Zhuang medicine composition for treating breast cancer as well as preparation method and application of Zhuang medicine composition

Abstract

The invention relates to the technical field of traditional Chinese medicine, in particular to a Zhuang medicine composition for treating breast cancer and a preparation method and application thereof. The oldenlandia diffusa and the corydalis saxicola are used as monarch drugs, have the effects of clearing away heat and toxic materials, clearing damp and resisting inflammation, and diminishing swelling and removing stasis, and are common drugs for treating cancers by Zhuang medicine; radix ranunculi ternati is used as a ministerial drug for reducing phlegm and The beautiful millettia root is used as an assistant to tonify qi, strengthen body resistance, activate blood The liquorice can harmonize all the medicines and is often a conductant medicine. Generally, the whole prescription has the effects of clearing damp and strengthening qi, clearing away heat and toxic materials, reducing phlegm and resolving masses, and for breast cancer patients, the first method is to detoxify and strengthen qi, and the effects of softening hardness to dissipate stagnation, clearing heat and expelling damp, reducing phlegm and diminishing swelling are also to eliminate evil. The traditional Chinese medicine composition can obviously inhibit the growth of breast cancer tumor tissues, regulate the content of cytokines in serum and improve the immune function, so that the effect of treating breast cancer can be achieved, and the traditional Chinese medicine composition has good practical application value.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and specifically to a Zhuang medicine composition for treating breast cancer, its preparation method and application. Background Art

[0002] Breast cancer is one of the most common malignant tumors among women globally. In recent years, its incidence has been continuously rising and showing a trend of younger age. It has become the malignant tumor with the highest incidence, and the mortality rate ranks fifth. Currently, there are many clinical treatment methods for breast cancer, such as surgical treatment, endocrine therapy, chemotherapy, and radiotherapy, among which chemotherapy and radiotherapy are often the main methods. However, while existing chemotherapy drugs play an anti-tumor role, they cause varying degrees of damage to the body's immune system. Long-term use of chemotherapy drugs leads to a decrease in the body's sensitivity, and further increases the risk of recurrence and metastasis, thus seriously threatening the quality of life and life of patients. Therefore, it is extremely urgent and important to find innovative adjuvant anti-tumor drugs that are safe, effective, and have low toxicity and side effects.

[0003] "Zhuang Medicine Formulary" states that the asynchronous movement of the three qi, blocked channels, toxin deficiency, and imbalance of yin-yang and qi-blood are all key pathogenic factors. Zhuang medicine believes that the occurrence and development of tumors are caused by the combination of various toxins such as cold, heat, phlegm, and stasis. Therefore, detoxification, tonifying deficiency, and regulating qi are the three major principles in the clinical treatment of tumors by Zhuang medicine. Removing dampness and strengthening the vital qi, tonifying deficiency and weakness, can inhibit the occurrence and development of tumors and improve the clinical symptoms of patients by enhancing the body's immune function and the ability to resist and expel pathogenic factors.

[0004] Therefore, finding a Zhuang medicine composition for treating breast cancer that conforms to the principles of Zhuang medicine, is safe, effective, and has low toxicity and side effects is the current key research direction. Summary of the Invention

[0005] In order to solve the above technical problems existing in the prior art, the present invention provides a Zhuang medicine composition for treating breast cancer, its preparation method and application. Specifically, it is achieved through the following technical solutions:

[0006] A Zhuang medicine composition for treating breast cancer, comprising the following raw materials: Oldenlandia diffusa, Ranunculus ternatus Thunb., Corydalis saxicola Bunting, Millettia speciosa Champ., and Glycyrrhiza uralensis Fisch. Further, the weight parts of each raw material are: Oldenlandia diffusa 5 - 30 parts, Corydalis saxicola Bunting 5 - 30 parts, Ranunculus ternatus Thunb. 3 - 15 parts, Millettia speciosa Champ. 3 - 15 parts, and Glycyrrhiza uralensis Fisch. 1 - 10 parts. Further, the weight ratio of each raw material is: Oldenlandia diffusa : Corydalis saxicola Bunting : Ranunculus ternatus Thunb. : Millettia speciosa Champ. : Glycyrrhiza uralensis Fisch. = 15 : 10 : 5 : 5 : 1.

[0007] The application of the above-mentioned Zhuang medicine composition in the aspect of being used as or preparing an anti-cancer drug. Especially a drug for treating breast cancer.

[0008] The above-mentioned Zhuang medicine composition can be prepared into granule, decoction or other dosage forms.

[0009] The decoction can be prepared as follows: Weigh Oldenlandia diffusa, Corydalis saxicola, Ranunculus ternatus, Millettia speciosa Champ., and Glycyrrhiza uralensis Fisch. according to the proportion. First, soak them in 10 times the amount of pure water for 30 minutes, reflux and decoct under normal pressure for 1.5 hours, and filter while it is hot through a 200-mesh gauze. For the second decoction, reflux and extract with 8 times the amount of pure water for 1 hour, then collect the filtrate. Combine the filtrates obtained twice and concentrate them to 2.0 g / mL in a rotary evaporator under reduced pressure, and that's it. The parameters for concentration in the rotary evaporator under reduced pressure are 65 °C and -0.095 Mpa. The 2.0 g / mL specifically refers to containing 2.0 g / mL of crude drug content.

[0010] Zhuang medicine classifies tumors into the category of "poison diseases". Its theoretical basis mainly includes the view of nature and man of "synchronization of three qi, with yin and yang as the foundation", the view of body functions of "three channels and two paths, internal organs, qi and blood, bones and muscles", the etiology and pathogenesis theory of "poison and deficiency causing diseases", and the fundamental treatment principles of "regulating qi, detoxifying, and tonifying deficiency". The "three channels" respectively refer to the airway, the digestive tract, and the waterway, and the "two paths" refer to the dragon path and the fire path. If the "three channels" and "two paths" are blocked or the regulation is imbalanced, it will cause the imbalance of qi and blood in the human body, and the normal regulatory functions of the zang-fu organs will be lost. Poison of insects, miasma poison will stagnate and accumulate, and cancer poison will accumulate in the body for a long time, ultimately leading to the occurrence and development of tumors. Among them, "poison diseases" is the most characteristic content of Zhuang medicine. It is believed that "poisonous pathogen" is the fundamental pathogenesis of the occurrence of malignant solid tumors. "Detoxifying" as the first major method of treating diseases in Zhuang medicine plays an important role in the prevention and treatment of tumors. By correcting the "root cause" of the disease, that is, "imbalance of three qi and weakness of vital qi" from "detoxifying", the occurrence and development of malignant solid tumors can be effectively controlled.

[0011] Zhuang medicine regards cancer as a special "poison disease". "Poison" and "deficiency" are the most important and fundamental causes of tumor occurrence. Therefore, Zhuang medicine emphasizes that "poison and deficiency lead to all diseases", "poison and deficiency lead to cancer", and "the most deficient place is where poison accumulates". When the human body is in a state of deficiency, the "poison" pathogen will take advantage of the deficiency to invade. The theory of poison has become one of the core theories of Zhuang medicine. Zhuang medicine has always attached importance to the basic roles of detoxification, vital energy, and tonifying deficiency in diseases. It is believed that by detoxifying and expelling toxins through the "three channels" and "two routes", dredging the upper airway to make it unobstructed, relieving the middle digestive tract to make it harmonious and smooth, promoting the lower urinary tract to make it unobstructed, and dredging the two routes to enable the poison pathogen to be overcome and discharged out of the body, achieving the effect of removing poison and restoring peace, and recovering from cancer with the restoration of vital energy. It emphasizes the importance of detoxification and vital energy in tumor treatment. It has been confirmed by research that there is a close relationship between the body's immune function and the occurrence, development, recurrence, and metastasis of tumors. Chemotherapy, as the main current treatment method for malignant tumors, is extremely likely to cause immune function suppression, resulting in poor treatment effects and prognosis, and seriously affecting the quality of life of patients. Zhuang medicine classifies the common side effects of chemotherapy clinically as the prevalence of poison pathogen, weakness of vital energy, and symptoms of dampness stagnation and internal heat. Chemotherapy drugs cause qi deficiency and yin deficiency in the body, removing pathogenic factors and stagnating heat, affecting the curative effect and recovery. Therefore, while using Western medical techniques to treat tumors, it is an important link in tumor treatment to supplement with traditional Chinese medicine and ethnic medicine to clear heat and detoxify, strengthen qi and remove dampness, and regulate yin and yang to enhance the body's immune level. Plant drugs participating in tumor immunotherapy often do not directly kill tumor cells, but play a role in enhancing immune function by activating the cellular and humoral immune responses of the body and acting as a physiological and biochemical regulator of the body, thereby improving the body's state and inhibiting tumors. The spleen, as one of the most important immune organs of the body, is the location where immune cells such as T lymphocytes and B lymphocytes survive, and also serves as the main organ for generating immune responses and immune active substances, reflecting to a certain extent the level of immune function.

[0012] Specific immune responses mediated by T lymphocytes and B lymphocytes are the core of tumor immunity, and their dynamic balance can ensure the stability of the body's immune system. IL-2 is at the central link in the body's immune regulation system. It can not only stimulate the proliferation and differentiation of T cells, activate lymphocytes to inhibit the activity of tumor cells, but also promote the secretion of other cytokines such as IL-6, IFN-γ, TNF-α, etc. As one of the most important cytokines regulating the body's immune level, IL-6 has the effect of activating T and B lymphocytes and regulates the proliferation, differentiation, migration and adhesion of tumor cells. TNF-α is the cytokine with the strongest inhibitory effect on tumor activity and can directly induce apoptosis of tumor cells. IFN-γ has a powerful and direct inhibitory effect on the occurrence and growth of tumors and can enhance the antitumor effect of TNF. The composition of the present application can significantly increase the spleen index, improve the proliferation ability of T and B lymphocytes in the spleen, regulate the abnormal white blood cell level in the peripheral blood of nude mice with transplanted tumors, increase the proportion of lymphocytes in the peripheral blood, and increase the levels of cytokines such as IL-2, IL-6, IFN-γ, TNF-α in the serum. It can improve the disordered immune system of nude mice with transplanted tumors and enhance the immune function.

[0013] Compared with the prior art, the technical effects of the present invention are reflected in:

[0014] (1) According to "Fangjixue of Zhuang Medicine", the asynchronous movement of the three qi, the blocked channels, toxin deficiency, and the imbalance of yin and yang and qi and blood are all key pathogenic factors. Zhuang medicine believes that the occurrence and development of tumors are caused by the combination of various toxins such as cold, heat, phlegm, and stasis. Therefore, detoxification, tonifying deficiency, and regulating qi are the three major principles for the clinical treatment of tumors in Zhuang medicine. Removing dampness and strengthening vital qi, tonifying deficiency and weakness, can inhibit the occurrence and development of tumors and improve the clinical symptoms of patients by strengthening the body's immune function and enhancing the ability to resist and eliminate pathogenic factors. Using Oldenlandia diffusa and Picrorhiza scrophulariiflora as the monarch drugs, both have the effects of clearing heat and detoxifying, removing dampness and anti-inflammatory, and dissipating swelling and nodules, and are commonly used drugs for treating cancer in Zhuang medicine; using Ranunculus ternatus as the minister drug to dissipate phlegm and nodules; using Millettia speciosa as the assistant drug to replenish qi and strengthen the healthy qi, activate blood circulation and nourish yin; licorice can harmonize all the drugs and is often used as the guiding drug. Looking at the whole formula, its function lies in removing dampness and strengthening vital qi, clearing heat and detoxifying, and dissipating phlegm and nodules. For breast cancer patients, detoxifying and strengthening vital qi should be the first major method, and in addition, softening and resolving hard masses, clearing heat and removing dampness, and dissipating phlegm and swelling should be used to dispel pathogenic factors.

[0015] (2) The present invention treats animal breast cancer with Zhuang medicine theory, that is, softening and resolving hard masses, clearing heat and detoxifying to inhibit cancer; tonifying qi and strengthening the healthy qi as the foundation, removing dampness and promoting dredging as the application. It has been proven by experiments that it can significantly inhibit the growth of breast cancer tumor tissues, regulate the content of cytokines in the serum, and at the same time improve the immune function of animals, so as to achieve the effect of treating breast cancer. Therefore, it has good practical application value.

[0016] (3) The composition of the present invention can significantly inhibit the growth of MDA-MB-231 breast cancer xenografts in nude mice, induce apoptosis of human breast cancer MDA-MB-231 cells, and enhance the immune function of xenografts in nude mice. The mechanism may be related to downregulating the PI3K / Akt / mTOR pathway and the apoptosis-related protein Bcl-2, and upregulating the expression levels of the pro-apoptotic proteins Bax, Caspase-9, and Caspase-7. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of the combination on the growth of MDA-MB-231 transplanted tumor in nude mice (±s, n=6).

[0018] Figure 2 The effect of the combination on the pathological changes of tumor tissue in nude mice with MDA-MB-231 transplanted tumor (HE, ×400).

[0019] Figure 3 The effect of the combination on the number of leukocytes and the proportion of lymphocytes in the peripheral blood of nude mice with MDA-MB-231 transplanted tumors (±s, n=6). DETAILED DESCRIPTION

[0020] The technical solution of the present invention is further defined below in conjunction with specific implementation methods, but the scope of protection required is not limited to the description.

[0021] Example 1

[0022] Weigh 15g of Hedyotis diffusa, 10g of Coptis chinensis, 5g of Herba Lycopodii, 5g of Herba Cynanchifoliae, and 1g of Radix Glycyrrhizae. Add 10 times the amount of pure water to the mixture and soak for 30 minutes. Reflux and decoct at normal pressure for 1.5 hours, then filter through 200-mesh gauze while hot. Then, decoct the mixture again with 8 times the amount of pure water and reflux for 1 hour. Collect the filtrate, combine the two filtrates, and concentrate them to 2.0g / mL (containing 2.0g / mL of crude drug) in a vacuum rotary evaporator (65°C, -0.095 MPa). Store in a -20°C refrigerator.

[0023] Example 2

[0024] Weigh 30g of Hedyotis diffusa, 30g of Coptis chinensis, 3g of Herba Lycopodii, 3g of Herba Cynanchifoliae, 3g of Herba Cynanchi, and 1g of Radix Glycyrrhizae. Add 10 times the amount of pure water to the mixture and soak for 30 minutes. Reflux and decoct at normal pressure for 1.5 hours, then filter through 200-mesh gauze while hot. Then, decoct the mixture a second time with 8 times the amount of pure water and reflux for 1 hour. Collect the filtrate, combine the two filtrates, and concentrate them to 2.0g / mL (containing 2.0g / mL of crude drug) in a vacuum rotary evaporator (65°C, -0.095 MPa). Store in a -20°C refrigerator.

[0025] Example 3

[0026] Weigh 5 g of *Hedyotis diffusa*, 5 g of *Corydalis saxicola*, 15 g of *Ranunculus ternatus*, 15 g of *Millettia speciosa*, and 10 g of licorice root. First, soak them in 10 times the amount of pure water for 30 min, reflux and decoct under normal pressure for 1.5 h, and filter while it is hot through a 200-mesh gauze. For the second decoction, reflux and extract with 8 times the amount of pure water for 1 h, then collect the filtrate. Combine the filtrates obtained twice and concentrate them in a rotary evaporator under reduced pressure (65 °C, -0.095 Mpa) to 2.0 g / mL (containing 2.0 g / mL of crude drug content), and store them in a refrigerator at -20 °C.

[0027] Study on the anti-breast cancer cell MDA-MB-231 xenograft tumor in nude mice by the Zhuang medicine composition

[0028] 1 Materials and instruments

[0029] 1.1 Cell line

[0030] Human breast cancer MDA-MB-231 cells were purchased from Wuhan Punosai Life Science Co., Ltd., with the number H9XX570KO8, and they were the second-generation cells. They were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibiotics, and placed in a cell incubator at 37 °C and 5% CO₂. When the cells grew to 90% of the bottom of the flask, they were passaged once.

[0031] 1.2 Animals

[0032] SPF-grade BALB / c-nu female nude mice, 7 - 8 weeks old, with a body weight of 20 - 22 g, were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd., and the use license number was SCXK (Guangdong) 2022 - 0063. The animal experiments were completed in an SPF-grade experimental animal room, at a room temperature of 23 - 27 °C, a relative humidity of 50 - 60%, and under a natural day-night rhythm light environment, with free access to food and water. After 1 week of adaptive feeding, the experiments were started.

[0033] 1.3 Medicinal materials

[0034] *Hedyotis diffusa* (Guangxi Xianzhu Traditional Chinese Medicine Technology Co., Ltd., batch number: 20221101); *Corydalis saxicola* medicinal materials (Guangxi Xianzhu Traditional Chinese Medicine Technology Co., Ltd., batch number 20190701); *Millettia speciosa* medicinal materials (Guangxi Xianzhu Traditional Chinese Medicine Technology Co., Ltd., batch number 20201101); *Ranunculus ternatus* (Guangxi Liuzhou Baicaotang Chinese Herbal Pieces Factory Co., Ltd., batch number 20200710), and licorice root (Anhui Jiayou Chinese Herbal Pieces Co., Ltd., batch number 210102).

[0035] 1.4 Drugs and reagents

[0036] Cyclophosphamide for injection (batch number 2C534A) was purchased from Baxter International Inc., USA. DMEM medium (batch number 20221208), phosphate buffer solution (batch number 20220110) were purchased from Jiangsu KeyGen Biotech Co., Ltd.; Trypsin digestion solution (batch number 20221223), penicillin-streptomycin mixture (batch number 20210929), hematoxylin-eosin (HE) staining kit (batch number 20221217), concanavalin A (ConA, batch number 512C0310), lipopolysaccharide (LPS, batch number 820F038) were provided by Beijing Solarbio Science & Technology Co., Ltd.; Fetal bovine serum (PAN Company, Germany, batch number ST200707); Interleukin-2 (IL-2) ELISA kit (batch number 20220310), interleukin-6 (IL-6) ELISA kit (batch number 20220310), interferon-γ (IFN-γ) ELISA kit (batch number 20220510), tumor necrosis factor-α (TNF-α) ELISA kit (batch number 20220410) were purchased from Wuhan Beinlai Biotechnology Co., Ltd.

[0037] 1.5 Main instruments

[0038] XE-2100 hematology analyzer (Sysmex, Japan); SW-CJ-2FD type ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd.); JY2002 electronic balance (Shanghai Hengping Scientific Instrument Co., Ltd.); TGD-20MC high-speed and low-temperature centrifuge (Xiangrui, Changsha, China); Multiskan Skyhigh enzyme-linked immunosorbent detector (Thermo Fisher Scientific, USA); MI52-N inverted fluorescence microscope (Guangzhou Mingmei Optoelectronic Technology Co., Ltd.); TD5A tabletop centrifuge (Huxi Instrument Equipment Co., Ltd., Hunan); 4590 embedding machine (SAKURA Precision Technology Co., Ltd., Japan); RM2235 pathological tissue slicer (Leica Microsystems GmbH, Germany); IMS-130 granular ice maker (Changshu Xueke Refrigeration Equipment Co., Ltd.); HH-8 type electrothermal constant temperature water bath (Shanghai Hanno Instrument Co., Ltd.); CLM-170B-8-CN carbon dioxide incubator (ESCO, Singapore); GWB-1 ultrapure water purifier (Beijing Purkinje General Instrument Co., Ltd.).

[0039] 2 Methods

[0040] 2.1 Preparation of combined water extract

[0041] Weigh appropriate amounts of Hedyotis diffusa, Coptis chinensis, Herba Lycopodii, Herba Cynanchifoliae, and Radix Glycyrrhizae according to the ratio (15:10:5:5:1), add 10 times the amount of pure water for the first time, soak for 30 minutes, reflux and decoct for 1.5 hours under normal pressure, and filter through 200-mesh gauze while hot; decoct for the second time with 8 times the amount of pure water, reflux and extract for 1 hour, collect the filtrate, combine the two filtrates, place them in a vacuum rotary evaporator (65°C, -0.095 MPa), concentrate to 2.0 g / mL (containing 2.0 g / mL of crude drug), and store in a refrigerator at -20°C.

[0042] 2.2 Establishment of breast cancer xenograft model in nude mice

[0043] Human breast cancer MDA-MB-231 cells in logarithmic growth phase were suspended in normal saline and the cell density was adjusted to 3.0×10 7 Pipette 0.2mL of MDA-MB-231 cell suspension and slowly inject it into the nude mouse's armpit. After the inoculation is completed, gently press it with a sterile cotton swab for a while to establish a human breast cancer nude mouse xenograft tumor model. Weigh the nude mouse with tumors every 3 days after inoculation and measure the size of the tumor. When the tumor volume reaches 100mm 3 When the number of cells was 0.1, it indicated that the MDA-MB-231 breast cancer tumor-bearing mouse model was successfully established and relevant experiments could be performed. Six normal mice were randomly selected and injected with 0.2 mL of normal saline at the corresponding sites as the normal control group.

[0044] 2.3 Grouping and Dosing

[0045] After the MDA-MB-23 breast cancer transplanted tumor mouse model was successfully established, they were randomly divided into the model group, the cyclophosphamide (CTX, 25 mg·kg -1 ·d -1 ) group and combination low and high dose (2, 8g·kg -1 ) groups, 6 mice per group. The dosage conversion method for mice was based on the human and animal dosage formulas in the "Pharmacological Experimental Methodology" and was verified through repeated preliminary experiments. Each dose group of the Zhuang medicine combination was orally administered with the corresponding Zhuang medicine combination aqueous extract. The normal and model groups were orally administered with an equal volume of sterile water for injection once daily. The CTX group was intraperitoneally injected with the drug once a week for 21 consecutive days. During the dosing period, the mice were observed for general indicators such as body weight, mental state, hair, and behavior.

[0046] 2.4 Detection of spleen index, tumor growth volume, and tumor inhibition rate

[0047] Starting from the first day of administration, the long diameter (L) and short diameter (d) of the tumor of the tumor-bearing mice were measured with a vernier caliper every 3 days, and the tumor volume was recorded and calculated and the tumor growth curve was drawn. Tumor volume V = (L × d 2) / 2. After 24 hours of the last administration, the nude mice with breast cancer were injected with phenobarbital for euthanasia. The tumors and spleens were dissected, blotted dry with filter paper, and weighed. The tumor inhibition rate and spleen index were calculated respectively. Tumor inhibition rate = (mean tumor weight of the model group - mean tumor weight of the experimental group) / mean tumor weight of the model group × 100%. Spleen index = spleen weight (mg) / body weight (g).

[0048] 2.5 Hematoxylin-eosin (HE) staining to observe the morphological changes of breast cancer tumor tissue cells

[0049] To observe the pathological changes of tumor tissue, the tumor tissues of mice in each group were placed in 4% paraformaldehyde solution for fixation for 48 hours, followed by tissue dehydration, paraffin embedding, 3-μm sectioning, HE staining, ethanol dehydration, xylene clearing, and mounting. Finally, the morphological changes of tumor tissue cells were observed under an optical microscope.

[0050] 2.6 Detection of the number of white blood cells and the proportion of lymphocytes in peripheral blood by hematology analyzer

[0051] After 21 days of administration, blood was collected from the fundus venous plexus of mice using an EDTA anticoagulant tube, and the number of white blood cells and the proportion of lymphocytes in the peripheral blood of mice were detected and analyzed using an automatic hematology analyzer.

[0052] 2.7 Detection of cytokine levels in serum by ELISA

[0053] After 24 hours of the last administration, blood was collected from the fundus venous plexus of mice and allowed to stand for 1 hour, then centrifuged at 3500 r·min -1 for 15 minutes to separate the serum. The levels of IL-2, IL-6, TNF-α, and IFN-γ in the serum of mice were measured respectively according to the instructions of the ELISA kit.

[0054] 2.8 Detection of the proliferative ability of spleen lymphocytes

[0055] The spleens of mice were dissected in a laminar flow hood. An appropriate amount of spleen tissue was passed through a 200-mesh nylon mesh, added with erythrocyte lysate, lysed at room temperature for 20 minutes, centrifuged at 1200 r / min for 5 minutes, and the cell layer was washed 3 times with RPMI1640 complete medium. After resuspending the cells, the density of spleen cells was adjusted to 6×10 6 cells / mL. The ConA stimulation group, LPS stimulation group, and blank control group were set up, and RPMI 1640 complete medium containing ConA (10 μg·mL -1 ), RPMI 1640 complete medium containing LPS (20 μg·mL -1The complete RPMI 1640 medium and 100 μL of the complete RPMI 1640 medium were used, with each group repeated 3 times. After culturing in an incubator at 37 °C and 5% CO2 for 48 h, 10 μL of CCK-8 solution was added to each well and incubated for 1 h. The absorbance value (A) was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. The proliferation ability of nude mouse lymphocytes was represented by the stimulation index (SI), and SI = A 实验 / A 空白 .

[0056] 2.10 Data statistics and analysis

[0057] Data statistical analysis was performed using SPSS 20.0 software. All data were subjected to normality test and homogeneity of variance test. The experimental data were expressed as x ± s, and the results were analyzed by one-way analysis of variance. ANOVA was used for comparison among multiple groups, and differences were considered statistically significant when P < 0.05

[0058] 3. Results

[0059] 3.1 Effects of the composition on the growth status, body weight, liver and spleen indices, and transplanted tumors of nude mice with transplanted tumors

[0060] Compared with the normal group, the nude mice in the model group were listless, had dull reactions, and their hair was dry, yellowish, and fluffy. After drug intervention, each administration group showed varying degrees of improvement. There were no significant differences in the body weight changes of nude mice in the normal group, model group, CTX group, and each dose group of the composition (P > 0.05); compared with the normal group, the spleen index of nude mice in the model group was significantly increased (P < 0.01); compared with the model group, the spleen index of nude mice in the CTX group was significantly decreased (P < 0.01), while the spleen indices of the high- and low-dose groups of the composition were significantly increased (P < 0.01); compared with the CTX group, the spleen indices of mice in each dose group of the composition were significantly increased (P < 0.01); the tumor weights of nude mice in the CTX group and the high- and low-dose groups of the composition were inhibited to varying degrees (P < 0.01), and the inhibitory effect of the high-dose group of the composition was equivalent to that of CTX (P > 0.05). The tumor inhibition rates of the CTX group and the high- and low-dose groups of the composition were 66.67%, 58.17%, and 35.95%, respectively. The inhibitory effect of the composition on the tumor growth of mice with breast cancer transplanted tumors showed an obvious dose-dependence, as Figure 1 , shown in Table 1

[0061] Table 1. Effects of the composition on the body weight, tumor inhibition, and spleen index of nude mice with MDA-MB-231 transplanted tumors( n = 6)

[0062]

[0063] Compared with the normal group, # p < 0.05, ##p < 0.01; compared with the model group, *p < 0.05,

[0064] **p < 0.01; compared with the CTX group, △ p < 0.05, △△ p < 0.01

[0065] 3.2 Effects of the composition on the tumor tissue morphology of nude mice with transplanted tumors

[0066] As Figure 2 shown, in the tumor tissue of nude mice in the model group, the tumor cells were densely and richly arranged disorderly, the tumor cells were in irregular mass or cord-like shapes and diffusely distributed, the atypia of the tumor cells was obvious, the cell nuclei were round or oval, the cell nuclei were enlarged and deeply stained, and no necrosis foci were observed; in the CTX group, the number of tumor cells was significantly reduced, the cytoplasm of the tumor cells ruptured, the cell nuclei dissolved, and the necrosis area of the tumor cells was larger; in the high- and low-dose groups of the composition, a large number of tumor cells were found to be significantly reduced in number, sparsely arranged, obvious apoptosis occurred, and damaged tissue structures were visible.

[0067] 3.3 Effects of the composition on the number of white blood cells and the proportion of lymphocytes in nude mice with transplanted tumors

[0068] As Figure 3 shown, compared with the normal group, the number of white blood cells in the peripheral blood of nude mice in the model group was significantly increased (p < 0.01), and the proportion of lymphocytes was significantly decreased (p < 0.01); compared with the model group, the number of white blood cells in the peripheral blood of each administration group was significantly decreased (p < 0.01), the proportion of lymphocytes in the CTX group was significantly decreased (p < 0.01), and the proportion of lymphocytes in the high- and low-dose groups of the composition was significantly increased (p < 0.05, 0.01); compared with the CTX group, the number of white blood cells and the proportion of lymphocytes in the peripheral blood of mice in each dose group of the composition were significantly increased (P < 0.01).

[0069] 3.4 Effects of the composition on the splenic lymphoproliferative ability of nude mice with transplanted tumors

[0070] As shown in Table 2, compared with the model group, the proliferative abilities of T and B lymphocytes in the spleens of nude mice in the CTX group were significantly decreased (p < 0.05, 0.01), and the proliferative abilities of T and B lymphocytes in the spleens of mice in the high- and low-dose groups of the composition were significantly enhanced (p < 0.05, 0.01); compared with the CTX group, the proliferative abilities of T and B lymphocytes in mice in each dose group of the composition were significantly increased (p < 0.01). Table 2 Effects of the composition on the number of white blood cells and the proportion of lymphocytes in the peripheral blood of nude mice with MDA-MB-231 transplanted tumors( n = 6)

[0071]

[0072] Compared with the normal group, #p < 0.05, ## p < 0.01; compared with the model group, *p < 0.05, **p < 0.01; compared with the CTX group, △ p < 0.05, △△ p < 0.01

[0073] 3.5 Effects of the composition on cytokine levels in the serum of nude mice with transplanted tumors

[0074] As shown in Table 3, compared with the control group, the levels of IL-2, IL-6, IFN-γ, and TNF-α in the serum of mice in the model group were significantly decreased (p < 0.05, 0.01); compared with the model group, the levels of IL-2, IL-6, IFN-γ, and TNF-α in the serum of mice in each dose group of the composition were significantly increased (p < 0.05, 0.01); compared with the CTX group, the levels of IL-2, IL-6, IFN-γ, and TNF-α in the serum of mice in each dose group of the composition were significantly increased (p < 0.05, 0.01).

[0075] Table 3 Effects of the composition on cytokine levels in the serum of nude mice with MDA-MB-231 transplanted tumors ( n = 6)

[0076]

[0077] Compared with the normal group, # p < 0.05, ## p < 0.01; compared with the model group, *p < 0.05, **p < 0.01; compared with the CTX group, △ p < 0.05, △△ p < 0.01

[0078] 4. Discussion

[0079] Zhuang medicine classifies tumors into the category of "poisonous diseases". Its theoretical basis mainly includes the view of nature and man of "synchronization of three qi and yin-yang as the root", the view of body functions of "three channels and two paths, internal organs, qi and blood, bones and muscles", the etiology and pathogenesis theory of "poison and deficiency causing diseases", and the fundamental treatment principle of "regulating qi, detoxifying, and tonifying deficiency".

[11] 。"Three channels" respectively refer to the airway, the grain channel, and the water channel, and "two paths" refer to the dragon path and the fire path. If the "three channels" and "two paths" are blocked or the regulation is imbalanced, resulting in the imbalance of qi and blood in the human body and the loss of normal regulatory functions of the zang-fu organs, the poisonous insects and miasma poisons will stagnate and accumulate, and the cancer poison will accumulate in the body for a long time, ultimately leading to the occurrence and development of tumors.

[12] Among them, "poison disease" is the most distinctive content of Zhuang medicine. It is believed that "poisonous pathogen" is the fundamental pathogenesis of malignant solid tumors. As the first major therapeutic method in Zhuang medicine, "detoxification" plays an important role in the prevention and treatment of tumors. By correcting the "root" of the disease, namely "disharmony of the three qi and weakness of healthy qi", through "detoxification", the occurrence and development of malignant solid tumors can be effectively controlled. The results of this study show that the water extract of the Zhuang medicine combination of this application can significantly inhibit the growth rate of tumors in nude mice transplanted with human breast cancer cells MDA-MB-231, reduce the tumor mass, and induce apoptosis of tumor tissue cells. It is suggested that the water extract of Dendrobium officinale has the effect of inhibiting tumor growth and inducing apoptosis of tumor cells.

[0080] The specific immune responses mediated by T lymphocytes and B lymphocytes are the core of tumor immunity, and their dynamic balance can ensure the stability of the body's immune system. IL-2 is in the central link of the body's immune regulation system. It can not only stimulate the proliferation and differentiation of T cells, activate lymphocytes to inhibit the activity of tumor cells, but also promote the secretion of other cytokines such as IL-6, IFN-γ, TNF-α, etc. As one of the most important cytokines regulating the body's immune level, IL-6 has the effect of activating T and B lymphocytes and regulates the proliferation, differentiation, migration and adhesion of tumor cells.

[24] TNF-α is the cytokine with the strongest inhibitory activity against tumors and can directly induce apoptosis of tumor cells.

[25] IFN-γ has a powerful and direct inhibitory effect on the occurrence and growth of tumors, and at the same time can enhance the antitumor effect of TNF. The results of this study show that compared with the model group, the spleen index of mice in the CTX group was significantly reduced, and the proliferation ability of spleen T and B lymphocytes was significantly reduced, suggesting that the chemotherapeutic drug CTX further damaged the immune function of nude mice transplanted with tumors. Compared with the model group and the CTX group, the water extract of the Zhuang medicine combination can significantly increase the spleen index, improve the proliferation ability of spleen T and B lymphocytes, regulate the abnormal white blood cell level in the peripheral blood of nude mice transplanted with tumors, increase the proportion of lymphocytes in the peripheral blood, and increase the levels of cytokines IL-2, IL-6, IFN-γ, TNF-α in the serum. It is suggested that the water extract of the Zhuang medicine combination can improve the disordered immune system of nude mice transplanted with tumors and enhance the immune function.

[0081] To sum up, the Zhuang medicine composition of "clearing heat and detoxifying, removing dampness and dissipating nodules" can significantly inhibit the growth of tumors in nude mice with MDA-MB-231 breast cancer xenograft model and induce their apoptosis. At the same time, it can improve the disordered immune system of nude mice transplanted with tumors and enhance their immune function. The mechanism may be related to its down-regulation of the expression of PI3K / Akt / mTOR signaling pathway in tumor tissues and regulation of the mitochondrial pathway.

[0082] Finally, it should be noted that the above embodiments are only relatively representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above embodiments and there can be many variations. All variations that can be directly derived or associated by those of ordinary skill in the art from the disclosed content of the present invention should be considered within the protection scope of the present invention.

Claims

1. A Zhuang medicine composition for treating breast cancer, characterized in that, It includes the following raw materials: Oldenlandia diffusa, Radix Ranunculi Ternati, Corydalis saxicola, Millettia speciosa Champ., and Liquorice Root.

2. The Zhuang medicine composition according to claim 1, characterized in that, The weight parts of each raw material are as follows: Oldenlandia diffusa 5 - 30 parts, Corydalis saxicola 5 - 30 parts, Radix Ranunculi Ternati 3 - 15 parts, Millettia speciosa Champ. 3 - 15 parts, and Liquorice Root 1 - 10 parts.

3. The Zhuang medicine composition according to claim 1, characterized in that, The weight parts of each raw material are as follows: Oldenlandia diffusa : Corydalis saxicola : Radix Ranunculi Ternati : Millettia speciosa Champ. : Liquorice Root = 15 : 10 : 5 : 5 :

1.

4. Application of the Zhuang medicine composition according to claim 1 in being or preparing an anti - cancer drug.

5. The Zhuang medicine composition according to claim 3, characterized in that, The anti - cancer drug is a drug for treating breast cancer.

6. Granules prepared by using the Zhuang medicine composition according to claim 1.

7. Decoction prepared by using the Zhuang medicine composition according to claim 1.

8. The preparation method of the decoction according to claim 6, characterized in that, Specifically, weigh Oldenlandia diffusa, Corydalis saxicola, Radix Ranunculi Ternati, Millettia speciosa Champ., and Liquorice Root in proportion, soak them in 10 times the amount of pure water for 30 min for the first time, reflux and decoct under normal pressure for 1.5 h, and filter while it is hot with a 200 - mesh gauze; for the second decoction, reflux and extract with 8 times the amount of pure water for 1 h and then collect the filtrate. Combine the filtrates obtained twice and concentrate them to 2.0 g / mL in a rotary evaporator under reduced pressure, and that's it.

9. The preparation method according to claim 7, characterized in that, The parameters for concentration in the rotary evaporator under reduced pressure are 65 °C and - 0.095 Mpa.

10. The preparation method according to claim 7, characterized in that, The 2.0 g / mL specifically means that the amount of crude drug contained is 2.0 g / mL.

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