Traditional Chinese medicine compound composition for inhibiting lung cancer metastasis and application thereof

Abstract

The invention relates to a traditional Chinese medicine compound composition for inhibiting lung cancer metastasis and application thereof. The traditional Chinese medicine compound composition is prepared from the following raw material medicines in parts by weight: 40-45 parts of radix ophiopogonis, 4-8 parts of pinellia ternate, 4-8 parts of liquorice, 7-11 parts of ginseng, 4-8 parts of polished round-grained rice, 12-20 parts of Chinese dates and 18-27 parts of paris polyphylla. Compared with the prior art, the traditional Chinese medicine composition has a remarkable inhibiting effect on lung cancer metastasis, can remarkably induce lung cancer cell apoptosis and effectively inhibits proliferation and migration of lung cancer cells; animal experiments show that the traditional Chinese medicine composition not only can effectively reduce the number of mouse lung metastatic tumors, but also remarkably reduces the lung tumor metastasis load. In addition, the traditional Chinese medicine composition also remarkably increases the level of NK cells and CD8 + T cells in peripheral blood.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine compositions, and particularly relates to a traditional Chinese medicine compound composition for inhibiting lung cancer metastasis and its application. Background Art

[0002] Lung cancer is the main cause of cancer-related deaths globally. In China, the incidence and mortality of lung cancer have been continuously increasing, and the number of people dying from lung cancer each year has exceeded 700,000. With the application of immune and targeted therapy drugs in clinical practice, the survival rate of lung cancer patients has been continuously improved, but the 5-year overall survival rate is still less than 20%. The reason is that most patients have metastases at the time of diagnosis, and metastasis is the main cause of death in lung cancer patients, and there is currently a lack of specific drugs for preventing and treating lung cancer metastasis. A large number of studies have found that the level of circulating tumor cells (CTC) is related to distant metastasis and poor prognosis of lung cancer. Therefore, targeted elimination of tumor cells in the peripheral circulation and metastatic target organs may be an important treatment strategy for preventing tumor metastasis.

[0003] In recent years, with the understanding of the pathogenesis of lung cancer in traditional Chinese medicine, certain progress has been made in the treatment of lung cancer with traditional Chinese medicine. Mai Men Dong Tang is a classic formula from "Synopsis of the Golden Chamber", composed of six traditional Chinese medicines (Ophiopogon japonicus, Pinellia ternata, Glycyrrhiza uralensis, Panax ginseng, Japonica rice, Jujube), and has various pharmacological effects, such as regulating the secretion of alveolar surfactant, relieving cough, promoting airway purification, and alleviating airway hyperresponsiveness. Mai Men Dong Tang has the effects of clearing the lung and moistening dryness, clearing the stomach and nourishing the stomach, and is mainly used for treating syndromes of yin deficiency of the lung and stomach, and can also be used for treating lung diseases such as radiation pneumonia, allergic asthma, chronic bronchitis of the lung yin deficiency type, and pulmonary fibrosis, and has the potential to be applied to inhibit lung cancer metastasis.

[0004] Therefore, the research and development of a traditional Chinese medicine compound composition based on Mai Men Dong Tang that can effectively inhibit lung cancer metastasis is of great significance for improving the survival period of lung cancer patients after surgery. Summary of the Invention

[0005] The purpose of the present invention is to provide a traditional Chinese medicine compound composition for inhibiting lung cancer metastasis and its application for developing the application of the prescription of Mai Men Dong Tang in inhibiting lung cancer metastasis.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] The present invention first provides a traditional Chinese medicine compound composition for inhibiting lung cancer metastasis, and the traditional Chinese medicine compound composition is composed of the following raw materials in parts by weight: 40 - 45 parts of Ophiopogon japonicus, 4 - 8 parts of Pinellia ternata, 4 - 8 parts of Glycyrrhiza uralensis, 7 - 11 parts of Panax ginseng, 4 - 8 parts of Japonica rice, 12 - 20 parts of Jujube, and 18 - 27 parts of Paris polyphylla.

[0008] Further, the traditional Chinese medicine compound composition is composed of the following raw materials by weight: 42 parts of Ophiopogon japonicus, 6 parts of Pinellia ternata, 6 parts of Glycyrrhiza uralensis, 9 parts of Panax ginseng, 6 parts of japonica rice, 16 parts of Ziziphus jujuba, and 18 - 27 parts of Paris polyphylla.

[0009] In the traditional Chinese medicine compound composition, Ophiopogon japonicus: sweet in taste, slightly bitter, slightly cold in nature. It belongs to the heart, lung, and stomach meridians. It moistens the lungs and relieves cough, promotes the production of body fluid to benefit the stomach, and clears away heart vexation; Pinellia ternata: pungent in taste, warm in nature, toxic. It belongs to the spleen, stomach, and lung meridians. It dries dampness and resolves phlegm, suppresses the adverse flow of qi and stops vomiting, and dissipates binds and nodules; Glycyrrhiza uralensis: sweet in taste, neutral in nature. It belongs to the heart, lung, spleen, and stomach meridians. It invigorates the spleen and replenishes qi, moistens the lungs and relieves cough, clears heat and detoxifies, and harmonizes all the drugs; Panax ginseng: sweet and slightly bitter in taste, slightly warm in nature. It belongs to the spleen, lung, heart, and kidney meridians. It greatly replenishes primordial qi, invigorates the spleen and benefits the lungs, promotes the production of body fluid and quenches thirst, and calms the mind and improves intelligence; Japonica rice: sweet in taste, neutral in nature. It belongs to the spleen, stomach, and lung meridians. It replenishes the middle energizer and benefits qi, strengthens the spleen and harmonizes the stomach, eliminates vexation and quenches thirst, and stops diarrhea; Ziziphus jujuba: sweet in taste, warm in nature. It belongs to the spleen, stomach, and heart meridians. It replenishes the middle energizer and benefits qi, nourishes blood and calms the mind, and alleviates the properties of the drugs; Paris polyphylla: bitter in taste, slightly cold in nature. It belongs to the liver meridian. It clears heat and detoxifies, reduces swelling and alleviates pain, and cools the liver and stops fright.

[0010] In the whole formula, the dosage of Ophiopogon japonicus is the heaviest and it is the monarch drug. Its sweet and cold nature is clear and moistening, entering the lung and stomach meridians to nourish the yin of the lung and stomach, and clear away deficiency heat, playing a major therapeutic role in the main syndrome of yin deficiency of the lung and stomach. Although Pinellia ternata is warm and dry in nature, when combined with a large amount of Ophiopogon japonicus, its dryness is restricted, and it can suppress the adverse flow of qi and lower the qi, resolve phlegm and relieve cough, assisting Ophiopogon japonicus in treating symptoms such as cough, asthma, and throat discomfort in consumptive lung atrophy, and it is the minister drug. Paris polyphylla is bitter and slightly cold, with the functions of clearing heat and detoxifying, reducing swelling and alleviating pain, assisting the monarch drug Ophiopogon japonicus in enhancing the effect of clearing away heat toxins in the body, and it is the minister drug. Panax ginseng supplements the qi of the spleen and stomach. The spleen and stomach are the foundation of acquired constitution and the source of qi and blood production. When the qi of the spleen and stomach is strong, body fluids can naturally ascend to the lung, assisting Ophiopogon japonicus in nourishing the yin of the lung and stomach, and it is the assistant drug. Japonica rice and Ziziphus jujuba benefit the spleen and stomach in the whole formula, generate yin fluid, assist Panax ginseng in tonifying the spleen and stomach, and can also restrict the dryness of Pinellia ternata, and they are both assistant drugs. Glycyrrhiza uralensis can not only benefit qi and harmonize the middle, but also moisten the lungs and soothe the throat, and can also harmonize all the drugs, and it is the envoy drug.

[0011] The present invention also provides an application of the traditional Chinese medicine compound composition in the preparation of a drug for inhibiting lung cancer metastasis.

[0012] Further, the drug is an oral preparation, which includes freeze-dried powder, decoction, tablets, capsules, granules, pills, oral liquid, or syrup.

[0013] Further, the preparation method of the freeze-dried powder is: mixing Ophiopogon japonicus, Pinellia ternata, Glycyrrhiza uralensis, Panax ginseng, japonica rice, Ziziphus jujuba, and Paris polyphylla according to the weight ratio and cold-soaking them in an ethanol solution; successively carrying out reflux extraction, filtration, concentration, and freeze-drying to obtain the freeze-dried powder containing the traditional Chinese medicine compound composition.

[0014] Further, the preparation method of the freeze-dried powder is:

[0015] S1: Weigh Ophiopogon japonicus, Pinellia ternata, Glycyrrhiza uralensis, Panax ginseng, Japonica rice, Chinese date, and Paris vietnamensis, add water according to a liquid medicine ratio of 1:(6 - 10), soak in water for 0.5 - 12 h, heat under reflux for 0.5 - 4 h, and filter to obtain the first decoction liquid and medicinal residues.

[0016] S2: Soak the medicinal residues in water according to a liquid medicine ratio of 1:(6 - 10), heat under reflux again for extraction, and filter to obtain the second decoction liquid.

[0017] S3: Combine the first decoction liquid and the second decoction liquid, and concentrate to obtain a concentrate.

[0018] S4: Add absolute ethanol to the concentrate to make its alcohol content 60% - 80%, and purify at 2 - 6 °C for 18 - 30 h.

[0019] S5: Filter and collect the supernatant, remove the excess ethanol to obtain a refined liquid; freeze the refined liquid and then perform freeze-drying to obtain the freeze-dried powder containing the traditional Chinese medicine compound composition.

[0020] Furthermore, the preparation method of the decoction is as follows: Mix Ophiopogon japonicus, Pinellia ternata, Glycyrrhiza uralensis, Panax ginseng, Japonica rice, Chinese date, and Paris vietnamensis with water, and after decocting, concentrating, and filtering, the obtained filtrate is the decoction containing the traditional Chinese medicine compound composition.

[0021] Furthermore, the preparation method of the decoction is as follows: Weigh Ophiopogon japonicus, Pinellia ternata, Glycyrrhiza uralensis, Panax ginseng, Japonica rice, Chinese date, and Paris vietnamensis and mix with water, soak for 30 - 60 min; bring to a boil over high heat and then switch to low heat to continue decocting, with the first decocting time being 30 - 45 min and the second decocting time being 20 - 30 min; filter and mix the two decocted liquid medicines to obtain the decoction containing the traditional Chinese medicine compound composition.

[0022] Furthermore, the drug includes conventional pharmaceutically acceptable excipients.

[0023] Furthermore, the drug can inhibit the proliferation and migration of lung cancer cells and induce their apoptosis, and can inhibit the growth of metastatic tumors by upregulating the levels of NK cells and CD8+ T cells in peripheral blood.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The traditional Chinese medicine composition of the present invention has a significant inhibitory effect on lung cancer metastasis, can significantly induce apoptosis of lung cancer cells, and effectively inhibit the proliferation and migration of lung cancer cells; in animal experiments, it was found that this traditional Chinese medicine composition can not only effectively reduce the number of metastatic tumors in the lungs of mice, but also significantly reduce the lung tumor metastasis burden.

[0026] (2) In the traditional Chinese medicine composition of the present invention, Ophiopogon japonicus is used as the monarch drug to nourish the yin of the lung and stomach. Pinellia ternata is combined as the minister drug to lower adverse qi and resolve phlegm, and Paris vietnamensis is used to clear heat and detoxify. Ginseng, rice, and Chinese date are used as assistant drugs to tonify the spleen and replenish qi, and Glycyrrhiza uralensis is used as the guiding drug to harmonize all the drugs. The whole prescription can treat both the principal and secondary aspects of the disease, and together they can nourish yin and clear heat, lower adverse qi and resolve phlegm, and detoxify and reduce swelling. On the basis of drugs for supplementing qi and nourishing yin, the traditional Chinese medicine composition of the present invention adds a drug for eliminating pathogenic factors. The implication is that there are a large number of sweet and moist drugs in the Decoction of Ophiopogon japonicus, lacking drugs for eliminating pathogenic factors. Therefore, Paris vietnamensis is added to treat both the excess and deficiency simultaneously, thereby enhancing the activation effect on immune cells and also reflecting the theoretical idea of "deficiency of healthy qi with latent toxin".

[0027] (3) In animal experiments, the traditional Chinese medicine composition of the present invention can significantly up-regulate the levels of NK cells and CD8+ T cells in peripheral blood, while the traditional Decoction of Ophiopogon japonicus can only up-regulate the level of NK cells. Therefore, the traditional Chinese medicine composition of the present invention can not only inhibit the proliferation and migration of lung cancer cells and induce their apoptosis, but also may inhibit the growth of metastatic tumors by up-regulating the levels of NK cells and CD8+ T cells in peripheral blood, and thus play its role in anti-lung cancer metastasis. Description of the Drawings

[0028] Figure 1 Schematic diagram of the effects of different prescriptions on the proliferation of CTC-TJH-01 and LLC cells.

[0029] Figure 2 Schematic diagram of the effects of different prescriptions on the migration of CTC-TJH-01 and LLC cells (×100μm); *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0030] Figure 3 Schematic diagram of the effects of different prescriptions on the apoptosis of CTC-TJH-01 and LLC cells; *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0031] Figure 4 Schematic diagram of the effects of different prescriptions on the body weight of mice.

[0032] Figure 5 Schematic diagram of the effects of different prescriptions on the number of LLC lung tumors metastasized; *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0033] Figure 6 Schematic diagram of the effects of different prescriptions on the tissue morphology and metastatic burden of mouse lung metastatic tumors.

[0034] Figure 7 Schematic diagram of the effects of different prescriptions on the proportion of NK cells in peripheral blood of mice; *: P < 0.05, **: P < 0.01, ***: P < 0.001.

[0035] Figure 8 Schematic diagram of the proportion of CD8+ T cells in the peripheral blood of mice for different formula combinations; *: P < 0.05, **: P < 0.01, ***: P < 0.001. Detailed implementation manners

[0036] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation procedures are given, but the protection scope of the present invention is not limited to the following embodiments.

[0037] Unless otherwise specified, the reagents, methods, instruments and equipment used in the present invention are conventional reagents, methods, instruments and equipment in the art. Unless otherwise specified, the reagents and materials used in the following embodiments are all commercially available. All the Chinese herbal medicine pieces of the present invention are provided by the Chinese medicine pharmacy of Shanghai Traditional Chinese Medicine Hospital.

[0038] Example 1:

[0039] This example provides a traditional Chinese medicine compound composition for inhibiting lung cancer metastasis. The traditional Chinese medicine compound composition is composed of the following raw materials in parts by weight: 42 parts of Ophiopogon japonicus, 6 parts of Pinellia ternata, 6 parts of Glycyrrhiza uralensis, 9 parts of Panax ginseng, 6 parts of Japonica rice, 16 parts of Chinese date, and 18 parts of Paris polyphylla.

[0040] Example 2:

[0041] This example provides a traditional Chinese medicine compound composition for inhibiting lung cancer metastasis. The traditional Chinese medicine compound composition is composed of the following raw materials in parts by weight: 42 parts of Ophiopogon japonicus, 6 parts of Pinellia ternata, 6 parts of Glycyrrhiza uralensis, 9 parts of Panax ginseng, 6 parts of Japonica rice, 16 parts of Chinese date, and 27 parts of Paris polyphylla.

[0042] Comparative Example 1:

[0043] This comparative example provides a traditional Chinese medicine compound composition. The traditional Chinese medicine compound composition is composed of the following raw materials in parts by weight: 42 parts of Ophiopogon japonicus, 6 parts of Pinellia ternata, 6 parts of Glycyrrhiza uralensis, 9 parts of Panax ginseng, 6 parts of Japonica rice, and 16 parts of Chinese date.

[0044] Comparative Example 2:

[0045] This comparative example provides a traditional Chinese medicine compound composition. The traditional Chinese medicine compound composition is composed of the following raw materials in parts by weight: 42 parts of Ophiopogon japonicus, 6 parts of Pinellia ternata, 6 parts of Glycyrrhiza uralensis, 9 parts of Panax ginseng, 6 parts of Japonica rice, 16 parts of Chinese date, and 9 parts of Paris polyphylla.

[0046] The traditional Chinese medicine compound composition of the above examples of the present invention can be prepared into oral preparations, including freeze-dried powder, decoction, tablets, capsules, granules, decoctions, pills, oral liquids or syrups.

[0047] Among them, the preparation method of the freeze-dried powder of the traditional Chinese medicine compound composition includes the following steps:

[0048] (1) Weigh Ophiopogon japonicus, Pinellia ternata, Glycyrrhiza uralensis, Panax ginseng, Japonica rice, Chinese date, and Paris polyphylla by weight parts, add distilled water according to the liquid medicine ratio of 1:8, soak in water for 1 hour, heat under reflux for 2 hours, filter to obtain the first decoction and medicinal residues;

[0049] (2) Add water to the obtained medicinal residues according to the liquid medicine ratio of 1:8, heat under reflux for 2 hours, filter to obtain the second decoction;

[0050] (3) Combine the first decoction and the second decoction, heat and concentrate to obtain a concentrate;

[0051] (4) Slowly add absolute ethanol to the concentrate while stirring to make the alcohol content reach 70%, place it in a refrigerator at 4°C for 24 hours for purification;

[0052] (5) Filter and collect the supernatant, remove the excess ethanol to obtain a refined liquid;

[0053] (6) Place the refined liquid in a refrigerator at -80°C for 12 hours, and then freeze-dry it in a freeze dryer for 24 hours to obtain the freeze-dried powder of the traditional Chinese medicine compound composition.

[0054] The preparation method of the decoction of the traditional Chinese medicine compound composition is as follows:

[0055] Weigh Ophiopogon japonicus, Pinellia ternata, Glycyrrhiza uralensis, Panax ginseng, Japonica rice, Chinese date, and Paris polyphylla by weight parts, add an appropriate amount of water. The amount of added water is usually preferably 2 - 3 cm higher than the surface of the medicinal materials, and soak for 30 - 60 minutes. First, quickly boil the soaked medicinal materials and water with strong fire (big fire), and then continue to decoct with slow fire (small fire). The first decoction time can be extended to 30 - 45 minutes, and the second decoction is 20 - 30 minutes. After decocting for the specified time, filter the decocted liquid with clean gauze or filter screen and pour it into a container. Finally, mix the liquids obtained from the two decoctions together and stir evenly to obtain the decoction of the traditional Chinese medicine compound composition.

[0056] Tablets, capsules, granules, decoctions, pills, oral liquids or syrups of the traditional Chinese medicine compound composition can be prepared from the above-mentioned freeze-dried powder or decoction plus conventional excipients acceptable in pharmacy by conventional processes, which will not be elaborated here.

[0057] The present invention first conducts the following in vitro experimental tests on the traditional Chinese medicine compositions of the above-mentioned examples and comparative examples.

[0058] (1) Detect the effects of different formulae of the present invention on the proliferation activities of CTC-TJH-01 and LLC cells by the CCK-8 method

[0059] (1.1) Cells: The human lung adenocarcinoma circulating tumor cell line CTC-TJH-01 was derived from the peripheral blood of a female patient with stage IIa lung adenocarcinoma and is a cell line that can be stably passaged. Mouse LLC cells were purchased from the Cell Bank of the Chinese Academy of Sciences. CTC-TJH-01 cells were placed in F12K medium containing 10% fetal bovine serum, and LLC cells were placed in DMEM medium containing 10% fetal bovine serum, and both were cultured at 5% CO 2 2, 37 °C under saturated humidity.

[0060] (1.2) Drugs: The traditional Chinese medicine compound compositions of Examples 1, 2 and Comparative Examples 1, 2 were used as test drugs. The Chinese herbal medicine pieces were provided by the Chinese Medicine Pharmacy of Shanghai Traditional Chinese Medicine Hospital. The traditional Chinese medicine compound composition was soaked in 10-fold volume of water for 30 min, decocted twice, 1 h each time, the two extracts were mixed, rotary evaporated and concentrated, filtered and dried into powder, and stored in a 4 °C refrigerator.

[0061] (1.3) Reagents: CCK-8 kit, purchased from Shanghai Yaen Biopharmaceutical Technology Co., Ltd.

[0062] (1.4) Experimental method: CTC-TJH-01 and LLC cell suspensions with a density of 2×10 4 cells / mL were inoculated into 96-well plates, 100 μL per well. After the cells adhered, the cells were treated with different concentrations (0, 62.5, 125, 250, 500, 1000 μg / mL) of different formulations (the powders of different formulations in 1.2 were prepared into different concentrations with cell culture medium). After 48 h of intervention, 20 μL of CCK-8 solution was added to each well, and the culture plate was incubated in an incubator for 1-2 h. The absorbance was measured with an enzyme-linked immunosorbent assay at a wavelength of 450 nm to observe the inhibitory effect of different formulations on the proliferation of CTC-TJH-01 and LLC cells.

[0063] (2) Transwell experiment to detect the effect of different formulations on the migration of CTC-TJH-01 and LLC cells

[0064] (2.1) Cells: The same as 1.1.

[0065] (2.2) Drugs: The same as 1.2.

[0066] (2.3) Reagents: Crystal violet staining solution, purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0067] (2.4) Experimental method: After treating cells with different formulations at different concentrations (0, 250, 500 μg / mL) for 48 h, the cells were digested with trypsin and harvested. After adding 800 μL of medium containing 10% FBS to each well of a 24-well plate, a Transwell chamber was placed in it. 200 μL of the cell suspension of each group was added to the upper chamber of the Transwell and cultured in an incubator for 24 h. Then, the Transwell chamber was taken out and carefully washed once with PBS. The cells were fixed with paraformaldehyde for 1 h, stained with crystal violet for 20 min, and rinsed three times with PBS. The cells were observed and photographed under a microscope, and the cells were counted.

[0068] (3) Detection of the effects of different formulations on the apoptosis of CTC-TJH-01 and LLC cells by flow cytometry

[0069] (3.1) Cells: The same as in 1.1.

[0070] (3.2) Drugs: The same as in 1.2.

[0071] (3.3) Reagents: Annexin V-FITC / PI cell apoptosis detection kit, purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0072] (3.4) Experimental method: CTC-TJH-01 and LLC cells were seeded into 6-well plates at a concentration of 1×10 5 cells / well. After 24 h, the cells were treated with different formulations at different concentrations (0, 250, 500 μg / mL) for 48 h. After the cells were treated for 48 h, they were stained with Annexin V-FITC and propidium iodide (PI) for 10 min respectively. The apoptosis of the cells was detected using a flow cytometer.

[0073] The results of the cell viability of different traditional Chinese medicine compound compositions on CTC-TJH-01 and LLC cells are as Figure 1 shown. The Ophiopogon japonicus Decoction without adding Paris polyphylla (Control 1) or with a small amount of Paris polyphylla added (Control 2) had no inhibitory effect on the above two kinds of cells, while the Ophiopogon japonicus Decoction in Examples 1 and 2 (+18 / 27 g of Paris polyphylla) could significantly inhibit the viability of CTC-TJH-01 and LLC cells. The IC 50 values of Examples 1 and 2 for CTC-TJH-01 cells were 45.99 and 52.72 μg / mL respectively, and the IC50 values for LLC cells were 165.9 and 222.1 μg / mL respectively.

[0074] The effects of different traditional Chinese medicine compound compositions on the migration of CTC-TJH-01 and LLC cells are as Figure 2As shown, the Ophiopogon japonicus Decoction (+18 / 27 g of Paris polyphylla Smith) in Examples 1 and 2 can significantly inhibit the migration of CTC-TJH-01 and LLC cells, and is significantly superior to Comparative Example 1 (pure Ophiopogon japonicus Decoction) and Comparative Example 2 (Ophiopogon japonicus Decoction + 9 g of Paris polyphylla Smith), and the difference is statistically significant (P<0.05). The traditional prescription Ophiopogon japonicus Decoction has the effects of nourishing the lung and stomach and descending adverse qi, and Paris polyphylla Smith has the functions of clearing heat and detoxifying and dissipating swelling and nodules. The compound composition composed of the two shows significant advantages in the treatment research of lung cancer. The research shows that the traditional Chinese medicine compound compositions in Examples 1 and 2 have a strong inhibitory effect on the migration of lung cancer. This composition is rationally formulated with a variety of traditional Chinese medicines, and each medicinal material synergizes with each other. It may effectively curb the migration ability of lung cancer cells and reduce the occurrence of lung cancer metastasis through various mechanisms such as regulating the biological behavior of lung cancer cells, interfering with the tumor microenvironment, and inhibiting related signaling pathways.

[0075] The effects of different traditional Chinese medicine compound compositions on the apoptosis of CTC-TJH-01 and LLC cells are as Figure 3 shown, the Ophiopogon japonicus Decoction (+18 / 27 g of Paris polyphylla Smith) can significantly promote the apoptosis of CTC-TJH-01 and LLC cells, and is superior to the Ophiopogon japonicus Decoction and the Ophiopogon japonicus Decoction + 9 g of Paris polyphylla Smith composition, and the difference is statistically significant (P<0.05). The Ophiopogon japonicus Decoction itself has the effects of nourishing the lung and stomach, etc., and Paris polyphylla Smith has the effects of clearing heat and detoxifying and detumescence and relieving pain, etc. The compound composition composed of the two shows excellent performance in the treatment research of lung cancer. The research shows that the traditional Chinese medicine compound compositions in Examples 1 and 2 have a strong pro-apoptotic effect on lung cancer. This composition integrates the characteristics of a variety of traditional Chinese medicines, and each component complements each other. It can induce cancer cells to initiate the programmed death mechanism and promote their apoptosis through various ways such as regulating the signal transduction pathway in lung cancer cells, affecting gene expression, and destroying mitochondrial function, thereby inhibiting the proliferation and development of lung cancer cells.

[0076] In view of the fact that the traditional Chinese medicine compound compositions in Examples 1 and 2 show obvious inhibitory effects on the proliferation, apoptosis and migration of lung cancer cells in vitro and have good application potential in inhibiting the recurrence and metastasis after lung cancer surgery, the present invention also conducts the following in-vivo animal experiments on the traditional Chinese medicine compound compositions of Examples 1 and 2.

[0077] (4) Effects of different formulations on lung metastasis in mice

[0078] (4.1) Reagents: Mouse FITC-CD3 antibody (product number: 100220), mouse APC-NK1.1 antibody (product number: 108710), and mouse PerCP / Cyanine5.5-CD8 (product number: 100734) antibody were all purchased from Biolegend, USA.

[0079] (4.2) Animals: C57BL / 6 male mice, 6 weeks old, weighing 18 - 20 g, raised under specific pathogen free (SPF) conditions. All bedding, drinking water, standard feed, and other items in contact with the animals were sterilized by autoclaving.

[0080] (4.3) Tumor strain: LLC cells.

[0081] (4.4) Modeling method: Collect LLC cells in the logarithmic growth phase and adjust the cell concentration to 3×10 6 cells / mL. After 7 days of adaptive feeding, a lung metastasis model was established by injecting 0.1 mL of cell suspension into the tail vein of C57BL / 6 mice. The C57BL / 6 mice were randomly divided into a normal saline control group, Example 1 group, Example 2 group, and paclitaxel group, with 6 mice in each group. One month later, the C57BL / 6 mice were sacrificed by cervical dislocation.

[0082] (4.5) Drug grouping: The intervention was expected to last for 1 month, with 6 mice in each group.

[0083] Normal saline control group: Administered normal saline (NS) by gavage once a day for 1 month continuously.

[0084] Example 1 group: Administered by gavage (2.6 g / kg), 0.2 mL per mouse, once a day for 1 month continuously.

[0085] Example 2 group: Administered by gavage (2.6 g / kg), 0.2 mL per mouse, once a day for 1 month continuously.

[0086] Paclitaxel group: Administered by intraperitoneal injection (5 mg / kg), 0.1 mL per mouse, 3 times a week for 1 month of intervention.

[0087] (4.6) Observation indicators:

[0088] Body weight: Measure the weight changes of mice in different groups, fixed at three times a week.

[0089] Lung tumor metastasis: Take out the lungs of mice, take pictures, and calculate the number of nodules on the surface of lung tissue.

[0090] H&E staining: Fix the lung tissue with 4% paraformaldehyde, embed in paraffin, and cut into 5 - μm thick serial sections. All sections were dewaxed in xylene, rehydrated in gradient ethanol, and washed with distilled water. Stained with H&E and dehydrated. After the sections were mounted, they were photographed and observed under an optical microscope. Metastatic foci were determined by large, deeply stained, densely and disorderly arranged cell nuclei.

[0091] Flow cytometry: Blood was taken from the eyeballs of mice, 100 μL of mouse peripheral blood was aspirated, 5 μL of blocking agent was added, and blocked at room temperature for 10 min; the corresponding FITC-CD3, APC-NK1.1, and PerCP / Cyanine5.5-CD8 mouse flow antibodies were added and stained at room temperature for 20 min; red blood cell lysate was added and lysed in the dark at room temperature for 10 min; finally, centrifuged at 1000 r / min for 5 min, the cells were washed twice, and then detected by flow cytometry.

[0092] The effects of the above different formulations on the body weight change of mice are as Figure 4 shown. Compared with the normal saline group, there were no significant changes in the body weight of mice in the Example 1 group, Example 2 group, and paclitaxel group.

[0093] The situation of lung tumor metastasis in mice with the above different formulations is as Figure 5 shown. Compared with the normal saline group, the combination of Ophiopogon japonicus Decoction + 18 g of Paris vietnamensis in Example 1 and the combination of Ophiopogon japonicus Decoction + 27 g of Paris vietnamensis in Example 2 could significantly inhibit the lung metastasis of LLC cells (P < 0.05).

[0094] The effects of the above different formulations on the pathological morphology of lung metastatic tumors in mice are as Figure 6 shown. In the lung tissue of mice, metastatic foci were determined as those with large and deeply stained cell nuclei, overlapping each other, arranged densely and disorderly, and growing diffusely. Compared with the normal saline group, the Example 1 group (combination of Ophiopogon japonicus Decoction + 18 g of Paris vietnamensis) and the Example 2 group (combination of Ophiopogon japonicus Decoction + 27 g of Paris vietnamensis) could significantly reduce the lung metastasis burden of mice (P < 0.05), and there was no significant difference in the inhibitory effect compared with the commonly used chemotherapy drug paclitaxel.

[0095] The regulatory effects of the above different formulations on NK cells in the peripheral blood of mice are as Figure 7 shown. Compared with the normal saline group, the Example 1 group (combination of Ophiopogon japonicus Decoction + 18 g of Paris vietnamensis) and the Example 2 group (combination of Ophiopogon japonicus Decoction + 27 g of Paris vietnamensis) could significantly up-regulate the proportion of NK cells in the peripheral blood of mice (P < 0.05), and were superior to the paclitaxel group.

[0096] The regulatory effects of the above different formulations on CD8+ T cells in the peripheral blood of mice are as Figure 8 shown. Compared with the normal saline group, the Example 1 group (combination of Ophiopogon japonicus Decoction + 18 g of Paris vietnamensis) and the Example 2 group (combination of Ophiopogon japonicus Decoction + 27 g of Paris vietnamensis) could significantly up-regulate the proportion of CD8+ T cells in the peripheral blood of mice (P < 0.05).

[0097] From the above in vitro and in vivo experimental results, it can be seen that the group of Example 1 (Maxing Decoction + Paris polyphylla 18g composition) and the group of Example 2 (Maxing Decoction + Paris polyphylla 27g composition) can effectively inhibit the proliferation, apoptosis and migration of lung cancer cells, and have the effect of inhibiting the recurrence and metastasis after lung cancer surgery. It can not only effectively reduce the number of pulmonary metastatic tumors in mice, but also up-regulate the levels of NK cells and CD8+ T cells in peripheral blood, promote the clearance of circulating tumor cells, which is of great significance for the prevention and treatment of postoperative lung cancer patients.

[0098] In summary, under the guidance of the theory of strengthening the healthy qi and eliminating pathogenic factors in Huangdi Neijing, combined with the theory of "treating cancer by strengthening the healthy qi" and the pathogenesis theory of "latent toxin due to deficiency of healthy qi" in the subclinical stage of cancer metastasis, based on the main effect of strengthening the healthy qi of Maxing Decoction, one commonly used and clinically effective pathogenic factor-eliminating traditional Chinese medicine, Paris polyphylla, is added to formulate a traditional Chinese medicine compound composition for treating lung cancer metastasis. The traditional Chinese medicine composition of the present invention has a significant inhibitory effect on lung cancer metastasis, can significantly induce apoptosis of lung cancer cells, and effectively inhibit the proliferation and migration of lung cancer cells; in animal experiments, it is found that this traditional Chinese medicine composition can not only effectively reduce the number of pulmonary metastatic tumors in mice, but also significantly reduce the pulmonary tumor metastasis burden. In addition, this traditional Chinese medicine composition also significantly up-regulates the levels of NK cells and CD8+ T cells in peripheral blood.

[0099] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative labor. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A Chinese medicinal compound composition for inhibiting lung cancer metastasis, characterized in that: The Chinese medicine compound composition is composed of the following raw materials in parts by weight: 40-45 parts of Radix Ophiopogonis, 4-8 parts of Rhizoma Pinelliae, 4-8 parts of Radix Glycyrrhizae, 7-11 parts of Radix Ginseng, 4-8 parts of Japonica Rice, 12-20 parts of Jujube, and 18-27 parts of Rhizoma Paris.

2. A Chinese medicinal compound composition for inhibiting lung cancer metastasis according to claim 1, characterized in that: The Chinese medicine compound composition is composed of the following raw materials in parts by weight: 42 parts of Radix Ophiopogonis, 6 parts of Rhizoma Pinelliae, 6 parts of Radix Glycyrrhizae, 9 parts of Radix Ginseng, 6 parts of Japonica Rice, 16 parts of Jujube, and 18-27 parts of Rhizoma Paris.

3. Use of the Chinese medicine compound composition according to claim 1 or 2 in the preparation of a drug for inhibiting lung cancer metastasis.

4. Use of the Chinese medicine compound composition according to claim 3 in preparing a drug for inhibiting lung cancer metastasis, characterized in that: The medicine is an oral preparation, which includes lyophilized powder, decoction, tablet, capsule, granule, pill, oral liquid or syrup.

5. Use of the Chinese medicinal compound composition according to claim 4 in preparing a drug for inhibiting lung cancer metastasis, characterized in that: The preparation method of the freeze-dried powder is: Ophiopogon japonicus, pinellia tuber, liquorice, ginseng, polished rice, jujube and parsley are mixed according to weight ratio and cold-soaked in ethanol solution; reflux extraction, filtration, concentration and freeze-drying are performed in sequence to obtain freeze-dried powder containing the traditional Chinese medicine compound composition.

6. Use of the Chinese medicinal compound composition according to claim 4 in preparing a drug for inhibiting lung cancer metastasis, characterized in that: The preparation method of the freeze-dried powder is: S1: Weigh Ophiopogon japonicus, Pinellia ternata, Licorice root, Ginseng, Japonica rice, Jujube and Paris polyphylla, add water at a drug-liquid ratio of 1:(6-10), soak in water for 0.5-12h, heat under reflux for 0.5-4h, and filter to obtain the first decoction and drug residue; S2: soaking the residue in water at a drug-liquid ratio of 1:(6-10), heating under reflux extraction again, and filtering to obtain a second decoction; S3: combining the first decoction and the second decoction to obtain a concentrate; S4: adding anhydrous ethanol to the concentrate to make the alcohol content of the concentrate 60%-80%, and purifying at 2-6°C for 18-30h; S5: filtering and collecting the supernatant, removing excess ethanol, and obtaining a refined liquid; freezing the refined liquid and freeze-drying it to obtain a freeze-dried powder containing the Chinese medicine compound composition.

7. Use of the Chinese medicinal compound composition according to claim 4 in preparing a drug for inhibiting lung cancer metastasis, characterized in that: The preparation method of the decoction is: The filtrate obtained by mixing ophiopogon japonicus, pinellia tuber, liquorice, ginseng, polished rice, jujube and parsley with water is decocted, concentrated and filtered to obtain a water decoction containing the traditional Chinese medicine compound composition.

8. Use of the Chinese medicinal compound composition according to claim 4 in preparing a drug for inhibiting lung cancer metastasis, characterized in that: The preparation method of the decoction is: Weigh ophiopogon japonicus, pinellia tuber, liquorice, ginseng, polished rice, jujube and parsley and mix with water, and soak for 30-60 minutes; boil over high heat and then continue to decoct over low heat, with the first decoction time being 30-45 minutes and the second decoction time being 20-30 minutes; filter the two decoctions and mix them, so as to prepare a water decoction containing the traditional Chinese medicine compound composition.

9. Use of the Chinese medicinal compound composition according to claim 3 in preparing a drug for inhibiting lung cancer metastasis, characterized in that: The medicine includes conventional pharmaceutically acceptable excipients.

10. Use of the Chinese medicinal compound composition according to claim 3 in preparing a drug for inhibiting lung cancer metastasis, characterized in that: The drug can inhibit the proliferation and migration of lung cancer cells and induce apoptosis thereof, and can inhibit the growth of metastatic tumors by upregulating the levels of NK cells and CD8+T cells in peripheral blood.

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