Preparation method of phosphorylated acanthopanax senticosus polysaccharide and application thereof
Phosphorylated Acanthopanax senticosus polysaccharide was prepared by ultrasound-assisted enzymatic and phosphate methods, which solved the problems of poor polysaccharide absorption and unevenness, and achieved efficient extraction of polysaccharide and the effect of improving the health status of nematodes.
Patent Information
- Application Number
- CN202411805274.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-10
- Publication Date
- 2025-12-12
- Estimated Expiration
- 2044-12-10
AI Technical Summary
Natural polysaccharides have complex structures and large molecular weights, making them difficult for the human body to absorb. Furthermore, the unevenness of phosphorylation-modified polysaccharides may affect their performance, resulting in poor bioactivity and solubility, making it difficult to exert significant effects in the short term.
Acanthopanax senticosus polysaccharide was extracted using an ultrasound-assisted enzymatic method, and then purified by separation using a DEAE-650M cellulose column. Phosphorylated Acanthopanax senticosus polysaccharide PASPS-Ⅰ was prepared by structural modification using the phosphate method.
It improved the bioactivity and solubility of polysaccharides, prolonged the lifespan of nematodes, reduced the paralysis rate of nematodes, improved the motility of nematodes, and enhanced their stress resistance.
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Figure CN119613582B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biotechnology, in particular to a preparation method and application of phosphorylated acanthopanax senticosus polysaccharide. BACKGROUND
[0002] In the high-pressure rhythm of modern society, people begin to pay more attention to health problems. Long hours of work, lack of exercise, irregular work and rest time, high oil and high salt dietary habits, excessive mental stress, etc. can cause the body to show persistent fatigue, poor sleep quality, decreased exercise capacity, memory loss, stress disorders, and even shortened life span. In order to solve this situation, people focus on the field of traditional Chinese medicine. Traditional Chinese medicine prevention not only focuses on symptomatic treatment, but also focuses on overall health conditioning and balance. Traditional Chinese medicine, as an important part of traditional Chinese medicine, its unique theoretical system, treatment methods and clinical effects, provide positive contributions in many ways for the treatment of diseases, improvement of life quality and promotion of health.
[0003] Natural polysaccharides have complex structures and large molecular weights, which are difficult to be absorbed by the human body and cannot fully exert their biological activity. In order to solve this problem, researchers have made structural modifications to polysaccharides. Currently, the methods for modifying the structure of polysaccharides include sulfation, phosphorylation, acetylation, carboxymethylation, and selenization. Studies have shown that phosphorylated polysaccharides have the characteristics of reducing viscosity, increasing solubility, and enhancing biological activity. The methods for phosphorylating polysaccharides include phosphorus oxychloride method, phosphoric acid-anhydride method, phosphorus pentoxide method, and phosphate method. The phosphate method has the advantages of simple preparation process, safety, less by-products, no organic reagents involved, and environmental friendliness. However, phosphorylation modification usually leads to the introduction of phosphate groups at different levels, and the phosphate groups may be introduced at different positions on the polysaccharide molecule. This unevenness may affect the performance of the modified polysaccharides, and excessive or insufficient phosphorylation may affect the solubility, plasticity, and biological activity of the polysaccharides.
[0004] Acanthopanax senticosus (Rupr. et Maxim.) Harms is the dried root and rhizome or stem of Acanthopanax senticosus, which has a long history of medicinal use and is recorded in the herbal works of all dynasties. The Bencao Gangmu considers that Acanthopanax senticosus has a pungent and warm nature and belongs to the heart, spleen and liver meridians. It can tonify qi, calm the five viscera, strengthen the bones and tendons, calm the spirit, and nourish the liver blood, and is suitable for treating qi deficiency, anorexia, and other symptoms. Modern research shows that Acanthopanax senticosus contains flavonoids, polysaccharides, glycosides and other active ingredients, and polysaccharides are one of the main active ingredients. Acanthopanax senticosus polysaccharides can enhance the immune function of the body, promote the activation and proliferation of immune cells, and also have strong antioxidant properties. As an antioxidant, it helps to slow down the aging process and protect cells from oxidative damage. Acanthopanax senticosus polysaccharides also have an anti-fatigue effect, which can help the body cope with long-term physical or psychological stress, relieve fatigue, and improve work and exercise endurance and efficiency. The molecular structure of Acanthopanax senticosus polysaccharides is relatively complex, and the molecular weight is relatively large. This makes its absorption and metabolism in the body relatively slow. Although its immune regulation and anti-fatigue functions are its advantages, the slow metabolism process may make it difficult to achieve obvious effects in the short term or require a large dose to work.
[0005] Therefore, it is an urgent problem for those skilled in the art to provide a preparation method of phosphorylated Acanthopanax senticosus polysaccharides and its application. SUMMARY
[0006] Therefore, it is an urgent problem for those skilled in the art to provide a preparation method of phosphorylated Acanthopanax senticosus polysaccharides and its application.
[0007] The present application adopts ultrasonic-assisted enzyme method to extract Acanthopanax senticosus polysaccharides ASPS, and separates and purifies ASPS-I through a DEAE-650M cellulose chromatographic column to obtain phosphorylated Acanthopanax senticosus polysaccharides PASPS-I through phosphate method. The PASPS-I provided by the present application can prolong the lifespan of CL4176 nematodes, reduce the paralysis rate of CL4176 nematodes, improve the movement ability of CL4176 nematodes and improve the stress resistance of CL4176 nematodes.
[0008] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions:
[0009] A preparation method of phosphorylated Acanthopanax senticosus polysaccharides, comprising the following steps:
[0010] (1) adding an ethanol solution to Acanthopanax senticosus medicinal materials for defatting, filtering, and air-drying to obtain defatted Acanthopanax senticosus; adding deionized water to the defatted Acanthopanax senticosus for ultrasonic extraction, and centrifuging the solution after cooling to obtain supernatant;
[0011] (2) taking the supernatant obtained in step (1) to add cellulase for enzymolysis, then performing enzyme inactivation, and centrifuging to obtain an enzymolysis solution;
[0012] (3) taking the enzymolysis solution obtained in step (2) to add anhydrous ethanol for alcohol precipitation, centrifuging to obtain a precipitate, re-dissolving, concentrating, and freeze-drying to obtain a crude acanthopanax senticosus polysaccharide ASPS; the ASPS is separated and purified by a DEAE-650M cellulose chromatographic column to obtain an acanthopanax senticosus polysaccharide ASPS-I;
[0013] (4) taking the ASPS-I obtained in step (3), adding a phosphate solution, adjusting the pH of the solution, and starting a phosphorylation reaction; after the reaction is completed, adding anhydrous ethanol for alcohol precipitation, centrifuging, re-dissolving the precipitate, dialyzing, and freeze-drying to obtain a phosphorylated acanthopanax senticosus polysaccharide PASPS-I.
[0014] Further, the volume ratio of the acanthopanax senticosus to the ethanol solution in step (1) is 1: (6-10) g·mL -1 ; and the concentration of the ethanol solution is 70%.
[0015] Further, the solid-liquid ratio of the defatted acanthopanax senticosus to the deionized water in step (1) is 1: (10-20) g·mL -1 , the ultrasonic power is 40-60 W, the ultrasonic temperature is 40-80℃, and the ultrasonic time is 40-60 min.
[0016] Further, the cellulase is added in step (2) in an amount of 8-12% to make the concentration reach 1500 U·mL -1 , and the enzymolysis condition is 37℃ for 60 min, and then enzyme inactivation at 95℃.
[0017] Further, the enzymolysis solution is added with anhydrous ethanol to make the ethanol concentration reach 60% in step (3); the alcohol precipitation temperature is 4℃, the time is 48 h; and the centrifugation condition is 4000 r·min -1 , and the centrifugation time is 10 min.
[0018] Further, the mass of the ASPS-I in step (4) is 100 mg, and the volume of the phosphate solution is 100 mL; the ratio of sodium tripolyphosphate to sodium trimetaphosphate in the phosphate solution is 4:3, 5:2, or 6:1; the pH of the solution is adjusted to 7-9; the phosphorylation reaction temperature is 50-70℃, and the phosphorylation reaction time is 3-5 h; the alcohol precipitation temperature is 4℃, the time is 48 h; the centrifugation condition is 4000 r·min -1 , and the centrifugation time is 10 min; the molecular weight of the dialysis bag is 3500 Da; and the freeze-drying pressure is 13 kPa, and the freeze-drying temperature is -40℃ for 24 h.
[0019] Further, the method prepares a phosphorylated acanthopanax senticosus polysaccharide.
[0020] Further, the application provides the phosphated acanthopanax senticosus polysaccharide in prolonging the lifespan of nematodes, reducing the paralysis rate of nematodes, improving the movement ability of nematodes and / or improving the stress resistance of nematodes.
[0021] The application adopts CL4176 model Caenorhabditis elegans as a model, and the PASPS-I prepared by the application is used for administration, and the results show that the PASPS-I can prolong the lifespan of CL4176 nematodes, reduce the paralysis rate of CL4176 nematodes, improve the movement ability of CL4176 nematodes and improve the stress resistance of CL4176 nematodes.
[0022] According to the technical scheme, compared with the prior art, the application provides a preparation method of phosphated acanthopanax senticosus polysaccharide and application thereof, and has the following beneficial effects:
[0023] (1) The application adopts ultrasonic-assisted enzyme method to extract acanthopanax senticosus polysaccharide, which can not only solve the problem of low extraction efficiency and purity of polysaccharide caused by the hard cell wall of acanthopanax senticosus, but also improve the biological activity of acanthopanax senticosus polysaccharide.
[0024] (2) The application adopts phosphate method to modify the structure to obtain phosphated acanthopanax senticosus polysaccharide, and the preparation process is simple and safe, has less by-products, and does not involve organic reagents, which is friendly to the environment.
[0025] (3) The PASPS-I prepared by the application can prolong the lifespan of CL4176 nematodes, reduce the paralysis rate of CL4176 nematodes, improve the movement ability of CL4176 nematodes and improve the stress resistance of CL4176 nematodes. DETAILED DESCRIPTION
[0026] In order to more clearly illustrate the technical scheme in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or the prior art description, and obviously, the drawings in the following description are only embodiments of the application, and those skilled in the art can obtain other drawings according to the provided drawings without any creative labor.
[0027] Figure 1 HPGPC spectrum of ASPS-I (A) and PASPS-I (B) prepared by the application;
[0028] Figure 2 Influence of ASPS-I (A) and PASPS-I (B) prepared by the application on the lifespan of CL4176 nematodes;
[0029] Figure 3 Influence of ASPS-I and PASPS-I prepared by the application on the paralysis rate of CL4176 nematodes; Figure 4Effects of ASPS-I and PASPS-I prepared by the present application on the movement ability of CL4176 nematodes
[0030] Figure 5 Effects of ASPS-I and PASPS-I prepared by the present application on the oxidative stress ability of CL4176 nematodes
[0031] Figure 6 Effects of ASPS-I and PASPS-I prepared by the present application on the heat stress ability of CL4176 nematodes. DETAILED DESCRIPTION
[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.
[0033] Embodiment 1
[0034] A preparation method of acanthopanax senticosus polysaccharide, comprising the following steps:
[0035] 1kg of dried roots of acanthopanax senticosus is weighed, and 70% concentration ethanol solution is added for soaking at a material-liquid ratio of 1:8 g·mL -1 for 12 hours, and then filtered, and the ethanol is volatilized, and the residue is taken and dried in the shade. Deionized water is added at an ultrasonic power of 50 W and a material-liquid ratio of 1:15 g·mL -1 , and ultrasonic extraction is performed at 60°C for 50 minutes, and then the solution is cooled, centrifuged at 4000 r·min -1 for 10 minutes, and the supernatant is taken. 10% cellulase is added to the supernatant to make the concentration reach 1500 U·mL -1 , and enzymolysis is performed at 37°C for 60 minutes, and then enzyme inactivation is performed at 95°C for 10 minutes, and then the solution is centrifuged at 4000 r·min -1 for 10 minutes to obtain an enzymolysis solution. Anhydrous ethanol is added to the enzymolysis solution to make the ethanol concentration reach 60%, and then stirred, and then placed at 4°C for 48 hours, and then centrifuged at 4000 r·min -1 for 10 minutes, and then the precipitate is redissolved in water, concentrated, and then freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 hours to obtain acanthopanax senticosus crude polysaccharide ASPS.
[0036] The ASPS solution is further separated by a DEAE-650M cellulose chromatographic column: 1 mg·mL -1 of the ASPS solution is prepared, and then centrifuged at 4000 r·min -1After centrifugation for 10 min, the supernatant was sampled, and distilled water, 0.1, 0.2, 0.3, 0.4, 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 the phenol-sulfuric acid method. The single-peak elution component was collected and concentrated. Then, a dialysis bag with a molecular weight cutoff of 3500 Da was selected for desalting dialysis. The freeze-drying was performed at a pressure of 13 kPa and a temperature of -40℃ for 24 h to obtain the acanthopanax senticosus polysaccharide ASPS-Ⅰ.
[0037] A phosphate solution with a ratio of sodium tripolyphosphate to sodium trimetaphosphate of 6:1 was prepared. 100 mg of ASPS-Ⅰ was added to 100 mL of the phosphate solution, and the pH was adjusted to 8 by sodium bicarbonate. Then, the solution was stirred at 60℃ for 4 h. After the reaction was completed, four times the amount of anhydrous ethanol was added. After standing at 4℃ for 48 h, the solution was centrifuged at 4000 r·min -1 for 10 min. The precipitate was redissolved in water and dialyzed in a dialysis bag with a molecular weight cutoff of 3500 Da for 48 h. The freeze-drying was performed at a pressure of 13 kPa and a temperature of -40℃ for 24 h to obtain the phosphorylated acanthopanax senticosus polysaccharide PASPS-Ⅰ.
[0038] Figure 1 The HPGPC chromatogram of the prepared ASPS-Ⅰ and PASPS-Ⅰ is shown in the figure. The molecular weight of ASPS-Ⅰ is 162862 Da, and the molecular weight of PASPS-Ⅰ is 114494 Da.
[0039] The content of polysaccharides in acanthopanax senticosus was determined by the phenol-sulfuric acid method, and the polysaccharide extraction rate was calculated. The ASPS extraction rate was 8.26%, and the sugar content was 73.45%. The sugar content of PASPS-Ⅰ was 57.23%.
[0040] The phosphate content in the phosphorylated acanthopanax senticosus polysaccharide was determined by the molybdenum blue colorimetric method. The phosphate content of PASPS-Ⅰ was 6.17%.
[0041] Example 2
[0042] A method for preparing a kind of acanthopanax senticosus polysaccharide, comprising the following steps:
[0043] 1 kg of dried root of acanthopanax senticosus was weighed, and 70% ethanol solution was added at a solid-liquid ratio of 1:6 g·mL -1 for 12 h. After filtration and ethanol evaporation, the residue was dried in the shade. Deionized water was added at a solid-liquid ratio of 1:10 g·mL -1 under ultrasonic power of 40 W. The solution was extracted at 40℃ for 40 min. After cooling, the solution was centrifuged at 4000 r·min -1 for 10 min, and the supernatant was taken. 8% cellulase was added to the supernatant to make the concentration reach 1500 U·mL-1 Enzymolysis for 60 min at 37℃, then inactivate the enzyme for 10 min at 95℃, centrifugal at 4000r·min -1 for 10 min, then add anhydrous ethanol to the enzyme solution to reach 60% ethanol concentration, stir well, and stand still at 4℃ for 48 h, then centrifugal at 4000r·min -1 for 10 min, re-dissolve the precipitate with water, concentrate, and freeze-dry at 13 kPa and -40℃ for 24 h to obtain the crude acanthopanax senticosus polysaccharide ASPS1.
[0044] Further separate the ASPS1 solution through a DEAE-650M cellulose chromatographic column: prepare a 1 mg·mL -1 of ASPS solution, and centrifugal at 4000r·min -1 for 10 min, then take the supernatant and load it, and use distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol·L -1 of NaCl to elute, respectively, at a flow rate of 1 mL·min -1 Collect the eluate, and detect the absorbance by the phenol-sulfuric acid method, collect and combine the single-peak elution components, concentrate, select a dialysis bag with a molecular weight cut-off of 3500 Da for desalting dialysis, and freeze-dry at 13 kPa and -40℃ for 24 h to obtain the acanthopanax senticosus polysaccharide ASPS1-Ⅰ.
[0045] Prepare a phosphate solution with a ratio of sodium tripolyphosphate to sodium trimetaphosphate of 4:3, add 100 mg of ASPS1-Ⅰ to 100 mL of the phosphate solution, adjust the pH to 7 with sodium bicarbonate, then stir and react at 50℃ for 3 h, add four times the amount of anhydrous ethanol after the reaction is completed, stand still at 4℃ for 48 h, and centrifugal at 4000r·min -1 for 10 min, re-dissolve the precipitate with water, place it in a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis for 48 h, and freeze-dry at 13 kPa and -40℃ for 24 h to obtain the phosphorylated acanthopanax senticosus polysaccharide PASPS1-Ⅰ.
[0046] Determine the polysaccharide content in acanthopanax senticosus by the phenol-sulfuric acid method, and calculate the polysaccharide extraction rate; the ASPS extraction rate is 6.35%, and the sugar content is 64.17%. The sugar content of PASPS-Ⅰ is 49.68%.
[0047] Determine the phosphate content in the phosphorylated acanthopanax senticosus polysaccharide by the molybdenum blue colorimetric method; the phosphate content of PASPS-Ⅰ is 5.32%.
[0048] Example 3
[0049] A preparation method of an acanthopanax senticosus polysaccharide, comprising the following steps:
[0050] Take 1 kg of dry root of acanthopanax senticosus, and prepare 1:10 g.mL -1 of solution by adding 70% ethanol solution, soak for 12 h, filter, dry ethanol, take the residue, and dry in the shade. Under the ultrasonic power of 60 W, prepare 1:20 g.mL -1 of solution by adding deionized water, and extract for 60 min at 80°C. After the solution is cooled, centrifuge at 4000 r.min -1 for 10 min, and take the supernatant. Add 12% cellulase to the supernatant to make the concentration reach 1500 U.mL -1 , and carry out enzymolysis at 37°C for 60 min, and then carry out enzyme inactivation at 95°C for 10 min. Centrifuge at 4000 r.min -1 for 10 min to obtain an enzymolysis solution. Add anhydrous ethanol to the enzymolysis solution to make the ethanol concentration reach 60%, stir, and stand still at 4°C for 48 h. Centrifuge at 4000 r.min -1 for 10 min, re-dissolve the precipitate in water, concentrate, and freeze-dry at a pressure of 13 kPa and a temperature of -40°C for 24 h to obtain acanthopanax senticosus crude polysaccharide ASPS2.
[0051] Further separate the ASPS2 solution by DEAE-650M cellulose chromatography column: prepare 1 mg.mL -1 of ASPS solution, and centrifuge at 4000 r.min -1 for 10 min. After centrifugation, take the supernatant, and use distilled water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol.L -1 NaCl for elution, respectively. The flow rate is 1 mL.min -1 . Collect the eluate, detect the absorbance by the phenol-sulfuric acid method, collect the single-peak elution component, concentrate, select a dialysis bag with a molecular weight cut-off of 3500 Da for dialysis desalination, and freeze-dry at a pressure of 13 kPa and a temperature of -40°C for 24 h to obtain acanthopanax senticosus polysaccharide ASPS2-I.
[0052] Prepare a phosphate solution with a ratio of sodium tripolyphosphate to sodium trimetaphosphate of 5:2, add 100 mg of ASPS2-I to 100 mL of the phosphate solution, adjust the pH to 9 by sodium bicarbonate, then stir and react at 70°C for 5 h. After the reaction is completed, add four times the amount of anhydrous ethanol, stand still at 4°C for 48 h, and centrifuge at 4000 r.min -1 for 10 min. Re-dissolve the precipitate in water, place it in a dialysis bag with a molecular weight of 3500 Da, dialyze for 48 h, and freeze-dry at a pressure of 13 kPa and a temperature of -40°C for 24 h to obtain phosphorylated acanthopanax senticosus polysaccharide PASPS2-I.
[0053] The polysaccharide content in Acanthopanax senticosus was determined by phenol-sulfuric acid method, and the polysaccharide extraction rate was calculated. The extraction rate of ASPS was 7.84%, and the sugar content was 69.56%. The sugar content of PASPS-I was 54.39%.
[0054] The phosphate content in phosphorylated Acanthopanax senticosus polysaccharide was determined by molybdenum blue colorimetry. The phosphate content of PASPS-I was 5.88%.
[0055] Comparative Example 1
[0056] Phosphorylated Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that cellulase was not added during the extraction process. The other steps were the same as those of Example 1 to prepare Acanthopanax senticosus polysaccharide (ASPS-A) and phosphorylated Acanthopanax senticosus polysaccharide (PASPS-I-A).
[0057] Using the method of Comparative Example 1, the extraction rate of ASPS-A was 5.74%, and the sugar content was 51.63%. The phosphate content of PASPS-I-A was 5.35%. The plant cell wall is mainly composed of polysaccharides such as cellulose, hemicellulose, and pectin. These polysaccharides can be effectively degraded and released by the action of cellulase. Cellulase can hydrolyze cellulose chains, thereby loosening the structure of the cell wall. After the cell wall is treated with cellulase, the contact surface area of polysaccharides with the extraction solvent increases, and the solubility significantly enhances. Therefore, cellulase helps to improve the solubility and extraction rate of polysaccharides.
[0058] Comparative Example 2
[0059] Phosphorylated Acanthopanax senticosus polysaccharide was prepared according to the method of Example 1, except that traditional water extraction and microwave extraction were used to prepare Acanthopanax senticosus polysaccharide (ASPS), respectively. The other steps were the same as those of Example 1 to prepare Acanthopanax senticosus polysaccharide (ASPS-B and ASPS-C) and phosphorylated Acanthopanax senticosus polysaccharide (PASPS-I-B and PASPS-I-C).
[0060] Water extraction: 1 kg of dried Acanthopanax senticosus root was taken, and 70% ethanol solution was added at a solid-liquid ratio of 1:8 g·mL -1 The mixture was soaked for 12 h, filtered, and the ethanol was evaporated. The residue was dried in the shade. Deionized water was added at a solid-liquid ratio of 1:15 g·mL -1 The mixture was extracted at 100°C for 2 h. After three extractions, the filtrate was combined and concentrated. Anhydrous ethanol was added to a concentration of 60% ethanol. The mixture was stirred, and then placed at 4°C for 48 h. The mixture was centrifuged at 4000 r·min -1 for 10 min. The precipitate was redissolved in water, concentrated, and freeze-dried at a pressure of 13 kPa and a temperature of -40°C for 24 h to obtain Acanthopanax senticosus polysaccharide (ASPS).
[0061] Microwave extraction: 1 kg of dried Acanthopanax senticosus root was taken, and 70% ethanol solution was added at a solid-liquid ratio of 1:8 g·mL-1 The solid-liquid ratio is 1:15 g·mL, and the microwave extraction is carried out for 5 min at a microwave power of 230 W. After the solution is cooled, 4000 r·min centrifugal separation is carried out for 10 min, and the supernatant is taken. Anhydrous ethanol is added to the supernatant to obtain an ethanol concentration of 60%, and the solution is stirred uniformly. After the solution is placed at 4 DEG C for 48 h, 4000 r·min centrifugal separation is carried out for 10 min. The precipitate is redissolved in water, concentrated, and freeze-dried at a pressure of 13 kPa and a temperature of -40 DEG C for 24 h to obtain the crude polysaccharide ASPS of Acanthopanax senticosus. -1 -1 -1
[0062] By the method of the Comparative Example 2, the extraction rate of ASPS-B is 5.34%, the sugar content is 48.36%, the extraction rate of ASPS-C is 5.58%, the sugar content is 50.34%, and the phosphate contents of PASPS-I-B and PASPS-I-C are 4.27% and 5.13%, respectively. It can be seen that the extraction rate and the sugar content of the polysaccharide of Acanthopanax senticosus prepared by the ultrasonic-assisted enzyme method are obviously higher than those prepared by the traditional water extraction and the microwave extraction. The ultrasonic wave can cause cavitation, diffusion of solvent molecules and turbulence of liquid interface in the liquid, so as to enhance the dissolution and mass transfer efficiency of the polysaccharide in the solvent and significantly improve the extraction rate of the polysaccharide.
[0063] In order to further illustrate the application of the phosphorylated polysaccharide of Acanthopanax senticosus prepared by the application in CL4176 nematodes, the following experiments are carried out, and the specific content is as follows.
[0064] The CL4176 type nematodes are placed in a 16 DEG C constant temperature incubator for culture. The ASPS-I and PASPS-I in the Example 1 are dissolved into 1.50 mg·mL mother liquor of ultrapure water, and after filtration by a sterile filter head, an Escherichia coli OP50 solution with an OD value of 0.5 is added to prepare a solution with a mass concentration of 0.50 mg·mL, 1.00 mg·mL, 1.50 mg·mL, which are recorded as low, medium and high dose groups, respectively. 100 μL of each solution is taken and placed in 10 mL of NGM solid culture medium for standby. -1 570 -1 -1 -1
[0065] Experiment 1
[0066] Life span experiment: blank group (Con) and low, medium and high dose groups of ASPS-I and PASPS-I prepared by Example 1 were set. The nematodes synchronized to L3 stage were cultured in the medium, 30 nematodes in each medium, at 16℃. In order to avoid the influence of egg and larva growth on counting, 5-fluorouracil (50 μM) was added to NGM to inhibit nematode egg laying. The surviving nematodes were picked to new corresponding NGM medium every two days until all nematodes died, and at the same time, the number of nematodes in the medium was counted every day to screen the appropriate concentration. The results showed that compared with the Con group, ASPS-I and PASPS-I both had the effect of prolonging the life span of CL4176 nematodes (P<0.05), and PASPS-I had more obvious effect on prolonging the life span of CL4176 nematodes, among which ASPS-I and PASPS-I (1.00 mg·mL -1 ) had the best effect, and the subsequent experiment used 1.00 mg·mL -1 of the drug concentration, and the results are shown in Table 1, Figure 2 .
[0067] Table 1
[0068]
[0069] Experiment 2
[0070] Paralysis experiment: blank group (Con) and medium dose groups (1.00 mg·mL -1 ) of ASPS-I and PASPS-I prepared by Example 1 were set. The nematodes synchronized to L3 stage were cultured in the medium, 30 nematodes in each medium, at 16℃ for 36h, and then the temperature was raised to 25℃ for further culture for 28h, and the paralysis of nematodes was observed under a microscope every 2h. When the body or head of the nematode was touched, the nematode was considered paralyzed if it had no response or only the head wiggled but the body could not move. The results showed that compared with the Con group, ASPS-I and PASPS-I could improve the paralysis of CL4176 nematodes (P<0.05), and PASPS-I was better than ASPS-I in improving the paralysis of CL4176 nematodes, and the results are shown in Figure 3 .
[0071] Experiment 3
[0072] Motor ability determination experiment: blank group (Con) and medium dose groups (1.00 mg·mL -1Ten nematodes synchronized to the L3 stage were cultured in NGM medium at 16°C, with the medium changed every 2 days. After 4 and 8 days of culture, the nematodes were transferred to blank NGM medium, and 100 μL of M9 buffer solution was added. The number of sinusoidal movements performed by the nematodes within 30 seconds was measured under a microscope to determine the motility of CL4176 nematodes. Results showed that compared with the Con group, both ASPS-I and PASPS-I improved the motility of CL4176 nematodes (P<0.05), with PASPS-I showing a stronger improvement. (See attached table). Figure 4 .
[0073] Experiment 4
[0074] Oxidative stress experiment: A blank group (Con) and a medium-dose group (1.00 mg / mL) of ASPS-I and PASPS-I prepared in Example 1 were set up. -1 Nematodes synchronized to the L3 stage were cultured in medium, with 20 nematodes per medium, at 16℃ for 2 h. They were then transferred to a medium containing 0.1% juglone, and nematode survival was assessed every 1 h. Results showed that compared to the Con group, both ASPS-I and PASPS-I improved the antioxidant stress resistance of CL4176 nematodes (P<0.05), with PASPS-I showing a stronger effect than ASPS-I. (See attached table). Figure 5 .
[0075] Experiment 5
[0076] Heat stress experiment: A blank group (Con) and a medium-dose group (1.00 mg / mL) of ASPS-I and PASPS-I prepared in Example 1 were set up. -1 Nematodes synchronized to the L3 stage were cultured in medium, with 20 nematodes per medium, and cultured at 16℃ for 3 days. They were then transferred to 37℃, and nematode survival was monitored hourly until all nematodes died. Results showed that compared with the Con group, both ASPS-Ⅰ and PASPS-Ⅰ improved the heat stress resistance of CL4176 nematodes (P<0.05), and PASPS-Ⅰ improved the heat stress resistance of CL4176 nematodes more strongly than ASPS-Ⅰ. (See attached table). Figure 6 .
[0077] In summary, the experimental results show that the phosphorylated Acanthopanax senticosus polysaccharide prepared in this invention has a high degree of substitution, and different concentrations of phosphorylated Acanthopanax senticosus polysaccharide can all prolong the lifespan of CL4176 nematodes to a certain extent, reduce the paralysis rate of CL4176 nematodes, improve the motility of CL4176 nematodes, and enhance their stress resistance.
[0078] The foregoing description of the disclosed embodiments enables a person skilled in the art to make or use the application. Modifications of these embodiments will occur to persons of skill in the art, and that the appended claims are intended to cover all such modifications that do not depart from the true spirit and scope of the application. Therefore, the application is not limited to the embodiments shown but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for preparing phosphorylated acanthopanax senticosus polysaccharides, characterized by, The method comprises the following steps: (1) adding ethanol solution to the acanthopanax medicine to soak and remove fat, filtering, and airing to dry to obtain defatted acanthopanax; adding deionized water to the defatted acanthopanax to ultrasonically extract, and centrifuging the solution after cooling to obtain supernatant; The feed liquid ratio of the defatted acanthopanax senticosus and deionized water is 1: (10-20) g·mL -1 , the ultrasonic power is 40-60 W, the ultrasonic temperature is 40-80 ℃, and the ultrasonic time is 40-60 min. (2) adding the supernatant obtained in step (1) to cellulase to perform enzymolysis, and then performing enzyme inactivation, and centrifuging to obtain an enzymolysis solution; (3) adding anhydrous ethanol to the enzymatic hydrolysate obtained in step (2) to perform alcohol precipitation, centrifuging to obtain a precipitate, redissolving, concentrating and freeze-drying to obtain a crude acanthopanax senticosus polysaccharide ASPS; the ASPS is separated and purified through a DEAE-650M cellulose chromatographic column to obtain acanthopanax senticosus polysaccharide ASPS-I: distilled water, 0.1, 0.2, 0.3, 0.4 and 0.5 mol·L -1 NaCl are used for elution at a flow rate of 1 mL·min -1 The eluate is collected, and the absorbance is detected by a tube-separation phenol-sulfuric acid method; single-peak elution components are collected and combined. (4) adding a phosphate solution to the ASPS-I obtained in step (3) to adjust the pH of the solution, and starting a phosphorylation reaction; after the reaction is completed, adding anhydrous ethanol to perform alcohol precipitation, centrifuging, redissolving the precipitate, dialyzing, and freeze-drying to obtain phosphorylated acanthopanax polysaccharide PASPS-I; The ratio of sodium tripolyphosphate to sodium trimetaphosphate in the phosphate solution is 4:3, 5:2, or 6:1; the pH of the solution is adjusted to 7-9; the phosphorylation reaction temperature is 50-70 DEG C, and the phosphorylation reaction time is 3-5 h.
2. The method for preparing phosphorylated polysaccharides of acanthopanax according to claim 1, characterized in that, The volume ratio of the acanthopanax senticosus and the ethanol solution in step (1) is 1: (6-10) g·mL -1 ; the concentration of the ethanol solution is 70%.
3. The method of claim 1, wherein the phosphatidylated acanthopanax senticosus polysaccharide is prepared by the steps of: The cellulase is added in an amount of 8-12% to make the concentration reach 1500 U·mL -1 The enzymolysis condition is 37℃ for 60 min, and then the temperature is raised to 95℃ for enzyme inactivation. 4. The method for preparing phosphorylated Acanthopanax senticosus polysaccharide according to claim 1, characterized in that, The enzymatic hydrolysate of step (3) was added with anhydrous ethanol to a concentration of 60% ethanol; the temperature of alcohol precipitation was 4°C, and the time was 48 h; the centrifugation condition was 4000 r·min -1 , and the centrifugation time was 10 min.
5. The method for preparing phosphorylated Acanthopanax senticosus polysaccharide according to claim 1, characterized in that, The alcohol precipitation temperature in step (4) is 4℃, and the time is 48h; the centrifugation condition is 4000r·min -1 , the centrifugation time is 10min; the dialysis bag molecular weight is 3500Da; the freeze-drying pressure is 13kPa, and the freeze-drying temperature is -40℃ for 24h.
6. The phosphorylated acanthopanax polysaccharide prepared by the method according to any one of claims 1-5.
7. The use of the phosphorylated acanthopanax polysaccharide according to claim 6 in the preparation of a drug for prolonging the lifespan of nematodes, reducing the paralysis rate of nematodes, improving the movement ability of nematodes, and / or improving the stress resistance of nematodes.