Use of raffinose and derivatives thereof in the treatment of glioblastoma

Abstract

The present application relates to the use of raffinose or its derivatives in the preparation of a medicament for treating glioma disease, and the specific mechanism is that raffinose or its derivatives mediate cell cycle G1 / G2 arrest, and then induce glioma cell senescence, promote cell apoptosis, and ultimately achieve the effect of treating glioma.

Description

Technical Field

[0001] This invention relates to the field of pharmaceuticals, and more specifically to the use of raffinose or its derivatives in the treatment of glioma. Background Technology

[0002] Gliomas are the most common primary malignant tumors of the brain, accounting for approximately 80% of all intracranial tumors, with a global incidence rate of about 7 per 100,000. Among them, glioblastoma (GBM; WHO grade IV) is the most malignant, accounting for 60% to 70% of all glioma cases. In my country, the annual incidence rate of GBM is approximately 3 to 3.5 per 100,000, characterized by high morbidity and high mortality. Although global research on GBM has made some progress in the past few decades, due to the complex pathogenesis and aggressive nature of GBM, there are still no effective and safe treatment options for GBM patients, resulting in a very poor prognosis, with a median survival of only about 15 months.

[0003] Currently, treatment for GBM mainly includes comprehensive therapies such as surgery, radiotherapy, and chemotherapy. However, 5-year clinical data from a European multicenter cancer combination therapy trial (EORTC–NCIC trial) shows that even with such comprehensive treatment, the 2-year overall survival for GBM patients is only 27.2%, and the 5-year overall survival is as low as 9.8%. Temozolomide, as a first-line drug for glioma treatment, has evidence that it can treat some MGMTs by inducing cellular senescence in GBM. 6 For patients with positive MGMT promoter methylation, this only improved the 2-year overall survival to 48.9% and the 5-year overall survival to 13.8%, a negligible improvement. Furthermore, the efficacy of temozolomide in patients with negative MGMT promoter methylation remains disappointing. In addition, the blood-brain barrier (BBB), a natural barrier of the central nervous system, possesses a strong "defense" against large molecules, making it difficult for many highly effective anti-tumor drugs to be fully utilized in the treatment of central nervous system tumors. Therefore, finding novel, safe, effective, and efficient anti-tumor drugs that can cross the blood-brain barrier is of great value in the treatment of GBM patients.

[0004] Cellular senescence refers to the transition of cells from an active growth state to an irreversible state of growth arrest. Studies have shown that cellular senescence is closely related to the occurrence, development, and treatment of tumors. Tumor cells often exhibit senescence disorders, displaying unlimited proliferative capacity, leading to malignant cell proliferation. Disruption of cell cycle regulation mechanisms is a major cause of senescence disorders, and the G1 / S phase of the cell cycle may be a key regulatory point for senescence. Research indicates that when intracellular DNA is damaged by ultraviolet radiation, chemicals, etc., cells remain in the G1 phase and do not enter the S phase until DNA repair is achieved. If DNA replication is incomplete in the S phase or spindle formation is poor in the G2 phase, cells cannot enter the M phase, resulting in cell proliferation arrest. P21, as a major regulator of the G1 / S phase, can mediate cell cycle arrest and participate in the occurrence and development of cellular senescence. Some researchers have pointed out that P21 can inhibit the phosphorylation of RB and transcription factors (E2F) by inhibiting cellular CDK activity, thereby inducing cell growth arrest and participating in the process of cellular senescence. Furthermore, P21 can also induce cellular senescence by binding to the cell proliferation antigen (PCNA) in the cell nucleus, thereby blocking DNA replication. Therefore, in addition to inhibiting tumor formation through programmed cell death, we can also inhibit tumor development by preventing cell division through senescence.

[0005] Natural products play a crucial role in the discovery, design, and synthesis of new drugs. They are also an important source of bioactive substances and innovative medicines. Approved drugs such as paclitaxel, docetaxel, vinorelbine, camptothecin, and artemisinin are all derivatives or analogues of natural products. The development of natural products as drug sources has a unique function, potentially allowing for the screening of highly effective and low-toxicity lead compounds, thereby developing novel drugs to treat diseases, alleviate patient suffering, and improve their quality of life.

[0006] Raffinose is an indigestible short-chain oligosaccharide composed of galactose, glucose, and fructose, and can be found in many plants. Raffinose (Melitose) can be hydrolyzed by α-galactosidase (α-GAL) into D-galactose and sucrose. Current research on raffinose and its pentahydrate, raffinose pentahydrate (D(+)-Raffinosepentahydrate, RP), mainly focuses on non-tumor-related areas. Its therapeutic effects and underlying mechanisms in tumor-related diseases, particularly gliomas, especially GBM, are rarely studied. Summary of the Invention

[0007] In response to the above problems, the inventors unexpectedly discovered that raffinose and its derivatives can mediate cell cycle G1 / G2 arrest, thereby inducing glioma cell senescence, promoting cell apoptosis, and ultimately achieving the effect of treating glioma disease.

[0008] On one hand, the present invention relates to the use of raffinose or its derivatives in the preparation of medicaments for treating glioma disease.

[0009] In another preferred embodiment, the present invention relates to the use of raffinose or a derivative thereof in the preparation of a medicament for treating glioma, wherein the structure of raffinose or a derivative thereof is shown in formula (I) or formula (II). Wherein, the compound represented by formula (I) is raffinose, and the compound represented by formula (II) is raffinose pentahydrate (D(+)-raffinosepentahydrate, RP).

[0010]

[0011] In another preferred embodiment, the present invention relates to the use of raffinose or its derivatives in the preparation of a medicament for treating glioma, wherein the glioma is classified as grade I, II, III, or IV glioma according to the WHO classification of tumors of the central nervous system. According to the WHO classification of tumors of the central nervous system, gliomas are graded as follows: Grade I, mainly pilocytic astrocytoma, accounting for approximately 5% of gliomas, benign and curable; Grade II, mainly astrocytoma and oligodendroglioma, accounting for approximately 25% of gliomas, low-grade malignancy; Grade III, mainly anaplastic astrocytoma, accounting for approximately 15%-25% of gliomas, mostly evolving from Grade II, moderately malignant; Grade IV specifically refers to glioblastoma, highly malignant, accounting for about one-third of gliomas. Grades I and II are malignant tumors with a tendency towards benignity, Grade III is a purely malignant tumor, and Grade IV is the most malignant tumor. Clinically, drugs that can be used to treat glioblastoma can also be used to treat the other three grades of glioma.

[0012] In another preferred embodiment, the present invention relates to the use of raffinose or a derivative thereof in the preparation of a medicament for treating glioma, wherein the raffinose or a derivative thereof is prepared as a pharmaceutical composition comprising a therapeutically effective amount of raffinose or a derivative thereof and a pharmaceutically acceptable carrier.

[0013] In another preferred embodiment, the present invention relates to the use of raffinose or its derivatives in the preparation of a medicament for treating glioma, wherein the raffinose or its derivatives, when used as a medicament, can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1%-99%, preferably 0.5%-90%, 1%-80%, or 5%-50% raffinose or its derivatives, with the remainder being a pharmaceutically acceptable, non-toxic, and inert pharmaceutically viable carrier for humans and animals.

[0014] The pharmaceutical compositions of the present invention can be prepared into capsules, tablets, powders, granules, syrups, or similar dosage forms for oral administration, or into injections, powder for injection, ointments, suppositories, or similar dosage forms for non-gastrointestinal administration. These pharmaceutical formulations can be generated by conventional methods using excipients well known in the art, such as binders, excipients, stabilizers, disintegrants, flavoring agents, lubricants, etc., and can also be prepared into controlled-release dosage forms, sustained-release dosage forms, and various particulate delivery systems.

[0015] Although the dosage varies with symptoms and the patient's age, the nature and severity of the disease or disorder, and the route and manner of administration, for oral administration to adult patients, the normal dosage of raffinose or its derivatives is 1 to 1000 mg per day, preferably 5 to 500 mg, more preferably 50 to 150 mg, which may be administered as a single dose or in divided doses, for example once, twice or three times daily; for intravenous administration, 0.1 to 100 mg per day, preferably 0.5 to 50 mg, may be divided into one to three doses per day.

[0016] In another preferred embodiment, the present invention relates to the use of raffinose or a derivative thereof in the preparation of a medicament for treating glioma disease, said use comprising administering a therapeutically effective amount of the raffinose or a derivative thereof of the present invention to a subject in need of treatment. The term "subject" includes humans and non-human mammals, such as non-human primates, sheep, dogs, cats, cattle, and horses, with human patients being the preferred subject.

[0017] Raffinose is a trisaccharide composed of galactose, fructose, and glucose, widely found in various natural crops such as legumes, cabbage, broccoli, and ginger. Furthermore, as a natural compound, raffinose has advantages such as low toxicity, easy availability, and the ability to cross the blood-brain barrier. Evidence suggests that raffinose has good medicinal value for various human diseases, including anti-allergic, anti-obesity, anti-diabetic, and non-alcoholic lipid accumulation prevention, as well as improving gut microbiota and inhibiting Pseudomonas aeruginosa biofilm formation. The chemical structural formula of raffinose is:

[0018]

[0019] The inventors discovered that raffinose also plays an important role in immunotherapy and cancer treatment. Evidence shows that raffinose can promote IL-12 expression in antigen-presenting cells and increase CD4+ expression. + T cells secrete IL-2 and upregulate INF-γ, playing a vital role in the body's immune system's defense and tumor suppression functions. Meanwhile, IL-12 expression has a positive promoting effect on the treatment of diseases such as colorectal cancer, lymphoma, melanoma, and glioma. Furthermore, raffinose can induce autophagic cell death in HaCaT cells through an mTOR-independent pathway, ultimately inhibiting cell proliferation. With the continuous advancement of medical chemistry technology, raffinose pentahydrate (D(+)-Raffinose pentahydrate, RP), as the pentahydrate of raffinose, exhibits better drug stability, can be stored for a long time, and maintains a high drug activity potency (its structural formula is shown in II), giving it a broader application prospect.

[0020]

[0021] Through systematic experimental design, the inventors unexpectedly discovered that raffinose or its pentahydrate compound RP significantly inhibits the in vitro proliferation of GBM cells, effectively suppressing tumor cell proliferation. RP can induce apoptosis in GBM cells in vitro. The inventors treated three GBM cell lines (A172, U251, and U87) with 175 μM RP and found that as the treatment time increased, RP significantly induced the expression of apoptosis-related proteins BAX and Cleaved-CASPASE9, while downregulating the expression of the anti-apoptosis-related protein BCL2. Tumor cell apoptosis significantly increased, with a marked increase in the proportion of early and late apoptotic cells in the overall cell count. The inventors also collected six human primary GBM tissue samples, isolated and cultured tumor tissues in vitro, and treated them with RP for 0-36 hours. The results showed that with prolonged drug treatment time, RP significantly reduced the cell viability of human primary GBM samples.

[0022] Animal experiments showed that RP drugs could inhibit the growth of glioblastoma in situ in the brain of nude mice, without significant organ damage caused by drug toxicity. This indicates that RP drugs can also inhibit the tumor proliferation of GBM cells in vivo, with low toxicity to other organs and tissues.

[0023] Further research revealed that RP drugs may further inhibit GBM cell activity by regulating the P21 / Cell cycle signaling pathway. The inventors treated three GBM cell lines (A172, U251, and U87) with 175 μM RP and performed high-throughput RNA sequencing analysis 48 hours after treatment. Venn diagram cross-sectional analysis of genes showing significant differential changes in the three GBM cell lines due to RP treatment revealed 1664 genes with differential expression before and after RP intervention. KEGG analysis of these 1664 genes identified 26 key genes involved in the regulation of the Cell Cycle signaling pathway (with the most significant changes in P21). This indicates that RP intervention had the most significant regulatory effect on the Cell Cycle signaling pathway in tumor cells, with a significant upregulation of P21 gene expression in all three GBM cell lines.

[0024] The inventors used siRNA intervention technology to downregulate the expression of P21 in three GBM cell lines, and confirmed the results using Western blotting experiments. The results showed that downregulating P21 expression inhibited GBM cell proliferation and partially reversed the effects of RP. This strongly demonstrates that RP induces cell cycle arrest in tumor cells by significantly promoting P21 protein expression and inhibiting the phosphorylation of cyclin RB.

[0025] Based on the above experimental results, this invention is the first to discover that raffinose or its derivatives induce glioblastoma senescence and promote glioblastoma apoptosis by mediating G1 / G2 cell cycle arrest, ultimately inhibiting glioblastoma. This discovery is unprecedented in the prior art, suggesting the potential use of raffinose or its derivatives in the preparation of drugs for treating glioblastoma. This has significant medical value in increasing alternative treatment options for GBM patients and improving their survival prognosis. Clinically, raffinose or its derivatives can be used to treat grade IV glioblastoma, and correspondingly, it can also be applied to treat grade I, II, or III gliomas with slightly lower malignancy.

[0026] This invention provides the use of raffinose or its derivatives in the preparation of a medicament for treating glioma. The technical solution of this invention is summarized below:

[0027] 1. Use of raffinose or its derivatives in the preparation of drugs for the treatment of glioma.

[0028] 2. According to the use described in technical solution 1, the raffinose or its derivative is selected from raffinose or raffinose pentahydrate (D(+)-Raffinose pentahydrate, RP), and its structural formula is shown below:

[0029]

[0030] 3. According to the use described in technical solution 1, the glioma is classified as grade I, grade II, grade III or grade IV glioma according to the WHO classification of central nervous system tumors.

[0031] 4. According to the use described in technical solution 3, the glioma is classified as a grade I pilocytic astrocytoma according to the WHO classification of central nervous system tumors.

[0032] 5. According to the use described in technical solution 3, the glioma is classified as grade II astrocytoma and oligodendroglioma according to the WHO classification of central nervous system tumors.

[0033] 6. According to the use described in technical solution 3, the glioma is classified as a grade III anaplastic astrocytoma according to the WHO classification of central nervous system tumors.

[0034] 7. The use according to technical solution 3 is characterized in that: the glioma is classified as grade IV glioblastoma according to the WHO classification of central nervous system tumors.

[0035] 8. The use according to any one of technical solutions 1-7, characterized in that: the raffinose or its derivative thereof is prepared into a pharmaceutical composition, the pharmaceutical composition comprising a therapeutically effective amount of raffinose or its derivative thereof and a pharmaceutically acceptable carrier.

[0036] 9. The use according to any one of technical solutions 1-7, characterized in that: when the raffinose or its derivative is used as a drug, it can be used directly or in the form of a pharmaceutical composition.

[0037] 10. The use according to technical solution 8, characterized in that: the pharmaceutical composition contains 0.1%-99% raffinose or its derivatives, the remainder being a pharmaceutically acceptable, non-toxic and inert pharmaceutical carrier for humans and animals.

[0038] 11. The use according to technical solution 10, characterized in that: the pharmaceutical composition contains 0.5%-90%, 1%-80% or 5%-50% raffinose or its derivatives, the remainder being a pharmaceutically acceptable, non-toxic and inert pharmaceutical carrier for humans and animals.

[0039] 12. According to the use described in technical solution 8, the pharmaceutical composition is characterized in that: it is prepared into capsules, tablets, powders, granules, syrups or similar dosage forms for oral administration, or prepared into injections, powder injections, ointments, suppositories or similar dosage forms for non-gastrointestinal administration, or prepared into controlled-release dosage forms, sustained-release dosage forms, and various microparticle delivery systems.

[0040] 13. The use according to any one of technical solutions 1-7, characterized in that: the raffinose or its derivative is administered in a total daily dose of 1 to 1000 mg, in the form of a single dose or in divided doses.

[0041] 14. The use according to technical solution 13 is characterized in that: the raffinose or its derivative is administered in a total daily dose of 5 to 500 mg, in the form of a single dose or in divided doses.

[0042] 15. The use according to technical solution 13 is characterized in that: the raffinose or its derivative is administered in a total daily dose of 50-150 mg, either as a single dose or in divided doses.

[0043] 16. The use according to any one of technical solutions 1-7, characterized in that: the raffinose or its derivative is administered by intravenous injection, in doses of 0.1 to 100 mg once or three times a day.

[0044] 17. The use according to technical solution 16 is characterized in that: the raffinose or its derivative is administered by intravenous injection, in doses of 0.5 to 50 mg divided into one to three daily doses.

[0045] 18. The use according to any one of technical solutions 1-7, characterized in that: the use includes administering a therapeutically effective amount of raffinose or its derivative to a subject in need of treatment.

[0046] 19. The use according to technical solution 18, characterized in that: the subject includes humans and non-human mammals.

[0047] 20. The use according to technical solution 19 is characterized in that: the subject is a non-human mammal, selected from non-human primates, sheep, dogs, cats, cattle and horses. Attached Figure Description

[0048] Appendix Figure 1 RP drugs significantly inhibited the in vitro proliferation of GBM cells. Among them:

[0049] (A) The inventors treated three GBM cell lines (A172, U251, and U87) with different concentrations of RP and performed CCK8 assays on cell viability 48 hours after intervention. The results showed that the toxic effect of RP on GBM cell lines gradually increased with increasing drug concentration, and the inhibitory effect on GBM cells changed in a concentration-dependent manner. Among them, the U251 cell line showed the most significant inhibitory effect.

[0050] (B) The inventors applied RP drug to three GBM cell lines (A172, U251, U87), and the drug concentrations at which the cell viability inhibition rate reached 50% were (A172, left, 174.1 μM; U251, middle, 165.5 μM; U87, right, 186.3 μM). The inventors will use this concentration in subsequent experiments.

[0051] (C) The inventors treated three GBM cell lines (A172, U251, and U87) with RP at a concentration of 175 μM and detected cell viability using the CCK8 assay at different time points. The results showed that the inhibitory effect of RP on the viability of GBM cell lines was time-dependent as the duration of drug treatment increased.

[0052] (D) The inventors treated three GBM cell lines (A172, U251, and U87) with different concentrations of RP and examined the cell monoclonal results 48 hours after treatment. The results showed that RP significantly inhibited the proliferation of GBM cell lines and effectively suppressed tumor cell proliferation. **P<0.01, ***P<0.001.

[0053] Appendix Figure 2 RP drugs can induce apoptosis in GBM cells in vitro. Among them:

[0054] (A) The inventors treated three GBM cell lines (A172, U251, and U87) with RP at a concentration of 175 μM and performed flow cytometry to detect cell apoptosis after 24-72 hours. The results showed that as the duration of RP treatment gradually increased, tumor cell apoptosis significantly increased, with a marked increase in the proportion of early and late apoptotic cells in the total cell count.

[0055] (B) The inventors treated three GBM cell lines (A172, U251, and U87) with 175 μM RP and analyzed the expression of apoptosis-related proteins using Western blotting experiments after 0-72 hours. The results showed that RP significantly induced the expression of apoptosis-related proteins BAX and Cleaved-CASPASE9 over time. Simultaneously, it downregulated the expression of the anti-apoptotic protein BCL2.

[0056] Appendix Figure 3 RP drugs can also inhibit the tumor proliferation of GBM cells in vivo, and have low toxicity to other organs and tissues. Among them:

[0057] (A) After in situ intracranial tumor formation in nude mice, the mice were treated with intraperitoneal injection of RP drug (25 mg / kg, every other day, for 5 times). In vivo imaging of the tumor tissue was performed on the mice after the drug injection. The results showed that RP drug could inhibit the growth of in situ glioblastoma in the nude mouse brain.

[0058] (B) After dissecting the tumor-bearing nude mice, HE staining experiments were performed on different tissues and organs. The results showed that no obvious organ damage caused by drug toxicity was observed in the heart, liver, spleen, lungs, kidneys, brain, stomach and intestines of the four groups of nude mice: negative control group (NC), negative control RP treatment group (NC+RP), tumor-bearing group without RP treatment (Tumor), and tumor-bearing group treated with RP (Tumor+RP).

[0059] Appendix Figure 4 RP drugs can inhibit the in vitro activity of human primary GBM cells.

[0060] (A) The inventors collected 6 human primary GBM tissue samples, isolated and cultured the tumor tissue samples in vitro, and simultaneously intervened with RP drug for 0-36 hours. The results showed that with the extension of drug treatment time, RP drug could significantly reduce the cell viability of human primary GBM samples. ***P<0.001.

[0061] Appendix Figure 5 RP drugs may further inhibit GBM cell activity by regulating the P21 / Cell cycle signaling pathway. Specifically:

[0062] (A) The inventors treated three GBM cell lines (A172, U251, and U87) with RP at a concentration of 175 μM and performed high-throughput RNA sequencing analysis 48 hours after drug treatment. The inventors analyzed genes showing significant differences (more than 2-fold fold) between the three cell lines treated with RP and the negative control group (cells not treated with RP), and used a volcano plot to statistically analyze these genes. Red represents genes upregulated after drug treatment, and green represents genes downregulated after drug treatment.

[0063] (B) The inventors performed Venn diagram cross-sectional analysis on genes that showed significant differential changes in three GBM cell lines due to RP drug intervention, attempting to explore key regulatory pathways with common functions in the three GBM cell lines. The results showed that a total of 1664 genes showed differential expression before and after RP intervention. KEGG analysis of these 1664 genes revealed that 26 key genes were involved in the regulation of the Cell Cycle signaling pathway, indicating that RP intervention had the most significant regulatory effect on the Cell Cycle signaling pathway in tumor cells.

[0064] (C)Detailed information on 26 genes that showed significant differences before and after RP intervention in the Cell Cycle signaling pathway.

[0065] (D) KEGG diagram of 26 differentially expressed genes in the Cell Cycle signaling pathway. Purple text indicates the sites of action of differentially expressed genes.

[0066] (EF) The inventors performed validation analysis on the cellular RNA and protein levels of 26 differentially expressed genes after RP drug intervention, with P21 showing the most significant changes. In the qPCR results (E), it was observed that RP drug intervention resulted in a significant upregulation of P21 gene expression in all three GBM cell lines. Similarly, in the Western blot experiment (F), it was also observed that RP intervention significantly upregulated P21 protein expression in all three GBM cell lines.

[0067] Appendix Figure 6 Downregulation of P21 expression can inhibit the proliferation of GBM cells and partially reverse the effects of RP.

[0068] (A) The inventors used siRNA intervention technology to downregulate the expression of P21 in three GBM cell lines, and confirmed the results through Western blotting. The results showed that, compared to the siControl (negative control) group, RP significantly promoted P21 protein expression, inhibited the phosphorylation of cyclin RB, and induced cell cycle arrest in tumor cells. Meanwhile, siP21 effectively downregulated P21 expression in GBM cell lines, partially reversing the RP-induced P21 expression-promoting effect, while simultaneously promoting RB phosphorylation, inducing cells to enter the cell cycle, escaping cell cycle arrest, and preventing cell senescence.

[0069] (B) The inventors used siRNA intervention technology to downregulate the expression of P21 in three GBM cell lines, partially reversing the RP drug's effect on promoting P21 expression. Cell viability after intervention was assessed using CCK-8 assays. The results showed that downregulating P21 expression partially reversed the inhibitory effect of RP on GBM cells and partially promoted the recovery of cell viability. Detailed Implementation

[0070] Example 1: Preparation of Raffinose

[0071] The defatted cottonseed meal was subjected to seven countercurrent extractions with 70% methanol at pH 8 to obtain a dephenolized solution. 5 L of this solution was passed through a nanofiltration membrane with a molecular weight cutoff of 800 Da to obtain the permeate. The permeate was then loaded onto a solid-phase extraction column using a 200 L column packed with 150 mL of neutral alumina packing material. The loading volume was 2 BV, and the flow rate was 1.0 BV / h. After column chromatography, the solution was eluted with 2 BV of 95% methanol, followed by elution with 1.5 BV of deionized water to obtain the eluent. 0.2% activated carbon was added to the eluent, the pH was adjusted to 5-6, and the solution was heated to 50°C and stirred for 30 min. The mixture was then filtered to obtain a decolorized solution. 45% ethanol was added to the decolorized solution, and the temperature was lowered to 10°C for crystallization for 14 h. The crystals were filtered to obtain raffinose crystals, which were then washed with a small amount of 95% ethanol and dried at 80°C to obtain raffinose.

[0072] Example 2: Preparation of raffinose pentahydrate

[0073] The defatted cottonseed meal was subjected to seven countercurrent extractions with 70% methanol at pH 8 to obtain a dephenolized solution. 5 L of this solution was passed through a nanofiltration membrane with a molecular weight cutoff of 800 Da to obtain the permeate. The permeate was then loaded onto a solid-phase extraction column using a 200 L column packed with 150 mL of neutral alumina packing material. The loading volume was 2 BV, and the flow rate was 1.0 BV / h. After column chromatography, the solution was eluted with 2 BV of 95% methanol, followed by elution with 1.5 BV of deionized water to obtain the eluent. 0.2% activated carbon was added to the eluent, the pH was adjusted to 5-6, and the solution was heated to 50°C and stirred for 30 min. After filtration, a decolorized solution was obtained. 45% ethanol was added to the decolorized solution, and the temperature was lowered to 10°C for crystallization for 14 h. The resulting raffinose pentahydrate crystals were obtained by filtration. The RP drug used in this experiment was recrystallized and dissolved in dimethyl sulfoxide (DMSO) to form a 10 mM stock solution, which was then aliquoted and stored at -80°C for later use.

[0074] Example 3: Culture of GBM cells.

[0075] Three GBM cell lines (A172, U251, U87) were cultured in complete Dulbecco's modified Eagle medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS) and 100 U / ml penicillin / streptomycin. The cells were then cultured in a humidified incubator at 37°C and 5% (v / v) CO2. After the cells entered the logarithmic growth phase, they were collected for subsequent experiments.

[0076] Example 4: CCK8 Experiment

[0077] Concentration-dependent: Cells in the logarithmic growth phase were used, and the concentration was adjusted to 10. 4100 μl of cells / cell were added to centrifuge tubes. Treatment was performed with RP at seven different concentrations (0 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM). Cells were then seeded into 96-well plates, 100 μl per well. The plates were incubated at 37°C with 5% (v / v) CO2 for 48 hours. After reaching the target incubation time, 90 ml of fresh DMEM complete medium and 10 ml of CCK8 solution were added, and the plates were incubated at 37°C with 5% (v / v) CO2 for another 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader. The relative cell proliferation toxicity was calculated using the formula, and a curve was plotted. Figure 1 A: As drug concentration increased, the toxic effect of RP on GBM cell lines gradually increased, and its inhibitory effect on GBM cells changed in a concentration-dependent manner. The inhibitory effect on the U251 cell line was the most significant.

[0078] Time-dependent: Cells in the logarithmic growth phase were used, and the concentration was adjusted to 10. 4 Cells were added to centrifuge tubes at a rate of 100 μl / cell. Cells were divided into an RP group (175 mM concentration) and a DMSO control group (same volume as RP). Both groups of cells were seeded into 96-well plates, with 100 μl added to each well. Cells were incubated at 37°C with 5% (v / v) CO2 for 0, 12, 24, 36, 48, 60, and 72 hours. After reaching the target incubation time, 90 ml of fresh DMEM complete medium and 10 ml of CCK8 solution were added, and the mixture was shaken well before incubating for another 2 hours at 37°C with 5% (v / v) CO2. The absorbance of each well was measured at 450 nm using a microplate reader, and the relative cell proliferation toxicity was calculated using the formula, and a curve was plotted. Figure 1 C: As the duration of drug action increases, the inhibitory effect of RP on the activity of GBM cell line has a time-dependent effect; Figure 6 B: Downregulation of P21 expression can partially reverse the inhibitory effect of RP on GBM cells and partially promote the recovery of cell activity.

[0079] Example 5: Cloning Experiment

[0080] Cells in the logarithmic growth phase were harvested and their concentration adjusted to 500 cells / 2 ml using DMEM complete medium. 2 ml of the culture was seeded into each well of a 6-well plate. The plates were incubated at 37°C with 5% (v / v) CO2 for 10-14 days. Once cells clustered, 175 mM RP or DMSO (the same volume as RP) was added as a control. After mixing, the plates were incubated at 37°C with 5% (v / v) CO2 for another 48 hours. Cells were then fixed with 4% paraformaldehyde for 10-15 minutes and stained with crystal violet for 15-30 minutes. After washing off the crystal violet, the plates were allowed to dry at room temperature before microscopic examination and photographing. Cell colonies were counted. Figure 1 D:RP significantly inhibits the proliferation of GBM cell lines. It can effectively suppress the expansion of tumor cells.

[0081] Example 6: Flow cytometry detection of cell apoptosis.

[0082] Cells in the logarithmic growth phase were placed in 6-well plates, with 2 ml of cell suspension added to each well. After mixing, the plates were incubated at 37°C in a humidified incubator with 5% (v / v) CO2 for 24 hours. RP (175 mM) or DMSO (same volume as RP) was added to the 6-well plates as controls, and the plates were incubated at 37°C in a humidified incubator with 5% (v / v) CO2 for 0, 24, 48, and 72 hours. After reaching the target time, the culture was terminated, and cells resuspended in 1× Binding Buffer were obtained. The cell concentration was then adjusted to 10-1. 6 Cells / ml. Transfer 100 μl of the cell suspension to a 5 ml flow cytometry tube. Incubate with 5 μl Annexin V-FITC and 5 μl PI staining solution at room temperature in the dark for 15 min. After incubation, add 400 μl of 1× Binding Buffer to each flow cytometry tube, gently shake, and perform flow cytometry analysis within 1 hour. Figure 2 A: As the duration of RP drug intervention in cells gradually increased, the apoptosis of tumor cells significantly increased, with a marked increase in the proportion of early and late apoptotic cells in the total cell count.

[0083] Example 7: Western blot assay to detect the expression of related proteins.

[0084] Cell lysates were extracted using cell lysis buffer, and the protein concentration in the lysates was quantified using an enhanced BCA protein assay kit. Approximately 30-50 μg of protein was loaded per well, and pre-stained protein molecular weight standards were used to determine molecular weight. Electrophoresis was performed at 80V on ice for 30 min. Once the sample reached the separating gel, the voltage was increased to 120V for 60 min, until bromophenol blue reached the bottom of the gel. Electrophoresis was then stopped, and the separating gel was cut off. A piece of PVDF membrane (pre-soaked in methanol for 15 s until translucent) and two sheets of filter paper were cut according to the gel size. These were immersed in transfer buffer to create a sandwich structure arranged as (-) pole / sponge pad / filter paper / gel / PVDF membrane / filter paper / sponge pad (+). This structure was placed in the transfer tank and electrophoresed at a constant current of 170 mA for 120 min. After transfer, the PVDF membrane was immersed in blocking buffer (TBST containing 5% skim milk) and incubated at room temperature for 2 hours. It was then washed three times with TBST for 10 minutes each time. Next, primary antibody diluted with 5% BSA at an appropriate ratio was added and incubated overnight at 4°C. The next day, the antibody was recovered, and the PVDF membrane was washed three times with TBST for 10 minutes each time. Secondary antibody was added, and the membrane was incubated at room temperature for 2 hours. It was then washed three times with TBST for 10 minutes each time before proceeding with the ECL colorimetric reaction. Figure 2 B: Over time, RP drugs significantly induced the expression of apoptosis-related proteins BAX and Cleaved-CASPASE9. Simultaneously, they downregulated the expression of the anti-apoptosis-related protein BCL2. Figure 5 After F:RP intervention, the protein expression of P21 was significantly upregulated in the three GBM cell lines; Figure 6 A: RP can significantly promote the expression of p21 protein, inhibit the phosphorylation of cyclin RB, and induce cell cycle arrest in tumor cells.

[0085] The antibody reagent is used as follows:

[0086]

[0087]

[0088] Example 8: In situ glioma modeling in nude mice.

[0089] U87 cells transfected with luciferase-GFP in the logarithmic growth phase were digested and centrifuged (1000 rpm, 5 min, room temperature). The cells were resuspended in a 1:1 mixture of Matrigel and PBS, counted, and the cell concentration was adjusted to 2 × 10⁶ cells / mL. 8 / ml. Prepare 6 6-week-old female BALB / c(nu / nu) athymic nude mice, anesthetize and fix their heads. Position the needle at the anterior fontanelle, move it 2cm backward and 2cm to the right, and drill a hole in the skull surface through the skull using a drilling tool to create an injection hole. Aspirate 5μl of cell suspension with the sterilized needle, insert it 3.5cm into the brain tissue, then withdraw it 0.5cm, and slowly inject a solution containing 10... 6 The cell suspension was injected over a period of 5 minutes. After injection, the cell suspension was allowed to stand for 5 minutes before the injection needle was slowly withdrawn. The wound was then disinfected and sealed. One week after the cell suspension was injected, the tumor site was visualized and the constructed human glioma orthotopic model was evaluated. Figure 3 A: RP drugs can inhibit the growth of glioblastoma in situ in the brain of nude mice.

[0090] Example 9: RP administration method and mouse tissue sample collection

[0091] After the evaluation of the human glioma orthotopic model, mice were intraperitoneally injected with RP solution at a dose of 25 mg / kg, every other day for 5 consecutive times. Control group mice were injected with the same dose of corn oil. After the 5 administrations, the tumor site was re-imagined, and the inhibitory effect of RP on GBM in vivo was evaluated.

[0092] After completing the experiment, the tissue perfusion procedure was as follows: Mice were anesthetized and their chests were opened to ensure adequate exposure for cardiac puncture and incision of the right atrial appendage. A perfusion needle was inserted into the apex of the heart, and the right atrial appendage was opened to allow venous blood flow. Approximately 100 ml of physiological saline was initially infused. Once the mice's forelimbs and lungs turned white, 4% paraformaldehyde was then infused. Successful perfusion was indicated by violent twitching of the forelimbs and stiffness of the forelimbs and neck at the beginning of perfusion.

[0093] Brain tissue harvesting: After perfusion, cut open the skin to expose the head and neck, and cut the spinal cord at the cervical vertebrae; separate and remove the muscles at the back of the neck; carefully remove the skull with curved forceps, taking care of the dura mater when separating the skull to avoid scratching the brain tissue; when harvesting the brain, start from the medulla oblongata and slowly separate the skull base tissue to reduce damage to the brain.

[0094] Heart and lung tissue harvesting: After perfusion, the chest skin was cut open to expose the sternum and ribs. The ribs were cut to expose the lung tissue, and the trachea of ​​the nude mouse was ligated. The trachea and blood vessels around the heart were severed distal to the ligation site, and the entire lung, heart, and ligated trachea were removed together.

[0095] Finally, liver, kidney, stomach, and intestinal tissues were extracted. The extracted tissues were then incubated overnight at 4°C in 4% paraformaldehyde, followed by dehydration in 30% sucrose solution until the tissues completely sank to the bottom, and then frozen at -80°C. 30 μm thick sections were prepared and stored at -80°C for later use.

[0096] Example 10 HE staining

[0097] Tissue sections were dewaxed four times in xylene, 5 minutes each time. After dewaxing, the sections were washed three times with anhydrous ethanol for 2 minutes each time; then dehydrated with 95%, 85%, and 75% ethanol sequentially for 2 minutes each time, followed by washing with water. After washing, the tissue sections were stained with hematoxylin staining solution for 15 minutes, followed by washing, differentiation, and washing with water. The staining depth was observed under a microscope to ensure it was appropriate. The sections were then rinsed with tap water for 5-10 minutes for bluing. After bluing, the tissue sections were stained with eosin staining solution for 20-60 seconds, followed by washing with 75%, 95%, and 95% ethanol sequentially for 2 minutes each time, and then washed three times with anhydrous ethanol for 2 minutes each time. After staining, the tissue sections were cleared four times with xylene or an environmentally friendly clearing agent, immersing for 2 minutes each time. Finally, the tissue sections were mounted with neutral resin and observed and photographed under a microscope. Figure 3 B: RP has no significant toxic side effects on organs such as the heart, liver, spleen, lungs, kidneys, brain, stomach, and intestines.

[0098] Example 11: Collection of tumor samples from GBM patients and acquisition of primary human GBM cells.

[0099] Tumor sample collection: This study was approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (Permit No. 2022-623). With the written informed consent of all patients, 6 GBM patients (Grade IV) who underwent surgery at the First Affiliated Hospital of Wenzhou Medical University between 2019 and 2021 were included in this retrospective study.

[0100] Tumor Sample Collection: With the approval of the ethics committee and after obtaining written informed consent from all patients, six GBM patients (level IV) were included in this retrospective study. Excised GBM tumor tissue was placed in 5 ml of physiological saline and incubated in a humidified incubator at 37°C with 5% (v / v) CO2. Human primary GBM cells were immediately prepared for extraction.

[0101] Human primary GBM cell extraction: Tumor tissue was placed in a culture dish containing 5 ml of PBS and rinsed 2-3 times with PBS to remove blood vessels and impurities from the tumor surface. After rinsing, the tissue was placed in a culture dish containing 5 ml of DMEM complete medium and cut into 2-3 mm pieces. 3Small tissue blocks were centrifuged (1000 rpm, 5 min, room temperature). Each small tissue block was placed in a culture dish containing 3-4 ml of trypsin solution and digested for 10 min. Once the trypsin solution became slightly turbid, the tissue blocks were removed and placed in a culture dish containing DMEM complete medium, and then ground on a filter. The resulting cell suspension was lysed with erythrocyte lysis buffer to remove red blood cells, yielding primary glioma cells. Finally, the primary cells were placed in 5 ml of DMEM medium and cultured in a humidified incubator at 37°C with 5% (v / v) CO2. Figure 4 A: With prolonged drug action, RP significantly reduced cell viability in primary human GBM samples.

[0102] Example 12 RNA sequencing.

[0103] 1 μg of target mRNA was collected and isolated using oligo(dT) beads according to the polyA selection method. First, fragmentation was performed using fragmentation buffer. Then, double-stranded cDNA was synthesized using the SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) and random hexamer primers (Illumina). The synthesized cDNA was then end-repaired, phosphorylated, and had an "A" base added according to Illumina's library construction protocol. The library size was selected by selecting a 300 bp cDNA target fragment on 2% low-range superagarose gel, followed by 15 cycles of PCR amplification using Phusion DNA polymerase (NEB). After quantification using a TBS380, the paired-end RNA-seq library was sequenced using an Illumina NovaSeq 6000 sequencer (2 × 150 bp read length). Figure 5 Following AC:RP intervention, the drug showed the most significant effect on the cell cycle signaling pathway in tumor cells.

[0104] Example 13 Cell RNA extraction and real-time quantitative PCR.

[0105] RNA extraction: Transfer logarithmically growing cells to an EP tube. Add 1 ml of Trizol reagent to the cells and allow them to lyse fully. Add 200 μl of chloroform, vortex to mix, and incubate at room temperature for 5 min. Centrifuge (10000 rpm, 15 min, 4℃). After centrifugation, carefully pipette the uppermost aqueous phase (approximately 500 μl) into a new 1.5 ml EP tube. Add 500 μl of isopropanol, vortex to mix, and incubate at room temperature for 10 min. Centrifuge (10000 rpm, 15 min, 4℃), and collect the white precipitate at the bottom of the EP tube. Wash the RNA with 1 ml of anhydrous ethanol, resuspend, and centrifuge (10000 rpm, 15 min, 4℃). Collect the white precipitate again and dry at room temperature for 5-10 min. Then, 20 μl of DEPC water was added to dissolve the RNA. After the RNA was dissolved, the RNA concentration and purity were measured. The extracted RNA sample was stored at -80°C for use in the next step of the experiment.

[0106] Real-time quantitative PCR: 1 μg RNA was transferred to an enzyme-free EP tube, and DNA digestion mixture was added. The tube was incubated at room temperature for 30 min. 1 μl stop solution was added to terminate digestion, and the tube was incubated at 65°C for 10 min. Then, 1 μl random primer (500 μg / ml) was added, and the tube was incubated at 70°C for 5 min. After the reaction was complete, the tube was removed and immediately placed on ice for 5 min. After the ice bath, reverse transcription mixture was added for reverse transcription, and the tube was incubated at room temperature for 1 hour. The cDNA obtained from the reverse transcription was used for quantitative PCR using a SYBR Premix Ex Taq (Perfect Real-Time) quantitative PCR kit. Finally, the reaction was performed according to procedure 2. -ΔΔCt The method calculates the relative expression level of the target gene. Figure 5 Following E:RP intervention, P21 gene expression was significantly upregulated in all three GBM cell lines.

[0107] The RNA primers are as follows:

[0108] P21 (positive): 5'-AGGTGGACCTGGAGACTCTCAG-3';

[0109] P21 (reverse): 5'-TCCTCTTGGAGAAGATCAGCCG-3'.

[0110] GAPDH (forward): 5'-CGAAATCCCATCACCATCTTCCAGG-3';

[0111] GAPDH (reverse): 5'-GAGCCCCAGCCTTCTCCATG-3'.

[0112] The DNA digestion mixture is shown below:

[0113]

[0114] The reverse transcription mixture is shown below:

[0115]

[0116] The quantitative system is as follows:

[0117]

[0118] Amplification procedure:

[0119]

[0120] 2 -ΔΔCt The calculation method is as follows:

[0121]

[0122] Example 14: siRNA transfection of cells.

[0123] To prepare the transfection complex, use 120 μl of 1×riboFECT. TM Dilute 5 μl siRNA (20 μM) with CP Buffer and store, then add 12 μl riboFECT. TM CP Reagent was incubated at room temperature for 15 min to prepare a transfection complex. Cells in the logarithmic growth phase were divided into two groups (siP21 group and negative control group). After cell counting, cells were seeded into 6-well plates, with 2 ml of cell suspension added to each well. After mixing, the plates were incubated at 37°C and 5% (v / v) CO2 for 24 hours. Then, 1863 μl of DMEM complete medium and the transfection complex were added to each well. After mixing, the plates were incubated at 37°C and 5% (v / v) CO2 for another 24-48 hours. Proteins were extracted from each group, and Western blotting was used to detect the downregulation level of the target gene to assess the transfection effect. Figure 6 ).

Claims

1. Use of raffinose pentahydrate (D(+)-Raffinose pentahydrate, RP) in the preparation of a medicament for treating glioma, wherein the structural formula of said raffinose pentahydrate is shown below: (II)。 2. The use according to claim 1, characterized in that: The glioma mentioned is glioblastoma.

3. The use according to any one of claims 1-2, characterized in that: The raffinose pentahydrate is prepared as a pharmaceutical composition comprising a therapeutically effective amount of raffinose pentahydrate and a pharmaceutically acceptable carrier.

4. The use according to any one of claims 1-2, characterized in that: The raffinose pentahydrate is used directly as a medicine or in the form of a pharmaceutical composition.

5. The use according to claim 3, characterized in that: The pharmaceutical composition described herein contains 0.1%-99% raffinose pentahydrate, with the remainder being a pharmaceutically acceptable, non-toxic, and inert pharmaceutical carrier for humans and animals.

6. The use according to claim 5, characterized in that: The pharmaceutical composition described herein contains 0.5%-90% raffinose pentahydrate, with the remainder being a pharmaceutically acceptable, non-toxic, and inert pharmaceutical carrier for humans and animals.

7. The use according to claim 5, characterized in that: The pharmaceutical composition described herein contains 1%-80% raffinose pentahydrate, with the remainder being a pharmaceutically acceptable, non-toxic, and inert pharmaceutical carrier for humans and animals.

8. The use according to claim 5, characterized in that: The pharmaceutical composition described herein contains 5%-50% raffinose pentahydrate, with the remainder being a pharmaceutically acceptable, non-toxic, and inert pharmaceutical carrier for humans and animals.

9. The use according to claim 3, characterized in that: The pharmaceutical composition is prepared into capsules, tablets, or syrups for oral administration, or into injectable dosage forms for non-gastrointestinal administration, or into controlled-release dosage forms, sustained-release dosage forms, or microparticle delivery systems.

10. The use according to any one of claims 1-2, characterized in that: The raffinose pentahydrate is administered at a total daily dose of 1 to 1000 mg, either as a single dose or in multiple doses.

11. The use according to claim 10, characterized in that: The raffinose pentahydrate is administered at a total daily dose of 5 to 500 mg, either as a single dose or in divided doses.

12. The use according to claim 10, characterized in that: The raffinose pentahydrate is administered at a total daily dose of 50-150 mg, either as a single dose or in divided doses.

13. The use according to any one of claims 1-2, characterized in that: The aforementioned raffinose pentahydrate is administered intravenously, in doses of 0.1 to 100 mg, divided into one to three daily doses.

14. The use according to claim 13, characterized in that: The aforementioned raffinose pentahydrate is administered intravenously, in doses of 0.5 to 50 mg, divided into one to three daily doses.

15. The use according to any one of claims 1-2, characterized in that: The intended use includes administering a therapeutically effective amount of raffinose pentahydrate to a subject requiring treatment.

16. The use according to claim 15, characterized in that: The subjects included humans and non-human mammals.

17. The use according to claim 16, characterized in that: The subjects were non-human mammals, selected from non-human primates, sheep, dogs, cats, cattle, and horses.

Images