Combination of flavonoid glycosides for treating chronic obstructive pulmonary disease

Abstract

The present invention discloses a flavonoid glycoside combination for treating chronic obstructive pulmonary disease, comprising a tricholoma glycoside-irisin composition. The tricholoma glycoside-irisin composition is prepared with normal saline into a suspension at a concentration of 0.5 to 1.5 g / kg. Also provided are a method for preparing the flavonoid glycoside combination for treating chronic obstructive pulmonary disease and use of the flavonoid glycoside combination in preparing a medicament for treating chronic obstructive pulmonary disease. The flavonoid glycoside combination of the present invention is easy to obtain and has a good effect in treating chronic obstructive pulmonary disease.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating lung diseases, and more particularly to a flavonoid glycoside combination for treating chronic obstructive pulmonary disease. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a preventable and treatable chronic airway disease characterized by persistent respiratory symptoms and airflow limitation.

[0003] Currently, the mainstream treatments for COPD are long-acting β2-agonists (LABAs) and long-acting anticholinergics (LAMAs). These drugs are not widely used in primary healthcare settings. Theophylline-based drugs and relatively inexpensive short-acting preparations are also in short supply at the grassroots level. Antiasthmatic medications available at the grassroots level are primarily oral and intravenous formulations, with a lack of nebulized inhalation medications. Furthermore, while these Western medications can quickly control the condition, they often worsen lung function with relapse, leading to a decline in quality of life and even death. Traditional Chinese Medicine (TCM) considers COPD to be a "wheezing syndrome" or "lung distension" syndrome, caused by internal dampness and toxins and spleen and kidney deficiency. Treatment focuses on drying dampness and resolving phlegm, regulating qi and strengthening the spleen, and relieving cough and clearing turbidity. Traditional Chinese medicine (TCM) and its extracts exert their therapeutic effects through integrated regulation of multiple pathways and targets. Compared to antibiotics, they offer fewer side effects, are suitable for long-term use, do not cause dysbiosis, and are inexpensive, offering promising clinical implications and broad market prospects. Summary of the Invention

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

[0005] Another object of the present invention is to provide a flavonoid glycoside combination for treating chronic obstructive pulmonary disease, which is easy to obtain and has a good effect on treating chronic obstructive pulmonary disease.

[0006] To achieve these objectives and other advantages according to the present invention, a flavonoid glycoside combination for treating chronic obstructive pulmonary disease is provided, comprising a tricholoma glycoside-tectoriusin composition, wherein the tricholoma glycoside-tectoriusin composition is formulated with physiological saline into a 0.5-1.5 g / kg suspension.

[0007] Preferably, the tricholomaside-irisin composition is mixed with physiological saline to form a 1.0 g / kg suspension.

[0008] Preferably, the weight ratio of tricholoma to icterin in the tricholoma-icterin composition is 2:5.

[0009] Provided is a method for preparing a flavonoid glycoside combination for treating chronic obstructive pulmonary disease, comprising the following steps:

[0010] S1. Weighing Radix Sophorae flavescentis and Radix Belamcandae respectively, crushing, extracting with alcohol, and concentrating to obtain an alcohol extract of Radix Sophorae flavescentis and an alcohol extract of Radix Belamcandae;

[0011] S2, respectively, adding ethyl acetate to the alcohol extract of the Radix Sophorae flavescentis and the alcohol extract of the Rhizoma Belamcandae, extracting three times, and concentrating under reduced pressure to obtain a fraction of the Radix Sophorae flavescentis and a fraction of the Rhizoma Belamcandae;

[0012] S3, separating, purifying and recrystallizing the bean root fraction and the total flavonoids fraction of the Belamcanda chinensis to obtain tricholoma rutin and irisin;

[0013] S4, mixing the tricholoma serrata and the icterin to obtain a tricholoma serrata-icterin composition;

[0014] S5. Add physiological saline to the tricholomaside-irisin composition to prepare a suspension.

[0015] Preferably, when the crushed rhizome of bean curd and belamcanda chinensis are subjected to alcohol extraction in step S1, the solvent for the alcohol extraction is 70-75% ethanol, and the volume of the ethanol is 6-10 times that of the crushed rhizome of bean curd and belamcanda chinensis, respectively.

[0016] Preferably, in step S2, the volume ratio of the ethyl acetate to the alcohol extract of the rhizome of soybean and the alcohol extract of the belamcanda chinensis is 1:1.

[0017] Preferably, the separation and purification method of the bean root fraction and the Belamcanda chinensis fraction in step S3 is:

[0018] Sa, respectively dissolving the bean root fraction and the belamcanda chinensis fraction in a low concentration ethanol solution of less than 30%, dissolving and filtering to obtain a bean root filtrate and a belamcanda chinensis filtrate;

[0019] Sb. The filtrate of the bean root and the filtrate of the Belamcanda chinensis are further separated and purified using a D101 macroporous resin column to obtain tricholin and irisin.

[0020] Provided is the use of a flavonoid glycoside combination in the preparation of a medicament for treating chronic obstructive pulmonary disease.

[0021] The present invention has at least the following beneficial effects:

[0022] Trifolioside and irisin are easy to obtain and extract, and they are also effective in treating chronic obstructive pulmonary disease.

[0023] Other advantages, objectives and features of the present invention will be reflected in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the structural diagram of trifolioside;

[0025] Figure 2 is the structural diagram of irisin;

[0026] Figure 3 Graph showing the effect of different concentrations of the drug solution of the composition on the viability of BEAS-2B cells;

[0027] Figure 4 The graph shows the half-maximal inhibitory concentration of a combination of tricholoma scutellariae and irisin at different ratios on RAW264.7 macrophages. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.

[0029] Extraction, purification and identification of flavonoid glycoside combination

[0030] 1. Extraction and extraction

[0031] 50 kg each of Sophora flavescens (SDG) and Belamcanda chinensis (SG) were pulverized and extracted twice with 6-10 times the volume of 70-75% ethanol, each time for 5-7 days. The extracts were filtered, combined, and concentrated under reduced pressure to obtain alcohol extract concentrates with extraction yields of 17.78% and 25.22%, respectively. The alcohol extract concentrates were extracted three times with ethyl acetate at a volume ratio of 1:1. The extracts were combined, concentrated under reduced pressure, and the extraction solvent was recovered. The extracts were concentrated to an extract form, yielding two fractions: Sophora flavescens ethyl acetate extract (SDGY) and Belamcanda chinensis ethyl acetate extract (SGY).

[0032] 2. Separation

[0033] The above two fractions were dissolved in a low-concentration ethanol solution of less than 30% (preferably between 15-30%), filtered, and the filtrates were further separated and purified using a D101 macroporous resin column, eluted with 3 times the column volume of distilled water and 75% ethanol solution, discarded the water-washed portion, collected the 75% ethanol eluate, and recovered the solvent to obtain two different fractions, SDG-A (the fraction obtained by eluting and concentrating the SDGY fraction with 75% ethanol) and SG-A (the fraction obtained by eluting and concentrating the SGY fraction with 75% ethanol) (when the ethanol in the eluate was recovered under reduced pressure, a large amount of white (SDG-A) and yellow solids (SG-A) were precipitated in the rotary evaporator, which were further purified and structurally identified as tricholin and irisin). Note: obtaining SDG-A and SG-A respectively means that SDG-A was obtained by separation and purification of SDGY, and SG-A was obtained by separation and purification of SGY. This description and understanding are applicable elsewhere in this application.

[0034] 3. Purification and recrystallization

[0035] Fractions SDG-A and SG-A were separated and purified by Sephadex G20 chromatography. The samples were dissolved in an appropriate amount of methanol, filtered, loaded, and eluted with a 1:1 mixture of chloroform and methanol to obtain 1, 2, 3, ..., 20 fractions. Among them, SDG-A (trifolioside) recovers parts 16 and 17. When the elution solvent is recovered, a white solid is precipitated and attached to the bottle wall when the concentration is almost completed. SG-A (irisin) recovers parts 13 and 14. When the elution solvent is recovered, a yellow solid is precipitated and attached to the bottle wall when the concentration is almost completed. The obtained solids are filtered respectively and washed with an appropriate amount of a mixed solution of chloroform: methanol = 1:1. The mother liquor is left overnight, and solids are slowly precipitated at the bottom of the bottle or substances are precipitated on the bottle wall. If detected by thin layer chromatography and then by high performance liquid chromatography, they are all impure substances. The monomer compounds can be recrystallized and precipitated using mixed solutions of chloroform: methanol in different proportions to purify trifolioside and irisin. The structures are as follows Figure 1-2 shown.

[0036] MTT assay to determine the effect of trifolioside-irisin combination on the proliferation of human lung (bronchial) epithelial cells BEAS-2B

[0037] 1. Selection of drug-liquid ratio

[0038] Setting the weight ratios of cloverleaf glycoside and irisin to be 1:10, 3:10, 2:5, 3:5, and 4:5, respectively, accurately weighing appropriate amounts of samples, adding appropriate amounts of dimethyl sulfoxide (DMSO) to cloverleaf glycoside and irisin to dissolve the corresponding cloverleaf glycoside and irisin, respectively, and ultrasonically dissolving, taking appropriate amounts of cloverleaf glycoside and irisin solutions after adding DMSO and mixing them to obtain mother liquors, wherein five mother liquors can be configured according to five different weight ratios of cloverleaf glycoside and irisin, and the total concentrations of cloverleaf glycoside and irisin in the five mother liquors are the same, but the weight ratios of cloverleaf glycoside and irisin in the five mother liquors are 1:10, 3:10, 2:5, 3:5, and 4:5, respectively;

[0039] Accurately measure 12 μL of the above mixed mother solution and dilute it to 2 mL with culture medium to obtain the drug solution culture medium;

[0040] Finally, 100 μL of drug culture medium was added to 100 μL of cell solution to determine the inhibition rate of the samples on RAW264.7 macrophages at different weight ratios of tricholin and irisin, and the half-maximal inhibitory concentration (IC) of each sample was calculated. 50 The result is as follows Figure 4 As shown, when the ratio of tricholoma to irisin was 2:5 by weight, the IC 50 The lowest value.

[0041] 2. Preparation of drug solution, preparation of MTT solution and cell culture

[0042] Preparation of drug solution: Dissolve cloverside and irisin with DMSO to a suitable concentration as a stock solution, store at -20°C, and dilute to the required concentration before use, that is, a mixed solution with a ratio of cloverside:irisin of 2:5.

[0043] Preparation of MTT solution: MTT is typically prepared to a final concentration of 5 mg / ml using phosphate buffered saline (PBS) or physiological saline. After preparation, filter the solution with a 0.22 μm filter to remove bacteria and store at 4°C in the dark. During preparation and storage, it is best to wrap the container with aluminum foil.

[0044] Cell culture:

[0045] Thaw a 1 mL cryovial of human lung (bronchial) epithelial BEAS-2B cell suspension by rapid shaking in a 37°C water bath. Add 5 mL of RPMI-1640 complete medium containing 10% fetal bovine serum and mix thoroughly. Centrifuge at 1000 RPM for 5 minutes, discard the supernatant, add 4-6 mL of complete medium, and pipette until the cell suspension is complete. Transfer the tube to a culture flask (or 6 cm dish) and culture overnight.

[0046] The next day, change the culture medium and check the cell density. If the cell density reaches 80%-90%, subculture can be performed. Discard the culture supernatant and rinse the cells 1-2 times with PBS without calcium and magnesium ions.

[0047] Add 1-2 mL of digestion solution (0.25% Trypsin-0.53 mM EDTA) to the culture flask and place it in a 37°C incubator for digestion for 1-2 minutes. Then observe the cell digestion under a microscope. If most of the cells become round and fall off, quickly take it back to the operating table, tap the culture flask several times, and add more than 5 mL of complete culture medium containing 10% serum to terminate the digestion.

[0048] Gently pipette the cells until completely detached, aspirate, and centrifuge at 1000 RPM for 8-10 minutes. Discard the supernatant, add 1-2 mL of culture medium, and pipette evenly. Add 5-6 mL of culture medium per bottle, and divide the cell suspension into new dishes or bottles containing 5-6 mL of culture medium at a ratio of 1:2 to 1:5.

[0049] 3. Experimental Grouping

[0050] Blank control group: 100 μL RPMI-1640 complete medium and 100 μL 0.1% DMSO solution.

[0051] Negative control group: 100 μL with a density of 2 × 10 4 The cells were suspended in 100 μL of RPMI-1640 medium containing 0.1% DMSO.

[0052] Experimental group: 100 μL with a density of 2 × 10 4 The cells were suspended in a volume of 100 μL / ml and 100 μL of a 2:5 cloverside-irisin composition solution with a concentration of 10, 20, 40, 80, 100, 150, and 200 μg / ml were added respectively (the 2:5 cloverside-irisin composition was configured into a solution with a concentration of 10, 20, 40, 80, 100, 150, and 200 μg / ml).

[0053] 4. Experiment

[0054] Take cells in logarithmic growth phase, digest and suspend them into a concentration of 2×10 4100 μL of single cells (100 μg / ml) were plated into 96-well plates according to the grouping described above and incubated in a 37°C, 5% CO2, saturated humidity incubator for 12 hours. Cell morphology was observed. If cells were in good condition, 10, 20, 40, 80, 100, 150, or 200 μg / mL of the mixed composition were administered to each group. Six replicate wells were set up and incubated in a 37°C, 5% CO2, saturated humidity incubator for 24 hours. 20 μL of 5 mg / mL MTT was added and incubated in a 37°C, 5% CO2, saturated humidity incubator for 4 hours. The supernatant was removed and 150 μL of DMSO was added. The cells were shaken on a microplate oscillator for 10 minutes to completely dissolve the crystals. The OD value was measured at 570 nm. The average value of the six replicate wells was the result of a single experiment. The experiment was repeated three times.

[0055] Cell proliferation rate (%) = (experimental group - blank group) / (negative group - blank group) × 100%

[0056] From Table 1 and Figure 3 It can be seen that when the extract concentration is in the range of 10 to 200 μg / mL and acts on BEAS-2B cells, the cell proliferation rate is above 90%. It can be considered that the composition drug solution has no obvious cytotoxicity to BEAS-2B cells within the set concentration range.

[0057] Table 1 Effects of different concentrations of the combined drug solution on BEAS-2B cell proliferation ( n=3)

[0058]

[0059]

[0060] Study on the pharmacological activity of the combination of tricholoma rutaecarpin and irisin on COPD

[0061] 1. Animal Experiment Grouping

[0062] 1.1 Experimental Animals 475 SPF-grade ICR female mice, 6–8 weeks old, weighing 18–20 g, were selected from the Medical Experimental Animal Center of Guangxi Medical University (Animal License No.: SCXK-Gui2014-0002).

[0063] 1.2 Experimental drugs

[0064] 1.2.1 Trifolioside was obtained by extraction, separation and identification in the laboratory, and prepared into suspensions with normal saline at dosages of 0.25 g / kg, 0.50 g / kg, 1.0 g / kg, 1.5 g / kg and 2.0 g / kg, and stored at 4°C until use.

[0065] 1.2.2 Iridoside was obtained by extraction, separation and identification in the laboratory, and prepared into suspensions with normal saline at dosages of 0.25 g / kg, 0.50 g / kg, 1.0 g / kg, 1.5 g / kg and 2.0 g / kg, and stored at 4°C until use.

[0066] 1.2.3 The tricholoma scutellariae-irisin combination was prepared in a ratio of 2:5 with normal saline to prepare suspensions of 0.25 g / kg, 0.50 g / kg, 1.0 g / kg, 1.5 g / kg, and 2.0 g / kg, respectively, and stored at 4°C until use.

[0067] 1.3 Experimental Grouping

[0068] A total of 475 ICR female mice were randomly divided into 17 groups after one week of adaptive feeding: blank control group (group A), model control group (group B), tricholoma strychnine high, relatively high, medium, relatively low, and low dose groups (groups C1-C5), irisin high, relatively high, medium, relatively low, and low dose groups (groups D1-D5), and combination high, relatively high, medium, relatively low, and low dose groups (groups E1-E5), with 25 mice in each group.

[0069] 2 Modeling and drug administration methods

[0070] 2.1 Rearing conditions

[0071] Before and after administration, the experimental animals were housed in separate cages and fed with complete pellet feed and free access to water. The room temperature was 20±2°C and the humidity was 50-60%.

[0072] 2.2 Model Group

[0073] (1) On days 1, 29, and 57, LPS solution (30 μg / 6 μL) was instilled into the respiratory tract of mice through the nasal cavity at a dose of 1.5 mg / kg.

[0074] (2) Continuous cigarette smoke exposure from day 2 to day 84 (except days 29 and 57):

[0075] ① Place mice in animal transport boxes (60 cm × 45 cm × 20 cm) with ventilation windows, 25 mice per box, and seal the box with tape.

[0076] ② Place the animal transport box in a glass fumigation box, and leave a circular ventilation hole with a diameter of about 2 cm on each diagonal wall of the fumigation box to regulate the oxygen and air pressure inside and outside the box.

[0077] ③ Light a cigarette and insert it into a homemade metal burner rack. Place it in a homemade glass fumigation box and cover it. Burn 10 cigarettes at a time, 10 minutes each time, twice a day, with a 15-minute break between each time. Open the fumigation box lid for ventilation, 7 days a week, for a total of 12 weeks.

[0078] ④ After each cigarette smoke exposure, the animal transport box was placed in the transfer window and disinfected by ultraviolet light for 15 minutes before the mice were returned to the breeding cage.

[0079] (3) After modeling, distilled water was orally administered starting from the 13th week, 0.2 ml / day / animal, for a total of 4 weeks.

[0080] 2.3 Dosage Group

[0081] The modeling operation was the same as that of the model group. From the 13th week onwards, the drugs were administered orally at the concentrations and dosages mentioned in 1.2 for a total of 4 weeks.

[0082] 2.4 Blank control group

[0083] On days 1, 29, and 57, mice were instilled with 0.9% saline solution (6 μL / mouse) through the nose and fed normally for the rest of the day. From week 13 onwards, mice were orally administered 0.9% saline solution (0.2 ml / day / mouse) for 4 weeks.

[0084] 3 Observation indicators and methods

[0085] 3.1 General status observation

[0086] The quality of life and body weight of mice.

[0087] 3.2 Collection of experimental animals and determination of experimental test indicators

[0088] 3.2.1 Collection of mouse bronchoalveolar lavage fluid, serum, and intestinal mucus samples

[0089] 3.2.1.1 Collection of bronchoalveolar lavage fluid

[0090] (1) Mice were anesthetized by intraperitoneal injection of 2% pentobarbital (0.1 ml / 10 g) and fixed on the operating table in the supine position.

[0091] (2) Use ophthalmic scissors to cut the fur on the neck to expose the subcutaneous tissue and bluntly separate the trachea.

[0092] (3) Cut a horizontal incision at the distal end of the trachea and insert the tracheal connector of the cannula into the trachea centripetally. Secure it with cotton thread and connect the ventilator to the tracheal connector. Connect a 1ml syringe filled with sterile PBS to the tracheal cannula and slowly push it in for lavage.

[0093] (4) After injecting 0.8 ml each time, slowly aspirate. Repeat this three times and place the lavage fluid into a 1.5 ml EP tube.

[0094] (5) Centrifuge the lavage fluid at 3000 rpm for 15 min, and store the supernatant at -80°C for testing.

[0095] 3.2.1.2 Collection of serum

[0096] (1) Cut the skin below the xiphoid process and the muscle layer along the bilateral costal arches to expose the chest cavity.

[0097] (2) Cut the ribs along the sternum to expose the heart and lungs. Insert the needle into the apex of the heart with a 1 ml syringe and draw blood from the four chambers of the heart, taking approximately 0.8-1.0 ml of blood from each chamber.

[0098] (3) Slowly inject the solution into a 1.5 ml EP tube, place in a 37°C water bath for 15 minutes, and refrigerate at 4°C for 15 minutes. The blood sample will be stratified.

[0099] (4) Centrifuge at 3000 rpm for 15 min, and store the upper serum at -80°C for testing.

[0100] 3.2.1.3 Collection of intestinal mucus

[0101] The abdominal cavity was opened, the pylorus and cecum were found, the mesentery and surrounding tissues were gently separated, and the entire small intestine between the pylorus and the cecum was obtained. 10 ml of 0.01 M sterile PBS solution (pH = 7.4) was slowly injected with a syringe to flush the intestinal cavity.

[0102] The obtained washing liquid was collected into a 5 ml centrifuge tube, centrifuged at 3000 rpm for 15 min, and the supernatant was taken and stored at -80°C for testing.

[0103] 3.2.2 Collection and processing of mouse lung tissue morphology specimens

[0104] 3.2.2.1 Collection of morphological specimens

[0105] Cut the distal end of the trachea, separate the surrounding adhesion tissues, remove the lungs with forceps, process the left lung lobe for morphological specimens, and store the right lung lobe in liquid nitrogen using a cryopreservation tube.

[0106] 3.2.2.2 Morphological specimen processing

[0107] Fixation: Cover the left lung lobe with gauze, secure with a paper clip, and place in 10% formalin solution for fixation. Ensure the specimen is completely immersed and does not float on the surface. After 24 hours of fixation, remove the tissue and place the left lung lobe in a dehydration box in a fume hood.

[0108] Gradient alcohol dehydration: 75% alcohol--85% alcohol--90% alcohol--95% alcohol--anhydrous ethanol I--anhydrous ethanol II--benzene--xylene I--xylene II--wax I--wax II--wax III.

[0109] Embedding: The tissue, after being dipped in wax, is embedded in an embedding machine. Once the wax melts, it is placed in an embedding frame. Before the wax solidifies, the left lung lobe is placed and labeled. The tissue is then placed in a -20°C freezer to cool. Once the wax solidifies, the block is separated from the embedding frame.

[0110] Sectioning: Set the slicer to 5 μm thick and float the slices on the surface of 43°C warm water on the spreader. After the tissue is flattened, pick it up with a slide and place it in a 60°C oven. After it is dry, remove it and store it at room temperature for later use.

[0111] 3.2.2.3 HE staining

[0112] Dewaxing to water: Place the mouse lung tissue sections in a 60℃ oven for 1 hour. After the wax is completely melted, place the sections in xylene I and II for 15 minutes each, anhydrous ethanol I and II--95% alcohol I and II--90% alcohol--80% alcohol--70% alcohol--60% alcohol--50% alcohol for 2 minutes each, and gently rinse with tap water for 5 minutes.

[0113] After 15 min of hematoxylin staining, the excess stain was washed off with tap water.

[0114] Differentiate with 1% hydrochloric acid in ethanol for 30 seconds and wash with tap water for 10 minutes.

[0115] Stain with 1% eosin solution for 10 min and wash away excess stain with tap water for 1 min.

[0116] Gradient alcohol dehydration: 50% alcohol--60% alcohol--70% alcohol--80% alcohol--90% alcohol--95% alcoholⅠ,

[0117] Ⅱ--100% anhydrous ethanol Ⅰ and Ⅱ for 2 minutes each. Xylene Ⅱ for 10 minutes each, then seal with neutral gum

[0118] Observe the changes in lung tissue and bronchial wall, as well as inflammatory cell infiltration under an optical microscope

[0119] 3.2.3 Isolation and purification of lymphocytes from lung tissue and small intestinal epithelial cells

[0120] 3.2.3.1 Intraperitoneal injection of heparin

[0121] Heparin preparation: Specification: 12500 IU / vial, working concentration: 100 IU / ml.

[0122] Add 1 vial of heparin to 125 ml of 0.9% NaCl solution and mix well.

[0123] After weighing the mice, heparin was extracted at 10 IU / ml / 10 g and injected into the abdominal cavity from the left groin.

[0124] 3.2.3.2 Intraperitoneal anesthesia

[0125] Prepare 1% sodium pentobarbital: weigh 100 mg of sodium pentobarbital, add it to a centrifuge tube containing 10 ml of distilled water, and dissolve and mix thoroughly.

[0126] A 1% sodium pentobarbital solution was drawn up using a 1 ml syringe and administered at a rate of 0.05 ml / 10 g body weight. Lung tissue was collected from mice after the eyelids showed no reflex after stimulation.

[0127] 3.2.3.3 Isolation of Lymphocytes from Mouse Lung Tissue

[0128] Use sterile ophthalmic forceps to place the lung tissue into a 50 ml centrifuge tube and add 50 ml PBS to rinse three times.

[0129] Place the washed lung tissue in a sterile small dish (35 mm diameter, 10 mm depth) and mince as much as possible with ophthalmic scissors. Place a 70 μm cell strainer on a centrifuge tube and transfer the tissue to the strainer using a pipette. Wash three times with 1× Hanks' balanced solution and filter out the wash solution.

[0130] Add 50 ml of RPMI1640 and 1 ml of PBS to a 50 ml centrifuge tube, add lung tissue fragments with a pipette, and then add 200 μL of 100 mg / ml Collagenase NB4 and 40 μL of 10 mg / ml DNASe to make working concentrations of 2 mg / ml and 40 μg / ml, respectively.

[0131] Incubate in a constant temperature water bath at 37°C for 90 min, shaking once every 10 min.

[0132] Filter through a 70 μm cell strainer to remove undigested tissue fragments and impurities.

[0133] Use a pipette to rinse the cell sieve with 15 ml of RPMI 1640. Transfer the filtrate and rinse solution to a 50 ml centrifuge tube. Centrifuge at 4°C for 10 min at 2000 rpm.

[0134] After centrifugation, remove the supernatant by gently aspirating with a pipette against the wall, being careful not to disturb the cell layer. Leave 5 ml in the centrifuge tube and flick with your fingers to disperse the cell clumps at the bottom of the tube to suspend the cells.

[0135] Add 3 ml of 100% Percoll to a 15 ml centrifuge tube, then add 5 ml of the cell suspension, and then add 5 ml of 1× Hanks' balanced solution containing 5% PBS. Mix until the Percoll content in the solution is 30%. Centrifuge at 4°C for 18 min at 1800 rpm.

[0136] After centrifugation, discard the supernatant and retain 1 ml of the solution (containing 0.3 ml of Percoll). Tap the bottom of the tube to break up the cell clumps, add 4.1 ml of 100% Percoll and mix thoroughly. Bring the volume to 10 ml with 1× Hanks solution containing 5% PBS. After mixing, the Percoll content is 44%.

[0137] Use a long pipette to slowly add 2 ml of 70% Percoll to the bottom of a 15 ml centrifuge tube. Gently operate to allow the two densities of Percoll liquid to form an interface between the layers. Centrifuge at 4°C for 18 min, 1800 rpm.

[0138] At the interface between the 44% Percoll and 70% Percoll levels, cells will be aggregated into a single layer. Carefully discard the upper 1 / 3 of the liquid in the centrifuge tube using a pipette placed against the tube wall, then aspirate the cells against the tube wall and transfer them to a prepared 50ml centrifuge tube.

[0139] Add 1× Hanks' balanced solution containing 5% PBS to a total volume of 10 ml, shake thoroughly to mix, and centrifuge at room temperature for 5 min at 1500 rpm.

[0140] After discarding the supernatant, add 2 ml of 1× Hanks' RPMI 1640 solution containing 10% PBS to prepare a lymphocyte suspension for testing. Stain with 4 g / L trypan blue and count the cells using a cell counter.

[0141] 3.2.3.4 Isolation of Lymphocytes from Mouse Intestinal Epithelial Cells

[0142] Place the entire section of small intestine from the pylorus to the cecum in pre-cooled PBS solution on tin foil, use a thin plastic tube to flip the intestinal tube from one end of the small intestine into the intestinal cavity, gradually pass the plastic tube out from the other end of the small intestine, and flip the entire intestinal cavity, with the mucosal layer facing outward and the visceral layer facing inward.

[0143] The intestinal tube was carefully transferred to a 50 ml centrifuge tube, and RPMI1640 containing 10% PBS, 200 μL of 100 mg / ral Collagenase NB4, and 40 μL of 10 mg / ml DNASe were added to make the working concentrations of the latter two reach 2 mg / ml and 40 μg / ml respectively.

[0144] The centrifuge tube was placed at 37°C and centrifuged at 200 r / min for 60 min.

[0145] Filter the digestion solution through a 70 μm cell strainer into a 50 ml centrifuge tube to remove undigested tissue fragments and impurities. Centrifuge at 4°C, 2000 rpm, for 10 min.

[0146] After centrifugation, remove the supernatant by gently aspirating it against the wall with a pipette, taking care not to disturb the cell layer and leaving 5 ml in the centrifuge tube. Use your fingers to flick the cell clumps at the bottom of the centrifuge tube to suspend the cells.

[0147] Add 3 ml of 100% Percoll to a 15 ml centrifuge tube, then add 5 ml of the cell suspension, and then add 2 ml of 1× Hanks solution containing 5% PBS. Mix until the Percoll content in the liquid is 30%. Centrifuge at 4°C for 18 min, 1800 rpm.

[0148] After centrifugation, discard the supernatant and retain 1 ml of the solution (containing 0.3 ml of Percoll). Flick to disperse the cell clumps at the bottom, mix thoroughly, and add 4.1 ml of 100% Percoll. Add 1× Hanks solution containing 5% PBS to bring the total volume to 10 ml. After mixing, the Percoll content is 44%.

[0149] Use a pipette to slowly add 2 ml of 70% Percoll to the bottom of the centrifuge tube, gently operate to allow the two density liquids to separate and form an interface, and centrifuge at 4°C for 18 min, 1800 rpm.

[0150] At the interface between the 44% Percoll and 70% Percoll liquids, cells will be seen clumping into a single layer. Carefully discard the upper 1 / 3 of the liquid in the centrifuge tube using a pipette placed against the tube wall, then aspirate the cells against the tube wall and transfer them to a prepared 50ml centrifuge tube.

[0151] Add 1× Hanks solution containing 5% PBS to increase the total volume of the liquid to 10 ml, mix well, and centrifuge at room temperature for 5 minutes at 1500 r / min.

[0152] After discarding the supernatant, resuspend the cells in 2 ml of 1× Hanks medium containing 5% PBS to prepare a lymphocyte suspension for testing. Add 4 g / L trypan blue for staining and count the cells using a cell counter.

[0153] 3.2.4 Detection of inflammatory factors in mouse serum and intestinal mucus

[0154] 3.2.4.1 IL-6 content detection

[0155] All samples and test kits were equilibrated to room temperature (18-25°C).

[0156] Prepare 50 ml of 1× Wash Buffer using 5 ml of 10× Wash Buffer Concentrate and 45 ml of distilled water. Prepare the antibody cocktail using Antibody Diluent, capture antibody, and detection antibody. Combine 300 μL of 10× Capture Antibody and 300 μL of 10× Detector Antibody with 2.4 ml of Antibody Diluent to make 3 ml of the antibody cocktail. Mix gently.

[0157] Preparation of standard products:

[0158] Dissolve the mouse IL-6 recombinant protein lyophilized powder in 500 μL of sample-diluting saline. Mix thoroughly and gently at room temperature for 10 minutes to obtain a 2000 pg / mL standard solution. Label eight EP tubes, 1-8, for standards. Add 150 μL of sample-diluting saline to tubes 1-8. Use the standard solution to prepare a serial dilution. Add 150 μL of the prepared 2000 pg / ml standard solution to tube 1 to prepare a 2000 pg / ml standard solution. Then add 150 μL of the prepared 1000 pg / ml standard solution to tube 1 to prepare a 500 pg / ml standard solution. Then add 150 μL of the prepared 500 pg / ml standard solution to tube 3 to prepare a 150 pg / ml standard solution. Prepare standard solutions of 125, 62.5, 31.3, and 15.6 pg / ml in tubes 4, 5, 6, and 7, respectively. Tube 8 is the blank control.

[0159] Sample addition: Add 50 μL of sample (serum, intestinal mucus) or standard to the appropriate wells, then add 50 μL of antibody mixture to each well, being careful not to create bubbles.

[0160] Incubation: Seal the plate with film and incubate at room temperature on a shaker set at 400 rpm for 1 hour.

[0161] Wash the plate: Discard the liquid in the well plate and wash each well with 350 μL 1× Wash Buffer for a total of 3 times. After completion, turn the 96-well plate upside down on a clean paper towel to remove excess liquid.

[0162] Color development: Add 350 μL of TMB matrix to each well and incubate in a shaker at 400 rpm for 10 min in the dark. The liquid in the well plate will turn blue.

[0163] Stop: Add 100 μL of stop solution to each well and mix until the blue liquid turns yellow.

[0164] The OD value was measured by a microplate reader set at 450 nm.

[0165] Calculation: Use the OD value as the dependent variable Y and the standard concentration as the independent variable X. Calculate the standard equation and then substitute the OD value of the measured sample into the equation to obtain the IL-6 concentration.

[0166] 3.2.4.2 IL-13 content detection The IL-13 concentration was measured in the same way.

[0167] 4 Statistical methods

[0168] SPSS13.0 software was used for data processing. The experimental data were expressed as mean plus or minus standard deviation (x±S). T test was used for pairwise comparison of means.

[0169] 5 Experimental results

[0170] 5.1 The drug-treated group improved the quality of life and body weight of COPD mice

[0171] Normal control group: There was no death in the blank control group, the fur was shiny and soft, the breathing was slow, the frequency was moderate, the rhythm was even, the activity was normal, and the weight gradually increased;

[0172] Model group: Ten mice died in the COPD model group. Their hair became dull and dry, and some mice lost their hair. They became agitated and jumped when exposed to smoke. In the later stage, they tended to curl up in groups, and even trembled all over. They breathed rapidly, with obvious fluctuations in the chest and abdomen. They sometimes nodded irregularly and even opened their mouths to breathe. Their body weight was significantly lower than that of the normal group (P<0.001).

[0173] The deaths of mice in the high, relatively high, medium, relatively low, and low-dose groups of tricholoma pratense were 3, 2, 1, 2, and 4, respectively. The deaths of mice in the high, relatively high, medium, relatively low, and low-dose groups of irisin were 4, 1, 3, 3, and 4, respectively. Compared with the model group, the fur of the mice became shiny and smooth, their movements became normal, their breathing difficulties were improved, and their body weight was significantly higher than that of the model group (P<0.001).

[0174] One mouse died in each of the high-dose and medium-dose groups of the composition, and three mice died in each of the high-dose, low-dose, and low-dose groups. The general conditions during the modeling period were the same as those of the COPD model group. After oral gavage, the hair was smoother, the dyspnea was improved, the frequency was reduced, and the body weight was significantly higher than that of the model group (P<0.001). The results are shown in Table 2.

[0175] Table 2 Composition of drugs can improve the survival rate and body weight of COPD model mice

[0176]

[0177]

[0178] Note: n represents the number of surviving mice.

[0179] 5.2 Morphological and pathological changes of lung tissue in COPD model mice

[0180] The pathological manifestations of COPD include degeneration, necrosis, and desquamation of the bronchial mucosal epithelium, shortened cilia, and adhesions. Goblet and mucus cell hyperplasia and hypertrophy, with extensive mucus retention, congestion and edema of the bronchial wall, and narrowing of the tracheal lumen. Alveoli atrophy and collapse, alveolar septa damage and rupture, and adjacent alveoli fuse into bullae. Numerous chronic inflammatory cells infiltrate the trachea and alveoli, with rupture and atrophy of the submucosal smooth muscle and fibrous tissue proliferation. Bronchial structural remodeling, increased collagen content, and scarring occur. HE staining in this experiment revealed normal airway and alveolar structure in the blank control group, with regular cilia, regular alveolar shape and size, and intact airway mucosal epithelium. In the model control group, bronchial mucosal folds and flaky desquamation occurred, narrowing or occluding the bronchial lumen. Alveolar walls were damaged, the alveolar spaces expanded irregularly, and some fused into bullae. Chronic inflammatory cell infiltration was observed around the airways and in the lung interstitium, consistent with COPD pathology.

[0181] Compared with the model control group, the high, medium and low groups treated with two single-drug groups and the combination drug group showed morphological improvements, which were reflected in the relatively complete bronchial and alveolar structures, reduced luminal stenosis, relatively intact airway mucosal epithelium, regular cilia arrangement and reduced shedding, relatively uniform alveolar size, reduced number of bullae, and reduced infiltration of inflammatory cells around the airway wall and in the pulmonary interstitium.

[0182] 5.3 αβT / γδT cell ratio in lung tissue of COPD model mice

[0183] γδT cells are key effector cells of the respiratory mucosal immune system, participating in both inflammation and repair in chronic inflammatory diseases. In normal mouse lungs, the majority of αβT cells reside in the lung parenchyma, while the majority of γδT cells are distributed in non-alveolar areas, excluding the mucosa. The relative density of γδT cells is highest near the respiratory tract, blood vessels, and visceral pleura. Although γδT cells are much smaller in number, their relative density in non-alveolar areas matches or nearly matches that of αβT cells. In contrast, in the lung parenchyma, the largest areas in terms of tissue mass or surface area, the relative density of γδT cells is much lower than that of αβT cells. This difference in the distribution of these two T cells may be related to different functional roles. The monotonic distribution of αβT cells in the normal lung may reflect a degree of functional homogeneity, while the widespread distribution of γδT cells reflects functional heterogeneity. Throughout the lung, γδT cells primarily interact with myeloid cells, while αβT cells more frequently interact with lymphocytes. This comparison showed that leukocyte contacts with lung γδ T cells primarily involved myeloid cells, including F4 / 80+ macrophages and I-A+ dendritic cells, whereas αβ T cells frequently contacted CD45R+ lymphocytes, including B cells but also some T cells and plasmacytoid dendritic cells.

[0184] In the lung tissue morphology immunohistochemical staining experiment, cells were labeled with αβTCR and γδTCR and counted. In the blank control group, a small amount of both cells were found in the lung tissue; almost no γδT was found in the model control group; in the high, upper, medium, lower, and low-dose drug groups, more γδT cells were found near the trachea, which was significantly higher than that in the model group. The ratio of αβT / γδT cells in the mouse lung tissue was higher in the model group than in the blank control group, and showed a downward trend in the drug-treated group compared with the model group, and was significantly lower in the combination-treated group than in the single-drug treated group, as shown in Table 3.

[0185] With reference to the isolation method of lymphocytes between intestinal epithelial cells, we explored the experimental method of separating lung tissue lymphocytes by density gradient centrifugation. The isolation method of lymphocytes between small intestinal epithelial cells is relatively mature and recognized by the academic community. In addition, there are fewer types of cells in the intestinal mucosal epithelium, which is conducive to the separation of different cell populations. Referring to the flow cytometry results of small intestinal sample cells, the lymphocyte population in the cell suspension obtained after digestion and separation of lung tissue can be determined, and specific antibodies can be used to label the αβT cells and γδT cells therein, respectively, as shown in Table 4. The proportion of mouse γδT cells in the model group tended to decrease compared with the blank control group, and the low-dose combination liquid group had a significant increase compared with the model group (P<0.05), suggesting that the combination liquid treatment helps to increase the proportion of γδT cells in mouse lung tissue.

[0186] Table 3 Immunohistochemical staining cell counts showed that the combined drug solution reduced the αβT / γδT cell ratio in the lung tissue of COPD model mice

[0187]

[0188]

[0189] Table 4 Flow cytometer analysis results show that the combined drug solution reduces the ratio of αβT / γδT cells in the lung tissue of COPD model mice

[0190]

[0191] 5.4 IL-6 and IL-13 Contents in Mouse Serum, Bronchoalveolar Lavage Fluid, and Small Intestinal Mucus

[0192] Comparison of IL-6 and IL-13 levels in the serum, bronchoalveolar lavage fluid, and small intestinal mucus of mice revealed that the IL-6 level in serum was higher in the COPD model control group than in the blank control group (P<0.05), lower in the drug-treated groups, and significantly lower in the medium-dose combination group than in the model control group (P<0.05). No significant differences were observed in serum IL-13 levels among the groups. The IL-6 level in bronchoalveolar lavage fluid was higher in the COPD model control group than in the blank control group (P<0.05). There were no statistically significant differences between the high- and low-dose drug-treated groups and the model control group. The medium-dose drug-treated group was lower than the blank control group, the model control group, and the low-dose drug-treated group, and the medium- and low-dose combination groups were significantly lower than the single-dose drug-treated group (P<0.05). The IL-13 level in bronchoalveolar lavage fluid was higher in the COPD model control group than in the blank control group (P<0.05), lower in the drug-treated groups than in the model control group, and significantly lower in the medium- and low-dose combination groups (P<0.05). There was no significant difference in the IL-6 content in intestinal mucus among the groups; the IL-13 content in intestinal mucus of the COPD model control group was higher than that of the blank control group (P<0.05), and the IL-13 content in the high, upper, medium, lower and low dose groups of single administration and combination administration was lower than that of the model control group, and the high, upper, medium, lower and low dose groups of the combination were significantly decreased (P<0.05).

[0193] In summary, the amount of inflammatory factors secreted by COPD model mice was significantly increased. The levels of IL-6 in serum and bronchoalveolar lavage fluid, as well as IL-13 in bronchoalveolar lavage fluid and intestinal mucus in the combination drug group were significantly reduced compared with those in the model control group (P<0.05), as shown in Table 5.

[0194] Table 5 IL-6 and IL-13 levels in mouse serum and bronchoalveolar lavage fluid

[0195]

[0196]

[0197] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A flavonoid glycoside composition for treating chronic obstructive pulmonary disease, characterized in that: The invention comprises a tricholoma glycoside-tectoriusin composition, which is prepared into a 0.5-1.5 g / kg suspension with physiological saline.

2. The flavonoid glycoside composition for treating chronic obstructive pulmonary disease according to claim 1, wherein The tricholoma rutaecarpin-irisin composition is mixed with physiological saline to form a 1.0 g / kg suspension.

3. The flavonoid glycoside composition for treating chronic obstructive pulmonary disease according to claim 2, wherein: The weight ratio of tricholoma to icterin in the tricholoma-icterin composition is 2:

5.

4. The method for preparing the flavonoid glycoside composition for treating chronic obstructive pulmonary disease according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Weighing Radix Sophorae flavescentis and Radix Belamcandae respectively, crushing, extracting with alcohol, and concentrating to obtain an alcohol extract of Radix Sophorae flavescentis and an alcohol extract of Radix Belamcandae; S2. Adding ethyl acetate to the alcohol extract of the Radix Sophorae flavescentis and the alcohol extract of the Radix Belamcandae, respectively, extracting three times, and concentrating under reduced pressure to obtain a fraction of the Radix Sophorae flavescentis and a fraction of the Radix Belamcandae; S3, separating, purifying and recrystallizing the bean root fraction and the total flavonoids fraction of the Belamcanda chinensis to obtain tricholoma rutin and irisin; S4, mixing the tricholoma serrata and the icterin to obtain a tricholoma serrata-icterin composition; S5. Add physiological saline to the tricholomaside-irisin composition to prepare a suspension.

5. The preparation method according to claim 4, wherein When the crushed rhizome of bean curd and belamcanda chinensis are subjected to alcohol extraction in step S1, the solvent for the alcohol extraction is 70-75% ethanol, and the volume of the ethanol is 6-10 times that of the crushed rhizome of bean curd and belamcanda chinensis, respectively.

6. The preparation method according to claim 4, wherein In step S2, the volume ratio of the ethyl acetate to the alcohol extract of the rhizome of soybean and the alcohol extract of the beamroot is 1:

1.

7. The preparation method according to claim 4, wherein The separation and purification method of the bean root fraction and the Belamcanda chinensis fraction in step S3 is as follows: Sa, respectively dissolving the bean root fraction and the belamcanda chinensis fraction in a low concentration ethanol solution of less than 30%, dissolving and filtering to obtain a bean root filtrate and a belamcanda chinensis filtrate; Sb. The filtrate of the bean root and the filtrate of the Belamcanda chinensis are further separated and purified using a D101 macroporous resin column to obtain tricholin and irisin.

8. Use of the flavonoid glycoside composition according to any one of claims 1 to 3 in the preparation of a medicament for treating chronic obstructive pulmonary disease.

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