Actinomycetes-derived polyketone compound as well as preparation method and application thereof

Abstract

The invention discloses a polyketone compound derived from actinomycetes as well as a preparation method and application thereof, and relates to the technical field of microbial natural product mining and biological medicine, and the key point of the technical scheme is that the invention discloses a novel polyketone compound Strepactone derived from actinomycetes Streptomyces sp. DP0001, and the molecular formula of the novel polyketone compound Strepactone is C25H38O5. The compound is obtained through strain fermentation, ethyl acetate extraction and multi-step chromatographic purification, and the structure is identified through ESI-MS, 1H NMR and 13C NMR. Experiments show that Strepactone has an inhibition effect on staphylococcus aureus and methicillin-resistant staphylococcus aureus and has remarkable inhibition activity on human non-small cell lung cancer A549 cells, the half inhibitory concentration IC50 of the Strepactone is 5.36 mu M, and the Strepactone has no obvious cytotoxic activity on human embryo kidney cells 293T cells at the concentration of 30 mu M. The compound provided by the invention can be used for preparing anti-drug-resistant bacteria drugs and anti-human non-small cell lung cancer drugs, and also provides a structural basis for the development of novel antibacterial and anti-tumor leading drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial natural product mining and biological medicine, and more particularly to a polyketide compound derived from actinomycetes and a preparation method and application thereof. BACKGROUND

[0002] Lung cancer is the malignant tumor with the highest incidence and mortality in the world. According to the statistics of the World Health Organization, in 2022, there were 2.48 million new cases of lung cancer worldwide, and more than 1.8 million deaths, accounting for 18.7% of the total number of cancer deaths in the world in 2022; in China, in 2020, there were 820,000 new cases of lung cancer per year, and about 710,000 deaths, and the incidence is increasing year by year. Lung cancer can be divided into non-small cell lung cancer (NSCLC, accounting for 80-85%) and small cell lung cancer (SCLC, accounting for 15-20%) according to pathological type, both of which have the characteristics of fast progression, early metastasis and poor prognosis - the 5-year survival rate of patients with advanced lung cancer is generally less than 10%, and there is an urgent and unmet need for safe, efficient and widely applicable lung cancer treatment drugs in clinical practice.

[0003] Natural products are the core source of innovative drugs. From 1939 to 2016, nearly 60% of the drugs approved for marketing by the FDA were directly derived from natural products or based on natural products for research and development. Their structural novelty, biocompatibility and functional diversity are optimized by natural evolution, which is significantly better than chemically synthesized small molecule drugs, and they are widely used in the treatment of major diseases such as antibiosis and antitumor. Among them, polyketides are a class of natural products with diverse chemical structures and biological activities (antibiosis, antitumor, antioxidant, etc.), which have been widely used in medicine, agriculture and food industry. Although polyketides have been extensively studied, specific polyketides with antibacterial and human non-small cell lung cancer A549 activity are still relatively scarce. SUMMARY

[0004] The purpose of the present application is to provide a polyketide compound derived from actinomycetes and a preparation method and application thereof. Streptomyces A new polyketide compound Strepactone is obtained by antibacterial activity tracking separation from the secondary metabolites of a strain of actinomycetes Streptomyces sp. DP0001, which is found to have significant inhibitory activity against MRSA and human non-small cell lung cancer A549 for the first time, providing a new idea for solving the research and development of antitumor drugs.

[0005] The above technical purpose of the present application is realized by the following technical scheme: a polyketide compound derived from actinomycetes, wherein the actinomycete strain is Streptomyces sp. DP0001, and the polyketide compound is Strepactone. Streptomyces Streptomyces ​Strepactone, the molecular formula of which is C 25 H 38 O5, the chemical structure of which is shown as follows: .

[0006] The application further provides a preparation method of the above-mentioned polyketide compound, comprising the following steps: (1) strain activation and seed liquid preparation Strain activation: 4.0 g of yeast extract powder, 10.0 g of malt extract powder, 4.0 g of glucose, 0.5 g of calcium carbonate, 0.5 mL of glycerol, 12.0 g of agar and 1000 mL of pure water are used to prepare a solid culture medium, and the pH is 7.3; 0.5 mL of the activated strain is inoculated into the solid culture medium, and the culture is carried out at 28°C for 3-5 days; Streptomyces sp. DP0001 is inoculated into the solid culture medium, and the culture is carried out at 28°C for 3-5 days; Seed liquid preparation: 6 g of yeast extract powder, 10 g of malt extract powder, 5 g of glucose, 30 g of sodium chloride and 1000 mL of pure water are used to prepare a seed liquid culture medium, and the pH is 7.3; the activated single colony is inoculated into 100 mL of the seed liquid culture medium, and the culture is carried out at 28°C and 180 rpm for 2-3 days; the OD 600 =0.8~1.0, and the seed liquid is obtained; (2) fermentation culture Fermentation medium preparation: 10 g of corn powder, 4 g of yeast extract powder, 5 g of soybean powder, 10 g of soluble starch, 4 g of glucose, 1 g of magnesium sulfate, 2 g of ammonium sulfate, 2 g of calcium carbonate, 30 g of sea salt and 1000 mL of pure water are used to prepare a fermentation culture medium, and the pH is 7.4; The seed liquid is inoculated into the fermentation culture medium at a volume ratio of 5%, and the culture is carried out at 28°C and 180 rpm for 7 days, so that the fermentation liquid is obtained; (3) crude extract preparation The fermentation liquid is extracted with an equal volume of ethyl acetate for three times, and the extraction liquids are combined; the extraction liquid is concentrated by a rotary evaporator at 40°C and 0.09 MPa under reduced pressure, so that the crude extract is obtained; (4) separation and purification First step: silica gel column chromatography purification The crude extract is loaded onto a silica gel column with a pore size of 100-200, and gradient elution is carried out with petroleum ether-ethyl acetate at a volume ratio of 1:0→0:1; the active fraction is collected; Second step: medium pressure reversed phase C18 column chromatography purification The active fraction was loaded onto a 40-60 μm medium pressure C18 column and eluted with a gradient of methanol-water (10:90→100:0, by volume), and active sub-fractions were collected; Third step: gel column chromatography purification The active sub-fractions were loaded onto a Sephadex LH-20 gel column and eluted with methanol, and main peak fractions were collected; Fourth step: semi-preparative HPLC purification The gel column purified product was purified by semi-preparative HPLC, wherein the column was an ODS-A semi-preparative column, the mobile phase was 85% CH3CN / H2O, the flow rate was 3 mL / min, the detection wavelength was 210 nm, main peak components were collected, and after freeze-drying, the monomeric compound Strepactone was obtained.

[0007] The application further provides use of the above-mentioned polyketide compound in preparation of a human non-small cell lung cancer drug, which is used for specifically inhibiting growth of human non-small cell lung cancer A549 cells.

[0008] The application further provides use of the above-mentioned polyketide compound in preparation of an antibiotic.

[0009] In summary, the application has the following beneficial effects: (1) Novel structure: Strepactone is a new polyketide compound first discovered, which enriches the structural resources of actinomycete-derived polyketide compounds with dual activity against A549 cells; (2) Clear targeting of anti-tumor: specifically inhibits human non-small cell lung cancer A549 cells, avoids broad-spectrum toxicity of traditional anti-tumor drugs, and reduces the risk of side effects; (3) Strong specificity of antibacterial activity: has antibacterial effects on Staphylococcus aureus and MRSA (a clinically important drug-resistant bacterium), and provides a new candidate drug for solving the problem of MRSA drug resistance; (4) Scalable preparation method: based on conventional microbial fermentation and chromatography technology, the steps are clear, the reproducibility is high, the yield of crude extract is stable (14.3 g / 25 L), and the method is suitable for industrial production; (5) Wide application prospect: can be directly used for developing anti-drug-resistant bacteria and anti-tumor drugs, and can also be used as a lead structure for derivatization to further improve activity or optimize pharmacokinetic properties. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 is the chemical structural formula of Strepactone in the application: showing the stereochemical configuration and the positions of each substituent; Figure 2This is the ESI-MS mass spectrum (anion mode) of Strepactone in this invention: showing a quasi-molecular ion peak. m / z 463.7 [M + HCOO] - ; Figure 3 This is Strepactone in the present invention. 1 1H NMR spectrum (700 MHz, Chloroform- d ): Marks the chemical shift of the main hydrogen signal; Figure 4 This is Strepactone in the present invention. 13 C NMR and DEPT135 spectrum (176 MHz, Chloroform- d ): Displays 25 carbon signals and carbon types (methimyl, methylene); Figure 5 The following are plate diagrams of the antibacterial activity of the crude extract and fraction in this invention: Figure (A) shows Staphylococcus aureus (SA), Figure (B) shows MRSA, number 1 is the crude extract, 2~11 are Fr.1~Fr.10, and only 11 (Fr.10) shows a transparent inhibition zone; Figure 6 The HPLC-UV chromatogram (210 nm) of Strepactone in this invention shows the main peak (retention time 6.0 min) and purity ≥89%. Detailed Implementation

[0011] The following is in conjunction with the appendix Figures 1-6 The present invention will be described in further detail below.

[0012] Example 1: Preparation of a polyketide compound derived from actinomycetes The polyketide compound is named Strepactone, and its IUPAC name is: (2) S 4 S 5 R 6 S 7 S )-4,6-dihydroxy-5-methoxy-7-(8-methylnonyl)-9-oxatricyclo[9.4.0.0 2,6 ]pentadeca-1(15),11,13-trien-8-one; Chemical structure: ; The preparation method of the polyketide compound is as follows: (1) Strain activation and seed culture preparation Strain activation: Prepare a solid culture medium with pH 7.3 by mixing 4.0 g yeast extract, 10.0 g malt extract, 4.0 g glucose, 0.5 g calcium carbonate, 0.5 mL glycerol, 12.0 g agar, and 1000 mL pure water. Streptomyces sp.DP0001 was inoculated into this solid medium and incubated at 28°C for 3-5 days. Small and dense colonies with a grayish-white, glossy, and thick texture were observed, confirming successful activation. Preparation of seed culture: Prepare a seed culture medium with pH 7.3 by mixing 6 g yeast extract powder, 10 g malt extract powder, 5 g glucose, 30 g sodium chloride, and 1000 mL pure water. Inoculate activated single colonies into 100 mL of the seed culture medium and incubate at 28℃ and 180 rpm for 2-3 days. Measure the OD. 600 =0.8~1.0, to obtain seed solution; (2) Large-scale fermentation Preparation of fermentation medium: Take 10 g corn flour, 4 g yeast extract powder, 5 g soybean flour, 10 g soluble starch, 4 g glucose, 1 g magnesium sulfate, 2 g ammonium sulfate, 2 g calcium carbonate, 30 g sea salt, and 1000 mL pure water to prepare a fermentation medium with a pH of 7.4. The seed culture was inoculated into the fermentation medium at a volume ratio of 5%, with a total fermentation volume of 25 L. The culture was carried out at 28°C and 180 rpm for 7 days to obtain the fermentation broth. (3) Preparation of crude extract The fermentation broth was extracted three times with an equal volume of ethyl acetate, and the extracts were combined. The extract was concentrated under reduced pressure at 40℃ and 0.09MPa using a rotary evaporator to obtain 14.3 g of brownish-yellow crude extract, which was sealed and stored at 4℃. Example 2: Isolation, purification and structural identification of Strepactone (1) Activity tracking separation Silica gel column chromatography: 14.3 g of crude extract was dissolved in methanol and loaded onto a silica gel column (100-200 mesh). The column was eluted with a gradient of petroleum ether and ethyl acetate (1:0→0:1, 5 column volumes per gradient). One fraction was collected for every 200 mL of the extract. Similar fractions were combined to obtain Fr.1~Fr.10. Antimicrobial activity screening (disk method): Staphylococcus aureus (SA) and MRSA were inoculated into LB liquid medium (10 g tryptone + 5 g yeast extract + 10 g sodium chloride + 1000 mL pure water, pH 7.0) and incubated at 37°C until OD500. 600=0.6~0.8; After sterilizing LB agar medium (with 1.5% agar), cool to 50℃, add 3% bacterial solution, mix well, and pour into plates; dissolve each fraction in methanol (10 mg / mL), and add 5 μL to sterile paper discs (6 mm in diameter). Use 5 μL of 2 mg / mL gentamicin sulfate / 0.1 mg / mL ciprofloxacin hydrochloride (positive control) and 5 μL of methanol (negative control) as controls; after incubation at 37℃ for 12 h, only Fr.10 showed a clear inhibition zone (about 12 mm in diameter), which was identified as the active fraction; Medium-pressure C18 column chromatography: Dissolve 5.6 g of Fr.10 in methanol, load the solution onto a medium-pressure C18 column, and elute with a methanol-water gradient (10:90→100:0, 3 column volumes per gradient). Collect 10 subfractions Fr.10.1~Fr.10.10. Gel column chromatography and HPLC preparation: 910 mg Fr.10.9 was dissolved in methanol and loaded onto a Sephadex LH-20 column. Methanol was used for elution (flow rate 1 mL / min), and one tube was collected every 5 mL. The main peak tubes (tubes 25-30) were combined. The combined solution was purified by semi-preparative HPLC (column: YMC-Pack ODS-A, 5 μm, 250×10 mm; mobile phase 85% CH3CN / H2O; flow rate 3 mL / min; detection wavelength 210 nm). The main peak with a retention time of 6.0 min was collected and freeze-dried to obtain a white powder of Strepactone (3.8 mg).

[0013] (2) Structural identification Mass spectrometry: Anion ESI-MS display m / z 463.7 [M + HCOO] - Based on molecular weight calculations, the molecular formula is determined to be C64. 25 H 38 O5; Nuclear magnetic resonance: 1 H NMR (700 MHz, Chloroform- d ) δ 7.54 (d, J = 7.9 Hz, 1H, H-5), 7.29 (t, J = 7.5 Hz, 1H, H-4), 7.24 (t, J = 7.4 Hz, 1H, H-3), 7.15 (d, J = 8.0 Hz, 1H, H-2), 5.44 (d, J= 13.5 Hz, 1H, H-24b), 5.23 (d, J = 13.5 Hz, 1H, H-24a), 4.29 (m, 1H,H-9), 4.25 (dd, J = 11.6, 4.9 Hz, 1H, H-7), 3.67 (s, 1H, H-10), 3.48 (s, 3H, H-25), 3.13 (dd, J = 11.5, 1.9 Hz, 1H, H-12), 2.62 (ddd, J = 15.2, 11.6, 5.8 Hz,1H, H-8b), 2.32 (dd, J = 15.2, 4.9 Hz, 1H, H-8a), 1.74 (m, 1H, H-13b), 1.50 (m,1H, H-20), 1.33 (overlapped, 1H, H-13a), 1.33-1.24 (overlapped, 10H), 1.14(q, J = 6.9 Hz, 2H, H-19), 1.14 (m, 2H, H-19), 0.85 (d, J = 6.7 Hz, 6H, H-22 / 23); 13 C NMR (176 MHz, Chloroform- d ) δ 175.5 (s, C-23), 137.3 (s, C-1), 134.9(s, C-6), 131.7 (d, C-5), 128.0 (d, C-2), 127.9 (d, C-4), 127.5 (d, C-3),90.0 (s, C-10), 83.5 (s, C-11), 74.0 (d, C-9), 69.3 (t, C-24), 58.1 (q, C-25), 49.5 (d, C-12), 47.7 (d, C-7), 40.2 (t, C-8), 39.2 (t, C-19), 30.0 (t,C-17), 29.6 (t, C-16), 29.6 (t, C-15), 28.1 (d, C-20), 27.8 (t, C-14), 27.5(t, C-18), 25.3 (t, C-13), 22.8 (q, C-21 / 22). Structural analysis: Based on the DEPT135 spectrum (showing 9 methines and 9 methylenes), the compound was determined to contain a three-ring system (benzene ring, cyclopentane ring, and oxecyclic octane ring). The final structure is shown in the attached figure. Figure 1 As shown, it is named Strepactone.

[0014] Example 3: Assay of the antitumor activity of Strepactone (IC50) 50 value) (1) Cell culture: Human non-small cell lung cancer A549 (ATCC CCL-185) cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS), and human embryonic kidney cells 293T (ATCC CRL-3216) were cultured in DMEM high glucose medium containing 10% FBS in an incubator at 37°C and 5% CO2. The cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for experiments. (2) Cell seeding: A549 and 293T cells were digested with 0.25% trypsin-EDTA, centrifuged at 1000 rpm for 5 min, resuspended, counted, and the concentration was adjusted to 5×10⁻⁶. 3 Cells / well, seeded in 96-well plates, 100 μL per well, and cultured for 24 h to allow cells to adhere; (3) Drug treatment: Discard the old culture medium. For A549 cells, add 100 μL of culture medium containing Strepactone to the experimental group (dissolved in DMSO, final concentration 0→60 μM, final concentration of DMSO ≤0.1%). For 293T cells, add 100 μL of culture medium containing Strepactone to the experimental group (dissolved in DMSO, final concentration 30 μM). Add 100 μL of fresh culture medium to the blank control group and add 100 μL of culture medium containing 0.1% DMSO to the negative control group. Each group was repeated 3 times. (4) Activity detection (CCK-8 method): After culturing for 24 h, add 10 μL of CCK-8 solution to each well, incubate in the dark for 4 h, and measure the OD value at 450 nm using an enzyme-linked immunosorbent assay reader; (5) IC 50 Calculation: Cell viability (%) = (Experimental group OD - Control group OD) / (Negative group OD - Control group OD) × 100%; The IC50 of Strepactone against A549 cells was calculated by fitting a concentration-effect curve using GraphPad Prism 9 software. 50 The concentration was 5.36 μM; while Strepactone at a concentration of 30 μM showed no significant cytotoxic activity against 293T cells, with a cell viability of 98.06%.

[0015] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A polyketide derived from Actinomyces, characterized by: The actinomycete strain is Streptomyces sp. DP0001, which is isolated from coral and has a biological preservation number CCTCC NO: M20252331; the polyketide compound is named Strepactone, and the molecular formula of the polyketide compound is C 25 H 38 O5, the chemical structure of which is shown below: 。 2. The method of claim 1, wherein the method comprises the following steps: (1) activation of the strain and preparation of the seed solution; (2) fermentation culture; (3) preparation of the crude extract; and (4) separation and purification. The method comprises the following steps: (1) Activation of the strain and preparation of the seed solution The solid culture medium is prepared by using 4.0 g of yeast extract powder, 10.0 g of malt extract powder, 4.0 g of glucose, 0.5 g of calcium carbonate, 0.5 mL of glycerol, 12.0 g of agar and 1000 mL of pure water, and the pH is 7.

3. Streptomyces sp. DP0001 is inoculated into the solid culture medium and cultured at 28°C for 3-5 days. Preparation of seed liquid: take yeast extract powder 6 g, malt extract powder 10 g, glucose 5 g, sodium chloride 30 g, pure water 1000 mL to make seed liquid medium, pH is 7.3; pick up the activated single colony and inoculate into 100 mL seed liquid medium, 28℃, 180 rpm shaking culture for 2-3 days, measure OD 600 =0.8~1.0, get seed liquid; (2) Fermentation culture The fermentation culture medium is prepared by using 10 g of corn powder, 4 g of yeast extract powder, 5 g of soybean powder, 10 g of soluble starch, 4 g of glucose, 1 g of magnesium sulfate, 2 g of ammonium sulfate, 2 g of calcium carbonate, 30 g of sea salt and 1000 mL of pure water, and the pH is 7.

4. The seed solution is inoculated into the fermentation culture medium at a volume ratio of 5%, and the fermentation culture is carried out at 28°C and 180 rpm for 7 days to obtain a fermentation broth. (3) Preparation of the crude extract The fermentation broth is extracted with an equal volume of ethyl acetate for 3 times, and the extracted solution is concentrated by a rotary evaporator at 40°C and 0.09 MPa under reduced pressure to obtain the crude extract. (4) Separation and purification First step: silica gel column chromatography purification The crude extract is loaded onto a silica gel column with a particle size of 100-200, and gradient elution is performed with petroleum ether-ethyl acetate at a volume ratio of 1:0→0:1, and the active fraction is collected. Second step: medium pressure reverse phase C18 column chromatography purification The active fraction is loaded onto a medium pressure C18 column with a particle size of 40-60 μm, and gradient elution is performed with methanol-water at a volume ratio of 10:90→100:0, and the active sub-fraction is collected. Third step: gel column chromatography purification The active sub-fraction is loaded onto a Sephadex LH-20 gel column, and methanol is used as the eluent, and the main peak fraction is collected. Fourth step: semi-preparative HPLC purification The gel column purification product is purified by semi-preparative HPLC, and the semi-preparative HPLC column is ODS-A semi-preparative column, the mobile phase is 85% CH3CN / H2O, the flow rate is 3 mL / min, the detection wavelength is 210 nm, and the main peak component is collected. After freeze-drying, the monomeric compound Strepactone is obtained.

3. The use of the polyketide compound derived from actinomycetes in the preparation of a drug for human non-small cell lung cancer.

4. The use of a polyketide compound derived from Actinomyces according to claim 3 in the preparation of a drug for human non-small cell lung cancer, characterized in that: The drug is used to specifically inhibit the growth of human non-small cell lung cancer A549 cells.

5. The use of the polyketide compound derived from actinomycetes in the preparation of an antibiotic.

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