Application of traditional Chinese medicine composition in preparation of medicine for treating liver cancer

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a traditional Chinese medicine composition in preparation of a medicine for treating liver cancer. The traditional Chinese medicine composition is prepared from 12-20 parts of herba epimedii, 2-8 parts of radix dipsaci, 1-5 parts of radix salviae miltiorrhizae, 1-5 parts of rhizoma anemarrhenae, 1-5 parts of fructus psoraleae and 1-5 parts of radix rehmanniae. According to the traditional Chinese medicine composition, expression of CD31 protein, Ki67 protein and Bcl-2 protein in liver cancer cells is reduced, expression of Bax protein, Fas protein and Fast protein in the liver cancer cells is improved, proliferation of the liver cancer cells is inhibited, apoptosis of the liver cancer cells is promoted, and the traditional Chinese medicine composition has a good intervention effect in the aspect of liver cancer treatment.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to an application of a traditional Chinese medicine composition in preparing a medicine for treating liver cancer. Background Art

[0002] At present, the commonly used treatments for liver cancer include surgical resection, liver transplantation, chemical embolization, and radiofrequency ablation. However, most patients diagnosed with liver cancer in the middle and late stages have unresectable liver cancer and cannot undergo surgery. They need to take liver cancer treatment drugs to effectively relieve liver cancer. Traditional Chinese medicine has the characteristics of multiple components, multiple targets, multiple pathways, and overall synergistic effects. It can be used throughout the process of tumor treatment, preventing cancer recurrence and metastasis, controlling the progression of the patient's disease, improving the patient's quality of life, and even improving the body's immune function. It is an important resource for the research and development of effective anti-liver cancer substances.

[0003] Xianling Gubao Capsule (XLGBJ) and Xianling Gubao Tablet (XLGBP) are included in the National Essential Medicines Catalog and are used in orthopedics. They are prepared from six medicinal materials: Epimedium, Dipsacus, Salvia miltiorrhiza, Anemarrhena, Psoralea corylifolia, and Rehmannia glutinosa. They are mainly used clinically for osteoporosis, fractures, osteoarthritis, aseptic necrosis of bone, and other diseases. However, in the prior art, there is no report on the pharmaceutical composition for treating liver cancer of the present invention. Summary of the invention

[0004] However, it is still unclear whether Xianlinggubao Capsule (XLGBJ) and Xianlinggubao Tablet (XLGBP) can play a good intervention role in the treatment of liver cancer in the prior art. Based on this, the main purpose of the present invention is to prove through animal experiments, etc. that Xianlinggubao Capsule (XLGBJ) and Xianlinggubao Tablet (XLGBP) have a good intervention effect in the treatment of liver cancer, and provide a new solution for the treatment of liver cancer.

[0005] In view of the above problems existing in the prior art, the present invention provides an application of a Chinese medicine composition in the preparation of a drug for treating liver cancer. The specific technical solution is as follows:

[0006] Technical Solution 1: Use of a traditional Chinese medicine composition in the preparation of a drug for treating liver cancer, characterized in that the traditional Chinese medicine composition comprises: 12-20 parts of epimedium, 2-8 parts of dipsaccharum, 1-5 parts of salvia miltiorrhiza, 1-5 parts of anemarrhena, 1-5 parts of psoralea corylifolia and 1-5 parts of rehmannia root.

[0007] Technical Solution 2: According to the application described in Technical Solution 1, the Chinese medicine composition also includes pharmaceutically acceptable excipients.

[0008] Technical Solution 3: According to the application described in Technical Solution 1 or 2, it is characterized in that the dosage form of the Chinese medicine composition includes one or more dosage forms selected from the group consisting of oral liquid, powder, tablet, capsule, granule, paste, powder and pill.

[0009] Technical Solution 4: The application according to any one of Technical Solutions 1-3 is characterized in that the dosage form of the Chinese medicine composition includes one or more dosage forms selected from the group consisting of tablets, capsules and granules.

[0010] Technical solution 5: The use according to any one of technical solutions 1-4, characterized in that the liver cancer is hepatocellular carcinoma, preferably, the hepatocellular carcinoma is H22 liver cancer cell,

[0011] Technical Solution 6: The use according to any one of Technical Solutions 1-5, characterized in that it is used in the preparation of liver cancer drugs that reduce the expression of CD31 protein, Ki67 protein and / or Bcl-2 protein.

[0012] Technical Solution 7: The use according to any one of Technical Solutions 1-6, characterized in that it is used in the preparation of liver cancer drugs that increase the expression of Bax protein, Fas protein and / or Fasl protein.

[0013] Technical Solution 8: The application according to any one of Technical Solutions 1-7 is characterized in that when the Chinese medicine composition is used to treat liver cancer, the clinical dosage for each administration is 9.89 mg / kg-23.97 mg / kg.

[0014] Technical Solution 9: The application according to any one of Technical Solutions 1-8 is characterized in that when the Chinese medicine composition is used to treat liver cancer, the total clinical dosage is 148.35 mg / kg-494.50 mg / kg.

[0015] Technical Solution 10: According to the application described in any one of Technical Solutions 1-9, the Chinese medicine composition is administered via the gastrointestinal tract.

[0016] Technical Solution 11: According to the application described in any one of Technical Solutions 1-10, the Chinese medicine composition has a significant inhibitory effect on the growth of H22 mouse tumors. Preferably, the inhibition rate of the Chinese medicine combination on the tumor is greater than or equal to 48%, and preferably, the inhibition rate is 48-55%.

[0017] Technical Solution 12: According to the use described in any one of Technical Solutions 1-11, the Chinese medicine composition reduces the AFP level in the serum of H22 mice, and / or increases the percentage of necrosis area of ​​tumor tissue of H22 mice.

[0018] Preferably, the AFP level in the serum of H22 mice is less than or equal to 1.665 mg / L, and / or the percentage of necrosis area of ​​tumor tissue of H22 mice is greater than or equal to 31%.

[0019] More preferably, the AFP level in the serum of H22 mice is 1.440-1.665 mg / L, and / or the percentage of necrosis area of ​​tumor tissue of H22 mice is 31%-45%.

[0020] Technical solution 13: The use according to any one of technical solutions 1-12 is characterized in that the positive expression rate of CD31 protein in H22 mouse tumor tissue by the traditional Chinese medicine composition is less than or equal to 1.5%, preferably 1%-1.5%,

[0021] And / or the positive expression rate of Ki67 protein is less than or equal to 6.5%, preferably, 2%-6.5%, and / or the positive expression rate of Bcl-2 protein is less than or equal to 6.5%, preferably, 1.5%-6.5%.

[0022] Technical solution 14: The use according to any one of technical solutions 1-13, characterized in that the positive expression rate of Bax protein in H22 mouse tumor tissue by the traditional Chinese medicine composition is less than or equal to 6.5%, preferably 3%-6.5%,

[0023] and / or the positive expression rate of Fas protein is less than or equal to 15%, preferably, 8%-15%,

[0024] And / or the positive expression rate of Fasl protein is less than or equal to 18%, preferably, 11%-18%. Beneficial effects of the present invention:

[0025] The Chinese medicine composition provided by the present invention has a good intervention effect in treating liver cancer, specifically: the Chinese medicine composition of the present invention has a significant inhibitory effect on tumor growth, can significantly reduce the AFP level in tumor-bearing serum, increase the necrotic area of ​​tumor cells, prolong the survival period, and has anti-liver cancer activity; by regulating key proteins, it intervenes in the expression levels of CD31, Ki67, Bcl-2, Bax, Fas and Fasl proteins in tumor tissues, thereby inhibiting tumor cell proliferation, inhibiting angiogenesis and promoting cell apoptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown are the changes in body weight of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group during the drug treatment.

[0027] Figure 2Shown are the tumor growth curves of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group on days 1, 3, 5, 7, 9, 11, 13 and 15 after administration. Figure 3 Shown are the tumor volumes of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group on the 15th day after administration.

[0028] Figure 4 Shown are the survival rates of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group within 60 days.

[0029] Figure 5 Shown are the AFP levels in the serum of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group.

[0030] Figure 6 Shown are the tumor weights of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group.

[0031] Figure 7 Shown are the spleen indexes of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group.

[0032] Figure 8 Shown are the liver coefficients of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group.

[0033] Fig. 9 Shown are tumor tissue sections of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group.

[0034] Fig.10 Shown are the percentages of tumor tissue necrosis area of ​​mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group.

[0035] Fig.11 Shown are the microscopic images of the expression of Ki67 protein, CD31 protein, Bax protein, BcL-2 protein, Fas protein or Fasl protein in the tumor tissues of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group.

[0036] Fig.12 Shown is the percentage of positive area for protein expression of Ki67 protein, CD31 protein, Bax protein, BcL-2 protein, Fas protein or Fasl protein in tumor tissues of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group. DETAILED DESCRIPTION

[0037] In view of the fact that it is still unclear whether Xianlinggubao Capsule (XLGBJ) and Xianlinggubao Tablet (XLGBP) can play a good role in the treatment of liver cancer in the prior art, the inventor has conducted a thorough study and proved through animal experiments that Xianlinggubao Capsule (XLGBJ) and Xianlinggubao Tablet (XLGBP) have a good role in the treatment of liver cancer. Among them, the Xianlinggubao Capsule and Xianlinggubao Tablet are a traditional Chinese medicine composition, which is prepared by main ingredients and auxiliary ingredients, wherein the main ingredients include Epimedium, Dipsacus asper, Salvia miltiorrhiza, Anemarrhena asphodeloides, Psoralea corylifolia and Rehmannia glutinosa. And the inventors further studied that when the main ingredients in the Chinese medicine composition include: 12-20 parts of Epimedium, 2-8 parts of Dipsacus asper, 1-5 parts of Salvia miltiorrhiza, 1-5 parts of Anemarrhena asphodeloides, 1-5 parts of Psoralea corylifolia and 1-5 parts of Rehmannia glutinosa, the Chinese medicine composition has a good intervention effect in the treatment of liver cancer, which is specifically reflected in: the Chinese medicine composition reduces the expression of CD31 protein, Ki67 protein and Bcl-2 protein, and increases the expression of Bax protein, Fas protein and Fasl protein. The Chinese medicine composition inhibits tumors in H22 liver cancer model mice, and the inhibition rate of tumors in H22 liver cancer model mice is greater than or equal to 49%, improves the spleen index and / or liver coefficient of H22 liver cancer model mice, and prolongs the survival period of H22 liver cancer model mice. The Chinese medicine composition makes the tumor cells of H22 liver cancer model mice loosely arranged, the intercellular matrix is ​​large, the cell nucleus is broken, and the area of ​​cell necrosis is increased.

[0038] In order to better understand the above technical solution, the technical solution of the present invention is clearly and completely explained in conjunction with the specific implementation method below. It should be noted that the content in the specific implementation method is only a specific implementation and explanation of the technical solution of the present invention, and should not be understood as limiting the scope of protection of the present invention.

[0039] In some specific embodiments, the present invention provides an application of a Chinese medicine composition in the preparation of a drug for treating liver cancer, characterized in that the Chinese medicine composition comprises: 12-20 parts of epimedium, 2-8 parts of dipsaci, 1-5 parts of salvia miltiorrhiza, 1-5 parts of anemarrhena, 1-5 parts of psoralea corylifolia and 1-5 parts of rehmannia glutinosa. The Chinese medicine composition and preparation method thereof provided by the present invention have been disclosed in patent publication number CN1781535A.

[0040] In some specific embodiments, the Chinese medicine composition further includes a pharmaceutically acceptable excipient or auxiliary material or carrier. It should be noted that the present invention has no particular limitation on the specific types of pharmaceutically acceptable auxiliary materials. As long as the Chinese medicine composition having a dosage form including oral liquid, powder, tablet, capsule, granule, paste, powder or pill can be prepared by conventional preparation methods using pharmaceutically acceptable auxiliary materials based on the technical common sense in the art, it can be used in the present invention.

[0041] Pharmaceutically acceptable excipients include one or more of fillers, binders, adhesives, humectants, flavoring agents, disintegrants, lubricants and stabilizers. Preferably, fillers include one or more of starch, lactose, sucrose, glucose, microcrystalline cellulose, mannitol and silicic acid. Binders include honey, sucrose, alginate, gelatin, polyvinyl pyrrolidone and gum arabic, humectants include glycerol, disintegrants include one or more of agar, calcium carbonate, potato and cassava starch and alginic acid. Lubricants include one or more of calcium stearate and magnesium stearate. Stabilizers include one or more of hydroxypropyl methylcellulose and chitosan.

[0042] In some specific embodiments, the administration method of the Chinese medicine composition for treating liver cancer is oral administration or non-oral administration. Preferably, in some specific embodiments, the drug is administered orally through the gastrointestinal tract.

[0043] In some specific embodiments, the Chinese medicine composition provided by the present invention is usually used as a test subject in mammals, such as humans, primates, sheep, horses, camels, pigs, dogs, cats, rats or mice. The Chinese medicine composition provided by the present invention can be used in vivo or in vitro in the test subject.

[0044] It should be noted that the dosages corresponding to different subjects are not exactly the same, and can be determined according to the expected biological endpoint, the disease to be treated, the method of administration, and the age, weight, health status, course of treatment, individual differences, and dosage forms and routes of administration of the subject. Those skilled in the art can determine the optimal range of the effective dosage of each component according to individual needs, wherein the dosage refers to an amount sufficient to induce the desired biological response.

[0045] In some specific embodiments, when the test subject is a mouse, when the Chinese medicine composition is used to treat liver cancer, the dosage for each administration is 90 mg / kg-300 mg / kg.

[0046] Preferably, in some specific embodiments, when the subject is a human, the above-mentioned mouse dosage is used as a benchmark, and after body surface area conversion (conversion coefficient is 9.1) is used as a clinical equivalent dosage, when the Chinese medicine composition is used to treat liver cancer, each clinical dosage is 9.89 mg / kg-32.97 mg / kg.

[0047] In some specific embodiments, when the test subject is a mouse, when the Chinese medicine composition is used to treat liver cancer, the total dosage is 1350 mg / kg-4500 mg / kg.

[0048] Preferably, in some specific embodiments, when the subject is a human, the above-mentioned mouse dosage is used as a benchmark, and after body surface area conversion (conversion coefficient is 9.1) is used as a clinical equivalent dosage, when the traditional Chinese medicine composition is used to treat liver cancer, the total clinical dosage is 148.35 mg / kg-494.50 mg / kg.

[0049] Explanation of terms

[0050] CD31 protein: a cell surface adhesion molecule, mainly expressed on the surface of vascular endothelial cells, platelets and certain immune cells, involved in cell-cell adhesion, angiogenesis, inflammatory response and transendothelial migration of immune cells. In pathology, it is often used as a marker of vascular endothelial cells to identify vascular tumors (such as hemangiomas and angiosarcomas).

[0051] Ki67 protein: It is a protein related to nuclear proliferation. It is expressed in the cell cycle (G1, S, G2, M phases) and not expressed in the quiescent phase (G0 phase). It reflects the activity of cell proliferation and is a direct marker of the degree of cell division activity. It is used to evaluate the proliferation index of tumors in pathology.

[0052] Bcl-2 protein: It is a mitochondrial membrane anti-apoptotic protein. It inhibits apoptosis mainly by inhibiting mitochondrial membrane permeability and preventing the release of cytochrome C. It plays a key role in maintaining cell survival and regulating programmed death. It is used as a marker for evaluating tumor apoptosis inhibition in pathology (for example, Bcl-2 overexpression in follicular lymphoma is associated with prognosis).

[0053] Bax protein: It is a pro-apoptotic protein that promotes the release of cytochrome C by forming mitochondrial membrane permeability pores, activates the Caspase cascade reaction, and induces cell apoptosis. In pathology, it is used as a marker for evaluating tumor apoptosis activation, antagonizes Bcl-2, and regulates the balance of apoptosis.

[0054] Fas protein: a death receptor, as an apoptosis signal receptor on the cell membrane surface, after binding to FasL (Fas ligand), it activates downstream Caspase-8 / 10 and triggers the death receptor apoptosis pathway. It has the functions of participating in the killing of target cells by cytotoxic T cells of the immune system and maintaining immune tolerance.

[0055] FasL protein: a death ligand belonging to the tumor necrosis factor superfamily (TNFSF6). As a ligand of Fas protein, it induces apoptosis of target cells after binding to Fas. It is mainly expressed by activated T cells and NK cells, and participates in immune killing and immune regulation. It exists in the form of a transmembrane protein and can also be cleaved by proteases into a soluble form (sFasL). In pathology, it can be used as a tumor immunotherapy target to enhance FasL signals to eliminate tumor cells.

[0056] H22 liver cancer cells: They belong to the mouse ascites-type liver cancer cells, which have high proliferation ability and tumorigenicity. They are usually cultured in mice in the form of ascites. H22 cells were originally derived from mouse liver cancer tissue induced by chemicals (such as diethylnitrosamine). H22 mouse liver cancer cells quickly form tumors, which are suitable for in vivo drug efficacy evaluation and are often used to study tumor growth, metastasis and drug efficacy. They are commonly seen in the exploration of the mechanisms of solid tumors such as liver cancer and lung cancer. Anti-tumor drug screening, immunotherapy research and tumor microenvironment analysis, among which, anti-tumor drug screening: evaluate the tumor inhibition effect of chemotherapy drugs, Chinese medicine extracts or biological agents (such as detecting tumor volume, prolonged survival and other indicators); immunotherapy research: study the killing effect of immune cells (such as CAR-T, NK cells) on tumors; tumor microenvironment analysis: explore microenvironment changes related to tumor progression such as angiogenesis and inflammatory factor secretion.

[0057] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0058] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercial products. The sources of information on the experimental reagents used in the present invention are shown in Table 1, and the sources of information on the experimental instruments used in the present invention are shown in Table 2.

[0059] Table 1. Experimental reagent information

[0060]

[0061]

[0062] Table 2. Experimental instrument information

[0063]

[0064]

[0065] 1. The sources of the experimental mice and H22 mouse liver cancer cell lines used in the examples are as follows:

[0066] SPF male ICR mice (18-22 g) were purchased from Suzhou Xishan Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010. The animals were kept in an environment with a relative temperature of 18-25 °C and a relative humidity of 50% to 70% before the experiment. The feeding and management were strictly in accordance with the requirements and rules of experimental animals. They were kept for one week to adapt to the environment before the experiment. H22 mouse hepatoma cell line was purchased from Wuhan Pronocell Life Science Technology Co., Ltd.

[0067] 2. The preparation of the solution used in the embodiment is as follows:

[0068] (2.1) Doxorubicin sodium hydrochloride (DOX) solution: 30 mg of DOX was dissolved in normal saline and the volume was adjusted to 100 mL after ultrasonic treatment for 30 min to obtain a DOX solution with a final concentration of 0.3 mg / mL.

[0069] (2.2) Xianling Gubao Capsule (hereinafter referred to as Xianling Gubao Capsule, XLGBJ) solution: 3.0 g of XLGBJ was uniformly ground and added with water for injection to make up to 50 mL. The suspension was then stirred using a vortex instrument and ultrasonicated for 10 min to obtain a mixed solution of XLGBJ with a final concentration of 60 mg / mL.

[0070] (2.3) Xianlinggubao Tablets (hereinafter referred to as Xianlinggubao Tablets, XLGBP) intragastrical solution: 1.80 g of XLGBP was uniformly ground and added with water for injection to make up to 50 mL. The suspension was then stirred using a vortex machine and ultrasonicated for 10 min to obtain a final concentration of 36 mg / mL of XLGBP.

[0071] 3. Cultivation of H22 liver cancer cells

[0072] H22 hepatoma cells were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO. 2 The cells were cultured and subcultured in a cell culture incubator at 4 °C, subcultured every 2 to 3 days, and subcultured twice in total. During subculture, the cells were collected into a centrifuge tube, centrifuged at 1200 r / min for 5 min, the cells at the bottom were collected, and transferred to a culture flask for culture. The cells in the logarithmic growth phase were collected and stained with trypan blue. When the cell survival rate was greater than 95%, the cell concentration was adjusted to 1×10 7 / mL, used to establish H22 liver cancer mouse model.

[0073] 4. Establishment of H22 liver cancer mouse model

[0074] After H22 liver cancer cells were cultured in vitro to an appropriate density, the cells were collected after centrifugation at 1200 r / min for 3 min and resuspended in PBS to obtain a density of 1×10 7 / mL of H22 liver cancer cell suspension. Then, 0.2mL of H22 liver cancer cell suspension was injected intraperitoneally into 3 mice and fed for 5-6 days. 4-8mL of pale yellow mouse ascites was collected by aseptic extraction or dissection. After washing with phosphate buffer solution, 0.2mL of the treated ascites was taken (the concentration of H22 liver cancer cells in the ascites was 1×10 7 / mL) and passaged twice in normal mice; after the second passage, the mice were kept for 5-6 days, and the ascites of the second generation of mice were collected and washed with phosphate buffer solution to obtain the treated ascites (the concentration of H22 liver cancer cells in the ascites was 1×10 7 / mL); then the right armpit of the mouse was routinely disinfected with 75% ethanol, and 0.2mL of the treated ascites was subcutaneously injected (shake well before each inoculation). Within 5 days after inoculation, there was no obvious change in the appearance and mental state of the mouse. One week later, a rice-sized mass was seen growing under the right armpit of the mouse, indicating that the model was successfully established.

[0075] Example 1

[0076] The H22 liver cancer model mice and normal mice of the same batch were experimented at the same time, with a total of 7 groups (10 mice in each group), namely: normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group. The weight of mice in each group was recorded every day, and the general physiological conditions of mice in each group were observed. The dosing method of mice in each group is as follows:

[0077] (1) Normal group: Normal mice were selected and gavaged with about 2.5 mL of normal saline once a day based on the weight of each normal mouse, for a total of 15 times.

[0078] (2) Model group: H22 liver cancer model mice were selected and gavaged with approximately 2.5 mL of normal saline once a day, based on the weight of each H22 liver cancer model mouse, for a total of 15 times.

[0079] (3) DOX group: H22 mice with H22 liver cancer model were selected, and about 2.5 mL of DOX solution with a final concentration of 0.3 mg / mL was injected once every two days through the tail vein based on the body weight of each H22 liver cancer model mouse (i.e., 0.75 mg of DOX was administered per kg of mouse each time), for a total of 7 injections.

[0080] (4) XLGBJ-L group: H22 liver cancer model mice were selected and gavaged once daily with about 2.5 mL of a mixture of XLGBJ with a final concentration of 60 mg / mL (i.e., 150 mg of XLGBJ per kg of mouse per gavage) based on the weight of each H22 liver cancer model mouse, for a total of 15 times.

[0081] (5) XLGBJ-H group: H22 liver cancer model mice were selected and gavaged once daily with about 5.0 mL of a mixture of XLGBJ with a final concentration of 60 mg / mL (i.e., 300 mg of XLGBJ per kg of mouse per gavage) based on the weight of each H22 liver cancer model mouse, for a total of 15 times.

[0082] (6) XLGBP-L group: H22 liver cancer model mice were selected and gavaged once daily with about 2.5 mL of a mixture of XLGBP with a final concentration of 36 mg / mL (i.e., 90 mg of XLGBP per kg of mouse per gavage) based on the weight of each H22 liver cancer model mouse, for a total of 15 times.

[0083] (7) XLGBP-H group: H22 liver cancer model mice were selected and gavaged once daily with about 5.0 mL of a mixture of XLGBP with a final concentration of 36 mg / mL (i.e., 180 mg of XLGBP per kg of mouse per gavage) based on the weight of each H22 liver cancer model mouse, for a total of 15 times.

[0084] Technical effect evaluation

[0085] It should be noted that the experimental data for the evaluation of the technical effect of the present invention are expressed as mean ± standard deviation (Mean ± SD), and the experimental data were analyzed using SPSS22.0 statistical analysis software. Independent sample T-test was used for inter-group comparison, and P < 0.05 was considered statistically significant.

[0086] 1. Observation of physiological condition

[0087] During the administration period, the mental state, activity level and food intake of the mice were observed every day, and the weight of each group of mice was measured and recorded, and a weight change curve of the mice was drawn. The results are shown in Figure 1 shown.

[0088] like Figure 1 As shown, Figure 1The weight changes of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group during the drug treatment (the sample size of each group of mice was 8). The results showed that the mice in the normal group were in good health, had a normal diet and smooth hair; the mice in the model group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group had a normal diet, but moved slowly and had sparse hair; the mice in the DOX group ate less, had a significant decrease in weight, moved slowly, had sparse and yellow hair, and the DOX group showed obvious DOX toxic side effects.

[0089] 2. Tumor growth curve

[0090] On days 1, 3, 5, 7, 9, 11, 13, and 15, the long diameter (a) and short diameter (b) of the solid tumors in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group were measured with a vernier caliper, and the formula V = (a × b 2 )×0.5 to calculate the volume of transplanted tumors and draw the tumor growth curve of mice. The results are as follows Figure 2 After 15 days of administration, the mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group were killed by cervical dislocation and the tumors were removed to measure the tumor volume of each group of mice. The results are shown in the figure. Figure 3 shown.

[0091] like Figure 2 As shown, Figure 2 Figure 2 is the tumor growth curve of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group after administration for 1, 3, 5, 7, 9, 11, 13 and 15 days (the sample size of each group of mice is 8). Figure 3 As shown, Figure 3 The figure shows the tumor volume of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group on the 15th day after drug administration (the sample size of each group of mice is 8). The results show that after 15 days of drug intervention, the tumor volume of the DOX group was significantly lower than that of the model group (P<0.01), and the tumor volume of the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group was statistically different between the DOX group and the model group, and compared with the model group (P<0.05), showing a tumor inhibitory effect.

[0092] 3. Survival of mice

[0093] The mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group were given the drug for 15 days and then stopped. The mice were observed for 60 days and the survival days of the mice in each group were counted. The average survival time, median survival time and life extension rate were calculated. The calculation formula of life extension rate was: life extension rate = (average survival days of treatment group / average survival days of model group-1) × 100%. The results are shown in Table 3 and Figure 4 shown.

[0094] Table 3. Mean survival time, median survival time and life extension rate of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the sample size of each group of mice was 8)

[0095]

[0096]

[0097] Note: Compared with the model group, *P<0.05, ***P<0.001

[0098] As shown in Table 3 and Figure 4 As shown, Figure 4 The survival rates of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group within 60 days (the sample size of each group is 8 mice). The average survival time of mice in the model group was 26.9 days, and the average survival time of mice in the DOX group was 50.7 days, with a life extension rate of 89%, which was significantly different from the model group (P<0.001); the average survival time of mice in the XLGBJ-L group and XLGBP-H group was 43 days and 41.6 days, respectively, which was significantly higher than that in the model group (P<0.05), and the life extension rates were 60% and 55%, respectively. It shows that XLGBL or XLGBP can prolong the survival of mice and has anti-liver cancer activity.

[0099] 4. Determination of biochemical indicators

[0100] Blood was collected from the eyeballs of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group 2 hours after the last administration. The whole blood was allowed to stand at room temperature for 30 minutes, centrifuged at 8000 r / min and 4°C for 15 minutes, and the supernatant was collected and stored at -80°C for use. The serum samples stored at -80°C were taken out and thawed at 4°C. The ELISA kit instructions were followed, and the absorbance was measured at the corresponding wavelength using a multifunctional microplate reader to calculate the AFP content level in the serum of each group of mice. The results are shown in Figure 5 shown.

[0101] like Figure 5 As shown, Figure 5 The AFP levels in the serum of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (n=6) (Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the normal group, &P<0.05, &&P<0.01, &&&P<0.001). The results showed that the serum AFP level of mice in the model group was significantly higher than that in the normal group (P<0.05). The serum AFP level of mice in the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group was significantly decreased (P<0.05), but still significantly higher than that in the normal group. It should be noted that AFP is a tumor marker with extremely low content in adult serum. It is one of the important indicators for diagnosing cancer in clinical practice and is often used for the diagnosis and efficacy monitoring of liver cancer.

[0102] 5. Tumor inhibition rate and organ index

[0103] The mice in each group were killed by cervical dislocation, and the tumor, liver, and spleen tissues were taken. After the residual blood was washed, the tumor inhibition rate and organ index of each group of mice were calculated using the formula. Among them, the calculation formula of tumor inhibition rate is: tumor inhibition rate (GRI) = (average tumor weight of model group - average tumor weight of drug administration group) / average tumor weight of model group × 100%. The results are shown in Table 4 and Figure 5 As shown in Table 5, the calculation formula of the organ index (spleen / liver index) is: organ index (spleen / liver index) = spleen / liver mass (mg) / body weight (g). The results are shown in Table 5. Figure 6 and Figure 7 shown.

[0104] Table 4 Tumor weight and tumor inhibition rate of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the sample size of each group of mice is 8)

[0105]

[0106] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the DOX group, # P<0.05, ## P<0.01, ### P<0.001.

[0107] As shown in Table 4 and Figure 6 As shown, Figure 6Figure 2 is the tumor weight of mice in the model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the sample size of each group was 8 mice), (Note: compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the DOX group, # P<0.05, ## P<0.01, ### P<0.001). The results showed that compared with the model group, the tumor weight of mice in the DOX group was significantly reduced (P<0.001); the tumor weight of mice in the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group was between the model group and the DOX group, and there were statistical differences compared with the model group and the DOX group (P<0.01); the tumor inhibition rate of the DOX group was 72.3%, and the tumor inhibition rates of the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group were 49.5%, 45.9%, 53.7% and 51.1%, respectively (P<0.01).

[0108] Table 5 Organ indexes of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (sample size of each group of mice is 8)

[0109]

[0110] Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the normal group, &P<0.05, &&P<0.01, &&&P<0.001.

[0111] As shown in Table 5, Figure 7 and Figure 8 As shown, Figure 7 The spleen index of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the sample size of each group was 8 mice), (Note: compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the normal group, &P<0.05, &&P<0.01, &&&P<0.001). The results showed that compared with the model group, the spleen index of the DOX group was significantly reduced (P<0.001), and the spleen index of mice in the normal group and the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group was between the model group and the DOX group, and was significantly lower than that of the model group (P<0.05).

[0112] Figure 8The liver coefficients of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the sample size of each group was 8 mice), (Note: compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the normal group, &P<0.05, &&P<0.01, &&&P<0.001). The results showed that compared with the normal group, the liver coefficients of mice in the model group and XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group were significantly increased (P<0.01). Compared with the model group, the liver coefficients of mice in the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group were significantly decreased (P<0.05), and there was no statistical difference in the liver coefficients between the DOX group and the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group.

[0113] 6. Pathological tissue observation

[0114] Four mice were randomly selected from each group. A small amount of tumor tissue was cut with a blade and cleaned with saline. Then, it was fixed with 4% paraformaldehyde. The fixed tumor tissue was dehydrated, embedded, and sliced ​​(the slice thickness was 4 microns), and then H&E staining was performed. The H&E staining method was as follows: (1) Paraffin sections were dewaxed to water and the sections were placed in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and distilled water for washing. (2) Hematoxylin staining of cell nuclei: The sections were stained with Harris hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, blued with 0.6% ammonia water, and rinsed with running water. (3) Eosin staining of cytoplasm: The sections were stained with eosin staining solution for 1-3 minutes. (4) Dehydration and sealing: The slices were placed in 95% alcohol I for 5 min-95% alcohol II for 5 min-absolute ethanol I for 5 min-absolute ethanol II for 5 min-xylene I for 5 min-xylene II for 5 min to dehydrate and make them transparent. The slices were taken out of the xylene and dried slightly, and then sealed with neutral gum. Then, a digital slice scanner was used to collect images of the slices. The results are shown in the figure. Fig. 9 As shown, the pathological changes of tumor tissue were observed and the percentage of tumor tissue necrosis area was calculated, wherein the calculation formula of the percentage of tumor tissue necrosis area was: percentage of tumor tissue necrosis area (%) = tumor tissue necrosis area / total tumor area × 100%. The results are shown in Fig.10 shown.

[0115] like Fig. 9 As shown, Fig. 9Tumor tissue sections of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group, and XLGBP-H group; Fig.10 This is the percentage of necrotic area of ​​tumor tissue of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the number of tumor tissue samples of mice in each group was 3), (Note: compared with the model group, *P<0.05, **P<0.01, ***P<0.001; compared with the normal group, &P<0.05, &&P<0.01, &&&P<0.001). The results showed that in the model group, the tumor cells were closely arranged, with clear boundaries, larger and darker nuclei, and fewer necrotic areas. In the XLGBJ-L, XLGBJ-H, XLGBP-L, and XLGBP-H groups, the tumor cells were loosely arranged, with larger intercellular matrix, ruptured nuclei, and enlarged necrotic areas, especially in the XLGBP-H group. Compared with the model group, the percentage of necrotic area was significantly increased (P<0.01). In the DOX group, the necrotic area of ​​tumor cells was the largest, and most necrotic cells had central necrosis and ruptured nuclei.

[0116] 7. Immunohistochemical analysis

[0117] To further clarify the efficacy and potential mechanism of XLGBJ and XLGBP in the treatment of liver cancer, this experiment selected the key proteins used in the clinic to evaluate tumor angiogenesis: CD31 protein and the protein used to mark cells in the proliferation cycle: Ki67 protein, as well as Bcl-2 protein, Bax protein, Fas protein and Fasl protein present in the pathway of inducing cell apoptosis for immunohistochemical detection.

[0118] The specific steps of immunohistochemical analysis are as follows: (1) Paraffin sections: dewax to water and place the sections in xylene I for 10 min-xylene II for 10 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-95% alcohol for 5 min-90% alcohol for 5 min-80% alcohol for 5 min-70% alcohol for 5 min-distilled water. 2. Antigen repair: Soak the sections in citrate buffer, heat in a microwave oven at high temperature for 10 min, stop the fire for 8 min, heat again at medium-high temperature for 10 min, and wash with PBS 3 times after cooling, each time for 5 min. 3. Block endogenous peroxidase: Place the sections in 3% hydrogen peroxide at room temperature for 10 min; wash with PBS 3 times, each time for 5 min. 4. Serum blocking: Add bovine serum blocking solution and react at room temperature for 20 min. 5. Add primary antibody: Gently shake off the blocking solution, add the primary antibody prepared in a certain proportion of PBS to the sections, and then place them flat in a humidified box and incubate overnight at 4°C. Among them, the primary antibodies are Ki67, CD31, Bax, BcL-2, Fas and Fasl6, and the secondary antibody is added: PBS is washed 3 times, 5 minutes each time; the secondary antibody is added, 37℃, 30 minutes; PBS is washed 3 times, 5 minutes each time. Among them, the secondary antibody is HRP-labeled goat anti-rabbit. 7. DAB color development: prepare fresh DAB color development solution, add it to the tissue, color at room temperature, control the color development time under the microscope, and wash the slices with distilled water to stop color development. 8. Counterstaining of cell nuclei: hematoxylin counterstaining for 3 minutes, washing with tap water, differentiation with hematoxylin differentiation solution for a few seconds, rinsing with tap water, hematoxylin blueing solution blueing, and rinsing with running water. 9. Dehydration and sealing: the slices are placed in 75%, 85%, 95%, anhydrous ethanol, and xylene in turn, soaked for 10 minutes respectively, and sealed with neutral gum. 10. Microscope examination, image acquisition and analysis.

[0119] Then, a microscopic camera system was used to collect images, and one microscopic image of Ki67 protein, CD31 protein, Bax protein, BcL-2 protein, Fas protein and Fasl protein at 200 times magnification and two microscopic images at 400 times magnification were collected respectively. The cell nucleus stained with hematoxylin was blue, and the protein positive expression was brown-yellow. The results are as follows: Fig.11 The Halo data analysis system was used to calculate the percentage of positive areas of Ki67 protein, CD31 protein, Bax protein, BcL-2 protein, Fas protein or Fasl protein expression in two microscopic images magnified 400 times, and a bar graph was drawn to compare the groups. The results are shown in Fig.12 shown.

[0120] like Fig.11 and 12 As shown, Fig.11Microscopic images of the expression of Ki67 protein, CD31 protein, Bax protein, BcL-2 protein, Fas protein and Fasl protein in the tumor tissues of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the number of samples of tumor tissue of mice in each group is 3) 。 like Fig.12 As shown, Fig.12 The percentage of positive areas of Ki67 protein, CD31 protein, Bax protein, BcL-2 protein, Fas protein and Fas1 protein l protein expression in tumor tissues of mice in the normal group, model group, DOX group, XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (the number of samples of tumor tissues of mice in each group was 3). (Note: Compared with the model group, *P<0.05, **P<0.01, ***P<0.001).

[0121] The results showed that the expression levels of CD31 protein, Ki67 protein and Bcl-2 protein were the highest in the model group, and the expression levels of CD31, Ki67 and Bcl-2 in the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group were significantly decreased (P<0.05). It should be noted that CD31 is mainly used to prove the existence of endothelial cell tissue, Ki67 is mainly used to mark cells in the proliferation cycle, and Bcl-2 is an apoptosis inhibitor that can inhibit cell apoptosis in a variety of cell systems; the higher its expression level, the more active the tumor cell proliferation and the higher the malignancy.

[0122] The results showed that the expression levels of Bax protein, Fas protein and Fasl protein were the lowest in the model group, and the expression levels of Bax protein, Fas protein and Fasl protein were significantly increased in the XLGBJ-L group, XLGBJ-H group, XLGBP-L group and XLGBP-H group (P<0.05). This indicates that the anti-tumor effects of XLGBJ and XLGBP are related to the inhibition of tumor cell proliferation, inhibition of angiogenesis and promotion of cell apoptosis by regulating certain key proteins. It should be noted that Bax protein is a pro-apoptotic protein in the Bcl-2 gene family. After the combination of Fas protein and Fasl protein, they will form a trimer, cause a Caspase cascade reaction, and induce cell death; the higher its expression level, the better the effect of inducing cancer cell apoptosis.

[0123] In summary, the XLGBJ-L group, XLGBJ-H group, XLGBP-L group or XLGBP-H group all had significant inhibitory effects on the tumor growth of mice with H22 liver cancer model, but the tumor inhibitory effects of the XLGBJ-L group, XLGBJ-H group, XLGBP-L group or XLGBP-H group were lower than those of the DOX group, while there was no significant difference between the tablets (i.e., XLGBP-L group or XLGBP-H group) and the capsules XLGBJ-L group or XLGBJ-H group, and there was no dose-dependency between different concentrations. Furthermore, the AFP level in the serum of mice in the XLGBJ-L group, XLGBJ-H group, XLGBP-L group or XLGBP-H group was significantly reduced, the necrotic area of ​​tumor cells was increased, and the survival of mice was prolonged. Furthermore, the protein expression levels of CD31, Ki67, Bcl-2, Bax, Fas and Fasl in the tumor tissues of mice in the XLGBJ-L group, XLGBJ-H group, XLGBP-L group or XLGBP-H group were statistically different from those in the model group (P<0.05). These results all prove that Xianling Gubao Capsule or Xianling Gubao Tablet has a good intervention effect in the treatment of liver cancer.

[0124] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not limitations of the present invention. Any non-prominent substantial features and non-significant improvements made by those skilled in the art on this basis should fall within the scope of protection of the present invention.

Claims

1. Use of a Chinese medicine composition in preparing a drug for treating liver cancer, characterized in that: The traditional Chinese medicine composition comprises: 12-20 parts of epimedium, 2-8 parts of dipsaci, 1-5 parts of salvia miltiorrhiza, 1-5 parts of anemarrhena, 1-5 parts of psoralea corylifolia and 1-5 parts of rehmannia root.

2. The use according to claim 1, wherein the Chinese medicine composition further comprises a pharmaceutically acceptable excipient.

3. The use according to claim 1 or 2, characterized in that: The dosage form of the traditional Chinese medicine composition includes one or more dosage forms selected from the group consisting of oral liquid, powder, tablet, capsule, granule, paste, powder and pill.

4. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the traditional Chinese medicine composition includes one or more dosage forms selected from the group consisting of tablets, capsules and granules.

5. The use according to any one of claims 1 to 4, characterized in that: The liver cancer is hepatocellular carcinoma.

6. The use according to any one of claims 1 to 5, characterized in that: Application in the preparation of liver cancer drugs for reducing the expression of CD31 protein, Ki67 protein and / or Bcl-2 protein.

7. The use according to any one of claims 1 to 6, characterized in that: Application of the invention in the preparation of liver cancer drugs for increasing the expression of Bax protein, Fas protein and / or Fasl protein.

8. The use according to any one of claims 1 to 7, characterized in that: When the traditional Chinese medicine composition is used to treat liver cancer, the clinical dosage for each administration is 9.89 mg / kg-32.97 mg / kg.

9. The use according to any one of claims 1 to 8, characterized in that: When the traditional Chinese medicine composition is used to treat liver cancer, the total clinical dosage is 148.35 mg / kg-494.50 mg / kg.

10. The use according to any one of claims 1 to 9, wherein the Chinese medicine composition is administered via the gastrointestinal tract.

11. The use according to any one of claims 1 to 10, wherein the Chinese medicine composition has a significant inhibitory effect on H22 mouse tumor growth.

12. The use according to any one of claims 1-11, wherein the Chinese medicine composition reduces the AFP level in the serum of H22 mice, and / or the Chinese medicine composition increases the percentage of necrosis area of ​​tumor tissue in H22 mice.

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