Application of annular Fokecycline in preparation of nasopharynx cancer resisting medicine

Abstract

The invention discloses an application of annular formicin in preparation of a nasopharynx cancer resisting medicine. On the basis of the first patent, the inventor finds that the cyclic formicin separated from streptomyces 5017 fermentation liquor shows an obvious inhibition effect on tested nasopharyngeal carcinoma cells 5-8F, TW03 and CNE1, the IC50 values are 26.21 mu g / mL, 19.80 mu g / mL and 23.10 mu g / mL respectively, and the cells can be induced to generate apoptosis in vitro. Therefore, the annular Fukamycin has the potential to become a lead compound of the nasopharyngeal carcinoma resistant medicine, and has potential development and application values in research and development of the nasopharyngeal carcinoma resistant medicine. As the tokecycline is a secondary metabolite derived from marine streptomyces, the discovery also has great significance on development of marine biological resources in China.

Description

Technical Field

[0001] This invention belongs to the field of natural product technology, and particularly relates to the application of cyclic fociclomycin in the preparation of anti-nasopharyngeal carcinoma drugs. Background Technology

[0002] Nasopharyngeal carcinoma is a malignant tumor of the head and neck, with a high incidence in southern my country, such as Guangxi, Guangdong, Hunan, and Fujian. Currently, the main treatment for nasopharyngeal carcinoma is radiotherapy combined with chemotherapy. Common chemotherapy drugs include paclitaxel, cisplatin, 5-fluorouracil, nedaplatin, and gemcitabine. However, these drugs have significant side effects and are prone to drug resistance, affecting patient treatment and recovery. Therefore, there is an urgent need to find new drugs for the treatment of nasopharyngeal carcinoma.

[0003] Cyclofaulknamycin (Cyc-fly) is a small polypeptide secondary metabolite produced by Streptomyces, first isolated from Streptomyces albus. Cyclofaulknamycin contains a cyclic guanidine group in its structure. Although known compounds containing cyclic guanidine groups exhibit significant pharmacological activity, such as the broad-spectrum antibacterial compound muraymycins, the anti-tuberculosis drugs viomycin and capreomycin IA, the in vitro antibacterial and antitumor activities of cyclofaulknamycin have not been reported.

[0004] Chinese patent “Streptomyces 5017 and its application in antagonizing plant pathogens” (patent number 201911027910.8, publication date December 31, 2019) reported a Streptomyces 5017 isolated from the rhizosphere soil of mangrove plants in Guangxi and disclosed the application of Streptomyces 5017 in antagonizing plant pathogens, but did not mention its secondary metabolites. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide the application of cyclic fociclomycin in the preparation of anti-nasopharyngeal carcinoma drugs.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] Application of cyclic fociclomycin in the preparation of anti-nasopharyngeal carcinoma drugs.

[0008] The anti-nasopharyngeal carcinoma effect is achieved by cyclic floccomycin in vitro by inhibiting the growth of nasopharyngeal carcinoma cells, arresting nasopharyngeal carcinoma cells in the G0 / G1 phase, and inducing apoptosis.

[0009] Nasopharyngeal carcinoma originates from nasopharyngeal carcinoma cell lines 5-8F, TW03, and CNE1.

[0010] The structural formula of cyclic foclomycin is as follows:

[0011] .

[0012] The preparation method of cyclic focimycin: cyclic focimycin was isolated and purified from the fermentation broth of Streptomyces 5017 from Guangxi mangrove forests; the preservation number of Streptomyces 5017 from Guangxi mangrove forests is CCTCC NO: M2019648.

[0013] The above preparation method is carried out according to the following steps:

[0014] 1) Streptomyces 5017 was fermented in a culture medium, and then the bacterial cells and fermentation supernatant were separated by centrifugation. The fermentation supernatant was extracted three times with equal volumes of ethyl acetate, and the organic phases were combined and concentrated under reduced pressure to obtain the crude extract of fermentation supernatant. The crude extract was dissolved and then subjected to gradient elution using a silica gel column.

[0015] 2) Collect the 100% methanol aqueous solution eluent and purify it using semi-preparative high performance liquid chromatography.

[0016] In step 1): the culture medium is the modified Streptomyces International No. 2 liquid medium, with 2 g / L glucose, 2 g / L yeast extract and 2 g / L malt extract in the medium; the culture conditions are 180 rpm and 28℃ shaking culture for 5 days.

[0017] In step 1), the centrifugation conditions were 8000 rpm, 25℃, and 10 min.

[0018] In step 1): gradient elution is performed by dissolving the crude extract and then separating it using a silica gel column packed with SelectCore 100 C18 (Nanochrom), followed by gradient elution with 200 mL of 20%, 40%, 60%, 80% and 100% methanol aqueous solution.

[0019] The conditions for the semi-preparative HPLC in step 2) are as follows:

[0020] Chromatographic column: ;

[0021] Washout phase: ;

[0022] Flow rate: .

[0023] Based on the aforementioned prior patent, the applicant, in its research on cyclic foclomycin isolated from the fermentation broth of Streptomyces 5017, discovered that it exhibited significant inhibitory effects on tested nasopharyngeal carcinoma cells 5-8F, TW03, and CNE1. 50 The values ​​are respectively It can induce apoptosis in these cells in vitro. Therefore, cyclic foclomycin has the potential to become a lead compound for anti-nasopharyngeal carcinoma drugs and has potential development and application value in the research and development of anti-nasopharyngeal carcinoma drugs. Since cyclic foclomycin is a secondary metabolite derived from marine streptomyces, this discovery is also of great significance for the development of my country's marine biological resources. Attached Figure Description

[0024] Figure 1 This is the structure and mass spectrum of the cyclic flocculin obtained by the present invention.

[0025] Figure 2 This describes the cytotoxic activity and IC50 of cyclic fociclomycin against various nasopharyngeal carcinoma carcinoma lines in vitro. 50 The results are shown in the figure. In the figure: a) Nasopharyngeal carcinoma cell line TW03, b) Nasopharyngeal carcinoma cell line 5-8F, c) Nasopharyngeal carcinoma cell line CNE1.

[0026] Figure 3 This is a graph showing the effects of cyclic fociclomycin on apoptosis in various nasopharyngeal carcinoma cell lines. In the graph: a 5-8F+DMSO, b TW03+DMSO, c CNE1+DMSO.

[0027] Figure 4 This is a graph showing the experimental results of cyclic fociclomycin in causing cell cycle arrest in nasopharyngeal carcinoma cell lines 5-8F, TW03, and CNE1.

[0028] Preservation Information

[0029] Streptomyces sp. 5017, accession number CCTCC NO: M2019648, accession date: August 19, 2019, accession address: Wuhan University, Wuhan, China, 430072, depositary institution: China Center for Type Culture Collection.

[0030] Preservation conditions: Streptomyces sp. 5017 was preserved in modified Streptomyces International No. 2 liquid medium containing 20% ​​glycerol (glucose 2.0 g / L, malt extract 2.0 g / L, yeast extract 2.0 g / L, pH 7.2-7.4) at -80°C. Detailed Implementation

[0031] Example 1: Preparation method of cyclic foclomycin

[0032] 1. Cyclofomycin was isolated from the fermentation broth of Streptomyces sp. 5017.

[0033] 2. Streptomyces sp. 5017 was fermented using modified International Streptomyces No. 2 liquid medium (2.0 g / L glucose, 2.0 g / L malt extract, 2.0 g / L yeast extract, pH 7.2-7.4) at 180 rpm and 28°C for 7 days.

[0034] The method for separating and purifying cyclic fociclomycin from fermentation broth is as follows: after centrifuging the fermentation supernatant and bacterial cells (8000 rpm, 10 min), the supernatant is extracted three times with an equal volume of ethyl acetate, and concentrated under reduced pressure at 45 °C to obtain the crude extract of fermentation broth.

[0035] The crude extract was dissolved in methanol and loaded into a chromatography column containing SelectCore 100 C18 (Nanochrom) packing material, and then eluted sequentially with 200 mL of 20%, 40%, 60%, 80% and 100% methanol aqueous solution.

[0036] The eluent of 100% methanol-water solution was collected and purified by semi-preparative high-performance liquid chromatography (HPLC). Cyclic fociclomycin was collected after 17 min (mass spectrometry analysis showed the results as follows). Figure 1 (As shown). The conditions for semi-preparative HPLC include: chromatographic column: ; Elution phase: .

[0037] The cyclic foclomycin obtained in Example 1 was used to evaluate its in vitro anti-nasopharyngeal carcinoma activity, as detailed below:

[0038] Example 2: Evaluation of the in vitro inhibitory activity of cyclic fociclomycin on various nasopharyngeal carcinoma cells.

[0039] 1) Experimental Methods: Cyclofomycin was dissolved in dimethyl sulfoxide (DMSO) and filtered through a 0.22 μm sterile filter to obtain a sterile sample solution. Nasopharyngeal carcinoma cell lines 5-8F, TW03, and CNE1 in logarithmic growth phase were respectively prepared into 3×10⁻⁶ cells / mL solutions using high-glucose DMEM medium (Thermo Fisher Scientific, USA). 5 Cells were seeded at a concentration of [number] cells / mL into 96-well plates, with 100 μL of cell suspension added to each well. The plates were incubated overnight at 37°C in a 5% CO2 incubator. After cell attachment, the old culture medium was discarded, and each well was incubated with [concentration of...]. Complete culture medium of cyclic foclomycin The culture was reacted under the same conditions for 24 hours, the old culture medium was discarded, and new culture medium was added to each well. Prepare CCK8 solution (CCK8 solution: high glucose DMEM medium = 1:10), continue the reaction for 1.5 h, then place the 96-well plate in a multi-functional microplate reader and measure the absorbance at 450 nm. ×100%, to add 100g of complete culture medium containing only DMSO. As a negative control, survival curves were plotted using GraphPad Prism 10, and the half-inhibitory concentration (WIC) of the drug was calculated. .

[0040] 2) Experimental results: The results are as follows Figure 2 As shown, cyclic foclomycin is effective against... ,right .

[0041] Example 3: Evaluation of apoptosis induction in various nasopharyngeal carcinoma cells by cyclic foclomycin in vitro.

[0042] 1) Experimental methods: An apoptosis kit was used, employing IC50 assays. 50 Value concentration of cyclic fociclomycin , Treatment of nasopharyngeal carcinoma cells Flow cytometry was used to detect apoptosis 24 hours later, with the treatment group receiving only an equal amount of DMSO serving as a control. Following the instructions of the apoptosis kit, cell supernatant was collected from each group, washed with PBS buffer, and digested with trypsin without EDTA. After digestion was terminated, the collected supernatant was added, and the cell suspension was centrifuged at 1500 rpm for 5 minutes. Cells (1-10) were collected. After centrifugation, the supernatant was discarded. Subsequently, the cells were washed three times with pre-cooled PBS, discarding the supernatant after each centrifugation. Resuspend the cell pellet in 1× binding buffer. Add [the appropriate amount of solution] to each tube of cell sample. After staining with reagents, the mixture was incubated at room temperature in the dark for 5 minutes. Cell samples were then filtered through a 300-mesh nylon filter and analyzed using a flow cytometer.

[0043] 2) Experimental results: such as Figure 3 As shown, cyclic foclomycin can induce apoptosis in three nasopharyngeal carcinoma cell lines: 5-8F, TW03, and CNE1. The proportion of early apoptosis induced by this compound in TW03 and CNE1 was significantly higher than that in late apoptosis, while the apoptosis-promoting effect in 5-8F cells was more pronounced in late apoptosis than in early apoptosis.

[0044] Example 4: Evaluation of the effect of cyclic foclofomine on the cell cycle of nasopharyngeal carcinoma cells

[0045] 1) Experimental methods: A cell cycle kit was used, employing IC50 assays. 50 Value concentration of cyclic fociclomycin Nasopharyngeal carcinoma carcinoma lines 5-8F, TW03, and CNE1 were treated, and cell cycle analysis was performed by flow cytometry 24 hours later. A control group treated with only an equal volume of DMSO was used. Cells were digested with 0.25% trypsin-EDTA solution according to the cell cycle kit instructions. After digestion was terminated, the cell suspension was centrifuged at 1000 g for 5 minutes and collected. Cells were centrifuged, and the supernatant was discarded. The cells were washed once with PBS buffer that had been pre-chilled on ice, centrifuged again, and the supernatant was discarded. Add... and Vortex for 10 seconds to mix, then incubate at room temperature in the dark for 30 minutes. Filter the cell samples using a 300-mesh nylon filter, select the lowest loading speed, and perform analysis on a flow cytometer. The cell cycle fitting model was the Watson (Pragmatic) model.

[0046] 2) Experimental results: such as Figure 4 As shown, cyclic fociclomycin can alter the cell cycle distribution of various nasopharyngeal carcinoma cells, arresting the cell cycle in the G0 / G1 phase.

[0047] In summary, this invention, through various in vitro antitumor activity experiments, has demonstrated that cyclic fociclomycin exhibits significant inhibitory activity against the proliferation of various nasopharyngeal carcinoma cells in vitro, arresting them in the G0 / G1 phase and inducing apoptosis. Therefore, cyclic fociclomycin possesses the potential to become a lead compound for anti-nasopharyngeal carcinoma drugs and has promising prospects for drug development.

Claims

1. Application of cyclic fociclomycin in the preparation of anti-nasopharyngeal carcinoma drugs.

2. The application according to claim 1, characterized in that: The anti-nasopharyngeal carcinoma agent is cyclic fociclomycin, which inhibits the growth of nasopharyngeal carcinoma cells, arrests them in the G0 / G1 phase, and induces apoptosis.

3. The application according to claim 1, characterized in that: The nasopharyngeal carcinoma is derived from nasopharyngeal carcinoma cell lines 5-8F, TW03, and CNE1.

4. The application according to claim 1, characterized in that: The structural formula of the cyclic foctocin is as follows: 。 5. The method for preparing the cyclic foclomycin according to claim 1, characterized in that: Cyclofomycin was isolated and purified from the fermentation broth of *Streptomyces 5017* from Guangxi mangrove forests; the preservation number of *Streptomyces 5017* from Guangxi mangrove forests is CCTCC NO: M2019648.

6. The preparation method according to claim 5, characterized in that... Follow these steps: 1) Streptomyces 5017 was cultured in a culture medium, and then the cells and fermentation supernatant were separated by centrifugation. The fermentation supernatant was extracted three times with equal volumes of ethyl acetate, and the organic phases were combined and concentrated under reduced pressure to obtain the crude extract of fermentation supernatant. The crude extract was dissolved and then subjected to gradient elution using a silica gel column. 2) Collect the 100% methanol aqueous solution eluent and purify it using semi-preparative high performance liquid chromatography.

7. The preparation method according to claim 6, characterized in that... In step 1): the culture medium is a modified Streptomyces International No. 2 liquid culture medium, with 2 g / L glucose, 2 g / L yeast extract and 2 g / L malt extract in the medium; the culture conditions are 180 rpm and 28℃ shaking culture for 5 days.

8. The preparation method according to claim 6, characterized in that... In step 1), the centrifugation conditions are 8000 rpm, 25℃, and 10 min.

9. The preparation method according to claim 6, characterized in that... In step 1): the gradient elution is performed after the crude extract has been dissolved, using a elution chamber packed with SelectCore 100 C. 18 Separation was performed by silica gel column chromatography using a Nanochrom apparatus, with gradient elution using 200 mL of 20%, 40%, 60%, 80%, and 100% methanol aqueous solutions.

10. The preparation method according to claim 6, characterized in that... The conditions for the semi-preparative HPLC described in step 2) are as follows: Chromatographic column: ; Washout phase: ; Flow rate: .

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