Isoliquiritigenin composition and use thereof in preparing a drug for treating thyroid cancer

Abstract

The present application belongs to the technical field of pharmaceutical preparation and use, and particularly relates to a composition of isofraxidin and application thereof in preparation of a medicine for treating thyroid cancer. The present application explores the role of isofraxidin in treating anaplastic thyroid cancer, and fully proves that isofraxidin can significantly inhibit the proliferation of anaplastic thyroid cancer cells, induce the apoptosis of anaplastic thyroid cancer cells, up-regulate the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-PARP of anaplastic thyroid cancer cells, and inhibit the migration of anaplastic thyroid cancer cells by using a model of cell line xenograft tumor nude mice and cell in-vitro and in-vivo combination.

Description

Technical Field

[0001] This invention belongs to the technical field of pharmaceutical preparations and uses, specifically relating to isoliquiritin compositions and their application in the preparation of drugs for treating thyroid cancer. Background Technology

[0002] Thyroid cancer is a malignant tumor originating from thyroid tissue. It is the most common thyroid malignancy, accounting for approximately 1% of all malignant tumors. It includes four pathological types: papillary carcinoma, follicular carcinoma, undifferentiated carcinoma, and medullary carcinoma. Traditional Chinese medicine classifies thyroid cancer into three types: qi stagnation and blood stasis type, internal accumulation of blood stasis and toxins type, and yin deficiency and fire excess type. Thyroid cancer can invade surrounding tissues and organs, such as cervical lymph nodes, trachea, and larynx, and can also metastasize to distant sites. Common symptoms of thyroid cancer include a neck mass, hoarseness, and difficulty swallowing. Early diagnosis and treatment are crucial for improving survival rates. Currently, treatment methods for thyroid cancer include surgical resection, radiotherapy, and drug therapy. Surgical resection is the primary treatment method, removing the tumor and surrounding tissues. Radiotherapy can be used to eliminate residual lesions or control metastases after surgery. Drug therapy mainly includes thyroid hormone replacement therapy and radioactive iodine therapy.

[0003] Isoliquiritin is a natural plant compound, primarily found in licorice root, and is an isoflavone compound found in licorice. Licorice root is a common traditional Chinese medicine widely used in medicine. Isoliquiritin possesses various biological activities and pharmacological effects, and its broad pharmacological activity has attracted considerable attention in recent years. Studies have found that it has antioxidant, anti-inflammatory, antibacterial, and antitumor properties. It can regulate the immune system through multiple pathways, reduce inflammatory responses, and inhibit cell proliferation and metastasis, thus showing potential therapeutic effects on various diseases. It can also dilate arteries, providing protection for the heart and brain.

[0004] Studies have shown that isoliquiritigenin possesses broad-spectrum and highly effective antitumor activity, exerting antitumor activity against cancers such as liver cancer, breast cancer, and colon cancer. It inhibits tumor cell invasion and metastasis through mechanisms such as regulating signaling pathways, reducing cell proliferation, and inducing apoptosis. However, due to its poor water solubility and weak polarity (its solubility in water is only 3.74 μg / mL), it is a hydrophobic drug and is unstable in aqueous solutions and when exposed to light, thus limiting its widespread clinical application.

[0005] Anaplastic thyroid cancer (ATC) is a very rare but dangerous malignant tumor, a type of thyroid cancer. Compared with other types of thyroid cancer, ATC grows faster, is more malignant, and usually responds poorly to treatment. Specifically, it has the following characteristics: (1) Rapid growth and invasiveness: Tumor cells in ATC have a tendency to proliferate rapidly and infiltrate surrounding tissues, causing the tumor to grow and spread rapidly in a short period of time. (2) High malignancy: This type of cancer cell is highly malignant, with high atypia and destructive power, making it difficult to control and treat. (3) Prone to metastasis: ATC is prone to distant metastasis, and distant metastatic lesions are usually present at the time of diagnosis. Currently, the effect of isoliquiritigenin on ATC cancer cells is still unclear. Summary of the Invention

[0006] To overcome the shortcomings of the prior art, the present invention provides a highly stable isoliquiritigenin composition and its application in the preparation of drugs for treating undifferentiated thyroid cancer.

[0007] The first objective of this invention is to provide the use of an isoliquiritigenin composition in the preparation of a medicament for treating thyroid cancer.

[0008] Furthermore, the thyroid cancer in question is undifferentiated thyroid cancer. Compared with other types of thyroid cancer, undifferentiated thyroid cancer grows faster, is more malignant, and is more difficult to treat.

[0009] Experiments have shown that the isoliquiritigenin composition can inhibit the activity of undifferentiated thyroid cancer cells.

[0010] Experiments have shown that the isoliquiritigenin composition can induce apoptosis in undifferentiated thyroid cancer cells.

[0011] Experiments have shown that the isoliquiritigenin composition can upregulate the expression of cleaved-caspase3 and cleaved-PARP, proteins associated with apoptosis in undifferentiated thyroid cancer cells.

[0012] Experiments have shown that the isoliquiritigenin composition can upregulate the expression of BAX, a pro-apoptotic protein in the mitochondria of undifferentiated thyroid cancer cells, and downregulate the expression of BCL2, a protein that inhibits apoptosis.

[0013] Experiments have shown that the isoliquiritigenin composition can inhibit the migration of undifferentiated thyroid cancer cells.

[0014] Each 1L of the isoliquiritigenin composition contains:

[0015] Isoliquiritin 5–50 μmol, agar 6–16 μmol, gum arabic 20–45 μmol, sodium chloride 2–12 μmol, polycrylene 0.5–1.0 μmol, water.

[0016] In one embodiment, each 1L of the isoglycyrrhizin composition contains:

[0017] Isoliquiritigenin 10 μmol, agar 6 μmol, gum arabic 20 μmol, sodium chloride 2 μmol, polycrylene 0.5 μmol, water;

[0018] In one embodiment, each 1L of the isoglycyrrhizin composition contains:

[0019] Isoliquiritigenin 15 μmol, agar 8 μmol, gum arabic 25 μmol, sodium chloride 4 μmol, polycrylene 0.6 μmol, water;

[0020] In one embodiment, each 1L of the isoglycyrrhizin composition contains:

[0021] Isoliquiritigenin 20 μmol, agar 10 μmol, gum arabic 30 μmol, sodium chloride 6 μmol, polycrylene 0.7 μmol, water;

[0022] In one embodiment, each 1L of the isoglycyrrhizin composition contains:

[0023] Isoliquiritigenin 25 μmol, agar 12 μmol, gum arabic 35 μmol, sodium chloride 8 μmol, polycrylene 0.8 μmol, water;

[0024] In one embodiment, each 1L of the isoglycyrrhizin composition contains:

[0025] Isoliquiritigenin 30 μmol, agar 14 μmol, gum arabic 40 μmol, sodium chloride 10 μmol, polycrylene 0.9 μmol, water;

[0026] In one embodiment, each 1L of the isoglycyrrhizin composition contains:

[0027] Isoliquiritigenin 50 μmol, agar 16 μmol, gum arabic 45 μmol, sodium chloride 12 μmol, polycrylene 1.0 μmol, water.

[0028] In several embodiments, the preparation method of the isoliquiritigenin composition is as follows:

[0029] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0030] Compared with the prior art, the technical advantages of the present invention are as follows:

[0031] This invention provides an isoliquiritigenin composition that overcomes the instability of isoliquiritigenin in aqueous solution and when exposed to light. The isoliquiritigenin composition of this invention exhibits good stability in aqueous solution under high temperature, high humidity and light exposure, and its isoliquiritigenin content remains essentially unchanged.

[0032] Based on the advantages of traditional Chinese medicine, this study proposes the role of isoliquiritigenin in the treatment of undifferentiated thyroid cancer. Using xenograft tumor models in nude mice and combined in vitro and in vivo cell models, it was fully demonstrated that isoliquiritigenin significantly inhibits the proliferation of undifferentiated thyroid cancer cells, induces apoptosis in undifferentiated thyroid cancer cells, upregulates the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-PARP in undifferentiated thyroid cancer cells, and inhibits the migration of undifferentiated thyroid cancer cells. Attached Figure Description

[0033] Figure 1 The effect of ISL on the viability of undifferentiated thyroid cancer cells was detected by CCK8 assay. (a) 8505C cells and (b) CAL62 cells.

[0034] Figure 2 The clonogenic assay was used to detect the effect of isoliquiritigenin on the proliferation of undifferentiated thyroid cancer cells (8505C and CAL62 cells).

[0035] Figure 3 Figure 1 shows the effect of isoliquiritigenin-induced apoptosis in undifferentiated thyroid cancer cells detected by the TUNEL / DAPI apoptosis detection kit. (a) shows 8505C cells and (b) shows CAL62 cells.

[0036] Figure 4 Flow cytometry was used to detect the apoptotic cell rate of isoliquiritigenin-induced apoptosis in undifferentiated thyroid cancer cells (8505C and CAL62 cells) (*p<0.05, **p<0.01, ***p<0.001 compared with the control group).

[0037] Figure 5 Effects of isoliquiritigenin on apoptosis-related proteins in undifferentiated thyroid cancer cells, (a) 8505C cells, (b) CAL62 cells.

[0038] Figure 6JC-1 assay was used to detect the effect of isoliquiritigenin on changes in mitochondrial membrane potential in undifferentiated thyroid cancer cells. (a) 8505C cells and (b) CAL62 cells.

[0039] Figure 7 Effects of isoliquiritigenin on cell proliferation of 8505C cell xenografts in nude mice. Figure A: Tumor volume growth curves of 8505C xenografts in BALB / c nude mice treated with intraperitoneal injection of ISL (0, 10, 25 and 50 mg / kg) every other day (n=5 mice / group); Figure B: Macroscopic view of the xenografts at the experimental endpoint; Figure C: Tumor weight in each group (n=5), and the inhibitory effect of ISL on the growth of 8505c tumors in vivo.

[0040] Figure 8 Effect of isoliquiritigenin on wound healing in undifferentiated thyroid cancer cells, (a) 8505C cells, (b) CAL62 cells.

[0041] Figure 9 Effect of isoliquiritigenin on the migration ability of undifferentiated thyroid cancer cells (8505C and CAL62 cells) (*p<0.05, **p<0.01, ***p<0.001 compared with the control group).

[0042] Figure 10 Stability of isoglycyrrhizin compositions in Examples 1-6 and Comparative Examples 1-3. Detailed Implementation

[0043] To make the objectives and technical solutions of this invention clearer, the following embodiments are provided for further explanation. However, the scope of protection of this invention is not limited to these embodiments; the embodiments are merely for illustrative purposes. Those skilled in the art should understand that any changes or equivalent substitutions that do not depart from the concept of this invention are included within the scope of protection of this invention.

[0044] Example 1: Isoliquiritin Composition

[0045] Each 1L of the isoliquiritigenin composition contains:

[0046] Isoliquiritigenin 10 μmol, agar 6 μmol, gum arabic 20 μmol, sodium chloride 2 μmol, polycrylene 0.5 μmol, water;

[0047] Preparation method:

[0048] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0049] Example 2: Isoliquiritin Composition

[0050] Each 1L of the isoliquiritigenin composition contains:

[0051] Isoliquiritigenin 15 μmol, agar 8 μmol, gum arabic 25 μmol, sodium chloride 4 μmol, polycrylene 0.6 μmol, water;

[0052] Preparation method:

[0053] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0054] Example 3: Isoliquiritin Composition

[0055] Each 1L of the isoliquiritigenin composition contains:

[0056] Isoliquiritigenin 20 μmol, agar 10 μmol, gum arabic 30 μmol, sodium chloride 6 μmol, polycrylene 0.7 μmol, water;

[0057] Preparation method:

[0058] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0059] Example 4: Isoliquiritin Composition

[0060] Each 1L of the isoliquiritigenin composition contains:

[0061] Isoliquiritigenin 25 μmol, agar 12 μmol, gum arabic 35 μmol, sodium chloride 8 μmol, polycrylene 0.8 μmol, water;

[0062] Preparation method:

[0063] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0064] Example 5: Isoliquiritin Composition

[0065] Each 1L of the isoliquiritigenin composition contains:

[0066] Isoliquiritigenin 30 μmol, agar 14 μmol, gum arabic 40 μmol, sodium chloride 10 μmol, polycrylene 0.9 μmol, water;

[0067] Preparation method:

[0068] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0069] Example 6: Isoliquiritin Composition

[0070] Each 1L of the isoliquiritigenin composition contains:

[0071] Isoliquiritigenin 50 μmol, agar 16 μmol, gum arabic 45 μmol, sodium chloride 12 μmol, polycrylene 1.0 μmol, water;

[0072] Preparation method:

[0073] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0074] Comparative Example 1: Isoliquiritin Composition

[0075] Each 1L of the isoliquiritigenin composition contains:

[0076] Isoliquiritigenin 25 μmol, agar 12 μmol, gum arabic 35 μmol, sodium chloride 8 μmol, water;

[0077] Preparation method:

[0078] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this solution, then add sodium chloride and stir until evenly mixed. Add water to the total volume and stir until well combined.

[0079] Comparative Example 2: Isoliquiritin Composition

[0080] Each 1L of the isoliquiritigenin composition contains:

[0081] Isoliquiritigenin 25 μmol, gum arabic 35 μmol, sodium chloride 8 μmol, polycrylene 0.8 μmol, water;

[0082] Preparation method:

[0083] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this solution, then add sodium chloride and stir until evenly mixed. Add water to the total volume and stir until well combined.

[0084] Comparative Example 3: Isoliquiritin Composition

[0085] Each 1L of the isoliquiritigenin composition contains:

[0086] Isoliquiritigenin 25 μmol, agar 3 μmol, gum arabic 35 μmol, sodium chloride 8 μmol, polycrylene 2 μmol, water;

[0087] Preparation method:

[0088] Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir evenly. Add water to the total volume and stir well to obtain the final product.

[0089] Pharmacological experiments

[0090] Laboratory animals: BALB / c-Nude mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All were 4-5 weeks old, male, and weighed 18-20g. They were housed at the Laboratory Animal Center of Zhejiang Cancer Hospital and placed in a specific pathogen-free (SPF) laminar flow rack and fed with special sterilized feed.

[0091] Experimental materials: Isoglycine (ISL) was purchased from Ron Reagents, catalog number: R018236, MW: 256.25 g / mol, and stored at 4℃. DMSO (D2650, Sigma); RPMI 1640 medium (SH3080909.01, Cytiva); fetal bovine serum (10099141C, Gibco); CCK8 reagent (GK10001, GLPBIO); BCA protein concentration assay kit (P0010, Beyotime); TUNEL apoptosis assay kit (C1090, Beyotime); β-Actin (66009-1-Ig, Proteintech); cleaved-caspase 3 (9664S, Cell Signaling Technology); cleaved-PARP (ET1608-10, Huaan Biotechnology); BAX (ET1603-34, Huaan Biotechnology); BCL-2 (ET1702-53, Huaan Biotechnology).

[0092] Isoliquiritigenin preparation: In vitro experiments: ISL was first dissolved in DMSO to prepare a 100mM stock solution, which was then aliquoted and stored at -20℃. In vivo experiments: The working concentration of isoliquiritigenin was prepared using a 5% DMSO / 30% PEG-400 / 5% Tween PBS solution and dissolved immediately before use.

[0093] I. Isoliquiritigenin inhibits the activity of undifferentiated thyroid cancer cells (8505C and CAL62 cells).

[0094] 1. The effect of ISL on the viability of undifferentiated thyroid cancer cells (8505C and CAL62 cells) was detected by CCK8 assay.

[0095] 1) Experimental protocol: Undifferentiated thyroid cancer cells (8505C and CAL62 cells) in logarithmic growth phase were seeded into 96-well plates (0.3 × 10⁻⁶ cells per well). 4 Cells were placed in wells (5, 10, 15, 20, 25, and 30 μM) and PBS solution was added around the outer edge. The cells were then placed in a cell culture incubator for 24 hours to allow adhesion. Different concentrations of ISL (5, 10, 15, 20, 25, and 30 μM) were added, and the cells were cultured for another 24 and 48 hours to observe cell morphology. The original culture medium was discarded, and serum-free culture medium and 10 μL CCK8 solution were added in the dark. After incubation for 2 hours, the absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated using the following formula. Results are shown below. Figure 1 .

[0096] Cell viability = [OD value of experimental group - OD value of blank group] / [OD value of control group - OD value of blank group]

[0097] Value] × 100%.

[0098] 2) Experimental results: such as Figure 1 As shown, the viability of 8505C and CAL62 cells decreased with increasing ISL concentration and duration of treatment. The IC50 values ​​of ISL for 8505C cells after 24 h and 48 h of treatment were also observed. 50 The IC50 values ​​of ISL at 22.68 and 15.15 μM, respectively, for CAL62 cells after 24 h and 48 h of treatment were [not specified]. 50 The concentrations were 21.75 and 16.80 μM, respectively, indicating that ISL could inhibit the viability of 8505C and CAL62 cells.

[0099] 2. Clonogenic assay to detect the effect of isoliquiritigenin on the proliferation of undifferentiated thyroid cancer cells (8505C and CAL62 cells).

[0100] 1) Experimental protocol: Undifferentiated thyroid cancer cells (8505C and CAL62 cells) in logarithmic growth phase were seeded into 6-well plates (500 cells / well) and cultured in a cell culture incubator for 48 hours. The culture medium was changed and different concentrations of ISL (0, 10, 20, and 30 μM) were added. Culture was continued, changing the medium every three days for two weeks. After fixation with 4% paraformaldehyde, the cells were stained with crystal violet and photographed. The results are shown in the figure. Figure 2 .

[0101] 2) Experimental results: such as Figure 2 As shown, ISL can reduce the clonogenic ability of 8505C and CAL62 cells in a dose-dependent manner.

[0102] II. Isoliquiritigenin-induced apoptosis in undifferentiated thyroid cancer cells (8505C and CAL62 cells)

[0103] 1. The TUNEL / DAPI apoptosis detection kit was used to detect isoliquiritigenin-induced apoptosis in 8505C and CAL62 cells.

[0104] 1) Experimental Protocol: Undifferentiated thyroid cancer cells (8505C and CAL62 cells) in logarithmic growth phase were seeded into 6-well plates and cultured in a cell culture incubator for 24 hours to allow them to adhere. Cells were then treated with ISL at final concentrations (0, 10, 20, and 30 μM) for 24 hours. Cells were gently rinsed once with PBS, fixed with 4% paraformaldehyde for 30 minutes, washed once with PBS, permeabilized with 0.3% Triton X-100 PBS, incubated at room temperature for 5 minutes, washed twice with PBS, covered with TUNEL assay solution (TdT enzyme: fluorescent labeling solution = 1:50), and incubated at 37°C in the dark for 1 hour. Cells were washed three times with PBS for 5 minutes each time. Cells were counterstained with DAPI solution and incubated at room temperature in the dark for 5 minutes. Cell fluorescence was photographed under a fluorescence microscope using red and blue light, and images were collected. The results are shown in the figure below. Figure 3 .

[0105] 2) Experimental results: such as Figure 3 As shown, the nuclei of viable cells stained blue by DAPI, while the nuclei of TUNEL-positive cells are stained red. The number of TUNEL-positive cells can reflect the degree of apoptosis. With increasing ISL concentration, the number of TUNEL-positive cells increases, and ISL can induce apoptosis in 8505C and CAL62 cells in a dose-dependent manner.

[0106] 2. Flow cytometry detection of apoptotic cell rate in isoliquiritigenin-induced apoptosis of 8505C and CAL62 cells

[0107] 1) Experimental Protocol: Undifferentiated thyroid cancer cells (8505C and CAL62 cells) in logarithmic growth phase were seeded into 6-well plates and cultured in a cell culture incubator for 24 hours to allow them to adhere. Cells were then treated with ISL at final concentrations (0, 10, 20, and 30 μM) for 24 hours. Cells were digested with EDTA-free trypsin, centrifuged, resuspended in 1× Binding Buffer, stained with Annexin V-FITC and PI, gently vortexed, and incubated at room temperature in the dark for 5 minutes. The number of apoptotic cells was statistically analyzed using flow cytometry. Results are shown in the figure below. Figure 4 .

[0108] 2) Experimental results: such as Figure 4 As shown, isoliquiritigenin (ISL) induced apoptosis in 8505C and CAL62 cells, with the number of apoptotic cells increasing with increasing ISL concentration. In 8505C cells, the number of apoptotic cells increased from 2.41% to 35.22%, while in CAL62 cells, the number of apoptotic cells increased from 6.61% to 22.83%.

[0109] III. Western blotting assay to detect the effect of isoliquiritigenin on apoptosis-related proteins in undifferentiated thyroid cancer cells (8505C and CAL62 cells).

[0110] 1) Experimental Protocol: Logarithmic growth phase 8505C and CAL62 cells were seeded into 6-well plates and cultured in a cell culture incubator for 24 hours to allow adherence. Cells were then treated with ISL at final concentrations (0, 10, 20, and 30 μM) for 24 hours. The culture medium was discarded, and the cells were slowly washed once with PBS. 100 μL of Western blotting and IP cell lysis buffer was added to each well of the 6-well plate, and the cells were lysed on ice for 10 min. Cells were scraped off with a cell scraper and collected in 1.5 ml EP tubes. The tubes were centrifuged at 15000 rpm for 10 min in a pre-chilled centrifuge at 4°C. The supernatant was carefully transferred to EP tubes. Protein concentration was quantified according to the BCA kit instructions, with a total protein concentration of 30 μg. The corresponding volume of 5× buffer was added to prepare the protein sample, which was then denatured by boiling in a metal bath at 100°C for 10 min. The samples were aliquoted and stored at -80°C. Protein samples were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes, and blocked on a shaker. The membrane was incubated overnight at 4°C with a protein-specific primary antibody, washed, and then incubated with anti-rabbit or anti-mouse secondary antibody at room temperature for 1 hour. After washing, the membrane was developed with ECL working solution and exposed for photographs. β-Actin was used as an internal control. The results are shown in the figure below. Figure 5 .

[0111] 2) Experimental results: such as Figure 5 As shown, in 8505C and CAL62 cells, compared with the control group, ISL treatment downregulated the expression level of the anti-apoptotic protein Bcl-2, upregulated the expression level of the pro-apoptotic protein BAX, and gradually upregulated the expression levels of the apoptosis-related proteins cleaved-caspase3 and cleaved-PARP. These results suggest that ISL may induce apoptosis by mediating mitochondrial-dependent proteins.

[0112] IV. Effects of JC-1 assay on mitochondrial membrane potential changes in 8505C and CAL62 cells

[0113] 1) Experimental Protocol: Logarithmically growing 8505C and CAL62 cells were seeded into 6-well plates and cultured in a cell culture incubator for 24 hours to allow adherence. Cells were then treated with ISL at final concentrations of 0, 10, 20, and 30 μM for 24 hours. The culture medium was discarded, and the cells were rinsed once with PBS. 1 mL of culture medium was added, followed by 1 mL of JC-1 working solution. The mixture was gently shaken and incubated at 37°C for 20 minutes. After incubation, the cells were washed twice with 1×JC-1 staining buffer. 1 mL of culture medium was added to each well, and the cells were photographed under a fluorescence microscope using red and green light to observe fluorescence. The results are shown in the figure below. Figure 6 .

[0114] 2) Experimental results: such as Figure 6As shown, in 8505C and CAL62 cells, with increasing isoliquiritigenin concentration, JC-1 monomers (green fluorescence) increased, multimers (red fluorescence) decreased, mitochondrial membrane potential decreased, and cells were in the early stage of apoptosis. ISL can induce mitochondrial-dependent apoptosis in 8505C and CAL62 cells.

[0115] V. Effects of isoliquiritigenin on the proliferation of tumor cells in nude mice with 8505c cell xenografts

[0116] 1) Experimental protocol: 8505c cells in logarithmic growth phase were used, and the concentration was adjusted to 2×10⁻⁶ cells / mL using serum-free medium. 7 8505c cells / mL were used to create a tumor model by subcutaneous injection of 0.1 mL of 8505c cells into BALB / c nude mice. The tumors were allowed to grow to 100 mm. 3 Under aseptic conditions, tumors were harvested and prepared into uniform tissue fragments. These fragments were then subcutaneously injected into the right axilla of each animal to establish a BALB / c nude mouse model of undifferentiated thyroid cancer cells (8505c). The mice were randomly divided into four groups: a control group, and low-, medium-, and high-dose isoglycyrrhizin groups, with five mice in each group. The experimental groups received intraperitoneal injections of low-dose (10 mg / kg), medium-dose (25 mg / kg), and high-dose (50 mg / kg) isoglycyrrhizin every other day. The control group received the same dose of isoglycyrrhizin-free solvent (0.2 ml). The long and short diameters of the tumors were measured simultaneously. Two weeks later, the mice were sacrificed, the tumor nodules were dissected, and weighed. The tumor volume was calculated using the formula: V = 1 / 2 × a × b. 2 , where a and b represent the length and width, respectively. See the result diagram below. Figure 7 .

[0117] 2) Experimental results: such as Figure 7 As shown in Figure A: Tumor volume growth curves of 8505C xenografts in BALB / c nude mice treated with intraperitoneal injections of ISL (0, 10, 25, and 50 mg / kg) every other day (n = 5 mice / group). Figure B: Macroscopic view of the xenografts at the experimental endpoint. Figure C: Recording of tumor weight in each group (n = 5). The results indicate that ISL has an inhibitory effect on the growth of 8505c tumors in vivo.

[0118] VI. Effects of isoliquiritigenin on the migration ability of 8505C and CAL62 cells

[0119] 1. Scratch assay to detect the effect of isoliquiritigenin on wound healing of 8505C and CAL62 cells.

[0120] 1) Experimental Protocol: Draw three horizontal lines evenly on the back of a 12-well plate using a marker pen. Spread logarithmic growth phase 8505c and CAL62 cells evenly in the wells and incubate for 24 hours to allow adhesion. Using a sterile 200μL pipette tip, make vertical scratches perpendicular to the black horizontal lines in each well. Wash the cells in the wells three times with PBS solution, then replace with serum-free 1640 medium, adding ISL to final concentrations of 0 and 10μM. Observe and photograph the scratch width at 0 hours under a microscope. Observe and photograph the scratch width again after 24 hours of incubation. See the results below. Figure 8 .

[0121] 2) Experimental results: such as Figure 8 As shown, in 8505c and CAL62 cells, the control group migrated to the scratch edge relatively quickly, and ISL treatment inhibited cell wound healing.

[0122] 2. Transwell assay to detect the effect of isoliquiritigenin on the migration ability of 8505C and CAL62 cells.

[0123] 1) Experimental protocol: 8505C and CAL62 cells in logarithmic growth phase were resuspended in serum-free 1640 blank medium, and the cell concentration was adjusted to 5 × 10⁻⁶ cells / year. 4 Cells / mL. Add 600 μL of 1640 complete culture medium to a 24-well plate, place the chamber at the top, and add 200 μL of cell suspension to each chamber of the control and experimental groups. Add ISL to the drug treatment group to a final concentration of 10 μM. Incubate for 48 h, then remove the 24-well plate, carefully aspirate the culture medium from the chamber and the lower layer, wash the chamber with PBS, fix the cells with 4% paraformaldehyde for 15 min, add crystal violet, stain for 30 min, wash away excess stain, gently wipe away the cells from the inner layer of the chamber with a cotton swab, and return to a clean 24-well plate for observation and photography under a microscope. Results are shown below. Figure 9 .

[0124] 2) Experimental results: such as Figure 9 As shown, in 8505c and CAL62 cells, compared with the control group, the number of cells crossing the chamber membrane decreased after ISL treatment, indicating that ISL has an inhibitory effect on cell migration ability.

[0125] In summary, isoliquiritigenin can inhibit the proliferation of undifferentiated thyroid cancer cells both in vitro and in vivo. It induces mitochondrial-dependent apoptosis in undifferentiated thyroid cancer cells, upregulates the expression of apoptosis-related proteins cleaved-caspase3 and cleaved-PARP, and inhibits the migration of undifferentiated thyroid cancer cells, demonstrating potential anti-tumor effects.

[0126] The above description is merely a technical solution of the present invention and is not intended to limit it. Any equivalent modifications to the foregoing embodiments or direct or indirect applications in related technical fields are similarly included within the scope of patent protection of the present invention.

[0127] Stability studies of isoglycyrrhizin compositions in Examples 1-6 and Comparative Examples 1-3

[0128] The isoglycyrrhizin compositions of Examples 1-6 and Comparative Examples 1-3 were placed under the conditions of illuminance of 4500lx±500lx, temperature of 40±2℃, and relative humidity of 75%±5% for 6 months. Samples were taken at the end of the 0th, 1st, 3rd and 6th months of the test period, and the isoglycyrrhizin content was determined by high performance liquid chromatography.

[0129] Figure 10 The stability of the isoglycyrrhizin compositions in Examples 1-6 and Comparative Examples 1-3 is shown. This invention overcomes the defects of instability in aqueous solutions and under light. The isoglycyrrhizin compositions in Examples 1-6 exhibit good stability in aqueous solutions under high temperature, high humidity, and light exposure, and their isoglycyrrhizin content remains essentially unchanged.

Claims

1. The use of isoliquiritigenin composition in the preparation of a drug for treating undifferentiated thyroid cancer, characterized in that, The isoliquiritigenin composition contains, per 1L: 5-50 μmol of isoliquiritigenin, 6-16 μmol of agar, 20-45 μmol of gum arabic, 2-12 μmol of sodium chloride, 0.5-1.0 μmol of polycrylene, and water.

2. The application according to claim 1, characterized in that, The isoliquiritigenin composition can inhibit the activity of undifferentiated thyroid cancer cells.

3. The application according to claim 1, characterized in that, The isoliquiritigenin composition can induce apoptosis in undifferentiated thyroid cancer cells.

4. The application according to claim 1, characterized in that, The isoliquiritigenin composition can upregulate the expression of cleaved-caspase3 and cleaved-PARP, proteins associated with apoptosis in undifferentiated thyroid cancer cells.

5. The application according to claim 1, characterized in that, The isoliquiritigenin composition can upregulate the expression of BAX, a pro-apoptotic protein in the mitochondria of undifferentiated thyroid cancer cells, and downregulate the expression of BCL2, a protein that inhibits apoptosis.

6. The application according to claim 1, characterized in that, The isoliquiritigenin composition can inhibit the migration of undifferentiated thyroid cancer cells.

7. The application according to claim 1, characterized in that, The isoliquiritin composition contains per 1L: Isoliquiritigenin 10 μmol, agar 6 μmol, gum arabic 20 μmol, sodium chloride 2 μmol, polycrylene 0.5 μmol, water; Alternatively, isoliquiritigenin 15 μmol, agar 8 μmol, gum arabic 25 μmol, sodium chloride 4 μmol, polycrylene 0.6 μmol, water; Alternatively, isoliquiritigenin 20 μmol, agar 10 μmol, gum arabic 30 μmol, sodium chloride 6 μmol, polycrylene 0.7 μmol, and water; Alternatively, isoliquiritigenin 25 μmol, agar 12 μmol, gum arabic 35 μmol, sodium chloride 8 μmol, polycrylene 0.8 μmol, and water; Alternatively, isoliquiritigenin 30 μmol, agar 14 μmol, gum arabic 40 μmol, sodium chloride 10 μmol, polycrylene 0.9 μmol, water; Alternatively, 50 μmol of isoliquiritigenin, 16 μmol of agar, 45 μmol of gum arabic, 12 μmol of sodium chloride, 1.0 μmol of polycrylene, and water.

8. The application according to claim 1, characterized in that, The preparation method of the isoliquiritigenin composition is as follows: Dissolve gum arabic in an appropriate amount of water and stir to form a gum arabic gel. Separately, dissolve agar and polycrylene in an appropriate amount of water at 30℃~40℃. After cooling to 25℃, add isoglycyrrhizin and stir. Add the gum arabic gel to this mixture, then add sodium chloride and stir well. Add water to the total volume and stir until smooth.

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