Traditional Chinese medicine composition for treating chronic obstructive pulmonary disease and its application

Abstract

The present invention discloses a traditional Chinese medicine composition for treating chronic obstructive pulmonary disease and its application, comprising the following raw materials: sophocarpine and irigenin. The content of sophocarpine is 0.1-2 parts by weight, and the content of irigenin is 1 part by weight. The preparation method of sophocarpine is as follows: take the crude powder of Sophora subprostrata, add acetone to the crude powder of Sophora subprostrata, reflux for 10-40 min, filter to obtain the first filtrate and concentrate it to obtain the first extract. The first extract is subjected to column chromatography using a silica gel column, and the eluent is gradient elution with a petroleum ether-chloroform, petroleum ether-ethyl acetate, and petroleum ether-acetone system in turn. Take the eighth sample, and then purify the eighth sample by gel column chromatography to obtain sophocarpine. By combining sophocarpine and irigenin, the present invention can effectively reduce the pathological damage of the lung tissue of model rats and improve their lung function.

Description

Technical Field

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

[0002] Chronic obstructive pulmonary disease (COPD) severely impairs respiratory and lung functions and even leads to heart failure. The incidence rate among people over 40 years old globally has reached as high as 9% - 10%. Its chronic progression process brings a heavy burden to individuals, families and even social medical resources. Currently, the purpose of drug treatment is to relieve the symptoms of patients, reduce the frequency and severity of acute attacks, and improve the health status and exercise tolerance of patients. The common types of drugs for treating COPD include bronchodilators, inhaled or oral glucocorticoids, phosphodiesterase-4 inhibitors, methylxanthine drugs, and other drugs such as vaccines and antibiotics for treatment, but they mainly focus on symptomatic treatment and will produce relatively large side effects such as increased infection and immunosuppression. Therefore, the research and development of drugs for treating the respiratory tract, especially chronic lung diseases, are particularly prominent, and it is very urgent to find new and effective treatment drugs. Summary of the Invention

[0003] An object of the present invention is to solve at least the above problems and provide at least the advantages described hereinafter.

[0004] Another object of the present invention is to provide a traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, which can effectively reduce the pathological damage of lung tissue in model rats and improve their lung function by combining sophocarpine with irigenin.

[0005] To achieve these objects and other advantages of the present invention, there is provided a traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, comprising the following raw materials: sophocarpine, irigenin.

[0006] Preferably, the amount of sophocarpine is 0.1 - 2 parts by weight, and the amount of irigenin is 1 part by weight.

[0007] Preferably, the preparation method of sophocarpine is as follows: take the crude powder of Sophora subprostrata, add acetone to the crude powder of Sophora subprostrata, reflux for 10 - 40 min, filter to obtain the first filtrate and concentrate it to obtain the first extract, subject the first extract to column chromatography using a silica gel column, and elute with a gradient of petroleum ether - chloroform, petroleum ether - ethyl acetate, and petroleum ether - acetone systems. Take the eighth sample, and then purify the eighth sample by gel column chromatography to obtain sophocarpine.

[0008] Preferably, the amount of acetone is 6 - 10 times the amount of the crude powder of Sophora subprostrata.

[0009] Preferably, the eluent is eluted successively with a volume ratio of petroleum ether to chloroform of 5:1, 3:1, 1:1, eluted successively with a volume ratio of petroleum ether to ethyl acetate of 20:1, 10:1, 5:1, 3:1, 1:1, and eluted successively with a volume ratio of petroleum ether to acetone of 20:1, 10:1, 5:1, 3:1, 1:1 and acetone.

[0010] Preferably, the preparation method of irigenin is as follows: take the crude powder of Belamcanda chinensis, add ethanol to the crude powder of Belamcanda chinensis for reflux extraction, filter to obtain the second filtrate and concentrate to obtain the second extract, add 1.5 times the amount of water to dissolve the second extract, then add ethyl acetate for extraction, take the ethyl acetate layer and concentrate to obtain the third extract, subject the third extract to column chromatography on a silica gel column, and the eluent is eluted successively with a gradient of petroleum ether - ethyl acetate and chloroform - methanol systems, take the eighth component, and then purify the eighth component by gel column chromatography and concentrate to obtain irigenin.

[0011] Preferably, in the preparation method of irigenin, the eluent is eluted successively with a volume ratio of petroleum to ethyl acetate of 3:1, 2:1, 1:1, and the volume ratio of chloroform to methanol is 1:1.

[0012] Preferably, when the eighth component is purified by gel column chromatography, the eluent is chloroform and methanol, and the volume ratio of chloroform to methanol is 1:1.

[0013] Preferably, the application of the traditional Chinese medicine composition in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0014] The present invention has at least the following beneficial effects:

[0015] In the present invention, it is shown that the expression levels of TNF-α, IL-8, IL-6, and IL-1β in the sera of rats in each administration group all show a downward trend, and the difference is statistically significant (P < 0.05); the number of white blood cells (WBC) and the number of neutrophils in the peripheral blood of rats in each administration group all show a decreasing trend, among which, the decrease in the dichroa rootin - irigenin composition group is obvious (P < 0.05). Thus, it is indicated that the dichroa rootin - irigenin composition can improve and inhibit inflammation, improve the alveolar structure, inhibit airway remodeling, and improve the symptoms of the COPD rat model; in the present invention, consistent results are obtained for the SOD activity and MDA level in the sera of COPD rats after intervention with the dichroa rootin - irigenin composition, which can preliminarily indicate that the dichroa rootin - irigenin composition may participate in the improvement of the symptoms of COPD rats by inhibiting the oxidative stress response. In summary, the dichroa rootin - irigenin composition can reduce the pathological damage of the lung tissue of COPD model rats and improve their lung function.

[0016] Other advantages, objects, and features of the present invention will be partly reflected by the following description, and partly will be understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0017] Figure 1 It is a HE staining diagram of rat lung tissue during the modeling period for one of the technical solutions described in the present invention. Among them, control is the normal control group, and Moodel(3w) is the third week of modeling;

[0018] Figure 2 It is a graph showing the change in the body weight of rats during the drug administration period for one of the technical solutions described in the present invention;

[0019] Figure 3 It is a graph showing the index change of the thymus and spleen of rats for one of the technical solutions described in the present invention. Among them, the ordinate Thymus in the left figure represents the thymus, and the ordinate Spleen in the right figure represents the spleen;

[0020] Figure 4 It is a H&E staining diagram of rat lung tissue after drug administration for one of the technical solutions described in the present invention;

[0021] Figure 5 It is the IC of the Sophora subprostrata rootin-Irisflorentin composition with different ratios on RAW264.7 cells for one of the technical solutions described in the present invention 50 ;

[0022] Figure 6 It is a graph showing the effect of different compositions on cell viability for one of the technical solutions described in the present invention. Detailed Embodiments

[0023] The following further describes the present invention in detail with reference to the drawings, so that those skilled in the art can implement it according to the description in the specification.

[0024] <Example 1>

[0025] A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, comprising the following raw materials: Sophora subprostrata rootin, Irisflorentin, 0.1 part by weight of Sophora subprostrata rootin, and 1 part by weight of Irisflorentin.

[0026] <Example 2>

[0027] A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, comprising the following raw materials: Sophora subprostrata rootin, Irisflorentin, 0.6 part by weight of Sophora subprostrata rootin, and 1 part by weight of Irisflorentin.

[0028] <Example 3>

[0029] A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, comprising the following raw materials: sophocarpine and irigenin, 2 parts by weight of sophocarpine and 1 part by weight of irigenin.

[0030] The sophocarpine and irigenin in Examples 1-3 were all purchased from the market.

[0031] <Example 4>

[0032] A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, comprising the following raw materials: sophocarpine and irigenin, 0.6 parts by weight of sophocarpine and 1 part by weight of irigenin.

[0033] The preparation method of sophocarpine is as follows: take the crude powder of Sophora subprostrata, add acetone to the crude powder of Sophora subprostrata, reflux for 10 min, filter to obtain the first filtrate and concentrate it to obtain the first extract, subject the first extract to column chromatography using a silica gel column, and elute with petroleum ether-chloroform, petroleum ether-ethyl acetate, and petroleum ether-acetone systems in a gradient manner. Take the eighth sample, and then purify the eighth sample by gel column (Sephadex LH-20 gel) chromatography to obtain sophocarpine.

[0034] The acetone is 6 times the amount of the crude powder of Sophora subprostrata.

[0035] In the eluent, the volume ratio of petroleum ether to chloroform is 5:1, 3:1, and 1:1 for sequential elution, the volume ratio of petroleum ether to ethyl acetate is 20:1, 10:1, 5:1, 3:1, and 1:1 for sequential elution, and the volume ratio of petroleum ether to acetone is 20:1, 10:1, 5:1, 3:1, 1:1, and acetone for sequential elution.

[0036] The preparation method of irigenin is as follows: take the crude powder of Belamcanda chinensis, add ethanol for reflux extraction, filter to obtain the second filtrate and concentrate it to obtain the second extract, dissolve it in 1.5 times the amount of water of the second extract, then extract with ethyl acetate, take the ethyl acetate layer and concentrate it to obtain the third extract, subject the third extract to column chromatography using a silica gel column, and elute with petroleum ether-ethyl acetate and chloroform-methanol systems in a gradient manner. Take the eighth component, and then purify the eighth component by gel column chromatography, and concentrate to obtain irigenin.

[0037] In the preparation method of irigenin, the volume ratio of petroleum to ethyl acetate in the eluent is 3:1, 2:1, and 1:1 for sequential elution, and the volume ratio of chloroform to methanol is 1:1.

[0038] When the eighth component is purified by gel column chromatography, the eluent is chloroform and methanol, and the volume ratio of chloroform to methanol is 1:1.

[0039] <Example 5>

[0040] A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, comprising the following raw materials: sophocarpine and irigenin, with 0.6 parts by weight of sophocarpine and 1 part by weight of irigenin.

[0041] The preparation method of sophocarpine is as follows: take the crude powder of Sophora subprostrata, add acetone to the crude powder of Sophora subprostrata, reflux for 25 min, filter to obtain the first filtrate and concentrate it to obtain the first extract. Subject the first extract to column chromatography using a silica gel column, and elute with a gradient of petroleum ether-chloroform, petroleum ether-ethyl acetate, and petroleum ether-acetone systems in sequence. Take the eighth sample, and then purify the eighth sample by gel column (Sephadex LH-20 gel) chromatography to obtain sophocarpine.

[0042] The amount of acetone is 8 times the amount of the crude powder of Sophora subprostrata.

[0043] In the eluent, the volume ratio of petroleum ether to chloroform is 5:1, 3:1, 1:1 for sequential elution, the volume ratio of petroleum ether to ethyl acetate is 20:1, 10:1, 5:1, 3:1, 1:1 for sequential elution, and the volume ratio of petroleum ether to acetone is 20:1, 10:1, 5:1, 3:1, 1:1 and acetone for sequential elution.

[0044] The preparation method of irigenin is as follows: take the crude powder of Belamcanda chinensis, add ethanol for reflux extraction, filter to obtain the second filtrate and concentrate it to obtain the second extract. Dissolve the second extract in 1.5 times the amount of water, then extract with ethyl acetate, take the ethyl acetate layer and concentrate it to obtain the third extract. Subject the third extract to column chromatography using a silica gel column, and elute with a gradient of petroleum ether-ethyl acetate and chloroform-methanol systems in sequence. Take the eighth component, and then purify the eighth component by gel column chromatography, and concentrate to obtain irigenin.

[0045] In the preparation method of irigenin, the volume ratio of petroleum to ethyl acetate in the eluent is 3:1, 2:1, 1:1 for sequential elution, and the volume ratio of chloroform to methanol is 1:1.

[0046] When the eighth component is purified by gel column chromatography, the eluent is chloroform and methanol, and the volume ratio of chloroform to methanol is 1:1.

[0047] <Example 6>

[0048] A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, comprising the following raw materials: sophocarpine and irigenin, with 0.6 parts by weight of sophocarpine and 1 part by weight of irigenin.

[0049] The preparation method of sophoraflavanone G is as follows: Take the crude powder of Sophora subprostrata, add acetone to the crude powder of Sophora subprostrata, reflux for 40 min, filter, obtain the first filtrate and concentrate it to obtain the first extract. Subject the first extract to column chromatography using a silica gel column, and elute it successively with a gradient of petroleum ether-chloroform, petroleum ether-ethyl acetate, and petroleum ether-acetone systems. Take the eighth sample, and then purify the eighth sample by gel column chromatography (Sephadex LH-20 gel) to obtain sophoraflavanone G.

[0050] The amount of acetone is 10 times the amount of the crude powder of Sophora subprostrata.

[0051] In the eluent, the volume ratio of petroleum ether to chloroform is eluted successively as 5:1, 3:1, 1:1, the volume ratio of petroleum ether to ethyl acetate is eluted successively as 20:1, 10:1, 5:1, 3:1, 1:1, and the volume ratio of petroleum ether to acetone is eluted successively as 20:1, 10:1, 5:1, 3:1, 1:1 and then acetone.

[0052] The preparation method of irigenin is as follows: Take the crude powder of Belamcanda chinensis, add ethanol for reflux extraction, filter to obtain the second filtrate and concentrate it to obtain the second extract. Dissolve it with 1.5 times the amount of water of the second extract, then extract it with ethyl acetate, concentrate the ethyl acetate layer to obtain the third extract. Subject the third extract to column chromatography using a silica gel column, and elute it successively with a gradient of petroleum ether-ethyl acetate and chloroform-methanol systems. Take the eighth component, and then purify the eighth component by gel column chromatography and concentrate it to obtain irigenin.

[0053] In the preparation method of irigenin, the volume ratio of petroleum to ethyl acetate in the eluent is eluted successively as 3:1, 2:1, 1:1, and the volume ratio of chloroform to methanol is 1:1.

[0054] When the eighth component is purified by gel column chromatography, the eluent is chloroform and methanol, and the volume ratio of chloroform to methanol is 1:1.

[0055] <Experimental Characterization>

[0056] I. Pharmacological and Pharmacodynamic Experiments

[0057] 1 Preparation of Experimental Drugs

[0058] 1.1 Preparation of Experimental Drug Suspensions

[0059] Weigh sophoraflavanone G and prepare sophoraflavanone G suspensions with three gradients of 0.25 g / kg, 0.50 g / kg, and 1.0 g / kg using normal saline, and store them at 4°C for later use;

[0060] Weigh irigenin and prepare suspensions with three gradients of 0.25 g / kg, 0.50 g / kg, and 1.0 g / kg using normal saline, and store them at 4°C for later use;

[0061] Weigh Sophora subprostrata rootin and Iris dichotoma Pall flavone with a mass ratio of 3:5 (i.e., the traditional Chinese medicine composition in Example 5), and prepare a mixed suspension with three gradients of 0.25 g / kg, 0.50 g / kg, and 1.0 g / kg for administration by mixing them with normal saline, and store it at 4°C for later use.

[0062] Both Sophora subprostrata rootin and Iris dichotoma Pall flavone were prepared by the method of Example 5. The Sophora subprostrata rootin prepared in Example 5 was a white solid powder, which was a monomeric compound detected by TLC and HPLC, and its structure was identified by NMR and mass spectrometry as follows:

[0063]

[0064] The Iris dichotoma Pall flavone prepared in Example 5 was a yellow solid powder, which was a monomeric compound detected by TLC and HPLC, and its structure was identified by NMR and mass spectrometry as follows:

[0065]

[0066] 1.2 Preparation of dexamethasone acetate tablet suspension

[0067] Take 1 dexamethasone acetate tablet, add normal saline to adjust the concentration to prepare a dexamethasone acetate tablet suspension of 0.0675 mg / kg, and store it at 4°C.

[0068] 1.3 Preparation of chloral hydrate injection

[0069] Weigh chloral hydrate crystals, add normal saline to prepare an injection containing 10% chloral hydrate (prepared and used immediately).

[0070] 2 Preparation and grouping of experimental animals

[0071] Select healthy male SD rats aged 7 - 9 weeks and weighing (220 ± 20) g. 120 male rats were raised one week before the experiment. Divide the 120 rats into 12 groups and raise them in 12 cages (10 rats in each cage). The breeding conditions are that the room temperature is 23 - 26°C, the humidity is 47 - 53%, change the clean drinking water daily, add feed, and change the bedding;

[0072] The twelve groups were respectively the normal control group (A), the model group (B), the low-dose sophocarpine group (C1, 0.25 g / kg), the medium-dose sophocarpine group (C2, 0.50 g / kg), the high-dose sophocarpine group (C3, 1.0 g / kg), the low-dose irigenin group (D1, 0.25 g / kg), the medium-dose irigenin group (D2, 0.50 g / kg), the high-dose irigenin group (D3, 1.0 g / kg), the low-dose sophocarpine-irigenin group (E1, 0.25 g / kg), the medium-dose sophocarpine-irigenin group (E2, 0.50 g / kg), the high-dose sophocarpine-irigenin group (E3, 0.50 g / kg), and the dexamethasone acetate group (F). Groups C, D, and E were the experimental groups.

[0073] 2 Experimental methods

[0074] 2.1 Establishment of a rat model of chronic obstructive pulmonary disease

[0075] An experimental animal model of chronic obstructive pulmonary disease (COPD) in rats was replicated by combining intratracheal instillation of lipopolysaccharide (LPS) and cigarette smoke (CS). Except for the normal control group, the other 11 groups were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.41 ml / 100 g) on the 1st and 21st days of the experiment. After depilation and disinfection of the neck, the trachea was exposed surgically, and 200 μl of LPS (1 mg / ml) was injected into the trachea. Immediately, the rats were made to stand upright and shaken left and right to ensure uniform distribution of LPS in the lungs. Then, the incision was sutured under aseptic conditions. In the normal control group, an equal volume of normal saline was injected into the trachea on the 1st and 21st days of the experiment. The surgical procedures were carried out in a laminar flow cabinet, and the wound was disinfected with iodophor after the operation. On the second day after LPS instillation, the rats were placed in a self-made smoking chamber (45×34×18 cm), which was prepared by modifying a rat cage, with a transparent PVC cover plate 0.5 cm thick placed on top for easy observation of the rats. There was a round hole with a diameter of about 1 cm on each side. Each cage contained 10 rats, and 4 cigarette tobacco strands were lit in each cage for smoking. The rats were smoked twice a day for 30 minutes each time, with a 4-hour interval between the two smoking sessions. The model replication period was 12 weeks, and drug intervention started from the ninth week. Smoking was carried out once a day. Finally, the success of model preparation was judged based on the general condition of the rats, changes in lung function, and pathological tissues (criteria for successful model replication: the rats showed dry and yellowish hair, rapid breathing, and moist nose; the lung function of the rats decreased; and there was an increase in inflammatory cell infiltration in the bronchial and lung tissue mucosa).

[0076] 2.2 Drug intervention treatment

[0077] The rats with COPD model were randomly grouped according to their body weights. All groups of rats started drug intervention on the first day of the 9th week of model replication. The normal control group (A) and the model group (B) were given an equal amount of normal saline by gavage, 10 ml / kg per day; the low-dose sophocarpine group (C1), the medium-dose sophocarpine group (C2), the high-dose sophocarpine group (C3), the low-dose irigenin group (D1), the medium-dose irigenin group (D2), the high-dose irigenin group (D3), the low-dose sophocarpine-irigenin group (E1), the medium-dose sophocarpine-irigenin group (E), and the high-dose sophocarpine-irigenin group (E3) were given the corresponding suspension; the suspension of dexamethasone acetate tablets group was given by gavage, 0.0675 mg / kg per day, which was 10 times the clinical conventional dosage of adults, once a day, and administered continuously for 4 weeks.

[0078] 2.3 Specimen collection

[0079] The nine experimental groups of C (C1, C2, C3), D (D1, D2, D3), and E (E1, E2, E3) were each randomly divided into two sub-groups. The first sub-group was used for pulmonary function measurement experiments, and the second sub-group took intact and undamaged lung tissues for pathological section analysis.

[0080] 2.4 Pulmonary function measurement

[0081] Rats in each experimental group were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.41 ml / 100 g) at the 9th week of model establishment and after the end of treatment. They were fixed supine on the operating table, the hair on the neck was depilated, disinfected, the neck skin was incised, the subcutaneous tissue was bluntly separated, the trachea was fully exposed, an inverted "T" incision was made on the trachea, a rat tracheal intubation was inserted, and sutured and fixed. The two ends of the intubation were respectively connected to a flow rate and pressure sensor, and the flow rate sensor was connected to a small animal ventilator. The tidal volume was set at 10 ml / Kg and the respiratory frequency was 70 times / min. The flow rate and pressure sensors were connected to a computer data recording and analysis system. After turning on the system, the instrument was first run for inspection. The plethysmograph box was opened, and the tracheal intubation port was pinched with fingers. When a regular and stable press waveform appeared, the rat tracheal intubation was connected to a "Y" - shaped connecting tube, and the plethysmograph box was closed tightly. After recording a period of quiet breathing, the vital capacity was detected. Negative pressure (-25 cm H2O) was connected to aspirate air to cause deep exhalation. When a complete respiratory peak appeared, the pause button was clicked, and the values of various pulmonary function parameters in the recording and analysis system were recorded.

[0082] 2.5 Detection and specimen collection

[0083] Collect blood from the abdominal aorta using a negative pressure blood collection needle. After coagulation, separate the serum, centrifuge at 3000 rpm for 5 minutes, take the supernatant and aliquot it into EP tubes, store at -20°C, and wait for experiments such as ELISA. Insert the front end of the tracheal cannula into a 5-ml syringe containing 3 ml of normal saline in the neck, tie it tightly with a suture, inject it into the lung tissue, wash it repeatedly 2 times, aspirate back and forth, and then recover the bronchoalveolar lavage fluid (BALF), inject it into a 5-ml EP tube, and the recovery rate is 50 - 70%. Centrifuge to take the supernatant and store at -20°C for ELISA. Take 0.5 ml of peripheral blood and place it in a micro blood collection tube containing EDTA anticoagulant, and detect the total number of white blood cells (×10 9 ) and neutrophil percentage (%) in the peripheral blood of rats on the same day. Open the chest cavity, take out the whole lung tissue, rinse it with normal saline and dry the excess water on the surface with filter paper, then take the left lung tissue and fix it in 10% tissue fixative for HE staining and immunohistochemical detection; the remaining lung tissue is placed in liquid nitrogen for freezing and then transferred to an -80°C refrigerator for storage for Western-blot detection. Weigh the removed thymus and spleen to calculate the index changes.

[0084] 2.6 Detection of rat peripheral blood white blood cell classification

[0085] Place the collected peripheral blood on ice and gently shake it on a shaker to prevent blood coagulation. Then, use an animal blood analyzer to measure the blood cells and save the data.

[0086] 2.7 Verification of the model establishment by HE staining experiment and observation of the effects of experimental drugs and their combinations on chronic obstructive pulmonary disease in rats

[0087] Observe the degree of pathological damage of rat lung tissue through HE staining experiment. Put the left lung tissue of rats into 4% tissue fixative for fixation and dehydration for 48 hours, and then embed it in paraffin by the conventional paraffin embedding method for wax sectioning. Dewax the cut lung tissue wax sections with four xylenes, 5 minutes each time, then dehydrate them successively with absolute ethanol for 4 minutes, 90% ethanol for 4 minutes, 80% ethanol for 4 minutes, and 70% ethanol for 5 minutes. After that, rinse the wax sections with slow running tap water 3 times and drain the excess water. Then perform staining. First, stain with hematoxylin for 1 - 2 minutes, rinse with clear water 3 times, 1 minute each time, differentiate the tissue with 1% hydrochloric acid alcohol, rinse with clear water 3 times, 1 minute each time. Then stain with 0.5% eosin solution for 1 minute, rinse with clear water 3 times, 1 minute each time, dehydrate with alcohol, air dry, and finally mount the sections with resin and observe the pathology of the lung tissue under a 100× microscope.

[0088] 2.8 ELISA experiment to detect the contents of TNF-α, IL-1β, IL-6, IL-8, MMP-2, MMP-9 and TIMP-1 proteins in rat serum

[0089] After collecting whole blood from the abdominal aorta of rats, let it stand at room temperature for one hour, centrifuge at 3000 rpm for 15 minutes, and transfer the supernatant to 500 μL centrifuge tubes. According to the standard dry powder provided by the kit, dilute the standard product into 6 groups of standard diluents with different concentration gradients in the centrifuge tube for preparing the standard curve. Set blank wells, zero wells, standard wells, and wells for testing samples respectively. Accurately add 50 μL of the standard product to the enzyme-coated plate, add 50 μL of the sample to be tested to the well for testing samples, and add 50 μL of biotin antigen to the zero wells, standard wells, and wells for testing samples. After adding the samples, gently shake the coated plate to mix evenly, seal the plate with a sealing film, and incubate in a 37 °C incubator for 30 minutes. After incubation, discard the liquid, drain, add 300 μL of the prepared washing solution to each well, discard after standing for 30 seconds, pat dry, and repeat this 5 times. Then, except for the blank wells, add 50 μL of biotin antigen marker to each well, seal the plate with a sealing film, and incubate in a 37 °C incubator for 30 minutes. After incubation, also discard the liquid, repeat washing the plate 5 times, and pat dry. When developing color, first add 50 μL of chromogenic agent A to each well, then add 50 μL of chromogenic agent B, gently shake and mix evenly, and develop color at 37 °C in the dark for 10 minutes. Add 50 μL of stop solution to each well to terminate the reaction. Zero with the blank well, measure the absorbance of each well at a wavelength of 450 nm. Finally, calculate the linear regression equation of the standard curve based on the absorbance values, substitute the sample absorbance values into the equation, calculate the actual concentrations of TNF-α, IL-1β, IL-6, IL-8, MMP-2, MMP-9, and TIMP-1 proteins in the samples, and perform statistical analysis.

[0090] 2.9 Detection of the contents of MMP-9 and TIMP-1 proteins in the bronchoalveolar lavage fluid of rats in each group by ELISA

[0091] The method is the same as 2.8

[0092] 2.10 Determination of the activities of SOD and the content of MDA in rat serum

[0093] 2.10.1 Determination of the content of SOD in rat serum

[0094] Use the WST-1 method kit to determine the activity of superoxide dismutase (SOD). In this reaction system, the enzyme amount corresponding to a 50% inhibition rate of SOD is one SOD activity unit (U).

[0095] 2.10.2 Determination of the content of MDA in rat serum

[0096] Use the WST-1 method kit to determine the activity of superoxide dismutase (SOD). In this reaction system, the enzyme amount corresponding to a 50% inhibition rate of SOD is one SOD activity unit (U).

[0097] 2.11 Detection of the protein expression levels of MMP-2, MMP-9, TIMP-1, HMGB1, TLR4, NF-κB p65, HIF-1α, VEGF, and EGF in the lung tissues of rats by Western blot

[0098] Weigh 100 mg of the lung tissues from each experimental group of rats. Put the tissues into a sterilized small mortar pre-cooled with liquid nitrogen in advance, and quickly grind them. Add an appropriate amount of liquid nitrogen during the process and continue grinding until the tissues are ground into fine powder. Then add 1 ml of protein lysate and lyse on ice for 10 min. Then centrifuge at 14,000 rpm for 25 min in an ultra-low temperature high-speed centrifuge. Take the supernatant for use.

[0099] According to the instructions in the protein concentration determination kit, mix reagent A and reagent B evenly at a ratio of 50:1 to prepare the protein determination working solution. At the same time, dilute the standard protein to 8 concentrations of 2000, 1500, 1000, 750, 500, 250, 125, 25, and 0 μg / mL according to the instructions. Dilute according to the ratio of rat lung tissue protein sample: normal saline = 1:19. Add the diluted standard protein and tissue protein samples to a 96-well plate, then add 200 μL of the protein determination working solution. Incubate the 96-well plate in a 37°C constant temperature incubator for 30 min, and then measure the OD value of each well at a wavelength of 562 nm. Draw a standard curve based on the standard protein concentration and its corresponding OD value, and calculate the concentration of the tissue protein sample according to the linear equation. Adjust the protein concentration to the same concentration with the lysate.

[0100] Sample preparation was carried out by adding protein loading buffer in a ratio of 1:4, and the protein was denatured by heating in boiling water at 100°C for 5 min. A 10% separating gel was prepared using the Yaenzyme Rapid Gel Preparation Kit, and electrophoresis buffer and transfer buffer were prepared. Finally, the protein loading amount per lane was adjusted to 50 - 100 μg according to the protein concentration. After electrophoresis of the protein at a constant voltage of 80 V until it reached the separating gel, the voltage was adjusted to 110 V and electrophoresis was continued until the bottom, followed by transfer. A transfer "sandwich" was made together with thick filter paper. Then, transfer was carried out in an ice bath at a constant current of 250 mA for 70 min. After the transfer was completed, the PVDF membrane was placed in TBST and washed on a shaker 3 times, 5 min each time. After the washing was completed, the PVDF membrane with protein bands was put into primary antibodies against GAPDH, β-actin, MMP-2, MMP-9, TIMP-1, HMGB1, TLR4, NF-κB p65, HIF-1α, VEGF, and EGF and incubated overnight at 4°C. The next day, it was washed 3 times with TBST, 5 min each time. Then it was transferred to the secondary antibody and incubated at room temperature for 1 h. Finally, it was placed in TBST and washed on a shaker 3 times, 5 min each time. Color development was carried out by chemiluminescence method, imaging and scanning the film through the Amersham Imager 680 system, and the gray value analysis of the target protein was performed using ImageJ software.

[0101] 2.12 Detection of the expression of TLR4, NF-κB p65, HIF-1α, and VEGF proteins in rats by immunohistochemistry

[0102] The antigen expression in rat lung tissue was observed by immunohistochemistry. The rat lung tissue was fixed and paraffin sections were prepared by the conventional paraffin embedding method. The cut lung tissue wax sections were dewaxed through four gradients of xylene, 5 min each time, and then dehydrated successively in 100% ethanol for 4 min, 90% ethanol for 4 min, 80% ethanol for 4 min, and 70% ethanol for 5 min. After that, it was rinsed 3 times with tap water. It was incubated with 3% H2O2 in deionized water at room temperature for 5 - 10 min to eliminate the activity of endogenous peroxidase, and rinsed 3 times with PBS, 5 min each time. Then the tissue sections were heat - repaired in 10 mM citrate buffer (pH 6.0) for 10 min. After cooling, it was washed 3 times with PBS, 5 min each time. After the repair, 5% BSA blocking solution was added dropwise and incubated at 37°C for 30 min. Then it was dried by shaking, without washing. Then it was incubated overnight at 4°C with primary antibodies against TLR4, NF - κB p65, HIF - 1α, and VEGF. The next day, the residual primary antibodies were washed away with PBS, 5 min each time, for a total of 3 times. Then it was incubated with secondary antibody at 37°C for 30 min. It was also washed three times with PBS. Then it was stained with DAB for 1 min, and the color development was observed under the microscope. The staining time was extended to 2 min appropriately. Finally, it was stained with hematoxylin for 2 min, dehydrated with alcohol - hydrochloric acid for 1 min, and decolorized with distilled water for 10 min. Finally, it was sealed with resin, and the antigen reaction was observed under a 200× microscope. Then the positive staining intensity was measured by IPP software (Image - Pro Plus), and the protein expression was evaluated by IOD value.

[0103] 2.13 Statistical methods

[0104] SPSS 24.0 and GraphPad Prism 7 were used for statistical analysis and drawing. The experimental results were expressed as Mean ± SD. One - way ANOVA was used for comparing the means of sample data among multiple groups, and the t - test was used for pairwise comparison between groups. P < 0.05 indicated statistical significance.

[0105] 3 Experimental results

[0106] 3.1 The rat model of chronic obstructive pulmonary disease was successfully induced by the LPS combined with smoking method

[0107] When LPS was injected into the trachea combined with smoking for 9 weeks, the general condition of the rats was poor, the body weight gain was slower than that of the normal control group, there was emphysema in the lung tissue, and white patch-like symptoms appeared on the surface. Through the detection of an animal pulmonary function analyzer, it was found that the pulmonary function of the rats decreased significantly. Compared with the normal control group, in the model group, the forced expiratory volume in 0.3 seconds (FVC 0.3), the ratio of FVC 0.3 to FVC (FVC 0.3 / FVC), the peak expiratory flow (PEF), the flow rate at 50% of FVC exhaled (Forced expiratory flow 50%, FEF50%), and the maximal midexpiratory flow curve (MMF) were all significantly reduced. Appearance inspection found that the color of the rat lung tissue became lighter and small white spots appeared. The results of HE staining pathological examination showed that with the increase of the modeling time, the alveolar wall of the rat lung tissue proliferated and thickened, more and more bronchial cilia fell off, and more and more inflammatory cells infiltrated the interstitium. As Figure 1 shown, the pulmonary function parameters at the 9th week of modeling are shown in Table 1;

[0108] Table 1 Pulmonary function parameters at the 9th week of modeling

[0109]

[0110] Note: Compared with the normal control group, # P<0.05, control is the normal control group, Model is the model group

[0111] 3.2 Effects of the sophocarpine-iridin composition on the general condition of COPD rats

[0112] 3.2.1 Changes in biological characteristics

[0113] The rats in the normal control group breathed evenly and smoothly, their mouths and noses were clean, there was no secretion, their hair was shiny, their activity was normal, they were sensitive in response, their body weight increased steadily, their food and water intake were normal, their urine and feces were normal, and their tongues were rosy. The rats in the model group breathed rapidly, and some even opened their mouths to breathe significantly, with moist rales during breathing, their mouths and noses were moist with secretions, their fur was dry, yellow and dull, the hair loss phenomenon was relatively serious, their activities were slow, they were listless and curled up in a crawling position, their reactions were dull, and their body weight gain was slower than that of other groups. As the modeling time increased, the food and water intake decreased, and there was occasional loose stools, and their tongues were dark purple. The general characteristics and respiratory symptoms of the rats in each experimental group and the dexamethasone tablet group were worse than those of the normal control group and better than those of the model group. The general characteristics and respiratory symptoms of the rats in each dose group of sophocarpine-iridin were better than those of the dexamethasone tablet group (compared with the normal control group, #P < 0.05; compared with the model group, *P < 0.05).

[0114] 3.2.2 Changes in body weight of rats after drug administration intervention

[0115] The body weights of rats in each group gradually increased with the increase of weeks of age. Among them, the normal control group had the fastest growth rate, and the body weight of rats in the model group increased slower than that in other groups; the growth trend of the body weight of rats in the lophocarpine-irisflorentin group accelerated compared with the model group after drug administration from the 9th week, but there was no significant difference among groups C and D. The body weight of rats in the dexamethasone tablet group decreased significantly, as Figure 2 shown.

[0116] 3.2.3 Changes in thymus and spleen indices of rats

[0117] Compared with the normal control group, the thymus index of rats in the model group decreased significantly (P < 0.05). Compared with the model group, the thymus indices of rats in each drug administration group showed an upward trend, and the thymus index of the lophocarpine-irisflorentin group increased significantly (P < 0.05), while that of the dexamethasone tablet group decreased. Compared with the normal control group, the spleen index of rats in the model group decreased significantly (P < 0.05). Compared with the model group, the spleen indices of rats in the lophocarpine-irisflorentin group drug administration group increased significantly (P < 0.05), as Figure 3 shown.

[0118] 3.3 Effects of lophocarpine-irisflorentin composition on lung function of COPD rats

[0119] Compared with the normal control group, FEV0.3, FEV0.3 / FVC, PEF, FEF50%, and MMF of rats in the model group decreased significantly (P < 0.05). Compared with the model group, all lung function parameters of the drug administration group increased, and the upward trends of the irisflorentin group and the lophocarpine-irisflorentin group were more significant (P < 0.05), as shown in Table 2.

[0120] Table 2 Lung function parameters of rats after drug administration

[0121]

[0122] Note: Compared with the normal control group, #P < 0.05; compared with the model group, *P < 0.0

[0123] 3.4 Effects of lophocarpine-irisflorentin on pathological changes of lung tissue in COPD rats

[0124] In the normal control group, the ciliated columnar epithelium of the bronchus at all levels in rats was intact, the respiratory tract and alveolar epithelium were structurally intact, the cilia were arranged neatly, the smooth muscle was intact, there was no fibrous tissue hyperplasia, and the morphological structure of the alveolar tissue was intact. In the model group, the alveolar walls of the lung tissue in rats were hyperplastic and thickened, there was more infiltration of chronic inflammatory cells in the interstitial tissue, some alveoli were atrophied, and some alveoli were dilated; the ciliated columnar epithelium of the bronchus at all levels was incomplete, part of the ciliated epithelium was exfoliated, goblet cells increased, and mucus glands were hyperplastic and hypertrophic; mucus plugs could be seen in the bronchioles; the alveolar lumen was irregularly enlarged, and some alveoli ruptured and fused into bullae. The above-mentioned pathology in each dose group of the lophanthin-tectorigenin group and the dexamethasone tablet group was improved to varying degrees. The improvement in the high-dose lophanthin-tectorigenin group and the dexamethasone tablet group was the most obvious, as Figure 4 shown.

[0125] 3.5 Effects of the lophanthin-tectorigenin composition on the inflammatory response in COPD rats

[0126] 3.5.1 Effects of the lophanthin-tectorigenin composition on the number of white blood cells in the peripheral blood of COPD rats

[0127] Compared with the normal control group, the number of white blood cells (WBC) and neutrophils in the peripheral blood of rats in the model group increased significantly (P < 0.05); compared with the model group, the number of WBC and neutrophils in each administration group of rats showed a decreasing trend. Among them, the curative effect of the lophanthin group and the tectorigenin group had no obvious relationship with the administration dose; the decrease in the lophanthin-tectorigenin group was obvious, and the effect was the best when the administration amount was 0.5 g / kg. As the administration dose increased, the number of white blood cells and neutrophils no longer decreased (P < 0.05), as shown in Table 3.

[0128] Table 3 The number of white blood cells and neutrophils in the peripheral blood of rats

[0129]

[0130]

[0131] Note: Compared with the normal control group, # P < 0.05; compared with the model group, *P < 0.05

[0132] 3.5.2 Effects of the lophanthin-tectorigenin composition on serum inflammatory factors in COPD rats

[0133] Compared with the normal control group, the concentrations of TNF-α, IL-1β, IL-6, and IL-8 in the serum of rats in the model group were significantly increased (P < 0.05); compared with the model group, the Sophora subprostrata rootin group (C1-C3) and Iris dichotoma flavone group (D1-D2) had certain therapeutic effects. The concentrations of TNF-α, IL-1β, IL-6, and IL-8 in the serum of rats in the Sophora subprostrata rootin-Iris dichotoma flavone group were significantly decreased, and the effect was most obvious when the administration dose was 0.5 g / kg. When the dose was increased again, the effect no longer increased (P < 0.05), as shown in Table 4.

[0134] Table 4 Expression of inflammatory factors in rat serum

[0135]

[0136] Note: Compared with the normal control group, # P < 0.05; compared with the model group, *P < 0.05

[0137] 3.6 Effect of Sophora subprostrata rootin-Iris dichotoma flavone composition on oxidative stress in COPD rats

[0138] Compared with the normal control group, the SOD activity value in the serum of rats in the model group was significantly decreased, and the MDA content was significantly increased (P < 0.05); compared with the model group, the SOD activity value in the serum of rats in the Sophora subprostrata rootin group and Iris dichotoma flavone group increased to a certain extent, but not significantly. The Sophora subprostrata rootin group had a certain degree of decrease in MDA content, and the Iris dichotoma flavone group had a significant decrease (P < 0.05). The SOD activity value in the serum of rats in the Sophora subprostrata rootin-Iris dichotoma flavone group was significantly increased (P < 0.05), and the concentration of MDA in the serum of rats was significantly decreased (P < 0.05). The effect was most obvious when the administration concentration of the Sophora subprostrata rootin-Iris dichotoma flavone group was 0.5 g / kg. When the dose was increased again, the effect no longer increased, as shown in Table 5.

[0139] Table 5 Expression of SOD and MDA in rat serum ( n = 5)

[0140]

[0141] Note: Compared with the normal control group, # P < 0.05; compared with the model group, *P < 0.05

[0142] 4 Experimental study on pulmonary cytotoxicity

[0143] 4.1 Selection of component ratio

[0144] The inhibitory rate of the cytisine - irigenin composition at different ratios on RAW264.7 cells was determined by the MTT method, and the IC 50 value was calculated.

[0145] Preparation of the liquid medicine: Weigh an appropriate amount of cytisine and irigenin samples precisely, and add appropriate DMSO according to the weight ratios of 1:10, 3:10, 2:5, 3:5, 4:5, 1:1, and 2:1 respectively to prepare a series of cytisine - irigenin compositions with different ratios. Precisely measure 12 μL of the above - mentioned mixed solution and dilute it to 2 mL with the culture medium.

[0146] Determination method: Set up a drug - adding group (cytisine - irigenin composition), a negative control group (culture medium and cell fluid), and a blank group (culture medium). Each group of test samples has 3 duplicate wells and is given the same culture conditions. The specific operation is as follows: Take monolayer - cultured RAW264.7 cells, digest them with 0.25% trypsin, and inoculate them into a 96 - well culture plate containing RPMI1640 with 10% fetal bovine serum (5×10 3 cells / well, 100 μL), and culture them at 37°C, 5% CO2, and saturated humidity for 24 h; Add 100 μL of the above - prepared cytisine - irigenin composition sample solution to the cell fluid and continue to culture under the same conditions; After 48 h, discard the culture solution, add 50 μL of MTT solution (1 mg / mL) to each well and continue to culture for 4 h to obtain purple - blue formazan products; Centrifuge, discard the supernatant, add 150 μL of DMSO to each well, and gently shake for 10 min until the formazan is completely dissolved; Use an enzyme - linked immunosorbent assay (ELISA) reader to measure the optical density (OD) value of each well at a wavelength of 490 nm. Calculate the cell inhibitory rate of each composition with different ratios according to the following formula. The above experiments were repeated 3 times. The results are shown in Figure 5. When the ratio of the cytisine - irigenin composition is 3:5, the IC 50 value for RAW264.7 macrophages is the lowest.

[0147] Cell inhibitory rate (%) = (experimental group - negative group) / (negative group - blank group)×100%

[0148] 4.2 Cytotoxicity experiment on lung cells

[0149] The effect of the cytisine - irigenin composition on the proliferation of human lung (bronchial) epithelial BEAS - 2B cells was determined by the MTT method, and the cytotoxicity experiment of the cytisine - irigenin composition on lung cells was detected.

[0150] 4.2.1 Preparation of the liquid medicine and cell culture

[0151] Preparation of the medicinal solution: Sophoraflavanone G and Irisflorentin were dissolved in DMSO at a suitable concentration to prepare a stock solution, which was stored at -20°C. Before use, it was diluted to the required concentration, that is, a mixed medicinal solution with a ratio of Sophoraflavanone G to Irisflorentin of 3:5.

[0152] Preparation of the MTT solution: Usually, the final concentration of MTT prepared is 5 mg / ml. Phosphate buffer (PBS) or normal saline must be used as the solvent. After preparation, it was 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 was preferably wrapped with aluminum foil.

[0153] Cell culture: Human lung (bronchial) epithelial cells BEAS-2B were cultured in RPMI-1640 complete medium containing 10% fetal bovine serum, placed in a constant temperature incubator at 37°C and 5% CO2 with saturated humidity, and the medium was changed once every 1 to 2 days. Subculture was carried out when the cell density reached 70% - 80%.

[0154] 4.2.2 Experimental grouping

[0155] Blank control group: 100 μL of RPMI-1640 complete medium and 100 μL of 0.1% DMSO solution.

[0156] 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

[0157] Experimental group: 100 μL of cell suspension with a density of 2×10 4 cells / ml and 100 μL of mixed composition medicinal solution with concentrations of 10, 20, 40, 80, 100, 150, and 200 μg / ml were added respectively.

[0158] 4.2.3 Experiment and results

[0159] Cells in the logarithmic growth phase were digested and resuspended to a concentration of 2×10 4Single cells at a density of 100 cells / ml. Take 100 μL and inoculate it into a 96-well plate according to the above grouping. Place it in an incubator with a saturated humidity of 37°C and 5% CO2 for 12 h. Observe the cell morphology. If the cell state is good, administer drugs at concentrations of 10, 20, 40, 80, 100, 150, and 200 μg / ml according to the grouping. Set up 6 replicate wells and incubate them in the incubator for 24 h. Add 20 μL of 5 mg / ml MTT. After incubating in an incubator with a saturated humidity of 37°C and 5% CO2 for 4 h, remove the supernatant, add 150 μL of DMSO, and oscillate on a micro oscillator for 10 min until the crystals are completely dissolved. Then measure the OD value at 570 nm. The average value of 6 parallel replicate wells is the result of one experiment. The experiment is independently repeated three times.

[0160] Cell viability (%) = (experimental group - blank group) / (negative group - blank group) × 100%

[0161] As shown in Table 1 and Figure 6 It can be seen that after the extract at a concentration range of 10 - 200 μg / mL acts on BEAS-2B cells, the cell proliferation rate is above 95%. It can be considered that the composition liquid medicine has no obvious cytotoxicity to BEAS-2B cells within the set concentration range.

[0162] Table 1 Effects of composition liquid medicine at different concentrations on the proliferation of BEAS-2B cells ( n = 3)

[0163]

[0164] 5. Conclusion

[0165] In this study, a classic modeling method of intratracheal injection of lipopolysaccharide combined with smoking was used. The success of model construction was evaluated based on the pathological morphology of lung tissue and changes in lung function. After 9 weeks of modeling, it was found that the alveolar walls in the lung tissue of the model rats were thickened, the local lumen of the bronchi was deformed, epithelial cells were shed, the secretion in the bronchi increased, and obvious inflammatory cell infiltration was visible. Moreover, the capillaries and small veins in the alveolar walls were dilated and filled, indicating congestion in the lung tissue and suggesting pathological damage in both the bronchi and lung tissue of the rats after model preparation. Through lung function tests, it was found that the FEV0.3 / FVC and PEF of the model group rats were significantly decreased, suggesting an increase in airway resistance, airflow limitation, and pulmonary ventilation dysfunction, as well as a decrease in airway compliance, that is, a decrease in lung function, which was in line with the basic characteristics of the COPD model, indicating successful modeling. After intervention and treatment with the sophocarpine-iridin composition, the pathological damage in the lung tissue of the rats in each dose group was improved to varying degrees, and lung function parameters such as FEV0.3, FEV0.3 / FVC, PEF, FEF50%, and MMF were significantly increased, indicating that sophocarpine-iridin has the effect of improving the pathological damage of lung tissue and lung function in COPD model rats. The general condition of the rats administered with sophocarpine-iridin was better than that of the dexamethasone tablet group. After administration intervention, the body weight growth rate of the rats in the sophocarpine-iridin combination group was faster than that of the model group, while the body weight of the dexamethasone tablet group showed a downward trend, which also reflected the problem of greater adverse reactions of glucocorticoids in the application of COPD. The thymus and spleen indices of each administration group were increased, and the sophocarpine-iridin combination group was significantly increased (P < 0.05), indicating that sophocarpine-iridin played a role in improving the overall function of COPD rats.

[0166] The results of this study showed that the expression levels of TNF-α, IL-8, IL-6, and IL-1β in the serum of the rats in each administration group showed a downward trend, and the difference was statistically significant (P < 0.05); the number of white blood cells (WBC) and neutrophils in the peripheral blood of the rats in each administration group showed a decreasing trend. Among them, the decrease in the sophocarpine-iridin composition group was obvious (P < 0.05). Thus, it was indicated that the sophocarpine-iridin composition could improve and inhibit inflammation, improve the alveolar structure, inhibit airway remodeling, and improve the symptoms of the COPD rat model.

[0167] In this study, consistent results were obtained for the SOD activity and MDA level in the serum of COPD rats after intervention with the sophocarpine-iridin composition, which could preliminarily indicate that the sophocarpine-iridin composition might be involved in the improvement of the symptoms of COPD rats by inhibiting the oxidative stress response.

[0168] In summary, the sophocarpine-iridin composition can reduce the pathological damage of lung tissue in COPD model rats and improve their lung function.

[0169] The number of devices and the processing scale described herein are used to simplify the description of the present invention. Applications, modifications, and variations of the traditional Chinese medicine composition for treating chronic obstructive pulmonary disease according to the present invention will be apparent to those skilled in the art.

[0170] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the illustrated examples described herein.

Claims

1. A traditional Chinese medicine composition for treating chronic obstructive pulmonary disease, characterized in that, It comprises the following raw materials: Sophora tonkinensis root flavone is 0.1 - 2 parts by weight, and Iris dichotoma pall flavone is 1 part by weight; The preparation method of Sophora tonkinensis root flavone is as follows: Take the crude powder of Sophora tonkinensis root, add acetone to the crude powder of Sophora tonkinensis root, the amount of acetone is 6 - 10 times the amount of the crude powder of Sophora tonkinensis root, reflux for 10 - 40 min, filter to obtain the first filtrate and concentrate it to obtain the first extract. Subject the first extract to column chromatography using a silica gel column, and the eluents are gradient elution with petroleum ether - chloroform, petroleum ether - ethyl acetate, and petroleum ether - acetone systems in sequence. Take the eighth sample, and then purify the eighth sample by gel column chromatography to obtain Sophora tonkinensis root flavone; In the eluent, the volume ratio of petroleum ether to chloroform is eluted in sequence as 5:1, 3:1, 1:1, the volume ratio of petroleum ether to ethyl acetate is eluted in sequence as 20:1, 10:1, 5:1, 3:1, 1:1, and the volume ratio of petroleum ether to acetone is eluted in sequence as 20:1, 10:1, 5:1, 3:1, 1:1; The preparation method of Iris dichotoma pall flavone is as follows: Take the crude powder of Belamcanda chinensis, add ethanol for reflux extraction, filter to obtain the second filtrate and concentrate it to obtain the second extract. Add water with a volume 1.5 times that of the second extract to dissolve it, then add ethyl acetate for extraction, take the ethyl acetate layer and concentrate it to obtain the third extract. Subject the third extract to column chromatography using a silica gel column, and the eluents are gradient elution with petroleum ether - ethyl acetate and chloroform - methanol systems in sequence. Take the eighth component, and then purify the eighth component by gel column chromatography and concentrate it to obtain Iris dichotoma pall flavone; In the preparation method of Iris dichotoma pall flavone, the volume ratio of petroleum to ethyl acetate in the eluent is eluted in sequence as 3:1, 2:1, 1:1, and the volume ratio of chloroform to methanol is 1:1; When the eighth component is purified by gel column chromatography, the eluent is chloroform and methanol, and among them, the volume ratio of chloroform to methanol is 1:

1.

2. Use of the traditional Chinese medicine composition according to claim 1, characterized in that Application of the traditional Chinese medicine composition in the preparation of a medicament for treating chronic obstructive pulmonary disease.

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