Flavonoid aglycone composition for treating chronic obstructive pulmonary disease and its application

Abstract

The present invention discloses a flavonoid aglycone composition for treating chronic obstructive pulmonary disease. The flavonoid aglycone composition includes maackiain and tectorigenin, and the mass ratio of maackiain to tectorigenin is 7:1 to 3:2. The present invention also discloses the application of the flavonoid aglycone composition in the medicine for treating chronic obstructive pulmonary disease. The present invention combines maackiain in the genuine medicinal material Sophora flavescens and tectorigenin in Belamcanda chinensis, and has the effect of treating chronic obstructive pulmonary disease. The contents of these two flavonoid aglycones, maackiain and tectorigenin, in their medicinal materials are very high and are easily obtained. Therefore, developing them into drugs for treating COPD has great clinical and market value and is worthy of vigorous promotion and application.

Description

Technical Field

[0001] The present invention belongs to the technical field of medicine. More specifically, the present invention relates to a flavonoid aglycone composition for treating chronic obstructive pulmonary disease and its application. Background Art

[0002] Chronic obstructive pulmonary disease (abbreviated as COPD) is a common and frequently-occurring disease in respiratory system diseases. The symptoms include dyspnea, cough and / or expectoration. Its etiological characteristics are progressive airflow obstruction and continuously aggravated airflow limitation, accompanied by abnormal and persistent inflammatory reactions of the lungs or airways to harmful gases and particles. Smoking and exposure to cigarette smoke are the main causes of COPD, but air pollution and other exposures also play important roles in the occurrence and development of the disease. The incidence rate of COPD in people over 40 years old in China reaches 13.7%, and the number of patients is about 100 million, ranking the second in the disease economic burden in China. Due to problems such as the insufficient total amount and uneven distribution of medical resources, and the insufficient understanding of COPD by doctors and patients, the current situation of COPD prevention and treatment at the grass-roots level is not optimistic. It is urgent to pay attention to the acute exacerbation of COPD, especially women and patients aged ≥65 are more likely to be affected by air pollutants and their conditions are aggravated. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2020 edition predicts that by 2060, more than 5.4 million people may die from COPD and its complications every year. Although COPD is a disease with high incidence, high mortality and high disability rate, COPD is still a disease that can be prevented and treated, and is divided into stable-phase treatment and exacerbation-phase treatment.

[0003] Current treatment methods for COPD, including physical therapy and drug intervention, aim to relieve symptoms and acute exacerbations, delay the decline of lung function, and improve quality of life. The main treatment drugs include antibiotics, glucocorticoids, theophyllines, anticholinergics, β2-receptor agonists, etc. COPD patients have multiple comorbidities. According to the patient's condition, combination drug use or combined non-drug treatment such as oxygen therapy can delay the progression of the disease and mortality. Modern medical treatment drugs can improve clinical symptoms, improve lung function, delay the progression of the disease, and improve quality of life, etc., but there are also a series of adverse reactions such as headache, nausea, vomiting, dry mouth, diarrhea, arrhythmia, rash, urinary retention, etc. Moreover, the abuse of antibiotics also leads to ecological imbalance or disorder of the intestinal flora or the release of endotoxin from bacterial cell lysis during the evolution of COPD, thus causing a series of pathological changes. Traditional Chinese medicine has a long history in the treatment of COPD and has the characteristics of definite curative effect, few adverse reactions, and long-lasting curative effect. Traditional Chinese medicine believes that the main pathological changes of COPD are deficiency in origin and excess in superficiality, with deficiency of healthy qi as the main pathogenesis. The deficiency of healthy qi is the deficiency of the lungs, spleen, and kidneys, and the excess in superficiality is phlegm turbidity, retained fluid, and blood stasis, and the three are intertwined. Therefore, the main treatment methods for the acute exacerbation period of COPD include relieving exterior cold, warming yang to resolve fluid retention, drying dampness and resolving phlegm, clearing heat and resolving phlegm, promoting blood circulation to remove stasis, etc. In the stable period, it is mainly to tonify the lungs, spleen, and kidneys or dispel pathogenic factors. In recent years, the use of traditional Chinese medicine compounds and effective components of traditional Chinese medicine to treat COPD has become the focus of attention of traditional Chinese medicine practitioners. Traditional Chinese medicine preparations can well improve the immune disorder of patients and improve the lung function of patients. Traditional Chinese medicine such as Cordyceps sinensis, Ginkgo biloba, Sophora flavescens, Belamcanda chinensis, Platycodon grandiflorum, Scutellaria baicalensis, etc. are often used as components in traditional Chinese medicine preparations for the treatment of chronic obstructive pulmonary disease. However, the components that play a role in chronic obstructive pulmonary disease in these traditional Chinese medicine preparations are not clear. In order to further explore the pharmacological mechanism of traditional Chinese medicine acting on COPD and conduct basic research to clarify the pharmacological mechanism of traditional Chinese medicine components acting on COPD will have great clinical and market value for the development of drugs for the treatment of COPD. Summary of the Invention

[0004] One object of the present invention is to solve at least the above problems and provide at least the advantages described hereinafter.

[0005] One object of the present invention is to provide a flavonoid aglycone composition for the treatment of chronic obstructive pulmonary disease, which has a good therapeutic effect on chronic obstructive pulmonary disease.

[0006] To achieve these objects and other advantages of the present invention, there is provided a flavonoid aglycone composition for the treatment of chronic obstructive pulmonary disease, and the flavonoid aglycone composition includes maackiain and tectorigenin.

[0007] Preferably, the mass ratio of maackiain to tectorigenin is 7:1 to 3:2.

[0008] Preferably, both maackiain and tectorigenin are extracted from Chinese medicinal materials.

[0009] Preferably, maackiain is extracted from Sophora tonkinensis, and tectorigenin is extracted from Belamcanda chinensis.

[0010] Preferably, the method for extracting maackiain from Sophora tonkinensis comprises the following steps:

[0011] Sophora tonkinensis is crushed, and an ethanol solution with a mass 6 - 7 times that of Sophora tonkinensis is added for extraction at least once, with each extraction lasting 5 - 7 days. After the extraction liquid is filtered, the filtrates are combined, concentrated, and a concentrated ethanol extract of Sophora tonkinensis is obtained;

[0012] The concentrated ethanol extract of Sophora tonkinensis is extracted with ethyl acetate 1 - 5 times, the extraction liquids are combined, and concentrated to a paste-like state to obtain an extract paste of Sophora tonkinensis;

[0013] The extract paste of Sophora tonkinensis is dissolved in an ethanol solution, filtered, and the filtrate is separated and purified using a macroporous resin column. It is eluted successively with 3 column volumes of distilled water and a 75% ethanol solution. The water-washed part is discarded, the 75% ethanol eluate is collected, concentrated, and a crude extract of maackiain is obtained;

[0014] The crude extract of maackiain is dissolved in methanol, filtered, and the filtrate is separated and purified using Sephadex gel chromatography. A mixed solution of chloroform:methanol is used as the eluent for elution. The later part is recovered, concentrated, and when white solids adhere to the bottle wall, it is taken out and filtered. The filter residue is washed with a mixed solution of chloroform:methanol, and the washing solution is left overnight. Solids slowly precipitate at the bottom of the bottle or substances precipitate on the bottle wall, and maackiain is obtained.

[0015] Preferably, the method for extracting tectorigenin from Belamcanda chinensis comprises the following steps:

[0016] Belamcanda chinensis is crushed, and an ethanol solution with a mass 6 - 7 times that of Belamcanda chinensis is added for extraction at least once, with each extraction lasting 5 - 7 days. After the extraction liquid is filtered, the filtrates are combined, concentrated, and a concentrated ethanol extract of Belamcanda chinensis is obtained;

[0017] The concentrated ethanol extract of Belamcanda chinensis is extracted with ethyl acetate 1 - 5 times, the extraction liquids are combined, and concentrated to a paste-like state to obtain an extract paste of Belamcanda chinensis;

[0018] The extract paste of Belamcanda chinensis is dissolved in an ethanol solution, filtered, and the filtrate is separated and purified using a macroporous resin column. It is eluted successively with 3 column volumes of distilled water and a 75% ethanol solution. The water-washed part is discarded, the 75% ethanol eluate is collected, concentrated, and a crude extract of tectorigenin is obtained;

[0019] The crude extract of tectorigenin is dissolved in methanol, filtered, and the filtrate is separated and purified by Sephadex gel chromatography. A mixed solution of chloroform and methanol is used as the eluent for elution. The later eluted part is recovered, concentrated, and when yellow solid adheres to the bottle wall, it is taken out and filtered. The filter residue is washed with a mixed solution of chloroform and methanol, and the washing solution is left overnight. Solids slowly precipitate at the bottom of the bottle or substances precipitate on the bottle wall to obtain tectorigenin.

[0020] Use of the flavonoid aglycone composition as described above in the medicine for treating chronic obstructive pulmonary disease.

[0021] The present invention has at least the following beneficial effects: The present invention combines maackiain in Sophora subprostrata, a genuine medicinal material, and tectorigenin in Belamcanda chinensis, and has the effect of treating chronic obstructive pulmonary disease. The contents of the two flavonoid aglycones, maackiain and tectorigenin, in their medicinal materials are very high and are easily obtained. Therefore, developing them into drugs for treating COPD has great clinical and market value and is worthy of vigorous promotion and application.

[0022] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Description of the Drawings

[0023] Figure 1 It is a HE staining diagram of mouse lung tissue (200×); among them, A is the blank control group, B is the model control group, C is the high-concentration maackiain, D is the high-concentration tectorigenin, and E is the medium-concentration composition. Detailed Embodiments

[0024] The following further detailed description of the present invention is provided to enable those skilled in the art to implement it according to the description in the specification.

[0025] It should be understood that the terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0026] It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0027] A flavonoid aglycone composition for treating chronic obstructive pulmonary disease, wherein the flavonoid aglycone composition includes maackiain and tectorigenin.

[0028] In another technical solution, the mass ratio of maackiain to tectorigenin is 7:1 to 3:2.

[0029] In another technical solution, both maackiain and tectorigenin are extracted from Chinese medicinal materials.

[0030] In another technical solution, maackiain is extracted from Sophora tonkinensis, and tectorigenin is extracted from Belamcanda chinensis. Maackiain can also be extracted from Chinese medicinal materials such as Millettia speciosa Champ., Sophora japonica L. branches, and Uncaria rhynchophylla. Tectorigenin can also be extracted from Chinese medicinal materials such as Puerariae flos and Wisteria sinensis.

[0031] In another technical solution, the method for extracting maackiain from Sophora tonkinensis includes the following steps:

[0032] Sophora tonkinensis is crushed, and an ethanol solution with a mass 6 - 7 times that of Sophora tonkinensis is added for extraction at least once, with each extraction lasting 5 - 7 days. After the extraction solution is filtered, the filtrates are combined, concentrated, and a concentrated ethanol extract of Sophora tonkinensis is obtained;

[0033] The concentrated ethanol extract of Sophora tonkinensis is extracted with ethyl acetate 1 - 5 times, the extraction solutions are combined, and concentrated to a paste-like state to obtain an extract paste of Sophora tonkinensis;

[0034] The extract paste of Sophora tonkinensis is dissolved in an ethanol solution, filtered, and the filtrate is separated and purified using a macroporous resin column. It is eluted successively with distilled water 3 times the column volume and 75% ethanol solution. The water-washed part is discarded, the 75% ethanol eluate is collected, concentrated, and a crude extract of maackiain is obtained;

[0035] The crude extract of maackiain is dissolved in methanol, filtered, and the filtrate is separated and purified using Sephadex gel chromatography. A mixed solution of chloroform:methanol is used as the eluent for elution. The later eluted part is recovered, concentrated, and when white solids adhere to the bottle wall, it is taken out and filtered. The filter residue is washed with a mixed solution of chloroform:methanol, and the washing solution is left overnight. Solids slowly precipitate at the bottom of the bottle or substances precipitate on the bottle wall, and maackiain is obtained.

[0036] In another technical solution, the method for extracting tectorigenin from Belamcanda chinensis includes the following steps:

[0037] Belamcanda chinensis is crushed, and an ethanol solution with a mass 6 - 7 times that of Belamcanda chinensis is added for extraction at least once, with each extraction lasting 5 - 7 days. After the extraction solution is filtered, the filtrates are combined, concentrated, and a concentrated ethanol extract of Belamcanda chinensis is obtained;

[0038] The concentrated ethanol extract of Belamcanda chinensis is extracted with ethyl acetate 1 - 5 times, the extraction solutions are combined, and concentrated to a paste-like state to obtain an extract paste of Belamcanda chinensis;

[0039] The extract paste of Belamcanda chinensis is dissolved in an ethanol solution, filtered, and the filtrate is separated and purified using a macroporous resin column. It is eluted successively with distilled water 3 times the column volume and 75% ethanol solution. The water-washed part is discarded, the 75% ethanol eluate is collected, concentrated, and a crude extract of tectorigenin is obtained;

[0040] The crude extract of tectorigenin is dissolved in methanol, filtered, and the filtrate is separated and purified by Sephadex gel chromatography. A mixed solution of chloroform and methanol is used as the eluent for elution. The later eluted part is recovered, concentrated, and when yellow solids adhere to the bottle wall, it is taken out and filtered. The filter residue is washed with a mixed solution of chloroform and methanol, and the washing solution is left overnight. Solids slowly precipitate at the bottom of the bottle or substances precipitate on the bottle wall, obtaining tectorigenin.

[0041] I. Extraction, Purification and Identification of Maackiain and Tectorigenin

[0042] (I). Extraction, Purification and Identification of Maackiain

[0043] 1. Extraction and Extraction

[0044] Take 50 kg of Sophora subprostrata (SDG) medicinal materials, crush them respectively, extract twice with 6 - 10 times the amount of 75% ethanol for 5 - 7 days each time. After filtering the extraction solutions, they are combined and concentrated under reduced pressure to obtain a concentrated solution of the ethanol extract. The extraction rate is 17.78%. The concentrated ethanol extract is extracted 3 times with ethyl acetate with a volume ratio of 1:1. The extraction solutions are combined and concentrated under reduced pressure to recover the extraction solvent, and concentrated to a paste-like state to obtain the extract of Sophora subprostrata (SDGY).

[0045] 2. Separation

[0046] The SDGY extract is dissolved in a low-concentration ethanol solution of 30% or less, filtered, and the filtrate is further separated and purified using a D101 macroporous resin column. It is eluted successively with 3 column volumes of distilled water and 75% ethanol solution. The water-washed part is discarded, and the 75% ethanol eluate is collected, concentrated and the solvent is recovered to obtain the crude extract of maackiain (SDGY-A fraction).

[0047] 3. Purification and Identification

[0048] The SDGY-A fraction is separated and purified by Sephadex gel chromatography (Sephadex G20). After an appropriate amount of the SDGY-A fraction is dissolved in methanol and filtered, the filtrate is loaded onto the column. A mixed solution of chloroform and methanol with a volume ratio of 1:1 is used as the eluent to obtain 1, 2, 3,..., 20 fractions. Among them, approximately the later eluted part is recovered. When the solvent is concentrated and recovered, white solids adhere to the bottle wall. It is taken out and filtered, and the filter residue is washed with an appropriate volume of a mixed solution of chloroform and methanol with a volume ratio of 1:1. The washing solution is left overnight. Solids slowly precipitate at the bottom of the bottle or substances precipitate on the bottle wall. Through thin-layer chromatography detection and then high-performance liquid chromatography detection, if they are all impure substances, the monomer compound can be recrystallized and precipitated with a mixed solution of chloroform and methanol with different volume ratios, and the precipitated monomer compound is identified. The extract in Sophora subprostrata is maackiain, and the structure is as shown below:

[0049]

[0050] (II) Extraction, Purification and Identification of Tectoridin

[0051] 1. Extraction and Extraction

[0052] Take 50 kg of Belamcanda chinensis (L.) DC. (SG) medicinal materials, crush them, and extract them twice with 6 - 10 times the amount of 70 - 75% ethanol for 5 - 7 days each time. After filtering the extraction solutions, combine and concentrate them under reduced pressure to obtain a concentrated ethanol extract solution, with an extraction rate of 25.22%. Extract the concentrated ethanol extract solution three times with ethyl acetate at a volume ratio of 1:1, combine each extraction solution, concentrate it under reduced pressure, recover the extraction solvent, and concentrate it to a paste-like state to obtain the Belamcanda chinensis (SGY 2) paste.

[0053] 2. Separation

[0054] Dissolve the SGY 2 paste with a low-concentration ethanol solution of 30% or less, filter it, and further separate and purify the filtrate using a D101 macroporous resin column. Elute it successively with 3 column volumes of distilled water and 75% ethanol solution, discard the water-washed part, collect the 75% ethanol eluate, recover the solvent, and obtain the crude extract of tectoridin (SGY-A fraction).

[0055] 3. Purification and Identification

[0056] Separate and purify the SGY-A fraction by Sephadex G20 gel chromatography. After dissolving the SGY-A fraction with an appropriate amount of methanol, filter it, load the filtrate, and use a chloroform:methanol mixed solution with a volume ratio of 1:1 as the eluent to obtain 1, 2, 3,..., 20 fractions; among them, when approximately recovering the later part, when there is a yellow solid adhering to the bottle wall during the concentration and solvent recovery, take it out and filter it, wash the filter residue with an appropriate volume ratio of 1:1 chloroform:methanol mixed solution, let the washing solution stand overnight, and slowly precipitate solids at the bottom of the bottle or substances precipitate on the bottle wall. Detect it by thin-layer chromatography and then by high-performance liquid chromatography. If they are all impure substances, the monomer compound can be recrystallized and precipitated with a chloroform:methanol mixed solution in different proportions, and the precipitated monomer compound is identified. The extract in Belamcanda chinensis is tectoridin, and the structure is as follows:

[0057]

[0058] II. Effects of Maackiain and Tectoridin Composition on the Proliferation of Human Lung (Bronchial) Epithelial BEAS-2B Cells

[0059] 1.1 Preparation of Medicinal Liquids, Preparation of MTT Solution and Cell Culture

[0060] Preparation of the medicinal solution: Sophoraflavanone G and tectorigenin were dissolved in DMSO to form a suitable concentration as the stock solution, which was stored at -20°C and diluted to the required concentration before use; a medicinal solution of the composition with a mass ratio of Sophoraflavanone G to tectorigenin of 3:2 was used to prepare medicinal solutions of different concentrations.

[0061] Preparation of the MTT solution: Usually, the final concentration of MTT prepared is 5 mg / ml. It must be dissolved in phosphate buffered saline (PBS), and after preparation, it is filtered through a 0.22 μm filter membrane to remove bacteria in the solution and stored at 4°C in the dark. During the preparation and storage process, the container is wrapped with aluminum foil.

[0062] Cell culture: A cryopreservation tube containing 1 mL of human lung (bronchial) epithelial cell BEAS-2B cell suspension was quickly shaken and thawed in a 37°C water bath, and 5 mL of 10% fetal bovine serum RPMI-1640 complete medium was added and mixed evenly. Centrifuge at 1000 RPM for 5 minutes, discard the supernatant, add 4 - 6 mL of fetal bovine serum RPMI-1640 complete medium and blow evenly; then add all the cell suspension into the culture flask and culture overnight; change the medium the next day and check the cell density. If the cell density reaches 80% - 90%, subculture can be carried out. The specific operation of subculture is as follows: Discard the culture supernatant, wash the cells 1 - 2 times with PBS without calcium and magnesium ions, add 1 - 2 mL of digestive solution (0.25% Trypsin - 0.53 mM EDTA) into the culture flask, place it in a 37°C incubator for digestion for 1 - 2 minutes, then observe the cell digestion situation under a microscope. If most of the cells become round and detached, quickly take it back to the operating table, gently tap the culture flask a few times and then add more than 5 mL of complete medium containing 10% serum to terminate digestion. Gently pipette the cells, aspirate them after complete detachment, centrifuge at 1000 RPM for 8 - 10 minutes, discard the supernatant, add 1 - 2 mL of culture medium and blow evenly. Add culture medium at 5 - 6 ml / bottle, and divide the cell suspension into new bottles containing 5 - 6 mL of culture medium at a ratio of 1:5.

[0063] 1.2. Experimental grouping

[0064] Blank control group: 100 μL of RPMI-1640 complete medium and 100 μL of 0.1% DMSO solution.

[0065] Negative control group: 100 μL of cell suspension with a density of 2×10 4 cells / ml, and 100 μL of RPMI-1640 medium containing 0.1% DMSO was added.

[0066] Experimental group: 100 μL of cell suspension with a density of 2×10 4A cell suspension of [number] / ml and 100 μL of the composition medicated solutions with concentrations of 10, 20, 40, 80, 100, 150, and 200 μg / ml were added respectively.

[0067] 1.3. Experiment

[0068] Cells in the logarithmic growth phase were digested and resuspended into single cells with a concentration of 2×10 4 / ml. 100 μL was taken and inoculated into a 96-well plate according to the above grouping, and cultured in an incubator with saturated humidity at 37°C and 5% CO2 for 12 h. The cell morphology was observed. If the cell state was good, drugs with concentrations of 10, 20, 40, 80, 100, 150, and 200 μg / ml were given according to the grouping. Six replicate wells were set up and co-incubated in the incubator for 24 h. 20 μL of 5 mg / ml MTT was added. After incubating for 4 h in an incubator with saturated humidity at 37°C and 5% CO2, the supernatant was removed, 150 μL of DMSO was added, and it was oscillated on a micro oscillator for 10 min until the crystals were completely dissolved, and then the OD value was measured at 570 nm. The average value of the six parallel replicate wells was the result of one experiment. The experiment was independently repeated three times.

[0069] Cell viability (%) = (experimental group - blank group) / (negative group - blank group) × 100%

[0070] As can be seen from Table 1, after the composition medicated solution of maackiain and irigenin with a concentration range of 10 - 200 μg / mL acted on BEAS-2B cells, the cell proliferation rate was above 90%. It can be considered that the composition medicated solution of maackiain and irigenin had no obvious cytotoxicity to BEAS-2B cells within the set concentration range.

[0071] Table 1 Effects of composition medicated solutions with different concentrations on the proliferation of BEAS-2B cells (x±s, n = 3)

[0072]

[0073] II. Pharmacological activity study of the maackiain-irigenin composition on COPD

[0074] 1. Animal feeding and grouping

[0075] 1.1. Experimental animals

[0076] 275 SPF-grade ICR female mice, 6 - 8 weeks old, weighing 18 - 20 g, from the Medical Experimental Animal Center of Guangxi Medical University (Animal License Number: SCXK Gui 2014-0002).

[0077] 1.2. Experimental drugs

[0078] Maackiain was isolated, identified and obtained from Sophora subprostrata by the laboratory, and formulated into suspensions with dosages of 0.25 g / kg, 0.50 g / kg, and 1.0 g / kg with normal saline for storage at 4°C for later use.

[0079] Tectorigenin was isolated, identified and obtained from Belamcanda chinensis by the laboratory, and formulated into suspensions with dosages of 0.25 g / kg, 0.50 g / kg, and 1.0 g / kg with normal saline for storage at 4°C for later use.

[0080] The maackiain-tectorigenin composition was mixed in a ratio of 3:2, and formulated into suspensions with dosages of 0.25 g / kg, 0.50 g / kg, and 1.0 g / kg with normal saline for storage at 4°C for later use.

[0081] 1.3 Experimental grouping

[0082] After 1 week of adaptive feeding, 275 ICR female mice were randomly divided into 11 groups: the blank control group was group A, the model control group was group B, the high, medium, and low dose groups of maackiain were groups C1, C2, and C3 respectively, the high, medium, and low dose groups of tectorigenin were groups D1, D2, and D3 respectively, and the high, medium, and low dose groups of the composition were groups E1, E2, and E3 respectively, with 25 mice in each group.

[0083] 2. Modeling and administration methods

[0084] 2.1 Breeding conditions

[0085] Before and after administration, the experimental animals were housed separately in cages, fed with complete pellet feed, and allowed free access to water. The room temperature was 20 ± 2°C, and the humidity was 50% - 60%.

[0086] 2.2 Model group

[0087] 1) On the 1st day, 29th day, and 57th day, LPS solution (30 μg / 6 μL) was instilled into the respiratory tract of mice through the nasal cavity, and the administration dose was 1.5 mg / kg.

[0088] 2) From the 2nd to 84th day (except the 29th day and 57th day), continuous exposure to cigarette smoke was carried out. The specific operation method was as follows:

[0089] ① The mice were placed in an animal transport box with a ventilation window (60 cm × 45 cm × 20 cm), 25 mice in each box, and sealed with tape.

[0090] ② The animal transport box was placed in a glass fumigation box, and there was a circular ventilation hole with a diameter of about 2 cm at each diagonal side wall of the fumigation box to adjust the oxygen and air pressure states inside and outside the box.

[0091] ③ Light a cigarette, insert it into the self-made metal combustion rack, place it in the self-made glass fumigation box, and cover it; burn 10 cigarettes each time until they are completely burned out, 10 minutes each time, twice a day, with a 15-minute interval in between. Open the fumigation box cover for ventilation during the interval, 7 days a week, for a total of 12 weeks.

[0092] ④ After each cigarette smoke exposure ends, place the animal transport box into the transfer window, disinfect it with ultraviolet light for 15 minutes, and then put the mice back into the breeding cage.

[0093] 3) After the modeling is completed, starting from the 13th week, orally administer distilled water, 0.2 ml per day per mouse, for a total of 4 weeks.

[0094] 2.3 Administration group

[0095] The modeling operation is the same as that of the model group. Starting from the 13th week, orally administer 1.2 experimental drugs, 0.2 ml per day per mouse, for a total of 4 weeks.

[0096] 2.4 Blank control group

[0097] On the 1st day, the 29th day, and the 57th day, instill 0.9% saline solution (6 μL per mouse) into the respiratory tract of the mice through the nasal cavity, and raise them normally at other times. Starting from the 13th week, orally administer 0.9% saline solution, 0.2 ml per day per mouse, for a total of 4 weeks.

[0098] 3. Observation indicators and methods

[0099] 3.1 General status observation

[0100] The quality of life and body weight of the mice, etc.

[0101] 3.2 Collection of experimental animal samples and determination of experimental detection indicators

[0102] 3.2.1 Collection of bronchoalveolar lavage fluid, serum, and intestinal mucus specimens from mice

[0103] The collection of bronchoalveolar lavage fluid specifically includes the following steps:

[0104] (1) Anesthetize the mice by intraperitoneal injection of 2% pentobarbital (0.1 ml / 10 g), and fix the mice in the supine position on the operating table.

[0105] (2) Use ophthalmic scissors to cut the neck fur to expose the subcutaneous tissue, and bluntly separate the trachea.

[0106] (3) Make an incision in the horizontal direction at the distal end of the trachea, obliquely insert the tracheal connector of the intubation into the trachea in the centripetal direction, fix it with cotton thread, and then connect the ventilator to the tracheal connector. Connect a 1 ml syringe filled with sterile PBS to the tracheal intubation, and slowly inject for lavage.

[0107] (4) Slowly aspirate after each injection of 0.8 ml, repeat 3 times, and then put the lavage fluid into a 1.5 ml EP tube.

[0108] (5) Centrifuge the lavage fluid at 3000 rpm for 15 min, take the supernatant and store it at -80 °C for further measurement.

[0109] Collection of serum specifically includes the following steps:

[0110] (1) Cut the skin under the xiphoid process, cut the muscular layer along the bilateral costal arches to expose the thoracic cavity.

[0111] (2) Cut the ribs along the sternal margin to expose the heart and lungs. Insert a 1 ml syringe into the apex of the heart and draw blood from the four chambers of the heart, about 0.8 - 1 ml from each.

[0112] (3) Slowly inject it into a 1.5 ml EP tube, place it in a 37 °C water bath for 15 min, and then refrigerate it at 4 °C for 15 min. It can be seen that the blood sample is stratified.

[0113] (4) Centrifuge at 3000 rpm for 15 min, take the upper layer of serum and store it at -80 °C for further measurement.

[0114] Collection of intestinal mucus specifically includes the following steps

[0115] (1) Open the abdominal cavity, find the pylorus and cecum, gently separate the mesentery and surrounding tissues, take the entire small intestine between the pylorus and cecum, and slowly inject and rinse the intestinal cavity with 10 ml of 0.01 M sterile PBS solution (pH = 7.4) using a syringe.

[0116] (2) Collect the obtained rinsing fluid into a 5 ml centrifuge tube, centrifuge at 3000 rpm for 15 min, take the supernatant and store it at -80 °C for further measurement.

[0117] 3.2.2 Collection and processing of morphological specimens of mouse lung tissue

[0118] The method for collecting morphological specimens is: Cut the distal end of the trachea, separate the surrounding adhered tissues, take out the lungs with forceps, process the left lung lobe as a morphological specimen, and store the right lung lobe in a cryopreservation tube in liquid nitrogen.

[0119] Processing of morphological specimens specifically includes the following steps:

[0120] 1) Fixation: Cover the left lung lobe with gauze, fix it with a paper clip, and place it in 10% formalin solution for fixation. Note that the specimen should be completely immersed to avoid floating on the liquid surface. After fixation for 24 h, take out the tissue and put the left lung lobe into a dehydration box in the fume hood.

[0121] 2) Gradient alcohol dehydration: 75% alcohol for 2 min; 85% alcohol for 2 min; 90% alcohol for 2 min; 95% alcohol for 2 min; absolute ethanol I for 2 min; absolute ethanol II for 2 min; alcohol-benzene for 2 min; xylene I for 2 min; xylene II for 2 min; paraffin I for 2 min; paraffin II for 2 min; paraffin III for 2 min.

[0122] 3) Embedding: The tissue after wax infiltration is embedded in an embedding machine. After the wax melts, it is poured into the embedding frame, and the left lung lobe is placed in it and labeled before solidification; then it is placed on a -20°C freezing table for cooling. After the wax solidifies, the wax block is separated from the embedding frame.

[0123] 4) Sectioning: The microtome is set to a section thickness of 5 μm. Let the sections float on the 43°C warm water surface of the microtome spreading table. After the tissue is flattened, pick it up with a glass slide and put it into an oven at 60°C for baking. Take it out after drying and store it at room temperature for later use.

[0124] HE staining, specifically including the following steps:

[0125] 1) Dewaxing to water: Place the mouse lung tissue sections in an oven at 60°C for 1 h. After the wax completely melts, place the sections in xylene I and II for 15 min each; absolute ethanol I and II for 2 min each; 95% alcohol I and II for 2 min each; 90% alcohol for 2 min; 80% alcohol for 2 min, 70% alcohol for 2 min, 60% alcohol for 2 min, 50% alcohol for 2 min, and gently rinse with tap water for 5 min.

[0126] 2) After hematoxylin staining for 15 min, wash off the excess stain with tap water.

[0127] 3) Differentiate with ethanol containing 1% hydrochloric acid for 30 s and wash with tap water for 10 min.

[0128] 4) Stain with 1% eosin solution for 10 min and wash off the excess stain with tap water for 1 min.

[0129] 5) Gradient alcohol dehydration: 50% alcohol for 2 min; 60% alcohol for 2 min; 70% alcohol; 80% alcohol; 90% alcohol for 2 min; 95% alcohol I for 2 min; 95% alcohol II for 2 min; 100% absolute ethanol I and II for 2 min each; xylene I and II for 10 min each; seal with neutral gum.

[0130] 6) Observe the changes in lung tissue and bronchial wall, as well as the infiltration of inflammatory cells under an optical microscope.

[0131] 3.2.3 Isolation and purification of lymphocytes between lung tissue and small intestinal epithelial cells

[0132] Intraperitoneal injection of heparin, specifically including the following steps:

[0133] 1) Heparin preparation: Specification: 12,500 IU / vial, working concentration: 100 IU / ml.

[0134] 2) Add 1 vial of heparin to 125 ml of 0.9% NaCl solution and mix well.

[0135] 3) After weighing the mice, draw heparin at a rate of 10 IU / ml / 10 g and inject it into the abdominal cavity from the left groin.

[0136] Intraperitoneal anesthesia, which specifically includes the following steps:

[0137] 1) Prepare 1% sodium pentobarbital: Weigh 100 mg of sodium pentobarbital and add it to a centrifuge tube containing 10 ml of distilled water, and dissolve and mix well.

[0138] 2) Use a 1 ml syringe to draw 1% sodium pentobarbital solution and administer it at a dose of 0.05 ml / 10 g body weight. Wait until there is no reflex when stimulating the mouse's eyelid, and collect the mouse lung tissue.

[0139] Isolation of lymphocytes from mouse lung tissue, which specifically includes the following steps:

[0140] 1) Use sterile ophthalmic forceps to put the lung tissue into a 50 ml centrifuge tube, and add 50 ml of PBS to wash it 3 times.

[0141] 2) Place the washed lung tissue in a sterile small petri dish (diameter 35 mm, depth 10 mm), and cut it into small pieces as much as possible with ophthalmic scissors. Place a 70 μm cell strainer on the centrifuge tube, and use a pipette to transfer the tissue to the strainer, and wash it 3 times with 1×Hanks balanced salt solution, and filter out the washing solution.

[0142] 3) Add 50 ml of RPMI1640 and 1 ml of PBS to a 50 ml centrifuge tube, add lung tissue fragments with a pipette, and then add 4200 μL of 100 mg / ml Collagenase NB and 40 μL of 10 mg / ml DNase to make the working concentrations 2 mg / ml and 40 μg / ml respectively.

[0143] 4) Incubate in a constant temperature water bath at 37°C for 90 min, and shake it with a shaker once every 10 min.

[0144] 5) Filter with a 70 μm cell strainer to filter out undigested tissue fragments and impurities.

[0145] 6) Use a pipette to take 15 ml of RPMI1640 to wash the cell strainer, transfer the filtrate and washing solution to a 50 ml centrifuge tube. Centrifuge at 4°C for 10 min at a speed of 2000 r / min.

[0146] 7) After centrifugation, gently aspirate and remove the supernatant with a pipette along the wall of the tube, taking care not to stir the cell layer. Leave 5 ml in the centrifuge tube, flick the bottom of the centrifuge tube with your finger to disperse the cell clumps at the bottom, and resuspend the cells.

[0147] 8) Add 3 ml of 100% percoll to a 15-ml centrifuge tube, then add 5 ml of the cell suspension, and then add 5 ml of 1×Hanks balanced salt solution containing 5% PBS. After mixing, the percoll content in the liquid is 30%. Centrifuge at 4°C for 18 min at 1800 r / min. After centrifugation, discard the supernatant, retain 1 ml of the solution (containing 0.3 ml of percoll), flick the bottom of the centrifuge tube to disperse the cell clumps, and add 4.1 ml of 100% percoll and mix well. Dilute to 10 ml with 1×Hanks solution containing 5% PBS. After mixing, the percoll content is 44%.

[0148] 9) Slowly add 2 ml of 70% percoll (by volume) to the bottom of a 15-ml centrifuge tube with a long pipette, and gently operate to form an interface between the two density percoll liquids. Centrifuge at 4°C, 1800 r / min for 18 min. A layer of cells can be seen aggregated at the interface between the 44% percoll and 70% percoll liquid levels. First, carefully discard the upper 1 / 3 of the liquid in the centrifuge tube along the wall with a pipette, and then aspirate the cells along the wall and transfer them into a prepared 50-ml centrifuge tube. Add 1×Hanks balanced salt solution containing 5% PBS to the centrifuge tube to make the total liquid volume 10 ml, and shake well. Centrifuge at 1500 r / min for 5 min at room temperature. After discarding the supernatant, add 2 ml of RPMI 1640 solution containing 10% PBS in 1×Hanks to prepare a lymphocyte suspension for testing. Add 4 g / L trypan blue for staining, and count with a cell counting chamber.

[0149] Isolation of intraepithelial lymphocytes in the small intestine of mice specifically includes the following steps:

[0150] 1) Place the entire small intestine between the pylorus and the cecum in a pre-cooled PBS solution on tin foil. Use a thin plastic tube to turn the intestinal tube into the intestinal lumen from one end of the small intestine, gradually pass the plastic tube out from the other end of the small intestine, and turn the entire intestinal lumen inside out, with the mucosal layer facing outwards and the serosal layer facing inwards.

[0151] 2) Carefully transfer the intestinal tube to a 50-ml centrifuge tube, and add RPMI 1640 containing 10% PBS and 100 mg / ml Collagenase NB4 200 μL, 10 mg / ml DNASe 40 μL, so that the working concentrations of Collagenase NB4 and DNASe reach 2 mg / ml and 40 μg / ml respectively.

[0152] 3) Place the centrifuge tube at 37 °C and centrifuge at 200 r / min for 60 min. Filter the digestion solution through a 70-μm cell strainer into a 50-ml centrifuge tube to remove undigested tissue fragments and impurities. Then centrifuge at 2000 r / min for 10 min at 4 °C. After centrifugation, gently aspirate the supernatant along the wall of the tube with a pipette, taking care not to stir the cell layer, and leave 5 ml in the centrifuge tube. Tap the bottom of the centrifuge tube with your finger to disperse the cell clumps and suspend the cells.

[0153] 4) Add 3 ml of 100% percoll to a 15-ml centrifuge tube, then add 5 ml of the cell suspension, and then add 2 ml of 1×Hanks solution containing 5% PBS. After mixing, the percoll content in the liquid is 30%. Centrifuge at 1800 r / min for 18 min at 4 °C. After centrifugation, discard the supernatant and leave 1 ml of the solution (containing 0.3 ml of percoll). Tap the bottom of the tube to disperse the cell clumps, mix well, and then add 4.1 ml of 100% percoll. Add 1×Hanks solution containing 5% PBS to make the total volume of the solution up to 10 ml. After mixing, the percoll content is 44%.

[0154] 5) Slowly add 2 ml of 70% percoll to the bottom of the centrifuge tube with a pipette. Gently operate to form an interface between the two density liquids by layering. Centrifuge at 1800 r / min for 18 min at 4 °C. At the junction of the 44% percoll and 70% percoll liquid surfaces, the cells can be seen to aggregate into a layer. First, carefully discard the upper 1 / 3 of the liquid in the centrifuge tube along the wall of the tube with a pipette, and then aspirate the cells along the wall of the tube and transfer them into a pre-prepared 50-ml centrifuge tube. Add 1×Hanks solution containing 5% PBS to make the total volume of the liquid up to 10 ml. After mixing, centrifuge at 1500 r / min for 5 min at room temperature. After discarding the supernatant, resuspend the cells with 2 ml of 1×Hanks solution containing 5% PBS to prepare a lymphocyte suspension for testing. Add 4 g / L trypan blue for staining and count with a cell counting chamber.

[0155] 3.2.4 Detection of the content of inflammatory factors in mouse serum, bronchoalveolar lavage fluid and intestinal mucus

[0156] Detection of the content of IL-6, which specifically includes the following steps:

[0157] 1) Equilibrate all samples to be tested and the detection kit to room temperature (18 - 25 °C).

[0158] 2) Prepare 50 ml of 1× Wash Buffer by mixing 5 ml of Wash Buffer concentrate (10×) with 45 ml of distilled water. Prepare an antibody mixture using antibody diluent, capture antibody, and detector antibody. Mix 300 μL of 10× Capture Antibody and 300 μL of 10× Detector Antibody with 2.4 ml of Antibody diluent to make 3 ml of antibody mixture, and gently mix well.

[0159] 3) Prepare standards: Add 500 μL of sample dilution saline to dissolve the lyophilized mouse IL-6 recombinant protein, dissolve and mix well, and gently mix at room temperature for 10 min to obtain a standard solution of 2000 pg / ml. Label 8 EP tubes as Standards 1 - 8. Add 150 μL of sample dilution saline to tubes numbered 1 - 8. Use the standard solution to prepare serial dilutions. Add 150 μL of the prepared 2000 pg / ml standard solution to tube No. 1 to make a 2000 pg / ml standard solution, then add 150 μL of the prepared 1000 pg / ml standard solution to tube No. 1 to make a 500 pg / ml standard solution, then add 150 μL of the prepared 500 pg / ml standard solution to tube No. 3 to make a 150 pg / ml standard solution, and so on to prepare 125, 62.5, 31.3, 15.6 pg / ml standard solutions in tubes 4, 5, 6, 7 in turn. Tube No. 8 is the blank control.

[0160] 4) Add samples: Add 50 μL of sample (serum, intestinal mucus) or standard to the appropriate wells, and then add 50 μL of antibody mixture to each well. Note to operate gently without generating bubbles.

[0161] 5) Incubate: Seal the plate with a film and incubate on a shaker set at 400 r / min at room temperature for 1 h.

[0162] 6) Wash the plate: Discard the liquid in the well plate, wash each well with 350 μL of 1× Wash Buffer, 3 times in total. After completion, invert the 96-well plate on a clean paper towel to remove excess liquid.

[0163] 7) Develop color: Add 350 μL of TMB substrate to each well and incubate on a shaker set at 400 r / min in the dark for 10 min. It can be observed that the liquid in the well plate turns blue.

[0164] 8) Terminate: Add 100 μL of stop solution to each well. After mixing, the blue liquid turns yellow.

[0165] 9) Set the microplate reader to measure the OD value at 450 nm.

[0166] 10) Calculation: Taking the OD value as the dependent variable Y and the standard product concentration as the independent variable X, calculate the standard equation, and then substitute the OD value of the measured sample into the equation to obtain the IL-6 concentration.

[0167] The detection of IL-13 content is the same as that of IL-6.

[0168] 4. Statistical methods

[0169] SPSS 13.0 software was used for processing. The experimental data were expressed as the mean plus or minus the standard deviation (x±S), and the pairwise comparison between means was performed using the T-test.

[0170] 5. Experimental results

[0171] 5.1 Observation results of general status

[0172] Blank control group: No mice died. The fur was shiny and smooth, the breathing was gentle, the frequency was moderate, the rhythm was uniform, the activities were normal, and the body weight gradually increased.

[0173] Model group: 10 mice in the chronic obstructive pulmonary disease (COPD) model group died. The hair was dull and dry, and some had hair loss. They were restless and jumped around during smoking. Later, most of them huddled together and even trembled all over. The breathing was rapid, the chest and abdomen fluctuated significantly, and there were irregular nodding movements from time to time. Even the breathing was open. The body weight was significantly lower than that of the normal group (P<0.001).

[0174] Drug administration group: 2, 4, and 5 mice died in the high, medium, and low dose groups of maackiain respectively, and 2 mice died in the high dose group of iriflavone and 4 mice died in each of the medium and low dose groups. Compared with the model group, the fur of the mice was shiny and smooth, the movement tended to be normal, the dyspnea was improved, and the body weight was significantly higher than that of the model group (P<0.001). 1 mouse died in each of the high and medium dose groups of the combination of maackiain and iriflavone, and 3 mice died in the low dose group. The general situation during modeling was the same as that of the COPD model group. After gavage, compared with the model group, the hair was smoother, the dyspnea was improved, the frequency slowed down, and the body weight of the high and low dose groups was significantly higher than that of the model group (P<0.001). The results are shown in Table 2.

[0175] Table 2 The combination drug can improve the survival rate and body weight of COPD model mice:

[0176]

[0177]

[0178] 5.2 Morphological pathological changes of lung tissue in COPD model mice

[0179] The pathological manifestations of the lung tissue in chronic obstructive pulmonary disease are as follows: degeneration, necrosis, and exfoliation of the bronchial mucosal epithelium, shortening and adhesion of cilia; hyperplasia and hypertrophy of goblet cells and mucous cells, massive mucus retention, congestion and edema of the tube wall, and narrowing of the tracheal lumen. Alveolar atrophy and collapse, rupture of the alveolar septum, and fusion of adjacent alveoli into large bullae. There is a large amount of chronic inflammatory cell infiltration around the trachea and alveoli, rupture and atrophy of the smooth muscle in the submucosal muscle layer, and proliferation of fibrous tissue; bronchial structural remodeling, increased collagen content, and scar formation. HE staining in this experiment showed that the airway and alveolar structures of the mice in the blank control group were normal, with neat cilia, regular shapes and sizes of alveoli, and intact airway mucosal epithelium( Figure 1 -A); in the model group, bronchial mucosal folds were formed and there were patchy exfoliations, causing the lumen to become narrow or occluded; the alveolar wall was damaged, the alveolar lumen was irregularly enlarged, and some were fused into large bullae, and chronic inflammatory cell infiltration was visible around the airway and in the lung interstitium, which was in line with the pathological manifestations of chronic obstructive pulmonary disease( Figure 1 -B).

[0180] When comparing the three groups of high-concentration maackiain, high-concentration tectorigenin, and medium-concentration combined drugs with the model control group, improvements in morphology were visible, manifested as relatively intact bronchial and alveolar structures, reduced degree of lumen stenosis, relatively intact airway mucosal epithelium, regular arrangement and reduced exfoliation of cilia, relatively uniform alveolar sizes, reduced number of large bullae, and reduced degree of inflammatory cell infiltration around the airway wall and in the lung interstitium( Figure 1 -C, D, E).

[0181] 5.3 Ratio of αβT / γδT cells in the lung tissue of chronic obstructive pulmonary disease model mice

[0182] γδT cells are key effector cells of the respiratory mucosal immune system and are involved in the inflammation and injury repair processes of chronic inflammatory diseases. In the normal mouse lung, most αβT cells are distributed in the lung parenchyma, while most γδT cells are distributed in the non-alveolar area except the mucosa. The relative density of γδT cells is the highest near the respiratory tract, blood vessels, and visceral pleura. Although the number of γδT cells is much smaller, their relative density in the non-alveolar area matches or is almost the same as that of αβT cells. On the contrary, in the lung parenchyma area, which is the largest in terms of tissue mass or tissue surface area, the relative density of γδT cells is much lower than that of αβT cells. The difference in the distribution of these two types of T cells may be related to their different functional roles. The single-region distribution of αβT cells in the normal lung may reflect a certain degree of functional homogeneity, while the extensive distribution of γδT cells reflects functional heterogeneity. Throughout the lung, γδT cells mainly contact myeloid cells, while αβT cells contact lymphocytes more. This comparison shows that the contact between leukocytes and lung γδT cells mainly involves myeloid cells, including F4 / 80 + macrophages and I-A + dendritic cells, while αβT cells often contact CD45R +Lymphocytes, including B cells, also have contact with some T cells and plasmacytoid dendritic cells.

[0183] In the immunohistochemical staining experiment of lung tissue morphology, cells were labeled and counted with αβTCR and γδTCR. In the blank control group, a small amount of both types of cells were found in the lung tissue; almost no γδT cells were seen in the model control group; in the high-, medium-, and low-dose drug groups, more γδT cells were found near the trachea, showing a significant increase compared with the model group. The ratio of αβT / γδT cells in the lung tissue of mice increased in the model group compared with the blank control group, showed a decreasing trend in the drug administration groups compared with the model group, and showed a significant decrease in the combined composition drug administration group compared with the single drug administration group, as shown in Table 3.

[0184] Referring to the method for separating intraepithelial lymphocytes of the intestine, we explored the experimental method for separating lung tissue lymphocytes by density gradient centrifugation. The method for separating intraepithelial lymphocytes of the small intestine is relatively mature and is recognized by the academic community. Moreover, the types of cells in the intestinal mucosal epithelium are relatively few, which is conducive to separating different cell populations. Referring to the flow cytometry analysis results of intestinal samples, the lymphocyte population in the cell suspension obtained after digestion and separation of lung tissue can be determined, and the αβT cells and γδT cells in it can be labeled with specific antibodies respectively, as shown in Table 4. The proportion of γδT cells in mice showed a decreasing trend in the model group compared with the blank control group, and showed a significant increase in the low-dose combined composition drug solution group compared with the model group (P<0.05), suggesting that the treatment with the combined composition drug solution helps to increase the proportion of γδT cells in the lung tissue of mice.

[0185] Table 3 Immunohistochemical staining cell count shows that the combined composition drug solution reduces the ratio of αβT / γδT cells in the lung tissue of mice with chronic obstructive pulmonary disease model (X±S)

[0186]

[0187]

[0188] Table 4 Flow cytometry analysis results show that the combined composition drug solution reduces the ratio of αβT / γδT cells in the lung tissue of mice with chronic obstructive pulmonary disease model (X±S)

[0189] Group αβ T cells (%) γδ T cells (%) αβT / γδT Group A 52.77±14.96 7.78±2.75 6.78 Group B 65.16±15.03 2.97±0.11 21.93 Group C1 53.62±9.89 3.94±1.62 13.61 Group C2 54.93±5.46 3.53±0.61 15.56 Group C3 56.12±6.01 2.98±0.39 18.83 Group D1 53.43±10.84 4.09±0.83 13.06 Group D2 54.93±4.12 3.68±0.46 14.92 Group D3 55.32±5.09 3.31±0.88 16.71 Group E1 52.57±0.51 5.39±1.37 9.75 Group E2 53.94±1.11 4.64±1.04 11.63 Group E3 54.02±3.50 4.217±0.62 12.95

[0190] 5.4 Contents of IL-6 and IL-13 in the serum, bronchoalveolar lavage fluid and intestinal mucus of mice

[0191] Compare the contents of IL-6 and IL-13 in the serum, bronchoalveolar lavage fluid and intestinal mucus of mice. The content of IL-6 in the serum of the chronic obstructive pulmonary disease (COPD) model control group was higher than that of the blank control group (P<0.05). The content of IL-6 in the administration groups was lower than that of the model control group, and the high- and medium-dose composition groups were significantly lower than the model control group (P<0.05). There was no significant difference in the content of IL-13 in the serum among the groups. The content of IL-6 in the bronchoalveolar lavage fluid of the COPD model control group was higher than that of the blank control group (P<0.05). There was no statistical difference between the low-dose administration group and the model control group. The high- and medium-dose administration groups were lower than the blank control group, the model control group and the low-dose administration group, and the high- and medium-dose composition groups were significantly lower than the single-drug administration group (P<0.05). The content of IL-13 in the bronchoalveolar lavage fluid of the COPD model control group was higher than that of the blank control group (P<0.05). The content of IL-13 in the administration groups was lower than that of the model control group, and the high- and medium-dose composition groups were significantly lower (P<0.05). There was no significant difference in the content of IL-6 in the intestinal mucus among the groups. The content of IL-13 in the intestinal mucus of the COPD model control group was higher than that of the blank control group (P<0.05). The high-, medium- and low-dose groups of single drug administration and composition administration were lower than the model control group, and the high-, medium- and low-dose composition groups were significantly lower (P<0.05).

[0192] In summary, the amount of inflammatory factors secreted by the COPD model mice increased significantly. The contents of IL-6 in the serum and bronchoalveolar lavage fluid and IL-13 in the bronchoalveolar lavage fluid and intestinal mucus in the composition liquid medicine group were significantly lower than those in the model control group (P<0.05), as shown in Table 5.

[0193] Table 5 Contents of IL-6 and IL-13 in the serum, bronchoalveolar lavage fluid and intestinal mucus of mice (X±S)

[0194]

[0195] When the mass ratio of maackiain and tectorigenin in the present invention is combined at 7:1 to 3:2, the test effects obtained are basically the same as the above results, and no further examples will be given here.

[0196] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.

Claims

1. A flavonoid aglycone composition for treating chronic obstructive pulmonary disease, characterized in that, The flavonoid aglycone composition comprises maackiain and tectorigenin, wherein the mass ratio of maackiain to tectorigenin is 7:1 to 3:

2.

2. The flavonoid aglycone composition for treating chronic obstructive pulmonary disease according to claim 1, wherein Both maackiain and tectorigenin are obtained by extraction from Chinese medicinal materials.

3. The flavonoid aglycone composition for treating chronic obstructive pulmonary disease according to claim 2, wherein Maackiain is extracted from Sophora subprostrata, and tectorigenin is extracted from Belamcanda chinensis.

4. The flavonoid aglycone composition for treating chronic obstructive pulmonary disease according to claim 3, wherein, The method for extracting maackiain from Sophora subprostrata comprises the following steps: Sophora subprostrata is pulverized, and an ethanol solution with a mass 6 - 7 times that of Sophora subprostrata is added for extraction at least once, with each extraction lasting 5 - 7 days. After the extraction liquid is filtered, the filtrates are combined and concentrated to obtain a concentrated Sophora subprostrata ethanol extract; The concentrated Sophora subprostrata ethanol extract is extracted with ethyl acetate 1 - 5 times, and the extraction liquids are combined and concentrated to a paste-like state to obtain a Sophora subprostrata extraction paste; The Sophora subprostrata extraction paste is dissolved in an ethanol solution, filtered, and the filtrate is separated and purified using a macroporous resin column. It is eluted successively with 3 column volumes of distilled water and a 75% ethanol solution. The water-washed part is discarded, the 75% ethanol eluate is collected and concentrated to obtain a crude maackiain extract; The crude maackiain extract is dissolved in methanol, filtered, and the filtrate is separated and purified using Sephadex gel chromatography. A mixed solution of chloroform:methanol is used as the eluent for elution. The later-eluted part is recovered and concentrated. When white solids adhere to the bottle wall, it is taken out and filtered. The filter residue is washed with a mixed solution of chloroform:methanol, and the washing solution is left standing overnight. Solids slowly precipitate at the bottom of the bottle or substances precipitate on the bottle wall to obtain maackiain.

5. The flavonoid aglycone composition for treating chronic obstructive pulmonary disease according to claim 3, characterized in that, The method for extracting tectorigenin from Belamcanda chinensis comprises the following steps: Belamcanda chinensis is pulverized, and an ethanol solution with a mass 6 - 7 times that of Belamcanda chinensis is added for extraction at least once, with each extraction lasting 5 - 7 days. After the extraction liquid is filtered, the filtrates are combined and concentrated to obtain a concentrated Belamcanda chinensis ethanol extract; The concentrated Belamcanda chinensis ethanol extract is extracted with ethyl acetate 1 - 5 times, and the extraction liquids are combined and concentrated to a paste-like state to obtain a Belamcanda chinensis extraction paste; The Belamcanda chinensis extraction paste is dissolved in an ethanol solution, filtered, and the filtrate is separated and purified using a macroporous resin column. It is eluted successively with 3 column volumes of distilled water and a 75% ethanol solution. The water-washed part is discarded, the 75% ethanol eluate is collected and concentrated to obtain a crude tectorigenin extract; The crude tectorigenin extract is dissolved in methanol, filtered, and the filtrate is separated and purified using Sephadex gel chromatography. A mixed solution of chloroform:methanol is used as the eluent for elution. The later-eluted part is recovered and concentrated. When yellow solids adhere to the bottle wall, it is taken out and filtered. The filter residue is washed with a mixed solution of chloroform:methanol, and the washing solution is left standing overnight. Solids slowly precipitate at the bottom of the bottle or substances precipitate on the bottle wall to obtain tectorigenin.

6. Use of the flavonoid aglycone composition according to any one of claims 1 - 5 in the preparation of a drug for treating chronic obstructive pulmonary disease.

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