Streptomyces sp., alkaloid compound produced by streptomyces sp., and preparation method and application of alkaloid compound

Abstract

The invention discloses streptomyces, alkaloid compounds produced by the streptomyces, a preparation method and application of the alkaloid compounds, the streptomyces is classified and named as Streptomyces labeckii W307, the Streptomyces labeckii W307 is preserved in the China General Microbiological Culture Collection Center on November 20, 2024, and the preservation number of the strain is CGMCC No.32721. The invention further discloses a preparation method of the alkaloid compounds produced by the streptomyces labeckii W307 and application of the alkaloid compounds produced by the streptomyces labeckii W307. The alkaloid compound is prepared by fermentation, extraction, separation and purification of the streptomyces lageri W307, and the 16S rRNA (Ribose Nucleic Acid) of the streptomyces lageri strain is as shown in SEQ ID NO. 1; the invention further provides application of the alkaloid compound in preparation of anti-tumor drugs and application of the alkaloid compound in preparation of antibacterial drugs, and a potential anti-tumor drug and a potential antibacterial drug are provided clinically.

Description

Technical Field

[0001] The present invention relates to the discovery of new skeleton compounds in the field of natural drug chemistry and the screening of new anti-tumor drugs and antibacterial drugs, and in particular to a streptomyces, alkaloid compounds produced by the streptomyces, and a preparation method and application thereof. Background Art

[0002] Actinomycetes, as a type of microbial resource, are closely related to human production and life, and therefore have important value. Among the antibiotics currently in common use, actinomycetes account for about 70%. However, due to climate change, human activities and other factors, the speed of discovering new metabolites from terrestrial actinomycetes is currently on a downward trend. Therefore, people are turning their attention to another new source of drugs with great development potential - marine actinomycetes.

[0003] Due to the special low temperature, low oxygen, low light and high pressure environmental characteristics of the deep sea, numerous marine microorganisms with unique biosynthetic pathways are bred, including unique actinomycete populations that can produce a variety of different secondary metabolites, such as Streptomyces and Nocardia. Nowadays, with the rapid development of new marine sampling, cultivation and identification technologies, more and more secondary metabolites with novel molecular structures and good physiological activities have been discovered. Actinomycetes from the ocean have become an important source for the discovery of drug lead compounds and drug development. Summary of the invention

[0004] The present invention aims to provide a streptomyces, an alkaloid compound produced by the streptomyces, and a preparation method and application thereof, in view of the shortcomings of low novelty rate of compounds derived from terrestrial actinomycetes and inactivity of compounds derived from natural sources.

[0005] The object of the present invention is achieved by the following technical solutions: As a first aspect, the present invention provides a Streptomyces, which is classified and named Streptomyces labedae W307, and is deposited in the China General Microbiological Culture Collection Center on November 20, 2024, with a culture collection number of CGMCC No. 32721. The Streptomyces is of marine origin.

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

[0007]

[0008] As a third aspect, a method for preparing the alkaloid compound is provided, wherein the alkaloid compound is prepared by fermentation, extraction, separation and purification of the marine-derived Streptomyces labedae W307, wherein the 16SrRNA gene sequence of the Streptomyces labedae is shown in SEQ ID No.1.

[0009] Furthermore, the extraction, separation and purification comprises the following steps: activating the streptomyces; fermenting the actinomycetes on a large scale using an MS medium shaker; separating and purifying the compound using ODS, LH-20 and HPLC; 1 H NMR, 13 C NMR, HSQC, COSY, and HMBC were used to analyze the planar structure of the compound, and the absolute configuration of the compound was determined by ECD calculation method.

[0010] As a fourth aspect, a use of the alkaloid compound in the preparation of anti-tumor drugs is provided.

[0011] Furthermore, the diseases targeted by the anti-tumor drug include: lung cancer, gastric cancer, colon cancer, cervical cancer, chronic myeloid 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, and rhabdomyosarcoma.

[0012] As a fifth aspect, there is provided a use of the alkaloid compound in the preparation of antibacterial drugs.

[0013] Furthermore, the pathogenic bacteria targeted by the antibacterial drug include: Stenotrophomonas maltophilia, Klebsiella pneumoniae, Acinetobacter baumannii, Acinetobacter pilosella, Bacillus subtilis, and Staphylococcus aureus.

[0014] The beneficial effects of the present invention are that the strain is Streptomyces of marine origin, which makes useful exploration and practical accumulation for the discovery and sustainable utilization of marine natural drug resources; 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 screened out good antibacterial activity and cytotoxic activity, and the inhibitory effect on some tumor cells is better than that of the positive drug cisplatin, and the cytotoxicity of normal cells is less than that of the positive drug cisplatin, which is of great significance for the screening of new anti-tumor drugs and antibacterial drugs. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0016] Figure 2 The structure diagram of the compound, where (A) is the planar structure of the compound, and (B) is a schematic diagram of the main two-dimensional NMR related signals;

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

[0018] An embodiment of the present invention provides a Streptomyces and an alkaloid compound with cytotoxic activity and antibacterial activity produced by the same. The bacterium is Streptomyces labedae W307, which was deposited in the China General Microbiological Culture Collection Center on November 20, 2024, with a culture collection number of CGMCC No.32721, and its 16SrRNA has the gene sequence shown in SEQ ID No.1.

[0019] The fermentation method of the bacteria and the extraction, separation and identification method of the compound include: A) taking seed liquid from a glycerol tube to a plate to activate the actinomycetes; B) using MS culture medium shaker to ferment the actinomycetes on a large scale; C) using ODS, LH-20, HPLC and other means to separate and purify the compound; C) using 1 H NMR, 13 The planar structure of the compound was analyzed by C NMR, HSQC, COSY, HMBC, etc., and the absolute configuration of the compound was determined by ECD calculation method.

[0020] The activity test results of the isolated compounds of the above embodiments of the present invention include A) 18 classical cells of mainstream cancer-causing organs in China (human lung cancer cells-A549, human gastric cancer cells-MKN-45, human colon cancer cells-HCT116, human cervical cancer cells-HeLa, human chronic myeloid leukemia cells-K-562, human renal 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) selected. -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 anti-tumor activity screening of compounds; B) 10 pathogenic bacteria including Stenotrophomonas maltophilia maltophiliaATCC13637), Enterococcus faecalis ATCC19433, Klebsiella Pneumoniae ATCC13833, Acinetobacter baumannii, Acinetobacter pylori, Providencia, Bacillus subtilis, Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213, Escherichia coli 25922, for the antibacterial activity screening of the compounds.

[0021] 1. Fermentation method of actinomycetes strains

[0022] (1) Pipette the seed solution from the 30% glycerol tube and apply it on SFM solid medium to revive the strain;

[0023] (2) Scrape the spores of the strain with a gun tip and inoculate them into LB liquid medium for seed culture;

[0024] (3) 1 mL of seed solution was inoculated into a 1 L conical flask containing 200 mL of MS liquid medium, and 10 L of the solution was fermented in a shaking incubator at 28°C and 180 rpm 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 culture medium (w / v)

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

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

[0031] 2. Treatment of fermentation broth

[0032] (1) extracting the fermentation broth three times with equal volumes of ethyl acetate;

[0033] (2) Evaporate the ethyl acetate extract to dryness, re-dissolve in methanol, centrifuge at 10,000 rpm and discard the precipitate;

[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 to obtain three components;

[0035] (4) 30% of the fraction was washed with 100% MeOH in LH-20 (Cytiva Sephadex TM LH-20) for separation and glass test tubes for collection;

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

[0037] 3. Structural identification of compounds

[0038] The planar structure of the compound is 1 H NMR, 13 The planar structure and main 2D NMR related signals of the compound determined by C NMR, HSQC, COSY, TOCXY, and HMBC are as follows: Figure 2 As shown, it is an alkaloid compound.

[0039] Table 1 Compound 423 1 H(600MHz) and 13C(151MHz) NMR information (DMSO-d6,δin ppm,J inHz).

[0040]

[0041] The absolute configuration of the compound was determined by ECD calculation. The calculation results 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 :215,245,280,310nm; 1 H and 13 C NMRspectral 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 Initial screening of compound cytotoxicity test

[0046] The initial screening concentration of the monomer compound's cytotoxic activity was 50 μM. The initial screening results are shown in the table below. The cytotoxic activity results showed that the compound had inhibitory activity against 20 types of cells, among which the inhibition rate against 14 types of cells was higher than 80%, and the inhibition rate against 7 types of cells was higher than that of the positive control cisplatin.

[0047] Table 2 Preliminary screening test results of the cytotoxic activity of compound 423 on 20 types of human cells (cell inhibition rate ± SD%)

[0048]

[0049] 5.2 Compound cytotoxicity test rescreening

[0050] The activity rescreening work was carried out on 14 cell types with compound inhibition rates higher than 80%. 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 Results of rescreening of the cytotoxic activity of compound 423 against 14 human cells (IC50±SDμM)

[0054]

[0055] 5.3 Antibacterial activity test of compounds

[0056] A total of 10 pathogenic bacteria were selected to conduct antibacterial experiments on the isolated monomer compounds. The information of the 10 strains and the positive drugs used are shown in Table 4.

[0057] 5.3.1 Antibacterial experiment operation process:

[0058] (1) The monomer compound and the positive drug were prepared to a starting concentration of 10 mM, and then diluted in a 96-well plate 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, for a total of 12 concentration gradients;

[0059] (2) After activating the 10 pathogenic bacteria, they were cultured in LB liquid medium at 37°C in a shaking incubator for 12 h;

[0060] (3) Pipette blank LB medium into row A of a 96-well plate, 100 μL / well;

[0061] (4) Dilute the grown bacterial solution with LB medium to an OD value between 0.01 and 0.02. Pipet the diluted bacterial solution into rows B and H of a 96-well plate at 100 μL per well.

[0062] (5) Use a gun to pipette 12 monomer compound solutions of concentration gradient D into rows B, C, and D of a 96-well plate, 1 μL / well. Pipet 12 positive drug solutions of concentration gradient D into rows E, F, and G of a 96-well plate, 1 μL / well.

[0063] (6) When the bacterial solution in row H grows to an OD value between 0.2 and 0.4, calculate the inhibitory concentration of the compound.

[0064] 5.3.2 ELISA reader parameter settings:

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

[0066] 5.3.3 Antibacterial test results

[0067] The results showed that the inhibitory activity of the compounds against Stenotrophomonas maltophilia, Klebsiella pneumoniae, Acinetobacter baumannii, Acinetobacter pilosulae, Bacillus subtilis, and Staphylococcus aureus was IC 50 <5μM, no significant inhibitory activity against Enterococcus faecalis and Providencia.

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

[0069]

[0070] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Without making creative work, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims

1. A Streptomyces, characterized in that It was classified and named Streptomyces labedae W307, and was deposited in the China General Microbiological Culture Collection Center on November 20, 2024, with the culture collection number CGMCC No.32721.

2. An alkaloid compound, characterized in that The molecular formula is C 24 H 26 N2O5 has the following structure:

3. A method for preparing the alkaloid compound according to claim 2, characterized in that: It is prepared by fermentation, extraction, separation and purification of the Streptomyces labedae W307 according to claim 1, wherein the 16SrRNA gene sequence of the Streptomyces labedae is shown in SEQ ID No.

1.

4. The preparation method according to claim 3, characterized in that: The extraction, separation and purification comprises the following steps: activating the streptomyces; fermenting the actinomycetes on a large scale using an MS medium shaker; separating and purifying the compound using ODS, LH-20 and HPLC; 1 H NMR, 13 C NMR, HSQC, COSY, and HMBC were used to analyze the planar structure of the compound, and the absolute configuration of the compound was determined by ECD calculation method.

5. Use of the alkaloid compound according to claim 2 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that: The anti-tumor drugs target the following diseases: lung cancer, gastric cancer, colon cancer, cervical cancer, chronic myeloid 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, and rhabdomyosarcoma.

7. Use of the alkaloid compound according to claim 2 in the preparation of antibacterial drugs.

8. The use according to claim 7, characterized in that: The pathogenic bacteria targeted by the antibacterial drug include: Stenotrophomonas maltophilia, Klebsiella pneumoniae, Acinetobacter baumannii, Acinetobacter pilosulae, Bacillus subtilis, and Staphylococcus aureus.

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