A streptomyces, alkaloid compounds produced by the same, and a preparation method and application thereof

Abstract

The application discloses streptomyces, alkaloid compounds produced by the streptomyces, and a preparation method and application of the alkaloid compounds. Streptomyces labedae The application discloses streptomyces, alkaloid compounds produced by the streptomyces, and a preparation method and application of the alkaloid compounds. Streptomyces labedae The application discloses streptomyces, alkaloid compounds produced by the streptomyces, and a preparation method and application of the alkaloid compounds. The 16S rRNA of the streptomyces strain W307 is shown as SEQ ID NO. 1; the application also provides application of the alkaloid compounds in preparation of antitumor drugs and in preparation of bacteriostatic drugs, and provides a potential antitumor drug and bacteriostatic drug for clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of discovery of new skeleton compounds in natural pharmaceutical chemistry and the field of screening of new antitumor drugs and antibacterial drugs, in particular to a Streptomyces, alkaloid compounds produced by the Streptomyces, and a preparation method and application thereof. BACKGROUND

[0002] Actinomycetes are a kind of microbial resources, which are closely related to human production and life, and thus have important value. Among the commonly used antibiotics, about 70% are derived from actinomycetes. However, due to changes in climate, human activities and other factors, the speed of discovering new metabolites from terrestrial actinomycetes is in a constant downward trend. Therefore, people turn their attention to another promising source of new drugs, i.e., marine actinomycetes.

[0003] Because of the special environmental characteristics of deep sea, such as low temperature, low oxygen, little light and high pressure, a large number of marine microorganisms with different biosynthetic pathways are bred, including unique actinomycete populations that can produce a variety of different secondary metabolites, such as Streptomyces and Nocardia. With the rapid development of new marine sampling technology, culture technology and identification technology, more and more secondary metabolites with novel molecular structures and good physiological activities have been discovered. Marine actinomycetes have become an important source for the discovery of lead compounds and the development of drugs. SUMMARY

[0004] The present application aims to solve the problems of low new rate of compounds from terrestrial actinomycetes and inactivity of natural source compounds, and provides a Streptomyces, alkaloid compounds produced by the Streptomyces, and a preparation method and application thereof.

[0005] The present application is achieved by the following technical solutions: as a first aspect, the present application provides a Streptomyces, which is classified and named as Streptomyces laverndulae W307 Streptomyces labedae , which was preserved in China General Microbiological Culture Collection Center on November 20, 2024, and the strain preservation number is CGMCC No.32721. The Streptomyces is of marine origin.

[0006] As a second aspect, the present application provides an alkaloid compound, which has a molecular formula of C 24 H 26 N2O5, and has the following structure:

[0007] .

[0008] As a third aspect, a preparation method of the alkaloid compound is provided, which is prepared by the above marine Streptomyces laverndulae W307 Streptomyces labedaeW307 is prepared by fermentation extraction separation and purification, and the 16S rRNA gene sequence of Streptomyces laverendae is shown as SEQ ID No. 1.

[0009] Further, the extraction separation and purification comprises the following steps: activating the Streptomyces; using MS culture medium to shake bed large-scale fermentation of actinomycetes; using ODS, LH-20, HPLC to separate and purify the compound; using 1 H NMR, 13 C NMR, HSQC, COSY, HMBC to analyze the planar structure of the compound, and using ECD calculation method to determine the absolute configuration of the compound.

[0010] As a fourth aspect, the application provides an application of the alkaloid compound in preparation of an anti-tumor drug.

[0011] Further, the anti-tumor drug is aimed at the diseases including lung cancer, gastric cancer, colon cancer, cervical cancer, chronic myelogenous leukemia, renal clear cell adenocarcinoma, esophageal cancer, bladder cancer, gallbladder cancer, breast cancer, liver cancer, brain tumor, prostate cancer, thyroid cancer, pancreatic cancer, osteosarcoma, malignant melanoma, rhabdomyosarcoma.

[0012] As a fifth aspect, the application provides an application of the alkaloid compound in preparation of an antibacterial drug.

[0013] Further, the antibacterial drug is aimed at the pathogenic bacteria including Stenotrophomonas maltophilia, Klebsiella pneumoniae, Acinetobacter baumannii, Acinetobacter pittii, Bacillus subtilis and Staphylococcus aureus.

[0014] The application has the beneficial effects that the strain is Streptomyces of marine origin, which makes beneficial exploration and practice accumulation for marine natural medicine resource discovery and sustainable utilization; the compound obtained by separation and purification is a new skeleton compound of alkaloids, which provides a research topic for subsequent biosynthesis research; the compound has good antibacterial activity and cytotoxicity, and the inhibition effect on part of tumor cells is better than that of the positive drug cisplatin, and the cytotoxicity on normal cells is less than that of the positive drug cisplatin, which has important significance for screening of new anti-tumor drugs and antibacterial drugs. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a high-resolution mass spectrum of the compound;

[0016] Figure 2 It is a structure diagram of the compound, wherein (A) is a planar structure of the compound, and (B) is a main two-dimensional nuclear magnetic correlation signal diagram;

[0017] Figure 3 It is an ECD calculation result diagram of the compound. DETAILED DESCRIPTION

[0018] An embodiment of the present invention provides a Streptomyces species and an alkaloid compound produced therefrom that has cytotoxic and antibacterial activity; the species is Streptomyces laureate. Streptomyces labedae W307 was deposited on November 20, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32721. Its 16S rRNA has the gene sequence shown in SEQ ID No. 1.

[0019] The fermentation method and the extraction, separation and identification methods of the compounds include: A) taking seed liquid from glycerol tubes and activating the actinomycetes on plates; B) large-scale fermentation of the actinomycetes on MS medium using a shaker; C) separation and purification of the compounds using ODS, LH-20, HPLC and other methods; D) using... 1 H NMR, 13 The planar structure of the compound was determined by methods such as C NMR, HSQC, COSY, and HMBC, and the absolute configuration of the compound was determined by ECD calculations.

[0020] The activity test results of the isolated compounds in the above embodiments of the present invention include A) 18 classic cell lines from the main cancer-causing organs in China (human lung cancer cells-A549, human gastric cancer cells-MKN-45, human colon cancer cells-HCT 116, human cervical cancer cells-HeLa, human chronic myeloid leukemia cells-K-562, human kidney clear cell adenocarcinoma cells-786-O, human esophageal cancer cells-TE-1, human bladder cancer cells-5637, human gallbladder cancer cells-GBC-SD, human breast cancer cells-MCF7, human liver cancer cells-HepG2, human brain tumor cells-SF126, human prostate cancer cells-DU145, human thyroid cancer cells-CAL-62, human pancreatic cancer cells-PATU8988T, human osteosarcoma cells-HOS, human malignant melanoma cells-A-375, human rhabdomyosarcoma cells-A-673) and 2 normal cell lines (human embryonic kidney cells-293T, human normal liver cells- L-02), for screening the antitumor activity of compounds; B) 10 pathogenic bacteria including Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ATCC13637), Enterococcus faecalis ( Enterococcus faecalis ATCC19433), Klebsiella pneumoniae ( Klebsiella Pneumoniae ATCC13833), Acinetobacter baumannii ( Acinetobacter baumannii Acinetobacter pylori ( ), Acinetobacter pittii Providencia ( Providencia Bacillus subtilis ( Bacillus subtilis ), Pseudomonas aeruginosa (Pseudomonas aeruginosa 27853), Staphylococcus aureus (ATCC 29213), Escherichia coli (ATCC 25922), for the screening of antibacterial activity of compounds. Staphylococcus aureus 29213), Escherichia coli (ATCC 25922), for the screening of antibacterial activity of compounds. Escherichia coli 25922), for the screening of antibacterial activity of compounds.

[0021] 1. Fermentation method of Actinomyces strains

[0022] (1) Seed liquid was taken from 30% glycerol tube and spread on SFM solid medium for strain recovery;

[0023] (2) Spores of the strain were scraped with a gun head and inoculated into LB liquid medium for seed liquid culture;

[0024] (3) 1 mL seed liquid was taken and inoculated into a 1 L conical flask containing 200 mL MS liquid medium, and fermentation was carried out at 28°C for 10 L with 180 rpm shaking for 7 days.

[0025] Culture medium and ingredients used:

[0026] Solid medium (w / v)

[0027] SFM: 2% soybean flour, 2% mannitol, 2% agar.

[0028] Liquid medium (w / v)

[0029] LB: 1% tryptone, 1% sodium chloride, 0.5% yeast extract powder;

[0030] MS: 2% soybean flour, 2% mannitol, 0.3% calcium carbonate, (PH 7.0).

[0031] 2. Processing method of fermentation broth

[0032] (1) The fermentation broth was extracted with an equal volume of ethyl acetate for 3 times;

[0033] (2) The ethyl acetate extraction phase was evaporated to dryness, redissolved with methanol, and the precipitate was discarded after centrifugation at 10000 rpm;

[0034] (3) The supernatant was added to an ODS (C18 filler YMC*GEL ODS-A-HG, 12 nm S-10 μm) column, and eluted with 30%, 70%, and 100% methanol-water system, respectively, to obtain 3 components;

[0035] (4) The 30% component was separated by LH-20 (Cytiva Sephadex TM LH-20) with 100% MeOH, and collected in a glass test tube;

[0036] (5) The compound-enriched part was spin-dried and separated by HPLC (HITACHI 5430), with mobile phase A being ultrapure water containing 0.1% formic acid and mobile phase B being acetonitrile containing 0.1% formic acid, and the liquid phase conditions being 20%-70%-100% acetonitrile, 35 min, C18 (YMC-Actus Triart C18 250 x 20.0, S-5 μm, 12 nm).

[0037] 3. Structure identification of the compound

[0038] The planar structure of the compound was determined by 1 H NMR, 13 C NMR, HSQC, COSY and HMBC, and the planar structure of the compound and the main two-dimensional nuclear magnetic correlation signals are shown in Figure 2 , which is an alkaloid compound.

[0039] Table 1. H (600 MHz) and 1 C (151 MHz) nuclear magnetic information (DMSO-d6, δ in ppm, J in Hz) of compound 423. 13

[0040]

[0041] The absolute configuration of the compound was determined by ECD calculation, and the calculation result showed that the absolute configuration of the compound was R type ( Figure 3 ).

[0042] 4. Compound characterization

[0043] Yellow amorphous powder; UV (MeOH) λ max : 215, 245, 280, 310 nm; 1 H and 13 C NMR spectral data, see Table 1; HR-ESI-MS: m / z 423.1902 [M + H] + (calcd.for C 24 H 27 N2O5, 423.1920)。

[0044] 5. Compound activity test results

[0045] 5.1 Compound cytotoxicity test primary screening

[0046] ​The monomer compound cytotoxicity activity primary screening concentration is 50 μM, and the primary screening results are shown in the following table. The cytotoxicity activity results show that the compound has inhibitory activity on 20 cells, of which the inhibition rate of 14 cells is higher than 80%, and the inhibition rate of 7 cells is higher than the positive control cisplatin.

[0047] Table 2. Compound 423 cytotoxicity activity primary screening test results on 20 human cells (cell inhibition rate ± SD%)

[0048]

[0049] 5.2 Compound cytotoxicity test rescreening

[0050] The 14 cells with an inhibition rate higher than 80% for the compound were subjected to activity rescreening work, and the activity rescreening results are shown in the following table:

[0051] A. The inhibitory effect of the compound on K-562 and PATU8988T cells is much better than that of the positive drug cisplatin;

[0052] B. The toxicity of the compound to normal human cells L-02 and 293T is significantly lower than that of the positive drug cisplatin.

[0053] Table 3. Compound 423 cytotoxicity activity rescreening results (IC50 ± SD μM) on 14 human cells

[0054]

[0055] 5.3 Compound antibacterial activity test

[0056] A total of 10 pathogenic bacteria were selected for antibacterial experiment test of the isolated monomer compound, and the information of the 10 strains and the positive drug used are shown in Table 4.

[0057] 5.3.1 Antibacterial experiment operation process:

[0058] (1) The monomer compound and the positive drug are prepared into a starting concentration of 10 mM, and are sequentially diluted to 5 mM, 2 mM, 1 mM, 0.5 mM, 0.2 mM, 0.1 mM, 0.05 mM, 0.02 mM, 0.01 mM, 0.005 mM, 0.002 mM, a total of 12 concentration gradients in a 96-well plate;

[0059] (2) After activation, the 10 pathogenic bacteria were placed in LB liquid medium and cultured at 37°C on a shaking table for 12 h;

[0060] (3) The blank LB medium was aspirated into row A of the 96-well plate, 100 μL / well;

[0061] (4) The grown bacteria liquid is diluted with LB medium to OD value of 0.01-0.02, and the diluted bacteria liquid is taken by a row gun to 96-well plates B-H row, 100 μL / well;

[0062] (5) The row gun is used to take 12 monomer compound solutions of concentration gradient D to 96-well plates B, C, D row, 1 μL / well, and 12 positive drug solutions of concentration gradient are added to 96-well plates E, F, G row, 1 μL / well;

[0063] (6) The bacteria liquid in H row is grown to OD value of 0.2-0.4, and the antibacterial concentration of the compound is calculated.

[0064] 5.3.2 Enzyme marker parameter setting:

[0065] End point method, detection thin wavelength 595 nm, no reference wavelength.

[0066] 5.3.3 Antibacterial experiment results

[0067] The results show that the compound has inhibitory activity IC 50 < 5 μM on Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Acinetobacter pittii, Bacillus subtilis, Staphylococcus aureus, and no obvious inhibitory activity on Enterococcus faecalis and Providencia.

[0068] Table 4 Antibacterial activity test results (IC50 μM) of compound 423 on 10 pathogenic bacteria

[0069]

[0070] It should be noted that the above only describes the preferred embodiments of the present application, and is not used to limit the present application. For those skilled in the art, the present application can have various changes and variations. Without creative labor, any modification, equivalent replacement, improvement, etc. should be included in the protection scope of the present application.

Claims

1. A Streptomyces, characterized in that, The classification name is Streptomyces ramosus Streptomyces labedae W307 was preserved in China General Microbiological Culture Collection Center on November 20, 2024, and the strain preservation number is CGMCC No. 32721.

2. An alkaloid compound, characterized in that, The molecular formula is C 24 H 26 N2O5, having the following structure: 。 3. A method for preparing the alkaloid compound according to claim 2, characterized in that, Streptomyces rimosus according to claim 1 Streptomyces labedae W307 was prepared by fermentation extraction, separation and purification, and the 16S rRNA gene sequence of the Streptomyces rimosus is shown as SEQ ID No.

1.

4. The production method according to claim 3, characterized by, The extraction, separation and purification include the following steps: activating the streptomyces; using MS medium to shake bed large-scale fermentation actinomycetes; using ODS, LH-20 and HPLC to separate and purify the compounds; using 1 H NMR, 13 C NMR, HSQC, COSY and HMBC to analyze the planar structure of the compound, and using ECD calculation method to determine the absolute configuration of the compound.

5. The use of the alkaloid compound of claim 2 in the preparation of an antitumor drug. The disease to which the antitumor drug is directed is one or more of lung cancer, stomach cancer, colon cancer, cervical cancer, chronic myelogenous leukemia, renal clear cell adenocarcinoma, esophageal cancer, bladder cancer, gallbladder cancer, breast cancer, liver cancer, brain tumor, prostate cancer, thyroid cancer, pancreatic cancer, osteosarcoma, malignant melanoma, rhabdomyosarcoma.

6. The use of the alkaloid compound of claim 2 in the preparation of an antibacterial medicament. The pathogenic bacteria targeted by the bacteriostatic drug are one or more of Stenotrophomonas maltophilia ( Stenotrophomonas maltophilia ), Klebsiella pneumoniae ( Klebsiella Pneumoniae ), Acinetobacter baumannii ( Acinetobacter baumannii ), Acinetobacter pittii ( Acinetobacter pittii ), Bacillus subtilis ( Bacillus subtilis ), Staphylococcus aureus ( Staphylococcus aureus ).

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