CN111956655A - Application of pulsatilla saponin B4 in preparation of medicine for treating chronic obstructive pulmonary disease - Google Patents
Application of pulsatilla saponin B4 in preparation of medicine for treating chronic obstructive pulmonary disease Download PDFInfo
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- CN111956655A CN111956655A CN202010840016.9A CN202010840016A CN111956655A CN 111956655 A CN111956655 A CN 111956655A CN 202010840016 A CN202010840016 A CN 202010840016A CN 111956655 A CN111956655 A CN 111956655A
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- saponin
- obstructive pulmonary
- pulsatilla
- chronic obstructive
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- OUHBKBTZUPLIIA-OTEDBJMHSA-N [(2s,3r,4s,5s,6r)-6-[[(2r,3r,4r,5s,6r)-3,4-dihydroxy-6-(hydroxymethyl)-5-[(2s,3r,4r,5r,6s)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxymethyl]-3,4,5-trihydroxyoxan-2-yl] (1r,3as,5ar,5br,7ar,8r,9s,11ar,11br,13ar,13br)-9-[(2s,3r,4s,5s)-4,5-dihydroxy- Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](CO)O[C@@H](OC[C@@H]2[C@H]([C@H](O)[C@@H](O)[C@H](OC(=O)[C@@]34[C@H]([C@@H](CC3)C(C)=C)[C@@H]3[C@@]([C@]5(C)CC[C@H]6[C@](C)(CO)[C@@H](O[C@H]7[C@@H]([C@@H](O)[C@@H](O)CO7)O[C@H]7[C@@H]([C@H](O)[C@@H](O)[C@H](C)O7)O)CC[C@]6(C)[C@H]5CC3)(C)CC4)O2)O)[C@H](O)[C@H]1O OUHBKBTZUPLIIA-OTEDBJMHSA-N 0.000 title claims abstract description 44
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
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- A—HUMAN NECESSITIES
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
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Abstract
The invention discloses application of pulsatilla saponin B4 in preparation of a medicine for treating chronic obstructive pulmonary disease. The invention has protective effect on COPD rats caused by dropping LPS into cigarettes in combination with trachea, the mechanism of the invention is probably related to the regulation of IL-12/STAT4 and IL-4/STAT6 signal channels, thereby regulating and regulating Th1/Th2 cell differentiation, having good treatment effect on COPD chronic inflammation and airway remodeling induced by Th1/Th2 cell differentiation imbalance, and providing theoretical basis for preventing and treating chronic obstructive pulmonary diseases.
Description
Technical Field
The invention relates to the technical field of medicines. More specifically, the invention relates to an application of pulsatilla saponin B4 in preparing a medicine for treating chronic obstructive pulmonary disease.
Background
Chronic Obstructive Pulmonary Disease (COPD) is a lung disease characterized by airflow limitation, which is not completely reversible, develops progressively and is associated with abnormal inflammatory responses of the lungs to harmful gases or particles such as cigarette smoke. The disease has high morbidity, mortality and disability rate, serious harm, difficult treatment and obviously shortened life expectancy, and is one of the main diseases harming the physical and mental health of the public. Smoking-stimulated T cell-mediated autoimmune imbalance is one of the possible pathogenesis of chronic obstructive pulmonary disease, and Th1/Th2 cell differentiation imbalance can induce COPD chronic inflammation and airway remodeling.
Pulsatillae radix is dried root of Pulsatilla chinensis (Bunge) Regel of Pulsatilla of Ranunculaceae, and has effects of clearing heat and detoxicating, cooling blood and relieving dysentery. Modern pharmacological studies show that the saponins in pulsatilla chinensis is the main active component of pulsatilla chinensis, and has the effects of enhancing immune function, resisting inflammation and the like. Pulsatillae saponin B4 is pentacyclic triterpenoid saponin extracted from Pulsatillae radix and has pharmacological effects of resisting inflammation, bacteria and virus, regulating immunity, resisting tumor, oxidation and virus, but Pulsatillae saponin B4 has no reported effect on treating chronic obstructive pulmonary disease.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
The invention also aims to provide the application of the pulsatilla saponin B4 in preparing the medicine for treating the chronic obstructive pulmonary disease, the pulsatilla saponin B4 has a protective effect on COPD rats caused by the intratracheal instillation of LPS in combination with cigarettes, the mechanism of the pulsatilla saponin B4 can be related to the regulation of IL-12/STAT4 and IL-4/STAT6 signal pathways, so that the regulation of Th1/Th2 cell differentiation is realized, a good treatment effect is realized on COPD chronic inflammation and airway remodeling induced by Th1/Th2 cell differentiation imbalance, and a theoretical basis is provided for preventing and treating the chronic obstructive pulmonary disease.
To achieve these objects and other advantages in accordance with the present invention, there is provided a use of pasqueoside B4 in the preparation of a medicament for the treatment of chronic obstructive pulmonary disease.
Preferably, the medicament contains effective amount of pulsatilla saponin B4 and pharmaceutically acceptable carrier.
Preferably, the medicament is formulated into a pharmaceutically acceptable dosage form.
Preferably, the medicament is formulated as an injection.
Preferably, the drug inhibits both the IL-12/STAT4 and IL-4/STAT6 signaling pathways.
Preferably, the dosage of the pulsatilla saponin B4 is not less than 2.5 mg/kg-d.
The invention at least comprises the following beneficial effects:
the invention uses ELISA kit to detect the content of relevant inflammatory cytokines in rat alveolar lavage fluid, real-time fluorescence quantitative method (RT-PCR) to detect the mRNA expression level of relevant genes in rat lung tissue, and Western blot to detect the expression level of relevant proteins in rat lung tissue. The results show that compared with a model control group, the Chinese pulsatilla saponin B4 low-dose group, the Chinese pulsatilla saponin B4 medium-dose group and the Chinese pulsatilla saponin B4 high-dose group have different increasing effects on the body weight of rats; the content of inflammatory cytokines in rat alveolar lavage fluid is improved to different degrees; the protein expression levels of IL-12/STAT4 signal pathway key protein T-beta, IL-12, STAT4mRNA and STAT4 in rat lung tissues have reducing effects to different degrees; the mRNA and protein expression levels of the key proteins GATA3, IL-4 and STAT6 of the IL-4/STAT6 signal pathway are increased to different degrees. The cigarette composition has a protective effect on COPD rats caused by the dropping of LPS into cigarettes in combination with trachea, the mechanism of the cigarette composition is probably related to the regulation of IL-12/STAT4 and IL-4/STAT6 signal pathways, so that the regulation of Th1/Th2 cell differentiation is realized, a good treatment effect is realized on COPD chronic inflammation and airway remodeling induced by Th1/Th2 cell differentiation imbalance, and a theoretical basis is provided for preventing and 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 graph showing the effect of pasqueoside B4 of the present invention on IL-12 in alveolar lavage fluid;
FIG. 2 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on IFN-. gamma.in alveolar lavage fluid;
FIG. 3 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on IL-4 in alveolar lavage fluid;
FIG. 4 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on T-beta/IL-12/STAT 4mRNA levels in lung tissue of COPD rats;
FIG. 5 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on the level of GATA3/IL-4/STAT6 mRNA in lung tissue of COPD rats;
FIG. 6 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on the levels of T-beta/IL-12/STAT 4 protein in lung tissue of COPD rats;
FIG. 7 is a graph showing the effect of Pulsatillae saponin B4 of the present invention on the level of GATA3/IL-4/STAT6 mRNA in lung tissue of COPD rats.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
Application of pulsatilla saponin B4 in preparing medicine for treating chronic obstructive pulmonary disease is provided.
The pharmaceutical preparation contains the pulsatilla saponin B4 with effective treatment amount, and the rest is pharmaceutically acceptable carriers and excipients which are nontoxic and inert to human and animals.
The pharmaceutically acceptable carrier or excipient is one or more selected from solid, semi-solid and liquid diluents, fillers and pharmaceutical adjuvants. The pharmaceutical preparation of the present invention is used in the form of a dose per unit body weight. The extract of the present invention can be administered to a patient in need of treatment by oral administration or injection. For oral administration, it can be made into tablet, sustained release tablet, controlled release tablet, capsule, dripping pill, pellet, suspension, emulsion, powder or granule (nanometer preparation), oral liquid, etc.; for injection, the composition can be made into sterilized aqueous or oily solution, sterile powder for injection, liposome or emulsion.
In the figures 1-7, the blank control group is abbreviated as a blank group, the model control group is abbreviated as a model group, the negative control group is abbreviated as DEX 5mg/kg, the low-dose group of pulsatilla saponin B4 is abbreviated as B42.5mg/kg, the dose group of pulsatilla saponin B4 is abbreviated as B45 mg/kg, and the high-dose group of pulsatilla saponin B4 is abbreviated as 10 mg/kg.
1. Material
1.1 Experimental animals
Healthy male SD rats, 60, SPF grade, body weight (180 ± 20) g, provided by the experimental animals center of the university of medical, guangxi, animal certification number: SCXK Gui 2009-. All experimental animals are raised in a controllable environment at the room temperature of 18-24 ℃ and the humidity of 40-50%, the animals eat and drink water freely during the experiment, and the circadian rhythm is normal.
1.2 major drugs and reagents
Pulsatillae saponin B4 (Jiangxi Bencao Tian Gong Tech science and technology, Inc., batch No. 2018042205); lipopolysaccharide/LPS (Sigma Co.); physiological saline (Sichuan Kelun pharmaceutical Co., Ltd., batch No. L219012211); ELISA assay kit (synbose); dexamethasone (Guangdong south China pharmaceutical industry group Co., Ltd., code: A14202002484).
1/1000 precision balance (Metler-Torledo instruments Shanghai Co., Ltd., model: ME 204E); low temperature high speed centrifuge (Eppendorf Co., model 5425R); 722sp visible spectrophotometer (Shanghai prism technologies, Inc.); an enzyme linked immunosorbent assay (BioTek company, USA, model: SYNERGYH 1).
2. Method of producing a composite material
2.1 establishment and grouping of animal models
60 healthy male SD rats are adaptively fed for 1 week and are randomly divided into 6 groups of 10 rats each, namely a blank control group, a model control group, a positive control group (DEX, 5mg/kg), pulsatilla saponin B4(2.5mg/kg), pulsatilla saponin B4(5mg/kg) and pulsatilla saponin B4(10 mg/kg). After grouping, marking and numbering the rats in each group, and feeding the rats in groups with feed and normal drinking water.
A rat model of chronic obstructive pulmonary disease is replicated by adopting a method of smoking cigarettes and instilling LPS in trachea. Molding a model control group, a positive control group (DEX, 5mg/kg), pulsatilla saponin B4(2.5mg/kg), pulsatilla saponin B4(5mg/kg) and pulsatilla saponin B4(10 mg/kg). In a normal room temperature environment, model rats are anesthetized on days 1 and 14 in an experiment, LPS (1mg/mL) is instilled through a trachea, on days 2-28 (no smoking is performed on the day of LPS instillation), and rats of a model control group, a negative control group (dexamethasone), pulsatilla saponin B4(2.5mg/kg), pulsatilla saponin B4(5mg/kg) and pulsatilla saponin B4(10mg/kg) are smoked by cigarettes once per day, and one cigarette is smoked for each rat for 1 hour. The passive smoking method comprises the following steps: the rat is put into an autonomous smoking box, and the cigarette is ignited from the smoke inlet hole at one side, so that the rat is passively smoked and infected. A proper amount of silica gel desiccant is placed at the bottom of the box to reduce the influence of water vapor generated by cigarette combustion on rats. The experiment is carried out on the 4 th day until the 3 rd day after the molding is finished, namely, each component is respectively treated by the medicine for 4 to 31 days, and the total time is 28 days. The tail vein of the blank control group and the model control group is given equal volume of physiological saline every day; the pulsatilla chinensis saponin B4 is prepared by carrying out tail vein injection on 2.5mg/kg, 5mg/kg and 10mg/kg of pulsatilla chinensis saponin B4 respectively; the positive control group was administered with Dexamethasone (DEX)5mg/kg intraperitoneally once daily for 28 consecutive days.
2.2 taking materials
(1) Alveolar lavage fluid (BLAF) collection: after abdominal aorta blood collection, the neck skin of a rat is cut open, organs are exposed, the thoracic cavity is continuously opened, the thymus and the pericardium covering lung tissues are stripped, the branch of the trachea is found, the right main bronchus is firstly tied, 6mL of sterile physiological saline with the concentration of 0.9 percent is used for injecting the left main bronchus and the left lung through the trachea, the suction is slowly and repeatedly carried out for 4 times, the centrifugation is carried out for 10min at4 ℃ and 3000r/min, supernatant is sucked, and the supernatant is placed in a 2mL cryovial and stored at the temperature of minus 80 ℃. After the cell pellet obtained after the alveolar lavage fluid centrifugation is resuspended by 1mL of sterilized PBS, the cell pellet is stored in a 2mL freezing tube and stored in a refrigerator at4 ℃ for counting and classifying white blood cells.
(2) Lung tissue fixation method: ligating the left lung, loosening the right lung, injecting 5mL of 4% paraformaldehyde solution through the trachea, fixing for 30s, taking out the middle lobe of the right lung, and storing in 4% paraformaldehyde. For HE staining.
(3) Collecting lung tissue, removing alveolar lavage fluid and fixing lung tissue, taking out the rest lung tissue, placing in a cryopreservation tube, storing at-80 ℃, and using for Western blotting and mRNA detection.
2.3 detection of IL-12, IFN-. gamma.and IL-4 in alveolar lavage fluid
Diluting alveolar lavage fluid supernatant by 10 times, measuring the contents of IL-12, IL-12R, IFN-gamma and IL-4 in BALF by an enzyme-linked immunosorbent assay (ELISA), and carrying out the experimental steps according to the instructions of an ELISA measuring kit.
2.4 detection of mRNA levels of T-beta/IL-12/STAT 4 and GATA3/IL-4/STAT6 in Lung tissue
Taking out lung tissues stored in an ultra-low temperature refrigerator at minus 80 ℃, adding 1mL Trizol reagent into every 50-100 mg of the tissues, and cracking the tissues in a homogenizer; centrifuging, removing the precipitate, taking the supernatant into a new EP tube, adding 0.2mL of chloroform ice, standing for 1-5 min, then 12000g, centrifuging for 10min at4 ℃, taking the upper aqueous phase into the new EP tube, adding isopropanol with the same volume, fully mixing uniformly, then placing on ice for 10min, 12000g, centrifuging for 10min at4 ℃, removing the supernatant, and adding 1mL of 75% ethanol for washing; 12000g, centrifuging for 5min at4 ℃, discarding the supernatant, absorbing the liquid on the tube wall as much as possible, drying for 5-10min at room temperature, adding DEPC water to dissolve RNA, and storing in a refrigerator at-80 ℃. The expression level of each mRNA was detected by a fluorescent real-time quantitative RT-PCR instrument.
2.5 detection of protein levels of T-beta/IL-12/STAT 4 and GATA3/IL-4/STAT6 in Lung tissue
Lung tissue was lysed with RIPA lysate and 1mL RIPA and 10. mu.L PMSF and 10. mu.L phosphatase inhibitor were added per 100mg of tissue. And (4) homogenizing, centrifuging and taking supernatant. Quantifying by using a BCA kit, diluting a sample, adding the sample, performing electrophoresis at 75V for 30min, at 110V for 1 h; transferring the membrane, keeping the pressure constant at 70V for 2h, sealing, incubating the primary antibody overnight, washing the membrane for 10 min/time by TBST (tertiary butyl ether sulfide) for 3 times; the secondary antibody was incubated at room temperature for 1h, and developed after TBST membrane washing.
2.6 statistical treatment
The single-factor analysis of variance and t-test data were performed using STATA8.0 statistical software, with P <0.05 as the difference, which was statistically significant.
3. Results
3.1 Effect of Pulsatillae saponin B4 on body weight in COPD rats
Compared with the blank group, the 14d and 28d of the model control group are obviously reduced after the model is subjected to weight modeling, and the anemonin B4 and the dexamethasone positive drug group can obviously improve the weight of the rat, so that the anemonin B4 has an improvement effect on the weight reduction of the COPD rat, and the details are shown in Table 1.
TABLE 1
P <0.05 compared to model control
3.2 Effect of Pulsatillae saponin B4 on detection of IL-12, IFN-gamma and IL-4 in COPD rat alveolar lavage fluid
After modeling, the IL-2 and IFN-gamma levels in the alveolar lavage fluid of the model control group are obviously increased, which shows that LPS is instilled by a cigarette smoking trachea to promote the secretion of Th1 type cell factors of a COPD rat model; pulsatillae radix saponin B4(5mg/kg), pulsatillae radix saponin B4(10mg/kg), and a positive control group (DEX, 5mg/kg) significantly reduced IL-1 and IFN- γ levels in alveolar lavage fluid, and pulsatillae radix saponin B4(10mg/kg) was more significant than the positive control group (DEX, 5mg/kg), indicating that pulsatillae radix saponin B4 has an effect of down-regulating Th1 type cytokines, as shown in fig. 1 and 2, wherein P <0.05, P <0.01, P <0.001, and P <0.05, and P <0.01 in fig. 2. The IL-4 level in the alveolar lavage fluid of the model control group is obviously reduced, and the IL-4 level in the alveolar lavage fluid is obviously increased by pulsatilla saponin B4(5mg/kg), pulsatilla saponin B4(10mg/kg) and a positive control group (DEX, 5mg/kg), and is detailed in figure 3, wherein P is less than 0.05 and P is less than 0.01 compared with the model control group in figure 3. The results indicate that the pulsatilla saponin B4 has a regulating effect on the expression of cytokines in alveolar lavage of rats with chronic obstructive pulmonary diseases, and the high-dose effect is obvious.
3.3 Effect of Pulsatillae saponin B4 on T-beta/IL-12/STAT 4mRNA levels in COPD rat lung tissue
Compared with a blank control group, the T-beta/IL-12/STAT 4mRNA level in the lung tissue homogenate of the model control group is obviously increased, the pulsatilla saponin B4(2.5mg/kg), the pulsatilla saponin B4(5mg/kg), the pulsatilla saponin B4(10mg/kg) and the positive control group (DEX, 5mg/kg) can obviously reduce the T-beta, IL-12 and STAT4mRNA level in the lung tissue of a COPD rat, and the detailed figure is shown in figure 4, wherein P is less than 0.05 and P is less than 0.01 in figure 4 compared with the model control group.
3.4 Effect of Pulsatillae saponin B4 on mRNA levels of GATA3/IL-4/STAT6 in lung tissue of COPD rats
Compared with a blank control group, the lung tissue homogenate of the model control group has obviously increased GATA3/IL-4/STAT6 mRNA level, and the pulsatilla saponin B4(2.5mg/kg), pulsatilla saponin B4(5mg/kg), pulsatilla saponin B4(10mg/kg) and positive control group (DEX, 5mg/kg) can obviously reduce the GATA3/IL-4/STAT6 mRNA level in the lung tissue of COPD rats, and detailed in figure 5, compared with the model control group, P is less than 0.05, P is less than 0.01, and P is less than 0.001.
3.5 Effect of Pulsatillae saponin B4 on levels of T-beta/IL-12/STAT 4 protein in COPD rat Lung tissue
Compared with a blank control group, the levels of T-beta/IL-12/STAT 4 in the lung tissue homogenate of the model group are obviously increased, and the levels of T-beta, IL-12 and STAT4 in the lung tissue of COPD rats can be obviously reduced by pulsatilla saponin B4(2.5mg/kg), pulsatilla saponin B4(5mg/kg), pulsatilla saponin B4(10mg/kg) and a positive control group (DEX, 5mg/kg), as shown in figure 6.
3.6 Effect of Pulsatillae saponin B4 on the levels of GATA3/IL-4/STAT6 protein in COPD rat Lung tissue
Compared with a blank control group, the levels of GATA3/IL-4/STAT6 in the lung tissue homogenate of the model group are obviously increased, and the levels of GATA3/IL-4/STAT6 in the lung tissue of a COPD rat can be obviously reduced by pulsatilla saponin B4(2.5mg/kg), pulsatilla saponin B4(5mg/kg), pulsatilla saponin B4(10mg/kg) and a positive control group (DEX, 5mg/kg), as shown in figure 7.
4. General theory of the invention
Experimental results show that the pulsatilla saponin B4 has a protective effect on COPD rats caused by LPS instillation in cigarettes in combination with trachea, the mechanism of the pulsatilla saponin B4 can regulate IL-12/STAT4 and IL-4/STAT6 signal pathways, so that the regulation and the regulation of Th1/Th2 cell differentiation are regulated, a good treatment effect is achieved on COPD chronic inflammation and airway remodeling induced by Th1/Th2 cell differentiation imbalance, and a theoretical basis is provided for preventing and treating chronic obstructive pulmonary diseases.
The number of apparatuses and the scale of the process described herein are intended to simplify the description of the present invention. Applications, modifications and variations of the present invention will be apparent to those skilled in the art.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (6)
1. Application of pulsatilla saponin B4 in preparing medicine for treating chronic obstructive pulmonary disease is provided.
2. The use of claim 1, wherein the medicament comprises a therapeutically effective amount of pasqueflower saponin B4 and a pharmaceutically acceptable carrier.
3. The use of claim 1, wherein the medicament is formulated into a pharmaceutically acceptable dosage form.
4. The use of claim 1, wherein the medicament is formulated as an injection.
5. The use of claim 3, wherein the medicament inhibits the IL-12/STAT4 and IL-4/STAT6 signaling pathways.
6. The use according to claim 1, wherein the pulsatillae radix saponin B4 is administered in an amount of not less than 2.5 mg/kg-d.
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