CN114044831B

CN114044831B - Rubus pure polysaccharide RCHP-S and purification and separation method and application thereof

Info

Publication number: CN114044831B
Application number: CN202111409303.5A
Authority: CN
Inventors: 孔媛芳; 邱渊皓; 陈朋; 董春红; 胡玉龙; 李杰明; 蔡军涛; 李晓飞
Original assignee: Henan University of Traditional Chinese Medicine HUTCM
Current assignee: Henan University of Traditional Chinese Medicine HUTCM
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-11-04
Anticipated expiration: 2041-11-25
Also published as: CN114044831A

Abstract

The invention belongs to the technical field of polysaccharide, and particularly discloses rubus corchorifolius pure polysaccharide RCHP-S as well as a purification and separation method and application thereof.

Description

Rubus pure polysaccharide RCHP-S and purification and separation method and application thereof

Technical Field

The invention belongs to the technical field of polysaccharides, and particularly relates to a rubus pure polysaccharide RCHP-S, and a purification and separation method and application thereof.

Background

Rubus corchorifolius, also called as "Rubus corchorifolius" in China, belongs to Rubus (also called as Rubus) of Rosaceae, and has various pharmacological activities such as anti-tumor, blood glucose and blood lipid lowering, anti-inflammatory, and antithrombotic effects, however, the research on the anti-inflammatory effect of Rubus corchorifolius pure polysaccharide is less at present, according to the research in literature, 2015, zhu et al have studied that the flavonoid glycoside component in Rubus corchorifolius shows the anti-inflammatory activity by inhibiting the activation of macrophage MAPKs; in the same year, the biological activity and the extraction process of extracting crude polysaccharide from fruits and leaves of rubus corchorifolius are optimized, the research on the dosage-dependent inhibition of Nitric Oxide (NO) generated by mouse macrophage RAW264.7 through inhibiting the expression of TNF-alpha, iNOS and IL-6 genes is carried out, and the polysaccharide from the fruits and leaves of rubus corchorifolius can be used as a potential anti-inflammatory drug. Therefore, the research on the anti-inflammatory activity of the rubus corchorifolius polysaccharide is only limited on the level of crude polysaccharide, in order to more clearly obtain the structure and the anti-inflammatory activity of the rubus corchorifolius pure polysaccharide, the application further purifies the crude polysaccharide, obtains the pure rubus corchorifolius polysaccharide structure through structure analysis, and deeply researches on colitis induced by Dextran Sodium Sulfate (DSS).

Disclosure of Invention

The invention aims to provide a rubus corchorifolius pure polysaccharide RCHP-S as well as a purification and separation method and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

a separation and purification method of Rubus pure polysaccharide RCHP-S comprises the following steps:

(1) Extracting and separating the crude polysaccharide of the raspberry: drying and crushing fresh fructus Rubi Corchorifolii Immaturus, adding ethanol, soaking for degreasing, filtering, centrifuging, extracting the drug residue with distilled water at 80 deg.C for 4 hr, repeating for three times, adding distilled water, filtering, centrifuging, mixing the filtrate and supernatant, concentrating, dialyzing with running water for 12 hr, dialyzing with distilled water for 24 hr, concentrating, adding ethanol into the concentrated solution, standing at 4 deg.C for 10 hr, centrifuging, and freezing at-80 deg.C to obtain fructus Rubi Corchorifolii Immaturus crude polysaccharide with yield of 14.32%, and naming as RCHP;

(2) The separation and purification of the crude raspberry polysaccharide comprises anion exchange column chromatography separation and gel column chromatography purification, and specifically comprises the following steps:

a. and (3) anion exchange column chromatography separation: dissolving rubus corchorifolius crude sugar RCHP in distilled water, then loading the sample along the inner wall of a chromatographic column, adjusting the flow of the chromatographic column until the sample completely permeates into an anion exchange filler, sequentially adopting distilled water with 20 times of column volume, 0.1M sodium chloride solution, 0.2M sodium chloride solution and 0.5M sodium chloride solution to wash at the flow rate of 3mL/min, combining the components of the same effluent, concentrating, dialyzing, and freeze-drying to obtain a polysaccharide sample;

b. gel column chromatography purification of Rubus Suavissimus polysaccharide: dissolving a polysaccharide sample purified by DEAE-52 column chromatography in 0.2M sodium chloride solution, loading along the inner wall of a chromatographic column, eluting with 0.2M sodium chloride solution, collecting combined samples, concentrating, dialyzing, and lyophilizing to obtain Rubus corchorifolius pure polysaccharide with yield of 3.46%, which is named as RCHP-S.

Further, the anion exchange column in the step a needs to be pretreated, specifically, anion exchange filler DEAE-52 is swelled in distilled water and then dried, then soaked and washed with hydrochloric acid, and then soaked, washed and activated with sodium hydroxide, and then the pretreated DEAE-52 filler is loaded into a chromatographic column, and the filler is eluted with 20 times of distilled water at a large flow rate to be tightly and uniformly distributed.

Further, in the step b, the number of the gel columns is two, and the packing materials are G-25sephadex and Sephacryl S300 respectively.

The pure polysaccharide RCHP-S obtained by the separation and purification method of the pure polysaccharide RCHP-S of the rubus corchorifolius has the molecular weight of 13.15kDa, contains 48.32 percent of uronic acid, and has the protein content of less than 1 percent.

Further, its chemical composition includes mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, in a molar ratio of 1.52.

Application of Rubus Suavissimus pure polysaccharide RCHP-S in preparing medicine for treating dextran sodium sulfate induced enteritis is provided.

The invention has the advantages that: the invention extracts the crude polysaccharide of the raspberry from the raspberry, purifies the crude polysaccharide by adopting a column chromatography to obtain pure polysaccharide, and performs structural analysis and characterization on the pure polysaccharide to obtain the structure of the pure polysaccharide, further researches the anti-inflammatory effect of the pure polysaccharide of the raspberry on Dextran Sodium Sulfate (DSS) -induced colitis mice, so that the relationship between the chemical structure and the biological activity of the pure polysaccharide of the raspberry is clearer.

Drawings

FIG. 1 is an HPGPC chromatogram of Rubus corchorifolius polysaccharide RCHP-S.

FIG. 2 is a graph of monosaccharide standard (A) and a PMP derivative of the Rubus polysaccharide RCHP-S sample (B).

FIG. 3 shows the preparation of Rubus corchorifolius polysaccharide RCHP-S ¹ H NMR chart.

FIG. 4 shows the preparation of Rubus corchorifolius polysaccharide RCHP-S ¹³ C NMR chart.

FIG. 5 is a drawing of Rubus polysaccharide RCHP-S ¹ H- ¹ H COSY spectrum.

FIG. 6 shows the preparation of Rubus corchorifolius polysaccharide RCHP-S ¹ H- ¹³ C HSQC spectrogram.

FIG. 7 shows polysaccharide RCHP-S of Rubus corchorifolius ¹ H- ¹³ C HMBC spectrum.

Fig. 8 is a diagram showing the evaluation results of DAI.

FIG. 9 is a graph showing the IL-6 (A) and TNF-. Alpha.B contents measured by Elisa.

Detailed Description

Examples

1. A separation and purification method of Rubus pure polysaccharide RCHP-S comprises the following steps:

(1) Extraction and separation of rubus corchorifolius crude polysaccharide

Drying fresh fructus Rubi Corchorifolii Immaturus at 60 deg.C, pulverizing into powder, sieving with 100 mesh sieve, soaking in 95% industrial ethanol for defatting, filtering, centrifuging, extracting the residue with distilled water at 80 deg.C for 4 hr, extracting for three times, adding distilled water, filtering, centrifuging, mixing the filtrate and supernatant, concentrating, dialyzing with running water for 12 hr, and dialyzing with distilled water for 24 hr. Concentrating, slowly adding industrial ethanol, standing at 4 deg.C overnight, centrifuging, freezing at-80 deg.C, and removing solvent to obtain crude Rubus coreanus polysaccharide, named as RCHP.

(2) Separation and purification of rubus corchorifolius crude polysaccharide

(a) Anion exchange column chromatography (DEAE-52) separation

Soaking newly purchased anion exchange filler DEAE-52 in distilled water overnight to fully swell the anion exchange filler DEAE-52, draining, soaking for 2 hours by using 0.5mol/L hydrochloric acid solution, washing for many times by using distilled water until effluent liquid is neutral, then soaking for 2 hours by using 0.5mol/L sodium hydroxide solution to fully activate hydroxide ions, and finally washing for many times by using distilled water until the effluent liquid is neutral. The pretreated DEAE-52 filler is uniformly filled into a clean chromatographic column (specification is 6 x 50 cm), and is eluted by 20 times of distilled water at a large flow rate, so that the filler is tightly and uniformly distributed.

Dissolving crude rubus corchorifolius sugar RCHP in a proper amount of distilled water, slowly loading along the inner wall of a chromatographic column, adjusting the flow rate of the chromatographic column, and sequentially adopting 20 times of column volume of distilled water, 0.1M sodium chloride solution, 0.2M sodium chloride solution and 0.5M sodium chloride solution to flush when a sample completely and slowly permeates into an anion exchange filler, wherein the flow rate is 3mL/min, and detecting the sugar content in an effluent liquid every 100mL by a sulfuric acid phenol method. Combining the same effluent components, concentrating, dialyzing, and freeze-drying.

(b) Gel column chromatography purification of Rubus corchorifolius polysaccharide

Further purifying the Rubus corchorifolius polysaccharide by gel column chromatography, respectively filling fully swollen gel filler G-25sephadex and Sephacryl S300 into two slender chromatographic columns (2.5 multiplied by 60 cm), lightly tapping the chromatographic columns during column filling to uniformly distribute the gel filler, dissolving a polysaccharide sample purified by DEAE-52 column chromatography into 0.2M sodium chloride solution, slowly loading the sample, eluting with 0.2M sodium chloride solution, tracking and monitoring the polysaccharide by a sulfuric acid phenol method, collecting and combining samples according to absorbance, concentrating and dialyzing, and freeze-drying to obtain the Rubus corchorifolius pure polysaccharide which is named as RCHP-S.

2. Structural analysis of Rubus Corchorifolius polysaccharide

(1) Measurement of molecular weight and homogeneity of Rubus Corchorifolius polysaccharide

The molecular weight and the uniformity of the rubus corchorifolius polysaccharide are measured through high performance gel permeation chromatography, and the content of neutral sugar, the content of acidic sugar and the content of protein are measured.

The high-efficiency gel permeation chromatograph is provided with three serially connected Ultrahydrogel (Waters, american) chromatographic columns (specifications are 250,1000and 2000, 30cm, 7.8mm and 6 mu mparticles respectively), the temperature of a column incubator is 40 ℃, the flow rate is 0.5mL/min, and the sample loading amount is 50 mu L. The column was calibrated using T-dextran (5.2X 103,4.86X 104,6.68X 105 Da).

(2) Sugar composition analysis of Rubus Corchorifolius polysaccharide

(a) Determination of neutral sugar content

The neutral sugar content of the polysaccharide was measured by the sulphuric acid phenol method and a standard curve was drawn using a glucose standard. And (3) taking 200 mu L of standard solution with gradient concentration, adding 200 mu L of 5% phenol solution, uniformly mixing, slowly adding 800 mu L of concentrated sulfuric acid into the system, shaking uniformly, standing at room temperature for 30min, fully cooling, measuring an absorption value at 490nm ultraviolet wavelength by taking a blank tube as a reference, and drawing a standard curve. And (3) taking 200 mu L of sample solution, operating according to the steps of the standard substance, and substituting the measured absorbance value into the standard curve to calculate the neutral sugar content of the sample.

(b) Determination of the acid sugar content

Preparing 1mg/mL galacturonic acid standard mother liquor, respectively taking 100 muL, 200 muL, 300 muL, 400 muL, 500 muL and 600 muL of galacturonic acid mother liquor, placing the galacturonic acid mother liquor in a 10mL volumetric flask, adding water to a constant volume, respectively sucking 1.0mL of standard solution, placing the standard solution in a 20mL test tube with a plug, slowly adding 6mL of sodium tetraborate-sulfuric acid solution into the system in an ice-water bath, uniformly mixing by using a vortex instrument, placing the system in a water bath boiling at 100 ℃ for heating for 5min, transferring the system into the ice-water bath for cooling, adding 100 muL of m-hydroxybiphenyl solution, uniformly shaking, measuring absorbance at the wavelength of 525nm, and drawing a standard curve by taking the concentration as the horizontal axis and the absorbance as the vertical axis. Weighing 1.0mg of rubus corchorifolius polysaccharide, placing in a 10mL volumetric flask, adding water to a constant volume, measuring an absorbance value according to the steps of a standard curve, and then substituting the absorbance value into the standard curve to calculate the content of uronic acid in a sample.

(c) Determination of protein content

Accurately weighing 100mg of bovine serum globulin, placing the bovine serum globulin in a 100mL volumetric flask, adding water to a constant volume, wherein the bovine serum globulin is a standard mother solution, respectively taking 100 mu L, 200 mu L, 300 mu L, 400 mu L, 500 mu L and 600 mu L of the standard mother solution, placing the standard mother solution in a 10mL volumetric flask, adding water to a constant volume, respectively sucking 1.0mL of the standard solution, respectively adding 5mL of Coomassie brilliant blue solution, shaking uniformly, and standing for 15min. After Coomassie brilliant blue is combined with protein, the solution changes from red to blue, the absorbance of the solution is measured at 595nm, and a standard curve is drawn by taking the concentration of the standard substance as a horizontal axis and the absorbance as a vertical axis. Weighing 1.0mg of sample, placing the sample in a 10mL volumetric flask, adding water to fix the volume, measuring the absorbance value according to the steps of a standard curve, and then substituting the standard curve to calculate the protein content in the sample.

(3) Monosaccharide composition analysis of Rubus Corchorifolius polysaccharide

The monosaccharide composition of the rubus polysaccharides was analyzed by High Performance Liquid Chromatography (HPLC) in combination with 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization.

(a) Derivatization of mixed monosaccharide standards

Weighing a proper amount of monosaccharide standard substances (glucose, galactose, rhamnose, mannose, glucuronic acid, galacturonic acid, xylose, arabinose and fucose), adding water to dissolve the monosaccharide standard substances to prepare a 5mg/mL solution, uniformly mixing 100 mu L of various monosaccharide standard solutions, adding 0.6M sodium hydroxide solution into the mixture, adding 200 mu L of 0.5mol/LPMP methanol solution (0.4355 g/5 mL) to seal, wrapping the mixture by tinfoil, uniformly mixing the mixture by vortex, reacting for 100min under the condition of 70 ℃ constant-temperature water bath, taking out the mixture to cool to room temperature, adding about 200 mu L of 0.3mol/L hydrochloric acid solution into the system to enable the solution to be neutral, adding water to 2mL, adding 2mL of chloroform to the solution, vortex and shaking, standing and layering, taking an upper aqueous phase solution, extracting the upper aqueous phase solution twice by using chloroform, filtering the aqueous phase solution by using a 0.22 mu M microporous filter membrane, and analyzing by HPLC.

(b) Sample preparation derivatization

A10 mg polysaccharide sample was weighed into a 20mL stoppered tube, and 3mL 2M trifluoroacetic acid (TFA) was added thereto, mixed uniformly, and then placed in a dry reactor to hydrolyze at 110 ℃ for 4 hours. The TFA solution was then removed by rotary evaporation under reduced pressure, and the acid-degraded residue was dissolved in a small amount of distilled water over multiple additions of water and rotary evaporation until the solution was not significantly sour. Derivatization of the acidolysis product of polysaccharide is carried out by adopting a standard PMP derivatization method.

(4) Methylation analysis of Rubus corchorifolius polysaccharide RCHP-S

(a) Reduction of uronic acids

30mg of acidic polysaccharide is weighed and dissolved in 10mL of 0.0432g/mL 75% aqueous solution of 1-cyclohexyl-2-morpholinoethylcarbodiimide p-toluenesulfonate, the pH value is adjusted to 4.75 by using 0.01M hydrochloric acid solution and 10% triethylamine and the mixture is left standing for two hours, 2M sodium borohydride solution is added dropwise into the system for 20mL while stirring is continued, and the pH value in the system is maintained at 7 by using 4M hydrochloric acid solution. Reducing for 2 hours, adding acetic acid to adjust the pH value to 6.75 to stop the reaction, dialyzing and freeze-drying the product to obtain the reduced raspberry polysaccharide.

(b) Methylation reaction

Weighing a rubus alceaefolius polysaccharide sample 5mg, placing the rubus alceaefolius polysaccharide sample in a vacuum drying oven, adopting anhydrous phosphorus pentoxide as a drying agent, carrying out vacuum drying for 10h, dissolving the rubus alceaefolius polysaccharide sample in 200 mu L dimethyl sulfoxide DMSO pre-dried by a molecular sieve, filling nitrogen into the system to replace air, adding 15mg of dry sodium hydroxide powder, filling nitrogen for protection, carrying out ultrasonic dissolution, then placing the reaction system in an ice water bath, solidifying the mixture, filling nitrogen again, slowly dropwise adding 100 mu L potassium iodide solution into the system, slowly raising the temperature of the system to 20 ℃, carrying out ultrasonic treatment for 30min while raising the temperature, then adding 1mL of 4mmol/L sodium sulfite aqueous solution into the system, uniformly mixing, adding 500 mu L chloroform, carrying out vortex oscillation, standing for layering, taking a chloroform layer, then carrying out repeated extraction for three times by using chloroform, combining the chloroform layer solution, adding anhydrous sodium sulfate into the chloroform phase for dewatering, carrying out centrifugal filtration, carrying out nitrogen blow-drying, carrying out infrared analysis on the methylation degree, and if hydroxyl in the polysaccharide is not completely replaced, repeating the process until the hydroxyl absorption peak in the infrared spectrum disappears.

(c) Hydrolysis

2mol/L of trifluoroacetic acid (1 mL) is added into the methylated product, the product is hydrolyzed at 120 ℃ for 3h, and methanol is added for rotary evaporation until the product is not sour.

(d) Reduction of

And adding 500 mu L of newly prepared 10mg/mL deuterated sodium borohydride into the system, reacting for 2h at room temperature, shaking the system up without interruption during the reaction, and then dropwise adding 4mol/L acetic acid until the system is neutral. Adding 200 mu L of methanol and blowing the mixture with nitrogen for drying, repeatedly adding the methanol and blowing the mixture with nitrogen for three times, and removing the excessive sodium borohydride as much as possible.

(e) Acetylation

Adding 500 mu L of newly prepared pyridine-acetic anhydride solution (1:1) into the system, reacting for half an hour at 120 ℃, then blowing dry with nitrogen, adding 500 mu L of methanol, blowing dry with nitrogen, repeatedly adding methanol and blowing dry with nitrogen for three times, adding 2mL of dichloromethane, whirling, shaking up, standing for layering, taking supernatant, concentrating to a proper volume, and analyzing by GC-MS (gas chromatograph-Mass spectrometer).

(f) GC-MS analysis of gas chromatography-mass spectrometer

By using Agilent 5975C equipment, carrier gas is high-purity helium, a chromatographic column is RXI-5SIL MS (300 multiplied by 0.25), the initial temperature is 140 ℃, the temperature rising speed is 5 ℃/min, the temperature rises to 240 ℃, the time is maintained for 10 minutes, the split ratio is 1.

(5) Nuclear magnetic resonance analysis of Rubus corchorifolius polysaccharide RCHP-S

Weighing 20mg of pure polysaccharide sample, dissolving in 2mL of deuterated water, lyophilizing, redissolving in 600 μ L of deuterated water, and performing polysaccharide extraction by using Brukeram 400 nuclear magnetic resonance spectrometer at room temperature ¹³ C NMR、 ¹ HNMR、 ¹ H-1H Cosy spectrum, ¹ H- ¹³ C HMBC Spectroscopy and ¹ H- ¹³ c HSQC spectrum scanning analysis.

3. In vivo anti-inflammatory Activity study of Rubus Coreanus polysaccharide

Female Kunming mice (5 weeks old) are about 25g, each group is divided into 6, and the stomach is respectively irrigated with (polysaccharide low dose group is 50mg/kg, high dose group is 200mg/kg and 50mg/kg positive drug sulfasalazine SSZ) for 1-13d, and pure water is used for comparison once a day. 4-8 days, 4% dextran sulfate (DSS, aladdin) aqueous solution is used instead, the mice are drunk freely for 5 days, different clinical symptoms such as diarrhea and hematochezia appear, the model establishment is shown to be successful, 9-13 days, the DSS is removed, all the mice are drunk freely after purified water is used, the experimental group continues to irrigate the stomach, and the mice are killed at 14 th day. The colon of mice of different groups is intercepted, the contents of IL-6 and TNF-alpha in intestinal tissues are measured by an ELISA kit, and are graded by a Disease Activity Index (DAI) for evaluation, the body weight of the mice is weighed every day for a fixed time from the first day of the experiment, and the color and the properties of the excrement of the mice are observed at the same time (table 1), wherein the normal excrement refers to formed hardness, the loose excrement refers to the shape-immaturity of the excrement but not adhered to the anus of the mice, and the loose excrement refers to the shape-immaturity of the excrement which can be adhered to the position near the anus of the mice.

TABLE 1 DAI scoring criteria

4. Characterization of the results

1. Extraction and separation of Rubus corchorifolius polysaccharide

As shown in Table 2, the crude polysaccharide RCHP was isolated from Rubus was obtained in a yield of 14.32%. After DEAE-52 column and gel chromatography, purified Rubus suavissimus polysaccharide was obtained, named RCHP-S with a yield of 3.46%.

TABLE 2 chemical characterization of Rubus corchorifolius polysaccharides

The molecular weight of the polysaccharide, the composition of the monosaccharides, the connection mode among the monosaccharides and other structural characteristics determine the type of the polysaccharide and have important influence on the biological activity of the polysaccharide. Uniformity and molecular weight of the raspberry polysaccharide were determined by HPGPC. HPGPC of Rubus corchorifolius polysaccharide shows singularity and pairThe peaks are called to indicate that the rubus corchorifolius polysaccharides are pure polysaccharides (figure 1). The standard curve of molecular weight-retention time measured by the standard glucan standard is log (Mw) = -0.1863T +12.13 ² =0.9876, the molecular weight of the rubus polysaccharide RCHP-S is calculated to be 13.15kDa based on HPGPC peak time. RCHP-S contains a large amount of uronic acid, the uronic acid content is 48.32%, and the protein content of RCHP-S is less than 1%.

2. Chemical composition analysis of Rubus Corchorifolius polysaccharide

As shown in fig. 2, the monosaccharide composition was measured by HPLC for pre-column PMP derivatization. The results show that RCHP-S consists of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, in a molar ratio of 1.52.

Methylation analysis is an indispensable experimental means for researching the types of glycosidic bonds and the linking mode in the polysaccharide. In this study, we performed methylation analysis of RCHP-S and combined with NMR spectroscopy to resolve the linkage pattern between monosaccharides in RCHP-S. For ease of description, the nine sugar residues analyzed by methylation are briefly described as residues A-I. As shown in Table 3, the raspberry polysaccharide RCHP-S was composed of 9 derivatives, namely 2,3,6-Me3-Gal,2,3,4,6-Me4-Gal,2,3-Me2-Gal,3,4-Me2-Rha,3-Me-Rha,2,3-Me2-Ara,2,3,5-Me3-Ara,2,3,6-Me3-Glc and 2,3,6-Me3-Man, in the monosaccharide composition results, the content of sugar residues Gal in RCHP-S is about one third of the content of sugar residues GalA, and since the linkage → 4,6) -Galp- (1 → this linkage is not necessarily Gal A, the ratio of the two linkages → 4) -Gal Ap- (1 → and → 4) -Galp- (1 → in the methylated sugar residues should be greater than 3:1.

TABLE 3 methylation analysis of RCHP-S after reduction

Method for preparing Rubus corchorifolius polysaccharide ¹ H NMR and ¹³ c NMR nuclear magnetic resonance spectrum, of RCHP-S ¹ In the H NMR nuclear magnetic resonance spectrum (FIGS. 3 and 4), a strong signal appears at the delta 3.78 positionNo. indicating that the sugar residue GalA was methyl-esterified, the proportion of methyl-esterification of the sugar residue GalA was about 74.67% as calculated from the ratio of the integration of the methyl-esterification hydrogen spectrum to the integration of the anomeric carbon point, and the signal at the δ 1.98 position indicating that the hydroxyl group at the 2-position of a part of the sugar residue GalA was acetylated, and the signal at the δ 1.25 position representing the methyl group at the 6-position on the Rha of the sugar residue.

In the HMBC spectrogram (FIG. 7), significant cross peaks of (A-C4, A-H1) and (A-C1, A-H4) are shown, indicating that most of the residues A are self-linked by the O-4 mode. The appearance of cross peaks (C-H1, E-C4) indicates that residue C is linked to residue E through O-4.

In RCHP-S ¹³ In the C NMR spectrum, we ascribed the signal appearing in the anomeric carbon region of the saccharide in combination with the results of the methylation analysis, the signal at 100.4ppm being C-1 for residue A. In addition, the signals at 170.8ppm and 175.2ppm are C-6 and acetyl for residue A, which also confirms that most of the carboxyl groups at position 6 of GalA in RCHP-S are methylated and some of the hydroxyl groups at position 2 are acetylated. The C-6 signal of residues D and E is present in the 16.5ppm region, which is the typical methyl signal peak for rhamnose. By HSQC (FIG. 6) and ¹ H- ¹ h COSY (FIG. 5) ascribes the chemical shifts of carbon and hydrogen on RCHP-S and the results are summarized in Table 4.

TABLE 4 chemical shift assignment of Rubus corchorifolius polysaccharide RCHP-S

The polysaccharide RCHP-S is analyzed by research to be a mixed pectin consisting of two structural regions of HG type and RG-I type. Wherein the HG type accounts for about 55%, the structure area is mainly a linear skeleton polymerized by 1,4-alpha-GalpA, and the esterification degree of carboxyl at C-6 position of galacturonic acid is about 50%. The RG-I type structural region, which accounts for about 45% of the polysaccharide RCHP-S, is a linear backbone consisting of → 2) - α -Rhap (1 → and → 4) - α -GalpA- (1 → alternate link and has a branch point at the C-4 position of the Rha sugar residue and a side chain consisting of galactose and arabinose, and has the following specific structure:

3. results of animal experiments

The control mice were fed with normal water and showed no significant weight loss and hematochezia. In the DSS group, mice developed obvious clinical symptoms after the third day by drinking DSS, mainly manifested by rough hair, weight loss, loose stools, blood stools, etc., but did not die, and the rubus polysaccharide high dose group was statistically significantly different from the model group in the DAI score after 10 days of treatment with the high and low dose of rubus polysaccharide and the positive drug sulfasalazine (fig. 8).

Inflammatory factors play a key role in inducing the progression of enteritis. We have found that the content of IL-6, TNF-alpha in colon tissues is obviously increased in a model group compared with a blank control group through detection of IL-6, TNF-alpha in colon tissues, compared with a DSS group, the polysaccharide group can obviously reduce IL-6 in colon tissues at high dose (figure 9A), and IL-6 has a very important effect on regulating inflammatory enteritis (IBD), and the result shows that the rubus corchorifolius polysaccharide can effectively inhibit cytokines, so that the symptoms of enteritis are improved. The detection of tumor necrosis factor TNF-alpha shows that the low-dose group of the Rubus corchorifolius polysaccharide can inhibit the secretion of the Rubus corchorifolius polysaccharide (figure 9B), and the result shows that the polysaccharide can improve the intestinal inflammation by inhibiting inflammatory factors.

In conclusion, the structure of the rubus corchorifolius pure polysaccharide is obtained by analyzing the chemical composition of the rubus corchorifolius pure polysaccharide RCHP-S; the anti-inflammatory effect of the rubus corchorifolius pure polysaccharide is evaluated by utilizing DSS to establish a colitis animal model experiment, and the result of treatment by giving the rubus corchorifolius pure polysaccharide RCHP-S shows that the rubus corchorifolius pure polysaccharide can reduce inflammatory factors generated by DSS-induced enteritis and relieve inflammatory reaction of DSS to intestinal tracts.

Claims

1. The application of Rubus idaeus pure polysaccharide RCHP-S in preparing a medicine for treating dextran sodium sulfate induced enteritis is characterized in that the separation and purification method of Rubus idaeus pure polysaccharide RCHP-S comprises the following steps:

(1) Extracting and separating the crude polysaccharide of the raspberry: drying and crushing fresh raspberry, adding ethanol for soaking and degreasing, filtering and centrifuging, extracting 4h from drug residues at 80 ℃ by using distilled water, repeating the three times of extraction, adding distilled water, filtering and centrifuging, combining filtrate and supernatant, concentrating, dialyzing 12h by using running water, dialyzing 24h by using distilled water, concentrating, adding ethanol into concentrated solution, placing 10h at 4 ℃, centrifuging and freezing at-80 ℃ to obtain crude raspberry polysaccharide, wherein the yield is 14.32 percent and the crude raspberry polysaccharide is named as RCHP;

a. and (3) anion exchange column chromatography separation: dissolving raspberry raw sugar RCHP in distilled water, then loading along the inner wall of a chromatographic column, adjusting the flow of the chromatographic column until the sample completely permeates into an anion exchange filler, sequentially washing with 20 times of column volume of distilled water, 0.1M sodium chloride solution, 0.2M sodium chloride solution and 0.5M sodium chloride solution at the flow rate of 3mL/min, combining the same effluent components, concentrating, dialyzing, and freeze-drying to obtain a polysaccharide sample, wherein the anion exchange column needs to be pretreated, specifically, an anion exchange filler DEAE-52 is soaked and washed with hydrochloric acid, then soaked and washed with sodium hydroxide for activation, then the pretreated DEAE-52 filler is loaded into the chromatographic column, and the filler is tightly and uniformly distributed by using 20 times of distilled water at a high flow rate for elution;

b. gel column chromatography purification of Rubus Suavissimus polysaccharide: dissolving a polysaccharide sample purified by DEAE-52 column chromatography in a sodium chloride solution of 0.2M, loading the sample along the inner wall of a chromatographic column, wherein the number of gel columns is two, and the fillers are G-25sephadex and Sephacryl S300 respectively, eluting with a sodium chloride solution of 0.2M, collecting and combining the samples, concentrating, dialyzing, and freeze-drying to obtain the rubus corchorifolius pure polysaccharide, wherein the yield is 3.46 percent and the name is RCHP-S.

2. The use according to claim 1, wherein the pure polysaccharide RCHP-S isolated by the method for isolating and purifying the pure polysaccharide RCHP-S has a molecular weight of 13.15kDa, and contains 48.32% uronic acid and less than 1% protein.

3. Use according to claim 2, characterized in that: the chemical composition of the raspberry pure polysaccharide RCHP-S comprises mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molar ratio of the raspberry pure polysaccharide RCHP-S to the galactose is 1.52:19.08:1.64:41.98:2.29:20.61:12.88.