A Chinese medicine composition for treating animal breast tumors and its application

Abstract

The present invention belongs to the technical field of traditional Chinese medicine, and is specifically related to a Chinese medicine composition for treating animal breast tumors and its application. The Chinese medicine composition for treating animal breast tumors includes the following raw materials in parts by weight: 6-150 parts of Astragalus, 3-75 parts of Angelica, 6-150 parts of Taraxacum, 3-75 parts of Hedyotis diffusa, 3-75 parts of Honeysuckle, 3-75 parts of Conch, 3-75 parts of Tripterygium wilfordii, 3-60 parts of Snake Six Valley, 2-50 parts of Beehive, 3-75 parts of Polygonum multiflorum, 3-75 parts of Bidens pilosa, and 3-75 parts of Licorice. The present invention treats animal breast tumors with traditional Chinese medicine thinking, i.e., soft dissolving and dispersing knots, clearing away heat and detoxicating to suppress cancer; strengthening the body as the basis, and removing evil as the use, and it has been proved by experiments that breast tumor growth can be significantly inhibited, the content of cytokines in serum is regulated, and the immune function of animals is improved at the same time, so that the effect of treating breast tumor diseases can be achieved, so there is a good value of practical application.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine composition for treating animal breast tumors and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] For animal breast tumors, surgery is currently the main treatment method in veterinary clinics, but there are still problems such as great harm to animals, complications and side effects, and difficulty in eradication, and the postoperative recurrence rate is still as high as 50% to 90%. Traditional Chinese medicine has significant advantages in inhibiting tumor growth, improving the body's immunity, and reducing complications and side effects after modern medical treatment. Therefore, traditional Chinese medicine is becoming more and more popular worldwide. The combination of traditional Chinese and Western medicine for breast cancer has become one of the key research directions in clinical and basic research at home and abroad. For people and animals who cannot benefit from conventional tumor treatments, traditional Chinese medicine treatment is an important choice. Summary of the invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides a Chinese medicine composition for treating animal breast tumors and its application. The present invention treats animal breast tumors with the thinking of traditional Chinese medicine, that is, softening and dissolving knots, clearing away heat and detoxifying to inhibit cancer; strengthening the body as the basis and removing evil as the purpose, so as to optimize the diagnosis and treatment of animal breast tumors. Based on the above research results, the present invention is completed.

[0005] One of the purposes of the present invention is to provide a Chinese medicine composition for treating animal breast tumors.

[0006] The second object of the present invention is to provide a method for preparing the above-mentioned Chinese medicine composition for treating animal breast tumors.

[0007] The third object of the present invention is to provide the application of the above-mentioned Chinese medicine composition for treating animal breast tumors.

[0008] A fourth object of the present invention is to provide a veterinary pharmaceutical preparation for treating breast tumors.

[0009] In order to achieve the above-mentioned invention object, the present invention discloses the following technical solutions:

[0010] The first aspect of the present invention provides a Chinese medicine composition for treating animal breast tumors, wherein the Chinese medicine composition for treating animal breast tumors comprises the following raw materials in parts by weight:

[0011] 6-150 parts of Astragalus, 3-75 parts of Angelica, 6-150 parts of Taraxacum, 3-75 parts of Oldenlandia Diffusa, 3-75 parts of Honeysuckle, 3-75 parts of Conch Shell, 3-75 parts of Tripterygium Wright, 3-60 parts of Snakegrass, 2-50 parts of Honeysuckle, 3-75 parts of Fructus Polygoni Multiflori, 3-75 parts of Bidens Piper, and 3-75 parts of Licorice.

[0012] The animal may be a non-human mammal, such as a mouse, a rat, a monkey, an ape, a dog, etc., preferably a dog.

[0013] It should be noted that, in the present invention, the above-mentioned traditional Chinese medicine composition for treating animal breast tumors is named Qingruhuayanjian.

[0014] The second aspect of the present invention provides a method for preparing the above-mentioned traditional Chinese medicine composition, which includes but is not limited to water decoction, water extraction and alcohol precipitation, enzymatic hydrolysis, acid-base extraction and ultrasonic extraction, preferably water decoction.

[0015] The third aspect of the present invention provides the use of the above-mentioned Chinese medicine composition in the preparation of a veterinary pharmaceutical preparation for treating breast tumors.

[0016] A fourth aspect of the present invention provides a veterinary pharmaceutical preparation for treating breast tumors, the pharmaceutical preparation comprising at least the above-mentioned Chinese medicine composition; further, the pharmaceutical preparation may also comprise pharmaceutically acceptable excipients.

[0017] Compared with the prior art, the above one or more technical solutions have achieved the following beneficial effects:

[0018] 1) The above technical scheme provides the anti-tumor effect of Qingruhuayanjian and its application in breast tumors. The Qingruhuayanjian uses Astragalus and Angelica as the main ingredients. Astragalus is effective in replenishing qi, lifting and lifting, supporting toxins and draining pus; Angelica is an important medicine for replenishing blood and activating blood circulation. The two herbs of Astragalus and Angelica are essential clinical medicines for the treatment of decreased biochemical indicators such as albumin and hemoglobin. Honeysuckle, dandelion, white flower oldenlandia, mountain conch, and three-leaf green are the auxiliary ingredients. They all have the effects of clearing heat and detoxifying, relieving pain and dispersing knots, and are commonly used medicines for cancer. Dandelion belongs to the liver meridian. In addition to clearing and dissolving knots, it can also soothe the liver and regulate qi. Mountain snail can nourish qi and yin, detoxify and reduce swelling, discharge pus and promote lactation. Tripterygium wilfordii can clear heat and detoxify, reduce swelling and resolve phlegm. With snake six grains, Bidens pilosa, and water safflower seeds as adjuvants, it can promote blood circulation and relieve pain, resolve phlegm and disperse accumulation, and remove blood stasis and reduce swelling. With beehive directly entering the collaterals as the messenger, it has excellent anti-inflammatory and swelling effects. It is a commonly used empirical medicine for acute mastitis and has a better effect on inflammatory changes around the breast. Licorice can clear heat and detoxify, and harmonize various medicines. It is a commonly used messenger. Among them, dandelion and angelica belong to the liver meridian and are commonly used targeted drugs for breast cancer. Looking at the whole prescription, it has the functions of strengthening the spleen and kidney, clearing heat and detoxifying, soothing the liver and relieving depression, and resolving phlegm and dispersing blood stasis. For breast cancer, the first method should be to strengthen the spleen and kidney and support the positive energy, and the other methods are to soften and disperse knots, clear heat and detoxify, soothe the liver and resolve depression, and resolve phlegm and disperse blood stasis to ward off evil.

[0019] 2) The Chinese medicine compound of the present application is simple and efficient, the raw materials are economical and affordable, and the preparation and extraction methods are simple, low-cost, universal, and easy to mass produce, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0021] Figure 1 The weight changes of mice in Example 2 of the present invention, wherein A. the weight change trend of mice during drug administration; B. the final weight of mice before sacrifice;

[0022] Figure 2 The changes of the mouse 4T1 transplanted tumor in Example 2 of the present invention, wherein A. mouse tumor growth curve; B. individual tumor growth curve; C. the final volume of the tumor measured after the mouse was dissected and the tumor was peeled off; D. the final weight of the tumor measured after the mouse was dissected and the tumor was peeled off;

[0023] Figure 3 The histopathological changes of breast cancer in mice in Example 2 of the present invention (HE staining, 200×) are the Qingruhuayanjian high-dose group (H), the medium-dose group (M), the low-dose group (L) and the model group (Model);

[0024] Figure 4 The histopathological changes of liver, spleen, lung, kidney, thymus and small intestine of mice in Example 2 of the present invention (HE staining, 200×);

[0025] Figure 5 The effect of the clear emulsified rock decoction in Example 2 of the present invention on apoptosis of transplanted tumor cells (TUNEL, 200×);

[0026] Figure 6 is the ratio of TUNEL positive cells in each group of transplanted tumors in Example 2 of the present invention;

[0027] Figure 7 The expression of MMP-9 in cancer tissues in Example 2 of the present invention, wherein: A. the expression of MMP-9 in transplanted tumor tissues of each group; B. the percentage of MMP-9 positive cells;

[0028] Figure 8 The expression of HSP70 in cancer tissues in Example 2 of the present invention, wherein: A. the expression of HSP70 in transplanted tumor tissues of each group; B. the percentage of HSP70 positive cells;

[0029] Fig. 9The changes in the levels of Th1 cytokines in serum in Example 2 of the present invention, wherein: A. changes in IL-2; B. changes in IFN-γ; C. changes in TNF-α;

[0030] Fig.10 The changes in the levels of Th2 cytokines in serum in Example 2 of the present invention, wherein A. changes in IL-4; B. changes in IL-10;

[0031] Fig.11 The effect of Qingruhuayanjian in Example 2 of the present invention on the proliferation of mouse spleen lymphocytes, wherein A. unstimulated group, B. LPS group, C. PHA group;

[0032] Fig.12 The effect of the clear emulsified rock decoction in Example 2 of the present invention on the proliferation of canine A72 cells (n=6);

[0033] Fig.13 This is the effect of the clear emulsified rock decoction in Example 2 of the present invention on the viability of CMT-U27 cells (n=6). DETAILED DESCRIPTION

[0034] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0035] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0036] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out according to conventional conditions or the conditions recommended by the sales company; if not particularly limited in the present invention, they can all be purchased through commercial channels.

[0037] In a typical embodiment of the present invention, a Chinese medicine composition for treating animal breast tumors is provided, wherein the Chinese medicine composition for treating animal breast tumors comprises the following raw materials in parts by weight:

[0038] 6-150 parts of Astragalus, 3-75 parts of Angelica, 6-150 parts of Taraxacum, 3-75 parts of Oldenlandia Diffusa, 3-75 parts of Honeysuckle, 3-75 parts of Conch Shell, 3-75 parts of Tripterygium Wright, 3-60 parts of Snakegrass, 2-50 parts of Honeysuckle, 3-75 parts of Fructus Polygoni Multiflori, 3-75 parts of Bidens Piper, and 3-75 parts of Licorice.

[0039] Specifically, 6-20 parts of Astragalus, 3-10 parts of Angelica, 6-20 parts of Taraxacum, 3-10 parts of Oldenlandia Diffusa, 3-10 parts of Honeysuckle, 3-10 parts of Conch Shell, 3-10 parts of Tripterygium Wright, 3-8 parts of Snakehead Millet, 2-8 parts of Apis cerana, 3-10 parts of Red Flower Fruit, 3-10 parts of Bidens pilosa, and 3-10 parts of Licorice.

[0040] Specifically, 6 parts of Astragalus, 3 parts of Angelica, 6 parts of Taraxacum, 3 parts of Oldenlandia Diffusa, 3 parts of Honeysuckle, 3 parts of Conch Shell, 3 parts of Tripterygium wilfordii, 3 parts of Snakehead Millet, 2 parts of Beehive, 3 parts of Red Flower Fructus, 3 parts of Bidens pilosa, and 3 parts of Licorice.

[0041] The animal may be a non-human mammal, such as a mouse, a rat, a monkey, an ape, a dog, etc., preferably a dog.

[0042] It should be noted that, in the present invention, the above-mentioned traditional Chinese medicine composition for treating animal breast tumors is named Qingruhuayanjian.

[0043] The formula characteristics of the Chinese medicine composition of the present invention are as follows:

[0044] Astragalus and Chinese angelica are the main ingredients. Astragalus is good at replenishing qi, lifting qi, expelling toxins and expelling pus; Chinese angelica is the main ingredient for replenishing blood and activating blood circulation. Chinese angelica and Chinese angelica are the main ingredients for the treatment of decreased biochemical indicators such as albumin and hemoglobin in cancer patients. Honeysuckle, dandelion, Hedyotis diffusa, conch and Tripterygium wilfordii are the main ingredients. They all have the effects of clearing away heat and detoxifying, relieving pain and dispersing nodules, and are commonly used drugs for cancer. Dandelion belongs to the liver meridian. In addition to clearing and dissolving knots, it can also soothe the liver and regulate qi. Mountain snail can nourish qi and yin, detoxify and reduce swelling, discharge pus and promote lactation. Tripterygium wilfordii can clear heat and detoxify, reduce swelling and resolve phlegm. With snake six grains, Bidens pilosa, and water safflower seeds as adjuvants, it can promote blood circulation and relieve pain, resolve phlegm and disperse accumulation, and remove blood stasis and reduce swelling. With beehive directly entering the collaterals as the messenger, it has excellent anti-inflammatory and swelling effects. It is a commonly used empirical medicine for acute mastitis and has a better effect on inflammatory changes around the breast. Licorice can clear heat and detoxify, and harmonize various medicines. It is a commonly used messenger. Among them, dandelion and angelica belong to the liver meridian and are commonly used targeted drugs for breast cancer. Looking at the whole prescription, it has the functions of strengthening the spleen and kidney, clearing heat and detoxifying, soothing the liver and relieving depression, and resolving phlegm and dispersing blood stasis. For breast cancer patients, strengthening the spleen and kidney and supporting the positive energy should be the first method, and softening and dispersing knots, clearing heat and detoxifying, soothing the liver and relieving depression, and resolving phlegm and dispersing blood stasis to ward off evil.

[0045] The experiments have proven that the above-mentioned Qingruhuayan Decoction has the following specific effects in treating breast tumors:

[0046] a) inhibiting breast tumor growth;

[0047] b) inhibiting the metastasis of breast cancer to liver and lung tissues;

[0048] c) Regulate the expression of MMP-9 and HSP70, enhance the secretion of Th1 cytokines, inhibit the release of Th2 cytokines, and thus enhance the body's immune function.

[0049] In another specific embodiment of the present invention, a method for preparing the above-mentioned traditional Chinese medicine composition is provided, which includes but is not limited to water decoction, water extraction and alcohol precipitation, enzymatic hydrolysis, acid-base extraction and ultrasonic extraction, preferably water decoction.

[0050] In another specific embodiment of the present invention, the preparation and extraction method comprises: soaking the above-mentioned medicinal materials in water, decocting the medicinal solution by water, and freeze-drying to obtain the clear emulsified Yanjian freeze-dried powder.

[0051] In another specific embodiment of the present invention, the specific steps of the water decoction method are: after the medicinal materials are soaked, boil over high heat, continue to decoct over low heat, pour out the supernatant and filter; continue to add water to the remaining medicinal residue until it covers the medicinal materials, boil over high heat, continue to decoct over low heat, pour out the supernatant and filter.

[0052] More specifically, the two decoctions are combined, concentrated, frozen and dried to obtain the product.

[0053] In another specific embodiment of the present invention, the use of the above-mentioned Chinese medicine composition in the preparation of a pharmaceutical preparation for veterinary treatment of breast tumors is provided. The present invention has been proved by mouse breast cancer transplant tumor model and canine breast cancer cell tumor inhibition experiment that it can induce apoptosis of breast cancer cells, improve the tumor inhibition rate, and inhibit the growth of breast cancer in the body; at the same time, by regulating the body's immune function, promoting the proliferation of lymphocytes, regulating the level changes of cytokines, exerting its immunoregulatory effect, enhancing the body's immunity, and thus exerting a good anti-tumor effect.

[0054] In another specific embodiment of the present invention, a pharmaceutical preparation for treating breast tumors for animals is provided, wherein the pharmaceutical preparation at least comprises the above-mentioned Chinese medicine composition; further, the pharmaceutical preparation may also comprise pharmaceutically acceptable excipients.

[0055] The pharmaceutical preparation can be prepared into various pharmaceutical dosage forms, preferably oral liquid, decoction, pill, capsule, tablet, powder or granule, etc., which are suitable for practical application.

[0056] In another specific embodiment of the present invention, the pharmaceutically acceptable excipients in the pharmaceutical preparation include, but are not limited to, one or more of diluents, fillers, disintegrants, lubricants, binders, flow agents, complex forming agents, plasticizers, colorants, sweeteners, viscosity enhancers, preservatives or antioxidants; further preferably, the excipients are acceptable for veterinary medicine.

[0057] In another specific embodiment of the present invention, the diluent includes but is not limited to sorbitol, mannitol, starch, lactose, powdered or microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, etc.;

[0058] The fillers include but are not limited to sodium carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, gelatinized starch, etc.;

[0059] The disintegrant includes but is not limited to dry starch, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, etc.;

[0060] The lubricant includes but is not limited to magnesium stearate, sodium lauryl sulfate or micro powder silica gel.

[0061] In another specific embodiment of the present invention, a method for treating breast tumors is provided, the method comprising: administering a therapeutically effective dose of the above-mentioned Chinese medicine composition or the above-mentioned pharmaceutical preparation to a subject.

[0062] The subject refers to an animal that has been the object of treatment, observation or experiment, preferably a non-human mammal, such as a mouse, rat, monkey, gorilla, dog, etc., more preferably a dog.

[0063] The "therapeutically effective amount" refers to the amount of active compound or pharmaceutical agent, including the compounds of the present invention, which can elicit the biological or medical response of the tissue system or animal sought by the researcher, veterinarian, doctor or other medical personnel, including the alleviation or partial alleviation of the symptoms of the disease, syndrome, condition or disorder being treated.

[0064] It must be recognized that the optimal dosage and interval of the active ingredients of the present invention are determined by their nature and external conditions such as the form, route and site of administration and the specific mammal to be treated, and this optimal dosage can be determined by conventional techniques.

[0065] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0066] Example 1

[0067] The specific preparation and extraction steps of a Chinese medicine composition for treating animal breast tumors are as follows:

[0068] Soaking: Mix 6g of Astragalus, 3g of Angelica, 6g of Taraxacum, 3g of Oldenlandia Diffusa, 3g of Honeysuckle, 3g of Conch Shell, 3g of Tripterygium, 3g of Snakehead Millet, 2g of Beehive, 3g of Red Flower Fructus Polygoni Multiflori, 3g of Bidens Bipinnata, and 3g of Licorice, mash them into a decoction pot, add 8 times the amount of drinking water, and soak for 60 minutes to unclog the capillaries of the medicinal materials and help the seepage of the effective ingredients.

[0069] Extraction of clear emulsified rock decoction: After the medicinal materials are soaked thoroughly, boil them over high heat, continue to boil them over low heat for 60 minutes, pour out the supernatant and filter; add 6 times the amount of drinking water to the remaining medicinal residue, boil them over high heat, then continue to boil them over low heat for 30 minutes, pour out the supernatant and filter.

[0070] Concentration: Combine the two decoctions and concentrate them using a rotary evaporator.

[0071] Freeze-drying: After the drug solution is cooled, freeze it in a -80℃ refrigerator for 48 hours, and then freeze-dry it in a freeze dryer for 48 hours.

[0072] Example 2

[0073] The application scope includes the following:

[0074] Direction 1, the effect of Qingruhuayanjian in Example 1 on breast tumors in mice;

[0075] Direction 2, the effect of the clear emulsified rock decoction in Example 1 on canine mammary tumor cells.

[0076] 1. Pharmacodynamics Experiment

[0077] The clear emulsified rock decoction in direction 1 is prepared into low (100 mg·kg -1 ), medium (200 mg kg -1 ), high (400 mg kg -1 ) doses were used for oral gavage of mice.

[0078] The clear emulsified rock decoction in direction 2 is respectively configured to a suitable concentration.

[0079] Mouse IL-2, IL-4, IL-10, TNF-α, IFN-γ ELISA kits (purchased from Hangzhou Lianke Biotechnology Co., Ltd.), tissue fixative (Beijing Solaibao Biotechnology Co., Ltd.), hematoxylin stain (purchased from Wuhan Bolfu Biotechnology Co., Ltd.), eosin stain (purchased from Wuhan Bolfu Biotechnology Co., Ltd.), LPS lipopolysaccharide, PHA lectin (Gibco, USA), anhydrous ethanol, n-butanol, xylene, hydrochloric acid aqueous solution, ammonia solution, neutral resin (Sinopharm Chemical Reagent Co., Ltd.), autofluorescence quencher (Beijing Pulilai Gene Technology Co., Ltd.), sodium citrate (EDTA) antigen retrieval solution, citric acid antigen retrieval buffer, BSA bovine serum albumin, DAPI stain, fluorescence quenching mounting medium, primary antibody, fluorescent secondary antibody, rabbit serum, DAB color development kit (Beijing Solebow Technology Co., Ltd.), Tunel kit (Roche, Switzerland), proteinase K (Biofroxx, Germany), membrane permeabilization solution, DAPI, anti-fluorescence quenching mounting medium (Wuhan Bolfu Biotechnology Co., Ltd.), MMP-9 antibody, HSP70 antibody (Abcam, UK), hydrogen peroxide (Sinopharm Chemical Reagent Co., Ltd., 0011218), DMEM high glucose medium, fetal bovine serum, 0.25% trypsin-EDTA, double antibody (penicillin 100.0U / mL-streptomycin 100.0mg / mL, Gibco, USA); DMSO (dimethyl sulfoxide, Beijing Solebow Biotechnology Co., Ltd.); phosphate buffer (PBS, Beijing Solebow Biotechnology Co., Ltd.); CCK kit (ZETA LIFE, USA), and other chemical reagents were of analytical grade.

[0080] Establishment of a mouse breast cancer transplant model

[0081] Fifty male and female Balb / c mice, 4-5 weeks old, 20±2 g, were purchased from the Comparative Medicine Center of Yangzhou University and were adaptively fed for one week before the experiment.

[0082] Take 4T1 cells in the logarithmic growth phase, wash them with PBS 1-2 times, add 0.25% trypsin for digestion, and observe the cells under a microscope. When the cells shrink and are about to detach from the wall but not detached, add culture medium to terminate digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at about 1000RPM for 5 minutes, discard the supernatant, resuspend the cells with 0.9% sterile saline, count them with a hemocytometer, and adjust the cell density to 1×10 7 / mL. To slow down the metabolic rate of cells, the prepared cell suspension needs to be placed in an ice box before inoculation. At the same time, to prevent cells from falling into a precipitate and clumping, and to increase the tumor formation rate, the cell suspension should be fully blown again during inoculation, and inoculation should be completed within half an hour after cell digestion as much as possible.

[0083] The vaccinator randomly grabs the mouse, fixes the mouse with one hand, uses pet-specific clippers to prepare the skin between the 4th and 5th pairs of nipples of the mouse, disinfects the inoculation site with 75% alcohol, and uses a 1mL syringe with the other hand to slowly inject 0.1mL of the prepared cell suspension into the subcutaneous fat pad below the nipple, and pulls out the needle after the inoculation is complete. The syringe needs to be replaced before each mouse is inoculated. After the injection, pay attention to observe whether the cell suspension flows out of the needle eye. After the inoculation is completed, give sufficient food and water, avoid touching the inoculation site, observe the growth rate of the mouse tumor every day, and determine the tumor positivity when the tumor diameter is ≥3mm. About one week after the inoculation, a small nodule the size of a rice grain can be touched subcutaneously at the inoculation site of the mouse. When the tumor volume reaches 100-200mm 3 The blank group was given a sham operation and inoculated with an equal amount of saline under the same conditions.

[0084] Experimental animal grouping and treatment

[0085] After the animal model was successfully constructed, it was treated according to the following groups and randomly divided into 5 groups, 10 mice in each group, a total of 50 mice. The normal group and the model group were gavaged with equal amounts of normal saline, and the high-dose group, the medium-dose group, and the low-dose group were gavaged with Qingru Huayan Jian respectively. Mice in each group were gavaged once every other day for 14 consecutive days, weighed every other day, and the tumor volume was measured every other day. After the last administration, fasting but not water was allowed for 24 hours, blood was collected after anesthesia, and samples were collected for the following index tests.

[0086] Cell culture

[0087] Cell recovery: Place the cryopreserved tubes of A72 (canine) breast cancer cells (third generation, adherent growth) and CMT-U27 (canine) breast cancer cells (third generation, adherent growth) in 37°C warm water and shake quickly to thaw for about 1 minute. After the cell cryopreserved solution is thawed, quickly transfer it to a centrifuge tube with 4-5mL of culture medium added in advance. Centrifuge at about 1000RPM for 5 minutes, discard the supernatant, add 1-2mL of complete culture medium and blow evenly, add the cell suspension to a T25 culture flask, add an appropriate amount of complete culture medium, and culture in a cell culture incubator at 37°C and 5% CO2.

[0088] Cell passaging: When the cell density reaches 80-90%, it can be passaged. Discard the supernatant culture medium and wash with PBS 2-3 times. Add 1mL of 0.25% trypsin to cover the entire culture bottle, cover it and put it into the incubator for digestion. Digest the cells for 0.5-2min, observe the cells under a microscope, and when the cells shrink and are about to detach from the wall but have not detached, add culture medium to terminate digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at about 1000RPM for 5min, and discard the supernatant. Add 2-3mL of fresh complete culture medium to the centrifuge tube to resuspend, and then transfer to a T25 culture flask. Divide the cells into two or three flasks for passaging, with a passaging ratio of 1:2 to 1:4, and finally add 6-8mL of complete culture medium to each flask.

[0089] 2. Experimental Methods

[0090] Direction 1, weight growth curve, tumor growth curve, drug inhibition rate, HE staining pathological examination, IL-2, IL-4, IL-10, TNF-α, IFN-γ content in cancer tissue and serum, apoptosis level of transplanted tumor cells, expression level of MMP-9 and HSP70 in cancer tissue, changes in spleen lymphocyte proliferation. (See Table 1 for specific experimental steps and operations)

[0091] Table 1 Experimental design table

[0092]

[0093]

[0094] Direction 2, take canine A72 cells and canine CMT-U27 cells in the logarithmic growth phase, add clear emulsified rock decoction in a safe concentration range (final concentration is 20mg / mL, 10mg / mL, 5mg / mL, 2.5mg / mL, 1.25mg / mL, 0.625mg / mL, 1640 complete medium dilution) to a 96-well cell culture plate, and incubate for 24h. After the incubation, take out the 96-well plate, discard the supernatant, rinse with PBS 1-2 times, then add 100μL DMEM complete medium to each well (containing 10μL CCK8 in each 100μL medium), and continue to incubate in a cell culture incubator for 1h. After the incubation, take out the 96-well plate, measure the absorbance of each well at a wavelength of 450nm on an ELISA reader, record the results, and calculate the cell survival rate at the same time.

[0095] 3. Results and Analysis

[0096] Data analysis methods

[0097] All experimental data were expressed as Mean ± SD. The data were preliminarily sorted and calculated using Excel, and the obtained data were analyzed using GraphPad Prism 6.0 software. SPSS 18.0 software was used for data variance analysis multiple comparisons, and P < 0.05 was set as statistically significant.

[0098] 3.1 Figure 1 Effect of Qingruhuayanjian on body weight of mice

[0099] like Figure 1 As shown in (A), during the treatment period, the body weight of mice in the normal group, high-dose and medium-dose groups showed a steady upward trend, while the body weight of mice in the low-dose group and model group showed a trend of first increasing and then decreasing to varying degrees, but the changes in body weight of mice in each group were small and not statistically significant (P>0.05). Figure 1 As shown in (B), there was no significant difference in the final body weight of mice in each group. The body weight of mice in the low-dose group and the model group showed a trend of decreasing, but the difference was not significant and not statistically significant (P>0.05).

[0100] 3.2 Effect of Qingruhuayanjian on the growth of transplanted tumors in mice

[0101] Effects of Qingruhuayanjian on 4T1 transplanted tumors in mice during treatment Figure 2 The mouse tumor growth curve is shown in Figure 2 As shown in A, the results showed that compared with the model group mice, the drug group significantly slowed down the tumor growth on the 9th day, and this inhibitory effect lasted until the end of the treatment cycle ( Figure 2 A).

[0102] After treatment, the mice were dissected and the tumors were removed, and the final volume of the tumors was measured ( Figure 2 C), weigh the final tumor weight ( Figure 2 D), the results showed that compared with the model group, the tumor volume of mice in each medication group was significantly reduced (P < 0.05), and the tumor weight was significantly reduced (P < 0.05), and the treatment effect of the medium-dose medication group was the most obvious. The results of mouse tumor inhibition rate showed that each medication group had a uniform and varying degree of inhibitory effect on mouse 4T1 breast cancer transplanted tumors, among which the medium-dose group had the most obvious inhibitory effect, reaching 49.63% (Table 2).

[0103] Table 2 Tumor inhibition rate of breast cancer in each group of mice after drug intervention (n=10)

[0104]

[0105] 3.3 Pathological changes in cancer tissue and liver, spleen, lung, kidney, thymus, and small intestine

[0106] 3.3.1 Histopathological changes of mouse breast cancer

[0107] like Figure 3 As shown in the figure, in the model group, tumor cells were distributed in an elliptical shape, with intact cell morphology, large nuclei, visible nucleoli, a large number of tumor cells, and a rare necrotic area. There were more interstitial vascular areas and abundant blood vessels, which could provide the nutrients required for the growth of tumor cells, indicating that the growth activity of tumor cells was good. Compared with the model group, the number of tumor cells in the medication group decreased, and the cell morphology was incomplete. Among them, the tumor cells in the high-dose group and the medium-dose group showed a greater degree of nuclear dissolution and deep nuclear staining, and nuclear fragments and nuclear division images appeared at the same time, and the necrotic area of ​​tumor cells was larger. It can be inferred that Qingruhuayanjian may induce apoptosis of breast tumor cells.

[0108] 3.3.2 Histopathological changes of liver, spleen, lung, kidney, thymus and small intestine of mice

[0109] like Figure 4 As shown in the figure, there were no obvious pathological changes in the spleen, kidney, and thymus of the model group, and the liver, lung, and small intestine showed different degrees of lesions. Among them, there were more cancer cells distributed around the central vein of the liver tissue, with larger nuclei and obvious nucleoli. The cancer cells lacked connection with the surrounding tissue cells, and some cancer cells had aggregated into clusters. The tissue boundaries were obvious, and there was a tendency to form cancerous tissue. The overall structure of the lung tissue was abnormal, the alveolar structure of the tissue was clear, and some alveoli were fused to form larger cysts (yellow arrows). The alveolar wall of the tissue was obviously thickened and solidified over a large area (green arrows). The interstitial lung tissue showed slight congestion (red arrows), and more inflammatory cell infiltration was seen in the tissue (black arrows). The overall structure of the small intestine was slightly abnormal, with obvious edema, and the intestinal villus structure was clear.

[0110] Except for mild abnormalities in liver and lung tissues, mice in the medication group had no obvious lesions in spleen, kidney, small intestine, and thymus tissues. In the high-dose and medium-dose groups, the overall structure of the lung tissue was slightly abnormal, the alveolar structure was clearly visible, a small amount of alveolar epithelial cells were seen to fall off in the tissue (indicated by the yellow arrow), some epithelial cells were seen to proliferate in the tissue, and the alveolar wall was slightly thickened (indicated by the red arrow). In the low-dose group, some epithelial cells were seen to fall off in the alveolar cavity (indicated by the yellow arrow), and the alveolar wall was seen to be significantly thickened and slightly substantialized (indicated by the red arrow). No obvious inflammatory cell infiltration was observed in the lung tissue of the high-dose and medium-dose groups, while a small amount of inflammatory cell infiltration was observed in the lung tissue of the low-dose group (indicated by the black arrow). In addition, a small amount of cancer cell infiltration was observed around the central vein of the liver tissue in the low-dose group, but the number of cancer cells was significantly reduced compared with the model group, and no cancer cells were seen in the liver tissue sections of the high-dose and medium-dose groups, indicating that Qingruhuayanjian can inhibit the metastasis of cancer cells.

[0111] 3.3.3 Effect of Qingruhuayanjian on apoptosis of transplanted tumor cells

[0112] like Figure 5 As shown, the cell nucleus is marked in blue, and apoptosis-positive tumor cells express green fluorescence. The blue fluorescence intensity of tumor cells in the model group and the drug group was basically the same. The model group occasionally had a very small amount of green fluorescence, the number of apoptotic cells was small, and the apoptosis rate was 6.69%. Compared with the model group, the green fluorescence intensity of breast tumor cells in the drug group was significantly enhanced, the tumor cells were broken into dots and flakes, and the number of apoptosis increased significantly. The differences were statistically significant (P < 0.05), among which the high-dose group and the medium-dose group were the most obvious, with average apoptosis rates of 71.91% and 74.85%, respectively. The average apoptosis rate in the low-dose group was 61.39%. Quantitative analysis results showed ( Figure 6 ), the model group had the lowest positive cell ratio, and the drug group had a significant increase in the positive cell ratio, among which the medium-dose group had the highest apoptotic cell ratio. The above results show that Qingruhuayanjian can inhibit breast cancer and promote apoptosis of breast cancer cells.

[0113] 3.4 Expression of MMP-9 and HSP70 in cancer tissues

[0114] 3.4.1 Expression of MMP-9 in cancer tissues

[0115] like Figure 7 As shown in A, the positive expression shown by DAB is brown-yellow. Compared with the model group, the positive expression of MMP-9 in the medication group was significantly reduced, and the cell coloring was weakened, indicating that Qingruhuayanjian can significantly reduce the expression of MMP-9 in breast tumor tissue (P < 0.05, Figure 7 B). Quantitative analysis results showed that the expression level of MMP-9 positive cells in the drug group was significantly different from that in the model group. The average percentages of MMP-9 positive cells in the high, medium and low dose groups were 37.22%, 29.20% and 39.57%, respectively, while the percentage of MMP-9 positive cells in the model group was 59.45%.

[0116] 3.4.2 Expression of HSP70 in cancer tissues

[0117] like Figure 8 As shown in the figure, compared with the model group, the positive expression and cell staining of HSP70 in the medication group tended to weaken, but the degree of reduction was not as significant as that of MMP-9. The quantitative analysis results showed that the expression level of HSP70 in the medication group was not as obvious as that of MMP-9, but it was still significantly different from the model group (P < 0.05). The average percentages of HSP70 positive cells in the high, medium and low dose groups were 33.95%, 33.30% and 39.80%, respectively, while the percentage of HSP70 positive cells in the model group was 52.22%.

[0118] 3.5 Effect of Clear Emulsified Rock Decoction on Serum Cytokines

[0119] 3.5.1 Changes in serum Th1 cytokine levels

[0120] like Fig. 9 As shown in the figure, compared with the blank group, the content of each cytokine in the model group did not change significantly (P>0.05), but the content of TNF-α showed an upward trend. Compared with the model group, the content of IL-2 in the medication group did not change significantly (P>0.05), the content of TNF-α in each medication group, and the content of IFN-γ in the high and medium dose groups increased significantly (P<0.05), and the content of IFN-γ in the low dose group did not change significantly. The above results show that Qingruhuayanjian can promote the secretion of Th1 cytokines in tumor mice.

[0121] 3.5.2 Changes in serum Th2 cytokine levels

[0122] like Fig.10 As shown in the figure, compared with the blank group, the expression level of IL-4 in the model group has an upward trend, but the difference is not significant (P>0.05), while the expression of IL-10 is significantly increased (P<0.05). Compared with the model group, the content of IL-4 has a downward trend, but the difference is not significant with the model group (P>0.05). The content of IL-10 in each medication group is significantly reduced (P<0.05), indicating that medication treatment can inhibit the secretion of Th2 cytokines.

[0123] 3.6 Effect of Qingruhuayanjian on spleen lymphocytes

[0124] The results of the effect of Qingru Huayan Jian on the proliferation of spleen lymphocytes in mice are as follows Fig.11 As shown. In the unstimulated group, there was no significant difference in the activity of spleen lymphocytes between the mice in each drug group and the model group and the blank group (P>0.05) ( Fig.11 A). In the LPS group, compared with the model group, the high-dose and medium-dose groups combined with LPS stimulated the proliferation of mouse spleen B lymphocytes significantly (P < 0.05), while the low-dose group combined with LPS stimulation had no significant effect, and there was no significant difference in the proliferation of mouse spleen B lymphocytes between the model group and the normal group (P > 0.05) ( Fig.11 B). In the PHA group, compared with the model group, there was no significant change in the activity of spleen T lymphocytes in the high-dose and low-dose groups (P>0.05) ( Fig.11 C), while the medium-dose group combined with PHA stimulation can significantly promote the proliferation of splenic T lymphocytes (P < 0.05). It can be seen that the medium-dose Qingruhuayanjian has the best effect on promoting the proliferation of mouse splenic lymphocytes. The above results show that Qingruhuayanjian can significantly promote the proliferation of splenic lymphocytes in tumor-bearing mice.

[0125] 3.7 Effect of Qingruhuayanjian on the proliferation of canine breast cancer cells

[0126] Effects of Qingruhuayanjian on A72 canine breast cancer cells Fig.12 Compared with the blank control group, when the concentration of Qingruhuayanjian was in the range of 0.625mg / mL-2.5mg / mL, the proliferation of A72 cells did not change significantly; however, when the concentration of Qingruhuayanjian was greater than or equal to 5mg / mL, it showed a significant inhibitory effect on the proliferation of A72 (P<0.05).

[0127] Effects of Qingruhuayanjian on CMT-U27 canine breast cancer cells Fig.13 Compared with the blank control group, when the concentration of clear emulsified rock decoction was in the range of 0.625mg / mL-5mg / mL, it had no significant effect on the proliferation of CMT-U27 cells; however, when the concentration of clear emulsified rock decoction was greater than or equal to 10mg / mL, it showed a significant inhibitory effect on the proliferation of CMT-U27 (P<0.05).

[0128] 4. Discussion

[0129] In this experiment, the mouse 4T1 breast tumor model was successfully constructed, and the weight and tumor growth of the mice were monitored during the treatment to evaluate the effect of the drug on breast tumors. When recording the weight of the mice, it was found that after 6 days of administration, except for the normal group of mice, the weight of the mice in the other groups decreased to varying degrees, among which the weight of the mice in the model group decreased the most, and the weight of the mice in the high-dose group and the medium-dose group remained almost unchanged, indicating that after the successful construction of the mouse breast tumor model, the tumor cell proliferation increased consumption, and the body intake was insufficient, resulting in metabolic disorders, which led to the generation of tumor cachexia and the downward trend of the mouse weight. Qingruhuayanjian improved the above symptoms and controlled the downward trend of the mouse weight. When drawing the tumor growth curve, it was found that after 6-9 days of administration, compared with the model group, the tumor volume of the mice in each medication group was significantly reduced, the tumor growth rate was significantly slowed down, and the final tumor weight was also significantly reduced, indicating that Qingruhuayanjian can effectively inhibit the growth of breast tumors. When calculating the tumor inhibition rate, it was found that each medication group had varying degrees of inhibitory effect on mouse breast cancer, among which the medium-dose group had the highest tumor inhibition rate, followed by the high-dose group. This suggests that the medium-dose Qingruhuayanjian may be most effective in inhibiting the growth of mouse breast cancer.

[0130] When observing the pathological sections of breast tumor tissue in this experiment, it was found that the tumor cells in the model group had normal morphology, large numbers, and rich blood vessels, which were conducive to the growth and proliferation of tumor cells. Compared with the model group, the number of tumor cells in the mice in the medication group was significantly reduced, the necrotic area increased, and the vascular area was less, suggesting that the drug may inhibit the growth of tumor cells by inducing the rupture of tumor cytoplasm and the dissolution of cell nuclei. At the same time, a large number of cancer cell metastases were seen around the central vein in the liver tissue sections of the model group, and the cancer cells had gradually aggregated into clusters, showing a trend of forming cancerous tissue, indicating that during the experimental period, breast cancer in mice had signs of liver metastasis. At the same time, more inflammatory cell infiltration and consolidation appeared in the lung tissue sections, which may indicate that breast cancer cells have a tendency to metastasize to the lungs. After drug treatment, the above symptoms were significantly improved, indicating that Qingruhuayanjian can significantly inhibit the metastasis of breast cancer to the liver and lung tissues.

[0131] This experiment found that compared with the model group, the expression levels of MMP-9 and HSP70 in breast tumor tissue of mice in the medication group were significantly reduced, suggesting that Qingruhuayanjian may control the growth and metastasis of breast cancer by regulating the expression of MMP-9 and HSP70 in the body.

[0132] Compared with the model group, the drug did not have a significant effect on the expression of IL-2 and IL-4 in serum, but the content of IL-2 in the medication group showed an upward trend, and the content of IL-4 showed a downward trend. At the same time, the content of IL-10 in the serum of the medication group was significantly reduced, and the content of TNF-α and INN-γ was significantly increased. The results of cancer tissue showed that there was no significant change in the content of IL-2 in the medication group mice, the expression levels of TNF-α and INN-γ increased significantly, and the content of IL-4 and IL-10 decreased significantly to varying degrees. It is suggested that within a certain concentration range, Qingruhuayanjian can enhance the secretion of Th1 cytokines, inhibit the release level of Th2 cytokines, and thus enhance the body's immune function.

[0133] In summary, Qingruhuayanjian can inhibit the growth of breast cancer by inducing apoptosis of 4T1 breast cancer cells and improving the tumor inhibition rate; it can also regulate the immune function of the body, promote the proliferation of lymphocytes, regulate the level of cytokines, exert its immunoregulatory effect, and enhance the body's immunity. In comparison, the medium dose of Qingruhuayanjian has the best anti-breast cancer effect.

[0134] It should be noted that the above examples are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention is described in detail with reference to the given examples, a person skilled in the art may modify or replace the technical solution of the present invention as needed without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A Chinese medicine composition for treating animal breast tumors, characterized in that: The Chinese medicine composition for treating animal breast tumors is prepared from the following raw materials in parts by weight: 6-150 parts of Astragalus, 3-75 parts of Angelica, 6-150 parts of Taraxacum, 3-75 parts of Oldenlandia Diffusa, 3-75 parts of Honeysuckle, 3-75 parts of Conch Shell, 3-75 parts of Tripterygium Wright, 3-60 parts of Snakegrass, 2-50 parts of Honeysuckle, 3-75 parts of Fructus Polygoni Multiflori, 3-75 parts of Bidens Piper, and 3-75 parts of Licorice.

2. The Chinese medicine composition according to claim 1, characterized in that The animal is a non-human mammal.

3. The Chinese medicine composition according to claim 2, characterized in that: The non-human mammals are mice, rats, monkeys, gorillas and dogs.

4. The Chinese medicine composition according to claim 3, characterized in that: The non-human mammal is a dog.

5. The method for preparing the Chinese medicine composition according to any one of claims 1 to 4, characterized in that: The method is a water decoction method.

6. The method for preparing the Chinese medicine composition according to claim 5, characterized in that: The preparation and extraction method comprises: soaking the above medicinal materials in water, decocting the medicinal liquid by water, and freeze-drying the medicinal materials to obtain freeze-dried powder.

7. The method for preparing the Chinese medicine composition according to claim 6, characterized in that: The specific steps of the water decoction method are: after the medicinal materials are soaked thoroughly, boil them over high heat, continue to decoct them over low heat, pour out the supernatant and filter; continue to add water to the remaining medicinal residue until it covers the medicinal materials, boil them over high heat, then continue to decoct them over low heat, pour out the supernatant and filter.

8. The method for preparing the Chinese medicine composition according to claim 7, characterized in that: The two decoctions are combined, concentrated, frozen and dried to obtain the product.

9. Use of the Chinese medicine composition according to any one of claims 1 to 4 in the preparation of a veterinary pharmaceutical preparation for treating breast tumors.

10. The use according to claim 9, characterized in that: The treatment of breast tumors is specifically manifested as: a) Inhibit breast tumor growth; b) Inhibit the metastasis of breast cancer to liver and lung tissues; c) Regulate the expression of MMP-9 and HSP70, enhance the secretion of Th1 cytokines, inhibit the release of Th2 cytokines, and thus enhance the body's immune function.

11. A veterinary pharmaceutical preparation for treating breast tumors, characterized in that: The pharmaceutical preparation comprises the traditional Chinese medicine composition according to any one of claims 1 to 4 and pharmaceutically acceptable excipients.

12. The pharmaceutical preparation according to claim 11, characterized in that The pharmaceutical preparation is an oral liquid, decoction, pill, capsule, tablet, powder or granule; The pharmaceutically acceptable excipients in the pharmaceutical preparation include one or more of diluents, fillers, disintegrants, lubricants, binders, flow agents, complex forming agents, plasticizers, colorants, sweeteners, viscosity enhancers, preservatives or antioxidants.

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