Napyradiomycins compound and application thereof in preparation of anti-inflammatory drugs

Abstract

The invention belongs to the technical field of bio-pharmaceuticals, and discloses a Napyradiomycin compound and application thereof, the compound is separated from a secondary metabolite of marine actinomycetes Streptomyces canus SJ-019, and the compound is named as Canumycin E. The invention further discloses a preparation method of the compound. An in-vitro anti-inflammatory activity test shows that under the concentration of 10 mu M, the inhibition effect on an inflammatory mediator NO is stronger than that of dexamethasone. The invention also discloses application of the compound in preparation of anti-inflammatory drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a Napyradiomycins compound and an application thereof in the preparation of an anti-inflammatory drug. Background Art

[0002] Terpenes are widely distributed in animals, plants, bacteria and fungi. They are a class of natural products produced in nature through a mixed pathway from a common terpene substrate and a variety of different synthons such as polyketides, phenols, alkaloids or amino acids. Therefore, terpene compounds in nature have amazing chemical diversity.

[0003] In the past few decades, some terpene natural products based on the polyketide 1,3,6,8-tetrahydroxynaphthalene (THN) skeleton have been found in Streptomyces in marine and soil samples. These natural products have extremely high molecular diversity and often have antibacterial, anti-tumor, anti-inflammatory and other biological activities, including the largest family of THN-derived terpene natural products, Napyradiomycins. In view of this, the continuous search for new structural Napyradiomycins compounds has great medicinal potential. Summary of the invention

[0004] The present invention aims at the deficiencies of the above-mentioned prior art and provides a Napyradiomycins compound and its application in the preparation of anti-inflammatory drugs. The compound has anti-inflammatory activity, can significantly inhibit the production of inflammatory mediator NO, and has the potential to be developed into a drug for inflammatory diseases.

[0005] The specific technical solutions are as follows:

[0006] One of the purposes of the present invention is to provide a Napyradiomycins compound, the chemical structure of which is shown in the following formula:

[0007]

[0008] The above compound is isolated from the secondary metabolite of marine actinomycete Streptomyces canus SJ-019 and is named Canumycin E. In vitro anti-inflammatory activity experiments found that Canumycin E at a concentration of 10 μM has a stronger inhibitory effect on NO production than the positive control dexamethasone.

[0009] The marine actinomycete Streptomyces canus SJ-019 was isolated from the seabed sediments of the intertidal zone in Yantai, China, and its preservation number is CGMCC No. PV065562, and it has been preserved in the General Microbiological Center of China Microbiological Culture Collection Administration.

[0010] Furthermore, the preparation method of the Napyradiomycins compound comprises: culturing the marine actinomycete Streptomyces canus SJ-019, harvesting the fermentation product; and purifying the fermentation product.

[0011] Furthermore, the culture medium used for culturing marine actinomycete Streptomyces canus SJ-019 is ISP2 culture medium or pure seawater ISP2 culture medium, preferably semi-seawater ISP2 culture medium.

[0012] Specifically, the culture method is as follows: streak the marine actinomycete Streptomyces canus SJ-019 on a solid culture medium and culture it until spores grow; then inoculate the spores into a liquid culture medium for fermentation and harvest the fermentation product.

[0013] Wherein, the culture is carried out on a solid culture medium at 25-28°C for 3-5 days.

[0014] The spores are cultured in a liquid medium at 25-28° C. for 7-10 days.

[0015] Furthermore, the purification includes: chromatographic purification of the fermentation product.

[0016] Specifically, the purification method is as follows: the fermentation product is filtered to obtain the fermentation liquid and mycelium, the fermentation liquid is adsorbed, eluted, concentrated under reduced pressure and extracted with an organic solvent by a macroporous adsorption resin column; the mycelium is directly extracted with an organic solvent under the action of ultrasound; the organic solvent extract is combined and concentrated under reduced pressure to obtain a crude extract. The crude extract is chromatographed on a silica gel column, eluted with dichloromethane-methanol, and the eluate is concentrated under reduced pressure to obtain a concentrated extract. The concentrated extract is chromatographed on an ODS gel column, eluted with a methanol-water gradient, and the eluted components are processed by thin layer chromatography detection, and then the eluted components are further purified by preparative HPLC to obtain the compound Canumycin E (5.0 mg, t R =48.8min).

[0017] After adsorption by the macroporous adsorption resin column, an organic alcohol is used for elution, and the organic alcohol is preferably methanol and / or ethanol, and methanol is most preferred.

[0018] Wherein, the macroporous adsorption resin column is a weakly polar or non-polar macroporous adsorption resin column, preferably a weakly polar macroporous adsorption resin column.

[0019] Among them, the organic solvent used for the extraction of the fermentation broth decompression concentrate and mycelium is preferably ethyl acetate.

[0020] In the silica gel column chromatography, dichloromethane-methanol (100:0, volume ratio) was used for elution.

[0021] In the ODS gel column chromatography, methanol-water (10:90 to 100:0, volume ratio) was used for gradient elution.

[0022] Among them, in HPLC purification, the HPLC column chromatography filler is preferably C-18 or C-8, and most preferably C-18.

[0023] Among them, in HPLC purification, the HPLC eluent is preferably methanol: water (70:30).

[0024] The second object of the present invention is to provide the use of the above compound Canumycin E in the preparation of anti-inflammatory drugs.

[0025] Furthermore, the use of the compound Canumycin E is characterized in that the compound can significantly inhibit the production of inflammatory mediator NO.

[0026]

Biological Deposit Instructions

[0027] China General Microbiological Culture Collection Center registration number: CGMCC No. PV065562;

[0028] Referenced biological material (strain): SJ-019;

[0029] The biological material (strain) requested for deposit is accompanied by a proposed taxonomic name: Streptomyces canus.

[0030] The biological material (strain) has been received and registered by the General Microbiology Center of China Microbiological Culture Collection Administration, and the survival test result is alive.

[0031] Collection address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

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

[0033] The present invention obtains a new Napyradiomycins compound Canumycin E from marine actinomycete Streptomyces canus SJ-019. In vitro anti-inflammatory activity tests show that its inhibitory effect on inflammatory mediator NO at a concentration of 10 μM is stronger than that of dexamethasone, and has the potential to be developed into a drug for inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the chemical structural formula of the compound Canumycin E;

[0035] Figure 2 Canumycin E 1 H NMR spectrum;

[0036] Figure 3 Canumycin E 13 C NMR spectrum;

[0037] Figure 4 Canumycin E 1 H- 1 H COSY spectrum;

[0038] Figure 5 is the HSQC spectrum of compound Canumycin E;

[0039] Figure 6 is the HMBC spectrum of compound Canumycin E;

[0040] Figure 7 It is the NOESY spectrum of compound Canumycin E;

[0041] Figure 8 This is the high-resolution mass spectrum of compound Canumycin E;

[0042] Fig. 9 The experimental ECD spectrum and calculated ECD spectrum of compound Canumycin E;

[0043] Fig.10 This is a graph showing the changes in NO concentration after the compound Canumycin E acted on mouse macrophage RAW 264.7 (****p<0.001). DETAILED DESCRIPTION

[0044] The principles and features of the present invention are described below in conjunction with examples, which are only used to explain the present invention and are not used to limit the scope of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0045] Example 1 Preparation of Compound Canumycin E

[0046] Marine actinomycete Streptomyces canus SJ-019 was cultured in semi-seawater ISP2 medium, and was streaked on a solid culture medium and cultured at 28°C for 3 days until spores grew. The spores were then inoculated into a liquid culture medium and cultured in a shaking incubator at 28°C for 10 days, and the fermentation product was harvested.

[0047] The fermentation product is filtered to obtain the fermentation broth and mycelium. The fermentation broth is adsorbed by a weakly polar macroporous adsorption resin column, then eluted with methanol, concentrated under reduced pressure and extracted with ethyl acetate; ethyl acetate is directly added to the mycelium for crushing and extraction; the ethyl acetate extract is combined and concentrated under reduced pressure to obtain a crude extract. The crude extract is chromatographed on a silica gel column, eluted with dichloromethane-methanol (100:0, volume ratio), and the eluate is concentrated under reduced pressure to obtain a concentrated extract. The concentrated extract is chromatographed on an ODS gel column, gradient eluted with methanol-water (10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0, volume ratio), and divided into 10 elution components according to thin layer chromatography detection, numbered as elution components 1 to 10 respectively. The eluted fractions 9 and 10 were combined and further purified by preparative HPLC system (Phenomenex C18 column, 250 mm×21.2 mm, 5 μm), methanol-water (70:30, volume ratio) to obtain compound Canumycin E (5.0 mg, t R =48.8min).

[0048] Example 2 Structure determination of Canumycin E

[0049] (1) Instruments and Materials

[0050] Autopol VI (Serial #91058) polarimeter, Waters ACQUITY UPLC I Class-Vion IMS QT high-resolution mass spectrometer, Applied photophysics 9798QB spectrometer, Waters 2489 UV / visible liquid chromatograph, Bruker AVANCE IIITM 600 nuclear magnetic resonance instrument.

[0051] (2) Chemical structure determination

[0052] Canumycin E: colorless powdery solid, [α] D 20 –46.02 (MeOH, c 0.083); UV (methanol): λ max(logε)195(3.4),263(2.9),372(2.7),412(2.0),412(1.9),420(1.8)nm; 1D and 2D-NMR (CDCl3) data are shown in Table 1 and Figure 2 to Figure 7 ; HRESIMS m / z: [MH] – 439.17596, see Figure 8 .

[0053] Table 1 NMR data of Canumycin E (in CDCl3) a

[0054]

[0055] a represent 1 HNMR 600MHz, 13 CNMR150MHz.

[0056] Based on the physicochemical parameters and spectral data of the above compounds ( Figures 2 to 8 ), the structure of the compound Canumycin E is determined as shown below:

[0057]

[0058] Example 3 Detection of anti-inflammatory activity of Canumycin E

[0059] The anti-inflammatory activity test method of compound Canumycin E is as follows:

[0060] Mouse macrophage RAW264.7 cells (ATCC, Manassas, VA, USA) were cultured in DMEM medium containing 10% FBS and 1% PS at 37°C in a 5% CO2 incubator. RAW 264.7 cells were plated at 5×10 5The cells were inoculated in a 96-well plate at a density of 10 μM / mL. The experimental group was pretreated with the compound Canumycin E at a final concentration of 10 μM for 3 h, and the control group was pretreated with the same concentration of dexamethasone (DEX) for the same time. Except for the blank group (containing only mouse macrophage RAW264.7 cells), the experimental group, the control group, and the LPS-treated group were incubated with 1 μg / mL LPS for 12 h. The amount of NO in the supernatant was measured using the Griess reaction. Specifically: 50 μL of supernatant was mixed with 50 μL of Griess reagent I in equal volumes and incubated at room temperature for 5 minutes in a dark environment; then 50 μL of Griess reagent II was added, and the absorbance at 540 nm was detected under an enzyme reader. At the same time, a sodium nitrite standard curve was prepared according to the instructions of the kit, with NO molar concentration as the horizontal axis and absorbance as the vertical axis, a standard curve was drawn, and the concentration of NO was calculated.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A Napyradiomycins compound, characterized in that: Its chemical structure is shown below:

2. The Napyradiomycins compound according to claim 1, characterized in that It is isolated from the secondary metabolites of the marine actinomycete Streptomyces canus SJ-019.

3. The Napyradiomycins compound according to claim 2, characterized in that: The preparation method comprises: culturing marine actinomycete Streptomyces canus SJ-019, harvesting fermentation products; and purifying the fermentation products.

4. The Napyradiomycins compound according to claim 3, characterized in that: The culture medium used for culturing marine actinomycete Streptomyces canus SJ-019 is semi-seawater ISP2 medium or pure seawater ISP2 medium.

5. The Napyradiomycins compound according to claim 3, characterized in that: The purification comprises: chromatographic purification of the fermentation product.

6. Use of the Napyradiomycins compound according to any one of claims 1 to 5 in the preparation of anti-inflammatory drugs.

7. The use according to claim 6, characterized in that The compound can significantly inhibit the production of inflammatory mediator NO.

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