Homogeneous polysaccharide from tussilago farfara, and preparation method therefor and use thereof

Abstract

A homogeneous polysaccharide from Tussilago farfara, and a preparation method therefor and a use thereof, relating to the technical field of medicine. The homogeneous polysaccharide from Tussilago farfara has a molecular weight ranging from 2.0×103 Da to 4.5×103 Da, and comprises fructose and glucose, wherein the molar ratio of the fructose to the glucose is 20.00-26.00:1.00. The homogeneous polysaccharide from Tussilago farfara has good effects in preventing and treating asthma, rhinitis, cough and chronic obstructive pulmonary diseases.

Description

A homogeneous polysaccharide from coltsfoot flower, its preparation method and uses 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, the dried flower bud of *Tussilago farfara* L., belongs to the genus *Tussilago* in the family Compositae and is a commonly used traditional Chinese medicine. Coltsfoot flower 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 types of epilepsy, 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 for this component recorded in the pharmacopoeia is coltsfoot ketone.

[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] 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.

[0006] CN106334030A discloses a traditional Chinese medicine composition containing coltsfoot flower that can fundamentally and effectively relieve lower respiratory tract symptoms from the inside out. The composition includes 5-9 parts of honeysuckle, 2-9 parts of schisandra, 5-8 parts of platycodon, 4-11 parts of puffball, 6-12 parts of datura, 3-8 parts of senecio scandens, 6-13 parts of oleaster leaf, 5-13 parts of coltsfoot flower, 3-12 parts of aster, and 2-7 parts of watercress.

[0007] However, there are currently no literature reports on in-depth research on the use of coltsfoot flower homogeneous polysaccharide for the treatment of respiratory diseases. Summary of the Invention

[0008] One object of the present invention is to provide a homogeneous polysaccharide from coltsfoot flower.

[0009] Another object of the present invention is to provide a method for preparing homogeneous polysaccharides from coltsfoot flowers.

[0010] Another object of the present invention is to provide a use for a homogeneous polysaccharide from coltsfoot flower.

[0011] This invention provides a homogeneous polysaccharide from coltsfoot flower, its preparation method, and its uses. Whole animal model experiments have confirmed that it has ideal effects in preventing and treating asthma, rhinitis, and cough. Cell experiments have also shown that it has a certain repairing effect in preventing and treating chronic obstructive pulmonary disease. It can be further used to develop and prepare innovative traditional Chinese medicines for the prevention and treatment of respiratory diseases.

[0012] In a first aspect, the present invention provides a homogeneous polysaccharide from coltsfoot flowers, wherein the structural units of the homogeneous polysaccharide from coltsfoot flowers include fructose and glucose; wherein the molar ratio of fructose to glucose is 20.00-26.00:1.00.

[0013] Preferably, the molecular weight of the coltsfoot flower homogeneous polysaccharide is selected from 2.0 × 10⁻⁶. 3 -4.5×10 3 Da.

[0014] Preferably, the molar ratio of fructose to glucose is 21.7:1.00;

[0015] Preferably, the molecular weight of the coltsfoot flower homogeneous polysaccharide is selected from 2.0 × 10⁻⁶. 3 -2.8×10 3 Da;

[0016] Preferably, the molar ratio of fructose to glucose is 25.9:1.00;

[0017] Preferably, the molecular weight of the coltsfoot flower homogeneous polysaccharide is selected from 3.6 × 10⁻⁶. 3 -4.5×10 3 Da;

[0018] Preferably, the structural units of the coltsfoot flower homogeneous polysaccharide include terminal glucose, 1,2-linked fructose, 1,2,6-linked fructose, 2,6-linked fructose and terminal fructose.

[0019] Preferably, the homogeneous polysaccharide from coltsfoot flower has the following primary structure:

[0020] Preferably, the total sugar content of the coltsfoot flower homogeneous polysaccharide is not less than 70%.

[0021] 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.

[0022] Preferably, the preparation method includes the following steps:

[0023] Step 1: Mix coltsfoot flowers with a solvent, heat to defatting, and obtain defatted medicinal material;

[0024] Step 2: Mix defatted medicinal materials with water, heat to extract, and combine the extracts;

[0025] Step 3: After concentrating the extract, perform alcohol precipitation (e.g., fractional alcohol precipitation) to obtain crude coltsfoot flower polysaccharide;

[0026] Step 4: The crude coltsfoot flower polysaccharide is eluted by an ion exchange column, and the eluent is dialyzed and dried to obtain refined coltsfoot flower polysaccharide;

[0027] Step 5: The refined coltsfoot flower polysaccharide was eluted by gel chromatography; the eluted fraction with the highest abundance was collected, dialyzed and dried to obtain homogeneous coltsfoot flower polysaccharide.

[0028] 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;

[0029] More preferably, in step 1, the solvent is ethanol;

[0030] More preferably, the ethanol has a mass fraction of 50%-95%;

[0031] Preferably, in step 1, the heating is performed 1-3 times; more preferably, in step 1, the heating is performed 2 times.

[0032] Preferably, for the first heating, the ratio of coltsfoot flower to solvent is 1g:9-11mL; the heating time is 2-2.5h; for the second heating, the ratio of coltsfoot flower to solvent is 1g:7-8mL; the heating time is 1-1.5h.

[0033] More preferably, in the first heating, the ratio of coltsfoot flower to solvent is 1g:10mL; the heating time is 2h; in the second heating, the ratio of coltsfoot flower to solvent is 1g:8mL; the heating time is 1.5h.

[0034] 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.

[0035] Preferably, in step 2, the heating extraction is performed 1-3 times; more preferably, in step 2, the heating extraction is performed 2 times.

[0036] Preferably, for the first heating, the ratio of defatted medicinal material to water is 1g:9-11mL; the heating time is 2-2.5h; for the second heating, the ratio of defatted medicinal material to water is 1g:7-8mL; the heating time is 1-1.5h.

[0037] 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.

[0038] Preferably, in step 3, the alcohol solution used for alcohol precipitation is an ethanol solution;

[0039] More preferably, the ethanol solution has a mass fraction of 85%-95%; more preferably, the ethanol solution has a mass fraction of 95%.

[0040] Preferably, in step 3, the final solution contains 30% alcohol by mass.

[0041] More preferably, in step 3, the mass fraction of alcohol in the final solution is 80%.

[0042] Preferably, in step 4, the ion exchange column is a weak anion exchange column;

[0043] Preferably, in step 4, the weak anion exchange column is a DEAE-Fast Flow.

[0044] Preferably, in step 4, the elution solvent is water.

[0045] Preferably, in step 5, the gel chromatography column is a dextran gel chromatography column.

[0046] Preferably, in step 5, the elution solvent is water or a 0.1 mmol / L-0.25 mol / L aqueous solution of sodium chloride.

[0047] Preferably, in step 5, the specific elution operation is as follows: the purified coltsfoot flower polysaccharide obtained in step 4 is dissolved in water, centrifuged, the supernatant is loaded onto a dextran gel chromatography column, eluted with a 0.1 mmol / L-0.25 mol / L sodium chloride aqueous solution, detected by a differential refractive index detector, the component with the highest abundance is collected, dialyzed, and then freeze-dried to obtain the homogeneous coltsfoot flower polysaccharide.

[0048] Preferably, in step 5, the dextran gel type is selected from G50, G25, Sephacryl S100 or Sephacryl S200.

[0049] Thirdly, the present invention provides a pharmaceutical composition comprising the above-described coltsfoot flower homogeneous polysaccharide.

[0050] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0051] Fourthly, the present invention provides the use of the above-described coltsfoot flower homogeneous polysaccharide or the pharmaceutical composition thereof in the preparation of a medicament for treating respiratory diseases. In other words, the present invention also provides a method for treating respiratory diseases, the method comprising administering to a subject commercially available coltsfoot flower homogeneous polysaccharide or the pharmaceutical composition thereof.

[0052] Preferably, the respiratory disease includes one or more of asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

[0053] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0054] 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

[0055] Figure 1 shows the molecular weight distribution of the homogeneous polysaccharide from Tussilago farfara prepared in Example 1 of this invention.

[0056] Figure 2 is a monosaccharide composition test spectrum of the homogeneous polysaccharide prepared by Tussilago farfara flower in Example 1 of the present invention.

[0057] Figure 3 is the infrared spectrum of the homogeneous polysaccharide from Tussilago farfara prepared in Example 1 of this invention.

[0058] Figure 4 shows the hydrogen spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0059] Figure 5 is the carbon spectrum of the homogeneous polysaccharide from Tussilago farfara prepared in Example 1 of this invention.

[0060] Figure 6 shows the DEPT135 carbon spectrum of the homogeneous polysaccharide from Tussilago farfara prepared in Example 1 of this invention.

[0061] Figure 7 shows the HSQC spectrum of the homogeneous polysaccharide from Tussilago farfara flower prepared in Example 1 of this invention.

[0062] Figure 8 shows the HMBC spectrum of the homogeneous polysaccharide from Tussilago farfara prepared in Example 1 of this invention.

[0063] Figure 9 is the HHCOSY spectrum of the homogeneous polysaccharide from Tussilago farfara prepared in Example 1 of this invention.

[0064] Figure 10 is a statistical chart of the number of times the rhinitis model of Embodiment 1 of the present invention scratches the nose.

[0065] Figure 11 is a statistical chart of IL-5 levels in the asthma model of Embodiment 1 of the present invention.

[0066] Figure 12 is a statistical chart of the cough latency and number of coughs in the cough model of Embodiment 1 of the present invention.

[0067] Figure 13 is a diagram showing the effect of Example 1 of the present invention on an in vitro model of CSE-induced chronic obstructive pulmonary disease. Detailed Implementation

[0068] 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.

[0069] In this invention, the terms "comprising" or "including" or similar terms as used mean that the element preceding the term covers the element listed after the term, but do not exclude the possibility of covering other elements as well. The term "about" as used in this invention has a meaning known to those skilled in the art, and preferably refers to the value modified by the term within the range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, or ±1%.

[0070] In this invention, the term "polysaccharide" refers to a compound consisting of at least ten monosaccharide units (or structural units) linked by glycosidic bonds or glycosidic bonds.

[0071] In this invention, the term "crude polysaccharides" or "crude polysaccharides" refers to the product obtained after water extraction and alcohol precipitation of coltsfoot flowers; it is sometimes also referred to as "total polysaccharides" in the art. Those skilled in the art will understand that the precipitate obtained from water extraction and ethanol precipitation of coltsfoot flowers certainly contains polysaccharides, but may also contain other non-sugar components. Those skilled in the art will understand that total polysaccharides do not refer to all the polysaccharides in coltsfoot flowers, because it is difficult to obtain all the polysaccharides from coltsfoot flowers regardless of the extraction method.

[0072] In this invention, the term "homogeneous polysaccharide" has a meaning known in the art, referring to a polysaccharide with uniform molecular weight and charge. Molecular weight uniformity and charge uniformity can be determined using methods known to those skilled in the art. For example, molecular weight uniformity can be determined by the symmetrical peak shape of an HPGPC spectrum; charge uniformity can be determined by ion-exchange column chromatography, typically producing a single symmetrical peak in the electrophoresis pattern.

[0073] The term "polysaccharide component" (sometimes also referred to as a fraction in specific experiments) refers to a mixture containing more than one homogeneous polysaccharide. In special cases, a polysaccharide component may contain only one homogeneous polysaccharide or may even be a single homogeneous polysaccharide.

[0074] The crude polysaccharide, homogeneous polysaccharide, and polysaccharide component in this invention are also referred to as coltsfoot flower polysaccharide, coltsfoot flower homogeneous polysaccharide, and coltsfoot flower polysaccharide component, respectively. This naming is based on the consideration that they can be extracted from the medicinal material coltsfoot flower, but does not exclude the possibility of obtaining them through other means or sources.

[0075] 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.

[0076] This invention provides a method for preparing homogeneous polysaccharides from coltsfoot flowers, the method comprising the following steps:

[0077] Step 1: Mix coltsfoot flowers with a solvent, heat to defatting, and obtain defatted medicinal material;

[0078] Step 2: Mix defatted medicinal materials with water, heat to extract, and combine the extracts;

[0079] Step 3: After concentrating the extract, alcohol precipitation was performed to obtain crude coltsfoot flower polysaccharide;

[0080] Step 4: The crude coltsfoot flower polysaccharide is eluted by an ion exchange column, and the eluent is dialyzed and dried to obtain refined coltsfoot flower polysaccharide;

[0081] Step 5: The refined coltsfoot flower polysaccharide was eluted by gel chromatography; the eluted fraction with the highest abundance was collected, dialyzed and dried to obtain homogeneous coltsfoot flower polysaccharide.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] Example 1:

[0087] Take the Coptis chinensis flower material, add 90wt% ethanol and heat under reflux for defatting twice, with material-to-liquid ratios of 1g:10mL and 1g:8mL respectively, and reflux times of 2h and 1.5h respectively. After defatting, dry the filter residue and extract it twice with water under heating, with material-to-liquid ratios of 1g:10mL and 1g:8mL respectively, and reflux times of 2h and 1.5h 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 in the solution was 30 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 DEAE Fast Flow weak anion exchange column (5 cm in diameter and 50 cm in length). The eluted phase was dialyzed and dried to obtain purified homogeneous coltsfoot flower polysaccharide. Then, the polysaccharide was dissolved in water and loaded onto a G50 dextran gel column (2 cm in diameter and 100 cm in length) and eluted with aqueous solution. The eluted fraction with the highest abundance was collected, dialyzed, and dried.

[0088] Example 2:

[0089] Take the Coptis chinensis flower material, add 90wt% ethanol and heat under reflux for defatting twice, with material-to-liquid ratios of 1g:10mL and 1g:8mL respectively, and reflux times of 2h and 1.5h respectively. After defatting, dry the filter residue and extract it twice with water under heating, with material-to-liquid ratios of 1g:10mL and 1g:8mL respectively, and reflux times of 2h and 1.5h 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 in 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 in 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 DEAE Fast Flow weak anion exchange column (5 cm in diameter and 50 cm in length). 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 (2 cm in diameter and 100 cm in length) and eluted with aqueous solution. The eluted fraction with the highest abundance was collected, dialyzed, and dried.

[0090] Example 3:

[0091] Take the Coptis chinensis flower material, add 90wt% ethanol and heat under reflux for defatting twice, with material-to-liquid ratios of 1g:10mL and 1g:8mL respectively, and reflux times of 2h and 1.5h respectively. After defatting, dry the filter residue and extract it twice with water under heating, with material-to-liquid ratios of 1g:10mL and 1g:8mL respectively, and reflux times of 2h and 1.5h 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 DEAE Fast Flow weak anion exchange column (5 cm in diameter and 50 cm in length). The eluted phase was dialyzed and dried to obtain purified homogeneous coltsfoot flower polysaccharide. Then, the polysaccharide was dissolved in water and loaded onto a Sephacryl-100 gel column (2 cm in diameter and 100 cm in length) and eluted with aqueous solution. The eluted fraction with the highest abundance was collected, dialyzed, and dried.

[0092] Examples 1, 2, and 3 all mainly contain homogeneous polysaccharides with the same repeating unit structure, and homogeneous polysaccharides with the same repeating unit structure can be separated and purified using gel packing.

[0093] Test Example 1: Chemical Structural Characteristics of Homogeneous Polysaccharides from Coltsfoot Flower

[0094] Determination of molecular weight in Examples 1-3

[0095] 1. Laboratory supplies:

[0096] Products obtained in Examples 1-3, sodium chloride, etc.

[0097] 2. Experimental Methods:

[0098] The molecular weights of the products obtained in Examples 1-3 were determined using HPSEC-MALLS-RID coupled technology. The instruments used included:

[0099] 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 (8×300mm); Mobile phase: 0.05M sodium chloride solution; Flow rate: 0.5mL / min; Column temperature: 40℃; Injection volume: 25μL; Detector: Waters 2414 differential detector, MALS detector. The wavelength of the MALS laser was 658.1nm, and the specific refractive index increment dn / dc value was 0.1380mg / L. The light scattering model was the Zimm model.

[0100] 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.

[0101] 3. Experimental Results:

[0102] The differential detection (dRI) and multi-angle laser light scattering (LS) overlay plot (dRI+LS) calculates the molecular weight distribution range, weight-average molecular weight (Mw, Da), and polydispersity index (Mw / Mn) of each component 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.

[0103] Table 1

[0104] Table 2

[0105] Determination of monosaccharide composition in Examples 1-2

[0106] 1. Laboratory supplies:

[0107] The solid samples obtained in Examples 1 and 2 contained trifluoroacetic acid (ACROS), 50% sodium hydroxide solution (Alfa Aesar), and sodium acetate (ThermoFishe). The equipment used included an ion chromatograph (ThermoFishe, ICS5000), an electrically heated constant-temperature drying oven (Lichen Technology, 101-1BS), a nitrogen evaporator (Lichen Technology, UGC-24M), and an electronic balance (Sartorius).

[0108] BS, 210S), centrifuge (Thermo Fisher, D-37520), 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.

[0109] 2. Experimental Methods:

[0110] 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.

[0111] 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.

[0112] Column: Dionex Carbopac™ PA20 (3*150mm);

[0113] Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaAc;

[0114] Flow rate: 0.3 mL / min;

[0115] Injection volume: 25 μL;

[0116] Column temperature: 30℃;

[0117] Elution gradient:

[0118] 0 min A phase / B phase / C phase (98.8:1.2:0, V / V);

[0119] 18min A phase / B phase / C phase (98.8:1.2:0, V / V);

[0120] 20 min A phase / B phase / C phase (50:50:0, V / V);

[0121] 30min A phase / B phase / C phase (50:50:0, V / V);

[0122] 30.1 min A phase / B phase / C phase (0:0:100, V / V);

[0123] 46min A phase / B phase / C phase (0:0:100, V / V);

[0124] 46.1 min A phase / B phase / C phase (0:100:0, V / V);

[0125] 50min A phase / B phase / C phase (0:100:0, V / V);

[0126] 50.1 min A phase / B phase / C phase (98.8:1.2:0, V / V);

[0127] 80 min A phase / B phase / C phase (98.8:1.2:0, V / V).

[0128] Detector: Electrochemical detector.

[0129] 3. Experimental Results:

[0130] The experimental results are shown in Tables 3 and 4, and the relevant spectra are shown in Figure 2. Table 3 represents the sample of Example 1, and Table 4 represents the sample of Example 2.

[0131] Table 3

[0132] Table 4

[0133] Determination of total sugar content in Example 1:

[0134] Instruments and reagents

[0135] 1. Instruments: Vortex mixer, pipette, microplate reader, water bath, graduated cylinder, electronic balance

[0136] 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.

[0137] Preparation of the reference solution:

[0138] 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.

[0139] Sample preparation:

[0140] 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.

[0141] Content determination:

[0142] 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.

[0143] 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.

[0144] Table 5. Correspondence between glucose content (μg / mL) and absorbance A

[0145] Table 6. Absorbance of homogeneous polysaccharide phenol by concentrated sulfuric acid method

[0146] Infrared spectral analysis of Example 1:

[0147] Five mg of the sample was vacuum-dried overnight in a phosphorus pentoxide-containing dryer, then pressed into a KBr pellet for infrared spectroscopy analysis. Figure 3 shows the infrared spectrum of the obtained homogeneous polysaccharide F1 from Tussilago farfara flower, with the absorption band in the 3600-3200 cm⁻¹ range. -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.

[0148] NMR spectroscopy analysis of Example 1:

[0149] 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).

[0150] Figures 4 and 5 show the F1 1H and 1C spectra of the obtained homogeneous polysaccharide from coltsfoot flower. First, its anomeric proton resonance region was analyzed, as shown in Figure 4. 1 H-NMR, F1 1The 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 δ 5.35 ppm should belong to the anomer of α-D-Glcp-1→. Stronger signal peaks were observed in the δ 3.5-4.2 range. 13 As shown in C-NMR figure 5, two signal peaks are observed 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. Based on the methylation analysis of F1, the signal peaks at δ104.61, 105.06, and 104.45 should belong to fructose residues, while 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.

[0151] DEPT135 spectrum analysis (Figure 6) shows that δ 62.15, 63.35, 63.42, 63.56, 64.82, 62.15, 62.12, and 61.17 are inverted peaks, which may be methylene, representing the C1 and C6 of fructose and the C6 of glucose.

[0152] The HSQC spectrum (Figure 7) shows an anomeric carbon signal of δ93.86 and a corresponding anomeric hydrogen signal of δ5.35. The HH-COSY spectrum (Figure 9) shows the signals for H1-2 as 5.35 / 3.46, H2-3 as 3.46 / 3.69, H3-4 as 3.69 / 3.39, H4-5 as 3.39 / 3.76, and H5-6a as 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→).

[0153] The HSQC spectrum (Figure 7) shows an anomeric carbon signal of 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, suggesting that this signal peak corresponds to C2. H3 is identified through HMBC, and combined with HH-COSY, the signals for H3-4 are 4.17 / 4.01; H4-5 is 4.01 / 3.78; and H5-6a is 3.78 / 3.68. We can deduce that H3, H4, H5, and H6a have δ values ​​of 4.17, 4.01, 3.78, and 3.68, respectively. The corresponding C3-C6 values ​​are δ 79.37, 75.56, 82.36, and 63.35. Using the DEPT135 spectrum, the C1 signal is found to be δ 62.15, while the corresponding H1a and b values ​​are δ 3.63 and 3.84, respectively. By analyzing the HMBC two-dimensional spectrum of the multi-bonded C-H relationship of F1 (Figure 8), the sequence of monosaccharides in its sugar chain was determined, and the assignment of the chemical shifts of the main residues of F1 by 1H NMR and 13C NMR based on the HSQC and 1H-1H COSY two-dimensional spectra was verified to be correct.

[0154] 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→.

[0155] In the HMBC spectrum, there is a strong cross-peak C2(→1-β-D-Fruf-2→)–H1(→1-β-D-Fruf-2→) in the C2 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→.

[0156] 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→.

[0157] 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→.

[0158] 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.

[0159] Test Example 2. Rhinitis Drug Efficacy Experiment

[0160] 1. Reagents and Instruments

[0161] 1.1 Reagents

[0162] Grade II ovalbumin (A5503-1G, Merk), Grade V ovalbumin (A5253-250G, Merk), dexamethasone (Xianju Pharmaceutical), aluminum hydroxide (Xilong Scientific), sodium carboxymethyl cellulose (Da Mao Chemical), sodium chloride (Da Mao Chemical).

[0163] Example 1: The homogeneous polysaccharide was prepared by the Natural Product Chemistry Group of the China Resources Jiangzhong Modern Chinese Medicine Research Center.

[0164] 1.2 Instruments

[0165] A 0.01 g balance (MSA2245CE, Sartorius) and a 10 μL pipette (Eppendorf) were used.

[0166] 1.3 Laboratory Animals

[0167] Balb / c mice, male, 6-8 weeks old.

[0168] 2 Experimental Methods

[0169] 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 given the drug starting on day 20. One hour after drug 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.

[0170] 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.

[0171] 3 Experimental Results

[0172] As shown in Figure 10, 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.

[0173] Test Example 3: Efficacy Experiment of Drugs for Treating Asthma

[0174] 1. Reagents and instruments, etc.

[0175] Reagents:

[0176] Grade II ovalbumin (A5503-1G, Merk), Grade V ovalbumin (A5253-250G, Merk), dexamethasone (Xianju Pharmaceutical), aluminum hydroxide (Xilong Scientific), sodium carboxymethyl cellulose (Da Mao Chemical), sodium chloride (Da Mao Chemical), Mouse IL-5 Uncoated ELISA Kit (88-7054-88, Invitrogen), etc.

[0177] instrument:

[0178] 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.

[0179] Laboratory animals:

[0180] Balb / c mice, male, 6-8 weeks old.

[0181] 2. Experimental Methods:

[0182] 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) once daily for seven days.

[0183] 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.

[0184] 3. Experimental Results:

[0185] As shown in Figure 11, 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.

[0186] Test Example 4. Antitussive Efficacy Experiment

[0187] 1. Reagents and instruments, etc.

[0188] Reagents:

[0189] Pentoxyverine citrate (Lisheng Pharmaceutical), concentrated ammonia (A112079, Aladdin), etc.

[0190] instrument:

[0191] A 0.01% balance (MSA2245CE, Sartorius) and a multi-functional cough and asthma induction device (model: YLS-8A).

[0192] Laboratory animals:

[0193] SD rats, male, 6-8 weeks old.

[0194] 2. Experimental Methods:

[0195] 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.

[0196] 3. Experimental Results:

[0197] The experimental results are shown in Figure 12. 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.

[0198] Test Example 5. Chronic Obstructive Pulmonary Disease (COPD) Efficacy Experiment

[0199] 1. Reagents and Instruments

[0200] 1.1 Reagents

[0201] 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.

[0202] 1.2 Instruments

[0203] 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.

[0204] 1.3 Experimental Cells

[0205] BEAS-2B human bronchial epithelial cells.

[0206] 2 Experimental Methods

[0207] 2.1 Preparation of Cigarette Extract

[0208] 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.

[0209] 2.2 Cell treatment methods

[0210] BEAS-2B cells were cultured to the logarithmic growth phase, and then cultured at a density of 5 × 10⁶ cells per well. 3 Cells were seeded into 96-well plates. After cell adhesion, CSE modeling was performed according to the experimental groups. After 24h and 48h of drug intervention, 10% CCK8 working solution (GlpBio commercial product) was added and incubated for 2-4h at the experimental endpoint, and the cell absorbance was measured.

[0211] Calculate cell viability based on absorbance values ​​of each cell group:

[0212] 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%.

[0213] 3 Experimental Results

[0214] As shown in Figure 13, cell survival was significantly reduced under CSE (0.5%) conditions. At 24 h and 48 h after administration, the group in Example 1 (100 μg / mL) showed a significant repair effect on CSE-induced cell damage (P<0.05).

[0215] The above results indicate that the coltsfoot flower homogeneous polysaccharide provided by this invention has a certain therapeutic effect on chronic obstructive pulmonary disease.

[0216] 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 homogeneous polysaccharide from coltsfoot flowers, wherein the structural units of the homogeneous polysaccharide from coltsfoot flowers comprise fructose and glucose; wherein, The molar ratio of fructose to glucose is 20-26:1.

00.

2. The coltsfoot flower homogeneous polysaccharide according to claim 1, wherein the molecular weight of the coltsfoot flower homogeneous polysaccharide is selected from 2.0 × 10⁻⁶. 3 -4.5×10 3 Da.

3. The coltsfoot flower homogeneous polysaccharide according to claim 1, wherein, The molar ratio of fructose to glucose is 21.7:1, and the molecular weight of the homogeneous polysaccharide from coltsfoot flower is selected from 2.0 × 10⁻⁶. 3 -2.8×10 3 Da; Alternatively, the molar ratio of fructose to glucose may be 25.9:1, and the molecular weight of the homogeneous polysaccharide from coltsfoot flowers may be selected from 3.6 × 10⁻⁶. 3 -4.5×10 3 Da.

4. The coltsfoot flower homogeneous polysaccharide according to claim 1, wherein the structural units of the coltsfoot flower homogeneous polysaccharide comprise terminal glucose, 1,2-linked fructose, 1,2,6-linked fructose, 2,6-linked fructose and terminal fructose.

5. The coltsfoot flower homogeneous polysaccharide according to claim 1, wherein the primary structure of the coltsfoot flower homogeneous polysaccharide is as follows:

6. The coltsfoot flower homogeneous polysaccharide according to claim 1, wherein the total sugar content of the coltsfoot flower homogeneous polysaccharide is not less than 70%.

7. The method for preparing the homogeneous polysaccharide of coltsfoot flower according to any one of claims 1-6, comprising the following steps: coltsfoot flower is defatted, extracted with water, precipitated with alcohol, eluted, and eluted twice to obtain the homogeneous polysaccharide of coltsfoot flower.

8. The preparation method according to claim 7, wherein, 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 was subjected to alcohol precipitation to obtain crude polysaccharide from Tussilago farfara. Step 4: The crude polysaccharide of coltsfoot flower is eluted by an ion exchange column, and the eluent is dialyzed and dried to obtain purified coltsfoot flower polysaccharide; Step 5: The refined coltsfoot flower polysaccharide was eluted by gel chromatography; the eluted fraction with the highest abundance was collected, dialyzed and dried to obtain homogeneous coltsfoot flower polysaccharide.

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

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

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

13. The preparation method according to claim 8, wherein, 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, wherein, 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, wherein, 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 comprising the coltsfoot flower homogeneous polysaccharide according to any one of claims 1-6 or the coltsfoot flower homogeneous polysaccharide prepared by any one 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, wherein, The respiratory diseases include one or more of asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

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