Application of leech dredging network preparation in preparation of anti-tumor drugs

Abstract

The application belongs to the field of traditional Chinese medicines, and particularly discloses application of a leech dredging collaterals preparation in preparation of an anti-tumor medicine. The leech dredging collaterals preparation is prepared from four raw medicinal materials of astragalus, ligusticum wallichii, leech and salvia miltiorrhiza, and has the effects of tonifying qi and activating blood and dredging qi and collaterals. Pharmacodynamic studies show that the leech dredging collaterals preparation has the effects of inhibiting growth of liver tumors and lung tumors, is safe and has a good effect, expands the clinical application range of the leech dredging collaterals preparation, and has a good popularization and application prospect.

Description

Technical Field

[0001] This invention relates to the application of leech-derived collaterals-clearing preparations in the preparation of antitumor drugs, belonging to the field of traditional Chinese medicine, specifically to the application of leech-derived collaterals-clearing preparations in the preparation of drugs for treating malignant tumors of the liver and lungs. Background Technology

[0002] A tumor is a new growth formed by the proliferation of local tissue cells under the influence of various carcinogenic factors. Because these growths often present as space-occupying, mass-like protrusions, they are also called growths. Based on the cellular characteristics of the growth and the degree of harm it causes to the body, tumors can be divided into two main categories: benign tumors and malignant tumors. Malignant tumors are a serious threat to human life and health worldwide, with an extremely high incidence rate, and are a leading cause of death globally. In China, malignant tumors rank second only to cerebrovascular diseases in the top ten disease mortality rates, with an incidence rate as high as 22.08% and a mortality rate of approximately 12.94%. Therefore, the prevention and treatment of malignant tumors is a common challenge in the life sciences field.

[0003] Currently, the most commonly used classic treatments in clinical practice include surgery, chemotherapy, and radiotherapy. However, surgery and radiotherapy are both local therapies and are ineffective for patients whose cancer has metastasized. Furthermore, while radiotherapy and chemotherapy kill tumor cells, they also damage normal cells and immune cells, leading to damage to various tissues and organs. In particular, many chemically synthesized anticancer drugs can cause serious side effects such as bone marrow suppression, weakened immunity, liver and kidney damage, and cardiotoxicity. Damage to the immune system can also lead to weakened immune function, preventing the immune surveillance system from functioning properly and potentially inducing other tumors. Additionally, patients develop drug resistance during chemotherapy, not only against the same type of drug but also against chemotherapy drugs with completely different chemical structures and mechanisms of action, significantly reducing efficacy and treatment effectiveness, resulting in unsatisfactory treatment outcomes. In short, the treatment of cancer has not yet achieved satisfactory results. Therefore, finding a highly effective, low-side-effect cancer treatment that can repair the immune system and enhance the body's immunity has become a new focus of international research.

[0004] In recent years, traditional Chinese medicine has made great strides in the treatment of tumors. It has the advantages of multi-component and multi-target synergistic effects, few toxic side effects and adverse reactions, as well as wide availability and low price. It plays an important synergistic role in the clinical treatment of tumors. It can improve the treatment effect of tumors by regulating immunity, reducing side effects and eliminating drug resistance. It has shown good efficacy in alleviating patient symptoms, prolonging survival time or in combination with radiotherapy and chemotherapy.

[0005] The formulation of this invention is named Chuanzhi Tongluo Capsules, produced by Lunan Houpu Pharmaceutical Co., Ltd., with approval number Z20090031. Clinically, it is used to treat ischemic stroke with blood stasis and qi deficiency as the main symptoms. During clinical use, it has been found to have good efficacy against tumors. The Chuanzhi Tongluo preparation is composed of four raw medicinal materials: leech, chuanxiong (Ligusticum striatum), astragalus, and danshen (Salvia miltiorrhiza). It has the effects of promoting blood circulation, removing blood stasis, invigorating qi, and unblocking collaterals. It was developed based on Buyang Huanwu Decoction. In the formula, leech is the principal ingredient, which effectively removes blood stasis and breaks up blood stasis; chuanxiong is the assistant ingredient, whose upward-moving nature allows it to work synergistically with leech to reach the brain collaterals, promoting blood circulation and unblocking collaterals, thus ensuring smooth flow of qi and blood and healthy organ function; astragalus and danshen are adjuvant ingredients. Astragalus invigorates qi to promote blood circulation and warm the meridians, while danshen invigorates blood, removes blood stasis, calms the mind, and nourishes the heart. The entire formula works synergistically to promote blood circulation, remove blood stasis, invigorate qi, and unblock collaterals. Summary of the Invention

[0006] The purpose of this invention is to provide an application of a leech-based meridian-clearing preparation in the preparation of anti-tumor drugs. This application was discovered during the clinical application of the company's drug and subsequently confirmed by relevant pharmacodynamic tests, demonstrating significant commercial value.

[0007] During the clinical application of leech-infused capsules, the inventors discovered that these capsules have therapeutic effects on malignant tumors. Pharmacodynamic tests showed that leech-infused capsules are an effective formula for treating malignant tumors of the liver and lungs. They can inhibit tumor cell growth, shrink tumors, enhance the body's immune function, and prolong the lives of cancer patients, providing a new option for clinical medication and demonstrating the holistic, multi-target, and multi-component synergistic therapeutic characteristics of compound traditional Chinese medicine.

[0008] It should be noted that the application described in this invention is based on the already marketed product, Hirudo Tongluo Capsule, but the application of this invention is not limited to the above-mentioned preparation. All Hirudo Tongluo preparations obtained using the following technical solutions of this invention can be used to treat malignant tumors.

[0009] The leech-based meridian-clearing preparation of this invention is made from the following traditional Chinese medicine components:

[0010] Leeches 300-800 parts by weight, Ligusticum striatum 300-800 parts by weight

[0011] Astragalus membranaceus 200-600 parts by weight, Salvia miltiorrhiza 200-600 parts by weight.

[0012] Preferably, the traditional Chinese medicine composition is made from the following components:

[0013] Leech 600 parts by weight, Ligusticum chuanxiong 600 parts by weight

[0014] Astragalus membranaceus 400 parts by weight, Salvia miltiorrhiza 400 parts by weight

[0015] In addition to its effects of promoting blood circulation, removing blood stasis, invigorating qi, and unblocking collaterals, the traditional Chinese medicine composition of this invention can be used clinically to treat ischemic stroke, including cerebral ischemia and hypoxia, cerebral thrombosis, cerebral infarction, and lacunar infarction. It can also treat tumors, with high safety and significant effects, opening up a new traditional Chinese medicine treatment method for tumors.

[0016] Using the traditional Chinese medicine described in this invention as raw material, and adding different excipients, the traditional Chinese medicine components of this invention can be prepared into different clinically acceptable oral drug preparations, such as granules, capsules, tablets, or pills, according to conventional processes.

[0017] In order for the above dosage forms to be realized, pharmaceutical excipients need to be added during the preparation of the above dosage forms. These pharmaceutical excipients may include, but are not limited to, fillers, binders, preservatives, flavoring agents, humectants, disintegrants, and lubricants.

[0018] The preparation process of the oral drug formulation includes the following steps:

[0019] A. Take the prescribed amount of leech medicinal material, crush it into coarse powder, add 4-8 times the amount of water, stir and extract at 37 ℃ for 10-30 h, centrifuge to discard the precipitate, filter the supernatant through a membrane filter, then filter it through an ultrafiltration device and concentrate it, freeze-dry the concentrate to obtain leech freeze-dried powder for later use.

[0020] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 4-8 times the amount of 65%-95% ethanol and reflux extract twice, each time for 2-3 hours, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.15-1.25 at 50-60 ℃, and set aside. Set aside the residue.

[0021] C. Take the dregs obtained in step B, add water in an amount of 3-5 times the weight of the original medicinal materials, decoct once for 1-2 hours, filter, and concentrate the filtrate to extract II with a relative density of 1.15-1.25 at 50-60 ℃, and set aside.

[0022] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0023] E. Mix the lyophilized leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize them into fine powder, and prepare oral drug formulations directly or by adding pharmaceutically acceptable excipients through conventional processes.

[0024] Preferably, for step A, take the prescribed amount of leech medicinal materials, add 6 times the amount of water, stir and extract at 37 °C and 100 r / min for 20 h, centrifuge at 6000 r / min for 30 min, discard the precipitate, filter the supernatant through a membrane with a pore size of 1 μm, then filter and sterilize through a membrane with a pore size of 0.2 μm, then filter through an ultrafilter with a molecular weight cut-off of 6000 - 10000 Da and concentrate to 600 mL, and freeze-dry the concentrated solution to obtain freeze-dried leech powder.

[0025] Preferably, for step B, add 6 times the amount of 80% ethanol, and concentrate the filtrate to an extract with a relative density of 1.20 at 50 - 60 °C.

[0026] Preferably, for step C, add 4 times the amount of water based on the weight of the crude drug, and concentrate the filtrate to an extract with a relative density of 1.20 at 50 - 60 °C.

[0027] To verify the efficacy of the Chuanzhi Tongluo preparation in the treatment of malignant tumors in the present invention, the inventor carried out a pharmacodynamic experimental study. It should be noted that the drugs selected for the pharmacodynamic experiment of the drugs in the present invention are the drugs obtained from the representative formula and its preparation method of the present invention. Due to space limitations, the experiments and results of the drugs obtained from other formulas and preparation methods included in the present invention are not enumerated here one by one.

[0028] Experimental Example 1 Study on the Effect of Chuanzhi Tongluo Capsule on H22 Hepatoma-bearing Mice

[0029] 1 Experimental Materials

[0030] 1.1 Experimental Animals

[0031] Kunming strain mice, female, 4 - 6 weeks old, weighing 18 - 22 g, experimental animal license number: SYXK(Shandong)-20180008, provided by Shandong Xinsdai Pharmaceutical Co., Ltd. Before the experiment, all mice were placed in an environment with a room temperature of 24 - 26 °C, a relative humidity of 50% - 60%, good ventilation and lighting, and were fed with standard mouse feed routinely, with free access to food and water.

[0032] 1.2 Instruments, Reagents and Drugs

[0033] Centrifuge 5415R high-speed refrigerated centrifuge (Eppendorf AG, USA); SZX-1LLB200 inverted microscope (Olympus, Japan); high-glucose DMEM culture medium (BI, USA, batch number: 0033617); fetal bovine serum (Gibco, batch number: 42Q7380K); trypsin (BI, USA, batch number: 0010218); cyclophosphamide (Baxter International Ltd., batch number: 6F125A, 0.2g / tablet); H22 tumor strain provided by Shandong Academy of Medical Sciences; test drug: Chuanzhi Tongluo Capsules (provided by Lunan Houpu Pharmaceutical Co., Ltd., National Drug Approval Number Z20090031); positive control drug: Pingxiao Tablets (Shenyang Kangda Pharmaceutical Group Co., Ltd., National Drug Approval Number Z21021484).

[0034] 2 Experimental Methods

[0035] 2.1 Cell Culture

[0036] Mouse hepatocellular carcinoma H22 cells were cultured in DMEM complete medium containing 10% fetal bovine serum in culture dishes and placed in a 37℃ 5% CO2 constant temperature incubator. The medium was changed every other day, and the growth status of the cells was observed under an inverted microscope. When the confluence of cells reached 70%-80%, they were passaged or used for experiments.

[0037] 2.2 Obtaining ascites fluid from mice

[0038] H22 cells in logarithmic growth phase were centrifuged at 800 r / min for 5 min, the supernatant was discarded, cells were counted, and the cell density was adjusted to 1×10⁻⁶. 7 Cells / mL. Two Kunming mice were randomly selected, and 0.2 mL of cell suspension was injected into the peritoneal cavity of each mouse using a 1 mL syringe. The abdominal condition was observed. If significant abdominal distension was observed after 7-10 days, the inoculation was considered successful, and ascites passage could be performed. The mice were euthanized by cervical dislocation, and milky white ascites was extracted in a clean bench. The ascites cell density was diluted with PBS to 1×10⁻⁶. 7 Cells per mL were collected and injected into the peritoneum of mice at a rate of 0.2 mL. After three passages, the ascites cells from the third generation were used for tumor-bearing experiments in Kunming mice.

[0039] 2.3 Subcutaneous tumor formation experiment in mice

[0040] Third-generation ascites H22 cells were collected, counted, and the cell density was adjusted to 5 × 10⁻⁶. 6 Cells were injected subcutaneously at a rate of 0.2 mL / mL into the midline of the abdomen of female Kunming mice. One to two days after inoculation, if the tumor diameter measured with calipers was 1 mm... 3Mice were randomly divided into four groups: a model group, a positive control group, a low-dose group (referred to as the "low-dose group"), a medium-dose group (referred to as the "medium-dose group"), and a high-dose group (referred to as the "high-dose group") of Hirudo Tongluo Capsules, with 10 mice in each group. Ten untreated mice were also included as a blank control group. The equivalent dose for mice was calculated according to the "Equivalent Dose Ratio Table Based on Human and Animal Body Surface Area." The low, medium, and high-dose groups of Hirudo Tongluo Capsules were administered via gavage at 0.11375 g / kg, 0.22750 g / kg, and 0.45500 g / kg, respectively. The positive control group received an intraperitoneal injection of Pingxiao tablets at 0.62790 g / kg. The blank and model groups were administered the same volume of physiological saline via gavage. All groups received the medication for 10 consecutive days. After the 10th day of administration, mice were kept NPO (no food but allowed water). The following day, blood was collected from the orbital cavity, and the mice were euthanized by cervical dislocation. The tumors were dissected, weighed, and recorded. The tumor inhibition rate for each group was calculated.

[0041] Tumor inhibition rate = (mean tumor weight in the model group - mean tumor weight in the sample group) / mean tumor weight in the model group × 100%.

[0042] 2.4 Statistical Analysis

[0043] SPSS 21.0 statistical software was used for analysis. Experimental data are expressed as mean ± standard deviation (%). x ± s The results were expressed in the form of “)”. Comparisons among multiple groups were performed using one-way ANOVA. P <0.05 indicates that the difference is statistically significant.

[0044] 3. Experimental Results

[0045] The results showed that, compared with the normal control group, the average tumor weight of mice in the model group was significantly increased ( P <0.05) indicates successful model establishment; compared with the model group, after treatment with different doses of leech to clear the meridians, the average tumor weight of mice was reduced to a certain extent, and the difference was statistically significant. P <0.05), indicating that different dosage groups of the leech-infused collaterals capsule of the present invention and the positive control group all inhibited tumor growth in H22 mice, and that the inhibitory effect of the leech-infused collaterals preparation of the present invention on tumors was dose-dependent. The results are shown in Table 1.

[0046] Table 1. Inhibitory effect of the traditional Chinese medicine composition of the present invention on H22 transplanted tumors in mice ( n =10, ` x ± s )

[0047]

[0048] Note: Compared with the normal control group, " #"express P <0.05; compared to the model group, "*" indicates P <0.05.

[0049] Experiment Example 2: Effects of Chuanzhi Tongluo Capsules on SMMC-7721 Hepatocellular Carcinoma-Bearing Mice

[0050] 1. Experimental Materials

[0051] 1.1 Laboratory Animals

[0052] Male BALB / c nude mice, 4-6 weeks old, weighing 18-22 g, were purchased from the Shanghai Laboratory Animal Center. Before the experiment, all mice were placed in an environment with a room temperature of 24-26 ℃, relative humidity of 50%-60%, good ventilation and lighting, and were fed standard mouse food with free access to food and water.

[0053] 1.2 Cell lines

[0054] The human liver cancer cell line SMMC-7721 was purchased from the cell bank of the Shanghai Institute of Biology, Chinese Academy of Sciences.

[0055] 1.3 Instruments, reagents and chemicals

[0056] DMEM culture medium was purchased from GIBCO; fetal bovine serum was purchased from Hangzhou Sijiqing Bioengineering Materials Co., Ltd.; trypsin was purchased from Flash Crystal Biotechnology Co., Ltd.; CO2 incubator (HERAce® 150, US-based KJ Instruments); Z-320K high-speed refrigerated centrifuge (HERMLE, Germany); U90-136002 fluorescence inverted microscope (Leica, Germany).

[0057] 2 Experimental Methods

[0058] 2.1 Cell Culture

[0059] SMMC-7721 cells were seeded at an appropriate concentration in cell culture flasks, and DMEM culture medium containing 10% fetal bovine serum was added. The flasks were then incubated in an incubator at 37 ℃, 5% CO2, and saturated humidity.

[0060] 2.2 Hepatocellular carcinoma mouse model experiment

[0061] Male BALB / c nude mice, weighing (20±2) g, were acclimatized for 3 days before tumor implantation. Under aseptic conditions, logarithmic growth phase SMMC-7721 hepatocellular carcinoma cells were collected for experiments. After digestion with 0.25% trypsin, the viable cell count was determined by trypan blue exclusion assay to be greater than 95%, and the cells were diluted with PBS to a concentration of 1×10⁻⁶ cells / mL. 7 A cell suspension of 0.2 mL was injected subcutaneously into the right axilla of mice, with each mouse receiving 2 × 10⁹ cells.6 The tumor formation in mice was observed. When nodules were palpable under the inoculated skin and continued to grow, the model was considered successful.

[0062] 2.3 Grouping, drug administration, and detection indicators

[0063] On days 7-9 after inoculation (tumor volume approximately 0.05 cm) 3 Mice were randomly divided into a model group, a positive control group, a low-dose group of Hirudo Tongluo capsules (hereinafter referred to as "low-dose group"), a medium-dose group of Hirudo Tongluo capsules (hereinafter referred to as "medium-dose group"), and a high-dose group of Hirudo Tongluo capsules (hereinafter referred to as "high-dose group"), with 10 mice in each group. In addition, 10 untreated mice were selected as a blank control group.

[0064] The equivalent dose for mice was calculated based on the "Equivalent Dose Ratio Table for Humans and Animals Based on Body Surface Area". The low, medium, and high dose groups of Chuanzhi Tongluo Capsules were administered by gavage at 0.11375 g / kg, 0.22750 g / kg, and 0.45500 g / kg, respectively. The positive control group received an intraperitoneal injection of Pingxiao Tablets at 0.62790 g / kg. The blank control group and the model group were administered the same volume of physiological saline by gavage. All groups were administered the medication continuously for 10 days. After the 10th day of administration, mice were kept NPO (nothing by mouth) but allowed free access to water. The following day, blood was collected from the orbital cavity, and the mice were euthanized by cervical dislocation. The tumors were dissected, weighed, and recorded. The tumor inhibition rate for each group was calculated.

[0065] Tumor inhibition rate = (mean tumor weight in the model group - mean tumor weight in the sample group) / mean tumor weight in the model group × 100%.

[0066] 2.4 Statistical Analysis

[0067] SPSS 21.0 statistical software was used for analysis. Experimental data are expressed as mean ± standard deviation (%). x ± s The comparisons among multiple groups were expressed in the form of ")". One-way ANOVA was used for comparisons among multiple groups. P <0.05 indicates that the difference is statistically significant.

[0068] 3. Experimental Results

[0069] The results showed that, compared with the normal control group, the average tumor weight of mice in the model group was significantly increased ( P <0.05) indicates successful model establishment; compared with the model group, after treatment with different doses of leech to clear the meridians, the average tumor weight of mice was reduced to a certain extent, and the difference was statistically significant. P <0.05), indicating that different dosage groups of the Chuanzhi Tongluo Capsules of the present invention and the positive control group all have an inhibitory effect on tumor growth in liver cancer mice.

[0070] Experiments showed that both the group of the present invention and the positive control group had inhibitory effects on the tumor growth of liver cancer mice, and the larger the dose of Chuanzhi Tongluo Capsule of the present invention, the more obvious the inhibitory effect on tumors. Compared with the model group, the low, medium, and high dose groups of Chuanzhi Tongluo Capsule of the present invention and the positive control group all had significant ( P <0.05) differences. The results are shown in Table 2.

[0071] Table 2 Inhibitory effect of the traditional Chinese medicine composition of the present invention on tumors of liver cancer mice ( n =10, ` x ± s )

[0072]

[0073] Note: Compared with the normal control group, " # " indicates P <0.05; compared with the model group, "*" indicates P <0.05.

[0074] Experimental Example 3 Study on the effect of Chuanzhi Tongluo Capsule on Lewis lung cancer-bearing mice

[0075] 1 Experimental materials

[0076] 1.1 Experimental animals

[0077] C57BL / 6 mice, male, 4 - 6 weeks old, weighing 18 - 22 g, experimental animal license number: SYXK(Shandong)-20180008, provided by Shandong Xinsdai Pharmaceutical Co., Ltd. Before the experiment, all mice were placed in an environment with a room temperature of 24 - 26 °C, a relative humidity of 50% - 60%, good ventilation and lighting, and were fed with standard mouse feed regularly, with free access to food and water.

[0078] 1.2 Instruments, reagents and drugs

[0079] Mouse Lewis lung cancer cell line (purchased from Shanghai Heyuan Biotechnology Co., Ltd.); cyclophosphamide for injection (CTX, Shanxi Pude Pharmaceutical Co., Ltd., national drug approval number H14023686). Electronic balance (Ohaus International Trade Co., Ltd.); CO2 incubator (Thermo3111).

[0080] 2 Experimental methods

[0081] 2.1 Construction of tumor-bearing mouse model

[0082] After the Lewis lung cancer cells were resuscitated and subcultured, the third-generation cells were inoculated under the right axilla of C57BL / 6 mice. After 8 days of culture, mice with good mental state and plump tumor bodies were selected. After cervical dislocation and death, they were disinfected with 75% alcohol, and the tumor tissues were separated and cut into about 1 mm3 Grind the tissue blocks of varying sizes. Prepare a tumor cell suspension with sterile saline to a concentration of 1×10⁻⁶. 7 The number of viable cells was >95% using the trypan blue counting method under a microscope. 0.2 mL of viable cells per mouse was subcutaneously injected into the right axilla to create a Lewis lung cancer tumor-bearing mouse model.

[0083] 2.2 Grouping, drug administration, and detection indicators

[0084] C57BL / 6 mice were randomly divided into four groups based on body weight: a blank control group, a model group, a positive control group (cyclophosphamide), and four groups (low-dose group, medium-dose group, and high-dose group, with 10 mice in each group). Except for the blank control group, all other groups were inoculated with 0.2 mL of tumor cell suspension subcutaneously in the axilla of the right upper limb of the mice. The entire process from preparing the tumor cell suspension to inoculation should be completed within 60 minutes. 24 h after inoculation, except for the positive control group which was intraperitoneally injected with 40 mg / kg CTX every other day, the corresponding concentration of the drug was administered by gavage to each dose group of the cyclophosphamide group, while the blank control group and the model group were administered distilled water by gavage. The administration was once a day for 14 consecutive days.

[0085] The equivalent dose for mice was calculated based on the "Equivalent Dose Ratio Table for Humans and Animals Based on Body Surface Area". The low, medium, and high dose groups of Chuanzhi Tongluo Capsules were administered via gavage at 0.11375 g / kg, 0.22750 g / kg, and 0.45500 g / kg, respectively. The positive control group received an intraperitoneal injection of Pingxiao Tablets at 0.62790 g / kg. The blank control group and the model group were administered the same volume of physiological saline via gavage. All groups were administered the medication continuously for 14 days. After the 14th day of administration, mice were kept NPO (no food, no water) but allowed to drink. The following day, blood was collected from the orbital cavity, and the mice were euthanized by cervical dislocation. The tumors were dissected, weighed, and recorded. The tumor inhibition rate for each group was calculated.

[0086] Tumor inhibition rate = (mean tumor weight in the model group - mean tumor weight in the sample group) / mean tumor weight in the model group × 100%.

[0087] 2.3 Statistical Analysis

[0088] SPSS 21.0 statistical software was used for analysis. Experimental data are expressed as mean ± standard deviation (%). x ± s The comparisons among multiple groups were expressed in the form of ")". One-way ANOVA was used for comparisons among multiple groups. P <0.05 indicates that the difference is statistically significant.

[0089] 3. Experimental Results

[0090] The results showed that, compared with the normal control group, the average tumor weight of mice in the model group was significantly increased ( P <0.05) indicates successful model establishment; compared with the model group, after treatment with different doses of leech to clear the meridians, the average tumor weight of mice was reduced to a certain extent, and the difference was statistically significant. P <0.05), indicating that different dose groups of the Chuanzhi Tongluo Capsules of the present invention and the positive control group all have an inhibitory effect on tumor growth in Lewis lung cancer-bearing mice.

[0091] Experiments showed that both the *Hirudo medicinalis* capsule group and the positive control group of this invention inhibited tumor growth in Lewis mice, and the higher the dose of the herbal composition in this invention, the more significant the tumor-inhibiting effect. Compared with the model group, the low, medium, and high dose groups of the *Hirudo medicinalis* capsule and the positive control group of this invention all showed significant (…). P The difference was <0.05. The results are shown in Table 3.

[0092] Table 3. Inhibitory effect of the traditional Chinese medicine composition of the present invention on Lewis transplanted tumors in mice. n =10, ` x ± s )

[0093]

[0094] Note: Compared with the normal control group, " # "express P <0.05; compared to the model group, "*" indicates P <0.05.

[0095] In summary, the leech-based preparation of this invention has the function of inhibiting the growth of liver and lung cancer tumors, and has a significant effect on the treatment of malignant tumors. Moreover, as a commercially available traditional Chinese medicine, it has the advantage of good safety. Detailed Implementation

[0096] To enable those skilled in the art to fully understand the present invention, the present invention is further illustrated below through specific embodiments. However, those skilled in the art should understand that the embodiments of the present invention do not limit the present invention in any way.

[0097] Example 1: Preparation of Granules

[0098] Leech 8 kg, Ligusticum chuanxiong 3 kg, Astragalus membranaceus 6 kg, Salvia miltiorrhiza 2 kg

[0099] A. Take the prescribed amount of leech medicinal material, add 4 times the amount of water, stir and extract for 20 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0100] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 5 times the amount of 75% ethanol and reflux extract twice, 3 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.17 at 50-60 ℃, and set aside for later use; keep the residue for later use.

[0101] C. Take the dregs obtained in step B, add water in 5 times the weight of the original medicinal materials, decoct once for 2 hours, filter, and concentrate the filtrate to extract II with a relative density of 1.22 at 50-60 ℃, and set aside.

[0102] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0103] E. Mix the freeze-dried leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize them into fine powder, add the sucrose powder:dextrin in the formula ratio of 3:2 by weight, mix well, make into granules, dry, and granulate to obtain the final product.

[0104] Example 2 Preparation of microspheres

[0105] Leech 3 kg, Ligusticum chuanxiong 8 kg, Astragalus membranaceus 2 kg, Salvia miltiorrhiza 6 kg

[0106] A. Take the prescribed amount of leech medicinal material, add 5 times the amount of water, stir and extract for 10 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0107] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 8 times the amount of 65% ethanol and reflux extract twice, 2 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.21 at 50-60 ℃, and set aside for later use; keep the residue for later use.

[0108] C. Take the dregs obtained in step B, add water in three times the weight of the original medicinal materials, decoct once for 1 hour, filter, and concentrate the filtrate to extract II with a relative density of 1.16 at 50-60 ℃, and set aside.

[0109] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0110] E. Mix the lyophilized leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize them into fine powder, add appropriate amounts of microcrystalline cellulose, chitosan, dextrin, micronized silica gel, and dextrin (the mass ratio of the leech extract: microcrystalline cellulose: chitosan: micronized silica gel: dextrin is 32:47:15:3:3), mix thoroughly, add 40% (by weight) of the prescribed amount of 30% ethanol solution as a wetting agent and knead continuously to form a soft material, and extrude it into strips through a sieve plate with an extruder aperture of 0.9 mm; turn on the rounding machine, select a speed of 1100 rpm, place the strips in the rounding machine, and round them for 6 minutes until the particles are rolled into pellets, take out the micro pellet semi-finished product, dry it at 65 ℃, and sieve to obtain 20-30 mesh micro pellets.

[0111] Example 3: Preparation of Capsules

[0112] Leech 6 kg, Ligusticum chuanxiong 6 kg, Astragalus membranaceus 4 kg, Salvia miltiorrhiza 4 kg

[0113] A. Take the prescribed amount of leech medicinal material, add 6 times the amount of water, stir and extract for 20 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0114] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 6 times the amount of 80% ethanol and reflux extract twice, 2 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.20 at 50-60 ℃, and set aside for later use; the residue is set aside for later use.

[0115] C. Take the dregs obtained in step B, add water in four times the weight of the original medicinal materials, decoct once for 2 hours, filter, and concentrate the filtrate to extract II with a relative density of 1.20 at 50℃, and set aside.

[0116] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0117] E. Mix the leech freeze-dried powder obtained in step A with the fine powder of leech meridian-clearing extract obtained in step D, pulverize into fine powder, add starch:microcrystalline cellulose in the formula ratio of 6:1 by weight, mix well, granulate, dry, granulate, fill, polish in a polishing machine, and remove damaged capsules to obtain the final product.

[0118] Example 4: Preparation of tablets

[0119] Leech 3 kg, Ligusticum chuanxiong 3 kg, Astragalus membranaceus 6 kg, Salvia miltiorrhiza 6 kg

[0120] A. Take the prescribed amount of leech medicinal material, add 8 times the amount of water, stir and extract for 30 h at 37℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0121] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 7 times the amount of 95% ethanol and reflux extract twice, 2 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.18 at 50-60 ℃, and set aside for later use; the residue is reserved for later use.

[0122] C. Take the dregs obtained in step B, add water in three times the weight of the original medicinal materials, decoct once for 2 hours, filter, and concentrate the filtrate to extract II with a relative density of 1.23 at 50℃, and set aside.

[0123] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0124] E. Mix the freeze-dried leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize into fine powder, add starch:dextrin:sucrose in the formula ratio of 5:1:1.5 by weight, mix well, make coarse granules, dry, pulverize, sieve, granulate, dry at low temperature, granulate, add 0.3% magnesium stearate, mix well, and compress into tablets to obtain the final product.

[0125] Example 5: Preparation of Capsules

[0126] Leech 8 kg, Ligusticum chuanxiong 8 kg, Astragalus membranaceus 2 kg, Salvia miltiorrhiza 2 kg

[0127] A. Take the prescribed amount of leech medicinal material, add 7 times the amount of water, stir and extract for 15 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0128] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 4 times the amount of 65% ethanol and reflux twice, 3 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.22 at 50-60 ℃, and set aside for later use; the residue is reserved for later use.

[0129] C. Take the dregs obtained in step B, add water in 5 times the weight of the original medicinal materials, decoct once for 2 hours, filter, concentrate the filtrate to extract II with a relative density of 1.17 at 50℃, and set aside.

[0130] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0131] E. Mix the leech freeze-dried powder obtained in step A with the fine powder of leech meridian-clearing extract obtained in step D, pulverize into fine powder, add starch:microcrystalline cellulose in the formula ratio of 6:1 by weight, mix well, granulate, dry, granulate, fill, polish in a polishing machine, and remove damaged capsules to obtain the final product.

[0132] Example 6: Preparation of Tablets

[0133] Leech 4 kg, Ligusticum chuanxiong 7 kg, Astragalus membranaceus 3 kg, Salvia miltiorrhiza 5 kg

[0134] A. Take the prescribed amount of leech medicinal material, add 6 times the amount of water, stir and extract for 10 h at 37 ℃ and 100 r / min, centrifuge at 6000 r / min for 30 min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0135] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 5 times the amount of 85% ethanol and reflux extract twice, 3 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.25 at 50-60 ℃, and set aside for later use; keep the residue for later use.

[0136] C. Take the dregs obtained in step B, add water in three times the weight of the original medicinal materials, decoct once for 1 hour, filter, and concentrate the filtrate to extract II with a relative density of 1.15 at 50-60 ℃, and set aside.

[0137] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0138] E. Mix the freeze-dried leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize into fine powder, add starch:dextrin:sucrose in the formula ratio of 5:1:1.5 by weight, mix well, make coarse granules, dry, pulverize, sieve, granulate, dry at low temperature, granulate, add 0.22% magnesium stearate, mix well, and compress into tablets to obtain the final product.

[0139] Example 7 Preparation of Granules

[0140] Leech 7 kg, Ligusticum chuanxiong 4 kg, Astragalus membranaceus 5 kg, Salvia miltiorrhiza 3 kg

[0141] A. Take the prescribed amount of leech medicinal material, add 4 times the amount of water, stir and extract for 25 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0142] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 6 times the amount of 95% ethanol and reflux twice, each time for 2 hours, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.19 at 50-60 ℃, and set aside for later use; the residue is set aside for later use.

[0143] C. Take the dregs obtained in step B, add water in four times the weight of the original medicinal materials, decoct once for 1 hour, filter, and concentrate the filtrate to extract II with a relative density of 1.25 at 50-60 ℃, and set aside.

[0144] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0145] E. Mix the freeze-dried leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize them into fine powder, add the sucrose powder:dextrin in the formula ratio of 3:2 by weight, mix well, make into granules, dry, and granulate to obtain the final product.

[0146] Example 8 Preparation of microspheres

[0147] Leech 5 kg, Ligusticum chuanxiong 7 kg, Astragalus membranaceus 4 kg, Salvia miltiorrhiza 3 kg

[0148] A. Take the prescribed amount of leech medicinal material, add 5 times the amount of water, stir and extract for 20 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0149] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 8 times the amount of 65% ethanol and reflux extract twice, 3 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.20 at 50-60 ℃, and set aside for later use; the residue is set aside for later use.

[0150] C. Take the dregs obtained in step B, add water in four times the weight of the original medicinal materials, decoct once for 1 hour, filter, and concentrate the filtrate to extract II with a relative density of 1.20 at 50-60 ℃, and set aside.

[0151] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0152] E. Mix the lyophilized leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize them into fine powder, add appropriate amounts of microcrystalline cellulose, chitosan, dextrin, micronized silica gel, and dextrin (the mass ratio of fine powder of leech extract to microcrystalline cellulose to chitosan to micronized silica gel to dextrin is 32:47:15:3:3), mix thoroughly, add 40% (by weight) of the prescribed amount of 30% ethanol solution as a wetting agent and knead continuously to form a soft material, and extrude it into strips through a sieve plate with an extruder aperture of 0.9 mm; turn on the rounding machine, select a speed of 1100 rpm, place the strips in the rounding machine, and round them for 6 minutes until the particles are rolled into pellets, take out the micro pellet semi-finished product, dry it at 65 ℃, and sieve to obtain 20-30 mesh micro pellets.

[0153] Example 9 Preparation of Capsules

[0154] Leech 6 kg, Ligusticum chuanxiong 4 kg, Astragalus membranaceus 3 kg, Salvia miltiorrhiza 5 kg

[0155] A. Take the prescribed amount of leech medicinal material, add 4 times the amount of water, stir and extract for 10 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0156] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 7 times the amount of 80% ethanol and reflux extract twice, 2 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.18 at 50-60 ℃, and set aside for later use; the residue is set aside for later use.

[0157] C. Take the dregs obtained in step B, add water in 5 times the weight of the original medicinal materials, decoct once for 2 hours, filter, and concentrate the filtrate to extract II with a relative density of 1.17 at 50-60 ℃, and set aside.

[0158] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0159] E. Mix the leech freeze-dried powder obtained in step A with the fine powder of leech meridian-clearing extract obtained in step D, pulverize into fine powder, add starch:microcrystalline cellulose in the formula ratio of 6:1 by weight, mix well, granulate, dry, granulate, fill, polish in a polishing machine, and remove damaged capsules to obtain the final product.

[0160] Example 10 Preparation of tablets

[0161] Leech 7 kg, Ligusticum chuanxiong 5 kg, Astragalus membranaceus 5 kg, Salvia miltiorrhiza 4 kg

[0162] A. Take the prescribed amount of leech medicinal material, add 7 times the amount of water, stir and extract for 20 h at 37 ℃ and 100 r / min, centrifuge for 30 min at 6000 r / min, discard the precipitate, filter the supernatant through a 1 μm pore membrane, then filter through a 0.2 μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000 Da and concentrate to 600 mL, freeze-dry the concentrate to obtain leech freeze-dried powder;

[0163] B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 6 times the amount of 70% ethanol and reflux extract twice, 2 hours each time, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.23 at 50℃, and set aside. Set aside the residue.

[0164] C. Take the dregs obtained in step B, add water in three times the weight of the original medicinal materials, decoct once for 2 hours, filter, and concentrate the filtrate to extract II with a relative density of 1.18 at 50℃, and set aside.

[0165] D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use;

[0166] E. Mix the freeze-dried leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize into fine powder, add starch:dextrin:sucrose in the formula ratio of 5:1:1.5 by weight, mix well, make coarse granules, dry, pulverize, sieve, granulate, dry at low temperature, granulate, add 0.2% magnesium stearate, mix well, and compress into tablets to obtain the final product.

Claims

1. The application of leech-infused collaterals-clearing preparation in the preparation of antitumor drugs, wherein the leech-infused collaterals-clearing preparation is made from the following components: 600 parts by weight of leech, 600 parts by weight of chuanxiong rhizome, 400 parts by weight of astragalus root, and 400 parts by weight of danshen root; wherein the tumor is liver cancer or lung cancer.

2. The application according to claim 1, characterized in that, The leech-based collateral-clearing preparation is an oral medication.

3. The application according to claim 2, characterized in that, The oral drug preparation is one of the following: granules, capsules, tablets, or pills.

4. The application according to claim 2, characterized in that, The preparation process of the oral drug formulation includes the following steps: A. Take the prescribed amount of leech medicinal material, crush it into coarse powder, add 4-8 times the amount of water, stir and extract at 37℃ for 10-30 hours, centrifuge to discard the precipitate, filter the supernatant through a membrane filter, then filter it through an ultrafiltration device and concentrate it, freeze-dry the concentrate to obtain leech freeze-dried powder for later use. B. Take the prescribed amount of Ligusticum chuanxiong, Astragalus membranaceus, and Salvia miltiorrhiza, add 4-8 times the amount of 65%-95% ethanol and reflux extract twice, each time for 2-3 hours, filter, combine the filtrates, concentrate the filtrate to extract I with a relative density of 1.15-1.25 at 50-60℃, and set aside for later use; the residue is reserved for later use. C. Take the dregs obtained in step B, add water in an amount of 3-5 times the weight of the original medicinal materials, decoct once for 1-2 hours, filter, and concentrate the filtrate to extract II with a relative density of 1.15-1.25 at 50-60℃, and set aside for later use; D. Combine the extract I obtained in step B with the extract II obtained in step C, spray dry, and obtain a fine powder of Hirudo medicinalis extract for later use; E. Mix the lyophilized leech powder obtained in step A with the fine powder of the leech extract obtained in step D, pulverize them into fine powder, and prepare oral drug formulations directly or by adding pharmaceutically acceptable excipients through conventional processes.

5. The application according to claim 4, characterized in that, Step A: Take the prescribed amount of leech medicinal material, add 6 times the amount of water, and stir and extract for 20 hours at 37℃ and 100r / min. Centrifuge at 6000r / min for 30 minutes, discard the precipitate, filter the supernatant through a 1μm pore membrane, then filter through a 0.2μm pore membrane for sterilization, then filter through an ultrafiltration device with a molecular weight cutoff of 6000-10000Da and concentrate to 600mL. Freeze-dry the concentrate to obtain leech freeze-dried powder.

6. The application according to claim 4, characterized in that, In step B, add 6 times the amount of 80% ethanol and concentrate the filtrate to extract I with a relative density of 1.20 at 50-60°C.

7. The application according to claim 4, characterized in that, In step C, add water in an amount four times the weight of the original medicinal material, and concentrate the filtrate to extract II with a relative density of 1.20 at 50-60℃.

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