Application of burdock polysaccharide in relieving motor ability decline and intestinal flora disorder caused by acute sleep deprivation

Abstract

The invention discloses application of burdock polysaccharide in relieving motor ability decline and intestinal flora disorder caused by acute sleep deprivation, and belongs to the field of medical biology. The invention verifies the influence of the burdock polysaccharide on motor ability reduction, intestinal flora homeostasis and function reduction caused by acute sleep deprivation, and also verifies the relationship of the burdock polysaccharide on motor ability, body energy metabolism and intestinal flora of an acute sleep deprivation individual and a potential mediation mechanism of the burdock polysaccharide in an intestinal-muscle axis. According to the invention, burdock polysaccharide is used on an acute sleep deprivation model, and the application of burdock polysaccharide in treating motor ability and intestinal injury after acute sleep deprivation is verified. Through athletic ability, energy metabolism, inflammatory factors in serum, oxidative stress in liver and Hamp of ileum tissue; e staining, expression of tight junction protein and determination of indexes such as intestinal flora and the like find that the burdock polysaccharide can effectively improve the athletic ability and the intestinal flora after acute sleep deprivation.

Description

Technical Field

[0001] The invention relates to application of burdock polysaccharide in alleviating decreased motor ability and intestinal flora disorder caused by acute sleep deprivation, and belongs to the field of medicine and biology. Background Art

[0002] Sleep plays a key role in cognitive function and physical health. However, when individuals experience sleep deprivation (SD), their health status faces severe challenges. This is mainly achieved by disrupting the body's energy metabolism, immune response and endocrine signaling.

[0003] Sleep deprivation can be divided into acute sleep deprivation and chronic sleep deprivation. Acute sleep deprivation usually lasts for several hours or even days; chronic sleep deprivation usually lasts for several hours a day for weeks or even months. Sleep deprivation is very common in work and daily life: sleep deprivation can occur when working night shifts, going on missions, or working overtime; physical or mental illnesses can also lead to sleep deprivation.

[0004] Sports events have extremely high requirements for the comprehensive qualities of athletes, especially in terms of brain alertness, physical reaction ability and cognitive decision-making ability. Athletes need to demonstrate good reaction ability and quick decision-making ability when facing sports. Studies have shown that athletes often suffer from acute sleep deprivation during training or before competition due to bad sleeping habits, high-intensity training, stress and environmental factors. Acute sleep deprivation may have a certain impact on athletes' cognitive decision-making ability and sports performance.

[0005] In addition, the stability of the microbial community is essential for human health, and microbial dysbiosis has been considered an important factor leading to sleep-related diseases. Recent studies have emphasized that the role of intestinal microbiota in SD is increasing, playing a key role in maintaining intestinal homeostasis (Peijun Tian, ​​Hou Yunfei, Wang Zheng, et al. Probiotics administration alleviates cognitive impairment and circadian rhythm disturbance induced by sleep deprivation [J]. Food Science and Human Wellness, 2024, 13 (4): 1951-1961; Z Wang, Chen WH, Li SX, et al. Gut microbiota modulates the inflammatory response and cognitive impairment induced by sleep deprivation [J]. Mol Psychiatry, 2021, 26 (11): 6277-6292.). There is convincing evidence that regular physical activity can effectively regulate the composition of intestinal microbiota, and athletes show significantly higher intestinal microbial diversity compared with sedentary people (Tian et al., 2024; Wang et al., 2021). In this context, the "gut-muscle axis" hypothesis provides a new perspective for understanding how the gut microbiota regulates skeletal muscle adaptation during exercise. However, long-term SD can lead to a decrease in probiotics and an increase in harmful bacteria, thereby disrupting the balance of the gut microbiota (LM Zheng, LiY. Modifications in the Composition of the Gut Microbiota in Rats Induced by Chronic Sleep Deprivation: Potential Relation to Mental Disorders[J]. Nat Sci Sleep, 2024, 161313-1325.).Studies have also found that transplanting intestinal microbiota regulated by sleep drugs or supplementing intestinal microbial metabolites (such as butyrate) can significantly improve the neurological and peripheral symptoms of SD mice (X Wang, Wang Z, Cao J, et al. Gut microbiota-derived metabolites mediate the neuroprotective effect of melatonin in cognitive impairment induced by sleep deprivation [J]. Microbiome, 2023, 11 (1): 17.). Therefore, using dietary intervention strategies to regulate the "SD group microbiota" has become an important way to enhance immunity and maintain motor function.

[0006] Burdock is a plant with both medicinal and edible properties. It belongs to the Asteraceae family and is also known as burdock, Oriental ginseng, etc. Its flowers, leaves, roots and fruits can all be used as medicine. Because of its multiple biological activities and pharmacological functions, it has a good development prospect. Among them, burdock polysaccharides (ALP, C 27 H 34 O 11 ) are important bioactive ingredients extracted from the natural plant burdock. They have a variety of significant health benefits, including immune enhancement, anti-tumor and antioxidant effects. They have good antioxidant, anti-inflammatory, hypoglycemic and other biological activities, are safe and stable to use, and have broad application prospects in the biomedical field. However, in clinical practice, there is no use of burdock polysaccharides to repair the decreased motor ability and intestinal flora disorders caused by acute sleep deprivation. Summary of the invention

[0007] Burdock polysaccharides increased the relative abundance of beneficial bacteria, such as Akkermansia and Muribaculaceae, while decreasing the abundance of opportunistic gut bacteria, such as Colidextribacter and Lachnoclostridium.

[0008] Burdock polysaccharides provide protection to the intestinal barrier, regulate intestinal microbiota, and change the intestinal muscle connection, thereby alleviating SD-induced exercise damage through immune and metabolic pathways.

[0009] The invention provides an application of burdock polysaccharide in preparing a product for resisting sleep deprivation damage.

[0010] The burdock polysaccharide described in the present invention can be a commercially available product or can be directly prepared by itself, both of which can achieve the technical solution of the present invention.

[0011] In one embodiment of the present invention, the product contains an effective dose of burdock polysaccharide, and the product is a food, a medicine, a dietary supplement or a health product.

[0012] In one embodiment of the present invention, the preparation method of burdock polysaccharide is:

[0013] (1) Dissolve burdock root powder in water, extract at 50°C to 70°C for 90 to 120 minutes, centrifuge, and collect the supernatant;

[0014] (2) After concentrating the supernatant obtained in step (1), adding trichloroacetic acid, stirring, centrifuging, and taking the supernatant;

[0015] (3) adding ethanol to the supernatant of step (2), precipitating with alcohol for 6 to 8 hours, centrifuging, discarding the supernatant, and freeze-drying the precipitate to obtain burdock polysaccharide.

[0016] In one embodiment of the present invention, the mass volume ratio of the burdock root powder to water in step (1) is 1:10-50 g / mL.

[0017] In one embodiment of the present invention, the centrifugation in step (1) is performed at 4000-6000 r / min for 15-20 min.

[0018] In one embodiment of the present invention, the burdock root powder in step (1) has a mesh size of 80 to 100.

[0019] In one embodiment of the present invention, the volume concentration of trichloroacetic acid in step (2) is 10-20%.

[0020] In one embodiment of the present invention, the centrifugation in step (2) is performed at 4000-6000 r / min for 15-20 min.

[0021] In one embodiment of the present invention, the alcohol precipitation in step (3) is carried out at 4° C.; the concentration of ethanol is 95%.

[0022] In one embodiment of the present invention, the freeze-drying conditions are: -40°C to -70°C, 80 to 100 Pa.

[0023] In one embodiment of the present invention, the centrifugation in step (3) is performed at 4000-6000 r / min for 10-20 min.

[0024] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0025] In one embodiment of the present invention, the food further comprises special dietary food.

[0026] In one embodiment of the present invention, the health care product further contains acceptable excipients.

[0027] In one embodiment of the present invention, the dosage form of the drug includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills or solutions.

[0028] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0029] In one embodiment of the present invention, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

[0030] The present invention also provides the use of the burdock polysaccharide in preparing a product having any one or more of the following effects or functions:

[0031] (a) Alleviate and / or treat the decline in motor performance caused by acute sleep deprivation in individuals;

[0032] (b) alleviating and / or treating a decrease in blood glucose levels in an individual caused by acute sleep deprivation injury;

[0033] (c) improving energy metabolism, inflammation and oxidative stress levels in individuals affected by acute sleep deprivation;

[0034] (d) Alleviating and / or treating intestinal barrier damage caused by acute sleep deprivation in an individual.

[0035] In one embodiment of the present invention, the athletic ability includes but is not limited to grip strength and muscle endurance; the energy metabolism includes but is not limited to glycogen and muscle glycogen levels; the oxidative stress level includes but is not limited to SOD and CAT levels in liver tissue and MDA content level; the inflammation level includes but is not limited to CRP level in serum, inflammatory factor IL-6 level, TNF-α level.

[0036] In one embodiment of the present invention, the product contains an effective dose of burdock polysaccharide, and the product is a food, a medicine, a dietary supplement or a health product.

[0037] In one embodiment of the present invention, the preparation method of the burdock polysaccharide is:

[0038] (1) Dissolve burdock root powder in water, extract at 50°C to 70°C for 90 to 120 minutes, centrifuge, and collect the supernatant;

[0039] (2) After concentrating the supernatant obtained in step (1), adding trichloroacetic acid, stirring, centrifuging, and taking the supernatant;

[0040] (3) adding ethanol to the supernatant of step (2), subjecting the mixture to alcohol precipitation for 6 to 8 hours, centrifuging, discarding the supernatant, and freeze-drying the precipitate to obtain burdock polysaccharide;

[0041] In one embodiment of the present invention, the mass volume ratio of the burdock root powder to water in step (1) is 1:10-50 g / mL.

[0042] In one embodiment of the present invention, the centrifugation in step (1) is performed at 4000-6000 r / min for 15-20 min.

[0043] In one embodiment of the present invention, the burdock root powder in step (1) has a mesh size of 80 to 100.

[0044] In one embodiment of the present invention, the volume concentration of trichloroacetic acid in step (2) is 10-20%.

[0045] In one embodiment of the present invention, the centrifugation in step (2) is performed at 4000-6000 r / min for 15-20 min.

[0046] In one embodiment of the present invention, the alcohol precipitation in step (3) is carried out at 4° C.; the concentration of ethanol is 95%.

[0047] In one embodiment of the present invention, the freeze-drying conditions are: -40°C to -70°C, 80 to 100 Pa.

[0048] In one embodiment of the present invention, the centrifugation in step (3) is performed at 4000-6000 r / min for 10-20 min.

[0049] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks.

[0050] In one embodiment of the present invention, the food further comprises special dietary food.

[0051] In one embodiment of the present invention, the health care product further contains acceptable excipients.

[0052] In one embodiment of the present invention, the dosage form of the drug includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills or solutions.

[0053] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0054] In one embodiment of the present invention, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

[0055] The present invention also provides a composition, food, medicine, dietary supplement or health product for resisting sleep deprivation damage, wherein the composition, food, medicine, dietary supplement or health product comprises burdock polysaccharide;

[0056] In one embodiment of the present invention, the preparation method of burdock polysaccharide is:

[0057] (1) Dissolve burdock root powder in water, extract at 50°C to 70°C for 90 to 120 minutes, centrifuge, and collect the supernatant;

[0058] (2) After concentrating the supernatant obtained in step (1), adding trichloroacetic acid, stirring, centrifuging, and taking the supernatant;

[0059] (3) adding ethanol to the supernatant of step (2), subjecting the mixture to alcohol precipitation for 6 to 8 hours, centrifuging, discarding the supernatant, and freeze-drying the precipitate to obtain burdock polysaccharide;

[0060] In one embodiment of the present invention, the mass volume ratio of the burdock root powder to water in step (1) is 1:10-50 g / mL.

[0061] In one embodiment of the present invention, the centrifugation in step (1) is performed at 4000-6000 r / min for 15-20 min.

[0062] In one embodiment of the present invention, the burdock root powder in step (1) has a mesh size of 80 to 100.

[0063] In one embodiment of the present invention, the volume concentration of trichloroacetic acid in step (2) is 10-20%.

[0064] In one embodiment of the present invention, the centrifugation in step (2) is performed at 4000-6000 r / min for 15-20 min.

[0065] In one embodiment of the present invention, the alcohol precipitation in step (3) is carried out at 4° C.; the concentration of ethanol is 95%.

[0066] In one embodiment of the present invention, the freeze-drying conditions are: -40°C to -70°C, 80 to 100 Pa.

[0067] In one embodiment of the present invention, the centrifugation in step (3) is performed at 4000-6000 r / min for 10-20 min.

[0068] Beneficial Effects

[0069] 1. The present invention explores the improving effect of burdock polysaccharide on motor ability and intestinal biota in sleep deprivation through animal experiments, providing ideas for the further application of burdock polysaccharide in anti-fatigue.

[0070] 2. The present invention uses burdock polysaccharide on an acute sleep deprivation model induced in mice to explore the use of burdock polysaccharide in the present invention for treating motor ability and intestinal damage after acute sleep deprivation. Through the determination of indicators such as mouse motor ability, energy metabolism, inflammatory factors in serum, oxidative stress in the liver, H&E staining of ileum tissue, expression of tight junction proteins, and intestinal flora, it was found that the burdock polysaccharide can effectively improve motor ability and intestinal flora after acute sleep deprivation.

[0071] 3. The present invention provides a new treatment method for people who suffer from acute sleep deprivation due to bad sleeping habits, high intensity training, stress and environmental factors, that is, the burdock polysaccharide of the present invention can effectively improve the motor ability and intestinal flora after acute sleep deprivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 : Effect of ALP on the motor ability of mice with acute sleep deprivation; (A) is the grip strength of the forelimbs of mice, and (B) is the motor coordination ability of mice in the rotating rod test.

[0073] Figure 2:Effects of ALP on energy metabolism, oxidative stress balance and immune regulation of SD mice; (A)-(C) are blood glucose, liver glycogen and muscle glycogen contents, (D)-(F) are liver CAT, MDA and SOD contents, (G)-(I) are serum CRP and inflammatory factors (IL-6 and TNF-α) contents, respectively.

[0074] Figure 3 :ALP protects the intestinal barrier by promoting the expression of tight junction proteins; (A) is the H&E staining and histopathological examination of ileum tissue, (B) is the villus height, crypt depth and the ratio of villus height to crypt depth of ileum tissue, (C) is the LPS level in ileum tissue, (D)-(E) are the mRNA expression levels of tight junction proteins ZO-1 and Occludin in ileum tissue, respectively.

[0075] Figure 4 : Effect of ALP on SD-related intestinal flora; where (A) is α diversity (Ace, Shannon, Chao index), (B) is β diversity of hierarchical clustering at the genus level, (C) is principal component analysis (PCA) at the OTU level, (D) is PCoA analysis at the genus level, (E) is the change of ALP of microbial flora at the genus level, and (F) is the relative abundance of microorganisms at the genus level.

[0076] Figure 5 :ALP regulates intestinal microbial functions and metabolic pathways; (A)-(B) are linear discriminant analysis effect size (LEfSe) analysis of microorganisms at the genus level, (C) is the phylogenetic tree and species relative abundance at the genus level, (D)-(E) are microbial network interaction diagrams of the SD+ALP group, and (F) is the Mantel correlation analysis between exercise performance (as well as antioxidant capacity and energy metabolism), inflammation-related biomarkers (CRP, IL-6, TNF-α and LPS) and intestinal microbiota composition. DETAILED DESCRIPTION

[0077] The detection methods involved in the following embodiments are:

[0078] Analysis of colon tissue

[0079] Colon tissue samples were collected from each group (n = 3 per group) and stored in a solution of 4% paraformaldehyde (BSA) / PBS (v / v) for histological evaluation. Tissue samples were dehydrated with 70% ethanol and then embedded in paraffin. They were cut into 4 μm slices and stained with hematoxylin and eosin (H&E). Digital images of colon tissues were acquired using a digital panoramic scanner (WS-10, provided by Jiangsu Zhiyue Medical Technology Co., Ltd.) and analyzed and processed in detail using CaseViewer v2.3 software.

[0080] Detection of inflammatory factors

[0081] Fresh anticoagulated whole blood was collected from the orbital vein and centrifuged at 3500 rpm for 15 minutes at 4°C to separate serum. The levels of oxidative stress indicators superoxide dismutase (SOD), glutathione peroxidase (glp), and malondialdehyde (MDA) in serum were detected by Elisa method. At the same time, the same method was used to determine the levels of inflammatory factors, especially interleukin-6 (IL-6), tumor necrosis factor (TNF-α), and c-reactive protein (CRP). The kits used in the experiment were operated according to the instructions provided by Xiamen Huijia Biotechnology Co., Ltd. and Beijing Sun Biotechnology Co., Ltd.

[0082] Immunoblot analysis

[0083] Colon tissue samples were homogenized with RIPA buffer (1:3, m / v), and 1% phosphatase inhibitors and protease inhibitors (purchased from Beyotime Biotechnology) were added. Proteins were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE, provided by EpiZyme Biotechnology, China) (conditions: 120V, 1 hour). Subsequently, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane at 350mA using a Bio-Rad laboratory electrophoresis instrument using 20% ​​methanol / tris-glycine transfer buffer (from Servicebio), and the blotting lasted for 1.5 to 2 hours. After blotting, the membrane was blocked with 5% skim dry milk in TBST for 2.5 hours, and then primary antibodies (including zonulaoccluden-1 (ZO-1), occludin and β-actin) were added at a ratio of 1:1000 v / v (using primary antibody dilution buffer) and incubated overnight at 4°C. Next, the PVDF membrane was incubated with the secondary antibody for 1 hour and washed 5 times. Finally, the blot was detected using an enhanced chemiluminescence detection kit (provided by Beyotime) on a fully automated gel imaging analysis system (Universal Hood III, Bio-rad, USA).

[0084] Microbial flora detection

[0085] Microbial DNA was extracted from mouse colonic contents aseptically and rapidly using the Genomic DNA Kit (Omega Bio-Tek). We used 16S rRNA gene sequencing specifically targeting the V3-V4 region to identify bacterial species. Amplification was performed using the whole genome 341F / 806R primer pair (341F: 5′-CCTAYGGGRBGCASCAG-3′ and 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). PCR conditions were set at 95°C for initial denaturation for 5 min, followed by 30 cycles of 95°C for 30 s, 60°C for 30 s, and 75°C for 30 s, and a final extension at 75°C for 10 min.

[0086] Subsequently, all amplicons were purified using the DNA Gel / PCR purification Miniprep kit (Biomiga, BW-DC3511-01, China) and quantified using the Quant-iT PicoGreen dsDNA assay kit (Life Technologies, Carlsbad, USA). Sequencing was then performed on the Illumina NovaSeq platform. Splicing was performed based on the direct overlap of PE, and bases with a quality value of ≤20 were filtered out using Flash (version 1.2.11). After this, a strict quality control process was implemented to remove unqualified sequences with a quality value of ≤20. Bacterial sequences with a similarity of ≥0.97 were divided into operational taxonomic units (otu) and clustered using USEARCH11-uparse based on the Unite database.

[0087] Grip strength test

[0088] In this study, the grip strength test was conducted using a Panlab dynamometer (model LE902, provided by Hong Kong Youcheng Biotechnology Co., Ltd., China) to assess the forelimb grip strength of each experimental mouse. The specific operation is: gently lift the mouse's tail, guide its front paws to grasp the metal wire of the dynamometer, and then slowly pull back while ensuring that the tail is parallel to the experimental table until the mouse releases the wire. To ensure the accuracy of the data, each mouse was tested three times in a row, and the average score was processed for subsequent statistical analysis.

[0089] Muscle endurance test

[0090] First, the mice were trained to use the rotarod. During the training, the mice were placed on an accelerating rotarod (model ZB-200, manufactured by Chengdu Taimeng Technology Co., Ltd.). The speed of the device was gradually increased from 5 rpm to 15 rpm within 5 minutes, and the mice needed to maintain balance on the rotating rod. Formal testing can only be carried out after 3 training sessions. In the formal test, the mice were placed on a rotarod with a fixed speed of 15 rpm for a duration test until the mice could no longer maintain balance and fell. The time each mouse persisted on the rotarod was recorded, and this length of time was used as a key indicator to assess their motor coordination.

[0091] Hematoxylin and eosin (HE) staining

[0092] Ileal tissue samples were collected from mice sacrificed in each group (n = 3 per group) and stored in 4% paraformaldehyde (BSA) / PBS (v / v) solution for histological evaluation. Tissue samples were dehydrated with 70%, 80%, 90%, and 100% ethanol, then embedded in paraffin and cut into 4 μm sections. Hematoxylin and eosin (H&E) staining was then performed. Finally, digital images of ileal tissues were obtained using a digital panoramic scanner (provided by Jiangsu Zhiyue Medical Technology Co., Ltd., WS-10), and detailed analysis and processing were performed using CaseViewer v2.3 software.

[0093] Detection of biomarkers in serum and liver

[0094] Fresh anticoagulated whole blood samples were collected from mouse eyeballs and centrifuged at 3500 rpm for 15 min at 4°C to separate serum from blood. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory factors including interleukin 6 (IL-6), tumor necrosis factor (TNF-α) and C-reactive protein (CRP) in serum.

[0095] Then, the oxidative stress indicators in the liver were measured, including the content of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA). The kits used in the experiment were operated according to the instructions provided by Xiamen Huijia Biotechnology Co., Ltd. and Beijing Solebow Technology Co., Ltd.

[0096] Ileal tissue mRNA extraction and qPCR detection

[0097] Mouse ileum RNA was isolated using TRIzol reagent. Reverse transcription was performed using the SuperRT cDNA synthesis kit. Quantitative analysis was performed using SYBR Green. Data were calculated using the comparative cycle threshold method (2-ΔΔct), and β-actin was used as the normalizer gene. All primers were synthesized by Thermo Fisher Scientific and the sizes were determined. Primers were designed using Primer 5 software. The specificity of the primers was tested using the blast program, and the PCR products were detected using agarose gel electrophoresis. The specific primers used for real-time quantitative polymerase chain reaction (qPCR) are shown in Table 1.

[0098] Total RNA from ileum tissue was extracted using the TRIzol method according to the instructions. Liquid nitrogen was placed in the collected fresh ileum tissue, ground into powder, 100 mg of tissue was mixed and dissolved with 1 mL of Trizol solution, 200 μL of chloroform was added and shaken, and the supernatant was centrifuged at 4°C. The white RNA precipitate was centrifuged again and washed twice with 75% ethanol and dissolved in DEPC (diethyl pyrocarbonate) water. Total RNA was reverse transcribed into cDNA using Super ScriptⅡ reverse transcriptase (Invitrogen, Carlsbad).

[0099] Real-time quantitative PCR analysis was performed using SYBR Green Supermix dye and specific primers of the gene to be tested. The reaction system was: 5 μL SYBR Green Supermix dye, 1 μL cDNA, 0.4 μL forward primer, 0.4 μL reverse primer, 3.2 μL dd water. The amplification program set for qPCR was: preheating at 50°C for 1 min, preheating at 95°C for 2 min, maintaining at 95°C for 5 s, maintaining at 60°C for 15 s, and reacting for 40 cycles. The change in mRNA expression of the target gene relative to the housekeeping gene was calculated according to the comparative cycle threshold method (2-ΔΔct).

[0100] Table 1 Specific primers used in q-PCR

[0101]

[0102] 16S rRNA analysis of intestinal microbiota

[0103] Microbial DNA was rapidly extracted from the colonic contents of mice in a sterile environment using a genomic DNA test kit (Omega Bio-Tek). However, 16S rRNA gene sequencing (V3-V4 region) detected only bacteria, which were amplified from the entire genome using a barcoded 341F / 806R primer pair (341F 5'-CCTAYGGGRBGCASCAG-3' and 806R, 5'-GGACTACHVGGGTWTCTAAT-3'). PCR conditions were set at 95°C for 5 minutes, followed by 95°C / 30s, 60°C / 30s, 75°C / 30s, and a final extension at 75°C for 10 minutes. All amplifications were purified using a DNA Gel / PCR Purification Mini prep kit (Biomiga, BW-DC3511-01, China), quantified using a QFEsnt-iT PicoGreen dsDNA As-say kit (LifeTechnologies, Carlsgood, USA), and then sequenced on the Illumina NovaSeq platform. Paired-end sequences were stitched based on the direct overlap relationship of PE (Paired-end) reads, and bases with read quality values ​​≤ 20 were screened using Flash (version 1.2.11). Subsequently, quality control was performed by screening unqualified sequences with quality values ​​≤ 20. Bacteria with sequence similarity ≥ 0.97 were exclusively assigned an operational taxonomic unit (OTU) and clustered using USEARCH11-uparse based on the Unite database.

[0104] Data processing

[0105] All results in this study were expressed as mean ± standard deviation (mean ± SD) to ensure the accuracy and reliability of the data. To study the differences between the groups, we used SPSS24.0 software (SPSS, Chicago, IL, USA) for one-way analysis of variance (ANOVA), and further determined the significant differences between the groups by Dunnett's multiple comparison test. In the statistical results, *p<0.05 and **p<0.01 indicated significant and extremely significant differences compared with the SD group, respectively. In order to visualize and analyze the data in depth, we used the ggpubr and ggplot2 packages in R software (v4.1.0) and GraphPad Prism 8.1.0 to generate statistical analysis and box plots. In addition, we used Mantel analysis to test the correlation between the factors and explore their degree of association.

[0106] Finally, to gain a more comprehensive understanding of the interactions and relationships among the factors, we constructed a network model using Cytoscape (v3.8.0).

[0107] The burdock polysaccharide described in the present invention can be a commercially available product or can be directly prepared by itself, both of which can achieve the technical solution of the present invention.

[0108] Example 1: Preparation of burdock polysaccharide

[0109] The specific steps are as follows:

[0110] The hot water extraction method is adopted: 200g of 100-mesh burdock powder is weighed, and it is extracted with deionized water at a solid-liquid ratio of 1:10 (g / mL) at 70°C for 90min, and then centrifuged at 4000r / min for 15min; the filter residue is taken to repeat the above process, and the two filtrates are combined and concentrated to 1 / 3 of the original volume by rotary evaporation at 70°C; then an equal volume of trichloroacetic acid with a mass concentration of 10% is added, magnetic stirring is performed for 30min, and centrifuged at 4000r / min for 15min, the precipitate is removed, and the supernatant is retained; repeat 3 to 5 times until there is no denatured protein layer at the junction of the two phases; the supernatant is combined, and finally 4 times the volume of 95% ethanol is added to the supernatant, and alcohol precipitation is performed at 4°C for 6h; then centrifuged at 4000r / min for 10min, the supernatant is discarded, and the precipitate is freeze-dried at -40°C and 100Pa to obtain ALP.

[0111] The results are shown in Table 2:

[0112] Table 2: Monosaccharide composition ratio in burdock polysaccharide ALP

[0113] sample Mannose glucose fructose Galactose ALP 2.14％ 62.54％ 34.03％ 1.29％

[0114] Example 2: Application of burdock polysaccharide in sleep deprivation

[0115] Thirty-two healthy adult male C57BL / 6 mice (Wei Tong Li Hua) weighing 20g-40g were acclimated for one week under specific pathogen-free (SPF) conditions at the Experimental Animal Center of Jiangnan University (Wuxi, China). The experimental protocol was approved by the Ethics Committee of the Experimental Animal Center of Jiangnan University. The burdock polysaccharide ALP prepared in Example 1 was used to intervene in acute sleep deprivation of mice.

[0116] The specific steps are as follows:

[0117] The experimental mice were randomly divided into four groups (8 mice in each group). From the 1st to the 7th day of the experiment, the mice in each group were free to eat ordinary maintenance feed (ordinary feed purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.) and drink water freely.

[0118] The specific groups are as follows:

[0119] Normally fed control group (Con group): From day 8 to day 14, mice were gavaged with normal saline at a dose of 0.1 mL / 10 g body weight every day and slept normally;

[0120] Model group (SD group): On days 8 to 12, mice were gavaged with normal saline at a dose of 0.1 mL / 10 g body weight every day and slept normally; on days 13 to 14, mice were sleep deprived for 48 h and gavaged with normal saline at a dose of 0.1 mL / 10 g body weight every day;

[0121] Caffeine-fed group (Pos group, caffeine was gavaged at a daily dose of 10 mg / kg body weight, purity ≥98%, purchased from Shanghai Chunyou Biological Co., Ltd., dissolved in physiological saline for later use): On days 8 to 12, mice were gavaged with caffeine solution at a dose of 0.1 mL / 10 g body weight (10 mg / kg) every day and slept normally; on days 13 to 14, mice were subjected to 48 h of sleep deprivation and gavaged with caffeine solution at a dose of 0.1 mL / 10 g body weight (10 mg / kg) every day;

[0122] The group fed with burdock polysaccharide (SD+ALP group, the daily gavage dose of burdock polysaccharide was 400 mg / kg body weight, prepared in Example 1 and dissolved in physiological saline for later use): on days 8 to 12, the mice were gavaged with burdock polysaccharide solution (ALP solution, burdock polysaccharide was dissolved in physiological saline according to the dosage requirements) at a dose of 0.1 mL / 10 g body weight (400 mg / kg) every day, and slept normally; on days 13 to 14, they were subjected to 48 h of sleep deprivation and gavage with ALP solution at a dose of 0.1 mL / 10 g body weight (400 mg / kg) every day.

[0123] The 48-hour sleep deprivation is to construct an acute SD model, and the specific operation method is as follows:

[0124] Mice were placed in an automatic sleep deprivation system (YAN-239, Shanghai Yuyan Scientific Instrument Co., Ltd.), and mice were free to take water and food. A stainless steel rod was firmly fixed in the center of each cage. The mice in the group that needed sleep deprivation were placed in the SD state for 4 hours before the experiment. Subsequently, a rotating metal rod with timed movement (5 rpm) was used to smoothly sweep across the bottom of the cage, thereby preventing the mice from falling asleep during the 48-hour SD period described above.

[0125] After the experiment, each mouse in each group was subjected to behavioral tests, and finally all the mice were killed and tissue samples were collected.

[0126] Example 3: Application of burdock polysaccharide in sleep deprivation

[0127] The effects of the motor ability, body energy metabolism, inflammation level and oxidative stress level, and bacterial species of the intestinal flora of each group of mice after the experiment in Example 2 were tested, as follows:

[0128] 1. ALP improves the exercise capacity of acute SD mice

[0129] In order to evaluate the effect of burdock polysaccharide on the motor ability of SD mice and other groups of mice, forelimb grip strength test and rotarod test were performed on SD mice and other groups of mice after the experiment in Example 2.

[0130] The specific steps are as follows:

[0131] Gently lift the mouse's tail and guide its front paws to grasp the metal wire of the grip dynamometer. Then, while ensuring that the tail is parallel to the experimental platform, slowly pull the mouse back until it releases the metal wire. To ensure the accuracy of the data, each mouse was tested three times in a row, and the scores were averaged for subsequent statistical analysis.

[0132] During the training phase, mice were exposed to an accelerating rotating rod apparatus (model ZB-200, provided by Chengdu Taimeng Technology Co., Ltd.). The speed of this apparatus gradually increased from 5 to 15 revolutions per minute over a five-minute period. Throughout the training, mice had to maintain balance on the rotating rod. In the formal assessment, mice were placed on a rotating rod at a constant speed of 15 rpm until they could no longer maintain balance and fell. The time each mouse stayed on the rod was recorded as a measure of their motor coordination ability.

[0133] The results showed that the grip strength of the control group (Con group) fed with normal diet, the control group (acute sleep deprivation SD group) fed with normal saline, the group fed with caffeine (Pos group), and the group fed with burdock polysaccharide (SD+ALP group) were 118.0N, 71.5N, 93.8N, and 96.8N, respectively; the muscle endurance was 1058.4s, 484.8s, 751.4s, and 734.9s, respectively;

[0134] It can be seen that ALP significantly improved the grip strength of mice ( Figure 1 A), indicating that ALP enhanced muscle strength compared with acute sleep deprivation SD mice (93.8±5.4 vs 71.5±12.3N, p<0.01, SD+ALP vs SD group).

[0135] At the same time, in the rotarod test, the muscle endurance of SD+ALP mice was significantly improved (751.4±76.3 vs 484.8±158 s, p<0.01, SD+ALP vs SD group, Figure 1B) These behavioral results verified that ALP significantly improved the motor ability of acute sleep deprived SD mice.

[0136] Exercise capacity is related to the body's metabolic utilization of energy substrates, so we further evaluated the glucose metabolism, oxidative stress and inflammation levels of mice.

[0137] 2. ALP improves the body's energy metabolism, inflammation levels and oxidative stress levels

[0138] ALP intervention for individuals exposed to acute sleep deprivation SD can improve their energy metabolism, inflammation level and oxidative stress level. After the experiment in Example 2, the blood glucose, liver glycogen and muscle glycogen levels in the blood, SOD and CAT levels in the liver tissue and MDA, and CRP, inflammatory factors IL-6 and TNF-α levels in the serum of each group of mice were tested. The results are as follows: Figure 2 shown.

[0139] The results show:

[0140] In the control group (Con group) fed normally, the blood glucose, liver glycogen and muscle glycogen levels were 7.1mmol / L, 7.8mg / g and 0.34mg / g, respectively; the SOD and CAT levels in liver tissue and the MDA content were 32.4nmol / mg prot, 23.6nmol / mg prot and 0.2nmol / mg prot, respectively; the CRP, inflammatory factor IL-6 and TNF-α levels in serum were 167.2μg / L, 109.1pg / mL and 6.6ng / mL, respectively;

[0141] In the control group (acute sleep deprivation model SD group) fed with normal saline, the blood glucose, liver glycogen and muscle glycogen levels were 4.5mmol / L, 3.4mg / g and 0.26mg / g, respectively; the SOD and CAT levels in liver tissue and the MDA content were 15.7nmol / mg prot, 14.5nmol / mg prot and 0.9nmol / mg prot, respectively; the CRP, inflammatory factor IL-6 and TNF-α levels in serum were 218.5μg / L, 160.1pg / mL and 8.8ng / mL, respectively;

[0142] The blood glucose, liver glycogen and muscle glycogen levels of the caffeine-fed group (Pos group) were 6.0mmol / L, 3.1mg / g and 0.34mg / g, respectively; the SOD and CAT levels in liver tissue and the MDA content were 34.6nmol / mg prot, 23.0nmol / mg prot and 0.35nmol / mg prot, respectively; the CRP, inflammatory factor IL-6 and TNF-α levels in serum were 176.7μg / L, 128.1pg / mL and 7.0ng / mL, respectively;

[0143] The blood glucose, liver glycogen and muscle glycogen levels of the group fed with burdock polysaccharide (SD+ALP group) were 6.2mmol / L, 9.3mg / g and 0.36mg / g, respectively; the SOD and CAT levels in liver tissue and the MDA content were 32.0nmol / mg prot, 21.1nmol / mg prot and 0.5nmol / mg prot, respectively; the CRP, inflammatory factor IL-6 and TNF-α levels in serum were 180.0μg / L, 129.5pg / mL and 6.7ng / mL, respectively;

[0144] It can be seen that Figure 2 As shown in the results, ALP pretreatment significantly increased the blood glucose and liver glycogen and muscle glycogen levels in SD mice with acute sleep deprivation model (p<0.01, SD+ALP vs SD). At the same time, ALP maintained the oxidative stress balance in SD mice with acute sleep deprivation by increasing the levels of SOD and CAT in liver tissue and reducing the content of MDA ( Figure 2 DF). In addition, acute sleep deprivation SD significantly increased the inflammatory level of mice, while ALP pretreatment significantly reduced the levels of CRP and inflammatory factors (IL-6 and TNF-α) in serum ( Figure 2 GI). In conclusion, 7-day preventive treatment with ALP can modulate energy metabolism, intracellular redox balance, and systemic inflammation levels, which are crucial for the homeostasis of the acute sleep-deprived SD mice.

[0145] 3. ALP protects the intestinal barrier by promoting the expression of tight junction proteins

[0146] In order to evaluate the effect of ALP on the intestinal barrier, we performed H&E staining on the ileum tissue of mice after the experiment in Example 2 ( Figure 3 ).

[0147] The results show:

[0148] The ileal mucosa of the normally fed control group (Con group) was intact and smooth, the epithelial cells were arranged in an orderly manner, and there was no inflammatory cell infiltration.

[0149] on the contrary;

[0150] The saline-fed control group (acute sleep deprivation model SD group) showed severe epithelial defects, crypt damage, and extensive inflammatory cell infiltration.

[0151] The group fed with caffeine (Pos group): The ileum epithelial structure was less damaged, the goblet cells, crypts and glandular structures were arranged more orderly, and showed a small amount of inflammatory cell infiltration.

[0152] The group fed with burdock polysaccharide (SD+ALP group) had milder ileal mucosal damage, reduced inflammatory cell infiltration, and more complete crypt glands.

[0153] 4. ALP affects the interaction of intestinal microorganisms

[0154] ALP significantly increased the expression of tight junction proteins ZO-1 and occludin in ileal tissue ( Figure 3 ), indicating that ALP may enhance intestinal barrier function by activating these proteins. ALP has a regulatory effect on the intestinal flora of acute sleep deprived SD mice.

[0155] In order to evaluate the effect of ALP on the composition of intestinal flora, after the end of the experiment in Example 2, the intestinal flora community was analyzed by 16s rRNA amplicon sequencing of colon contents obtained from the Con, model group SD and SD+ALP groups.

[0156] The results show:

[0157] (1) Compared with the control group (Con group) fed with normal diet and the model group (acute sleep deprivation model SD group) fed with normal saline, ALP increased the α diversity (Ace and Chao index) of the microbiota ( Figure 4 A).

[0158] (2) Diversity results showed that the intestinal microbiota of the group fed with burdock polysaccharides (SD+ALP group) may show microbial community characteristics comparable to those of the control group (Con group) fed with normal saline. Cluster forest analysis showed that there were significant differences between the Con and SD+ALP groups compared with the model group (acute sleep deprivation model SD group) fed with normal saline ( Figure 4 B).

[0159] (3) The Adonis test was used to evaluate the diversity of bacterial community structure. The experimental results in the principal component analysis (PCA) score diagram revealed that there was a significant difference in the OTU level between the normally fed control group (Con group) and the saline fed model group (acute sleep deprivation model SD group) ( Figure 4 C).

[0160] (4) This study also conducted enterotype analysis at the genus level of the dominant microbial community. Figure 4 D) in order to group different samples with similar dominant microbial community structures according to the relative abundance of microbial groups.

[0161] (5) Through the above cluster analysis, the microbial groups with significant differences between the control group (Con group) fed with normal feeding, the group fed with burdock polysaccharide (SD+ALP group) and the model group fed with normal saline (acute sleep deprivation model SD group) were identified as Lactobacillus enteric microbiota. From the results, burdock polysaccharide optimized the intestinal flora structure of the acute sleep deprivation model SD mice, making it different from other groups ( Figure 4 E).

[0162] (6) At the genus level, burdock polysaccharides significantly increased the abundance of Alistipes, Ruminococcus, and Muribaculum, while inhibiting harmful bacteria such as Blautia and Ligilactobacillus ( Figure 4 F).

[0163] 5. ALP regulates intestinal microbial function and metabolic pathways

[0164] Example 2 After the experiment, the colon contents obtained from the Con, model group SD and SD+ALP groups were sequenced, and a linear discriminant effect size (LEfSe) analysis was performed on the Con, SD and SD+ALP groups from the phylum to the genus level ( Figure 5 AB in ); further study the role of key microbial groups and explore their interrelationships within the community ( Figure 5 AB in).

[0165] We performed a linear discriminant analysis (LDA) and found that the main representative microbial community branches of the SD+ALP group were: Tannerellaceae (LDA value = 4.25), Hungatella (LDA value = 4.05), and Staphylococcus_sciuri (LDA value = 4.01).

[0166] To confirm the evolutionary relationship of the intestinal flora, a phylogenetic tree was generated at the genus level and the relative abundance of species at the genus level for each sample ( Figure 5The results showed that ALP optimized the intestinal flora composition, making it different from the intestinal flora composition of the SD group and the Con group. By establishing a network interaction diagram of the microorganisms in the burdock polysaccharide SD+ALP group, it was found that Actinobacteriota, Bacteroidota, Deferribacterota, Firmicutes, and Verrucomicrobiotas had a strong correlation ( Figure 5 DE in ).

[0167] According to Mantel correlation analysis, strong correlations were observed between inflammatory factors, exercise performance, oxidative stress, and energy metabolism ( Figure 5 At the same time, systemic inflammation is also closely related to the composition of intestinal flora.

[0168] In conclusion, our results suggest a reciprocal relationship between the gut-muscle axis and that the microbiota may represent a viable therapeutic target for enhancing motility and intestinal barrier function through administration of ALP.

[0169] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. Application of burdock polysaccharide in the preparation of products for resisting sleep deprivation damage.

2. The use according to claim 1, characterized in that: The product contains an effective dose of burdock polysaccharide and is a food, a medicine, a dietary nutritional supplement or a health product.

3. The use according to claim 1 or 2, characterized in that: A preparation method of the burdock polysaccharide is: (1) Dissolve burdock root powder in water, extract at 50°C to 70°C for 90 to 120 minutes, centrifuge, and collect the supernatant; (2) After concentrating the supernatant obtained in step (1), adding trichloroacetic acid, stirring, centrifuging, and taking the supernatant; (3) adding ethanol to the supernatant of step (2), subjecting the mixture to alcohol precipitation for 6 to 8 hours, centrifuging, discarding the supernatant, and freeze-drying the precipitate to obtain burdock polysaccharide; Preferably, in step (1), the mass volume ratio of the burdock root powder to water is 1:10-50 g / mL; Preferably, in step (1), the centrifugation is performed at 4000-6000 r / min for 15-20 min; Preferably, in step (1), the burdock root powder is 80-100 mesh; Preferably, in step (2), the volume concentration of trichloroacetic acid is 10-20%; Preferably, in step (2), the centrifugation is performed at 4000-6000 r / min for 15-20 min; Preferably, in step (3), the alcohol precipitation is carried out at 4°C; the concentration of ethanol is 95%; Preferably, in step (3), the freeze-drying conditions are: -40°C to -70°C, 80 to 100 Pa.

4. The use according to claim 2 or 3, characterized in that: The food includes, but is not limited to, cereal products, vegetable products, fruit products, fermented foods, meat products, egg products, dairy products, beverages, and snacks; Preferably, the food also includes special dietary food; Preferably, the health care product further contains acceptable excipients.

5. The use according to claim 2 or 3, characterized in that: The dosage form of the drug includes any one of drops, oral liquid, tablets, capsules, granules, films, gels, powders, emulsions, pills or solutions; Preferably, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

6. Application of burdock polysaccharide in the preparation of products having any one or more of the following effects: (a) Alleviate and / or treat the decline in motor performance caused by acute sleep deprivation in individuals; (b) alleviating and / or treating a decrease in blood glucose levels in an individual caused by acute sleep deprivation injury; (c) improving energy metabolism, inflammation and oxidative stress levels in individuals affected by acute sleep deprivation; (d) Alleviating and / or treating intestinal barrier damage caused by acute sleep deprivation in an individual.

7. The use according to claim 6, characterized in that: The athletic ability includes but is not limited to grip strength and muscle endurance; the energy metabolism includes but is not limited to liver glycogen level and muscle glycogen level; the oxidative stress level includes but is not limited to SOD content level, CAT content level and MDA content level in liver tissue; the inflammation level includes but is not limited to CRP level, inflammatory factor IL-6 level and TNF-α level in serum.

8. The use according to claim 6 or 7, characterized in that: The product contains an effective dose of burdock polysaccharide and is a food, a medicine, a dietary nutritional supplement or a health product.

9. The use according to any one of claims 6 to 8, characterized in that: The preparation method of the burdock polysaccharide is: (1) Dissolve burdock root powder in water, extract at 50°C to 70°C for 90 to 120 minutes, centrifuge, and collect the supernatant; (2) After concentrating the supernatant obtained in step (1), adding trichloroacetic acid, stirring, centrifuging, and taking the supernatant; (3) adding ethanol to the supernatant of step (2), subjecting the mixture to alcohol precipitation for 6 to 8 hours, centrifuging, discarding the supernatant, and freeze-drying the precipitate to obtain burdock polysaccharide; Preferably, in step (1), the mass volume ratio of the burdock root powder to water is 1:10-50 g / mL; Preferably, in step (1), the centrifugation is performed at 4000-6000 r / min for 15-20 min; Preferably, in step (1), the burdock root powder is 80-100 mesh; Preferably, in step (2), the volume concentration of trichloroacetic acid is 10-20%; Preferably, in step (2), the centrifugation is performed at 4000-6000 r / min for 15-20 min; Preferably, in step (3), the alcohol precipitation is carried out at 4°C; the concentration of ethanol is 95%; Preferably, in step (3), the freeze-drying conditions are: -40°C to -70°C, 80 to 100 Pa.

10. A composition, food, medicine, dietary supplement or health product for preventing sleep deprivation damage, characterized in that: The composition, food, medicine, dietary supplement or health product includes burdock polysaccharide; Preferably, the preparation method of the burdock polysaccharide is: (1) Dissolve burdock root powder in water, extract at 50°C to 70°C for 90 to 120 minutes, centrifuge, and collect the supernatant; (2) concentrating the supernatant obtained in step (1) to 1 / 3 of the original volume, then adding trichloroacetic acid, stirring, centrifuging, and taking the supernatant; (3) adding 95% ethanol to the supernatant of step (2), precipitating with alcohol for 6 hours, centrifuging, discarding the supernatant, and freeze-drying the precipitate to obtain burdock polysaccharide; Preferably, in step (1), the mass volume ratio of the burdock root powder to water is 1:10-50 g / mL; Preferably, in step (1), the centrifugation is performed at 4000-6000 r / min for 15-20 min; Preferably, in step (1), the burdock root powder is 80-100 mesh; Preferably, in step (2), the volume concentration of trichloroacetic acid is 10-20%; Preferably, in step (2), the centrifugation is performed at 4000-6000 r / min for 15-20 min; Preferably, in step (3), the alcohol precipitation is carried out at 4°C; the concentration of ethanol is 95%; Preferably, in step (3), the freeze-drying conditions are: -40°C to -70°C, 80 to 100 Pa.

Images