CN116327754A - Flavonoid aglycone composition for treating chronic obstructive pulmonary disease and application thereof - Google Patents

Abstract

The invention discloses a flavonoid aglycone composition for treating chronic obstructive pulmonary disease, which comprises Gao Lihuai elements and tectorigenin, wherein the mass ratio of Gao Lihuai elements to tectorigenin is 7:1-3:2. The invention also discloses application of the flavonoid aglycone composition in medicines for treating chronic obstructive pulmonary disease. The invention combines Gao Lihuai element in the radix sophorae tonkinensis and tectorigenin of blackberry lily, and has the effect of treating chronic obstructive pulmonary disease. The Gao Lihuai element and tectorigenin have high content in medicinal materials and are easy to obtain, so that the composition can be developed into a medicament for treating COPD, has great clinical and market values, and is worthy of great popularization and application.

Description

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

Technical Field

The invention belongs to the technical field of medicines. More particularly, the invention relates to a flavonoid aglycone composition for treating chronic obstructive pulmonary disease and application thereof.

Background

Chronic Obstructive Pulmonary Disease (COPD) is a common and frequently occurring disease in respiratory diseases, symptoms including dyspnea, cough and/or expectoration, whose etiology is characterized by progressive airflow obstruction and continuously aggravated airflow limitation, with abnormally sustained inflammatory reactions of the lungs or airways to noxious gases and particulates, smoking and cigarette smoke exposure are the primary causes of COPD, but air pollution and other exposures also play a significant role in the development and progression of the disease. The incidence rate of chronic lung obstruction in China over 40 years reaches 13.7%, the number of patients is about 1 hundred million, and the economic burden of diseases in China is 2 nd. Due to the problems of insufficient total amount of medical resources, uneven distribution, insufficient knowledge of doctors and patients on COPD and the like, the current situation of basic COPD prevention and treatment is not optimistic, and importance is attached to the fact that acute exacerbation of COPD is not slow, especially women and patients more than or equal to 65 years old are more easily affected by air pollutants to exacerbate the illness. Chronic obstructive pulmonary disease global initiative (GOLD) 2020 predicts that over 540 tens of thousands of people die annually from chronic obstructive pulmonary disease and complications by 2060, and COPD is a disease of high morbidity, mortality, and disability rate, but COPD is still a disease that can be prevented and treated, and is classified as stationary phase treatment and exacerbation treatment.

Current methods of treatment for COPD, including physical therapy and pharmaceutical intervention, aim to alleviate symptoms and acute exacerbations, delay lung function reduction, improve quality of life. The main therapeutic drugs include antibiotics, glucocorticoids, theophyllines, anticholinergic agents, beta 2 receptor agonists and the like, and COPD patients have various complications, and can be used for combined therapy or combined non-drug therapy oxygen therapy treatment according to the conditions of the patients, so that the exacerbation of illness and death rate can be delayed. Modern medical treatment drugs can improve clinical symptoms, improve lung functions, delay disease progression, improve quality of life and the like, but also have a series of adverse reactions such as headache, nausea, vomiting, dry mouth, diarrhea, arrhythmia, rash, urine retention and the like, and abuse of antibiotics also leads to physiological unbalance or disturbance of intestinal flora or release of bacterial cell dissolved endotoxin in the evolution process of COPD, thereby causing a series of pathological changes. The traditional Chinese medicine has long history of treating COPD, has the characteristics of clear curative effect, less adverse reaction, lasting curative effect and the like, and the traditional Chinese medicine considers that the main pathological changes of slow-blocking lung are principal deficiency and secondary excess, the healthy qi deficiency is the main pathogenesis, the healthy qi deficiency is deficiency of lung, spleen and kidney, and secondary excess is phlegm turbidity, water retention and blood stasis, and the three are mutually combined. Therefore, in the acute exacerbation stage of chronic obstructive pulmonary disease, it is mainly indicated for the treatment of relieving exterior syndrome, dispelling cold, warming yang, resolving fluid, eliminating dampness, resolving phlegm, clearing heat, resolving phlegm, promoting blood circulation, and removing blood stasis, and in the stationary stage, it is mainly indicated for the treatment of invigorating lung, spleen and kidney or both eliminating pathogenic factors. In recent years, the treatment of COPD by using Chinese herbal compounds and Chinese herbal active ingredients is the focus of attention of middle doctors, and the Chinese herbal preparation can well improve the immune disorder condition of patients and improve the lung function of the patients. Chinese medicines such as cordyceps sinensis, ginkgo leaves, subprostrate sophora, blackberry lily, platycodon grandiflorum, baical skullcap root and the like are traditional Chinese medicine components for treating chronic obstructive pulmonary disease frequently in traditional Chinese medicine preparations, but the components of the traditional Chinese medicine preparations acting on the chronic obstructive pulmonary disease are not clear. In order to further develop the pharmacological mechanism of the traditional Chinese medicine acting on the COPD, basic research is carried out to determine the pharmacological mechanism of the traditional Chinese medicine component acting on the COPD, and the traditional Chinese medicine has great clinical and market value for developing the medicine for treating the COPD.

Disclosure of Invention

It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.

It is an object of the present invention to provide a flavonoid aglycone composition for treating chronic obstructive pulmonary disease, which has a better therapeutic effect on chronic obstructive pulmonary disease.

To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a flavonoid aglycone composition for treating chronic obstructive pulmonary disease, comprising Gao Lihuai element and tectorigenin.

Preferably, the mass ratio of Gao Lihuai element to tectorigenin is 7:1-3:2.

Preferably, gao Lihuai element and iris Huang Sujun are extracted from Chinese medicinal materials.

Preferably, gao Lihuai extract is extracted from radix Sophorae Tonkinensis, and the rhizoma Belamcandae is extracted from rhizoma Iridis Tectori Huang Sucong.

Preferably, the method for extracting Gao Lihuai element from the subprostrate sophora comprises the following steps:

pulverizing radix Sophorae Tonkinensis, adding 6-7 times of ethanol solution, leaching for at least 1 time, each for 5-7 days, filtering the extractive solution, mixing filtrates, and concentrating to obtain radix Sophorae Tonkinensis ethanol extract concentrate;

extracting the concentrated solution of the alcohol extract of the subprostrate sophora with ethyl acetate for 1-5 times, combining the extracting solutions, and concentrating the extracting solutions into an extract to obtain the extract of the subprostrate sophora;

dissolving radix Sophorae Tonkinensis extract with ethanol solution, filtering, separating and purifying the filtrate with macroporous resin column, sequentially eluting with 3 times of column volume distilled water and 75% ethanol solution, discarding water washing part, collecting 75% ethanol eluate, and concentrating to obtain Gao Lihuai element crude extract;

dissolving Gao Lihuai crude extract with methanol, filtering, separating and purifying filtrate with sephadex chromatography, and purifying with chloroform: eluting with methanol mixed solution as eluent, recovering the later part, concentrating, taking out and filtering when white solid is attached to the bottle wall, and using chloroform: and (3) cleaning filter residues by using a mixed solution of methanol, standing the cleaning solution overnight, and slowly precipitating solids at the bottom of the bottle or separating substances out from the wall of the bottle to obtain Gao Lihuai elements.

Preferably, the method for extracting tectorigenin from blackberry lily comprises the following steps:

pulverizing rhizoma Belamcandae, adding 6-7 times of ethanol solution, extracting for at least 1 time, each for 5-7 days, filtering the extractive solution, mixing filtrates, and concentrating to obtain rhizoma Belamcandae ethanol extract concentrate;

extracting the concentrated solution of the blackberry lily ethanol extract with ethyl acetate for 1-5 times, mixing the extracts, concentrating into extract, and obtaining blackberry lily extract;

dissolving rhizoma Belamcandae extract with ethanol solution, filtering, separating and purifying the filtrate with macroporous resin column, sequentially eluting with 3 times of column volume distilled water and 75% ethanol solution, discarding water washing part, collecting 75% ethanol eluate, and concentrating to obtain rhizoma Iridis Tectori Huang Sucu extract;

dissolving the iris Huang Sucu extract with methanol, filtering, separating and purifying the filtrate by sephadex chromatography, and purifying by chloroform: eluting with methanol mixed solution as eluent, recovering the later part, concentrating, taking out and filtering when yellow solid is adhered to the bottle wall, and using chloroform: and (3) cleaning filter residues by using a methanol mixed solution, standing the cleaning solution overnight, and slowly precipitating solids or substances separated from the bottle bottom or the bottle wall to obtain tectorigenin.

The application of the flavonoid aglycone composition in medicines for treating chronic obstructive pulmonary disease is provided.

The invention at least comprises the following beneficial effects: the invention combines Gao Lihuai element in the radix sophorae tonkinensis and tectorigenin of blackberry lily, and has the effect of treating chronic obstructive pulmonary disease. The Gao Lihuai element and tectorigenin have high content in medicinal materials and are easy to obtain, so that the composition can be developed into a medicament for treating COPD, has great clinical and market values, and is worthy of great popularization and application.

Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.

Drawings

FIG. 1 is a graph of HE staining of lung tissue of mice (200X); wherein A is blank control group, B is model control group, C is Gao Lihuai element with high concentration, D is tectorigenin with high concentration, and E is medium concentration composition.

Detailed Description

The present invention is described in further detail below to enable those skilled in the art to practice the invention by reference to the specification.

It will be understood that terms, such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.

The experimental methods described in the following embodiments are conventional methods unless otherwise indicated, and the reagents and materials are commercially available.

A flavonoid aglycone composition for treating chronic obstructive pulmonary disease, comprising Gao Lihuai element and tectorigenin.

In another technical scheme, the mass ratio of Gao Lihuai element to tectorigenin is 7:1-3:2.

In another technical scheme, gao Lihuai element and iris Huang Sujun are extracted from traditional Chinese medicinal materials.

In another embodiment, gao Lihuai is extracted from Sophora subprostrata, and the iris Huang Sucong is extracted from blackberry lily. Gao Lihuai the extract can also be extracted from radix Millettiae Dillecae, ramulus Sophorae, ramulus Uncariae cum Uncis, etc. Tectorigenin can also be extracted from Chinese medicinal materials such as flos Puerariae Lobatae, caulis Sinomenii, etc.

In another embodiment, the method for extracting Gao Lihuai from radix Sophorae Tonkinensis comprises the following steps:

pulverizing radix Sophorae Tonkinensis, adding 6-7 times of ethanol solution, leaching for at least 1 time, each for 5-7 days, filtering the extractive solution, mixing filtrates, and concentrating to obtain radix Sophorae Tonkinensis ethanol extract concentrate;

extracting the concentrated solution of the alcohol extract of the subprostrate sophora with ethyl acetate for 1-5 times, combining the extracting solutions, and concentrating the extracting solutions into an extract to obtain the extract of the subprostrate sophora;

dissolving radix Sophorae Tonkinensis extract with ethanol solution, filtering, separating and purifying the filtrate with macroporous resin column, sequentially eluting with 3 times of column volume distilled water and 75% ethanol solution, discarding water washing part, collecting 75% ethanol eluate, and concentrating to obtain Gao Lihuai element crude extract;

dissolving Gao Lihuai crude extract with methanol, filtering, separating and purifying filtrate with sephadex chromatography, and purifying with chloroform: eluting with methanol mixed solution as eluent, recovering the later part, concentrating, taking out and filtering when white solid is attached to the bottle wall, and using chloroform: and (3) cleaning filter residues by using a mixed solution of methanol, standing the cleaning solution overnight, and slowly precipitating solids at the bottom of the bottle or separating substances out from the wall of the bottle to obtain Gao Lihuai elements.

In another technical scheme, the method for extracting tectorigenin from blackberry lily comprises the following steps:

pulverizing rhizoma Belamcandae, adding 6-7 times of ethanol solution, extracting for at least 1 time, each for 5-7 days, filtering the extractive solution, mixing filtrates, and concentrating to obtain rhizoma Belamcandae ethanol extract concentrate;

extracting the concentrated solution of the blackberry lily ethanol extract with ethyl acetate for 1-5 times, mixing the extracts, concentrating into extract, and obtaining blackberry lily extract;

dissolving rhizoma Belamcandae extract with ethanol solution, filtering, separating and purifying the filtrate with macroporous resin column, sequentially eluting with 3 times of column volume distilled water and 75% ethanol solution, discarding water washing part, collecting 75% ethanol eluate, and concentrating to obtain rhizoma Iridis Tectori Huang Sucu extract;

dissolving the iris Huang Sucu extract with methanol, filtering, separating and purifying the filtrate by sephadex chromatography, and purifying by chloroform: eluting with methanol mixed solution as eluent, recovering the later part, concentrating, taking out and filtering when yellow solid is adhered to the bottle wall, and using chloroform: and (3) cleaning filter residues by using a methanol mixed solution, standing the cleaning solution overnight, and slowly precipitating solids or substances separated from the bottle bottom or the bottle wall to obtain tectorigenin.

1. Extraction, purification and identification of Gao Lihuai element and tectorigenin

Extraction, purification and identification of Gao Lihuai element

1. Extraction and extraction

Taking 50kg of radix sophorae tonkinensis (SDG) medicinal materials, respectively crushing, leaching with 6-10 times of 75% ethanol for 2 times, leaching for 5-7 days each time, filtering the extracting solution, merging and concentrating under reduced pressure to obtain an alcohol extract concentrated solution, extracting the alcohol extract concentrated solution with ethyl acetate with the volume ratio of 1:1 for 3 times, merging the extracting solutions, concentrating under reduced pressure, recovering the extracting solvent, and concentrating to obtain radix sophorae tonkinensis (SDGY) extractum.

2. Separation

Dissolving SDGY extract with 30% low concentration ethanol solution, filtering, further separating and purifying the filtrate with D101 macroporous resin column, eluting with 3 times of column volume distilled water and 75% ethanol solution sequentially, discarding water washing part, collecting 75% ethanol eluate, concentrating, and recovering solvent to obtain Gao Lihuai element crude extract (SDGY-A fraction).

3. Purification and identification

Separating and purifying SDGY-A fraction by Sephadex G20, dissolving SDGY-A fraction with appropriate amount of methanol, filtering, loading filtrate, and mixing with the filtrate at volume ratio of 1: chloroform of 1: methanol mixed solution is used as eluent to obtain 1, 2, 3, 20 parts; wherein, when the solvent is recovered by concentration, white solid is attached to the bottle wall, and the proper volume ratio for filtration is 1: chloroform of 1: the mixed solution of methanol is used for cleaning filter residues, the cleaning solution is placed overnight, the bottle bottom slowly precipitates out solids or substances precipitated on the bottle wall, the substances are detected by thin layer chromatography, and the substances are detected by high performance liquid chromatography, if the substances are all impure substances, chloroform with different volume ratios can be used: recrystallizing the methanol mixed solution to separate out a monomer compound, and identifying the separated monomer compound, wherein the extract of the subprostrate sophora is Gao Lihuai element, and the structure is shown as follows:

(II) extraction, purification and identification of tectorigenin

1. Extraction and extraction

50kg of blackberry lily (SG) medicinal material is taken, crushed, leached with 6-10 times of 70-75% ethanol for 2 times, each time for 5-7 days, and the extracting solution is filtered, combined and decompressed and concentrated to obtain an alcohol extract concentrated solution, wherein the extraction rate is 25.22%. Extracting the concentrated solution of the alcohol extract with ethyl acetate with volume ratio of 1:1 for 3 times, mixing the extractive solutions, concentrating under reduced pressure, recovering the extraction solvent, and concentrating to obtain rhizoma Belamcandae (SGY 2) extract.

2. Separation

Dissolving SGY 2 extract with 30% low concentration ethanol solution, filtering, further separating and purifying the filtrate with D101 macroporous resin column, eluting with 3 times of column volume distilled water and 75% ethanol solution sequentially, discarding water washing part, collecting 75% ethanol eluate, and recovering solvent to obtain rhizoma Iridis Tectori Huang Sucu extract (SGY-A fraction).

3. Purification and identification

Separating and purifying SGY-A fraction by Sephadex G20, dissolving SGY-A fraction with a proper amount of methanol, filtering, loading filtrate, and using a volume ratio of 1: chloroform of 1: methanol mixed solution is used as eluent to obtain 1, 2, 3, 20 parts; wherein, when the solvent is recovered by concentration, the yellow solid is adhered to the bottle wall, and the mixture is taken out and filtered, and the volume ratio of the mixture to the bottle wall is 1: chloroform of 1: the mixed solution of methanol is used for cleaning filter residues, the cleaning solution is placed overnight, the bottle bottom slowly precipitates out solids or substances precipitated on the bottle wall, the substances are detected by thin layer chromatography, and the substances are detected by high performance liquid chromatography, if the substances are all impure substances, chloroform can be used for the detection: recrystallizing the mixed solution with different proportions of methanol to separate out a monomer compound, and identifying the separated monomer compound, wherein the extract in the blackberry lily is tectorigenin, and the structure is shown as follows:

2. effects of Gao Lihuai and tectorigenin compositions on proliferation of human lung (bronchus) epithelial cells BEAS-2B cells

1.1 preparation of liquid medicine, preparation of MTT solution, and cell culture

Preparing a liquid medicine: dissolving Gao Lihuai element and tectorigenin with DMSO to a proper concentration as a stock solution, preserving at-20deg.C, and diluting to a desired concentration before use; gao Lihuai: the mass ratio of tectorigenin is 3:2, preparing the composition liquid medicine with different concentrations.

Preparation of MTT solution: usually MTT is prepared to a final concentration of 5mg/ml, phosphate Buffer (PBS) is used as solvent, and the solution is prepared and filtered by a 0.22 μm filter membrane to remove bacteria, and the solution is stored at 4 ℃ in a dark place. During the preparation and storage process, the containers were wrapped with aluminum foil paper.

Cell culture: a freezing tube of 1mL human lung (bronchus) epithelial cells BEAS-2B cell suspension was rapidly thawed by shaking in a 37℃water bath, and 5mL10% fetal bovine serum RPMI-1640 complete medium was added and mixed well. Centrifuging for 5 minutes at 1000RPM, removing supernatant, supplementing 4-6 mL of complete culture medium of fetal bovine serum RPMI-1640, and uniformly blowing; then adding all cell suspensions into a culture flask for culturing overnight; the next day the fluid was changed and the cell density was checked. If the cell density reaches 80% -90%, subculturing can be performed. The specific operation of subculture is as follows: the culture supernatant is discarded, the cells are rinsed for 1-2 times by PBS without calcium and magnesium ions, 1-2 mL of digestive juice (0.25% Trypsin-0.53mM EDTA) is added into a culture flask, the culture flask is placed into a 37 ℃ incubator for digestion for 1-2 min, then the cell digestion condition is observed under a microscope, if most of the cells are rounded and fall off, the operation table is quickly returned, and after tapping the culture flask, more than 5mL of complete culture medium containing 10% serum is added for stopping the digestion. Gently blowing the cells, sucking out after complete shedding, centrifuging for 8-10 minutes at 1000RPM, discarding the supernatant, adding 1-2 mL of culture solution, and uniformly blowing. The culture solution is added according to 5-6 ml/bottle, and the cell suspension is added according to 1: the ratio of 5 is divided into new bottles containing 5-6mL of culture solution.

1.2, experimental grouping

Blank control group: 100. Mu.L of RPMI-1640 complete medium and 100. Mu.L of 0.1% DMSO solution.

Negative control group: 100 mu L density of 2X 10 ⁴ mu.L of 0.1% DMSO in RPMI-1640 medium was added to each/ml of cell suspension.

Experimental group: 100 mu L density of 2X 10 ⁴ Cell suspensions at a concentration of 10, 20, 40, 80, 100, 150, 200. Mu.g/ml, respectively, and 100. Mu.L of the composition liquid medicine were added, respectively.

1.3 experiments

Taking logarithmic growth phase cells, and digesting and suspending to obtain a concentration of 2×10 ⁴ Inoculating 100 μl of single cells per ml into 96-well plate according to above groups, placing into 37 deg.C and 5% CO ₂ Culturing in a saturated humidity incubator for 12 hours, observingIf the cell morphology is good, administering drugs of 10, 20, 40, 80, 100, 150 and 200 mug/ml in groups, setting 6 compound holes, placing into an incubator for CO-incubation for 24h, adding 20 mug of 5mg/ml MTT, and adding 5% CO at 37 DEG C ₂ After 4h incubation in a saturated humidity incubator, the supernatant was removed, 150. Mu.L DMSO was added, and after 10min shaking with a micro-shaker to completely dissolve the crystals, the OD was measured at 570nm, and the average of 6 parallel wells was the result of one experiment. Experiments were independently repeated three times.

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

As can be seen from Table 1, when the concentration of the pharmaceutical composition of Gao Lihuai and tectorigenin was in the range of 10 to 200. Mu.g/mL, the cell proliferation rate was 90% or more, and it was considered that the pharmaceutical composition of Gao Lihuai and tectorigenin had no significant cytotoxicity to BEAS-2B cells in the set concentration range.

TABLE 1 Effect of different concentrations of composition liquid on BEAS-2B cell proliferation (x+ -s, n=3)

2. Research on pharmacological activity of Gao Lihuai element-tectorigenin composition on COPD

1. Animal feeding group

1.1, laboratory animals

275 SPF-grade ICR female mice were selected, which were 6-8 weeks old, 18-20 g in weight, and were from the university of Guangxi medical science medical laboratory animal center (animal license number: SCXK cassia 2014-0002).

1.2 Experimental drugs

Gao Lihuai the extract is prepared by extracting radix Sophorae Tonkinensis from laboratory, separating, and identifying, and making into suspension with administration amount of 0.25g/kg,0.50g/kg, and 1.0g/kg with physiological saline, and preserving at 4deg.C.

Tectorigenin is obtained by extracting, separating and identifying rhizoma Belamcandae in laboratory, and is prepared into suspension with administration amount of 0.25g/kg,0.50g/kg and 1.0g/kg with physiological saline, and stored at 4deg.C for use.

Gao Lihuai A-tectorigenin composition, according to 3:2, preparing into suspension with dosage of 0.25g/kg,0.50g/kg and 1.0g/kg by normal saline, and preserving at 4deg.C for use.

1.3 Experimental grouping

275 ICR female mice were randomly divided into 11 groups after 1 week of adaptive feeding: the blank control group is A group, the model control group is B group, the Gao Lihuai high, medium and low dose groups are C1, C2 and C3 groups respectively, the iris Huang Sugao, the medium and low dose groups are D1, D2 and D3 groups respectively, and the composition high, medium and low dose groups are E1, E2 and E3 groups respectively, and 25 groups each.

2. Modeling and administration method

2.1 feeding conditions

Before and after administration, the experimental animals are fed in separate cages, full price pellet feed is fed, free drinking water is carried out, the room temperature is 20+/-2 ℃, and the humidity is 50% -60%.

2.2 model groups

1) LPS solution (30 ug/6 uL) was instilled into the respiratory tract of mice from the nasal cavity on days 1, 29 and 57, respectively, at a dose of 1.5mg/kg.

2) The cigarette smoke exposure is continuously carried out on days 2-84 (except for 29 th and 57 th), and the specific operation method comprises the following steps:

(1) mice were placed in animal transport cases (60 cm. Times.45 cm. Times.20 cm) with ventilation windows, 25 cases each, sealed with tape.

(2) The animal transport case is placed in a glass fumigating case, and 1 circular ventilation holes with the diameter of about 2cm are respectively reserved on the diagonal side walls of the fumigating case for adjusting the oxygen and air pressure states inside and outside the case.

(3) Igniting cigarettes, inserting the cigarettes on a self-made metal combustion frame, placing the cigarettes in a self-made glass smoking box, and covering the cigarettes; 10 cigarettes are burned out each time, 10 min/time, 2 times/day, 15min intervals in the middle, and the fumigating box cover is opened for ventilation at intervals of 7 days/week for 12 weeks.

(4) After each cigarette smoke exposure, the animal transport case is placed in a delivery window, sterilized by ultraviolet lamp for 15 minutes, and then the mice are put back into the rearing cage.

3) After the molding was completed, distilled water was orally administered at 0.2 ml/day for 4 weeks from week 13.

2.3 dosing groups

The molding operation was carried out in the same manner as in the model group, and 1.2 experimental medicines were orally administered from week 13, 0.2 ml/day for 4 weeks.

2.4 blank control group

0.9% physiological saline solution (6 uL/mouse) was instilled into the respiratory tract of mice from nasal cavity on day 1, 29 and 57, respectively, and the rest of the time was fed normally. From week 13, 0.9% saline solution, 0.2 ml/day was administered orally for a total of 4 weeks.

3. Observation index and method

3.1 general State viewing

Quality of life and weight of mice.

3.2 determination of Experimental animal Material and Experimental detection index

3.2.1 collecting samples of bronchoalveolar lavage fluid, serum, and intestinal mucus from mice

The collecting method of bronchoalveolar lavage fluid specifically comprises the following steps:

(1) 2% pentobarbital was intraperitoneally injected into anesthetized mice (0.1 ml/10 g), and the mice were fixed to the console in a supine position.

(2) The neck fur is sheared by the ophthalmology scissors to expose subcutaneous tissue, and the air outlet pipe is passively separated.

(3) And cutting an incision in the horizontal direction of the distal end of the trachea, inserting the tracheal joint of the intubation tube into the trachea in a centripetal and oblique way, and connecting the breathing machine with the tracheal joint after the cotton thread is fixed. A1 ml syringe containing sterile PBS was connected to the endotracheal tube and lavage was performed by slow bolus injection.

(4) After each injection of 0.8ml, the aspiration was slow and after 3 repetitions the lavage was placed in a 1.5ml EP tube.

(5) The rotational speed of the centrifuge is 3000rpm, the lavage liquid is centrifuged for 15min, and the supernatant liquid is taken and stored at the temperature of minus 80 ℃ to be measured.

The serum collection specifically comprises the following steps:

(1) Skin was cut under the xiphoid process, and the myometrium was cut along the bilateral rib arches, exposing the chest.

(2) The ribs were cut along the sternum edge, the heart and lung were exposed, a needle was inserted from the apex of the heart using a 1ml syringe, and blood was taken from the four chambers of the heart, each about 0.8-1ml.

(3) Slowly pouring into 1.5ml EP tube, placing into 37deg.C water bath for 15min, and refrigerating at 4deg.C for 15min to visualize delamination of blood sample.

(4) The centrifuge rotates at 3000rpm, centrifugates for 15min, and the upper serum is taken and stored at-80 ℃ to be measured.

The intestinal mucus collection comprises the following steps

(1) The abdominal cavity was opened, the pylorus and cecum were found, the mesentery and surrounding tissues were gently separated, the entire small intestine between pylorus and cecum was taken, and 10ml (ph=7.4) of 0.01M sterile PBS solution was slowly injected by syringe to flush the intestinal lumen.

(2) The obtained flushing liquid is collected into a 5ml centrifuge tube, the rotation speed of the centrifuge is 3000rpm for 15min, and the supernatant liquid is taken and stored at the temperature of minus 80 ℃ to be measured.

3.2.2 collecting and processing Lung tissue morphology samples of mice

The morphological specimen collection method comprises the following steps: cutting off the end of the distal end of the trachea, separating the adhered tissues around, taking out the lung by forceps, performing morphological sample treatment on the left lung lobe, and storing the right lung lobe in liquid nitrogen by using a freezing tube.

Morphological specimen processing, specifically comprising the following steps:

1) Fixing: the left lung lobes are covered by gauze, and are fixed by paper clips and then are fixed in 10% formalin solution, and the specimens are completely immersed to avoid floating on the liquid surface. After 24h fixation the tissue is removed and the left lung lobes are placed in a dehydration box in a fume hood.

2) Gradient alcohol dehydration: 75% alcohol for 2min;85% alcohol for 2min;90% alcohol for 2min;95% alcohol for 2min; absolute ethanol I2 min; absolute ethanol II for 2min; benzene alcohol for 2min; xylene I2 min; xylene II for 2min; paraffin wax I2 min; paraffin II for 2min; paraffin III for 2min.

3) Embedding: embedding the waxed tissue in an embedding machine, spreading the waxed tissue into an embedding frame after wax is melted, and putting left lung lobes into the embedding frame and marking the left lung lobes before solidification; then placing the mixture in a freezing table at the temperature of minus 20 ℃ for cooling, and separating the wax block from the embedding frame after the wax is solidified.

4) Slicing: the slicing machine is set to have the slice thickness of 5Pm, so that slices float on the warm water surface of the slice spreading machine at 43 ℃, after the tissues are flattened, the slices are fished up by using glass slides, and the glass slides are placed into a 60 ℃ oven for baking. Taking out the mixture after drying and preserving the mixture at normal temperature for standby.

HE staining, comprising the following steps:

1) Dewaxing to water: placing the mouse lung tissue slices in an oven at 60 ℃ for 1h, and placing the slices in xylene I, II for 15min each after the wax is completely melted; absolute ethyl alcohol I, II for 2min each; 95% alcohol I, II for 2min each; 90% alcohol for 2min;80% alcohol 2min,70% alcohol 2min,60% alcohol 2min,50% alcohol 2min tap water gently rinsed for 5min.

2) After hematoxylin staining for 15min, excess dye liquor was washed away with tap water.

3) Differentiation was performed for 30s with ethanol containing 1% hydrochloric acid, and washing was performed for 10min with tap water.

4) 1% eosin solution is used for dyeing for 10min, and tap water is used for washing off redundant dye liquor for 1min.

5) Gradient alcohol dehydration: 50% alcohol for 2min;60% alcohol for 2min;70% alcohol; 80% alcohol; 90% alcohol for 2min;95% alcohol I2 min;95% alcohol II for 2min;100% absolute ethanol I, II for 2min each; xylene I, II for 10min each; and (5) sealing the neutral resin.

6) Changes in lung tissue and bronchial wall were observed under an optical microscope and inflammatory cell infiltration.

3.2.3 isolation and purification of Lung tissue and Small intestine epithelial intercellular lymphocytes

The intraperitoneal injection of heparin specifically comprises the following steps:

1) Heparin preparation: specification 12500 IU/branch, working concentration: 100IU/ml.

2) 1 branch of heparin was added to 125ml of 0.9% NaCl solution and mixed well.

3) After weighing the mice, heparin was extracted at a rate of 10IU/ml/10g, and the mice were injected into the abdominal cavity from the left inguinal region.

The abdominal anesthesia specifically comprises the following steps:

1) 1% sodium pentobarbital is prepared: 100mg of sodium pentobarbital is weighed, added into a centrifuge tube containing 10ml of distilled water, fully dissolved and uniformly mixed.

2) A1% solution of sodium pentobarbital was withdrawn from the syringe by 1ml and administered at a rate of 0.05ml/10g body weight. The eyelid of the mice to be stimulated is not reflected, and the lung tissues of the mice are collected.

The method for separating the mouse lung tissue lymphocytes specifically comprises the following steps:

1) The lung tissue was placed into a 50ml centrifuge tube with sterile ophthalmic forceps and rinsed 3 times with 50ml PBS.

2) The washed lung tissue was placed in a sterile small dish (diameter 35mm, depth 10 mm) and minced as much as possible with ophthalmic scissors. The 70um cell filter screen was placed on a centrifuge tube, the tissue was transferred to the filter screen with a pipette, and 1 XHanks equilibration solution was washed 3 times and the wash solution was filtered off.

3) 50ml of RPM 1640 and 1ml of PBS were added to a 50ml centrifuge tube, and the lung tissue fragments were added with a pipette, followed by 100mg/ml Collagenase NB 4200. Mu.L and 10mg/ml DNASE 40. Mu.L to give working concentrations of 2mg/ml and 40ug/ml, respectively.

4) The incubation was carried out for 90min at 37℃in a constant temperature water bath, and the shaking was carried out 1 time every 10min with a shaker.

5) Filtering with 70um cell filter screen to remove undigested tissue fragments and impurities.

6) 15ml of RPM 1640 was pipetted onto the cell sieve and the filtrate and rinse were transferred to a 50m1 centrifuge tube. Centrifuging at 4 ℃ for 10min at a rotating speed of 2000r/min.

7) After centrifugation, the supernatant was removed by gently pipetting with a pipette, taking care not to agitate the cell layer, leaving 5ml in the centrifuge tube, and floating the cells by flicking the bottom of the centrifuge tube with a finger.

8) To a 15ml centrifuge tube, 3ml of 100% percoll was added, followed by 5ml of cell suspension, and then 5ml of 1 XHanks equilibration solution with 5% PBS, and after mixing the solution had a 30% percoll content. Centrifuge at 4℃for 18min and 1800r/min. After centrifugation, the supernatant was removed, 1ml of solution (containing 0.3ml of percoll) was retained, the bottom of the tube was flicked to break up the cell mass, and 4.1ml of 100% percoll was added and mixed well. The volume was increased to 10ml with a 1 Xhanks solution containing 5% PBS and the content of percoll after blending was 44%.

9) Slowly adding 2ml of percoll with the volume fraction of 70% into the bottom of a 15ml centrifuge tube by using a long straw, layering the two types of percoll liquid with the density to form an interface by a gentle operation, centrifuging for 18min at the temperature of 4 ℃ under the speed of 1800r/min, gathering visible cells at the interface of 44% percoll liquid level and 70% percoll liquid level into a layer, carefully discarding the upper 1/3 of the liquid in the centrifuge tube by using the straw to stick to the tube wall, sucking the cells out by sticking to the tube wall, and transferring the cells into the prepared 50ml centrifuge tube. To the centrifuge tube, 1 XHanks equilibration solution containing 5% PBS was added to give a total volume of 10ml, and the mixture was thoroughly shaken. Centrifugation was performed at 1500r/min for 5min at room temperature, and after discarding the supernatant, 2ml of 1 XHanks RPMI1640 solution containing 10% PBS was added to prepare a lymphocyte suspension to be measured. 4g/L trypan blue staining was added and counted with a cell counter plate.

The method for separating the mouse intestinal epithelial inter-cell lymphocytes specifically comprises the following steps:

1) Placing the whole section of small intestine between pylorus and cecum in PBS solution precooled on tinfoil, turning over the intestinal tube from one end of small intestine into intestinal cavity with thin plastic tube, gradually making the plastic tube pass out from the other end of small intestine, turning over the whole intestinal cavity, and making mucosa layer outwards and viscera layer inwards.

2) The intestinal tube was carefully transferred to a 50ml centrifuge tube, and 10% PBS in RPMI1640 and 100mg/ral Collagenase NB 4200. Mu.L, 10mg/ml DNASE in 40. Mu.L were added to give working concentrations of Collagenase NB4 and DNASE of 2mg/ml,40ug/ml, respectively.

3) Centrifuging the centrifuge tube at 37 ℃ for 60min at 200r/min, filtering the digestive juice into a 50ml centrifuge tube by using a 70um cell filter screen, and filtering undigested tissue fragments and impurities; then centrifuging at 2000r/min for 10min at 4 ℃, removing supernatant by suction pipe adherence, taking out 5ml cell mass at the bottom of the centrifuge tube without stirring, and suspending cells by flicking with fingers.

4) 3ml of 100% percoll is added into a 15ml centrifuge tube, 5ml of cell suspension is added, then 2ml of 1 XHanks solution containing 5% PBS is added, after uniform mixing, the content of percoll in the liquid is 30%, the liquid is centrifuged for 18min at 1800r/min at 4 ℃, the supernatant is removed after centrifugation, 1ml of solution (containing 0.3m1 of percoll) is remained, cell clusters at the bottom of the dispersion are flicked, after uniform mixing, 4.1ml of 100% percoll is added, 1 XHanks solution containing 5% PBS is added, the total amount of the solution is increased to 10mi, and after uniform mixing, the content of percoll is 44%.

5) Slowly adding 2mL of 70% percoll into the bottom of a centrifuge tube by using a straw, layering the two liquids with different densities to form an interface by using a gentle operation, and centrifuging for 18min at a temperature of 1800r/min at 4 ℃; the visible cells at the junction of the 44% percoll liquid level and the 70% percoll liquid level are gathered into a layer, the upper 1/3 of the liquid in the centrifuge tube is firstly carefully discarded by a suction tube, then the cells are sucked out by the tube wall, the liquid is transferred into a prepared 50ml centrifuge tube, 1 XHanks containing 5% PBS is added to compatibilize the total liquid amount to 10ml, after uniform mixing, the cells are centrifuged for 5min at room temperature at 1500r/min, after the supernatant is discarded, the cells are resuspended by 2ml 1 XHanks containing 5% PBS, and the lymphocyte suspension is prepared for measurement, and is stained by adding 4g/L trypan blue and counted by a cell counting plate.

3.2.4 detection of inflammatory factor content in mouse serum, alveolar lavage fluid and intestinal mucus

The IL-6 content detection method specifically comprises the following steps:

1) All samples and test kits to be tested were equilibrated to room temperature (18-25 ℃).

2) 50ml of 1 XWash Buffer was prepared from 5ml of Wash Buffer concentrate (10X) and 45ml of distilled water. Antibody mixtures were prepared with antibody dilutions, capture antibodies and detection antibodies. 300uL 10 XCapture Antibody and 300ul 10X Detector Antibody were mixed with 2.4ml Antibody dilutions to make 3ml Antibody mixture, which was gently mixed.

3) Preparing a standard product: adding 500uL of sample diluted normal saline to dissolve mouse IL-6 recombinant protein freeze-dried powder, fully dissolving and uniformly mixing, and lightly mixing for 10min at room temperature to obtain 2000pg/ml standard solution. The 8 EP tubes are marked and are standard 1-8. 150. Mu.L of sample diluted physiological saline was added to tube Nos. 1-8. Serial dilutions were made using standard solutions, 150 μl of the prepared 2000pg/ml standard solution was added to tube 1 to prepare 2000pg/ml standard solution, 150 μl of the prepared 1000pg/ml standard solution was added to tube 1 to prepare 500pg/ml standard solution, and 150 μl of the prepared 500pg/ml standard solution was added to tube 3 to prepare 150pg/ml standard solution, so 125, 62.5, 31.3, 15.6pg/ml standard solution was sequentially prepared in tubes 4, 5, 6, 7, with 8 being blank control.

4) Sample adding: mu.L of sample (serum, intestinal mucus) or standard is added to the appropriate wells, and then 50. Mu.L of antibody mixture is added to each well, taking care of the gentle handling, without generating bubbles.

5) Incubation: plates were sealed with a film and incubated for 1h at room temperature on a shaker set at 400 r/min.

6) Washing the plate: the wells were discarded and each well was washed 3 times with 350 μl of 1 xWash Buffer and after completion the 96 well plates were back-buckled on a clean paper towel to remove excess liquid.

7) Color development: a350. Mu.L TMB matrix was added to each well and incubated for 10min in a shaker at 400r/min in the absence of light, allowing blue coloration of the liquid in the well plate to be observed.

8) And (3) terminating: 100. Mu.L of stop solution was added to each well and after mixing the blue liquid turned yellow.

9) The microplate reader was set to 450nm for OD measurements.

10 Calculation: and calculating a standard equation by taking the OD value as a dependent variable Y and the standard concentration as an independent variable X, and substituting the OD value of the measured sample into the equation to obtain the IL-6 concentration.

The detection of the IL-13 content was the same as the detection method of IL-6.

4. Statistical method

The experimental data are expressed by mean plus minus standard deviation (x+/-S) and are processed by SPSS13.0 software, and the comparison between the mean values is performed by T test.

5. Experimental results

5.1 general State observations

Blank control group: the mice have no death, smooth fur, smooth breath, moderate frequency, uniform rhythm, normal activity and gradual weight increase.

Model group: the mice in the model group of chronic obstructive pulmonary disease die 10, hair is dark, part of the mice has unhairing phenomenon, the mice are rested and leaped during smoking, the mice are curled by multiple binding piles in the later period, even tremble whole body, shortness of breath, obvious chest and abdomen fluctuation, irregular nodding movement, even open breath, and the weight is obviously lower than that of the mice in the normal group (P is less than 0.001).

Drug administration group: gao Lihuai the mice had 2 deaths in the high, medium and low dose groups, 4 deaths in the 5-dose group and 2 deaths in the iris Huang Sugao-dose group, and 4 deaths in the medium and low dose groups, respectively, and compared with the model group, the mice had smooth fur, tended to normal behavior, improved dyspnea, and significantly higher body weight than the model group (P < 0.001). The Gao Lihuai and tectorigenin compositions were 1 in each of the high-dose and medium-dose groups and 3 in the low-dose group, the slow pulmonary resistance model group was the same as the general cases during molding, the hair was smoother after administration of the composition to the stomach, the dyspnea was improved, the frequency was slowed down, the weight of the high-dose and low-dose groups was significantly higher than that of the model group (P < 0.001), and the results are shown in Table 2.

Table 2 the composition drug can improve survival and body weight of mice with slow-blocking lung model:

5.2 model of chronic obstructive pulmonary disease mouse pulmonary histomorphometric pathological changes

The lung tissue pathology of chronic obstructive pulmonary disease is manifested by degeneration, necrosis, shedding of bronchial mucosa epithelium, shortening cilia, and adhesion; goblet cells and mucus cells proliferate and hypertrophy, a large amount of mucus is retained, the vessel wall is engorged with blood, the edema, and the tracheal lumen is narrowed. Alveoli collapse, the alveolar spaces break apart, and adjacent alveoli fuse into a lung bleb. A large amount of chronic inflammatory cells infiltrate around the trachea and alveoli, smooth muscle rupture and atrophy of submucosa and fibrous tissue hyperplasia; and (3) remodelling the bronchus structure, increasing the collagen content and forming scars. The HE staining of the experiment shows that the airway and alveoli of the mice in the blank control group are normal in structure, the cilia are regular, the shape and the size of the alveoli are regular, and the airway mucosa epithelium is complete (figure 1-A); forming the folds of the bronchus mucosa of the model group, and flaking off to narrow or block the lumen; the alveolar wall is destroyed, the alveolar space is irregularly enlarged, and the alveolar wall is partially fused into lung bullae, and chronic inflammatory cell infiltration is visible around the airway and in the lung interstitium, so that the chronic inflammatory cell infiltration accords with the pathological manifestations of chronic obstructive pulmonary disease (figure 1-B).

The three groups of high-concentration Gao Lihuai element, high-concentration tectorigenin and medium-concentration combined medicament are compared with a model control group, so that morphological improvement is seen, and the improvement is reflected in that bronchus and alveolus structures are complete, the narrow degree of a tube cavity is reduced, airway mucous membrane epithelium is relatively complete, cilia arrangement rules and shedding are reduced, the alveolus size is uniform, the number of lung bullae is reduced, and inflammatory cell infiltration degree around the tube wall of the airway and in the lung interstitium is reduced (figures 1-C, D, E).

5.3 Lung tissue alpha beta T/gamma delta T cell ratio in mice with Slow resistance Lung model

Gamma delta T cells are key effector cells of the respiratory tract mucosal immune system and are involved in the inflammatory and injury repair process of chronic inflammatory diseases. In normal mouse lungs, most αβt cells are distributed in the lung parenchyma, while most γαβ0t cells are distributed in non-alveolar regions other than the mucosa. The relative density of γαβ2t cells is highest in locations near the respiratory tract, blood vessels and visceral pleura. Although the γδ T cell numbers are much smaller in scale, they match or nearly match αβ1T cells in the relative density of the non-alveolar region. In contrast, in the region of the lung parenchyma where the tissue mass or tissue surface area is greatest, the relative density of γδ T cells is much lower than αβ T cells. The difference in the distribution of these two T cells may be related to the difference in the functional roles. The single regional distribution of αβt cells in the lung of normal lungs may reflect some degree of functional homogeneity, while the broad distribution of γδ T cells reflects functional heterogeneity. γδ T cells are mainly in contact with myeloid cells and αβt is more in contact with lymphocytes throughout the lung. This comparison shows that contact of leukocytes with pulmonary γδ T cells is primarily involved in myeloid lineage cells, including F4/80 ⁺ Macrophages and I-A ⁺ Dendritic cells, while αβt cells are often associated with CD45R ⁺ Lymphocytes, including B cells, also have some T cells in contact with plasmacytoid dendritic cells.

In the lung tissue morphological immunohistochemical staining experiment, cells are marked and counted by alpha beta TCR and gamma delta TCR, and a blank control group can see that two cells exist in lung tissue in a small amount; the model control group showed little γδt presence; the high, medium and low dose drug groups showed that more γδ T cells were present near the trachea, significantly increased compared to the model group, the αβt/γδ T cell ratio in the lung tissue of mice was increased in the model group compared to the blank control group, the drug administration group showed a decreasing trend compared to the model group, and the composition drug administration group showed a significant decrease compared to the single drug administration group, as shown in table 3.

Referring to the isolation method of intestinal epithelial inter-cell lymphocytes, we explored an experimental method of density gradient centrifugation to isolate lung tissue lymphocytes. The method for separating the lymphocyte among the intestinal epithelial cells is mature, is accepted by academia, has fewer cell types of intestinal mucosa epithelium, and is favorable for separating different cell groups. Referring to the results of flow analysis of small intestine sample cells, lymphocyte populations in the cell suspension obtained after digestion and separation of lung tissue can be determined, and the specific antibodies can be used to label the αβ T cells and γδ T cells therein, respectively, as shown in table 4. The proportion of the gamma delta T cells of the mice is reduced in the model group compared with the blank control group, and the liquid medicine group of the low-dose composition is obviously increased (P is less than 0.05) compared with the model group, which indicates that the liquid medicine treatment of the composition is beneficial to improving the proportion of the gamma delta T cells of the lung tissues of the mice.

TABLE 3 immunohistochemical staining cell count shows that the composition liquid reduces the αβT/γδ T cell ratio (X.+ -. S) of lung tissue in mice with slow resistance lung model

Table 4 flow cytometer analysis results show that the composition liquid medicine reduces the alpha beta T/gamma delta T cell ratio (X+ -S) of the lung tissue of a mouse model of slow resistance lung

Group of	Alpha beta T cells (%)	Gamma delta 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
				C1 group	53.62±9.89	3.94±1.62	13.61
C2 group	54.93±5.46	3.53±0.61	15.56
				Group C3	56.12±6.01	2.98±0.39	18.83
D1 group	53.43±10.84	4.09±0.83	13.06
				D2 group	54.93±4.12	3.68±0.46	14.92
D3 group	55.32±5.09	3.31±0.88	16.71
				E1 group	52.57±0.51	5.39±1.37	9.75
E2 group	53.94±1.11	4.64±1.04	11.63
				E3 group	54.02±3.50	4.217±0.62	12.95

5.4 IL-6 and IL-13 content in mouse serum, alveolar lavage fluid and small intestine mucus

Comparing the IL-6 and IL-13 content in serum, alveolar lavage fluid and small intestine mucus of the mice, wherein the IL-6 content in serum is higher than that in a blank control group (P < 0.05), the administration groups are lower than that in the model control group, and the high and medium dose composition groups are obviously lower than that in the model control group (P < 0.05); no significant differences were seen between the groups for the IL-13 content in serum. The IL-6 content in the alveolar lavage fluid is higher than that in the blank control group (P < 0.05); the low-dose administration group and the model control group have no statistical difference in comparison, the high-dose administration group and the medium-dose administration group are lower than the blank control group, the model control group and the low-dose administration group, and the high-dose administration group and the medium-dose administration group of the composition are obviously reduced (P is less than 0.05) compared with the single-dose administration group; the IL-13 content in the alveolar lavage fluid is higher than that in a blank control group (P < 0.05), the administration group is lower than that in the model control group, and the high and medium dose groups of the composition are obviously reduced (P < 0.05). No significant differences were seen between the groups of IL-6 content in intestinal mucus; the IL-13 content in intestinal mucus was higher in the slow-blocking lung model control than in the blank (P < 0.05), the high, medium, and low doses of the individual and composition were lower than in the model control, and the high, medium, and low doses of the composition were significantly lower (P < 0.05).

In conclusion, the amount of inflammatory factors secreted by mice with slow-blocking lung model is obviously increased, and the content of IL-6 in serum and alveolar lavage fluid of the composition liquid medicine group and IL-13 in alveolar lavage fluid and intestinal mucus of the composition liquid medicine group is obviously reduced compared with that of a model control group (P is less than 0.05), and the composition liquid medicine group is shown in Table 5.

TABLE 5 IL-6 and IL-13 content (X.+ -. S) in mouse serum, alveolar lavage fluid and small intestine mucus

When the Gao Lihuai element and tectorigenin are combined in a mass ratio of 7:1-3:2, the obtained test effect is basically consistent with the above results, and is not illustrated here.

Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.

Claims (7)

1. A flavonoid aglycone composition for treating chronic obstructive pulmonary disease, comprising Gao Lihuai element and tectorigenin.

2. The flavonoid aglycone composition for treating chronic obstructive pulmonary disease according to claim 1, wherein the mass ratio of Gao Lihuai element to tectorigenin is 7:1-3:2.

3. The flavonoid aglycone composition for treating chronic obstructive pulmonary disease according to claim 1, wherein Gao Lihuai element and iris Huang Sujun are extracted from Chinese medicinal materials.

4. A flavonoid aglycone composition for the treatment of chronic obstructive pulmonary disease according to claim 3, wherein Gao Lihuai element is extracted from subprostrate sophora and said iris Huang Sucong blackberry lily is extracted.

5. A flavonoid aglycone composition according to claim 3 for the treatment of chronic obstructive pulmonary disease, wherein the method of extracting Gao Lihuai element from subprostrate sophora comprises the steps of:

pulverizing radix Sophorae Tonkinensis, adding 6-7 times of ethanol solution, leaching for at least 1 time, each for 5-7 days, filtering the extractive solution, mixing filtrates, and concentrating to obtain radix Sophorae Tonkinensis ethanol extract concentrate;

extracting the concentrated solution of the alcohol extract of the subprostrate sophora with ethyl acetate for 1-5 times, combining the extracting solutions, and concentrating the extracting solutions into an extract to obtain the extract of the subprostrate sophora;

dissolving radix Sophorae Tonkinensis extract with ethanol solution, filtering, separating and purifying the filtrate with macroporous resin column, sequentially eluting with 3 times of column volume distilled water and 75% ethanol solution, discarding water washing part, collecting 75% ethanol eluate, and concentrating to obtain Gao Lihuai element crude extract;

dissolving Gao Lihuai crude extract with methanol, filtering, separating and purifying filtrate with sephadex chromatography, and purifying with chloroform: eluting with methanol mixed solution as eluent, recovering the later part, concentrating, taking out and filtering when white solid is attached to the bottle wall, and using chloroform: and (3) cleaning filter residues by using a mixed solution of methanol, standing the cleaning solution overnight, and slowly precipitating solids at the bottom of the bottle or separating substances out from the wall of the bottle to obtain Gao Lihuai elements.

6. A flavonoid aglycone composition for the treatment of chronic obstructive pulmonary disease according to claim 3, wherein the method of extracting tectorigenin from blackberry lily comprises the steps of:

pulverizing rhizoma Belamcandae, adding 6-7 times of ethanol solution, extracting for at least 1 time, each for 5-7 days, filtering the extractive solution, mixing filtrates, and concentrating to obtain rhizoma Belamcandae ethanol extract concentrate;

extracting the concentrated solution of the blackberry lily ethanol extract with ethyl acetate for 1-5 times, mixing the extracts, concentrating into extract, and obtaining blackberry lily extract;

dissolving rhizoma Belamcandae extract with ethanol solution, filtering, separating and purifying the filtrate with macroporous resin column, sequentially eluting with 3 times of column volume distilled water and 75% ethanol solution, discarding water washing part, collecting 75% ethanol eluate, and concentrating to obtain rhizoma Iridis Tectori Huang Sucu extract;

dissolving the iris Huang Sucu extract with methanol, filtering, separating and purifying the filtrate by sephadex chromatography, and purifying by chloroform: eluting with methanol mixed solution as eluent, recovering the later part, concentrating, taking out and filtering when yellow solid is adhered to the bottle wall, and using chloroform: and (3) cleaning filter residues by using a methanol mixed solution, standing the cleaning solution overnight, and slowly precipitating solids or substances separated from the bottle bottom or the bottle wall to obtain tectorigenin.

7. Use of a flavonoid aglycone composition according to any one of claims 1 to 6 in a medicament for the treatment of chronic obstructive pulmonary disease.

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