Saccharum alhagi acidic polysaccharide as well as extraction and separation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly relates to saccharum alhagi acidic polysaccharide as well as an extraction and separation method and application thereof. The method for separating the polysaccharide in the saccharum alhagi comprises the following steps: degreasing the saccharum alhagi, removing impurities, then carrying out water extraction and alcohol precipitation to obtain crude saccharum alhagi polysaccharide, deproteinizing and decoloring the crude saccharum alhagi polysaccharide, and separating the decolored crude polysaccharide by a dialysis method to obtain high-molecular-weight polysaccharide and low-molecular-weight oligosaccharide, performing activity screening on the high-molecular-weight polysaccharide and the low-molecular-weight oligosaccharide through a mouse suffering from ulcerative colitis; or carrying out DEAE cellulose column chromatography separation on the decolored crude polysaccharide to obtain various gradient polysaccharides, and respectively carrying out sephadex column chromatography to obtain the homogeneous acidic polysaccharide. The obtained acidic polysaccharide is subjected to activity verification through an adult zebra fish model and a young zebra fish model suffering from ulcerative colitis, and results prove that the obtained acidic polysaccharide has the effects of relieving zebra fish intestinal injury, recovering mucus secretion, inflammatory cell aggregation and other symptoms.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and in particular relates to a spinosa acid polysaccharide and an extraction and separation method and application thereof. Background Art

[0002] Alhagi Honey is a light yellow granule formed by the evaporation of sugar juice secreted by the branches and leaves of Alhagi camelorum Fisch. in hot and dry weather. The main component of Alhagi Honey is sugar, and it tastes sweet. It is a commonly used medicinal material in traditional medicine. After being boiled in water, it has the effects of astringing the intestines, relieving pain, and nourishing and strengthening the body.

[0003] Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is mainly manifested by diarrhea and abdominal pain, and in severe cases, bloody stools. It is difficult to cure, has a high recurrence rate, and carries the risk of cancer. Existing first-line drugs have limited efficacy, and 5-aminosalicylic acid (5-ASA) is controversial because of its obvious hepatotoxicity. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a spinosa acidic polysaccharide and its extraction and separation method and application. The acidic polysaccharide extracted and separated by the present invention belongs to a traditional Chinese medicine plant polysaccharide, has high safety, few adverse reactions, and good therapeutic effect on ulcerative colitis.

[0005] The present invention provides a method for extracting and separating polysaccharides from thorny sugar, comprising the following steps:

[0006] Deproteinizing the crude polysaccharide of the spinosa truncatula to obtain the deproteinized crude polysaccharide;

[0007] Decolorizing the deproteinized crude polysaccharide to obtain decolorized crude polysaccharide;

[0008] The decolorized crude polysaccharide is subjected to DEAE cellulose column chromatography separation, wherein the DEAE cellulose column chromatography separation comprises: cellulose activation, column loading, sample loading and elution; the elution comprises water washing and salt washing, and the eluate obtained by the water washing is concentrated and dried to obtain a neutral sugar component CAH-0, and the salt washing comprises sequentially eluting with a first NaCl solution, a second NaCl solution, a third NaCl solution and a fourth NaCl solution, wherein the concentrations of the first NaCl solution, the second NaCl solution, the third NaCl solution and the fourth NaCl solution are 0.06-0.14, 0.16-0.24, 0.26-0.34 and 0.36-0.44 mol / L, respectively; and each eluate obtained by the salt washing is dialyzed, concentrated and dried to obtain multiple groups of polysaccharide components CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4;

[0009] Alternatively, the decolorized crude polysaccharide is dialyzed to obtain high molecular weight polysaccharides and low molecular weight oligosaccharides, respectively; the molecular weight cutoff of the dialysis bag used for the dialysis is 2000Da;

[0010] CAH-0, CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4 were subjected to dextran gel column chromatography to obtain neutral sugar, the first acidic polysaccharide, the second acidic polysaccharide, the third acidic polysaccharide and the fourth acidic polysaccharide.

[0011] Preferably, the crude polysaccharide of the thorn sugar is obtained by defatting and removing impurities from the thorn sugar, then extracting with water and precipitating with alcohol.

[0012] Preferably, the water extraction temperature is 65-75° C., and the time is 1-2 hours; and the volume concentration of ethanol in the mixed solution during the alcohol precipitation is 75-85%.

[0013] Preferably, the decolorization adopts macroporous resin decolorization, and the macroporous resin decolorization uses AB-8 macroporous adsorption resin.

[0014] Preferably, the DEAE cellulose column chromatography separation uses DEAE-52 cellulose.

[0015] Preferably, the concentration of the sample solution during loading is 5 mg / mL; and the elution rate is 1 mL / min.

[0016] Preferably, the dextran gel column chromatography uses dextran gel G-100.

[0017] Preferably, the dextran gel column chromatography further includes ultrafiltration, and the molecular weight cutoff of the ultrafiltration is 100 KDa.

[0018] The present invention also provides a spinosa polysaccharide obtained by the extraction and separation method described in the above technical scheme, wherein the spinosa polysaccharide includes a high molecular weight polysaccharide and an acidic polysaccharide, and the acidic polysaccharide includes a first acidic polysaccharide, a second acidic polysaccharide, a third acidic polysaccharide and a fourth acidic polysaccharide.

[0019] The present invention also provides the use of the spinosa polysaccharide in preparing medicine for treating ulcerative colitis.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The invention provides a method for extracting and separating polysaccharides from thorn sugar. The method comprises the following steps: defatting and removing impurities from thorn sugar powder, and then extracting with water and precipitating with alcohol to obtain crude polysaccharides; then removing protein from the crude polysaccharides, decolorizing with macroporous resin, collecting the decolorized crude polysaccharides, passing through an anion exchange column (DEAE cellulose column), gradient eluting with sodium chloride eluents of different concentrations, collecting the eluents for dialysis and desalting, and because many oligosaccharides are lost in the enrichment process, firstly separating the decolorized crude polysaccharides through a dialysis bag with a molecular weight cutoff of 2000Da, and performing activity screening, and finally determining that the high molecular weight polysaccharides in the crude polysaccharides have therapeutic effects, and then separating CAH-0.4 obtained by DEAE through a gel column to obtain a quaternary acidic polysaccharide with a relatively uniform molecular weight (molecular weight range of 158000-165000), and performing structural analysis on the uniform polysaccharide. The homogeneous acidic polysaccharide obtained in the present invention is verified for activity by using an adult zebrafish model and a juvenile zebrafish model suffering from ulcerative colitis. The results show that the homogeneous acidic polysaccharide obtained has the effects of alleviating intestinal damage in zebrafish, restoring mucus secretion, inflammatory cell aggregation and other symptoms. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 is the elution curve of DEAE;

[0024] Figure 2 Figure 2 is the molecular weight determination result of each DEAE gradient polysaccharide (a-e) by HPGPC method; Figure 3 is the molecular weight determination result of the macromolecule (CAH-1) obtained after dialysis of the decolorized crude polysaccharide;

[0025] Figure 3 and Figure 4 The structural identification results of low molecular weight oligosaccharides and high molecular weight polysaccharides (UV, infrared, monosaccharide composition, scanning);

[0026] Figure 5 This is the result of LC-MS detection of small molecular weight oligosaccharide samples;

[0027] Figure 6 The results of the mouse ulcerative colitis experiment and the concentrations of the pro-inflammatory factors IL-6 and TNF-α in the mice;

[0028] Figure 7 The results of each gradient polysaccharide dextran gel column chromatography;

[0029] Figure 8The results of molecular weight determination, infrared absorption spectrum determination, and monosaccharide composition determination of the spinosa polysaccharide CAH-0.4-1 are as follows;

[0030] Fig. 9 This is the result of scanning electron microscopy analysis of the spinosa polysaccharide CAH-0.4-1;

[0031] Figures 10 to 16 The results of one-dimensional nuclear magnetic resonance 1H-NMR, 13C-NMR, DEPT-135 and two-dimensional nuclear magnetic resonance COSY, HSQC, HMBC, and NOESY determination of the spinosa polysaccharide CAH-0.4-1;

[0032] Fig.17 This is the possible structural model of CAH-0.4-1;

[0033] Fig.18 and Fig.19 The effect of CAH-0.4-1 homogeneous polysaccharide on neutrophil migration in the zebrafish model of ulcerative colitis;

[0034] Fig. 20 Results of HE staining and AB-PAS staining of the intestine of adult zebrafish;

[0035] Fig.21 The present invention is a flow chart for preparing acidic polysaccharides. DETAILED DESCRIPTION

[0036] The present invention provides a method for extracting and separating polysaccharides from thorny sugar, comprising the following steps:

[0037] Deproteinizing the crude polysaccharide of the spinosa truncatula to obtain the deproteinized crude polysaccharide;

[0038] Decolorizing the deproteinized crude polysaccharide to obtain decolorized crude polysaccharide;

[0039] The decolorized crude polysaccharide is subjected to DEAE cellulose column chromatography separation, wherein the DEAE cellulose column chromatography separation comprises: cellulose activation, column loading, sample loading and elution; the elution comprises water washing and salt washing, and the eluate obtained by the water washing is concentrated and dried to obtain a neutral sugar component CAH-0, and the salt washing comprises sequentially eluting with a first NaCl solution, a second NaCl solution, a third NaCl solution and a fourth NaCl solution, wherein the concentrations of the first NaCl solution, the second NaCl solution, the third NaCl solution and the fourth NaCl solution are 0.06-0.14, 0.16-0.24, 0.26-0.34 and 0.36-0.44 mol / L, respectively; and each eluate obtained by the salt washing is dialyzed, concentrated and dried to obtain multiple groups of polysaccharide components CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4;

[0040] Alternatively, the decolorized crude polysaccharide is dialyzed to obtain high molecular weight polysaccharide (CAH-1) and low molecular weight oligosaccharide (CAH-2), respectively, and the molecular weight cutoff of the dialysis bag used for the dialysis is 2000Da;

[0041] CAH-0, CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4 were subjected to dextran gel column chromatography to obtain neutral sugar, the first acidic polysaccharide, the second acidic polysaccharide, the third acidic polysaccharide and the fourth acidic polysaccharide.

[0042] In the present invention, unless otherwise specified, the materials and equipment used are commercially available products in the art.

[0043] The method comprises defatting and impurity-removing the thorn sugar, and then performing water extraction and alcohol precipitation to obtain the thorn sugar crude polysaccharide.

[0044] In the present invention, the defatting and impurity removal preferably further comprises crushing and sieving the thorn sugar (medicinal material) to obtain thorn sugar powder; the sieving is preferably through a 100-mesh sieve.

[0045] In the present invention, the degreasing and impurity removal preferably include: using petroleum ether reflux degreasing and using ethanol solution leaching and impurity removal. The temperature of the petroleum ether reflux degreasing is preferably 55-65°C, more preferably 60°C, and the time is preferably 1-2h, more preferably 1.5h. The temperature of the ethanol solution leaching and impurity removal is preferably 65-75°C, more preferably 70°C, and the time is preferably 1-2h, more preferably 1.5h; the volume fraction of ethanol in the ethanol solution is preferably 95-100%; the role of the ethanol solution leaching and impurity removal is to remove pigments and small molecules.

[0046] In the present invention, the water extraction and alcohol precipitation preferably include: using water extraction, concentrating the obtained extract and using ethanol for alcohol precipitation, and the obtained precipitate is the crude polysaccharide of Saccharum officinale. The temperature of the water extraction is preferably 65-75°C, more preferably 70°C, and the time is preferably 1-2h, more preferably 1.5h; after the water extraction, it is preferably further filtered to remove the drug residue, and the number of filtrations is preferably 3-4 times. During the alcohol precipitation, the volume concentration of ethanol in the mixed solution is preferably 75-85%, more preferably 80%, and the alcohol precipitation time is preferably 24h; after the alcohol precipitation, it is preferably further washed to obtain the precipitate, and the washing reagent is preferably anhydrous ethanol.

[0047] After obtaining the crude polysaccharide of the thorny sorghum, the present invention sequentially removes protein and decolorizes the crude polysaccharide of the thorny sorghum to obtain decolorized crude polysaccharide; the decolorized crude polysaccharide is subjected to DEAE cellulose column chromatography separation to obtain a neutral sugar component CAH-0 and multiple groups of polysaccharide components CAH-0.1, CAH-0.2, CAH-0.3, and CAH-0.4.

[0048] In the present invention, the protein removal is preferably carried out by the Sevage method, and the Sevage method preferably includes the following steps: dissolving the crude polysaccharide of the thorny spruce in water to obtain a crude polysaccharide solution of the thorny spruce, mixing it with a chloroform-n-butanol mixture, separating the solid and the liquid, concentrating the obtained clear liquid, and drying it to obtain crude polysaccharide with protein removed. The mass concentration of the crude polysaccharide of the thorny spruce in the crude polysaccharide solution of the thorny spruce is preferably 10-20%; the volume ratio of chloroform and n-butanol in the chloroform-n-butanol mixture is preferably 4:1; the volume ratio of the crude polysaccharide solution of the thorny spruce to the chloroform-n-butanol mixture is preferably 4:1; the solid-liquid separation method is preferably centrifugation, the speed of the centrifugation is preferably 5000r / min, the temperature is preferably 4°C, the time is preferably 10min, and the number of times is preferably not less than 5 times; the concentration is preferably reduced pressure concentration, and the drying is preferably freeze drying.

[0049] In the present invention, the decolorization preferably adopts macroporous resin decolorization, and the macroporous resin decolorization preferably uses AB-8 macroporous adsorption resin. The macroporous resin decolorization preferably includes: activation, column loading, sample loading and elution of the macroporous resin, and the obtained eluate is concentrated and dried to obtain the decolorized crude polysaccharide. The activation is preferably soaked in anhydrous ethanol with a volume of 5 times that of the macroporous resin for 2 hours; the column loading is preferably wet column loading; the concentration of the sample solution during the sample loading is preferably 0.05g / mL, and the adsorption is preferably performed for 2 hours after the sample loading; the elution reagent is preferably water.

[0050] In the present invention, the DEAE cellulose column chromatography separation includes: cellulose activation, column loading, sample loading and elution; the elution includes water washing and salt washing, the eluate obtained by the water washing is concentrated and dried to obtain the neutral sugar component CAH-0, the salt washing includes sequentially eluting with a first NaCl solution, a second NaCl solution, a third NaCl solution and a fourth NaCl solution, and the concentrations of the first NaCl solution, the second NaCl solution, the third NaCl solution and the fourth NaCl solution are 0.06-0.14, 0.16-0.24, 0.26-0.34 and 0.36-0.44 respectively; each eluate obtained by the salt washing is dialyzed, concentrated and dried to obtain multiple groups of polysaccharide components CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4. The DEAE cellulose column chromatography separation preferably uses DEAE-52 cellulose, and the cellulose activation preferably uses water to swell the cellulose; the column packing preferably uses wet column packing; the concentration of the sample solution during loading is preferably 5 mg / mL; the elution rate is preferably 1 mL / min; the molecular weight cutoff of the dialysis bag used for dialysis is preferably 3500Da, the time is preferably 48h, and the function of the dialysis is to remove salt.

[0051] The concentrations of the first NaCl solution, the second NaCl solution, the third NaCl solution and the fourth NaCl solution are preferably 0.1, 0.2, 0.3 and 0.4 mol / L, respectively. The CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4 are acidic crude polysaccharide components obtained by separating crude polysaccharides by DEAE-52.

[0052] The present invention dialyzes the decolorized crude polysaccharide to obtain high molecular weight polysaccharides and low molecular weight oligosaccharides respectively; the molecular weight cutoff of the dialysis bag used in the dialysis is 2000Da.

[0053] In the present invention, the dialysis preferably includes: mixing the decolorized C. argentea crude polysaccharide with water to obtain a polysaccharide solution; placing the polysaccharide solution in a dialysis bag, and then placing the dialysis bag in water for dialysis. The concentration of the polysaccharide solution is preferably 0.05 g / mL water; the dialysis time is preferably 48 hours, and the water is preferably changed every 3 hours during the dialysis process; after the dialysis, it is preferably further reduced pressure concentration and freeze drying. The high molecular weight C. argentea polysaccharide (CAH-1) corresponds to the dialysate in the bag, and the low molecular weight C. argentea oligosaccharide (CAH-2) corresponds to the solution outside the dialysis bag.

[0054] After obtaining CAH-0, CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4, the present invention respectively performs dextran gel column chromatography on CAH-0, CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4 to obtain neutral sugar, first acidic polysaccharide, second acidic polysaccharide, third acidic polysaccharide and fourth acidic polysaccharide.

[0055] In the present invention, the dextran gel column chromatography preferably uses dextran gel G-100. The dextran gel column chromatography preferably includes: filler activation, column loading, sample loading and elution, and the obtained eluate is concentrated and dried to obtain acidic polysaccharides; the filler activation is preferably swelling the filler with water; the column loading is preferably wet column loading; the concentration of the sample solution during the sample loading is preferably 5 mg / mL; the elution reagent is preferably water.

[0056] In the present invention, the dextran gel column chromatography preferably further includes ultrafiltration, and the ultrafiltration preferably includes: dissolving the polysaccharide obtained by the dextran gel column chromatography in water, placing it in a 100KDa ultrafiltration centrifuge tube, centrifuging, dissolving and drying the non-permeated sample; the centrifugation speed is preferably 8000r / min, the temperature is preferably 4°C, the time is preferably 15min, and the number of times is preferably 3 times.

[0057] The present invention also provides a spinosa polysaccharide obtained by the extraction and separation method described in the above technical scheme, wherein the spinosa polysaccharide includes a high molecular weight polysaccharide and an acidic polysaccharide, and the acidic polysaccharide includes a first acidic polysaccharide, a second acidic polysaccharide, a third acidic polysaccharide and a fourth acidic polysaccharide.

[0058] In the present invention, the high molecular weight polysaccharide preferably includes mannose, rhamnose, galactose, galacturonic acid, glucose, glucuronic acid and arabinose; the mass ratio of the first acidic polysaccharide, the second acidic polysaccharide, the third acidic polysaccharide and the fourth acidic polysaccharide is preferably 1:1.3:1.35:1.35; the fourth acidic polysaccharide preferably includes galacturonic acid, galactose, rhamnose, glucuronic acid, glucose, mannose and arabinose, and the mass ratio of galacturonic acid (GalA), galactose (Gal), rhamnose (Rha), glucuronic acid (GlcA), glucose (Glc), mannose (Man) and arabinose (Ara) is preferably 73:12:7:4:3:1.

[0059] The present invention also provides the use of the acidic polysaccharide described in the above technical solution in the preparation of a medicine for treating ulcerative colitis.

[0060] In order to further illustrate the present invention, the spinosa acid polysaccharide and its extraction and separation method and application provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] 1. Extraction of crude polysaccharides from Saccharum officinale

[0063] The crushed thorn sugar medicinal material is subjected to water bath reflux degreasing treatment with petroleum ether (temperature: 60°C, time: 1.5h), and then the extract is filtered to obtain a precipitate and dried. The above-mentioned dried precipitate is extracted with 95% ethanol solution (temperature: 70°C, time: 1.5h) to remove pigments and small molecules, and the extract is filtered to obtain a precipitate and dried. The medicinal material precipitate after impurity removal with 95% ethanol solution is extracted with pure water in a constant temperature water bath at 70°C for 1.5h, the extract is filtered (removing the drug residue), and the filtrate is collected. Repeat three times until the solution is colorless, combine the filtrate and concentrate, then add anhydrous ethanol to 80% (v / v) alcohol precipitation concentration, and precipitate for 24h. Filter and collect the precipitate, wash it with anhydrous ethanol, and obtain a light yellow dry powder, which is the thorn sugar crude polysaccharide.

[0064] 2. Separation and purification of crude polysaccharides from Saccharum officinale

[0065] 1) Sevage method to remove protein

[0066] After the crude polysaccharide of the spinosa obtained in step 1 is dissolved in water (mass concentration is 10%), it is mixed with a chloroform: n-butanol mixture (volume ratio 4:1) at a volume ratio of 4:1, vortexed and mixed, and then centrifuged (centrifugation conditions: 5000r / min, 4°C, 10min), the white precipitate is removed, and the chloroform: n-butanol mixture, vortexed and centrifuged are repeatedly added 5 times to ensure that the protein component is completely removed. The supernatant after the protein is removed is concentrated under reduced pressure and vacuum freeze-dried to obtain the crude polysaccharide without protein.

[0067] 2) AB-8 macroporous adsorption resin decolorization

[0068] Activation of macroporous resin: Soak in anhydrous ethanol 5 times the volume of the resin for 2 hours, and stir from time to time to allow the resin to swell fully. When the macroporous resin is rinsed with anhydrous ethanol, the ethanol becomes clear. After alcohol treatment, rinse the resin continuously with deionized water to displace the ethanol and use it to pack the column.

[0069] Column packing, sample loading, and elution: wet column packing, using deionized water as the mobile phase, flush the column until the dripping liquid is clear, then weigh 5 g of crude polysaccharide except for protein, dissolve it with 100 mL of deionized water, draw the sample with a rubber-tipped dropper and slowly drip it onto the column, adsorb for 2 hours, elute with distilled water, collect the eluate, and use a-naphthol reaction to detect whether the elution is complete, concentrate the eluate under reduced pressure, and freeze-dry to obtain the decolorized crude polysaccharide.

[0070] 3) The decolorized crude polysaccharide is subjected to DEAE cellulose column chromatography

[0071] DEAE-52 cellulose activation: directly weigh the cellulose and swell it with excess deionized water. After swelling, it can be loaded into the column.

[0072] Column loading, sample loading, and elution: Directly wet-load the swollen cellulose into a glass chromatography column, weigh 500 mg of decolorized crude polysaccharide powder, dissolve it in 100 mL of ultrapure water, centrifuge to obtain the supernatant (3000 r / min, 4°C, 5 min), add the sample with a sterile dropper, and perform gradient elution with ultrapure water, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl solution, respectively, at an elution rate of 1.0 mL / min, and collect 10 mL of eluate in each tube. Detect the absorbance of each tube at 490 nm using the phenol-sulfuric acid method, and plot the DEAE elution curve based on the absorbance value versus the number of tubes (e.g. Figure 1 ). The eluates of each gradient were collected according to the elution curve, wherein the eluates of salt washing were placed in dialysis bags with a molecular weight cutoff of 3500Da, dialyzed with ultrapure water for 48 hours, concentrated under reduced pressure, and freeze-dried; the eluates of water washing were directly concentrated under reduced pressure and freeze-dried to obtain polysaccharides of each gradient (CAH-0, CAH-0.1, CAH-0.2, CAH-0.3, CAH-0.4).

[0073] 4) Alternatively, the decolorized crude polysaccharide is separated by dialysis: oligosaccharides and polysaccharides of different molecular weights in the crude polysaccharide of the spinosa truncatula are obtained by dialysis.

[0074] First, take 5g of the dried and decolorized crude polysaccharide of the spiny sugar cane and dissolve it in 100mL of water. Add the obtained polysaccharide solution to a pre-activated dialysis bag with a molecular weight cutoff of 2000Da, and then put the dialysis bag into a beaker filled with pure water for dialysis for 48 hours. Change the water every 3 hours, collect the dialysate containing high molecular weight polysaccharides in all bags, concentrate under reduced pressure, and freeze-dry to obtain high molecular weight spiny sugar cane polysaccharide (CAH-1). The solution outside the dialysis bag is also concentrated under reduced pressure and freeze-dried to obtain low molecular weight spiny sugar cane oligosaccharide (CAH-2).

[0075] Test Example 1

[0076] 1. Molecular weight determination

[0077] The HPGPC method was used, and high-performance gel permeation chromatography tandem columns were used for detection. A standard curve was made with reference to dextran standards (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 Da) (with the logarithmic value of the standard dextran Mw as the ordinate and the retention time as the abscissa), the regression equation was calculated, and the relative molecular weight of the polysaccharides in each gradient was obtained by substituting the polysaccharides in each gradient, as shown in FIG. Figure 2 The molecular weight of the water elution part is below 1000. The molecular weight of the polysaccharide of about 20000 (5%) and a large number of oligosaccharides with molecular weight below 2000 may be in the 0.1 gradient elution part; the molecular weight of the polysaccharide of about 80000 (20%) and 20000 (80%) in the 0.2 gradient elution part; a small amount of polysaccharide of about 10000 (8%) and oligosaccharide below 10000 in the 0.3 gradient elution part; the molecular weight of the polysaccharide of 150000 (80%) and a small amount of oligosaccharide of 6000 (20%) in the 0.4 gradient elution part. By calculating the yield of each component (as shown in Table 1 below), the yield of DEAE column chromatography is low. Most of the oligosaccharides may be adsorbed on the fiber column during elution. Moreover, after the polysaccharide is eluted by DEAE, it needs to be dialyzed for desalting. This process will also remove a large amount of oligosaccharides.

[0078] Table 1 Polysaccharide yields of each gradient

[0079]

[0080] Considering whether the active substance in the thorn sugar is an oligosaccharide or a polysaccharide, the next step is to segment it with a dialysis bag before DEAE elution, and then characterize each part and perform activity analysis.

[0081] 2. Separation and identification of different molecular weight sugars, namely high molecular weight spiny sugar polysaccharide (CAH-1) and low molecular weight spiny sugar oligosaccharide (CAH-2)

[0082] 1) Determination of total sugar content

[0083] After the polysaccharide content standard curve was established by phenol-sulfuric acid method (y = 0.0069x-0.0282, R 2 =0.9967), take 0.5mL of polysaccharide / oligosaccharide sample (1mg / mL), add water to 2mL, then add 1mL of 6% phenol and 5mL of concentrated sulfuric acid, shake well and let stand for 30 minutes, then measure the absorbance at 490nm. The total sugar content of each component is thus obtained. The total sugar content of low molecular weight oligosaccharide (CAH-2) is 96.4%, and the total sugar content of high molecular weight polysaccharide (CAH-1) is 46.1% (this method has limitations in measuring acidic polysaccharides, and the uronic acid content of high molecular weight polysaccharides is measured below).

[0084] 2) Determination of uronic acid content

[0085] The standard curve of uronic acid content (y = 0.0014x-0.0011, R 2 =0.9807), and the prepared polysaccharide solution was subjected to uronic acid content determination, and the uronic acid content in the high molecular weight polysaccharide was measured to be 53.8%.

[0086] 3) CAH-1 molecular weight determination

[0087] HPGPC method was used to detect using high performance gel permeation chromatography tandem columns. The results are as follows Figure 2 As shown in f, the main molecular weight of the crude polysaccharide macromolecules (CAH-1) is above 2000 Da and is unevenly distributed, among which there are polysaccharides with higher molecular weights.

[0088] 4) UV absorption spectrum measurement

[0089] Dissolve the macromolecular polysaccharide (CAH-1) and oligosaccharide sample (CAH-2) in distilled water to prepare an aqueous solution with a concentration of 1 mg / mL. Scan the UV absorption spectrum in the range of 200-800 nm to observe whether there are UV absorption peaks at 260 nm and 280 nm (characteristic absorption regions of nucleic acids and proteins) to determine the content of nucleic acids and proteins in the small molecular weight oligosaccharide and macromolecular weight polysaccharide samples. Figure 3 As shown, small molecular weight oligosaccharide and large molecular weight polysaccharide samples have no obvious ultraviolet absorption at 260nm and 280nm.

[0090] 5) Infrared absorption spectrum measurement

[0091] Weigh 5.0 mg of polysaccharide / oligosaccharide powder, add KBr powder and grind in an agate mortar under infrared light. Mix well, put the sample in a tablet pressing mold, and press the tablet under vacuum with a hydraulic press at a pressure of 27 MPa to obtain sample tablets and KBr tablets. First, use KBr tablets to detect once, subtract air background, and then 4000 cm -1 Up to 400cm -1 Scan within the range and observe the corresponding functional group peaks ( Figure 3 c, d), CAH-1 and CAH-2 are both between 3200-3500cm -1 A broad and blunt stretching peak appears at 2900 cm, which is the OH stretching vibration peak of the hydroxy compound; -1 There is an absorption peak around 1400cm, which is the CH stretching vibration peak; -1 There are absorption peaks around 1130cm -1 There are absorption peaks around 750-900cm -1 The presence of an absorption peak indicates the presence of an asymmetric epoxy ring; 800 cm -1 There are absorption peaks around 900cm -1 There are absorption peaks around, indicating that it contains β-glycosidic bonds. Among them, the absorption peak of CAH-2 is more obvious than that of CAH-1, which may be because CAH-1 contains a large number of polysaccharides with uneven molecular weight, while CAH-2 contains fewer oligosaccharides with relatively uniform molecular weight.

[0092] 6) Determination of monosaccharide composition

[0093] Weigh 5 mg (±0.05 mg) of polysaccharide sample accurately, add 1 mL of 2M TFA acid solution, and heat at 121°C for 2 hours. Blow dry with nitrogen. Add 3 mL of methanol to wash, blow dry again, and repeat the methanol washing twice. Add 5 mL of sterile water to dissolve, and transfer to a chromatographic bottle for testing.

[0094] Take the supernatant after dissolving with sterile water, and rotate to concentrate or blow dry with nitrogen. Add 1ml 2MTFA solution and heat at 121℃ for 2 hours. Pass nitrogen and blow dry. Add methanol to wash, blow dry again, and repeat the methanol washing twice. Add sterile water to dissolve and transfer to a chromatographic bottle for testing. Use the PMP derivatization method to analyze the monosaccharide composition of the treated polysaccharide / oligosaccharide through a liquid chromatography system to obtain the following Figure 4 The results shown in a and b show that the large molecular weight acidic polysaccharides are mainly composed of mannose, rhamnose, galactose, galacturonic acid, glucose, glucuronic acid, and arabinose, while the small molecular weight oligosaccharides are only composed of glucose.

[0095] 7) Scanning electron microscopy analysis

[0096] Place the polysaccharide / oligosaccharide sample on the black gel of the sample stage, spray gold, and observe the polysaccharide morphology in the field of view of the scanning electron microscope (SEM). Figure 4 In c), both the low molecular weight oligosaccharides and the high molecular weight acidic polysaccharides in the crude polysaccharides are in the form of tightly connected fragments with relatively stable structures.

[0097] 8) HPLC-MS analysis

[0098] Chromatographic conditions: Chromatography: Dinonex Ultimate 3000UHPLC; Chromatographic column: Thermo HypersilGOLD HILIC 250mm×4.6mm, 5.0μm; Column temperature: 30°C; Injection volume: 1.0μL; Mobile phase: A: 0.1% formic acid; B: acetonitrile; Gradient elution conditions are shown in Table 2:

[0099] Table 2 Gradient elution conditions

[0100] Time / min Flow rate (mL / min) A％ B％ 0 1.0 25 75 20 1.0 25 75

[0101] Mass spectrometry conditions: mass spectrometer: Thermo Scientific Q Exactive ion source: HESI; sheath gas rate: 40arb; auxiliary gas rate: 10arb; spray voltage: positive ion 3.8kV, negative ion 3.0kV; capillary temperature: 320℃; auxiliary gas temperature: 300℃; S-lens: 50%; scan mode: FullMs / dd-ms2 top10; scan range: primary scan: resolution 70000, range 100~1500m / z; secondary scan: resolution 17500, starting ion 50m / z; collision voltage: NCE15, 30, 45.

[0102] The results of LC-MS detection of small molecular weight oligosaccharide samples are as follows Figure 5 As shown, the relative molecular masses of low molecular weight oligosaccharides are 342 and 504 (387 and 549 are both [M+HCOO] - ), all produced characteristic fragment ions such as m / z 143, m / z 161, and m / z 179. Figure 5 In b, m / z 341 is the fragment ion obtained by secondary mass spectrometry scanning of the parent ion, and m / z 179 is the fragment ion of m / z 341 losing C6H 10 O5 obtained ion fragment, m / z 161 is m / z 341 lost C6H 12 The fragment ion obtained by O6, m / z 143 is the fragment ion obtained by the loss of H2O from m / z161; Figure 5 In c, m / z 503 is the fragment ion obtained by secondary mass spectrometry scanning of the parent ion, and m / z 323 is the fragment ion of m / z 503 losing C6H 10O6 obtained ion fragment. In summary, there are mainly two types of oligosaccharides: disaccharides and trisaccharides, and the monosaccharide composition is glucose. It can be inferred that the small molecular weight oligosaccharides in crude polysaccharides are disaccharides and trisaccharides composed of glucose.

[0103] 3. Activity screening

[0104] 1) Animal modeling, grouping and drug administration

[0105] The animal model used was a mouse model of ulcerative colitis induced by dextran sulfate sodium (DSS), 30 male Kunming mice, weighing 18-22g, 6 mice in each group. The grouping and administration methods were as follows: blank control group (Control): free drinking water every day, and normal saline was given by gavage from the eighth day of modeling; model group (DSS): 4% DSS was continuously given by gavage until the end of the experiment, and free drinking water was given during the experiment; positive control group (5-ASA): 4% DSS was given by gavage every day, and 5-aminosalicylic acid (200 mg / kg) was given from the eighth day of modeling; crude polysaccharide macromolecule group (CAH-1): 4% DSS was given by gavage every day, and CAH-1 (200 mg / kg) was given from the eighth day of modeling; crude polysaccharide small molecule group (CAH-2): 4% DSS was given by gavage every day, and CAH-2 (200 mg / kg) was given from the eighth day of modeling. The experiment lasted for 14 days. After the experiment, all mice were sacrificed for sampling. Blood samples were collected from the eyeballs and placed in 2 mL centrifuge tubes at -4 ° C for 20 min. Serum was separated by centrifugation (3000 rpm, 4 ° C, 10 min) and stored at -80 ° C. The spleen tissue of each group of mice was weighed and the spleen index was calculated. The intestinal tissue from the ileum to 2 cm above the anus of the mouse was separated. After taking pictures and measuring the length, the intestinal tube was rinsed with PBS, part of which was stored in a -80 ° C refrigerator, and part was fixed in 4% paraformaldehyde solution for subsequent HE staining analysis.

[0106] 2) Disease Activity Index (DAI) Assessment

[0107] During the experiment, the weight of the mice, the viscosity of the feces, and the blood in the feces need to be recorded every day, and then the scores are assigned according to the scoring criteria and proportions. The average value is the DAI of the mice. The severity of the disease is proportional to the DAI score. The scoring table is shown in the following 3:

[0108] Table 3 Disease Activity Index (DAI) Scoring Table

[0109] Stool consistency Blood in stool Percentage of weight loss Fraction normal none 0 0 Molded but soft and sticky Black stools 1-5％ 1 Loose Slight bloodshot 5-10％ 2 Loose stools Naked bloody stool 10-15％ 3 diarrhea Heavy blood in the stool >15 4

[0110] 3) Histopathological evaluation

[0111] First, the colon fixed in 4% paraformaldehyde was dehydrated, wax-impregnated, and embedded. The embedded wax block was then cut into 4-5 μm slices, fixed on a slide, and then stained with hematoxylin-eosin.

[0112] 4) Determination of inflammatory cytokines

[0113] The results of the mouse ulcerative colitis experiment are as follows Figure 6 , the UC mouse model was induced by DSS, and then oligosaccharides and acidic polysaccharides of different molecular weights were given for treatment. It was finally found that the therapeutic effect of high molecular weight crude polysaccharides was much better than that of low molecular weight oligosaccharides. Colon shortening is an important phenotypic symptom of colitis. After the experiment, the colon length of each group of mice was measured. Compared with the Control group, DSS caused the colon of mice to be significantly shortened and twisted, and the feces in the colon were unformed and accompanied by blood. Treatment with CAH-1 significantly improved the colon shortening caused by DSS, and the colon length of the CAH-2 group was not much different from that of the model group. In terms of body weight, compared with the mice in the control group, the weight of the mice in the DSS group continued to decrease, while the weight of the mice in the CAH-1 group began to gradually increase, and the weight of the mice in the CAH-2 group did not change significantly; the same trend was shown in the spleen index and DAI score. Histological evaluation further analyzed the therapeutic effect on mice with DSS-induced colitis. H&E staining showed that the colon tissue structure of the mice in the Control group was basically normal, the colon mucosa was intact, and the goblet cells were arranged in an orderly manner in the crypts. Compared with the control group, the mice in the DSS group showed severe mucosal ulcers, inflammatory cell infiltration of mucosal tissue, disappearance of goblet cells, crypt damage and other histopathological changes in the colon tissue, and the pathological score increased significantly. After CAH-1 treatment, the colon lesions were improved and the pathological score decreased, but the CAH-2 group still had severe mucosal ulcers, inflammatory cell infiltration, disappearance of goblet cells, and the degree of decrease in pathological score was not obvious. The above experiments all show that the active role in the crude polysaccharide of the spinosa truncatula is the macromolecular polysaccharide.

[0114] The severity of inflammation in mice is related to the concentration of pro-inflammatory factors IL-6 and TNF-α. The levels of pro-inflammatory factors in the collected mouse serum were measured by enzyme-linked immunosorbent assay (ELISA), such as Figure 6 As shown in h, i, and j.

[0115] The present invention firstly crushes and sieves the thorn sugar to obtain the thorn sugar powder, defattes and removes impurities through petroleum ether and ethanol, and then extracts and precipitates the crude polysaccharide through water extraction and alcohol precipitation. The crude polysaccharide is decolorized by macroporous resin, and the decolorized crude polysaccharide is collected and gradient eluted by anion exchange column with sodium chloride eluents of different concentrations, and the eluent is collected and dialyzed for desalination. After the above steps, the thorn sugar is found to contain a large amount of oligosaccharides (oligosaccharides) after ion exchange, and these oligosaccharides are often removed in the anion exchange column chromatography process and the dialysis process, and the obtained acidic polysaccharides of different gradients are all macromolecules with a molecular weight of more than 2000. Considering that so many oligosaccharides are removed, a dialysis bag with a molecular weight cutoff of 2000Da is used for separation before the anion exchange column chromatography, and the dialysate outside the dialysis bag is decompressed and concentrated, and freeze-dried to obtain oligosaccharides with a molecular weight of less than 2000. The obtained oligosaccharide powder is then analyzed by liquid chromatography-mass spectrometry (LC-MS), and the obtained macromolecular polysaccharide is subjected to a series of characterizations such as molecular weight, monosaccharide composition, infrared chromatography, etc. Finally, the obtained macromolecular polysaccharide and low molecular weight oligosaccharide are screened for activity on a mouse model suffering from ulcerative colitis. The present invention uses dialysis to separate the oligosaccharide part and the polysaccharide part of the spiny sugar polysaccharide for the first time. The separated oligosaccharide is structurally identified by liquid chromatography-mass spectrometry technology, and at the same time, combined with infrared spectroscopy, ultraviolet spectroscopy, monosaccharide composition analysis and other technologies, the oligosaccharide part of the crude polysaccharide is analyzed by liquid chromatography-mass spectrometry for the first time, and the structure of most of the sugar substances discarded during the purification of the spiny sugar crude polysaccharide is clarified. On this basis, the activity is verified using an ulcerative colitis model, and it is determined that the macromolecular polysaccharide plays a major role in ulcerative colitis, and has the effect of alleviating symptoms such as shortened colon, loose stools, and bloody stools caused by ulcerative colitis. And according to the GPC molecular weight results, the molecular weight of neutral sugars obtained in the water elution part is all below 1000. It is inferred that neutral sugars are contained in CAH-2, and it is inferred that acidic polysaccharides are the main active substances. The next goal is to continue to purify acidic polysaccharides in each gradient.

[0116] Example 2 Structural analysis of homogeneous polysaccharides

[0117] In Example 1, the yield of polysaccharides eluted by 0.4 mol NaCl solution was the highest among acidic sugars, and when the effective components were screened by using the ulcerative colitis mouse model, it was concluded that the high molecular weight polysaccharide (CAH-1) had a good repair effect. Subsequently, the high molecular weight polysaccharides eluted preferentially were enriched during G100 separation, and the polysaccharides in the gradient were enriched for structural analysis and activity verification.

[0118] 1. After obtaining polysaccharides of various gradients (CAH-0, CAH-0.1, CAH-0.2, CAH-0.3, CAH-0.4) according to the method of Example 1, dextran gel column chromatography was performed

[0119] Activation of dextran gel G-100: The filler was swelled in excess deionized water for 24 h, and the upper floating matter was removed.

[0120] Column packing, sample loading, and elution: The swollen column filler is wet packed into the glass chromatography column. Weigh 50 mg of each component after gradient elution with DEAE cellulose column, dissolve it completely with 10 mL ultrapure water, and slowly add the sample along the wall of the tube with a sterile dropper. Use ultrapure water for elution, 10 mL per tube. At the same time, use the phenol-sulfuric acid method to measure the absorbance of each tube at 490 nm, collect the eluate, concentrate it under reduced pressure, and freeze-dry it to obtain high-purity polysaccharides. Use gel columns to purify all gradients. The results are as follows: Figure 7 , Figure 8 As shown in a (in the figure, CAH-0, CAH-0.1, CAH-0.2, CAH-0.3, and CAH-0.4 are all components after dextran gel column chromatography), CAH-0.4 is easier to obtain purer high molecular weight polysaccharides than other gradients.

[0121] 2. Ultrafiltration

[0122] Using Millipore 15mL 100KDa ultrafiltration centrifuge tube, the polysaccharide sample CAH-0.4 was dissolved in ultrapure water and centrifuged (8000r / min, 4°C, centrifugation for 15min), repeated three times. Ultrapure water was added to dissolve the unpermeated sample, and freeze-dried to obtain the spiny acid polysaccharide CAH-0.4-1.

[0123] Test Example 2

[0124] 1. CAH-0.4-1 molecular weight, infrared absorption spectrum and monosaccharide composition determination, scanning electron microscopy analysis

[0125] HPGPC method was used to detect the molecular weight of CAH-0.4-1 using high performance gel permeation chromatography columns. Infrared absorption spectrometry and monosaccharide composition of CAH-0.4 were performed. Figure 8 shown. Figure 8 In b, CAH-0.4-1 is a single and symmetrical peak with a relative molecular weight of approximately 163491 Da. Its infrared spectrum conforms to the characteristic absorption of polysaccharides at 3406 cm -1 A broad and blunt stretching peak appears at 2923cm, which is the OH stretching vibration peak of the hydroxy compound; -1 There is an absorption peak around 1417cm -1 There are absorption peaks on the left and right, which are the OH in-plane bending vibration peaks; 1145cm -1 There are absorption peaks around 750-900cm -1The presence of an absorption peak indicates the presence of an asymmetric epoxy ring; 800 cm -1 There are absorption peaks around 900cm -1 There are absorption peaks around, indicating that it contains β-glycosidic bonds. The monosaccharide composition results show that the polysaccharide contains a large amount of galacturonic acid, a small amount of galactose, rhamnose, glucuronic acid and glucose, and may contain very small amounts of mannose and arabinose (GalA:Gal:Rha:GlcA:Glc:Man:Ara mass ratio = 73:12:7:4:3:1).

[0126] The results of scanning electron microscopy analysis are as follows Fig. 9 As shown, the polysaccharide has a relatively stable structure and is in the form of tightly connected fragments.

[0127] 2. Methylation analysis

[0128] (1) Polysaccharide pretreatment: ① Weigh 5 mg of polysaccharide sample, add 1 mL of ultrapure water to dissolve, add 200 μL of 0.2 M MES hydrate, and then add 500 μL of 200 mg / mL carbodiimide, and react at room temperature for 2 h. Add 1 mL of 2 M imidazole, add 1 ml of 70 mg / mL sodium borodeuteride (NaBD4), and react for 3 h. ② Add 300 μL of glacial acetic acid to terminate the reaction. Dialyze the sample for 48 h, freeze-dry the sample after dialysis, and perform methylation treatment. ③ Add 1 mL of DMSO to the freeze-dried sample to dissolve. Add 30 mg of NaOH and incubate for 30 min. Add 250 μL of iodomethane solution, fill with nitrogen, react for 1 h in the dark, and add 250 μL of iodomethane solution again to react for 1 h. Add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. ④ Absorb the lower dichloromethane phase and blow dry with nitrogen. Add 1mL 2M TFA and react at 121℃ for 120min. Blow dry with nitrogen at 30℃. ⑤ Add 1mL freshly prepared 1MNaBD4 (in ammonia water). Incubate with magnetic stirring at room temperature for 2.5 hours. Add 300μL acetic acid to terminate the reaction and blow dry with nitrogen. Dissolve with 2mL 5% (vol / vol) acetic acid in methanol, dry twice in nitrogen at 40℃, then dissolve with 2mL methanol, and dry twice in nitrogen at 40℃. Add 1.5mL acetic anhydride, vortex to mix, and react at 100℃ for 2.5h. Add 2mL water and let stand for 10min. Add 1mL dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the lower dichloromethane phase and detect it on the machine.

[0129] (2) Chromatographic parameters: The chromatographic system used an Agilent gas chromatography system (Agilent 7890A; Agilent Technologies, USA), an HP-5MS capillary column (30 m×0.25 mm×0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), a carrier gas of high-purity helium (purity not less than 99.999%), a flow rate of 1.0 mL / min, and an injection port temperature of 260°C. The injection volume was 1 μL, split injection, split ratio 10:1, and solvent delay 2.2 min.

[0130] Temperature program: 50°C for 1.0 min, increase the temperature to 130°C at 50°C / min, increase the temperature to 230°C at 3°C / min, and maintain for 2 min.

[0131] (3) Mass spectrometry parameters: The mass spectrometry system used a quadrupole mass spectrometry detection system (Agilent5977B; Agilent Technologies, USA) from Aiglent, USA, equipped with an electron impact ion source (EI) and a MassHunter workstation. The electron impact ion source (EI), injection port temperature was 230°C, quadrupole temperature was 150°C, and electron energy was 70 eV. The scanning mode was full scan mode (SCAN), and the mass scanning range (m / z): 30-600.

[0132] (4) Qualitative and quantitative analysis: Based on the relative retention time and mass spectrum of each chromatographic peak and the ion fragment mass spectrum of the Complex Carbohydrate Research Center database of the University of Georgia (https: / / glygen.ccrc.uga.edu / ccrc / specdb / ms / pmaa / pframe.html), the type of sugar residue corresponding to each chromatographic peak was analyzed, and the content of each was calculated using the chromatographic peak area of ​​the corresponding sugar residue type. The results are shown in Table 4.

[0133] Table 4 Methylation analysis results

[0134]

[0135]

[0136] 3. Nuclear Magnetic Resonance Analysis

[0137] The structural characteristics of the polysaccharide were further obtained by nuclear magnetic resonance analysis. One-dimensional nuclear magnetic resonance 1H-NMR, 13C-NMR, DEPT-135 and two-dimensional nuclear magnetic resonance COSY, HSQC, HMBC, NOESY were measured. The spectra are shown in Figures 10 to 16 As shown, all H and C chemical shift information of each major sugar residue is obtained to infer the connection order between each sugar residue.

[0138] 1H-NMR spectrum of polysaccharide samples ( Fig.10 ) are concentrated in the δ3.0-5.5ppm region, with serious signal overlap. In the anomeric region of δ4.4-5.5ppm, multiple anomeric hydrogen signals are found, indicating that there are multiple sugar residues, and there is a certain overlap, which is difficult to distinguish. Other hydrogen signals are concentrated in the δ4.4-3.0ppm region, with serious signal overlap and difficult to attribute. Compared with the 1H-NMR spectrum, the 13C-NMR spectrum of the polysaccharide sample ( Fig.11 ), there are fewer spectral lines, and there are multiple anomeric carbons in the δ90-110ppm anomeric carbon region, indicating that there are multiple sugar residues in the polysaccharide sample.

[0139] From the 1H-NMR and 13C-NMR spectra, some characteristic signal peaks were found: (1) The resonance signal located in the high field region of δ1.10-1.30 ppm is a typical chemical shift of the methyl proton H-6 of the rhamnose (Rha) residue. The special deoxygenated structure of the methyl carbon C-6 of rhamnose is at δ16.58 ppm. Fig.13 ) found a cross peak of δ1.16 / 16.58ppm on the H-NMR spectrum. The peak signal intensity on the hydrogen spectrum and carbon spectrum was weak, indicating the presence of a small amount of rhamnose residues, which was consistent with the monosaccharide composition results; (2) The δ1.90-2.20ppm region in the 1H-NMR spectrum was the methyl proton signal of the O-acetyl group, and there were multiple signal peaks, indicating that the acetyl substitution position occurred at different positions of the sugar residue in the sugar chain. The carbon spectrum was located near δ20.00ppm and the methyl signal within the acetyl group had a very small peak signal intensity. Based on this, it can be inferred that this polysaccharide sample has a low degree of acetylation; (3) The monosaccharide composition of the polysaccharide sample contains a large amount of galacturonic acid. The characteristic absorption peak of the C-6 position of the unesterified galacturonic acid residue is near δ175.27ppm, and the characteristic absorption peak of the C-6 position of the esterified galacturonic acid residue is near δ170.99ppm. The peak signal intensity of the unesterified peak is higher than that of the esterified peak. These characteristic signals indicate the presence of a large amount of unesterified galacturonic acid.

[0140] There is a strong signal near δ97~99ppm in the anomeric region, which can be inferred to be the anomeric carbon signal of the sugar residue α-GalpA. There is a strong signal near δ102~106ppm in the anomeric region, which can be inferred to be the anomeric carbon signal of the sugar residue β-Galp. There is a signal at δ106~110ppm in the anomeric region, which is inferred to be the anomeric carbon signal of the sugar residue α-Araf. Through monosaccharide composition, methylation analysis and nuclear magnetic resonance analysis, it was found that there were multiple significant anomeric region signal peaks in the polysaccharide samples, which can be used for structural analysis. The chemical shifts of these anomeric region signals were δ4.97 / 98.93ppm, δ4.93 / 99.02ppm, δ5.08 / 98.07ppm, δ4.88 / 99.02ppm, δ4.98 / 98.90ppm, δ5.17 / 98.46ppm, δ5.17 / 98.46ppm, δ5.16 / 109.02ppm, δ5.00 / 107.38ppm, δ4.44 / 102.69ppm, δ4.38 / 103.15ppm, δ4.58 / 103.62ppm and δ4.41 / 102.69ppm. After the attribution of the anomeric signal was determined, COSY, HSQC, HMBC, NOESY and HSQC-TOCSY were used, combined with 1H-NMR, 13C-NMR spectra, monosaccharide composition and methylation analysis results, and compared with the chemical shift data of similar sugar residue substitutions in related literature. The above sugar residues were marked as GA1,4, GA1,3,4, GA1,2,4, GE1,4, GAt, Rha1,2 and Rha1,2,4, At, A1,5, G1,6, G1,3,6, G1,3, Gt in turn, and the 1H and 13C chemical shift signals of the main types of sugar residues in the polysaccharide samples were assigned. The results are shown in Table 5. The NMR analysis of the main sugar residue structures is as follows:

[0141] Sugar residue GA1,4: According to HSQC and COSY, the anomeric signals of the sugar residue were δ4.97ppm (H-1) and δ98.93ppm (C-1), indicating that the sugar residue was in α configuration. Based on the results of monosaccharide composition test and methylation analysis, it was inferred that the residue was α-GalpA sugar residue. The H-1 chemical shift of the sugar residue was determined to be δ4.97ppm by HSQC and COSY, and then by COSY( Fig.14 ) spectrum cross peaks can be deduced H-2, H-3, H-4 signals, the H-2, H-3, H-4 chemical shifts of the sugar residue are assigned to δ3.67ppm, δ3.90ppm, δ4.33ppm respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the HSQC correlation spectrum ( Fig.13) The chemical shifts of C-1 to C-4 on the sugar ring were assigned to δ98.93ppm, δ67.95ppm, δ68.65ppm, and δ77.57ppm, respectively, and the chemical shift of C-6 was δ175.27ppm, which is the characteristic absorption peak at the C-6 position in the unesterified galacturonic acid residue. The chemical shifts of C-1 and C-4 shifted to the low field, indicating that the residue was substituted at the C-1 and C-4 positions of the sugar ring. Combined with the methylation results, it was inferred that the sugar residue was →4)-α-GalpA-(1→, marked as GA1,4, and its chemical shift assignment is shown in Table 5.

[0142] Table 5 1H and 13C chemical shift assignments of each sugar residue in polysaccharide samples

[0143]

[0144]

[0145] According to a similar method, combined with the results of monosaccharide composition determination and methylation analysis, the hydrogen and carbon signals of other residues were deduced, and GA1,3,4 were inferred to be →3,4)-α-D-GalpA-(1→, GA1,2,4 were inferred to be →2,4)-α-D-GalpA-(1→, GE1,4 were inferred to be →4)-α-D-6-OMe-GalpA-(1→, GAt were inferred to be α-D-GalpA-(1→, Rha1,2 were inferred to be →2)-α-L-Rhap-(1→, Rha1, 2,4 is →2,4)-α-L-Rhap-(1→, At is inferred to be α-L-Araf-(1→, A1,5 is inferred to be →5)-α-L-Araf-(1→, G1,6 is inferred to be →6)-β-D-Galp-(1→, G1,3,6 is inferred to be →3,6)-β-D-Galp-(1→, G1,3 is inferred to be →3)-β-D-Galp-(1→, Gt is inferred to be β-D-Galp-(1→, The chemical shift assignments of the main sugar residues in the polysaccharide samples are summarized in Table 5.

[0146] According to the results of methylation analysis, the polysaccharide samples also contain 1,2-Xylp, 1,4-Xylp and other connection modes, and the signals in NMR are very weak, and it is impossible to attribute their 1H and 13C.

[0147] Through the coupling signals of the anomeric hydrogen and the carbon on each sugar residue, or the coupling signals of the anomeric carbon and the hydrogen on each sugar residue in the HMBC long-range correlation spectrum, and the two protons at the connection sites of adjacent sugar residues are close in space, they are easy to produce strong NOE signals. The HMBC long-range correlation spectrum and NOESY spectrum can further infer the mutual connection order between each sugar residue. HMBC correlation spectrum of polysaccharide sample ( Fig.15 ) and NOESY spectra ( Fig.16), the following coupling signals can be found in the figure: (1) In the NOESY spectrum, H-1 (δ4.97ppm) of sugar residue GA1,4 and H-4 (δ4.33ppm) of sugar residue GA1,4 have a cross peak (GA1,4H-1 / GA1,4H-4), indicating the existence of a →4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→ connection; (2) In the NOESY spectrum, sugar residue R There is a cross peak (Rha1,2,4H-1 / GA1,4H-4) between H-1 (δ5.17ppm) of ha1,2,4 and H-4 (δ4.33ppm) of sugar residue GA1,4, indicating the existence of →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→ connection, and the connection site is located at O-4; (3) H-1 (δ4.3 8ppm) and H-3 (δ3.86ppm) of sugar residue G1,3 have a cross peak (G1,3,6H-1 / G1,3H-3), indicating the existence of →3,6)-β-D-Galp-(1→3)-β-D-Galp-(1→ cross-linking, and the linking site is located at the O-3 position; (4) In the NOESY spectrum, H-1 (δ4.44ppm) of sugar residue G1,6 and H-6 ( δ3.88ppm) has a cross peak (G1,6H-1 / G1,3,6H-6), indicating the existence of →6)-β-D-Galp-(1→3,6)-β-D-Galp-(1→ connection, and the connection site is located at the O-6 position; (5) In the NOESY spectrum, there is a cross peak between H-6 of sugar residue G1,6 (δ3.96ppm) and H-1 of sugar residue Gt (δ4.41ppm) (G1,6H-6 / Gt H-1), indicating the existence of β-D-Galp-(1→6)-β-D-Galp-(1→6) linkage, with the linkage site located at O-6; (6) in the HMBC spectrum, there is a cross peak between the H-1 (δ5.00ppm) of sugar residue A1,5 and the C-5 (δ66.77ppm) of A1,5 (A1,5H-1 / A1,5C-5), and in the NOESY spectrum, there is a cross peak between the H-1 (δ5.00ppm) of sugar residue A1,5 and the H-5 (δ3.71ppm) of A1,5 (A1,5H-1 / A1,5H-5), indicating the existence of →5)-α-L-Araf-(1→5)-α-L-Araf-(1→ connection, and the connection site is located at O-5; (7) In the NOESY spectrum, there is a cross peak (A1,5H-1 / Rha1,2,4H-4) between the H-1 (δ5.00ppm) sugar residue of the sugar residue A1,5 and the H-4 (δ3.62ppm) of Rha1,2,4, indicating the existence of →5)-α-L-Araf-(1→2,4)-α-L-Rhap-(1→ connection, and the connection site is located at O-4.(8) In the NOESY spectrum, there is a cross peak (G1,3H-1 / Rha1,2,4H-4) between the H-1 (δ4.58ppm) of the sugar residue G1,3 and the H-4 (δ3.62ppm) of Rha1,2,4, indicating the existence of a →3)-β-D-Galp-(1→2,4)-α-L-Rhap-(1→) linkage, with the linkage site located at O-4.

[0148] The analysis of the monosaccharide composition, methylation results, and one-dimensional and two-dimensional NMR information of the polysaccharide samples showed that the polysaccharide sample was a complex polysaccharide, which was mainly composed of HG-type domains and contained RG-I-type domains. The side chains were mainly composed of AG-I arabinogalactan and galactan polysaccharide composed of →3)-β-D-Galp-(1→. In the methylation results, the ratio of the three sugar residues of 4-GalA(p), 2,4-Rha(p), and 2-Rha(p) was close to 61:1:4. Its possible structural model is as follows: Fig.17 shown.

[0149] 4. CAH0.4-1 homogeneous polysaccharide activity verification

[0150] 1) Experiment on ulcerative colitis in juvenile zebrafish

[0151] Juvenile zebrafish modeling, grouping and drug administration: Select healthy Tg (lyzc: dsred) type 3dpf zebrafish, 6 tails / group were placed in a 24-well plate, a total of 8 groups, with duplicate wells, grouping and drug administration as follows: blank control group (Control): Normally raised in E3 medium every day; model group (DSS) was continuously induced with 0.25% DSS until the end of the experiment; positive control group (5-ASA): 100μg / mL 5-ASA solution was given from the fourth day after 0.25% DSS induction until the end of the experiment; polysaccharide administration group: polysaccharide concentrations were divided into five groups according to 5, 25, 50, 100, and 200μg / mL. Polysaccharide aqueous solution was given from the fourth day after 0.25% DSS induction until the end of the experiment. After the experiment, the aggregation of neutrophils was observed under a fluorescence microscope.

[0152] The results are as follows Fig.18 , Fig.19 There was almost no aggregation of neutrophils in the intestine of the normal group. After DSS modeling, neutrophils aggregated significantly in the intestine. Compared with the positive drug 5-ASA, the treatment effects of 5 and 25 μg / mL were not obvious. There was a slight improvement after 50 μg / mL treatment. After 100 and 200 μg / mL polysaccharide treatment, the effect was similar to that of the positive drug 5-ASA.

[0153] 2) Adult zebrafish ulcerative colitis experiment

[0154] 60 adult wild-type AB zebrafish were randomly divided into a blank control group, a DSS model group (0.5% DSS solution), a 5-ASA group, and a CAH-0.4-1 treatment group (50, 100, 200 μg / mL), with 10 fish in each group. Except for the blank control group, the other groups were first fed with 0.5% DSS solution. After 3 days, the blank control group and the DSS group were given fresh water, and the 5-ASA group and the polysaccharide treatment group were given 5-ASA and polysaccharide treatment, respectively. On the 6th day, the zebrafish were euthanized. The zebrafish intestines were taken for HE staining and AB-PAS staining analysis.

[0155] The results are as follows Fig. 20 HE staining of zebrafish intestine showed that the colon tissue structure of zebrafish in the Control group was basically normal, the colon mucosa was intact, and the goblet cells were arranged in an orderly manner in the crypts. Compared with the Control group, the colon tissue of zebrafish in the DSS group showed severe mucosal ulcers, inflammatory cells infiltrated into the mucosal tissue, goblet cells disappeared, crypt damage and other histopathological changes. After treatment with CAH-0.4-1, the colon lesions were improved. The 50μg / mL group still had mild mucosal ulcers, reduced inflammatory cell infiltration, goblet cells reappeared, and crypt damage. In the 200μg / mL group, the mucosal ulcers were better improved, the inflammatory cell infiltration was reduced, the goblet cells reappeared, and the crypt damage recovery effect was close to that of the 5-ASA group.

[0156] The results of Alcian blue staining show that the acidic protein staining of the intestinal cavity of zebrafish in the Control group showed a clear blue color, indicating that the secretory function of the intestinal goblet cells of zebrafish in the blank control group was normal. The intestinal cavity of zebrafish in the DSS group was severely damaged, and the cell secretion function was severely impaired. Compared with the DSS group, the acidic protein staining of the intestinal cavity of zebrafish in the 50μg / mL group showed a small amount of blue, indicating that the secretory function of the intestinal goblet cells of zebrafish was improved, but the cells were still damaged. The acidic protein staining of the intestinal cavity of zebrafish in the 100μg / mL group showed a clear blue treatment effect. The acidic protein staining of the intestinal cavity of zebrafish in the 200μg / mL group showed that its effect was not as good as that of the 100μg / mL group, but its goblet cell structure was relatively complete and the secretory function was also improved.

[0157] The present invention purifies acidic polysaccharides of different gradients obtained by anion exchange column chromatography by gel column chromatography, collects four acidic polysaccharides, further purifies by ultrafiltration, and finally obtains a homogeneous acidic polysaccharide with relatively uniform molecular weight. The purified homogeneous polysaccharide is structurally analyzed, and the structure of the polysaccharide is solved by means of monosaccharide composition determination, methylation analysis, one-dimensional and two-dimensional nuclear magnetic resonance analysis, etc., to determine that it is a homogeneous polysaccharide separated and purified from thorny sugar for the first time. Then, the obtained homogeneous polysaccharide is verified for activity by a juvenile zebrafish model and an adult zebrafish model suffering from ulcerative colitis. The results show that the obtained homogeneous polysaccharide has the effect of alleviating symptoms such as intestinal damage in zebrafish, restoring mucus secretion, and aggregation of inflammatory cells.

[0158] Although the above-mentioned embodiments have made a detailed description of the present invention, they are only some embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on the embodiments of the present invention without creative work, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A method for extracting and separating polysaccharides from thorny sugar, characterized in that: The following steps are involved: Deproteinizing the crude polysaccharide of Artemisia scoparia to obtain deproteinized crude polysaccharide; Decolorizing the deproteinized crude polysaccharide to obtain decolorized crude polysaccharide; The decolorized crude polysaccharide is subjected to DEAE cellulose column chromatography separation, wherein the DEAE cellulose column chromatography separation comprises: cellulose activation, column loading, sample loading and elution; the elution comprises water washing and salt washing, and the eluate obtained by the water washing is concentrated and dried to obtain a neutral sugar component CAH-0, and the salt washing comprises sequentially eluting with a first NaCl solution, a second NaCl solution, a third NaCl solution and a fourth NaCl solution, wherein the concentrations of the first NaCl solution, the second NaCl solution, the third NaCl solution and the fourth NaCl solution are 0.06-0.14, 0.16-0.24, 0.26-0.34 and 0.36-0.44 mol / L, respectively; and each eluate obtained by the salt washing is dialyzed, concentrated and dried to obtain multiple groups of polysaccharide components CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4; Alternatively, the decolorized crude polysaccharide is dialyzed to obtain high molecular weight polysaccharides and low molecular weight oligosaccharides, respectively; the molecular weight cutoff of the dialysis bag used for the dialysis is 2000Da; CAH-0, CAH-0.1, CAH-0.2, CAH-0.3 and CAH-0.4 were subjected to dextran gel column chromatography to obtain neutral sugar, the first acidic polysaccharide, the second acidic polysaccharide, the third acidic polysaccharide and the fourth acidic polysaccharide.

2. The extraction and separation method according to claim 1, characterized in that: The crude thorn sugar polysaccharide is obtained by defatting and removing impurities from the thorn sugar, then extracting with water and precipitating with alcohol.

3. The extraction and separation method according to claim 2, characterized in that: The water extraction temperature is 65-75° C. and the time is 1-2 hours; the volume concentration of ethanol in the mixed solution during the alcohol precipitation is 75-85%.

4. The extraction and separation method according to claim 1, characterized in that: The decolorization adopts macroporous resin decolorization, and the macroporous resin decolorization uses AB-8 macroporous adsorption resin.

5. The extraction and separation method according to claim 1, characterized in that: The DEAE cellulose column chromatography separation adopts DEAE-52 cellulose.

6. The extraction and separation method according to claim 1 or 5, characterized in that: The concentration of the sample solution during loading was 5 mg / mL; the elution rate was 1 mL / min.

7. The extraction and separation method according to claim 1, characterized in that: The dextran gel column chromatography adopts dextran gel G-100.

8. The extraction and separation method according to claim 1 or 7, characterized in that: The dextran gel column chromatography further includes ultrafiltration, and the molecular weight cutoff of the ultrafiltration is 100 KDa.

9. The spinosa polysaccharide obtained by the extraction and separation method according to any one of claims 1 to 8, wherein the spinosa polysaccharide comprises high molecular weight polysaccharides and acidic polysaccharides, and the acidic polysaccharides comprise first acidic polysaccharides, second acidic polysaccharides, third acidic polysaccharides and fourth acidic polysaccharides.

10. Use of the spinosa polysaccharide according to claim 9 in the preparation of a medicament for treating ulcerative colitis.

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