Kunlun chrysanthemum bud polysaccharide with effects of controlling sugar and reducing lipid and preparation method of Kunlun chrysanthemum bud polysaccharide

Abstract

The invention provides a Kunlun chrysanthemum bud polysaccharide with sugar controlling and lipid lowering effects and a preparation method thereof, and relates to the technical field of biological medicine, the main chain of the Kunlun chrysanthemum bud polysaccharide is formed by alternation of-> 4)-beta-D-Galp-(1-> and-> 4)-beta-D-Glcp-(1->; a side chain of the Kunlun chrysanthemum bud polysaccharide is composed of-> 5)-alpha-L-Araf-(1->,-> 3, 6)-beta-D-Galp, alpha-L-Araf, beta-D-Glcp / Galp and alpha-D-Rhap. The technical problems that the Kunlun chrysanthemum bud polysaccharide prepared in the prior art is complex in component and low in purity, and application of the Kunlun chrysanthemum bud polysaccharide in the field of sugar control and lipid reduction is seriously limited are solved, the effects of reducing blood sugar and blood lipid are achieved, the use dosage of a blood sugar reducing medicine metformin is reduced, and the sugar control and lipid reduction effects are enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a Kunlun fetal chrysanthemum polysaccharide with blood sugar control and lipid lowering effects and a preparation method thereof. Background Art

[0002] Diabetes mellitus is a chronic disease characterized by insufficient insulin secretion or impaired insulin utilization, with persistently elevated blood glucose levels as its primary hallmark. It is often accompanied by multiple complications, such as hyperlipidemia, which pose a life-threatening threat to patients. In recent years, the incidence and prevalence of diabetes have increased globally, posing a major challenge to public health. This disease causes chronic, progressive damage to tissues and organs, potentially leading to functional decline or even failure, and is associated with the risk of acute metabolic disorders. Therefore, coordinated treatment of hyperglycemia and hyperlipidemia is necessary.

[0003] Over the past few decades, metformin has become an important drug for the treatment of hyperglycemia and hyperlipidemia worldwide due to its high cost-effectiveness and significant glucose-lowering effects. Although metformin can improve the symptoms of hyperglycemia and hyperlipidemia by enhancing insulin sensitivity, inhibiting gluconeogenesis, promoting cellular glucose uptake, and lowering cholesterol, long-term use of metformin may cause gastrointestinal discomfort. Although metformin plays an important role in the treatment of diabetes, it is still necessary to explore new treatment strategies to enhance the efficacy of metformin and reduce its dosage, thereby improving patient prognosis.

[0004] Currently, combination therapy is a method frequently used in clinical treatment, which aims to maximize efficacy and reduce side effects through the rational use of multiple drugs. Studies have shown that polysaccharides can not only enhance the immunomodulatory effects of drugs, but also enhance the efficacy of anti-diabetic drugs (such as acarbose, metformin and sitagliptin). Polysaccharides can regulate glucose metabolism and improve pancreatic cell function, thereby enhancing the therapeutic effect of anti-diabetic drugs. For example, the combination therapy of low-dose metformin and Sargassum polysaccharides has a positive effect on diabetic rats induced by high-fat diet and streptozotocin.

[0005] Kunlun chrysanthemum can be used as medicine and food. The polysaccharides in Kunlun chrysanthemum have the effect of inhibiting carbohydrate enzymes in vitro and can significantly inhibit the activity of α-glucosidase. However, the Kunlun chrysanthemum polysaccharides prepared in the prior art have complex components and low purity, which seriously limits their application in the fields of lowering blood sugar and blood lipids. Therefore, there is an urgent need to develop a new Kunlun chrysanthemum polysaccharide with blood sugar and lipid-lowering effects and its preparation method to reduce the dosage of metformin and enhance the blood sugar and lipid-lowering effects.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] One of the purposes of the present invention is to provide a Kunlun fetal chrysanthemum polysaccharide, which has a good blood sugar control and lipid-lowering effect, can reduce the dosage of the hypoglycemic drug metformin and enhance the blood sugar control and lipid-lowering effect.

[0008] The second object of the present invention is to provide a method for preparing Kunlun fetal chrysanthemum polysaccharide, which has a simple process, is easy to operate, and is suitable for industrial production.

[0009] The third purpose of the present invention is to provide an application of Kunlun fetal chrysanthemum polysaccharide, which is beneficial to enhance the blood sugar and lipid-lowering effect of metformin on diabetic patients and reduce side effects.

[0010] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0011] In the first aspect, a Kunlun fetal chrysanthemum polysaccharide mainly consists of arabinose, galactose, galacturonic acid, rhamnose and glucose;

[0012] The main chain of the Kunlun fetal chrysanthemum polysaccharide is composed of →4)-β-D-Galp-(1→ and →4)-β-D-Glcp-(1→ alternately;

[0013] The side chain of the Kunlun fetus chrysanthemum polysaccharide is composed of →5)-α-L-Araf-(1→, →3,6)-β-D-Galp, α-L-Araf, β-D-Glcp / Galp and α-D-Rhap.

[0014] Furthermore, the molar percentages of arabinose, galactose, galacturonic acid, rhamnose and glucose are 26.52:37.96:26.43:5.38:3.71.

[0015] Furthermore, the structural formula of the repeating unit in the Kunlun fetal chrysanthemum polysaccharide is as follows:

[0016]

[0017] The uronic acid content of the Kunlun fetal chrysanthemum polysaccharide is 26.48±0.11%;

[0018] The Kunlun chrysanthemum polysaccharide is an acidic polysaccharide with a molecular weight of 3192Da.

[0019] In a second aspect, a method for preparing the Kunlun fetal chrysanthemum polysaccharide according to any one of the above items comprises the following steps:

[0020] (a) hydrolyzing Kunlun fetal chrysanthemum under acidic conditions, and then filtering to obtain a filtrate;

[0021] (b) concentrating the filtrate obtained in step (a), then subjecting it to alcohol precipitation, and filtering it to obtain a precipitate;

[0022] (c) mixing the precipitate obtained in step (b) with an alkaline solution, filtering the mixture, and adjusting the pH of the filtrate to neutral to obtain an extract;

[0023] (d) ultrafiltration of the extract obtained in step (c) to obtain a filtrate and drying the filtrate to obtain a crude extract;

[0024] (e) ultrafiltration of the crude extract obtained in step (d) to obtain a retentate and drying the retentate to obtain crude Kunlun fetal chrysanthemum polysaccharide;

[0025] (f) The obtained crude Kunlun Chrysanthemum polysaccharide is treated by anion exchange chromatography and dialysis purification to obtain the Kunlun Chrysanthemum polysaccharide.

[0026] Furthermore, in step (a), the hydrolysis method comprises the following steps:

[0027] After soaking Kunlun chrysanthemum in acid solution, hydrolyzing it under the conditions of cellulase synergistic magnetic field and electric field coupling to obtain hydrolyzate;

[0028] Preferably, the stirring condition for the hydrolysis is stirring at 85° C. for 2 h;

[0029] Preferably, the cellulase is a cellulase having an enzyme activity of 800 U / g after addition and a pH value of 5.3;

[0030] Preferably, the magnetic induction intensity of the magnetic field is 0.3T-0.5T;

[0031] Preferably, the alternating voltage frequency used in the electric field is 50 Hz.

[0032] Furthermore, the filtering method includes centrifugation.

[0033] Furthermore, in step (c), the stirring conditions for mixing the precipitate and the alkali solution are 40° C.-60° C. and 2 h-3 h.

[0034] Furthermore, in step (d), the ultrafiltration molecular cut-off is 10000Da;

[0035] Preferably, in step (e), the molecular cut-off of the ultrafiltration is 5000Da;

[0036] Preferably, in step (e), the molecular weight of the crude polysaccharide of Kunlun chrysanthemum is 5000Da-10000Da.

[0037] Furthermore, the eluent used in the anion exchange chromatography includes 0.01 mol / L NaOH solution and 3 volumes of 0 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L and 1.0 mol / L NaCl solutions.

[0038] In a third aspect, a use of the Kunlun fetal chrysanthemum polysaccharide described in any one of the above items in the preparation of drugs for lowering blood sugar and / or blood lipids.

[0039] Compared with the prior art, the present invention has at least the following beneficial effects:

[0040] The Kunlun chrysanthemum polysaccharide provided by the present invention has high purity, clear structural characteristics, and has the effects of lowering blood sugar and blood lipids; the Kunlun chrysanthemum polysaccharide can better prevent pancreatic cell atrophy by inhibiting the cleavage of Caspase-3 and the inflammatory factor NLRP-3, and can enhance the blood sugar-lowering effect of metformin on diabetic patients and reduce side effects, which is conducive to reducing the dosage of the blood sugar-lowering drug metformin.

[0041] The preparation method of Kunlun fetal chrysanthemum polysaccharide provided by the present invention has a simple process, is easy to operate, and is suitable for industrial production. The present invention can increase the yield of Kunlun fetal chrysanthemum polysaccharide and the release of effective ingredients through acid-enzyme treatment and magnetic field and electric field coupling.

[0042] The application of the Kunlun fetal chrysanthemum polysaccharide provided by the present invention is beneficial to enhancing the blood sugar control and lipid-lowering effect of metformin on diabetic patients and reducing side effects, and provides a new direction for hypoglycemic and hypolipidemic drugs or drug adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 A graph showing the gradient elution curve of anion exchange chromatography of a packing column of Kunlun Fetal Chrysanthemum polysaccharide provided in Example 1 of the present invention;

[0045] Figure 2 This is a comparison of the IC50 values ​​of Kunlun fetal chrysanthemum polysaccharide in Example 1 of the present invention, whether the extraction process includes enzyme treatment and magnetic field and electric field coupling;

[0046] Figure 3 This is a graph showing the inhibition of α-glucosidase by the purified fraction obtained in Test Example 1 of the present invention;

[0047] Figure 4 This is a graph showing the determination results of the monosaccharide composition (A) and molecular weight (B) of Kunlun Fetal Chrysanthemum polysaccharide obtained in Test Example 2 of the present invention;

[0048] Figure 5This is the infrared spectrum of Kunlun fetal chrysanthemum polysaccharide obtained in Test Example 3 of the present invention;

[0049] Figure 6 The Kunlun fetal chrysanthemum polysaccharide obtained in Test Example 5 of the present invention 1 H spectrum (A), 13 C spectrum (B), 1 H- 1 H COSY spectrum (C) and HSQC spectrum (D);

[0050] Figure 7 This is a schematic diagram of the animal experiment design scheme obtained in Experimental Example 6 of the present invention;

[0051] Figure 8 Effects of Kunlun Fetal Chrysanthemum polysaccharide (denoted as AI) obtained in Experimental Example 6 of the present invention on mouse body weight (A), final body weight (B), pancreas index (C), liver index (D) and kidney index (E);

[0052] Figure 9 Effects of Kunlun Fetal Chrysanthemum polysaccharide obtained in Experimental Example 6 of the present invention on HbA1c (A), insulin content (B), liver glycogen content (C), and muscle glycogen content (D) in diabetic mice;

[0053] Figure 10 Schematic diagrams of H&E staining of the mouse pancreas (A), immunofluorescence staining of the mouse pancreas, and co-staining of insulin (red) with cleaved Caspase-3 (B) or NLRP-3 (green) (C) obtained in Experimental Example 6 of the present invention;

[0054] Figure 11 Glycogen staining (A) and glycogen signal (B) of nematodes obtained in Experimental Example 7 of the present invention;

[0055] Figure 12 Oil Red O staining (A) and Oil Red O signal (B) of nematodes obtained in Experimental Example 7 of the present invention. DETAILED DESCRIPTION

[0056] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0057] According to a first aspect of the present invention, there is provided a Kunlun fetal chrysanthemum polysaccharide, which is mainly composed of arabinose, galactose, galacturonic acid, rhamnose and glucose;

[0058] The main chain of the Kunlun fetus chrysanthemum polysaccharide is composed of →4)-β-D-Galp-(1→ and →4)-β-D-Glcp-(1→ alternately;

[0059] The side chain of the Kunlun fetus chrysanthemum polysaccharide is composed of →5)-α-L-Araf-(1→, →3,6)-β-D-Galp, α-L-Araf, β-D-Glcp / Galp and α-D-Rhap.

[0060] The Kunlun fetal chrysanthemum polysaccharide of the present invention has good hypoglycemic and hypolipidemic effects. By inhibiting the cleavage of Caspase-3 and the inflammatory factor NLRP-3, it can better prevent pancreatic islet cell atrophy, enhance the hypoglycemic effect of metformin on diabetic patients and reduce side effects, which is conducive to reducing the dosage of the hypoglycemic drug metformin.

[0061] In the present invention, the molar percentages of arabinose, galactose, galacturonic acid, rhamnose and glucose are 26.52:37.96:26.43:5.38:3.71.

[0062] It should be noted that the structural formula of the repeating unit in the Kunlun fetal chrysanthemum polysaccharide is as follows:

[0063]

[0064]

[0065] In the present invention, the uronic acid content of Kunlun Fetal Chrysanthemum polysaccharide is 26.48±0.11%, and it is an acidic polysaccharide with a molecular weight of 3192Da.

[0066] In summary, the Kunlun fetus chrysanthemum polysaccharide of the present invention is composed of arabinose, galactose, galacturonic acid, rhamnose and glucose in a molar ratio (%) of 26.52:37.96:26.43:5.38:3.71, and is an acidic polysaccharide with a molecular weight of 3192 Da. Methylation and nuclear magnetic resonance results show that the Kunlun fetus chrysanthemum polysaccharide is mainly composed of residues such as →4)-β-D-Galp-(1→, →4)-β-D-Glcp-(1→, →5)-α-L-Araf-(1→, →3,6)-β-D-Galp, α-L-Araf, β-D-Glcp / Galp and α-D-Rhap.

[0067] According to a second aspect of the present invention, there is provided a method for preparing the Kunlun fetal chrysanthemum polysaccharide described in any one of the above, comprising the following steps:

[0068] (a) hydrolyzing Kunlun fetal chrysanthemum under acidic conditions, and then filtering to obtain a filtrate;

[0069] (b) concentrating the filtrate obtained in step (a), then subjecting it to alcohol precipitation, and filtering it to obtain a precipitate;

[0070] (c) mixing the precipitate obtained in step (b) with an alkaline solution, filtering the mixture, and adjusting the pH of the filtrate to neutral to obtain an extract;

[0071] (d) ultrafiltration of the extract obtained in step (c) to obtain a filtrate and drying the filtrate to obtain a crude extract;

[0072] (e) ultrafiltration of the crude extract obtained in step (d) to obtain a retentate and drying the retentate to obtain crude Kunlun fetal chrysanthemum polysaccharide;

[0073] (f) The obtained crude polysaccharide of Kunlun Fetus Chrysanthemum is treated by anion exchange chromatography and dialysis purification to obtain polysaccharide of Kunlun Fetus Chrysanthemum.

[0074] The preparation method of Kunlun chrysanthemum polysaccharide provided by the invention has simple process, easy operation and is suitable for industrial production.

[0075] In a preferred embodiment, in step (a), the hydrolysis method comprises the following steps:

[0076] The Kunlun chrysanthemum is soaked in acid solution and then hydrolyzed under the conditions of cellulase synergistically coupled with magnetic field and electric field to obtain a hydrolyzate.

[0077] In the present invention, the stirring conditions for hydrolysis can be stirring at 85°C for 2 hours, but is not limited thereto; the cellulase can be a cellulase with an enzyme activity of 800 U / g after addition, and the pH value is 5.3, but is not limited thereto; the magnetic induction intensity of the magnetic field can be 0.3T-0.5T, for example, 0.3T, 0.4T, 0.5T, but is not limited thereto; and the alternating voltage frequency used in the electric field can be 50Hz, but is not limited thereto.

[0078] The present invention adopts cellulase to cooperate with magnetic field and electric field coupling to prepare Kunlun fetal chrysanthemum polysaccharide. Under the coordinated cooperation of various process parameters, the yield of Kunlun fetal chrysanthemum polysaccharide is 5.63±0.37%, the total sugar content is 91.29±1.03%, and the uronic acid content is 26.48±0.11%.

[0079] In a preferred embodiment, the filtration method includes but is not limited to centrifugation, which is more conducive to the complete separation of the product.

[0080] In a preferred embodiment, in step (c), the stirring conditions for mixing the precipitate and the alkali solution can be stirring at 40° C.-60° C. for 2 h-3 h, which is more conducive to sufficient mixing of the precipitate and the alkali solution.

[0081] In a preferred embodiment, in step (d), the molecular cut-off of ultrafiltration can be 10,000 Da, but is not limited thereto.

[0082] In a preferred embodiment, in step (e), the molecular cut-off of ultrafiltration can be 5000 Da, but is not limited thereto.

[0083] In the present invention, in step (e), the molecular weight of the crude polysaccharide of Kunlun fetus chrysanthemum can be 5000Da-10000Da, for example, 5000Da, 6000Da, 7000Da, 8000Da, 9000Da, 10000Da, but not limited thereto.

[0084] In a preferred embodiment, the eluent used in anion exchange chromatography can be composed of 0.01 mol / L NaOH solution and 0 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L and 1.0 mol / L NaCl solutions in a volume ratio of 1:3.

[0085] Two components are collected in the anion exchange chromatography, namely the AI-1 component eluted by 0 mol / L NaCl solution and the AI-2 component eluted by 0.2 mol / L NaCl solution. The AI-2 component is dialyzed and purified to obtain Kunlun fetal chrysanthemum polysaccharide.

[0086] According to a third aspect of the present invention, there is provided a use of any of the above-mentioned Kunlun Fetal Chrysanthemum polysaccharides in the preparation of drugs for lowering blood sugar and / or blood lipids.

[0087] The application of the Kunlun fetal chrysanthemum polysaccharide provided by the present invention is beneficial to enhancing the blood sugar and lipid-lowering effect of metformin on diabetic patients and reducing side effects, providing a new direction for hypoglycemic drugs or drug adjuvants.

[0088] The present invention is further described below by way of examples. Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0089] Example 1

[0090] A Kunlun fetal chrysanthemum polysaccharide is prepared by the following method:

[0091] (a) soaking Kunlun chrysanthemum powder in an acid solution and then stirring it for a first time, and hydrolyzing it under the synergistic magnetic and electric field coupling of cellulase to obtain a hydrolyzate;

[0092] The ratio of Kunlun chrysanthemum powder to acid solution is 1g:10mL; the acid solution used is 0.1mol / L HCl solution;

[0093] The first stirring condition was stirring at 85°C for 2 h;

[0094] The enzyme activity after adding cellulase was 800 U / g, and the pH of the system was adjusted to 5.3 (to ensure enzyme activity);

[0095] At the same time, the magnetic field and electric field coupling conditions are: magnetic induction intensity 0.3T-0.5T at 50°C, alternating voltage frequency 50Hz, and hydrolysis for 3h-5h;

[0096] The obtained hydrolyzate is subjected to a first centrifugation to remove the precipitate and collect the filtrate;

[0097] The first centrifugation is performed at a speed of 6000-7000 rpm and for a time of 20-30 min.

[0098] (b) concentrating the filtrate obtained in step (a) to obtain a concentrate (solid content of 10 wt% to 20 wt%), subjecting the obtained concentrate to alcohol precipitation and then performing a second centrifugation to collect the precipitate;

[0099] Among them, the alcohol precipitation condition is to add 4 times the volume of 95% ethanol to the concentrate and place it at 4°C for 12 hours;

[0100] The second centrifugation speed is 6000 rpm-7000 rpm, and the time is 10 min-20 min;

[0101] (c) adding an alkaline solution to the precipitate obtained in step (b), performing a second stirring, and a third centrifugation to collect a filtrate, and adjusting the pH of the filtrate to neutral (7.0-7.5) to obtain an extract;

[0102] The second stirring condition is stirring at 40°C-60°C for 2h-3h;

[0103] The third centrifugation speed is 6000 rpm-7000 rpm, and the time is 10 min-20 min;

[0104] (d) subjecting the extract obtained in step (c) to a first ultrafiltration (repeated 2-3 times), obtaining a filtrate and drying it to obtain a crude extract;

[0105] The molecular cut-off of the first ultrafiltration was 10,000 Da, and the filtrate was collected;

[0106] (e) subjecting the mixed solution containing the crude extract to a second ultrafiltration (repeated 3-5 times) to obtain a retentate and drying the retentate to obtain a dried product, which is a crude polysaccharide from Kunlun fetus chrysanthemum;

[0107] The molecular cut-off of the second ultrafiltration was 5000 Da, and the retentate was collected;

[0108] After the retentate is dried, low molecular weight Kunlun fetal chrysanthemum crude polysaccharide with a molecular weight of 5000Da-10000Da is obtained;

[0109] (f) subjecting the solution containing the crude polysaccharide of Kunlun fetus chrysanthemum to anion exchange chromatography and dialysis purification to obtain the polysaccharide of Kunlun fetus chrysanthemum;

[0110] It should be noted that two components were collected in the anion exchange chromatography, namely, component AI-1 eluted by 0 mol / L NaCl solution and component AI-2 eluted by 0.2 mol / L NaCl solution. The AI-2 component was dialyzed and purified to obtain Kunlun fetal chrysanthemum polysaccharide.

[0111] Specifically, the separation and purification method of Kunlun fetal chrysanthemum crude polysaccharide is as follows:

[0112] (1) Activation and column loading of the filler: When the filler is used for the first time, it needs to be activated with acid and alkali solutions. It should be rinsed with 0.1M sodium hydroxide solution, deionized water and 0.01M hydrochloric acid in sequence, and then rinsed with a large amount of deionized water until it is neutral. Then, it should be slowly and evenly poured into the chromatography column (3.5cm×30cm). After the filler settles naturally, the excess water in the upper layer should be discharged from the lower water outlet to ensure that there is no stratification and air bubbles in the filler in the column.

[0113] (2) Sample loading: Prepare 30 mL of sample (10 mg / mL) solution, mix well, centrifuge (8000 rpm, 4°C, 10 min), take the supernatant for sample loading, and use 0, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L, and 1.0 mol / L NaCl solutions as eluents for elution. The elution rate is controlled at 1 mL / min. Collect one tube for every 6 mL, and elute 600 mL for each solubility.

[0114] The total sugar content of the collected solution was detected by phenol-sulfuric acid method, and the elution profile of Kunlun fetal chrysanthemum polysaccharide was drawn. Figure 1 , it can be seen that two components were collected during the elution process, namely AI-1 component eluted by 0 mol / L NaCl solution and AI-2 component eluted by 0.2 mol / L NaCl solution;

[0115] According to the main peak distribution of the elution spectrum, the components were combined and collected, and after rotary concentration, they were dialyzed for 36 hours using a dialysis bag with a molecular weight cutoff of 500 Da, and freeze-dried to obtain purified components.

[0116] In this example, the effects of enzyme treatment and magnetic field-electric field coupling on the α-glucosidase inhibitory activity of Kunlun fetal chrysanthemum polysaccharide were investigated:

[0117] 40 μL of 4 mg / mL Kunlun fetal chrysanthemum polysaccharide sample (extraction process includes enzyme treatment, magnetic field coupling and non-enzyme treatment, magnetic field coupling) was incubated with 30 μL of α-glucosidase dilution solution (37°C, 10 min), and then 30 μL of substrate p-NPG solution was added for reaction (37°C, 15 min). Finally, 100 μL of Na2CO3 solution (0.1 M) was added to terminate the reaction, and the absorbance was detected at 405 nm.

[0118] The control group was treated with no enzyme and magnetic field coupling during the extraction process, and the experimental group was treated with enzyme and magnetic field coupling during the extraction process. IC 50 value;

[0119] The results are as follows Figure 2 As shown, the IC 50 The value was 670.02 μg / mL, which was lower than the IC 50 The value was (533.15 μg / mL), which indicates that adding enzyme treatment and magnetic field-electric field coupling process during the extraction process can increase the yield of Kunlun fetal chrysanthemum polysaccharide and the release of effective ingredients, which is beneficial to enhance the hypoglycemic and hypolipidemic effects.

[0120] Test Example 1

[0121] Determination of the inhibitory activity of Kunlun fetal chrysanthemum polysaccharide on α-glucosidase:

[0122] The purified fractions (30 μL) corresponding to the two fractions (AI-1 fraction and AI-2 fraction) in Example 1 were incubated with 30 μL of α-glucosidase dilution solution (37°C, 10 min), and then 30 μL of substrate p-NPG solution was added for reaction (37°C, 15 min). Finally, 100 μL of Na2CO3 solution (0.1 M) was added to terminate the reaction. The absorbance was measured at 405 nm, and the inhibition rate was calculated by the following formula: Figure 3 ;

[0123]

[0124] Among them, A 样品 It represents the absorbance value of Kunlun fetal chrysanthemum polysaccharide, α-glucosidase and p-NPG. 样品空白 It represents the absorbance value of Kunlun fetal chrysanthemum polysaccharide and PBS, A 空白对照 Represents the absorbance values ​​of PBS, α-glucosidase and p-NPG.

[0125] from Figure 3It can be seen that the AI-1 purified component did not show inhibitory activity on α-glucosidase within the test concentration range, and the AI-2 purified component did not show inhibition on α-glucosidase at the test concentration of 0.5 mg / mL-1 mg / mL; however, with the increase of the concentration of the AI-2 purified component, at the test concentration of 2 mg / mL-8 mg / mL, the AI-2 purified component showed inhibition on α-glucosidase, and showed a dose-dependent manner, that is, the AI-2 purified component was selected as Kunlun fetal chrysanthemum polysaccharide.

[0126] Test Example 2

[0127] The purification yield and basic composition of Kunlun fetal chrysanthemum polysaccharide are as follows:

[0128]

[0129] Among them, W1 represents the mass of the lyophilized purified component, and W2 represents the mass of the sample;

[0130] Total sugar content detection: Using D-glucose as the standard, the phenol-sulfuric acid method was used to determine the total sugar content in Kunlun fetal chrysanthemum polysaccharide;

[0131] Uronic acid content detection: Using galacturonic acid as the standard, the m-hydroxybiphenyl colorimetric method was used to determine the uronic acid content in Kunlun fetal chrysanthemum polysaccharide;

[0132] Determination of monosaccharide composition: 5 mg of Kunlun fetal chrysanthemum polysaccharide and 2 mL of TFA (3M) were added to an ampoule and hydrolyzed (120°C, 3 h). The hydrolyzed polysaccharide solution was nitrogen-purged and dissolved in 5 mL of water. After dilution, the solution was centrifuged (12000 rpm, 5 min). The preparation method of the standard was the same as above. The sample was taken for ion chromatography analysis. Figure 4 Figure A in the middle;

[0133] Molecular weight determination: 5 mg / mL CTBPs solution was prepared with 0.1 mol / L sodium nitrate (containing 0.05% sodium azide) solution. After passing through the membrane, the molecular weight of Kunlun fetal chrysanthemum polysaccharide was analyzed by size exclusion gel chromatography, 18-angle laser light scattering and differential detection. Figure 4 Figure B in the middle.

[0134] The determination results are: the yield of Kunlun chrysanthemum polysaccharide is 5.63±0.37%, the total sugar content is 91.29±1.03%, and the uronic acid content is 26.48±0.11%; Kunlun chrysanthemum polysaccharide is composed of arabinose, galactose, galacturonic acid, rhamnose and glucose with a molar ratio (%) of 26.52, 37.96, 26.43, 5.38 and 3.71, and is an acidic polysaccharide with a molecular weight of 3192Da.

[0135] Test Example 3

[0136] Infrared spectroscopic determination of Kunlun fetal chrysanthemum polysaccharide:

[0137] The infrared spectrum analysis of Kunlun fetal chrysanthemum polysaccharide was carried out by KBr tablet method, and the scanning range of Fourier infrared spectrometer was 4000cm -1 -400cm -1 , infrared spectrum see Figure 5 .

[0138] Depend on Figure 5 It can be seen that Kunlun chrysanthemum polysaccharide is at 4000cm -1 -400cm -1 There is a characteristic absorption peak of polysaccharide in the range of 3370 cm -1 The strong absorption peak at 2925cm is caused by the stretching vibration of OH. -1 The absorption peak at 1610 cm is due to the asymmetric stretching vibration of CH. -1 The absorption peak that appears is the asymmetric stretching vibration of the non-ester carbonyl C=O, indicating the presence of uronic acid; 1417cm -1 The absorption peak at 1089cm is the angular vibration of CH or OH; -1 and 1026cm -1 The absorption peak at 891 cm -1 and 634cm -1 The absorption peak at is attributed to the β-glycosidic bond.

[0139] Test Example 4

[0140] Methylation determination of Kunlun fetal chrysanthemum polysaccharide:

[0141] First, carry out the methylation reaction. Weigh 3 mg of Kunlun fetal chrysanthemum polysaccharide and place it in a glass reaction bottle. Quickly add 1 mL of anhydrous DMSO and anhydrous alkaline solution, seal the bottle, dissolve it by ultrasonication, add iodomethane solution to react (30 ° C, 60 min), then add ultrapure water to terminate the reaction, add chloroform to extract, and then transfer the methylated Kunlun fetal chrysanthemum polysaccharide to an ampoule and add 1 mL 2MTFA, sealed, placed in an oven for hydrolysis (110 ° C, 90 min), after the hydrolysis is completed, TFA is removed, 2 mL of distilled water and 60 mg of sodium borohydride are added, reduced for 8 h, glacial acetic acid is added for neutralization, spin-dried, and then 1 mL of acetic anhydride is added for acetylation reaction (100 ° C, 1 h). After cooling, 3 mL of toluene is added and spin-dried. Repeat the operation until the residual acetic anhydride is removed, add 3 mL of dichloromethane to dissolve and transfer to a separating funnel. After adding a small amount of distilled water and shaking thoroughly, the upper aqueous solution is removed and repeated 4 times. Sufficient anhydrous sodium sulfate is added for drying, and the mixture is filtered through a 0.22 μm filter membrane and then analyzed by GC-MS. The operating parameters are shown in Table 1.

[0142] Table 1

[0143]

[0144] The main glycosidic bonds and connection modes of Kunlun fetal chrysanthemum polysaccharide are shown in Table 2. A total of 11 glycosidic bonds were detected, including non-reducing end (T-), consisting of T-Araf (20.50%), 1,5-Araf (11.50%), 1,4-Galp (12.20%), T-Galp (9.80%), 1,3,6-Galp (9.60%), 1,4-Glcp (8.40%), T-Glcp (8.30%), 1,6 -Galp (6.80%), 1,3-Galp (6.40%), T-Rhap (3.00%) and 1,3,5-Araf (3.60%); According to the methylation results, the terminal arabinose (20.50%), galactose (9.80%), glucose (8.30%) and rhamnose (3.00%) constitute the side chain ends of the polysaccharide, and the main chain is composed of →4)-Galp-(1→ and →4)-Glcp-(1→ ; The main chain structure is further inferred from the ratio and glycosidic bond connection mode to be →4)-Galp-(1→ and →4)-Glcp-(1→, with galactose as the main component and glucose as the auxiliary component, forming a 1→4 connected linear skeleton; the branching point of the polysaccharide is mainly →3,6)-Galp-(1→, indicating that galactose is substituted at the 3rd and 6th positions at the same time, with →3,5)-Araf-(1→ as the secondary branching point; the side chain end is dominated by Araf-(1→, indicating that arabinofuranoside is the main component. The main side chain terminal is arabinose, and there are also short chains or side chains in the form of →5)-Araf-(1→; secondary branches in the form of →3)-Galp-(1→ and →6)-Galp-(1→; the terminal residues account for 41.60%, indicating a high degree of branching, belonging to a highly branched polysaccharide; at the same time, the main chain residues account for 20.60%, combined with the branches to form a network structure, and contain a high proportion of T-Araf and 1,5-Araf, which is consistent with the characteristics of arabinogalactan.

[0145] Table 2 Analysis of methylation results of Kunlun fetal chrysanthemum polysaccharide

[0146]

[0147] Test Example 5

[0148] Nuclear magnetic resonance determination of Kunlun fetal chrysanthemum polysaccharide:

[0149] Weigh 30 mg of Kunlun fetal chrysanthemum polysaccharide, dissolve it in heavy water and freeze-dry it. After freeze-drying, dissolve it in heavy water again and freeze-dry it. Repeat this three times to fully exchange the active hydrogen in the sample. Dissolve the treated sample in 0.5 mL of heavy water and scan the one-dimensional and two-dimensional spectra on a nuclear magnetic resonance instrument. The results are shown in the figure. Figure 6 .

[0150] from Figure 6 As shown in Figure A, in the proton nuclear magnetic resonance spectrum, there are five characteristic resonance peaks in the 4.3ppm-5.8ppm region, among which the anomeric hydrogen signals follow the typical configuration distribution law: the β-anomeric configuration (δ4.48ppm, 4.66ppm) is located in the 4.3ppm-5ppm range, while the α-anomeric configuration (δ5.12ppm, 5.26ppm, 5.80ppm) is distributed in the 5ppm-5.8ppm high field region; Figure 6 Synchronous acquisition of B 13 C spectrum, and Figure 6 The HSQC two-dimensional correlation spectrum of D further verified the configuration characteristics of the anomeric carbon. The anomeric carbon signal showed five characteristic peaks in the range of 95ppm-110ppm, which were clearly correlated with the proton signal: δH / δC 4.48 / 101.19 (A), 4.66 / 103.59 (B), 5.12 / 99.47 (C), 5.26 / 109.70 (D) and 5.80 / 107.89 (E);

[0151] The assignment of the H and C signals of each residue is shown in Table 3. The δH 4.48ppm and 4.66ppm in residue A / B are consistent with the anomeric hydrogen characteristics of β-pyranose, and the δC 101ppm and 103ppm are consistent with the anomeric carbon range of β-pyranose. The C4 connection of →4)-Glcp-(1→ leads to an increase in C4 displacement (~78.4ppm), which is consistent with the glycosidic bond (2,3,6-Me3-Glcp) after polysaccharide methylation. Therefore, residue B is assigned to →4)-β-D-Glcp-(1→[ 5] ; The C2 / C3 / C4 at the terminal of Galp-(1→ is not connected to other sugars and has a low displacement, so the residue A is classified as β-D-Galp-(1→[ 80]; δH>5.0ppm and δC>99.0ppm are consistent with arabinofuranosyl; C5 is connected to other sugars, resulting in an increase in C5 displacement. Residue C is classified as →5)-α-L-Araf-(1→, α-configuration anomeric hydrogen (5.26ppm) and C1 (109.70ppm) are consistent with furanose α-arabinose, and residue D is classified as α-L-Araf-(1→. δH 5.80ppm is the characteristic peak of anomeric hydrogen of α-rhamnose (6-deoxy). Due to the deoxygenation effect and ring strain, it is slightly higher than that of typical pyranose, and the chemical shift is higher than that of other pyranose. Residue E is classified as α-D-Rhap-(1→. According to the methylation data, anomeric region signals and chemical shift information, it can be seen that the main chain of Kunlun fetus chrysanthemum polysaccharide is composed of →4)-β-D-Galp-(1→ and →4)-β-D-Glcp-(1→) alternating (20.60%). Embedded in →5)-α-L-Araf-(1→ flexible unit (11.50%); branch point located at →3,6)-β-D-Galp (9.60%), connecting two side chains - C3 position extends the terminal α-L-Araf (20.50%), C6 position connects the β-D-Glcp / Galp terminal (8.30% / 9.80%); the terminal α-D-Rhap (3.00%) modifies the side chain to form a highly branched heteropolysaccharide;

[0152] From this, we can know that the structure of Kunlun fetal chrysanthemum polysaccharide is as follows:

[0153]

[0154] Table 3 Sugar residues in Kunlun fetal chrysanthemum polysaccharide 1 H and 13 C signal ownership

[0155]

[0156] Test Example 6

[0157] To verify that Kunlun Fetal Chrysanthemum polysaccharide enhances the hypoglycemic effect of metformin, the experimental arrangement is as follows Figure 7 As shown:

[0158] Specifically, the experimental mice were divided into four groups, with 10 mice in each group; the diabetic mice induced by streptozotocin were divided into four groups: diabetic control group (denoted as DC group), metformin group (denoted as MET group), low-dose metformin and Kunlun fetal chrysanthemum polysaccharide group (denoted as LMET+AI group); the remaining 10 mice were assigned to the normal control group (NC);

[0159] The gavage procedures of each group were as follows: the NC and DC groups received an equal volume of normal saline, the MET group received 250 mg / kg metformin, and the LMET+AI group received 125 mg / kg metformin and 200 mg / kg Kunlun fetal chrysanthemum polysaccharide.

[0160] Throughout the experiment, fasting blood glucose (FBG) levels were measured and body weights were recorded weekly, and bedding was changed daily to keep the environment clean. After four weeks of treatment, all mice were fasted for 12 hours, weighed, and feces were collected. Blood samples were collected by orbital bleeding, placed in an ice bath for 1 hour, and then centrifuged at 4°C and 4000g for 10 minutes to collect serum. The serum was quickly frozen in liquid nitrogen and stored at -80°C. Some tissues and organs were fixed with 10% paraformaldehyde, and the remaining samples were stored at -80°C for further analysis.

[0161] Effects of LMET+AI on body weight and organs of diabetic mice:

[0162] Organ index: After autopsy, the pancreas, liver, and kidneys were extracted, washed, dried, and weighed. Organ index = (organ weight) / (body weight);

[0163] Results: Streptozotocin-induced diabetic mice lost weight, had swelling of organs such as the pancreas, increased organ index, and damaged organs, affecting their functions, such as Figure 8 As shown in Figures A and B, the NC group gradually gained weight throughout the experiment; in contrast, after STZ induction, the model group experienced a significant weight loss; however, after intervention with MET or LMET+AI, the weight loss in these groups was significantly alleviated, and the differences were statistically significant compared with the DC group (p<0.05). Figure 8 As shown in Figures C, D, and E, the pancreas, liver, and kidney indices of the NC group remained at the lowest level (p<0.05); in contrast, the pancreas, liver, and kidney indices of the DC group increased due to STZ induction, resulting in organ enlargement and increased organ indices; however, after intervention with MET or LMET+AI, the organ indices returned to normal; these results indicate that MET or LMET+AI intervention can effectively alleviate weight loss in diabetic mice and reduce increased organ indices.

[0164] Effects of LMET+AI on the blood glucose index of diabetic mice:

[0165] Oral glucose tolerance test (OGTT): On the 26th day of treatment, mice were fasted for 12 hours and subjected to an oral glucose tolerance test (OGTT). Fasting blood glucose (FBG) levels of each group were measured using a Yuwell blood glucose meter (Danyang, China). Subsequently, each group was gavaged with a glucose solution at a dose of 2 g / kg. Blood glucose levels were monitored 30, 60, 90, and 120 minutes after administration.

[0166] Biochemical analysis: Glycated hemoglobin (HbA1c content) was determined using a kit from Beijing Bosi Biotechnology Co., Ltd., serum insulin level was determined using a kit from Nanjing Jiancheng Bioengineering Institute, and liver and muscle glycogen content was assessed using a glycogen detection kit from Nanjing Jiancheng Bioengineering Institute.

[0167] Results: FBG reflects the blood glucose level of non-fasting mice and is an indicator of hyperglycemia. As shown in Table 4, the blood glucose level of the NC group was stable within the normal range (5.41±0.31mmol / L to 6.33±0.85mmol / L), confirming the absence of diabetes. In contrast, the FBG levels of the other groups were elevated, indicating that diabetes was successfully induced in these mice. After four weeks of intervention, the FBG levels of the MET group and the LMET+AI group were significantly lower than those of the DC group (P<0.05), indicating that these interventions were effective in alleviating hyperglycemia.

[0168] OGTT is an indicator of the body's ability to process glucose. The hallmark of diabetes is that when pancreatic function is impaired, glucose intolerance can lead to elevated blood sugar. As shown in Table 5, the experimental mice underwent OGTT, and the results showed that the NC group exhibited normal glucose tolerance, while the DC group had a significantly elevated blood sugar level within 120 minutes of the test (p<0.05), reflecting severe pancreatic damage. After treatment with MET or LMET+AI, the glucose tolerance of diabetic mice was improved. It is worth noting that LMET+AI is more effective than MET alone in improving glucose tolerance, which indicates that Kunlun fetal chrysanthemum polysaccharide can enhance the therapeutic effect of MET.

[0169] Glycated hemoglobin (HbA1c) is often used as an indicator for diabetes monitoring in clinical practice, such as Figure 8 As shown in Figure A, there were significant differences in HbA1c levels among the groups (p<0.05). The NC group had the lowest HbA1c level, indicating stable blood sugar, while the DC group had a significant increase in HbA1c (p<0.05). After treatment with MET and LMET+AI, the HbA1c levels of diabetic mice decreased, and the improvement in the LMET+AI group was better than that in the MET group. Figure 8 As shown in Figure B, there were significant differences in insulin levels among the groups (P<0.05). The DC group had the lowest insulin level. Both MET and LMET+AI treatments were able to increase the insulin level of diabetic mice, and LMET+AI was more effective than MET.

[0170] Figure 9Figures C and D show the results of glycogen content in mice. Glycogen synthesis involves converting glucose molecules into glycogen, which helps regulate blood sugar and provide energy during fasting or strenuous exercise. The NC group had higher liver and muscle glycogen contents, indicating normal organ function. STZ-induced damage reduced glucose utilization, increased blood sugar, and reduced glycogen content; the muscle glycogen level in the LMET+AI group was higher than that in the MET group (P < 0.05), indicating that Kunlun fetal chrysanthemum polysaccharide can enhance the hypoglycemic effect of metformin. Kunlun fetal chrysanthemum polysaccharide can serve as an adjuvant to improve the efficacy of metformin and reduce its dosage.

[0171] Table 4 Effects of Kunlun Fetal Chrysanthemum Polysaccharide on FBG in Diabetic Mice

[0172]

[0173] Table 5 Effects of Kunlun Fetal Chrysanthemum Polysaccharide on OGTT in Diabetic Mice

[0174]

[0175] Effects of LMET+AI on pancreatic tissue in diabetic mice:

[0176] Pancreatic H&E staining and immunofluorescence: Pancreatic tissue was excised and immersed in fixative, processed into paraffin blocks and sliced, then the sections were cleared of paraffin, stained, dehydrated, and mounted, followed by microscopic examination and image capture. For frozen sections of pancreatic tissue, the tissue was first fixed and dehydrated (rinsed overnight in PBS and sucrose solution), then embedded in OCT medium and stored at -80°C. Sections were cut using a cryostat and stored at -80°C in preparation for immunofluorescence analysis.

[0177] Results: H&E staining was used to evaluate pancreatic histopathology, e.g. Figure 10 As shown in middle A, the DC group showed obvious pancreatic islet atrophy, irregular shape, and reduced islet cell number; the pancreatic tissue atrophy of diabetic mice in the ME group and LMET+AI group was significantly improved, indicating a significant repair effect on the pancreas; compared with the DC group, the pancreatic tissue of the other groups showed a more regular and clear morphology, indicating that Kunlun fetal chrysanthemum polysaccharide can enhance the ability of MET to improve pancreatic damage in diabetic mice;

[0178] Cleaved Caspase-3 is the activated form of this cysteine ​​protease and a key mediator of apoptosis. Anti-apoptotic proteins can inhibit this process, such as Figure 10As shown in middle B, immunofluorescence analysis showed that cleaved Caspase-3 co-localized extensively with insulin in STZ-induced mouse islets. MET intervention reduced this phenomenon. The situation in the LMET+AI group was further improved, and the co-staining of Caspase-3 and insulin was reduced, indicating that compared with MET alone, Kunlun fetal chrysanthemum polysaccharide can enhance the ability of MET to promote insulin secretion and reduce the expression of apoptosis-related proteins, thereby better protecting pancreatic tissue.

[0179] The NLRP-3 inflammasome is involved in chronic inflammation associated with diabetes, especially in pancreatic islet cells, such as Figure 10 As shown in middle C, compared with other groups, the DC group showed stronger NLRP-3 expression but weaker insulin localization. In the LMET+AI group, Kunlun fetal chrysanthemum polysaccharide assisted MET treatment alleviated the inflammatory response of diabetic mice and increased insulin secretion. In both the LMET+AI and MET groups, the co-staining of NLRP-3 and insulin was reduced, but the improvement of pancreatic inflammation in the LMET+AI group was more obvious. These results indicate that Kunlun fetal chrysanthemum polysaccharide can enhance the effect of low-dose MET by inhibiting Caspase-3 and inflammatory factor NLRP-3, which cleaves apoptotic proteins, thereby alleviating apoptosis and inflammation in diabetic mice.

[0180] Test Example 7

[0181] Further verification of the blood sugar and blood lipid lowering effects of Kunlun Fetal Chrysanthemum Polysaccharide:

[0182] Construction of C.elegans high-sugar and high-fat model:

[0183] (1) C. elegans genotypes and culture conditions: The following nematode genotypes were selected: N2Bristol (wild type), ldrls1[dhs-3p::dhs-3::GFP] (Caenorhabditis Genetics Center); C. elegans were synchronized using standard nematode growth medium (NGM) agar plates and fed with Escherichia coli OP50;

[0184] All tests were carried out at 20°C (unless otherwise stated);

[0185] (2) Establishment of a high-sugar, high-fat model: C. elegans were exposed to NGM containing 2% glucose; NGM plates served as the control group (Control), NGM plates supplemented with 2% glucose and different doses of AI-2 (0.1, 1, and 10 mg / mL), and NGM plates supplemented with 2% glucose (Model); synchronized L1 C. elegans were grown at 20°C for 48 h under different dietary conditions until they reached the L4 stage;

[0186] Glycogen and Oil Red O staining analysis: C. elegans cultured under a high-sugar and high-fat model were thoroughly rinsed with M9 buffer in each group. Glycogen staining was performed using iodine staining to evaluate the hypoglycemic effect of AI-2 (i.e., Kunlun fetal chrysanthemum polysaccharide), and iodine vapor staining was performed on L4 nematodes to observe the hypoglycemic effect. Oil Red O staining was used to evaluate the lipid-lowering effect of AI-2, and Oil Red O staining was performed on L4 nematodes to observe lipid accumulation.

[0187] Results: Glycogen staining and Oil Red O staining can show the glucose and fat content in the body. The expression intensity of the staining signal was analyzed to further evaluate the hypoglycemic and lipid-lowering effects of AI-2. Figure 11 As shown in (A), the glycogen staining results showed that the Model group had serious glycogen accumulation in the body, indicating that the glucose content in the body was high. After AI-2 intervention, the glycogen content in the body of the nematodes was reduced, which is consistent with the detection of glucose content. Due to the decrease in glucose content in the body, glycogen synthesis is reduced. The signal intensity of glycogen staining was analyzed using ImageJ. Figure 11 (B) It can be seen that there are significant differences in glycogen signals between the groups (p<0.05), among which the control group and the 10.0mg / mL AI-2 group have the lowest glycogen signal expression. In addition, low, medium and high concentrations of AI-2 all show hypoglycemic effects on nematodes. The fat-soluble dye Oil Red O has specificity and can color neutral fats such as triglycerides in tissues. Figure 12 As shown in (A), the oil red staining of nematodes in the Model group corresponds to the triglyceride measured in the early stage, indicating that a large amount of fat has accumulated in the body. At this time, the nematodes remain in a high-fat state. After intervention with AI-2, all concentrations of AI-2 have the effect of reducing fat accumulation in the nematodes; Figure 12 (B) As can be seen, the signal after Oil Red O staining is highly expressed in the model. After AI-2 intervention, this trend was reversed, and the expression of Oil Red O signal in the high-fat model nematode was reduced. These results show that AI-2 can play a role in lowering blood sugar and lipids.

[0188] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Kunlun fetal chrysanthemum polysaccharide, characterized in that It is mainly composed of arabinose, galactose, galacturonic acid, rhamnose and glucose; The main chain of the Kunlun fetal chrysanthemum polysaccharide is composed of →4)-β-D-Galp-(1→ and →4)-β-D-Glcp-(1→ alternately; The side chain of the Kunlun fetus chrysanthemum polysaccharide is composed of →5)-α-L-Araf-(1→, →3,6)-β-D-Galp, α-L-Araf, β-D-Glcp / Galp and α-D-Rhap.

2. The Kunlun chrysanthemum polysaccharide according to claim 1, characterized in that The molar percentages of arabinose, galactose, galacturonic acid, rhamnose and glucose are 26.52:37.96:26.43:5.38:3.

71.

3. The Kunlun fetal chrysanthemum polysaccharide according to claim 1 or 2, characterized in that The structural formula of the repeating unit in the Kunlun fetal chrysanthemum polysaccharide is as follows: The uronic acid content of the Kunlun fetal chrysanthemum polysaccharide is 26.48±0.11%; The Kunlun chrysanthemum polysaccharide is an acidic polysaccharide with a molecular weight of 3192Da.

4. A method for preparing Kunlun chrysanthemum polysaccharide according to any one of claims 1 to 3, characterized in that: The following steps are involved: (a) hydrolyzing Kunlun fetal chrysanthemum under acidic conditions, and then filtering to obtain a filtrate; (b) concentrating the filtrate obtained in step (a), then subjecting it to alcohol precipitation, and filtering it to obtain a precipitate; (c) mixing the precipitate obtained in step (b) with an alkaline solution, filtering the mixture, and adjusting the pH of the filtrate to neutral to obtain an extract; (d) ultrafiltration of the extract obtained in step (c) to obtain a filtrate and drying the filtrate to obtain a crude extract; (e) ultrafiltration of the crude extract obtained in step (d) to obtain a retentate and drying the retentate to obtain crude Kunlun fetal chrysanthemum polysaccharide; (f) The obtained crude Kunlun Chrysanthemum polysaccharide is treated by anion exchange chromatography and dialysis purification to obtain the Kunlun Chrysanthemum polysaccharide.

5. The preparation method according to claim 4, characterized in that In step (a), the hydrolysis method comprises the following steps: After soaking Kunlun chrysanthemum in acid solution, hydrolyzing it under the conditions of cellulase synergistic magnetic field and electric field coupling to obtain hydrolyzate; Preferably, the stirring condition for the hydrolysis is stirring at 85° C. for 2 h; Preferably, the cellulase is a cellulase having an enzyme activity of 800 U / g after addition and a pH value of 5.3; Preferably, the magnetic induction intensity of the magnetic field is 0.3T-0.5T; Preferably, the alternating voltage frequency used in the electric field is 50 Hz.

6. The preparation method according to claim 4, characterized in that The filtering method includes centrifugation.

7. The preparation method according to claim 4, characterized in that In step (c), the stirring condition for mixing the precipitate and the alkali solution is stirring at 40° C.-60° C. for 2 h-3 h.

8. The preparation method according to claim 4, characterized in that In step (d), the molecular cut-off of the ultrafiltration is 10000Da; Preferably, in step (e), the molecular cut-off of the ultrafiltration is 5000Da; Preferably, in step (e), the molecular weight of the crude polysaccharide of Kunlun embryo chrysanthemum is 5000Da-10000Da.

9. The preparation method according to claim 4, characterized in that The eluent used in the anion exchange chromatography includes 0.01 mol / L NaOH solution and 3 volumes of 0 mol / L, 0.2 mol / L, 0.4 mol / L, 0.6 mol / L, 0.8 mol / L and 1.0 mol / L NaCl solutions.

10. Use of the Kunlun Fetal Chrysanthemum polysaccharide according to any one of claims 1 to 3 in the preparation of drugs for lowering blood sugar and / or blood lipids.

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