Astragalus polysaccharide for improving gait as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of bioactive polysaccharides, and particularly relates to an astragalus polysaccharide capable of improving gaits as well as a preparation method and application of the astragalus polysaccharide. The preparation method comprises the following steps: firstly, preparing astragalus polysaccharide by using a hot water extraction method, and then degrading the astragalus polysaccharide by using weak acid to prepare the low-molecular astragalus polysaccharide. Furthermore, the obtained astragalus polysaccharide and low-molecular astragalus polysaccharide are taken as objects, caenorhabditis elegans are taken as a model for behavioral research, the steering ratio of the caenorhabditis elegans is taken as a gait detection index, and both the astragalus polysaccharide and the low-molecular astragalus polysaccharide are proved to have the effect of improving gaits. The astragalus polysaccharide and the low-molecular astragalus polysaccharide provided by the invention can be further applied to development of products such as foods, medicines and health-care products, and have wide application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of bioactive polysaccharides, and in particular relates to an astragalus polysaccharide for improving gait, and a preparation method and application thereof. Background Art

[0002] Movement is one of the basic behaviors of humans and animals. The movement speed is usually used to characterize the movement ability of the body. However, the speed of movement cannot accurately reflect the health of the body. Gait is the posture and behavior characteristics that appear during walking. Gait can indicate the direction, stability, periodicity, rhythmicity, etc. of the body, which can well reflect the health of the body. For example, as a model animal recognized in the international biomedical field, the change of gait of Caenorhabditis elegans is also one of the important characteristics to measure its movement ability, including the weakening of its directionality, which is similar to humans.

[0003] At present, the main methods for improving gait include exercise intervention and nerve stimulation, and there are very few reports on polysaccharides that can improve gait. For example, crude mulberry polysaccharide has the ability to restore behavioral abnormalities caused by knee osteoarthritis, thus having the potential to indirectly improve gait. Astragalus is a traditional tonic Chinese medicine in my country. Its main ingredient, astragalus polysaccharide, has biological activities such as anti-aging and neuroprotection, but the role of astragalus polysaccharide in improving gait has not been reported.

[0004] Compared with the starting polysaccharide, the low molecular weight polysaccharide prepared by degradation has a smaller molecular weight, which is conducive to absorption and utilization. Studies have shown that low molecular weight astragalus polysaccharide has good anti-inflammatory and antioxidant activities, but there are very few studies on the behavioral aspects of low molecular weight astragalus polysaccharide, especially in terms of gait improvement. Summary of the invention

[0005] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a method for preparing astragalus polysaccharide for improving gait.

[0006] Another object of the present invention is to provide an astragalus polysaccharide for improving gait prepared by the above preparation method, wherein the astragalus polysaccharide can improve the body's gait abnormalities, specifically including the gait of neurodegenerative diseases and the gait of normal conditions.

[0007] Another object of the present invention is to provide a low-molecular-weight astragalus polysaccharide, which can not only improve the abnormal gait of the body, but also increase the movement speed of the body.

[0008] The fourth object of the present invention is to provide a method for preparing the above-mentioned low-molecular-weight astragalus polysaccharide.

[0009] The fifth object of the present invention is to provide applications of the above-mentioned astragalus polysaccharides and low-molecular-weight astragalus polysaccharides.

[0010] The purpose of the present invention is achieved through the following technical solutions: A method for preparing astragalus polysaccharide for improving gait comprises the following steps: (1) Degreasing water extraction: After crushing the Astragalus, add ethanol solution and reflux degreasing at 70-80 °C for 2-3 h. Reflux degreasing 1-3 times to obtain defatted Astragalus. Then add water and extract at 80-90 °C for 2-3 h. Repeat the extraction 1-3 times and collect the extract. (2) Enzymatic protein removal: The extract obtained in step (1) is concentrated by rotary evaporation, and then the pH of the system is adjusted to 6.0-7.0, and papain is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the pH of the system is adjusted to 8.0-8.5, and trypsin is added for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the extract is boiled and inactivated; (3) Ultrafiltration and freeze-drying: The system in step (2) is filtered to remove the precipitate, and then ultrafiltration is performed using a 1000 Da ultrafiltration membrane, and the retentate is collected and freeze-dried to obtain crude astragalus polysaccharide; (4) Separation and purification: using DEAE Sepharose TM Fast Flow anion exchange chromatography column was used for separation and purification, water and NaCl solution were used as eluents, and the sugar-containing eluate after water washing and the sugar-containing eluate after salt washing were collected; (5) Dialysis freeze-drying: Use a 1000 Da dialysis bag for dialysis treatment, collect the dialyzed solution and freeze-dry it to obtain neutral and acidic astragalus polysaccharides.

[0011] The astragalus can be astragalus capsulatus, astragalus mongolica and the like.

[0012] The mass volume ratio (g:mL) of astragalus to ethanol in step (1) is preferably 1:5-1:10.

[0013] The mass volume ratio (g:mL) of the astragalus to water in step (1) is preferably 1:10-1:20.

[0014] The volume fraction of the ethanol solution in step (1) is preferably 95%.

[0015] The papain enzymatic hydrolysis conditions in step (2) are preferably 60°C for 3 h.

[0016] The trypsin enzymatic hydrolysis conditions in step (2) are preferably 45°C for 3 h.

[0017] Astragalus polysaccharide for improving gait is prepared by the above preparation method.

[0018] A low-molecular-weight astragalus polysaccharide for improving gait is obtained by further acid degradation, washing, dialysis and ultrafiltration classification of the above-mentioned astragalus polysaccharide for improving gait.

[0019] The method for preparing the low-molecular-weight astragalus polysaccharide for improving gait comprises the following steps: (1) Acid degradation: Weigh astragalus polysaccharide and dissolve it in water, add trifluoroacetic acid to acid-degrade it in a water bath, and then evaporate the solution under reduced pressure; (2) Washing and dialysis: The product obtained in step (1) is washed with anhydrous methanol and subjected to rotary evaporation to remove trifluoroacetic acid. The product after rotary evaporation is dissolved in water, dialyzed with a 200 Da dialysis bag, and freeze-dried to obtain a freeze-dried powder; (3) Ultrafiltration classification: The freeze-dried powder obtained in step (2) is dissolved in water, placed in a filter centrifuge tube with a molecular weight cutoff of 10 kDa, and the filtrate is collected by centrifugation; the filtrate is placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, centrifuged, the retained portion is collected and freeze-dried to obtain low molecular weight astragalus polysaccharides with a molecular weight of 3-10 kDa.

[0020] The final concentration of trifluoroacetic acid after addition in step (1) is preferably 0.5 M.

[0021] The acid hydrolysis in step (1) is preferably carried out in a water bath at 95°C for 4 h.

[0022] The temperature of the reduced pressure evaporation in step (1) is preferably 45°C.

[0023] The number of times of washing and rotary evaporation in step (2) is preferably 3 times.

[0024] The dialysis described in step (2) is preferably performed using a 200 Da dialysis bag under tap water for 48 h or under ultrapure water for 24 h.

[0025] The centrifugation condition in step (3) is preferably centrifuged at 6500 rpm for 20 min.

[0026] The application of the astragalus polysaccharide or low-molecular astragalus polysaccharide in improving the gait of nematodes.

[0027] The gait includes the gait of neurodegenerative diseases and the gait of normal state.

[0028] The application of the astragalus polysaccharide or low-molecular astragalus polysaccharide in the preparation of products for preventing and treating neurodegenerative diseases.

[0029] A product for preventing and treating neurodegenerative diseases, comprising at least one of the above-mentioned astragalus polysaccharides and low-molecular astragalus polysaccharides.

[0030] Compared with the prior art, the present invention has the following advantages and effects: (1) The present invention prepares an astragalus polysaccharide with gait-improving effect by hot water extraction. The astragalus polysaccharide can be obtained from astragalus capsulatus, astragalus mongolicus, etc., with a molecular weight of 25000-31000 Da, and the molar percentage of monosaccharide composition is: arabinose 19%, rhamnose 3%, galactose 18%, galacturonic acid 17%, and glucose 43%.

[0031] (2) There are no reports on the effective improvement of gait by Astragalus polysaccharide. The present invention uses the turning ratio of C. elegans as an indicator to detect the improvement of gait by Astragalus polysaccharide. It is confirmed that Astragalus polysaccharide can reduce the turning ratio of C. elegans, enhance the directionality of C. elegans, and has the effect of improving gait.

[0032] (3) The present invention uses astragalus polysaccharide as a raw material and uses trifluoroacetic acid or the like to degrade it under weak acid conditions to obtain a low molecular weight astragalus polysaccharide with the effect of improving gait. The molecular weight of the astragalus polysaccharide is 3000-10000 Da, and the molar percentage of the monosaccharide composition is: arabinose 24%, rhamnose 7%, galactose 23%, galacturonic acid 6%, and glucose 40%.

[0033] (4) There are no reports on the efficacy of low molecular weight astragalus polysaccharides in improving gait. In behavioral experiments using Caenorhabditis elegans as a model, the present invention confirms that low molecular weight astragalus polysaccharides have the efficacy of improving gait and can also increase movement speed.

[0034] (5) The astragalus polysaccharides and low-molecular-weight astragalus polysaccharides provided by the present invention can be further used in the development of products such as food, medicine and health care products, and have broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a flow chart for the preparation of astragalus polysaccharide.

[0036] Figure 2 This is the elution curve of Astragalus polysaccharide.

[0037] Figure 3 This is the normal distribution graph of the effect of Astragalus polysaccharide on the number of turns of N2 Caenorhabditis elegans.

[0038] Figure 4 This is an analysis chart of the effects of Astragalus polysaccharides on the turning ratios of N2, AM141, and HA759 Caenorhabditis elegans.

[0039] Figure 5 This is an analysis chart of the effects of Astragalus polysaccharides on the movement speed of N2, AM141, and HA759 Caenorhabditis elegans.

[0040] Figure 6 This is an analysis of the effects of low molecular weight astragalus polysaccharides on the turning ratio and movement speed of wild-type Caenorhabditis elegans. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0042] The biological materials involved in the embodiments are: The N2 model nematode used in the Caenorhabditis elegans refers to the wild-type Caenorhabditis elegans.

[0043] AM141 (rmIs133 [unc-54p:: Q40:: YFP]) and HA759 (rtIs11 [osm-10p:: GFP + osm-10p:: HtnQ150 + dpy-20 (+)]) model nematodes are both Huntington's disease model Caenorhabditis elegans, and the above nematodes can be purchased from the Caenorhabditis Genetics Center of the University of Minnesota. Huntington's disease is a typical neurodegenerative disease, which usually presents symptoms of chorea and other abnormal movements, causing significant changes in the patient's gait. Therefore, wild-type and Huntington's disease model Caenorhabditis elegans were selected to develop the efficacy of astragalus polysaccharide in improving gait.

[0044] Example 1 Preparation, separation and purification of astragalus polysaccharide

[0045] (1) Ethanol defatting: 100 g of Astragalus membranaceus was crushed and passed through a 20-mesh sieve; then, 500 mL of 95% ethanol was added at a mass-to-volume ratio of 1:5, and the mixture was refluxed at 70 °C for 2 h. The reflux was repeated twice, and the ethanol was evaporated after filtration to obtain defatted Astragalus membranaceus.

[0046] (2) Hot water extraction: Add 1000 mL of deionized water at a mass volume ratio of 1:10 to the defatted Astragalus obtained in step (1), extract at 80 °C for 2 h, repeat the extraction twice, remove the residue, combine the extracts, and obtain an Astragalus polysaccharide extract.

[0047] (3) Enzymatic protein removal: The astragalus polysaccharide extract obtained in step (2) was concentrated to 200 mL by rotary evaporation at 40°C; then, the pH of the extract was adjusted to 6.0 with HCl solution, and 20 mg of papain (2000 U / g) was added at a dosage of 3000 U / g astragalus polysaccharide. After enzymatic hydrolysis at 60°C for 3 h, the pH of the extract was adjusted to 8.0 with NaOH solution, and 16 mg of trypsin (2500 U / g) was added at a dosage of 3000 U / g astragalus polysaccharide. After enzymatic hydrolysis at 45°C for 3 h, the extract was inactivated by boiling.

[0048] (4) Ultrafiltration and freeze-drying: The system after protein removal in step (3) is filtered to remove the precipitate, and ultrafiltration is performed using a 1000 Da ultrafiltration membrane. The retentate is collected and freeze-dried to obtain crude astragalus polysaccharide.

[0049] (5) Chromatographic column separation and purification: using DEAE Sepharose TM The crude Astragalus polysaccharide obtained in step (4) was separated using a Fast Flow anion exchange chromatography column, and the specific method is as follows: ① Pre-wash and sample loading: All solutions used must be filtered through a 0.45 μm microporous filter membrane. Pre-wash the chromatography column with 2 times the column volume of ultrapure water, 2 M NaCl solution, and ultrapure water in sequence. Control the pump speed to 5 rpm, that is, the sample loading flow rate is about 1 mL / min; then load the sample.

[0050] ② Water elution: After loading, use ultrapure water for elution first, control the pump speed to 5 rpm, set the automatic fraction collector to 5 min / tube, and collect 100 tubes.

[0051] ③ Salt elution: Prepare 900 mL of 3.4 mol / L NaCl solution for continuous elution. Control the rotation speed of pump 1 and pump 2 to 5 rpm. Pump 1 pumps the NaCl solution into 500 mL of ultrapure water to obtain a NaCl solution with a concentration increasing from 0 mol / L to 2 mol / L. At the same time, pump 2 pumps the mixed NaCl solution into the chromatography column for continuous elution. The automatic fraction collector is set to 5 min / tube, and 150 tubes are collected.

[0052] (6) Determination of sugar content: The sugar content of the eluate was determined using the phenol-sulfuric acid method. The specific method was as follows: 50 μL of each tube was taken into a 96-well plate, 150 μL of concentrated sulfuric acid was added, and then 30 μL of 5% phenol was immediately added. The plate was incubated at 90 °C for 5 min, and the absorbance was measured at 492 nm. The elution curve was plotted (e.g. Figure 2 ), the sugar-containing eluates of water wash and salt wash were collected separately according to the elution curve.

[0053] (7) Dialysis and freeze drying: The obtained water-washed sugar-containing eluate and salt-washed sugar-containing eluate were dialyzed and freeze-dried respectively. The specific method is as follows: use a 1000 Da dialysis bag, dialyze with tap water for 48 hours, and then dialyze with ultrapure water for 24 hours, collect the dialyzed solution and freeze-dry, and obtain neutral astragalus polysaccharide (water-washed) and acidic astragalus polysaccharide (salt-washed) respectively. Among them, the yield of neutral astragalus polysaccharide is lower, and the yield of acidic astragalus polysaccharide is higher. The ratio of the yield of neutral astragalus polysaccharide to that of acidic astragalus polysaccharide is about 1:5, so the subsequent experiments were carried out using acidic astragalus polysaccharide.

[0054] Example 2 Preparation of low molecular weight astragalus polysaccharide

[0055] (1) Acid degradation: 100 mg of the acidic astragalus polysaccharide prepared in Example 1 was weighed and dissolved in 100 mL of distilled water. Trifluoroacetic acid was added to make the final concentration 0.5 M. The mixture was acid-hydrolyzed in a 95 °C water bath for 4 h, and then the solution was evaporated under reduced pressure at 45 °C.

[0056] (2) Washing and dialysis: Wash the product obtained in step (1) with 5 mL of anhydrous methanol and remove trifluoroacetic acid under rotary evaporation at 45 °C. Repeat the washing step three times. Dissolve the product in distilled water, dialyze it in tap water for 48 h using a 200 Da dialysis bag, dialyze it in ultrapure water for 24 h, and lyophilize it to obtain a lyophilized powder.

[0057] (3) Ultrafiltration classification: Weigh the lyophilized powder to make a 10 mg / mL solution, pipette the solution into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10k Da, centrifuge at 6500 rpm for 20 min, and collect the filtrate. Repeat this process until all the solution is ultrafiltered. Add the collected filtrate to an ultrafiltration centrifuge tube with a molecular weight cutoff of 3k Da, centrifuge at 6500 rpm for 20 min, and collect the retained fraction. After freeze-drying, low molecular weight astragalus polysaccharides with a molecular weight of 3-10k Da are obtained.

[0058] Example 3 Analysis of Monosaccharide Composition of Astragalus Polysaccharide

[0059] The monosaccharide composition of the acidic astragalus polysaccharide and the low molecular weight astragalus polysaccharide prepared in Examples 1 and 2 was analyzed by gas chromatography (GC) after pre-column derivatization with trimethylsilane (TMS). The specific method is as follows: 1. Sample treatment: Add 2 mg of acidic astragalus polysaccharide and low molecular weight astragalus polysaccharide to two brown vials respectively, and then add 100 μL of inositol as internal standard, and freeze in a -80 ℃ refrigerator. After freeze drying, add 1 mL of hydrochloric acid methanol solution, seal the tube with nitrogen, and tighten the cap.

[0060] 2. Methanolysis: Methanolysis was carried out at 80 °C for 24 h. After the methanolysis was completed, the hydrochloric acid methanol was blown dry with nitrogen. 1 mL of anhydrous methanol was added to thoroughly wash the sample and blown dry with nitrogen. This was repeated three times.

[0061] 3. Gas chromatography analysis: Add 200 μL of TMS derivatization reagent to the reaction bottle, mix well, let stand for 30 min, and centrifuge at 6000 rpm for 2 min at room temperature. Take 1 μL of supernatant for gas chromatography analysis. Among them, the chromatographic conditions are: detector: FID; chromatographic column: DB-5 capillary column (30 m×0.25 mm×0.25 µm). Temperature program: 1 ℃ / min from 140 ℃ to 170 ℃, then increase the temperature to 250 ℃ at 6 ℃ / min, then increase the temperature to 260 ℃ at 30 ℃ / min, and keep it for 3 min; injection temperature: 260 ℃; detector temperature: 260 ℃; nitrogen flow rate: 0.9 mL / min, separation ratio is 20:1.

[0062] The results are shown in Table 1, where the molar percentages of monosaccharide composition of Astragalus polysaccharide are: arabinose 19%, rhamnose 3%, galactose 18%, galacturonic acid 17%, and glucose 43%.

[0063] The molar percentages of monosaccharide composition of low molecular weight astragalus polysaccharide are: arabinose 24%, rhamnose 7%, galactose 23%, galacturonic acid 6%, and glucose 40%.

[0064] Table 1 Monosaccharide composition of acidic astragalus polysaccharides and low molecular weight astragalus polysaccharides Polysaccharides Arabinose Rhamnose Galactose Galacturonic acid glucose Astragalus polysaccharide 19% 3% 18% 17% 43% Low molecular weight astragalus polysaccharide 24% 7% 23% 6% 40%

[0065] Example 4 Effects of acidic astragalus polysaccharides on the gait of Caenorhabditis elegans

[0066] 1. Preparation of nematodes: After synchronizing the N2, AM141, and HA759 model Caenorhabditis elegans cultured to the egg-laying stage, culture them in a constant temperature culture shaker at 120 rpm and 20 °C for 23 h to the L1 stage. At this time, feed them with NA22 concentrated bacterial solution and continue to culture for 42 h to the L4 stage. Transfer the worm solution to a 1.5 mL EP tube and wash the nematodes 2-3 times with S Medium solution until the supernatant is clear.

[0067] 2. Drug administration: In a 48-well plate (total volume of 500 μL per well), 10 μL AMP, 7.5 μL 5-FUdR and 50 μL NA22 were added to make the final concentrations 100 μg / mL, 75 μg / mL and about 0.6 (OD 570 nm), respectively. Astragalus polysaccharide powder was weighed and added to S Medium solution to dissolve, and then sterilized by 0.22 μm filter membrane to prepare 20 mg / mL acidic astragalus polysaccharide solution. 50 μL was added to the above 48-well plate containing AMP, 5-FUdR and NA22 as the acidic astragalus polysaccharide administration group, wherein the administration concentration of acidic astragalus polysaccharide was 2.0 mg / mL. The wells added with S Medium were used as blank control groups, and the administration concentration of the blank control group was 0 mg / mL. Then 100 μL of nematodes washed in step 1 were added, and the volume of each well was filled to 500 μL with S Medium to control the same volume of each well. Three replicate wells were set for each group.

[0068] 3. Sports ability test: The 48-well plate was placed in a constant temperature incubator at 120 rpm and 20°C for 24 h, which was recorded as Day 1. On Day 5 of the adult worms, C. elegans was taken to test the motility (crawling analysis). The specific method is as follows: Wash the nematodes 2-3 times with S Medium solution until the supernatant is clear, take 20 μL of the worm solution on a 3.5 cm NGM plate, wait for 10 min for the nematodes to crawl away and adapt to the new environment, and shoot a 30 s AVI crawling analysis video.

[0069] 4. Data Analysis: The crawling analysis video was converted into an AVI video with a resolution of 2000 × 1500, 8-bit grayscale, and uncompressed using VideoMach software. The video was analyzed using MatLab software to obtain data such as the number of turns and movement speed of the nematodes, and the direction rate was calculated based on the number of turns, where the direction rate = the number of nematodes with a turn number greater than the average turn number of N2 nematodes / the total number of nematodes.

[0070] The results are as follows Figure 3 It can be seen that the average number of turning times of N2 nematodes will be improved when acidic astragalus polysaccharide is administered to culture until Day 5, that is, the number of nematodes with too many turning times will be reduced after administration; Figure 4 As shown in the figure, when acidic APS was administered to culture until Day 5, the turning ratios of N2, AM141 and HA759 nematodes were all reduced, proving that acidic APS can enhance the directionality of C. elegans, that is, it has the effect of improving gait. In addition, Figure 5As shown in the figure, the movement speed of N2, AM141 and HA759 nematodes increased when acidic APS was administered to culture until Day 5.

[0071] Example 5 Effects of low molecular weight astragalus polysaccharides on the gait of Caenorhabditis elegans

[0072] 1. Preparation of nematodes: After synchronizing the N2 model Caenorhabditis elegans cultured to the egg-laying stage, culture them in a constant temperature culture shaker at 120 rpm and 20 °C for 23 h to the L1 stage. At this time, feed them with NA22 concentrated bacterial solution and continue to culture for 42 h to the L4 stage. Transfer the worm solution to a 1.5 mL EP tube and wash the nematodes 2-3 times with S Medium solution until the supernatant is clear.

[0073] 2. Drug administration: In a 48-well plate (total volume of 500 μL per well), 10 μL AMP, 7.5 μL 5-FUdR and 50 μL NA22 were added to make the final concentrations 100 μg / mL, 75 μg / mL and about 0.6 (OD 570 nm), respectively. Low molecular weight astragalus polysaccharide powder was weighed and added to S Medium solution to dissolve, and then sterilized by 0.22 μm filter membrane to prepare a 10 mg / mL low molecular weight astragalus polysaccharide solution. 50 μL was added to the above 48-well plate containing AMP, 5-FUdR and NA22 as the low molecular weight astragalus polysaccharide administration group, wherein the low molecular weight astragalus polysaccharide administration concentration was 1 mg / mL. The wells added with S Medium were used as blank control groups, and the administration concentration of the blank control group was 0 mg / mL. Then 100 μL of nematodes washed in step 1 were added, and the volume of each well was filled to 500 μL with S Medium to control the same volume of each well. Three replicate wells were set for each group.

[0074] 3. Sports ability test: The 48-well plate was placed in a constant temperature culture shaker at 120 rpm and 20°C for 24 h, which was recorded as Day 1. On Day 5 after the drug treatment, the Caenorhabditis elegans was taken out for experiment and a crawling analysis video was filmed. The specific method was the same as that in Example 4.

[0075] 4. Data Analysis: After the video is converted using VideoMach software, the video is analyzed using MatLab software to obtain data such as the turning ratio and movement speed. The specific method is the same as in Example 4.

[0076] The results are as follows Figure 6As shown, the administration of low molecular weight astragalus polysaccharides to Day 5 can reduce the turning ratio of wild-type Caenorhabditis elegans, proving that 3-10k Da low molecular weight astragalus polysaccharides can also enhance the directionality of Caenorhabditis elegans, improve gait, and increase movement speed; and compared with acidic astragalus polysaccharides, after the administration of low molecular weight astragalus polysaccharides, the turning ratio of N2 nematodes is lower and the movement speed is higher, which has a better effect of improving gait.

[0077] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing astragalus polysaccharide for improving gait, characterized in that The following steps are included: (1) Degreasing water extraction: After crushing the Astragalus, add ethanol solution and reflux degreasing at 70-80°C for 2-3 h. Repeat reflux degreasing 1-3 times to obtain defatted Astragalus. Then add water and extract at 80-90°C for 2-3 h. Repeat the extraction 1-3 times and collect the extract. (2) Enzymatic protein removal: The extract obtained in step (1) is concentrated by rotary evaporation, and then the pH of the system is adjusted to 6.0-7.0, and papain is added for enzymatic hydrolysis; after the enzymatic hydrolysis, the pH of the system is adjusted to 8.0-8.5, and trypsin is added for enzymatic hydrolysis, and after the enzymatic hydrolysis, the extract is boiled and inactivated; (3) Ultrafiltration and freeze-drying: The system in step (2) is filtered to remove the precipitate, and then ultrafiltration is performed using a 1000 Da ultrafiltration membrane, and the retentate is collected and freeze-dried to obtain crude astragalus polysaccharide; (4) Separation and purification: using DEAE Sepharose TM The separation and purification was performed using a Fast Flow anion exchange chromatography column, using water and NaCl solution as eluents, and collecting the sugar-containing eluate after water washing and the sugar-containing eluate after salt washing; (5) Dialysis freeze-drying: Use a 1000 Da dialysis bag for dialysis treatment, collect the dialyzed solution and freeze-dry it to obtain neutral and acidic astragalus polysaccharides.

2. The method for preparing astragalus polysaccharide for improving gait according to claim 1, characterized in that: The papain enzymatic hydrolysis conditions in step (2) are 60°C for 3 h; The trypsin enzymatic hydrolysis conditions described in step (2) are 45°C for 3 h.

3. An astragalus polysaccharide for improving gait, characterized in that It is prepared by the preparation method according to claim 1 or 2.

4. A low molecular weight astragalus polysaccharide for improving gait, characterized in that The gait-improving astragalus polysaccharide according to claim 3 is obtained by further acid degradation, washing, dialysis and ultrafiltration classification.

5. The method for preparing the low molecular weight astragalus polysaccharide for improving gait according to claim 4, characterized in that The following steps are included: (1) Acid degradation: Weigh astragalus polysaccharide and dissolve it in water, add trifluoroacetic acid to acid-degrade it in a water bath, and then evaporate the solution under reduced pressure; (2) Washing and dialysis: The product obtained in step (1) is washed with anhydrous methanol and subjected to rotary evaporation to remove trifluoroacetic acid. The product after rotary evaporation is dissolved in water, dialyzed with a 200 Da dialysis bag, and freeze-dried to obtain a freeze-dried powder; (3) Ultrafiltration classification: The freeze-dried powder obtained in step (2) is dissolved in water, placed in a filter centrifuge tube with a molecular weight cutoff of 10 kDa, and the filtrate is collected by centrifugation; the filtrate is placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, centrifuged, the retained portion is collected and freeze-dried to obtain low molecular weight astragalus polysaccharides with a molecular weight of 3-10 kDa.

6. The method for preparing low-molecular-weight astragalus polysaccharides for improving gait according to claim 5, characterized in that: The final concentration of trifluoroacetic acid after addition in step (1) is 0.5 M.

7. The method for preparing low-molecular-weight astragalus polysaccharides for improving gait according to claim 5, characterized in that: The acid hydrolysis in step (1) was carried out in a water bath at 95°C for 4 h.

8. Use of the astragalus polysaccharide according to claim 3 or the low-molecular-weight astragalus polysaccharide according to claim 4 in improving the gait of nematodes.

9. Use of the astragalus polysaccharide according to claim 3 or the low-molecular-weight astragalus polysaccharide according to claim 4 in the preparation of a product for preventing and treating neurodegenerative diseases.

10. A product for preventing and treating neurodegenerative diseases, comprising at least one of the astragalus polysaccharide according to claim 3 and the low-molecular-weight astragalus polysaccharide according to claim 4.

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