 mL of JC-1 staining working solution, mix well, and incubate at 37 °C for 20 min. After that, the supernatant was removed by centrifugation and washed twice with JC-1 staining buffer (1X). After resuspending in 2 mL of cell culture medium, the collected images were observed under a laser confocal microscope.

(6) Detection of intracellular reactive oxygen species

The content of intracellular ROS was detected by reactive oxygen species ROS detection kit.

The polyketide compound to be tested, pyoluteorin, was diluted with DMEM medium to the experimental design concentration (0.1, 0.3, 1, 3, 10 μM). The cultured MDA-MB-231 cells were trypsinized and then seeded in a 6-well plate (100,000/2mL/well), and placed in a 37°C incubator for 24h. For the experimental group, the polyketide compound pyoluteorin that has been prepared at different concentrations to be tested was added, and for the blank control group, an equal volume of DMEM medium was added, and it was placed in a 37°C incubator for 24 hours. After the incubation, the cell culture medium was removed, the cells were resuspended in DMEM medium (containing 10 μM 2', 7' fluorescein ethylene dichloride), and incubated at 37°C for 20 min. After washing twice with PBS, the content of ROS was detected by flow cytometry.

The intracellular ROS production aggregation images were observed by confocal laser microscopy. The polyketide compound to be tested, pyoluteorin, was diluted with DMEM medium to the experimental design concentration (0.1, 0.3, 1, 3, 10 μM). The cultured MDA-MB-231 cells were trypsinized and then seeded in a 6-well plate (100,000/2mL/well), and placed in a 37°C incubator for 24h. For the experimental group, the polyketide compound pyoluteorin that has been prepared at different concentrations to be tested was added, and for the blank control group, an equal volume of DMEM medium was added, and it was placed in a 37°C incubator for 24 hours. After the incubation, the cell culture medium was removed, the cells were resuspended in DMEM medium (containing 10 μM 2', 7' fluorescein ethylene dichloride), and incubated at 37°C for 20 min. Cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells. DAPI was added dropwise and incubated in the dark for 5 min to stain the specimens. After gently rinsing 4 times with serum-free cell culture medium to remove excess DAPI, ROS production aggregation images were collected under a laser confocal microscope.

3. Experimental results

The cytotoxic activity of the polyketide pyoluteorin to be tested against different tumor cells was tested by CTG method, and the results are shown in Table 1. ) of the screening results for cytotoxic activity (doxorubicin was a positive control, _IC50 =mean±standard error, n=3).

Table 1

From the data in Table 1, it can be seen that the polyketide pyoluteorin has good cytotoxic activity on human lung adenocarcinoma cells (A549), human colon cancer cells (HCT116), and human breast cancer cells (MCF-7), and has good cytotoxic activity against human cells. Breast cancer cells are highly active. Therefore, the cytotoxic activity of polyketide pyoluteorin on other breast cancer cell lines (MDA-MB-231, MDA-MB-468, BT474, HCC1954) was studied. Results of screening for cytotoxic activity in breast cancer cells and normal mammary epithelial cells ( _IC50 =mean ± SE, n=3).

Table 2

It can be seen from the data in Table 2 that the polyketide compound pyoluteorin has a significant inhibitory effect on human triple-negative breast cancer cells MDA-MB-231 (IC ₅₀ =0.975 μM), and has toxic and side effects on human normal breast epithelial cells MCF-10A Smaller (IC ₅₀ >50 μM), indicating that the compound has better selective cytotoxicity.

The results of the drug time-dose dependence experiment are shown in Figure 1. Figure 1 is a graph showing the inhibitory effect of the polyketide compound pyoluteorin on the growth of MDA-MB-231 cells. The inhibition rate of polyketide pyoluteorin on MDA-MB-231 cells after 24, 48, 72 h. The results showed that the inhibitory effect of polyketide pyoluteorin on MDA-MB-231 cells was concentration-time dependent. Different doses of 0.032, 0.16, 0.8, 4, 20, and 100 μM of the polyketide pyoluteorin acted on MDA-MB-231 cells for 24 hours, the survival rate of MDA-MB-231 cells decreased significantly, and the effect was the most effective when the effect time was 72 hours. Well, the results show that the polyketide compound pyoluteorin can inhibit the growth of tumor cells in a dose-time-dependent manner.

Continue to use MDA-MB-231 cells as a model to study the mechanism of the polyketide pyoluteorin inhibiting the growth of human triple-negative breast cancer cells. After PI staining, the effect of polyketide pyoluteorin on cell growth cycle was detected by flow cytometry. As shown in Fig. 2, Fig. 2 is a schematic diagram showing that the polyketide compound pyoluteorin can induce cell G ₂ /M arrest. The experimental data are expressed as mean ± standard error, using two-way ANOVA, Tukey test, P<0.05 is significant difference (*,P<0.05;**,P<0.01vs.Vehicle) . After treating MDA-MB-231 cells with different concentrations of polyketide pyoluteorin for 24h, the flow cytometry results showed that polyketide pyoluteorin could dose-dependently induce an increase in the proportion of cells in G ₂ phase and a decrease in the proportion of cells in S phase. The above experimental results showed that the polyketide compound pyoluteorin could induce MDA-MB-231 cell cycle arrest in G ₂ /M phase.

Whether the polyketide compound pyoluteorin can induce apoptosis was detected by Annexin V-FITC/PI double staining method. As shown in Figure 3 and Figure 4, Figure 3 is a schematic diagram of the induction of apoptosis of MDA-MB-231 cells by different doses of polyketide pyoluteorin, Figure 4 is the induction of apoptosis of MDA-MB-231 cells by different doses of polyketide pyoluteorin Schematic diagram of the trend statistics. The experimental data are expressed as the mean ± standard error, using one-way ANOVA, Duncan test, P<0.05 is significant difference (*, P<0.05; **, P<0.01, ***, P<0.001 vs. Vehicle). When MDA-MB-231 cells were treated with different concentrations of polyketide pyoluteorin for 24 hours, the cells showed different degrees of apoptosis, and with the increase of polyketide pyoluteorin concentration, the cell apoptosis rate increased from 5.57% to 5.57%. 40.84%. The above results indicated that the polyketide pyoluteorin could effectively induce the apoptosis of triple-negative breast cancer MDA-MB-231 cells in a dose-dependent manner.

Using JC-1 fluorescent probe staining, the effect of polyketide pyoluteorin on mitochondrial membrane potential of MDA-MB-231 cells was detected and analyzed by laser confocal microscopy. When the mitochondrial membrane potential is high, JC-1 aggregates in the mitochondrial matrix to form polymers, which can produce red fluorescence. When the mitochondrial membrane potential is reduced, JC-1 cannot aggregate in the mitochondrial matrix, and JC-1 exists in the form of monomer at this time, which can produce green fluorescence. As shown in Figure 5, in Figure 5, A is the schematic diagram of the confocal fluorescence image of mitochondrial membrane potential of MDA-MB-231 cells after the action of different concentrations of polyketide compound pyoluteorin (a. blank control; b. 0.1 μM pyoluteorin; c 0.3 μM pyoluteorin; d. 1 μM pyoluteorin; e. 3 μM pyoluteorin; f. 10 μM pyoluteorin); 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. After treating MDA-MB-231 cells with different concentrations of the polyketide pyoluteorin for 24 h, the fluorescence generated by JC-1 polymer gradually decreased while the fluorescence generated by JC-1 monomer gradually increased, indicating that the intracellular mitochondrial membrane potential is at the continuously decreasing. In addition, the ratio of the fluorescence intensity of JC-1 monomer to the fluorescence intensity of JC-1 polymer can directly represent the change of mitochondrial membrane potential. It can be seen from the experiment that the ratio increases with the increase of the compound concentration, indicating that the mitochondrial membrane potential has depolarized. The above experimental results indicate that the polyketide pyoluteorin may induce apoptosis by inducing the depolarization of the mitochondrial membrane potential in cells.

Finally, it was tested whether the polyketide pyoluteorin could affect the intracellular reactive oxygen species ROS content. After MDA-MB-231 cells were treated with different concentrations of polyketide pyoluteorin for 24 hours, DCFH-DA was added for staining. The reactive oxygen species in the cells could oxidize non-fluorescent DCFH to generate green fluorescent DCF. The fluorescence intensity of DCF was detected by flow cytometry, as shown in Figure 6. Figure 6 is a schematic diagram of the content of reactive oxygen species ROS in MDA-MB-231 cells after the action of different concentrations of polyketide compound pyoluteorin; A is the detection of flow cytometry Schematic diagram of the fluorescence intensity of DCFH-DA; B is the schematic diagram of the content of reactive oxygen species ROS in MDA-MB-231 cells. The experimental data are expressed as the mean ± standard error, using one-way analysis of variance (One-Way ANOVA), Duncan test, P<0.05 was considered significant difference (*, P<0.05; **, P<0.01, *** , P<0.001vs.Vehicle), the polyketide pyoluteorin can increase the content of reactive oxygen species ROS in MDA-MB-231 cells. And it can be seen from the results of laser confocal detection (as shown in Figure 7, Figure 7 is a schematic diagram of the laser confocal detection images of reactive oxygen species ROS in MDA-MB-231 cells after the action of different concentrations of polyketide compound pyoluteorin; A blank control; B is 0.1 μM pyoluteorin; C is 0.3 μM pyoluteorin; D is 1 μM pyoluteorin; E is 3 μM pyoluteorin; F is 10 μM pyoluteorin.), after the drug concentration increases, the intracellular fluorescence intensity is increasing, that is, the content of intracellular ROS is constantly increasing Increase. The above two experimental results indicated that the polyketide pyoluteorin induced apoptosis of MDA-MB-231 cells may be related to the ROS pathway.

The results of in vitro cell experiments showed that the polyketide pyoluteorin could inhibit the growth of triple-negative breast cancer MDA-MB-231 cells by inducing cell G ₂ /M phase arrest and apoptosis. Further mechanism studies found that apoptosis was related to the decrease of mitochondrial membrane potential (MMP) and the increase of intracellular reactive oxygen species (ROS).

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention in any form. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Within the scope of the technical solution of the present invention, personnel can make some changes or modifications to equivalent examples of equivalent changes by using the above-mentioned technical content, but any content that does not depart from the technical solution of the present invention is based on the technical solution of the present invention. Substantially any simple modifications, equivalent changes and modifications made to the above embodiments still fall within the scope of the solutions of the present invention.

Claims (6)

1. a preparation method of polyketide pyoluteorin, is characterized in that, comprises the following steps: The Aspergillus niger fermentation broth was filtered to remove mycelium, ethyl acetate was added to carry out liquid-liquid extraction, and ethyl acetate fractions were obtained after concentration; the extracted fermentation broth was passed through macroporous resin, and eluted with methanol to obtain macropolar fractions ; Merge the ethyl acetate part and the large polar part, separate and purify to obtain the target compound polyketide pyoluteorin; The separation and purification include the following steps: Through medium and low pressure reversed-phase column chromatography, with ODS and Sephadex LH-20 as stationary phase, and MeOH-H ₂ O 10%-100% according to different elution gradients, 32 components were obtained: E1-E16 10%-50% ; E17-E28 50%-100%; E29-E32 100%; Component E21 was collected by Sephadex LH-20 with MeOH-H ₂ O 70% isocratic elution to obtain 13 components E21-1-E21-13; Among them, component E21-13 is the target compound pyoluteorin; The chemical structure of the polyketide pyoluteorin is shown in formula I:

The polyketide pyoluteorin is isolated and prepared from Aspergillus niger derived from wetland soil. 2. the preparation method of polyketide pyoluteorin according to claim 1, is characterized in that, the preparation method of described Aspergillus niger fermentation broth comprises the following steps: The purified Aspergillus niger strain was inoculated into the modified Martin solid medium, and cultured at a temperature of 28°C for 7 days. After the cultivation, the agar plate containing the strain was cut into small pieces and inoculated into a 2L conical flask. , each conical flask was added with sterilized 1L modified Martin liquid medium, and fermented for 28 days at a temperature of 28°C to obtain Aspergillus niger fermentation broth. 3. the preparation method of polyketide pyoluteorin according to claim 2, is characterized in that, the preparation method of described Aspergillus niger bacterial strain comprises the following steps: Add fresh wetland soil into distilled water to dilute and mix evenly at a ratio of 1:100, take 100 μL of soil dilution and spread it on the separation medium, cultivate it for 4 days at a temperature of 28 °C, and pick and purify after the strain grows. A strain of Aspergillus niger was obtained. 4. the preparation method of polyketide pyoluteorin according to claim 3, is characterized in that, the composition of described separation medium is as follows: improved 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; add 50mg/L potassium dichromate and 20mg/L naphthyridine to the above modified Martin solid medium Keto acids as bacteriostatic agents. 5. The application of the polyketide compound pyoluteorin prepared by the method of any one of claims 1 to 4 in the preparation of antitumor drugs, wherein the tumor is breast cancer, triple-negative breast cancer, lung adenocarcinoma, and colon cancer. 6. The application of the polyketide pyoluteorin according to claim 5 in the preparation of an antitumor drug, wherein the antitumor drug is a drug or a pharmaceutical composition composed of the polyketide pyoluteorin as an active ingredient .

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