Bufogenin composition as well as preparation and application thereof

Abstract

The invention relates to the technical field of new application of bufogenin compositions, and discloses a bufogenin composition as well as a preparation and application thereof. The bufogenin composition is one or more of bufalin, cinobufogenin and resibufogenin, and the purity of the bufogenin composition is gt; 90%. The bufogenin composition remarkably inhibits tumor growth in vitro and in vivo, Hepa1-6 cells are subjected to immunogenic cell death through apoptosis, stimulated maturation of dendritic cells is further activated, corresponding immunosuppressive cells are depleted at the same time, and therefore the tumor microenvironment of liver cancer immunosuppression is improved, and the liver cancer immunosuppression effect is improved. The killing and inhibiting effects on Hepa1-6 tumor-bearing mice are enhanced. The bufogenin composition has important research significance and application significance in developing medicines or health-care products for treating or synergistically treating liver cancer by using the bufogenin composition.

Description

Technical Field

[0001] The present invention relates to the technical field of new applications of bufotoxin compositions, and more particularly to the application of bufotoxin compositions in the preparation of medicines or health products for treating liver cancer. Background Art

[0002] Hepatocellular carcinoma (HCC) is the leading cause of cancer death in the world, accounting for approximately 760,000 deaths in 2022. Emerging immunotherapeutic strategies, such as cancer vaccines, adoptive cell therapy, and immune checkpoint inhibitors, have shown great promise in the treatment of various cancers. However, these strategies often fail in HCC treatment, mainly due to the immunosuppressive properties of the tumor microenvironment (TME). The TME has developed multiple mechanisms to promote tumor immune escape, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and other immunosuppressive vectors. Therefore, strategies to transform the immunosuppressive “cold” TME into an immunostimulatory “hot” TME will provide new avenues for HCC immunotherapy.

[0003] Immunogenic cell death (ICD) refers to a form of regenerative cell death that can initiate adaptive immune responses against endogenous (cellular) or exogenous (viral) antigens provided by the dying cell. ICD is often accompanied by the activation of damage-associated molecular patterns (DAMPs). For example, three important ICD-related DAMPs are calreticulin (CRT), adenosine triphosphate (ATP), and high-mobility group protein B1 (HMGB1). CRT is exposed on the cell membrane surface and acts as an "eat me" signal to activate dendritic cells (DCs) and subsequently tumor-associated antigens (TAAs). ATP is secreted into the environment outside the cell membrane and acts as a "find me" signal to recruit DCs to the tumor. HMGB1 is released into the extracellular environment and promotes the production of cytotoxic CD8 TAAs. + Increasing evidence suggests that the triggering of ICD may be a potential strategy to reshape the TME of immunosuppressive agents. Therefore, during the treatment of HCC, effectively inducing ICD in liver cancer cells may play a tumor vaccine-like role, triggering a strong antitumor immune response and immune memory, providing a promising strategy for eradicating HCC.

[0004] Some chemotherapy drugs can trigger ICD, but the number of such drugs is very small, such as traditional drugs oxaliplatin (OxP), bortezomib (BTZ), mitoxantrone (MIT), etc. These chemotherapy drugs can not only cause chemotherapy-mediated cytotoxicity, but also restore the immunogenicity of tumor cells, and jointly achieve chemotherapeutic immunotherapy of tumors. Therefore, the development of new ICD drugs has attracted widespread attention.

[0005] Bufacine (B), cinobufagin (C) and resibufagin (R) are the main components of bufotoxin (BU), a natural cardiotonic steroid isolated from the traditional Chinese medicine toad venom, which has significant anti-tumor activity and shows certain anti-tumor activity against colorectal cancer, non-small cell lung cancer, breast cancer, pancreatic cancer, melanoma, osteosarcoma and other cell lines. Its efficacy and anti-tumor molecular mechanisms mainly include anti-proliferation, induction of cell apoptosis and anti-angiogenesis. Regarding the anti-tumor molecular mechanism of bufotoxin-based compositions, different research teams have revealed different molecular mechanisms in different tumors and cell lines, which may be related to inducing mitochondrial apoptosis, inhibiting the expression of anti-apoptotic proteins such as Bcl-2, and affecting Notch, GSK-3β, NF-kB and other signaling pathways.

[0006] Therefore, it is of great significance to study whether bufotoxin-based compositions can be used as a new type of ICD drug to treat liver cancer. Summary of the invention

[0007] In view of this, the present invention provides a bufotoxin composition and a preparation thereof, and provides the use of the bufotoxin composition in the preparation of drugs or health products for treating liver cancer, which can solve the above-mentioned defects in the prior art.

[0008] One of the purposes of the present invention is to provide a bufotoxin composition, wherein the bufotoxin composition is one or more of bufalin, cinobufogenin and resibufogenin.

[0009] Preferably, the chemical structural formula of the bufotoxin composition is:

[0010]

[0011] The molecular formula of Bufalin is C 24 H 34 O4:

[0012]

[0013] The molecular formula of resibufogenin is C 24 H 32 O4:

[0014]

[0015] The molecular formula of Cinobufagin is C 26 H 34 O6.

[0016] Preferably, the purity of the bufotoxin composition is greater than 90%.

[0017] Furthermore, the bufotoxin composition also contains tautomers and / or optical isomers of the active ingredients of bufalin, cinobufogenin and resibufogenin.

[0018] Furthermore, the bufotoxin composition mainly comprises bufalin, cinobufogenin and resifobufogenin.

[0019] Preferably, the mass ratio of bufalin, cinobufogenin and resifobufogenin is 1-5:3-7:1-4.

[0020] Preferably, the mass ratio of bufalin, cinobufogenin and resifobufogenin is 2-4:4-6:1-3.

[0021] The second object of the present invention is to provide a bufotoxin composition preparation, comprising a solvate, a pharmaceutically acceptable salt or ester, or a pharmaceutically acceptable prodrug or derivative of the bufotoxin composition.

[0022] Furthermore, the bufotoxin composition preparation is to prepare the bufotoxin composition or its pharmaceutically acceptable salt into injection, tablet, powder injection, granule, capsule, nano preparation.

[0023] Among them, nano preparations are preferred, such as nanocrystals, nano suspensions, nano particles, etc. The nano preparations may contain corresponding pharmaceutical excipients to achieve the purpose of sustained release, and the particle size of the nano preparations is 200-250 nm.

[0024] The third object of the present invention is to provide an application of a bufotoxin composition for use in the preparation of tumor treatment drugs or health products.

[0025] Furthermore, the tumor is liver cancer.

[0026] The anti-tumor molecular mechanism of the present invention is that the bufotoxin composition can induce apoptosis of tumor cells, further cause immunogenic cell death of tumor cells, activate the immunogenicity of tumor cells, reshape the immunosuppressive microenvironment of the tumor, and is used for the treatment of liver cancer.

[0027] The bufotoxin composition can induce apoptosis of Hepa1-6 tumor cells, further cause immunogenic cell death of Hepa1-6 tumor cells, release ATP, CRT, HMGB1; further, stimulate dendritic cell maturation, deplete Treg, MDSc, ​​increase CD4 + T, CD8 + T infiltration to reshape the tumor microenvironment; further, the toad venom ligand composition can reshape the immunosuppressive tumor microenvironment of liver cancer into an immune-activated microenvironment to inhibit tumor growth.

[0028] It can be seen from the above technical solution that, compared with the prior art, the beneficial effects achieved by the present invention are:

[0029] 1. The present invention confirms for the first time the ability of the bufotoxin composition to induce immunogenic cell death effects on Hepa1-6 cells in vitro. In the Hepa1-6 tumor-bearing mouse model, the bufotoxin composition can induce ICD effects in tumor tissues and successfully release damage-related molecules mainly composed of calreticulin, adenosine triphosphate, and high-mobility group protein B1. It has important research and application significance for using the bufotoxin composition to develop drugs or health products for the treatment or synergistic treatment of liver cancer.

[0030] 2. Experiments have shown that the ICD effect induced by the bufotoxin composition can effectively stimulate the activation of dendritic cells and increase CD4 + T and CD8 + The infiltration of T cells and the simultaneous depletion of MDSc and Treg cells successfully reshaped the immunosuppressive tumor microenvironment of hepatocellular carcinoma into an immune-activated microenvironment, effectively inhibited Hepa1-6 tumor growth, exerted a tumor vaccine-like effect, triggered a strong antitumor immune response and immune memory, and provided a promising strategy for the eradication of HCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0032] Figure 1 The proliferation inhibition and apoptosis effect of the bufotoxin composition on Hepa1-6 cells. (A is the cytotoxicity of Hepa1-6 cells at 24 hours; B is the cytotoxicity of Hepa1-6 cells at 48 hours; C is the cytotoxicity of Hepa1-6 cells at 72 hours; D is the apoptosis rate of Hepa1-6 cells)

[0033] Figure 2 The bufotoxin-based composition induces ICD effect in Hepa1-6 cells in vitro. (A is a laser confocal image of calreticulin, B is a semi-quantitative image; C is a laser confocal image of high-mobility group protein, D is a semi-quantitative image; E is an ATP release image.)

[0034] Figure 3Evaluation of the anti-tumor effect of the bufotoxin ligand combination in vivo. (A is the construction of the weight-bearing mouse model and the dosing plan; B is the average weight change curve of mice during treatment; C is the individual growth curve corresponding to Hepa1-6 tumor-bearing mice; D is the tumor growth curve of mice in different treatment groups.)

[0035] Figure 4 The induction of ICD effect by bufoveolin-based compositions in vivo. (A is a laser confocal image of calreticulin, B is a semi-quantitative image; C is a laser confocal image of high-mobility group protein, D is a semi-quantitative image; E and F are CD11c + CD80 + Cell expression; G and H are CD11c + CD86 + Cellular expression.

[0036] Figure 5 The activation of immune response of bufotoxin composition in tumor-bearing mice. (A and C are CD3 + CD4 + Expression of T cells; B and D are CD3 + CD8 + The expression of T cells; E and F are the expression of MDSc cells; G and H are the expression of Treg cells; I is the expression of IFN-β; J is the expression of IFN-γ. ) DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0039] The materials and methods used in the embodiments of the present invention are as follows:

[0040] 1.1 Reagents and grouping: The bufotoxin composition (BU) was homemade (the raw material mass ratio used was: bufotoxin: cinobufogenin: resibufogenin = 3:5:2, and the purity was greater than 90%), wherein Free BU represented the free bufotoxin group, HBNCs represented the bufotoxin nanosuspension group, and HBE NCs represented the bufotoxin enteric-coated nanocrystal group.

[0041] The preparation method of the above-mentioned bufotoxin enteric-coated nanocrystals:

[0042] Bufonogen nanocrystals modified with chitosan quaternary ammonium salt (HACC) were prepared by solvent evaporation method. Briefly, HACC aqueous solution (0.2 mg / mL) was the aqueous phase. Bufonogen, Eudragit L-100, and sorbitan oleate in a weight ratio of 10:10:1 were dissolved in 2 mL of methanol as the organic phase. The organic phase was then rapidly injected into the aqueous phase and sonicated in an ice bath for 10 min to obtain HBE NCs. Finally, methanol was evaporated under reduced pressure at 30 °C for 5 min using a rotary evaporator. In addition, bufonogen nanosuspensions (HB NCs) without Eudragit L-100 and sorbitan oleate in the organic phase were prepared by the above method.

[0043] 1.2 Cells: Hepa1-6 cells were purchased from Starfish Biotechnology Company.

[0044] 1.3 Experimental animals: C57 mice weighing 18±2 g were provided by the Animal Center of Ningxia Medical University and were kept in an SPF-level breeding environment with free access to food and water.

[0045] Example 1: Effects of different groups of bufotoxin compositions on proliferation inhibition and apoptosis of Hepa1-6 cells

[0046] Hepa1-6 cells in the logarithmic growth phase were taken, digested, centrifuged, and the cell density was adjusted to 2×10 5 cells / mL, inoculated in a 96-well plate, cultured until the cells adhered, and then added culture medium containing toad venom ligand raw material (Free BU), toad venom ligand nanosuspension group (HB NCs), toad venom ligand enteric nanocrystals (HBE NCs), and the drug-free culture medium was used as a control. After administration, the culture was continued for 24h, 48h, and 72h. At the end of the culture, the culture medium was discarded, 10μL CCK-8 solution was added to each well and incubated at 37℃ for 30-60min. After incubation, the results were analyzed using an ELISA instrument. Figure 1 .

[0047] Hepa1-6 cells in the logarithmic growth phase were taken, digested, centrifuged, and the cell density was adjusted to 2×10 5 cells / mL, inoculated in a 6-well plate, cultured until the cells adhered to the wall, and then added culture medium containing toad venom ligand raw material (Free BU), toad venom ligand nanosuspension group (HB NCs), toad venom ligand enteric nanocrystals (HBE NCs), and the drug-free culture medium was used as a control. After administration, the culture was continued for 24 hours. At the end of the culture, the cells were digested, collected by centrifugation, resuspended, and stained with PI and Annexin-V. After staining, the cells were analyzed by flow cytometry. The results are shown in Figure 1 .

[0048] Depend on Figure 1It can be seen that under the same culture time and dosage, the three groups of toad venom ligand compositions all had an inhibitory effect on the proliferation of Hepa1-6 cells. Among them, HB NCs had a stronger inhibitory effect on the proliferation of Hepa1-6 cells than free BU, and HBE NCs had a stronger inhibitory effect on the proliferation of Hepa1-6 cells than HB NCs. With the increase of culture time and dosage, the inhibition of Hepa1-6 cell proliferation by different groups of toad venom ligands was time- and dose-dependent ( Figure 1 AC). This may be related to the enhanced cell uptake of BU after nanoformulation. We further studied the effects of different preparations on inducing apoptosis in Hepa1-6 cells. Figure 1 As shown in D, the apoptosis rate of Hepa1-6 cells in the free group was 6.15%, that in the Free BU group was 19.32%, that in the HB NCS group was 29.37%, and that in the HBE NCs group was 36.93%. These results indicate that the bufotoxin composition can increase the apoptosis of cancer cells and exert an anti-tumor effect.

[0049] Example 2: Induction of ICD effect of different groups of bufotoxin compositions on Hepa1-6 cells in vitro

[0050] Hepa1-6 cells in the logarithmic growth phase were taken, digested, centrifuged, and the cell density was adjusted to 2×10 5 cells / mL, inoculated in a 12-well plate, cultured until the cells adhered, and then added culture medium containing toad venom ligand raw material (Free BU), toad venom ligand nanosuspension group (HB NCs), toad venom ligand enteric nanocrystals (HBE NCs), and the drug-free culture medium was used as a control. The culture was continued for 24 hours after administration. At the end of the culture, the culture medium was discarded, and room temperature PBS was slowly added to the cells, washed twice, 5 seconds each time; covered the cells with 4% neutral formaldehyde fixative (prepared with TBS buffer), placed at 4°C, and fixed for 15 minutes; removed the fixative, rinsed 3 times with 4°C pre-cooled PBS buffer, 1 minute each time; 0.5% Triton 100 (dissolved in PBS), permeabilized for 20 minutes, and washed 3 times with PBS; covered the sample completely with 5% blank goat serum, and incubated in a constant temperature and humidity incubator at 37°C for 30 minutes; incubated with the corresponding primary and secondary antibodies, and sealed after incubation. Laser confocal analysis was performed, and the results are shown in the results. Figure 2 .

[0051] Laser confocal microscopy showed that the fluorescence signals of calreticulin in Hepa1-6 treated with the three groups of toad venom ligand combinations were stronger than those in the control group, confirming that toad venom ligands triggered the translocation of tectorial reticular protein during ICD. The fluorescence signals of high mobility group protein B1 in Hepa1-6 treated with the three groups of toad venom ligand combinations were weaker than those in the control group. The cells released HMGB1 from the nucleus to the extracellular space as a damage-related molecular pattern, which significantly weakened the fluorescence signal of HMGB1 in the nucleus.

[0052] Example 3: Evaluation of the anti-tumor effects of different groups of bufotoxin compositions in vivo

[0053] Digest the logarithmically growing Hepa1-6 cells, centrifuge, resuspend, count, and adjust the cell concentration to 6×10 7 cells / mL, wipe the armpit of the mouse with alcohol for disinfection, and inoculate about 6×10 6 The liver cancer tumor-bearing mouse model was established by adding 100 mL of cells (about 0.1 mL). Two days after inoculation, the size of the mouse tumor was measured every day. 3 After about 24 hours, the mice were randomly assigned and the experiment was carried out. The liver cancer-bearing mice were randomly divided into 4 groups, including the Free BU group, the HB NCs group, the HBE NCs group and the saline group. The dosage was 6 mg / kg, and the drug was administered by gavage once every 2 days for a total of 7 times. The body weight and tumor volume were recorded every two days. The results are shown in Figure 3 .

[0054] like Figure 3 As shown in B, the tumor growth of animals treated with normal saline was uncontrolled, and the tumor volume reached 1777.01 mm 3 ( Figure 3 D). The effect of free BU on tumor volume was not obvious, which may be because free BU is highly hydrolyzed when exposed to gastric acid, thus reducing its activity. However, HB NCs had a certain inhibitory effect on tumors. The average tumor volume on day 14 was 824.07 mm 3 ( Figure 3 E). This may be due to the formation of HB NC nanocrystals after BU nanoparticle treatment, which increases the solubility and bioavailability of BU. In contrast, animals treated with HBE NCs showed a significant reduction in tumor volume, with an average tumor volume of 277.45 mm on day 14. 3 This stronger inhibitory effect on tumors may be due to the improved stability and intestinal absorption of BU in the gastrointestinal tract by HBE NCs. These results indicate that bufotoxin has a significant antitumor effect.

[0055] Example 4: Different groups of bufotoxin compositions induce ICD effect in tumor-bearing mice

[0056] Tumor tissues from tumor-bearing mice in different groups were collected and frozen sections were made. After sectioning, 3% hydrogen peroxide was added, and the tissues were placed in a humidified box at room temperature for 10 minutes, and then placed in distilled water for 1 minute; placed in 1x CB, 40% heat for 8 minutes, and cooled to room temperature; permeabilized with 0.3% Triton at room temperature for 15 minutes; the liquid around the tissues was aspirated, 10% NGS was added, and the tissues were placed in a humidified box at 37°C for 30 minutes; the corresponding primary and secondary antibodies were incubated, and the sections were sealed after incubation. Laser confocal analysis was performed, and the results are shown in the figure. Figure 4 .

[0057] like Figure 4 As shown in A to D, the fluorescence signal intensity of calreticulin in tumor tissue sections of the three groups of bufotoxin combination was significantly higher than that of the saline group, indicating the successful expression of CER. Similarly, high mobility group protein B1 was also significantly expressed. The high expression of CRT and HMGB-1 in DAMPs proved that bufotoxin successfully induced the ICD effect in tumor-bearing mice.

[0058] Example 5: Activation of immune response in tumor-bearing mice by different groups of bufotoxin compositions

[0059] The tumor tissue was peeled off, weighed, and appropriate amount of tumor tissue was taken into a 60mm culture dish and minced with sterile scissors. Then, it was added to the culture medium containing collagenase and deoxynucleotidase and placed in a 37°C incubator for digestion for 30 minutes, with shaking 3 times during the period. The digested tumor tissue was transferred to a 200-mesh sieve and ground with a 1mL sterile syringe piston to prepare a cell suspension. The filtered cell suspension was collected in a 15mL centrifuge tube and centrifuged at 300g for 5 minutes. 3mL of red blood cell lysis solution was added to the precipitate. After lysis on ice for 5 minutes, it was washed twice with PBS containing 2% fetal bovine serum and the precipitate was collected by centrifugation. An appropriate amount of D-Hanks solution was added to the cell precipitate to adjust the cell concentration to 1×10 6cells / mL and then divided into 4 parts for staining. Staining: ① First add CD16 / 32 antibody, incubate at 4℃ in dark for 10min, then add CD3, CD4, CD8 flow cytometry antibodies, incubate at 4℃ in dark for 30min, rinse with PBS and resuspend, and detect the proportion of T lymphocyte subsets by flow cytometry; ② First add CD16 / 32 antibody, incubate at 4℃ in dark for 10min, then add CD11b and Gr-1 flow cytometry antibodies, incubate at 4℃ in dark for 30min, rinse with PBS and resuspend, and detect MDSC by flow cytometry; ③ According to the requirements of Treg detection kit, first add CD4 and CD25 antibodies, incubate at 4℃ in dark for 30min, rinse with PBS, add 1mL membrane permeabilization solution to permeabilize for 30min-1h, and add 1×Permeabilization After washing twice with Buffer, add CD16 / 32 antibody and incubate at room temperature in the dark for 15 minutes, then add Foxp-3 flow cytometry antibody, incubate at room temperature in the dark for 30 minutes, rinse with 1×Permeabilization Buffer and resuspend, add 300μL FlowCytometry Staining Buffer to resuspend the cells, and then detect the proportion of Tregs cells by flow cytometry; ③ First add CD16 / 32 antibody, incubate at 4℃ in the dark for 10 minutes, then add CD11, CD80, and CD86 flow cytometry antibodies, incubate at 4℃ in the dark for 30 minutes, rinse with PBS and resuspend, and detect the proportion of DC cell subsets by flow cytometry.

[0060] Depend on Figure 4 Results from E to H show that mature dendritic cells (CD11 + CD80 + and CD11 + CD86 + The proportion of cells in the HBE NCs group was low, indicating that its immunogenicity was weak. HB NCs increased the number of moderately mature dendritic cells. As expected, CD11 + CD80 + and CD11 + CD86 + The proportion of cells was much higher than that of other groups, proving that bufotoxin can induce and promote the maturation of dendritic cells.

[0061] HBE NCs group CD3 + CD4 + The T cell content was the highest (23.3%), which was 2.9 times and 1.8 times higher than that of the saline group (7.6%) and the HB NCs group (13.9%), respectively. Figure 5 A) CD3 + CD8+ The T cell content was the highest (13.2%), which was 1.6 times and 1.2 times higher than that of the saline group and HB NCs group, respectively. Figure 5 B). This should be attributed to the activation of immune response by ICD effector and the promotion of T cell priming by ICD effector.

[0062] like Figure 5 As shown in IJ, the three groups of bufotoxin compositions significantly increased pro-inflammatory cytokines, including interferon-β (IFN-β) and interferon-γ (IFN-γ).

[0063] The MDSCs content in the HBE NCs group was the lowest (2.0%), which was 3.8-fold and 2.3-fold lower than that in the saline group (7.7%) and the HB NCs group (4.6%), respectively. Figure 5 E). The ICD effect resulted in a decrease in the number of MDSCs cells, indicating an improved immunosuppressive microenvironment. In addition, we investigated the number of another type of immunosuppressive cells (Treg) in tumor tissues. As expected, the number of Treg cells was the lowest in the HBE NCs group (2.51%), which was 3.0-fold and 1.7-fold less than that in the saline group and HB NCs group, respectively ( Figure 5 G).

[0064] Taken together, these results demonstrate that bufotoxin can reshape the “cold” tumor immune microenvironment of HCC into a “hot” tumor immune microenvironment, at least in part due to the ICD-mediated chemoimmunotherapeutic effect.

[0065] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

Claims

1. A bufotoxin composition, characterized in that: The bufotoxin composition is one or more of bufalin, cinobufogenin and resibufogenin, and the purity of the bufotoxin composition is greater than 90%.

2. A bufotoxin composition according to claim 1, characterized in that: The bufotoxin composition comprises tautomers and / or optical isomers of the active ingredients of bufalin, cinobufogenin and resibufogenin.

3. A bufotoxin composition according to claim 1, characterized in that: The bufotoxin composition mainly comprises bufalin, cinobufagin and resibufagin.

4. A bufotoxin composition according to claim 3, characterized in that: The mass ratio of bufalin, cinobufogenin and resibufogenin is 1-5:3-7:1-4.

5. A bufotoxin composition according to claim 4, characterized in that: The mass ratio of bufalin, cinobufogenin and resibufogenin is 2-4:4-6:1-3.

6. A bufotoxin composition preparation, characterized in that: A solvate, a pharmaceutically acceptable salt or ester, a pharmaceutically acceptable prodrug or a derivative comprising the bufotoxin composition according to any one of claims 1 to 5.

7. The bufotoxin composition preparation according to claim 6, characterized in that: The bufotoxin composition or its pharmaceutically acceptable salt is prepared into injection, tablet, powder injection, granule, capsule and nano preparation.

8. The bufotoxin composition preparation according to claim 7, characterized in that: The nano preparation is nano crystal, nano suspension or nano particle, and the particle size of the nano preparation is 200-250 nm.

9. An application of a bufotoxin composition, characterized in that: The use of any bufotoxin composition according to any one of claims 1 to 5 or any bufotoxin composition preparation according to any one of claims 6 to 8 in the preparation of tumor treatment drugs or health products.

10. The use of a bufotoxin composition according to claim 9, characterized in that: The tumor is liver cancer.

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