Astragalus polysaccharide for improving gait, and preparation method and application thereof

Abstract

The present application belongs to the technical field of bioactive polysaccharides, and particularly relates to astragalus polysaccharide for improving gait, and a preparation method and application thereof. The astragalus polysaccharide is prepared by hot water extraction, and low-molecular astragalus polysaccharide is prepared by degrading the astragalus polysaccharide with weak acid. Further, the obtained astragalus polysaccharide and low-molecular astragalus polysaccharide are taken as objects, and behavioral study is carried out on Caenorhabditis elegans as a model, and the turning ratio of Caenorhabditis elegans is taken as a gait detection index, so as to confirm that the astragalus polysaccharide and low-molecular astragalus polysaccharide both have the effect of improving gait. The astragalus polysaccharide and low-molecular astragalus polysaccharide provided by the present application can be further used for the development of food, medicine and health products, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioactive polysaccharide technology, specifically relating to an Astragalus polysaccharide for improving gait, its preparation method, and its application. Background Technology

[0002] Movement is one of the fundamental behaviors of humans and animals. While movement speed is typically used to characterize an organism's motor ability, it doesn't accurately reflect overall health. Gait, the posture and behavioral characteristics displayed during walking, indicates an organism's directionality, stability, periodicity, and rhythm, thus providing a good reflection of its health. For example, changes in the gait of *C. elegans*, an internationally recognized model organism in biomedicine, are also an important indicator of its motor ability, including a decrease in directionality, similar to those observed in humans.

[0003] Currently, methods for improving gait mainly include exercise intervention and nerve stimulation, but reports on polysaccharides with gait-improving effects are extremely rare. For example, crude mulberry polysaccharide has the ability to restore behavioral abnormalities caused by knee osteoarthritis, thus possessing the potential to indirectly improve gait. Astragalus is a traditional Chinese tonic, and its main component, astragalus polysaccharide, has biological activities such as anti-aging and neuroprotection; however, its effects on gait improvement have not been reported.

[0004] The low-molecular-weight polysaccharides prepared through degradation have a smaller molecular weight compared to their starting polysaccharides, which is beneficial for absorption and utilization. Studies have shown that low-molecular-weight Astragalus polysaccharides possess good anti-inflammatory and antioxidant activities, but research on their behavioral effects is extremely limited, especially regarding gait improvement. Summary of the Invention

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a method for preparing Astragalus polysaccharides that improve gait.

[0006] Another objective of this invention is to provide an Astragalus polysaccharide prepared by the above-described method that improves gait. This Astragalus polysaccharide can improve gait abnormalities in the body, specifically including gait in neurodegenerative diseases and gait in a normal state.

[0007] Another objective of this invention is to provide a low molecular weight astragalus polysaccharide that can not only improve gait abnormalities but also increase the body's movement speed.

[0008] The fourth objective of this invention is to provide a method for preparing the aforementioned low-molecular-weight astragalus polysaccharide.

[0009] The fifth objective of this invention is to provide applications of the above-mentioned Astragalus polysaccharide and low molecular weight Astragalus polysaccharide.

[0010] The objective of this invention is achieved through the following technical solution:

[0011] A method for preparing Astragalus polysaccharide to improve gait includes the following steps:

[0012] (1) Degreasing and water extraction: After pulverizing Astragalus membranaceus, add ethanol solution and reflux at 70~80 ℃ for 2~3 h. Reflux and defatting is repeated 1~3 times to obtain defatted Astragalus membranaceus. Then add water and extract at 80~90 ℃ for 2~3 h. Repeat the extraction 1~3 times and collect the extract.

[0013] (2) Enzymatic hydrolysis of protein: 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. 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 protein is boiled to inactivate it.

[0014] (3) Ultrafiltration freeze drying: The system in step (2) was filtered to remove the precipitate, and then ultrafiltration was performed using a 1000 Da ultrafiltration membrane. The retentate was collected and freeze-dried to obtain crude Astragalus polysaccharide.

[0015] (4) Separation and purification: DEAE Sepharose was used TM The sugar-containing eluent was separated and purified using a Fast Flow anion exchange chromatography column, with water and NaCl solution as eluents. The sugar-containing eluent after water washing and the sugar-containing eluent after salt washing were collected.

[0016] (5) Dialysis freeze-drying: Dialysis was performed using a 1000 Da dialysis bag, and the dialysis solution was collected and freeze-dried to obtain neutral and acidic Astragalus polysaccharides.

[0017] The Astragalus mentioned can be Astragalus membranaceus, Astragalus mongholicus, etc.

[0018] The preferred mass-to-volume ratio (g:mL) of Astragalus membranaceus to ethanol in step (1) is 1:5 to 1:10.

[0019] The preferred mass-to-volume ratio (g:mL) of Astragalus membranaceus to water in step (1) is 1:10 to 1:20.

[0020] The volume fraction of the ethanol solution in step (1) is preferably 95%.

[0021] The preferred conditions for papain hydrolysis in step (2) are 60 °C for 3 h.

[0022] The preferred conditions for trypsin hydrolysis in step (2) are 45 °C for 3 h.

[0023] An Astragalus polysaccharide for improving gait was prepared by the above-described preparation method.

[0024] A low-molecular-weight Astragalus polysaccharide for improving gait is obtained by further acid degradation, washing and dialysis, and ultrafiltration fractionation of the aforementioned Astragalus polysaccharide for improving gait.

[0025] The method for preparing the low-molecular-weight Astragalus polysaccharide for improving gait includes the following steps:

[0026] (1) Acid degradation: Weigh out Astragalus polysaccharide and dissolve it in water, add trifluoroacetic acid and acid hydrolyze in a water bath, then evaporate the solution under reduced pressure;

[0027] (2) Washing and dialysis: The product obtained in step (1) was washed with anhydrous methanol and rotary evaporated to remove trifluoroacetic acid. The product after rotary evaporation was dissolved in water, dialyzed with a 200 Da dialysis bag, and freeze-dried to obtain freeze-dried powder.

[0028] (3) Ultrafiltration fractionation: Dissolve the freeze-dried powder obtained in step (2) in water, place it in a centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuge and collect the filtrate; place the filtrate in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, centrifuge, collect the cut-off portion and freeze-dry it to obtain low molecular weight Astragalus polysaccharide with a molecular weight of 3-10 kDa.

[0029] The final concentration of trifluoroacetic acid added in step (1) is preferably 0.5 M.

[0030] The acid hydrolysis described in step (1) is preferably carried out in a water bath at 95 ℃ for 4 h.

[0031] The preferred temperature for vacuum drying in step (1) is 45 °C.

[0032] The number of times the washing and rotary evaporation is performed in step (2) is preferably 3 times.

[0033] The preferred method for dialysis in step (2) is to use a dialysis bag with 200 Da for dialysis under tap water for 48 h and under ultrapure water for 24 h.

[0034] The preferred centrifugation conditions in step (3) are centrifugation at 6500 rpm for 20 min.

[0035] The application of the aforementioned Astragalus polysaccharide or low molecular weight Astragalus polysaccharide in improving the gait of nematodes.

[0036] The gait mentioned includes the gait of neurodegenerative diseases and the gait of normal conditions.

[0037] The application of the aforementioned Astragalus polysaccharide or low molecular weight Astragalus polysaccharide in the preparation of products for the prevention and treatment of neurodegenerative diseases.

[0038] A product for preventing and treating neurodegenerative diseases, comprising at least one of the above-mentioned Astragalus polysaccharides and low molecular weight Astragalus polysaccharides.

[0039] The present invention has the following advantages and effects compared with the prior art:

[0040] (1) The present invention prepares an Astragalus polysaccharide with gait improvement by hot water extraction. The source of the Astragalus polysaccharide can be Astragalus capsulatum, Astragalus mongholicus, etc., with a molecular weight of 25,000-31,000 Da and a molar percentage of monosaccharide composition of 19% arabinose, 3% rhamnose, 18% galactose, 17% galacturonic acid, and 43% glucose.

[0041] (2) There are no reports on the effective improvement of gait by Astragalus polysaccharide. This invention uses the turning ratio of Caenorhabditis elegans as an indicator to detect the improvement of gait by Astragalus polysaccharide. It confirms that Astragalus polysaccharide can reduce the turning ratio of Caenorhabditis elegans and enhance the directionality of Caenorhabditis elegans, thus having the effect of improving gait.

[0042] (3) The present invention uses Astragalus polysaccharide as raw material and degrades it under weak acid conditions with trifluoroacetic acid to obtain a low molecular weight Astragalus polysaccharide with gait improvement effect. Its molecular weight is 3000-10000 Da and the molar percentage of monosaccharide composition is: arabinose 24%, rhamnose 7%, galactose 23%, galacturonic acid 6%, glucose 40%.

[0043] (4) The effect of low molecular weight Astragalus polysaccharide on gait improvement has not been reported. In the behavioral experiment using Caenorhabditis elegans as a model, this invention confirms that low molecular weight Astragalus polysaccharide has the effect of improving gait and can also increase movement speed.

[0044] (5) The Astragalus polysaccharide and low molecular weight Astragalus polysaccharide provided by the present invention can be further used in the development of food, pharmaceuticals and health products, and have broad application prospects. Attached Figure Description

[0045] Figure 1 This is a flowchart of the preparation process of Astragalus polysaccharides.

[0046] Figure 2 This is the elution curve of Astragalus polysaccharide.

[0047] Figure 3 This is a normal distribution diagram of the number of turns of N2 elegans nematodes affected by Astragalus polysaccharide.

[0048] Figure 4 This is a graph showing the results of the analysis of the effects of Astragalus polysaccharide on the turning ratio of N2, AM141, and HA759 nematodes.

[0049] Figure 5 This is a graph showing the results of the analysis of the effects of Astragalus polysaccharide on the movement speed of N2, AM141, and HA759 nematodes.

[0050] Figure 6 This is a graph showing the results of the analysis of the effects of low molecular weight Astragalus polysaccharide on the turning ratio and movement speed of wild-type Caenorhabditis elegans. Detailed Implementation

[0051] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0052] Biomaterials involved in the embodiments:

[0053] The N2 model nematode used represents the wild-type nematode.

[0054] The AM141 (rmIs133[unc-54p::Q40::YFP]) and HA759 (rtIs11[osm-10p::GFP + osm-10p::HtnQ150 + dpy-20(+)]) model nematodes both represent *C. elegans*, a model of Huntington's disease. These nematodes were all obtained from the *C. elegans* Genetics Center at the University of Minnesota. Huntington's disease is a typical neurodegenerative disease, typically presenting with choreiform and other abnormal motor symptoms, leading to significant gait changes. Therefore, wild-type and *C. elegans*, a model of Huntington's disease, were selected to explore the gait-improving effects of Astragalus polysaccharides.

[0055] Example 1: Preparation, separation and purification of Astragalus polysaccharides

[0056] (1) Ethanol defatting: 100g of Astragalus membranaceus was pulverized and passed through a 20-mesh sieve; then 500 mL of 95% ethanol was added at a mass-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.

[0057] (2) Hot water extraction: 1000 mL of deionized water was added to the defatted Astragalus membranaceus obtained in step (1) at a mass-to-volume ratio of 1:10. The mixture was extracted at 80 °C for 2 h. The extraction was repeated twice. The residue was removed, and the extracts were combined to obtain Astragalus polysaccharide extract.

[0058] (3) Enzymatic protein removal: The Astragalus polysaccharide extract obtained in step (2) was concentrated to 200 mL by rotary evaporation at 40℃; 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℃ 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℃ for 3 h, the extract was boiled to inactivate the protein.

[0059] (4) Ultrafiltration freeze drying: After removing the protein in step (3), the system was filtered to remove the precipitate, ultrafiltration was performed using a 1000 Da ultrafiltration membrane, the retentate was collected, and the crude polysaccharide of Astragalus membranaceus was obtained by freeze drying.

[0060] (5) Chromatographic column separation and purification: DEAE Sepharose was used. TM The crude polysaccharide of Astragalus membranaceus obtained in step (4) was separated using a Fast Flow anion exchange chromatography column, as follows:

[0061] ① Pre-washing and sample loading: All solutions used must be filtered through a 0.45 μm microporous membrane. The chromatography column should be pre-washed with 2 column volumes of ultrapure water, 2 M NaCl solution, and ultrapure water in sequence. The pump speed should be controlled at 5 rpm, i.e., the sample loading flow rate is about 1 mL / min. Then the sample is loaded.

[0062] ② Water elution: After loading the sample, use ultrapure water to elute, control the pump speed at 5 rpm, set the automatic fraction collector to 5 min / tube, and collect 100 tubes.

[0063] ③ Salt elution: Prepare another 900 mL of 3.4 mol / L NaCl solution for continuous elution. Control the 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, collecting 150 tubes.

[0064] (6) Sugar content determination: The sugar content of the eluent was determined using the phenol-sulfuric acid method. The specific method was as follows: 50 μL was taken from each tube and placed in a 96-well plate. 150 μL of concentrated sulfuric acid was added, followed immediately by 30 μL of 5% phenol. The plate was incubated in a 90 ℃ water bath for 5 min, and the absorbance was measured at 492 nm. An elution curve was plotted (e.g., ...). Figure 2 Based on the elution curves, sugar-containing eluents were collected from water washing and salt washing, respectively.

[0065] (7) Dialysis freeze-drying: The obtained sugar-containing eluents washed with water and sugar-containing eluents washed with salt were dialyzed and freeze-dried separately. The specific method is as follows: using a 1000 Da dialysis bag, dialyzing with tap water for 48 h, and then dialyzing with ultrapure water for 24 h, the dialysis solutions were collected and freeze-dried to obtain neutral astragalus polysaccharide (washed with water) and acidic astragalus polysaccharide (washed with salt). Among them, the yield of neutral astragalus polysaccharide was lower and the yield of acidic astragalus polysaccharide was higher. The ratio of the yields of neutral astragalus polysaccharide to acidic astragalus polysaccharide was about 1:5. Therefore, acidic astragalus polysaccharide was used in subsequent experiments.

[0066] Example 2 Preparation of low molecular weight Astragalus polysaccharide

[0067] (1) Acid degradation: Weigh 100 mg of the acidic astragalus polysaccharide prepared in Example 1, dissolve it in 100 mL of distilled water, add trifluoroacetic acid to make the final concentration 0.5 M, acid hydrolyze it in a water bath at 95 °C for 4 h, and then evaporate the solution under reduced pressure at 45 °C.

[0068] (2) Washing and dialysis: Wash the product obtained in step (1) with 5 mL of anhydrous methanol and remove trifluoroacetic acid by 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 with a 200 Da dialysis bag, dialyze it in ultrapure water for 24 h, and freeze-dry it to obtain freeze-dried powder.

[0069] (3) Ultrafiltration fractionation: Weigh the lyophilized powder to prepare a 10 mg / mL solution, add the solution to an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuge at 6500 rpm for 20 min and collect the filtrate. Repeat this process until all solutions have been ultrafiltered. Add the collected filtrate to an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, centrifuge at 6500 rpm for 20 min and collect the cutoff fraction. After lyophilization, low molecular weight Astragalus polysaccharides with a molecular weight of 3-10 kDa are obtained.

[0070] Example 3 Monosaccharide composition analysis of Astragalus polysaccharides

[0071] The monosaccharide composition of the acidic and low molecular weight astragalus polysaccharides prepared in Examples 1 and 2 was analyzed by gas chromatography (GC) after pre-column derivatization with trimethylsilane (TMS). The specific methods are as follows:

[0072] 1. Sample preparation: Add 2 mg of acidic astragalus polysaccharide and low molecular weight astragalus polysaccharide to two separate brown vials, and add 100 μL of inositol as an internal standard. Freeze at -80 ℃. After freeze-drying, add 1 mL of hydrochloric acid-methanol solution, seal the vials with nitrogen gas, and tighten the caps.

[0073] 2. Methanololysis: Methanololysis was performed at 80 °C for 24 h. After the alcoholysis was completed, the hydrochloric acid and methanol were dried with nitrogen. The sample was then thoroughly washed with 1 mL of anhydrous methanol and dried with nitrogen. This process was repeated three times.

[0074] 3. Gas Chromatography Analysis: Add 200 μL of TMS derivatization reagent to the reaction flask, mix well, let stand for 30 min, and centrifuge at 6000 rpm for 2 min at room temperature. Take 1 μL of the supernatant for gas chromatography analysis. Chromatographic conditions: Detector: FID; Column: DB-5 capillary column (30 m × 0.25 mm × 0.25 µm). Temperature program: 1 ℃ / min from 140 ℃ to 170 ℃, then 6 ℃ / min to 250 ℃, then 30 ℃ / min to 260 ℃, hold for 3 min; Injection temperature: 260 ℃; Detector temperature: 260 ℃; Nitrogen flow rate: 0.9 mL / min; Resolution ratio: 20:1.

[0075] The results are shown in Table 1. The molar percentages of monosaccharides in Astragalus polysaccharides are: arabinose 19%, rhamnose 3%, galactose 18%, galacturonic acid 17%, and glucose 43%.

[0076] The molar percentage of monosaccharides in low molecular weight Astragalus polysaccharides is as follows: arabinose 24%, rhamnose 7%, galactose 23%, galacturonic acid 6%, and glucose 40%.

[0077] Table 1. Monosaccharide composition of acidic astragalus polysaccharide and low molecular weight astragalus polysaccharide

[0078] polysaccharides Arabic sugar Rhamnose Galactose Galacturonic acid glucose Astragalus polysaccharides 19% 3% 18% 17% 43% Low molecular weight Astragalus polysaccharide 24% 7% 23% 6% 40%

[0079] Example 4: Effects of Acidic Astragalus Polysaccharide on the Gait of *C. elegans*

[0080] 1. Nematode preparation:

[0081] After synchronizing the N2, AM141, and HA759 model nematodes that had reached the oviposition stage, they were cultured in a constant temperature shaker at 120 rpm and 20 ℃ for 23 h to reach the L1 stage. At this time, they were fed with concentrated NA22 bacterial solution and cultured for another 42 h to reach the L4 stage. The nematode solution was then transferred to 1.5 mL EP tubes and the nematodes were washed 2-3 times with S Medium solution until the supernatant was clear.

[0082] 2. Drug administration:

[0083] In a 48-well plate (total volume 500 μL per well), 10 μL of pre-prepared AMP, 7.5 μL of 5-FUdR, and 50 μL of NA22 were added to make their final concentrations approximately 100 μg / mL, 75 μg / mL, and 0.6 (OD 570 nm), respectively. Astragalus polysaccharide powder was weighed, dissolved in S Medium solution, and then filtered through a 0.22 μm filter membrane for sterilization to prepare an acidic astragalus polysaccharide solution of 20 mg / mL. 50 μL of this solution was added to the 48-well plate containing AMP, 5-FUdR, and NA22 as the acidic astragalus polysaccharide administration group, with an administration concentration of 2.0 mg / mL. The wells containing S Medium served as the blank control group, with an administration concentration of 0 mg / mL. 100 μL of nematodes washed in step 1 was added, and the volume of each well was brought up to 500 μL with S Medium to ensure that the volume of each well was the same. Three replicates were set up for each group.

[0084] 3. Athletic ability assessment:

[0085] The 48-well plates were incubated in a constant-temperature shaker at 120 rpm and 20 ℃ for 24 h, which was recorded as Day 1. On Day 5 of the adult worms, the motility (crawling analysis) of *C. elegans* was assessed using the following method:

[0086] Wash the nematodes 2-3 times with S Medium solution until the supernatant is clear. Take 20 μL of the nematode solution and place it on a 3.5 cm NGM plate. Wait 10 min for the nematodes to crawl away and adapt to the new environment. Record a 30 s AVI crawling analysis video.

[0087] 4. Data Analysis:

[0088] The crawling analysis video was converted to an uncompressed AVI video with a resolution of 2000×1500, 8-bit grayscale, using VideoMach software. The video was then analyzed using MatLab software to obtain data such as the number of turns and movement speed of the nematodes. Based on the number of turns, the direction rate was calculated, where the direction rate = the number of nematodes with a turn count greater than the average number of turns for N2 nematodes / the total number of nematodes.

[0089] The results are as follows Figure 3 It can be seen that the average number of turning times of N2 nematodes after administration of acidic Astragalus polysaccharide to Day 5 was improved, meaning that the number of nematodes with excessive turning times decreased after administration; for example Figure 4 As shown, the turning rates of N2, AM141, and HA759 nematodes decreased after Day 5 of culture treated with acidic astragalus polysaccharide, demonstrating that acidic astragalus polysaccharide can enhance the directionality of *C. elegans*, i.e., it has the effect of improving gait. Furthermore, as... Figure 5 As shown, the motility of N2, AM141, and HA759 nematodes increased after being cultured with acidic Astragalus polysaccharide until Day 5.

[0090] Example 5: Effects of low molecular weight Astragalus polysaccharide on the gait of *C. elegans*

[0091] 1. Nematode preparation:

[0092] After synchronizing the N2 model nematodes that had reached the oviposition stage, they were cultured in a constant temperature shaker at 120 rpm and 20 ℃ for 23 h to reach the L1 stage. At this time, they were fed with concentrated NA22 bacterial solution and cultured for another 42 h to reach the L4 stage. The nematode solution was then transferred to 1.5 mL EP tubes and the nematodes were washed 2-3 times with S Medium solution until the supernatant was clear.

[0093] 2. Drug administration:

[0094] 10 μL of pre-prepared AMP, 7.5 μL of 5-FUdR, and 50 μL of NA22 were added to a 48-well plate (total volume of 500 μL per well) to achieve final concentrations of approximately 100 μg / mL, 75 μg / mL, and 0.6 (OD 570 nm), respectively. Low molecular weight astragalus polysaccharide powder was weighed, dissolved in S Medium solution, and then filtered through a 0.22 μm filter membrane for sterilization to prepare a 10 mg / mL low molecular weight astragalus polysaccharide solution. 50 μL of this solution was added to the 48-well plate containing AMP, 5-FUdR, and NA22 as the low molecular weight astragalus polysaccharide administration group, with a concentration of 1 mg / mL. The wells containing S Medium served as the blank control group, with an administration concentration of 0 mg / mL. 100 μL of nematodes washed in step 1 was added, and S Medium was used to bring the volume of each well to 500 μL to ensure the same volume. Three replicates were set for each group.

[0095] 3. Athletic ability assessment:

[0096] The 48-well plate was placed in a constant temperature shaker at 120 rpm and 20 ℃ and cultured for 24 h. This was recorded as Day 1. On Day 5 after drug treatment, the nematodes were removed for experiments, and crawling analysis videos were taken. The specific method was the same as in Example 4.

[0097] 4. Data Analysis:

[0098] After converting the video using VideoMach software, the video is analyzed using MatLab software to obtain data such as steering ratio and motion speed. The specific method is the same as in Example 4.

[0099] The results are as follows Figure 6 As shown, low molecular weight astragalus polysaccharide administration to Day 5 can reduce the turning ratio of wild-type nematodes, demonstrating that 3-10 kDa low molecular weight astragalus polysaccharide can also enhance the directionality of nematodes, improve gait, and increase movement speed. Compared with acidic astragalus polysaccharide, low molecular weight astragalus polysaccharide administration resulted in a lower turning ratio and higher movement speed in N2 nematodes, demonstrating a better effect on improving gait.

[0100] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of Astragalus polysaccharide in the preparation of drugs for the prevention and treatment of Huntington's disease, characterized in that: The aforementioned Astragalus polysaccharide was prepared by the following method: (1) Degreasing and water extraction: After pulverizing Astragalus membranaceus, add ethanol solution and reflux at 70~80 ℃ for 2~3 h. Reflux and defatting is repeated 1~3 times to obtain defatted Astragalus membranaceus. Then add water and extract at 80~90 ℃ for 2~3 h. Repeat the extraction 1~3 times and collect the extract. (2) Enzymatic hydrolysis of protein: 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. 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 protein is boiled to inactivate it. (3) Ultrafiltration freeze drying: The system in step (2) was filtered to remove the precipitate, and then ultrafiltration was performed using a 1000 Da ultrafiltration membrane. The retentate was collected and freeze-dried to obtain crude Astragalus polysaccharide. (4) Separation and purification: DEAE Sepharose was used TM The separation and purification were performed using a Fast Flow anion exchange chromatography column, with water and 2 mol / L NaCl solution used as eluents in sequence, and the sugar-containing eluent after salt washing was collected. (5) Dialysis freeze-drying: Dialysis was performed using a 1000 Da dialysis bag, and the dialysis solution was collected and freeze-dried to obtain acidic Astragalus polysaccharide; The molar percentage of monosaccharide composition of the acidic Astragalus polysaccharide is as follows: arabinose 19%, rhamnose 3%, galactose 18%, galacturonic acid 17%, and glucose 43%.

2. The application according to claim 1, characterized in that: The conditions for papain hydrolysis in step (2) are 60 °C for 3 h. The conditions for trypsin hydrolysis in step (2) are 45 °C for 3 h.

3. The application of a low molecular weight Astragalus polysaccharide in the preparation of drugs for the prevention and treatment of Huntington's disease, characterized in that: The low molecular weight Astragalus polysaccharide is obtained by further acid degradation, washing and dialysis and ultrafiltration fractionation of the Astragalus polysaccharide described in claim 1.

4. The application according to claim 3, characterized in that: The low molecular weight astragalus polysaccharide was prepared by the following method: (1) Acid degradation: Weigh the Astragalus polysaccharide described in claim 1, dissolve it in water, add trifluoroacetic acid for acid hydrolysis in a water bath, and then evaporate the solution under reduced pressure; (2) Washing and dialysis: The product obtained in step (1) was washed with anhydrous methanol and rotary evaporated to remove trifluoroacetic acid. The product after rotary evaporation was dissolved in water, dialyzed with a 200 Da dialysis bag, and freeze-dried to obtain freeze-dried powder. (3) Ultrafiltration fractionation: Dissolve the freeze-dried powder obtained in step (2) in water, place it in a centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuge and collect the filtrate; place the filtrate in an ultrafiltration centrifuge tube with a molecular weight cutoff of 3 kDa, centrifuge, collect the cut-off portion and freeze-dry it to obtain low molecular weight Astragalus polysaccharide with a molecular weight of 3-10 kDa.

5. The application according to claim 4, characterized in that: The final concentration of trifluoroacetic acid after addition in step (1) is 0.5 M.

6. The application according to claim 4, characterized in that: The acid hydrolysis described in step (1) is carried out in a 95 ℃ water bath for 4 h.

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