A kind of radix taraxaci polysaccharide, preparation method and use

Abstract

This invention discloses a coltsfoot flower extract, its preparation method, and its uses, belonging to the field of pharmaceutical technology. The coltsfoot flower extract is a coltsfoot flower polysaccharide, comprising fructose, glucose, arabinose, galactose, and glucosamine, with a molecular weight of 1.21 × 10⁻⁶. 2 -6.58×10 6 Da; The deproteinized polysaccharide obtained from coltsfoot flower extract contains fructose, arabinose, glucose, and galactose, with a molecular weight of 4.86 × 10⁻⁶. 3 -4.34×10 6 Da; The decolorized polysaccharide obtained from coltsfoot flower extract contains fructose, arabinose, glucose, and galactose, with a molecular weight of 1.42 × 10⁻⁶. 2 -1.05×10 7 The above-mentioned polysaccharides have 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 coltsfoot flower polysaccharide extract, its preparation method, and its uses. Background Technology

[0002] Coltsfoot flower, the dried flower bud of the traditional Chinese medicine *Tussilago farfara* L., belongs to the genus *Tussilago* in the family Compositae and is a commonly used herb in 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 coltsfoot ketone is recorded in the pharmacopoeia.

[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 invention 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 extract for the treatment of respiratory diseases. This invention discloses a coltsfoot flower extract, a deproteinized polysaccharide from coltsfoot flower extract, and a decolorized polysaccharide from coltsfoot flower extract, as well as their preparation methods. Whole animal model experiments have confirmed its effectiveness in preventing and treating asthma, cough, and rhinitis, and cell experiments have demonstrated its repairing effect on damage caused by chronic obstructive pulmonary disease. It can be further used to develop innovative traditional Chinese medicine drugs for the prevention and treatment of respiratory diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a coltsfoot flower polysaccharide extract, its preparation method, and its use in the preparation of medicines for respiratory diseases.

[0008] To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

[0009] A coltsfoot flower extract, characterized in that the preparation method of the coltsfoot flower extract includes the following steps: coltsfoot flowers are heated to defatted, then extracted with water, then precipitated with alcohol to obtain a precipitate, and then dried to obtain the coltsfoot flower extract.

[0010] Preferably, the coltsfoot flower extract is a coltsfoot flower polysaccharide.

[0011] Preferably, the coltsfoot flower polysaccharide includes fructose, glucose, arabinose, galactose, and glucosamine.

[0012] Preferably, the molar ratio of fructose, glucose, arabinose, galactose, and glucosamine is 0.7-0.9:0.1-0.2:0.005-0.01:0.001-0.003:0.001-0.003; more preferably, the molar ratio of fructose, glucose, arabinose, galactose, and glucosamine is 0.75-0.9:0.1-0.15:0.006-0.009:0.001-0.002:0.001-0.002; more preferably, the molar ratio of fructose, glucose, arabinose, galactose, and glucosamine is 0.861:0.131:0.008:0.002:0.001.

[0013] Preferably, the molecular weight of the coltsfoot flower extract is 1.21 × 10⁻⁶. 2 -6.58×10 6 Da.

[0014] More preferably, the molecular weight of the coltsfoot flower extract is 1.21 × 10⁻⁶. 2 -1.63×10 3 Da.

[0015] Preferably, the coltsfoot flower extract contains sugars and proteins.

[0016] Preferably, the coltsfoot flower extract contains not less than 20% sugar by weight.

[0017] Preferably, the coltsfoot flower extract contains no more than 42% protein by weight.

[0018] Preferably, the preparation method of the coltsfoot flower extract includes the following steps:

[0019] Step 1-1: Mix coltsfoot flowers with solvent, heat to degrease, filter, and air dry to obtain degreased medicinal material;

[0020] Steps 1-2: Mix defatted medicinal materials with water, heat to extract, combine the extracts, concentrate to obtain concentrated solution;

[0021] Steps 1-3: Mix the concentrated liquid with the alcohol solution to obtain a precipitate, then dry it to obtain the coltsfoot flower extract.

[0022] Preferably, in step 1-1, the solvent is selected from at least one of ethanol, petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether, and methanol;

[0023] Preferably, in step 1-1, the heating is performed 1-3 times;

[0024] Preferably, in step 1-1, the heating is performed twice;

[0025] Preferably, for the first heating, the ratio of coltsfoot flower to solvent is 1:9-11 g / mL, and the heating time is 2-2.5 h; for the second heating, the ratio of coltsfoot flower to solvent is 1:7-9 g / mL, and the heating time is 1-1.5 h.

[0026] 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.

[0027] Preferably, in steps 1-2, the water is selected from at least one of ultrapure water, deionized water, and distilled water; more preferably, in steps 1-2, the water is selected from ultrapure water.

[0028] Preferably, in steps 1-2, the heating is performed 1-3 times;

[0029] Preferably, in steps 1-2, the heating is performed twice;

[0030] 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 1:7-9 g / mL; the heating time is 1-1.5 h.

[0031] Preferably, in steps 1-2, the concentration is to concentrate the raw medicinal material to a concentration of 0.5-1.5 mg / mL; more preferably, in steps 1-2, the concentration is to concentrate the raw medicinal material to a concentration of 1 mg / mL.

[0032] Preferably, in steps 1-3, the alcohol solution is an ethanol solution;

[0033] Preferably, in steps 1-3, the final solution obtained by mixing the concentrate and the alcohol solution has an ethanol mass fraction of 60%-95%; more preferably, in steps 1-3, the final solution obtained by mixing the concentrate and the alcohol solution has an ethanol mass fraction of 80%.

[0034] Preferably, in steps 1-3, the mixing is performed by slowly adding the concentrated liquid to the alcohol solution and allowing it to stand for 10-16 hours.

[0035] In another aspect, the present invention provides a deproteinized polysaccharide, which is obtained by deproteinizing any of the above-mentioned coltsfoot flower extracts, and the molecular weight of the deproteinized polysaccharide is 4.86 × 10⁻⁶. 3 -4.34×10 6 Da.

[0036] Preferably, the deproteinized polysaccharide comprises fructose, arabinose, glucose, and galactose.

[0037] Preferably, the deproteinized polysaccharide comprises sugar and protein.

[0038] Preferably, the deproteinized polysaccharide contains at least 50% sugar by mass.

[0039] Preferably, the deproteinized polysaccharide contains no more than 6% protein by weight.

[0040] Preferably, the molar ratio of fructose, arabinose, glucose and galactose is 0.8-0.9:0.07-0.085:0.06-0.072:0.01-0.025.

[0041] More preferably, the molar ratio of fructose, arabinose, glucose and galactose is 0.82-0.88:0.075-0.080:0.062-0.070:0.012-0.020.

[0042] More preferably, the molar ratio of fructose, arabinose, glucose and galactose is 0.841:0.077:0.066:0.016.

[0043] Furthermore, the present invention also provides a method for preparing the aforementioned deproteinized polysaccharide, comprising the following steps:

[0044] Step 2-1: Dissolve and enzymatically hydrolyze the coltsfoot flower extract;

[0045] Step 2-2: The supernatant after enzymatic hydrolysis in Step 2-1 is concentrated to obtain a concentrated solution;

[0046] Step 2-3: The concentrated solution from Step 2-2 is mixed with the alcohol solution to obtain a precipitate;

[0047] Step 2-4: The precipitate from step 2-3 is dried to obtain the deproteinized polysaccharide.

[0048] Preferably, in step 2-1, the solvent used for dissolution is water;

[0049] Preferably, the water is selected from at least one of ultrapure water, deionized water, and distilled water; more preferably, the water is selected from ultrapure water.

[0050] Preferably, in step 2-1, the enzyme is papain, and the concentration of the papain is 8-12 g / L; more preferably, in step 2-1, the concentration of the papain is 10 g / L.

[0051] Preferably, in step 2-1, the enzymatic hydrolysis temperature is 40℃-60℃ and the time is 3h-5h; more preferably, in step 2-1, the enzymatic hydrolysis temperature is 50℃ and the time is 4h.

[0052] Preferably, in step 2-2, the concentration is to concentrate the raw medicinal material to a concentration of 0.5-1.5 mg / mL; more preferably, in step 2-2, the concentration is to concentrate the raw medicinal material to a concentration of 1 mg / mL.

[0053] Preferably, in steps 2-3, the alcohol solution is an ethanol solution.

[0054] More preferably, the ethanol solution has a mass fraction of 95%.

[0055] Preferably, in step 2-3, the final solution obtained by mixing the concentrate and the alcohol solution has an ethanol mass fraction of 60%-95%; more preferably, in step 2-3, the final solution obtained by mixing the concentrate and the alcohol solution has an ethanol mass fraction of 80%.

[0056] Preferably, in steps 2-3, the mixing involves slowly adding the concentrated liquid to the alcohol solution and allowing it to stand for 10-16 hours.

[0057] Furthermore, the present invention also provides a decolorized polysaccharide, which is obtained by decolorizing any of the above-described coltsfoot flower extracts, and the decolorized polysaccharide has a molecular weight of 1.42 × 10⁻⁶. 2 -1.05×10 7 Da.

[0058] Preferably, the decolorized polysaccharide comprises fructose, arabinose, glucose, and galactose.

[0059] Preferably, the molar ratio of fructose, arabinose, glucose, and galactose is 0.8-0.9:0.07-0.085:0.06-0.072:0.01-0.022; more preferably, the molar ratio of fructose, arabinose, glucose, and galactose is 0.82-0.86:0.07-0.080:0.06-0.070:0.01-0.020. More preferably, the molar ratio of fructose, arabinose, glucose, and galactose is 0.840:0.078:0.066:0.016.

[0060] In another aspect, the present invention also provides a method for preparing the aforementioned decolorized polysaccharide, comprising the following steps: adsorbing coltsfoot flower extract using macroporous resin to obtain decolorized polysaccharide.

[0061] Preferably, the coltsfoot flower extract, deproteinized polysaccharide, and decolorized polysaccharide do not contain coltsfoot ketone.

[0062] In another aspect, the present invention also provides a pharmaceutical composition comprising at least one of the aforementioned coltsfoot flower extract, the aforementioned deproteinized polysaccharide, and the aforementioned decolorized polysaccharide.

[0063] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0064] On the other hand, the present invention also provides the use of at least one of the aforementioned coltsfoot flower extract, the aforementioned deproteinized polysaccharide, and the aforementioned decolorized polysaccharide in the preparation of medicaments for respiratory diseases.

[0065] Preferably, the respiratory diseases include asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

[0066] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0067] The coltsfoot flower extract, deproteinized polysaccharide, and decolorized polysaccharide provided by this invention have good effects in preventing and treating asthma, rhinitis, cough, and chronic obstructive pulmonary disease, providing a new option for preparing innovative traditional Chinese medicines for the prevention and treatment of respiratory diseases. Attached Figure Description

[0068] Figure 1 This is the molecular weight distribution spectrum of Example 1 of the present invention.

[0069] Figure 2 This is the molecular weight distribution spectrum of Example 2 of the present invention.

[0070] Figure 3 This is the molecular weight distribution spectrum of Example 3 of the present invention.

[0071] Figure 4 This is a monosaccharide composition test spectrum of Example 1 of the present invention.

[0072] Figure 5 This is a monosaccharide composition test spectrum of Example 2 of the present invention.

[0073] Figure 6 The monosaccharide composition test spectrum of Example 3 of the present invention.

[0074] Figure 7 The chromatograms are comparison diagrams of Example 1 of the present invention and the coltsone sample (a is the chromatogram of coltsone standard, b is the chromatogram of Example 1, and c is the superimposed chromatogram of the two).

[0075] Figure 8 This is a bar chart showing the IL-5 level and inflammatory cell count levels in the asthma efficacy tests of Examples 1-3 of this invention.

[0076] Figure 9 This is a statistical chart showing the number of coughs during the antitussive efficacy tests in Examples 1-3 of the present invention.

[0077] Figure 10 This is a statistical chart of the cough latency period in the antitussive efficacy tests of Examples 1-3 of the present invention.

[0078] Figure 11 This is a statistical chart showing the number of times the nose was scratched during the rhinitis efficacy test in Examples 1-2 of the present invention.

[0079] Figure 12 The following are effect graphs of the in vitro model of CSE-induced chronic obstructive pulmonary disease in Examples 1-2 of the present invention (a is a 24-hour statistical graph, b is a 48-hour statistical graph).

[0080] Figure 13 This is a diagram illustrating the effect of Example 3 of the present invention on an in vitro model of CSE-induced chronic obstructive pulmonary disease. Detailed Implementation

[0081] 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.

[0082] 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%.

[0083] In this invention, the term "total polysaccharide" refers to the sum of a class of complex and large carbohydrate substances formed by the condensation and dehydration of multiple monosaccharide molecules. Total polysaccharides include various types of polysaccharides, which may be composed of the same monosaccharide molecules (called homopolysaccharides) or different types of monosaccharide units (called heteropolysaccharides). Total polysaccharides can be further subdivided into various types, including but not limited to proteoglycans, dextran, mucopolysaccharides, and xylan. These polysaccharides may differ in structure and function, but all fall under the category of total polysaccharides.

[0084] 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.

[0085] This invention provides a method for extracting coltsfoot flower extract, the method comprising the following steps:

[0086] Step 1-1: Mix coltsfoot flowers with an alcohol solution, heat 1-3 times to defatting, filter, and air dry to obtain defatted medicinal material;

[0087] Steps 1-2: Mix defatted medicinal materials with water, heat and extract 1-3 times, combine the extracts, concentrate to obtain concentrated solution;

[0088] Steps 1-3: Mix the concentrated liquid with the alcohol solution to obtain a precipitate, and dry it to obtain the coltsfoot flower extract.

[0089] Preferably, the method includes the following steps:

[0090] Step 1-1: Mix coltsfoot flowers with a 50-95 wt% ethanol solution for defatting. The defatting solvent is not limited to ethanol solution; petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether, and methanol can also be used. The mixture is heated twice, with the material-to-liquid ratio being 1:10 g / mL and 1:8 g / mL, respectively. The reflux times are 2 h and 1.5 h, respectively. After filtration, the medicinal material is dried.

[0091] Steps 1-2: Mix defatted medicinal materials with water and heat to extract twice. During the two heating processes, the material-to-liquid ratios are 1:10 g / mL and 1:8 g / mL, respectively, and the reflux times are 2 h and 1.5 h, respectively. Combine the extracts and concentrate them to a crude drug concentration of 1 mg / mL to obtain a concentrated solution.

[0092] Steps 1-3: Mix the concentrated solution with the ethanol solution until the concentration of ethanol in the solution is 60%-95%; obtain the precipitate, dry it; and obtain the coltsfoot flower polysaccharide.

[0093] This invention provides a method for preparing deproteinized polysaccharides, the method comprising the following steps:

[0094] Step 2-1: Dissolve coltsfoot flower polysaccharide, add papain, and enzymatically hydrolyze;

[0095] Step 2-2: The supernatant after enzymatic hydrolysis in Step 2-1 is concentrated to obtain a concentrated solution;

[0096] Step 2-3: The concentrated solution from Step 2-2 is mixed with the alcohol solution to obtain a precipitate;

[0097] Step 2-4: The precipitate from step 2-3 is dried to obtain the deproteinized polysaccharide.

[0098] Preferably, the method includes the following steps:

[0099] Step 2-1: Dissolve coltsfoot flower polysaccharide in water, add papain, and perform enzymatic hydrolysis. The concentration of papain is 8-12 g / L, the hydrolysis temperature is 40℃-60℃, and the time is 3-5 h.

[0100] Step 2-2: Centrifuge and collect the supernatant of the product obtained in Step 2-1, and concentrate it to obtain a concentrated solution with a crude drug concentration of 1 mg / mL;

[0101] Step 2-3: Add 95% ethanol to the concentrate obtained in step 2-2 until the concentration of ethanol in the solution is 60%-95%;

[0102] Step 2-4: Dry the precipitate obtained in step 2-3 to obtain the deproteinized polysaccharide.

[0103] In this invention, the concentration in steps 1-2 and 2-2 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Example

[0108] Example 1: Preparation of Coltsfoot Flower Extract

[0109] Coltsfoot flower medicinal material (Jiangxi Guodu Traditional Chinese Medicine Pieces Co., Ltd., originating from Hebei, product batch number: 20231201) was added to 90% ethanol and heated 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, the medicinal material 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 concentration of the solution was 80%. The solution was allowed to stand overnight, and the precipitate was dried to obtain coltsfoot flower extract (i.e., coltsfoot flower polysaccharide).

[0110] Example 2: Preparation of deproteinized polysaccharides

[0111] For the sample obtained in Example 1, it was reconstituted with ultrapure water, and papain at a concentration of 10 g / L was added. The mixture was enzymatically hydrolyzed in a water bath at 50°C for 4 hours. The supernatant was collected by centrifugation and concentrated to a concentration of 1 mg / mL of crude drug. Ethanol was slowly added until the final concentration of the solution was 80%. The mixture was allowed to stand overnight, and the precipitate was dried to obtain the deproteinized polysaccharide of Tussilago farfara.

[0112] Example 3: Preparation of decolorized polysaccharides

[0113] For the sample obtained in Example 1, it was reconstituted with ultrapure water, centrifuged, and the supernatant was collected; D101 macroporous resin was taken and washed with water, the sample solution obtained in Example 1 was loaded onto the sample, and the loading liquid was collected; after loading, it was eluted with 3BV pure water; the eluents were combined and concentrated; the concentrated sample was freeze-dried to obtain the decolorized polysaccharide of Tussilago farfara.

[0114] Test Example 1: Determination of sugar and protein content in coltsfoot flower extract and deproteinized polysaccharide

[0115] 1. Laboratory supplies:

[0116] The products obtained in Examples 1 and 2.

[0117] 2. Experimental Methods:

[0118] Sugar content was determined using the sulfuric acid-phenol method; protein content was determined using the BCA protein concentration assay kit; and the products obtained in Examples 1 and 2 were each tested three times.

[0119] 3. Experimental Results:

[0120] The sugar content of different batches of the coltsfoot flower extract obtained in Example 1 was 52.8%, 50.2%, and 42.3%, respectively; the sugar content of different batches of the deproteinized polysaccharide obtained in Example 2 was 68.6%, 76.4%, and 73.8%, respectively; the protein content of different batches of the coltsfoot flower extract obtained in Example 1 was 41.3%, 41.6%, and 41.2%, respectively; and the protein content of different batches of the deproteinized polysaccharide obtained in Example 2 was 5.4%, 5.2%, and 4.8%, respectively.

[0121] Test Example 2: Determination of the molecular weight of coltsfoot flower extract, deproteinized polysaccharide, and decolorized polysaccharide

[0122] 1. Laboratory supplies:

[0123] The products obtained in Examples 1-3, sodium chloride, etc.

[0124] 2. Experimental Methods:

[0125] The molecular weights of the products obtained in Examples 1-3 were determined using HPSEC-MALLS-RID coupled technology (DynaPro NanoStar dynamic laser light scattering instrument: Wyatt DynaPro NanoStar, USA; high performance liquid chromatograph: Shimadzu LC-10A; differential detector: Shimadzu RI-10A; column: BRT105-103-101; tandem gel column: BoRui Saccharide, BRT105-103-101; centrifuge: Eppendorf 5424). Prepare a 0.05M sodium chloride solution, filter through a 0.45μm filter 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. The flow rate is 0.7 mL / min, the column temperature is 40℃, and the injection volume is 50 μL. The detector is a Waters 2414 differential detector with a Malls detector. The wavelength of the Malls laser is 661.0 nm, and the specific refractive index increment dn / dc value is 0.1380 mg / L. The light scattering model is the Zimm model.

[0126] 3. Experimental Results:

[0127] The molecular weight distribution spectra of Examples 1-3 are as follows: Figure 1-3 As shown in Table 1, differential detection (dRI) and multi-angle laser light scattering (LS) were used to calculate the molecular weight distribution range, weight-average molecular weight (Mw, Da), and polydispersity index (Mw / Mn) of each component sample.

[0128] Table 1

[0129]

[0130] Combination Figure 1-3 As can be seen, the coltsfoot flower extract has a relatively wide molecular weight distribution (1.21×10⁻⁶). 2 -6.58×10 6 The molecular weight of the main peak (Da) is 1.21 × 10⁻⁶. 2 -1.63×10 3 Da (corresponding to peak3); while the molecular weight of the deproteinized polysaccharide is 4.86 × 10⁻⁶. 3 -4.34×10 6 The molecular weight of the decolorized polysaccharide is 1.42 × 10⁻⁶. 2 -1.05×10 7 Da.

[0131] Test Example 3: Determination of the monosaccharide composition of coltsfoot flower extract, deproteinized polysaccharide, and decolorized polysaccharide

[0132] 1. Laboratory supplies:

[0133] The solid samples obtained in Examples 1-3 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), an electronic balance (Sartorius BS, 210S), 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.

[0134] 2. Experimental Methods:

[0135] 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 with nitrogen, add deionized water and vortex mix to prepare a standard stock solution.

[0136] 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.

[0137] Chromatographic column: Dionex Carbopac™ PA20 (3*150mm); Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH & 100mM NaAc; Flow rate: 0.3mL / min; Injection volume: 25μL; Column temperature: 30℃; Elution gradient: 0min A / B / C (98.8:1.2:0, V / V), 18min A / B / C (98.8:1.2:0, V / V), 20min A / B / C (50:50:0, V / V), 30min A / B / C (50:50:0, V / V), 30.1min A / B Phase A / B / C (0:0:100, V / V), 46 min A / B / C (0:0:100, V / V), 46.1 min A / B / C (0:100:0, V / V), 50 min A / B / C (0:100:0, V / V), 50.1 min A / B / C (98.8:1.2:0, V / V), 80 min A / B / C (98.8:1.2:0, V / V). Detector: Electrochemical detector.

[0138] 3. Experimental Results:

[0139] The results of the monosaccharide composition tests in Examples 1-3 are shown in Tables 2-4, and the relevant chromatograms are shown in [reference needed]. Figure 4-6 .

[0140] Table 2

[0141]

[0142]

[0143] Table 3

[0144] Name Peak area RT Mole ratio ug / mg Arabic sugar 8.412 10.525 0.077 21.62 Galactose 1.45 12.975 0.016 5.39 glucose 5.916 14.7 0.066 22.17 fructose 32.032 19.167 0.841 283.80

[0145] Table 4

[0146] Name Peak area RT Mole ratio ug / mg Arabic sugar 8.449 10.509 0.078 21.71 Galactose 1.423 12.95 0.016 5.29 glucose 5.899 14.675 0.066 22.10 fructose 31.699 19.134 0.840 280.85

[0147] Test Example 4: Determination of coltsfoot ketone content in coltsfoot flower extract

[0148] 1. Laboratory supplies:

[0149] The product obtained in Example 1.

[0150] 2. Experimental Methods:

[0151] The HPLC method was used for determination. The chromatographic column was a YMC Triart C18 column, 4.6 × 250 mm, with a particle size of 5 μm. The mobile phase was methanol-water (85:15), with isocratic elution. The detection wavelength was 190-450 nm (220 nm). The column temperature was 30℃. The injection volume was 10 μL. An appropriate amount of styraxone reference standard was weighed and dissolved in the mobile phase to prepare the reference solution.

[0152] 3. Experimental Results:

[0153] like Figure 7 As shown, no coltsfoot ketone was detected in the coltsfoot flower extract obtained in Example 1. It is understood that the deproteinized polysaccharide prepared in Example 2 and the decolorized polysaccharide prepared in Example 3 also do not contain coltsfoot ketone.

[0154] Test Example 5: Efficacy Experiment of Drugs for Treating Asthma

[0155] 1. Reagents and instruments, etc.

[0156] Reagents:

[0157] 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.

[0158] instrument:

[0159] 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 (VICTORNIVO, PerkinElmer), surgical instruments.

[0160] Laboratory animals:

[0161] Balb / c mice, male, 6-8 weeks old

[0162] 2. Experimental Methods:

[0163] Balb / c mice were used in the experiment and divided into 6 groups: normal group, model group, positive group (dexamethasone 2 mg / kg), Example 1 group (450 mg / kg), Example 2 group (450 mg / kg), and Example 3 group (450 mg / kg). The test drug was prepared to the required concentration using 5‰ sodium carboxymethyl cellulose. 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, the mice were administered the drug on day 20. One hour after administration, nebulization challenge (2% Grade II OVA solution, 100 mL) was performed for one hour each time, once daily, for 7 consecutive days.

[0164] The day after the last nebulization, the mice were anesthetized and euthanized, and the bronchoalveolar lavage fluid was collected to determine the number of inflammatory cells and the IL-5 level.

[0165] 3. Experimental Results:

[0166] See relevant statistical results Figure 8 Compared with the model group, the levels of the inflammatory factor IL-5 in the bronchoalveolar lavage fluid (BALF) of the three test substances were significantly reduced after administration (P<0.05). The number of leukocytes, neutrophils, lymphocytes and monocytes in the bronchoalveolar lavage fluid (BALF) of the coltsfoot extract obtained in Example 1 was significantly reduced after administration (P values ​​were all less than 0.05). The number of leukocytes, neutrophils, lymphocytes and monocytes in the bronchoalveolar lavage fluid (BALF) of the deproteinized polysaccharide obtained in Example 2 and the decolorized polysaccharide obtained in Example 3 showed a decreasing trend after administration. It is inferred from this that the three test substances in Examples 1-3, especially Example 1, have a relieving effect on the OVA-induced asthma model.

[0167] Test Example 6:

[0168] Cough suppressant efficacy experiment

[0169] 1. Reagents and instruments, etc.

[0170] Reagents:

[0171] Pentoxyverine citrate (Lisheng Pharmaceutical), concentrated ammonia (A112079, Aladdin), etc.

[0172] instrument:

[0173] 0.01% balance (MSA2245CE, Sartorius), multi-functional cough and asthma induction device (model: YLS-8A), etc.

[0174] Laboratory animals:

[0175] SD rats, male, 6-8 weeks old

[0176] 2. Experimental Methods:

[0177] Healthy SD rats were randomly divided into three groups: a model group, a pentoxyverine citrate (50 mg / kg) group, and three groups (Examples 1-3, 400 mg / kg). The drugs and pentoxyverine citrate for each experimental group (Examples 1-3) were diluted with 5‰ CMC-Na to the corresponding concentrations and administered by gavage. Preventative administration was given 3 days prior. 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.

[0178] 3. Experimental Results:

[0179] Experimental results are as follows Figure 9-10 As shown, compared with the model group, all treatment groups significantly reduced the number of coughs in experimental rats (P<0.01 or P<0.001) and prolonged the cough latency after administration, indicating that the samples in Examples 1-3 could relieve cough.

[0180] Test Example 7:

[0181] Rhinitis drug efficacy experiment

[0182] 1. Reagents and instruments, etc.

[0183] Reagents:

[0184] 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).

[0185] instrument:

[0186] 0.01% balance (MSA2245CE, Sartorius), 10 μL pipette (Eppendorf), etc.

[0187] Laboratory animals:

[0188] Balb / c mice, male, 6-8 weeks old

[0189] 2. Experimental Methods:

[0190] Healthy Balb / c mice were randomly divided into a model group and a positive control group (dexamethasone 2 mg / kg), and were given the following treatments: Coltsfoot flower extract (350 mg / kg) obtained in Example 1, deproteinized polysaccharide (350 mg / kg) obtained in Example 2, and decolorized polysaccharide (350 mg / kg) obtained in Example 3. The test drugs were prepared to the required concentrations 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, 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.

[0191] 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.

[0192] 3. Experimental Results:

[0193] Experimental results are as follows Figure 11 As shown, the mice in the model group exhibited obvious nose scratching and sneezing after modeling, indicating successful modeling. Compared with the model group, the positive drug group and the coltsfoot flower extract obtained in Example 1 and the deproteinized polysaccharide obtained in Example 2 significantly reduced the number of nose scratchings in mice, indicating that the samples in Examples 1-2 could alleviate rhinitis symptoms.

[0194] Test Example 8: Efficacy Experiment of Pharmacotherapy for Chronic Obstructive Pulmonary Disease

[0195] 1. Reagents and Instruments

[0196] 1.1 Reagents

[0197] BEAS-2B human bronchial epithelial cell culture medium (Kunming Institute of Botany, Chinese Academy of Sciences), DMEM medium (C11995500BT, Gibco), FBS (10099-141, Gibco), CCK8 (RM02823, Abclonal), and cigarettes (Seven Wolves). Samples in Examples 1-3 were prepared by the Natural Product Chemistry Group of the China Resources Jiangzhong Modern Traditional Chinese Medicine Research Center.

[0198] 1.2 Instruments

[0199] 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.

[0200] 1.3 Experimental Cells

[0201] BEAS-2B human bronchial epithelial cells

[0202] 2. Experimental Methods

[0203] 2.1 Preparation of Cigarette Extract

[0204] 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.

[0205] 2.2 Cell treatment methods

[0206] BEAS-2B cells were cultured to the logarithmic growth phase and seeded into 96-well plates at a density of 5 × 10³ cells per well. After cell adhesion, cigarette extract (CSE) was prepared according to literature methods. CSE modeling was performed according to experimental groups, followed by drug intervention. After 24 hours or 48 hours, CCK8 working solution was added and incubated for 2-4 hours at the experimental endpoint, and the cell absorbance was measured.

[0207] Calculate cell viability based on absorbance values ​​of each cell group:

[0208] 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%.

[0209] 3. Experimental Results

[0210] like Figure 12 As shown, cell viability was significantly reduced under CSE (0.5%) conditions, 24 h after drug administration ( Figure 12 -a) and 48h ( Figure 12 -b) The cell viability of the 100 μg / mL Coltsfoot Flower Extract group (Example 1), the 100 μg / mL Deproteinized Polysaccharide group (Example 2), the 200 μg / mL Coltsfoot Flower Extract group (Example 1), and the 200 μg / mL Deproteinized Polysaccharide group (Example 2) was significantly higher than that of the CSE (0.5%) group, suggesting that it has a certain repair effect on cell damage caused by CSE; Figure 13 As shown, the 100 μg / mL and 200 μg / mL decolorized polysaccharide groups in Example 3 also significantly improved cell survival rate after 48 hours of administration, suggesting that it has a certain repair effect on cell damage caused by CSE.

[0211] The above results indicate that the coltsfoot flower extract, deproteinized polysaccharide, and decolorized polysaccharide provided by this invention can all alleviate the symptoms of chronic obstructive pulmonary disease (COPD) and have certain therapeutic effects on COPD.

[0212] 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 coltsfoot flower extract, characterized in that, The preparation method of the coltsfoot flower extract includes the following steps: coltsfoot flowers are heated and defatted, then extracted with water, and then precipitated with alcohol to obtain a precipitate, which is then dried to obtain the coltsfoot flower extract.

2. The coltsfoot flower extract according to claim 1, characterized in that, The coltsfoot flower extract is a coltsfoot flower polysaccharide.

3. The coltsfoot flower extract according to claim 2, characterized in that, The coltsfoot flower polysaccharide includes fructose, glucose, arabinose, galactose, and glucosamine.

4. The coltsfoot flower extract according to claim 3, characterized in that, The molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.7-0.9: 0.1-0.2: 0.005-0.01: 0.001-0.003: 0.001-0.

003.

5. The coltsfoot flower extract according to claim 4, characterized in that, The molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.75-0.9: 0.1-0.15: 0.006-0.009: 0.001-0.002: 0.001-0.

002.

6. The coltsfoot flower extract according to claim 1, characterized in that, The coltsfoot flower extract contains no less than 20% sugar by weight.

7. The coltsfoot flower extract according to claim 1, characterized in that, The molecular weight of the coltsfoot flower extract is 1.21 × 10⁻⁶. 2 -6.58×10 6 Da.

8. The coltsfoot flower extract according to claim 7, characterized in that, The molecular weight of the coltsfoot flower extract is 1.21 × 10⁻⁶. 2 -1.63×10 3 Da.

9. The coltsfoot flower extract according to any one of claims 1-8, characterized in that, The preparation method of the coltsfoot flower extract includes the following steps: Step 1-1: Mix coltsfoot flowers with solvent, heat to degrease, filter, and air dry to obtain degreased medicinal material; Steps 1-2: Mix defatted medicinal materials with water, heat to extract, combine the extracts, concentrate to obtain concentrated solution; Steps 1-3: Mix the concentrated liquid with the alcohol solution to obtain a precipitate, then dry it to obtain the coltsfoot flower extract.

10. The coltsfoot flower extract according to claim 9, characterized in that, In step 1-1, the solvent is selected from at least one of ethanol, petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether, and methanol.

11. The coltsfoot flower extract according to claim 9, characterized in that, In step 1-1, the heating is performed 1-3 times.

12. The coltsfoot flower extract according to claim 11, characterized in that, In step 1-1, the heating is 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 1:7-9 g / mL; the heating time is 1-1.5 h.

13. The coltsfoot flower extract according to claim 9, characterized in that, In steps 1-2, the heating is performed 1-3 times.

14. The coltsfoot flower extract according to claim 13, characterized in that, In steps 1-2, the heating is performed twice; for 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; for the second heating, the ratio of defatted medicinal material to water is 1:7-9 g / mL; the heating time is 1-1.5 h.

15. The coltsfoot flower extract according to claim 9, characterized in that, In steps 1-2, the concentration refers to concentrating the raw medicinal material to a concentration of 0.5-1.5 mg / mL.

16. The coltsfoot flower extract according to claim 9, characterized in that, In steps 1-3, the alcohol solution is ethanol; in the final solution obtained by mixing the concentrate and the alcohol solution, the mass fraction of ethanol is 60%-95%.

17. The coltsfoot flower extract according to claim 1, characterized in that, The coltsfoot flower extract contains no more than 42% protein by weight.

18. A deproteinized polysaccharide, characterized in that, The deproteinized polysaccharide is prepared from the coltsfoot flower extract according to any one of claims 1-17 by deproteinization, and the molecular weight of the deproteinized polysaccharide is 4.86 × 10⁻⁶. 3 -4.34×10 6 Da.

19. The deproteinized polysaccharide according to claim 18, characterized in that, The deproteinized polysaccharide contains fructose, arabinose, glucose, and galactose.

20. The deproteinized polysaccharide according to claim 19, characterized in that, The deproteinized polysaccharide contains not less than 50% sugar and not more than 6% protein by mass.

21. The deproteinized polysaccharide according to claim 19, characterized in that, The molar ratio of fructose, arabinose, glucose and galactose is 0.8-0.9:0.07-0.085:0.06-0.072:0.01-0.

025.

22. The deproteinized polysaccharide according to claim 21, characterized in that, The molar ratio of fructose, arabinose, glucose and galactose is 0.82-0.88:0.075-0.080:0.062-0.070:0.012-0.

020.

23. The method for preparing deproteinized polysaccharide according to any one of claims 18-22, characterized in that, Includes the following steps: Step 2-1: Dissolve and enzymatically hydrolyze the coltsfoot flower extract; Step 2-2: The supernatant after enzymatic hydrolysis in Step 2-1 is concentrated to obtain a concentrated solution; Step 2-3: The concentrated solution from Step 2-2 is mixed with the alcohol solution to obtain a precipitate; Step 2-4: The precipitate from step 2-3 is dried to obtain the deproteinized polysaccharide.

24. The preparation method according to claim 23, characterized in that, In step 2-1, the solvent used for dissolution is water, the enzyme is papain, the concentration of the papain is 8-12 g / L, the enzymatic hydrolysis temperature is 40℃-60℃, and the time is 3h-5h.

25. The preparation method according to claim 23, characterized in that, In step 2-2, the concentration is to concentrate the raw drug to a concentration of 0.5-1.5 mg / mL.

26. The preparation method according to claim 23, characterized in that, In steps 2-3, the alcohol solution is ethanol; in the final solution obtained by mixing the concentrate and the alcohol solution, the mass fraction of ethanol is 60%-95%.

27. A decolorized polysaccharide, characterized in that, The decolorized polysaccharide was obtained by decolorizing the coltsfoot flower extract according to any one of claims 1-17, and the molecular weight of the decolorized polysaccharide was 1.42 × 10⁻⁶. 2 -1.05×10 7 Da.

28. The decolorized polysaccharide according to claim 27, characterized in that, The decolorized polysaccharide contains fructose, arabinose, glucose, and galactose.

29. The decolorized polysaccharide according to claim 28, characterized in that, The molar ratio of fructose, arabinose, glucose and galactose is 0.8-0.9:0.07-0.085:0.06-0.072:0.01-0.

022.

30. The decolorized polysaccharide according to claim 29, characterized in that, The molar ratio of fructose, arabinose, glucose and galactose is 0.82-0.86:0.07-0.080:0.06-0.070:0.01-0.

020.

31. The method for preparing the decolorized polysaccharide according to any one of claims 27-30, characterized in that, Includes the following steps: Coltsfoot flower extract was adsorbed using macroporous resin to obtain decolorized polysaccharides.

32. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the following: the coltsfoot flower extract according to any one of claims 1-17, the deproteinized polysaccharide according to any one of claims 18-26, or the decolorized polysaccharide according to any one of claims 27-31.

33. Use of the coltsfoot flower extract according to any one of claims 1-17, the deproteinized polysaccharide according to any one of claims 18-26, the decolorized polysaccharide according to any one of claims 27-31, or the pharmaceutical composition according to claim 32 in the preparation of a medicament for respiratory diseases.

34. The use according to claim 33, characterized in that, The respiratory diseases mentioned include asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

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