A uniform radix trichosanthis polysaccharide, a preparation method and use thereof

Abstract

This invention discloses a homogeneous polysaccharide from coltsfoot flower, its preparation method, and its uses, relating to the field of pharmaceutical technology. The homogeneous polysaccharide from coltsfoot flower has a molecular weight of 2.0 × 10⁻⁶. 3 -4.5×10 3 Da contains fructose and glucose; wherein the molar ratio of fructose to glucose is 20.00-26.00:1.00. The homogeneous polysaccharide from Tussilago farfara prepared by this invention has good effects in preventing and treating asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a homogeneous polysaccharide from Tussilago farfara, its preparation method, pharmaceutical composition, and uses. Background Technology

[0002] Coltsfoot flower, also known as coltsfoot in traditional Chinese medicine Tussilago farfara L. The dried flower buds of Tussilago farfara, belonging to the genus Tussilago in the family Compositae, are a commonly used traditional Chinese medicine. Tussilago farfara was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica): "It has a pungent and warm taste. It treats cough, shortness of breath, wheezing, sore throat, various convulsions, and chills and fever." Based on its medicinal value, researchers have systematically studied its chemical composition using ultraviolet, infrared, mass spectrometry, nuclear magnetic resonance, and X-ray diffraction techniques, identifying approximately 175 chemical components, including terpenes, organic acids, flavonoids, alkaloids, and chromones. Currently, the content index component recorded in the pharmacopoeia is coltsone.

[0003] Respiratory infectious diseases are a class of illnesses caused by bacteria, viruses, and atypical pathogens, such as pneumonia, bronchiectasis, and acute exacerbations of chronic obstructive pulmonary disease. Clinically, they present with symptoms such as chills and fever, cough with sputum, chest tightness, and shortness of breath. The pathogenesis of respiratory infectious diseases is complex, mainly manifested as inflammatory responses and immune damage. Traditional Chinese medicine views respiratory infectious diseases as a dynamic pathological process. Treatment should combine strengthening the body's resistance and eliminating pathogenic factors, based on the different clinical manifestations in the early and late stages of the disease. Strengthening the body's resistance involves regulating the body's immunity, while eliminating pathogenic factors involves antiviral, anti-inflammatory, and antibacterial agents.

[0004] Currently, Western medicine treatment primarily focuses on anti-infection, which is highly effective, but it also has problems such as antibiotic overuse, bacterial resistance, and immune damage. In the early stages of the disease, the causative microorganism is not yet clear, and Western medicine treatment mainly relies on empirical anti-infection with broad-spectrum antibiotics, which can severely impact the body's immune system, potentially accelerating disease progression and affecting prognosis. In contrast, the advantages of traditional Chinese medicine treatment include syndrome differentiation and treatment, holistic view, combined use of multiple methods, and comprehensive synergy.

[0005] Chinese patent application CN111097921A discloses a method for preparing anti-colon cancer silver nanoparticles using coltsfoot flower polysaccharide, which includes the extraction step of coltsfoot flower polysaccharide powder and the preparation process of anti-colon cancer silver nanoparticles. Chinese invention patent application CN106334030A discloses a traditional Chinese medicine composition containing coltsfoot flower that can fundamentally and effectively alleviate lower respiratory tract symptoms from the inside out. It contains 5-9 parts honeysuckle, 2-9 parts schisandra, 5-8 parts platycodon, 4-11 parts puffball, 6-12 parts datura, 3-8 parts senecio scandens, 6-13 parts oleaster leaf, 5-13 parts coltsfoot flower, 3-12 parts aster, and 2-7 parts watercress.

[0006] However, there are currently no patent publications or literature reports on in-depth research on the use of coltsfoot flower homogeneous polysaccharide for the treatment of respiratory diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a homogeneous polysaccharide from coltsfoot flower, its preparation method, and its uses. This invention discloses a homogeneous polysaccharide from coltsfoot flower, its preparation method, and its uses. Whole-animal model experiments have confirmed its ideal effects in preventing and treating asthma, rhinitis, and relieving cough. Cellular experiments have demonstrated its certain repairing effect in preventing and treating chronic obstructive pulmonary injury. It can be further used to develop and prepare innovative traditional Chinese medicines for the prevention and treatment of respiratory diseases.

[0008] To achieve the above-mentioned objectives, the technical solution of this invention is as follows: In a first aspect, the present invention provides a homogeneous polysaccharide from coltsfoot flowers, the homogeneous polysaccharide from coltsfoot flowers comprising fructose and glucose; wherein the molar ratio of fructose to glucose is 20.00-26.00:1.00.

[0009] Preferably, the molecular weight of the coltsfoot flower homogeneous polysaccharide is selected from 2.0 × 10⁻⁶. 3 -4.5×10 3 Da.

[0010] Preferably, the molar ratio of fructose to glucose is 21.7:1.00; Preferably, the molecular weight of the coltsfoot flower homogeneous polysaccharide is selected from 2.0 × 10⁻⁶. 3 -2.8×10 3 Da; Preferably, the molar ratio of fructose to glucose is 25.9:1.00; Preferably, the molecular weight of the coltsfoot flower homogeneous polysaccharide is selected from 3.6 × 10⁻⁶. 3 -4.5×10 3 Da; Preferably, the coltsfoot flower homogeneous polysaccharide comprises terminal glucose, 1,2-linked fructose, 1,2,6-linked fructose, 2,6-linked fructose and terminal fructose.

[0011] Preferably, the homogeneous polysaccharide from coltsfoot flower has the following primary structure: .

[0012] Preferably, the total sugar content of the coltsfoot flower homogeneous polysaccharide is not less than 70%. Secondly, the present invention provides a method for preparing the above-mentioned homogeneous polysaccharide from coltsfoot flowers, comprising the following steps: coltsfoot flowers are defatted, extracted with water, precipitated with alcohol, eluted, and eluted twice to obtain homogeneous polysaccharide from coltsfoot flowers.

[0013] Preferably, the preparation method includes the following steps: Step 1: Mix coltsfoot flowers with a solvent, heat to defatting, and obtain defatted medicinal material; Step 2: Mix defatted medicinal materials with water, heat to extract, and combine the extracts; Step 3: After concentration, the extract is subjected to fractional alcohol precipitation to obtain crude homogeneous polysaccharide from Tussilago farfara. Step 4: The crude homogeneous polysaccharide from coltsfoot flower is eluted by an ion exchange column, and the eluent is dialyzed and dried to obtain purified homogeneous polysaccharide from coltsfoot flower. Step 5: The purified coltsfoot flower homogeneous polysaccharide was eluted by gel chromatography; the eluted fraction with the highest abundance was collected, dialyzed and dried to obtain the coltsfoot flower homogeneous polysaccharide.

[0014] Preferably, in step 1, the solvent is selected from at least one of ethanol, petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether, and methanol; More preferably, in step 1, the solvent is ethanol; More preferably, the ethanol has a mass fraction of 50%-95%; Preferably, in step 1, the heating is performed 1-3 times; more preferably, in step 1, the heating is performed 2 times.

[0015] Preferably, for the first heating, the ratio of coltsfoot flower to solvent is 1:9-11 g / mL; the heating time is 2-2.5 h; for the second heating, the ratio of coltsfoot flower to solvent is 1:7-8 g / mL; the heating time is 1-1.5 h.

[0016] More preferably, in the first heating, the ratio of coltsfoot flower to solvent is 1:10 g / mL; the heating time is 2 hours; in the second heating, the ratio of coltsfoot flower to solvent is 1:8 g / mL; the heating time is 1.5 hours.

[0017] Preferably, in step 2, the water is selected from at least one of ultrapure water, deionized water, and distilled water; more preferably, in step 2, the water is selected from ultrapure water.

[0018] Preferably, in step 2, the heating extraction is performed 1-3 times; more preferably, in step 2, the heating extraction is performed 2 times.

[0019] Preferably, in the first heating, the ratio of defatted medicinal material to water is 1:9-11 g / mL; the heating time is 2-2.5 h; in the second heating, the ratio of defatted medicinal material to water is 1:7-8 g / mL; the heating time is 1-1.5 h.

[0020] Preferably, in step 3, the concentration is to concentrate the raw medicinal material to a concentration of 0.5-1.5 mg / mL; more preferably, in step 3, the concentration is to concentrate the raw medicinal material to a concentration of 1 mg / mL.

[0021] Preferably, in step 3, the alcohol solution used for alcohol precipitation is an ethanol solution; More preferably, the ethanol solution has a mass fraction of 85%-95%; more preferably, the ethanol solution has a mass fraction of 95%.

[0022] Preferably, in step 3, the mass fraction of alcohol in the final solution is 30%. More preferably, in step 3, the final solution contains 80% alcohol by mass.

[0023] Preferably, in step 4, the ion exchange column is a weak anion exchange column; Preferably, in step 4, the weak anion exchange column is a DEAE-Fast Flow.

[0024] Preferably, in step 4, the elution solvent is water.

[0025] Preferably, in step 5, the gel chromatography column is a dextran gel chromatography column.

[0026] Preferably, in step 5, the elution solvent is water or a 0.1 mmol / L-0.25 mol / L aqueous solution of sodium chloride.

[0027] Preferably, in step 5, the specific elution operation is as follows: the purified coltsfoot flower homogeneous polysaccharide obtained in step 4 is dissolved in water, centrifuged, the supernatant is loaded onto a dextran gel chromatography column, eluted with 0.1 mmol / L-0.25 mol / L sodium chloride aqueous solution, detected by differential refractive index detector, the component with the highest abundance is collected, dialyzed and freeze-dried to obtain the coltsfoot flower homogeneous polysaccharide.

[0028] Preferably, in step 5, the dextran gel type is selected from G50, G25, Sephacryl S100 or Sephacryl S200.

[0029] Thirdly, the present invention provides a pharmaceutical composition comprising the above-described coltsfoot flower homogeneous polysaccharide.

[0030] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0031] Fourthly, the present invention provides the use of the above-described coltsfoot flower homogeneous polysaccharide in the preparation of medicaments for treating respiratory diseases.

[0032] Preferably, the respiratory diseases include asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows: The homogeneous polysaccharide from coltsfoot flower provided by this invention has good effects in preventing and treating asthma, rhinitis, cough and chronic obstructive pulmonary disease, providing a new option for the preparation of innovative traditional Chinese medicine for the prevention and treatment of respiratory diseases. Attached Figure Description

[0034] Figure 1 The molecular weight distribution spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0035] Figure 2 This is a monosaccharide composition test spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of the present invention.

[0036] Figure 3 The infrared spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0037] Figure 4 The hydrogen spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0038] Figure 5 The carbon spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0039] Figure 6 The DEPT135 carbon spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0040] Figure 7 HSQC spectrum of homogeneous polysaccharide from coltsfoot flower prepared in Example 1 of this invention. Figure 8 The HMBC pattern of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0041] Figure 9 The HHCOSY spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0042] Figure 10 This is a statistical chart showing the number of times the rhinitis model in Embodiment 1 of the present invention scratches the nose.

[0043] Figure 11 This is a statistical chart of IL-5 levels in an asthma model according to Embodiment 1 of the present invention.

[0044] Figure 12 This is a statistical chart showing the cough latency and number of coughs in the cough model of Embodiment 1 of the present invention.

[0045] Figure 13This is a diagram illustrating the effect of Example 1 of the present invention on an in vitro model of CSE-induced chronic obstructive pulmonary disease. Detailed Implementation

[0046] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the invention based on the disclosed content, which should also fall within the scope of protection claimed in this application.

[0047] In this invention, the terms "comprising" or "including," and similar terms, mean that the element preceding the term encompasses the element listed after it, and do not exclude the possibility of encompassing other elements. The terms "inner," "outer," "upper," "lower," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. When the absolute position of the described object changes, the relative positional relationship may also change accordingly. In this invention, unless otherwise explicitly specified and limited, the term "attached," etc., should be interpreted broadly. For example, it can refer to a fixed connection, a detachable connection, or an integral part; it can refer to a direct connection or an indirect connection through an intermediate medium; it can refer to the internal communication of two elements or the interaction relationship between two elements. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances. The term “about” as used in this invention has a meaning known to those skilled in the art, and preferably refers to the numerical value modified by the term within the range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, or ±1%.

[0048] In this invention, the term "pharmaceuticalally acceptable excipient" refers to all pharmaceutical materials, other than the active pharmaceutical ingredient, added to the formulation to address the formation properties, efficacy, stability, and safety of the drug product during manufacturing and formulation preparation. These substances have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as excipients, carriers, and improving stability, pharmaceutically acceptable excipients also possess important functions such as solubilization, co-solubilization, and sustained-release. They are crucial components that may affect the quality, safety, and efficacy of the drug. The pharmaceutically acceptable excipients described in this application can be suitable carriers or excipients, emulsifiers, wetting agents, preservatives, stabilizers, antioxidants, adjuvants (e.g., aluminum hydroxide adjuvants, oil adjuvants, Freund's complete adjuvants, and Freund's incomplete adjuvants), etc.

[0049] This invention provides a method for preparing homogeneous polysaccharides from coltsfoot flowers, the method comprising the following steps: Step 1: Mix coltsfoot flowers with a solvent, heat to defatting, and obtain defatted medicinal material; Step 2: Mix defatted medicinal materials with water, heat to extract, and combine the extracts; Step 3: After concentration, the extract is subjected to fractional alcohol precipitation to obtain crude homogeneous polysaccharide from Tussilago farfara. Step 4: The crude homogeneous polysaccharide from coltsfoot flower is eluted by an ion exchange column, and the eluent is dialyzed and dried to obtain purified homogeneous polysaccharide from coltsfoot flower. Step 5: The purified coltsfoot flower homogeneous polysaccharide was eluted by gel chromatography; the eluted fraction with the highest abundance was collected, dialyzed and dried to obtain the coltsfoot flower homogeneous polysaccharide.

[0050] In this invention, the concentration used in the preparation method can be achieved by any method known in the art. There are no particular limitations on this concentration, provided that the active ingredient is not destroyed. A common concentration method is evaporation, such as atmospheric pressure evaporation, reduced pressure evaporation, thin-film evaporation, etc., but it is not limited thereto.

[0051] In this invention, the drying process involved can be any one of atmospheric pressure drying, vacuum drying, spray drying, freeze drying, etc., and is not limited thereto.

[0052] The pharmaceutical compositions involved in this invention can be used in various dosage forms, often depending on the route of administration. The pharmaceutical compositions involved in this invention can be administered via multiple routes, such as oral, parenteral, etc. The dosing regimen and dosage depend on various factors, such as the route of administration, the patient's health condition, etc., and can be determined by a physician. Dosage ranges can be determined by those skilled in the art through routine experiments.

[0053] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention were obtained through conventional commercial means. Unless otherwise specified, all contents mentioned below are mass contents. Unless otherwise specified, it is understood that the process was carried out at room temperature.

[0054] Example 1: Coltsfoot flower medicinal material was taken and heated under reflux with 90wt% ethanol for defatting twice, with material-to-liquid ratios of 1:10 g / mL and 1:8 g / mL, and reflux times of 2 h and 1.5 h, respectively. After defatting, the residue was dried and extracted twice with water under heating, with material-to-liquid ratios of 1:10 g / mL and 1:8 g / mL, and reflux times of 2 h and 1.5 h, respectively. The extracts were combined and concentrated to a crude drug concentration of 1 mg / mL. Ethanol was slowly added until the final ethanol concentration of the solution was 30wt%. The mixture was allowed to stand overnight to obtain a precipitate and a supernatant. The precipitate was dried to obtain crude coltsfoot flower polysaccharide. After the crude coltsfoot flower polysaccharide was dissolved in water, it was eluted through a weak anion exchange column. The eluent was dialyzed and dried to obtain purified homogeneous coltsfoot flower polysaccharide. Then, it was dissolved in water and loaded onto a G50 dextran gel column and eluted with aqueous solution. The eluted fraction with the highest abundance was collected, dialyzed, and dried.

[0055] Example 2: Take the Coptis chinensis flower material, add 90wt% ethanol and heat under reflux for defatting twice, with material-to-liquid ratios of 1:10 g / mL and 1:8 g / mL, and reflux times of 2 h and 1.5 h, respectively. After defatting, dry the filter residue and extract it twice with water under heating, with material-to-liquid ratios of 1:10 g / mL and 1:8 g / mL, and reflux times of 2 h and 1.5 h, respectively. The extracts were combined and concentrated to a crude drug concentration of 1 mg / mL. Ethanol was slowly added until the final concentration of the solution was 30 wt%. The mixture was allowed to stand overnight to obtain a precipitate and a supernatant. Ethanol was slowly added to the supernatant until the final ethanol concentration of the solution was 80 wt%. The mixture was allowed to stand overnight to obtain a precipitate and a supernatant. The precipitate was dried to obtain crude coltsfoot flower polysaccharide. After the crude coltsfoot flower polysaccharide was dissolved in water, it was eluted through a weak anion exchange column. The eluted phase was dialyzed and dried to obtain purified homogeneous coltsfoot flower polysaccharide. The polysaccharide was then dissolved in water and loaded onto a G25 dextran gel column and eluted with aqueous solution. The eluted fraction with the highest abundance was collected, dialyzed, and dried.

[0056] Example 3: Take the Coptis chinensis flower material, add 90wt% ethanol and heat under reflux for defatting twice, with material-to-liquid ratios of 1:10 g / mL and 1:8 g / mL, and reflux times of 2 h and 1.5 h, respectively. After defatting, dry the filter residue and extract it twice with water under heating, with material-to-liquid ratios of 1:10 g / mL and 1:8 g / mL, and reflux times of 2 h and 1.5 h, respectively. The extracts were combined and concentrated to a crude drug concentration of 1 mg / mL. Ethanol was slowly added until the final ethanol concentration of the solution was 30 wt%. The mixture was allowed to stand overnight to obtain a precipitate and a supernatant. Ethanol was slowly added to the supernatant until the final concentration of the solution was 80 wt%. The mixture was allowed to stand overnight to obtain a precipitate and a supernatant. The precipitate was dried to obtain crude coltsfoot flower polysaccharide. After the crude coltsfoot flower polysaccharide was dissolved in water, it was eluted through a weak anion exchange column. The eluted phase was dialyzed and dried to obtain purified homogeneous coltsfoot flower polysaccharide. The purified polysaccharide was then dissolved in water and loaded onto a Sephacryl-100 gel column and eluted with aqueous solution. The eluted fraction with the highest abundance was collected, dialyzed, and dried.

[0057] Examples 1, 2, and 3 all mainly contain the same homogeneous polysaccharide, which can be separated and purified using gel packing.

[0058] Test Example 1: Chemical Structural Characteristics of Homogeneous Polysaccharides from Coltsfoot Flower Determination of molecular weight in Examples 1-3 1. Laboratory supplies: The products obtained in Examples 1-3, sodium chloride, etc.

[0059] 2. Experimental Methods: The molecular weights of the products obtained in Examples 1-3 were determined using HPSEC-MALLS-RID coupled technology. The instruments used included: DynaPro NanoStar dynamic laser light scattering system: Wyatt DynaPro NanoStar (USA); High-performance liquid chromatograph: Shimadzu LC-10A; Differential detector: Shimadzu RI-10A; Column: BRT105-103-101; Tandem gel column: BRT103 gel column (8 × 300 mm); Mobile phase: 0.05M sodium chloride solution; Flow rate: 0.5 mL / min; Column temperature: 40℃; Injection volume: 25 μL; Detector: Waters 2414 differential detector, MALS detector. The wavelength of the MALS laser was 658.1 nm, and the specific refractive index increment dn / dc value was 0.1380 mg / L. The light scattering model was the Zimm model.

[0060] Centrifuge: Eppendorf 5424. Prepare 0.05M sodium chloride solution, filter through a 0.45μm membrane, and sonicate for 10 min. Accurately weigh the sample, prepare a 10 mg / mL solution with the mobile phase, centrifuge at 12000 rpm for 10 min, and filter the supernatant through a 0.22μm microporous membrane for later use. Flow rate: 0.7 mL / min; column temperature: 40℃; injection volume: 50 μL; detector: Waters 2414 differential detector, MALLS detector. The wavelength of the MALLS laser was 661.0 nm, and the specific refractive index increment dn / dc value was 0.1380 mg / L; the light scattering model was the Zimm model.

[0061] 3. Experimental Results: The overlay plot of differential detection (dRI) and multi-angle laser light scattering (LS) (dRI+LS) calculates the molecular weight distribution range, weight-average molecular weight (Mw, Da), and polydispersity index (Mw / Mn) for each component of the sample. The data are shown in Table 1 (multi-angle laser light scattering (LS)) and Table 2 (differential detection (dRI)). The molecular weight distribution spectrum is shown in... Figure 1 As shown: Table 1

[0062] Table 2

[0063] Determination of monosaccharide composition in Examples 1-2 1. Laboratory supplies: The solid samples obtained in Examples 1-2 contained trifluoroacetic acid (ACROS), 50% sodium hydroxide solution (Alfa Aesar), and sodium acetate (ThermoFishe). Equipment included an ion chromatograph (ThermoFishe, ICS5000), an electrically heated constant-temperature drying oven (Lichen Technology, 101-1BS), a nitrogen evaporator (Lichen Technology, UGC-24M), an electronic balance (Sartorius BS, 210 S), a centrifuge (ThermoFishe, D-37520), and a pipette (DRAGONLAB, 19050983). Mannose (C17D9H77586), rhamnose (H10S9Z69863), galacturonic acid (K02A9B66077), galactose (E1927035), glucose (Q18F10N80946), glucuronic acid ((K14M10S82777), arabinose (S15A10G85850)), xylose (A22S6X3606), fucose (X29D7Y27768)), hydrochloric acid Glucosamine (A22S6X3606), N-acetyl-D-glucosamine (A21J8X40372), D-fructose (J01J10R89818), D-ribose (H26F10Z81556), galactosamine hydrochloride (B01J8S37079), L-guluronic acid (S200115AG1), D-mannuronic acid (S200108AM1); all monosaccharide standards were obtained from Borui Sugar Biotechnology.

[0064] 2. Experimental Methods: Take appropriate amounts of 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid) and add 2 mL of 3M TFA. Hydrolyze at 80℃ for 2 h, blow dry under nitrogen, add deionized water and vortex mix to prepare standard stock solutions.

[0065] Precisely prepare concentration standards from the standard solutions of each monosaccharide to form a mixed standard. Determine the mass of different monosaccharides using an absolute quantification method, and calculate the molar ratio based on the molar mass of the monosaccharides.

[0066] Column: Dionex Carbopac™ PA20 (3*150 mm); Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaAc; Flow rate: 0.3 mL / min; Injection volume: 25µL; Column temperature: 30℃; Elution gradient: 0min A phase / B phase / C phase (98.8:1.2:0, V / V); 18min A phase / B phase / C phase (98.8:1.2:0, V / V); 20min A phase / B phase / C phase (50:50:0, V / V); 30min A phase / B phase / C phase (50:50:0, V / V); 30.1 min A phase / B phase / C phase (0:0:100, V / V); 46min A phase / B phase / C phase (0:0:100, V / V); 46.1 min A phase / B phase / C phase (0:100:0, V / V); 50 min A phase / B phase / C phase (0:100:0, V / V); 50.1 min A phase / B phase / C phase (98.8:1.2:0, V / V); 80 min A phase / B phase / C phase (98.8:1.2:0, V / V).

[0067] Detector: Electrochemical detector.

[0068] 3. Experimental Results: The experimental results are shown in Tables 3-4, and the relevant spectra are shown in [the table]. Figure 2 Table 3 represents the samples of Example 1, and Table 4 represents the samples of Example 2.

[0069] Table 3

[0070] Table 4

[0071] Determination of total sugar content in Example 1: Instruments and reagents 1. Instruments: Vortex mixer, pipette, microplate reader, water bath, graduated cylinder, electronic balance 2. Reagents: D-anhydrous glucose (110833-202109, China National Institutes for Food and Drug Control), phenol (Xilong Chemical, Q / STXH229-2009), concentrated sulfuric acid (Chengdu Kelong Chemical, GB / T625-2007, 2024041602), and pure water.

[0072] Preparation of the reference solution: Accurately weigh 10.79 mg of D-anhydrous glucose reference standard, dissolve it in pure water to prepare 10 mg / mL, take 200 μL and dilute it with pure water to 2 mL to obtain 1 mg / mL stock solution, shake well and use it for later use.

[0073] Sample preparation: Accurately weigh 5.53 mg of homogeneous polysaccharide, dissolve it in pure water to prepare 10 mg / mL solution, take 100 μL and dilute it with pure water to 1 mL to obtain 1 mg / mL stock solution, shake well and use for later use.

[0074] Content determination: To construct a standard curve: Accurately measure 0.05 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, and 0.5 ml of 1 mg / mL reference stock solution into stoppered test tubes, and add water to make up to 1 mL. Accurately add 1 mL of 6% phenol (2.99 g / 50 mL) solution and 5 mL of concentrated sulfuric acid to each tube, shake well, and place in a boiling water bath (100℃) for 25 minutes. After removal, immediately cool to room temperature in an ice-water bath. At the same time, set up the corresponding solution as a blank. Transfer 200 μL of the standard working solution after each reaction and measure the absorbance at a wavelength of 486 nm. Perform three parallel measurements and plot the standard curve with absorbance as the ordinate and concentration as the abscissa.

[0075] Assay: Accurately measure 250 μL of 1 mg / mL sample stock solution into a stoppered test tube, add water to make up to 1 mL, and accurately add 1 mL of 6% phenol (2.99 g / 50 mL) solution and 5 mL of concentrated sulfuric acid to each tube. Shake well, place in a boiling water bath (100 °C) for 25 minutes, remove and immediately cool to room temperature in an ice water bath. Transfer 200 μL of the working sample solution after each reaction and measure the absorbance at a wavelength of 486 nm. Perform three parallel measurements.

[0076] Table 5. Correspondence between glucose content (μg / mL) and absorbance A

[0077] Table 6. Absorbance of homogeneous polysaccharide phenol by concentrated sulfuric acid method

[0078] Infrared spectral analysis of Example 1: Take 5 mg of sample and place it in a dryer containing phosphorus pentoxide for vacuum drying overnight. Press it into a KBr pellet and perform infrared spectroscopy analysis. Figure 3 To obtain the infrared spectrum of the homogeneous polysaccharide F1 obtained from coltsfoot flower, the absorption band is located at 3600-3200 cm⁻¹. -1 This is the absorption peak of the stretching vibration of -OH, and the absorption peak in this region is a characteristic peak of carbohydrates. Specifically: 3359 cm⁻¹ -1 The peak at 2931 cm⁻¹ is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates.-1 2888cm -1 The peak at 1643 cm⁻¹ is the absorption peak of the stretching vibration of CH, a characteristic peak of carbohydrates. -1 An absorption peak is observed at 1423 cm⁻¹, which may be attributed to the C=O stretching vibration. -1 Location, 1128cm -1 An absorption peak is observed at 1384 cm⁻¹, which may be attributed to the CO stretching vibration. -1 1334cm -1 An absorption peak is observed at 1029 cm⁻¹, which may be attributed to the C=O symmetric stretching vibration. -1 An absorption peak is observed at 989 cm⁻¹, which may be attributed to the OH-angle vibration. The peak is also observed at 989 cm⁻¹. -1 An absorption peak is observed at 935 cm⁻¹, which may be attributed to the transverse vibration of the terminal methine. -1 An absorption peak is observed at 873 cm⁻¹, which may be attributed to the asymmetric stretching vibration of the pyran ring. -1 The absorption peak at 817 cm⁻¹ may be attributed to the CH-angle vibration of the equatorial bond, which is different from the terminal CH of the pyran ring. -1 The presence of an absorption peak at this point may be attributed to the stretching vibration of the pyran ring symmetry.

[0079] NMR spectroscopy analysis of Example 1: Weigh 50 mg of homogeneous polysaccharide sample from Tussilago farfara flower, dissolve it in 0.5 mL of heavy water, and freeze-dry. Then, dissolve the freeze-dried powder again in 0.5 mL of heavy water and continue freeze-drying, repeating the above process to ensure sufficient exchange of active hydrogens. Next, dissolve the sample in 0.5 mL of heavy water and measure its 1H NMR, 13C NMR, DEPT135, and two-dimensional spectra at 600 MHz at room temperature (25 °C).

[0080] Figure 4 and Figure 5 To obtain the F1 1H and 1C spectra of the homogeneous polysaccharide from coltsfoot flower, its anomeric proton resonance region was first analyzed, such as... Figure 4 1 H-NMR, F1 1 The 1H-NMR spectrum showed a weak signal peak at δ 5.35 ppm. Combined with the methylation analysis results of F1, it can be concluded that the δ 5.35 ppm peak belongs to the anomer of α-D-Glcp-1→. Stronger signal peaks were observed in the δ 3.5-4.2 ppm range. 13 C-NMR Figure 5As shown, there are two signal peaks in the anodic carbon signal region: δ104.61, 105.06, 104.45, 94.82, and δ93.78. Therefore, the signal peaks at δ93.78 and 94.82 belong to the major anodic carbon signals of F1. According to the methylation analysis results of F1, the signal peaks at δ104.61, 105.06, and 104.45 should belong to fructose residues, and the signal peaks at δ93.78 and 94.82 should belong to glucose residues. Furthermore, the anodic signal of F1 shows a significant downward shift, indicating that F1 contains β-glycosidic bonds.

[0081] DEPT135 spectrum ( Figure 6 Analysis showed that δ 62.15, 63.35, 63.42, 63.56, 64.82, 62.15, 62.12, and 61.17 were inverted peaks, indicating that they may be methylene groups, which are C1 and C6 of fructose and C6 of glucose.

[0082] Through HSQC map ( Figure 7 The anomeric carbon signal was observed to be δ93.86, and the corresponding anomeric hydrogen signal in the HSQC spectrum was δ5.35. This was confirmed by HH-COSY (…). Figure 9 The signals for H1-2 are 5.35 / 3.46; for H2-3, 3.46 / 3.69; for H3-4, 3.69 / 3.39; for H4-5, 3.39 / 3.76; and for H5-6a, 3.76 / 3.63. We can deduce that the δ values ​​for H1, H2, H3, H4, H5, and H6a are 5.35, 3.46, 3.69, 3.39, 3.76, and 3.63, respectively. The corresponding C1-C6 values ​​are δ 93.78, 72.51, 73.74, 70.46, 75.92, and 62.15. Therefore, this signal should be attributed to the glycosidic bond α-D-Glcp-(1→).

[0083] Through HSQC map ( Figure 7The anomeric carbon signal is observed to be δ 104.61, with a high integral ratio and no corresponding proton. Combined with monosaccharide composition analysis, this polysaccharide contains a large amount of fructose, so this signal peak can be inferred to be C2. H3 is found through HMBC, and combined with HH-COSY, the signals of H3-4 are 4.17 / 4.01; the signal of H4-5 is 4.01 / 3.78; and the signal of H5-6a is 3.78 / 3.68. We can infer that H3, H4, H5, and H6a are δ 4.17, 4.01, 3.78, and 3.68, respectively. The corresponding C3-C6 are δ 79.37, 75.56, 82.36, and 63.35. With the help of DEPT135 spectrum, the C1 signal is obtained as δ 62.15, and the corresponding H1a and b are δ 3.63 and 3.84, respectively. Through the HMBC two-dimensional spectrum of the multi-bond carbon-hydrogen relationship of F1 ( Figure 8 Analysis determined the sequence of monosaccharides in the sugar chain and verified the accuracy of the previous assignment of the chemical shifts of the major F1 residues by 1H NMR and 13C NMR based on HSQC and 1H-1H COSY two-dimensional spectra.

[0084] One characteristic of inulin-type fructans is the presence of glucose residues at their terminals. In the HMBC spectrum, the anomeric hydrogen of α-D-Glcp-1→ has a correlation peak with the C1 of →1-β-D-Fruf-2→, indicating the presence of α-D-Glcp-1→1-β-D-Fruf-2→.

[0085] In the HMBC spectrum, there is a strong cross-peak C2 (→1-β-D-Fruf-2→) – H1 (→1-β-D-Fruf-2→) with its own H1 resonance region of the glycosidic bond →1-β-D-Fruf-2→, indicating the presence of →2-β-D-Fruf-1→2-β-D-Fruf-1→.

[0086] In the HMBC spectrum, there is a strong cross-peak C2 (→1-β-D-Fruf-2→) – H1 (→1,6-β-D-Fruf-2→) in the resonance region of the glycosidic bond →1-β-D-Fruf-2→ and →1,6-β-D-Fruf-2→, indicating the presence of →2-β-D-Fruf-1→1,6-β-D-Fruf-2→.

[0087] A strong cross-peak exists between the C2 resonance region of the glycosidic bond →1,6-β-D-Fruf-2→ and the H1 resonance region of →1,6-β-D-Fruf-2→, namely C2 (→1,6-β-D-Fruf-2→) – H1 (→1,6-β-D-Fruf-2→), indicating the presence of →1,6-β-D-Fruf-2→1,6-β-D-Fruf-2→. Similarly, a strong cross-peak exists between the C2 resonance region of the β-D-Fruf-2→ glycosidic bond and the H6 resonance region of →1,6-β-D-Fruf-2→, namely C2 (β-D-Fruf-2→) – H6 (→1,6-β-D-Fruf-2→), indicating the presence of β-D-Fruf-2→1,6-β-D-Fruf-2→.

[0088] A strong cross-peak exists in the resonance region between C2 of the glycosidic bond →1,6-β-D-Fruf-2→ and H1 of →1-β-D-Fruf, namely C2 (β-D-Fruf-2→) – H1 (→1-β-D-Fruf), indicating the presence of →1,6-β-D-Fruf-2→1-β-D-Fruf.

[0089] Test Example 2. Rhinitis Drug Efficacy Experiment 1. Reagents and Instruments 1.1 Reagents Grade II ovalbumin (A5503-1G, Merk), Grade V ovalbumin (A5253-250G, Merk), dexamethasone (Xianju Pharmaceutical), aluminum hydroxide (Xilong Scientific), sodium carboxymethyl cellulose (Daomao Chemical), sodium chloride (Daomao Chemical). Example 2: Homogeneous polysaccharide prepared by the Natural Product Chemistry Group of the China Resources Jiangzhong Modern Traditional Chinese Medicine Research Center.

[0090] 1.2 Instruments A 0.01 g balance (MSA2245CE, Sartorius) and a 10 μL pipette (Eppendorf).

[0091] 1.3 Experimental Animals Balb / c mice, male, 6-8 weeks old.

[0092] 2 Experimental Methods Balb / c mice were used in the experiment, which was divided into three groups: a model group, a positive control group (dexamethasone 2 mg / kg), and Example 1 group (350 mg / kg). The test drug was prepared to the required concentration using 5‰ sodium carboxymethyl cellulose. All mice were sensitized three times (0.5 mg / mL of Grade V OVA and 1.6 mg / mL Al(OH)3 suspension, 0.2 mL / mouse, i.p.), on days 0, 7, and 14. After sensitization, the mice were administered the drug starting on day 20. One hour after administration, a challenge was performed by intranasal instillation (5% Grade II OVA solution), with 20 μL (10 μL / nostril) instilled into each mouse once daily for 5 days.

[0093] After the last nasal drop was administered, wait 1 minute and then record the number of times the mouse scratched its nose within 5 minutes.

[0094] 3 Experimental Results like Figure 10 As shown, after modeling, the mice in the model group exhibited obvious nose scratching and sneezing, indicating successful modeling. Compared with the model group, intervention with the homogeneous polysaccharide (350 mg / Kg) prepared in Example 1 significantly reduced the number of nose scratchings in the mice, indicating that the homogeneous polysaccharide provided by this invention has a significant therapeutic effect on rhinitis.

[0095] Test Example 3: Efficacy Experiment of Drugs for Treating Asthma 1. Reagents and instruments, etc. Reagents: Grade II ovalbumin (A5503-1G, Merk), Grade V ovalbumin (A5253-250G, Merk), dexamethasone (Xianju Pharmaceutical), aluminum hydroxide (Xilong Scientific), sodium carboxymethyl cellulose (Daomao Chemical), sodium chloride (Daomao Chemical), MouseIL-5 Uncoated ELISA Kit (88-7054-88, Invitrogen), etc.

[0096] instrument: 0.01% balance (MSA2245CE, Sartorius), ultrasonic nebulizer (402AI, Yuwell), animal blood cell analyzer (BC-5000VEI, Mindray), benchtop refrigerated centrifuge (ST1R Plus, Thermoscientific), multi-functional microplate reader (VICTOR NIVO, PerkinElmer), surgical instruments.

[0097] Laboratory animals: Balb / c mice, male, 6-8 weeks old.

[0098] 2. Experimental Methods: Balb / c mice were used in the experiment and divided into four groups: a normal group, a model group, and a group receiving dexamethasone 1 mg / kg and Example 1 group receiving 350 mg / kg. Mice in the model group were sensitized with OVA for three weeks, once a week; nebulized challenge was administered once daily for one week, and the modeling process lasted for four weeks. Drug administration was synchronized with nebulized challenge, with the drug administered one hour before nebulized challenge. Mice other than the normal group were sensitized three times (0.5 mg / mL of Grade V OVA and 1.6 mg / mL Al(OH)3 suspension, 0.2 mL / mouse), on days 0, 7, and 14. After sensitization, mice began receiving the drug on day 20, followed by one hour of nebulized challenge (100 mL of 2% Grade II OVA solution) one hour after drug administration, once daily for seven days.

[0099] The day after the last nebulization, the mice were anesthetized and euthanized, and the bronchoalveolar lavage fluid was collected to determine the IL-5 level.

[0100] 3. Experimental Results: like Figure 11 As shown, compared with the model group, the administration of the homogeneous polysaccharide (350 mg / kg) prepared in Example 1 to the asthmatic mouse model significantly reduced the level of the inflammatory factor IL-5 in the bronchoalveolar lavage fluid (P<0.05), indicating that the homogeneous polysaccharide provided by the present invention has the effect of relieving asthma.

[0101] Test Example 4. Antitussive Efficacy Experiment 1. Reagents and instruments, etc. Reagents: Pentoxyverine citrate (Lisheng Pharmaceutical), concentrated ammonia (A112079, Aladdin), etc.

[0102] instrument: 0.01% balance (MSA2245CE, Sartorius), multi-functional cough and asthma induction device (model: YLS-8A).

[0103] Laboratory animals: SD rats, male, 6-8 weeks old.

[0104] 2. Experimental Methods: Healthy SD rats were divided into a model group, a positive control group (pentoxyverine citrate 60 mg / kg), and an Example 1 group (400 mg / kg). The sample from Example 1 and pentoxyverine citrate were diluted with 5‰ CMC-Na to the corresponding concentrations and administered by gavage, with preventative administration for 3 days prior. The rats were fasted for 24 hours before the last administration, but allowed free access to water. One hour after the last administration, the SD rats were placed in a multifunctional cough-inducing and asthma-inducing device, where 15% concentrated ammonia was continuously nebulized for 25 seconds. The number of coughs and the cough latency were recorded within 4 minutes. Cough symptoms included significant abdominal contraction or distension and wide-open mouth.

[0105] 3. Experimental Results: Experimental results are as follows Figure 12 As shown, compared with the model group, the administration of the homogeneous polysaccharide (400 mg / kg) prepared in Example 1 significantly reduced the number of coughs and prolonged the cough latency in rats, indicating that the homogeneous polysaccharide provided by the present invention has the effect of relieving cough.

[0106] Test Example 5. Chronic Obstructive Pulmonary Disease (COPD) Efficacy Experiment 1. Reagents and Instruments 1.1 Reagents BEAS-2B human bronchial epithelial cell culture medium (Kunming Institute of Physics, Chinese Academy of Sciences), DMEM medium (C11995500BT, Gibco), FBS (10099-141, Gibco), CCK8 (RM02823, Abclonal), and cigarettes (Seven Wolves). F1 corresponds to the homogeneous polysaccharide in the sample of Example 1, prepared by the Natural Product Chemistry Group of the China Resources Jiangzhong Modern Chinese Medicine Research Center.

[0107] 1.2 Instruments Biosafety cabinet (HFsafe1200LC, Shanghai Lishen Scientific Instruments Co., Ltd.), CO2 incubator (D180, Shenzhen Ruiwode Life Technology Co., Ltd.), inverted biological microscope (ECLIPSE Ts2, Nikon), hemocytometer (MF3543, Shanghai Qiujing), multi-functional microplate reader (VICTOR, NIVO), etc.

[0108] 1.3 Experimental Cells BEAS-2B human bronchial epithelial cells 2 Experimental Methods 2.1 Preparation of Cigarette Extract After lighting a cigarette, place it on the inhalation device and allow the smoke to pass through serum-free DMEM culture medium. Each cigarette should burn for 1-2 minutes. Once the smoke has passed through the medium, stop inhaling, adjust the pH to 7.4, and remove impurities and bacteria using a 0.22 μm filter to obtain the cigarette extract (CSE). Measure the absorbance (OD value) at 320 nm to establish an experimental curve, ensuring that the OD values ​​of each independent experiment are similar. Finally, dilute the CSE to the required concentration as needed for the experiment.

[0109] 2.2 Cell treatment methods BEAS-2B cells were cultured to the logarithmic growth phase, and then cultured at a density of 5 × 10⁶ cells per well. 3Cells were seeded into 96-well plates. After cell adhesion, cigarette extract (CSE) was prepared. CSE modeling was performed according to experimental groups. After drug intervention for 24 h and 48 h, CCK8 working solution was added and incubated for 2-4 h at the experimental endpoint, and the cell absorbance was measured.

[0110] Calculate cell viability based on absorbance values ​​of each cell group: Cell viability (%) = (OD value of experimental group - OD value of blank culture medium) / (OD value of control group - OD value of blank culture medium) × 100%.

[0111] 3 Experimental Results like Figure 13 As shown, cell viability was significantly reduced under CSE (0.5%) conditions. At 24h and 48h after administration, the group in Example 1 (100 μg / mL) showed a significant repair effect on CSE-induced cell damage (P<0.05).

[0112] The above results indicate that the coltsfoot flower homogeneous polysaccharide provided by this invention has a certain therapeutic effect on chronic obstructive pulmonary disease.

[0113] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A uniform polysaccharide of Coltsfoot flower, characterized in that, The homogeneous polysaccharide from the coltsfoot flower contains fructose and glucose; wherein the molar ratio of fructose to glucose is 20-26:1.

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2. The uniform polysaccharide of Coltsfoot according to claim 1, characterized in that, The molecular weight of the uniform polysaccharide of the Flos Farfarae is selected from the group consisting of 2.0 x 10 3 -4.5 x 10 3 Da.

3. The uniform polysaccharide of Coltsfoot according to claim 1, characterized in that, The molar ratio of fructose to glucose is 21.7:1, the molecular weight of the uniform polysaccharide of Flos Farfugii is selected from 2.0 x 10 3 -2.8 x 10 3 Da; or the molar ratio of said fructose, glucose is 25.9:1, the molecular weight of the uniform polysaccharide of said Fructus Chrysanthemi Indici is selected from 3.6 x 10 3 -4.5 x 10 3 Da.

4. The uniform polysaccharide of Coltsfoot according to claim 1, characterized in that, The homogeneous polysaccharide from Coltsfoot flower comprises terminal glucose, 1,2-linked fructose, 1,2,6-linked fructose, 2,6-linked fructose, and terminal fructose.

5. The uniform polysaccharide of Coltsfoot according to claim 1, characterized in that, The homogeneous polysaccharide from coltsfoot flower has the following primary structure: 。 6. The coltsfoot flower homogeneous polysaccharide according to claim 1, characterized in that, The total sugar content of the homogeneous polysaccharide from the coltsfoot flower is not less than 70%.

7. The method for preparing homogeneous polysaccharide from coltsfoot flower according to any one of claims 1-6, characterized in that, The process includes the following steps: Coltsfoot flower is defatted, extracted with water, precipitated with alcohol, eluted, and eluted twice to obtain a homogeneous coltsfoot flower polysaccharide.

8. The preparation method according to claim 7, characterized in that, Includes the following steps: Step 1: Mix coltsfoot flowers with a solvent, heat to defatting, and obtain defatted medicinal material; Step 2: Mix defatted medicinal materials with water, heat to extract, and combine the extracts; Step 3: After concentration, the extract is subjected to fractional alcohol precipitation to obtain crude homogeneous polysaccharide from Tussilago farfara. Step 4: The crude homogeneous polysaccharide from coltsfoot flower is eluted by an ion exchange column, and the eluent is dialyzed and dried to obtain purified homogeneous polysaccharide from coltsfoot flower. Step 5: The purified coltsfoot flower homogeneous polysaccharide was eluted by gel chromatography; the eluted fraction with the highest abundance was collected, dialyzed and dried to obtain the coltsfoot flower homogeneous polysaccharide.

9. The preparation method according to claim 8, characterized in that, In step 1, the solvent is selected from at least one of ethanol, petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether, and methanol; The solvent is ethanol; the mass fraction of the ethanol is 50%-95%. The heating process is repeated 1-3 times.

10. The preparation method according to claim 9, characterized in that, The heating extraction was performed twice. For the first heating, the ratio of coltsfoot flower to solvent is 1:9-11 g / mL; the heating time is 2-2.5 h. For the second heating, the ratio of coltsfoot flower to solvent is 1:7-8 g / mL; the heating time is 1-1.5 h.

11. The preparation method according to claim 8, characterized in that, In step 2, the heating extraction is performed 1-3 times.

12. The preparation method according to claim 11, characterized in that, The heating extraction was performed twice. For the first heating, the ratio of defatted medicinal materials to water is 1:9-11 g / mL; the heating time is 2-2.5 h. For the second heating, the ratio of defatted medicinal materials to water is 1:7-8 g / mL; the heating time is 1-1.5 h.

13. The preparation method according to claim 8, characterized in that, In step 3, the concentration refers to concentrating the raw medicinal material to a concentration of 0.5-1.5 mg / mL; The alcohol solution used for alcohol precipitation is an ethanol solution; the final ethanol solution after alcohol precipitation has a mass fraction of 30%-95%.

14. The preparation method according to claim 8, characterized in that, In step 4, the ion exchange column is a DEAE-Fast Flow; the elution solvent is water.

15. The preparation method according to claim 8, characterized in that, In step 5, The gel chromatography column is a dextran gel chromatography column; The elution solvent is water or an aqueous solution of sodium chloride; The dextran gel model is selected from G50, G25, Sephacryl S100 or Sephacryl S200.

16. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the coltsfoot flower homogeneous polysaccharide according to any one of claims 1-6 or the coltsfoot flower homogeneous polysaccharide prepared by any one of the preparation methods of claims 7-15.

17. Use of the coltsfoot flower homogeneous polysaccharide according to any one of claims 1-6, the coltsfoot flower homogeneous polysaccharide prepared by any one of the preparation methods of claims 7-15, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for treating respiratory diseases.

18. The use according to claim 17, characterized in that, The respiratory diseases mentioned include asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

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