Application of prodigiosin in preparation of medicine for treating brain glioma

Abstract

The invention provides application of prodigiosin in preparation of a medicine for treating brain glioma, and belongs to the technical field of biological medicine. The invention provides application of prodigiosin in treatment of brain glioma, and prodigiosin and temozolomide can synergistically inhibit proliferation capacity of brain glioma cells, induce apoptosis of tumor cells, reduce healing and migration capacity of cell wounds, reduce formation of cell adhesion spots and block autophagy tumors of tumor cells, so that the prodigiosin and temozolomide can be used for treating brain glioma. The treatment effect on brain glioma is remarkably improved, the dosage of temozolomide is reduced, and the pharmaceutical composition has important significance on improvement of the lifetime and the life quality of a patient.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a styraxin in the preparation of a drug for treating glioma. Background Technology

[0002] Malignant gliomas (WHO grade 3-4) are highly heterogeneous and aggressive, difficult to completely remove surgically, prone to recurrence, have a poor prognosis, and an average survival of no more than 2 years.

[0003] In clinical practice, surgical treatment combined with radiotherapy and chemotherapy is the core treatment regimen for gliomas. Temozolomide, an alkylating agent, is currently recognized internationally as a first-line standard chemotherapy drug and plays a crucial role in comprehensive glioma treatment. However, the molecular heterogeneity of tumors and complex drug resistance mechanisms significantly limit its efficacy: these include epigenetic dysregulation mediated by aberrant methylation of long non-coding RNA promoter regions, and overactivation of the epidermal growth factor receptor signaling pathway. The methylation status of methylguanine-DNA methyltransferase repair proteins, along with the altered biological behavior associated with 1p / 19q chromosomal co-deletion, and other molecular and cellular mechanisms, collectively contribute to the development of primary or acquired drug resistance in approximately 50% of patients during treatment, manifesting as inadequate response to temozolomide or disease progression. Therefore, the current field of glioma treatment urgently needs to overcome the limitations of traditional chemotherapy and find drugs that can synergistically enhance the efficacy of temozolomide, increasing its effectiveness or reducing its dosage, providing new strategies and hope for prolonging patient survival and improving quality of life.

[0004] Prodigiosin (PG) is a secondary metabolite produced by microorganisms. In recent years, it has attracted scientific attention due to its various potential beneficial properties, such as antifungal, antibacterial, antiprotozoal, antimalarial, UV-protective anticancer, and immunosuppressive effects. Studies have shown that prodigiosin and its derivatives are effective pro-apoptotic factors that can act on various cancer cell lines, with many cellular targets showing little or no toxicity to normal cell lines. Therefore, some of its derivatives have been tested in clinical trials as candidate drugs for cancer treatment. However, research on the efficacy of prodigiosin alone or in combination with other drugs in the treatment of glioma is scarce. Summary of the Invention

[0005] The purpose of this invention is to provide an application of levofloxacin in the preparation of a drug for treating glioma. It has been found that levofloxacin can be used directly to treat glioma and, when used in combination with temozolomide, can exert a synergistic effect to enhance the anti-glioma activity of temozolomide and reduce the dosage of temozolomide.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of strychnine in the preparation of a drug for treating glioma.

[0007] Preferably, the erythromycin is extracted from Serratia marcescens.

[0008] The present invention also provides a pharmaceutical composition for treating glioma, comprising styraxin and temozolomide as used in the present invention.

[0009] Preferably, pharmaceutically acceptable carriers and excipients are also included.

[0010] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a drug for treating glioma.

[0011] Preferably, the concentration of the styraxin is 0.125~2 μg / mL; and the concentration of the temozolomide is 200~1200 μM.

[0012] Beneficial effects

[0013] The drug for treating glioma provided by this invention contains the natural product strychnine, extracted from Serratia marcescens. As a widely distributed natural compound, it has high biocompatibility and low toxicity. It can be used directly to treat glioma, and when used in combination with temozolomide, it significantly improves the treatment effect and reduces the dosage, which is of great significance for improving patient survival and quality of life. Attached Figure Description

[0014] Figure 1 The diagram shows the results of PG and TMZ inhibiting glioma cell growth in Example 1. Figure 2 This is a diagram showing the results of PG synergistically inhibiting glioma cell growth in Example 1; Figure 3 This is a diagram showing the results of PG synergistically inhibiting glioma cell proliferation in Example 1; Figure 4 This is a graph showing the detection results of PG synergistic TMZ-induced apoptosis in glioma cells in Example 2; Figure 5 This is a diagram showing the results of PG synergistically enhancing TMZ's inhibition of glioma cell wound healing and migration in Example 3; Figure 6 This is a diagram showing the results of PG synergistically enhancing TMZ to reduce glioma cell adhesion spots in Example 4; Figure 7 This is a graph showing the effect of the synergistic effect of PG and TMZ on LC3II and P62 expression in Example 5; Figure 8This is a diagram showing the results of PG and TMZ synergistically blocking autophagic flux to inhibit glioma cell growth in Example 5. Detailed Implementation

[0015] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] Example 1: The synergistic effect of styracil-enhanced temozolomide on inhibiting glioma cell growth.

[0017] 1. Cells and drugs

[0018] Both T98G and A172 are human gliomas, both cultured in DMEM + 10% FBS medium.

[0019] Temozolomide (TMZ) was purchased from MCE and prepared as a 100 mM stock solution using DMSO. Prodigiosin (PG) was prepared in this experiment (preparation method is described in patent 202011319766.8) and prepared as a 10 mg / ml stock solution using DMSO.

[0020] 2. Experimental Procedure

[0021] 2.1 Detection of the inhibitory effects of styraxin (PG) and temozolomide (TMZ) on glioma cell growth

[0022] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 96-well plates with 5000 cells / 100 μL DMEM + 10% serum medium per well. The plates were then incubated at 37°C and 5% C. Incubate overnight in a cell culture incubator.

[0023] (2) Preparation of PG gradient concentration solutions: Add 2 μL of the stock solution to 1 mL of DMEM serum-free medium and then perform serial dilution. Add 10 μL of PG with different concentrations to each well of a 96-well plate to make the working concentration gradients 2, 1, 0.5, 0.25 and 0.125 μg / ml. Incubate at 37℃ in a 5% CO2 cell culture incubator for 48 h.

[0024] Preparation of TMZ gradient concentration solutions: Add the stock solution to DMEM serum-free medium according to the required volume. Then, add 10 μL of TMZ containing different concentrations to each well of a 96-well plate to create working concentration gradients of 1200, 1000, 800, 600, 400, and 200 μM. Incubate at 37°C in a 5% CO2 cell culture incubator for 48 h. Add an equal volume of DMEM serum-free medium containing DMSO to the control wells.

[0025] (3) CCK8 detection: After 48 h of culture, discard the culture supernatant. Add 10 μL of CCK-8 solution to DMEM + 10% serum medium per well, mix well, and then add 100 μL of CCK8-containing medium to each well. Blank wells are cell-free but contain an equal amount of CCK8. Incubate at 37℃, 5% C Incubate in a cell culture incubator for 1 hour. Measure the absorbance of each well at 450 nm using a microplate reader.

[0026] (4) Data Analysis: After subtracting the absorbance value of the blank well from the absorbance value of each well, the cell survival rate under different drug gradients was calculated with the survival rate of the untreated group being 100%. The results are as follows: Figure 1 As shown.

[0027] Depend on Figure 1 It can be seen that in T98G and A172 cells, cell survival rate decreases with increasing PG concentration. Similarly, cell survival rate also decreases with increasing TMZ concentration.

[0028] 2. Detection of PG synergistic TMZ inhibition of glioma cell growth

[0029] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 96-well plates with 5000 cells / 100 μL DMEM + 10% serum medium per well. The plates were then incubated at 37°C and 5% C. Incubate overnight in a cell culture incubator.

[0030] (2) Preparation of PG gradient concentration solution: Add 2 μL of the stock solution to 1 mL of DMEM serum-free medium and then dilute it. Add 10 μL of medium containing PG to each well of the 96-well plate to make the working concentration gradient 0.5 μg / ml.

[0031] Preparation of TMZ gradient concentration solutions: Add the stock solution to DMEM serum-free medium according to the required volume. Then, add 10 μL of TMZ containing different concentrations to each well of a 96-well plate to create working concentration gradients of 1000, 800, 600, and 400 μM. Incubate at 37°C in a 5% CO2 cell culture incubator for 48 h. Add an equal volume of DMEM serum-free medium containing DMSO to the control wells.

[0032] (3) CCK8 detection: After 48 h of culture, discard the culture supernatant. Add 10 μL of CCK-8 solution to DMEM + 10% serum medium per well, mix well, and then add 100 μL of CCK8-containing medium to each well. Blank wells are cell-free but contain an equal amount of CCK8. Incubate at 37℃, 5% C Incubate in a cell culture incubator for 1 hour. Measure the absorbance of each well at 450 nm using a microplate reader.

[0033] (4) Data Analysis: After subtracting the absorbance value of the blank well from the absorbance value of each well, the cell survival rate under different drug gradients was calculated with the survival rate of the untreated group being 100%. The results are as follows: Figure 2 As shown.

[0034] Depend on Figure 2 The results show that in T98G and A172 cells, the combined use of PG and TMZ (TP group) effectively inhibited tumor cell growth, showing a significant difference compared to treatment with PG or TMZ alone. These results indicate that PG can synergistically inhibit glioma cell growth with TMZ.

[0035] 2.3 Detection of PG synergistic TMZ inhibition of glioma cell proliferation

[0036] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 96-well plates with 40,000 cells / 500 μL DMEM + 10% serum medium per well. The plates were then incubated at 37°C and 5% C. Incubate overnight in a cell culture incubator.

[0037] (2) The preparation of PG and TMZ was the same as above: the working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. After adding PG and TMZ to the cells, they were incubated at 37°C in a 5% CO2 cell culture incubator for 48 h.

[0038] (3) EdU detection: After culturing for 48 hours, EdU working solution was added and cultured for another 2 hours. Cells were then fixed with 4% paraformaldehyde and finally stained with DAPI staining solution.

[0039] (4) Data Analysis: The number of DAPI-positive cells and EdU-positive cells were counted separately in the same field of view. The EdU-positive cell rate was calculated by dividing the number of EdU-positive cells by the number of DAPI-positive cells. The results are as follows: Figure 3 As shown.

[0040] Depend on Figure 3 The results show that in T98G and A172 cells, the combined use of PG and TMZ (TP group) effectively inhibited tumor cell proliferation, showing a significant difference compared to treatment with PG or TMZ alone. These results indicate that PG can synergistically inhibit glioma cell proliferation with TMZ.

[0041] Example 2: Synergistic effect of styracil-based erythromycin on enhancing temozolomide-induced apoptosis in glioma cells.

[0042] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 7 × 10⁶ cells per well in a 12-well plate. 4 1.5 × 10⁶ cells / 1 mL DMEM + 10% serum medium, or seed 1.5 × 10⁶ cells / well in a 6-well plate. 5 10 cells / 2 mL LDM + 10% serum medium, or seed 8.5 × 10⁸ cells / 10 cm cell culture dish. 5 10 cells / 10mL DMEM + 10% serum medium, incubated at 37°C, 5% C Incubate overnight in a cell culture incubator.

[0043] (2) The preparation of PG and TMZ was the same as above: the working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. After adding PG and TMZ to the cells, they were placed at 37°C and 5% C. Incubate in a cell culture incubator for 48 hours.

[0044] (3) PI detection: After culturing for 48 h, PI staining solution was added to the culture medium of 6-well plate to a working concentration of 2 μg / mL, and the apoptosis of cells was observed under a fluorescence microscope; Apoptosis detection: After 48 h of culture, cells were collected from 12-well plates, and apoptosis detection reagents PI and 7-AAD were added. The cell apoptosis status was detected by flow cytometry. Western blot assay: After 48 h of culture, cells were collected from 10 cm cell dishes, subjected to SDS-PAGE electrophoresis and membrane transfer, and the cleavage activity of PARP and Caspase 3 was detected.

[0045] The results are as follows Figure 4 As shown: In the PI assay, compared with the Con group and the single-drug group, the TP combined with drug group showed a significantly higher number of apoptotic cells (see...). Figure 4 (A and 4B); The same effect was observed in flow cytometry, with the percentage of apoptotic and necrotic cells in the TP combined with drug group being significantly higher than the other three groups (see A and 4B). Figure 4 C and 4D); In Western blot analysis, it was found that the TP combined with the drug group could induce the cleavage of PARP and Caspase 3 and the activity was significantly higher than that of the other three groups (see C and 4D). Figure 4 E). The results showed that PG could synergistically induce apoptosis in glioma cells with TMZ.

[0046] Example 3: The synergistic effect of styracil-based erythromycin on enhancing temozolomide's inhibition of glioma cell wound healing and migration.

[0047] 1. The synergistic effect of lecithin on enhancing temozolomide's inhibition of glioma cell wound healing.

[0048] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 1.5 × 10⁶ cells per well in a 12-well plate. 5 Cells / 1 mL DMEM + 10% serum medium, incubated at 37°C, 5% C Incubate overnight in a cell culture incubator.

[0049] (2) Use the tip of a pipette to gently draw lines in the cell well plate, then remove the remaining cells with culture medium and take a picture under a fluorescence microscope.

[0050] (3) The preparation of PG and TMZ was the same as above: the working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. After adding PG and TMZ to the cells, they were placed at 37°C and 5% C. Incubate in a cell culture incubator for 48 hours.

[0051] (4) Photograph detection: After 24 hours of culture, photographs of the same treatment location were taken under a fluorescence microscope, and the cell wound healing rate was calculated using ImageJ software.

[0052] like Figure 5 A and Figure 5 As shown in B: In T98G and A172 cells, compared with the Con group and the TMZ single drug group, the cell wound healing rate in the TP combined drug group was significantly slower. Although there was no significant difference compared with the PG group, the cell wound healing rate was still slower than that in the PG group.

[0053] 2. The synergistic effect of lecithin on enhancing the inhibition of glioma cell migration by temozolomide.

[0054] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and 2×10⁶ cells were added to the transwell wells. 4 The upper chamber is filled with 200 μL of serum-free DMEM medium, and the lower chamber is filled with 600 μL of DMEM + 10% serum medium.

[0055] (2) The preparation of PG and TMZ was the same as above: the working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. After adding PG and TMZ to the cells, they were placed at 37°C and 5% C. Incubate in a cell culture incubator for 48 hours.

[0056] (3) Photo detection: After culturing for 24 hours, the migration of cells was planned under a microscope, and the cell migration rate was calculated using ImageJ software.

[0057] like Figure 5 As shown in Figure C: In A172 cells, the TP combined with PG significantly inhibited cell migration compared to the Con group and the single-drug group. In T98G cells, the TP combined with PG significantly inhibited cell migration compared to the Con group and the TMZ single-drug group. Although there was no significant difference between the TP group and the PG group, it was still evident that TP inhibited cell migration more effectively than PG. These results indicate that the combined use of TP can significantly inhibit wound healing and migration of glioma cells.

[0058] Example 4: The synergistic effect of styraxin on temozolomide to reduce glioma cell adhesion spots.

[0059] (1) Place the cell slides: Place the 24-well plate slides into the 24-well plate.

[0060] (2) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 7 × 10⁶ cells per well in a 24-well plate. 4 Cells / 0.5 mL DMEM + 10% serum medium, incubated at 37°C, 5% C Incubate overnight in a cell culture incubator.

[0061] (3) The preparation of PG and TMZ was the same as above: the working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. After adding PG and TMZ to the cells, they were placed at 37°C and 5% C. Incubate in a cell culture incubator for 48 hours.

[0062] (4) Sample processing: After 48 h of culture, cells were fixed with 4% paraformaldehyde for 15 min at room temperature, then permeabilized with PBS containing 0.1% Triton X-100 for 15 min at room temperature, and then blocked with 3% BSA at room temperature for 1 h. Cells were co-incubated overnight at 4 °C with rabbit anti-Paxillin antibody diluted 1:100 in PBST buffer containing 1% BSA. Pre-adsorbed Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) was used as the secondary antibody, diluted 1:500 in PBS buffer containing 5% BSA, and incubated for 1.5 h in the dark. Phalloidin (488) was used for staining, diluted 1:200 in PBS buffer, and incubated for 30 min in the dark. A secondary antibody-only control was set up, i.e., PBS was used instead of the primary antibody. Nuclear DNA was labeled with blue fluorescence using DAPI. Cell adhesion spots were examined under a confocal microscope.

[0063] The results are as follows Figure 6 As shown: In T98G ( Figure 6 A) and A172 ( Figure 6 B) In cells, comparing the Con group and the single-drug group, it can be clearly seen that the TP combined with the drug group can significantly reduce the number of cell adhesion plaques.

[0064] Example 5: The synergistic effect of styracil and temozolomide in blocking autophagic flux and inhibiting glioma cell growth.

[0065] 1. Observation under a fluorescence microscope: synergistic enhancement of LC3II expression by styracil and temozolomide.

[0066] (1) Place the cell slides: Place the 24-well plate slides into the 24-well plate.

[0067] (2) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 7 × 10⁶ cells per well in a 24-well plate. 4 Cells / 0.5 mL DMEM + 10% serum medium, incubated at 37°C, 5% C Incubate overnight in a cell culture incubator.

[0068] (3) The plasmid pLV3-GFP-LC3 was transduced into T98G and A172 cells using lipo3000.

[0069] (4) PG and TMZ were prepared as above: the working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. After adding PG and TMZ to the cells, they were placed at 37°C and 5% C. Incubate in a cell culture incubator for 48 h.

[0070] (5) Photo detection: After 48 hours of culture, the expression of GFP was observed under a fluorescence microscope.

[0071] The results are as follows Figure 7 A and Figure 7 As shown in Figure B, after autophagy induction, GFP-LC3 is converted to its phosphatidylethanolamine-coupled form (LC3-II) and recruited to the autophagosome membrane, forming clear green fluorescent spots. The results indicate that TP and PG can significantly promote the formation of green fluorescent spots, demonstrating that TP can activate autophagy.

[0072] 2. Western blot analysis of the effects of styraxin and temozolomide on LC3II and P62 expression.

[0073] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 8.5 × 10⁸ cells per 10 cm cell culture dish. 5 10 cells / 10mL DMEM + 10% serum medium, incubated at 37°C, 5% C Incubate overnight in a cell culture incubator.

[0074] (2) The preparation of PG and TMZ was the same as above: the working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. After adding PG and TMZ to the cells, they were placed at 37°C and 5% C. Incubate in a cell culture incubator for 48 hours.

[0075] (3) Western blot was used to detect the expression of LC3II and P62.

[0076] LC3-II is a marker of autophagosome membrane, and its increased content usually indicates an increased number of autophagosomes. However, the underlying cause of this increase requires p62 for "differential diagnosis": when autophagosomes carrying "cargo" fuse with lysosomes to form autolysosomes, the p62 within them, along with its bound "cargo," is completely degraded by lysosomal enzymes. Therefore, when autophagy is activated and autophagic flux is unimpeded, autophagosomes can promptly fuse with lysosomes and degrade their contents (including p62), resulting in reduced p62 expression. When autophagy is activated but downstream lysosomal function is impaired, p62 expression increases.

[0077] Western blot results showed that in T98G cells, the TP combined with PG significantly upregulated the expression of LC3II and P62 compared to the Con group and the TMZ monotherapy group. Although there was no significant difference between the TP group and the PG group, it was still evident that TP upregulated the expression of LC3II and P62 more strongly than PG (see [link to data]). Figure 7 C). In A172 cells, compared with the Con group and the single-drug group, the TP combined with the drug group significantly upregulated the expression of LC3II and p62 (see C). Figure 7 D). The above results indicate that the combined use of TP drugs blocks autophagic flux, which impairs autolysosomal degradation and leads to cell death.

[0078] 3. The autophagy inhibitor CQ enhances the synergistic effect of sclerotinum and temozolomide.

[0079] To further verify that the combined use of TP drugs blocks autophagic flux, thereby impairing autolysosomal degradation and leading to cell death, the experiment used the autophagy activator rapamycin (Rapa) and the autophagy inhibitor chloroquine (CQ) for verification.

[0080] (1) Cell seeding: After T98G and A172 cells reached the logarithmic growth phase, the cells were digested and seeded into 5 × 10⁶ cells per well in a 96-well plate. 3 Cells per 0.1 mL DMEM + 10% serum medium, incubated at 37°C, 5% C Incubate overnight in a cell culture incubator.

[0081] (2) PG and TMZ drug preparations were as above: The working concentration of PG was 0.5 μg / ml in T98G and A172 cells; the working concentration of TMZ was 800 μM in T98G cells; and the working concentration of TMZ was 600 μM in A172 cells. Rapamycin and CQ powders were dissolved in DMSO to prepare drug formulations with working concentrations of 20 μM and 30 μM, respectively. After adding the corresponding drugs to the cells, they were incubated at 37°C and 5% C. Incubate in a cell culture incubator for 48 hours.

[0082] (3) CCK8 detection: After 48 h of culture, discard the culture supernatant. Add 10 μL of CCK-8 solution to DMEM + 10% serum medium per well, mix well, and then add 100 μL of CCK8-containing medium to each well. Blank wells are cell-free but contain an equal amount of CCK8. Incubate at 37℃, 5% C Incubate in a cell culture incubator for 1 hour. Measure the absorbance of each well at 450 nm using a microplate reader.

[0083] like Figure 8 As shown, in T98G and A172 cells, the combined use of PG and TMZ effectively inhibited tumor cell growth, showing a significant difference compared to treatment with PG or TMZ alone. The addition of Rapamycin had no significant effect on the growth of cells in the TP combined group; however, the addition of CQ significantly reduced the growth of cells in the TP combined group. These results further indicate that the combined use of TP drugs blocks autophagic flux, impairing autolysosomal degradation and leading to cell death.

[0084] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a styracin in the preparation of a drug for the treatment of glioma.

2. The application as described in claim 1, characterized in that, The erythromycin is extracted from Serratia marcescens.

3. A pharmaceutical composition for treating glioma, characterized in that, It contains styraxin and temozolomide as used in the application of claim 1 or 2.

4. The pharmaceutical composition according to claim 3, characterized in that, It also includes pharmaceutically acceptable carriers and excipients.

5. Use of the pharmaceutical composition of claim 3 in the preparation of a medicament for treating glioma.

6. The application as described in claim 5, characterized in that, The concentration of the styraxin is 0.125~2 μg / mL; the concentration of the temozolomide is 200~1200 μM.

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