Preparation of a polysaccharide from coltsfoot flower by ultrafiltration and use thereof

Abstract

This invention relates to the field of pharmaceutical technology, specifically disclosing a coltsfoot flower polysaccharide and its uses. The coltsfoot flower polysaccharide is prepared by ultrafiltration and comprises at least one of coltsfoot flower polysaccharide fraction one, coltsfoot flower polysaccharide fraction two, and coltsfoot flower polysaccharide fraction three; the molecular weight of coltsfoot flower polysaccharide fraction one is 1.46 × 10⁻⁶. 3 -1.02×10 7 Da, composed of fructose, glucose, and glucosamine, has a molecular weight of 1.02 × 10⁻⁶ for the second polysaccharide fraction of coltsfoot flower. 2 -4.02×10 6 Da, composed of fructose and glucose, has a molecular weight of 5.57 × 10³ for the third polysaccharide fraction of coltsfoot flower. 2 -1.38×10 7 Da is composed of fructose, glucose, arabinose, galactose, and glucosamine. These polysaccharides have shown 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 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.

[0006] Chinese invention patent application 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 patent publications or literature reports on in-depth research on the use of coltsfoot flower polysaccharides for the treatment of respiratory diseases. Summary of the Invention

[0008] The purpose of this invention is to provide a coltsfoot flower polysaccharide and its uses. Whole animal model experiments have confirmed its effectiveness in preventing and treating asthma, rhinitis, cough, and chronic obstructive pulmonary disease (COPD). Cell experiments have demonstrated its repairing effect on COPD damage, and it can be further used to develop innovative traditional Chinese medicines for the prevention and treatment of respiratory diseases.

[0009] To achieve the above-mentioned objectives, the technical solution of this invention is as follows:

[0010] On one hand, the present invention provides a coltsfoot flower polysaccharide, wherein the coltsfoot flower polysaccharide comprises at least one of coltsfoot flower polysaccharide fraction one, coltsfoot flower polysaccharide fraction two, and coltsfoot flower polysaccharide fraction three; wherein the molecular weight of coltsfoot flower polysaccharide fraction one is 1.46 × 10⁻⁶. 3 -1.02×10 7 Da, the molecular weight of the second polysaccharide fraction of the coltsfoot flower is 1.02 × 10⁻⁶. 2 -4.02×10 6 Da, the molecular weight of the third polysaccharide fraction of the coltsfoot flower is 5.57 × 10⁻⁶. 2 -1.38×10 7 Da.

[0011] Preferably, the molecular weight of the main peak of the first polysaccharide fraction of the coltsfoot flower is selected from 1.46 × 10⁻⁶. 3 -1.06×10 4 Da.

[0012] Preferably, the molecular weight of the main peak of the second polysaccharide fraction of coltsfoot flower is selected from 1.02 × 10⁻⁶. 2 -1.50×10 5 Da.

[0013] Preferably, the molecular weight of the main peak of the third fraction of the coltsfoot flower polysaccharide is selected from 5.57 × 10⁻⁶. 2 -1.10×10 3 Da.

[0014] Preferably, the first polysaccharide fraction of coltsfoot flower contains fructose, glucose, and glucosamine;

[0015] Preferably, the molar ratio of fructose, glucose, and glucosamine is 0.80-0.90:0.10-0.20:0.001-0.005;

[0016] More preferably, the molar ratio of fructose, glucose, and glucosamine is 0.82-0.86:0.10-0.18:0.002-0.005;

[0017] More preferably, the molar ratio of fructose: glucose: glucosamine is 0.842:0.154:0.004.

[0018] Preferably, the second polysaccharide fraction of the coltsfoot flower contains fructose and glucose;

[0019] The molar ratio of fructose to glucose is 0.80-0.95:0.10-0.20;

[0020] More preferably, the molar ratio of fructose to glucose is 0.83-0.92:0.10-0.15;

[0021] More preferably, the molar ratio of fructose to glucose is 0.877:0.123.

[0022] Preferably, the third polysaccharide fraction of Coltsfoot flower contains fructose, glucose, arabinose, galactose, and glucosamine;

[0023] The molar ratio of fructose, glucose, arabinose, galactose, and glucosamine is 0.80-0.90:0.05-0.15:0.02-0.035:0.004-0.010:0.001-0.003;

[0024] More preferably, the molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.82-0.90: 0.08-0.15: 0.02-0.030: 0.005-0.010: 0.001-0.002.

[0025] More preferably, the molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.854:0.108:0.029:0.007:0.002.

[0026] On the other hand, the present invention provides a method for preparing the above-mentioned coltsfoot flower polysaccharide, the method comprising the following steps:

[0027] Step 1: Mix coltsfoot flowers with an alcohol solution, heat to defatt the mixture, filter, and air dry to obtain defatted medicinal material;

[0028] Step 2: Mix the defatted medicinal materials obtained in Step 1 with water, heat and extract, combine the extracts, concentrate to obtain concentrated solution;

[0029] Step 3: Mix the concentrated solution obtained in Step 2 with the alcohol solution to obtain a precipitate, dry it, and obtain the coltsfoot flower extract;

[0030] Step 4: Dissolve the coltsfoot extract from Step 3 and ultrafilter it using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a filtrate containing coltsfoot polysaccharide fraction one and a remaining solution containing coltsfoot polysaccharide fraction two and coltsfoot polysaccharide fraction three.

[0031] Preferably, step 4 is followed by step 5:

[0032] Step 5: Use an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to ultrafilter the remaining solution obtained in Step 4 for 20-60 minutes to obtain a filtrate containing the second polysaccharide fraction of Coltsfoot and a solution containing the third polysaccharide fraction of Coltsfoot.

[0033] Preferably, step 5 is followed by step 6:

[0034] Step 6: Concentrate and dry the filtrate containing the first polysaccharide fraction of coltsfoot flower described in Step 4, the filtrate containing the second polysaccharide fraction of coltsfoot flower described in Step 5, and the solution containing the third polysaccharide fraction of coltsfoot flower, respectively, to obtain the first polysaccharide fraction of coltsfoot flower, the second polysaccharide fraction of coltsfoot flower, and the third polysaccharide fraction of coltsfoot flower.

[0035] Preferably, in step 1, the alcohol solution is an ethanol solution;

[0036] Preferably, the ethanol solution has a mass fraction of 50%-95%; more preferably, the ethanol solution has a mass fraction of 85%-95%; and even more preferably, the ethanol solution has a mass fraction of 95%.

[0037] Preferably, in step 1, the heating is performed 1-3 times;

[0038] Preferably, in step 1, the heating is performed twice;

[0039] Preferably, in the first heating, the ratio of defatted medicinal material to water is 1:9-11 g / mL; the heating time is 2-2.5 h; in the second heating, the ratio of defatted medicinal material to water is 1:7-8 g / mL; the heating time is 1-1.5 h.

[0040] More preferably, in the first heating, the ratio of defatted medicinal material to water is 1:10 g / mL; the heating time is 2 hours; in the second heating, the ratio of defatted medicinal material to water is 1:8 g / mL; the heating time is 1.5 hours.

[0041] Preferably, in step 2, the heating is performed 1-3 times;

[0042] Preferably, in step 2, the heating is performed twice;

[0043] Preferably, in the first heating, the ratio of defatted medicinal material to water is 1:9-11 g / mL; the heating time is 2-2.5 h; in the second heating, the ratio of defatted medicinal material to water is 1:7-8 g / mL; the heating time is 1-1.5 h.

[0044] More preferably, in the first heating, the ratio of defatted medicinal material to water is 1:10 g / mL; the heating time is 2 hours; in the second heating, the ratio of defatted medicinal material to water is 1:8 g / mL; the heating time is 1.5 hours.

[0045] Preferably, in step 2, the solvent is water; more preferably, the water is selected from at least one of ultrapure water, deionized water, and distilled water; even more preferably, the water is selected from ultrapure water.

[0046] Preferably, in step 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, the concentration is to concentrate the raw medicinal material to a concentration of 1 mg / mL.

[0047] Preferably, in step 3, the alcohol solution is an ethanol solution;

[0048] Preferably, the ethanol solution has a mass fraction of 50%-95%; more preferably, the ethanol solution has a mass fraction of 85%-95%; and even more preferably, the ethanol solution has a mass fraction of 95%.

[0049] Preferably, in step 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 3, the final solution obtained by mixing the concentrate and the alcohol solution has an ethanol mass fraction of 80%.

[0050] In another aspect, the present invention provides a pharmaceutical composition comprising at least one of the above-described coltsfoot polysaccharide fraction one, coltsfoot polysaccharide fraction two, and coltsfoot polysaccharide fraction three.

[0051] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0052] In another aspect, the present invention provides the use of the above-mentioned coltsfoot flower polysaccharide in the preparation of medicaments for treating respiratory diseases.

[0053] Preferably, the respiratory diseases include asthma, rhinitis, cough, and chronic obstructive pulmonary disease (COPD). The beneficial effects of this invention are: the coltsfoot flower extract provided by this invention has good effects in preventing and treating asthma, rhinitis, cough, and COPD, providing a new option for preparing innovative traditional Chinese medicines for the prevention and treatment of respiratory diseases. Attached Figure Description

[0054] Figure 1 This is the molecular weight distribution spectrum of Example 1 of the present invention.

[0055] Figure 2 This is the molecular weight distribution spectrum of Example 2 of the present invention.

[0056] Figure 3 This is the molecular weight distribution spectrum of Example 3 of the present invention.

[0057] Figure 4 This is a monosaccharide composition test spectrum of Example 1 of the present invention.

[0058] Figure 5 This is a monosaccharide composition test spectrum of Example 2 of the present invention.

[0059] Figure 6 This is a monosaccharide composition test spectrum of Example 3 of the present invention.

[0060] Figure 7 The bar chart shows the IL-5 level and inflammatory cell count levels in the asthma efficacy tests of Examples 1-3 of the present invention.

[0061] Figure 8 This is a statistical chart showing the number of coughs and the cough latency period in the antitussive efficacy test of Example 1 of the present invention.

[0062] Figure 9 This is a statistical chart showing the number of coughs and the cough latency period in the antitussive efficacy test of Example 2 of the present invention.

[0063] Figure 10 This is a statistical chart showing the number of coughs and the cough latency period in the antitussive efficacy test of Example 3 of the present invention.

[0064] Figure 11 This is a statistical chart showing the number of times the nose was scratched during the rhinitis efficacy test in Example 3 of the present invention.

[0065] Figure 12 This is a diagram showing the effect of Examples 1-3 of the present invention on an in vitro model of CSE-induced chronic obstructive pulmonary disease. Detailed Implementation

[0066] 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.

[0067] 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%.

[0068] 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.

[0069] This invention provides a method for producing coltsfoot flower polysaccharide, the method comprising the following steps:

[0070] Step 1: Mix coltsfoot flowers with an ethanol solution and heat to defatt the mixture 1-3 times;

[0071] Step 2: After defatting, the defatted medicinal materials are mixed with solvent and heated for extraction 1-3 times. The extracts are combined and concentrated to obtain a concentrated solution.

[0072] Step 3: Add ethanol to the concentrate obtained in Step 2, precipitate and dry to obtain coltsfoot flower extract;

[0073] Step 4: Dissolve the coltsfoot flower extract in water and ultrafilter for 20-60 minutes using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a filtrate containing coltsfoot flower polysaccharide fraction one, and a residual solution containing coltsfoot flower polysaccharide fraction two and coltsfoot flower polysaccharide fraction three; wherein, the molecular weight of coltsfoot flower polysaccharide fraction one is 1.46 × 10⁻⁶. 3 -1.02×10 7 Da, the molecular weight of the main peak is selected from 1.46 × 10. 3 -1.06×10 4 Da.

[0074] Step 5: Use an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to ultrafilter the remaining solution obtained in Step 4 for 20-60 minutes to obtain a filtrate containing the second polysaccharide fraction of Coltsfoot and a remaining solution containing the third polysaccharide fraction of Coltsfoot; wherein the molecular weight of the second polysaccharide fraction of Coltsfoot is 1.02 × 10⁻⁶. 2 -4.02×10 6 Da, the molecular weight of the main peak is selected from 1.02×10. 2 -1.50×10 5 The molecular weight of the third polysaccharide fraction of coltsfoot flower is 5.57 × 10⁻⁶. 2 -1.38×10 7 Da, the molecular weight of the main peak is selected from 5.57 × 10. 2 -1.10×10 3 Da.

[0075] Step 6: Concentrate and dry the filtrate containing the first polysaccharide fraction of Coltsfoot, the filtrate containing the second polysaccharide fraction of Coltsfoot, and the remaining solution containing the third polysaccharide fraction of Coltsfoot to obtain the first, second, and third polysaccharide fractions of Coltsfoot. The first polysaccharide fraction of Coltsfoot contains fructose:glucose:glucosamine (molar ratio: 0.842:0.154:0.004); the second polysaccharide fraction of Coltsfoot contains fructose and glucose (molar ratio: 0.877:0.123); the third polysaccharide fraction of Coltsfoot contains fructose:glucose:arabinose:galactose:glucosamine; the molar ratio of fructose:glucose:arabinose:galactose:glucosamine is 0.854:0.108:0.029:0.007:0.002.

[0076] Preferably, in step 1, the concentration of the ethanol solution is 50%-95%; and the heating is performed twice, with the material-to-liquid ratios being 1:10 g / mL and 1:8 g / mL respectively, and the reflux times being 2 h and 1.5 h respectively; in step 2, the heating is performed twice; with the material-to-liquid ratios being 1:10 g / mL and 1:8 g / mL respectively, and the reflux times being 2 h and 1.5 h respectively; in step 2, the concentration is to concentrate to a crude drug concentration of 1 mg / mL; in step 3, the concentration of the added ethanol is 95%, and the concentration of ethanol in the final solution of step 3 is 60%-95%.

[0077] 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.

[0078] 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 to this.

[0079] 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.

[0080] 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.

[0081] Example

[0082] Examples 1 to 3:

[0083] The instruments used in Examples 1 to 3 included an ultrafiltration machine (SIEMENS, KM-3838-2), an ultrafiltration membrane (SpiralMembrane element, Kaimitech, USA), and an oven (Being, BO-200FL).

[0084] Take the Coptis chinensis flower herb, add 90% ethanol, heat and reflux twice for defatting, 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, air-dry the defatted herb, then extract twice with water by heating, with material-to-liquid ratios of 1:10 g / mL and 1:8 g / mL, and extraction times of 2 h and 1.5 h, respectively. The extracts were combined and concentrated to a concentration of 1 mg / mL 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 a total polysaccharide sample from Tussilago farfara. The total polysaccharide sample from Tussilago farfara was then ultrafiltered for 30 min using a 10 kDa ultrafiltration membrane to obtain a solution containing components with a molecular weight less than 10 kDa (Example 1, i.e., Tussilago farfara polysaccharide fraction 1). The remaining solution was ultrafiltered for 30 min using a 50 kDa ultrafiltration membrane to obtain a solution containing components with a molecular weight of 10 kDa-50 kDa (Example 2, i.e., Tussilago farfara polysaccharide fraction 2) and a solution containing components with a molecular weight greater than 50 kDa (Example 3, i.e., Tussilago farfara polysaccharide fraction 3). The solutions were concentrated and dried to obtain the samples of Examples 1-3.

[0085] Test Example 1: Determination of molecular weight in Examples 1-3

[0086] 1. Laboratory supplies:

[0087] Samples obtained in Examples 1-3, sodium chloride, etc.

[0088] 2. Experimental Methods:

[0089] The molecular weights of the products obtained in Examples 1-4 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.

[0090] 3. Experimental Results:

[0091] 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. The data are shown in Table 1, and the molecular weight distribution spectrum is shown in the figure. Figure 1-3 As shown, where Figure 1 , Figure 2 , Figure 3 The molecular weight distribution spectra of Examples 1, 2, and 3 are shown in sequence.

[0092] Table 1

[0093]

[0094] Test Example 2: Determination of Monosaccharide Composition in Examples 1-3

[0095] 1. Laboratory supplies:

[0096] The samples obtained in Examples 1-3 contained trifluoroacetic acid (ACROS), 50% sodium hydroxide solution (AlfaAesar), 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)), salt The following monosaccharide standards were obtained: N-acetylglucosamine (A22S6X3606), N-acetyl-D-glucosamine (A21J8X40372), D-fructose (J01J10R89818), D-ribose (H26F10Z81556), galactosamine hydrochloride (B01J8S37079), L-guluronic acid (S200115AG1), and D-mannuronic acid (S200108AM1). All monosaccharide standards were sourced from Borui Sugar Biotechnology.

[0097] 2. Experimental Methods:

[0098] 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.

[0099] 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.

[0100] 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 Phase A / B / C (50:50:0, V / V), 30.1 min Phase A / B / C (0:0:100, V / V), 46 min Phase A / B / C (0:0:100, V / V), 46.1 min Phase A / B / C (0:100:0, V / V), 50 min Phase A / B / C (0:100:0, V / V), 50.1 min Phase A / B / C (98.8:1.2:0, V / V), 80 min Phase A / B / C (98.8:1.2:0, V / V). Detector: Electrochemical detector.

[0101] 3. Experimental Results:

[0102] The experimental results are shown in Tables 2-4, and the relevant spectra are shown in [the table]. Figure 4-6 .

[0103] Table 2

[0104] Name Peak area RT Mole ratio ug / mg Glucosamine 1.043 11.792 0.004 2.70 glucose 20.917 14.608 0.154 78.37 fructose 48.365 18.983 0.842 428.51

[0105] Table 3

[0106] Name Peak area RT Mole ratio ug / mg glucose 13.945 14.558 0.123 52.25 fructose 41.968 18.917 0.877 371.84

[0107] Table 4

[0108] Name Peak area RT Mole ratio ug / mg Arabic sugar 3.55 10.434 0.029 9.12 Glucosamine 0.419 11.759 0.002 1.09 Galactose 0.68 12.85 0.007 2.53 glucose 10.755 14.55 0.108 40.30 fructose 36.009 18.925 0.854 319.04

[0109] Test Example 3: Efficacy Experiment of Drugs for Treating Asthma

[0110] 1. Reagents and instruments, etc.

[0111] Reagents:

[0112] 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.

[0113] instrument:

[0114] A 0.01% analytical balance (MSA2245CE, Sartorius), an ultrasonic nebulizer (402AI, Yuwell), an animal hematology analyzer (BC-5000VEI, Mindray), a benchtop refrigerated centrifuge (ST1R Plus, Thermoscientific), a multi-functional microplate reader (VICTORNIVO, PerkinElmer), and surgical instruments were used. Laboratory animals:

[0115] Balb / c mice, male, 6-8 weeks old.

[0116] 2. Experimental Methods:

[0117] Balb / c mice were used in the experiment and divided into 6 groups: normal group, model group, positive group (dexamethasone 2 mg / kg), and solid sample obtained in Examples 1-3 (350 mg / kg). The test drug was prepared to the required concentration using 5‰ sodium carboxymethyl cellulose. Mice other than the normal group were sensitized 3 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, respectively. After sensitization, the mice were given the drug starting on day 20. One hour after drug administration, they were challenged by nebulization (2% grade II OVA solution, 100 mL), each time lasting 1 hour, once a day, for 7 days.

[0118] 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.

[0119] 3. Experimental Results:

[0120] like Figure 7As shown, 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, eosinophils, lymphocytes and monocytes in the bronchoalveolar lavage fluid (BALF) of the test substance Example 1 was significantly reduced after administration (P values ​​were all less than 0.05). This confirms that the three test substances of Examples 1-3, especially Example 1, have a relieving effect on the OVA-induced asthma model.

[0121] Test Example 4:

[0122] Cough suppressant efficacy experiment

[0123] 1. Reagents and instruments, etc.

[0124] Reagents:

[0125] Pentoxyverine citrate (Lisheng Pharmaceutical), concentrated ammonia (A112079, Aladdin), etc.

[0126] instrument:

[0127] 0.01% balance (MSA2245CE, Sartorius), multi-functional cough and asthma induction device (model: YLS-8A), etc.

[0128] Laboratory animals:

[0129] SD rats, male, 6-8 weeks old.

[0130] 2. Experimental Methods:

[0131] Healthy SD rats were divided into a model group, a positive control group (pentoxyverine citrate 60 mg / kg), and a treatment group (400 mg / kg) containing the samples from Examples 1-3. The samples from Examples 1-3 and the positive control were diluted with 5‰ CMC-Na to the corresponding concentrations and administered by gavage, with preventative administration for 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.

[0132] 3. Experimental Results:

[0133] Experimental results are as follows Figure 8-10 As shown, compared with the model group, after administration, the treatment groups of samples 1-3 all significantly reduced the number of coughs and prolonged the cough latency in rats, indicating that samples 1-3 could alleviate cough.

[0134] Test Example 5:

[0135] Rhinitis drug efficacy experiment

[0136] 1. Reagents and instruments, etc.

[0137] Reagents:

[0138] 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).

[0139] instrument:

[0140] A 0.01 g balance (MSA2245CE, Sartorius) and a 10 μL pipette (Eppendorf).

[0141] Laboratory animals:

[0142] Balb / c mice, male, 6-8 weeks old

[0143] 2. Experimental Methods:

[0144] Healthy Balb / c mice were randomly divided into a model group and a positive control group (dexamethasone 2 mg / kg) and the sample obtained in Examples 1-3 (350 mg / kg). 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), 20 μL per nostril per mouse, 10 μL / nostril, once daily for 5 days.

[0145] 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.

[0146] 3. Experimental Results:

[0147] Experimental results showed that mice in the model group exhibited obvious nose scratching and sneezing after modeling, indicating successful modeling. Meanwhile, as... Figure 11 As shown, compared with the model group, the positive drug group and the sample of test drug Example 3 significantly reduced the number of times mice scratched their noses after intervention, indicating that the sample of Example 3 can relieve rhinitis.

[0148] Test Example 6: Efficacy Experiment of Pharmacotherapy for Chronic Obstructive Pulmonary Disease

[0149] 1. Reagents and Instruments

[0150] 1.1 Reagents

[0151] 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). The samples obtained in Examples 1-3 were prepared by the Natural Product Chemistry Group of the China Resources Jiangzhong Modern Traditional Chinese Medicine Research Center.

[0152] 1.2 Instruments

[0153] 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.

[0154] 1.3 Experimental Cells

[0155] BEAS-2B human bronchial epithelial cells

[0156] 2 Experimental Methods

[0157] 2.1 Preparation of Cigarette Extract

[0158] 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.

[0159] 2.2 Cell treatment methods

[0160] 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, cigarette extract (CSE) was prepared according to literature methods. CSE modeling was performed according to experimental groups. After drug intervention for 48 hours, CCK8 working solution was added and incubated for 2-4 hours at the experimental endpoint, and the cell absorbance was measured.

[0161] Calculate cell viability based on absorbance values ​​of each cell group:

[0162] 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%.

[0163] 3 Experimental Results

[0164] CCK8 cell viability test results are as follows: Figure 11 As shown, cell viability was significantly reduced under CSE (0.5%) conditions. After 48 hours of drug administration, the cell viability of the samples obtained in Examples 1-3 was higher than that of the CSE (0.5%) group at concentrations of 100 μg / mL and 200 μg / mL.

[0165] The above results demonstrate that the samples of Examples 1-3 provided by this invention all exhibit certain therapeutic effects on chronic obstructive pulmonary disease.

[0166] 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 polysaccharide, characterized in that, The coltsfoot polysaccharide comprises at least one of coltsfoot polysaccharide fraction one, coltsfoot polysaccharide fraction two, and coltsfoot polysaccharide fraction three; the molecular weight of coltsfoot polysaccharide fraction one is 1.46 × 10⁻⁶. 3 -1.02×10 7 Da, the molecular weight of the second polysaccharide fraction of the coltsfoot flower is 1.02 × 10⁻⁶. 2 -4.02×10 6 Da, the molecular weight of the third polysaccharide fraction of the coltsfoot flower is 5.57 × 10⁻⁶. 2 -1.38×10 7 Da.

2. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The molecular weight of the main peak of the first polysaccharide fraction of the coltsfoot flower was selected from 1.46 × 10⁻⁶. 3 -1.06×10 4 Da.

3. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The molecular weight of the main peak of fraction two of the coltsfoot flower polysaccharide was selected from 1.02 × 10⁻⁶. 2 -1.50×10 5 Da.

4. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The molecular weight of the main peak of fraction 3 of the coltsfoot flower polysaccharide was selected from 5.57 × 10⁻⁶. 2 -1.10×10 3 Da.

5. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The first polysaccharide fraction of the coltsfoot flower contains fructose, glucose, and glucosamine.

6. The coltsfoot flower polysaccharide according to claim 5, characterized in that, The molar ratio of fructose, glucose and glucosamine in the first polysaccharide fraction of the coltsfoot flower is 0.80-0.90:0.10-0.20:0.001-0.

005.

7. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The second polysaccharide fraction of the coltsfoot flower contains fructose and glucose.

8. The coltsfoot flower polysaccharide according to claim 7, characterized in that, The molar ratio of fructose and glucose in the polysaccharide fraction of the coltsfoot flower is 0.80-0.95:0.10-0.

20.

9. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The third polysaccharide fraction of the coltsfoot flower contains fructose, glucose, arabinose, galactose, and glucosamine.

10. The coltsfoot flower polysaccharide according to claim 9, characterized in that, The molar ratio of fructose, glucose, arabinose, galactose and glucosamine in the three polysaccharide fractions of the coltsfoot flower is 0.80-0.90: 0.05-0.15: 0.02-0.035: 0.004-0.010: 0.001-0.

003.

11. The method for preparing coltsfoot flower polysaccharide according to any one of claims 1-10, characterized in that, Includes the following steps: Step 1: Mix coltsfoot flowers with an alcohol solution, heat to defatt the mixture, filter, and air dry to obtain defatted medicinal material; Step 2: Mix the defatted medicinal materials obtained in Step 1 with water, heat and extract, combine the extracts, concentrate to obtain concentrated solution; Step 3: Mix the concentrated solution obtained in Step 2 with the alcohol solution to obtain a precipitate, dry it, and obtain the coltsfoot flower extract; Step 4: Dissolve the coltsfoot extract from Step 3 and ultrafilter it using an ultrafiltration membrane with a molecular weight cutoff of 10 kDa to obtain a filtrate containing coltsfoot polysaccharide fraction one and a remaining solution containing coltsfoot polysaccharide fraction two and coltsfoot polysaccharide fraction three.

12. The preparation method according to claim 11, characterized in that, Step 4 is followed by: Step 5: Use an ultrafiltration membrane with a molecular weight cutoff of 50 kDa to ultrafilter the remaining solution obtained in Step 4 for 20-60 minutes to obtain a filtrate containing the second polysaccharide fraction of Coltsfoot and a solution containing the third polysaccharide fraction of Coltsfoot.

13. The preparation method according to claim 12, characterized in that, Step 5 is followed by step 6: the filtrate containing the first polysaccharide part of coltsfoot flower in step 4, the filtrate containing the second polysaccharide part of coltsfoot flower in step 5, and the solution containing the third polysaccharide part of coltsfoot flower are concentrated and dried respectively to obtain the first polysaccharide part of coltsfoot flower, the second polysaccharide part of coltsfoot flower, and the third polysaccharide part of coltsfoot flower.

14. The preparation method according to claim 11, characterized in that, In step 1, the alcohol solution is an ethanol solution; the mass fraction of the ethanol solution is 50%-95%.

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

16. The preparation method according to claim 15, characterized in that, In step 1, 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 1:7-8 g / mL; the heating time is 1-1.5 h.

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

18. The preparation method according to claim 17, characterized in that, In step 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 1:7-8 g / mL; the heating time is 1-1.5 h.

19. The preparation method according to claim 11, characterized in that, In step 2, the concentration refers to concentrating the raw medicinal material to a concentration of 0.5-1.5 mg / mL.

20. The preparation method according to claim 11, characterized in that, In step 3, the alcohol solution is ethanol; in the final solution obtained by mixing the concentrated solution and the alcohol solution, the mass fraction of ethanol is 60%-95%.

21. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises at least one of the following: Coltsfoot polysaccharide fraction one, coltsfoot polysaccharide fraction two, and coltsfoot polysaccharide fraction three as described in claims 1-20.

22. The pharmaceutical composition according to claim 21, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients.

23. Use of the coltsfoot flower polysaccharide according to any one of claims 1-20 and the pharmaceutical composition according to any one of claims 21-22 in the preparation of a medicament for treating respiratory diseases.

24. The use according to claim 23, characterized in that, The respiratory diseases mentioned include asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

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