A drug for treating breast cancer with pilose antler and ginseng as the monarch drug, its preparation method and application

Abstract

The present invention discloses a drug for treating breast cancer with pilose antler and ginseng as the monarch drugs, and its preparation method and application. The drug uses pilose antler and ginseng as the monarch drugs, angelica sinensis and white peony root as the ministerial drugs, and is formulated with bupleurum root, scutellaria baicalensis, cyperus rotundus and fritillaria thunbergii. According to weight parts, the contents of each component in the aforementioned drug are: 12 parts of pilose antler, 3 parts of ginseng, 10 parts of angelica sinensis, 10 parts of white peony root, 5 parts of bupleurum root, 5 parts of scutellaria baicalensis, 10 parts of cyperus rotundus and 10 parts of fritillaria thunbergii. The preparation method is as follows: Step 1, soak pilose antler and ginseng separately; Step 2, add angelica sinensis, white peony root, bupleurum root, scutellaria baicalensis, cyperus rotundus and fritillaria thunbergii and add water to fully submerge them; Step 3, conduct decoction; Step 4, filter with gauze and combine the filtrates three times; Step 5, compress to a paste state, freeze-dry, and store in a sealed manner for later use. Beneficial effects: Reduce the types and frequencies of western medicine used, reduce the curative effect of metastasis, and can intervene in the occurrence and development of breast cancer by acting on cancer-related targets and related signaling pathways.

Description

Technical Field

[0001] The invention relates to a medicine, a preparation method and an application thereof, and in particular to a medicine for treating breast cancer with antler ginseng as a main drug, a preparation method and an application thereof. Background Art

[0002] At present, according to statistics from relevant organizations, breast cancer has surpassed lung cancer and become the world's largest cancer. At present, the mainstream treatment methods for breast cancer include chemotherapy, endocrine, targeted, immunotherapy and other treatment methods based on Western medicine. However, Western medicine has two problems: first, the side effects are large, patients are difficult to tolerate, and the compliance is poor, and the patient's own immune ability is reduced, thus affecting the treatment effect. Endocrine therapy is the treatment method with the mildest side effects in Western medicine, but only 60% of patients can complete the treatment, and most patients will relapse and metastasize due to interruption of treatment; second, the treatment mechanism of Western medicine is to kill the malignant cell subpopulations that proliferate vigorously or carry a certain target, which lacks systemicity. Because tumor cells have heterogeneity and high-frequency gene mutations in time and space, no matter which Western medicine is used, it will eventually lead to drug resistance, so there is an unsolvable bottleneck in the development of Western medicine. Traditional Chinese medicine treatment is an important supplement to breast cancer treatment, which has the effect of increasing efficacy and reducing toxicity, especially reducing the toxicity and side effects of perioperative and chemotherapy periods, and helping to improve the quality of life of patients during treatment. At present, the Chinese herbal prescriptions that have been proven effective by small-sample clinical evidence-based medicine and are used clinically include: Sanyin prescription after breast cancer surgery, Weici Sanjie prescription, Huaier granules, compound Tubeimu preparation, compound Banyan capsule, compound Hongshu prescription, etc. There are also certain problems: First, the treatment effect of Chinese herbal prescriptions applied independently is not significant. Their combined use with western medicine can increase the effect, but cannot reduce the duration and frequency of western medicine use; second, most of the prescriptions used in clinical practice are from classic prescriptions, that is, prescriptions recorded in ancient Chinese medicine books, which are added or subtracted according to syndrome differentiation and treatment, and innovative prescriptions are relatively rare. Summary of the invention

[0003] The main purpose of the present invention is to solve the problem of large side effects and drug resistance produced by existing western medicines for treating breast cancer during the treatment process;

[0004] Another purpose of the present invention is to solve the problem that the Chinese medicines currently used in the treatment of breast cancer are all from the prescriptions recorded in ancient Chinese medicine books, which leads to uncertain efficacy and relatively rare innovative prescriptions;

[0005] Another purpose of the present invention is to solve the problem that the Chinese medicine currently used in the treatment of breast cancer is combined with Western medicine, and the application dosage and frequency of Western medicine cannot be reduced.

[0006] The present invention provides a drug for treating breast cancer with pilose antler and ginseng as the monarch drugs, as well as a preparation method and application thereof, so as to achieve the above-mentioned purposes and solve the above-mentioned problems.

[0007] The drug for treating breast cancer provided by the present invention uses pilose antler and ginseng as the monarch drugs, angelica and white peony as the minister drugs, and is formulated with bupleurum, scutellaria, cyperus rotundus and fritillaria thunbergii. The content of each component in the above-mentioned drugs according to weight parts is: 12 parts of pilose antler, 3 parts of ginseng, 10 parts of angelica, 10 parts of white peony, 5 parts of bupleurum, 5 parts of scutellaria, 10 parts of cyperus rotundus and 10 parts of fritillaria thunbergii.

[0008] The preparation method of the drug for treating breast cancer with pilose antler and ginseng as the monarch drugs provided by the present invention includes the following steps:

[0009] Step 1: Take 12 parts of pilose antler and 3 parts of ginseng according to weight parts and soak them separately.

[0010] Step 2: Take 10 parts of angelica, 10 parts of white peony, 5 parts of bupleurum, 5 parts of scutellaria, 10 parts of cyperus rotundus and 10 parts of fritillaria thunbergii according to weight parts and add 8 times the total weight of the above components of water to fully submerge them.

[0011] Step 3: First decoct the soaked 12 parts of pilose antler in Step 1 for 0.5 hours, and then add the soaked 10 parts of angelica, 10 parts of white peony, 5 parts of bupleurum, 5 parts of scutellaria, 10 parts of cyperus rotundus and 10 parts of fritillaria thunbergii and the solution in Step 2 and continue to decoct.

[0012] Step 4: Decoct the soaked 3 parts of ginseng in Step 1 separately, and pour the aqueous solution of the decocted ginseng into the decocted medicinal decoction in Step 3; complete the decoction 3 times, 2 hours each time, and filter with gauze and combine the 3 filtrates.

[0013] Step 5: Use a rotary evaporator to reduce the pressure of the filtrate obtained in Step 4 to compress it into a paste, place the extract in a freeze dryer, pre-freeze at -20°C for 12 hours, sublime and dry at -80°C for 24 hours, then put it into a vacuum dryer for 48 hours. After drying, turn off the vacuum pump, collect the dried powder sample, and store it sealed at -20°C for later use.

[0014] The application of the drug (hereinafter referred to as pilose antler compound preparation) prepared with pilose antler and ginseng as the monarch drugs provided by the present invention in the treatment of breast cancer.

[0015] The mechanism of action of each component of the above-mentioned prepared drug is as follows:

[0016] Antlers can restore the normal function of the disordered sex hormone axis. The disorder of sex hormones is an important cause of breast cancer. On this basis, cell experiments and animal experiments have shown that antler discs can inhibit the proliferation and metastasis of breast cancer and have a certain preventive effect on the formation of hormone receptor-positive breast cancer. Mechanism research shows that antler discs exert anti-tumor effects through extracellular matrix TENASCIN and NF-κB.

[0017] Ginseng also has certain curative effects on cancer patients: Ginseng is a traditional Chinese medicine with a long history. Its active ingredient, ginsenoside, can inhibit the rapid proliferation of tumor cells to a certain extent, promote the apoptosis of tumor cells, restore the effect of chemotherapy drugs on tumor cells, inhibit tumor metastasis and invasion, and when used in combination with chemotherapy drugs, it can increase its efficacy and reduce side effects. Multiple extracts of ginseng block certain signal transduction pathways in cells. For example, ginsenoside CK has an obvious inhibitory effect on the proliferation of MCF-7 cells and epithelial-mesenchymal transition (EMT); ginsenoside Rh1 is an extract of protopanaxatriol-type ginsenoside, which can inhibit the ROS-mediated PI3K / AKT signal transduction pathway, promote the tumor cell cycle to arrest in the prophase, and promote cell apoptosis, showing certain anti-tumor activity; ginsenoside Rh4 induces cell apoptosis by reducing Bcl-2, increasing Bax, activating Caspase-9, Caspase-3 and PARP. Ginseng can change the sensitivity to chemotherapy drugs, promote the apoptosis of cancer cells, and thus control the progression of breast cancer.

[0018] The ministerial drugs Bupleurum and Scutellaria regulate the unfavorable pivot of Shaoyang. Shaoyang is a meridian in the half-exterior and half-interior, which is responsible for the opening and closing of the pivot and the communication between yin and yang. The qi movement of the human body, both inside and outside, between the surface and the interior, and among the zang-fu organs and up and down, all depends on the pivot rotation of the qi mechanism of Shaoyang. For diseases of the Shaoyang meridian, sweating, purging, and warming therapies cannot be used, and only the harmonizing therapy can be adopted. The combination of Bupleurum, Scutellaria, and Ginseng in the aforementioned drugs follows the meaning of Xiao Chaihu Decoction to harmonize Shaoyang. Bupleurum has a pungent taste, soothes the liver and relieves depression, and promotes the yang qi of Shaoyang; the gallbladder of Shaoyang belongs to wood, and damp-heat is likely to accumulate and stagnate in the gallbladder. Scutellaria is bitter and cold, clears heat and dries dampness, and removes the damp-heat in the gallbladder. Ginseng supplements the middle jiao and supports the healthy qi to prevent damage to the middle jiao. When the qi movement of Shaoyang, both on the surface and in the interior, is normal, the damp-heat will be cleared and removed. Combined with Bupleurum, it forms a prescription similar to Xiaoyao Powder to soothe the liver, relieve depression, nourish blood, and strengthen the spleen. Angelica is sweet, pungent, and warm, nourishes blood and harmonizes blood, and its pungent taste can disperse, so it is a qi medicine in blood. White Peony Root is sour, bitter, and slightly cold, nourishes blood and astringes yang, and soothes the liver and relieves spasm; the combination of Angelica, White Peony Root, and Bupleurum nourishes the liver body and helps the liver function, enabling the liver to be harmonious when the blood is harmonious and the liver to be nourished when the blood is sufficient. They are all ministerial drugs. Cyperus rotundus enters the liver meridian with a pungent taste and is a sacred medicine for gynecology, being good at soothing the liver, promoting qi movement, and relieving pain. Fritillaria thunbergii is cold and purgative, and can dissipate nodules. Clinically, it is often used for scrofula, goiter, sores, carbuncles, and lung abscesses, etc. The combination of these two drugs can purge heat and remove toxins, and relieve depression and dissipate nodules, treating scrofula and phlegm nodules. Among the drugs, the monarch drug antler is a product with vital essence of flesh and blood, which supplements the congenital essence, and is combined with ginseng to greatly tonify the vital qi and strengthen the spleen and stomach to assist the source of the generation of qi and blood in the acquired. Assisted by Bupleurum, Scutellaria, Angelica, White Peony Root, etc., it disperses the unfavorable pivot of Shaoyang and simultaneously soothes the liver and nourishes blood, and is combined with various drugs to remove stasis and dissipate nodules.

[0019] Advantages of the present invention:

[0020] The drug provided by the present invention, which uses pilose antler and ginseng as the monarch drug to treat breast cancer, can be clinically used in the prescriptions for the treatment of breast cancer. Some clinical case observations have been carried out, including 30 women with high-risk breast cancer factors and 11 breast cancer patients undergoing neoadjuvant therapy and salvage therapy. In combination with western medicine, good curative effects have been achieved. During the period when some patients stopped western medicine treatment due to drug resistance or side effects of western medicine, the compound pilose antler preparation effectively controlled the development of the tumor, and no adverse reactions occurred in all patients. Pilose antler has the effects of warming and tonifying the liver and kidneys, strengthening the muscles and bones, and promoting blood circulation to reduce swelling. Therefore, it can be determined that using pilose antler as the monarch drug to form a prescription for treating breast cancer has a definite curative effect. The ginsenosides in the drug of the present invention can significantly adjust the immune regulation function in the human body, change the direct drug resistance of tumor cells in the body to certain chemical drugs, reduce the chronic liver and kidney toxicity caused by some chemotherapy drugs, protect the human central nervous system, regulate the system function, resist fatigue, repair wounds, etc. Finally, animal experiments were carried out again for verification, and observations on clinical patients confirmed that it can reduce the types and frequencies of western medicine used, and has obvious effects of reducing the progression of the primary breast cancer focus and reducing metastasis. During the chemotherapy period, it can also effectively reduce the toxic and side effects of chemotherapy and inhibit the metastatic focus.

[0021] The main active ingredients of the pilose antler compound preparation provided by the present invention for treating triple-negative breast cancer can tightly bind to the corresponding target proteins and have good binding energy, thereby exerting a pharmacodynamic effect. In addition, the molecular docking results show that the docked targets are mainly closely related to cancer pathways, such as TP53, AKT1, SRC, IL6, ESR1, etc., mainly involving oncogenes, tumor suppressor genes, signal transduction, tumor immunity, chromosomal mutations, etc., further proving that the active ingredients of the pilose antler compound preparation can intervene in the occurrence and development of breast cancer by acting on cancer-related targets and related signal pathways.

[0022] Typical cases are as follows:

[0023] Typical case 1: A patient with advanced breast cancer with liver metastasis. After 3 days of treatment with the targeted drug abemaciclib and the endocrine drug fulvestrant, the liver function showed that the transaminase increased by 10 times. The targeted drug abemaciclib was stopped, and the pilose antler compound preparation provided by the present invention was used for treatment. After 2 months of evaluation, the liver metastasis focus was stable and did not progress, and the liver function returned to normal.

[0024] Typical Case 2: For breast cancer with bone metastasis treated by endocrine therapy and CDK4 / 6 targeted therapy, the breast lesions progressed twice. In the third cycle of chemotherapy using the TX regimen for the third-line treatment, the lesions progressed again. The compound pilose antler preparation provided by the present invention was used for three weeks of treatment. The color Doppler ultrasound showed that the lesions significantly shrank, from 9.2X20 mm to 27.9X18.1 mm.

[0025] Typical Case 3: For advanced breast cancer with bone and liver metastases, in the fourth course of TX chemotherapy for the first-line treatment, the target breast lesions progressed. The compound pilose antler preparation provided by the present invention was used for treatment. The color Doppler ultrasound showed that the target breast lesions significantly shrank, from 53.9X17.6 mm to 43.3X17.4 mm. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of the body weights of mice in each group in the experiment described in the present invention.

[0027] Figure 2 It is a schematic diagram of the external appearance of excised tumors of mice in each group described in the present invention.

[0028] Figure 3 It is a schematic diagram of the tumor growth curve and tumor volume of mice described in the present invention. Among them: A in the figure is the tumor growth curve of mice in each group, B in the figure is the average volume of excised tumors of mice in each group, *P<0.05, **P<0.01, ***P<0.001.

[0029] Figure 4 It is a schematic diagram of the tumor mass and tumor inhibition rate of mice in each group described in the present invention. Among them: A in the figure is the tumor mass of mice in each group, B in the figure is the tumor inhibition rate of each treatment group, *P<0.05, **P<0.01, ***P<0.001.

[0030] Figure 5 It is a schematic diagram of the "drug-active ingredient-target" interaction network described in the present invention.

[0031] Figure 6 It is a Venn diagram of the component targets and disease targets described in the present invention.

[0032] Figure 7 It is a schematic diagram of the PPI network topology analysis described in the present invention.

[0033] Figure 8Schematic diagram of GO functional enrichment of potential targets for treating triple-negative breast cancer with the antler compound preparation of the present invention. Among them: A in the figure: Biological Process (BP), B in the figure: Cellular Component (CC), C in the figure: Molecular Function (MF), and D in the figure: Bar chart of the expression of biological processes, cellular components, and molecular functions related to proteins in A, B, and C above.

[0034] Figure 9 Schematic diagram of KEGG enrichment of potential targets for treating breast cancer with the antler compound preparation of the present invention. In the figure: A: The top 20 enrichment pathways, B in the figure: KEGG top 20 enrichment bubble chart.

[0035] Figure 10 Thermal diagram of docking of the core targets and active compounds of the present invention.

[0036] Figure 11 Schematic diagram of the docking results of the present invention. In the figure: LYS: Lysine; ASP: Aspartic acid; GLU: Glutamic acid.

[0037] Figure 12 Schematic diagram of the antler ginseng formula of the present invention reducing the expression of oncogenes and increasing the expression of tumor suppressor genes. In the figure: A: Detection of the expression of oncogenes AKT1, SRC1, pro-inflammatory and cancer cytokines IL-6 and the expression of tumor suppressor gene TP53 and the gene ESR1 affecting breast cancer drug resistance by Western blot; B in the figure: Bar chart showing the mean and variance of the expression of each protein in different samples of each group, intuitively showing the expression trends of each protein in the control group, low-dose antler ginseng formula group, medium-dose antler ginseng formula group, and high-dose antler ginseng formula group. Detailed implementation method

[0038] Please refer to Figures 1 to 12 as shown in

[0039] The drug for treating breast cancer with antler and ginseng as the monarch drugs provided by the present invention is prepared with antler and ginseng as the monarch drugs, angelica and white peony as the ministerial drugs, and is formulated with bupleurum, scutellaria, cyperus rotundus and fritillaria thunbergii. According to weight parts, the content of each component in the above-mentioned drugs is: 12 parts of antler, 3 parts of ginseng, 10 parts of angelica, 10 parts of white peony, 5 parts of bupleurum, 5 parts of scutellaria, 10 parts of cyperus rotundus and 10 parts of fritillaria thunbergii.

[0040] The preparation method of the drug for treating breast cancer with antler and ginseng as the monarch drugs provided by the present invention includes the following steps:

[0041] Step 1: Weigh 12 parts of antler and 3 parts of ginseng according to weight parts and soak them separately.

[0042] Step 2: Take 10 parts by weight of Angelica sinensis, 10 parts of Paeonia lactiflora, 5 parts of Bupleurum chinense, 5 parts of Scutellaria baicalensis, 10 parts of Cyperus rotundus and 10 parts of Fritillaria thunbergii, and add 8 times the total weight of the above components of water to fully immerse them;

[0043] Step 3: First decoct the soaked antler (12 parts) in Step 1 for 0.5 hours, and then add the soaked Angelica sinensis (10 parts), Paeonia lactiflora (10 parts), Bupleurum chinense (5 parts), Scutellaria baicalensis (5 parts), Cyperus rotundus (10 parts) and Fritillaria thunbergii (10 parts) in Step 2 and the solution to continue decocting;

[0044] Step 4: Decoct the soaked ginseng (3 parts) in Step 1 separately, and add the aqueous solution of the decocted ginseng into the decocted medicinal decoction in Step 3; complete the decoction 3 times, each time for 2 hours, filter with gauze and combine the filtrates of the 3 times;

[0045] Step 5: Use a rotary evaporator to decompress and compress the filtrate obtained in Step 4 into a paste. Place the extract in a freeze dryer, pre-freeze at -20°C for 12 hours, sublimate and dry at -80°C for 24 hours, then put it into a vacuum dryer for 48 hours. After the drying is completed, turn off the vacuum pump, collect the dried powder sample, and store it sealed at -20°C for later use.

[0046] The application of the drug prepared with antler and ginseng as the monarch drugs provided by the present invention in the treatment of breast cancer.

[0047] The mechanism of action of each component of the above-prepared drug is as described below:

[0048] Antler can restore the normal disordered sex hormone axis. The disorder of sex hormones is an important cause of breast cancer. On this basis, cell experiments and animal experiments show that antler disk can inhibit the proliferation and metastasis of breast cancer and has a certain preventive effect on the formation of hormone receptor-positive breast cancer. Mechanism research shows that antler disk exerts anti-tumor effects through extracellular matrix TENASCIN and NF-KB.

[0049] Ginseng also has certain curative effects on cancer patients: Ginseng is a traditional Chinese medicine with a long history. Its active ingredient, ginsenoside, can, to a certain extent, inhibit the rapid proliferation of cancer cells, promote cancer cell apoptosis, restore the effect of chemotherapy drugs on cancer cells, inhibit cancer metastasis and invasion. When used in combination with chemotherapy drugs, it can increase the drug effect and reduce the toxic and side effects. Multiple extracts of ginseng can block certain signal transduction pathways in cells. For example, ginsenoside CK has an obvious inhibitory effect on the proliferation of MCF-7 cells and epithelial-mesenchymal transition (EMT); ginsenoside Rh1 is an extract of protopanaxatriol-type ginsenoside, which can inhibit the ROS-mediated PI3K / AKT signal transduction pathway, promote the cancer cell cycle to arrest in the prophase, and promote cell apoptosis, showing certain anti-tumor activity; ginsenoside Rh4 induces cell apoptosis by reducing Bcl-2, increasing Bax, activating Caspase-9, Caspase-3 and PARP, etc. Ginseng can change the sensitivity to chemotherapy drugs, promote cancer cell apoptosis, and thus control the progression of breast cancer.

[0050] The ministerial drugs Bupleurum and Scutellaria regulate the unfavorable pivot of Shaoyang. Shaoyang is a meridian in the half-exterior and half-interior region, which is responsible for the opening and closing of the pivot and the communication between yin and yang. The qi movement of the human body, including the interior and exterior, the zang-fu organs, and the upper and lower parts, all depends on the pivot rotation of the qi mechanism of Shaoyang. For diseases of the Shaoyang meridian, sweating, purging, and warming therapies cannot be used, and only the harmonizing therapy can be adopted. The combination of Bupleurum, Scutellaria, and Ginseng in the aforementioned drugs is based on the meaning of the Minor Bupleurum Decoction to harmonize Shaoyang. Bupleurum has a pungent taste, soothes the liver and relieves depression, and promotes the yang qi of Shaoyang; the gallbladder of Shaoyang belongs to wood, and damp-heat is prone to accumulate in the gallbladder. Scutellaria is bitter and cold, clears heat and dries dampness, and eliminates damp-heat in the gallbladder. Ginseng supplements the middle-jiao and strengthens the healthy qi to prevent damage to the middle-jiao. When the qi movement of Shaoyang in the interior and exterior is normal, damp-heat is cleared and reduced. Combined with Bupleurum, it forms a prescription similar to the Free and Easy Wanderer Powder, soothes the liver and relieves depression, nourishes blood and strengthens the spleen. Angelica is sweet, pungent, and warm, nourishes blood and harmonizes blood, and its pungent taste can disperse, so it is a qi drug in blood. White Peony Root is sour, bitter, and slightly cold, nourishes blood and astringes yang, and soothes the liver and relieves spasm; the combination of Angelica, White Peony Root and Bupleurum nourishes the liver body and aids the liver function, so that when the blood is harmonious, the liver is harmonious, and when the blood is sufficient, the liver is nourished. They are all ministerial drugs. Cyperus rotundus enters the liver meridian with a pungent taste and is a sacred drug for women's diseases. It is good at soothing the liver, promoting qi and relieving pain. Fritillaria thunbergii is cold and purgative, and can dissipate nodules. Clinically, it is often used for scrofula, goiter, sores, carbuncles, lung abscess, etc. The combination of the two drugs can purge heat and resolve toxins, and relieve depression and dissipate nodules, and treat scrofula and phlegm nodes. The monarch drug Cornu Cervi Pantotrichum in the drug is a product with vital essence of flesh and blood, which supplements the congenital essence, and is combined with Ginseng to greatly tonify the primordial qi and strengthen the spleen and stomach, so as to assist the source of qi and blood transformation of the acquired nature. It is assisted by Bupleurum, Scutellaria, Angelica, White Peony Root, etc. to disperse the unfavorable pivot of Shaoyang and at the same time soothe the liver and nourish blood, and combined with other drugs to dissipate stasis and nodules.

[0051] The specific experiments on the above-prepared drugs are as follows:

[0052] 1. Drug dose setting:

[0053] According to the corresponding pharmacopoeia, the adult dosage of the compound preparation of antler provided by the present invention is 65 g / d. Based on the average human body weight of 60 kg and the conversion coefficient between mice and humans being 0.0026, the clinically equivalent dosage of the compound preparation of antler for mice is 8.45 g / kg / d. The dosage obtained by the above method is the medium dosage, from which the low dosage is obtained as 4.23 g / kg / d and the high dosage is 16.90 g / kg / d.

[0054] 2. Establishment of 4T1 triple-negative mouse breast cancer model:

[0055] After culturing the required amount of 4T1 breast cancer cells, they were digested with trypsin, centrifuged, and resuspended in PBS. The cells were counted and diluted to a cell concentration of 1×10 7 cells / ml. All mice were intraperitoneally injected with pentobarbital sodium at 50 mg / kg and 0.5% lidocaine solution for infiltration anesthesia. Under anesthesia, 0.1 ml of the cell suspension was inoculated into the left third pair of mammary fat pads of each mouse. After the inoculation, the cotton swab was pressed for a moment to prevent the cells from flowing out. During the inoculation process, the cell suspension was pipetted to ensure uniformity.

[0056] 3. Grouping of the first batch of animal experiments:

[0057] BALB / c mice were randomly divided into 4 groups: PBS control group, low-dose group at 0.42 mg / ml, medium-dose group at 0.85 mg / ml, and high-dose group at 1.70 mg / ml, with 6 mice in each group. Starting from the second day after tumor inoculation, the mice were gavaged at different dosages of 0.2 ml / mouse and continuously administered for 21 days.

[0058] 4. Grouping of the second batch of animal experiments:

[0059] The experimental BALB / c mice were divided into 3 groups, with 10 mice in each group, namely PBS group, DTX group, and DTX + medium-dose (0.85 mg / kg) group.

[0060] Gavage started on the second day after inoculation. In the DTX group, 0.2 ml of DTX was intraperitoneally injected once a week. In the DTX + medium-dose group, DTX was intraperitoneally injected in the first and fourth weeks after inoculation and combined with gavage of the medium-dose antler formula. Measurement of tumor volume (L×W2×0.4) and mouse body weight started on the seventh day after inoculation. The total duration was 31 days.

[0061] The experimental results are as follows:

[0062] 1. Changes in body weight of mice in each group:

[0063] The changes in body weight of mice in each group before and after medication are as Figure 1As shown, there were no significant differences in the body weights of each group of mice before medication. After medication, the body weights of each group gradually increased over time. The low-concentration group of the antler compound preparation at 0.42 mg / kg had a relatively fast growth rate, while the high-concentration group of the antler compound preparation at 1.7 mg / kg had a relatively gentle increase in body weight. After 21 days of medication, there were no significant differences in the body weights among the control group, the low-concentration group, and the medium-concentration group.

[0064] 2. Appearance of the excised tumors of each group of mice:

[0065] After 21 days of intragastric administration to the tumor-bearing mice, the mice were anesthetized and euthanized. After blunt dissection of the tumor tissue, pictures were taken to record the appearance, as Figure 2 shown. Macroscopically, the tumor tissue of the control group was significantly larger than that of the medium- and high-concentration medication groups. The size of the tumors in the medication groups gradually decreased with the increase in the drug dose.

[0066] 3. Tumor growth curves and tumor volumes of each group of mice:

[0067] The tumor growth curves of each group of mice were as Figure 3 shown in Figure A. Starting from the 7th day after tumor inoculation, the tumor volumes of each group showed a continuous increasing trend. There were no significant differences in the growth rates between the Con group and the DACP-L group in the first 17 days. Starting from the 17th day, the tumor growth in the Con group increased significantly faster. The tumor growth rate of the DACP-M group was at a medium level, and the DACP-H group had the slowest growth rate. That is, the larger the medication dose, the smaller the increase in tumor volume, and the growth rate was less than that of the Con group.

[0068] After the mice were euthanized, the tumor volumes were bluntly separated as Figure 3 shown in Figure B. The tumor volumes of each drug treatment group were significantly smaller than those of the Con group (P<0.05). Among them, compared with the control group, the tumor volume of the DACP-L group was significantly smaller than that of the Con group (P<0.05), and the tumor volumes of the DACP-M group and the DACP-H group were extremely significantly smaller than that of the Con group (P<0.001). When comparing the tumor volumes among the medication groups, the tumor volume of the DACP-L group was significantly smaller than that of the DACP-M group (P<0.01), extremely significantly smaller than that of the DACP-H group (P<0.001), and the tumor volume of the DACP-M group was significantly smaller than that of the DACP-H group (P<0.05).

[0069] 4. Tumor masses and tumor inhibition rates of each group of mice:

[0070] The tumor masses of each group of mice are shown in Table 1 below and Figure 4As shown in Figure A, intragastric administration of Cervus elaphus compound preparations at different doses effectively reduced the tumor mass. The final tumor mass of the mice in the Con group was 1.2 ± 0.15 g, that of the mice in the DACP-L group was 1.04 ± 0.10 g, that of the mice in the DACP-M group was 0.69 ± 0.18 g, and that of the mice in the DACP-H group was 0.44 ± 0.14 g. Compared with the Con group, the tumor mass in the DACP-L group of the drug treatment group decreased, but the difference was not significant (P > 0.05), while the tumor mass in the DACP-M group and the DACP-H group decreased extremely significantly (P < 0.001). At the same time, when comparing the tumor mass among the treatment groups, the tumor mass in the DACP-M group and the DACP-H group decreased extremely significantly compared with the DACP-L group (P < 0.001), and the tumor mass in the DACP-H group decreased compared with the DACP-M group, but the difference was not significant (P > 0.05).

[0071] The tumor inhibition rates of each treatment group are shown in Table 1 and Figure 4 Figure B. Intragastric administration of Cervus elaphus compound preparations at different concentration doses could inhibit tumor growth, and with the increase of the drug dose, the tumor inhibition rates of the DACP-L, DACP-M, and DACP-H groups gradually increased, reaching 12.93%, 42.14%, and 63.14% respectively. The tumor inhibition rate of the DACP-H group was significant, reaching more than 50%.

[0072] Table 1 Effects of Cervus elaphus compound preparation on tumor growth in mice (x±s, n = 6)

[0073]

[0074]

[0075] 5. Effect of Cervus elaphus formula on reducing metastasis in tumor-bearing mice:

[0076] Multiple metastatic foci were visible in the lung HE sections of the PBS group, and the metastatic foci in the Cervus elaphus formula group were significantly lower than those in the control group.

[0077] 6. The Cervus elaphus recurrence preparation reduced the course of chemotherapy and the frequency of drug use, and its anti-tumor efficacy was superior to that of single-agent chemotherapy. In the PBS group, in the chemotherapy group, DTX (docetaxel once a week), in the chemotherapy + traditional Chinese medicine group (chemotherapy drugs once every two weeks combined with traditional Chinese medicine), the dose and frequency of chemotherapy drugs in the chemotherapy + traditional Chinese medicine group were reduced by half compared with the chemotherapy group.

[0078] The antler and ginseng formula can effectively increase the tumor weight of tumor-bearing mice, reduce the side effects of chemotherapy, and maintain the body weight of animals. The body weight of the experimental animals in the chemotherapy group decreased significantly. In the chemotherapy combined with traditional Chinese medicine treatment group, the body weight was higher than that in the chemotherapy group and the PBS group; for the tumor volume, DTX in the chemotherapy group and chemotherapy + traditional Chinese medicine were significantly lower than those in the PBS group (P < 0.05), and there was a tendency for the chemotherapy + traditional Chinese medicine group to be lower than the chemotherapy group; for the tumor weight, DTX in the chemotherapy group and chemotherapy + traditional Chinese medicine were significantly lower than those in the PBS group (P < 0.05), and there was a tendency for the chemotherapy + traditional Chinese medicine group to be lower than the chemotherapy group.

[0079] Explore the action targets of the compound preparation of antler in the treatment of breast cancer by network pharmacology, analysis docking technology and Western blot hybridization, as follows:

[0080] 1. Prediction of active ingredients and targets of the compound preparation of antler:

[0081] Screen the active ingredients of the compound preparation of antler and delete the active ingredients without predicted targets and duplicates, such as Figure 5 shown. A total of 96 active ingredients were obtained. At the same time, after predicting the target proteins of the active ingredients in the TCMSP database and deleting the duplicate values, 1776 human target proteins and 300 target genes were obtained.

[0082] 2. Targets of breast cancer represented by triple-negative:

[0083] 4113 disease gene targets related to triple-negative breast cancer were obtained through the database. According to the correlation score, the median was intercepted multiple times, and finally 650 disease gene targets were obtained. A Venn diagram was made with the 300 gene targets predicted by the active ingredients of the compound preparation of antler, and the intersection was taken to obtain 92 final intersection targets, which are the potential action targets of the compound preparation of antler on triple-negative breast cancer, as Figure 6 shown.

[0084] 3. Results of construction of PPI network and screening of core targets for 92 intersection targets:

[0085] The PPI network contains 92 nodes and 354 edges. One free node was deleted, indicating that there were 91 targets in this network that produced 354 interaction relationships in total. Visual analysis was performed by Cytoscape software, and the topological parameters obtained were: BC > 150.901, CC > 0.004, DC > 15.560. Among them, 19 core targets were screened out by meeting the three numerical values.

[0086] 4. GO enrichment analysis and KEGG pathway annotation analysis:

[0087] Perform GO functional enrichment analysis on the roles of 92 targets related to the treatment of triple-negative breast cancer by the compound preparation of antler in gene functions, as Figure 11As shown, there are 530 BP-related entries, mainly involving positive regulation of gene expression, negative regulation of apoptotic process, signal transduction, positive regulation of cell proliferation, protein autophosphorylation, negative regulation of gene expression, etc.; 120 MF-related entries, mainly involving protein binding, enzyme binding, DNA binding, protein kinase binding, ubiquitin-protein ligase binding, protein serine / threonine / tyrosine kinase activity transcription factor activity, sequence-specific DNA binding, etc.; 56 CC-related entries, mainly involving cell nucleus, cytoplasm, nucleoplasm, macromolecular complex, mitochondrion, extracellular exosome, chromatin, etc.

[0088] The KEGG pathway enrichment function was used to explore the roles of 92 potential gene targets of the antler compound preparation in the treatment of triple-negative breast cancer in the signal pathway. The enrichment results showed that there were 160 related pathways. The top 20 enriched pathways were selected according to the FDR value and P value, as Figure 11 shown, and the most prominent pathway among them was the cancer pathway.

[0089] 5. Molecular docking results:

[0090] According to the degree value, the top 10 main active pharmaceutical ingredients were selected, and the molecular docking verification results were carried out with the top 5 targets among the 19 core targets. The docking results were evaluated by the docking binding energy, as shown in Table 2. A heat map of the docking scores between the core targets and the active compounds was drawn, as shown in Figure 11 . The top 6 with good docking results were visualized, and all of them had good active pocket structures, as Figure 11 shown. Through the visualization analysis of the molecular docking results, it can be seen that different active ingredient ligands and the target protein receptors are well combined through hydrogen bonds. There are 47 that meet the docking binding energy ≤ -5 kJ / mol, indicating that the main active ingredients of the antler compound preparation for the treatment of triple-negative breast cancer can tightly bind to the corresponding target proteins and have good binding energy, so as to exert the pharmacological effects. In addition, the molecular docking results show that the docked targets are mainly closely related to the cancer pathway, such as TP53, AKT1, SRC, IL6, ESR1, etc., mainly involving oncogenes, tumor suppressor genes, signal transduction, tumor immunity, chromosomal mutations, etc., further proving that the active ingredients of the antler compound preparation can intervene in the occurrence and development of triple-negative breast cancer by acting on cancer-related targets and related signal pathways.

[0091] Table 2 Molecular docking results (unit: kJ / mol)

[0092]

[0093] 6. Molecular verification by Western blot hybridization: The antler formula inhibits breast cancer by inhibiting oncogenes or pro-cancer factors AKT, IL-6, SRC, and at the same time inhibits breast cancer by enhancing the expression of the tumor suppressor gene P53, specifically asFigure 11 and Figure 12 as shown

Claims

1. A drug for treating breast cancer with pilose antler and ginseng as the monarch drug, characterized in that: It is made with pilose antler and ginseng as the monarch drugs, angelica sinensis and white peony root as the minister drugs, and is formulated with bupleurum, scutellaria baicalensis, cyperus rotundus and thunberg fritillary bulb. According to weight parts, the content of each component in the aforementioned drugs is: 12 parts of pilose antler, 3 parts of ginseng, 10 parts of angelica sinensis, 10 parts of white peony root, 5 parts of bupleurum, 5 parts of scutellaria baicalensis, 10 parts of cyperus rotundus and 10 parts of thunberg fritillary bulb.

2. A preparation method of a drug for treating breast cancer with pilose antler and ginseng as the monarch drug, characterized in that: The method it includes is as follows: Step 1: Take 12 parts of pilose antler and 3 parts of ginseng by weight and soak them separately. Step 2: Take 10 parts of angelica sinensis, 10 parts of white peony root, 5 parts of bupleurum, 5 parts of scutellaria baicalensis, 10 parts of cyperus rotundus and 10 parts of thunberg fritillary bulb by weight and add 8 times the total weight of the aforementioned components of water to fully submerge them. Step 3: Decoct the 12 parts of pilose antler soaked in Step 1 for 0.5 hour first, and then add the 10 parts of angelica sinensis, 10 parts of white peony root, 5 parts of bupleurum, 5 parts of scutellaria baicalensis, 10 parts of cyperus rotundus and 10 parts of thunberg fritillary bulb soaked in Step 2 and the solution to continue decocting. Step 4: Decoct the 3 parts of ginseng soaked in Step 1 separately, and add the aqueous solution of ginseng after decoction to the decocted medicinal decoction in Step 3; complete decocting 3 times, 2 hours each time, filter with gauze and combine the 3 filtrates. Step 5: Use a rotary evaporator to reduce the pressure of the filtrate obtained in Step 4 to compress it into a paste, place the extract in a freeze dryer, pre-freeze at -20°C for 12 hours, sublime and dry at -80°C for 24 hours, then put it into a vacuum dryer for 48 hours. After drying is completed, turn off the vacuum pump, collect the dried powder sample, and store it sealed at -20°C for later use.

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