Application of ginkgetin in treatment of chronic pulmonary obstruction

Abstract

The invention discloses application of ginkgetin in treatment of chronic pulmonary obstruction, and relates to the field of medical technical methods of ginkgetin. According to the invention, by constructing a model of cigarette extract on airway epithelial cell injury, the biflavone compound is found to have a protective effect on epithelial cell injury caused by smoking; through transcriptomics research, biflavone compounds such as ginkgetin and the like are found to have pharmacological activity of inhibiting CEBP beta protein high expression caused by cigarette extracts, so that the effect of protecting airway inflammation caused by smoking is achieved. More importantly, the ginkgetin and the glucocorticoid are jointly used, so that the treatment effect on chronic pulmonary obstruction is remarkably improved compared with that of independently using the ginkgetin and the glucocorticoid, and the ginkgetin and the glucocorticoid have wide medical application.

Description

TECHNICAL FIELD

[0001] The present application relates to the medical technical method field of ginkgo biloba biflavones, in particular to the application of ginkgo biloba biflavones in the treatment of chronic pulmonary obstruction. BACKGROUND

[0002] Chronic obstructive pulmonary disease (COPD) is a chronic bronchitis or emphysema with airflow obstruction, which can further develop into pulmonary heart disease and respiratory failure. Chronic obstructive pulmonary disease is related to abnormal inflammatory response of harmful gas and harmful particles, and has high morbidity and mortality. The global incidence rate of patients over 40 years old has reached 9-10%.

[0003] Smoking is the main environmental risk factor for chronic obstructive pulmonary disease and other airway inflammatory diseases. Many studies have shown that the incidence of chronic obstructive pulmonary disease and other diseases is directly related to smoking. Compared with non-smokers, smokers have a higher prevalence of respiratory symptoms and abnormal lung function, a faster annual decline in FEV1 (forced expiratory volume in one second), and a higher mortality rate. It has good practical significance to develop a compound that has protective effect on airway inflammation caused by smoking.

[0004] Although chronic obstructive pulmonary disease (COPD) has similar symptoms to asthma, the pathogenesis and clinical diagnosis of the two are completely different. According to the definition of the Global Initiative for Chronic Obstructive Pulmonary Disease (GOLD) in 2020, COPD is characterized by persistent respiratory symptoms and airflow limitation, which is the result of airway and / or alveolar abnormalities caused by excessive exposure to toxic particles or gases, and is irreversible. Asthma is an inflammatory disease of the airway mediated by various inflammatory cells, leading to reversible airflow obstruction. The main drug for treating asthma is glucocorticoids, but this drug has a general effect on COPD. Most studies, such as the tool book Respiratory Diseases (People's Medical Publishing House, 3rd edition, 2022; Chapter 4, page 43), or the literature "Association of Inhaled Corticosteroids With All-Cause Mortality Risk in Patients With COPD: A Meta-analysis of 60 Randomized Controlled Trials" (Chen, H., et al., https: / / doi.org / 10.1016 / j.chest.2022.09.144.), found that regular use of glucocorticoids alone cannot change the long-term downward trend of FEV1 or mortality in COPD patients. Therefore, long-acting bronchodilators combined with glucocorticoids are usually used in clinical treatment. However, bronchodilators are mostly β2 receptor agonists, and have no therapeutic effect on airway inflammation in COPD.

[0005] Biflavones are a class of compounds with a series of physiological activities such as anti-inflammatory, antioxidant, antimicrobial, anticancer, and neuroprotective effects. Studies have shown that biflavones in ginkgo biloba can inhibit airway mucus overexpression caused by elastase, thereby inhibiting airway inflammation. Amentoflavone can resist Staphylococcus aureus infection and inhibit lung inflammation caused by it, and also has a protective effect on acute lung injury in rats caused by lipopolysaccharide, and can inhibit the expression of key proteins TLR4 and NF-κB in the inflammation process and the levels of downstream inflammatory factors TNF-α, IL-6 and IL-1β. Amentoflavone can activate PPAR-α / γ, restore the expression of Arg-1 and Fizz1 genes, and inhibit the differentiation of macrophages M1 induced by lipopolysaccharide combined with recombinant human interferon-γ. Amentoflavone can inhibit the NF-κB and STAT3 inflammation pathways by regulating Nrf 2, to reduce the pulmonary inflammatory response induced by carrageenan, and has clinical potential for the treatment of non-infectious acute pleurisy and lung injury.

[0006] However, the above studies on biflavones are not based on a model corresponding to COPD, and do not essentially clarify whether biflavones have an effect on lung injury and inflammation caused by smoking-induced chronic obstructive pulmonary disease. In summary, no study has shown whether biflavones have a protective effect on airway inflammation caused by smoking-induced chronic obstructive pulmonary disease. SUMMARY

[0007] The present application first uses a mouse smoking model to find that biflavone extract has a significant therapeutic effect on airway inflammation and lung function decline caused by cigarette fumigation. Through a model of damage to airway epithelial cells by cigarette extract, it is found that biflavone compounds have a protective effect on epithelial cell damage caused by smoking. Through transcriptomic research, it is found that biflavone compounds such as ginkgetin have a pharmacological activity of inhibiting high expression of CEBPβ protein caused by cigarette extract, thereby having an effect of protecting airway inflammation caused by smoking. Based on the above research results, and based on the pathogenesis of COPD (caused by a large amount of exposure to toxic particles or gases), the present application provides a use of ginkgo biflavones in the treatment of chronic pulmonary obstruction, which is specifically implemented through the following technologies.

[0008] A use of ginkgo biflavones for preparing a drug for treating chronic pulmonary obstruction, or a use of ginkgo biflavones for treating chronic pulmonary obstruction without the purpose of disease diagnosis and treatment.

[0009] Further, the ginkgo biflavones include at least one of 7-demethylginkgetin, ginkgetin, isoginkgetin, and sciadopitysin.

[0010] Further, the ginkgo biflavones and glucocorticoids are used in combination.

[0011] Further, the glucocorticoids are at least one of dexamethasone, budesonide, beclometasone, and fluticasone.

[0012] Still further, the ginkgo biflavones and glucocorticoids are used in a ratio of (1-3):1.

[0013] Preferably, the ginkgo biflavones and glucocorticoids are used in a ratio of 2:1.

[0014] The present application also provides a combination of drugs for treating chronic pulmonary obstruction, which is composed of ginkgo biflavones and glucocorticoids.

[0015] Compared with the prior art, the present application has the following advantages:

[0016] 1、The application discloses a mechanism of ginkgo biloba biflavones, which is one of the mechanisms of the biflavones, that is, the biflavones can reduce airway inflammation caused by cigarette extract by down-regulating the CEBP / β inflammation signal pathway, thereby achieving the effect of treating chronic pulmonary obstruction. The mechanism is completely different from the mechanism of existing drugs. Therefore, after the biflavones and the glucocorticoid are used together, the airway inflammation and chronic pulmonary obstruction caused by smoking have better effects due to the complementary mechanism of the biflavones and the glucocorticoid.

[0017] 2、Compared with the glucocorticoid, the lung function index (0.1 second forced expiratory volume / mouse forced vital capacity, FEV0.1 / FVC) of a mouse with chronic obstructive pulmonary disease is significantly increased after the biflavones are administered, while the glucocorticoid has no such effect. The biflavones and the glucocorticoid are used together, so that the lung function decrease caused by smoking can be improved, and the airway inflammation of chronic pulmonary obstruction can be improved from different mechanisms by acting on different signal pathways.

[0018] 3、The biflavones have a protective effect on airway epithelial injury caused by smoking, while the glucocorticoid has no such effect. The biflavones and the glucocorticoid are used together, so that the airway epithelial injury caused by smoking can be better inhibited. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The figure is the evaluation result of the treatment effect of the biflavones on the airway inflammation and lung function decrease caused by cigarette fumigation in Example 1. Among them, Figure 1 A-C are respectively the changes of IL-6, IL-17A and TNF-α indexes of the mice in each group before and after modeling; Figure 1 D is the change result of the lung function index (0.1 second forced expiratory volume / mouse forced vital capacity, FEV0.1 / FVC) of the COPD mouse after the biflavones and the glucocorticoid are administered.

[0020] Figure 2 The figure is the tissue photo of the lung tissue of the mice in each group after the slices are dyed in Example 1.

[0021] Figure 3 The figure is the research result of the influence of the biflavones on the inflammation signal pathway in Example 2. The left graph is a gene heat map of the significant changes in transcriptomics before and after the biflavones are administered, and the right graph is a WB detection graph of the CEBP / β signal pathway before and after the biflavones are administered.

[0022] Figure 4 The figure is the determination result of the protective effect of the biflavones and the glucocorticoid on the airway epithelial cell injury caused by smoking in Example 3. Among them, Figure 4 A is the detection result of the protective effect of different biflavones on the CSE-treated airway epithelial cells, Figure 4B is the detection result of the protective effect of 4 glucocorticoids (dexamethasone, budesonide, beclomethasone and fluticasone) alone on CSE-treated airway epithelial cells, Figure 4 C is the detection result of the protective effect of ginkgetin combined with dexamethasone on CSE-treated airway epithelial cells.

[0023] Figure 5 is the detection result of the therapeutic effect of ginkgetin combined with glucocorticoids on airway inflammation and lung function decline caused by cigarette fumigation. Among them, Figure 5 A is the detection result of the lung function index (0.1 second forced expiratory volume / mouse forced vital capacity, FEV0.1 / FVC); Figure 5 B-D are the detection results of 3 airway inflammation factor indexes IL-6, IL-17A and TNF-α, respectively. DETAILED DESCRIPTION

[0024] The technical solutions of the present application will be described below in a clear and complete manner. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0025] Example 1: Ginkgetin has a therapeutic effect on airway inflammation and lung function decline caused by cigarette fumigation

[0026] 1. Establishment of test animal model

[0027] (1) After the purchased 6-week-old C57BL / 6 mice arrived, they were caged, 8 mice per cage; and were raised in an IVC independent air supply isolation cage.

[0028] During the entire modeling period, the mice were given water and feed at regular intervals every two days; and the bedding was replaced twice a week to ensure the cleanliness of the mouse cages.

[0029] The temperature in the breeding room was maintained at 20-25℃, and the humidity was maintained between 50-60%.

[0030] (2) After the mice were adaptively fed for one week, modeling began. The mice were placed in a specially designed cigarette fumigation box together with the mouse cages, and cigarettes (Hongjinlong brand cigarettes, tar content 10mg, smoke nicotine content 0.9mg, smoke carbon monoxide content 12mg, procurement source: Hubei China Tobacco Industry Co., Ltd.) were lit. 6 cigarettes were lit each time, and the fumigation time was 30min, and the fumigation was performed twice, a total of 1h; each day morning and evening, a total of 10 weeks.

[0031] The airway instillation of lipopolysaccharide was performed on the 1st, 14th, 28th and 42nd days of fumigation, and the concentration of the instilled lipopolysaccharide was 2 mg / ml, 30 μl per mouse. The specific operation method was as follows: the mouse was anesthetized by intraperitoneal injection of 3% sodium pentobarbital, and after the mouse was observed to be weak and muscle relaxed, the mouse was made to stand straight with the upper jaw teeth hung on a silk thread, and the mouse was fixed by pulling the tail; the lipopolysaccharide solution was instilled into the trachea by using a microsyringe, and the mouse was considered to be successfully instilled into the airway when the mouse showed a coughing phenomenon. The four days of instillation (i.e. the 1st, 14th, 28th and 42nd days) did not perform cigarette fumigation.

[0032] 2. Test of the treatment effect of ginkgo acetate extract on COPD mice

[0033] (1) Grouping

[0034] The blank group was not treated and was bred in a normal environment, and was recorded as the "NC group";

[0035] The model group was modeled by cigarette fumigation and airway instillation of lipopolysaccharide (LPS), and was recorded as the "M group";

[0036] The positive group was intraperitoneally injected with dexamethasone sodium phosphate 0.2 ml (specification 5 mL: 5 mg, i.e. the dosage was about 10 mg / kg), and the administration was started from the 40th day and lasted for one month until the end of modeling, and was recorded as the "PC group";

[0037] The biflavone mixed extract group was administered by gavage, and the drug was a biflavone mixed extract, and the biflavone content in the extract was 92.84% after pre-detection; among them, the 7-demethyl ginkgol content was 7.51%, the ginkgol content was 18.62%, the isoginkgol content was 26.96%, the taxifolin biflavone content was 39.25%, and the amentoflavone content was 0.5%. The dosage was 10 mg / kg, and the administration was started from the 40th day and lasted for one month until the end of modeling, and was recorded as the "EA group".

[0038] The biflavone monomer low-dose group was modeled by the same steps as the model group. Ginkgol monomer (HPLC≥98%) was administered by gavage, and the dosage was 10 mg / kg, and the administration was started from the 40th day and lasted for one month until the end of modeling, and was recorded as the "LBF group".

[0039] The biflavone mixed high-dose group was modeled by the same steps as the model group. Ginkgol monomer was administered by gavage, and the dosage was 40 mg / kg, and the administration was started from the 40th day and lasted for one month until the end of modeling, and was recorded as the "HBF group".

[0040] (2) Determination of the lung function of the mouse

[0041] Mouse lung function was measured using a small animal pulmonary function tester (FlexiVent; manufacturer: SCIREQ, Canada). After anesthesia, the mice were fixed on the pulmonary function tester, and the instrument software was activated to measure the mouse's lung function and obtain various lung function indicators.

[0042] (3) Determination of inflammatory factors in mouse alveolar lavage fluid

[0043] 24 hours after the completion of the pulmonary function test, mice were intraperitoneally injected with 3% sodium pentobarbital. After anesthesia, the trachea and lung tissues of the mice were dissected and alveolar lavage was performed.

[0044] The steps for alveolar lavage are as follows: 1.5ml of PBS solution is taken, 0.5ml of which is used for each alveolar lavage, and lavage is performed three times. During lavage, the lavage fluid is injected into the lung tissue along the trachea using a microsyringe to expand the lung, and the lavage fluid is then aspirated.

[0045] The lavage fluid was collected three times and centrifuged in a refrigerated centrifuge at 1500 rpm / min for 10 min. The supernatant was collected after centrifugation for cytokine detection. The remaining lung tissue was used for HE staining. ELISA kits (Wuhan Sewell Biotechnology Co., Ltd.) were used to detect various cytokines. The detection principle was a double antibody sandwich method. The specific operation steps were carried out according to the kit instructions. The OD value was determined using an enzyme-linked microplate reader, and the corresponding detection standard curve was established for the concentration calculation of each sample. The measured indicators included IL-6, IL-17A, and TNF-α.

[0046] (4) Mouse lung tissue staining

[0047] Mouse lung tissue was obtained, dehydrated and transparentized using a biological tissue dehydrator, embedded in paraffin, sliced ​​with a microtome, dewaxed, stained with hematoxylin solution for 2 minutes, stained with 1% water-soluble eosin solution for 3 minutes, washed, air-dried, sealed, and finally examined under a microscope.

[0048] (5) Test results

[0049] like Figure 1 As shown in AC, by detecting inflammatory factors in the alveolar lavage fluid after modeling and drug administration, it was found that the mouse IL-6, IL-17A, TNF-α and other indicators all increased significantly after modeling, proving that the modeling was successful. After dexamethasone administration, inflammatory factors decreased significantly, indicating that glucocorticoids have the effect of inhibiting airway inflammation. The administration of different ginkgo leaf extracts had a good inhibitory effect on airway inflammation in COPD mice. Both the mixed extract of biflavonoids and the single biflavonoids significantly reduced the inflammatory factor indicators in the alveolar lavage fluid, and the therapeutic effect of the biflavonoid extract showed a certain dose-dependent effect.

[0050] By comparing the results of the M group, PC group, EA group, LBF group and HBF group, we can see that Figure 1 As shown in Figure D, the lung function of COPD mice was measured, and the ratio of forced vital capacity (FVC) and forced expiratory volume in 0.1 seconds (FEV0.1), namely peak expiratory flow (PEF), and other parameters were used to evaluate their lung function. The experimental results showed that the ratio of FEV0.1 / FVC was significantly reduced after modeling, proving that long-term cigarette induction combined with airway instillation of lipopolysaccharide can significantly reduce the lung function of mice. After the administration of dexamethasone, the increase in lung function indicators did not increase significantly, indicating that intraperitoneal injection of glucocorticoids cannot improve the lung function of mice. After the administration of biflavonoid monomer, both the low-dose LBF group and the high-dose HBF group had a significant increase in lung function indicators, indicating that biflavonoids have the effect of improving the lung function of COPD model mice.

[0051] like Figure 2 As shown in the figure, different staining methods were used to evaluate inflammation and mucus secretion in the lung tissue of COPD mice after sectioning the mouse lung tissue. HE staining revealed that after modeling, the alveolar tissue of COPD model mice was significantly denser, indicating that the alveolar tissue wall thickness increased and the alveolar space became dense and narrow. After the administration of glucocorticoids, the density of the alveolar space was slightly restored, and a small amount of inflammatory infiltration was still visible ( Figure 2 (Indicated by the black arrow in the middle). However, after administration of ginkgo biloba flavonoids monomer, alveolar density was almost completely restored, comparable to that of the blank control group. This suggests that administration of different ginkgo biloba flavonoids during the final month of modeling has an inhibitory effect on lung pathological lesions.

[0052] Example 2: Effects of biflavonoids on the expression of pro-inflammatory transcription factor CEBPβ ​​protein induced by cigarette extract

[0053] Transcriptomics and Western-Blot were used to find out the mechanism by which biflavonoid compounds inhibited the effects of cigarette extracts.

[0054] 1. Cell modeling

[0055] A549 cells were incubated in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% anti-antibody. Cells were seeded at a density of 5,000 cells / well in appropriate culture plates and cultured for 24 hours before drug administration or modeling. For modeling, cells were treated with 10% CSE, and cell viability was measured 24 hours after treatment.

[0056] 2. Experimental Grouping

[0057] (1) Blank group: cells were inoculated and cultured in culture bottles for 24 h, and then starved with serum-free medium for 27 h. The cells were then detached with a small amount of trypsin, centrifuged, and the precipitate was washed with PBS solution for 3 times. After centrifugation, the PBS washing solution was poured out, and the cell precipitate was added to the cell RNA protection solution, which was stored at -20°C. The sample was sent for detection of the transcriptome. The blank group sample was made in 4 parallel.

[0058] (2) Model group: cells were inoculated and cultured in culture bottles for 24 h, and then starved with serum-free medium for 24 h. CSE was then added for modeling treatment, and the CSE concentration was 10%. After 3 h, the cells were detached with a small amount of trypsin, centrifuged, and the precipitate was washed with PBS solution for 3 times. After centrifugation, the PBS washing solution was poured out, and the cell precipitate was added to the cell RNA protection solution, which was stored at -20°C. The sample was sent for detection of the transcriptome. The model group sample was made in 4 parallel.

[0059] (3) Drug group: cells were inoculated and cultured in culture bottles for 24 h, and then starved with serum-free medium for 22 h. 250 μg / ml of biflavonoid extract was then added, and 2 h later, CSE was added for modeling treatment, and the CSE concentration was 10%. After 3 h, the cells were detached with a small amount of trypsin, centrifuged, and the precipitate was washed with PBS solution for 3 times. After centrifugation, the PBS washing solution was poured out, and the cell precipitate was added to the cell RNA protection solution, which was stored at -20°C. The sample was sent for detection of the transcriptome. The drug group sample was made in 4 parallel.

[0060] 2. Test process

[0061] In order to detect whether the biflavonoids inhibit the high expression of CEBPβ protein caused by smoking, Western-Blot experiment was carried out. The cell model was the same as in Example 2. The specific steps are as follows:

[0062] Before the cells were stimulated with 10% CSE for 2 h, the cells were treated with different concentrations of biflavonoid mixture (biflavonoid content was 92.84%, among which 7-demethyl ginkgoid content was 7.51%, ginkgoid content was 18.62%, isoginkgoid content was 26.96%, taxifolin biflavonoid content was 39.25%, and agathisflavone content was 0.5%) and ginkgoid (HPLC≥98%) at 50, 100 and 250 μg / ml.

[0063] Then the cells were treated with cell lysis solution RIPA buffer added with protease inhibitor PMSF, and the lysis solution was collected after being placed on ice for 30 min.

[0064] Finally, the protein concentration in the lysate was determined using a BCA protein assay kit. After protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The PVDF membrane was blocked with blocking solution at room temperature for 2 h. The primary antibody was incubated with the PVDF membrane at 4°C overnight. The membrane was washed with TBST (TWEEN-20 containing tri-phase buffered saline) three times for 5 min each time. The secondary antibody was added and incubated at 37°C for 2 h. The protein bands were detected using a dual-color infrared laser system (Odyssey CLx, LI-COR Inc., USA).

[0065] The experimental results are shown in Table 1. Figure 3 Using cell transcriptome analysis, it was found that after administration of biflavone extract, the high expression of inflammatory protein CEBPβ caused by cigarette extract was significantly reduced, and the expression of subsequent inflammatory chemokine CCL2 was down-regulated, thereby inhibiting the generation of airway inflammation. Western-Blot experiments further proved that after administration of ginkgo flavone monomers and biflavone mixtures, the high expression of pro-inflammatory transcription factor CEBPβ caused by cigarette extract could be inhibited, thereby reducing airway inflammation and playing a protective role. The mechanism of this inflammation inhibition is completely different from that of glucocorticoids, and there is no literature report that glucocorticoids can regulate airway inflammation through CEBPβ. Therefore, the combination of the two drugs has a synergistic effect in reducing airway inflammation.

[0066] Example 3: Determination of the protective effect of biflavone and glucocorticoid combination on airway epithelial cell damage caused by smoking

[0067] 1. Preparation of cigarette extract (CSE)

[0068] Take 3 cigarettes (Hongjinlong brand cigarettes, tar content 10 mg, smoke nicotine content 0.9 mg, smoke carbon monoxide content 12 mg, company: Hubei China Tobacco Industry Co., Ltd.), ignite at the cigarette combustion chamber, open the gas cylinder, and allow compressed air to flow in. The smoke of the fully burned cigarette is collected through the gas collection chamber, which is equipped with 300 ml of PBS solution. Ethanol is used to collect excess waste gas. After the PBS solution in the collection chamber is removed, the pH is adjusted to 7.4, and the 0.22 μm filter membrane is used to filter in the clean bench. The prepared cell culture medium is used for dilution and addition.

[0069] 2. Cell modeling and biflavone pharmacological activity research

[0070] The modeling method was the same as in Example 2. A549 cells were incubated in RPMI-1640 medium containing 10% fetal bovine serum and 1% bispecific antibody. Cells were seeded into appropriate culture plates at a density of 5,000 cells / well and cultured for 24 hours before dosing or modeling. Cells were treated with 10% CSE for modeling, and cell viability was measured 24 hours after treatment.

[0071] 3. Protective effects of different ginkgo biloba flavonoids and glucocorticoids on CSE-treated cells

[0072] The cell modeling process is as described in step 2 above. The drug was administered 2 h before CSE modeling.

[0073] The administration groups were as follows: different types of Ginkgo biloba flavonoids were administered alone: ​​the administration concentration range was 0-250 μg / ml (specifically 0, 10, 20, 50, 100 and 250 μg / ml).

[0074] Different types of glucocorticoids were administered alone: ​​the administration concentration range was 0-250 μg / ml (specifically 0, 10, 20, 50, 100 and 250 μg / ml).

[0075] Combination of Ginkgo biloba flavonoids and glucocorticoids: Ginkgo biloba flavonoids and dexamethasone were selected for combination use, with the dosage ratios being 1:1, 2:1 and 3:1.

[0076] The experimental results are attached. Figure 4 As shown. Treatment of airway epithelial cells with cigarette smoke extract had a damaging effect on the cells. However, administration of 100 or 250 μg / ml of biflavonoids significantly prevented the decrease in cell viability induced by CSE ( Figure 4 A), with good therapeutic effect. Administering glucocorticoids alone cannot prevent the decrease in cell viability induced by CSE ( Figure 4 B). When the two are used together at a ratio of 1:1, 2:1 and 3:1, they all have a better protective effect ( Figure 4 C).

[0077] Example 4: Ginkgo biloba flavonoids combined with glucocorticoids have therapeutic effects on airway inflammation and decreased lung function caused by cigarette fumigation

[0078] 1. Establishment of experimental animal model

[0079] Cigarette fumigation experiments were conducted using C57BL / 6 mice. Mice and their cages were placed in a custom-made cigarette fumigation chamber and the cigarettes were lit. Six cigarettes were lit per fumigation session, lasting 30 minutes, with two sessions totaling 1 hour. This was repeated daily, morning and evening, for 10 weeks. During this time, lipopolysaccharide was administered. The modeling method was the same as in Example 1.

[0080] 2. Effects of Ginkgo Biflavonoids on Airway Inflammation and Lung Function Indexes in COPD Mice (1) Grouping Blank group: No treatment was performed and the mice were raised under normal conditions, designated as the "NC group"; Model group: Cigarette fumigation plus airway instillation of lipopolysaccharide (LPS) was used to establish the model, designated as the "M group"; Glucocorticoids alone group: Dexamethasone sodium phosphate 0.2 ml (specification 5 mL: 5 mg, i.e., the dosage was approximately 10 mg / kg) was administered intraperitoneally, starting from the 40th day and lasting for 1 month until the end of the model; Ginkgo biloba combined with glucocorticoids group (1:1): Dexamethasone sodium phosphate 10 mg / kg was administered intraperitoneally, and ginkgo biloba (HPLC ≥ 98%) was administered orally at a dose of 10 mg / kg, starting from the 40th day and lasting for 1 month until the end of the model;

[0081] Ginkgetin and glucocorticoid combined administration group (2:1): dexamethasone sodium phosphate 10 mg / kg was intraperitoneally injected, and ginkgetin (HPLC ≥ 98%) was administered orally at a dose of 20 mg / kg, starting from the 40th day until the end of modeling, for 1 month;

[0082] Ginkgo biloba and glucocorticoid combined administration group (3:1): dexamethasone sodium phosphate 10 mg / kg was injected intraperitoneally, and ginkgo biloba (HPLC ≥ 98%) was administered by gavage at a dose of 30 mg / kg, starting from the 40th day and lasting for 1 month until the end of modeling.

[0083] (2) Determination of mouse lung function

[0084] Mouse lung function was measured using a small animal pulmonary function tester (FlexiVent; manufacturer: SCIREQ, Canada). After anesthesia, the mice were fixed on the pulmonary function tester, and the instrument software was activated to measure the mouse's lung function and obtain various lung function indicators.

[0085] (3) Determination of inflammatory factors in mouse bronchoalveolar lavage fluid

[0086] Twenty-four hours after the pulmonary function test, mice were anesthetized with an intraperitoneal injection of 3% sodium pentobarbital. Trachea and lung tissue were removed and bronchoalveolar lavage was performed. The OD value of the bronchoalveolar lavage fluid was measured using a microplate reader, and a standard curve was established for the calculation of the concentration of each sample. The assay indicators included IL-6, IL-17A, and TNF-α.

[0087] (4) Test results

[0088] Lung function indicators such as Figure 5As shown in A, it can be seen that the lung function of COPD mice cannot be significantly improved by glucocorticoid alone, but the lung function index of COPD mice can be significantly increased by combined administration of ginkgetin. When the administration ratio of ginkgetin to glucocorticoid is 2:1, the lung function recovery is the best, and the ratio is considered to be the optimal ratio.

[0089] The airway inflammation factor indexes are as follows: Figure 5 As shown in B-D, it can be seen that the airway inflammation of COPD mice can be significantly reduced by glucocorticoid alone, and the airway inflammation index of COPD mice can be further reduced and returned to the normal level by combined administration of ginkgetin. Compared with glucocorticoid alone, the combined administration of ginkgetin and glucocorticoid has a synergistic effect on the reduction of airway inflammation of COPD mice, which is related to the down-regulation of different inflammatory signaling pathways by the two. According to the experimental data, when the administration ratio of ginkgetin to glucocorticoid is 2:1, the airway inflammation index decreases more, and the ratio is considered to be the optimal administration ratio.

[0090] The above specific embodiments describe the implementation of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Whether the biflavones have a protective effect on inflammation caused by high expression of CEBPβ caused by other factors, whether other types of biflavones have the effects described in the patent, and whether all the embodiments need to be exhausted or not, cannot be determined here. Within the scope of the claims and technical concepts of the present application, the technical solutions of the present application can be modified and changed in many ways, and these simple modifications all belong to the protection scope of the present application.

Claims

1. An application of ginkgo biloba flavonoids, characterized in that: Used to prepare drugs for treating chronic pulmonary obstructive pulmonary disease, or for the treatment of chronic pulmonary obstructive pulmonary disease for purposes other than disease diagnosis and treatment.

2. Use according to claim 1, characterized in that, The ginkgo biloba flavonoids include at least one of 7-demethylginkgylcatechin, ginkgetin, isoginkgetin and ginkgylcatechin.

3. The use according to claim 1, characterized in that Combined use of ginkgo biloba flavonoids and glucocorticoids.

4. Use according to claim 1, characterized in that, The glucocorticoid is at least one of dexamethasone, budesonide, beclomethasone and fluticasone.

5. Use according to claim 3 or 4, characterized in that, The dosage of ginkgo biloba flavonoids and glucocorticoid is (1-3):

1.

6. Use according to claim 5, characterized in that, The dosage of ginkgo biloba flavonoids and glucocorticoid is 2:

1.

7. A pharmaceutical combination for the treatment of chronic pulmonary obstruction, characterized in that, It is composed of ginkgo biloba flavonoids and glucocorticoids.

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