Use of compound merulin a in the preparation of drugs for resisting brain glioma

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a compound Merulin A in preparation of an anti-glioma drug. The application provides application of the compound Merulin A in preparation of the anti-glioma drug. Experiments prove that the compound Merulin A exhibits significant anti-glioma activity, can effectively promote early apoptosis and cell necrosis of glioma cells, and further inhibit proliferation and migration of the glioma cells. Moreover, due to advantages of wide source, simple extraction process, short cycle and low cost, the compound Merulin A has a wide application prospect as the anti-glioma drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine. More specifically, it relates to the application of compound Merulin A in the preparation of anti-glioma drugs. BACKGROUND

[0002] Glioma, a malignant tumor originating from glial cells, is the main type of primary malignant heterogeneous tumors in the central nervous system. Due to its high incidence and high mortality, glioma poses a major challenge to human health. The treatment of glioma is particularly difficult because glioma often grows in an infiltrative manner, with unclear boundaries with the surrounding brain tissue, which poses a great challenge to surgical resection and drug treatment. Currently, the drug options for treating glioma are relatively limited, among which temozolomide and paclitaxel are relatively common drugs. Temozolomide, as a chemotherapy drug, has been widely used in the treatment of neuroglioma. It produces significant anti-tumor effect by regulating cell cycle, inhibiting cell proliferation and inducing apoptosis, etc. While paclitaxel, as a broad-spectrum anticancer drug, mainly exerts its anticancer effect by inhibiting the synthesis and repair of tumor cell DNA, and inducing tumor cell apoptosis. Although the application of paclitaxel in the treatment of brain glioma is limited by the blood-brain barrier, it can still be used as an adjunctive therapy to provide some therapeutic effect for patients.

[0003] However, in clinical treatment, the treatment drugs for glioma patients are still relatively scarce. Therefore, the medical community needs to increase research and development efforts and actively explore and develop more efficient new drugs against brain glioma, with the aim of providing patients with more diversified and effective treatment options to improve the quality of life and prognosis of patients. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the defects and deficiencies of the existing drugs for treating glioma, and to provide the application of compound Merulin A in the preparation of anti-glioma drugs.

[0005] The above-mentioned purposes of the present application are achieved by the following technical solutions:

[0006] The present application protects the application of compound Merulin A in the preparation of anti-glioma drugs, and the structure of the compound Merulin A is as follows:

[0007] .

[0008] Marine-derived fungal natural products constitute one of the important sources of marine natural products. In the process of searching for compounds with anti-glioma activity, it is of great significance to further study the secondary metabolites of marine fungi. The inventors successfully isolated a meroterpenoid compound Merulin A from the fermentation products of the fungus Pseudolagarobasidium acaciicola SYSU-MS9908 isolated from sea squirts. Studies have shown that the compound Merulin A has a significant inhibitory effect on the proliferation and migration of glioma cells, and can induce early apoptosis and cell necrosis of glioma cells. Therefore, the compound Merulin A shows good application prospects as an anti-glioma drug.

[0009] Further, the compound Merulin A can be obtained by various means, including commercial channels, extraction from natural sources, and chemical synthesis methods. Regardless of the means of obtaining, the effect of the compound Merulin A is not affected.

[0010] Further, as an optional embodiment, the compound Merulin A is obtained by isolation and purification from the mycelium of a marine sea squirt-derived fungal strain.

[0011] Preferably, the marine sea squirt-derived fungal strain is Pseudolagarobasidium acaciicola SYSU-MS9908, which was deposited with the Guangdong Microbial Culture Collection Center on December 24, 2024, and has the accession number GDMCC NO: 65664.

[0012] More preferably, the isolation and purification specifically comprises the following steps:

[0013] S1. Fermenting the fungus Pseudolagarobasidium acaciicola SYSU-MS9908, and obtaining a fermentation product by static culture;

[0014] S2. Isolating the broth from the fermentation product obtained in step S1, purifying and concentrating the broth to obtain an extract, and obtaining the compound Merulin A by isolation and purification of the extract.

[0015] Further, in step S1, the fermentation is performed in a sterilized potato culture medium.

[0016] Preferably, the volume of the potato culture medium is 10-30 L.

[0017] Preferably, the temperature of the fermentation is 110-130 ℃.

[0018] More preferably, the fermentation time is 10-30 min.

[0019] Specifically, in step S1, the fermentation is sterilizing 10-30 L of potato medium at 110-130 ℃ for 10-30 min.

[0020] Preferably, in step S1, the standing culture time is 5-10 days.

[0021] Further, in step S2, the separation is filtration separation.

[0022] Further, in step S2, the purification is extraction.

[0023] Still further, in step S2, the solvent for extraction is ethyl acetate and water.

[0024] Further, the separation and purification includes column chromatography, reverse phase chromatography and reverse phase high performance liquid chromatography steps.

[0025] Still further, the column chromatography is silica gel column chromatography of the extract using gradient concentrations of petroleum ether / ethyl acetate as the mobile phase.

[0026] Still further, the reverse phase chromatography is reverse phase chromatography of the column chromatography product using ODS-C18 to obtain the crude product of compound Merulin A.

[0027] Still further, as a preferred method, the conditions of the reverse phase high performance liquid chromatography are using a chromatographic column with a size of 4.6×250 mm and a packing particle size of 5 µm, the mobile phase is a methanol / water system (volume ratio of 2:3), and the flow rate is set to 3 mL / min. Under these conditions, the retention time of compound Merulin A ranges from 12 to 15 min.

[0028] Further, the compound Merulin A is replaced by a pharmaceutically acceptable salt or solvate thereof.

[0029] Further, the anti-glioma is promoting apoptosis of glioma cells.

[0030] Still further, the promoting apoptosis of glioma cells is promoting early apoptosis of glioma cells.

[0031] Further, the anti-glioma is promoting necrosis of glioma cells.

[0032] Further, the anti-glioma is inhibiting proliferation of glioma cells.

[0033] Further, the anti-glioma is inhibiting migration of glioma cells.

[0034] Further, the brain glioma cells include one or more of U251, T98G, U87-MG.

[0035] Further, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0036] Preferably, the dosage form of the drug is an oral agent, an injection agent or a microneedle agent.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] The present application provides the application of compound Merulin A in the preparation of an anti-brain glioma drug. Experiments have proved that compound Merulin A exhibits significant anti-brain glioma activity, can effectively promote the early apoptosis and cell necrosis of brain glioma cells, and further inhibit the proliferation and migration of brain glioma cells. Moreover, due to the advantages of wide source, simple extraction process, short cycle and low cost, compound Merulin A has a broad application prospect as an anti-brain glioma drug. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Figure 4 is a result statistical diagram of the proliferation inhibition rate of different human brain glioma cell lines (U87-MG (a), T98G (b), U251 (c)) of different concentrations of compound Merulin A and paclitaxel in Example 2; wherein, NC is a negative control group (0.1 % DMSO), and * represents p<0.05, ** represents p<0.01, and *** represents p<0.001 compared with the drug-added group.

[0040] Figure 2 Figure 5 is a result statistical diagram of the migration influence of different human brain glioma cell lines (U87-MG (a), T98G (b), U251 (c)) of different concentrations of compound Merulin A and paclitaxel in Example 3, (a)~(c) are micrographs of cell lines U87-MG, U251 and T98G, respectively, and (d)~(f) are data statistical diagrams of the migration rate of cell lines U87-MG, U251 and T98G, respectively; wherein, NC is a negative control group (0.1 % DMSO), and * represents p<0.05, ** represents p<0.01, and *** represents p<0.001 compared with the drug-added group.

[0041] Figure 3Statistical chart of flow cytometry results of the apoptosis influence of different concentrations of compound Merulin A, paclitaxel and temozolomide on different human glioma cell lines (U87-MG (a), T98G (b)) in Example 4; wherein, Normal represents normal cells, Early Apoptosis represents early apoptosis, Later Apoptosis represents late apoptosis, Nercrosis represents necrotic cells, NC is the negative control group (0.5% DMSO), TMZ is the 87 μg / mL temozolomide treatment group (temozolomide at this concentration has good brain glioma treatment effect), PTX is the 0.8 μg / mL paclitaxel treatment group (paclitaxel at this concentration has good brain glioma treatment effect).

[0042] Figure 4 Statistical chart of flow cytometry data of the apoptosis influence of different concentrations of compound Merulin A, paclitaxel and temozolomide on different human glioma cell lines (U87-MG (a), T98G (b)) in Example 4; Normal represents normal cells, Early Apoptosis represents early apoptosis, Later Apoptosis represents late apoptosis, Nercrosis represents necrotic cells, NC is the negative control group (0.5% DMSO), TMZ is the 87 μg / mL temozolomide treatment group (temozolomide at this concentration has good brain glioma treatment effect), PTX is the 0.8 μg / mL paclitaxel treatment group (paclitaxel at this concentration has good brain glioma treatment effect); * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. DETAILED DESCRIPTION

[0043] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0045] Figure 1 (a) represents the (a) graph in FIG. 1, Figure 1 (b) represents the (b) graph in FIG. 1, and the order of the other graphs is sequentially continued. Figure 1 Figure 1 Example 1 Preparation and characterization of compound Merulin A

[0046] Example 1 Preparation and characterization of compound Merulin A

[0047] 1. Preparation of compound Merulin A

[0048] ​A fungus Pseudolagarobasidium acaciicola SYSU-MS7908 derived from sea squirts was inoculated into 20 L of liquid potato medium, which had been sterilized at 121 ℃ for 20 minutes. The fungus was fermented at room temperature for 7 days. Subsequently, the mycelium was separated from the fermentation broth using filter cloth. The obtained fermentation broth was subjected to ethyl acetate / water extraction treatment, and then the ethyl acetate layer was concentrated to obtain an extract. Next, the extract was subjected to silica gel column chromatography using gradient concentrations of petroleum ether / ethyl acetate as the mobile phase system. During the chromatography process, the component with a petroleum ether to ethyl acetate volume ratio of 3:2 was collected. Through ODS-C18 reverse phase chromatography, a crude component containing the natural product Merulin A was obtained from the component. In order to further purify, the crude component was separated by RP-HPLC (using a C18 chromatographic column with a specification of 4.6 × 250 mm, 5 µm; the mobile phase was methanol / water with a volume ratio of 40 / 60; the flow rate was 3 mL / min; the retention time was 13 minutes for separation preparation, and finally about 1.1 grams of the target compound Merulin A was obtained.

[0049] 2. Characterization of Compound Merulin A

[0050] The compound Merulin A obtained in Step 1 was subjected to nuclear magnetic resonance and mass spectrometry detection, and analysis confirmed that the compound Merulin A obtained in Step 1 had been successfully isolated from the fungus. The physicochemical properties of the compound are as follows:

[0051] Nuclear magnetic resonance hydrogen spectrum data: 1 HNMR (400 MHz, CDCl3) δH 4.11 (m, 1H, H-2), 3.75(ddd, J = 9.0, 8.4, 3.8 Hz, 1H, H-3), 2.68 (td, J = 14.9, 6.6 Hz, 1H, H-9a), 2.43 (ddd, J = 15.6, 4.9, 2.2 Hz, 1H, H-9b), 2.15 (m, 2H, H-4), 2.02 (m, 3H, H-1a, 5a, 10a), 1.55 (m, 3H, H-1b, 5b, 10b), 1.40 (s, 3H, H-14), 1.25 (s, 3H, H-13), 0.97 (s, 3H, H-12).

[0052] Nuclear magnetic resonance carbon spectrum data: 13CNMR (100 MHz, CDCl3,) δ 208.1, 90.3, 79.2, 69.5, 41.4, 37.5, 35.9, 35.7, 32.3, 30.7, 26.3, 25.7, 24.8, 21.6.

[0053] Mass spectrometry data: HR-ESIMS m / z 277.1414, [M+Na]+ (calcd. for C 14 H 22 O4Na, 277.1416).

[0054] Example 2: Test of the inhibitory effect of compound Merulin A on the proliferation of glioma cells.

[0055] 1. Experimental Methods

[0056] First, U251, T98G, and U87-MG glioma cells were retrieved from liquid nitrogen, revived, cultured, and passaged. When the cells were in the logarithmic growth phase, they were diluted to a concentration of approximately 4 × 10⁻⁶ cells / mL using DMEM complete medium. 4 Cells / mL. Then, 100 μL of cell suspension was added to each well of a 96-well plate, and the plate was incubated in a CO2 incubator for 24 h. Next, Merulin A was diluted to concentrations of 50 μM, 25 μM, 12.5 μM, 6.25 μM, and 3.125 μM using DMEM complete medium. After 24 h of incubation, the 96-well plates were removed, the old culture medium was aspirated with a pipette, and 200 μL of the above-mentioned culture medium containing different concentrations of Merulin A was added. Simultaneously, wells with DMSO as a negative control and paclitaxel solution as a positive control were set up, with three parallel wells for each test sample. In addition, a set of blank wells (cell-free) was set up as a control for each 96-well plate. The prepared 96-well plates were placed back into the CO2 incubator and incubated for 12 h, 24 h, 48 h, and 72 h, respectively. After incubation, the 96-well plates were removed, and the culture medium was slowly aspirated with a pipette. Next, the MTT reagent was diluted 10-fold with serum-free DMEM medium, and 100 μL of the diluted MTT solution was added to each well. The 96-well plate was returned to the CO2 incubator and incubated for another 4 h to allow the blue crystals to fully form. Afterward, the liquid in the plate was carefully aspirated using a pipette, and 100 μL of DMSO was added to each well to fully dissolve the blue precipitate. Finally, the absorbance (OD value) of each well was read at 570 nm using a microplate reader. The inhibition rate of glioma cells at different concentrations of compound Merulin A was calculated using the following formula:

[0057] Inhibition rate = (OD 实验组 - OD 阴性对照组 ) / OD 阴性对照组 x 100%.

[0058] 2. Experimental results

[0059] From Figure 1 it can be observed that when human glioma cells are treated with different concentrations of compound Merulin A, the compound Merulin A has good cell proliferation inhibition activity on human glioma cells U87-MG, U251 and T98G at 48 h and 72 h after treatment, and shows concentration dependence.

[0060] Experimental Example 3: Test of the migration ability of compound Merulin A on glioma cells

[0061] 1. Experimental method

[0062] First, the well-conditioned U251, T98G and U87-MG glioma cells are treated with trypsin digestion, and then inoculated in 6-well plates at a cell number of 5 x 10 5 After inoculation, the cells are cultured at 37°C, 5% CO2 and 95% relative humidity. When the cell density reaches about 90%, a 200 μL pipette tip is used to make a vertical scratch on the cell layer from top to bottom in the super-clean bench. The scratched cells are then gently washed away with PBS buffer to ensure the clarity of the scratch. After the scratch is made, the initial scratch distance is immediately photographed and recorded as a reference for subsequent analysis. Next, the old culture solution is removed, and fresh culture solution containing different concentrations (10 μM, 5 μM, 2 μM) of compound Merulin A is added to each well, and cultured for 48 h. For comparison, a negative control group is set up in the experiment, which only adds 0.5% DMSO (the same solvent as used for dissolving Merulin A), and a positive control group is set up, which adds a common anti-tumor drug paclitaxel (1 μM). After 48 hours of action of compound Merulin A, the cell migration in each well is observed using an inverted microscope, and the monolayer cell images are taken. By measuring the movement distance of the cell front and the change in the scratch width, the effect of different concentrations of compound Merulin A on the migration ability of glioma cells is analyzed.

[0063] 2. Experimental results

[0064] From Figure 2It can be seen that the compound Merulin A has a significant inhibitory effect on the migration of human glioma cells, and this inhibitory effect shows obvious concentration dependence. With the increase of the concentration of Merulin A, the inhibitory effect on the migration of glioma cells also correspondingly increases.

[0065] Example 4 Test of the effect of compound Merulin A on apoptosis of human glioma cells

[0066] 1. Experimental method

[0067] The well-conditioned T98G and U87-MG glioma cells were first subjected to trypsin digestion, and then were inoculated into 6-well plates at a cell number of 5 x 10 5 The cells were cultured at 37°C, 5% CO2 concentration and 95% relative humidity. When the cell density was observed to be about 90%, the next step was performed.

[0068] First, the old culture solution was removed, and fresh culture solution containing different concentrations of Merulin A (1 μg / mL, 3 μg / mL, 5 μg / mL) was added to each well, and the action time was 48 hours. In order to set up the control group, a part of the cell wells were added with culture solution containing 0.5% DMSO as negative control (NC), and another part of the cell wells were added with culture solution containing paclitaxel (PTX) (0.8 μg / mL) and temozolomide (TMZ) (87 μg / mL) as positive control. After 48 hours, the original culture solution in the wells was aspirated and stored in a centrifuge tube for standby. Then, the cells in each group were washed with PBS solution, and then an appropriate amount of trypsin was added for digestion. After the digestion process was completed, the original culture solution was added to terminate the digestion, and the cells were carefully blown off from the bottom of the culture plate with a pipette. The washed PBS solution and trypsin-digested cells were collected into the same centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After centrifugation, the supernatant in the centrifuge tube was carefully aspirated, and 1.5 mL of pre-cooled PBS was added to resuspend the cells. Centrifugation was performed again at 1000 rpm for 5 minutes to remove the cell fragments in the PBS and cell suspension.

[0069] Next, the apoptosis detection was performed. 100 μL of Annexin-V FITC / PI binding solution, 5 μL of Annexin-V FITC solution and 10 μL of PI solution were added to each sample tube, respectively, and mixed well. The mixed sample was incubated at room temperature for 30 minutes in the dark. In order to adjust the cell gain and compensation, a non-stained cell sample, an Annexin-V FITC stained cell sample and a PI stained cell sample were also set. When using the flow cytometer for detection, the cell gain was first adjusted using the non-stained cell sample, and then the compensation of the fluorescence channel was adjusted using the Annexin-V FITC single stained and PI single stained cell samples, respectively. Finally, the Annexin-V FITC and PI double stained cell sample was analyzed, the cells were divided into four quadrants according to the fluorescence intensity, and the percentage of cells in each quadrant was calculated, so as to obtain the apoptosis rate of the sample.

[0070] 2. Experimental results

[0071] From Figure 3 It can be seen that in the two brain glioma cell lines, with the increase of the concentration of the compound Merulin A, the flow cytometry analysis showed that the proportion of early apoptosis (Q3 region) and cell necrosis (Q1 region) showed an increasing trend. This indicates that the compound Merulin A has a toxic effect on brain glioma cells in vitro, mainly by triggering early apoptosis and cell necrosis mechanism to inhibit the proliferation and migration of brain glioma cells. Further, from the statistical chart of Figure 4 , the results of the above flow cytometry analysis can be more intuitively observed. Therefore, the compound Merulin A has good potential as an anti-brain glioma drug.

[0072] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. Use of the compound Merulin A or a pharmaceutically acceptable salt thereof for the manufacture of a medicament against brain glioma, characterized in that, The structure of the compound Merulin A is as follows: 。 2. Use according to claim 1, characterized in that, The anti-glioma is to promote apoptosis of glioma cells.

3. Use according to claim 2, characterized in that, The promoting apoptosis of glioma cells is to promote early apoptosis of glioma cells.

4. The use according to claim 1, characterized in that, The anti-glioma is to promote necrosis of glioma cells.

5. The use according to claim 1, characterized in that, The anti-glioma is to inhibit proliferation of glioma cells.

6. The use according to claim 1, characterized in that, The anti-glioma is to inhibit migration of glioma cells.

7. Use according to any one of claims 2 to 6, characterized in that, The glioma cells include one or more of U251, T98G, U87-MG.

8. The use according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier or adjuvant.

9. Use according to claim 8, characterized in that, The dosage form of the drug is oral, injection or microneedle.

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