Streptomyces AH1901-2, application thereof, preparation method and application of naphthomycin compounds

Abstract

The present application relates to the technical field of biological medicine, in particular to streptomyces AH1901-2 and application thereof, preparation method and application of naphthymycin compounds, and belongs to the field of biological medicine. The streptomyces AH1901-2 is isolated from rhizosphere soil of Ligularia sibirica in Tianshan region (5.8°E, 28.0°N) in Xinjiang, and naphthymycin compounds are isolated and purified from liquid fermentation liquor of the streptomyces AH1901-2, and subsequent studies show that the naphthymycin compounds have obvious inhibitory effect on NO production of mouse mononuclear macrophages, and can be used for preparing anti-inflammatory drugs and treating inflammation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to Streptomyces AH1901-2 and its applications, and to methods for preparing and applying naphthycin compounds. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Microbial bioactive secondary metabolites, especially those from actinomycetes, play a crucial role. Studies have shown that nearly ten thousand bioactive compounds have been discovered in actinomycetes, with the majority of these compounds originating from the genus Streptomyces (Streptomyces). Streptomyces The discovery of new compounds in actinomycetes derived from basic soil has decreased over the past two decades. Consequently, scientists have increasingly shifted their research focus to actinomycetes from unique habitats such as glaciers, oceans, plateaus, and the endophytic environments of plants and animals. To adapt to harsh environments such as low temperature, low oxygen, high pressure, and high salinity, microorganisms in these special habitats have developed unique new genes related to bioactive substance synthesis pathways during long-term evolution, resulting in the production of many new secondary metabolites.

[0004] Naphthylmycins belong to the 29-membered aniline macrolide antibiotic family and possess the largest ring in the anilinemycin family to date. Structurally, they not only have the characteristic naphthalene ring and C12-13 shifted double bond, but also the characteristic triene. Naphthylmycin compounds exhibit significant anti-inflammatory, antibacterial, and antitumor biological activities, demonstrating great potential for drug development. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a Streptomyces AH1901-2 and its application, a method for preparing naphthycin compounds and their applications.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a Streptomyces ( Streptomyces The Streptomyces sp.) AH1901-2, the preservation number of which is CGMCC No. 32711.

[0008] A second aspect of the present invention provides the use of Streptomyces AH1901-2 described in the first aspect in the preparation of naphthycin compounds, said naphthycin compounds comprising compound 1, compound 2, compound 3, and compound 4; the structural formulas of compound 1, compound 2, compound 3, and compound 4 are shown sequentially as formulas I to IV:

[0009]

[0010] .

[0011] Among them, compound 1 is named Naphthomycin R, compound 2 is named Naphthomycin S, compound 3 is named Naphthomycin T, and compound 4 is the known compound Naphthomycin E.

[0012] A third aspect of the present invention provides a compound, said compound being a naphthycin derivative, said compound having a chemical structure as shown in Formulas I to III below, or a stereoisomer thereof, or a physiologically acceptable salt thereof:

[0013] .

[0014] A fourth aspect of the present invention provides a method for preparing the naphthycin-like compounds described in the second aspect, comprising fermenting Streptomyces AH1901-2 as described in the first aspect to obtain a liquid fermentation culture; extracting, separating, and obtaining the naphthycin-like compounds.

[0015] In one or more embodiments, a method for obtaining a liquid fermentation culture by fermentation culture of Streptomyces AH1901-2 includes:

[0016] Streptomyces AH1901-2 was inoculated into TSB medium, and a seed culture was obtained after the first culture. The seed culture was then inoculated into ISP-7 liquid medium, and a second culture was conducted to obtain mycelium and liquid fermentation products.

[0017] Preferably, the ISP-7 liquid culture formulation is as follows: 15 g glycerol, 0.5 g L-tyrosine, 1 g L-aspartic acid, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 2 g NaCl, 0.01 g FeSO4·7H2O, 1 mL trace element solution, and 1000 mL distilled water; pH 7.2~7.4; wherein the trace element solution is formulated as follows: 0.1 g FeSO4·7H2O, 0.1 g MnCl2·4H2O, 0.1 g ZnSO4·7H2O, plus 100 mL distilled water.

[0018] In one or more embodiments, the extraction includes the following steps:

[0019] The liquid fermentation culture was subjected to macroporous resin solid-phase extraction, and the macroporous resin solid-phase extract was eluted with methanol to obtain a liquid fermentation extract. The crude extract was obtained by vacuum distillation.

[0020] In one or more embodiments, the separation includes the following steps:

[0021] The crude extract was separated by normal-phase silica gel column chromatography, and eluted sequentially with dichloromethane-methanol systems of varying concentrations (volume ratios of 100:0, 100:1, 100:2, 100:3, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1, respectively). The elution fractions Fr. 2 (dichloromethane to methanol volume ratio of 100:2 to 100:3) and Fr. 6 (dichloromethane to methanol volume ratio of 100:3 to 20:1) were collected.

[0022] The elution fractions Fr. 2 and Fr. 6 were subjected to preparative high-performance liquid chromatography (HPLC) with water and acetonitrile as the mobile phase. The purification conditions for elution fractions Fr. 2 and Fr. 6 were 60% acetonitrile and 63% acetonitrile, respectively, to obtain the naphthycin compounds.

[0023] Preferably, compound 4 is obtained by preparative high performance liquid chromatography (HPLC) of elution fraction Fr. 2 with a retention time of 7.5 min, and compound 3 is obtained with a retention time of 9.5 min.

[0024] The elution fraction Fr. 6 was prepared by high performance liquid chromatography (HPLC) to obtain compound 2 with a retention time of 3.7 min and compound 1 with a retention time of 8.2 min.

[0025] A fifth aspect of the present invention provides the use of the Streptomyces AH1901-2 or naphthycin compounds described in the first aspect in the preparation of anti-inflammatory drugs;

[0026] The naphthycin compounds include compound 1, compound 2, compound 3, and compound 4; the structural formulas of compound 1, compound 2, compound 3, and compound 4 are shown in formulas I to IV respectively:

[0027]

[0028] .

[0029] A sixth aspect of the present invention provides an anti-inflammatory drug comprising an active ingredient and pharmaceutically acceptable excipients; said active ingredient comprising a naphtholic cytokine compound, said naphtholic cytokine compound comprising compound 1, compound 2, compound 3 and compound 4; the structural formulas of compound 1, compound 2, compound 3 and compound 4 are shown sequentially as shown in Formulas I to IV:

[0030]

[0031] .

[0032] The beneficial effects of this invention are as follows:

[0033] This invention isolates Streptomyces ( ) from the rhizosphere soil of Ligustrum lucidum in the Tianshan region of Xinjiang (5.8°E, 28.0°N). Streptomyces Naphthycin compounds were isolated and purified from the liquid fermentation broth of Streptomyces AH1901-2. Subsequent studies have shown that naphthycin compounds have a significant inhibitory effect on NO production in mouse mononuclear macrophages and can be used to prepare anti-inflammatory drugs for the treatment of inflammation. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 This image shows the growth morphology of Streptomyces AH1901-2 on ISP-7 medium.

[0036] Figure 2 Phylogenetic tree analysis for Streptomyces AH1901-2;

[0037] Figure 3 The HPLC chromatogram of the fermentation products of Streptomyces AH1901-2 is shown.

[0038] Figure 4 The HRESI-MS spectrum of naphthycin compound 1;

[0039] Figure 5 For naphthycin compound 1 1 H-NMR (600 MHz, CDCl3) spectrum;

[0040] Figure 6 For naphthycin compound 1 13 C-NMR (150 MHz, CDCl3) spectrum;

[0041] Figure 7 The HRESI-MS spectrum of naphthycin compound 2;

[0042] Figure 8 For naphthycin compound 2 1 H-NMR (600 MHz, CD3OD) spectrum;

[0043] Figure 9 For naphthycin compound 2 13 C-NMR (150 MHz, CD3OD) spectrum;

[0044] Figure 10 The HRESI-MS spectrum of naphthycin compound 3;

[0045] Figure 11 For naphthycin compound 3 1 H-NMR (600 MHz, CDCl3) spectrum;

[0046] Figure 12 For naphthycin compound 3 13 C-NMR (150 MHz, CDCl3) spectrum;

[0047] Figure 13 For naphthycin compound 4 1 H-NMR (600 MHz, CDCl3) spectrum;

[0048] Figure 14 For naphthycin compound 4 13 C-NMR (150 MHz, CDCl3) spectrum. Detailed Implementation

[0049] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0050] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0051] Preservation instructions:

[0052] Biological material: Streptomyces AH1901-2, classification and nomenclature: Streptomyces sp. AH1901-2 was deposited on November 19, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 32711.

[0053] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0054] The culture media used in the following examples include:

[0055] The MS medium (mannitol-soybean agar medium) formula is: 20 g soybean flour, 20 g mannitol, 20 g agar powder, and 1000 mL distilled water; the pH value is left unadjusted. Before use, add 2.5 mol / L MgCl2 solution to a final concentration of 10 mmol / L. The solid medium preparation process is as follows: weigh, dissolve, and stir the ingredients according to the above formula, and autoclave at 115 ℃ for 30 min.

[0056] The ISP-7 liquid culture formulation is as follows: 15 g glycerol, 0.5 g L-tyrosine, 1 g L-aspartic acid, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 2 g NaCl, 0.01 g FeSO4·7H2O, 1 mL trace element solution, and 1000 mL distilled water; pH 7.2~7.4; wherein, the trace element solution formulation is: 0.1 g FeSO4·7H2O, 0.1 g MnCl2·4H2O, 0.1 g ZnSO4·7H2O, plus 100 mL distilled water.

[0057] TSB medium is commercially available.

[0058] Example 1

[0059] Isolation, solid culture and preservation of Streptomyces strain AH1901-2:

[0060] (1) Isolation and solid-state culture of strains:

[0061] Streptomyces was isolated from the rhizosphere soil of Ligustrum lucidum in the Tianshan region of Xinjiang (5.8°E, 28.0°N). Streptomyces Streptomyces sp. AH1901-2 was inoculated into MS medium plates and cultured at 30 ℃. The Streptomyces sp. AH1901-2 grew well in the 30 ℃ incubator, and the colony morphology after 5-7 days of culture was as follows. Figure 1 As shown. Initially, the colonies are white, small, compact, and opaque. As they grow, the surface gradually becomes velvety, dark brown, and generally round with spiral edges, making them difficult to pick up.

[0062] (2) Preservation of strains:

[0063] A single colony of *Streptomyces* AH1901-2, activated on MS medium, was inoculated into 50 mL of TSB medium containing glass beads. After 24 h, 500 μm of the bacterial culture was transferred to a 1.5 mL centrifuge tube, and an equal volume of 50% glycerol was added. The mixture was then stored at -80 °C. The strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on November 19, 2024, with accession number CGMCC No. 32711.

[0064] Example 2

[0065] Species identification of Streptomyces AH1901-2 strain:

[0066] (1) A single colony of the purified Streptomyces AH1901-2 cultured in Example 1 was picked and inoculated into 50 mL of TSB medium with glass culture, and cultured in a shaker at 30 ℃ and 200 rpm for 24 ~ 30 h. Then the bacterial culture was transferred to a 50 mL centrifuge tube, centrifuged at 8000 rpm at room temperature for 10 min, the supernatant was discarded and the bacterial cells were collected. Finally, the bacterial cells were sent to a sequencing company for whole genome sequencing.

[0067] (2) Transfer the fermented bacterial solution from (1) to a 50 mL centrifuge tube and centrifuge at 8000 rpm for 10 min at room temperature to remove the supernatant as much as possible;

[0068] (3) Resuspend the bacterial cells in 30 mL of sterile water, mix well, centrifuge at 8000 rpm for 10 min at room temperature, and discard the supernatant;

[0069] (4) Repeat step (3) twice more to remove the supernatant as much as possible;

[0070] (5) Take 8 mL of the prepared SET solution (75 mM NaCl, 25 mM EDTA, 20 mM Tris, pH 8.0) into a 50 mL centrifuge tube and resuspend the bacterial cells;

[0071] (6) Then add 100 μL of 25 mg / mL lysozyme, mix by inverting, and incubate in a 37 ℃ water bath for 1 h, inverting and mixing once every 20 min during the period;

[0072] (7) Add 500 μL of 20 mg / mL proteinase K, invert and mix well, then add 1 mL of 10% SDS, and continue to invert and mix well.

[0073] (8) Then incubate in a 50℃ water bath for 2 hours, inverting and mixing once every 30 minutes until the bacterial solution becomes clear (if the bacterial solution does not become clear after 2 hours, the incubation time needs to be extended appropriately).

[0074] (9) Add 3.5 mL of 5 mol / L NaCl solution to the clear bacterial solution and mix by inverting.

[0075] (10) Then add 5 mL of sterile water, and then add 15 mL of phenol-chloroform-isoamyl alcohol (volume ratio of 25:24:1), and mix thoroughly by inverting until the solution turns milky white;

[0076] (11) Centrifuge at 8000 rpm for 10 min at room temperature;

[0077] (12) Use a 1 mL de-sharpened pipette tip to draw 500 μL of the supernatant and transfer it to a 2 mL Ep tube until the supernatant is completely transferred.

[0078] (13) Add 35 μL of 3 mol / L sodium acetate (NaAC, pH 7.5) to each 2 mL Ep tube, then add 1.2 mL of anhydrous ethanol, gently invert and mix until white flocculent DNA appears;

[0079] (14) Prepare a new 2 mL Eppendorf tube, add 1.5 mL of 75% ethanol to each tube, and then use a 1 mL blue pipette tip to pick up the white flocculent DNA into the 75% ethanol.

[0080] (15) After centrifuging at 10,000 rpm for 5 min at room temperature, pour off the alcohol, invert the container onto absorbent paper, drain excess liquid, and then place it in an oven to dry.

[0081] (16) Dissolve the DNA in 500 μL of sterile water and store at 4 °C for later use;

[0082] (17) The 16S rRNA gene was amplified by polymerase chain reaction (PCR), and the PCR product was sequenced by a sequencing company. After sequencing, the 16S rRNA gene sequence was compared using BLAST on the National Center for Biotechnology Information (NCBI) website, and the sequence was analyzed using a constructed phylogenetic tree (e.g., Figure 2 As shown in the image, strain AH1901-2 and... Streptomyces reticuli Within the same evolutionary clade, it was identified as belonging to the genus *Streptomyces*. Streptomyces sp.).

[0083] Example 3

[0084] Preparation of naphthycin compounds:

[0085] (1) First, Streptomyces AH1901-2 was inoculated into MS medium and incubated in a 30 ℃ constant temperature incubator for 5-7 days;

[0086] (2) Seed culture preparation: Use an inoculation loop to scoop out a single colony of Streptomyces AH1901-2 in good growth condition from (1) above and inoculate it into 50 mL of TSB medium with glass beads (250 mL Erlenmeyer flask), and incubate at 30 ℃ and 200 rpm for 24 h.

[0087] (3) Preparation of fermentation broth: Take 1 mL of the seed liquid obtained in (2) above and inoculate it into the fermentation medium ISP-7. Shake and culture at 200 rpm in a shaker at 30℃ for 7 days. On the 6th day, add 1 mL of non-ionic macroporous resin XAD-16 to adsorb secondary metabolites.

[0088] (4) Post-fermentation treatment: Transfer the fermentation broth to a 50 mL centrifuge tube, centrifuge at 25 ℃ and 8000 rpm for 10 min, discard the supernatant, and invert it onto absorbent paper to drain; then add 30 mL of methanol to the centrifuge tube, vortex to mix, and extract by shaking on a homogenizer for 4 h; then filter the extract and distill under reduced pressure to obtain crude extract.

[0089] (5) The crude extract obtained in (4) above was mixed with dry method and packed into a column by wet method, and then eluted in a silica gel column of 200-300 mesh. Dichloromethane and methanol were used as elution solvents. The elution was carried out in sequence according to the volume ratio of dichloromethane to methanol of 100:0, 100:1, 100:2, 100:3, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1 and 0:1. The elution volume of each gradient was about 2L. After evaporation, the fractions were combined to obtain 6 fractions (Fr. 1-Fr. 6). The elution fractions Fr. 2 (120 mg) with the volume ratio of dichloromethane to methanol of 100:2 to 100:3 and Fr. 6 (180 mg) with the volume ratio of dichloromethane to methanol of 100:3 to 20:1 were collected.

[0090] (6) The fractions Fr. 2 and Fr. 6 obtained in step (5) above were separated and purified by HPLC. Water and acetonitrile were used as the mobile phase. The elution conditions were 60% acetonitrile and 63% acetonitrile, respectively. The mobile phase flow rate was 4.7 mL / min. Fractions 1 (5.6 mg) and 2 (3.3 mg) with retention times of 8.2 min and 3.7 min for Fr. 6 were collected. Fractions 3 (5.5 mg) and 4 (1.6 mg) with retention times of 9.5 min and 7.5 min for Fr. 2 were collected. Finally, the four naphthycin compounds with structural formulas I to IV were obtained by vacuum distillation.

[0091] Example 4: HPLC chromatographic analysis of crude extract and structural identification of oligomycin compounds

[0092] 4.1 HPLC chromatographic analysis of the crude extract:

[0093] The crude extract obtained in step (4) of Example 3 was dissolved in chromatographic methanol and transferred to a 1.5 mL centrifuge tube. It was centrifuged at 14600 rpm for 20 min. The supernatant was then aspirated with a 1 mL syringe, and the liquid was filtered through a 0.22 μm organic phase filter membrane into a 1.5 mL liquid chromatography vial for high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. The instrument used for HPLC analysis was a UtiMate™ 3000-Bruker impact HD, with a Thermo Scientific™ Acclaim™ C18 column (2.1 × 100 mm, 2.2 μm, 0.3 mL / min). The UV absorption wavelength was 190–400 nm, the column temperature was 25 ℃, and the mobile phase consisted of phase A (ddH2O and 0.1% formic acid) and phase B (acetonitrile and 0.1% formic acid). The detection wavelengths were 210 nm, 254 nm, and 290 nm, and the elution program was 0–3 min. The infusion rates were 5% B, 3-18 min 5%-95% B, 18-22 min 95% B, 22-25 min 5% B, at a flow rate of 0.3 mL / min. The resulting HPLC-MS chromatogram of the liquid fermentation product is shown below. Figure 3 As shown.

[0094] 4.2 Structural identification of naphthycin compounds:

[0095] Structural identification of compound 1

[0096] The physicochemical properties and spectral data of compound 1 are as follows:

[0097] Yellow amorphous solid; HRESI(+)MS spectrum ( Figure 4 ) showed m / z [M+H] + 748.3122、[M+NH4] + 765.3394 and [M-H2O+H] + From 730.3015, the molecular formula of compound 1 is deduced to be C. 41 H 49 NO 10 S. Compound 1 1 H-NMR (600 MHz) and 13The C-NMR (150 MHz) spectra are shown below. Figure 5 and Figure 6 Analysis of compound 1 1 H-NMR spectrum ( Figure 5 Compound 1 was found to contain six methyl hydrogens (δ-hydroxyl groups). H 0.82, 0.96, 1.20, 1.70, 2.04, 2.34), 4 hydroxyl hydrogens (δ H 3.21, 3.58, 4.05, 4.29), 10 aromatic or olefinic hydrogens (δ H 5.52, 5.52, 5.58, 5.96, 6.25, 6.36, 6.51, 6.70, 6.81, 7.88); analysis of compound 1 13 C-NMR spectrum ( Figure 6 The structure of compound 1 was found to contain 41 carbons, including 7 methyl carbons (δ-carbons). C 10.9, 11.4, 12.6, 16.5, 16.6, 17.5, 18.8), 20 aromatic or olefinic carbons (δ C 120.2, 122.3, 123.7, 126.9, 131.1, 132.8, 132.9, 133.1, 134.5, 134.9, 136.0, 136.4, 137.3, 137.9, 138.2, 138.7, 141.4, 142.9, 147.0, 160.7) and 5 carbonyl carbons (δ C 167.6, 178.7, 182.1, 202.4, 204.0).

[0098] Based on the above physicochemical properties 1 H-NMR and 13 The structure of compound 1 was determined by C-NMR spectroscopy. Compound 1 was named Naphthomycin R, and its structure is shown below:

[0099] .

[0100] Structural identification of compound 2

[0101] Yellow amorphous solid; HRESI(+)MS spectrum ( Figure 7 ) showed m / z [M+H] + 792.3372, [M+NH4] + 809.3653 and [M-H2O+H] + From 774.3270, the molecular formula of compound 2 is deduced to be C.43 H 53 NO 11 S. Compound 2 1 H-NMR (600 MHz) and 13 The C-NMR (150 MHz) spectra are shown below. Figure 8 and Figure 9 .

[0102] Based on the above physicochemical properties 1 H-NMR and 13 The structure of compound 2 was determined by C-NMR spectroscopy. Compound 2 was named Naphthomycin S, and its structure is shown below:

[0103] .

[0104] Structural identification of compound 3

[0105] Yellow amorphous solid; HRESI(+)MS spectrum ( Figure 10 ) showed m / z [M+H] + 732.3178, [M+NH4] + 749.3440 and [M-H2O+H] + From 714.3069, the molecular formula of compound 3 is deduced to be C. 41 H 49 NO9S. Compound 3 1 H-NMR (600 MHz) and 13 The C-NMR (150 MHz) spectra are shown below. Figure 11 and Figure 12 .

[0106] Based on the above physicochemical properties 1 H-NMR and 13 The structure of compound 3 was determined by C-NMR spectroscopy. Compound 3 was named Naphthomycin T, and its structure is shown below:

[0107] .

[0108] Structural identification of compound 4

[0109] Yellow amorphous solid; HRESI(+)MS spectrum shows m / z [M+H] + From 686.3295, the molecular formula of compound 4 is deduced to be C. 40 H 47 NO 10 Analysis of compound 4 1 H-NMR and13 C-NMR spectrum ( Figure 13 and Figure 14 It was discovered that compound 4 has a highly similar structure to the above-mentioned compounds, belonging to the naphthycin class of compounds, and later it was found to be similar to compounds in the literature. 1 H-NMR, 13 Comparison of C-NMR and HRESI(+) MS data confirmed that compound 4 is Naphthomycin E, and its structure is shown below:

[0110] .

[0111] Example 5

[0112] Evaluation of the anti-inflammatory activity of naphthycin compounds 1-4

[0113] The test cells were mouse mononuclear macrophages RAW264.7.

[0114] RAW264.7 cells were seeded in 96-well plates at a cell density of 3–4 × 10⁻⁶ cells / well. 3 / well. Incubate in a cell culture incubator at 37°C with 5% CO2. After cell attachment, remove the culture medium from the 96-well plate and pretreat RAW264.7 cells with the specified concentrations of compounds (5, 10, 20, and 40 μM) for 30 min. Add LPS to a final concentration of 10 μg / mL. No treatment is given to the blank control group. Continue incubation for 24 h. Collect 50 μL of the supernatant for nitric oxide detection. Then, remove the kit and place it at room temperature. Add 50 μL of sample to each well of the 96-well plate, followed by 50 μL of Griess Reagent I and 50 μL of Griess Reagent II. Measure the absorbance at 540 nm using a microplate reader. Calculate the IC50 using GraphPad Prism 5.0 software. 50 Values ​​were calculated by repeating the experiment three times and expressed as mean ± standard deviation (SD).

[0115] Each cell line was tested in triplicate. The results are shown in Table 1.

[0116] Table 1. Effects of compounds 1-4 on NO production rate in RAW264.7 cell line (IC50, 100%) 50 (μM)

[0117]

[0118] The experimental results above indicate that compounds 3 and 4 significantly inhibit NO production, greatly suppressing its generation. Furthermore, none of the compounds showed cytotoxicity against mouse monocyte / macrophage RAW264.7 cells, suggesting that any inhibition of NO production was not due to the compounds' cytotoxic activity.

[0119] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Streptomyces ( Streptomyces The application of sp.) AH1901-2 in the preparation of naphthycin compounds, characterized in that, The Streptomyces AH1901-2 strain has the accession number CGMCC No. 32711; The naphthomycin-like compound is compound 3; the structural formula of compound 3 is shown in Formula III: 。 2. The method for preparing the naphthycin-like compound according to claim 1, characterized in that, The Streptomyces AH1901-2 strain described in claim 1 was fermented to obtain a liquid fermentation culture; the naphthycin compounds were extracted, separated, and obtained.

3. The preparation method according to claim 2, characterized in that, Methods for obtaining liquid fermentation cultures from Streptomyces AH1901-2 include: Streptomyces AH1901-2 was inoculated into TSB medium, and a seed culture was obtained after the first culture. The seed culture was then inoculated into ISP-7 liquid medium, and a second culture was conducted to obtain mycelium and liquid fermentation products.

4. The preparation method according to claim 3, characterized in that, The ISP-7 liquid culture formulation is as follows: 15 g glycerol, 0.5 g L-tyrosine, 1 g L-asparagine, 0.5 g K2HPO4, 0.5 g MgSO4·7H2O, 2 g NaCl, 0.01 g FeSO4·7H2O, 1 mL trace element solution, and 1000 mL distilled water; pH 7.2~7.4; wherein, the trace element solution is formulated as follows: 0.1 g FeSO4·7H2O, 0.1 g MnCl2·4H2O, 0.1 g ZnSO4·7H2O, plus 100 mL distilled water.

5. The preparation method according to claim 2, characterized in that, The extraction includes the following steps: The liquid fermentation culture was subjected to macroporous resin solid-phase extraction, and the macroporous resin solid-phase extract was eluted with methanol to obtain a liquid fermentation extract. The crude extract was obtained by vacuum distillation.

6. The preparation method according to claim 5, characterized in that, The separation includes the following steps: The crude extract was separated by normal-phase silica gel column chromatography, and then eluted sequentially with a gradient concentration of dichloromethane-methanol system. The volume ratios of the dichloromethane-methanol system were 100:0, 100:1, 100:2, 100:3, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, and 0:1, respectively. The elution fractions Fr. 2 with a dichloromethane to methanol volume ratio of 100:2 to 100:3 were collected. The elution fraction Fr. 2 was purified by preparative high-performance liquid chromatography using water and acetonitrile as the mobile phase and acetonitrile with a volume concentration of 60% as the purification condition to obtain the naphthycin compounds.

7. The preparation method according to claim 6, characterized in that, Compound 3 was obtained at a retention time of 9.5 min.

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