Application of berberine in preparation of STAT3 inhibitor

Abstract

The application discloses application of berberine in preparation of STAT3 inhibitors. The application proves through experiments that berberine can directly combine with STAT3 protein, and can play an anti-tumor role of inhibiting cell proliferation and migration by inhibiting phosphorylation of a Tyr705 site of STAT3, down-regulating expression of downstream c-Myc protein, and inducing double blockage of MCF-7 cells in G1 and G2 phases. The application can provide a candidate molecule and a theoretical basis for new drug development with STAT3 as a target, and has important theoretical significance and potential application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of berberine in the preparation of STAT3 inhibitors. Background Technology

[0002] Activator of transcription 3 (STAT3) is an important member of the STAT family, playing a crucial role in regulating biological processes such as cell proliferation, apoptosis, differentiation, angiogenesis, and immune responses. Under normal physiological conditions, STAT3 signaling is strictly regulated to maintain a transiently activated state; however, in various malignant tumors, STAT3 exhibits persistent abnormal activation. Numerous studies have shown that STAT3 is persistently abnormally activated in breast cancer, promoting malignant proliferation, invasion, metastasis, and chemotherapy resistance of breast cancer cells by upregulating the expression of anti-apoptotic proteins (such as Survivin, Bcl-xL, and Mcl-2), cell cycle regulators (such as Cyclin D1 and c-Myc), and matrix metalloproteinases (such as MMP-2 and MMP-9). Furthermore, abnormal activation of STAT3 is closely related to tumor immune escape and the maintenance of tumor stem cell characteristics, further exacerbating tumor progression and treatment resistance. Therefore, STAT3 is widely recognized as an important potential target for breast cancer treatment, and the development of small-molecule inhibitors targeting STAT3 has become a hot topic in anti-breast cancer drug development.

[0003] In recent years, significant progress has been made in research on STAT3 inhibitors. Studies have shown that the PARP inhibitor olaparib can induce elevated IL-6 levels in triple-negative breast cancer (TNBC) cells, thereby activating the STAT3 signaling pathway and leading to PARP inhibitor resistance. Furthermore, the combined use of the novel STAT3 inhibitor LLL12B significantly enhances the anti-tumor effect of olaparib, suggesting a synergistic effect between STAT3 inhibitors and chemotherapy or targeted therapies. Zheng Dongning's research found that Pulsatilla chinensis saponin E2, a novel STAT3 inhibitor, has a significant inhibitory effect on metastasis of human triple-negative breast cancer xenografts, further confirming the potential of STAT3 targeting in breast cancer treatment. In addition, Li Xiaojie et al.'s research showed that chronic stress can promote angiogenesis and mediate the progression of triple-negative breast cancer by regulating the β2-AR / IL-6 / STAT3 pathway, suggesting that the STAT3 signaling pathway also plays an important role in the regulation of the tumor microenvironment. These studies fully demonstrate the enormous potential of STAT3 targeting in breast cancer treatment. Summary of the Invention

[0004] The purpose of this invention is to provide a new use for berberine, namely, as a STAT3 inhibitor and for its use in the preparation of a therapeutic drug for STAT3-abnormally activated breast cancer.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: A novel use of berberine: its application in the preparation of STAT3 inhibitors.

[0006] Furthermore, berberine was used to prepare therapeutic drugs for diseases caused by abnormal activation of STAT3.

[0007] Furthermore, the diseases caused by abnormal STAT3 activation include STAT3-abnormally activated breast cancer.

[0008] Furthermore, berberine can be used as the sole active ingredient, combined with pharmaceutically acceptable excipients to formulate a pharmaceutically acceptable dosage form. The pharmaceutically acceptable dosage form is an injection.

[0009] Furthermore, the therapeutic drug can also be prepared by combining berberine with other antitumor active ingredients.

[0010] The significant advantages of this invention are: This invention experimentally demonstrates that berberine, a small molecule compound derived from traditional Chinese medicine, directly binds to the STAT3 protein. The interactions are primarily hydrogen bonding, electrostatic interactions, and hydrophobic interactions, with static quenching as the binding mechanism and hydrophobic interaction being the main driving force. Berberine can induce dual G1 and G2 phase arrest in MCF-7 breast cancer cells by inhibiting STAT3 phosphorylation and downregulating the expression of its downstream target protein c-Myc, thereby inhibiting cell proliferation and migration and exerting an anti-tumor effect. Therefore, berberine can be used as an inhibitor targeting STAT3 and for the treatment of STAT3-abnormally activated breast cancer. Attached Figure Description

[0011] Figure 1 The graph (A) shows the changes in the response signal of different concentrations of berberine to STAT3 protein in the affinity assay, and the binding curve obtained by fitting it (B).

[0012] Figure 2 This is a diagram showing the molecular docking of berberine with STAT3 protein.

[0013] Figure 3 The fluorescence spectra of berberine and STAT3 protein at 30℃ (A) and 40℃ (B) are shown.

[0014] Figure 4 Stern-Volmer curves of berberine-STAT3 fluorescence quenching at different temperatures.

[0015] Figure 5 The curves of lg[(F0-F) / F] versus lg[Q] for berberine-STAT3 at different temperatures are shown.

[0016] Figure 6 Figure showing the effect of berberine on the proliferation of MCF-7 cells (compared to the 0 µmol / L concentration group) **** P <0.0001).

[0017] Figure 7 This is a comparison of the wound healing rate of MCF-7 cells after treatment with berberine for different time periods.

[0018] Figure 8 Figure showing the effect of berberine on the expression levels of related proteins in MCF-7 cells (compared to the 0 μmol / L control group) **** P <0.0001; *** P <0.001;** P <0.01;* P <0.05). Detailed Implementation

[0019] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0020] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods. Example

[0021] 1 Experimental Methods 1.1 Determination of the affinity between STAT3 and berberine STAT3 protein was immobilized on the surface of a CM5 sensor chip using a manual coupling method. A mixture of EDC and NHS (1:1 volume ratio) was injected into the chip surface at a flow rate of 10 μL / min for 7 min to activate the protein. The STAT3 protein was diluted to 100 μg / mL with 10 mM sodium acetate buffer (pH 4.5) and then injected into the activated chip surface at a flow rate of 10 µL / min until the target immobilization level was reached. Subsequently, 1 mol ethanolamine hydrochloride solution was injected at a flow rate of 10 μL / min to block unreacted active groups. A blank reference channel was set up for background subtraction and refractive index correction. Berberine was dissolved and serially diluted to five concentrations (1.2, 3.7, 11, 33, and 100 µM). A multi-cycle kinetic mode was used, with injection at a flow rate of 30 μL / min, a binding time of 60 s, and a dissociation time of 60 s. The equilibrium dissociation constant (KD) was calculated from the obtained sensor map using steady-state affinity fitting.

[0022] 1.2 Molecular docking of STAT3 with berberine The 3D structure of the STAT3 protein (PDB, PDB ID 1BG1) was downloaded from the Protein Database (PDB). The 3D structure of berberine was downloaded from the Pubchem database. Pre-treatment of the STAT3 protein and berberine was performed using Discovery Studio molecular docking software. For the STAT3 protein, surrounding water molecules and ligand molecules were removed, and hydrogen atoms were added to simulate the in vivo state of STAT3. The STAT3 protein was prepared using the Prepare Protein program, and binding sites were generated from the STAT3 receptor structure using Discovery Studio. Molecular docking was performed at site1.

[0023] 1.3 Fluorescence spectra of STAT3 and berberine Berberine standard was dissolved in DMSO to prepare a 5 mmol / L stock solution, which was then serially diluted with 1×PBS to 0, 4, 8, 16, and 20 μmol / L. These gradient solutions were then added to opaque black 96-well plates. STAT3 protein was prepared into a 500 μg / mL stock solution with purified water, and then added to the berberine gradient solutions to ensure a final STAT3 concentration of 5 μg / mL in each sample. After thorough mixing, the mixture was incubated at 25°C for 30 min. Fluorescence spectra were recorded using a multi-mode microplate reader at 30°C and 40°C (excitation wavelength set to 280 nm, emission spectrum scanning range 290–450 nm, excitation and emission slit widths both 2 nm, measurement mode: top reading).

[0024] 1.4 Cellular Experiments with STAT3 and Berberine (1) Cell culture Remove the MCF-7 cell cryovials from liquid nitrogen and rapidly thaw them in a 37°C water bath. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in 1 mL of complete culture medium. Transfer the resulting cell suspension to a culture dish containing 6 mL of complete culture medium and incubate at 37°C, 5% CO2, and saturated humidity. When the cell density reaches 80%–90% confluence, passage the cells, aliquoting the cell suspension into new culture dishes at a 1:2 ratio for subsequent experiments.

[0025] (2) CCK-8 assay for cell proliferation MCF-7 cells in good logarithmic growth phase were collected, counted, and then 1.5 × 10⁻⁶ cells were harvested. 4Cell suspension was seeded into 96-well plates at a seeding rate of 1 cell / well and cultured for 24 h to allow cell adhesion. When the cell density reached 70%–80%, the 96-well plates were removed, and cell adhesion and morphology were observed under a microscope. The old culture medium was then discarded, and 100 μL of complete culture medium containing different concentrations of berberine (0, 25, 50, 75, 100 μM) was added to each well, with three replicates for each concentration. A blank control group (containing only complete culture medium) was also included, and the plates were cultured for another 24 h. CCK-8 working solution was prepared at a 1:10 ratio, and 100 μL was added to each well, followed by incubation for 1 h. The absorbance at 450 nm was measured using a microplate reader, and the OD values ​​of each well were recorded for data analysis.

[0026] (3) Cell scratch test MCF-7 cells in the logarithmic growth phase were digested, centrifuged, resuspended, and counted to prepare a density of 3 × 10⁻⁶ cells / cells. 5 Single-cell suspension of cells / mL was prepared. Horizontal lines were evenly drawn on the back of a 6-well plate with a marker (at least three lines per well). 2 mL of cell suspension was seeded into each well, and the plates were incubated at 37°C with 5% CO2 for 24 h. When cell confluence reached approximately 90%, a 200 μL sterile pipette tip was used to scratch the cells perpendicularly to the marked lines on the back. The cells were gently washed twice with PBS to remove detached cells. Then, 2 mL of the corresponding culture medium was added to each well: blank control medium, medium containing 0.5% DMSO, and medium containing 18 μM berberine. Microscopic images were taken at 24 and 48 h after scratching to record the wound healing progress. The scratch area was measured using ImageJ software, and the wound healing rate was calculated using the following formula: Scratch healing rate (%) = [(0 h scratch area - scratch area at the corresponding time point) / 0 h scratch area] × 100%.

[0027] (4) Cell cycle experiment Take MCF-7 cells in the logarithmic growth phase and in good condition, and suspend the cells at 2.5 × 10⁻⁶. 5Cells were seeded at a density of 1 / mL in 6-well plates and cultured for 24 h. Blank medium, medium containing 0.5% DMSO, medium containing 9 μM berberine, and medium containing 18 μM berberine were added to each well, and cultured for another 24 h. Cell supernatant (containing detached cells) was collected from each well and transferred to the corresponding centrifuge tube. Cells were washed once with PBS, and the washes were collected together. 0.5 mL of EDTA digestion solution was added to each well, and the cells were digested at 37°C until they became rounded and detached. The digestion solution was discarded, and the collected complete culture medium was added to terminate the digestion. The cells were gently pipetted to completely detach. The cell suspension was transferred to the corresponding centrifuge tube, centrifuged, washed, and then 1 mL of pre-chilled 70% ethanol was added while gently vortexing to mix. The cells were fixed overnight at 4°C. After centrifugation and washing, 500 μL of freshly prepared PI staining working solution was added and the cells were gently resuspended. The cells were incubated at room temperature in the dark for 30 min, then filtered and placed on ice in the dark for analysis. Flow cytometry was used for detection.

[0028] (5) Western blotting MCF-7 cells in the logarithmic growth phase were harvested and treated at a concentration of 2.5 × 10⁻⁶. 5 Cells were seeded at 2 mL / well in 6-well plates and cultured for 24 h. Afterward, complete culture medium containing different concentrations of berberine (0, 9, 18, 36, 54 μM) was added to each well, while the control group received complete culture medium containing 0.5% DMSO. Cells were cultured for another 24 h, then the culture medium was discarded, and the cells were washed twice with pre-chilled PBS. 100 μL of RIPA lysis buffer (containing PMSF and a phosphatase inhibitor) was added to each well, and the cells were lysed on ice for 1 h. The lysis buffer was then collected using a cell scraper, sonicated (200 W, ice bath), and centrifuged at 12000 rpm for 5 min at 4 °C. The supernatant was used to determine protein concentration using the BCA method. Protein concentrations were standardized across groups using RIPA lysis buffer, and 5×SDS loading buffer was added. The cells were then heated in a 100 °C metal bath for 10 min to denature the proteins.

[0029] Protein samples were separated by SDS-PAGE gel electrophoresis (150 V, 1 h), and then transferred to a methanol-activated PVDF membrane using a wet transfer method (400 mA, 30 min). After transfer, the membrane was blocked with rapid blocking buffer for 15 min and washed three times with PBST (3 min each time). The membrane was cut according to the molecular weight of the target protein and incubated overnight at 4 °C with the corresponding primary antibody (p-STAT3, c-Myc, GAPDH, etc.). The next day, the membrane was washed three times with TBST (10 min each time), and then incubated at room temperature for 1 h with secondary antibody. After washing three more times with TBST (10 min each time), the membrane was developed using ECL chemiluminescence buffer and images were acquired.

[0030] 2. Results and Discussion 2.1 Determination of the affinity between STAT3 and berberine Different concentrations (1.2, 3.7, 11, 33, 100 µM) of berberine were selected, and the affinity between berberine and STAT3 protein was determined using SPR technology. Figure 1 The results showed that the binding response signal (RU) increased in a concentration-dependent manner with increasing berberine concentration, and the signal rose rapidly during the binding phase and decreased rapidly during the dissociation phase, indicating rapid binding and dissociation with STAT3 protein (A). Binding curves obtained by steady-state (1:1) affinity fitting (B) showed that the equilibrium dissociation constant KD for the interaction between berberine and STAT3 protein was 5.235 × 10⁻⁶. -5 M (52.35 μM).

[0031] SPR affinity assay results showed that berberine could bind directly and specifically to STAT3 protein, with a KD value in the micromolar range (52.35 μM). The binding kinetics were fast binding and fast dissociation, suggesting its potential as a small molecule inhibitor of STAT3.

[0032] 2.2 Molecular docking of STAT3 with berberine Figure 2 Molecular docking results showed that berberine forms stable interactions with multiple key amino acid residues in the A chain of the STAT3 protein, with a CDOCKER binding energy of -26.3608 kJ / mol. Specifically, berberine forms π-anionic interactions with ASPA:570 and ASPA:566 residues, π-alkyl stacking interactions with PRO A:471 residue, π-cation interactions with LYS A:573 residue, hydrogen bonding interactions with THR A:515 residue, and amide-π stacking interactions with ILE A:467 residue. The van der Waals interactions marked in green in the figure further enhance the binding stability of berberine to the STAT3 protein. These multiple interaction types collectively suggest that berberine has a good binding affinity to the STAT3 protein and can serve as a potential inhibitor of STAT3.

[0033] 2.3 Fluorescence spectra of STAT3 and berberine Fluorescence spectroscopy, with its advantages of high sensitivity and real-time monitoring, is a classic technique for studying protein-small molecule interactions. Proteins possess natural fluorescence properties due to the presence of tryptophan, tyrosine, and phenylalanine residues, with tryptophan exhibiting the highest quantum yield and being the primary source of protein fluorescence. When a protein binds to a ligand, its conformation or microenvironment may change, leading to alterations in the intrinsic fluorescence signal. By analyzing the fluorescence signal, information such as the binding constant, binding site, and thermodynamic parameters of small molecules can be obtained. Figure 3The fluorescence spectra of berberine and STAT3 protein at different temperatures show that, at 30℃ and 40℃, STAT3 protein exhibits maximum fluorescence intensity in the emission wavelength range of 320–340 nm. Furthermore, the fluorescence intensity of STAT3 decreases with increasing berberine concentration, indicating that the interaction between berberine and STAT3 induces fluorescence quenching of STAT3 protein, forming a weakly fluorescent berberine-STAT3 complex. When the temperature increases to 40℃, the fluorescence intensity of STAT3 protein significantly decreases compared to 30℃, demonstrating that temperature affects the interaction between berberine and STAT3; the higher the temperature, the stronger the fluorescence quenching effect of berberine on STAT3.

[0034] The bimolecular quenching constant Ksv was calculated based on the Stern-Volmer equation, and the fluorescence lifetime τ0 = 10 was used. -8 s by K SV Further calculations yielded the fluorescence quenching rate constant Kq, and the results are shown in Table 1. As can be seen from Table 1, the Kq values ​​at different temperatures are 3.784 × 10⁻⁶. 12 L·mol -1 ·s -1 (30℃), 3.634×10 12 L·mol -1 ·s -1 (40℃), all are much greater than the maximum rate constant of diffusion-controlled quenching in biological macromolecular systems (2.0×10). 10 L·mol -1 ·s -1 This indicates that the quenching process is not simply caused by intermolecular diffusion and collision; and as the temperature increases, both the Ksv and Kq values ​​decrease, indicating that the fluorescence quenching mechanism between berberine and STAT3 protein is static quenching, and the two interact by forming a ground-state complex.

[0035] Table 1. Stern-Volmer fluorescence quenching rate constant Kq and bimolecular quenching constant Ksv at different temperatures

[0036] According to the formula (F0 / F=1+Kqτ0[Q]=1+Ksv[Q]), where F0 and F represent the fluorescence intensity of the STAT3 protein solution and the complex after adding berberine, respectively, [Q] is the concentration of berberine, Kq is the rate constant in the quenching process, Ksv is the Stern-Volmer quenching constant, and τ0 is the average lifetime of the fluorescent polymer, which is 10. -8 s) Plotting F0 / F against [Q] yields Stern-Volmer curves for fluorescence quenching at different temperatures. Figure 4It can be seen that the curves at different temperatures are all linear and have good fit.

[0037] Plotting lg[(F0-F) / F] against lg[Q] yielded the lg[(F0-F) / F] vs. lg[Q] curves for berberine-STAT3 at different temperatures. The binding constant Ka and binding site n were then calculated based on the double logarithmic equation for static quenching. The study showed that when Ka is in the range of 10... 5 ~10 7 L·mol -1 This indicates a strong binding force between the protein and small molecules, when Ka is in the range of 10. 2 ~10 4 L·mol -1 This indicates a moderate binding force between the protein and the small molecule. Table 2 shows that the binding site n between berberine and STAT3 protein is less than 1 at different temperatures, indicating that there is only one binding site between berberine and STAT3 protein, and the complex formed by berberine and STAT3 is of the 1:1 type. Simultaneously, the Ka value between berberine and STAT3 protein is consistently within 10 at different temperatures. 2 ~10 4 L·mol -1 The presence of these parameters indicates a moderate binding affinity between berberine and the STAT3 protein.

[0038] Table 2. Binding sites n and binding constant Ka of berberine-STAT3 at different temperatures

[0039] Thermodynamic parameters were calculated using the Van't Hoff equation and thermodynamic formulas. In Table 3, ΔG < 0 indicates that the binding reaction between berberine and STAT3 protein is spontaneous. Both ΔH and ΔS > 0 indicate that the interaction between berberine and STAT3 protein is primarily hydrophobic. This suggests that hydrophobic interaction is the main force driving the binding process between berberine and STAT3.

[0040] Table 3 Thermodynamic parameters of berberine and STAT3

[0041] 2.4 CCK-8 assay for cell proliferation The effect of berberine on the proliferation of human breast cancer MCF-7 cells was detected using the CCK-8 assay. Figure 6 As shown, berberine inhibited the proliferation of MCF-7 cells in a concentration-dependent manner within the range of 0–100 µM (A); the IC50 of berberine on MCF-7 cells after 24 h was obtained by nonlinear regression fitting. 50The value was 18.96 µM (B). These results indicate that berberine has significant in vitro anti-proliferative activity against MCF-7 cells.

[0042] 2.5 Cell Scratch Assay The ImageJ software was used to quantitatively calculate the change in the area of ​​the scratch region, and the results are as follows: Figure 7 As shown, compared with the blank control group, the scratch healing rate of MCF-7 cells was significantly reduced after treatment with 18 µM berberine for 24 h and 48 h, indicating that berberine can significantly inhibit the migration of MCF-7 cells. Its inhibitory effect was already apparent at 24 h and lasted until 48 h.

[0043] 2.6 Cell Cycle The effect of berberine on the cell cycle distribution of MCF-7 cells was detected by flow cytometry combined with PI staining. Cells were treated with different concentrations of berberine (9, 18 μM) for 24 h, fixed and stained, and the percentage of each cell cycle phase (G1, S, G2) was analyzed by flow cytometry. The results are shown in Table 4.

[0044] Table 4 shows that, compared with the control group, there were no statistically significant differences in the proportion of cells in each phase in the DMSO group. P >0.05), indicating that 0.5% DMSO had no significant effect on the cell cycle distribution of MCF-7 cells; however, after treatment with 9 μM berberine for 24 h, the proportion of cells in the G1 phase significantly increased to 59.07±1.90% ( P <0.01), the proportion of S-phase cells decreased significantly to 14.70±0.35% ( P <0.01), the proportion of cells in G2 phase significantly increased to 27.20±1.87% ( P <0.05%, indicating that 9 μM berberine can induce G1 phase arrest in MCF-7 cells, accompanied by G2 phase cell accumulation; simultaneously, after treatment with 18 μM berberine for 24 h, the proportion of G1 phase cells further increased to 61.17±3.58% ( ). P <0.05), the proportion of S phase cells decreased to 9.44±3.57% ( P <0.01), the proportion of cells in G2 phase increased to 30.40±0.17% ( P <0.01). Compared with the 9 μM group, the 18 μM group showed a stronger G1 phase arrest effect, a lower proportion of cells in the S phase, and more significant accumulation in the G2 phase, which was concentration-dependent. This indicates that berberine exerts its anti-tumor effect by inhibiting the proliferation of MCF-7 cells by arresting cell cycle progression.

[0045] Table 4. Effects of berberine on the cell cycle of MCF-7 cells (x±s)

[0046] 2.7 Western blotting Figure 8 Western blot results showed that berberine treatment significantly affected the phosphorylation level of STAT3 and the expression of its downstream target protein c-Myc in MCF-7 cells. Compared with the DMSO control group, after 24 h of treatment with different concentrations (9, 18, 36, 54 μM) of berberine, the protein expression level of p-STAT3 (Tyr705) decreased in a concentration-dependent manner, indicating that the inhibitory effect of berberine on STAT3 phosphorylation was clearly concentration-dependent. Simultaneously, the expression level of c-Myc, the downstream target protein of STAT3, also showed a similar concentration-dependent downregulation trend, which was largely consistent with that of p-STAT3. These results indicate that the inhibitory effect of berberine on c-Myc expression may be closely related to its inhibition of STAT3 phosphorylation. That is, berberine can effectively inhibit the phosphorylation activation of STAT3, thereby downregulating the expression of c-Myc protein and ultimately affecting cell cycle progression and cell proliferation activity. This suggests that berberine may exert its anti-tumor effect by inhibiting the STAT3 / c-Myc signaling pathway, providing direct protein-level evidence for berberine as a potential anti-breast cancer drug targeting STAT3.

[0047] The above experiments confirm that berberine, derived from traditional Chinese medicine, can directly bind to STAT3 protein. The main binding modes are hydrogen bonding, electrostatic interaction, and hydrophobic interaction. This binding is a spontaneous, statically quenched process, with hydrophobic interaction being the primary driving force. Berberine exerts its antitumor mechanism by inhibiting STAT3 Tyr705 phosphorylation and downregulating downstream c-Myc protein expression, thereby inducing dual arrest of MCF-7 cells in both G1 and G2 phases.

[0048] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. Application of berberine in the preparation of STAT3 inhibitors.

2. The application of berberine in the preparation of therapeutic drugs for diseases caused by abnormal activation of STAT3, characterized in that, Diseases caused by abnormal STAT3 activation include STAT3-abnormally activated breast cancer.

3. The application according to claim 1 or 2, characterized in that, Berberine was used as the sole active ingredient and formulated into a pharmaceutically acceptable dosage form with pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable dosage form is an injection.

5. The application according to claim 2, characterized in that, The therapeutic drug was prepared by combining berberine with other antitumor active ingredients.

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