Acanthopanax senticosus residue polysaccharide for reducing Aβ deposition in Caenorhabditis elegans, and preparation method and application thereof

Abstract

The present invention discloses a method for preparing a polysaccharide from Acanthopanax senticosus medicinal residue that reduces Aβ deposition in Caenorhabditis elegans. The method comprises the following steps: using waste generated in the elegans glycoside extraction process as a raw material, drying and crushing the waste, adding water and stirring evenly, subjecting the waste to dynamic high-pressure microfluidization homogenization, adding a surfactant to the treated liquid for ultrasonic extraction, centrifuging the obtained polysaccharide extract to obtain the supernatant, concentrating the supernatant under reduced pressure, and precipitating the polysaccharide from the Acanthopanax medicinal residue with alcohol. The present invention uses Acanthopanax medicinal residue as a raw material, recovers and reuses the active ingredients in the Acanthopanax medicinal residue, and promotes the sustainable development of the Acanthopanax medicinal residue industry. The preparation method disclosed in the present invention has the advantages of being easy to operate, time-saving, efficient, and pollution-free. The Acanthopanax medicinal residue polysaccharide prepared by the present invention has the efficacy of reducing Aβ deposition in Caenorhabditis elegans.

Description

Technical Field

[0001] The invention relates to the technical field of polysaccharide extraction, in particular to an Acanthopanax senticosus medicinal residue polysaccharide capable of reducing Aβ deposition in Caenorhabditis elegans, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is a degenerative disorder of the central nervous system that poses a serious threat to the health and life of the elderly. It presents with insidious onset, difficult diagnosis, and a high mortality rate. β-amyloid protein deposition and neurofibrillary tangles are the primary pathological hallmarks of AD, but its pathogenesis is complex, and many aspects remain under investigation. Currently, clinically used drugs for AD primarily include cholinesterase inhibitors and NMDA (N-methyl-D-aspartic acid receptor) blockers, which can partially inhibit AD progression but require long-term medication and are associated with adverse reactions such as nausea, vomiting, and diarrhea. Therefore, the search for safe and effective medications to prevent and treat AD is of great importance. Traditional Chinese Medicine (TCM) classifies AD as a "dementia," "forgetfulness," or "dementia." While the disease is located in the brain, it is closely associated with dysfunction of the heart, liver, spleen, and kidneys, resulting in a mental illness caused by insufficient kidney essence and a void in the brain marrow. Therefore, TCM often uses herbs with kidney-tonifying and essence-replenishing properties to treat dementia.

[0003] Acanthopanax senticosus (Rupr. et Maxim.) Harms, a plant of the Araliaceae family, is acrid, slightly bitter, and warm in nature. It enters the spleen, kidney, and heart meridians, and has the effects of invigorating qi and strengthening the spleen, tonifying the kidneys, and calming the mind. It is commonly used to treat symptoms such as spleen and lung qi deficiency, lung and kidney deficiency, and heart and spleen deficiency, and has broad development prospects. In industrial production, 75% ethanol is often used to extract eleutherosides, while other macromolecular active ingredients such as polysaccharides remain in the waste residue and waste liquid and are not effectively utilized. Polysaccharides are one of the main active ingredients in Acanthopanax senticosus medicinal residues and have multiple pharmacological activities such as protecting the central nervous system, anti-aging, and enhancing the body's immunity. Currently, traditional methods for extracting Acanthopanax senticosus polysaccharides mostly use Acanthopanax senticosus medicinal materials as raw materials and adopt a water extraction and alcohol precipitation method for extraction. This method has the disadvantages of being time-consuming, consuming a large amount of solvent, having a low extraction rate, and wasting resources.

[0004] Dynamic high-pressure microfluidization is a novel pretreatment and high-pressure homogenization technology. It disrupts plant cell walls through instantaneous high-speed collisions, intense shear, and instantaneous pressure release, promoting the dissolution of active ingredients, increasing mass transfer rates, and ultimately improving extraction yields. It has been widely used in the extraction of active plant polysaccharides. Surfactants are a novel solvent that offers advantages such as environmental friendliness, safety, ease of degradation, and a lack of toxic side effects. They hold great potential for the extraction of natural active substances. However, the application of dynamic high-pressure microfluidization in conjunction with surfactants to the extraction of polysaccharides from Acanthopanax senticosus residues has not been reported.

[0005] Therefore, how to promote the maximum utilization of Acanthopanax senticosus resources and provide an Acanthopanax senticosus residue polysaccharide with high extraction rate and purity while reducing Aβ deposition in Caenorhabditis elegans and a preparation method thereof are technical problems that technicians in this field urgently need to solve. Summary of the Invention

[0006] In view of this, the present invention provides a polysaccharide from Acanthopanax senticosus residues for reducing Aβ deposition in Caenorhabditis elegans, as well as a preparation method and application thereof.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A method for preparing a polysaccharide from Acanthopanax senticosus residues for reducing Aβ deposition in Caenorhabditis elegans comprises the following steps:

[0009] (1) taking the residue of Acanthopanax senticosus and drying it, then crushing it, adding water, stirring it evenly, and then performing dynamic high-pressure microfluidization homogenization;

[0010] (2) taking the treated liquid, adding a surfactant and performing ultrasonic extraction to obtain a polysaccharide extract;

[0011] (3) centrifuging the obtained polysaccharide extract, taking the supernatant, and concentrating under reduced pressure to obtain a concentrate;

[0012] (4) adding anhydrous ethanol to the concentrated solution for alcohol precipitation;

[0013] (5) centrifuging the alcohol solution, re-dissolving the precipitate, concentrating under reduced pressure, and freeze-drying to obtain the Acanthopanax senticosus residue polysaccharide APSP-50;

[0014] (6) The polysaccharide ASPS-50 from Acanthopanax senticosus residue was separated and purified using DEAE-650M filler to obtain ASPS-50-Ⅰ, ASPS-50-Ⅱ and ASPS-50-Ⅲ.

[0015] Furthermore, the drying temperature in step (1) is 45-50° C., and the drying time is 2-2.5 h;

[0016] The crushing particle size is 90 to 110 mesh;

[0017] Furthermore, the mass ratio of the Acanthopanax senticosus residue to water in step (1) is 1:20-40;

[0018] The stirring speed is 300-500 r / min, and the stirring time is 3-5 min;

[0019] The micro-jet homogenization pressure is 120-160 MPa, the time is 60-90 seconds, and the micro-jet homogenization treatment is performed 2-3 times.

[0020] Furthermore, the surfactant type in step (2) is sodium dodecyl sulfate;

[0021] The amount of the surfactant used is 2-3% of the mass of the treated liquid;

[0022] Furthermore, the ultrasonic extraction power in step (2) is 500-700W, the extraction temperature is 60-90°C, and the extraction time is 30-50min.

[0023] Furthermore, the centrifugal speed in step (3) is 4500-5000 r·min -1 , the centrifugation time is 10 to 15 minutes.

[0024] Furthermore, the alcohol precipitation temperature in step (4) is 4° C. and the alcohol precipitation time is 48 h;

[0025] The amount of anhydrous ethanol added is such that the volume concentration of ethanol in the solution is 50%.

[0026] Furthermore, the ultrasonic decompression in step (3) and step (5) is carried out at a pressure of 12 kPa, a temperature of 50° C., and a rotation speed of 30 r / min.

[0027] The freeze drying in step (5) is further carried out at a pressure of 13 Pa and a temperature of -40°C for 24 hours.

[0028] Furthermore, the centrifugal speed in step (5) is 4500-5000 r·min -1 , the centrifugation time is 10 to 15 minutes.

[0029] The present invention also provides a polysaccharide from Acanthopanax senticosus residues for reducing Aβ deposition in Caenorhabditis elegans, which is prepared by the above method.

[0030] The present invention has the beneficial effects of using Acanthopanax senticosus residue as raw material for the first time, conducting in-depth research on its chemical composition and pharmacological effects. The polysaccharide from Acanthopanax senticosus residue provided by the present invention is extracted for the first time using dynamic high-pressure microfluidics in conjunction with a surfactant. This method has the advantages of being easy to operate, time-saving, efficient, and pollution-free. Furthermore, the polysaccharide from Acanthopanax senticosus residue provided by the present invention has the effect of reducing Aβ deposition in Caenorhabditis elegans.

[0031] This invention uses Acanthopanax senticosus residue as raw material to recover and reuse the active ingredients in the residue, promoting the sustainable development of the Acanthopanax industry. The disclosed preparation method has the advantages of being easy to operate, time-saving, efficient, and pollution-free. The polysaccharide prepared from the Acanthopanax residue has the efficacy of reducing Aβ deposition in Caenorhabditis elegans.

[0032] The present invention also provides the use of the aforementioned polysaccharide from Acanthopanax senticosus medicinal residues in reducing Aβ deposition in C. elegans. This study used the CL4176 transgenic nematode strain, an Aβ model for C. elegans, and administered ASPS-50 prepared by the present invention. Results showed that ASPS-50 can prevent paralysis in nematodes, prolong nematode lifespan, alleviate heat stress damage, and improve nematode motility. However, there are relatively few reports on polysaccharides from Acanthopanax senticosus medicinal residues in this regard, suggesting that polysaccharides from Acanthopanax senticosus medicinal residues have significant potential in reducing Aβ deposition in C. elegans.

[0033] The CL4176 transgenic nematode strain used in the present invention was purchased from CGC (Caenorhabditis Genetics Center). The content of the present invention does not involve biological deposit content. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 HPGPC spectra of ASPS-50-Ⅰ, ASPS-50-Ⅱ and ASPS-50-Ⅲ prepared in the present invention;

[0035] Figure 2 The effects of different concentrations of ASPS-50 on paralysis of the CL4176 transgenic nematode strain of the present invention are shown;

[0036] Figure 3 The effect of different concentrations of ASPS-50 on the lifespan of the CL4176 transgenic nematode strain of the present invention;

[0037] Figure 4 The effect of different concentrations of ASPS-50 on the heat stress resistance of the CL4176 transgenic nematode strain of the present invention is shown;

[0038] Figure 5 The figure shows the effects of different concentrations of ASPS-50 on the motility of the CL4176 transgenic nematode strain of the present invention. DETAILED DESCRIPTION

[0039] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0040] Example 1

[0041] Acanthopanax senticosus residue polysaccharide for reducing Aβ deposition in Caenorhabditis elegans

[0042] (1) 500 g of Acanthopanax senticosus residue was dried at 50°C for 2 h and then crushed to 100 mesh. Water was then added, with the mass ratio of Acanthopanax senticosus residue to water being 1:30. The mixture was stirred at 400 r / min for 4 min and then subjected to dynamic high-pressure microfluidization homogenization at a microfluidization pressure of 140 MPa for 60 s. The microfluidization homogenization was repeated three times.

[0043] (2) taking the treated liquid, adding 2.5% sodium dodecyl sulfate, and ultrasonically extracting at 500W and 60°C for 30 minutes to obtain a polysaccharide extract;

[0044] (3) The obtained polysaccharide extract was heated at 5000 r·min -1 After centrifugation for 10 min, the supernatant was collected and concentrated under reduced pressure at 12 kPa, 50 °C, and 30 r / min to obtain a concentrate.

[0045] (4) adding anhydrous ethanol to the concentrated solution until the ethanol concentration in the solution is 50%, and subjecting the solution to alcohol precipitation at 4°C for 48 hours;

[0046] (5) The alcohol solution was heated at 5000 r·min -1 The mixture was centrifuged for 10 minutes, and the precipitate was redissolved, concentrated under reduced pressure at 12KPa, 50°C, and 30r / min, and freeze-dried at 13Pa and -40°C for 24 hours to obtain the Acanthopanax senticosus residue polysaccharide APSP-50.

[0047] The extraction rate of ASPS-50 was 8.25% and the sugar content was 72.54%.

[0048] In order to further demonstrate the technical effect of the present invention, the present invention optimizes the ratio of acupuncture Acanthopanax slag to deionized water, microjet pressure, microjet times, surfactant type and surfactant dosage, and the specific contents are as follows:

[0049] Test Example 1

[0050] Acanthopanax senticosus residue polysaccharide for reducing Aβ deposition in Caenorhabditis elegans

[0051] (1) 500 g of Acanthopanax senticosus residue was dried at 50°C for 2 h and then crushed to 100 mesh. Water was then added and three parallel treatments were set up. The mass ratios of Acanthopanax senticosus residue to water were 1:20, 1:30, and 1:40, respectively. After stirring at 400 r / min for 4 min, dynamic high-pressure microfluidization homogenization was performed. The microfluidization homogenization pressure was 120 MPa, the time was 60 s, and the microfluidization homogenization treatment was repeated three times.

[0052] (2) taking the treated liquid, adding 2.5% sodium dodecyl sulfate, and ultrasonically extracting at 500W and 60°C for 30 minutes to obtain a polysaccharide extract;

[0053] (3) The obtained polysaccharide extract was heated at 5000 r·min -1 After centrifugation for 10 min, the supernatant was collected and concentrated under reduced pressure at 12 kPa, 50 °C, and 30 r / min to obtain a concentrate.

[0054] (4) adding anhydrous ethanol to the concentrated solution until the ethanol concentration in the solution is 50%, and subjecting the solution to alcohol precipitation at 4°C for 48 hours;

[0055] (5) The alcohol solution was heated at 5000 r·min -1 The mixture was centrifuged for 10 minutes, and the precipitate was redissolved, concentrated under reduced pressure at 12KPa, 50°C, and 30r / min, and freeze-dried at 13Pa and -40°C for 24 hours to obtain Acanthopanax senticosus polysaccharide 1, Acanthopanax senticosus polysaccharide 2, and Acanthopanax senticosus polysaccharide 3, respectively.

[0056] Optimization of the ratio of Acanthopanax senticosus residue to deionized water resulted in an extraction rate of 5.32% for Acanthopanax senticosus polysaccharide 1 and a sugar content of 49.43%; an extraction rate of 6.88% for Acanthopanax senticosus polysaccharide 2 and a sugar content of 57.4%; and an extraction rate of 6.13% for Acanthopanax senticosus polysaccharide 3 and a sugar content of 53.67%. Therefore, the optimal ratio of Acanthopanax senticosus residue to deionized water is 1:30.

[0057] Test Example 2

[0058] (1) 500 g of Acanthopanax senticosus residue was dried at 50°C for 2 h and then crushed to 100 mesh. Water was then added, with the mass ratio of Acanthopanax senticosus residue to water being 1:30. After stirring at 400 r / min for 4 min, dynamic high-pressure microfluidization homogenization was performed. Three parallel microfluidization schemes were set with microfluidization pressures of 120, 140, and 160 MPa, respectively, for 60 s, and the microfluidization homogenization treatment was repeated three times.

[0059] (2) taking the treated liquid, adding 2.5% sodium dodecyl sulfate, and ultrasonically extracting at 500W and 60°C for 30 minutes to obtain a polysaccharide extract;

[0060] (3) The obtained polysaccharide extract was heated at 5000 r·min -1 After centrifugation for 10 min, the supernatant was collected and concentrated under reduced pressure at 12 kPa, 50 °C, and 30 r / min to obtain a concentrate.

[0061] (4) adding anhydrous ethanol to the concentrated solution until the ethanol concentration in the solution is 50%, and subjecting the solution to alcohol precipitation at 4°C for 48 hours;

[0062] (5) The alcohol solution was heated at 5000 r·min -1 After centrifugation for 10 minutes, the precipitate was redissolved, concentrated under reduced pressure at 12KPa, 50°C, and 30r / min, and freeze-dried at 13Pa and -40°C for 24 hours to obtain Acanthopanax senticosus polysaccharide 4, Acanthopanax senticosus polysaccharide 5, and Acanthopanax senticosus polysaccharide 6.

[0063] Optimization of the microfluidization pressure resulted in an extraction yield of 7.16% for Acanthopanax senticosus polysaccharide 4 and a sugar content of 62.48%; an extraction yield of 7.83% for Acanthopanax senticosus polysaccharide 5 and a sugar content of 70.41%; and an extraction yield of 6.89% for Acanthopanax senticosus polysaccharide 6 and a sugar content of 58.72%. Therefore, a microfluidization pressure of 140 MPa was preferred for preparation.

[0064] Test Example 3

[0065] Acanthopanax senticosus residue polysaccharide for reducing Aβ deposition in Caenorhabditis elegans

[0066] (1) 500 g of Acanthopanax senticosus residue was dried at 50°C for 2 h and then crushed to 100 mesh. Water was then added, with the mass ratio of Acanthopanax senticosus residue to water being 1:30. After stirring at 400 r / min for 4 min, dynamic high-pressure microfluidization homogenization was performed. The microfluidization homogenization pressure was 140 MPa and the time was 60 s. Three parallel schemes were set up, with the microfluidization treatment times of 2, 3, and 4 times.

[0067] (2) taking the treated liquid, adding 2.5% sodium dodecyl sulfate, and ultrasonically extracting at 500W and 60°C for 30 minutes to obtain a polysaccharide extract;

[0068] (3) The obtained polysaccharide extract was heated at 5000 r·min -1 After centrifugation for 10 min, the supernatant was collected and concentrated under reduced pressure at 12 kPa, 50 °C, and 30 r / min to obtain a concentrate.

[0069] (4) adding anhydrous ethanol to the concentrated solution until the ethanol concentration in the solution is 50%, and subjecting the solution to alcohol precipitation at 4°C for 48 hours;

[0070] (5) The alcohol solution was heated at 5000 r·min -1 After centrifugation for 10 minutes, the precipitate was redissolved, concentrated under reduced pressure at 12KPa, 50°C, and 30r / min, and freeze-dried at 13Pa and -40°C for 24 hours to obtain Acanthopanax senticosus polysaccharide 7, Acanthopanax senticosus polysaccharide 8, and Acanthopanax senticosus polysaccharide 9.

[0071] Optimization of the number of microfluidization treatments resulted in an extraction yield of 7.41% and a sugar content of 65.87% for Acanthopanax senticosus polysaccharide 7; an extraction yield of 8.17% and a sugar content of 71.95% for Acanthopanax senticosus polysaccharide 8; and an extraction yield of 7.95% and a sugar content of 70.93% for Acanthopanax senticosus polysaccharide 9. Therefore, a microfluidization treatment of three times is preferred for preparation.

[0072] Test Example 4

[0073] Acanthopanax senticosus residue polysaccharide for reducing Aβ deposition in Caenorhabditis elegans

[0074] (1) 500 g of Acanthopanax senticosus residue was dried at 50°C for 2 h and then crushed to 100 mesh. Water was then added, with the mass ratio of Acanthopanax senticosus residue to water being 1:30. The mixture was stirred at 400 r / min for 4 min and then subjected to dynamic high-pressure microfluidization homogenization at a microfluidization pressure of 140 MPa for 60 s. The microfluidization homogenization was repeated three times.

[0075] (2) Taking the treated liquid, adding 2.5% surfactant, setting up three parallel schemes with surfactants of Tween 80, sodium dodecyl sulfate and sucrose ester respectively, and ultrasonically extracting at 500W and 60℃ for 30min to obtain polysaccharide extract;

[0076] (3) The obtained polysaccharide extract was heated at 5000 r·min -1 After centrifugation for 10 min, the supernatant was collected and concentrated under reduced pressure at 12 kPa, 50 °C, and 30 r / min to obtain a concentrate.

[0077] (4) adding anhydrous ethanol to the concentrated solution until the ethanol concentration in the solution is 50%, and subjecting the solution to alcohol precipitation at 4°C for 48 hours;

[0078] (5) The alcohol solution was heated at 5000 r·min -1 The mixture was centrifuged for 10 minutes, and the precipitate was redissolved, concentrated under reduced pressure at 12KPa, 50°C, and 30r / min, and freeze-dried at 13Pa and -40°C for 24 hours to obtain Acanthopanax senticosus polysaccharide 10, Acanthopanax senticosus polysaccharide 11, and Acanthopanax senticosus polysaccharide 12.

[0079] By optimizing the surfactant types, the extraction rate of Acanthopanax senticosus polysaccharide 10 was 7.34% and the sugar content was 63.77%; the extraction rate of Acanthopanax senticosus polysaccharide 11 was 8.23% and the sugar content was 72.34%; and the extraction rate of Acanthopanax senticosus polysaccharide 12 was 7.51% and the sugar content was 64.23%. Therefore, sodium dodecyl sulfate is the preferred surfactant for the preparation.

[0080] Test Example 5

[0081] Acanthopanax senticosus residue polysaccharide for reducing Aβ deposition in Caenorhabditis elegans

[0082] (1) 500 g of Acanthopanax senticosus residue was dried at 50°C for 2 h and then crushed to 100 mesh. Water was then added, with the mass ratio of Acanthopanax senticosus residue to water being 1:30. The mixture was stirred at 400 r / min for 4 min and then subjected to dynamic high-pressure microfluidization homogenization at a microfluidization pressure of 140 MPa for 60 s. The microfluidization homogenization was repeated three times.

[0083] (2) taking the treated liquid, setting up three parallel schemes, adding 2.0, 2.5, and 3.0% sodium dodecyl sulfate respectively, and ultrasonically extracting at 500W and 60°C for 30 minutes to obtain polysaccharide extracts;

[0084] (3) The obtained polysaccharide extract was heated at 5000 r·min -1 After centrifugation for 10 min, the supernatant was collected and concentrated under reduced pressure at 12 kPa, 50 °C, and 30 r / min to obtain a concentrate.

[0085] (4) adding anhydrous ethanol to the concentrated solution until the ethanol concentration in the solution is 50%, and subjecting the solution to alcohol precipitation at 4°C for 48 hours;

[0086] (5) The alcohol solution was heated at 5000 r·min -1 After centrifugation for 10 minutes, the precipitate was redissolved, concentrated under reduced pressure at 12KPa, 50°C, and 30r / min, and freeze-dried at 13Pa and -40°C for 24 hours to obtain Acanthopanax senticosus polysaccharide 13, Acanthopanax senticosus polysaccharide 14, and Acanthopanax senticosus polysaccharide 15.

[0087] By optimizing the surfactant dosage, the extraction rate of Acanthopanax senticosus polysaccharide 13 was 7.78% and the sugar content was 68.35%; the extraction rate of Acanthopanax senticosus polysaccharide 14 was 8.25% and the sugar content was 72.54%; and the extraction rate of Acanthopanax senticosus polysaccharide 15 was 7.62% and the sugar content was 66.97%. Therefore, the preferred surfactant dosage was 2.5%.

[0088] Comparative Example 1:

[0089] Acanthopanax senticosus residue polysaccharide was prepared according to the method of Example 1, except that the dynamic high-pressure microfluidization treatment was not performed before extraction. Other steps were the same as in Example 1 to prepare Acanthopanax senticosus residue polysaccharide (ASPS-50-A).

[0090] Comparative Example 2:

[0091] Acanthopanax senticosus residue polysaccharide was prepared according to the method of Example 1, except that no surfactant (sodium dodecyl sulfate) was added during the extraction process. Other steps were the same as in Example 1 to prepare Acanthopanax senticosus residue polysaccharide (ASPS-50-B).

[0092] Comparative Example 3:

[0093] Acanthopanax senticosus residue polysaccharides were prepared according to the method of Example 1, except that traditional water extraction and ultrasonic extraction were used respectively. Other steps were the same as in Example 1 to prepare Acanthopanax senticosus residue polysaccharides (ASPS-50-C and ASPS-50-D).

[0094] The polysaccharide content of the Acanthopanax senticosus residues in each comparative example was determined using the phenol-sulfuric acid method, and the polysaccharide extraction rate was calculated. The polysaccharide extraction rate and sugar content determination results of each Acanthopanax senticosus residue are shown in Table 1.

[0095] ASPS-50 ASPS-50-A ASPS-50-B ASPS-50-C ASPS-50-D Extraction rate 8.25％ 5.98％ 6.97％ 5.03％ 5.22％ Sugar content 72.54％ 52.73％ 60.53％ 45.23％ 48.55％

[0096] Results showed that dynamic high-pressure microfluidization technology can, through multiple effects, thoroughly mix the material and solvent, promoting the dissolution of active ingredients and increasing polysaccharide yield. The addition of surfactants can reduce the interfacial tension between the solvent and the material, making it easier for the solvent to penetrate plant cells, further increasing the yield and content of polysaccharides from Acanthopanax senticosus residues. Compared with traditional water extraction and ultrasonic extraction, both significantly improved the extraction rate and content of polysaccharides from Acanthopanax senticosus residues. Using dynamic high-pressure microfluidization in conjunction with surfactants for extraction combines the advantages of both, optimizing the extraction process and achieving synergistic efficiency.

[0097] Experimental Example 1

[0098] The pretreated DEAE-650M filler was packed into the column using the wet method to prepare 1 mg·mL -1 ASPS-50 solution prepared in Example 1, 4000 r·min -1 After centrifugation for 10 min, the supernatant was taken and loaded with distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol·L -1 NaCl was used for elution at a flow rate of 1 mL min -1 The eluate was collected, and the absorbance was detected by phenol-sulfuric acid method. The single peak elution components were collected and combined. After concentration, a dialysis bag with a molecular weight cutoff of 3500Da was selected for dialysis desalination and freeze-dried to obtain three polysaccharides, which were recorded as ASPS-50-Ⅰ, ASPS-50-Ⅱ, and ASPS-50-Ⅲ.

[0099] The relative molecular weight of ASPS-50 was distributed in the range of 1.6×10 3 ~2.8×10 6 Among them, ASPS-50-Ⅰ, ASPS-50-Ⅱ and ASPS-50-Ⅲ all showed single symmetrical peaks, with molecular weights of 1645Da, 1827KDa and 2768KDa respectively.

[0100] Experimental Example 2

[0101] The nematodes of strain CL4176 were cultured at a constant temperature of 16°C and fed with Escherichia coli OP50. The Acanthopanax senticosus polysaccharide prepared in Example 1 was dissolved in ultrapure water to a concentration of 2 mg·mL -1 The mother liquor was sterilized and filtered, and E. coli OP50 was added to prepare the concentrations of 0.125, 0.25, and 0.5 mg mL -1 Take 100 μL of each solution and spread it on NGM plates, with 3 plates for each mass concentration.

[0102] The paralysis experiment was conducted with a blank group and low-, medium- and high-dose groups of the polysaccharide from the Acanthopanax senticosus residue prepared in Example 1. The synchronized CL4176 eggs were placed in the blank group and the dosing groups at each concentration, 30 eggs per plate, and cultured at 16°C for 24 hours. The plates were then transferred and cultured at 16°C for 12 hours, then transferred to a 25°C incubator for 36 hours of temperature culture. After that, the eggs were observed under a microscope every 2 hours, and the number of paralyzed nematodes was recorded (the head and body of the nematode were touched lightly with a pick several times, and the body parts other than the head were considered paralyzed if they did not move). The results showed that compared with the blank group, the low-, medium- and high-dose groups of the polysaccharide from the Acanthopanax senticosus residue prepared in Example 1 had certain anti-nematode paralysis effects (P<0.05), among which ASPS-50 (0.5 mg·mL -1 ) group had the best effect. Figure 2 .

[0103] Experimental Example 3 Lifespan Experiment

[0104] A blank group and low, medium and high dose groups of the polysaccharide of Acanthopanax senticosus medicinal residue prepared in Example 1 were set up. The synchronized CL4176 eggs were placed in the blank group and the drug administration groups at each concentration, 30 eggs per plate, and the survival of the nematodes was recorded every 3 days until all died. The head and tail of the nematodes were touched lightly with a pick. If there was no reaction within 30 seconds, it was determined to be dead. The results showed that compared with the blank group, the low, medium and high dose groups of the polysaccharide of Acanthopanax senticosus medicinal residue prepared in Example 1 could prolong the lifespan of the nematodes to a certain extent (P<0.05), among which ASPS-50 (0.5 mg·mL -1 ) group had the best effect. Figure 3 .

[0105] Experimental Example 4 Heat stress experiment

[0106] A blank group and low-, medium- and high-dose groups of the polysaccharide from the Acanthopanax senticosus medicinal residue prepared in Example 1 were set up. The synchronized CL4176 eggs were placed in the blank group and the drug-dosing groups at each concentration, with 30 eggs per plate. After culturing in a 16°C incubator for 3 days, the temperature was raised to 35°C. The survival of the nematodes was observed once every 1 hour until all the nematodes died. The results showed that compared with the blank group, the low-, medium- and high-dose groups of the polysaccharide from the Acanthopanax senticosus medicinal residue prepared in Example 1 could weaken the heat stress damage to the nematodes (P<0.05), among which ASPS-50 (0.5 mg·mL -1 ) group had the best effect. Figure 4 .

[0107] Experimental Example 5: Exercise Ability Measurement Experiment

[0108] A blank group and low-, medium- and high-dose groups of polysaccharides from the Acanthopanax senticosus residue prepared in Example 1 were set up. The synchronized CL4176 eggs were placed in the blank group and the groups of each concentration, 10 eggs per plate, and cultured in a 16°C incubator, with the culture dish changed every 2 days. When the worm body grew to 10 days, an appropriate amount of M9 buffer solution was added, and the number of sinusoidal movements of the worm body within 30 seconds was detected under a microscope to determine the changes in the movement ability of CL4176 nematodes. The results showed that compared with the blank group, the low-, medium- and high-dose groups of polysaccharides from the Acanthopanax senticosus residue prepared in Example 1 could improve the movement ability of nematodes (P<0.05), among which ASPS-50 (0.5 mg·mL -1 ) group had the best effect. Figure 5 .

[0109] The above experimental results show that the polysaccharide extraction rate and sugar content of the Acanthopanax senticosus residue prepared by the present invention are high, and different concentrations of the drug administration groups can resist nematode paralysis to a certain extent, prolong nematode lifespan, alleviate heat stress damage, improve movement ability, and reduce Aβ deposition in Caenorhabditis elegans.

[0110] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a polysaccharide from Acanthopanax senticosus residues for reducing aβ deposition in Caenorhabditis elegans, characterized in that: The following steps are involved: (1) Take the Acanthopanax senticosus residue and dry it at 45-50℃ for 2-2.5h, then crush it into 90-110 mesh. Then add water, the mass ratio of Acanthopanax senticosus residue to water is 1:20-40, stir at 300-500 r / min for 3-5 min, and then perform dynamic high-pressure micro-jet homogenization. The micro-jet homogenization pressure is 120-160MPa, the time is 60-90s, and the micro-jet homogenization treatment is repeated 2-3 times. (2) Take the treated liquid, add a surfactant, and perform ultrasonic extraction at 500-700W and 60-90℃ for 30-50min to obtain a polysaccharide extract; the surfactant type is sodium dodecyl sulfate, and the amount of the surfactant is 2-3% of the mass of the treated liquid; (3) Centrifuging the obtained polysaccharide extract, taking the supernatant, and concentrating under reduced pressure to obtain a concentrated solution; (4) adding anhydrous ethanol to the concentrated solution for alcohol precipitation; (5) The alcohol solution was centrifuged, the precipitate was redissolved, concentrated under reduced pressure, and freeze-dried to obtain the Acanthopanax senticosus residue polysaccharide APSP-50; (6) The polysaccharide ASPS-50 from Acanthopanax senticosus residue was separated and purified using DEAE-650M filler to obtain ASPS-50-Ⅰ, ASPS-50-Ⅱ and ASPS-50-Ⅲ.

2. The method for preparing a polysaccharide from Acanthopanax senticosus residues for reducing aβ deposition in Caenorhabditis elegans according to claim 1, characterized in that: The centrifugal speed in step (3) is 4500~5000 r·min -1 , the centrifugation time is 10~15 min.

3. The method for preparing a polysaccharide from Acanthopanax senticosus residues for reducing Aβ deposition in Caenorhabditis elegans according to claim 1, characterized in that: The alcohol precipitation temperature in step (4) is 4°C and the alcohol precipitation time is 48 h.

4. The method for preparing a polysaccharide from Acanthopanax senticosus residues for reducing Aβ deposition in Caenorhabditis elegans according to claim 1, characterized in that: The centrifugal speed in step (5) is 4500~5000 r·min -1 , the centrifugation time is 10~15 min.

5. The method for preparing a polysaccharide from Acanthopanax senticosus residues for reducing aβ deposition in Caenorhabditis elegans according to claim 1, characterized in that: The reduced pressure concentration in step (3) and step (5) is carried out at a pressure of 12 kPa, a temperature of 50° C., and a rotation speed of 30 r / min.

6. The method for preparing a polysaccharide from Acanthopanax senticosus residues for reducing Aβ deposition in Caenorhabditis elegans according to claim 1, characterized in that: The freeze drying in step (5) is freeze drying at a pressure of 13 Pa and a temperature of -40°C for 24 hours.

7. A polysaccharide from Acanthopanax senticosus residues for reducing aβ deposition in Caenorhabditis elegans, characterized in that: Prepared by the method according to any one of claims 1 to 6.

8. Use of the polysaccharide from Acanthopanax senticosus residue as claimed in claim 7 in the preparation of a drug for reducing Aβ deposition in Caenorhabditis elegans.

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