Application of compound Colletochin in preparation of anti-glioma drug

Abstract

The invention relates to the technical field of biological medicines, in particular to application of a compound Colletochin to preparation of a drug for resisting brain glioma. The invention provides an application of a compound Colletochin in preparation of a drug for resisting brain glioma. Experiments prove that the compound Colletochin shows remarkable anti-brain glioma activity, and proliferation, migration and invasion of brain glioma cells can be inhibited by retarding the cell cycle of the brain glioma. Moreover, the compound Colletochin is wide in source, simple in extraction process, short in preparation period and low in cost, so that the compound Colletochin shows a wide application prospect in the development of anti-glioma drugs.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, and in particular to the use of the compound Colletochin in the preparation of anti-glioma drugs. Background Technology

[0002] Gliomas are malignant tumors originating from glial cells. As the most common primary malignant heterogeneous tumors of the central nervous system, their incidence and mortality rates have remained consistently high, making them one of the major diseases threatening human health. The treatment of these tumors faces numerous severe challenges: the core issue lies in their invasive growth pattern, with indistinct boundaries between the tumor tissue and surrounding normal brain tissue, making radical surgical resection difficult and resulting in a very high risk of postoperative recurrence. Simultaneously, the blood-brain barrier, as a natural intracranial protective barrier, severely hinders the penetration of most drugs to the lesion site, significantly reducing drug bioavailability, posing a dual challenge to clinical treatment.

[0003] To achieve safe and effective treatment, chemotherapy drugs remain the primary treatment option in clinical practice. Temozolomide, a classic chemotherapy drug, inhibits tumor proliferation and induces apoptosis by interfering with the DNA replication and repair process of tumor cells. It is often used in combination with radiotherapy to treat various gliomas, such as glioblastoma. Although paclitaxel is a broad-spectrum anticancer drug that can exert its anti-tumor effect by inhibiting tumor cell division, its efficacy is limited by the significant restriction of the blood-brain barrier and can only be used as an adjunct in the treatment of gliomas.

[0004] Although the existing drugs mentioned above have alleviated the condition of some patients and improved their short-term prognosis to some extent, overall, there is still a relative lack of drugs suitable for gliomas with different gene mutation types and different clinical stages. There is an urgent need in the clinic to further explore and develop more effective new anti-glioma drugs to provide patients with diversified treatment options, thereby fundamentally improving the overall treatment effect of gliomas and the quality of life of patients. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiency and inadequacy of existing drugs for treating glioma, and to provide the application of the compound Colletochin in the preparation of anti-glioma drugs.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution: This invention protects the use of the compound Colletochin in the preparation of an anti-glioma drug, the structure of which is shown below: .

[0007] Marine-derived fungal natural products constitute one of the important sources of marine natural products. In the search for compounds with anti-glioma activity, in-depth research on the secondary metabolites of marine fungi is of great significance. The inventors isolated fungi from sea snails... Tolypocladium Colletochin was successfully isolated from the fermentation products of sp. SYSU-F00117. Studies have shown that Colletochin significantly inhibits the proliferation, migration, and invasion of glioma cells. Therefore, Colletochin shows promising potential as an anti-glioma drug.

[0008] Furthermore, the compound Colletochin can be obtained through various means, including commercial purchases, extraction from natural sources, and chemical synthesis. Regardless of the method of acquisition, the function of the compound Colletochin remains unaffected.

[0009] Furthermore, as an optional implementation, the compound Colletochin is isolated and purified from the cells of a fungal strain derived from sea cucumber.

[0010] Preferably, the fungal strain from which the sea cucumber is derived is... Tolypocladium sp. SYSU-F00117 was deposited on August 12, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 66833, and address: No. 100 Xianlie Road, Yuexiu District, Guangzhou City, Guangdong Province.

[0011] More preferably, the separation and purification specifically includes the following steps: S1. Fungi Tolypocladium Fermentation was carried out using sp. SYSU-F00117, and the fermentation product was obtained by static culture. S2. Solid-liquid separation is performed on the fermentation product obtained in step S1 to obtain bacterial liquid and bacterial cells. The extracts of the obtained bacterial liquid and bacterial cells are extracted and concentrated to obtain bacterial liquid extract and bacterial cell extract, respectively. The two extracts are mixed and then separated and purified to obtain the compound Colletochin.

[0012] Furthermore, the fermentation is carried out in a sterilized potato culture medium.

[0013] Furthermore, the sterilization temperature of the potato culture medium is 110~130 ℃.

[0014] Furthermore, the sterilization time of the potato culture medium is 10-30 min.

[0015] Preferably, the static incubation period is 5 to 10 days.

[0016] Further, the extract of the bacterial cells is obtained through the following steps: The bacterial cells were extracted with methanol several times, and the resulting extract was concentrated to obtain the bacterial extract.

[0017] Furthermore, the methanol extraction is performed 3 to 5 times.

[0018] Furthermore, the solvent used for extraction is a combination of ethyl acetate and water.

[0019] Furthermore, the volume ratio of ethyl acetate to water is 1:(0.5~2).

[0020] Preferably, the volume ratio of ethyl acetate to water is 1:(0.8~1.2).

[0021] More preferably, the volume ratio of ethyl acetate to water is 1:1.

[0022] Furthermore, the concentration is a vacuum concentration.

[0023] Furthermore, the separation and purification includes column chromatography, gel column chromatography, and normal-phase silica gel column chromatography steps.

[0024] Furthermore, the column chromatography method involves using a mixture of petroleum ether and ethyl acetate as the mobile phase to perform silica gel column chromatography on the aforementioned mixed extract.

[0025] Preferably, the volume ratio of petroleum ether to ethyl acetate is 1:(0.1~1).

[0026] More preferably, the volume ratio of petroleum ether to ethyl acetate is 1:(0.25~0.75).

[0027] Furthermore, the gel column chromatography is performed using a mixture of dichloromethane and methanol as the mobile phase to perform gel column chromatography on the aforementioned column chromatography product to obtain the crude product of the compound Colletochin.

[0028] Preferably, the volume ratio of dichloromethane to methanol is 1:(0.5~1.5).

[0029] More preferably, the volume ratio of dichloromethane to methanol is 1:1.

[0030] Preferably, the gel column is Sephadex LH-20.

[0031] Furthermore, the normal-phase silica gel column chromatography uses a mixture of dichloromethane and methanol as the mobile phase to perform normal-phase silica gel column chromatography on the crude product of the aforementioned compound Colletochin.

[0032] Preferably, the volume ratio of dichloromethane to methanol is 1:(0.01~0.02).

[0033] Furthermore, the compound Colletochin can be replaced with its pharmaceutically acceptable salts or solvates. Both the salts and solvates of Colletochin retain the core nucleus structure and bioactive sites of the compound, and therefore can be used as equivalent alternatives to Colletochin in the preparation of anti-glioma drugs.

[0034] Furthermore, the anti-glioma method involves arresting the glioma cell cycle.

[0035] Furthermore, the cell cycle arrest of glioma cells refers to arresting glioma cells in the S phase.

[0036] Furthermore, the anti-glioma method involves inhibiting the proliferation of glioma cells.

[0037] Furthermore, the anti-glioma method involves inhibiting the migration of glioma cells.

[0038] Furthermore, the anti-glioma treatment involves inhibiting the invasion of glioma cells.

[0039] Furthermore, the glioma cells include one or more of U251, T98G, and U87-MG.

[0040] Preferably, the glioma cells include U251 and / or U87-MG.

[0041] Furthermore, the drug also includes pharmaceutically acceptable carriers or excipients.

[0042] Furthermore, the dosage form of the drug is an oral dosage form, an injection, or a microneedle.

[0043] Compared with the prior art, the present invention has the following beneficial effects: This invention provides the application of the compound Colletochin in the preparation of anti-glioma drugs. Experiments have demonstrated that Colletochin exhibits significant anti-glioma activity, inhibiting the proliferation, migration, and invasion of glioma cells by arresting their cell cycle. Furthermore, Colletochin shows broad application prospects in the development of anti-glioma drugs due to its wide availability, simple extraction process, short preparation cycle, and low cost. Attached Figure Description

[0044] Figure 1The graph shows the statistical results of the inhibition rate of different concentrations (3.125~50 μM) of the compound Colletochin on the proliferation of different cell lines (U87-MG, U251, T98G) in Example 2. Among them, NC is the negative control group (0.5 % DMSO) and PC is the positive control group (10 μM paclitaxel treatment group). Compared with the drug-treated group, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0045] Figure 2 The following are statistical graphs showing the effects of different concentrations (1-5 μM) of the compounds Colletochin, 45 μM temozolomide, and 1 μM paclitaxel on the migration of human glioma cells in Example 3. Figures (a) and (b) are micrographs of cell lines U87-MG and U251, respectively; figures (c) and (d) are statistical graphs showing the migration rates of cell lines U87-MG and U251, respectively. NC is the negative control group (0.1% DMSO), PTX is the 1 μM paclitaxel treatment group, and TMZ is the 45 μM temozolomide treatment group. Compared with the drug-treated groups, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0046] Figure 3 The following are flow cytometry diagrams showing the effects of different concentrations (1-5 μM) of the compounds Colletochin, 1 μM paclitaxel, and 450 μM temozolomide on the cell cycle of human glioma cells in Example 4. Among them, (a) is a statistical diagram of the cell cycle data of cell line U87-MG, and (b) is a statistical diagram of the cell cycle data of cell line U251. NC is the negative control group (0.5% DMSO), PTX is the 1 μM paclitaxel treatment group, and TMZ is the 450 μM temozolomide treatment group.

[0047] Figure 4 The following are flow cytometry percentage statistics of the effects of different concentrations (1~5 μM) of the compounds Colletochin, 1 μM paclitaxel, and 450 μM temozolomide on the cell cycle of human glioma cells in Example 4; wherein, (a) is the percentage statistics of the cell cycle of cell line U87-MG, and (b) is the percentage statistics of the cell cycle of cell line U251; NC is the negative control group (0.5% DMSO), PTX is the 1 μM paclitaxel treatment group, and TMZ is the 450 μM temozolomide treatment group. Detailed Implementation

[0048] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0049] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0050] Figure 2 (a) indicates Figure 2 Figure (a) in the middle, Figure 2 (b) indicates Figure 2 The order of the other figures follows the same pattern as in figure (b).

[0051] Example 1: Preparation and characterization of compound Colletochin 1. Preparation of the compound Colletochin S1. Fungi derived from sea cucumbers and preserved at the Guangdong Provincial Center for Microbial Culture Collection. Tolypocladium sp. SYSU-F00117 (preservation date: August 28, 2025, preservation number: GDMCC NO: 66833) was inoculated into 20 L of liquid potato medium sterilized at 120 ℃ for 20 min and fermented at room temperature for 7 days to obtain fungal fermentation product; S2. The fungal fermentation product obtained in step S1 is separated using filter cloth, and the bacterial broth and bacterial cells are processed separately. The separated bacterial broth is extracted with an ethyl acetate-water system (volume ratio 1:1), the ethyl acetate layer is collected and concentrated under reduced pressure to obtain a bacterial broth extract. The filtered bacterial cells are extracted four times with methanol, and the extract is concentrated and evaporated to obtain a crude extract. The crude extract is extracted with an ethyl acetate-water system (volume ratio 1:1), the ethyl acetate layer is collected and concentrated under reduced pressure to obtain a bacterial cell extract. The bacterial broth extract and bacterial cell extract are combined and subjected to column chromatography, using a gradient concentration of petroleum ether-ethyl acetate mixture as the mobile phase (volume ratios of 4:1, 7:3, and 1.5:1, respectively). The eluent fraction with a petroleum ether-ethyl acetate volume ratio of 1.5:1 is collected. This eluent fraction is subjected to Sephadex analysis. LH-20 gel column chromatography was performed using a methanol-dichloromethane mixture (1:1 v / v) as the mobile phase. Based on the gel molecular sieve effect, the larger molecular weight fraction was eluted first, followed by the smaller molecular weight fraction. Five fractions were collected sequentially. Each fraction was compared with Colletochin standard by TLC, and the second collected fraction was identified as the crude fraction containing the target product Colletochin. Finally, the crude fraction was subjected to normal-phase silica gel column chromatography, using a gradient concentration of dichloromethane-methanol mixture as the mobile phase (1:0.01 v / v, 1:0.02 v / v). The eluent fraction with a dichloromethane:methanol v / v ratio of 1:0.02 was collected, dried, and yielded approximately 1 gram of the target compound Colletochin.

[0052] 2. Characterization of the compound Colletochin The compound Colletochin obtained in step 1 was analyzed by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The analysis confirmed that Colletochin had been successfully isolated from the fungus. The specific physicochemical properties of this compound are as follows: 1H NMR spectroscopy data: 1H NMR (600 MHz, CDCl3) δH 5.28 (m, 1H, H-13), 4.86 (m, 1H, H-18b), 4.5 (m, 1H, H-18a), 3.5 (m, 1H, H-3), 3.24 (s, 3H, H-6'), 2.65 (dd, J = 12.8, 3.6 Hz, 1H, H-19b), 2.63 (m, 1H, H-7b), 2.37 (m, 1H, H-19a), 2.34 (m, 1H, H-9), 2.17 (m, 1H, H-7a, 12), 1.99 (m, 1H, H-1b), 1.85 (s,4H, H-5, 7'), 1.72 (s, 3H, H-16), 1.67(s, 3H, H-15), 1.64 (m, 2H, H-2), 1.61(m, 1H, H-6b), 1.47 (s, 3H, H-8'), 1.44 (m, 2H, H-1a, 6a), 1.41 (m, 2H, H-11), 0.95 (s, 3H, H-20), 0.87 (s, 3H, H-17).

[0053] Carbon NMR spectroscopy data: 13 C NMR (600 MHz, CDCl3,) δC 182.4, 165,1 157.8,149.8, 130.5, 126.0, 119.3, 110.3, 104.2, 73.6, 55.6, 55.4, 41.3, 39.4, 38.1,38.0, 33.9, 28.4, 25.9, 23.4, 23.4, 21.4, 20.27, 18.2, 17.6, 17.0, 10.1.

[0054] Mass spectrometry data: HR-ESIMS m / z 442.3083, [M+Na] + (calcd. for C 14 H 22 O4Na, 465.6400).

[0055] Example 2: Test of the inhibitory effect of compound Colletochin on the proliferation of glioma cells. 1. Experimental Methods 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, Colletochin 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 Colletochin 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 150 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 Colletochin was calculated using the following formula: Inhibition rate = (OD) 实验组 -OD 阴性对照组 ) / OD 阴性对照组 ×100%.

[0056] 2. Experimental Results from Figure 1 It was observed that when human glioma cells were treated with different concentrations of the compound Colletochin, Colletochin showed good cell proliferation inhibitory activity against human glioma cells U87-MG, U251, and T98G at 48 h and 72 h of treatment, and the activity was concentration-dependent.

[0057] Experiment Example 3: Testing the migration ability of the compound Colletochin on glioma cells. 1. Experimental Methods First, healthy U251 and U87-MG glioma cells were digested with trypsin and then seeded into 6-well plates at a density of 5 × 10⁶ cells per well. 5 Cells were cultured at 37 °C, 5% CO2, and 95% relative humidity after seeding. When the cell density reached approximately 90%, scratches were created vertically from top to bottom on the cell layer using a 200 μL pipette tip in a clean bench. The scratched cells were then gently rinsed with PBS buffer to ensure the clarity of the scratches. Immediately after scratch creation, the initial scratch distance was photographed and recorded as a baseline for subsequent analysis. Next, the old culture medium was removed, and fresh culture medium containing different concentrations of the compound Colletochin (5 μM, 2 μM, and 1 μM) was added to each well, and the cells were incubated for 36 h. For comparison, a negative control group was set up, which received only 0.5% DMSO (the same solvent used to dissolve Colletochin), and a positive control group was set up, which received the common antitumor drug paclitaxel (1 μM) and the anti-glioma drug temozolomide (45 μM), respectively. After treatment with the compound Colletochin for 36 hours, cell migration in each well was observed using an inverted microscope, and monolayer cell images were captured. The effects of different concentrations of Colletochin on the migration ability of glioma cells were analyzed by measuring changes in the migration distance of the cell leading edge and the width of the scratch.

[0058] 2. Experimental Results from Figure 2 It was found that the compound Colletochin significantly inhibited the migration of human glioma cells, and this inhibitory effect showed a clear concentration-dependent effect. The inhibitory effect on glioma cell migration increased with increasing Colletochin concentration.

[0059] Example 4: Test of the effect of compound Colletochin on the cell cycle of human glioma. 1. Experimental Methods Healthy U87-MG and U251 glioma cells were first digested with trypsin and then seeded into 6-well plates at a density of 5 × 10⁶ cells per well. 5 These cells were cultured at 37 °C, 5% CO2 concentration, and 95% relative humidity. The next step was performed when the cell density reached approximately 90%.

[0060] First, the old culture medium was removed, and fresh culture medium containing different concentrations of Colletochin (1 μM, 2 μM, 5 μM) was added to each well, with an incubation period of 48 hours. To set up control groups, some wells were filled with culture medium containing 0.5% DMSO as a negative control (NC), and another group of wells were filled with culture medium containing paclitaxel (PTX) (1 μM) and temozolomide (TMZ) (450 μM) as a positive control. After 48 hours, the original culture medium was aspirated from the wells, and the cells were stored in centrifuge tubes for later use. Next, the cells in each group were washed with PBS solution, and then digested with an appropriate amount of trypsin. After digestion, the original culture medium was added to stop the digestion, and the cells were carefully separated from the bottom of the culture plate using a pipette. 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, the supernatant in the centrifuge tube was carefully aspirated, and then 1.5 mL of pre-chilled PBS was added to resuspend the cells. Centrifuge again at 1000 rpm for 5 minutes to remove cell debris from PBS and cell suspension.

[0061] Next, cell fixation was performed. Cells were resuspended in 0.5 mL of pre-chilled PBS, and 1.5 mL of pre-chilled anhydrous ethanol was slowly added dropwise to the cell suspension. The cells were fixed overnight at -20 °C. The fixed cell suspension was centrifuged at 1000 r / min for 5 min, excess ethanol was carefully removed, and the cells were washed with PBS and centrifuged again at 1000 r / min for 5 min. The supernatant was carefully removed. Next, cell cycle analysis was performed. 0.5 mL of staining buffer, 25 μL of propidium iodide staining solution (20X), and 10 μL of RNase A (50X) were added to each sample tube. The cell pellet was slowly and thoroughly resuspended and incubated at 37 °C in the dark for 30 minutes. Red fluorescence was detected at an excitation wavelength of 488 nm using flow cytometry. Analysis was performed using Flowjo software, and the percentage of cells in each cell cycle was calculated.

[0062] 2. Experimental Results from Figure 3As can be seen, in both glioma cell lines, with increasing concentrations of the compound Colletochin, flow cytometry analysis showed a continuous increase in its proportion in the S phase of the cell cycle (the yellow portion in the fitted graph), significantly higher than the control group. It is important to clarify that the S phase is a critical period for DNA replication in the cell cycle. The increased proportion in this phase directly indicates that Colletochin can arrest the cell cycle in glioma cells at the S phase. S-phase arrest further activates the apoptosis pathway, preventing tumor cells from completing DNA replication and subsequent division, thus hindering proliferation. This result suggests that the in vitro toxicity of Colletochin to glioma cells is mainly achieved by inducing S-phase arrest, thus affecting cell cycle progression and ultimately inhibiting glioma cell proliferation and migration. Furthermore, from... Figure 4 The results of the flow cytometry analysis described above can be observed more intuitively in the statistical graph. Therefore, the compound Colletochin has the potential to be used as an anti-glioma drug.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of compound Colletochin in the preparation of anti-glioma drugs, characterized in that, The structure of the compound Colletochin is shown below: 。 2. The application according to claim 1, characterized in that, The compound Colletochin is replaced by its pharmaceutically acceptable salt or solvate.

3. The application according to claim 1 or 2, characterized in that, The anti-glioma method involves arresting the cell cycle of glioma cells.

4. The application according to claim 3, characterized in that, The cell cycle arrest in glioma refers to arresting glioma cells in the S phase.

5. The application according to claim 1 or 2, characterized in that, The anti-glioma treatment involves inhibiting the proliferation of glioma cells.

6. The application according to claim 1 or 2, characterized in that, The anti-glioma treatment involves inhibiting the migration of glioma cells.

7. The application according to claim 1 or 2, characterized in that, The anti-glioma treatment involves inhibiting the invasion of glioma cells.

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

9. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable carriers or excipients.

10. The application according to claim 9, characterized in that, The drug is available in oral, injectable, or microneedle form.

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