Application of codonopsis pilosula extract in preparation of anti-pulmonary fibrosis medicine

Abstract

The invention discloses an application of a codonopsis pilosula extract in preparation of an anti-pulmonary fibrosis medicine, relates to the technical field of traditional Chinese medicine compositions, and solves the problem that in the prior art, an anti-pulmonary fibrosis medicine generates large side effects in application. According to the invention, water extract components, small molecule components and polysaccharide components in the codonopsis pilosula are separated and extracted to obtain different component extracts of the codonopsis pilosula, and through screening, when the mass ratio of the small molecule components to the polysaccharide components in the codonopsis pilosula is 1: 8, the synergistic effect between the two effective components is optimal; the lung tissue pathological injury degree, the hydroxyproline content, the collagen level and the fibronectin level of the pulmonary fibrosis rats are effectively reduced. The anti-pulmonary fibrosis medicine prepared from the codonopsis pilosula extract is small in side effect in the application process, low in preparation cost, easy to operate and suitable for large-scale production and clinical application, and a new thought is provided for preparation of the anti-pulmonary fibrosis medicine.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine compositions, and in particular to application of a codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug. Background Art

[0002] Pulmonary fibrosis is a serious chronic lung disease characterized by the gradual replacement of lung tissue with fibrotic connective tissue, leading to a gradual decline in lung function. The causes of the disease are complex and diverse, including environmental factors (such as long-term exposure to harmful substances), genetic background, drug effects, viral infections, and immune diseases. Patients with pulmonary fibrosis often experience difficulty breathing, dry coughs, and decreased exercise tolerance. As the disease progresses, these symptoms become more and more severe until they may eventually lead to respiratory failure. Although the course of pulmonary fibrosis progresses relatively slowly, due to its irreversible fibrosis process, once diagnosed, the patient's lung damage is usually difficult to repair and extremely difficult to treat.

[0003] In the preparation system of Western medicine, the main treatments for pulmonary fibrosis currently include anti-fibrotic drugs, such as pirfenidone and nintedanib. These drugs delay further deterioration of lung function by inhibiting the process of fibrosis. However, there are obvious shortcomings in the application of Western medicine to prepare anti-fibrotic drugs. First, these drugs are often accompanied by many adverse reactions. Patients may experience side effects such as gastrointestinal discomfort, fatigue, nausea, skin reactions, and liver damage, which affect the quality of life. Secondly, the preparation cost of anti-fibrotic drugs is high, and patients need to take them continuously for a long time, which is a heavy burden on patients with poor economic conditions and their families. In addition, although the application of Western medicine to prepare anti-fibrotic drugs can slow down the progression of the disease to a certain extent, it is mainly symptomatic treatment, lacks the means to fundamentally reverse the disease, and there is no drug that can completely cure pulmonary fibrosis. Therefore, finding a preparation scheme with fewer side effects and better efficacy has become the focus of research.

[0004] Traditional Chinese medicine has gradually shown important potential in the preparation and treatment of anti-pulmonary fibrosis drugs. The theory of traditional Chinese medicine emphasizes the holistic concept, focuses on regulating the body's functions, and achieves disease relief and cure by enhancing the body's self-repair ability. The application advantage of traditional Chinese medicine in the preparation of anti-pulmonary fibrosis drugs is that it usually has fewer side effects and can effectively improve patients' clinical symptoms and improve their quality of life. At the same time, modern medical research has found that some ingredients in traditional Chinese medicine, such as antioxidants, anti-inflammatory substances, and immunomodulators, can play a positive role in different stages of pulmonary fibrosis. With the continuous in-depth study of the effective ingredients and mechanisms of traditional Chinese medicine, this provides a scientific basis and theoretical support for the application of traditional Chinese medicine in the preparation of anti-pulmonary fibrosis drugs. In particular, some traditional Chinese medicines, such as Codonopsis pilosula, Astragalus membranaceus, and Licorice, have been proven to have positive effects in the treatment of lung diseases due to their effects of invigorating qi and strengthening the spleen, nourishing the lungs and nourishing yin.

[0005] As one of the important medicinal materials in traditional Chinese medicine, Codonopsis pilosula is widely used in the treatment of various debilitating diseases for its effects of nourishing the spleen and stomach, replenishing qi and nourishing yin. In recent years, the research on Codonopsis pilosula extract in the field of anti-fibrosis has made significant progress, especially in the preparation of anti-pulmonary fibrosis drugs, showing potential application value. The active ingredients in Codonopsis pilosula, such as Codonopsis pilosula polysaccharides and acetylene glycosides, have antioxidant, anti-inflammatory, immunomodulatory and tissue repair promoting effects. These characteristics have made Codonopsis pilosula extract widely concerned in anti-fibrosis treatment. Relevant clinical studies have shown that Codonopsis pilosula extract can inhibit the excessive deposition of collagen in the fibrosis process and slow down the progression of fibrosis by regulating cytokine levels. Codonopsis pilosula extract is not only effective in inhibiting fibrosis, but also can improve the patient's respiratory function by enhancing the repair ability of lung tissue. It can be seen that with the continuous development of traditional Chinese medicine research, the application of Codonopsis pilosula extract in the preparation of anti-pulmonary fibrosis drugs has shown broad prospects. Summary of the invention

[0006] In order to solve the problem of large side effects of anti-pulmonary fibrosis drugs in the prior art, the present invention proposes an application of Codonopsis pilosula extract in anti-pulmonary fibrosis drugs, separates and extracts different components in the Codonopsis pilosula extract, and provides a composition that effectively improves pulmonary fibrosis without side effects. The technical solution of the present invention is as follows:

[0007] Application of a Codonopsis pilosula extract in preparing an anti-pulmonary fibrosis drug.

[0008] The above-mentioned Codonopsis pilosula extract is composed of Codonopsis pilosula water extract component, small molecule component and polysaccharide component;

[0009] Furthermore, the mass ratio of the small molecule component to the polysaccharide component is (16-1): (1-128).

[0010] The preparation method of the Codonopsis pilosula extract comprises the following steps:

[0011] S1: crush the Codonopsis pilosula into powder, soak it in distilled water, heat it under reflux, combine the extracts and concentrate them by rotary evaporation to obtain a concentrated extract;

[0012] S2: dividing the concentrated extract obtained in step S1 into two parts, freeze-drying one part of the concentrated solution to obtain a water extract component, and adding ethanol to the other part of the concentrated solution to precipitate overnight, and then filtering to obtain a supernatant and a precipitate;

[0013] S3: The supernatant component obtained in step S2 is concentrated by rotary evaporation, added to a column filled with AB-8 macroporous resin, retained and adsorbed, eluted with 5 column volumes of distilled water, and then eluted with 8 column volumes of 80% alcohol, the 80% alcohol eluate is collected, and rotary evaporation and freeze-dried to obtain a small molecule component; the precipitate is dried under reduced pressure to obtain a crude polysaccharide component;

[0014] Further, the Codonopsis pilosula slices in S1 are crushed into powder and sieved with 30-50 mesh;

[0015] Furthermore, the volume of the distilled water in S1 is 7 to 9 times that of the Codonopsis pilosula powder, and the soaking time is 0.5 to 1.5 hours;

[0016] Furthermore, the temperature of heating reflux in S1 is 100° C.;

[0017] Furthermore, the heating reflux times in S1 is 2 to 4 times;

[0018] Furthermore, the final concentration of alcohol in the alcohol precipitation in S2 is 70% to 90%;

[0019] Furthermore, the adsorption time in S3 is retained for 24 hours.

[0020] Compared with the prior art, the present invention solves the problem of large side effects of the anti-pulmonary fibrosis drugs in the prior art during use, and the specific beneficial effects are:

[0021] 1. New anti-fibrosis components: The small molecule components and polysaccharide components in Codonopsis pilosula can show synergistic effects when combined in a certain proportion. They can reverse the proliferation and migration of MRC-5 cells induced by TGF-B1, effectively improve inflammatory infiltration and destruction of alveolar tissue structure and collagen deposition in lung tissue, inhibit the expression of Fibronectin and Col-I proteins, significantly enhance the anti-pulmonary fibrosis effect, and provide new ideas for the preparation of anti-pulmonary fibrosis drugs.

[0022] 2. Small side effects: The Codonopsis pilosula extract prepared by the present invention has small side effects during the application of anti-pulmonary fibrosis, can effectively reduce the uneven feeling of patients during use, improve medication compliance, and enhance the continuity of treatment.

[0023] 3. Promote the modernization of traditional Chinese medicine: Separate and extract the extracts of Codonopsis pilosula (such as polysaccharides and small molecules), and explore the optimal ratio between these components, which provides a new method for the scientific research of traditional Chinese medicine components, promotes the modernization and refinement of traditional Chinese medicine, and at the same time, provides more effective drug options for the clinical treatment of pulmonary fibrosis.

[0024] 4. Economical and practical: The preparation method adopted by the present invention has the advantages of low cost and simple operation, and is suitable for large-scale production and clinical application. This economical and practical characteristic enables the Codonopsis pilosula extract to increase the market supply in the process of preparing anti-pulmonary fibrosis drugs, and can meet the clinical demand for effective, safe and affordable drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the process for preparing the components of Codonopsis pilosula extract;

[0026] Figure 2 Schematic diagram of the evaluation of the drug combination index of small molecules and polysaccharides based on MRC-5 fibroblasts;

[0027] Figure 3 Schematic diagram of the inhibition rate of TGF-β1-induced MRC-5 cells by using Codonopsis pilosula small molecules, polysaccharides alone, and the combination of small molecules and polysaccharides at four different mass ratios;

[0028] Figure 4 Schematic diagram of the Edu fluorescence experiment to detect the effects of different components of Codonopsis pilosula extract on TGF-β1-induced MRC-5 cell proliferation;

[0029] Figure 5 Schematic diagram of the scratch test to detect the effects of different components of Codonopsis pilosula extract on TGF-β1-induced MRC-5 cell proliferation;

[0030] Figure 6 Schematic diagram of the effects of different components of Codonopsis pilosula extract on HE staining of rat lung tissue;

[0031] Figure 7 Schematic diagram of the effects of different components of Codonopsis pilosula extract on Masson staining of rat lung tissue;

[0032] Figure 8 This is a schematic diagram of the test results of different components of Codonopsis pilosula extract on the hydroxyproline content in rat lung tissue;

[0033] Fig. 9 Schematic diagram of immunohistochemical experiments to detect the effects of different components of Codonopsis pilosula extract on FN and COL-I proteins in rat lung tissue;

[0034] Fig.10 Schematic diagram of the Westren blot experiment to detect the effects of different components of Codonopsis pilosula extract on FN and COL-I proteins in rat lung tissue. DETAILED DESCRIPTION

[0035] In order to make the technical solution of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the specification of the present invention. It should be noted that the following embodiments are only used to better understand the technical solution of the present invention and should not be understood as a limitation to the present invention.

[0036] Example 1.

[0037] Preparation of effective components of Codonopsis pilosula:

[0038] S1: Take 1 kg of Codonopsis pilosula slices, add distilled water and soak for 1 hour, wherein the volume ratio of distilled water to Codonopsis pilosula slices is 1:8, then heat and reflux at 100°C for 2 times, each time for 1 hour, and combine the extracts and evaporate them to a concentration of 0.6 g / mL to obtain a concentrated extract;

[0039] S2: freeze-drying a portion of the concentrated solution obtained in step S1 to obtain a water extract component, adding ethanol to another portion of the concentrated solution until the ethanol concentration is 80%, subjecting the mixture to alcohol precipitation overnight, and filtering to obtain a supernatant and a precipitate;

[0040] S3: The supernatant obtained in step S2 is concentrated by rotary evaporation and added to a column filled with AB-8 macroporous resin. After adsorption for 24 hours, it is first eluted with 5 column volumes of pure water, and then eluted with 8 column volumes of 80% alcohol. The 80% alcohol eluate is collected, and then lyophilized by rotary evaporation to obtain a small molecule component; the precipitate is dried under reduced pressure to obtain a crude polysaccharide component. The schematic diagram of the process for preparing the components of Codonopsis pilosula is shown in the figure. Figure 1 shown.

[0041] Effect example 1.

[0042] Evaluation of the drug combination index and efficacy of Codonopsis pilosula small molecules and polysaccharides based on TGF-β1-induced human lung fibroblasts MRC-5:

[0043] Human lung fibroblasts MRC-5 were purchased from Shanghai Fuheng Biotechnology Co., Ltd. (Product No.: FH0903); recombinant human TGF-β1 was purchased from Suzhou Jinan Protein Technology Co., Ltd. (Product No.: GMP-CA59). The following are the specific implementation steps:

[0044] G1: Human fibroblast MRC-5 cells in logarithmic growth phase were selected with a cell density of 80% (cell number of 4×10 6), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 1 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 16:1. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 2.6281, indicating that the mass ratio of the small molecule component to the polysaccharide component showed an antagonistic effect at 16:1.

[0045] Table 1

[0046]

[0047]

[0048] G2: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, and the cell density was 80% (cell number was 4×10 6 ), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 2 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 8:1. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at different concentrations with the same mass ratio was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 3.2092, indicating that the mass ratio of the small molecule component to the polysaccharide component was 8:1 and showed an antagonistic effect.

[0049] Table 2

[0050]

[0051]

[0052] G3: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 3 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 4:1. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 3.2416, indicating that the mass ratio of the small molecule component to the polysaccharide component was 4:1 and showed an antagonistic effect.

[0053] Table 3

[0054]

[0055]

[0056] G4: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 4 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 2:1. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 2.4419, indicating that the mass ratio of the small molecule component to the polysaccharide component was 2:1 and showed an antagonistic effect.

[0057] Table 4

[0058]

[0059]

[0060] G5: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and 2 mL of a mixture of 2 mg / mL of small molecule components and 2 mg / mL of polysaccharide components (the mass ratio of small molecule components to polysaccharide components was 1:1) was added thereto. When TGF-β1 was added for 24 hours, the cell activity was detected by CCK-8, and the inhibition rate was calculated to be 34.22%. In addition, small molecules and polysaccharide components of different concentrations as shown in Table 5 were added thereto, wherein the mass ratio of small molecule components to polysaccharide components was 1:1. When TGF-β1 was added for 24 hours, the cell activity was detected by CCK-8, and the inhibition rate at different concentrations of small molecules and polysaccharide components at the same mass ratio was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 0.3257, indicating that the mass ratio of the small molecule component to the polysaccharide component showed a synergistic effect when it was 1:1.

[0061] Table 5

[0062]

[0063]

[0064] G6: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 6 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 1:2. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 0.8409, indicating that the mass ratio of the small molecule component to the polysaccharide component showed a synergistic effect at 1:2.

[0065] Table 6

[0066]

[0067]

[0068] G7: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 7 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 1:4. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 0.6876, indicating that the mass ratio of the small molecule component to the polysaccharide component showed a synergistic effect at 1:4.

[0069] Table 7

[0070]

[0071]

[0072] G8: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, and the cell density was 80% (cell number was 4×10 6 ), followed by digestion, passage, counting, and then inoculation into 96-well plates and continued cultivation in a cell culture incubator. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 8 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 1:8. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 0.2560, indicating that the mass ratio of the small molecule component to the polysaccharide component showed a synergistic effect at 1:8.

[0073] Table 8

[0074]

[0075]

[0076] G9: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6), then digested, passaged, counted, and then inoculated into a 96-well plate and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 9 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 1:16. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 2.1599, indicating that the mass ratio of the small molecule component to the polysaccharide component showed an antagonistic effect at 1:16.

[0077] Table 9

[0078]

[0079]

[0080] G10: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), then digested, passaged, counted, and then inoculated into a 96-well plate and placed in a cell culture incubator for further culture. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 10 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 1:32. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 2.2326, indicating that the mass ratio of the small molecule component to the polysaccharide component showed an antagonistic effect at 1:32.

[0081] Table 10

[0082]

[0083]

[0084] G11: Human fibroblast MRC-5 cells in logarithmic growth phase were taken, with a cell density of 80% (cell number of 4×10 6), then digested, passaged, counted, and then inoculated into a 96-well plate and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 11 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 1:64. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 2.2856, indicating that the mass ratio of the small molecule component to the polysaccharide component showed an antagonistic effect at 1:64.

[0085] Table 11

[0086]

[0087]

[0088] G12: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), then digested, passaged, counted, and then inoculated into a 96-well plate and placed in a cell culture incubator for further culture. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and small molecules and polysaccharide components of different concentrations as shown in Table 12 were added thereto, respectively, wherein the mass ratio of the small molecule component to the polysaccharide component was controlled to be 1:128. When the time of adding TGF-β1 was 24 h, the cell activity was detected by CCK-8, and the inhibition rate of the small molecule and polysaccharide components at the same mass ratio and different concentrations was calculated. The above concentrations and inhibition rates were imported into CompuSyn software to evaluate the drug combination index CI value, and it was finally concluded that Fa was 0.5 and CI was 3.0979, indicating that the mass ratio of the small molecule component to the polysaccharide component showed an antagonistic effect at 1:128.

[0089] Table 12

[0090]

[0091]

[0092] According to the above effect examples 1G5 to G8, when the mass ratio of the small molecule to the polysaccharide mixture is 1:1, 1:2, 1:4 and 1:8, as shown in Table 13, the CI values ​​are all less than 1, proving that there is a synergistic effect between the small molecule and the polysaccharide mixture. Figure 2Shown is a schematic diagram of the evaluation of the drug combination index of small molecules and polysaccharides based on MRC-5 fibroblasts as described in Effect Examples 1G1 to G12.

[0093] Table 13

[0094]

[0095]

[0096] Effect example 2.

[0097] Evaluation of the efficacy of various components of Codonopsis pilosula and the synergistic effects between the components based on TGF-β1-induced human lung fibroblasts MRC-5 cells:

[0098] BeyoClick TM The EdU-594 cell proliferation detection kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd. (Product No.: C0071L). The specific implementation steps are as follows:

[0099] P1: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for further culture. When the cell density reached 70% (6000 cells inoculated in each well), 10ng / mL TGF-β1 was used to stimulate the cells, and 2mL of a small molecule group with a concentration of 0.4mg / mL was added to intervene in the cells with a crude drug amount (20mg / mL), which was the small molecule group (CS). When TGF-β1 was added for 24h, cell activity was detected by CCK-8, and the calculated inhibition rate was 25.53%.

[0100] P2: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for further culture. When the cell density reached 70% (6000 cells inoculated in each well), 10ng / mL TGF-β1 was used to stimulate the cells, and 2mL of a polysaccharide group with a concentration of 3.2mg / mL was added to intervene in the cells with a crude drug amount (20mg / mL), which was the polysaccharide group (CP). When TGF-β1 was added for 24h, the cell activity was detected by CCK-8, and the calculated inhibition rate was 2.63%.

[0101] P3: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6000 cells inoculated in each well), 10 ng / mL TGF-β1 was used to stimulate the cells, and 2 mL of 11 mg / mL Codonopsis pilosula water extract was added to the cells. The crude drug amount (20 mg / mL) intervened in the cells. This group was the water extract group (CWE). When TGF-β1 was added for 24 hours, the cell activity was detected by CCK-8, and the calculated inhibition rate was 31.96%.

[0102] P4: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6,000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and 2 mL of a mixture of 2 mg / mL of small molecule components and 2 mg / mL of polysaccharide components was added (the mass ratio of small molecule components to polysaccharide components was 1:1). When TGF-β1 was added for 24 hours, cell activity was detected by CCK-8, and the calculated inhibition rate was 34.22%.

[0103] P5: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, with a cell density of 80% (cell number of 4×10 6 ), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6,000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and 2 mL of a mixture of a small molecule component with a concentration of 1.33 mg / mL and a polysaccharide component with a mass ratio of 1:2 was added thereto. When TGF-β1 was added for 24 hours, the cell activity was detected by CCK-8, and the calculated inhibition rate was 26.25%.

[0104] P6: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, and the cell density was 80% (cell number 4×10 6), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6,000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and 2 mL of a mixture of 0.8 mg / mL small molecule components and 3.2 mg / mL polysaccharide components (the mass ratio of small molecule components to polysaccharide components was 1:4) was added to the cells. When TGF-β1 was added for 24 hours, the cell activity was detected by CCK-8, and the calculated inhibition rate was 31.40%.

[0105] P7: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, and the cell density was 80% (cell number 4×10 6 ), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6000 cells were inoculated in each well), 10 ng / mL of TGF-β1 was used to stimulate the cells, and 2 mL of a mixture of 0.44 mg / mL small molecule components and 3.56 mg / mL polysaccharide components (the mass ratio of small molecule components to polysaccharide components was 1:8) was added to the cells. This group was the CPS group. When TGF-β1 was added for 24 hours, the cell activity was detected by CCK-8, and the calculated inhibition rate was 37.93%.

[0106] P8: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, and the cell density was 80% (cell number 4×10 6 ), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6,000 cells inoculated per well), the cells were stimulated with dimethyl sulfoxide, and this group was the blank group.

[0107] P9: Human fibroblast MRC-5 cells in logarithmic growth phase were selected, and the cell density was 80% (cell number 4×10 6 ), then digested, passaged, counted, and then inoculated into 96-well plates and placed in a cell culture incubator for continued culture. When the cell density reached 70% (6,000 cells inoculated per well), 10 ng / mL TGF-β1 was used to stimulate the cells, and this group was the model group.

[0108] According to Effect Example 2P1 to P9, as shown in Table 14, CS, CP components and components with different mass ratios of small molecules and polysaccharides (1:1, 1:2, 1:4, 1:8) can reverse the proliferation of MRC-5 cells induced by TGF-β1. Figure 3As shown in the results, CS, CP, CWE and CPS components were able to reverse the proliferation of MRC-5 cells induced by TGF-β1, and in the CPS component, a significant synergistic effect was shown when the mass ratio of small molecules to polysaccharides was 1:8. At the same time, the actual ratio of small molecules to polysaccharides in the water extract was close to 1:8, so we chose a mixture composed of a mass ratio of small molecules to polysaccharides of 1:8 for subsequent experiments.

[0109] Table 14

[0110]

[0111] Cell proliferation detection (Edu):

[0112] After human fibroblast MRC-5 cells were treated with the drugs and other stimulations described in Effect Example 2P1 to P9, Edu working solution was added to make its concentration in the culture medium 1x, and the cells were incubated for 2 hours. After Edu labeling was completed, the culture medium was discarded, and 4% paraformaldehyde was added to fix at room temperature for 15 minutes. Then, the cells were washed 3 times with washing solution, each time for 5 minutes. The washing solution was discarded, PBS containing 0.3% TritonX-100 was added, and the cells were incubated at room temperature for 15 minutes. The click reaction solution was added and incubated for 30 minutes. The results are shown in the figure. Figure 4 As shown, compared with the blank group, the model group could significantly promote cell proliferation after administration of TGF-β1, while the CS, CP, CWE and CPS groups could reverse the cell proliferation induced by TGF-β1, among which the inhibitory effect of the CPS group was more significant, proving that the mixture group with a mass ratio of small molecule components to polysaccharide components of 1:8 showed excellent synergistic effect.

[0113] Cell scratch assay:

[0114] Human fibroblast MRC-5 cells in logarithmic growth phase were selected, and the cell density was 80% (cell number was 4×10 6 Then, the cells were digested, passaged, counted, and inoculated into 96-well plates and placed in a cell culture incubator for further culture. When the cell density reached 70% (3×10 cells were inoculated per well), 5 ), use a 200 μL pipette tip to draw a line in the central area of ​​cell growth to remove the cells in the central part, and then treat with drugs and other stimulations as described in Effect Example 2P1 to P9, culture for 24 hours, take out the cells, and observe under a microscope whether the surrounding cells migrate to the central scratch area. Figure 5Schematic diagram of the scratch experiment to detect the effects of different components of Codonopsis pilosula on TGF-β1-induced MRC-5 cell proliferation. The results of the scratch experiment showed that compared with the blank group, the model group could significantly promote the migration rate of cells after administration of TGF-β1, while the CS, CP, CWE and CPS groups could reverse the migration of cells induced by TGF-β1, among which the inhibitory effect of the CPS group was more significant.

[0115] Effect example 4.

[0116] Evaluation of the improving effects of different components of Codonopsis pilosula on bleomycin-induced pulmonary fibrosis in rats:

[0117] Male SD rats, weighing 220 ± 20 g, were purchased from Liaoning Changsheng Biotechnology Co., Ltd., with experimental animal license number: SCXK (Liao) 2020-0001; hydroxyproline (Hyp) ELISA kit was purchased from Shanghai Meike Biotechnology Co., Ltd., with product number: MK15110A.

[0118] R1: Before the operation, the rats were fasted for 12 hours and anesthetized with 20% urethane. The rats were fixed on a mouse board, the skin and fascia were cut longitudinally to expose the trachea, and 200 μL of bleomycin (5 mg / kg) was injected. 200 μL of normal saline was added to the blank control group. After the drug was injected, the mouse board was shaken upright from side to side for 1 to 2 minutes so that the drug could be evenly distributed after entering the lungs. The wound was sutured and the rat was waited for natural awakening.

[0119] R2: Rats were randomly divided into 6 groups, namely sham operation group (Control), model group (Model), water extract group (CWE), total polysaccharide group (CP), small molecule group (CS), small molecule: polysaccharide (1:8) mixture group (CPS); 7 days after film formation, oral administration began, and the dosage was converted according to the equivalent dose coefficient (the rat equivalent dose is 6.25 times that of adults, and the adult body weight is calculated as 70 kg). Referring to the maximum high dose of 2 times in the "Chinese Pharmacopoeia" (2010), the daily equivalent amount of Codonopsis pilosula raw medicine is 5.4g / kg, and each component of Codonopsis pilosula is administered according to its content in 5.4g of raw medicine. According to the yield conversion, the dosage of each group is: CWE group 0.54g / mL, CP group 0.17g / mL, CS group 0.02g / mL, CPS group (CS: 0.02g / mL, CP: 0.16g / mL), and the administration volume is 0.5 ml per 100g of rat.

[0120] R3: Use a blood collection tube to collect blood from the abdominal aorta, keep it at 4°C for 24 hours, then centrifuge it at 4°C (4000 rpm) for 10 minutes, collect the serum, and store it in a -80°C refrigerator.

[0121] R4: The rat lung tissue was removed and cleaned with normal saline, and the water was absorbed with filter paper. The upper lobe of the right lung was fixed in 4% paraformaldehyde for HE and Masson staining, and the remaining lung tissue was stored in a -80°C refrigerator for future use.

[0122] R5: Determination of hydroxyproline content in rat lung tissue. Hydroxyproline is unique to collagen fibers when pulmonary fibrosis occurs. The content of hydroxyproline reflects the degree of pulmonary fibrosis. Prepare lung tissue and use the hydroxyproline (Hyp) ELISA kit to determine the content of hydroxyproline in lung tissue. Follow the instructions.

[0123] R6: Fix the lung tissue in 4% paraformaldehyde for 72 hours, rinse the fixative with distilled water, dehydrate with different gradients of alcohol, and embed the tissue after transparentization with xylene. After embedding, slice the tissue with a semi-automatic rotary slicer, with a slice thickness of 4 μm, and bake the slide at 70°C for 1 hour.

[0124] R7: Lung tissue sections are dewaxed to water, and then stained after the printing is completed. The sections are placed in xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, 70% alcohol for 5 minutes, and distilled water for washing. Then add hematoxylin to stain for 8 minutes, rinse with tap water, differentiate with 1% hydrochloric acid alcohol for 10 seconds, rinse with tap water, return to blue with 0.6% ammonia water, and rinse with running water. Add eosin staining solution for staining for 3 minutes, and place the sections in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, toluene I for 5 minutes, and xylene II for 5 minutes to dehydrate and make them transparent. Take the sections out of xylene, dry them slightly, and seal them with neutral gum.

[0125] R8: Lung tissue sections were dewaxed and stained with Masson's Ponceau acid red solution for 10 minutes, immersed in 2% acetic acid aqueous solution for a while, differentiated in 1% phosphoric acid aqueous solution for 5 minutes, and then directly stained with aniline blue aqueous solution for 5 minutes after drying, dehydrated with alcohol gradient, transparentized with xylene I and II, and sealed with neutral gum.

[0126] R9: Dewax lung tissue slices to water, soak the tissue slices in 3% H2O2 for 20 minutes, rinse with PBS for 3 minutes, wash 3 times. Soak the slices in 0.3% Triton X-100 (diluted with PBS) for 15 minutes to break the membrane, rinse with PBS for 3 minutes, wash 3 times. Put the tissue slices in sodium citrate repair solution, heat and boil in a water bath for 15 minutes, cool to room temperature. Rinse with PBS for 3 minutes, wash 3 times. Add blocking solution for 30 minutes, add 25 μL of primary antibody (FN, COL-I; dilution ratio (1:200)) and incubate overnight at 4°C, add corresponding secondary antibody (secondary antibody is goat anti-rabbit 1:500), incubate at room temperature for 30 minutes, mix DAB colorimetric solution with 1:19 ratio, add it dropwise to the tissue, and let it stand for 10 seconds. If the nucleus is over-stained, use 1% hydrochloric acid alcohol differentiation solution for differentiation, and then rinse the slices with distilled water (anti-blue in water); dehydrate and dry with gradient alcohol from high to low, use xylene for transparent treatment for 20 minutes, and finally seal the slices with neutral resin glue.

[0127] R10: Weigh 0.1g of lung tissue, add 900μL of RIPA lysis buffer to extract the protein in the lung tissue, and use the kit for protein quantification. Add Loading Buffer and boil for 5min to denature the protein. Take 30μg for electrophoresis, react at 80V for 40min, then react at 120V for 50min, then wet transfer at 350mA for 90min, dilute GAPDH, Fibronectin and Col-I antibodies at a ratio of 1:1000, and incubate at 4℃ overnight. Dilute the secondary antibody at a ratio of 1:10000. The secondary antibody corresponding to GAPDH is goat anti-mouse, and the secondary antibody corresponding to Fibronectin and Col-I antibodies is goat anti-rabbit. Develop after incubation with the secondary antibody for 1 hour.

[0128] In summary, after 7 days of bleomycin-induced rat pulmonary fibrosis model, oral administration began, and after 4 weeks of continuous administration, the pathology of lung tissue, pulmonary fibrosis and related proteins were evaluated. The results of HE staining showed that pathological changes, inflammatory infiltration and alveolar tissue structure destruction occurred in rat tissues with pulmonary fibrosis. After the CS, CP, CWE and CPS components of Codonopsis pilosula were used to treat rat tissues with pulmonary fibrosis, corresponding improvements were obtained. Among them, after the CPS and CWE groups were used to treat rat tissues with pulmonary fibrosis, the pathological changes of rat lung tissue, inflammatory infiltration and alveolar tissue structure destruction were significantly improved, which was similar to the sham operation group ( Figure 6 ). Masson staining results showed that a large number of collagen fibers appeared in the lung tissue of rats with pulmonary fibrosis. After intervention with Codonopsis pilosula CS, CP, CWE and CPS components, the deposition of collagen in the lung tissue was significantly improved. Among them, CS, CWE and CPS components had a more significant effect on improving the lung tissue of rats with pulmonary fibrosis ( Figure 7). ELISA results showed that the hydroxyproline content in rats with pulmonary fibrosis was increased. After intervention with Codonopsis pilosula CS, CP, CWE and CPS components, the hydroxyproline content was reduced, and the CPS group showed a synergistic effect, which was consistent with the results of Massson ( Figure 8 ). The results of immunohistochemistry showed that the expression of fibronectin and collagen Col-I in rats with pulmonary fibrosis was significantly increased, and was significantly decreased after intervention with Codonopsis pilosula CS, CP, CWE and CPS components ( Fig. 9 ). In order to further verify the expression of fibrosis-related proteins in lung tissue, the expression of Fibronectin and Col-I proteins in lung tissue was detected by Westren blot. The results showed that the intervention of different components of Codonopsis pilosula could inhibit the expression of Fibronectin and Col-I proteins, and the small molecule and polysaccharide mixture group (1:8) showed a synergistic effect ( Fig.10 ).

Claims

1. Use of a Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug.

2. The use of the Codonopsis pilosula extract according to claim 1 in the preparation of an anti-pulmonary fibrosis drug, characterized in that: The codonopsis pilosula extract consists of a codonopsis pilosula water extract component, a small molecule component and a polysaccharide component.

3. The use of the Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug according to claim 2, characterized in that: The mass ratio of the small molecule component to the polysaccharide component is 1:(1-8).

4. The use of the Codonopsis pilosula extract according to claim 1 in the preparation of an anti-pulmonary fibrosis drug, characterized in that: The preparation method of the Codonopsis pilosula extract comprises the following steps: S1: crush the Codonopsis pilosula into powder, soak it in distilled water, heat it under reflux, combine the extracts and concentrate them by rotary evaporation to obtain a concentrated extract; S2: dividing the concentrated extract obtained in step S1 into two parts, freeze-drying one part of the concentrated solution to obtain a water extract component, and adding ethanol to the other part of the concentrated solution to precipitate overnight, and then filtering to obtain a supernatant and a precipitate; S3: The supernatant component obtained in step S2 is concentrated by rotary evaporation, added to a column filled with AB-8 macroporous resin for retention and adsorption, eluted with 5 times the column volume of distilled water, and then eluted with 8 times the column volume of 80% alcohol, the 80% alcohol eluate is collected, and rotary evaporation and freeze-dried to obtain a small molecule component; the precipitate is dried under reduced pressure to obtain a polysaccharide component.

5. The use of the Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug according to claim 4, characterized in that: The particle size of the powder in S1 is 30-50 mesh.

6. The use of the Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug according to claim 4, characterized in that: The volume of the distilled water in S1 is 7 to 9 times that of the Codonopsis pilosula decoction slice powder, and the soaking time is 0.5 to 1.5 hours.

7. The use of the Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug according to claim 4, characterized in that: The heating reflux temperature in S1 is 100°C.

8. The use of the Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug according to claim 4, characterized in that: The heating reflux times in S1 is 2 to 4 times.

9. The use of the Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug according to claim 4, characterized in that: The final concentration of alcohol in the alcohol precipitation in S2 is 70% to 90%.

10. The use of the Codonopsis pilosula extract in the preparation of an anti-pulmonary fibrosis drug according to claim 4, characterized in that: The adsorption time in S3 is retained for 24 hours.

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