Use of beta-sitosterol glycoside in the preparation of a product for the treatment of lung cancer

Abstract

This invention relates to the application of β-sitosterol glycoside in the preparation of products for treating lung cancer. This invention creatively provides a new use for the active monomer β-sitosterol glycoside, demonstrating that β-sitosterol glycoside can significantly inhibit the migration ability of lung cancer cells at doses without cell proliferation inhibitors, clarifying the specificity of its anti-metastatic effect, and laying the foundation for further new drug development of this monomer component. Through an in vivo mouse lung metastasis model, this invention demonstrates that β-sitosterol glycoside effectively inhibits the formation of lung metastases while exhibiting no significant toxic side effects in experimental animals, demonstrating high safety, and providing a new candidate molecule for the development of low-toxicity, highly effective anti-lung cancer metastasis drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of β-sitosterol glycoside in the preparation of products for the treatment of lung cancer. Background Technology

[0002] Cancer is a major threat to human life and health, and the treatment of lung cancer metastasis still faces many challenges. First, commonly used anti-tumor metastasis drugs, such as chemotherapy drugs and targeted drugs, generally have problems such as large toxic side effects and easy development of drug resistance. Moreover, most drugs exert their effects by directly killing tumor cells, and have limited ability to specifically inhibit cell migration, a key link in tumor metastasis.

[0003] Secondly, although the traditional Chinese medicine compound Jinfukang oral liquid has shown potential in anti-lung cancer metastasis in clinical applications, as a compound preparation, its components are complex and its pharmacodynamic material basis is unclear, making it difficult to conduct in-depth research on quality control and mechanism of action, which limits its further development and promotion as an anti-metastasis drug.

[0004] Finally, existing research lacks systematic reports on the pharmacological activities of the monomeric components in Jinfukang oral liquid, especially β-sitosterol glycoside, a steroidal compound. Currently, only studies have been found on its anti-inflammatory, antioxidant, or inhibitory effects on the proliferation of some tumor cells. No studies have been found on its specific inhibitory effect on lung cancer cell migration and its in vivo anti-lung cancer metastasis effect. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of β-sitosterol glycoside in the preparation of products for the treatment of lung cancer.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of β-sitosterol glycoside in the preparation of products for the treatment of lung cancer.

[0007] This invention creatively provides a novel use for the active monomer β-sitosterol glycoside, demonstrating that β-sitosterol glycoside can significantly inhibit the migration ability of lung cancer cells at doses without cell proliferation inhibitors, clarifying the specificity of its anti-metastatic effect, and laying the foundation for further drug development of this monomer component. Using an in vivo mouse lung metastasis model, this invention confirms that β-sitosterol glycoside effectively inhibits the formation of lung metastases while exhibiting no significant toxic side effects in experimental animals, demonstrating high safety, and providing a new candidate molecule for the development of low-toxicity, highly effective anti-lung cancer metastasis drugs.

[0008] Preferably, the β-sitosterol glycoside involved in this invention can be prepared by methods already disclosed in the art, or it can be prepared by the following method: (1) Extract the raw material of *Hemibarbus scutellariae* using ultrasound at 20-30℃ (e.g., 20℃, 22℃, 24℃, 26℃, 28℃, 30℃, etc.) for 0.5-2 h (e.g., 0.5 h, 1 h, 1.5 h, 2 h, etc.), for a total of 3-6 times (e.g., 3 times, 4 times, 5 times, 6 times, etc.), collect the filtrate, and obtain crude extract of *Hemibarbus scutellariae*. (2) The crude extract of *Hemiberlesia lingua* was separated by column chromatography and eluted, and then purified by gel chromatography to obtain the β-sitosterol glycoside.

[0009] All other specific point values ​​not listed above within the numerical ranges mentioned above can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.

[0010] Preferably, the β-sitosterol glycoside exerts its therapeutic effect on lung cancer by targeting and binding to the PA2G4 protein.

[0011] Preferably, the product further includes pharmaceutically acceptable excipients.

[0012] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0013] Secondly, the present invention provides the use of β-sitosterol glycoside in the preparation of products for inhibiting lung cancer metastasis.

[0014] Preferably, the product further includes pharmaceutically acceptable excipients.

[0015] Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0016] Thirdly, the present invention provides the use of β-sitosterol glycoside in the preparation of products for inhibiting the proliferation and migration of lung cancer cells.

[0017] Preferably, the product further includes pharmaceutically acceptable excipients.

[0018] Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0019] Fourthly, this invention provides the application of β-sitosterol glycoside in the preparation of PA2G4 protein-targeting binders.

[0020] Preferably, the product further includes pharmaceutically acceptable excipients.

[0021] Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

[0022] Compared with the prior art, the present invention has the following beneficial effects: This invention creatively provides a novel use for the active monomer β-sitosterol glycoside, demonstrating that β-sitosterol glycoside can significantly inhibit the migration ability of lung cancer cells at doses without cell proliferation inhibitors, clarifying the specificity of its anti-metastatic effect, and laying the foundation for further drug development of this monomer component. Using an in vivo mouse lung metastasis model, this invention confirms that β-sitosterol glycoside effectively inhibits the formation of lung metastases while exhibiting no significant toxic side effects in experimental animals, demonstrating high safety, and providing a new candidate molecule for the development of low-toxicity, highly effective anti-lung cancer metastasis drugs. Attached Figure Description

[0023] Figure 1 This invention investigates the migration-inhibiting effect of the extract Fr.6 of *Hemiberlesia lataniae* on CTC-TJH-01 cells. Figure 2 The nuclear magnetic resonance spectrum (top of the image) and mass spectrometry detection image (bottom of the image) of the extract component Fr.6 of *Sedum aizoon* in this invention are shown. Figure 3 The figure shows the results of the metastasis inhibition effect of β-sitosterol glycoside on a lung cancer metastasis model mouse in Example 1 of the present invention. The left side of the figure is the tumor volume, and the right side of the figure is the quantitative analysis result. Figure 4 The results of the DARTS experiment; Figure 5 This is a banding result from the CETSA verification experiment of the β-sitosterol glycoside target protein in Example 1 of the present invention. Figure 6 This is a statistical result graph of the CETSA verification experiment of β-sitosterol glycoside target protein in Example 1 of the present invention. From left to right in the graph, the statistical analysis results of PA2G4 protein, SERPINB1 protein and DNAJA1 protein are shown. Figure 7 The results of the comparison of the cell migration inhibitory activities of β-sitosterol glycoside and β-sitosterol in Example 1 of the present invention are shown. Figure 8 The results of CETSA analysis show the binding interaction between β-sitosterol glycoside and the PA2G4 protein of β-sitosterol in Example 1 of this invention. Detailed Implementation

[0024] To further illustrate the technical means and effects of the present invention, the following describes the technical solution of the present invention in conjunction with preferred embodiments of the present invention. However, the present invention is not limited to the scope of the embodiments.

[0025] Example 1 This embodiment provides a β-sitosterol glycoside, prepared by the following method: (1) Crush 50 g of *Ipomoea aquatica* raw material, place it in an Erlenmeyer flask, add 1 L of anhydrous methanol, and perform ultrasonic extraction at 25℃ for 1 h. Repeat the extraction 5 times in total, collect the filtrate, and obtain crude extract of *Ipomoea aquatica*. (2) The crude extract of *Ipomoea aquatica* was initially separated using 400-mesh silica gel as the stationary phase. The mobile phase consisted of four solvents: petroleum ether, dichloromethane, ethyl acetate, and anhydrous methanol. The solvents were eluted in a gradient order of increasing polarity and concentrated by rotary evaporation under reduced pressure at 45°C to obtain *Ipomoea aquatica* extract. (3) The extract of *Ipomoea aquatica* was separated using a Sephadex LH-20 gel chromatography column. Anhydrous methanol was used as the mobile phase for isocratic elution, and the elution flow rate was controlled at 1.0 mL / min. One tube of eluent was collected every 5 mL. Thin-layer chromatography (TLC) was used to detect the eluent of each tube. Dichloromethane-methanol (9:1 v / v) was used as the developing solvent, and 10% sulfuric acid ethanol solution was used for color development. Similar components were combined according to the spot position and color development, and finally 6 fractions were obtained. They were numbered Fr.1 to Fr.6 in the elution order, where Fr.6 is the β-sitosterol glycoside.

[0026] Test Example 1 This test case investigated the inhibitory effects of CTC-TJH-01s on the proliferation and migration of six extracts from *Hemiberlesia lingua*.

[0027] IC50 determination: The half-maximal inhibitory concentration (IC50) of six extracts of *Hemiberlesia lingua* on CTC-TJH-01 cells was determined by the MTT assay.

[0028] CTC-TJH-01 cells in logarithmic growth phase were harvested and spaced at 5 × 10⁶ cells per well. 3Cells were seeded at a density of [number] cells per well in 96-well plates and incubated overnight at 37°C with 5% CO2. After cell attachment, extracts of various concentrations (100, 50, 20, 12.5, 6.25, 3.125, 1.5, 0.75, 0.35, 0 μg / mL) were added to each well, with three replicates per concentration. Cells were incubated for another 48 hours. After incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for another 4 hours. The supernatant was discarded, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The mixture was shaken to dissolve the formazan crystals, and the absorbance of each well was measured at 570 nm using a microplate reader. Cell viability and IC50 values ​​were calculated.

[0029] Scratch assay: The scratch assay was used to detect the effects of six extracts of *Scutellaria baicalensis* on the migration ability of CTC-TJH-01 cells.

[0030] CTC-TJH-01 cells in logarithmic growth phase were harvested and spaced at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 μL in 6-well plates and cultured at 37°C in a 5% CO2 incubator until cell confluence reached 90% or higher. Using a 200 μL sterile pipette tip, uniform straight lines were drawn vertically on the cell monolayer in each well. The cells were gently washed three times with phosphate-buffered saline (PBS) to remove detached cells, and then serum-free culture medium was added. Based on the IC50 values ​​of each extracted component against CTC-TJH-01 cells obtained from the aforementioned MTT assay, and to avoid interference from cell proliferation inhibition on migration assay results, the maximum concentration of the drug administered was set not to exceed its IC50 value.

[0031] Four groups were set up for each extract: a control group (with an equal volume of solvent added) and three drug-treated groups (9, 17.5, and 35 μM). After drug administration, the 6-well plates were placed in an incubator at 37°C and 5% CO2 for further incubation. Images of the same scratch location were observed and photographed under an inverted microscope at 0 h and 24 h, respectively. The scratch area or scratch width of each group was measured, and the scratch healing rate was calculated.

[0032] Scratch healing rate (%) = (0 h scratch area - 24 h scratch area) / 0 h scratch area × 100% Each experiment was conducted in triplicate, with each independent experiment repeated three times. The inhibitory effect of each extracted component on the migration ability of CTC-TJH-01 cells was evaluated by comparing the scratch healing rate of each treatment group with that of the control group.

[0033] Statistical analysis: The experiment was independently repeated three times, and data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed on the obtained data, and one-way ANOVA was used for comparisons among multiple groups. A p-value < 0.05 was considered statistically significant.

[0034] Test results showed that among the six extracts of *Ipomoea aquatica* (Fr.1-Fr.6), only Fr.6 had an inhibitory effect on the migration of CTC-TJH-01 cells (p<0.05). Figure 1 As shown.

[0035] Test Example 2 This test case describes the structural identification of Fr.6, an extract of *Hemiberlesia lingua*.

[0036] NMR detection: Approximately 1 mg of the Fr.6 fraction purified by gel chromatography was placed in a clean, dry NMR tube. 0.5 mL of deuterated methanol (CD3OD) was added, and the solution was shaken to dissolve. The solution was then transferred to a 5 mm NMR tube, ensuring the solution height was at least 4 cm and that there were no bubbles or suspended particles on the surface. The NMR tube was then smoothly placed in the Bruker Avance NEO NMR spectrometer and kept at a constant temperature of 25°C. Detection was performed after the probe temperature stabilized. Standard pulse sequence acquisition was used. 1 H-NMR spectrum and 13 C-NMR spectrum.

[0037] Mass spectrometry analysis: Approximately 1 mg of Fr.6 fractional extract was dissolved in methanol to prepare a sample solution with a concentration of 10 μg / mL. The solution was filtered through a 0.22 μm microporous membrane and transferred to a vial. Detection was performed using an Agilent 6545 high-resolution mass spectrometer in positive ion mode with an electrospray ionization (ESI) source. The mass spectrometry parameters were set as follows: spray voltage 3.5 kV, sheath gas temperature 350 °C, sheath gas flow rate 11 L / min, nebulizer pressure 35 psig, fragmentation voltage 175 V, and scan range m / z 100–1500. The sample was directly injected at a flow rate of 10 μL / min using a syringe pump for analysis.

[0038] Structural identification: Fr.6 flowed as a white powder, mp 285-287°C, and showed a positive Liebermann-Burchard reaction. 13C-NMR (150 MHz, CD3OD) δ: 140.45 (C-5), 121.18 (C-6), 100.81 (C-1'), 76.94 (C-3'), 76.77 (C-5'), 73.46 (C-2'), 70.08 (C-4'), 61.09 (C-6'), 56.18 (C-14), 55.44 (C-17), 49.61 (C-9), 45.15 (C-24), 41.85 (C-13), 38.32 (C-4), 36.84 (C-1), 36.21 (C-10), 35.49 (C-20), 33.36 (C-22), 31.42 (C-2), 29.27 (C-25), 28.71 (C-16), 27.79 (C-7), 25.45 (C-23), 23.86 (C-28), 20.60 (C-26), 19.70 (C-27), 19.09 (C-21), 18.93 (C-11), 18.61 (C-19), 11.78 (C-29), 11.66 (C-18). HR-MS (ESI) shows [M+Na]. + m / z 599.4284 (calculated value 599.4282, C) 35 H 60 O6Na), the molecular formula is determined to be C. 35 H 60 O6. NMR data are consistent with those reported in the literature for β-sitosterol glycosides.

[0039] The NMR and mass spectrometry results for the Fr.6 fraction are as follows: Figure 2 As shown, the detection results indicate that Fr.6 fractions are β-sitosterol glycosides.

[0040] Test Example 3 This test case investigates the metastasis inhibitory effect of β-sitosterol glycoside from Example 1 on a lung cancer metastasis model mouse.

[0041] luc lentivirus was purchased from Jiman Biotechnology Co., Ltd.; clean-grade 18-20 g C57BL / 6J male mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd.; carrageenan was purchased from Sigma-Aldrich; dimethyl sulfoxide (DMSO), anhydrous ethanol, and castor oil were purchased from Adamas; isoflurane was produced by RWD; and the in vivo imaging system was produced by PerkinElmer.

[0042] Lentiviral transfection and cell preparation: LLC (Lewis lung carcinoma) cells in logarithmic growth phase were transfected at a concentration of 3 × 10⁻⁶ cells / cells.5 Cells were seeded per well in 6-well plates and cultured overnight. When cell confluence reached 60%, a lentiviral suspension containing the luciferase gene (MOI=10) was added. The plates were incubated at 37°C in a 5% CO2 incubator for 24 h, then the medium was replaced and cultured for another 48 h. Stable luciferase-expressing LLC-Luc cell lines were obtained through puromycin selection (0.5 μg / mL). After transfection and expansion culture, the cells were washed once with sterile phosphate-buffered saline (PBS), trypsinized to collect the cells, counted, resuspended in PBS, and the cell density was adjusted to 5 × 10⁶ cells / well. 6 Quantity / mL, keep on ice for later use.

[0043] Preparation of matrix solution: Take 10 μL of DMSO solution, and then add 40 μL of ethanol, 500 μL of castor oil and 900 μL of physiological saline in sequence to prepare 1 mL of matrix solution.

[0044] Animal grouping and administration regimen: The above LLC-Luc cell suspension was injected into C57BL / 6J mice via the tail vein, with each mouse receiving 100 μL (containing 5 × 10⁻⁶ cells). 5 (cells). The day of inoculation was recorded as day 0. The day after inoculation (day 1) the mice were randomly divided into 5 groups of 5 mice each, with the following grouping and administration regimens: control group, low-dose β-sitosterol glycoside group (1.25 mg / kg), medium-dose group (2.5 mg / kg), high-dose group (5 mg / kg), once a day; positive control group (cDDP): 1.5 mg / kg, once every 3 days. β-sitosterol glycoside was prepared into suspensions of the corresponding concentrations using the above matrix solution, and used after sonication. Cisplatin was prepared with physiological saline. All administration was by gavage, with an administration volume of 100 μL per mouse.

[0045] In vivo imaging: The first in vivo imaging was performed on day 1 after modeling, once a week. From the third week onwards, it was performed every 3 days until the end of the experiment on day 30. The specific procedure was as follows: Each mouse was injected intraperitoneally with fluorescein potassium salt solution (3 mg / mouse, prepared with sterile PBS, concentration 15 mg / mL, injection volume 200 μL). Ten minutes after injection, the mice were anesthetized in an isoflurane anesthesia induction chamber (induction concentration 4%, maintenance concentration 1.5%~2%), and then transferred to the dark chamber of the in vivo imaging system. Images were acquired using bioluminescence mode.

[0046] Sampling and Lung Metastasis Counting: All mice were euthanized after the final in vivo imaging was completed on day 30 post-modeling. The thoracic cavity was opened, and the lungs were completely removed. The lungs were rinsed with physiological saline to remove surface blood and observed under a dissecting microscope. The number of metastatic nodules (white or grayish-white nodules, ≥0.5 mm in diameter) on the lung surface of each mouse was counted. The lung tissue was then fixed in 4% paraformaldehyde for subsequent HE staining and pathological analysis.

[0047] Statistical analysis: Data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed on the obtained data. One-way ANOVA was used for comparisons among multiple groups. A p < 0.05 was considered statistically significant.

[0048] Lung tissue and nodule number in different groups of mice as follows Figure 3 As shown in the figure. The control group had pulmonary nodules, indicating that the model was successfully established; compared with the control group, the number of pulmonary metastatic nodules in the medium- and high-dose drug groups was significantly reduced (P<0.05).

[0049] Test Example 4 This test case uses the DARTS experiment to identify potential target proteins for β-sitosterol glycoside.

[0050] RIPA lysis buffer, protease inhibitors, Coomassie brilliant blue staining solution, and BCA protein quantification kit were purchased from Beyotime Biotechnology Co., Ltd.; TNC buffer was purchased from Shanghai Yuanye Technology Co., Ltd.; and Pronase was purchased from Shanghai Huiyi Biotechnology Co., Ltd.

[0051] The DARTS (Drug Affinity Responsive Target Stability) experiment is based on the principle that drugs can protect target proteins from protease degradation after binding to them. By comparing the differences in protein bands between the drug-treated group and the control group after protease treatment, potential target proteins of drug action can be identified.

[0052] Cell lysis buffer preparation: CTC-TJH-01 cells in logarithmic growth phase were harvested, the culture medium discarded, and washed twice with pre-chilled phosphate-buffered saline (PBS). 100 μL of pre-chilled RIPA lysis buffer (containing 1‰ PMSF serine protease inhibitor) was added, and the cells were incubated on ice for 20 minutes, vortexing every 5 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit, and the concentration was adjusted to 2 mg / mL. The cells were then aliquoted and stored at -80°C for later use.

[0053] Experimental Procedure: The above lysate was divided into six groups: a Control group, a Pronase-only group, and groups with concentrations of 50, 100, 500, and 1000 μM. Except for the Control group, each group received 0.06% Pronase. The specific procedure was as follows: Equal volumes of cell lysate (each containing 30 μg of total protein) were added to 50, 100, 500, and 1000 μM β-sitosterol glycoside solution (prepared with DMSO, ensuring a consistent final DMSO concentration across groups) and an equal volume of control solvent. The mixture was incubated at 25°C for 60 minutes to allow for complete drug-protein binding. Subsequently, except for the Control group, Pronase was added to each group to a final concentration of 0.06%, mixed thoroughly, and incubated at 25°C for 30 minutes. After the reaction, 5×SDS-PAGE loading buffer was immediately added to each tube, mixed well, and then heated at 100°C for 10 minutes to inactivate and denature the protease.

[0054] SDS-PAGE and Coomassie Brilliant Blue Staining: Take 10 μL of each of the above-treated samples and perform SDS-PAGE gel electrophoresis. Use a 10% separating gel and a 5% stacking gel, and perform electrophoresis under constant voltage conditions (stacking gel 80 V, 30 min; separating gel 120 V, 1 h). After electrophoresis, place the gel in Coomassie Brilliant Blue staining solution and stain on a shaker at 25°C for 1 hour. After staining, discard the staining solution, add destaining solution (50% pure water, 40% methanol, 10% glacial acetic acid), and shake on a shaker to destain. Change the destaining solution every 30 minutes until the background is clear and the protein bands are clearly visible.

[0055] Image acquisition and analysis: The decolorized gel was photographed to observe the differences in protein bands among the groups. Compared with the Pronase control group (group 2), the specific protein bands appearing in the β-sitosterol glycoside administration group due to drug protection are the potential target protein bands.

[0056] The gel in the DARTS experiment, such as Figure 4 As shown, the 42Kd molecular weight band exhibits a significant protein band difference compared to the pronase enzyme group alone, and this difference intensifies with increasing drug concentration. This suggests that the 42Kd molecular weight band contains a potential target protein of β-sitosterol glycoside. Differential bands were selected for gel excision, enzymatic digestion, and mass spectrometry identification to determine the potential target protein.

[0057] Test Example 5 This test case is a CETSA validation experiment for the β-sitosterol glycoside target protein.

[0058] RIPA lysis buffer, protease inhibitor, BCA protein quantification kit, rabbit secondary antibody, and mouse secondary antibody were purchased from Beyotime Biotechnology Co., Ltd.; PA2G4 antibody, SERPINB1 antibody, and DNAJA1 antibody were purchased from Ibotek Biotechnology Co., Ltd.; GAPDH was purchased from Sewell Biotechnology Co., Ltd.

[0059] The CETSA (Cellular Thermal Shift Assay) experiment is based on the principle that drug binding to target proteins enhances the thermal stability of those proteins. It verifies the interaction between the drug and candidate target proteins by detecting changes in protein solubility in solution at different temperatures. This experiment can further validate candidate targets screened by the DARTS experiment.

[0060] Cell lysis buffer preparation: CTC-TJH-01 cells in logarithmic growth phase were harvested, the culture medium discarded, and washed twice with pre-chilled phosphate-buffered saline (PBS). 50 μL of pre-chilled RIPA lysis buffer (containing phosphatase and protease inhibitors) was added, and the cells were incubated on ice for 20 minutes, vortexing every 5 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit, and the concentration was adjusted to 2 mg / mL for later use.

[0061] Drug incubation and temperature gradient treatment: The cell lysates were divided into two groups: a control group and a drug treatment group. An equal volume of DMSO was added to the control group, while a β-sitosterol glycoside solution with a final concentration of 35 μM was added to the drug treatment group. Both groups were incubated at 25℃ for 1 hour to allow the drug to fully bind to the protein. After incubation, each group was divided into six equal aliquots and placed in PCR tubes. A temperature gradient was then applied for heating. The heating conditions were as follows: 25℃, 35℃, 45℃, 50℃, 55℃, and 60℃. Heating time at each temperature was 3 minutes. After heating, the tubes were immediately placed on ice for 5 minutes to allow protein renaturation. The treated samples were centrifuged at 12,000 rpm for 15 minutes at 4℃, and the supernatant was collected. Each supernatant was mixed with 5×SDS-PAGE loading buffer and heated at 100℃ for 10 minutes to denature the protein. The tubes were then cooled on ice for later use.

[0062] Western blot analysis: Take 20 μL of each of the prepared samples and perform SDS-PAGE gel electrophoresis. Use a 10% separating gel and a 5% stacking gel, and perform electrophoresis under constant voltage conditions (stacking gel 80 V, 30 min; separating gel 120 V, 1 h). After electrophoresis, transfer the protein to a PVDF membrane using a wet transfer method. Transfer conditions: constant current 300 mA, transfer at ice bath for 60 min. After transfer, place the PVDF membrane in 5% skim milk blocking buffer (prepared by TBST) and block on a shaker at 25°C for 1 hour. After blocking, incubate the membrane with the following primary antibodies: PA2G4 antibody (rabbit), SERPINB1 antibody (rabbit), DNAJA1 antibody (rabbit), and GAPDH antibody (mouse), as internal controls. Incubate overnight on a shaker at 4°C. The next day, wash the PVDF membrane three times with TBST buffer for 10 minutes each time. Subsequently, the corresponding secondary antibodies, goat anti-rabbit IgG and goat anti-mouse IgG, were added and incubated at 25°C for 1 hour. The gel was then washed three times with TBST buffer, 10 minutes each time. The gel was then placed in a gel imaging system for development and photography.

[0063] Statistical analysis: The experiment was independently repeated three times, and the data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed on the obtained data. One-way ANOVA was used for comparisons among multiple groups, and a p-value < 0.05 was considered statistically significant.

[0064] The band development results are as follows Figure 5 As shown, the statistical results for PA2G4 protein, SERPINB1 protein, and DNAJA1 protein are as follows, from left to right: Figure 6 As shown, PA2G4 protein remained detectable as a distinct band at higher temperatures in the treatment group, while the band was significantly weakened at the same temperature in the control group (P<0.05). Compared with the control group, there were no significant differences in the protein bands of SERPINB1 and DNAJA1 proteins at higher temperatures in the treatment group (P>0.05). These results indicate that PA2G4 protein is a target protein of β-sitosterol glycoside.

[0065] Test Example 6 This test case compares the inhibitory cell migration activities of β-sitosterol and β-sitosterol glycoside.

[0066] F12K culture medium was purchased from Shanghai Yuanye Biotechnology Co., Ltd., the sonicator was manufactured by FUJIFILM VisualSonics, and the inverted microscope was manufactured by Nikon.

[0067] To compare the inhibitory effects of β-sitosterol and β-sitosterol glycoside on the migration ability of lung cancer cells, in order to clarify the influence of glycoside structure on drug efficacy.

[0068] Scratch assay: CTC-TJH-01 cells in logarithmic growth phase were used, and 5 × 10⁶ cells were injected into each well. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and cultured at 37°C in a 5% CO2 incubator until cell confluence reached over 90%. Using a 200 μL sterile pipette tip, uniform straight lines were drawn vertically on the cell monolayer in each well. Cells were gently washed three times with PBS to remove detached cells, followed by the addition of serum-free culture medium. Based on the effective concentration of β-sitosterol glycoside determined in previous experiments, two treatment groups were established: β-Sitosterol group: β-sitosterol was added to a final concentration of 35 μM; β-sitosterol glycoside group: β-sitosterol glycoside was added to a final concentration of 35 μM. Each group had three replicates. After drug administration, the 6-well plates were incubated at 37℃ in a 5% CO2 incubator. Images of the same scratch location were observed and photographed under an inverted microscope at 0 h and 24 h, and the scratch area of ​​each group was measured to calculate the scratch healing rate.

[0069] Scratch healing rate (%) = (0 h scratch area – 24 h scratch area) / 0 h scratch area × 100% Statistical analysis: The experiment was independently repeated three times, and the data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed on the obtained data. One-way ANOVA was used for comparisons among multiple groups, and a p-value < 0.05 was considered statistically significant.

[0070] Scratching results as follows Figure 7 As shown, compared with the β-sitosterol group, β-sitosterol glycoside significantly increased the inhibition of CTC-TJH-01 cell migration (P<0.05), indicating that the glycoside structure of β-sitosterol glycoside is the key to its inhibitory activity on migration.

[0071] Test Example 7 This test case was used to detect the CETSA effect of β-sitosterol and β-sitosterol glycosides on the binding of PA2G4 protein.

[0072] RIPA lysis buffer, protease inhibitor, BCA protein quantification kit, rabbit secondary antibody, and mouse secondary antibody were purchased from Beyotime Biotechnology Co., Ltd.; PA2G4 antibody was purchased from Ibotek Biotechnology Co., Ltd.; and GAPDH was purchased from Sewell Biotechnology Co., Ltd.

[0073] The CETSA (Cellular Thermal Shift Assay) method was used to compare the binding ability of β-sitosterol (aglycone) and β-sitosterol glycoside (glycoside) to PA2G4 protein in CTC-TJH-01 cells, in order to clarify the effect of glycoside structure on target interaction.

[0074] Preparation of cell lysis buffer: Take 8 × 10⁸ cells cultured in a six-well plate.5 CTC-TJH-01 cells / well were collected, the culture medium was discarded, and the cells were washed twice with pre-chilled phosphate-buffered saline (PBS). 50 μL of pre-chilled RIPA lysis buffer (containing a mixture of phosphatase and protease inhibitors) was added, and the cells were incubated on ice for 20 minutes, vortexing every 5 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein quantification kit, and the concentration was adjusted to 2 mg / mL for later use.

[0075] Drug incubation and temperature gradient treatment: The cell lysates were divided into two groups: β-sitosterol group: β-sitosterol was added to a final concentration of 35 μM; β-sitosterol glycoside group: β-sitosterol glycoside was added to a final concentration of 35 μM. The final concentration of DMSO was kept consistent across all groups. The samples were incubated at 25℃ for 1 hour to allow the drug to fully bind to the protein. After incubation, each group of samples was divided into 6 equal parts and placed in PCR tubes. A temperature gradient was set for heating treatment. The heating conditions were as follows: 25℃, 35℃, 45℃, 50℃, 55℃, and 60℃. The heating time at each temperature point was 3 minutes. After heating, the samples were immediately placed on ice for 5 minutes to allow the protein to renature. The treated samples were centrifuged at 4℃ and 12,000 rpm for 15 minutes, and the supernatant was collected. Each supernatant was taken, 5×SDS-PAGE loading buffer was added, mixed well, and heated at 100℃ for 10 minutes to denature the protein. The samples were then cooled on ice for later use.

[0076] Western blot analysis: 20 μL of each of the prepared samples were subjected to SDS-PAGE gel electrophoresis. A 10% separating gel and a 5% stacking gel were used, and electrophoresis was performed under constant voltage conditions (stacking gel 80 V, 30 min; separating gel 120 V, 1 h). After electrophoresis, the proteins were transferred to a PVDF membrane using a wet transfer method under the following conditions: constant current 300 mA, transfer in an ice bath for 60 minutes. After transfer, the PVDF membrane was placed in 5% skim milk blocking buffer (prepared by TBST) and blocked on a shaker at 25°C for 1 hour. After blocking, PA2G4 antibody was added and incubated overnight at 4°C. The next day, after washing, rabbit antibody was added and incubated at 25°C for 1 hour. The membrane was then developed and photographed using a gel imaging system. GAPDH antibody was then added, and the same steps were followed for development and photographing.

[0077] Statistical analysis: The experiment was independently repeated three times, and the data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed on the obtained data. One-way ANOVA was used for comparisons among multiple groups, and a p-value < 0.05 was considered statistically significant.

[0078] WB results are as follows Figure 8As shown, in the β-sitosterol glycoside group, significant protein bands of PA2G4 protein were detected at 50℃, 55℃, and 60℃. The band intensity decreased dose-dependently with increasing temperature, but was significantly stronger than that in the β-sitosterol group (P<0.05). These results indicate that β-sitosterol glycosides can bind to PA2G4 protein and enhance its thermal stability, while β-sitosterol does not bind to PA2G4 protein at the same concentration (35 μM), suggesting that the glycoside structure is crucial for the interaction between β-sitosterol glycosides and the PA2G4 target.

[0079] The applicant declares that the technical solution of this invention is illustrated by the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of raw materials for the products of this invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0080] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0081] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. Application of β-sitosterol glycoside in the preparation of products for the treatment of lung cancer.

2. The application according to claim 1, characterized in that, The β-sitosterol glycoside exerts its therapeutic effect on lung cancer by targeting and binding to the PA2G4 protein.

3. The application according to claim 1 or 2, characterized in that, The product also includes pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable excipients include any one or a combination of at least two of the following: carriers, diluents, excipients, fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, antibacterial agents, or buffers.

5. Application of β-sitosterol glycoside in the preparation of products for inhibiting lung cancer metastasis.

6. The application according to claim 5, characterized in that, The product also includes pharmaceutically acceptable excipients; Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

7. Application of β-sitosterol glycoside in the preparation of products for inhibiting the proliferation and migration of lung cancer cells.

8. The application according to claim 7, characterized in that, The product also includes pharmaceutically acceptable excipients; Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

9. Application of β-sitosterol glycoside in the preparation of PA2G4 protein-targeting binders.

10. The application according to claim 9, characterized in that, The product also includes pharmaceutically acceptable excipients; Preferably, the excipients include any one or a combination of at least two of the following: carrier, diluent, excipient, filler, binder, wetting agent, disintegrant, emulsifier, cosolvent, solubilizer, osmotic pressure regulator, surfactant, coating material, colorant, pH adjuster, antioxidant, antibacterial agent, or buffer.

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