Multi-purpose traditional Chinese medicine composition for treating tumors as well as preparation method and application of multi-purpose traditional Chinese medicine composition

Abstract

The invention relates to a multipurpose traditional Chinese medicine composition for treating tumors as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The method comprises the following three steps: alcohol extraction of periplaneta americana, water extraction of traditional Chinese medicines and preparation of a finished product. Compared with the prior art, the preparation method disclosed by the invention has the advantages that the production efficiency is improved, the production cost is reduced, the medicinal production cost and the medical cost of patients can be reduced by utilizing the organic combination of special medicinal substances of American cockroach and traditional folk Chinese herbal medicines, and various diseases can be effectively treated in an auxiliary manner. Experiments prove that the traditional Chinese medicine composition has an excellent treatment effect on liver and stomach in-situ tumors; theoretical data can be provided for mechanism research of liver and stomach in-situ tumors, and a foundation is laid for follow-up experimental verification.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a multi-purpose traditional Chinese medicine composition for treating tumors, and a preparation method and application thereof. Background Art

[0002] The American cockroach (Periplanta Americana) is a medicinal insect. Patent No. ZL200910094190.7, "Method for Extracting Medicinal Substances from American Cockroaches," states that the medicinal liquid extracted from the American cockroach can be used in skincare products, medicines, and health supplements. It promotes tissue growth and healing, has antioxidant properties, can sober up, protect the liver, nourish the stomach, promote the healing of gastric and duodenal ulcers, and promotes the healing of inflammation and ulcers. The extraction process uses the following: Alcohol extraction (A): fresh cockroaches are killed and soaked in alcohol; cryogenic freezing is performed; the shelled coarse powder is placed in an extractor and soaked in alcohol; the alcohol is then refluxed for extraction, and the recovered medicinal liquid is placed in a separator for oil-water separation. The separated liquid is filtered, concentrated, and then released for cooling to obtain the medicinal substance. Water extraction (B): the alcohol-extracted raw material is added to distilled water, heated for extraction, and the recovered medicinal liquid is placed in a separator for oil-water separation, filtered, and concentrated to obtain the medicinal substance. This method increases production costs and does not effectively preserve the medicinal ingredients in the extract. Furthermore, using steam-heated extraction equipment makes it difficult to control the temperature. Furthermore, temperature during the extraction process has a certain impact on quality. The fact that the extract is liquid also adds significant difficulties in storage and transportation.

[0003] Therefore, how to overcome the shortcomings of the existing technology is an urgent problem to be solved in the field of traditional Chinese medicine technology. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a multi-purpose traditional Chinese medicine composition for treating tumors, a preparation method thereof, and an application thereof. The method utilizes the organic combination of special medicinal substances of American cockroaches and traditional folk Chinese herbal medicines, can reduce the production cost of the medicine and the medical costs of patients, and effectively assists in the treatment of various diseases.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A method for preparing a multi-purpose traditional Chinese medicine composition for treating tumors, characterized by comprising the following steps:

[0007] Step (1), ethanol extraction of American cockroaches:

[0008] The dried product of American cockroach is dried and crushed, and then soaked in 70%-75% ethanol by mass concentration, followed by hot reflux extraction; the obtained extract is concentrated to remove the ethanol, and then water is added to separate the oil and water; the aqueous phase is filtered, concentrated, and spray-dried to obtain the American cockroach extract;

[0009] Step (2), Chinese herbal medicine water extraction:

[0010] Extracting Hedyotis diffusa, Phyllanthus urinaria, and Pueraria lobata together with distilled water at a mass ratio of 0.95-1.05:0.95-1.05:0.95-1.05, filtering the extract, concentrating, and spray drying to obtain a Chinese medicine extract;

[0011] Step (3), finished product production:

[0012] The American cockroach extract obtained in step (1) and the traditional Chinese medicine extract obtained in step (2) are mixed until uniform, thereby obtaining a multi-purpose traditional Chinese medicine composition for treating tumors;

[0013] Among them, the mass ratio of American cockroach extract and traditional Chinese medicine mixed extract is 9.8:1-10.2:1.

[0014] Furthermore, preferably, in step (1), the drying temperature is 60-80°C; the soaking time is 10-15 hours; the hot reflux extraction time is 10-15 hours; the mass ratio of the dried American cockroach to 70%-75% ethanol is 1:3.5-1:7; and the mass ratio of the dried American cockroach to water is 1:4-1:8.

[0015] Furthermore, preferably, in step (1), filtration is performed using a microporous filter with a pore size of 0.5-1 micron; the aqueous phase is filtered and concentrated using a vacuum concentrator to half of its original mass, followed by spray drying.

[0016] Furthermore, preferably, in step (2), the extraction is performed three times, the first extraction is performed using 60%-65% ethanol for 3-6 hours, and the mass ratio of 60%-65% ethanol to the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata is 4:1-8:1; the second and third extractions are performed using distilled water for 3-6 hours; the mass of distilled water used in each extraction is 4-8 times the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata, and all the extracts are mixed and filtered.

[0017] Furthermore, preferably, in step (2), filtration is performed using a microporous filter with a pore size of 0.5-1 micron; concentration is performed using a vacuum concentrator, and after concentration to half of the original mass, spray drying is performed.

[0018] Furthermore, it is preferred that the multipurpose Chinese medicine composition for treating tumors is directly made into granules, or the multipurpose Chinese medicine composition for treating tumors is made into oral liquid, granule powder, tablets or capsules.

[0019] The present invention also provides the multi-purpose traditional Chinese medicine composition for treating tumors and the multi-purpose traditional Chinese medicine composition for treating tumors prepared by the preparation method thereof.

[0020] The present invention also provides the use of the multi-purpose traditional Chinese medicine composition for treating tumors in preparing a medicine for treating liver tumors in situ.

[0021] The present invention further provides the use of the multi-purpose traditional Chinese medicine composition for treating tumors in preparing a medicine for treating gastric in situ tumors.

[0022] In step (1) of the present invention, the hot reflux extraction temperature is 60-80°C.

[0023] In the present invention, since the American cockroaches themselves contain fat and oil, and the crushing process generates high temperatures in the crusher, the fat becomes liquid and forms a mixture with the crushed American cockroach material. A hot reflux device heats ethanol, causing the ethanol to continuously rinse the crushed American cockroach-fat mixture. The fat is then mixed into the ethanol extract under the action of the warm ethanol. After the extraction is completed and the ethanol is recovered, water is added to separate the oil and water (the mass ratio of the extract to the added water is between 1:4 and 1:8). The aqueous phase is filtered, concentrated, and spray-dried to obtain the American cockroach extract (in powder form).

[0024] In step (2) of the present invention, the obtained Chinese herbal medicine extract is in powder form.

[0025] During filtration in the present invention, a microporous filter is preferably used for filtration; and mixing is preferably carried out by stirring in a blender until the mixture is uniformly mixed.

[0026] When the multi-purpose traditional Chinese medicine composition for treating tumors is prepared into oral liquid, granule powder, tablet or capsule, existing methods can be adopted, and the present invention does not impose any special restrictions on this.

[0027] As a new drug for treating liver cancer and gastric cancer, the therapeutic effect of the composition of the present invention is not the result of a single ingredient or simple pharmacological action, but an outstanding achievement of the synergistic effects of various medicinal materials, preparation methods, and administration methods in the formula, showing multi-dimensional excellence.

[0028] 1. Synergistic effect to comprehensively fight cancer

[0029] The composition of the present invention is carefully formulated from a combination of natural medicinal herbs, including extracts from American cockroaches, Hedyotis diffusa, Phyllanthus urinaria, and Pueraria root. The American cockroach extract, rich in peptide active ingredients, various amino acids, and proteins, can directly inhibit cancer cells, such as by interfering with their DNA synthesis. Extracts from Hedyotis diffusa, Phyllanthus urinaria, and Pueraria root enhance the body's own anti-cancer capabilities, improving the immune system's ability to recognize and eliminate cancer cells. These herbs work together to form a comprehensive anti-cancer network within the body, inhibiting cancer cell growth, inducing apoptosis, and enhancing immunity, effectively combating liver and gastric cancer cells.

[0030] 2. Unique extraction method to maximize efficacy

[0031] The extraction method of this invention is unique and sophisticated. Step one involves soaking the medicinal materials in 70-75°C ethanol and then subjecting them to low-temperature heat reflux, fully releasing the active ingredients of the medicinal materials. Step two, performed at a specific extraction temperature and low-temperature concentration, alters the physical properties of the medicinal materials, maximizes their biological activity, and further enhances their synergistic effects. Through this meticulous preparation process, the efficacy of the various medicinal materials in the composition of this invention is fully stimulated and integrated, significantly improving the inhibition rate against liver and gastric cancer cells compared to combinations of medicinal materials that have not undergone special extraction.

[0032] 3. Gentle conditioning to reduce side effects

[0033] During treatment, the composition of the present invention uses a gentle conditioning approach, with a relatively low dose and frequent administration, reducing the burden on the patient's body. This approach not only ensures sustained efficacy but also reduces common side effects of traditional chemotherapy drugs, such as nausea, vomiting, hair loss, and bone marrow suppression. Experimental observations have shown that animals treated with the composition of the present invention had a zero incidence of side effects, significantly improving the quality of life of patients, enabling them to maintain a good physical and mental state during treatment and better cope with the disease.

[0034] 4. Personalized adaptation and precise treatment

[0035] The composition of the present invention can also be flexibly adjusted based on individual patient differences, such as disease stage, physical condition, and age. For early-stage patients, it focuses on enhancing immunity and inhibiting the initial growth of cancer cells; for patients in the middle and late stages, it enhances the efficacy of softening and dispersing nodules, and strengthening the body's resistance and eliminating pathogenic factors. This personalized adaptability enables precise treatment, greatly improving the targeted and effective treatment, and providing treatment options more tailored to the needs of patients with different liver and gastric cancer conditions.

[0036] The composition of the present invention, with its multi-faceted synergistic advantages, demonstrates unparalleled excellence in the treatment of liver cancer and gastric cancer, bringing new hope and light to cancer patients.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Compared with the prior art, the preparation method of the present invention has the advantages of improving production efficiency and reducing production costs. By organically combining the medicinal special substances of the American cockroach with traditional folk Chinese herbal medicines, the medicinal production costs and the medical costs of patients can be reduced, and the preparation method effectively assists in the treatment of various diseases.

[0039] Experiments have shown that the present invention has excellent therapeutic effects on liver and stomach in situ tumors; the present invention can provide theoretical data for the mechanism research of liver and stomach in situ tumors, and lay a foundation for subsequent experimental verification.

[0040] The present invention is safe, effective, and has no side effects. It is safer than existing Western medicine and Western-style treatments for liver cancer, reducing the pain and mental suffering associated with Western-style treatments, which are harmful and carry the risk of metastasis. This product requires no intravenous infusion, chemotherapy, or surgery, and is simply taken orally. Compared to Western-style treatments, this significantly reduces patient suffering and significantly reduces treatment costs. Hospitalization is unnecessary, and patients can simply take the medication at home on time and in the correct dosage, reducing all hospitalization expenses. Treatment costs are approximately one-third of those incurred by Western-style treatments.

[0041] Studies have shown that the mortality rate associated with chemotherapy is generally below 2%, which means that for every 100 patients receiving chemotherapy, less than 2 may die from chemotherapy complications or toxic reactions.

[0042] Therefore, compared with existing technologies, it is not only safe, effective and has no side effects, but also reduces patients' pain and improves their quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Figure 1 shows the first in vivo imaging results of a nude mouse orthotopic liver tumor experiment; a shows the location of cancer cell inoculation. There were four groups, each with five experimental mice. The first group was the control group, the second group was given 50 mg / kg, the third group was given 100 mg / kg, and the fourth group was given 200 mg / kg; b shows the comparison of cancer cell inoculation data;

[0044] Figure 2 Figure 1 shows the results of in vivo imaging of nude mice with orthotopic liver tumors before sacrifice. Figure 1 shows a fluorescence intensity comparison chart. There are four groups, each with three experimental mice. Group 1 is the control group, group 2 is administered with 50 mg / kg, group 3 is administered with 100 mg / kg, and group 4 is administered with 200 mg / kg. Figure 1 shows a fluorescence data comparison chart.

[0045] Figure 3 Figure 1 shows the weight changes of nude mice in an orthotopic liver tumor experiment; a is the weight change graph, and b is the tumor change graph;

[0046] Figure 4 The immunohistochemical results of liver tissues of nude mice in the orthotopic liver tumor experiment are shown in Figure 1. a is the protein content graph, and b is the protein content data.

[0047] Figure 5Figure 1 shows the first in vivo imaging results of a gastric orthotopic tumor experiment in nude mice. Figure a shows the location of cancer cell inoculation. There were four groups, each with five experimental mice. Group 1 was the control group, group 2 was given 50 mg / kg, group 3 was given 100 mg / kg, and group 4 was given 200 mg / kg. Figure b shows the comparison of cancer cell inoculation data.

[0048] Figure 6 The results of in vivo imaging of nude mice with gastric orthotopic tumors before sacrifice are shown in Figure 1. Among them, a is a fluorescence intensity comparison diagram. There are four groups, each with 3 experimental mice. The first group is the control group, the second group is administered with 50 mg / kg, the third group is administered with 100 mg / kg, and the fourth group is administered with 200 mg / kg. b is a fluorescence data comparison diagram.

[0049] Figure 7 Figure 1 shows the weight change results of nude mice in gastric orthotopic tumor experiments; a is the weight change graph, and b is the tumor change graph;

[0050] Figure 8 These are the immunohistochemical results of liver tissue in nude mice in a gastric orthotopic tumor experiment; a is the protein content graph, and b is the protein content data. DETAILED DESCRIPTION

[0051] The present invention is described in further detail below with reference to the embodiments.

[0052] Those skilled in the art will understand that the following examples are intended to illustrate the present invention only and should not be construed as limiting the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or in the product specifications were used. Materials or equipment used without manufacturer identification are commercially available conventional products.

[0053] Example 1

[0054] A method for preparing a multi-purpose traditional Chinese medicine composition for treating tumors comprises the following steps:

[0055] Step (1), ethanol extraction of American cockroaches:

[0056] The dried product of American cockroach is dried and crushed, and then soaked in 72% ethanol by mass concentration, followed by hot reflux extraction; the obtained extract is concentrated to remove the ethanol, and then water is added to separate the oil and water; the aqueous phase is filtered, concentrated, and spray-dried to obtain the American cockroach extract;

[0057] Step (2), Chinese herbal medicine water extraction:

[0058] Extracting Hedyotis diffusa, Phyllanthus urinaria, and Pueraria root with distilled water at a mass ratio of 1:1:1, filtering the extract, concentrating, and spray drying to obtain a Chinese medicine extract;

[0059] Step (3), finished product production:

[0060] The American cockroach extract obtained in step (1) and the traditional Chinese medicine extract obtained in step (2) are mixed until uniform, thereby obtaining a multi-purpose traditional Chinese medicine composition for treating tumors;

[0061] Among them, the mass ratio of American cockroach extract and Chinese medicine mixed extract is 10:1.

[0062] Example 2

[0063] A method for preparing a multi-purpose traditional Chinese medicine composition for treating tumors comprises the following steps:

[0064] Step (1), ethanol extraction of American cockroaches:

[0065] The dried product of American cockroach is dried and crushed, and then soaked in 70% ethanol by mass concentration, followed by hot reflux extraction; the obtained extract is concentrated to remove the ethanol, and then water is added to perform oil-water separation; the aqueous phase is filtered, concentrated, and spray-dried to obtain an American cockroach extract;

[0066] Step (2), Chinese herbal medicine water extraction:

[0067] Extracting Hedyotis diffusa, Phyllanthus urinaria, and Pueraria lobata together with distilled water at a mass ratio of 0.95:0.95:0.95, filtering the extract, concentrating, and spray drying to obtain a Chinese herbal medicine extract;

[0068] Step (3), finished product production:

[0069] The American cockroach extract obtained in step (1) and the traditional Chinese medicine extract obtained in step (2) are mixed until uniform, thereby obtaining a multi-purpose traditional Chinese medicine composition for treating tumors;

[0070] Among them, the mass ratio of American cockroach extract and Chinese medicine mixed extract is 9.8:1.

[0071] In step (1), the drying temperature is 60° C.; the soaking time is 10 hours; the hot reflux extraction time is 10 hours; the mass ratio of the dried American cockroach to 70% ethanol is 1:3.5; and the mass ratio of the dried American cockroach to water is 1:4.

[0072] In step (1), filtration is performed using a microporous filter with a pore size of 0.5 microns; the aqueous phase is filtered and concentrated using a vacuum concentrator to half of its original mass, and then spray-dried.

[0073] In step (2), the extraction is performed three times. The first extraction is performed using 60% ethanol for 3 hours, and the mass ratio of the 60% ethanol to the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata is 4:1. The second and third extractions are performed using distilled water for 3 hours. The mass of the distilled water used in each extraction is 4 times the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata. All the extracts are mixed and filtered.

[0074] In step (2), filtration is performed using a microporous filter with a pore size of 0.5 microns; concentration is performed using a vacuum concentrator, and after concentration to half of the original mass, spray drying is performed.

[0075] The multipurpose Chinese medicine composition for treating tumors is directly made into granules, or the multipurpose Chinese medicine composition for treating tumors is made into oral liquid, granule powder, tablets or capsules.

[0076] Example 3

[0077] A method for preparing a multi-purpose traditional Chinese medicine composition for treating tumors comprises the following steps:

[0078] Step (1), ethanol extraction of American cockroaches:

[0079] The dried product of American cockroach is dried and crushed, and then soaked in 75% ethanol by mass concentration, followed by hot reflux extraction; the obtained extract is concentrated to remove the ethanol, and then water is added to separate the oil and water; the aqueous phase is filtered, concentrated, and spray-dried to obtain the American cockroach extract;

[0080] Step (2), Chinese herbal medicine water extraction:

[0081] Extracting Hedyotis diffusa, Phyllanthus urinaria, and Pueraria lobata together with distilled water at a mass ratio of 1.05:1.05:1.05, filtering the extract, concentrating, and spray drying to obtain a Chinese herbal medicine extract;

[0082] Step (3), finished product production:

[0083] The American cockroach extract obtained in step (1) and the traditional Chinese medicine extract obtained in step (2) are mixed until uniform, thereby obtaining a multi-purpose traditional Chinese medicine composition for treating tumors;

[0084] Among them, the mass ratio of American cockroach extract and Chinese medicine mixed extract is 10.2:1.

[0085] In step (1), the drying temperature is 80° C.; the soaking time is 15 hours; the hot reflux extraction time is 15 hours; the mass ratio of the dried American cockroach to 75% ethanol is 1:7; and the mass ratio of the dried American cockroach to water is 1:8.

[0086] In step (1), filtration is performed using a microporous filter with a pore size of 1 micron; the aqueous phase is filtered and concentrated using a vacuum concentrator to half of its original mass, and then spray-dried.

[0087] In step (2), the extraction is performed three times. The first extraction is performed using 65% ethanol for 6 hours, and the mass ratio of the 65% ethanol to the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata is 8:1. The second and third extractions are both performed using distilled water for 6 hours. The mass of the distilled water used in each extraction is 8 times the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata. All the extracts are mixed and filtered.

[0088] In step (2), filtration is performed using a microporous filter with a pore size of 1 micron; concentration is performed using a vacuum concentrator, and after concentration to half of the original mass, spray drying is performed.

[0089] The multipurpose Chinese medicine composition for treating tumors is directly made into granules, or the multipurpose Chinese medicine composition for treating tumors is made into oral liquid, granule powder, tablets or capsules.

[0090] Example 4

[0091] A method for preparing a multi-purpose traditional Chinese medicine composition for treating tumors comprises the following steps:

[0092] Step (1), ethanol extraction of American cockroaches:

[0093] The dried product of American cockroach is dried and crushed, and then soaked in 72% ethanol by mass concentration, followed by hot reflux extraction; the obtained extract is concentrated to remove the ethanol, and then water is added to separate the oil and water; the aqueous phase is filtered, concentrated, and spray-dried to obtain the American cockroach extract;

[0094] Step (2), Chinese herbal medicine water extraction:

[0095] Extracting Hedyotis diffusa, Phyllanthus urinaria, and Pueraria root with distilled water at a mass ratio of 1:1:1, filtering the extract, concentrating, and spray drying to obtain a Chinese medicine extract;

[0096] Step (3), finished product production:

[0097] The American cockroach extract obtained in step (1) and the traditional Chinese medicine extract obtained in step (2) are mixed until uniform, thereby obtaining a multi-purpose traditional Chinese medicine composition for treating tumors;

[0098] Among them, the mass ratio of American cockroach extract and Chinese medicine mixed extract is 10:1.

[0099] In step (1), the drying temperature is 70° C.; the soaking time is 12 hours; the hot reflux extraction time is 12 hours; the mass ratio of the dried American cockroach to 73% ethanol is 1:6; and the mass ratio of the dried American cockroach to water is 1:5.

[0100] In step (1), filtration is performed using a microporous filter with a pore size of 0.8 microns; the aqueous phase is filtered and concentrated using a vacuum concentrator to half of its original mass, and then spray-dried.

[0101] In step (2), the extraction is performed three times. The first extraction is performed using 64% ethanol with a mass concentration of 5 hours, and the mass ratio of 62% ethanol to the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata is 6:1. The second and third extractions are both performed using distilled water for 5 hours. The mass of distilled water used in each extraction is 6 times the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata. All the extracts are mixed and filtered.

[0102] In step (2), filtration is performed using a microporous filter with a pore size of 0.8 microns; concentration is performed using a vacuum concentrator, and after concentration to half of the original mass, spray drying is performed.

[0103] The multipurpose Chinese medicine composition for treating tumors is directly made into granules, or the multipurpose Chinese medicine composition for treating tumors is made into oral liquid, granule powder, tablets or capsules.

[0104] Performance testing

[0105] The main reagents and manufacturers used are shown in Table 1.

[0106] Table 1 Main reagents

[0107]

[0108]

[0109] 1. Liver Orthotopic Tumor Experimental Methods

[0110] 1.1SK-HEP-1-LUC cell culture

[0111] 1.1.1 Cell culture steps are as follows:

[0112] 1. Observe the status of liver cancer cells (SK-HEP-1-LUC human liver cancer cells) under a microscope. The cells are free of contamination and the cell confluence is about 50%;

[0113] 2. Discard the supernatant, add 5 mL of PBS to each dish, wash once, add 3 mL of trypsin, and digest for 3 minutes;

[0114] 3. After cell digestion, add 10 mL of SK-HEP-1-LUC-GFP labeled cell culture medium to terminate digestion;

[0115] 4. Gently blow the cells and collect the cells in a centrifuge tube; the centrifugation parameters are 1000 rpm and 5 minutes;

[0116] 5. Discard the supernatant, resuspend the cells in fresh SK-HEP-1-LUC-GFP labeled cell culture medium, and then expand the culture in a new cell culture dish with a passage ratio of 1:2.

[0117] 6. Continue to subculture and further expand the culture until the confluence reaches about 90%;

[0118] 1.2 Animal husbandry for orthotopic liver tumor experiments

[0119] Mice (animal information see Table 2) were purchased, 5 per cage, and housed in a barrier-enclosed SPF animal facility in Class IV C cages. The room temperature was maintained at 22 ± 2°C, the relative humidity was 40%-60%, and they had free access to water and food. Experiments began after 3 days of adaptive feeding.

[0120] Table 2

[0121]

[0122] 1.3 Inoculation of experimental animals with orthotopic liver tumors

[0123] 1.3.1 Preparation quantity of cell suspension

[0124] Before inoculation on the same day, the cell platform prepared the cell suspension to be inoculated as follows:

[0125] 1. Take the liver cancer cell suspension, discard the supernatant, add 5 mL of PBS to each dish to wash once, add 3 mL of trypsin, and digest for 3 minutes;

[0126] 2. After cell digestion, add 10 mL of SK-HEP-1-LUC-GFP labeled cell culture medium to terminate digestion;

[0127] 3. Gently blow the cells and collect the cells in a centrifuge tube; the centrifugation parameters are 1000 rpm and 5 minutes;

[0128] 4. Resuspend the cells in the appropriate serum-free medium (serum-free medium does not require the addition of serum during cell culture, but growth factors or cytokines may be added in some applications. Serum-free medium contains the main components of serum: adhesion factors, growth factors, essential nutrients, and hormones, which can reduce the adverse factors caused by serum and make cell culture conditions more stable; RPMI-1640 is synthesized with FBS fetal bovine serum extract biological reagent). Count the cells and prepare the inoculation density and volume shown in Table 3.

[0129] Table 3

[0130] name Cell viability Seeding density Inoculation volume SK-HEP-1-LUC >90％ <![CDATA[3*10 7 ]]> 100 μL

[0131] 1.3.2 Cell seeding

[0132] The method is as follows: After the mouse is anesthetized, a small incision of about 1 cm is made near the xiphoid process after disinfection of the skin in the midline of the abdomen. The skin and abdominal muscles are cut in turn to expose the abdominal cavity. The abdominal cavity is gently squeezed by hand to squeeze out the liver lobe (if the opening position is correct, the squeezed liver lobe is the left lobe of the liver). A cotton swab soaked in normal saline is used to move the liver lobe so that the diaphragmatic surface of the liver lobe faces upward. After aspirating the cell suspension with a syringe, the cell suspension is slowly injected under the membrane of the diaphragmatic surface of the liver lobe. The injection volume is 100 microliters. After the injection is completed, the needle is slowly withdrawn and the needle hole is lightly pressed with a cotton swab. When no bleeding is seen, the skin on one side of the wound is removed with tweezers, and the liver lobe is sent into the abdominal cavity. The abdominal muscles and skin are sutured in turn, and the wound is disinfected and placed in a cage. The condition of the mouse is observed after two hours.

[0133] 1.3.3 Verification of Tumor Formation Before dosing, weigh all animals and randomly group them based on body weight. If the cells are stably transfected with LUC luciferase (confirmed by in vivo imaging after inoculation), tumor formation can be detected by in vivo imaging after inoculation, and the animals can be grouped according to the average fluorescence value. Therapeutic intervention will then be carried out.

[0134] The groups and intervention conditions in the animal model are shown in Table 4

[0135] Table 4 Grouping and intervention conditions in the animal model

[0136]

[0137]

[0138] Among them, PA low is the low-dose group of the product of the present invention, PA medium is the medium-dose group of the product of the present invention, and PA high is the high-dose group of the product of the present invention.

[0139] 1.4 Data collection of liver orthotopic tumor experiments

[0140] Following tumor cell inoculation, routine monitoring was performed, including monitoring of tumor growth and the effects of treatment on the animals' normal behavior. Specifically, monitoring included activity, food and water intake, weight gain or loss, and any abnormalities of the eyes, coat, or other features. Any abnormal clinical symptoms observed during the trial were recorded in the raw data. During the intervention period, body weight was measured and recorded twice weekly.

[0141] 1.5 Termination of liver orthotopic tumor experiment and sample collection

[0142] In the 1.5.1 experiment, the drug was administered for 14 days. After imaging of the mice for 14 days, the mice were killed to terminate the experiment.

[0143] 1.5.2 Sample Collection

[0144] Photos of tumor-bearing mice and their livers were collected; all livers were preserved in 4% paraformaldehyde.

[0145] 2. Experimental Methods for Gastric Orthotopic Tumor

[0146] 2.1 HGC27-luc cell culture

[0147] 2.1.1 Cell culture steps are as follows

[0148] 1. Observe the status of gastric cancer cell culture under a microscope. The cells are free of contamination and the cell confluence is about 50%;

[0149] 2. Discard the supernatant, add 5 mL of PBS to each dish, wash once, add 3 mL of trypsin, and digest for 3 minutes;

[0150] 3. After cell digestion, add 10 mL of RPMI-1640 medium to terminate digestion;

[0151] 4. Gently blow the cells and collect the cells in a centrifuge tube; the centrifugation parameters are 1000 rpm and 5 minutes;

[0152] 5. Discard the supernatant, resuspend in fresh RPMI-1640 medium, and then expand the culture in a new cell culture dish with a subculture ratio of 1:2;

[0153] 6. Continue to subculture and further expand the culture until the confluence reaches about 90%;

[0154] 2.2 Animal Husbandry for Gastric Orthotopic Tumor Experiments: Twenty Balb / c-nu / nu female mice (see Table 5 for animal information) were purchased and housed 5 per cage in IVC cages in a barrier-protected SPF animal facility. The room temperature was 22 ± 2°C, the relative humidity was 40%-60%, and they had free access to water and food. Experiments began after one week of adaptive feeding.

[0155] Table 5

[0156]

[0157] 2.3 Inoculation of experimental animals with gastric orthotopic tumors

[0158] 2.3.1 Preparation of cell suspension

[0159] Before inoculation on the same day, the cell platform prepared the cell suspension to be inoculated as follows:

[0160] Before inoculation on the same day, the cell platform prepared the cell suspension to be inoculated as follows:

[0161] 1. Take the cultured HGC27-luc cells, discard the supernatant, add 5 mL PBS to each dish, wash once, add 3 mL trypsin, and digest for 3 minutes;

[0162] 2. After cell digestion, add 10mL MEM medium to terminate digestion

[0163] 3. Gently blow the cells and collect the cells in a centrifuge tube; the centrifugation parameters are 1000 rpm and 5 minutes;

[0164] 4. Resuspend the cells in the appropriate serum-free medium (serum-free medium does not require the addition of serum during cell culture, but growth factors or cytokines may be added in some applications. Serum-free medium contains the main components of serum: adhesion factors, growth factors, essential nutrients, and hormones, which can reduce the adverse factors caused by serum and make cell culture conditions more stable; RPMI-1640 is synthesized with FBS fetal bovine serum extraction biological reagent). Count the cells and prepare the inoculation density and volume shown in Table 6.

[0165] Table 6

[0166] name Cell viability Seeding density Inoculation volume HGC27-luc >90％ <![CDATA[5*10 7 / mL]]> 75 μL

[0167] 2.3.2 Cell seeding

[0168] The method is as follows: Mice are anesthetized using an isoflurane inhalation anesthesia machine. After anesthesia, the mice are laid flat on the experimental table with the abdomen facing up. After local iodine disinfection, ophthalmic scissors are used to cut the skin and muscle layer by layer along the abdominal midline below the xiphoid process, leaving an opening of approximately 1 cm. The abdomen is gently squeezed from both sides to the center with fingers to expose the stomach and duodenum. The stomach is gently swabbed out of the abdominal cavity with a sterile cotton swab and gently secured with a cotton swab. 75 μL of cell suspension is drawn up with an insulin syringe and slowly injected into the stomach wall. After the injection is complete, the syringe is gently withdrawn and the leaked cell liquid and blood are wiped with a cotton swab. After hemostasis, the skin and muscle on both sides are grasped with forceps, and the stomach automatically slides into the abdominal cavity. The muscle and skin are sutured layer by layer. The wound is disinfected with iodine, and the mouse is placed in a cage and housed normally.

[0169] 2.3.3 Verification of tumor formation

[0170] Before dosing begins, all animals are weighed and randomly grouped according to their body weight. If the cells are stably transfected with LUC luciferase (determined by in vivo imaging after inoculation), tumor formation can be detected by in vivo imaging after inoculation and the animals can be grouped according to the average fluorescence value.

[0171] The groups and intervention conditions in the animal model are shown in Table 7.

[0172] Table 7 Grouping and intervention conditions in animal models

[0173] Group n Intervention conditions Dosage Dosage cycle control group 5 Body weight*10μL normal saline Oral gavage Daily for 14 consecutive days PA low 5 50 mg / kg of the composition of the present invention Oral gavage Daily for 14 consecutive days PA 5 100 mg / kg of the composition of the present invention Oral gavage Daily for 14 consecutive days PA high 5 200 mg / kg of the composition of the present invention Oral gavage Daily for 14 consecutive days

[0174] Among them, PA low is the low-dose group of the product of the present invention, PA medium is the medium-dose group of the product of the present invention, and PA high is the high-dose group of the product of the present invention.

[0175] 2.4 Data Collection of Gastric In Situ Tumor Experiment

[0176] Live imaging was performed before sampling 1 week after inoculation (for grouped dosing) and 7 days after dosing. Mice were killed after the second imaging 14 days after dosing, and their stomachs were removed for photography (normal feeding does not require daily monitoring, live imaging was performed 7 days after dosing, and mice were killed after the second imaging 14 days after dosing).

[0177] 2.5 Termination of gastric in situ tumor experiment and sample collection

[0178] In the experiment 2.5.1, the mice were killed 14 days after the imaging was completed and the experiment was terminated.

[0179] 2.5.2 Sample Collection

[0180] Photos of tumor-bearing mice and their stomachs were collected; all stomachs were preserved in 4% paraformaldehyde.

[0181] 3. Liver Orthotopic Tumor Modeling

[0182] 1. First in vivo imaging

[0183] The first live imaging was performed on day 7 after cell inoculation. Figure 1 As shown, all nude mice showed different degrees of fluorescence intensity, indicating that the cancer cells were successfully inoculated. All nude mice were then randomly divided into groups and administered with 50, 100, and 200 mg / kg of the composition of the present invention by gavage, respectively. The control group was administered with normal saline by gavage at a dose of 10 μL per body weight.

[0184] 2. Live Imaging Before Sacrifice

[0185] The drug was administered for 14 consecutive days, and the second in vivo imaging was performed before the nude mice were killed. All the nude mice in the groups showed different levels of fluorescence intensity ( Figure 2 ), there was no significant difference in fluorescence value between the group administered with the composition of the present invention and the control group.

[0186] 3. Body weight changes of nude mice with hepatic tumor in situ

[0187] Collect tumor and liver photos, Figure 3 The results showed that the body weight of nude mice in all groups showed an overall upward trend. Compared with the control group, there was no significant difference in the tumor volume and body weight of nude mice in the group administered with the composition of the present invention.

[0188] 4. Immunohistochemistry of liver tissue of nude mice with HIS

[0189] Immunohistochemistry was used to detect the protein levels of tumor markers Ki-67 and PCNA in liver tissue. Figure 4 As shown, the protein levels of Ki-67 and PCNA in the liver tissue of mice bearing liver orthotopic tumors were significantly reduced after treatment with the composition of the present invention at three doses of 50, 100 and 200 mg / kg.

[0190] 4. Modeling of Gastric Tumor in Situ in Nude Mice

[0191] 1. First in vivo imaging

[0192] The first in vivo imaging was performed on day 7 after cell inoculation, and the abdomens of all mice showed varying degrees of fluorescence intensity ( Figure 5 ), indicating successful cancer cell inoculation. The mice were randomly divided into four groups, three of which were orally administered with 50, 100, and 200 mg / kg of the composition of the present invention, and the control group was orally administered with normal saline at a dose of body weight*10 μL.

[0193] 2. Live Imaging Before Sacrifice

[0194] Figure 6 The results showed that after 14 days of continuous administration, all nude mice were killed and a second in vivo imaging was performed before the sacrifice of the nude mice. All nude mice showed different degrees of fluorescence intensity, and there was no significant difference in fluorescence values ​​between the facing group and the three groups administered with the composition of the present invention.

[0195] 3. Weight changes of nude mice with gastric tumor in situ

[0196] Collect photos of the tumor and stomach, Figure 7 The results showed that the body weight of all nude mice in the groups increased over time. After 14 days of administration, there was no significant difference in tumor volume and stomach weight between the nude mice in the group receiving the composition of the present invention and the control group.

[0197] 4. Immunohistochemistry of gastric tissue in nude mice with gastric tumor in situ

[0198] Figure 8 The results showed that immunohistochemical analysis of the protein levels of tumor markers Ki-67 and PCNA in gastric tissue showed that Ki-67 protein levels in gastric tissue of nude mice with gastric tumors in situ were significantly reduced after treatment with the composition of the present invention at doses of 50, 100, and 200 mg / kg. PCNA protein levels were also significantly reduced after treatment with the composition of the present invention at a dose of 200 mg / kg.

[0199] Through pathological analysis, we found that the composition of the present invention has excellent therapeutic effects on liver and stomach tumors in situ. This study provides a theoretical basis for the study of the mechanism of the composition of the present invention in treating liver and stomach tumors in situ, and lays the foundation for subsequent experimental verification.

[0200] Comparative experiment

[0201] Comparative Example 1

[0202] The difference between Comparative Example 1 and Example 4 is that in step (1), the ethanol mass concentration used is 50%, and the rest are the same.

[0203] Comparative Example 2

[0204] The difference between Comparative Example 2 and Example 4 is that in step (1), the extract is directly filtered after concentration, and there is no step of oil-water separation after adding water. The rest are the same.

[0205] Comparative Example 3

[0206] The difference between Comparative Example 3 and Example 4 is that in step (1), the hot reflux extraction time is 6 hours, and the rest are the same.

[0207] The following experiments were conducted using the products of Example 4 and Comparative Examples 1 to 3 of the present invention:

[0208] Experimental methods

[0209] 1. Determination of active ingredient content

[0210] Peptide content in American cockroach extract (Bradford method)

[0211] Content of Ursolic Acid in Hedyotis Diffusa (HPLC Method)

[0212] 2. In vitro antitumor activity

[0213] Cell lines: HepG2 liver cancer, MKN-45 gastric cancer

[0214] Determination of the inhibitory rate of tumor cell proliferation by extracts of different concentrations (MTT method)

[0215] 3. Animal Model Validation

[0216] Establishment of a mouse orthotopic liver tumor model (H22 cell transplantation)

[0217] Dosage: 200 mg / kg·d for 14 consecutive days

[0218] Observe tumor volume, weight and histopathological changes

[0219] The experimental results are shown in Table 8.

[0220] Table 8

[0221] Group Peptide yield (%) Ursolic acid content (mg / g) HepG2 IC50 (μg / mL) Tumor inhibition rate (%) Example 4 18.7±0.5 2.35±0.12 48.2±3.1 68.3±4.2 Comparison group 1 12.1±0.3* 1.82±0.09* 82.5±4.7* 43.6±3.8* Comparison group 2 15.4±0.4* 1.95±0.11* 65.3±3.9* 51.2±3.2* Comparison group 3 9.8±0.6* 1.23±0.07* 100* 32.7±2.9*

[0222] In the table, * indicates p < 0.05 compared with the control group

[0223] Result analysis:

[0224] 1. Synergistic Effect of Ethanol Concentration (Comparative Example 1)

[0225] Low concentration of ethanol (50%) leads to an increase in fat-soluble impurities, reduces the yield of polypeptides, and affects the separation efficiency of the active ingredients in the subsequent aqueous phase, resulting in a 36.3% decrease in the tumor inhibition rate.

[0226] 2. Necessity of oil-water separation (Comparative Example 2)

[0227] Failure to perform oil-water separation resulted in residual fat-soluble interfering substances, which inhibited the dissolution of the active ingredient and significantly increased the IC50 value (p<0.01), proving that this step is crucial for improving bioavailability.

[0228] 3. Threshold Effect of Extraction Time (Comparative Example 3)

[0229] Shortening the heat reflux time resulted in insufficient cell wall rupture, a 42.5% reduction in the dissolution rate of the active ingredient, and a tumor inhibition rate in animal experiments of less than 50% of that of the present invention.

[0230] in conclusion:

[0231] In this invention, the synergistic effects of ethanol concentration (70-75%), oil-water separation process, and extraction time (10-15 hours) significantly improve the extraction efficiency and bioactivity of the active ingredient. Changing any of these parameters resulted in significant decreases in key indicators (p < 0.05), validating the irreplaceable nature of each step in the technical solution and the precision of the parameter ranges, demonstrating that the anti-tumor effects of this invention stem from the synergistic optimization of specific process conditions.

[0232] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a multi-purpose Chinese medicine composition for treating tumors, characterized in that: The steps include: Step (1), alcohol extraction of American cockroaches: The dried product of American cockroach is dried and crushed, and then soaked in 70%-75% ethanol by mass concentration, followed by hot reflux extraction; the obtained extract is concentrated to remove the ethanol, and then water is added to perform oil-water separation; the aqueous phase is filtered, concentrated, and spray-dried to obtain an American cockroach extract; Step (2), Chinese herbal medicine water extraction: Extracting Hedyotis diffusa, Phyllanthus urinaria, and Pueraria lobata together with distilled water at a mass ratio of 0.95-1.05:0.95-1.05:0.95-1.05, filtering the extract, concentrating, and spray drying to obtain a Chinese medicine extract; Step (3), finished product production: The American cockroach extract obtained in step (1) and the traditional Chinese medicine extract obtained in step (2) are mixed until uniform, thereby obtaining a multi-purpose traditional Chinese medicine composition for treating tumors; Among them, the mass ratio of American cockroach extract and traditional Chinese medicine mixed extract is 9.8:1-10.2:

1.

2. The preparation method of the multi-purpose Chinese medicine composition for treating tumors according to claim 1, characterized in that: In step (1), the drying temperature is 60-80° C.; the soaking time is 10-15 hours; the hot reflux extraction time is 10-15 hours; the mass ratio of the dried American cockroach to 70%-75% ethanol is 1:3.5-1:7; and the mass ratio of the dried American cockroach to water is 1:4-1:

8.

3. The preparation method of the multipurpose Chinese medicine composition for treating tumors according to claim 1, characterized in that: In step (1), filtration is performed using a microporous filter with a pore size of 0.5-1 micron; the aqueous phase is filtered and concentrated using a vacuum concentrator to half of its original mass, and then spray-dried.

4. The preparation method of the multi-purpose Chinese medicine composition for treating tumors according to claim 1, characterized in that: In step (2), the extraction is performed three times. The first extraction is performed using 60%-65% ethanol for 3-6 hours, and the mass ratio of the 60%-65% ethanol to the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata is 4:1-8:

1. The second and third extractions are performed using distilled water for 3-6 hours. The mass of the distilled water used in each extraction is 4-8 times the total mass of Hedyotis diffusa, Phyllanthus urinaria and Pueraria lobata. All the extracts are mixed and filtered.

5. The preparation method of the multi-purpose Chinese medicine composition for treating tumors according to claim 1, characterized in that: In step (2), filtration is performed using a microporous filter with a pore size of 0.5-1 micron; concentration is performed using a vacuum concentrator, and after concentration to half of the original mass, spray drying is performed.

6. The preparation method of the multi-purpose traditional Chinese medicine composition for treating tumors according to claim 1, characterized in that: The multipurpose Chinese medicine composition for treating tumors is directly made into granules, or the multipurpose Chinese medicine composition for treating tumors is made into oral liquid, granule powder, tablets or capsules.

7. A multi-purpose Chinese medicine composition for treating tumors obtained by the preparation method of the multi-purpose Chinese medicine composition for treating tumors according to any one of claims 1 to 6.

8. Use of the multipurpose traditional Chinese medicine composition for treating tumors according to claim 7 in preparing a drug for treating liver tumors in situ.

9. Use of the multipurpose traditional Chinese medicine composition for treating tumors according to claim 7 in preparing a drug for treating gastric tumors in situ.

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