3,29-dibenzoyl-triterpene triol in the preparation of a medicine for preventing and treating liver cancer

Abstract

The application relates to application of 3,29-dibenzoyl-trilone in preparation of a medicine for preventing and treating liver cancer, and belongs to the technical field of biological medicine. In order to solve the problem of limited treatment effect of the existing liver cancer treatment medicine, the application provides application of 3,29-dibenzoyl-trilone in preparation of a medicine used for preventing and treating liver cancer, and the medicine used for preventing and treating liver cancer contains at least one of 3,29-dibenzoyl-trilone or a derivative thereof. Through in-vitro experiments and in-vivo experiments, it is found for the first time that 3,29-dibenzoyl-trilone has a strong inhibiting effect on liver cancer cells and liver tumors. 3,29-dibenzoyl-trilone is a kind of natural medicine, so the development and utilization prospect of 3,29-dibenzoyl-trilone for liver cancer treatment is good. The application provides a new application of 3,29-dibenzoyl-trilone, and provides a new medicine selection for liver cancer clinical treatment.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of 3,29-dibenzoyltriacyltriol in the preparation of drugs for the prevention and treatment of liver cancer. Background Technology

[0002] Primary liver cancer is a malignant tumor with a high mortality rate. Its incidence rate ranks fourth among malignant tumors, and it is a major malignant tumor of the digestive system.

[0003] Early-stage liver cancer can be treated with surgical resection, radiofrequency ablation, and anhydrous ethanol injection. However, over 80% of liver cancer patients are diagnosed at an advanced stage, resulting in very limited treatment options and a poor overall prognosis. Commonly used chemotherapy drugs for liver cancer include cisplatin, gemcitabine, and fluorouracil, but their efficacy is relatively low. Targeted therapies commonly used for liver cancer include sorafenib, lenvatinib, regorafenib, and cabozantinib, but their effectiveness needs improvement. Therefore, developing highly effective drugs for liver cancer treatment remains a pressing challenge.

[0004] 3,29-Dibenzoyl rarounitriol is a polychlorinated triterpenoid ester active compound extracted from Trichosanthes kirilowii seeds. Currently, there are no research reports on its anti-liver cancer effects. Summary of the Invention

[0005] To address the limited efficacy of existing liver cancer treatments, this invention provides the application of 3,29-dibenzoyltriacyltriol in the preparation of drugs for the prevention and treatment of liver cancer.

[0006] The technical solution of this invention:

[0007] Application of 3,29-dibenzoyltrichosanthesinol in the preparation of drugs for the prevention and treatment of liver cancer.

[0008] Furthermore, the drug for preventing and treating liver cancer contains at least one of 3,29-dibenzoyltriacyltriol or its derivatives.

[0009] Furthermore, the effective concentration of 3,29-dibenzoyltriacyltriol or its derivative in the drug for preventing and treating liver cancer is not less than 3 μM.

[0010] Furthermore, the drug for preventing and treating liver cancer also contains one or more pharmaceutically acceptable excipients.

[0011] Furthermore, the excipients are at least one of the following: sustained-release agent, filler, binder, humectant, disintegrant, absorption promoter, surfactant, antibacterial agent, fragrance, antioxidant, pH adjuster, protectant, diluent, lubricant, or solvent.

[0012] Furthermore, the drug for preventing and treating liver cancer is administered via injection.

[0013] Furthermore, when the drug for preventing and treating liver cancer is administered by injection, the dosage of 3,29-dibenzoyltriacyltriol or its derivative is not less than 10 mg / kg.

[0014] Furthermore, the dosage form of the drug for preventing and treating liver cancer is an injection.

[0015] Furthermore, the drug for preventing and treating liver cancer is administered orally.

[0016] Furthermore, the dosage form of the drug for preventing and treating liver cancer is capsule, pill, tablet, granule, tincture, inhalation preparation, oral solution, oral suspension or oral emulsion.

[0017] The beneficial effects of this invention are:

[0018] This invention, through in vitro experiments, discovered that 3,29-dibenzoyltriacyltriol has a strong inhibitory effect on liver cancer cells, inhibiting their proliferation, promoting apoptosis, and inhibiting migration. In vivo experiments confirmed that treatment with 3,29-dibenzoyltriacyltriacyltriol reduced the tumor burden of orthotopic liver tumors and significantly inhibited the growth of subcutaneous xenografts in nude mice.

[0019] 3,29-Dibenzoyltrichosanthesinol, as a natural drug with broad biological activity, has the advantages of few side effects, high safety, and easy clinical translation, providing a new drug option for the clinical treatment of liver cancer. Attached Figure Description

[0020] Figure 1 This is a comparison of cell viability of liver cancer cells in different groups during the CCK8 experiment in Example 1;

[0021] Figure 2 These are stained photographs of liver cancer cells in each group during the cell colony formation experiment in Example 1.

[0022] Figure 3 Scatter plot of liver cancer cells in each group during the flow cytometry apoptosis experiment in Example 1;

[0023] Figure 4 This is a comparison of the apoptosis rate of liver cancer cells in each group during the flow cytometry apoptosis experiment in Example 1.

[0024] Figure 5 This is a comparison image of the number of migrating HCCLM3 cells in each group during the Transwell experiment in Example 1.

[0025] Figure 6These are stained images of migrating cells and a comparison of the number of migrating cells in each group of MHCC97-H cells in the Transwell experiment of Example 1;

[0026] Figure 7 These are stained images of migrating SNU449 cells in each group and a comparison of the number of migrating cells in the Transwell experiment of Example 1.

[0027] Figure 8 These are in vivo images of mice from each group in Example 2;

[0028] Figure 9 This is a comparison of the fluorescence intensity of the livers of mice in each group on days 8, 17, 24, and 31 after drug administration in Example 2.

[0029] Figure 10 These are photographs of liver tumors in mice from each group 31 days after administration in Example 2.

[0030] Figure 11 This is a comparison chart of liver tumor weight, alanine aminotransferase activity, and L-lactate dehydrogenase activity in mice of different groups after 31 days of drug administration in Example 2.

[0031] Figure 12 These are photographs of the subcutaneous xenografts of HCCLM3 cells from each group of mice in Example 3.

[0032] Figure 13 This is a comparison of the volume and weight of subcutaneous HCCLM3 cell xenografts in each group of mice in Example 3. Detailed Implementation

[0033] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0034] Example 1

[0035] This embodiment demonstrates, through in vitro cell experiments, that 3,29-dibenzoyltrichosanthesinol has in vitro inhibitory activity against liver cancer cells.

[0036] 1. CCK8 Experiment

[0037] HCCLM3, MHCC97-H, and Huh7 hepatocellular carcinoma cells were cultured in DMEM medium containing 10% fetal bovine serum at a concentration of 3 × 10⁻⁶ mg / L. 3 Cells were seeded at a density of 100 cells / mL in 96-well plates and cultured for 24 hours. The cells were then divided into control and experimental groups. The experimental groups were cultured in DMEM medium with 3,29-dibenzoyltriol added to achieve final concentrations of 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM and 32 μM, respectively. The control group was cultured in DMEM medium with an equal amount of DMSO. Each group was divided into 6 replicates. After 48 hours of culture, cell viability was analyzed by CCK8 assay.

[0038] The results are as follows Figure 1 As shown, 3,29-dibenzoyltrichosanthesinol can inhibit the proliferation of liver cancer cells, and the half-maximal inhibitory concentration (IC50) of 3,29-dibenzoyltrichosanthesinol against HCCLM3 was determined. 50 The concentration was 10.63 μM, which is suitable for the IC of MHCC97-H. 50 The IC value for Huh7 is 5.042 μM. 50 The value is 3.331 μM.

[0039] 2. Cell clone formation experiment

[0040] MHCC97-H and HCCLM3 cells were digested with trypsin, resuspended in DMEM containing 10% fetal bovine serum, and counted to achieve a cell count of 4 × 10⁶ cells per well. 5 Cells were cultured in two batches. An equal volume of DMSO was added to the DMEM medium in the control group. 3,29-dibenzoyltriacylglycerol was added to the DMEM medium in the MHCC97-H experimental group to achieve a final concentration of 5 μM. 3,29-dibenzoyltriacylglycerol was added to the DMEM medium in the HCCLM3 experimental group to achieve a final concentration of 11 μM. Cells were cultured for two more days, with the medium being replaced with an equal volume of DMSO and the same concentration of the drug every three days. Several days later, cells were fixed with 4% paraformaldehyde, and 1 mL of 0.4% crystal violet solution was added to each well for staining for 5 minutes. Cells were then slowly washed with PBS.

[0041] The results are as follows Figure 2 As shown, 3,29-dibenzoyltrichosanthesinol can significantly inhibit the proliferation of liver cancer cells.

[0042] 3. Flow cytometry apoptosis assay

[0043] HCCLM3 cells were seeded in DMEM medium containing 10% fetal bovine serum and cultured at 37°C and 5% CO2 for 24 hours. On the second day, the cells were divided into six groups: blank control, FITC single-positive control, PI single-positive control, negative control, positive control, and experimental group. The experimental group received 11 μM of 3,29-dibenzoyltriol, and the negative control group received an equal volume of DMSO. After 48 hours of culture, the cells were washed with sterile PBS and digested with EDTA-free trypsin. Apoptosis was induced in the Annexin V-FITC single-positive control, PI single-positive control, negative control, and positive control cells using an apoptosis induction kit. Annexin V-FITC staining was performed on Annexin V-FITC single-positive control cells, PI staining on PI single-positive control cells, and Annexin V-FITC and PI staining on positive control cells. After incubation at room temperature in the dark for 5 minutes, the cells were analyzed by flow cytometry. For cells in the experimental group treated with 3,29-dibenzoyltriacyltriol, DMEM culture medium was collected, adherent cells were washed with sterile PBS, and cells were digested with EDTA-free trypsin. After cell collection, Annexin V-FITC and PI staining were performed, and cells were incubated at room temperature in the dark for 5 minutes before flow cytometry analysis. Based on Annexin V-FITC single-positive control, PI single-positive control, blank control, and positive control, compensation regulation and apoptosis analysis were performed using Flowjo flow cytometry data analysis software. The proportions of late apoptotic cells in Q2 and early apoptotic cells in Q3 were statistically analyzed.

[0044] The results are as follows Figure 3 and Figure 4 As shown, 3,29-dibenzoyltrichosanthesinol can promote apoptosis in liver cancer cells.

[0045] 4. Transwell experiment

[0046] HCCLM3, MHCC97-H, and SNU449 were resuspended in serum-free DMEM medium, and 200 μL of DMEM containing 3 × 10⁻⁶ cells / mL was added. 4 Serum-free DMEM of cell suspension was added to the upper chamber of a Transwell chamber, while 20% fetal bovine serum-containing medium was added to the lower chamber. 11 μM, 5 μM, and 11 μM of 3,29-dibenzoyltriol were added to the lower chambers of the HCCLM3, MHCC97-H, and SNU449 experimental groups, respectively, while an equal volume of DMSO was added to the control group. Cells were cultured at 37°C and 5% CO2 for 36 hours. After fixation with 4% paraformaldehyde and crystal violet staining, cells in the upper chamber were wiped away with a cotton swab and photographed under a microscope.

[0047] The results are as follows Figure 5 , Figure 6 and Figure 7 As shown, 3,29-dibenzoyltrichosanthesinol can significantly inhibit the migration of HCCLM3, MHCC97-H and SNU449.

[0048] Example 2

[0049] This embodiment demonstrates through animal experiments that 3,29-dibenzoyltrichosanthesinol has an inhibitory effect on orthotopic liver tumors in mice.

[0050] Thirty-two 6-8 week old C57BL / 6 mice were acclimatized in an SPF environment for approximately one week. An orthotopic xenograft model was established by directly injecting the liver with luciferase-labeled Hepa1-6 hepatocellular carcinoma cells. First, mice were intraperitoneally injected with tribromoethanol at a dose of 0.2 mL / 10 g. The skin was disinfected with povidone-iodine, and a longitudinal incision was made below the xiphoid process along the midline of the abdomen. After cutting open the skin and peritoneum, the liver lobe was fully exposed. 30 μL of insulin containing 3 × 10⁻⁶ cells was drawn using a 0.3 mL disposable insulin syringe. 5 A suspension of Hepa1-6 cells was injected into the liver via a slow needle insertion. After injection, the needle was withdrawn, and the liver was covered with sterile gauze until bleeding ceased. The peritoneum was then closed layer by layer. Mice were housed in an SPF-grade environment for one week post-surgery and randomly divided into four groups. One group served as a control group without treatment, while the other three groups received 3,29-dibenzoyltriacylglycerol treatment, receiving three different doses of 3,29-dibenzoyltriacylglycerol via intraperitoneal injection twice weekly: 15 mg / kg, 30 mg / kg, and 60 mg / kg. During the treatment period, luciferase substrate was injected intraperitoneally weekly at a dose of 150 mg / kg for small animal in vivo imaging analysis.

[0051] The results are as follows Figure 8 and Figure 9 As shown, liver tumors in mice treated with 30 mg / kg and 60 mg / kg of 3,29-dibenzoyltricholate were significantly reduced at 24 and 31 days, and liver tumors in mice treated with 15 mg / kg, 30 mg / kg and 60 mg / kg of 3,29-dibenzoyltricholate were significantly reduced at 31 days.

[0052] Liver samples were taken from mice on day 31, and the results were as follows: Figure 10 and Figure 11 As shown, the tumor weight in the 3,29-dibenzoyltriacyltriol treatment group was significantly lower than that in the control group. Serum alanine aminotransferase (ALT) and L-lactate dehydrogenase (LDH) activities in mice were measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the results are as follows: Figure 11As shown, the alanine aminotransferase (ALT) and L-lactate dehydrogenase (LDH) activities in mice treated with 15 mg / kg, 30 mg / kg, and 60 mg / kg of 3,29-dibenzoyltrichosanthesinol were significantly lower than those in the control group, demonstrating that treatment with 3,29-dibenzoyltrichosanthesinol reduced tumor burden and was effective.

[0053] Example 3

[0054] This embodiment demonstrates through animal experiments that 3,29-dibenzoyltrichosanthesinol has an inhibitory effect on subcutaneous xenografts in nude mice.

[0055] Thirty-two 3-4 week old BALB / c Nude mice were acclimatized in an SPF environment for one week. HCCLM3 cells were resuspended in PBS to a concentration of 2.5 × 10⁶ cells / mL. 7 HCCLM3 cells were subcutaneously inoculated into the back of nude mice using a 1 mL syringe at a concentration of 0.2 mL / mL. Mice were then housed in an SPF-grade environment for one week post-surgery until the xenograft reached a size of 200 mm². 3 Students were randomly divided into four groups. One group served as a control group without medication, while the other three groups were 3,29-dibenzoyltriacyltriol treatment groups, receiving three different intraperitoneal injections twice weekly at three different concentrations: 10 mg / kg, 20 mg / kg, and 50 mg / kg, for three consecutive weeks. During the treatment period, the long and short diameters of the transplanted tumors were measured using calipers, and the tumor volume was calculated. Tumor volume = 0.5 × a × b 2 Let 'a' represent the longest diameter and 'b' represent the longest shortest diameter in the vertical direction. The tumor tissue of nude mice after the drug administration cycle is shown below. Figure 12 As shown. Figure 13 As shown, 3,29-dibenzoyltriacyltriol at concentrations of 10 mg / kg, 20 mg / kg and 50 mg / kg significantly inhibited the growth of subcutaneous xenografts in nude mice.

Claims

1. Application of 3,29-dibenzoyltriacyltriol in the preparation of drugs for the prevention and treatment of liver cancer.

2. The application of 3,29-dibenzoyltriacyltriol according to claim 1 in the preparation of drugs for the prevention and treatment of liver cancer, characterized in that, The drug for preventing and treating liver cancer also contains one or more pharmaceutically acceptable excipients.

3. The application of 3,29-dibenzoyltriacyltriol according to claim 2 in the preparation of drugs for the prevention and treatment of liver cancer, characterized in that, The excipients are at least one of the following: sustained-release agent, binder, wetting agent, disintegrant, absorption promoter, surfactant, antibacterial agent, fragrance, antioxidant, pH adjuster, diluent, lubricant, or solvent.

4. The application of 3,29-dibenzoyltriacyltriol according to claim 3 in the preparation of drugs for the prevention and treatment of liver cancer, characterized in that, The drug for preventing and treating liver cancer is in the form of an injection.

5. The application of 3,29-dibenzoyltriacyltriol according to claim 3 in the preparation of drugs for the prevention and treatment of liver cancer, characterized in that, The dosage forms of the drugs for preventing and treating liver cancer are capsules, pills, tablets, granules, tinctures, inhaled preparations, oral solutions, oral suspensions, or oral emulsions.

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