CN116098912A - Flavonoid glycoside combinations for the treatment of chronic obstructive pulmonary disease - Google Patents

Flavonoid glycoside combinations for the treatment of chronic obstructive pulmonary disease Download PDF

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CN116098912A
CN116098912A CN202310161961.XA CN202310161961A CN116098912A CN 116098912 A CN116098912 A CN 116098912A CN 202310161961 A CN202310161961 A CN 202310161961A CN 116098912 A CN116098912 A CN 116098912A
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ext
glycoside
tectoridin
red clover
blackberry lily
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宋志军
吴云秋
闫炳雄
姚彩云
阮丽君
刘喜慧
宋梦新
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Guangxi Botanical Garden of Medicinal Plants
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Guangxi Botanical Garden of Medicinal Plants
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07H17/07—Benzo[b]pyran-4-ones
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Abstract

The invention discloses a flavonoid glycoside combination for treating chronic obstructive pulmonary disease, which comprises a red clover glycoside-tectoridin combination, wherein the red clover glycoside-tectoridin combination and physiological saline are configured into a suspension of 0.5-1.5g/kg, and also provides a preparation method of the flavonoid glycoside combination for treating chronic obstructive pulmonary disease and application of the flavonoid glycoside combination in preparing a medicament for treating chronic obstructive pulmonary disease.

Description

Flavonoid glycoside combinations for the treatment of chronic obstructive pulmonary disease
Technical Field
The invention relates to the technical field of medicines for treating lung diseases. More particularly, the present invention relates to a flavonoid glycoside combination for use in the treatment of chronic obstructive pulmonary disease.
Background
Chronic Obstructive Pulmonary Disease (COPD) is a preventable, treatable chronic tracheal disease characterized by persistent respiratory symptoms and airflow limitation.
Currently, the main drugs for treating COPD are long-acting β2 receptor agonists (LABA), long-acting anticholinergic drugs (LAMA), which are not commonly used in basic medical and health institutions, theophylline drugs and relatively inexpensive short-acting preparations are lacking in basic layers, antiasthmatic drugs prepared in basic layers are mainly oral and intravenous injection formulations, and atomized inhalation drugs are lacking. In addition, although the western medicines can rapidly control the illness state in a short time, the damage of the lung function is aggravated along with the repetition of the illness state, so that the life quality of patients is reduced and even the patients die. The traditional Chinese medicine considers that the COPD belongs to the category of 'asthmatic syndrome' and 'lung distension', and the etiology is internal damp toxin and deficiency of spleen and kidney, and the treatment is based on the main principles of eliminating dampness and resolving phlegm, regulating qi and strengthening spleen, and relieving cough and eliminating turbidity. The traditional Chinese medicine and the extract thereof play a role in treatment through multi-path and multi-target integrated regulation in the process of treating diseases, have small side effect compared with antibiotics, are suitable for long-term administration, do not cause dysbacteriosis, are low in cost, and have positive clinical significance and broad market prospect.
Disclosure of Invention
It is an object of the present invention to solve at least the above problems and to provide at least the advantages to be described later.
It is still another object of the present invention to provide a flavonoid glycoside combination for use in the treatment of chronic obstructive pulmonary disease that is readily available and has good efficacy for the treatment of chronic obstructive pulmonary disease.
To achieve these objects and other advantages and in accordance with the purpose of the invention, there is provided a flavonoid glycoside combination for treating chronic obstructive pulmonary disease, including a red clover glycoside-tectoridin composition, which is formulated in a suspension of 0.5-1.5g/kg with physiological saline.
Preferably, the red clover glycoside-tectoridin composition is formulated with physiological saline as a suspension of 1.0 g/kg.
Preferably, the weight ratio of the red clover glycoside to the tectoridin in the red clover glycoside-tectoridin composition is 2:5.
a method for preparing a flavonoid glycoside combination for treating chronic obstructive pulmonary disease is provided, comprising the steps of:
s1, respectively weighing the bean roots and the blackberry lily, and carrying out crushing, alcohol extraction and concentration to obtain an alcohol extract of the bean roots and an alcohol extract of the blackberry lily;
s2, ethyl acetate is respectively added into the alcohol extract of the bean roots and the alcohol extract of the blackberry lily for three times, and the extraction is carried out under reduced pressure and concentrated to obtain a fraction of the bean roots and a fraction of the blackberry lily;
s3, respectively separating, purifying and recrystallizing the fraction of the bean roots and the total flavone fraction of the blackberry lily to obtain red clover glycoside and tectoridin;
s4, mixing the red clover glycoside and the tectoridin to obtain a red clover glycoside-tectoridin composition;
s5, adding physiological saline into the red clover glycoside-tectoridin composition to prepare a suspension.
Preferably, when the crushed bean roots and the blackberry lily in the step S1 are subjected to alcohol extraction, the alcohol extraction solvent is 70-75% ethanol, and the volume of the ethanol is 6-10 times of that of the crushed bean roots and the crushed blackberry lily respectively.
Preferably, in step S2, the volume ratio of the ethyl acetate to the alcohol extract of the bean root to the alcohol extract of the blackberry lily is 1:1.
preferably, in step S3, the separation and purification method of the fraction of the bean root and the fraction of the blackberry lily is as follows:
sa, respectively dissolving the fraction of the bean roots and the fraction of the blackberry lily in a low-concentration ethanol solution with concentration below 30%, dissolving and filtering to obtain filtrate of the bean roots and filtrate of the blackberry lily;
sb, further separating and purifying the filtrate of the root of red bean and the filtrate of the blackberry lily by using a D101 macroporous resin column respectively to obtain the red clover glycoside and the tectoridin.
Provides an application of a flavonoid glycoside combination in preparing a medicament for treating chronic obstructive pulmonary disease.
The invention at least comprises the following beneficial effects:
the red clover glycoside and the tectoridin are easy to obtain and convenient to extract, and the red clover glycoside and the tectoridin have good effect of treating chronic obstructive pulmonary diseases.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a block diagram of red clover glycoside;
FIG. 2 is a diagram showing the structure of tectoridin;
FIG. 3 is a graph showing the effect of different concentrations of composition drug solution on BEAS-2B cell viability;
FIG. 4 is a graph showing half-maximal inhibitory concentration of red clover glycoside and tectoridin compositions in varying proportions on RAW264.7 macrophages.
Detailed Description
The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.
Extraction, purification and identification of flavonoid glycoside combination
1. Extraction and extraction
Taking 50kg of radix sophorae tonkinensis (SDG) and blackberry lily (SG) respectively, respectively crushing, leaching with 6-10 times of 70-75% ethanol for 2 times, each time for 5-7 days, filtering the extracting solution, merging and concentrating under reduced pressure to obtain alcohol extract concentrated solutions, wherein the extraction rates are 17.78% and 25.22% respectively. The volume ratio of the ethanol extract concentrated solution is 1:1, mixing the extracts, concentrating under reduced pressure, recovering the extraction solvent, concentrating to obtain extract, and respectively obtaining 2 fractions of extract (SDGY) of radix Sophorae Tonkinensis extracted and concentrated with ethyl acetate, and extract (SGY) of rhizoma Belamcandae extracted and concentrated with ethyl acetate.
2. Separation
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3. Purification and recrystallization
Sephadex chromatography (Sephadex G20) was performed on the fraction SDG-A, SG-a, and after dissolving the sample with an appropriate amount of methanol, the sample was filtered and loaded with chloroform: methanol 1: the mixed solution 1 was used as an eluent to elute 1, 2, 3, and 20 sites. Ext>ext> whereinext>ext>,ext>ext> atext>ext> theext>ext> partsext>ext> ofext>ext> recoveringext>ext> 16ext>ext> andext>ext> 17ext>ext> ofext>ext> SDGext>ext> -ext>ext> Aext>ext> (ext>ext> redext>ext> cloverext>ext>)ext>ext>,ext>ext> whenext>ext> recoveringext>ext> theext>ext> elutingext>ext> solventext>ext>,ext>ext> solidext>ext> precipitatedext>ext> whiteext>ext> solidext>ext> isext>ext> adheredext>ext> toext>ext> theext>ext> bottleext>ext> wallext>ext> whenext>ext> theext>ext> quickext>ext> concentrationext>ext> isext>ext> finishedext>ext>,ext>ext> andext>ext> atext>ext> theext>ext> partsext>ext> ofext>ext> recoveringext>ext> 13ext>ext> andext>ext> 14ext>ext> ofext>ext> SGext>ext> -ext>ext> Aext>ext> (ext>ext> tectoridinext>ext>)ext>ext>,ext>ext> whenext>ext> recoveringext>ext> theext>ext> elutingext>ext> solventext>ext>,ext>ext> solidext>ext> precipitatedext>ext> yellowext>ext> solidext>ext> isext>ext> adheredext>ext> toext>ext> theext>ext> bottleext>ext> wallext>ext> whenext>ext> theext>ext> quickext>ext> concentrationext>ext> isext>ext> finishedext>ext>,ext>ext> theext>ext> obtainedext>ext> solidext>ext> isext>ext> filteredext>ext> respectivelyext>ext>,ext>ext> andext>ext> thenext>ext> aext>ext> properext>ext> amountext>ext> ofext>ext> chloroformext>ext> isext>ext> usedext>ext>:ext>ext> Methanol=1: 1, the mother solution is placed overnight, the solid is slowly precipitated at the bottom of the bottle or substances separated out from the wall of the bottle, for example, the substances are detected by thin layer chromatography, and then the substances are all impure substances through high performance liquid chromatography detection, and chloroform can be used for the following steps: and recrystallizing the mixed solution with different proportions of methanol to separate out a monomer compound, and purifying to obtain the red clover glycoside and the tectoridin, wherein the structures are shown in figures 1-2.
MTT assay Effect of Red clover glycoside-Iris glycoside composition on proliferation of human lung (bronchus) epithelial cells BEAS-2B cells
1. Liquid medicine proportion selection
Providing red clover glycoside and tectoridin, precisely weighing a proper amount of samples according to the weight ratio of 1:10, 3:10, 2:5, 3:5 and 4:5, respectively adding a proper amount of dimethyl sulfoxide (DMSO) into the red clover glycoside and the tectoridin to dissolve the corresponding red clover glycoside and tectoridin, ultrasonically dissolving, and mixing a proper amount of red clover glycoside added with DMSO and tectoridin solution added with DMSO to obtain mother liquor, wherein five mother liquor can be prepared according to five different weight ratios of red clover glycoside and tectoridin, and the total concentration of red clover Zhou Caogan and tectoridin of the five mother liquor is the same, but the weight ratio of red clover Zhou Caogan to tectoridin in the five mother liquor is 1:10, 3:10, 2:5, 3:5 and 4:5 respectively;
precisely measuring 12 mu L of the mixed mother solution, and diluting to 2mL by using a culture medium to obtain a liquid medicine culture medium;
finally, 100 mu L of liquid medicine culture medium is added into 100 mu L of cell liquid, the inhibition rate of samples on RAW264.7 macrophages under the condition of different weight ratios of red-blood axin and tectoridin is measured, and half inhibition concentration IC of each sample is calculated 50 Values. The results are shown in FIG. 4, which shows IC for RAW264.7 macrophages when the ratio of red clover glycoside to tectoridin is 2:5 by weight 50 The value is the lowest.
2. Preparation of liquid medicine, preparation of MTT solution and cell culture
Preparing a liquid medicine: dissolving red clover glycoside and tectoridin in DMSO to a proper concentration as stock solution, storing at-20deg.C, and diluting to the required concentration before use, namely red clover glycoside: the ratio of tectoridin is 2:5.
Preparation of MTT solution: usually MTT is prepared to a final concentration of 5mg/ml, phosphate Buffer (PBS) or physiological saline is used as solvent, and the solution is prepared and filtered by a 0.22 μm filter membrane to remove bacteria, and the solution is stored at 4 ℃ in a dark place. The containers are preferably wrapped in aluminum foil during the preparation and storage process.
Cell culture:
1mL of a frozen tube of human lung (bronchus) epithelial cells BEAS-2B cell suspension was thawed by shaking in a water bath at 37℃and 5mL of 10% fetal bovine serum RPMI-1640 complete medium was added and mixed well. Centrifugation is carried out at 1000RPM for 5 minutes, the supernatant is discarded, 4-6 mL of complete medium is added and then the mixture is blown up to a cell suspension and transferred to a flask (or 6cm dish) for overnight incubation.
The next day the fluid was changed and the cell density was checked. If the cell density reaches 80% -90%, subculturing is carried out, namely, the culture supernatant is discarded, and the cells are rinsed for 1-2 times by PBS without calcium and magnesium ions.
Adding 1-2 mL of digestive juice (0.25% Trypsin-0.53mM EDTA) into a culture flask, placing the culture flask into a 37 ℃ incubator for digestion for 1-2 min, observing the digestion condition of cells under a microscope, if most of the cells become round and fall off, rapidly taking the cells back to an operating table, tapping the culture flask, and adding more than 5mL of complete culture medium containing 10% serum to stop digestion.
Gently blowing the cells, sucking out after complete shedding, centrifuging for 8-10 minutes at 1000RPM, discarding the supernatant, adding 1-2 mL of culture solution, and uniformly blowing. Supplementing culture solution according to 5-6 ml/bottle, and adding cell suspension and culture solution according to 1:2 to 1: the ratio of 5 is divided into a new dish or bottle containing 5-6mL of culture solution.
3. Experimental grouping
Blank control group: 100. Mu.L of RPMI-1640 complete medium and 100. Mu.L of 0.1% DMSO solution.
Negative control group: 100. Mu.L of density 2X 10 4 Cell suspension per ml and 100. Mu.L of RPMI-1640 medium in 0.1% DMSO.
Experimental group: 100. Mu.L of density 2X 10 4 Cell suspensions at a concentration of 100. Mu.L each and 100. Mu.L each10. 20, 40, 80, 100, 150, 200. Mu.g/ml of a red clover glycoside-tectoridin 2:5 composition liquid (the red clover glycoside-tectoridin 2:5 composition is prepared into 10, 20, 40, 80, 100, 150, 200. Mu.g/ml liquid).
4. Experiment
Taking logarithmic growth phase cells, and digesting and suspending to obtain a concentration of 2×10 4 Inoculating 100 μl of single cells per ml into 96-well plate according to above groups, placing into 37 deg.C and 5% CO 2 Culturing in saturated humidity incubator for 12 hr, observing cell morphology, if cell state is good, administering 10, 20, 40, 80, 100, 150, 200 μg/ml mixed composition liquid medicine in groups, setting 6 multiple holes, incubating in incubator for 24 hr, adding 20 μl 5mg/ml MTT, and incubating at 37deg.C and 5% CO 2 After 4h incubation in a saturated humidity incubator, the supernatant was removed, 150. Mu.L DMSO was added, and after 10min shaking with a micro-shaker to completely dissolve the crystals, the OD was measured at 570nm, and the average of 6 parallel wells was the result of one experiment. Experiments were independently repeated three times.
Cell proliferation rate (%) = (experimental group-blank group)/(negative group-blank group) ×100%
As can be seen from Table 1 and FIG. 3, the cell proliferation rate was 90% or more after the extract was applied to BEAS-2B cells at a concentration ranging from 10 to 200. Mu.g/mL, and it was considered that the composition liquid medicine had no significant cytotoxicity to BEAS-2B cells in the set concentration range.
TABLE 1 Effect of composition solutions of different concentrations on BEAS-2B cell proliferation
Figure BDA0004094493930000051
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Figure BDA0004094493930000061
Pharmacological activity research of red clover glycoside and tectoridin composition on COPD
1. Animal experiment group
1.1 laboratory animals SPF grade ICR female 475 mice were selected, 6-8 weeks old, and 18-20 g weight, medical laboratory animal center at university of Guangxi medical science (animal license number: SCXK cassia 2014-0002).
1.2 Experimental drugs
1.2.1 Red clover glycoside is obtained by extraction, separation and identification in laboratory, and is prepared into suspension with administration of 0.25g/kg,0.50g/kg, 1.0g/kg, 1.5g/kg and 2.0g/kg by using physiological saline, and stored at 4 ℃ for standby.
1.2.2 tectoridin is obtained by laboratory extraction, separation and identification, and is prepared into suspension with administration of 0.25g/kg,0.50g/kg, 1.0g/kg, 1.5g/kg and 2.0g/kg by physiological saline, and stored at 4deg.C for use.
1.2.3 Red clover glycoside-tectoridin combination, which is prepared by physiological saline according to the proportion of 2:5, and the suspensions with the administration of 0.25g/kg,0.50g/kg, 1.0g/kg, 1.5g/kg and 2.0g/kg are prepared respectively, and are preserved at 4 ℃ for standby.
1.3 Experimental grouping
475 ICR female mice were randomly divided into 17 groups after 1 week of adaptive feeding: the blank control group is A group, the model control group is B group, the red clover glycoside high, medium, low and low dose groups are C1-C5 groups, the tectoridin high, medium, low and low dose groups are D1-D5 groups and the composition high, medium, low and low dose groups are E1-E5 groups, each group comprises 25.
2 molding and administration method
2.1 feeding conditions
Before and after administration, the experimental animals are fed in separate cages, full price pellet feed is fed, free drinking water is carried out, the room temperature is 20+/-2 ℃, and the humidity is 50-60%.
2.2 model groups
(1) LPS solution (30. Mu.g/6. Mu.L) was instilled into the respiratory tract of mice from the nasal cavity on days 1, 29 and 57, respectively, at a dose of 1.5mg/kg.
(2) Cigarette smoke exposure was continued on days 2-84 (except for days 29, 57):
(1) mice were placed in animal transport cases (60 cm. Times.45 cm. Times.20 cm) with ventilation windows, 25 cases each, sealed with tape.
(2) The animal transport case is placed in a glass fumigating case, and 1 circular ventilation holes with the diameter of about 2cm are respectively reserved on the diagonal side walls of the fumigating case for adjusting the oxygen and air pressure states inside and outside the case.
(3) The cigarettes are lighted and inserted on a self-made metal burning frame, and then put in a self-made glass smoking box and capped. 10 cigarettes are burned out each time, 10 min/time, 2 times/day, 15min intervals in the middle, and the fumigating box cover is opened for ventilation at intervals of 7 days/week for 12 weeks.
(4) After each cigarette smoke exposure, the animal transport case is placed in a delivery window, sterilized by ultraviolet lamp for 15 minutes, and then the mice are put back into the rearing cage.
(3) After the molding was completed, distilled water was orally administered at 0.2 ml/day for 4 weeks from week 13.
2.3 dosing groups
The molding operation was carried out in the same manner as in the molding group, and the administration amount of the drug described in 1.2 was administered by intragastric administration for 4 weeks from week 13.
2.4 blank control group
0.9% physiological saline solution (6. Mu.L/mouse) was instilled into the respiratory tract of mice from the nasal cavity on days 1, 29 and 57, respectively, and the rest of the time was kept normally. From week 13, 0.9% saline solution, 0.2 ml/day was administered orally for a total of 4 weeks.
3 observation index and method
3.1 general State viewing
Quality of life and weight of mice.
3.2 determination of Experimental animal Material and Experimental detection index
3.2.1 collecting samples of bronchoalveolar lavage fluid, serum, and intestinal mucus from mice
3.2.1.1 collection of bronchoalveolar lavage fluid
(1) 2% pentobarbital was intraperitoneally injected into anesthetized mice (0.1 ml/10 g), and the mice were fixed to the console in a supine position.
(2) The neck fur is sheared by the ophthalmology scissors to expose subcutaneous tissue, and the air outlet pipe is passively separated.
(3) And cutting an incision in the horizontal direction of the distal end of the trachea, inserting the tracheal joint of the intubation tube into the trachea in a centripetal and oblique way, and connecting the breathing machine with the tracheal joint after the cotton thread is fixed. A1 ml syringe containing sterile PBS was connected to the endotracheal tube and lavage was performed by slow bolus injection.
(4) After each injection of 0.8ml, the aspiration was slow and after 3 repetitions the lavage was placed in a 1.5ml EP tube.
(5) The rotational speed of the centrifuge is 3000rpm, the lavage liquid is centrifuged for 15min, and the supernatant liquid is taken and stored at the temperature of minus 80 ℃ to be measured.
3.2.1.2 collection of serum
(1) Skin was cut under the xiphoid process, and the myometrium was cut along the bilateral rib arches, exposing the chest.
(2) The ribs were cut along the sternum edge, the heart and lung were exposed, a needle was inserted from the apex of the heart using a 1ml syringe, and blood was taken from the four chambers of the heart, each about 0.8-1.0ml.
(3) Slowly pouring into 1.5ml EP tube, placing into 37deg.C water bath for 15min, and refrigerating at 4deg.C for 15min to visualize delamination of blood sample.
(4) The centrifuge rotates at 3000rpm, centrifugates for 15min, and the upper serum is taken and stored at-80 ℃ to be measured.
3.2.1.3 collection of intestinal mucus
The abdominal cavity was opened, the pylorus and cecum were found, the mesentery and surrounding tissues were gently separated, the entire small intestine between pylorus and cecum was taken, and 10ml (ph=7.4) of 0.01M sterile PBS solution was slowly injected by syringe to flush the intestinal lumen.
The obtained flushing liquid is collected into a 5ml centrifuge tube, the rotation speed of the centrifuge is 3000rpm for 15min, and the supernatant liquid is taken and stored at the temperature of minus 80 ℃ to be measured.
3.2.2 collecting and processing Lung tissue morphology samples of mice
3.2.2.1 morphological sample collection
Cutting off the end of the distal end of the trachea, separating the adhered tissues around, taking out the lung by forceps, performing morphological sample treatment on the left lung lobe, and storing the right lung lobe in liquid nitrogen by using a freezing tube.
3.2.2.2 morphological sample processing
Fixing: the left lung lobes are covered by gauze, and are fixed by paper clips and then are fixed in 10% formalin solution, and the specimens are completely immersed to avoid floating on the liquid surface. After 24h fixation the tissue is removed and the left lung lobes are placed in a dehydration box in a fume hood.
Gradient alcohol dehydration: 75% alcohol-85% alcohol-90% alcohol-95% alcohol-absolute alcohol I-absolute alcohol II-alcohol benzene-xylene I-xylene II-wax I-wax II-wax III.
Embedding: embedding the waxed tissue in an embedding machine, spreading the melted wax into an embedding frame, and placing left lung lobes and marking before solidification. Then placing the mixture in a freezing table at the temperature of minus 20 ℃ for cooling, and separating the wax block from the embedding frame after the wax is solidified.
Slicing: the slicing machine is set to have the slice thickness of 5Pm, so that slices float on the warm water surface of the slice spreading machine at 43 ℃, after the tissues are flattened, the slices are fished up by using glass slides, and the glass slides are placed into a 60 ℃ oven for baking. Taking out the mixture after drying and preserving the mixture at normal temperature for standby.
3.2.2.3HE dyeing
Dewaxing to water: the mouse lung tissue slices are placed in an oven at 60 ℃ for 1h, the slices are placed in xylene I and II for 15min respectively after wax is completely melted, absolute ethyl alcohol I, II-95% alcohol I, II-90% alcohol-80% alcohol-70% alcohol-60% alcohol-50% alcohol are respectively placed in the oven for 2min, and tap water is gently rinsed for 5min.
After hematoxylin staining for 15min, excess dye liquor was washed away with tap water.
Differentiation was performed for 30s with ethanol containing 1% hydrochloric acid, and washing was performed for 10min with tap water.
1% eosin solution is used for dyeing for 10min, and tap water is used for washing off redundant dye liquor for 1min.
Gradient alcohol dehydration: 50% alcohol-60% alcohol-70% alcohol-80% alcohol-90% alcohol-95% alcohol I,
II- -100% absolute ethanol I and II for 2min each. Xylene II transparent for 10min, neutral resin sealing piece
Observation of changes in pulmonary tissue and bronchial wall under optical microscope and infiltration of inflammatory cells
3.2.3 isolation and purification of Lung tissue and Small intestine epithelial intercellular lymphocytes
3.2.3.1 intraperitoneal injection of heparin
Heparin preparation: specification 12500 IU/branch, working concentration: 100IU/ml.
1 branch of heparin was added to 125ml of 0.9% NaCL solution and mixed well.
After weighing the mice, heparin was extracted at a rate of 10IU/ml/10g, and the mice were injected into the abdominal cavity from the left inguinal region.
3.2.3.2 Abdominal anesthesia
1% sodium pentobarbital is prepared: 100mg of sodium pentobarbital is weighed, added into a centrifuge tube containing 10ml of distilled water, fully dissolved and uniformly mixed.
A1% solution of sodium pentobarbital was withdrawn from the syringe by 1ml and administered at a rate of 0.05ml/10g body weight. The eyelid of the mice to be stimulated is not reflected, and the lung tissues of the mice are collected.
3.2.3.3 mouse lung tissue lymphocyte isolation
The lung tissue was placed into a 50ml centrifuge tube with sterile ophthalmic forceps and rinsed 3 times with 50ml PBS.
The washed lung tissue was placed in a sterile small dish (diameter 35mm, depth 10 mm) and minced as much as possible with ophthalmic scissors. The 70 μm cell filter was placed on a centrifuge tube, the tissue was transferred to the filter with a pipette, and 1 XHanks equilibration solution was washed 3 times and the wash solution was filtered off.
50ml of RPM 1640 and 1ml of PBS were added to a 50ml centrifuge tube, and the lung tissue fragments were added with a pipette, followed by 100mg/ml Collagenase NB of 200. Mu.L and 10mg/ml of DNASE of 40. Mu.L, to give working concentrations of 2mg/ml and 40. Mu.g/ml, respectively.
The incubation was carried out for 90min at 37℃in a constant temperature water bath, and the shaking was carried out 1 time every 10min with a shaker.
The undigested tissue fragments and impurities were filtered off with a 70 μm cell strainer.
15ml of RPM 1640 was pipetted onto the cell sieve and the filtrate and rinse were transferred to a 50ml centrifuge tube. Centrifuging at 4 ℃ for 10min at a rotating speed of 2000r/min.
After centrifugation, the supernatant was removed by gently pipetting with a pipette, taking care not to agitate the cell layer, leaving 5ml in the centrifuge tube, and floating the cells by flicking the bottom of the centrifuge tube with a finger.
3ml of 100% percoll and then 5ml of cell suspension were added to a 15ml centrifuge tube, and then 5ml of 1 XHanks equilibration solution containing 5% PBS was added, and after mixing, the content of percoll in the solution was 30%. Centrifuge at 4℃for 18min and 1800r/min.
After centrifugation, the supernatant was removed, 1ml of solution (containing percoll 0.3 ml) was retained, the bottom of the centrifuge tube was flicked to break up the cell mass, and 4.1ml 100%percoll was added and mixed well. The volume was increased to 10ml with a 1 Xhanks solution containing 5% PBS and the content of percoll after blending was 44%.
70% of percoll 2ml is slowly added to the bottom of a 15ml centrifuge tube by using a long suction tube, and the mixture is gently operated to form an interface between two types of percoll liquid with different densities, and the mixture is centrifuged for 18min at 4 ℃ and 180 r/min.
Cells were visible as a layer at the interface of the 44% percoll and 70% percoll levels. The upper 1/3 of the liquid in the centrifuge tube is carefully discarded by sticking the tube wall to the tube wall, and then the cells are sucked out by sticking the tube wall and transferred into a prepared 50ml centrifuge tube.
1 XHanks equilibration solution containing 5% PBS was added to give a total volume of 10ml, and the mixture was thoroughly shaken. Centrifuge for 5min at room temperature, 1500r/min.
After discarding the supernatant, 2ml of a 1 XHanks RPMI1640 solution containing 10% PBS was added to prepare a lymphocyte suspension to be measured. 4g/L trypan blue staining was added and counted with a cell counter plate.
Isolation of lymphocytes between mouse intestinal epithelium of 3.2.3.4
Placing the whole section of small intestine between pylorus and cecum in PBS solution precooled on tinfoil, turning over the intestinal tube from one end of small intestine into intestinal cavity with thin plastic tube, gradually making the plastic tube pass out from the other end of small intestine, turning over the whole intestinal cavity, and making mucosa layer outwards and viscera layer inwards.
The intestinal tube was carefully transferred to a 50ml centrifuge tube, and 200. Mu.L of RPMI1640 and 100mg/ral Collagenase NB containing 10% PBS and 40. Mu.L of 10mg/ml DNASE were added to give working concentrations of 2mg/ml and 40. Mu.g/ml, respectively.
The tube was centrifuged at 37℃for 60min at 200r/min.
The digestate was filtered through a 70 μm cell strainer into a 50ml centrifuge tube, and undigested tissue fragments and impurities were removed. Centrifuging at 4 ℃ for 10min and 2000r/min.
After centrifugation, the supernatant was removed by pipetting with a pipette, taking care that 5ml of cell-layer was left in the centrifuge tube without agitation, and the pellet at the bottom of the centrifuge tube was flicked with a finger to suspend the cells.
3ml of 100% percoll and then 5ml of cell suspension were added to a 15ml centrifuge tube, followed by 2ml of 1 Xhanks solution in 5% PBS, and the content of percoll in the liquid after mixing was 30%. Centrifuge at 4℃for 18min and 1800r/min.
After centrifugation, the supernatant was removed, 1ml of solution (containing percoll 0.3 ml) was left, the cell mass at the bottom of the dispersion was flicked off, and 4.1ml 100%percoll was added after mixing. The total amount of the solution was increased to 10ml by adding 1 Xhanks solution containing 5% PBS, and the content of percoll after mixing was 44%.
70% percoll 2ml was slowly added to the bottom of the centrifuge tube with a pipette, and the two densities of liquid were layered to form an interface by gentle manipulation, and centrifuged at 4℃for 18min and 180 r/min.
Cells were visible as a layer at the interface of the 44% percoll and 70% percoll levels. The upper 1/3 of the liquid in the centrifuge tube is carefully discarded by sticking the tube wall to the tube wall, and then the cells are sucked out by sticking the tube wall and transferred into a prepared 50ml centrifuge tube.
The total amount of liquid was made up to 10ml by adding 1 XHanks containing 5% PBS, and after mixing, centrifuged at room temperature for 5min at 1500r/min.
After discarding the supernatant, the cells were resuspended in 2ml of 1 Xhanks containing 5% PBS and made into lymphocyte suspensions for measurement. 4g/L trypan blue staining was added and counted with a cell counter plate.
3.2.4 detection of inflammatory factor content in mouse serum and intestinal mucus
3.2.4.1IL-6 content detection
All samples and test kits to be tested were equilibrated to room temperature (18-25 ℃).
50ml of 1 XWash Buffer was prepared from 5ml of Wash Buffer concentrate (10X) and 45ml of distilled water. Antibody mixtures were prepared with antibody dilutions, capture antibodies and detection antibodies. mu.L of 10 XCapture Antibody and 300. Mu.L of 10X Detector Antibody were mixed with 2.4ml of Antibody dilution to prepare 3ml of Antibody mixture, which was gently mixed.
Preparing a standard product:
adding 500 mu L of sample diluted normal saline to dissolve mouse IL-6 recombinant protein freeze-dried powder, fully dissolving and uniformly mixing, and lightly mixing for 10min at room temperature to obtain 2000pg/ml standard solution. The 8 EP tubes are marked and are standard 1-8. 150. Mu.L of sample diluted physiological saline was added to tube Nos. 1-8. Serial dilutions were made using standard solutions, 150 μl of the prepared 2000pg/ml standard solution was added to tube 1 to prepare 2000pg/ml standard solution, 150 μl of the prepared 1000pg/ml standard solution was added to tube 1 to prepare 500pg/ml standard solution, and 150 μl of the prepared 500pg/ml standard solution was added to tube 3 to prepare 150pg/ml standard solution, so 125, 62.5, 31.3, 15.6pg/ml standard solution was sequentially prepared in tubes 4, 5, 6, 7, with 8 being blank control.
Sample adding: mu.L of sample (serum, intestinal mucus) or standard is added to the appropriate wells, and then 50. Mu.L of antibody mixture is added to each well, taking care of the gentle handling, without generating bubbles.
Incubation: plates were sealed with a film and incubated for 1h at room temperature on a shaker set at 400 r/min.
Washing the plate: the wells were discarded and each well was washed 3 times with 350 μl of 1 xWash Buffer and after completion the 96 well plates were back-buckled on a clean paper towel to remove excess liquid.
Color development: a350. Mu.L TMB matrix was added to each well and incubated for 10min in a shaker at 400r/min in the absence of light, allowing blue coloration of the liquid in the well plate to be observed.
And (3) terminating: 100. Mu.L of stop solution was added to each well and after mixing the blue liquid turned yellow.
The microplate reader was set to 450nm for OD measurements.
And (3) calculating: and calculating a standard equation by taking the OD value as a dependent variable Y and the standard concentration as an independent variable X, and substituting the OD value of the measured sample into the equation to obtain the IL-6 concentration.
3.2.4.2IL-13 content detection IL-13 concentration was measured in the same manner.
4 statistical method
The experimental data are expressed by mean plus minus standard deviation (x+/-S) and are processed by SPSS13.0 software, and the comparison between the mean values is performed by T test.
5 experimental results
5.1 administration group improving quality of life and body weight of COPD mice
Normal control group: mice in the blank control group have no death, smooth fur, moderate respiration, moderate frequency, uniform rhythm, normal activity and gradual weight increase;
model group: the mice in the slow lung-blocking model group die 10 mice, hair is dark, part of hair is unhairing, the hair jumps during smoking, the later stage is curled by multiple binding piles, even the whole body is trembled, the respiratory depression, the chest and abdomen undulation are obvious, irregular nodding movement is caused, even the hair is opened and breathed, and the weight is obviously lower than that of the normal group (P < 0.001).
The red clover glycoside is high, medium, low and low dose groups respectively kill 3, 2, 1, 2 and 4, the iris glycoside is high, medium, low and low dose groups respectively kill 4, 1, 3 and 4, compared with the model group, the mice skin and hair are smooth, the actions tend to be normal, the dyspnea is improved, and the weight is obviously higher than that of the model group (P < 0.001).
The composition had 1 death in the higher and middle dose groups and 3 deaths in the higher, lower and lower dose groups, and the model group had a smooth hair and improved dyspnea after administration of the composition to the stomach, and the weight was significantly higher than that of the model group (P < 0.001), and the results are shown in Table 2.
Table 2 composition can improve survival rate and body weight of mice with slow pulmonary disease model
Figure BDA0004094493930000131
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Figure BDA0004094493930000132
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Note that: wherein n represents the number of surviving mice.
5.2 model of Lung with Slow resistance model mouse Lung histomorphology pathological changes
The lung tissue pathology of the slow-blocking lung is shown by degeneration, necrosis and shedding of bronchial mucosa epithelium, shortening cilia and adhesion; goblet cells and mucus cells proliferate and hypertrophy, a large amount of mucus is retained, the vessel wall is engorged with blood, the edema, and the tracheal lumen is narrowed. Alveoli collapse, the alveolar spaces break apart, and adjacent alveoli fuse into a lung bleb. A large amount of chronic inflammatory cells infiltrate around the trachea and alveoli, smooth muscle rupture and atrophy of submucosa and fibrous tissue hyperplasia; and (3) remodelling the bronchus structure, increasing the collagen content and forming scars. The experiment HE staining shows that the air passage and the alveoli of the mice in the blank control group are normal in structure, cilia are orderly, the shape and the size of the alveoli are regular, and the airway mucosa epithelium is complete; the model control group bronchus mucosa fold is formed and has flakiness to fall off, so that the lumen is narrowed or blocked; the alveolar wall is destroyed, the alveolar space is irregularly enlarged, the alveolar wall is partially fused into lung large bubbles, and chronic inflammatory cell infiltration is visible around the airway and in the lung interstitium, so that the chronic inflammatory cell infiltration accords with the pathological manifestations of chronic obstructive pulmonary disease.
Compared with a model control group, the high, medium and low groups treated by the two single drug groups and the composition drug can improve morphology, which is reflected in the fact that bronchus and alveolus structures are complete, the narrow degree of a tube cavity is reduced, airway mucous membrane epithelium is relatively complete, cilia arrangement rules and shedding are reduced, the alveolus size is uniform, the number of lung bullae is reduced, and inflammatory cell infiltration degree around the tube wall of the airway and in the lung interstitium is reduced.
5.3 Lung tissue alpha beta T/gamma delta T cell ratio in mice with Slow resistance Lung model
Gamma delta T cells are key effector cells of the respiratory tract mucosal immune system and are involved in the inflammatory and injury repair process of chronic inflammatory diseases. In normal mouse lungs, most αβt cells are distributed in the lung parenchyma, while most γδ T cells are distributed in non-alveolar regions other than the mucosa. The relative density of γδ T cells is highest in the vicinity of the respiratory tract, blood vessels and visceral pleura. Although the γδ T cell numbers are much smaller in scale, they match or nearly match αβ T cells in the relative density of the non-alveolar region. In contrast, in the region of the lung parenchyma where the tissue mass or tissue surface area is greatest, the relative density of γδ T cells is much lower than αβ T cells. The difference in the distribution of these two T cells may be related to the difference in the functional roles. The single regional distribution of αβt cells in the lung of normal lungs may reflect some degree of functional homogeneity, while the broad distribution of γδ T cells reflects functional heterogeneity. γδ T cells are mainly in contact with myeloid cells and αβt is more in contact with lymphocytes throughout the lung. This comparison shows that leukocyte contact with lung γδ T cells is primarily involved in myeloid lineage cells, including f4/80+ macrophages and I-a+ dendritic cells, while αβ T cells are often contacted with cd45r+ lymphocytes, including B cells as well as some T cells and plasmacytoid dendritic cells.
In the lung tissue morphological immunohistochemical staining experiment, cells are marked and counted by alpha beta TCR and gamma delta TCR, and a blank control group can see that two cells exist in lung tissue in a small amount; the model control group showed little γδt presence; the high, higher, medium, lower, and low dose drug groups showed that more γδ T cells were present near the trachea, significantly increased compared to the model group, the αβt/γδ T cell ratio in the lung tissue of mice was increased in the model group compared to the placebo group, the drug administration group showed a decreasing trend compared to the model group, and the composition drug administration group showed a significant decrease compared to the single drug administration group, as shown in table 3.
Referring to the isolation method of intestinal epithelial inter-cell lymphocytes, we explored an experimental method of density gradient centrifugation to isolate lung tissue lymphocytes. The method for separating the lymphocyte among the intestinal epithelial cells is mature, is accepted by the academia, has fewer cell types of intestinal mucosa epithelium, and is favorable for separating different cell groups. Referring to the results of flow analysis of small intestine sample cells, lymphocyte populations in the cell suspension obtained after digestion and separation of lung tissue can be determined, and the specific antibodies can be used to label the αβ T cells and γδ T cells therein, respectively, as shown in table 4. The proportion of the gamma delta T cells of the mice is reduced in the model group compared with the blank control group, and the liquid medicine group of the low-dose composition is obviously increased (P is less than 0.05) compared with the model group, which indicates that the liquid medicine treatment of the composition is beneficial to improving the proportion of the gamma delta T cells of the lung tissues of the mice.
TABLE 3 immunohistochemical staining cell count shows compositionsLiquid medicine for reducing alpha beta T/gamma delta T cell proportion of lung tissue of slow-resistance lung model mouse
Figure BDA0004094493930000151
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Figure BDA0004094493930000152
Figure BDA0004094493930000161
Table 4 flow cytometer analysis results show that the composition liquid medicine reduces the proportion of alpha beta T/gamma delta T cells in lung tissue of a slow-resistance lung model mouse
Figure BDA0004094493930000162
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Figure BDA0004094493930000163
5.4 IL-6 and IL-13 content in mouse serum, alveolar lavage fluid and small intestine mucus
Comparing the IL-6 and IL-13 content in serum, alveolar lavage fluid and small intestine mucus of the mice, wherein the IL-6 content in serum is higher than that in a blank control group (P < 0.05), the administration groups are lower than that in the model control group, and the medium-dose composition group is obviously lower than that in the model control group (P < 0.05); no significant differences were seen between the groups for the IL-13 content in serum. The IL-6 content in the alveolar lavage fluid is higher than that in the blank control group (P < 0.05); the dosing groups with high and low doses are not statistically different from the control group of the model group, the doses in the dosing groups are lower than those in the blank control group, the model control group and the low-dose dosing group, and the doses in the middle and lower dose groups of the composition are obviously reduced (P is less than 0.05) compared with the single dosing group; the IL-13 content in the alveolar lavage fluid is higher than that in a blank control group (P < 0.05), the administration groups are lower than that in the model control group, and the medium and lower dosage groups of the composition are obviously reduced (P < 0.05). No significant differences were seen between the groups of IL-6 content in intestinal mucus; the slow-blocking lung model control group had a higher IL-13 content in the intestinal mucus than the blank group (P < 0.05), and the high, higher, medium, lower, low dose groups of the individual and composition administration were all lower than the model group control group, and the high, higher, medium, lower, low dose groups of the composition were significantly lower (P < 0.05).
In conclusion, the amount of inflammatory factors secreted by mice with slow-blocking lung model is obviously increased, and the content of IL-6 in serum and alveolar lavage fluid of the composition liquid medicine group and IL-13 in alveolar lavage fluid and intestinal mucus of the composition liquid medicine group is obviously reduced compared with that of a model control group (P is less than 0.05), and the composition liquid medicine group is shown in Table 5.
TABLE 5 IL-6 and IL-13 content in mouse serum and alveolar lavage fluid
Figure BDA0004094493930000171
Figure BDA0004094493930000172
Figure BDA0004094493930000181
Although embodiments of the present invention have been disclosed above, it is not limited to the details and embodiments shown and described, it is well suited to various fields of use for which the invention would be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the specific details and illustrations shown and described herein, without departing from the general concepts defined in the claims and their equivalents.

Claims (8)

1. A flavonoid glycoside combination for use in the treatment of chronic obstructive pulmonary disease comprising a red clover glycoside-tectoridin composition in a suspension of 0.5-1.5g/kg with physiological saline.
2. The flavonoid glycoside combination for the treatment of chronic obstructive pulmonary disease according to claim 1, wherein said red clover glycoside-tectoridin composition is formulated in a suspension of 1.0g/kg in physiological saline.
3. The flavonoid glycoside combination for the treatment of chronic obstructive pulmonary disease according to claim 2, wherein the weight ratio of red clover glycoside to tectoridin in the red clover glycoside-tectoridin composition is 2:5.
4. a method of preparing a flavonoid glycoside combination for use in the treatment of chronic obstructive pulmonary disease according to any one of claims 1-3, comprising the steps of:
s1, respectively weighing the bean roots and the blackberry lily, and carrying out crushing, alcohol extraction and concentration to obtain an alcohol extract of the bean roots and an alcohol extract of the blackberry lily;
s2, ethyl acetate is respectively added into the alcohol extract of the bean roots and the alcohol extract of the blackberry lily for three times, and the extraction is carried out under reduced pressure and concentrated to obtain a fraction of the bean roots and a fraction of the blackberry lily;
s3, respectively separating, purifying and recrystallizing the fraction of the bean roots and the total flavone fraction of the blackberry lily to obtain red clover glycoside and tectoridin;
s4, mixing the red clover glycoside and the tectoridin to obtain a red clover glycoside-tectoridin composition;
s5, adding physiological saline into the red clover glycoside-tectoridin composition to prepare a suspension.
5. The method according to claim 4, wherein the alcohol extraction solvent is 70-75% ethanol when the crushed bean roots and blackberry lily in step S1 are subjected to alcohol extraction, and the volume of the ethanol is 6-10 times of the volume of the crushed bean roots and blackberry lily respectively.
6. The method according to claim 4, wherein in step S2, the volume ratio of the ethyl acetate to the alcoholic extract of the bean roots and the alcoholic extract of blackberry lily is 1:1.
7. the method according to claim 4, wherein the separation and purification method of the fraction of the bean roots and the fraction of the blackberry lily in the step S3 comprises the following steps:
sa, respectively dissolving the fraction of the bean roots and the fraction of the blackberry lily in a low-concentration ethanol solution with concentration below 30%, dissolving and filtering to obtain filtrate of the bean roots and filtrate of the blackberry lily;
sb, further separating and purifying the filtrate of the root of red bean and the filtrate of the blackberry lily by using a D101 macroporous resin column respectively to obtain the red clover glycoside and the tectoridin.
8. Use of a flavonoid glycoside combination according to any one of claims 1-3 for the preparation of a medicament for the treatment of chronic obstructive pulmonary disease.
CN202310161961.XA 2023-02-24 2023-02-24 Flavonoid glycoside combinations for the treatment of chronic obstructive pulmonary disease Pending CN116098912A (en)

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