Application of pinocembrin derivative in preparation of antitumor drugs

Abstract

The invention relates to an application of pinocembrin 7-O-[3 ''-O-galloyl-4 '', 6 ''-hexahydroxybiphenyl dicarbonyl]-beta-D-glucoside in preparation of a medicine for preventing and treating liver cancer, and the medicine comprises any one medicine preparation form which can be taken, or any one medicine preparation form which can be taken, or any one medicine preparation form which can be taken, or any one medicine preparation form which can be taken, or any one medicine preparation form which can be taken. The pharmaceutical preparation can be used for treating liver cancer, such as an injection, a capsule, a pill, a tablet, a granule, a tincture, an inhalation preparation, an oral solution, an oral suspension or an oral emulsion, the form of the pharmaceutical preparation is selected from oral and injection forms, the liver cancer comprises primary liver cancer and secondary liver cancer, the primary liver cancer comprises hepatocellular carcinoma, intrahepatic cholangiocarcinoma and mixed liver cancer, and the secondary liver cancer comprises hepatocellular carcinoma, intrahepatic cholangiocarcinoma and mixed liver cancer. The secondary liver cancer comprises a giant block type, a nodule type and a diffuse type.

Description

Technical Field

[0001] This invention relates to a novel pharmaceutical use of a gerbinin derivative, and particularly to the use of gerbinin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside in the preparation of antitumor drugs. Background Technology

[0002] Primary liver cancer is a highly prevalent malignant tumor of the digestive system in my country. Due to its often subtle early symptoms, approximately 80% of patients are already out of the surgical field by the time of diagnosis, resulting in a very poor overall prognosis. Treatment for advanced liver cancer primarily relies on chemotherapy and targeted therapies. However, chemotherapy generally suffers from low efficacy, while targeted therapy is characterized by significant side effects and a high risk of developing drug resistance. Currently, existing liver cancer treatments still fall short of clinical needs. Therefore, exploring and developing novel anti-liver cancer drugs with novel mechanisms of action and superior efficacy is a crucial and urgent task requiring breakthroughs.

[0003] This invention aims to discover lead compounds with anti-liver cancer activity from natural products, providing candidate molecules for drug development and combination therapy strategies for anti-liver cancer.

[0004] 5,7-Dihydroxyflavanone is a flavonoid compound extracted from the pine tree. It has several derivatives, such as pinocembrin 7-O-β-D glucoside, pinocembrin dihydrochalcone-7-O-[3''-O-galloyl-4'',6''-hexahydroxydiphenoyl]-β-D-glucoside, and pinocembrin-7-O-(3''-O-galloyl-4'',6''-hexahydroxydiphenoyl)-β-glucoside.

[0005] In the course of our research on 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside, we unexpectedly discovered its inhibitory effect on the proliferation, migration, and growth of liver cancer cells. Summary of the Invention

[0006] This invention provides the application of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside in the preparation of anticancer drugs. During the research of this compound, the invention unexpectedly discovered its inhibitory effect on the proliferation and migration of liver cancer cells and tumor growth.

[0007] Therefore, the present invention further provides the application of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside in the preparation of a drug for treating liver cancer.

[0008] The drugs described in this invention include any form of oral pharmaceutical preparation, such as injections, capsules, pills, tablets, granules, tinctures, inhaled preparations, oral solutions, oral suspensions, or oral emulsions.

[0009] The pharmaceutical formulations of the present invention are preferably oral and injectable forms, most preferably tablets, capsules, and injections.

[0010] The liver cancer described in this invention includes primary liver cancer and secondary liver cancer, wherein primary liver cancer includes hepatocellular carcinoma, intrahepatic bile duct carcinoma and mixed liver cancer, and secondary liver cancer includes massive type, nodular type and diffuse type.

[0011] This invention further includes a method for treating liver cancer using a pharmaceutical preparation made from 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside, comprising taking an effective amount of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside, said effective amount including taking it once or three times daily, once or three times weekly, once or three times monthly, or any other optional method of administration, based on the principle of effective treatment of liver cancer.

[0012] The pharmaceutical preparation prepared by the present invention includes a certain dose of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside, wherein the certain dose includes 1 mg to 5000 mg, preferably 5-1000 mg, and more preferably 10-200 mg.

[0013] The pharmaceutical formulations of the present invention may, when necessary, incorporate at least one of the following pharmaceutically acceptable carriers: sustained-release agents, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, antibacterial agents, fragrances, antioxidants, pH adjusters, protectants, diluents, lubricants, natural colorants, and solvents.

[0014] The beneficial effects of this invention are: 1. Currently, there are no relevant literature reports on the inhibitory effects of jugacin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on the proliferation, migration, and tumor growth of liver cancer cells. This invention, combining in vitro and in vivo experiments, is the first to discover that jugacin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside has a strong inhibitory effect on liver cancer cells and tumors in vivo.

[0015] 2. Gorbinin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside is a natural drug with broad biological activity. It is a complex natural derivative formed by structurally modifying gorbinin by adding glucose, hexahydroxybiphenyldicarboxyl, and galloyl groups. The flavonoid-tannin hybrid formed by gorbinin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside gives it the potential for multi-target and high activity. The novel application of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside provides a new drug option for the clinical treatment of liver cancer. Attached Figure Description

[0016] Figure 1 3,29-Gorcinol 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside inhibited the proliferation and migration of hepatocellular carcinoma cells in vitro. A: CCK8 assay for the IC50 of 3,29-girhodin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside against HCCLM3. 50 B: CCK8 assay for the IC50 response of 3,29-goldenin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside to SNU449 50 C: IC50 of 3,29-Gorcinol 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside against MHCC97-H 50 D: Colony formation assay to detect the inhibitory effect of 3,29-gesinolide 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on the proliferation of HCCLM3, SNU449 and MHCC97-H cells. E: Flow cytometry analysis to detect the promoting effect of 3,29-gaborin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on apoptosis in MHCC97-H cells. F: Transwell assay to detect the inhibitory effect of 3,29-goldenin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside on the migration of MHCC97-H cells. G: Transwell assay to detect the inhibitory effect of 3,29-goldenin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside on HCCLM3 cell migration. H: Transwell assay to detect the inhibitory effect of 3,29-goldenin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside on SNU449 cell migration. Figure 2 Inhibitory effect of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on orthotopic liver tumors in mice. A: Small animal in vivo imaging assay to detect the inhibitory effect of 3,29-goldenin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on orthotopic liver tumors in mice. B: Liver samples from mouse orthotopic tumors following administration of 3,29-goldenin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside. C: Fluorescence intensity of orthotopic liver transplanted tumors in mice on day 8 after tumor implantation, detected by small animal in vivo imaging. D: Fluorescence intensity of orthotopic liver transplanted tumors in mice on day 31 post-implantation, detected by small animal in vivo imaging. E: Liver-to-body weight ratio in mice after administration of 3,29-germinol 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside F: Serum GPT activity in mice after administration of 3,29-gallotinib 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside Figure 3 Inhibitory effect of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on subcutaneous hepatocellular carcinoma xenografts in nude mice A: Subcutaneous hepatocellular carcinoma xenografts in nude mice after administration of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside B: Tumor volume growth curve, tumor weight, and serum L-LDH level of subcutaneous hepatocellular carcinoma xenografts in nude mice after administration of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the invention are not limited thereto.

[0018] Example 1: In vitro inhibitory activity of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside against hepatocellular carcinoma cells. 1. CCK8 Experiment Three types of liver cancer cells, HCCLM3, SNU449, and MHCC97-H, were respectively treated with 3×10⁻⁶ cells / mL. 3 Cells were seeded at a density of [number] cells / mL in 96-well plates. After 24 hours of adhesion, the culture medium was replaced with different concentration gradients of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside: 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, and 32 μM. An equal volume of DMSO was added to the control group's DMEM medium. Six replicates were set up for each group. After 48 hours of culture, cell viability was analyzed using a CCK8 assay. Figure 1 As shown in AC, 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside significantly inhibited the proliferation of three types of hepatocellular carcinoma cells in a dose-dependent manner. Its half-maximal inhibitory concentration (IC50) was [data missing]. 50 The values ​​were: HCCLM3 cells, 2.348 μM; SNU449 cells, 2.275 μM; the inhibitory activity against MHCC97-H cells was the strongest, with an IC50 value of 1.022 μM.

[0019] 2. Cell clone formation experiment To further evaluate the antitumor and proliferative effects of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside, a colony formation assay was performed using crystal violet staining. HCCLM3, SNU449, and MHCC97-H cells were digested with trypsin, resuspended in DMEM containing 10% fetal bovine serum, and counted. Cells were then arranged at 4 × 10⁶ cells per well. 5The density of inoculation was determined. The control group received an equal volume of DMSO in DMEM medium. The HCCLM3 experimental group received 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside in DMEM medium to achieve a final concentration of 2.5 μM. The SNU449 experimental group received 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside in DMEM medium to achieve a final concentration of 2.5 μM. The MHCC97H experimental group received 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside in DMEM medium to achieve a final concentration of 1.5 μM. Culture was continued for two days, with the medium replaced every three days with an equal volume of DMSO and the same concentration of the drug in DMEM medium. After several days of culture, the cells were fixed with 4% paraformaldehyde for 10 minutes and stained with 0.4% crystal violet solution for 10 minutes. The results are as follows: Figure 1 As shown in Figure D, compared with the control group, the clonogenic ability of cells in each group treated with geraniol derivatives was significantly inhibited.

[0020] 3. Flow cytometry apoptosis assay To investigate the effect of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on apoptosis in hepatocellular carcinoma cells, Annexin V-FITC / PI double staining combined with flow cytometry was used for analysis. MHCC97-H cells were first divided into six groups according to experimental requirements, including a blank control, a single-positive control (Annexin V-FITC and PI for fluorescence compensation), a negative control, a positive control, and an experimental group. The experimental group received 1.5 μM 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside for 72 hours, while the positive control received an apoptosis-inducing agent for 30 minutes. The culture supernatant was collected, and adherent cells were gently washed with pre-cooled PBS. Cells were digested with trypsin without EDTA, and after digestion, the cells were resuspended in binding buffer. Subsequently, Annexin V-FITC and PI dye were added, and the cells were incubated at room temperature in the dark for 5 minutes. Flow cytometry analysis was performed using FlowJo software, and a gating strategy was used to distinguish between live cells, early apoptotic cells, and late apoptotic cell populations. Results are as follows: Figure 1 As shown in E, compared with the negative control group, the proportion of early and late apoptotic cells in the experimental group was significantly increased, indicating that 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside can effectively induce and promote apoptosis of liver cancer cells.

[0021] 4. Transwell experiment MHCC97-H, HCCLM3, and SNU449 cells were resuspended in 200 μL of serum-free medium and seeded into the upper chamber of a Transwell cell line (3 × 10⁶ cells per chamber). 4 Cells were cultured in a medium containing 20% ​​fetal bovine serum in the lower chamber. Specific concentrations of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside (2.5 μM for HCCLM3 and SNU449, 1.5 μM for MHCC97-H) were added to the lower chamber of each cell line, while an equal volume of DMSO was added to the control group. After culturing at 37°C and 5% CO2 for 36 hours, cells were fixed with 4% paraformaldehyde for 10 minutes, followed by crystal violet staining for 10 minutes. Unmigrated cells from the upper chamber were carefully removed, and the cells that migrated to the lower chamber were counted. Results are as follows: Figure 1 As shown in FH, compared with the control group, 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside significantly inhibited the migration ability of liver cancer cells.

[0022] See results Figure 1 3,29-Gorcinol 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside inhibited the proliferation and migration of hepatocellular carcinoma cells in vitro. Example 2: Inhibitory effect of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside on orthotopic liver xenografts in mice. To evaluate the in vivo anti-hepatocellular carcinoma efficacy of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside, a mouse orthotopic hepatocellular carcinoma model was established. In this study, 32 6-8 week old C57BL / 6 mice were acclimatized in an SPF-grade environment for one week, and then a xenograft model was established by orthotopic injection of luciferase-labeled Hepa1-6 hepatocellular carcinoma cells into the liver. After anesthesia with tribromoethanol, a midline abdominal incision was made to expose the liver, and 3×10⁻⁶ glucoside was injected into the liver lobe. 5 A suspension of 30 μL of cells was administered post-surgery, followed by suturing and continuous observation until recovery. One week after modeling, mice were randomly divided into four groups (n=8). Figure 2C): One group served as the DMSO control group, while the other three groups were treatment groups for jugazosin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside, administered intraperitoneally at doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg, respectively, twice weekly. During the treatment period, luciferase substrate was injected intraperitoneally weekly at a dose of 150 mg / kg, and tumor growth was monitored using a small animal imaging system. The results showed that, compared with the control group, the 5 mg / kg, 10 mg / kg, and 20 mg / kg dose groups of jugazosin 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside exhibited significant tumor-suppressing effects at treatment doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg after day 24 of treatment. Figure 2 D). At the end of the experiment, the mice and their liver tissues were weighed, further confirming that the tumor weight in each treatment group was significantly lower than that in the control group (D). Figure 2 E).

[0023] To assess drug safety, serum alanine aminotransferase (ALT) activity was measured. For example... Figure 2 As shown in Figure F, the GPT levels in mice treated with 5 mg / kg, 10 mg / kg, and 20 mg / kg doses were significantly lower than those in the control group, demonstrating that 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside has the function of inhibiting tumor growth without causing significant liver damage, further confirming its therapeutic efficacy and safety.

[0024] See results Figure 2 Inhibitory effect of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxyl]-β-D-glucoside on orthotopic liver tumors in mice. Example 3: Inhibitory effect of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside on subcutaneous hepatocellular carcinoma xenografts in nude mice. 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. 3At that time, the patients were randomly divided into four groups. One group was a control group without medication, and the other three groups received 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside at three different concentrations, administered intraperitoneally twice weekly: 5 mg / kg, 10 mg / kg, and 20 mg / kg, for three consecutive weeks. During the administration 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 3 As shown in C. Figure 3 As shown in A and 3B, 5 mg / kg, 10 mg / kg and 20 mg / kg of gersinolide 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside significantly inhibited the growth of subcutaneous xenografts in nude mice.

[0025] See results Figure 3 Inhibitory effect of 7-O-[3''-O-galloyl-4'',6''-hexahydroxybiphenyldicarboxylic acid]-β-D-glucoside on subcutaneous hepatocellular carcinoma xenografts in nude mice.

Claims

1. Use of pinocembrin 7-O-[3"-O-galloyl-4",6"-hexahydroxydiphenyl dicarboxyl]-β-D-glucoside for the preparation of a medicament for preventing and treating liver cancer.

2. Use according to claim 1, characterized in that: The medicament includes any one of the forms of orally administrable pharmaceutical preparations, such as injections, capsules, pills, tablets, granules, tinctures, inhalation preparations, oral solutions, oral suspensions or oral emulsions.

3. Use according to claim 2, characterized in that: The form of the pharmaceutical preparation is selected from oral and injection forms.

4. Use according to claim 2, characterized in that: The form of the pharmaceutical preparation is selected from tablets, capsules and injections.

5. The use according to claim 1, characterized in that: The liver cancer includes primary liver cancer and secondary liver cancer, wherein the primary liver cancer includes hepatocellular carcinoma, intrahepatic cholangiocarcinoma and mixed type liver cancer, and the secondary liver cancer includes massive type, nodular type and diffuse type.

6. Use according to claim 2, characterized in that: The form of the pharmaceutical preparation includes a dose of pinocembrin 7-O-[3"-O-galloyl-4",6"-hexahydroxydiphenyl dicarboxyl]-β-D-glucoside, wherein the dose includes from 1 mg to 5000 mg, preferably 5-1000 mg, and more preferably 10-200 mg.

7. Use according to claim 2, characterized in that: The form of the pharmaceutical preparation can be added with a pharmaceutically acceptable carrier, such as at least one of sustained release agents, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, antibacterial agents, fragrances, antioxidants, pH regulators, protective agents, diluents, lubricants, natural colorants and solvents, as necessary.

8. The use according to claim 1, characterized in that: The effective concentration of pinocembrin 7-O-[3"-O-galloyl-4",6"-hexahydroxydiphenyl dicarboxyl]-β-D-glucoside for inhibiting the proliferation of liver cancer cells is at least 1 μM.

9. The use according to claim 1, characterized in that: The administration dose of pinocembrin 7-O-[3"-O-galloyl-4",6"-hexahydroxydiphenyl dicarboxyl]-β-D-glucoside for inhibiting the growth of mouse tumors is at least 5 mg / kg.

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