CN112778430A

CN112778430A - Sulfated modified cyclocarya paliurus polysaccharide and preparation method and application thereof

Info

Publication number: CN112778430A
Application number: CN202110033668.6A
Authority: CN
Inventors: 王文君; 韩艺; 欧阳克蕙; 李景恩; 陈凌利; 张清峰; 赵猛; 张阳
Original assignee: Jiangxi Agricultural University
Current assignee: Jiangxi Agricultural University
Priority date: 2021-01-12
Filing date: 2021-01-12
Publication date: 2021-05-11

Abstract

The invention provides sulfated and modified cyclocarya paliurus polysaccharide and a preparation method and application thereof, and belongs to the technical field of modified polysaccharide. According to the invention, sulfuric acid groups are introduced into cyclocarya paliurus polysaccharide, so that the water solubility of the polysaccharide is improved; the sulfation modification site of the sulfation modified cyclocarya paliurus polysaccharide is → 4) -alpha-D-Glcp- (1 → C6, and the sulfation modification does not damage the main chain of the polysaccharide, and can remove the side chain which is unstable under the acidic and high-temperature conditions, thereby improving the stability of the polysaccharide, further improving the bioactivity of the polysaccharide and reducing IC₅₀The application range of the polysaccharide can be increased and the application amount can be reduced. The invention finds the modification site effective for improving the antioxidant activity by combining the structural characteristics of sulfated and modified cyclocarya paliurus polysaccharide, provides theoretical basis and possibility for revealing the structure-activity relationship and effective positioning and modification of sulfation, and simultaneously provides theoretical basis for developing functional food and health care medicine taking cyclocarya paliurus as a raw material.

Description

Sulfated modified cyclocarya paliurus polysaccharide and preparation method and application thereof

Technical Field

The invention relates to the technical field of modified polysaccharides, in particular to sulfated modified cyclocarya paliurus polysaccharide and a preparation method and application thereof.

Background

Sulfation modification can improve various biological activities of polysaccharide by changing the structure and conformation of polysaccharide, and cyclocarya paliurus has been attracting attention as a raw material of health care medicine. Along with the increasing pursuit of people for health, the immunological activity of cyclocarya paliurus polysaccharide is deeply excavated, and the further improvement of the polysaccharide activity by using a chemical method becomes a research hotspot. However, the primary structure of the sulfated cyclocarya paliurus polysaccharide and the mechanism of action of the sulfated cyclocarya paliurus polysaccharide against oxidation have not been reported.

Disclosure of Invention

The invention aims to provide sulfated and modified cyclocarya paliurus polysaccharide and a preparation method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides sulfated and modified cyclocarya paliurus polysaccharide, which has a structure shown in a formula I:

wherein X + Y is 10, X and Y are both not 0 and are both positive integers;

the → [3) - β -D-Galp- (1)]2→3)-β-D-Galp-(1→3)-β-D-Galp-(1→3)-β-D-Galp-(1→3)-β-D-Galf-(1→[4)--D-Glcp-(1]_X→[4)-α-D-Glcp-(1]_Y→ is the main chain;

the alpha-D-Glcp-1 →, alpha-L-Araf- (1 → and alpha-L-Araf- (1 → 4) -beta-D-Galp- (1 → is a side chain;

the α -D-Glcp-1 is located at the C6 position of the side chain α -L-Araf- (1 → 4) - β -D-Galp- (1 → upper Galp;

the α -L-Araf- (1 → 4) - β -D-Galp- (1 → the C4 site located at the corresponding position Galp on the backbone;

said α -L-Araf- (1 → C6 site located at the corresponding position on the backbone Galp, C6 site and C5 site of Galf, respectively;

said HSO₄ ^-C6 site at the backbone at the corresponding position Glcp;

chain segments of the main chain are connected to form a closed-loop structure;

the sulfated and modified cyclocarya paliurus polysaccharide is of a primary structure.

The invention provides a preparation method of sulfated and modified cyclocarya paliurus polysaccharide, which comprises the following steps:

mixing cyclocarya paliurus leaf powder with water, and performing ultrasonic extraction to obtain cyclocarya paliurus polysaccharide;

mixing the formamide solution of the cyclocarya paliurus polysaccharide with a sulfation reagent, and performing sulfation modification to obtain a sulfation modified cyclocarya paliurus polysaccharide;

the sulfating agent is a mixture of chlorosulfonic acid and pyridine, and the volume ratio of the chlorosulfonic acid to the pyridine is 1: 6.

Preferably, the preparation process of the cyclocarya paliurus leaf powder comprises the following steps: and sequentially cleaning cyclocarya paliurus leaves, drying for the first time, crushing, sieving, degreasing and drying for the second time to obtain cyclocarya paliurus leaf powder.

Preferably, the dosage ratio of the cyclocarya paliurus leaf powder to water is 1:10 mL.

Preferably, the temperature of the ultrasonic leaching is 70-80 ℃, and the time is 1-1.5 h.

Preferably, after the ultrasonic extraction is completed, the method further comprises the steps of sequentially carrying out first concentration, alcohol precipitation, dialysis, first drying, cellulose column adsorption, elution, second concentration and second drying on the obtained product to obtain cyclocarya paliurus polysaccharide.

Preferably, the reagent for alcohol precipitation is ethanol with the volume fraction of 95%.

Preferably, the concentration of the formamide solution of the cyclocarya paliurus polysaccharide is 10-30 mg/mL; the volume ratio of the sulfating reagent to the formamide solution of the cyclocarya paliurus polysaccharide is 1: 2.

Preferably, the temperature of the sulfation modification is 60 ℃ and the time is 2 h.

The invention provides an application of sulfated and modified cyclocarya paliurus polysaccharide prepared by the technical scheme or sulfated and modified cyclocarya paliurus polysaccharide prepared by the preparation method in the technical scheme in preparing an antioxidant drug.

The invention provides sulfated and modified cyclocarya paliurus polysaccharide, which introduces sulfate groups into the cyclocarya paliurus polysaccharide, so that the water solubility of the polysaccharide is improved; the sulfation modification site of the sulfation modified cyclocarya paliurus polysaccharide is → 4) -alpha-D-Glcp- (1 → C6, the sulfation modification does not damage the main chain of the polysaccharide, the introduction of a sulfate group can improve the capability of the polysaccharide for providing hydrogen ions, and the hydrogen ions can be combined with radical ions to form a stable complex to terminate the radical chain reaction, so the sulfation modified cyclocarya paliurus polysaccharide has better oxidation resistance; meanwhile, the sulfating modification can improve the water solubility of cyclocarya paliurus polysaccharide, increase the utilization rate of organisms, remove unstable side chains under acidic and high-temperature conditions, improve the stability of the polysaccharide, further improve the bioactivity of the polysaccharide and reduce IC₅₀The application range of the polysaccharide can be increased and the application amount can be reduced.

The invention finds the modification site effective for improving the antioxidant activity by combining the structural characteristics of sulfated and modified cyclocarya paliurus polysaccharide, provides theoretical basis and possibility for revealing the structure-activity relationship and effective positioning and modification of sulfation, and simultaneously provides theoretical basis for developing functional food and health care medicine taking cyclocarya paliurus as a raw material.

Drawings

FIG. 1 is a CPP prepared in example 1_0.05And S-CPP_0.05HPGPC chromatogram and monosaccharide composition profile of (a);

FIG. 2 is a CPP prepared in example 1_0.05And S-CPP_0.05FT-IR spectrum of (1);

FIG. 3 shows preparation of example 1CPP of_0.05And S-CPP_0.05Scanning electron micrographs at different magnifications;

FIG. 4 is a CPP prepared in example 1_0.05The methylation chromatographic analysis spectrogram;

FIG. 5 is a CPP prepared in example 1_0.05And S-CPP_0.05Is/are as follows¹H NMR spectra (A, B),¹³C NMR spectrum (C, D) and Dept135 spectrum (E);

FIG. 6 is a CPP prepared in example 1_0.052D spectrogram of (a);

FIG. 7 is the S-CPP prepared in example 1_0.052D spectrogram of (a);

FIG. 8 is a CPP_0.05And S-CPP_0.05The antioxidant effect diagram of (1);

FIG. 9 is a CPP_0.05And S-CPP_0.05To H₂O₂Graphs and control plots of the effect on survival of induced DCs;

FIG. 10 is a graph showing data on the expression levels (ratio to control group) of MDA (A), SOD (B) and CAT (C);

FIG. 11 is a graph showing data of relative protein expression amounts (ratio to control group) of Nrf2(A) and Keap1 (B).

Detailed Description

wherein X + Y is 10, X and Y are both not 0 and are both positive integers;

said HSO₄ ^-C6 site at the backbone at the corresponding position Glcp;

chain segments of the main chain are connected to form a closed-loop structure;

In the invention, the sulfation modification site of the sulfation modified cyclocarya paliurus polysaccharide is → 4) -alpha-D-Glcp- (1 → C6, and the sulfation modification does not damage the main chain of the polysaccharide, but removes the side chain which is unstable under acidic and high-temperature conditions, improves the stability of the polysaccharide, further improves the biological activity of the polysaccharide and reduces IC₅₀The application range of the polysaccharide can be increased and the application amount can be reduced.

In the present invention, unless otherwise specified, all the starting materials required for the preparation are commercially available products well known to those skilled in the art.

The cyclocarya paliurus polysaccharide is obtained by mixing cyclocarya paliurus leaf powder with water and carrying out ultrasonic extraction. In the present invention, the preparation process of the cyclocarya paliurus leaf powder preferably comprises: and sequentially cleaning, drying, crushing, sieving, degreasing and drying the cyclocarya paliurus leaves to obtain cyclocarya paliurus leaf powder. The method for obtaining the cyclocarya paliurus leaves is not particularly limited, and the cyclocarya paliurus leaves can be obtained according to a method known in the field. The process of the washing, the first drying, the pulverizing and the second drying is not particularly limited, and may be performed according to a process well known in the art; the screening is preferably a 60 mesh screen. In the present invention, the reagent for degreasing is preferably petroleum ether; the amount of the petroleum ether used in the present invention is not particularly limited, and degreasing can be achieved by using an amount known in the art.

The process of mixing the cyclocarya paliurus leaf powder and water is not particularly limited, and the cyclocarya paliurus leaf powder and water can be mixed according to the process well known in the art.

In the invention, the dosage ratio of the cyclocarya paliurus leaf powder to water is preferably 1:10 mL; the temperature of ultrasonic leaching is preferably 70-80 ℃, and more preferably 75 ℃; the time is preferably 1 to 1.5 hours, and more preferably 1.2 to 1.3 hours; the power of the ultrasound is preferably 1400W. The invention preferably adopts ultrasonic extraction circulation to extract twice, so that the polysaccharide is fully extracted.

In the invention, after the ultrasonic extraction is completed, preferably, the method further comprises the steps of sequentially carrying out first concentration, alcohol precipitation, dialysis, first drying, cellulose column adsorption, elution, second concentration and second drying on the obtained product to obtain cyclocarya paliurus polysaccharide. In the present invention, the first concentration preferably concentrates the resulting product system to 10% of the original volume; the reagent used for alcohol precipitation is preferably ethanol with the volume fraction of 95%. The present invention does not specifically limit other parameters of the first concentration and the alcohol precipitation, and the concentration and the alcohol precipitation may be performed according to a process well known in the art.

After the alcohol precipitation is finished, the interference protein of the obtained material is preferably removed by sequentially adopting papain and Sevag reagents, the obtained material is dialyzed by adopting ultrapure water, and crude polysaccharide is obtained after the first drying. In the invention, the dosage ratio of the papain to the polysaccharide solution obtained by alcohol precipitation is preferably 10g of papain/L of the polysaccharide solution. In the invention, the Sevag reagent is preferably a mixture of chloroform and n-butanol, and the volume ratio of the chloroform to the n-butanol is preferably 4: 1; the invention has no special limit on the dosage of the Sevag reagent and can adjust the dosage according to the actual requirement. In the present invention, the reagent for dialysis is preferably ultrapure water; the first drying method is preferably freeze drying. The present invention is not particularly limited with respect to other parameters of the dialysis and the first drying, and may be performed according to a procedure well known in the art.

After the crude polysaccharide is obtained, the crude polysaccharide is preferably subjected to cellulose column adsorption, elution, second concentration and second drying in sequence to obtain cyclocarya paliurus refined polysaccharide; the cellulose column is preferably a DEAE cellulose column, and the elution reagent is preferably 0.05mol/mLNaCl aqueous solution; the second drying means is preferably freeze drying. Other conditions for the adsorption and elution of the cellulose column are not particularly limited in the present invention, and the processes of the second concentration and the second drying are not particularly limited in the present invention, and may be performed according to a process well known in the art.

After the cyclocarya paliurus polysaccharide is obtained, the formamide solution of the cyclocarya paliurus polysaccharide is mixed with a sulfating reagent for sulfating modification, and the sulfating modified cyclocarya paliurus polysaccharide is obtained. In the invention, the concentration of the formamide solution of the cyclocarya paliurus polysaccharide is preferably 10-30 mg/mL, and more preferably 15-25 mg/mL; the volume ratio of the sulfating reagent to the formamide solution of the cyclocarya paliurus polysaccharide is preferably 1: 2; the sulfating agent is a mixture of chlorosulfonic acid and pyridine, and the volume ratio of the chlorosulfonic acid to the pyridine is 1: 6. In the present invention, the sulfating agent is preferably prepared by adding chlorosulfonic acid dropwise to pyridine, and stirring for 30min under ice-water bath condition and room temperature condition, respectively, to obtain the sulfating agent. The process of the dropwise addition and stirring is not particularly limited in the present invention, and may be performed according to a process well known in the art. The invention shortens the sulfation modification time by controlling the ratio of chlorosulfonic acid to pyridine and the ratio of sulfation reagent to formamide solution of polysaccharide, and avoids the damage of excessive chlorosulfonic acid to polysaccharide while improving efficiency.

In the present invention, the process of mixing the formamide solution of the cyclocarya paliurus polysaccharide with the sulfating agent is preferably to add the sulfating agent dropwise into the formamide solution of the cyclocarya paliurus polysaccharide, and the process of adding dropwise is not particularly limited in the present invention and may be performed according to a process well known in the art.

In the present invention, the temperature of the sulfation modification is preferably 60 ℃ and the time is preferably 2 h; the sulfation modification is preferably carried out under stirring conditions, and the stirring process is not particularly limited in the present invention and may be carried out according to a process well known in the art.

After the sulfation modification is completed, the invention preferably sequentially performs neutralization, alcohol precipitation, dialysis and drying on the obtained product system to obtain the sulfation modified cyclocarya paliurus polysaccharide. In the invention, the reagent for neutralization is preferably a 5M NaOH aqueous solution, the reagent for alcohol precipitation is preferably absolute ethyl alcohol, and the time for alcohol precipitation is preferably 12 h. After the alcohol precipitation is finished, the obtained product is preferably redissolved and dialyzed; the drying is preferably freeze drying. The present invention does not specifically limit the other specific conditions for the neutralization, alcohol precipitation, reconstitution, dialysis and drying, and the procedures well known in the art may be followed.

The invention provides an application of sulfated and modified cyclocarya paliurus polysaccharide in the technical scheme or sulfated and modified cyclocarya paliurus polysaccharide prepared by the preparation method in the technical scheme in preparation of dendritic cell antioxidant drugs. The present invention is not particularly limited to the specific method for the application, and the method may be applied according to a method known in the art. The invention cultures dendritic cells DCs in vitro and establishes H through measuring the free radical scavenging capacity and the iron ion reducing capacity of sulfated and modified cyclocarya paliurus polysaccharide₂O₂The induced oxidative stress model researches the antioxidation and molecular mechanism of cyclocarya paliurus polysaccharide and sulfated derivatives thereof (sulfated and modified cyclocarya paliurus polysaccharide) on DCs, and finds out a modification site effective in improving the antioxidation activity by combining the structural characteristics of the sulfated polysaccharide, thereby providing a theoretical basis for disclosing the structure-activity relationship and developing functional foods and health-care medicines taking cyclocarya paliurus as a raw material.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Cleaning fresh cyclocarya paliurus leaves, drying, crushing, sieving (60 meshes), soaking in petroleum ether for degreasing, drying, mixing the obtained cyclocarya paliurus leaf powder with water (the dosage ratio of the cyclocarya paliurus leaf powder to the water is 1:10(W/v)), and performing ultrasonic extraction twice (the ultrasonic power is 1400W) at 75 ℃ for 1h each time; after extraction, concentrating the obtained product to 10% of the original volume, treating the product with 95% (v/v) ethanol to precipitate polysaccharide, removing interference proteins from the obtained product with papain and Sevag reagents respectively (the dosage ratio of the papain to the polysaccharide solution obtained by alcohol precipitation is 10g of papain/L of the polysaccharide solution, the Sevag reagent is a mixture of chloroform and n-butyl alcohol, the volume ratio of the chloroform to the n-butyl alcohol is 4:1), dialyzing with ultrapure water, and freeze-drying to obtain crude polysaccharide; adsorbing the crude polysaccharide by a DEAE cellulose column, eluting with 0.05mol/mLNaCl, concentrating, and freeze drying to obtain Cyclocarya Paliurus Polysaccharide (CPP)_0.05；

Dropwise adding chlorosulfonic acid (the volume ratio of chlorosulfonic acid to pyridine is 1:6) into pyridine, and stirring in ice-water bath at room temperature for 30min to obtain sulfating reagent; dropwise adding the sulfation reagent (10mL) into 20mL formamide solution (10mg/mL) of cyclocarya paliurus polysaccharide, magnetically stirring for 2h at 60 ℃, performing sulfation modification, neutralizing the obtained product with 5M NaOH solution, precipitating with absolute ethanol for 12h, redissolving, dialyzing and freeze-drying the obtained product to obtain sulfation modified cyclocarya paliurus polysaccharide, which is recorded as S-CPP_0.05。

Characterization and testing

1. CPP prepared for example 1_0.05And S-CPP_0.05And (5) carrying out structure verification:

1) degree of Substitution (DS), total sugar, protein and uronic acid content determination:

in order to analyze the degree of substitution of sulfate group for polysaccharide hydroxyl, the barium chloride-gelatin method is adopted to determine S-CPP_0.05Degree of substitution DS: will S-CPP_0.05(3mg) hydrochloric acid hydrolysis (1mL) at 100 ℃ for 6h, cooling and then supplementing 1mL with hydrochloric acid; 0.2mL of the sample solution was added with 1mL of barium chloride (1%) and 3.8mL of trichloroacetic acid (3%), followed by shaking thoroughly and incubation at room temperature for 20min, absorbance was measured at 360nm, and the blank control was replaced with 0.2mL of hydrochloric acid.

The DS is calculated according to the following formula:

wherein S% is the mass fraction of the element S.

The total sugar content, the protein content and the uronic acid content are respectively determined by adopting a phenol-sulfuric acid colorimetric method, a Coomassie brilliant blue G-250 method and a sulfuric acid-carbazole method, and the experiments are repeated at least three times.

2) Determination of molecular weight (Mw)

CPP_0.05And S-CPP_0.05The molecular weights of (A) were all determined by means of a High Performance Gel Permeation Chromatography (HPGPC) system (LC-10A, Shimadzu, Japan). Chromatographic conditions are as follows: BRT 105-104-; polysaccharide concentration: 5 mg/mL; mobile phase: 0.02% sodium azide (NaN 3); flow rate: 0.6 mL/min; temperature: 35 ℃; sample introduction amount: 20 μ L. Polysaccharide Mw was calculated using dextran standards (Mw 1152, 11600, 23800, 48600, 80900, 148000, 273000, 409800) to create calibration curves.

3) Determination of monosaccharide composition:

the acetylated derivatives are prepared by polysaccharide hydrolysis, reduction and acetylation, and the obtained acetylated derivatives are subjected to GC-MS analysis. The method comprises the following specific steps: taking 2mg polysaccharide sample to be tested, hydrolyzing the polysaccharide sample with 1mL trifluoroacetic acid (2M) for 90min, evaporating the polysaccharide sample to dryness by a rotary evaporator, adding 2mL double distilled water and 100mg sodium borohydride into the obtained mixture for reduction, then adding glacial acetic acid for neutralization, carrying out rotary evaporation, drying the mixture in a 110 ℃ oven, then adding 1mL acetic anhydride for acetylation at 100 ℃ for reaction for 1h, cooling, then adding 3mL toluene, carrying out reduced pressure concentration and evaporation to dryness, and repeating the steps for 4 times to remove redundant acetic anhydride. The acetylated product was dissolved in 3mL of chloroform and transferred to a separatory funnel, and after adding a small amount of distilled water and shaking sufficiently, the upper aqueous solution was removed, and this was repeated 5 times. Drying the chloroform layer by using a proper amount of anhydrous sodium sulfate, fixing the volume to 10mL, and analyzing by using a Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer to determine an acetylation product sample;

GC-MS conditions: RXI-5SILMS chromatographic column 30m × 0.25mm × 0.25 mm; the temperature programming conditions are as follows: the initial temperature is 120 ℃, and the temperature is increased to 250 ℃/min at the speed of 3 ℃/min; keeping for 5 min; the temperature of the sample inlet is 250 ℃, the temperature of the detector is 250 ℃/min, the carrier gas is helium, and the flow rate is 1 mL/min.

Specific results obtained in the above 1) to 3) are shown in Table 1 and FIG. 1.

TABLE 1S-CPP prepared in example 1_0.05Chemical analysis, molecular weight and monosaccharide composition

As can be seen from Table 1, sulfated-modified cyclocarya paliurus polysaccharide (S-CPP)_0.05) The degree of substitution is 0.32 +/-0.02, which indicates that the structure change of the polysaccharide causes easier connection between sulfate groups and polysaccharide chains; with respect to molecular weight, S-CPP_0.05Mw and CPP of (36.307kDa)_0.05(30.160kDa) compared to a significant increase.

FIG. 1 is a CPP prepared in example 1_0.05And S-CPP_0.05FIG. 1 shows the HPGPC chromatogram (A) and monosaccharide composition (B) of (A)_0.05And S-CPP_0.05Mainly composed of arabinose, glucose and galactose, and the molar ratio of each monosaccharide is shown in table 1.

2. CPP prepared for example 1_0.05And S-CPP_0.05Carrying out infrared test, which comprises the following steps: fully grinding and uniformly mixing a polysaccharide powder sample and dry KBr in a mass ratio of 1:100, pressing into a sheet, analyzing a polysaccharide basic characteristic peak and a characteristic absorption peak of a sulfated group by using a Nicolet iS5 Fourier transform infrared spectrometer, wherein the frequency range iS 4000-400cm^-1The results are shown in FIG. 2.

FIG. 2 is a CPP prepared in example 1_0.05And S-CPP_0.05The FT-IR spectrum of (A) was as shown in FIG. 2, which shows that CPP is_0.05And S-CPP_0.05Has a similar spectrum profile and is located at 3410cm^-1、2930cm^-1、1630cm^-1、1420cm^-1And 1070cm^-1The typical signal peaks of the polysaccharide are clearly visible, which are due to intermolecular hydrogen bonds, CH, respectively₂C-H stretching of the group, C ═ O asymmetric stretching vibration of the carboxylic acid (-COO-) group, deformation vibration of the C-H bond, C-O-C asymmetric stretching vibration, and symmetric stretching vibration. Sulfated and modified cyclocarya paliurus polysaccharide S-CPP_0.05The specific absorption peak of sulfate radical is detected in 1250cm separately due to S ═ O stretching vibration and C ═ O ═ S symmetric stretching vibration^-1And 820cm^-1The absorption peak appeared.

3. CPP prepared for example 1_0.05And S-CPP_0.05Performing scanning electron microscope test, and performing CPP by using FEI Quanta 250 scanning electron microscope with 15kV acceleration voltage_0.05And S-CPP_0.05The surface topography of (a) was scanned at 2000 x and 10000 x image magnification, respectively, and the results are shown in fig. 3.

FIG. 3 is a CPP prepared in example 1_0.05And S-CPP_0.05Scanning electron micrographs at different magnifications. As can be seen from FIG. 3, CPP_0.05(A and B in FIG. 3) show a relatively incomplete but smooth morphology. In the image of sulfated polysaccharide, S-CPP_0.05(C and D in FIG. 3) are in a flaky and smooth structure, and the modification of sulfation causes the change of the form of the polysaccharide to increase the contact area.

4. CPP prepared for example 1_0.05And S-CPP_0.05Performing methylation analysis, and analyzing CPP_0.05After methylation, hydrolysis and acetylation are carried out in sequence, the methylation, hydrolysis and acetylation are measured by GC-MS and compared with a standard mass spectrum library, and the method specifically comprises the following steps: weighing polysaccharide sample (3mg) and placing in a glass reaction flask, adding 1mL of anhydrous DMSO, rapidly adding NaOH powder, sealing, dissolving under ultrasound, and adding 1mL of iodomethane (CH)₃I) Reacting for 60min at 30 ℃ in a magnetic stirring water bath, and then adding 2mL of ultrapure water into the mixture to terminate the methylation reaction to obtain methylated polysaccharide; taking methylated polysaccharide, adding 1mL trifluoroacetic acidHydrolyzing for 90min (2M, TFA), evaporating to dryness by a rotary evaporator, adding 2mL of double distilled water and 60mg of sodium borohydride into the obtained residue, reducing for 8h, then adding glacial acetic acid for neutralization, performing rotary evaporation, drying in an oven at 101 ℃, then adding 1mL of acetic anhydride for acetylation, reacting for 1h at 100 ℃, cooling, then adding 3mL of toluene into the obtained product, performing reduced pressure concentration and evaporation to dryness, and repeating for 4 times to remove redundant acetic anhydride to obtain an acetylation product; the resulting acetylated product was consumed 3mL of CH₂Cl₂Transferring to a separating funnel after dissolving, adding a small amount of distilled water, fully shaking, removing the upper-layer aqueous solution, and repeating the steps for 4 times; CH (CH)₂Cl₂The layer was dried over an appropriate amount of anhydrous sodium sulfate, and the volume was adjusted to 10mL, and the mixture was placed in a liquid phase vial.

Measuring an acetylation product sample by using a Shimadzu GCMS-QP 2010 gas chromatography-mass spectrometer; GC-MS conditions: RXI-5SILMS chromatographic column 30m × 0.25mm × 0.25 mm; the temperature programming conditions are as follows: the initial temperature is 120 ℃, and the temperature is increased to 250 ℃/min at the speed of 3 ℃/min; keeping for 5 min; the temperature of the sample inlet is 250 ℃, the temperature of the detector is 250 ℃/min, the carrier gas is helium, and the flow rate is 1 mL/min. The specific results are shown in FIG. 4.

FIG. 4 is a CPP prepared in example 1_0.05FIG. 4 shows the spectrum of the methylation Chromatogram of (CPP)_0.05Contain eight types of glycosidic linkages, and the unsubstituted residues are predominantly → 4) -Glcp- (1 → and → 3) -Galp- (1 →; from which the CPP can be inferred_0.05Is a polysaccharide with a certain degree of branching, wherein → 4) -Glcp- (1 → and → 3) -Galp- (1 →) may be the backbone unit thereof.

5. CPP prepared in example 1_0.05And S-CPP_0.05Performing Nuclear Magnetic Resonance (NMR) analysis, which comprises the following steps: respectively weighing CPP_0.05And S-CPP_0.0550mg each, dissolved in 0.5mLD₂In O and freeze-dried, and the resulting lyophilized powder was then redissolved in 0.5mL of D₂And continuously freezing and drying, and repeating the above processes to fully exchange active hydrogen. The resulting sample was then dissolved in 0.5mL of heavy water and placed in a 600MHz NMR spectrometer at room temperature and 25 ℃ to record a one-dimensional spectrum (¹H NMR、¹³C NMR and DEPT-135), and two-dimensional mapping using Bruker software (C NMR and DEPT-135) ((C NMR and DEPT-135)¹H-¹H COSY、¹H-¹³C HSQC, HMBC, and NOESY), the results are shown in FIGS. 5-7 and Table 2.

FIG. 5 is a CPP prepared in example 1_0.05And S-CPP_0.05Is/are as follows¹H NMR spectra (A and B in FIG. 5),¹³C NMR spectrum (C and D in FIG. 5) and Dept135 spectrum (E in FIG. 5). As can be seen from the figure 5 of the drawings,¹the signals in the H NMR spectra (A and B in FIG. 5) are mainly concentrated between delta 3-5.5ppm, where the delta 3.2-4.0ppm region belongs to the sugar ring proton signal; 8 major signal peaks were identified, δ 5.35, 5.32, 5.26, 5.17, 4.45, 4.41, 4.4 and 4.39ppm respectively. Residues are labeled from a to H in descending order of their abnormal proton chemical shifts. By¹³C NMR spectra (C and D in FIG. 5) showed that the nuclear magnetic carbon spectrum signals were mainly concentrated at 60 to 120ppm, and by observing the carbon spectrum, it was found that the main anomeric carbon signal peaks δ 101.14, 101.33, 104.47, 104.48, 104.69, 105.23, 110.62, and 110.78ppm were mainly located between δ 93 to 105. The main signal peaks at δ 85.22, 85.12, 83.22, 82.90, 82.62, 82.62, 81.70, 81.58, 81.57, 81.50, 78.41, 78.35, 77.97, 76.40, 76.40, 76.18, 74.81, 74.58, 74.34, 74.10, 74.03, 72.88, 72.53, 71.97, 71.96, 71.94, 71.68, 71.31, 70.76, 70.61, 70.48, 70.26, 70.25, 69.82, 62.64, 62.30, 62.26 and 61.95ppm are distributed in the region of 60-85 ppm. In the Dept135 spectrum (E in FIG. 5), δ 68.53, 61.95, 62.3, 62.26, 70.76, 70.48, 62.64, 70.26ppm are peak inverses, indicating that C6 has undergone a chemical shift. Whereas δ 68.53, 70.76, 70.48, 70.26ppm shifted to low field, respectively, indicating that substitution occurred.

FIG. 6 is a CPP prepared in example 1_0.052D spectrum of (a), wherein¹H-¹H COSY(A)；¹H-¹³C HSQC (B); HMBC (C); NOESY (D); FIG. 7 is the S-CPP prepared in example 1_0.052D spectrum of (a), wherein¹H-¹H COSY(A)；¹H-¹³C HSQC (B); HMBC (C); NOESY (D); as can be inferred from the combination of FIGS. 6 and 7, S-CPP_0.05The main chain connecting structure of (1) is → [3) -beta-D-Galp- (1)]2→3)-β-D-Galp-(1→3)-β-D-Galp-(1→3)-β-D-Galp-(1→3)-β-D-Galf-(1→[4)--D-Glcp-(1]_X→[4)-α-D-Glcp-(1]_YThe terminal unit is α -L-Araf- (1 →, a D-Glcp- (1 →, attached to the backbone via O-6, O-5, O-4 linkages the sulfation modification site is → 4) - α -D-Glcp- (1 → C6.

TABLE 2S-CPP prepared in example 1_0.05Is/are as follows¹H and¹³c Signal attribution

As can be seen from the combination of FIGS. 5-7 and Table 2, the modification site of the sulfated and modified cyclocarya paliurus polysaccharide is C6 of → 4) -alpha-D-Glcp- (1 → of the polysaccharide backbone.

6. CPP prepared in example 1_0.05And S-CPP_0.05Determination of antioxidant Activity:

measurement of radical scavenging Activity and reducing Capacity the antioxidant activity of polysaccharide samples was evaluated using DPPH, ABTS, hydroxyl radical scavenging Capacity and reducing Capacity, respectively. Ascorbic acid (Vc) served as a positive control. The cleaning effect calculation formula is as follows:

wherein A is₀: ultrapure Water instead of sample, A₁: polysaccharides or Vc, A₂: sample only (ethanol instead of DPPH solution).

Wherein A is₀: ultrapure Water instead of sample, A₁: polysaccharides or Vc, A₂: sample only (PBS instead of ABTS).

Wherein A is₀: ultrapure Water instead of sample, A₁: polysaccharides or Vc, A₂: sample only (ultrapure water instead of H)₂O₂)

FIG. 8 is a CPP_0.05And S-CPP_0.05The antioxidant effect of (A) (DPPH (FIG. 5 (A)); ABTS (FIG. 5 (B)); hydroxyl radical (FIG. 5 (C)); reducing power (FIG. 5 (D))); wherein, A-C: different letters indicate that different samples differ significantly at the same concentration (P)<0.05); a-f: different letters indicate significant differences between different concentrations (P) for the same samples<0.05). As can be seen from FIG. 8, CPP_0.05And S-CPP_0.05All have good cleaning effect. CPP at 4mg/mL_0.05And S-CPP_0.05The clearance rate of DPPH reaches 54.10 percent and 61.00 percent respectively. At the same time, two polysaccharides CPP_0.05And S-CPP_0.05semi-Inhibitory Concentration (IC) for scavenging DPPH free radicals₅₀) 2.71mg/mL and 1.43mg/mL, respectively.

7. Determination of cell viability: a dendritic cell oxidative stress model is established, and whether the polysaccharide pretreatment can slow down the oxidative stress is researched to evaluate the antioxidant activity of the dendritic cell.

1) Culture and treatment of mouse dendritic cells (DC cells):

preparation of a complete culture medium: adding 10% Fetal Bovine Serum (FBS) (FBS: medium is 1: 9(v/v)) into RPMI-1640 medium, adding penicillin 100U/mL and streptomycin 100 μ g/mL, and storing at 4 deg.C;

preparing a cyclocarya paliurus polysaccharide solution and a sulfated and modified cyclocarya paliurus polysaccharide solution: dissolving the two polysaccharide powders in incomplete culture medium to prepare a solution of 10mg/mL, filtering and sterilizing by using a microporous filter head (0.22 mu m), and diluting the solution by using the culture medium according to the required final concentration when in test;

freezing and storing cells: frozen stock solution to complete medium: serum: mixing dimethyl sulfoxide (DMSO) at a volume ratio of 7:2:1, standing at low temperature in dark before use, and preparing for use; freezing at 4 deg.C for 0.5 hr, -20 deg.C for 2 hr, -80 deg.C for 8 hr, and storing in liquid nitrogen;

recovery of cells: placing the cryopreservation tube containing the cells in water at 37 ℃, and quickly dissolving the cells in parallel by marking an 8 shape without the water passing through the tube opening;

fifthly, taking out the culture bottle for culturing adherent cells from the incubator, placing the culture bottle on a clean bench, discarding the old culture medium, washing the culture bottle with PBS for 2 times, cleaning the PBS, adding 2mL of pancreatin for digestion, placing the culture bottle at 37 ℃ for 5min, slightly shaking the culture bottle to completely drop the cells, collecting the digested cells in a centrifuge tube for centrifugation for 5min at 1000r/min, discarding the supernatant, adding fresh complete culture medium to resuspend the cells, adjusting the cell density, paving the culture bottle in a new culture bottle, placing the culture bottle at 37 ℃ with 5% CO, and placing the culture bottle₂The culture is carried out in a constant temperature incubator.

2) Mouse dendritic cell DCs were subjected to the following grouping:

blank control group (normal group): adding only culture medium;

(II) H₂O₂Group (2): only H was added to a final concentration of 1mM₂O₂A solution;

(III) cyclocarya paliurus polysaccharide + H₂O₂Group (2): adding cyclocarya paliurus polysaccharide (37.5. mu.g/mL, 75. mu.g/mL, 150. mu.g/mL and 300. mu.g/mL) at different concentrations, culturing for 24H, and adding H with final concentration of 1mM₂O₂Reacting the solution for 3 hours;

(tetra) sulfated-modified cyclocarya paliurus polysaccharide + H₂O₂Group (2): adding sulfated modified cyclocarya paliurus polysaccharide (37.5. mu.g/mL, 75. mu.g/mL, 150. mu.g/mL and 300. mu.g/mL) at different concentrations, culturing for 24H, and adding H with final concentration of 1mM₂O₂And (6) reacting for 3 hours.

At 1 × 10⁶And (3) inoculating the DC cells into a 96-well plate at the density of one cell per mL, and treating the cells in groups according to the step 2) after the cells are attached to the wall, wherein each group is provided with 3 multiple wells. After being treated according to the groups, the cell culture plate is placed in an incubator to be cultured for 24 hours, and the solution is changed and added with H₂O₂The medium of (4) was treated for 3 hours, then 10% of CCK-8 reagent (CCK-8 reagent: medium: 1: 9(v/v)) was added to each well, and after standing in an incubator for 2 hours,the absorbance was measured at a wavelength of 450nm, and the results are shown in FIG. 9.

FIG. 9 is a CPP_0.05And S-CPP_0.05To H₂O₂Graph and control of the effect on survival of induced DCs (M ± SD, n ═ 3) in which the mean values represented by the different letters a to d were significantly different (P)<0.05)。

From FIG. 9, it can be seen that the channel H₂O₂After 3h treatment, the survival rate of DCs is obviously reduced (P)<0.05), which is only 43.89% of the normal group, indicating successful molding. CPP_0.05And S-CPP_0.05The group has obvious protective effect on dendritic cells, and the cell survival rate is increased along with the increase of the concentration of the polysaccharide, and the group and the polysaccharide are dose-dependent. At a concentration of 300. mu.g/mL, CPP_0.05The survival rate of the group was 79.62%, while S-CPP_0.05The survival rate of the group was 86.02%, 6.40% higher than that of the CPP0.05 group. The results show that two polysaccharides CPP_0.05And S-CPP_0.05To H₂O₂The induced dendritic cells have protective effect, S-CPP_0.05The protection effect is better.

8. Measuring the MDA content and the SOD and CAT activities in DCs:

at 1 × 10⁶DCs cells are inoculated on a 96-well plate at the density of one cell per mL, and after the cells are attached to the wall, the cells are treated by 10 groups according to the method in the 2), and 3 multiple holes are arranged in each group. After being treated according to the groups, the cell culture plate is placed in an incubator to be cultured for 24 hours, and the solution is changed and added with H₂O₂The cells were harvested with PBS and disrupted with a sonicator, and the levels of Malondialdehyde (MDA), superoxide dismutase (SOD) and Catalase (CAT) were measured as described in the kit, and the results are shown in FIG. 10.

Fig. 10 is a data graph showing the expression levels (ratio to control group) of MDA (a in fig. 10), SOD (B in fig. 10), and CAT (C in fig. 10) (M ± SD, n ═ 3). The mean values for the different letters a-f are significantly different (P < 0.05).

As seen in FIG. 10, 1mM H was compared with that of the blank control group₂O₂The SOD activity after treatment is obviously reduced. And two polysaccharides (CPP)_0.05And S-CPP_0.05) SOD Activity and H of the treated cells₂O₂The group ratio was significantly higher. When the concentration reaches 300 mug/mL, the CPP_0.05Group and S-CPP_0.05The SOD activity values of the groups were 82.16% and 90.31% of the normal group, respectively. S-CPP_0.05The group is obviously higher than the CPP_0.05And (4) grouping. Similar to the SOD results, H₂O₂The CAT activity is obviously reduced after the treatment. Polysaccharide pretreatment reverses H₂O₂Induced CAT downregulation. Notably, S-CPP_0.05The activity value of the group in the concentration range of 37.5-150 mu g/mL is higher than that of CPP_0.05And (4) grouping.

9. Determination of Keap1 and Nrf2 protein levels in DCs:

total protein was extracted from DCs with a protein extraction reagent on ice, and Nrf2, Keap1 and GAPDH proteins were determined, in the same grouping procedure as step 2) of item 7 above. An equal amount of protein (12. mu.L/lane) was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. After blocking, the membranes were incubated with primary and secondary antibodies for 3 hours and 1.5 hours, followed by imaging in the Gene Genius bioimaging system (SYNGENE co., MD, USA). The Image was processed using Image J software, the results of which are shown in FIG. 11.

Fig. 11 is a data graph of relative protein expression amounts (ratio to control group) of Nrf2(a in fig. 11) and Keap1(B in fig. 11) (M ± SD, n ═ 3). The mean values for the different letters a-e differ significantly (P < 0.05).

As can be seen from FIG. 11, H without polysaccharide pretreatment was compared with the normal group₂O₂Group Nrf2 expression was significantly increased. In the CPP_0.05And S-CPP_0.05In the polysaccharide-treated group, the expression of Keap1 was significantly reduced, and the expression of Nrf2 was significantly increased (P<0.05). Thus, the polysaccharide pretreatment effectively activates the signal channel and starts the expression of a plurality of downstream antioxidant proteins, thereby weakening the oxidative stress and regulating the redox balance in the organism. In addition, S-CPP_0.05Exhibits specific CPP_0.05Stronger regulating capacity. Taken together, the sulfation modification may be through the Keap1-Nrf2 signaling pathway to improve CPP_0.05Protecting DCs against oxidative stress.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A sulfated modified cyclocarya paliurus polysaccharide is characterized by having a structure shown in a formula I:

wherein X + Y is 10, X and Y are both not 0 and are both positive integers;

said HSO₄ ^-C6 site at the backbone at the corresponding position Glcp;

chain segments of the main chain are connected to form a closed-loop structure;

2. The method for preparing sulfated and modified cyclocarya paliurus polysaccharide as claimed in claim 1, which comprises the following steps:

3. The method for preparing cyclocarya paliurus leaf powder according to claim 2, wherein the cyclocarya paliurus leaf powder is prepared by the following steps: and sequentially cleaning cyclocarya paliurus leaves, drying for the first time, crushing, sieving, degreasing and drying for the second time to obtain cyclocarya paliurus leaf powder.

4. The preparation method of claim 2, wherein the dosage ratio of cyclocarya paliurus leaf powder to water is 1:10 mL.

5. The preparation method according to claim 2, wherein the temperature of the ultrasonic leaching is 70-80 ℃ and the time is 1-1.5 h.

6. The preparation method of claim 2, wherein after the ultrasonic extraction is completed, the method further comprises sequentially performing first concentration, alcohol precipitation, dialysis, first drying, cellulose column adsorption, elution, second concentration and second drying on the obtained product to obtain cyclocarya paliurus polysaccharide.

7. The method according to claim 6, wherein the alcohol precipitation reagent is 95% ethanol by volume.

8. The preparation method according to claim 2, wherein the concentration of the formamide solution of the cyclocarya paliurus polysaccharide is 10-30 mg/mL; the volume ratio of the sulfating reagent to the formamide solution of the cyclocarya paliurus polysaccharide is 1: 2.

9. The method according to claim 2 or 8, wherein the temperature of the sulfation modification is 60 ℃ and the time is 2 h.

10. The sulfated and modified cyclocarya paliurus polysaccharide of claim 1 or the sulfated and modified cyclocarya paliurus polysaccharide prepared by the preparation method of any one of claims 2 to 9 is applied to the preparation of antioxidant drugs.