A coltsfoot flower polysaccharide prepared by fractional precipitation and its uses

Abstract

This invention relates to the field of pharmaceutical technology, specifically disclosing a coltsfoot flower polysaccharide and its uses. The polysaccharide is prepared by ethanol precipitation and fractionation, and comprises fructose, glucose, arabinose, galactose, and glucosamine; wherein the molar ratio of fructose, glucose, arabinose, galactose, and glucosamine is 0.2-1.2:0.05-0.3:0.01-0.035:0.001-0.006:0.001-0.005. The above polysaccharide has good effects in preventing and treating asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a 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 patent 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. This composition 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 ethanol precipitation and fractionation polysaccharides for the treatment of respiratory diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a coltsfoot flower polysaccharide and its uses. This invention discloses a graded coltsfoot flower polysaccharide, which has been shown to have ideal cough-preventing effects in whole-animal model experiments, and cell experiments have demonstrated its repairing effect on damage caused by chronic obstructive pulmonary disease. It can be further used to develop and prepare innovative traditional Chinese medicines for the prevention and treatment of respiratory diseases.

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

[0009] On one hand, the present invention provides a coltsfoot flower polysaccharide, which comprises fructose, glucose, arabinose, galactose and glucosamine; wherein the molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.2-1.2:0.05-0.3:0.01-0.035:0.001-0.006:0.001-0.005.

[0010] Preferably, the molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.5-1.0: 0.10-0.20: 0.01-0.020: 0.001-0.005: 0.001-0.004.

[0011] More preferably, the molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.802:0.173:0.018:0.004:0.003.

[0012] Preferably, the coltsfoot flower polysaccharide comprises graded polysaccharide fraction one and / or graded polysaccharide fraction two, wherein graded polysaccharide fraction one and graded polysaccharide fraction two comprise fructose, glucose, arabinose, galactose and glucosamine.

[0013] Preferably, in the graded polysaccharide fraction one, the molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.5-0.82: 0.15-0.20: 0.01-0.02: 0.003-0.006: 0.002-0.004.

[0014] Preferably, in the graded polysaccharide fraction two, the molar ratio of arabinose, galactose, glucose and galacturonic acid is 0.5-0.9: 0.1-0.15: 0.005-0.015: 0.001-0.005: 0.0005-0.0015.

[0015] Preferably, the molecular weight of the first fraction of the graded polysaccharide is selected from 7.05 × 10⁻⁶. 2 -6.55×10 6 Da.

[0016] Preferably, the molecular weight of the major peak of the graded polysaccharide fraction is 7.05 × 10⁻⁶. 2 -1.40×10 3 Da.

[0017] Preferably, the molecular weight of the second graded polysaccharide fraction is selected from 1.90 × 10⁻⁶. 3 -5.71×10 6 Da.

[0018] Preferably, the molecular weight of the two main peaks of the graded polysaccharide fraction is 1.90 × 10⁻⁶. 3 -2.45×10 3 Da.

[0019] On the other hand, the present invention provides a method for preparing the above-mentioned coltsfoot flower polysaccharide, comprising the following steps:

[0020] Coltsfoot flowers were defatted, extracted with water, and precipitated with alcohol to obtain the precipitate, which was then dried to obtain coltsfoot flower polysaccharides.

[0021] Preferably, the preparation method includes the following steps:

[0022] Step 1: Mix coltsfoot flowers with an alcohol solution, heat to defatt the mixture, filter, and obtain defatted medicinal material;

[0023] Step 2: Mix the defatted medicinal materials from Step 1 with water, heat to extract, combine the extracts, concentrate to obtain a concentrated solution;

[0024] Step 3: Mix the concentrated solution from Step 2 with the alcohol solution, remove the solvent, until the mass fraction of alcohol in the final solution is 25%-35%, obtain the precipitate, dry it, and fractionate the polysaccharide fraction 1.

[0025] More preferably, the mass fraction of alcohol in the final solution is 30%.

[0026] Preferably, step 3 is followed by:

[0027] Step 4: Mix the supernatant after removing the precipitate in Step 3 with the alcohol solution to remove the solvent until the alcohol concentration in the final solution is 75%-85%, obtain the precipitate, dry it, and fractionate the polysaccharide fraction II.

[0028] Further, preferably, the concentration of alcohol in the final solution is 80%.

[0029] Preferably, in step 1, the alcohol solution is an aqueous solution of ethanol. The degreasing solvent is not limited to an alcohol solution, and can also be petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether, and methanol.

[0030] Preferably, the aqueous solution of ethanol has a mass fraction of 50%-95%;

[0031] Preferably, the mixture is degreased.

[0032] Preferably, the heating is performed twice;

[0033] Preferably, for the first heating, the ratio of coltsfoot flower to alcohol solution is 1:9-11 g / mL; the heating time is 2-2.5 h; for the second heating, the ratio of coltsfoot flower to alcohol solution is 1:7-9 g / mL; the heating time is 1-1.5 h.

[0034] More preferably, in the first heating, the ratio of coltsfoot flower to alcohol solution is 1:10 g / mL, and the heating time is 2 hours; in the second heating, the ratio of coltsfoot flower to alcohol solution is 1:8 g / mL, and the heating time is 1.5 hours.

[0035] Preferably, in step 2, the water is selected from at least one of ultrapure water, deionized water, and distilled water; more preferably, in step 2, the water is selected from ultrapure water.

[0036] Preferably, in step 2, the heating is performed twice;

[0037] 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.

[0038] 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.

[0039] Preferably, in step 3, the alcohol solution is an ethanol solution;

[0040] More preferably, the ethanol solution has a mass fraction of 95%.

[0041] Preferably, in step 3, the final solution contains 30% alcohol by mass.

[0042] Preferably, in step 4, the alcohol solution is an ethanol solution;

[0043] More preferably, the ethanol solution has a mass fraction of 95%.

[0044] Preferably, in step 4, the mass fraction of alcohol in the final solution is 80%.

[0045] In another aspect, the present invention provides a pharmaceutical composition comprising the above-described coltsfoot polysaccharide.

[0046] Preferably, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0047] 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.

[0048] Preferably, the respiratory diseases include asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] The coltsfoot flower polysaccharide provided by this invention has good effects in preventing and treating asthma, rhinitis, cough and chronic obstructive pulmonary disease, and provides a new option for preparing innovative traditional Chinese medicine for the prevention and treatment of respiratory diseases. Attached Figure Description

[0051] Figure 1 This is a molecular weight distribution map of the graded polysaccharides in Example 1 of the present invention.

[0052] Figure 2 This is a molecular weight distribution map of the graded polysaccharides in Example 2 of the present invention.

[0053] Figure 3 This is a monosaccharide composition test spectrum of Example 1 of the present invention.

[0054] Figure 4 This is a monosaccharide composition test spectrum of Example 2 of the present invention.

[0055] Figure 5 Bar charts showing IL-5 levels and inflammatory cell counts in asthma efficacy trials of Examples 1-2 of this invention.

[0056] Figure 6 This is a statistical chart showing the number of coughs during the antitussive efficacy test in Examples 1-2 of the present invention.

[0057] Figure 7 This is a statistical chart showing the number of times the nose was scratched during the rhinitis efficacy test in Example 2 of the present invention.

[0058] Figure 8 This is a diagram illustrating the effect of Example 1 of the present invention on an in vitro model of CSE-induced chronic obstructive pulmonary disease.

[0059] Figure 9 This is a diagram illustrating the effect of Embodiment 2 of the present invention on an in vitro model of CSE-induced chronic obstructive pulmonary disease. Detailed Implementation

[0060] 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.

[0061] 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%.

[0062] 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.

[0063] This invention provides a method for producing coltsfoot flower polysaccharide, the method comprising the following steps:

[0064] Step 1: Mix coltsfoot flower with alcohol solution, heat 1-3 times to defatting (the defatting solvent is not limited to alcohol solution, petroleum ether, ethyl acetate, cyclohexane, acetone, dichloromethane, chloroform, diethyl ether and methanol can also be used), filter to obtain defatted medicinal material;

[0065] Step 2: Mix the defatted medicinal materials from Step 1 with water, heat and extract 1-3 times, combine the extracts, concentrate to obtain concentrated solution;

[0066] Step 3: Mix the concentrated solution from Step 2 with the alcohol solution until the mass fraction of alcohol in the final solution is 25%-35%, to obtain a precipitate, dry it, and fractionate the polysaccharide fraction 1.

[0067] Step 4: Mix the supernatant from Step 3 with the alcohol solution until the concentration of ethanol in the final solution is 75%-85%, to obtain a precipitate, dry it, and fractionate the polysaccharide fraction II.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] Example 1:

[0073] Take the Tussilago farfara flower herb, add 90% ethanol, and heat under reflux for defatting twice, with material-to-liquid ratios of 1:10 and 1:8, and reflux times of 2 h and 1.5 h, respectively. After defatting, dry the defatted herb and extract it twice with water under heat, with material-to-liquid ratios of 1:10 and 1:8, and reflux times of 2 h and 1.5 h, respectively. Combine the extracts and concentrate to a crude drug concentration of 1 mg / mL. Slowly add ethanol until the final concentration of the solution is 30%. Let it stand overnight to obtain a precipitate and supernatant. Dry the precipitate to obtain the 30% graded polysaccharide fraction of Tussilago farfara flower (i.e., graded polysaccharide fraction one) of Example 1.

[0074] Example 2:

[0075] Take the supernatant after removing the precipitate in Example 1, slowly add ethanol to the supernatant until the final concentration of the solution is 80%, let it stand overnight to obtain the precipitate, and dry the precipitate to obtain the 80% graded polysaccharide fraction of Tussilago farfara flower in Example 2 (i.e. graded polysaccharide fraction two).

[0076] Determination of molecular weight in Examples 1-2

[0077] 1. Laboratory supplies:

[0078] The products obtained in Examples 1-2, sodium chloride, etc.

[0079] 2. Experimental Methods:

[0080] 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.

[0081] 3. Experimental Results:

[0082] 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 1-2 As shown.

[0083] Table 1

[0084]

[0085] Test Example 1: Determination of Monosaccharide Composition in Examples 1-2

[0086] 1. Laboratory supplies:

[0087] The solid samples obtained in Examples 1-2 contained trifluoroacetic acid (ACROS), 50% sodium hydroxide solution (Alfa Aesar), and sodium acetate (ThermoFishe). The equipment used included an ion chromatograph (ThermoFishe, ICS5000), an electrically heated constant-temperature drying oven (Lichen Technology, 101-1BS), a nitrogen evaporator (Lichen Technology, UGC-24M), 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.

[0088] 2. Experimental Methods:

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 3. Experimental Results:

[0093] The experimental results are shown in Tables 2-5, and the relevant spectra are shown in [the table]. Figure 3-4 Table 2 represents the sample from Example 1, and Table 2 represents the sample from Example 2.

[0094] Table 2

[0095]

[0096]

[0097] Table 3

[0098] Name Peak area RT Moor ratio ug / mg Arabic sugar 2.157 10.592 0.012 5.54 Glucosamine 0.333 11.942 0.001 0.86 Galactose 0.437 13.059 0.003 1.62 glucose 18.148 14.792 0.126 68.00 fructose 52.253 19.259 0.857 462.96

[0099] Test Example 2: Efficacy Experiment of Asthma Treatment

[0100] 1. Reagents and instruments, etc.

[0101] Reagents:

[0102] 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.

[0103] instrument:

[0104] 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.

[0105] Laboratory animals:

[0106] Balb / c mice, male, 6-8 weeks old.

[0107] 2. Experimental Methods:

[0108] Balb / c mice were used in the experiment and divided into 5 groups: normal group, model group, positive group (dexamethasone 2 mg / kg), sample group of Example 1, and sample group of Example 2. 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 starting 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.

[0109] 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.

[0110] 3. Experimental Results:

[0111] like Figure 5As shown, compared with the normal group, the concentration of the inflammatory factor IL-5 and the number of inflammatory cells in the bronchoalveolar lavage fluid of the model group were significantly increased after modeling. Compared with the model group, at a dosage of 350 mg / kg, the product obtained in Example 1 showed no statistically significant difference in improving the efficacy of inflammatory factors and inflammatory cells. However, the product obtained in Example 2 significantly reduced the concentration of the inflammatory factor IL-5 and the number of inflammatory cells in the bronchoalveolar lavage fluid (P < 0.001 or < 0.05), indicating that the product obtained in Example 2 has a therapeutic effect on asthma.

[0112] Test Example 3:

[0113] Cough suppressant efficacy experiment

[0114] 1. Reagents and instruments, etc.

[0115] Reagents:

[0116] Pentoxyverine citrate (Lisheng Pharmaceutical), concentrated ammonia (A112079, Aladdin), etc.

[0117] instrument:

[0118] A 0.01% balance (MSA2245CE, Sartorius) and a multi-functional cough and asthma induction device (model: YLS-8A).

[0119] Laboratory animals:

[0120] SD rats, male, 6-8 weeks old.

[0121] 2. Experimental Methods:

[0122] Healthy SD rats were divided into a model group, a positive control group (pentoxyverine citrate 60 mg / kg), Example 1 (200 mg / kg), and Example 2 (200 mg / kg). Rats in the model group were nebulized with 15% ammonia for 25 seconds. The modeling period was 1 day, and prophylactic administration was given for 3 days. The sample from Example 1 and the positive control drug pentoxyverine were diluted with 5‰ CMC-Na to the corresponding concentrations and administered by gavage, with prophylactic administration given 3 days in advance. 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, and continuously nebulized with 15% concentrated ammonia for 25 seconds. The number of coughs in the SD rats within 4 minutes was recorded. Cough symptoms included significant abdominal contraction or distension and wide-open mouth.

[0123] 3. Experimental Results:

[0124] Experimental results are as follows Figure 6As shown, compared with the model group, after administration, both Example 1 (200 mg / kg) and Example 2 (200 mg / kg) reduced the number of coughs in rats, and Example 2 had a better therapeutic effect, indicating that the products obtained in Example 1 and Example 2 can both exert an antitussive effect.

[0125] Test Example 4. Rhinitis Drug Efficacy Experiment

[0126] 1. Reagents and Instruments

[0127] 1.1 Reagents

[0128] 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). The polysaccharides in Examples 1-2 were prepared by the Natural Product Chemistry Group of the China Resources Jiangzhong Modern Traditional Chinese Medicine Research Center.

[0129] 1.2 Instruments

[0130] A 0.01 g balance (MSA2245CE, Sartorius) and a 10 μL pipette (Eppendorf).

[0131] 1.3 Experimental Animals

[0132] Balb / c, male, 6-8 weeks old

[0133] 2 Experimental Methods

[0134] Balb / c mice were used in the experiment, which was divided into three groups: a model group, a positive control group (dexamethasone 2 mg / kg), Example 2 (350 mg / kg), and Example 2 (175 mg / kg). The test drug was prepared to the required concentration using 5‰ sodium carboxymethyl cellulose. All mice were sensitized three times (0.5 mg / mL of Grade V OVA and 1.6 mg / mL Al(OH)3 suspension, 0.2 mL / mouse, i.p.), on days 0, 7, and 14. After sensitization, the mice were given the drug starting on day 20. One hour after drug administration, a challenge was performed by intranasal instillation (5% Grade II OVA solution), with 20 μL (10 μL / nostril) instilled into each mouse once daily for 5 days.

[0135] 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.

[0136] 3 Experimental Results

[0137] like Figure 7As shown, after modeling, the mice in the model group exhibited obvious nose scratching and sneezing, indicating successful modeling. Intervention with the products obtained in Example 2 (350mg / Kg, 175mg / Kg) significantly reduced the number of nose scratching and sneezing in the mice, indicating that Example 2 has a significant therapeutic effect on rhinitis.

[0138] Test Example 5. Efficacy Experiment of Pharmacotherapy for Chronic Obstructive Pulmonary Disease

[0139] 1. Reagents and Instruments

[0140] 1.1 Reagents

[0141] 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), cigarettes (Seven Wolves). KDHP 30 Corresponding to the 30% fractionated polysaccharide of Sample 1 in Example 1, KDHP 80 Corresponding to the 80% fractionated polysaccharide of Sample 2, KDHP 30 KDHP 80 All were prepared by the Natural Product Chemistry Group of the China Resources Jiangzhong Modern Traditional Chinese Medicine Research Center.

[0142] 1.2 Instruments

[0143] 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.

[0144] 1.3 Experimental Cells

[0145] BEAS-2B human bronchial epithelial cells

[0146] 2 Experimental Methods

[0147] 2.1 Preparation of Cigarette Extract

[0148] 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.

[0149] 2.2 Cell treatment methods

[0150] 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. CSE modeling was performed according to experimental groups, followed by drug intervention. At the experimental endpoint, CCK8 working solution was added and incubated for 2-4 hours, and cell absorbance was measured.

[0151] Calculate cell viability based on absorbance values ​​of each cell group:

[0152] 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%.

[0153] 3 Experimental Results

[0154] like Figure 8-9 As shown, cell survival rate was significantly reduced under CSE (0.5%) conditions. At 24h and 48h after administration, Examples 1-2 (100μg / mL) had a certain repair effect on cell damage caused by CSE. The cell survival rate of Examples 1-2 (200μg / mL) was higher than that of the CSE (0.5%) group.

[0155] The above results indicate that the products obtained from the graded polysaccharides of Coltsfoot flower in Examples 1-2 provided by this invention have a significant repair effect on the CSE-induced chronic obstructive pulmonary disease model and have a certain therapeutic effect on chronic obstructive pulmonary disease.

[0156] 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 flower polysaccharide comprises fructose, glucose, arabinose, galactose, and glucosamine; wherein the molar ratio of fructose, glucose, arabinose, galactose, and glucosamine is 0.2-1.2:0.05-0.3:0.01-0.035:0.001-0.006:0.001-0.

005.

2. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The molecular weight of the coltsfoot flower polysaccharide was selected from 7.05 × 10⁻⁶. 2 -6.55×10 6 Da.

3. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The molar ratio of fructose, glucose, arabinose, galactose and glucosamine is 0.5-1.0: 0.10-0.20: 0.01-0.020: 0.001-0.005: 0.001-0.

004.

4. The coltsfoot flower polysaccharide according to claim 1, characterized in that, The coltsfoot flower polysaccharide comprises graded polysaccharide fraction one and / or graded polysaccharide fraction two, wherein graded polysaccharide fraction one and graded polysaccharide fraction two comprise fructose, glucose, arabinose, galactose and glucosamine.

5. The coltsfoot flower polysaccharide according to claim 4, characterized in that, The molar ratio of fructose, glucose, arabinose, galactose and glucosamine in the graded polysaccharide fraction is 0.802:0.173:0.018:0.004:0.

003.

6. The coltsfoot flower polysaccharide according to claim 4, characterized in that, The molar ratio of fructose, glucose, arabinose, galactose, and glucosamine in the graded polysaccharide fraction II is 0.857:0.126:0.012:0.003:0.

001.

7. The coltsfoot flower polysaccharide according to claim 4, characterized in that, The molecular weight of the first fraction of the hierarchical polysaccharide is selected from 7.05 × 10⁻⁶. 2 -6.55×10 6 Da.

8. The coltsfoot flower polysaccharide according to claim 7, characterized in that, The molecular weight of the major peak in the fractional polysaccharide fraction is 7.05 × 10⁻⁶. 2 -1.40×10 3 Da.

9. The coltsfoot flower polysaccharide according to claim 4, characterized in that, The molecular weight of the second fraction of the hierarchical polysaccharide is selected from 1.90 × 10⁻⁶. 3 -5.71×10 6 Da.

10. The coltsfoot flower polysaccharide according to claim 9, characterized in that, The molecular weight of the two main peaks in the fractionated polysaccharide fraction is 1.90 × 10⁻⁶. 3 -2.45×10 3 Da.

11. The method for preparing coltsfoot flower polysaccharide according to any one of claims 1-10, characterized in that, Includes the following steps: Coltsfoot flowers were defatted, extracted with water, and precipitated with alcohol to obtain the precipitate. The precipitate was then dried to obtain coltsfoot flower ethanol precipitate fractionated polysaccharide.

12. The preparation method according to claim 11, characterized in that, Includes the following steps: Step 1: Mix coltsfoot flowers with an alcohol solution, heat to defatt the mixture, filter, and obtain defatted medicinal material; Step 2: Mix the defatted medicinal materials from Step 1 with water, heat to extract, combine the extracts, concentrate to obtain a concentrated solution; Step 3: Mix the concentrate from Step 2 with the alcohol solution, remove the solvent, until the mass fraction of alcohol in the final solution is 25%-35%, obtain the precipitate, dry it, and fractionate the polysaccharide fraction 1.

13. The preparation method according to claim 12, characterized in that, Step 3 is followed by: Step 4: Mix the supernatant after precipitation in Step 3 with the alcohol solution until the alcohol concentration in the final solution is 75%-85%, to obtain the precipitate, dry it, and fractionate the polysaccharide fraction II.

14. The preparation method according to claim 12, characterized in that, In step 1, the alcohol solution is ethanol; the mass fraction of the ethanol is 50%-95%; the heating is performed twice; for the first heating, the ratio of coltsfoot flower to alcohol solution is 1:9-11 g / mL; the heating time is 2-2.5 h; for the second heating, the ratio of coltsfoot flower to alcohol solution is 1:7-9 g / mL; the heating time is 1-1.5 h.

15. The preparation method according to claim 12, characterized in that, In step 2, the heating is performed twice; for the first heating, the ratio of coltsfoot flower to aqueous solution is 1:9-11 g / mL; the heating time is 2-2.5 h; for the second heating, the ratio of coltsfoot flower to aqueous solution is 1:7-9 g / mL; the heating time is 1-1.5 h; the concentration is to concentrate to a crude drug concentration of 0.5-1.5 mg / mL.

16. The preparation method according to claim 12, characterized in that, In step 3, the alcohol solution is an ethanol solution; in the final solution, the mass fraction of alcohol is 30%.

17. The preparation method according to claim 12, characterized in that, In step 4, the alcohol solution is an ethanol solution; in the final solution, the mass fraction of alcohol is 80%.

18. A pharmaceutical composition comprising the coltsfoot flower polysaccharide according to any one of claims 1-10.

19. Use of the coltsfoot flower polysaccharide according to any one of claims 1-10, the coltsfoot flower polysaccharide prepared by any one of claims 11-17, or the pharmaceutical composition according to claim 18 in the preparation of a medicament for treating respiratory diseases.

20. The use according to claim 19, characterized in that, The respiratory diseases mentioned include asthma, rhinitis, cough, and chronic obstructive pulmonary disease.

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