Application of compound Merulin A in preparation of anti-glioma drug

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a compound Merulin A in preparation of anti-glioma medicines. The invention provides an application of a compound Merulin A in preparation of a drug for resisting brain glioma. Experiments prove that the compound Merulin A shows remarkable anti-glioma activity, can effectively promote early apoptosis and necrosis of glioma cells, and further inhibits proliferation and migration of the glioma cells. Moreover, due to the advantages of being wide in source, simple in extraction process, short in period, low in cost and the like, the compound Merulin A has a wide application prospect as the anti-glioma drug.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and more specifically, relates to the application of the compound Merulin A in the preparation of anti-glioma drugs. Background Art

[0002] Glioma, a malignant tumor originating from glial cells, is the main type of primary malignant heterogeneous tumors of the central nervous system. Due to its high incidence and high mortality rate, glioma poses a major challenge to human health. The treatment of glioma is particularly difficult because gliomas often grow in an infiltrative manner and have unclear boundaries with surrounding brain tissue, which makes surgical resection and drug treatment face great challenges. At present, the drug options for the treatment of glioma are relatively limited, among which temozolomide and paclitaxel are more commonly used drugs. Temozolomide, as a chemotherapy drug, has been widely used in the treatment of glioma. It produces significant anti-tumor effects through mechanisms such as regulating the cell cycle, inhibiting cell proliferation and inducing cell apoptosis. Paclitaxel, as a broad-spectrum anticancer drug, mainly exerts its anti-cancer 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 subject to certain limitations, such as the obstruction of the blood-brain barrier, it can still be used as an adjuvant therapy drug to a certain extent to provide patients with certain therapeutic effects.

[0003] However, in clinical treatment, there is still a relative lack of therapeutic drugs for glioma patients. Therefore, the medical community urgently needs to increase research and development efforts, actively explore and develop more new drugs that are highly effective against brain gliomas, aiming to provide patients with more diverse and more effective treatment options to improve their quality of life and prognosis. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing drugs for treating gliomas, which are still relatively scarce, and to provide the use of the compound Merulin A in the preparation of anti-glioma drugs.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The invention discloses a method for preparing an anti-glioma drug using the protected compound Merulin A. The structure of the compound Merulin A is as follows: .

[0006] Fungal natural products of marine origin constitute one of the important sources of marine natural products. In the process of exploring compounds with anti-glioma activity, in-depth research on the secondary metabolites of marine fungi is of great significance. The inventors successfully isolated the terpene compound Merulin A from the fermentation product of the fungus Pseudolagarobasidium acaciicola SYSU-MS9908 isolated from the sea squirt. 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.

[0007] Furthermore, the compound Merulin A can be obtained through a variety of ways, including commercial channels, extraction from natural sources, and chemical synthesis methods. Regardless of the method of obtaining, the effect of the compound Merulin A is not affected.

[0008] Furthermore, as an optional embodiment, the compound MerulinA is isolated and purified from the fungus strain derived from marine ascidian.

[0009] Preferably, the marine ascidian-derived fungal strain is Pseudolagarobasidium acaciicola SYSU-MS9908 was deposited in Guangdong Provincial Microbiological Culture Collection Center on December 24, 2024, with the deposit number GDMCC NO: 65664.

[0010] More preferably, the separation and purification specifically comprises the following steps: S1. fermenting the fungus Pseudolagarobasidium acaciicola SYSU-MS9908, and statically culturing to obtain a fermentation product; S2. Separating the bacterial liquid from the fermented product obtained in step S1, purifying and concentrating the bacterial liquid to obtain an extract, and separating and purifying the obtained extract to obtain the compound Merulin A.

[0011] Furthermore, in step S1, the fermentation is carried out in a sterilized potato culture medium.

[0012] Preferably, the volume of the potato culture medium is 10-30 L.

[0013] Preferably, the fermentation temperature is 110-130°C.

[0014] More preferably, the fermentation time is 10 to 30 min.

[0015] Specifically, in step S1, the fermentation is performed by sterilizing 10 to 30 L of potato culture medium at 110 to 130° C. for 10 to 30 min.

[0016] Preferably, in step S1, the static culture time is 5 to 10 days.

[0017] Furthermore, in step S2, the separation is filtration separation.

[0018] Furthermore, in step S2, the purification is extraction.

[0019] Furthermore, in step S2, the extraction solvents are ethyl acetate and water.

[0020] Furthermore, the separation and purification includes column chromatography, reverse phase chromatography and reverse phase high performance liquid chromatography steps.

[0021] Furthermore, the column chromatography method uses gradient concentrations of petroleum ether / ethyl acetate as mobile phase to perform silica gel column chromatography on the extract.

[0022] Furthermore, the reverse phase chromatography is to use ODS-C18 to perform reverse phase chromatography on the product after column chromatography to obtain a crude product of the compound Merulin A.

[0023] Furthermore, as a preferred method, the reverse HPLC method is carried out using a 4.6×250 mm column with a filler particle size of 5 µm, a mobile phase of a methanol / water system (volume ratio of 2:3), and a flow rate of 3 mL / min. Under these conditions, the retention time of the compound Merulin A ranges from 12 to 15 min.

[0024] Furthermore, the compound Merulin A is substituted with a pharmaceutically acceptable salt or solvate thereof.

[0025] Furthermore, the anti-glioma effect is to promote apoptosis of glioma cells.

[0026] Furthermore, the promoting apoptosis of glioma cells is promoting early apoptosis of glioma cells.

[0027] Furthermore, the anti-glioma effect is to promote glioma cell necrosis.

[0028] Furthermore, the anti-glioma effect is to inhibit the proliferation of glioma cells.

[0029] Furthermore, the anti-glioma effect is to inhibit the migration of glioma cells.

[0030] Furthermore, the brain glioma cells include one or more of U251, T98G, and U87-MG.

[0031] Furthermore, the drug also includes a pharmaceutically acceptable carrier or excipient.

[0032] Preferably, the drug is in the form of an oral preparation, an injection or a microneedle.

[0033] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides the use of the compound Merulin A in the preparation of an anti-glioma drug. Experiments have shown that the compound Merulin A exhibits significant anti-glioma activity, can effectively promote early apoptosis and cell necrosis of glioma cells, and thus inhibit the proliferation and migration of glioma cells. In addition, due to the advantages of wide sources, simple extraction process, short cycle and low cost, the compound Merulin A has broad application prospects as an anti-glioma drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The results are statistical graphs showing the proliferation inhibition rates of different human glioma cell lines (U87-MG (a), T98G (b), and U251 (c)) with different concentrations of the compound Merulin A and paclitaxel in Example 2; among them, compared with the drug-treated group, the NC negative control group (0.1% DMSO) has *p<0.05, **p<0.01, and ***p<0.001.

[0035] Figure 2 The results of the effects of different concentrations of the compound Merulin A and paclitaxel on the migration of different human glioma cell lines (U87-MG (a), T98G (b), U251 (c)) in Example 3 are statistical graphs, (a) to (c) are microscopic images of cell lines U87-MG, U251, and T98G, respectively, and (d) to (f) are statistical graphs of the migration rates of cell lines U87-MG, U251, and T98G, respectively; wherein NC is the negative control group (0.1% DMSO), and compared with the drug-treated group, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001 in the NC group.

[0036] Figure 3The results of flow cytometry analysis of the effects of different concentrations of the compound Merulin A, paclitaxel and temozolomide on apoptosis of different human glioma cell lines (U87-MG (a), T98G (b)) in Example 4 are statistical graphs; wherein Normal represents normal cells, Early Apoptosis represents early apoptosis, Later Apoptosis represents late apoptosis, Nercrosis represents necrotic cells, NC represents the negative control group (0.5% DMSO), TMZ represents the 87 μg / mL temozolomide treatment group (temozolomide at this concentration has a good therapeutic effect on gliomas), and PTX represents the 0.8 μg / mL paclitaxel treatment group (paclitaxel at this concentration has a good therapeutic effect on gliomas).

[0037] Figure 4 The flow cytometry data of the effects of different concentrations of the compound Merulin A, paclitaxel and temozolomide on apoptosis of different human glioma cell lines (U87-MG (a), T98G (b)) in Example 4 are statistical graphs; Normal represents normal cells, Early Apoptosis represents early apoptosis, Later Apoptosis represents late apoptosis, Nercrosis represents necrotic cells, NC represents the negative control group (0.5% DMSO), TMZ represents the 87 μg / mL temozolomide treatment group (temozolomide at this concentration has a good therapeutic effect on gliomas), and PTX represents the 0.8 μg / mL paclitaxel treatment group (paclitaxel at this concentration has a good therapeutic effect on gliomas); * represents p<0.05, ** represents p<0.01, and *** represents p<0.001. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Figure 1 (a) means Figure 1 In Figure (a), Figure 1 (b) indicates Figure 1 The order of the other figures is similar.

[0041] Example 1 Preparation and characterization of compound Merulin A 1. Preparation of compound Merulin A The fungus Pseudolagarobasidium acaciicola SYSU-MS7908 from the sea squirt was inoculated into 20L of liquid potato culture medium, which had been sterilized at 121 °C for 20 minutes. The fungus was fermented and cultured at room temperature for 7 days. Subsequently, the bacteria were separated from the fermentation broth using a filter cloth. The resulting fermentation broth was extracted with ethyl acetate / water, and then the ethyl acetate layer was concentrated to obtain an extract. Next, the extract was subjected to silica gel column chromatography using a gradient concentration of petroleum ether / ethyl acetate as the mobile phase system. During the chromatography process, components with a volume ratio of petroleum ether to ethyl acetate of 3:2 were collected. A crude component containing the natural product Merulin A can be obtained from this component by ODS-C18 reverse phase chromatography. For further purification, the crude fraction was separated and prepared by RP-HPLC (using a C18 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), and finally about 1.1 g of the target compound Merulin A was obtained.

[0042] 2. Characterization of compound Merulin A The compound Merulin A obtained in step 1 was subjected to nuclear magnetic resonance and mass spectrometry detection. After analysis and detection, it was confirmed that the compound Merulin A obtained in step 1 was successfully isolated from fungi. The specific data of the physicochemical properties of the compound are as follows: H NMR 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).

[0043] C NMR 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.

[0044] Mass spectral data: HR-ESIMS m / z 277.1414, [M+Na]+ (calcd. for C 14 H 22 O4Na,277.1416).

[0045] Example 2 Testing of the ability of the compound Merulin A to inhibit the proliferation of brain glioma cells 1. Experimental Methods First, U251, T98G, and U87-MG glioma cells were taken out of liquid nitrogen for recovery culture and passage. When the cells were in the logarithmic growth phase, the cells were diluted to a concentration of about 4×10 4 / mL. Then, 100 μL of cell suspension was added to each well of the 96-well plate, and the 96-well plate was placed in a CO2 incubator for 24 h. Next, the compound 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. The 96-well plate that had been cultured for 24 h was taken out, the old culture medium was aspirated with a pipette, and then 200 μL of the above-mentioned culture medium containing different concentrations of the compound Merulin A was added. At the same time, wells with DMSO as a negative control and paclitaxel solution as a positive control were set, and 3 parallel test wells were set for each test sample. In addition, a set of blank wells (no cells) was set for each 96-well plate as a control. The set 96-well plate was placed in a CO2 incubator again and cultured for 12 h, 24 h, 48 h and 72 h, respectively. After the culture was completed, the 96-well plate was taken out and the culture medium was slowly aspirated with a pipette. Then, the MTT reagent was diluted 10 times 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 for 4 h to allow the blue crystals to fully form. After that, the liquid in the well 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 a wavelength of 570 nm on a microplate reader. The inhibitory rate of the compound Merulin A at different concentrations of brain glioma cells was calculated according to the following formula: Inhibition rate = (OD 实验组 -OD 阴性对照组 ) / OD阴性对照组 ×100%.

[0046] 2. Experimental results from Figure 1 It can be observed that when different concentrations of the compound Merulin A were used to treat human glioma cells, at 48 h and 72 h of treatment, the compound Merulin A had good cell proliferation inhibitory activity on human glioma cells U87-MG, U251, and T98G, and showed concentration dependence.

[0047] Experimental Example 3 Testing of the compound Merulin A on the migration ability of brain glioma cells 1. Experimental Methods First, the U251, T98G, and U87-MG glioma cells in good condition were digested with trypsin and then inoculated in 6-well plates, with 5×10 cells in each well. 5 The inoculated cells were cultured at 37 °C, 5% CO2 and 95% relative humidity. When the cell density reached about 90%, a 200 μL pipette tip was used in the clean bench to vertically scratch the cell layer from top to bottom. The scratched cells were then gently rinsed and removed with PBS buffer to ensure the clarity of the scratch. After the scratch was made, the initial scratch distance was immediately recorded by taking a photo as a benchmark for subsequent analysis. Next, the old culture medium was removed, and fresh culture medium containing different concentrations of the compound Merulin A (10 μM, 5 μM, 2 μM) was added to each well and cultured for 48 h. For comparison, a negative control group was set up in the experiment, in which only 0.5% DMSO (the same solvent used to dissolve Merulin A) was added, and a positive control group, in which the common anti-tumor drug paclitaxel (1 μM) was added. After the compound Merulin A was acted on for 48 hours, the cell migration in each well was observed using an inverted microscope, and the monolayer cell image was taken. The effects of different concentrations of the compound Merulin A on the migration ability of glioma cells were analyzed by measuring the movement distance of the cell leading edge and the change in scratch width.

[0048] 2. Experimental results from Figure 2 It can be seen that the compound Merulin A has a significant inhibitory effect on the migration of human brain glioma cells, and this inhibitory effect shows a significant concentration dependence. As the concentration of Merulin A increases, its inhibitory effect on the migration of glioma cells also increases accordingly.

[0049] Example 4 Testing of the Effect of Compound Merulin A on Apoptosis of Human Glioma Cells 1. Experimental Methods T98G and U87-MG glioma cells in good condition were first digested with trypsin and then seeded into 6-well plates, with 5×10 cells seeded per well. 5 The cells were cultured at 37°C, 5% CO2 concentration and 95% relative humidity. When the cell density reached about 90%, the next step was performed.

[0050] First, the old culture medium was removed and fresh culture medium containing different concentrations of Merulin A (1 μg / mL, 3 μg / mL, 5 μg / mL) was added to each well for 48 hours. In order to set up the control group, a culture medium containing 0.5% DMSO was added to some of the cell wells as a negative control (NC), and a culture medium containing paclitaxel (PTX) (0.8 μg / mL) and temozolomide (TMZ) (87 μg / mL) was added to the other part of the cell wells as a positive control. After 48 hours, the original culture medium in the well was aspirated and stored in a centrifuge tube for later use. Next, each group of cells was washed with PBS solution, and then an appropriate amount of trypsin was added for digestion. After the digestion process was completed, the original culture medium was added to stop the digestion, and the cells were carefully blown with a pipette to separate them from the bottom of the culture plate. The washed PBS solution and trypsin-digested cells were collected in the same centrifuge tube and centrifuged at 1000 rpm for 5 minutes. After centrifugation, carefully remove the supernatant in the centrifuge tube, then add 1.5 mL of pre-cooled PBS to resuspend the cells. Centrifuge again at 1000 rpm for 5 minutes to remove cell debris from the PBS and cell suspension.

[0051] Next, perform apoptosis detection. Add 100 μL Annexin-V FITC / PI conjugate, 5 μL Annexin-V FITC solution, and 10 μL PI solution to each tube of sample and mix thoroughly. Incubate the mixed sample at room temperature in the dark for 30 minutes. In order to adjust the cell gain and compensation, set up an unstained cell sample, a cell sample with only Annexin-V FITC staining, and a cell sample with only PI staining solution. When using a flow cytometer for detection, first use the unstained cell sample to adjust the cell gain, and then use the Annexin-V FITC single-stained and PI single-stained cell samples to adjust the compensation of the fluorescence channel. Finally, analyze the Annexin-V FITC and PI double-stained cell samples, divide the cells into four quadrants according to the fluorescence intensity, and calculate the percentage of cells in each quadrant to obtain the apoptosis rate of the sample.

[0052] 2. Experimental results from Figure 3It can be seen that in the two glioma cell lines, as the concentration of the compound Merulin A increases, flow cytometry analysis shows that the sum of the proportions of early apoptosis (Q3 region) and cell necrosis (Q1 region) shows a trend of increasing. This indicates that the compound Merulin A has a toxic effect on glioma cells in vitro, mainly by triggering early apoptosis and cell necrosis mechanisms to inhibit the proliferation and migration of glioma cells. Figure 4 The results of the above flow cytometry analysis can be observed more intuitively in the statistical graph. Therefore, the compound Merulin A has good potential as an anti-glioma drug.

[0053] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of the compound Merulin A in the preparation of an anti-glioma drug, characterized in that: The structure of the compound Merulin A is as follows:

2. The application according to claim 1, characterized in that: The compound Merulin A is substituted with a pharmaceutically acceptable salt or solvate thereof.

3. The use according to claim 1 or 2, characterized in that: The anti-glioma effect is to promote the apoptosis of glioma cells.

4. The use according to claim 3, characterized in that: The promoting apoptosis of brain glioma cells is promoting early apoptosis of brain glioma cells.

5. The use according to claim 1 or 2, characterized in that: The anti-glioma effect is to promote glioma cell necrosis.

6. The use according to claim 1 or 2, characterized in that: The anti-glioma effect is to inhibit the proliferation of glioma cells.

7. The use according to claim 1 or 2, characterized in that: The anti-glioma effect is to inhibit the migration of glioma cells.

8. The use according to any one of claims 3 to 7, characterized in that: The brain glioma cells include one or more of U251, T98G, and U87-MG.

9. The use according to any one of claims 1 to 8, characterized in that: The medicine also includes a pharmaceutically acceptable carrier or excipient.

10. The use according to any one of claims 1 to 9, characterized in that: The dosage form of the drug is oral preparation, injection or micro-injection.

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