Traditional Chinese medicine composition for treating liver cancer and preparation method thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating liver cancer and a preparation method thereof, the traditional Chinese medicine composition takes turtle shell as a monarch drug, astragalus membranaceus, curcuma zedoary, oyster and nightshade as ministerial drugs, radix pseudostellariae for tonifying qi and nourishing yin, lucid ganoderma for nourishing qi and blood and enhancing the effect of astragalus membranaceus for tonifying healthy qi, oldenlandia diffusa for clearing away heat and toxic materials, akebia fruit for regulating qi, activating blood and removing stasis, and the traditional Chinese medicine composition has the effects of clearing heat and removing toxicity. The capacities of the monarch drug and the ministerial drug for removing stasis toxin and dissipating abdominal mass are enhanced; raw liquorice is used as a conductant drug for regulating the drug property. The traditional Chinese medicine composition has the effects of soothing the liver, strengthening the spleen, tonifying qi and activating blood for liver cancer patients, and can also soften hard lumps, dissipate stagnation, clear away heat and toxic materials, nourish yin, promote salivation, clear deficiency heat and improve the life quality of the patients. The Chinese herbal medicine composition disclosed by the invention is simple in preparation method, easy to popularize, high in compliance in the aspect of anti-liver cancer use, high in tumor inhibition rate, exact in curative effect and safe in clinical application, and has important significance in treatment of liver cancer.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine technology, and in particular to a traditional Chinese medicine composition for treating liver cancer and its preparation method. Background Technology

[0002] Liver cancer is a malignant tumor that occurs in the liver, including primary liver cancer and secondary liver cancer. Currently, common clinical treatments for liver cancer include early diagnosis, surgical resection, liver transplantation, ablation, molecular targeted therapy, and radiotherapy and chemotherapy. However, because liver cancer has an insidious onset, often presenting with few or no symptoms in the early stages, and progresses rapidly, most patients are already in locally advanced stages or have distant metastases at the time of diagnosis. Given the severe side effects of chemotherapy and the limited availability of liver transplantation, the search for safer, more effective, and widely available drugs or treatments for liver cancer is particularly urgent and important.

[0003] Traditional Chinese medicine (TCM) possesses unique advantages in the treatment of liver cancer. It can significantly reduce the adverse reactions of Western medicine treatments such as surgery, radiotherapy, chemotherapy, and interventional therapy, while also noticeably improving the quality of life, enhancing immunity, and prolonging survival for liver cancer patients. Furthermore, it can inhibit the growth and metastasis of liver cancer, significantly improving treatment outcomes. Therefore, combining TCM's understanding of liver cancer treatment with the active development of anti-tumor TCM compound formulas will have significant practical implications. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a traditional Chinese medicine composition for treating liver cancer and a method for preparing the same.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a traditional Chinese medicine composition, said composition being made from the following traditional Chinese medicine raw materials in parts by weight: 10-30 parts of vinegar-processed turtle shell, 30-50 parts of Astragalus membranaceus, 10-20 parts of Codonopsis pilosula, 30-50 parts of Hedyotis diffusa, 10-15 parts of Solanum nigrum, 10-15 parts of Curcuma zedoaria, 30-50 parts of oyster shell, 10-20 parts of Citrus aurantium, 10-20 parts of Ganoderma lucidum, and 5-15 parts of raw licorice.

[0006] In some embodiments of the present invention, the traditional Chinese medicine composition is made from the following traditional Chinese medicine raw materials in parts by weight: 15-30 parts of vinegar-processed turtle shell, 30-45 parts of astragalus, 15-20 parts of codonopsis, 30-45 parts of oldenlandia diffusa, 12-15 parts of black nightshade, 12-15 parts of turmeric, 30-45 parts of oyster shell, 15-20 parts of eucommia ulmoides, 15-20 parts of ganoderma lucidum, and 5-10 parts of raw licorice.

[0007] In some embodiments of the present invention, the vinegar-processed turtle shell is the principal ingredient; the astragalus, turmeric, oyster shell, and black nightshade are the assistant ingredients; the codonopsis, ganoderma, oldenlandia diffusa, and eucommia ulmoides are the adjuvant ingredients; and the raw licorice is the guiding ingredient.

[0008] In a second aspect, the present invention provides a method for preparing the traditional Chinese medicine composition described above, the method comprising the following steps: boiling vinegar-processed turtle shell and oyster shell in water, then adding astragalus, codonopsis, oldenlandia diffusa, black nightshade, turmeric, eucommia ulmoides, ganoderma lucidum and raw licorice, continuing to heat and boil, filtering to obtain the first decoction and dregs, boiling the dregs in water, filtering to obtain the second decoction, and mixing the first and second decoctions to obtain the traditional Chinese medicine composition.

[0009] In some embodiments of the present invention, the step of adding water to the dregs includes adding water to the dregs at a weight of 8-10 times the total weight of the dregs.

[0010] In some embodiments of the present invention, the step of boiling the vinegar-processed turtle shell and oyster shell with water includes 10-15 times the total weight of the Chinese medicinal materials in water.

[0011] In some embodiments of the present invention, the vinegar-processed turtle shell is obtained by scalding clean turtle shell with sand until the surface turns light yellow, removing it, extracting it with vinegar, and drying it.

[0012] In some embodiments of the present invention, the vinegar extraction includes soaking the treated cleaned turtle shell in vinegar.

[0013] In some embodiments of the present invention, 20 kg of vinegar is used for every 100 kg of cleaned turtle shell.

[0014] In some embodiments of the present invention, the preparation method includes the following steps: taking vinegar-processed turtle shell and oyster shell by weight, adding water at a weight of 10-15 times the total weight of the Chinese herbal raw materials, boiling and continuously heating for 30-60 minutes; adding the aforementioned weight parts of Astragalus membranaceus, Codonopsis pilosula, Hedyotis diffusa, Solanum nigrum, Curcuma zedoaria, Citrus aurantium, Ganoderma lucidum, and Glycyrrhiza uralensis, continuing to heat for 30-60 minutes, filtering to obtain the first decoction and the remaining solid residue; adding 8-10 times the weight of water to the remaining solid residue, boiling and continuously heating for 30-45 minutes, filtering to obtain the second decoction; mixing the first and second decoctions to obtain the Chinese herbal composition preparation.

[0015] A third aspect of the present invention provides the use of the herbal composition described above in the preparation of a medicament for the prevention and / or treatment of liver cancer.

[0016] In some embodiments of the present invention, the dosage form of the drug includes injections, powders, capsules, tablets, ointments, suppositories, aerosols, pills, sustained-release tablets, suspensions, granules, lozenges, powders, drops, pills, powders, solutions, creams, patches, tablets, or films.

[0017] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

[0018] In some embodiments of the present invention, the excipients include at least one of a diluent, a stabilizer, an osmotic pressure regulator, a pH regulator, a preservative, and an antioxidant.

[0019] In some embodiments of the present invention, the preservative includes at least one selected from benzoic acid, p-hydroxybenzoic acid, sorbitol, phenoxyethanol, and benzalkonium bromide.

[0020] In some embodiments of the present invention, the osmotic pressure regulator includes at least one of lactose, fructose, glucose, mannitol, sorbitol, and sodium chloride.

[0021] In some embodiments of the present invention, the pH adjuster includes at least one of sodium hydroxide, potassium hydroxide, and acetic acid.

[0022] In some embodiments of the present invention, the antioxidant includes at least one of sodium metabisulfite, vitamin C, and vitamin E.

[0023] In some embodiments of the present invention, the dosage of the traditional Chinese medicine composition is to divide the traditional Chinese medicine preparation prepared according to the above preparation method into two portions, and take them once in the morning and once in the evening.

[0024] A fourth aspect of the present invention provides a method for preparing a drug, the method comprising administering the traditional Chinese medicine composition of claim 1 to an animal by gavage, and then collecting blood and separating serum to obtain the drug.

[0025] In some embodiments of the present invention, the animal includes at least one of mice, rats, and rabbits.

[0026] A fifth aspect of the present invention provides a product comprising a drug prepared by the preparation method described in the above aspects.

[0027] In some embodiments of the present invention, the product includes pharmaceuticals.

[0028] A sixth aspect of the present invention provides the use of a medicament prepared by the preparation method described above in the preparation of products for the prevention and / or treatment of liver cancer.

[0029] In some embodiments of the present invention, the product includes a drug that inhibits the proliferation of liver cancer cells.

[0030] In some embodiments of the present invention, the liver cancer cells include at least one of HCC-lm3 cells, Hep1-6 cells, Hep3B cells, and HepG2 cells.

[0031] In some embodiments of the present invention, the prevention and / or treatment of liver cancer includes at least one of the following: inhibiting hepatocyte proliferation, inhibiting the activation of the mTOR pathway in liver cancer cells, inhibiting hypoxic energy metabolism in liver cancer cells, promoting necrosis and edema of liver cancer tumors, inhibiting the proliferation and growth of liver cancer tumors, and reducing the malignancy of tumors.

[0032] The beneficial effects of this invention are: The principal ingredient in the traditional Chinese medicine composition of this invention is vinegar-processed turtle shell, which softens and disperses nodules and eliminates masses, targeting tangible lumps (tumor tissue) formed by the accumulation of qi, blood, phlegm, and toxins. It is combined with Astragalus membranaceus, Curcuma zedoaria, oyster shell, and Solanum nigrum as assistant ingredients. Astragalus membranaceus strengthens the spleen and replenishes qi, not only eliminating the source of the disease and helping the principal ingredient eliminate tangible lumps, but also aiding the spleen in digestion and replenishing qi, blood, and body fluids, thus restoring the body's resistance and eliminating pathogenic factors. Curcuma zedoaria breaks up blood stasis, promotes qi circulation, eliminates accumulations, and relieves pain, helping the principal ingredient disperse qi and blood stagnation in tangible lumps and also alleviating the pain caused by tangible lumps. Oyster softens hard masses, dissipates nodules, nourishes yin and subdues yang, helping the principal drug disperse phlegm and fluid accumulation in tangible masses, and also nourishes liver yin while suppressing liver yang, aiding in the recovery of liver physiological function; Black nightshade detoxifies and invigorates blood, helping the principal drug disperse the toxic blood stasis in tangible masses; Codonopsis pilosula tonifies qi and nourishes yin, Ganoderma lucidum tonifies qi and blood, enhancing the effect of Astragalus membranaceus in tonifying vital energy; Hedyotis diffusa clears heat and detoxifies, and Anemarrhena asphodeloides regulates qi, invigorates blood, and dissipates nodules, enhancing the ability of the principal and assistant drugs to remove blood stasis and toxins and dissipate masses; Raw licorice root serves as the guiding drug, harmonizing the properties of the drugs and also having the effect of clearing heat and detoxifying.

[0033] Oyster shell is slightly cold in nature and salty in taste. It enters the liver, gallbladder, and kidney meridians. It has a calming and sedative effect, nourishes yin and yang, softens hardness and dissipates nodules. It is used to treat palpitations, insomnia, dizziness, tinnitus, scrofula, phlegm nodules, and abdominal masses. When oyster shell is combined with turtle shell, its effects of nourishing yin and yang, softening hardness and dissipating nodules are enhanced.

[0034] Curcuma zedoaria is warm in nature and bitter and pungent in taste. It enters the liver and spleen meridians. It breaks up blood stasis, promotes qi circulation, eliminates stagnation, and relieves pain. It is used to treat masses and accumulations caused by qi stagnation and blood stasis, amenorrhea, and abdominal pain due to blood stasis. Curcuma zedoaria breaks up blood stasis and can clear blood stasis in the larger meridians of the liver meridian. When combined with Solanum nigrum and oyster shell, it can specifically eliminate qi stagnation, blood stasis, phlegm, and toxins in tangible masses.

[0035] The traditional Chinese medicine composition of this invention is primarily used to treat primary and secondary liver cancer. This composition for treating liver cancer exhibits significant effects in soothing the liver and strengthening the spleen, invigorating qi and promoting blood circulation in liver cancer patients. It also softens and disperses masses, clears heat and detoxifies, nourishes yin and generates fluids, and clears deficiency heat, thus improving the quality of life for patients. It can significantly improve symptoms and signs in liver cancer patients such as hypochondriac pain, fatigue, emotional distress, poor appetite, abdominal distension, splenomegaly, dark tongue, and thready pulse, and can improve liver function in liver cancer patients.

[0036] The herbal composition of the present invention can be combined with Western medicine treatment plans for liver cancer and effectively improve the treatment effect and quality of life of liver cancer patients. In the treatment of liver cancer, it can achieve the technical effects of high operability, high compliance, high tumor inhibition rate, definite curative effect, and low clinical application safety and adverse reactions. Attached Figure Description

[0037] Figure 1 The figure shows the experimental results of the effect of the drug-containing serum prepared by the traditional Chinese medicine composition of this invention on the expression level of PCNA protein in HCC-lm3 cells.

[0038] Figure 2 This is a bar chart showing the effect of the drug-containing serum prepared from the traditional Chinese medicine composition of this invention on the proliferation rate of HCC-lm3 cells.

[0039] Figure 3 The figure shows the experimental results of the effect of the drug-containing serum prepared by the traditional Chinese medicine composition of the present invention on the expression levels of mTOR and p-mTOR proteins in HCC-lm3 cells.

[0040] Figure 4 The figure shows the experimental results of the effect of the drug-containing serum prepared by the traditional Chinese medicine composition of the present invention on the expression levels of AKT, p-AKT and PI3K proteins in HCC-lm3 cells.

[0041] Figure 5 The figure shows the experimental results of the effect of the drug-containing serum prepared by the traditional Chinese medicine composition of the present invention on the expression level of HIF-1α protein in HCC-lm3 cells.

[0042] Figure 6 The figure shows the experimental results of the effect of the drug-containing serum prepared by the traditional Chinese medicine composition of the present invention on the pathological state of subcutaneous xenograft tumors in mice with liver cancer cells. The scale bar is 100 μm.

[0043] Figure 7 The figure shows the experimental results of the effect of the drug-containing serum prepared by the traditional Chinese medicine composition of the present invention on the malignancy of subcutaneous xenograft tumors in mice with liver cancer cells. The scale bar is 100 μm. Detailed Implementation

[0044] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art. The term "oyster" in traditional Chinese medicine specifically refers to oyster shells.

[0045] Example 1 This embodiment provides the preparation of the traditional Chinese medicine composition for treating liver cancer according to the present invention, as detailed below: According to the medicinal material standards of the Pharmacopoeia of the People's Republic of China (2020 Edition), the following Chinese medicinal materials were selected: 15g of vinegar-processed turtle shell, 30g of Astragalus membranaceus, 15g of Codonopsis pilosula, 30g of Hedyotis diffusa, 12g of Solanum nigrum, 12g of Curcuma zedoaria, 30g of oyster shell, 15g of Citrus aurantium, 15g of Ganoderma lucidum, and 5g of raw licorice.

[0046] Place the vinegar-processed turtle shell and oyster shell into a clay pot, add water equal to 15 times the total weight of the raw materials to cover the herbs, bring to a boil and continue heating for 30 minutes; add the remaining Chinese herbs into the clay pot and continue heating for 45 minutes, filter to obtain the first decoction and the remaining solid dregs; add 10 times the weight of water to the remaining solid dregs, bring to a boil and continue heating for 45 minutes, filter to obtain the second decoction; mix the first and second decoctions to obtain the Chinese herbal composition.

[0047] Example 2 This embodiment provides the preparation of the traditional Chinese medicine composition for treating liver cancer according to the present invention, as detailed below: According to the medicinal material standards of the Pharmacopoeia of the People's Republic of China (2020 Edition), the following Chinese medicinal materials were selected: 30g of vinegar-processed turtle shell, 30g of Astragalus membranaceus, 20g of Codonopsis pilosula, 45g of Hedyotis diffusa, 15g of Solanum nigrum, 15g of Curcuma zedoaria, 45g of oyster shell, 20g of Citrus aurantium, 20g of Ganoderma lucidum, and 10g of raw licorice root. The preparation method is the same as in Example 1.

[0048] Example 3 This embodiment provides the preparation of the traditional Chinese medicine composition for treating liver cancer according to the present invention, as detailed below: According to the medicinal material standards of the Pharmacopoeia of the People's Republic of China (2020 Edition), the following Chinese medicinal materials were selected: 15g of vinegar-processed turtle shell, 45g of Astragalus membranaceus, 20g of Codonopsis pilosula, 45g of Hedyotis diffusa, 12g of Solanum nigrum, 12g of Curcuma zedoaria, 30g of oyster shell, 15g of Citrus aurantium, 20g of Ganoderma lucidum, and 10g of raw licorice root. The preparation method is the same as in Example 1.

[0049] Example 4 This embodiment provides the effect of the drug-containing serum prepared from the traditional Chinese medicine composition of Example 1 on the proliferation of HCC-lm3 cells (human liver cancer cells) and the changes in the expression levels of proliferation-related proteins. The dosage of the traditional Chinese medicine composition used was to divide the traditional Chinese medicine composition prepared in Example 1 into two portions, which were taken once in the morning and once in the evening. The specific experimental steps are as follows.

[0050] Preparation of drug-containing serum: Rats were randomly divided into a high-dose group of the herbal composition (KAF-H group), a sorafenib positive drug group (Sora group), and a saline negative control group (NC group). The KAF-H group received twice the standard human treatment dose in Example 1 (standard treatment dose is 1 dose / day), and the Sora group received the standard human treatment dose of sorafenib. The dosage for rats (animal dose) was calculated based on the adult (60kg) clinical dose (200mg twice daily, 400mg once). K m =37, rat K m =6. The NC group used the same volume of physiological saline instead of the treatment drug. The drug was administered at 8:00 AM and 8:00 PM daily for 3 consecutive days. On the 4th day, the drug was administered once at 8:00 AM, and rat serum was collected 2 hours later. The KAF-H rat serum group was prepared by diluting the NC group rat serum by 1 / 2 and 1 / 4 to obtain the KAF-M group serum and KAF-L group serum, respectively.

[0051] Experimental steps: Western Blot experiment: HCC-lm3 cells were routinely plated into 6cm dishes and divided into Control (normal fetal bovine serum group, fetal bovine serum purchased from Wuhan Pronosei Life Science Technology Co., Ltd.), NC group, KAF-L group, KAF-M group, KAF-H group and Sora group. Each group was treated with 3mL of serum (containing 10% (v / v) of the corresponding group's serum + 90% (v / v) DMEM high glucose basal medium) for 24h.

[0052] Cells were lysed using RIPA lysis buffer (radioimmunoprecipitation lysis buffer) after treatment, and proteins were extracted. Protein concentration was quantified using a BCA protein assay kit (Guangzhou Jiebes Biotechnology Co., Ltd., G3522). An equal volume of protein sample was mixed with 5×SDS-PAGE protein loading buffer (Guangzhou Jiebes Biotechnology Co., Ltd.), and heated at 95°C for 10 minutes to denature the proteins. SDS-PAGE gel electrophoresis was prepared using 10% (v / v) acrylamide, with 10 μg loaded into each well. After electrophoresis, the gel was transferred to a membrane and blocked with 5% BSA at room temperature for 2 hours. Anti-PCNA antibody (Wuhan Sanying Biotechnology Co., Ltd.) was used as the primary antibody, and anti-GAPDH antibody (Wuhan Saiweier Biotechnology Co., Ltd.) was used as the internal control. The membrane was incubated overnight at 4°C, washed three times with TBST buffer, and then incubated with secondary antibody (goat anti-mouse antibody, Proteintech) at room temperature for 2 hours. After washing three times with TBST buffer, the membrane was exposed using a high-sensitivity ECL chemiluminescence reagent (Guangzhou Jiebes Biotechnology Co., Ltd.).

[0053] CCK-8 assay: HCC-lm3 cells were seeded in 96-well plates with 5000 cells per well. After 12 hours of culture and cell adhesion, the cells were divided into four groups: Control (normal fetal bovine serum), NC, KAF-L, KAF-M, and KAF-H. Each group was treated with 10% (v / v) drug-containing serum and 90% (v / v) basal medium for 24 hours. 10 μL of CCK-8 assay reagent (Guangzhou Jiebesi Biotechnology Co., Ltd.) was added to each well, and the cells were incubated at 37°C for 1-2 hours. The OD value at 450 nm was measured using a microplate reader, and the cell proliferation inhibition rate was calculated.

[0054] Western Blot experimental results are as follows: Figure 1 As shown, compared with the Control and NC groups, the KAF-L, KAF-M, KAF-H, and Sora groups significantly reduced the expression of PCNA protein in HCC-lm3 cells in a dose-dependent manner, indicating that the herbal composition can significantly inhibit the proliferation of HCC-lm3 cells.

[0055] CCK-8 test results are as follows Figure 2 As shown, compared with the Control and NC groups, the KAF-L, KAF-M, and KAF-H groups significantly reduced the proliferation of HCC-lm3 cells, indicating that the herbal composition can significantly inhibit the proliferation of HCC-lm3 cells.

[0056] Example 5 This embodiment provides the effect of the drug-containing serum prepared by the traditional Chinese medicine composition in Example 1 on the expression of mTOR, p-mTOR, AKT, p-AKT and PI3K in HCC-lm3 cells, with GAPDH as an internal control.

[0057] The preparation of the herbal composition containing serum, cell plating, and drug treatment regimen are the same as in Example 4.

[0058] The Western Blot experimental procedure is the same as in Example 4, except that the antibodies used in this example are: anti-mTOR antibody (Wuhan Sanying Biotechnology Co., Ltd.), anti-p-mTOR antibody (Wuhan Sanying Biotechnology Co., Ltd.), anti-AKT antibody (Wuhan Sanying Biotechnology Co., Ltd.), anti-p-AKT antibody (Wuhan Sanying Biotechnology Co., Ltd.), and anti-PI3K antibody (Wuhan Sanying Biotechnology Co., Ltd.). The secondary antibody used is goat anti-mouse antibody (Proteintech).

[0059] Experimental results are as follows Figure 3-4 As shown, Figure 3 The experimental results showed that, compared with the Control and NC groups, the KAF-L, KAF-M, KAF-H, and Sora groups significantly reduced the p-mTOR / mTOR ratio in HCC-lm3 cells, indicating that the herbal composition can significantly inhibit the activation of the mTOR pathway in HCC-lm3 cells. Figure 4 The experimental results showed that compared with the Control and NC groups, the drug KAF-L, KAF-M, KAF-H and Sora groups could significantly reduce the p-AKT / AKT ratio in HCC-lm3 cells and reduce PI3K expression, indicating that the traditional Chinese medicine composition could significantly inhibit the amplification of upstream regulatory factors of the mTOR pathway in HCC-lm3 cells.

[0060] Example 6 This embodiment provides an assessment of the effect of the drug-containing serum prepared by the traditional Chinese medicine composition in Example 1 on the expression of HIF-1α in HCC-lm3 cells, and evaluates its effect on the mTOR pathway in HCC-lm3 cells, with GAPDH as an internal control.

[0061] The preparation of the herbal composition containing serum, cell plating, and drug treatment regimen are the same as in Example 4.

[0062] Experimental methods: The Western Blot experimental procedure is the same as in Example 4, except that the antibody used in this example is: anti-HIF-1α antibody (Wuhan Sanying Biotechnology Co., Ltd.).

[0063] Experimental results are as follows Figure 5 As shown, compared with the Control and NC groups, the KAF-L, KAF-M, KAF-H, and Sora groups significantly reduced the expression of HIF-1α in HCC-lm3 cells, indicating that the herbal composition can significantly inhibit hypoxic energy metabolism in HCC-lm3 cells.

[0064] Example 7 This embodiment provides the effect of the traditional Chinese medicine composition of Example 2 on the pathological state of subcutaneous xenograft tumors in a mouse model of subcutaneous liver cancer cells. The specific experimental steps are as follows: C57 mice were randomly divided into four groups: a medium-dose group of the herbal medicine composition (KAF-M group), a high-dose group of the herbal medicine composition (KAF-H group), a sorafenib positive drug group (Sora group), and a saline negative control group (model group). The dosage and administration time for each group were the same as those in the preparation of drug-containing serum in Example 4. The KAF-H group was given twice the standard human treatment dose in Example 2, the KAF-M group was given the standard human treatment dose in Example 2, the Sora group was given the standard human treatment dose of sorafenib, and the dosage for mice was converted to the adult (60 kg) clinical dose. The model group was given an equal volume of saline instead of the treatment drug.

[0065] Experimental steps: Take a suspension of Hep1-6 cells (Wuhan Saiweier Biotechnology Co., Ltd.) in the logarithmic growth phase, with a concentration of approximately 1×10⁻⁶. 7 Cells per mL were injected into the right axilla of C57 mice, 100 μL per mouse. One week after inoculation, mice that failed to establish the model were removed, and the remaining mice with subcutaneous hepatocellular carcinoma xenografts were randomly divided into KAF-M, KAF-H, Sora, and model groups. The mice received the corresponding drug treatment daily, with the dosage and timing as in Example 4, for a total of 3 weeks. Mice were sacrificed 24 hours after the last drug treatment, and the subcutaneous xenograft tissue was dissected for subsequent experiments.

[0066] Subcutaneous xenograft tissue was fixed in 4% (v / v) neutral paraformaldehyde for 48 h, washed with running water, dehydrated with graded ethanol, cleared with xylene, embedded in paraffin, sectioned, and sectioned to a thickness of 4 μm. H&E staining was performed using a hematoxylin-eosin (H&E) high-resolution constant staining kit (Wuhan Saive Biotechnology Co., Ltd.).

[0067] Experimental results are as follows Figure 6 As shown, compared with the model group, the KAF-M, KAF-H, and Sora groups significantly promoted necrosis of subcutaneous xenografts in mice with hepatocellular carcinoma (the pink area indicated by the blue arrow is the necrotic area) and promoted tumor cell edema in the subcutaneous xenografts of mice with hepatocellular carcinoma (the area indicated by the red arrow), and this was in a dose-dependent manner. This indicates that the herbal composition can significantly promote necrosis of subcutaneous xenografts and edema of tumor cells in mice with hepatocellular carcinoma.

[0068] Example 8 This embodiment describes the effect of the traditional Chinese medicine composition of Example 2 on the malignancy of subcutaneous xenografts in a mouse model of hepatocellular carcinoma. The expression changes of KI67, a marker of cell proliferation activity, in the subcutaneous xenografts were detected after treatment with the traditional Chinese medicine composition of Example 2 in the mice.

[0069] The preparation of the subcutaneous xenograft mouse model of liver cancer cells, the drug treatment regimen, and the preparation of paraffin sections of tissue were the same as in Example 7.

[0070] Experimental methods: Paraffin sections of subcutaneous xenograft tumor tissue were dewaxed and hydrated. Antigen retrieval was performed using EDTA antigen retrieval solution (pH 9.0, Wuhan Saiwei Biotechnology Co., Ltd.). Endogenous peroxidase and biotin were inactivated with 3% (w / w) hydrogen peroxide solution. The sections were blocked at room temperature for 30 min and incubated overnight at 4°C with anti-KI67 antibody (Wuhan Sanying Biotechnology Co., Ltd., Cat No. 84192-4-RR). The sections were then treated with GTVision. TM III. Detection System / Mo&Rb (Including DAB) Immunohistochemical Detection Kit (Gene Technology Shanghai Co., Ltd.): Incubate with secondary antibody at room temperature for 1 hour and develop DAB color according to the instructions. Counterstain with hematoxylin solution, dehydrate and mount the slide, and scan and photograph the slide.

[0071] Experimental results are as follows Figure 7 As shown, compared with the model group, the KAF-M, KAF-H, and Sora groups significantly inhibited the expression of KI67 in the subcutaneous xenografts of hepatocellular carcinoma in mice in a dose-dependent manner. This indicates that the herbal composition can significantly inhibit the proliferation of tumor cells in the subcutaneous xenografts of hepatocellular carcinoma in mice, inhibit the growth of the subcutaneous xenografts, and reduce the malignancy of the subcutaneous xenografts.

[0072] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, The traditional Chinese medicine composition is made from the following traditional Chinese medicine raw materials in parts by weight: 10-30 parts of vinegar-processed turtle shell, 30-50 parts of astragalus, 10-20 parts of codonopsis, 30-50 parts of oldenlandia diffusa, 10-15 parts of black nightshade, 10-15 parts of turmeric, 30-50 parts of oyster shell, 10-20 parts of eucommia ulmoides, 10-20 parts of ganoderma lucidum, and 5-15 parts of raw licorice.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The traditional Chinese medicine composition is made from the following traditional Chinese medicine raw materials in parts by weight: 15-30 parts of vinegar-processed turtle shell, 30-45 parts of astragalus, 15-20 parts of codonopsis, 30-45 parts of oldenlandia diffusa, 12-15 parts of black nightshade, 12-15 parts of turmeric, 30-45 parts of oyster shell, 15-20 parts of eucommia ulmoides, 15-20 parts of ganoderma lucidum, and 5-10 parts of raw licorice.

3. The method for preparing the traditional Chinese medicine composition according to any one of claims 1-2, characterized in that, The preparation method includes the following steps: boil vinegar-processed turtle shell and oyster shell in water, then add astragalus, codonopsis, oldenlandia diffusa, black nightshade, turmeric, eucommia ulmoides, ganoderma lucidum and raw licorice, continue heating and boiling, filter to obtain the first decoction and dregs, boil the dregs in water, filter to obtain the second decoction, mix the first decoction and the second decoction to obtain the Chinese herbal composition.

4. The method for preparing the traditional Chinese medicine composition according to claim 3, characterized in that, The process of adding water to the medicinal residue includes adding water at a weight of 8-10 times the total weight of the medicinal residue.

5. The method for preparing the traditional Chinese medicine composition according to claim 3, characterized in that, The step of boiling the vinegar-processed turtle shell and oyster shell in water includes 10-15 times the total weight of the medicinal materials in water.

6. The use of the herbal composition according to any one of claims 1-2 in the preparation of a medicament for the prevention and / or treatment of liver cancer.

7. A method for preparing a drug, characterized in that, The preparation method includes administering the traditional Chinese medicine composition of claim 1 to animals by gavage, collecting blood and separating serum.

8. A product characterized in that, The product includes a drug prepared using the preparation method of claim 7.

9. The use of the medicament as described in claim 8 in the preparation of products for the prevention and / or treatment of liver cancer.

10. The application according to claim 9, characterized in that, The product includes a drug that inhibits the proliferation of liver cancer cells; preferably, the liver cancer cells include at least one of HCC-lm3 cells, Hep1-6 cells, Hep3B cells, and HepG2 cells.

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