A poly-saccharide degraded from amomum tsao-ko with neuroprotective activity and a preparation method thereof

Abstract

The application discloses a kind of Amomum tsao-ko degradation polysaccharides with neuroprotective activity and a preparation method thereof, and specifically relates to the technical field of Amomum tsao-ko polysaccharide degradation.The preparation method uses a degrading agent H2O2-Vc to be mixed with 1-5 mg / mL Amomum tsao-ko polysaccharide solution until the final concentration of H2O2-Vc is 12-18 mmol / L, then ultrasonic power is 170-240 W, ultrasonic temperature is 40-60°C, and reaction time is 30-90 min.After the reaction is completed, Amomum tsao-ko polysaccharide degradation solution is obtained, and Amomum tsao-ko degradation polysaccharides are prepared through dialysis, purification, drying and grinding processes.The application uses ultrasonic wave assisted combination H2O2-Vc free radical degradation technology to degrade Amomum tsao-ko polysaccharides.This method not only effectively reduces the molecular weight of Amomum tsao-ko polysaccharides, but also successfully retains the original monosaccharide type and biological activity of Amomum tsao-ko polysaccharides, and experiments prove that it also has significant neuroprotective effect, and can be developed into a drug with neuroprotective effect as an active ingredient.

Description

Technical Field

[0001] The present invention relates to the technical field of polysaccharide degradation, and in particular to a degradable amomum villosum polysaccharide with neuroprotective activity and a preparation method thereof. Background Art

[0002] The Chinese medicinal herb Amomum tsao-ko Crevostet Lemarie is one of the nationally approved medicinal and edible substances. It is derived from the Amomum tsao-ko plant of the Zingiberaceae family. The medicinal part is the dried mature fruit. It is a variety included in the various editions of the Chinese Pharmacopoeia and has the traditional effects of drying dampness, warming the middle, stopping malaria and removing phlegm. Studies have shown that polysaccharides are one of the main functionally active components in Amomum tsao-ko. Its research has mainly focused on content determination, extraction and purification, and anti-complement activity. However, the presence of larger molecular weight fragments in Amomum tsao-ko polysaccharides seriously affects their bioavailability, limiting the development of the biological properties of Amomum tsao-ko polysaccharides and their applications. Therefore, finding a method to effectively reduce the larger molecular weight fragments in Amomum tsao-ko polysaccharides is the basis for fully realizing their potential medical and health value and developing new products.

[0003] The degradation methods of polysaccharides mainly include physical degradation, chemical degradation, biodegradation and the coordinated use of multiple degradation methods. The chemical degradation method is relatively simple to operate, but it has the problem of complex separation and purification and greater environmental pollution. Although the physical degradation method can reduce environmental pollution, it has the problem of long degradation time. The biodegradation method has mild reaction conditions, but the production cost is high, the reaction conditions must be strict, and the screening of enzymes and microorganisms is difficult. The coordinated use of multiple degradation methods can make up for the shortcomings of a single degradation method, and at the same time can improve the degradation efficiency of natural polysaccharides and avoid excessive degradation of polysaccharides. Ultrasonic-assisted combined H2O2-Vc method has significant advantages as an efficient, rapid, green, environmentally friendly, low-cost and controllable polysaccharide degradation strategy. However, there are still no relevant research reports on the degradation of Amomum villosum polysaccharides using ultrasonic-assisted combined H2O2-Vc method.

[0004] Ischemic stroke (IS) is a neurological disorder characterized by brain tissue necrosis due to insufficient blood and oxygen supply to the brain. It accounts for over 80% of all strokes and is a serious health hazard with a complex pathogenesis. Ischemia / reperfusion (I / R) injury is a key component of the pathological process of ischemic cerebrovascular disease. Studies have shown that cerebral ischemia-reperfusion can trigger neuronal apoptosis, inflammatory responses, and oxidative stress, which in turn affect neurological function. However, current drugs for the treatment and prevention of I / R injury remain insufficient to meet clinical needs.

[0005] In recent years, studies have found that polysaccharide components derived from traditional Chinese medicine can exert significant neuroprotective effects on ischemic brain injury by regulating oxidative stress, inhibiting cell apoptosis and alleviating neuroinflammatory pathways, showing good development potential.

[0006] In summary, developing an efficient, green and controllable degradation technology for the preparation of degradable amomum villosum polysaccharides with neuroprotective effects is of great significance for expanding the medicinal value of amomum villosum polysaccharides and developing new neuroprotective drugs or functional health products. Summary of the Invention

[0007] The present invention utilizes ultrasound-assisted H2O2-Vc combined method to degrade Amomum villosum polysaccharide, and optimizes the degradation conditions using response surface methodology with degradation rate as an indicator, thereby solving the problem that the existing Amomum villosum polysaccharide has a large molecular weight, which limits its application in neuroprotection efficiency.

[0008] To achieve the above object, the present invention provides a method for preparing degradable polysaccharides from Amomum villosum, comprising the following steps:

[0009] Step S1: adding a degradation agent H2O2-Vc to a 1-5 mg / mL amomum tsaoko polysaccharide solution to a final H2O2-Vc concentration of 12-18 mmol / L, sealing and mixing thoroughly to obtain a mixed solution of H2O2-Vc and amomum tsaoko polysaccharide;

[0010] Step S2: placing the mixed solution obtained in step S1 in a heated ultrasonic extractor at an ultrasonic power of 170-240W and an ultrasonic temperature of 40-60°C for 30-90 minutes for degradation. After the reaction is completed, a degradation solution of amomum villosum polysaccharide is obtained;

[0011] Step S3: The degradation solution of the amomum villosum polysaccharide obtained in step S2 is dialyzed, dried, and then ground into powder to obtain degradation polysaccharide of the amomum villosum.

[0012] Step S4: The degraded amomum villosum polysaccharide obtained in step S3 is dissolved in purified water and loaded onto a Sephadex G-25 chromatography column at a loading concentration of 20-30 mg / mL and a loading volume of 0.02-0.04 BV for separation and purification.

[0013] Preferably, the preparation of the amomum tsaoko polysaccharide solution comprises: placing amomum tsaoko polysaccharide powder in a reaction kettle, adding distilled water, and ultrasonicating for 10 to 15 minutes to remove obvious particles and insoluble matter.

[0014] Preferably, the method for preparing the degradation agent H2O2 solution used in step S1 includes: taking a certain concentration of H2O2 solution into a volumetric flask, adding distilled water, and mixing to a fixed volume to obtain a H2O2 solution with a concentration of 800-1000 mmol / L.

[0015] Preferably, the method for preparing the degradation agent Vc solution used in step S1 includes: taking Vc powder and dissolving it in a volumetric flask, adding distilled water, and mixing it to a fixed volume to prepare a Vc solution with a concentration of 800-1000 mmol / L.

[0016] Preferably, the dialysis in step S3 is as follows: the degraded amomum villosum polysaccharide solution in step S2 is transferred into a dialysis bag with a molecular weight cutoff of 1000Da, dialyzed with running water for 24 hours to remove residual H2O2 and VC, the solution in the dialysis bag is dried in a water bath, and ground into powder to obtain the degraded amomum villosum polysaccharide.

[0017] Preferably, the drying temperature in step S3 is 50-55°C.

[0018] Preferably, the separation and purification in step S4 is as follows: take the degraded amomum villosum polysaccharide in step S3, add purified water to dissolve it, and load it onto a Sephadex G-25 chromatography column with a loading concentration of 20~30 mg / mL and a loading volume of 0.02~0.04BV. Pure water is used as the elution solvent, the flow rate is 0.25mL / min, 2mL is collected in each tube, the elution is constant, and the elution process is monitored by the phenol-sulfuric acid method. The eluate is collected according to the color reaction, and the purified degraded amomum villosum polysaccharide (D-ATP) is obtained by concentration and freeze-drying.

[0019] The degraded polysaccharide of Amomum villosum prepared by the above-mentioned preparation method has a content of 92.51±2.49%, i.e., a purity of >90%; the molecular weight of the degraded polysaccharide of Amomum villosum is 1.16×10 5 Da, with a distribution index of 1.15; the degradable polysaccharides from Amomum villosum are composed of eight monosaccharides, including mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 2.4:1.0:1.6:2.0:74.9:8.4:1.3:5.6; the degradable polysaccharides from Amomum villosum can significantly improve the survival rate of PC12 cells after oxygen glucose deprivation and reperfusion (OGD / R) treatment in the concentration range of 12.5-100µg / mL.

[0020] The present invention discloses a degradable amomum villosum polysaccharide with neuroprotective activity and a preparation method thereof. The process conditions of the optimal degradation agent H2O2-Vc concentration, ultrasound time, ultrasound power and ultrasound temperature are optimized. The preparation method has a degradation rate of about 60%, and the larger molecular weight fragments in the obtained amomum villosum degradable polysaccharide are significantly reduced, and the polysaccharide has a good neuroprotective effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the single-factor experiment for optimizing the degradation process of Amomum villosum polysaccharide are shown in Figure 1; (a) is the degradation rate at different H2O2-Vc concentrations, (b) is the degradation rate at different ultrasonic times, (c) is the degradation rate at different ultrasonic temperatures, and (d) is the degradation rate at different ultrasonic powers.

[0022] Figure 2 It is a response surface high-line diagram for the optimization of the degradation process of amomum villosum polysaccharide; wherein, (a) is the effect of the degradation agent H2O2-Vc concentration X1 and the ultrasonic time X2 on the degradation rate of amomum villosum polysaccharide, (b) is the effect of the degradation agent H2O2-Vc concentration X1 and the ultrasonic temperature X3 on the degradation rate of amomum villosum polysaccharide, (c) is the effect of the degradation agent H2O2-Vc concentration X1 and the ultrasonic power X4 on the degradation rate of amomum villosum polysaccharide, (d) is the effect of the ultrasonic time X2 and the ultrasonic temperature X3 on the degradation rate of amomum villosum polysaccharide, (e) is the effect of the ultrasonic time X2 and the ultrasonic power X4 on the degradation rate of amomum villosum polysaccharide, and (f) is the effect of the ultrasonic temperature X3 and the ultrasonic power X4 on the degradation rate of amomum villosum polysaccharide;

[0023] Figure 3 It is a three-dimensional response surface diagram for the optimization of the degradation process of amomum villosum polysaccharide; among them, (a) is the effect of the degradation agent H2O2-Vc concentration X1 and ultrasonic time X2 on the degradation rate of amomum villosum polysaccharide, (b) is the effect of the degradation agent H2O2-Vc concentration X1 and ultrasonic temperature X3 on the degradation rate of amomum villosum polysaccharide, (c) is the effect of the degradation agent H2O2-Vc concentration X1 and ultrasonic power X4 on the degradation rate of amomum villosum polysaccharide, (d) is the effect of ultrasonic time X2 and ultrasonic temperature X3 on the degradation rate of amomum villosum polysaccharide, (e) is the effect of ultrasonic time X2 and ultrasonic power X4 on the degradation rate of amomum villosum polysaccharide, and (f) is the effect of ultrasonic temperature X3 and ultrasonic power X4 on the degradation rate of amomum villosum polysaccharide;

[0024] Figure 4 This is the separation and elution curve of the degraded polysaccharide of Amomum villosum on Sephadex G-25 column;

[0025] Figure 5 The high performance gel permeation chromatograms of tsaoko polysaccharide (a) and tsaoko degraded polysaccharide (b);

[0026] Figure 6 Liquid chromatograms of monosaccharide composition of standard monosaccharide (a), Amomum villosum polysaccharide (b) and Amomum villosum degraded polysaccharide (c);

[0027] Figure 7 The cytotoxicity test of Amomum villosum polysaccharide (a) and Amomum villosum degraded polysaccharide (b) on PC12 cells;

[0028] Figure 8 The protective effects of Amomum villosum polysaccharide (a) and Amomum villosum degraded polysaccharide (b) on PC12 cells damaged by OGD / R; Ctrl represents the blank control group; OGD / R represents the model group; ### Indicates significant difference compared with the Ctrl group; ns indicates no significant difference compared with the model (P>0.05); * indicates moderately significant difference compared with the model (P<0.05); ** indicates significant difference compared with the model (P<0.01); *** indicates extremely significant difference compared with the model (P<0.001); DETAILED DESCRIPTION

[0029] The technical solutions of the present invention are described systematically and accurately in conjunction with the embodiments and accompanying drawings of the present invention. It should be noted that the described embodiments are only part of the invention. In-depth description is given in conjunction with specific embodiments, aiming to thoroughly demonstrate the purpose, technical solutions and advantages of the present invention. These embodiments are used to analyze the principles rather than limit the scope. Unless otherwise specified in the experiment, the experimental methods adopted are conventional methods, and experimental equipment, materials, and reagents can be purchased from conventional commercial stores in this field. When the data is statistically analyzed, the experiment is repeated three times to ensure reliability, and the results are presented as mean ± standard deviation. The DesignExpert software is then used to perform variance analysis to explore the rules, and finally the processed data is plotted into charts using the Origin2021 software to intuitively present complex information and make the technical content clear and easy to understand.

[0030] The tsaoko used in the examples is the dried mature fruit of the tsaoko (Amomumtsao-ko Crevostet Lemarie) of the Zingiberaceae family, purchased from the Bozhou Traditional Chinese Medicine Wholesale Market in Anhui Province.

[0031] Example 1;

[0032] Preparation of degradable polysaccharides from Amomum villosum and its process optimization;

[0033] Preparation of Amomum villosum polysaccharide;

[0034] The dried tsaoko was crushed and passed through a 10-mesh sieve. Ultrapure water was added at a ratio of 1:30 (m / v), and refluxed at 85°C twice. The tsaoko polysaccharide extract was filtered to obtain the tsaoko polysaccharide extract. The aqueous extract was concentrated to 1 / 5 of the original volume, and 98% ethanol was added to a final ethanol concentration of 85%. After standing for 24 hours, the extract was centrifuged, the precipitate was collected, evaporated to dryness in a water bath, and ground into powder to obtain the tsaoko polysaccharide (ATP).

[0035] Single-factor experimental design;

[0036] A single-factor experiment was conducted with degradation rate as an indicator. The effects of different H2O2-Vc concentrations, ultrasonic time, ultrasonic temperature, and ultrasonic power on the degradation rate of Amomum villosum polysaccharide were studied. The method for determining the degradation rate is as follows. 50 mL of 5 mg / mL polysaccharide solution was taken and degraded under different conditions to obtain polysaccharide degradation solutions under different conditions. 1 mL of polysaccharide degradation solution under different conditions was taken, added with 4 mL of ultrapure water, mixed thoroughly, and 1 mL of 3,5-dinitrosalicylic acid (DNS) reagent was added and shaken, then placed in a boiling water bath for 5 minutes. After cooling to room temperature, 4 mL of ultrapure water was added, and after mixing, 200 μL was taken to measure the absorbance of the solution at a wavelength of 540 nm to calculate the reducing sugar content of the Amomum villosum polysaccharide degradation product. Take 1 mL of 5 mg / mL polysaccharide solution, add 19 mL of ultrapure water, and mix thoroughly to obtain the polysaccharide solution dilution. Take 100 μL of the polysaccharide solution dilution, add 200 μL of phenol solution (5%) and mix thoroughly. Then add 700 μL of concentrated sulfuric acid and react in a 37°C water bath for 1 hour. Measure the absorbance of the solution at a wavelength of 490 nm to calculate the total sugar content of the Amomum villosum polysaccharide. The degradation rate is calculated according to formula (1):

[0037] Degradation rate (%) = (R2-R2-R0) / T × 100%;

[0038] Wherein R0 is the reducing sugar content in H2O2-Vc solution; R1 is the reducing sugar content in Amomum villosum polysaccharide; R2 is the reducing sugar content in degradation product; T is the total sugar content of Amomum villosum polysaccharide.

[0039] Effect of H2O2-Vc concentration: Take 5 mg / mL polysaccharide solution, and investigate the effect of different H2O2-Vc concentrations (9, 12, 15, 18, 21, 24 mmol / L) on the degradation efficiency of Amomum villosum polysaccharide under the conditions of ultrasonic time 60 min, ultrasonic temperature drop 50℃, and ultrasonic power 240 W.

[0040] Effect of ultrasonic time: Take 5 mg / mL polysaccharide solution, and investigate the effect of different ultrasonic times (30, 60, 90, 120, 150 min) on the degradation efficiency of Amomum villosum polysaccharide under the conditions of H2O2-Vc concentration of 15 mmol / L, degradation temperature of 50°C, and ultrasonic power of 240 W.

[0041] Effect of ultrasonic temperature: Take 5 mg / mL polysaccharide solution, and investigate the effect of different ultrasonic temperatures (40, 50, 60, 70, 80℃) on the degradation efficiency of Amomum villosum polysaccharide under the conditions of H2O2-Vc concentration of 15 mmol / L, ultrasonic time of 60 min, and ultrasonic power of 240 W.

[0042] Effect of ultrasonic power: Take 5 mg / mL polysaccharide solution, and investigate the effect of different ultrasonic powers (30, 100, 170, 240, 310 W) on the degradation rate under the conditions of H2O2-Vc concentration of 15 mmol / L, ultrasonic time of 60 min, and degradation temperature of 50°C.

[0043] The degradation rate was calculated using the method of Example 1 to determine the optimal degradation conditions under the single-factor experiment.

[0044] The results of single factor experiment on degradation of Amomum villosum polysaccharide are as follows Figure 1 As shown in the results, the optimal degradation conditions of Amomum villosum polysaccharide under single factor experiment were H2O2-Vc concentration of 15 mmol / L, ultrasonic time of 60 min, ultrasonic temperature of 50℃ and ultrasonic power of 240 W.

[0045] 1.3 Response surface experimental design;

[0046] Based on a single-factor experiment and the Box-Behnken experimental design principle, 29 response surface experiments were designed to optimize the degradation process of polysaccharides from Amomum villosum. The factors and levels are shown in Table 1. The independent variables were H2O2-Vc concentration (X1), ultrasound time (X2), ultrasound temperature (X3), and ultrasound power (X4), with the polysaccharide degradation rate as the response value Y.

[0047] Table 1 Factors and levels of response surface experimental design;

[0048]

[0049] Model building and analysis of variance;

[0050] The results of the response surface experimental design are shown in Table 2. Using Design-Expert13 software, a quadratic polynomial regression analysis was performed on the data in Table 2 to obtain a quadratic polynomial regression equation. The quadratic multivariate regression equation for the degradation rate (Y) and H2O2-Vc concentration (X1), ultrasonic time (X2), ultrasonic temperature (X3), and ultrasonic power (X4) is:

[0051] ;

[0052] The variance analysis results of the response surface quadratic model for the optimization of the degradation process of tsaoko polysaccharide are shown in Table 3. The P value of the regression model (Model) is less than 0.0001, indicating that the model is extremely significant. The P value of the lack of fit term (Lack of Fit) is 0.8007, which is greater than 0.05 and is not significant, indicating that the lack of fit is not related to the pure error, which fully proves the reliability of the model. In addition, the coefficient of determination (R 2 ) is 0.9368, and the adjusted coefficient of determination (Adj-R 2 ) is 0.8736, which is consistent with R 2The results are close, indicating that the model has high accuracy and can be used to predict the response value. The coefficient of variation (CV%) is 2.77, indicating that the regression model is feasible within the variable range and has high experimental reliability and precision. According to the F value, it can be seen that the ultrasonic power has the greatest effect on polysaccharide degradation, and the ultrasonic time has the least effect. X1X4, X3 2 and X4 2 The effect on polysaccharide degradation was extremely significant (P<0.001); X1, X4 and X2X3 had a very significant effect on it (P<0.01); X2, X3, X3X4 and X1 2 The effect was significant (P<0.05); the other factors had no significant effect.

[0053] Response surface 3D plots and high-line plots can intuitively reflect the impact of various factors on polysaccharide degradation and the significance of the interactions between factors. A greater slope in the response surface 3D plot and a closer ellipse in the response surface high-line plot indicate a more significant interaction between the two factors on the degradation of amomum villosum polysaccharides. The experimental results showed that the interaction between H2O2-Vc concentration and ultrasonic power, ultrasonic time and ultrasonic temperature, and ultrasonic temperature and ultrasonic power significantly affected the degradation of amomum villosum polysaccharides.

[0054] Table 2 Response surface experimental design and results;

[0055]

[0056] Table 3 ANOVA results of the response surface quadratic model for the optimization of the degradation process of amomum villosum polysaccharide;

[0057]

[0058] Note: * indicates P < 0.05, significant effect; ** indicates P < 0.01, very significant effect; *** indicates P < 0.001, extremely significant effect.

[0059] Preparation of degradable polysaccharides from Amomum villosum;

[0060] According to the optimal degradation conditions obtained by single factor and response surface methodology, the amomum villosum polysaccharide was degraded under these conditions, and then the degraded amomum villosum polysaccharide was prepared by dialysis, water bath drying and grinding into powder.

[0061] Validation experiments;

[0062] The optimal values ​​of each variable were obtained using Design-Expert software. The theoretical optimal degradation process for amomum villosum polysaccharide was as follows: H2O2-Vc concentration of 15.82 mmol / L, ultrasonic time of 30.00 min, ultrasonic temperature of 51.23°C, and ultrasonic power of 246.05 W, with a predicted degradation rate of 59.64%. To account for actual operation, the conditions were modified to H2O2-Vc concentration of 16 mmol / L, ultrasonic time of 30 min, ultrasonic temperature of 51°C, and ultrasonic power of 240 W. The actual degradation rate of amomum villosum polysaccharide was measured to be 63.60 ± 2.02% (n = 3). The actual value was close to the theoretical prediction, indicating that the optimal degradation process conditions for amomum villosum polysaccharide obtained by this design model are stable and reliable.

[0063] Example 2;

[0064] Isolation and purification of degraded polysaccharides from Amomum villosum;

[0065] Take the degraded polysaccharide of Amomum villosum, add purified water to dissolve it again, and use Sephadex G-25 chromatography column for separation and purification. Load the sample into Sephadex G-25 chromatography column with a loading concentration of 20~30 mg / mL and a loading volume of 0.02~0.04 BV. Use purified water as the elution solvent, the flow rate is 0.25 mL / min, collect 2 mL in each tube, elute for about 800 minutes, keep the elution constant, and number them according to the collection order. According to the phenol-sulfuric acid color reaction, use the bottle number as the horizontal axis and the absorbance value as the vertical axis to draw the chart as shown in the attached figure. Figure 4 As shown in the elution curve, a single elution peak appeared during the separation process. All the eluates of the elution peak were collected, concentrated, and freeze-dried to obtain the purified amomum villosum degraded polysaccharide D-ATP.

[0066] Example 3;

[0067] Structural characterization of degraded polysaccharides from Amomum villosum;

[0068] Determination of total sugar content;

[0069] The total sugar content was determined by the phenol-sulfuric acid method: the content of degraded polysaccharides in Amomum villosum was 92.51±2.49%, that is, the purity was >90%.

[0070] Molecular weight determination;

[0071] High-performance gel permeation chromatography (HPGPC) was used to determine the molecular weights of polysaccharides and degraded polysaccharides from Amomum villosum. The following conditions were used: detector: differential refractive index detector (RID-20A); column: Shodex SUGARKS-804 (8.0 mm × 300 mm); column temperature: 40°C; mobile phase: ultrapure water; flow rate: 1.0 mL / min; injection volume: 50 μL. Dextran standards of varying molecular weights were prepared and analyzed by HPGPC. A standard curve (logMw = -0.5800348X + 8.862232) was constructed using retention time as the abscissa and logarithm of molecular weight as the ordinate. A 1 mg / mL polysaccharide solution was prepared and filtered through a 0.22 μm microporous filter before injection. Data were reanalyzed using LabSolutions software to calculate the molecular weights of the polysaccharides.

[0072] The high performance gel permeation chromatography of tsaoko polysaccharide and tsaoko degraded polysaccharide is shown in Figure 2. Figure 5 The results showed that the polysaccharide of Amomum villosum was composed of two molecular weight fragments, 5.85×10 5 Da and 1.69×10 5 Da, the degraded polysaccharide of Amomum villosum only consists of one molecular weight fragment of 1.16×10 5 Da, and the distribution index is 1.15. It can be seen that the ultrasound-assisted H2O2-Vc combined method for degrading tsaoko polysaccharides developed in the present invention can degrade high molecular weight fragments above 500,000 Da in tsaoko polysaccharides to below 120,000 Da, successfully preparing low molecular weight tsaoko degraded polysaccharides.

[0073] Monosaccharide composition determination;

[0074] Monosaccharide components were determined using a pre-column derivatization method with 1-phenyl-3-methyl-5-pyrazolone. The following conditions were used: a detector wavelength of 245 nm, a Supersil ODS2 column (5 μm, 4.6 mm × 250 mm), a column temperature of 35°C, a mobile phase of acetonitrile:0.2 mol / mL phosphate buffer (pH 6.8) (v / v) of 83:17, a flow rate of 0.8 mL / min, and an injection volume of 20 μL.

[0075] Accurately weigh 10 mg of degraded amomum villosum polysaccharide and mix with 2 mL of 2 mol / L trifluoroacetic acid. Hydrolyze in a sealed reaction chamber at 120°C for 6 h. Add 200 μL of 0.5 mol / L PMP methanol solution and 0.3 mol / L NaOH solution to the hydrolyzate, in turn, incubate in a 70°C water bath for 1 h, add 200 μL of 0.3 mol / L HCl, extract three times with chloroform, and collect the aqueous layer.

[0076] Mannose (Man), glucosamine (GlcN), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), glucose (Glc), galactose (Gal), xylose (Xyl), arabinose (Ara), and fucose (Fuc) were used as standard controls. The derivatization procedure was the same as above. The pretreated samples and standards were filtered through a 0.22 μm filter, injected, and analyzed according to the above conditions.

[0077] The test results showed that Figure 6 As shown, both the tsaoko polysaccharide and the degraded tsaoko polysaccharide are composed of eight monosaccharides: Man, Rha, GlcA, GalA, Glc, Gal, Xyl, and Ara, with molar ratios of 2.6:1.0:1.5:2.9:77.1:8.4:1.1:5.4 and 2.4:1.0:1.6:2.0:74.9:8.4:1.3:5.6, respectively, indicating that degradation does not alter the basic structure of the polysaccharide. After degradation, the contents of Man, GalA, and Glc decreased, while those of GlcA, Xyl, and Ara increased, suggesting that the ultrasound-assisted H2O2-Vc degradation of tsaoko polysaccharide may preferentially target glycosidic bonds near Man, GalA, and Glc, while glycosidic bonds near other monosaccharides are more stable.

[0078] Example 4;

[0079] Effects of Amomum villosum polysaccharides on PC12 cell viability;

[0080] cell culture;

[0081] PC12 cells were cultured in DMEM (DMEM) supplemented with 1% penicillin (100 mg / mL) and streptomycin (1%) and 10% fetal bovine serum (FBS) in a 5% CO2-spared, 37°C incubator, with the medium replaced every two days. When cells entered the logarithmic growth phase and reached 80%–90% confluency, they were digested with 0.25% trypsin and harvested by centrifugation at 1000 rpm for 5 minutes. The cells were then resuspended in DMEM supplemented with 10% FBS and plated onto culture dishes.

[0082] Effects of different concentrations of amomum villosum polysaccharides on PC12 cell viability;

[0083] The cytotoxicity and protective effects of polysaccharides on PC12 cells were determined by CCK8 assay. PC12 cells were plated at 1×10 4The PC12 cells were inoculated in the 96-well plate at a density of 1 x 104 cells per well, and different concentrations of polysaccharide solution were added to the PC12 cells for incubation for 24 h. 10 μL of CCK8 reagent was added to each well, and the cells were incubated in a 37°C incubator for 1-2 h. After continuous shaking for 10 min, the OD value was detected at 450 nm by an enzyme-labeled instrument to determine the PC12 cell viability. Subsequently, the construction of the PC12 cell oxygen-glucose deprivation / reperfusion (OGD / R) model was performed to simulate the reperfusion process after ischemia. The PC12 cells pretreated with Alpinia oxyphylla polysaccharide for 2 h were placed in a hypoxic environment (5% CO2, 1% O2, 94% N2) for 10 h of hypoxic and anoxic treatment. Then, the PC12 cells were subjected to reoxygenation and reperfusion treatment, and were continuously cultured under normal conditions for 24 h. The PC12 cell viability was determined according to the above steps to evaluate the effect of different concentrations of Alpinia oxyphylla degraded polysaccharide on the PC12 cell viability.

[0084] The results of the cytotoxicity experiment are shown in Figure 7 The survival rates of the PC12 cells treated with Alpinia oxyphylla polysaccharide (ATP) and Alpinia oxyphylla degraded polysaccharide (D-ATP) were greater than 85%, indicating that the Alpinia oxyphylla polysaccharide and the Alpinia oxyphylla degraded polysaccharide had no cytotoxicity to the PC12 cells within the set concentration range.

[0085] The CCK8 method was used to detect the effect of 12.5, 25, 50 and 100 μmol / mL Alpinia oxyphylla polysaccharide and Alpinia oxyphylla degraded polysaccharide on the viability of the OGD / R damaged PC12 cells. The results are shown in Figure 8 The results show that the survival rate of the PC12 cells after OGD / R treatment was significantly reduced, but the Alpinia oxyphylla degraded polysaccharide pretreatment at a concentration of 12.5 μg / mL to 100 μg / mL significantly increased the survival rate of the PC12 cells in a concentration-dependent manner, while the undegraded Alpinia oxyphylla polysaccharide only significantly increased the survival rate of the PC12 cells at a concentration of 50 μg / mL, indicating that the Alpinia oxyphylla degraded polysaccharide had a better neuroprotective effect on the OGD / R damaged PC12 cells than the undegraded Alpinia oxyphylla polysaccharide. Therefore, the neuroprotective effect of the Alpinia oxyphylla polysaccharide can be effectively improved by the degradation technology, and the Alpinia oxyphylla degraded polysaccharide with a strong neuroprotective effect can be prepared.

[0086] The technical solutions described in the above specific embodiments are only part of the protection scope of the present application. Any technical solution obtained by reasonable substitution, optimization or derivation without deviating from the core idea of the present application is within the protection scope of the present application.

Claims

1. A use of a degraded polysaccharide of Amomum villosum in the preparation of a drug for protecting brain nerve cell damage caused by oxygen-glucose deprivation / reperfusion, characterized in that: The preparation method of the degradable amomum tsaoko polysaccharide comprises the following steps: degrading the amomum tsaoko polysaccharide extracted from the amomum tsaoko by using an ultrasonic-assisted combined H2O2-Vc method to obtain the degradable amomum tsaoko polysaccharide; Step S1: adding a degradation agent H2O2-Vc to a 1-5 mg / mL amomum tsaoko polysaccharide solution to a final H2O2-Vc concentration of 12-18 mmol / L, sealing and mixing thoroughly to obtain a mixed solution of H2O2-Vc and amomum tsaoko polysaccharide; Step S2: placing the mixed solution obtained in step S1 in a heated ultrasonic extraction instrument with an ultrasonic power of 170-240W and an ultrasonic temperature of 40-60°C for 30-90 minutes for degradation. After the reaction is completed, a degradation solution of amomum villosum polysaccharide is obtained; Step S3: The degraded amomum tsaoko polysaccharide solution obtained in step S2 is dialyzed, dried in a water bath, and then ground into powder to obtain degraded amomum tsaoko polysaccharide; Step S4: The degraded amomum villosum polysaccharide obtained in step S3 was dissolved in purified water and loaded onto a Sephadex G-25 chromatography column at a loading concentration of 20-30 mg / mL and a loading volume of 0.02-0.04 BV for separation and purification.

2. The use according to claim 1, characterized in that The polysaccharide powder of Amomum villosum is placed in a reactor, and distilled water is added and ultrasonicated for 10 to 15 minutes to remove obvious particles and insoluble matter.

3. The use according to claim 1, characterized in that The preparation process of the degradation agent H2O2-Vc includes: taking a certain concentration of H2O2 solution in a volumetric flask, adding distilled water, and mixing to a constant volume to obtain an H2O2 solution with a concentration of 800~1000mmol / L; taking Vc powder and placing it in a volumetric flask, adding distilled water, and mixing to a constant volume to obtain a Vc solution with a concentration of 800~1000mmol / L.

4. The use according to claim 1, characterized in that: In step S3, the molecular weight cut-off for dialysis is 1000 Da, the dialysis time is 24 h, and the drying temperature is 50-55° C.

5. A degradable polysaccharide of Amomum villosum used in the application of claim 1, characterized in that: The total sugar content of the degraded polysaccharides of Amomum villosum was 92.51±2.49%, i.e. the purity was >90%; The molecular weight of the degraded polysaccharide from Amomum villosum is 1.16×10 5 Da, distribution index is 1.15; The degradable polysaccharides of Amomum villosum are composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, with a molar ratio of 2.4:1.0:1.6:2.0:74.9:8.4:1.3:5.6.

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