Preparation method and application of polygonatum cyrtonema acidic polysaccharide

Abstract

The invention provides a preparation method and application of polygonatum cyrtonema acidic polysaccharide, and belongs to the technical field of polygonatum cyrtonema polysaccharide extraction. According to the invention, a separation and purification method combining anion exchange chromatography with gel chromatography is adopted, and finally the polygonatum cyrtonema acidic homogeneous polysaccharide (PCPA) with relatively high purity is obtained. An in-vivo pharmacodynamic experiment shows that the prepared PCPA can remarkably relieve intestinal mucosa injury and has relatively high intestinal immune regulation activity. The preparation method provided by the invention is stable in process, and the prepared polygonatum cyrtonema acidic polysaccharide is high in purity, is a homogeneous polysaccharide and has the effect of regulating intestinal immunity.

Description

Technical Field

[0001] The present invention belongs to the technical field of extraction of Polygonatum cyrtonema Hua polysaccharide, and particularly relates to a preparation method and application of acidic polysaccharide from Polygonatum cyrtonema Hua. Background Art

[0002] Polygonatum cyrtonema Hua, as a traditional Chinese medicine with both medicinal and edible properties, has the effects of invigorating the spleen and replenishing qi, nourishing the kidney and moistening the lung, etc. Polygonatum cyrtonema Hua contains various bioactive components, such as polysaccharides, steroidal saponins, flavonoids, etc. As one of the main pharmacodynamic components in Polygonatum cyrtonema Hua, polysaccharides have various pharmacological effects, such as lowering blood lipid and blood sugar, enhancing immunity, antioxidation, anti-tumor, etc. Polygonatum cyrtonema Hua is derived from nature, is cheap and easy to obtain, has less side effects on the human body, has obvious advantages, and is an excellent natural immune regulator.

[0003] At present, most of the research focuses on the water-washed neutral polysaccharide of Polygonatum cyrtonema Hua. There are few relevant reports on the separation, purification and activity of acidic polysaccharide from Polygonatum cyrtonema Hua, and the yield of acidic polysaccharide from Polygonatum cyrtonema Hua is low, and the separation method is complex. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a preparation method of acidic polysaccharide from Polygonatum cyrtonema Hua. The method provided by the present invention has simple and stable process, the prepared acidic polysaccharide from Polygonatum cyrtonema Hua has high purity and has high intestinal immune regulation activity.

[0005] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0006] A preparation method of acidic polysaccharide from Polygonatum cyrtonema Hua, comprising the following steps:

[0007] Debase fat of Polygonatum cyrtonema Hua root with ethanol and perform water extraction in sequence to obtain a water extract;

[0008] Mix the water extract with α-amylase, perform enzymatic hydrolysis and alcohol precipitation to obtain an alcohol precipitate;

[0009] Redissolve the alcohol precipitate to obtain a redissolved solution, deproteinize the redissolved solution and then perform dialysis, retain the fraction with a molecular weight cut-off greater than 3500 Da to obtain crude polysaccharide from Polygonatum cyrtonema Hua;

[0010] Perform preliminary separation on the crude polysaccharide from Polygonatum cyrtonema Hua through an anion exchange column, elute with an NaCl solution to obtain a salt-washed polysaccharide from Polygonatum cyrtonema Hua;

[0011] Purify the salt-washed polysaccharide from Polygonatum cyrtonema Hua through an agarose gel column, elute with ultrapure water to obtain an eluate containing acidic polysaccharide from Polygonatum cyrtonema Hua.

[0012] Preferably, the volume concentration of the ethanol solution used in ethanol degreasing is 70% - 90%, and the mass-to-volume ratio of the Polygonatum cyrtonema Hua roots to the ethanol solution is 1 g : (5 - 15) mL.

[0013] Preferably, the water extraction includes mixing the product of ethanol degreasing with water at a ratio of 1 g : (15 - 30) mL and then extracting. The temperature of the water extraction is 60 - 100°C, and the time of the water extraction is 1 - 3 h.

[0014] Preferably, the water extract is concentrated to 0.2 - 2 g / mL to obtain a concentrated solution. The concentrated solution and α-amylase are mixed at a mass-to-volume ratio of (2000 - 5000) g : 1 mL for enzymatic hydrolysis. The temperature of the enzymatic hydrolysis is 60 - 70°C, and the time is 0.5 - 3 h.

[0015] Preferably, the final concentration of the ethanol solution used in alcohol precipitation is 60% - 90%.

[0016] Preferably, the concentration of the NaCl solution is 0.1 - 0.5 moL / mL.

[0017] Preferably, the Polygonatum cyrtonema Hua salt-washed polysaccharide is the component obtained from the 0.2 M NaCl eluate.

[0018] Another object of the present invention is to provide the Polygonatum cyrtonema Hua acidic polysaccharide prepared by the preparation method described above. The molecular weight of the Polygonatum cyrtonema Hua acidic polysaccharide is 18.594 - 25.755 kDa.

[0019] Preferably, the Polygonatum cyrtonema Hua acidic polysaccharide includes galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose, and mannose. The molar ratio of galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose, and mannose is 41.9 : 16.55 : 11.01 : 11.7 : 8.51 : 8.21 : 2.11.

[0020] Another object of the present invention is to provide the application of the preparation method or the Polygonatum cyrtonema Hua acidic polysaccharide in the preparation of drugs for intestinal immune regulation.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a method for preparing polygonatum cyrtonema hua acidic polysaccharide. First, the dried rhizome of polygonatum cyrtonema hua is defatted with ethanol solution, and then the water extract containing polygonatum cyrtonema hua polysaccharide is obtained by water extraction. After removing the starch in the water extract with α-amylase, alcohol precipitation is carried out to obtain an alcohol precipitate containing protein and crude polygonatum cyrtonema hua polysaccharide; then the protein in the alcohol precipitate is removed; finally, it is eluted and separated by an anion exchange column and purified by an agarose gel column to obtain a highly pure polygonatum cyrtonema hua acidic homogeneous polysaccharide. The polysaccharide is mainly composed of galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose and mannose, and the molar ratio is 41.9:16.55:11.01:11.7:8.51:8.21:2.11. The preparation method provided by the present invention has the advantages of simple and stable process. The prepared polygonatum cyrtonema hua acidic polysaccharide has high purity, is a homogeneous polysaccharide, and has the effect of intestinal immune regulation. Description of the Drawings

[0023] Figure 1 DEAE elution curve of polygonatum cyrtonema hua polysaccharide;

[0024] Figure 2 6FF elution curve of polygonatum cyrtonema hua acidic polysaccharide;

[0025] Figure 3 Ultraviolet spectrum of polygonatum cyrtonema hua acidic homogeneous polysaccharide;

[0026] Figure 4 Infrared spectrum of polygonatum cyrtonema hua acidic homogeneous polysaccharide;

[0027] Figure 5 Absolute molecular weight distribution map of polygonatum cyrtonema hua acidic homogeneous polysaccharide;

[0028] Figure 6 Spatial conformation analysis diagram of polygonatum cyrtonema hua acidic homogeneous polysaccharide;

[0029] Figure 7 In vitro intestinal mucosal immune activity evaluation of different elution parts of polygonatum cyrtonema hua polysaccharide; *p<0.05, **p<0.01 compared with the control group;

[0030] Figure 8 Effect of polygonatum cyrtonema hua acidic homogeneous polysaccharide on the body weight and food intake of immunosuppressed mice; among them, A is the change in the body weight of mice; B is the change in the food intake of mice; #p<0.05, ##p<0.01 compared with the Ctrl group; *p<0.05, compared with the Cy group;

[0031] Figure 9Results of the effects of acidic homogeneous polysaccharides from Polygonatum cyrtonema Hua on the immune organ indices of immunosuppressed mice; among them, A is the spleen index of mice; B is the thymus index of mice; #p < 0.05, ##p < 0.01 compared with the Ctrl group; *p < 0.05, **p < 0.01, ***p < 0.001 compared with the Cy group;

[0032] Figure 10 Are HE staining diagrams of the duodenum, jejunum, ileum and colon. Detailed implementation manners

[0033] The present invention provides a preparation method of acidic polysaccharides from Polygonatum cyrtonema Hua, comprising the following steps: successively performing ethanol degreasing and water extraction on the roots of Polygonatum cyrtonema Hua to obtain a water extract; mixing the water extract with α-amylase, performing enzymatic hydrolysis and alcohol precipitation to obtain an alcohol precipitate; redissolving the alcohol precipitate to obtain a redissolved solution, deproteinizing the redissolved solution and then performing dialysis, intercepting the fraction with a molecular weight cut-off greater than 3500 Da to obtain crude polysaccharides from Polygonatum cyrtonema Hua; preliminarily separating the crude polysaccharides from Polygonatum cyrtonema Hua through an anion exchange column, eluting with an NaCl solution to obtain salt-washed polysaccharides from Polygonatum cyrtonema Hua; purifying the salt-washed polysaccharides from Polygonatum cyrtonema Hua through an agarose gel column, eluting with ultrapure water to obtain an eluate containing acidic polysaccharides from Polygonatum cyrtonema Hua.

[0034] In the present invention, the roots of Polygonatum cyrtonema Hua are preferably dried and then pulverized, and the pulverization is preferably sieved through a 45-mesh sieve to obtain a root powder of Polygonatum cyrtonema Hua.

[0035] In the present invention, the ethanol degreasing includes mixing the root powder of Polygonatum cyrtonema Hua with an ethanol solution at a mass-to-volume ratio of 1 g:(5 - 15) mL, performing degreasing at room temperature, the volume concentration of the ethanol solution is 70% - 90%, preferably 80%, the degreasing time is 20 - 30 h, preferably 24 h, and after degreasing, a degreased root powder of Polygonatum cyrtonema Hua is obtained.

[0036] In the present invention, the water extraction includes performing hot extraction after mixing the degreased root powder of Polygonatum cyrtonema Hua with water to obtain a water extract. The mass-to-volume ratio of the degreased root powder of Polygonatum cyrtonema Hua to water in the present invention is 1 g:(15 - 30) mL, preferably 1 g:(20 - 30) mL, more preferably 1 g:30 mL; the temperature of the water extraction is preferably 60 - 100°C, further preferably 70 - 80°C, more preferably 80°C; the number of times of water extraction is preferably 1 - 4 times, more preferably 2 times; the time for each water extraction is preferably 1 - 3 h, more preferably 2 h.

[0037] After the water extraction in the present invention, the water extract is concentrated to obtain a concentrated solution, and the concentration of the concentrated solution is 0.2 - 2 g / mL, preferably 1.7 g / mL.

[0038] After obtaining the concentrated solution in the present invention, the concentrated solution is mixed with α-amylase for enzymatic hydrolysis. After enzymatic hydrolysis, the enzyme is inactivated by boiling water bath, and an appropriate amount of absolute ethanol is added for alcohol precipitation to obtain an alcohol precipitate. In the present invention, the concentrated solution and α-amylase are mixed at a mass-to-volume ratio of (2000-5000) g: 1 mL, preferably at a ratio of 3000 g: 1 mL; the enzyme activity of the α-amylase is preferably 46 U / mL; the temperature of the enzymatic hydrolysis is 60-70 °C, preferably 65 °C; the time of the enzymatic hydrolysis is preferably 0.5-3 h, more preferably 1 h; the time for inactivating the enzyme by boiling water bath is preferably 10-30 min, more preferably 20 min. The final concentration of the ethanol solution used in the alcohol precipitation is preferably 60%-90%, further preferably 70%-85%, and more preferably 80%. The concentration of the ethanol solution in the alcohol precipitation refers to the volume concentration of ethanol.

[0039] In the present invention, after obtaining the alcohol precipitate, pure water is added to the alcohol precipitate and stirred in a water bath for re-dissolution to obtain a re-dissolved solution. In the present invention, the temperature of the water bath is preferably 40-80 °C, more preferably 65 °C; the mass ratio of the alcohol precipitate to water is preferably 1 g: (30-70) mL, preferably 1 g: (40-60) mL, more preferably 1 g: 50 mL.

[0040] In the present invention, after obtaining the re-dissolved solution, the re-dissolved solution is deproteinized by the Sevag method and then dialyzed to retain the components with a molecular weight cut-off greater than 3500 Da to obtain crude polysaccharide from Polygonatum cyrtonema Hua. In the present invention, the re-dissolved solution, chloroform, and n-butanol are mixed at a volume ratio of 30: 5: 1. After mixing, it is stirred for 2 h and then centrifuged (centrifuged at a speed of 4500 rpm for 15 min) to remove protein. The protein removal is repeated 5-8 times until there is no obvious protein layer. The residual organic solvent is removed by concentration under reduced pressure at 60 °C. After dialysis with a dialysis bag with a molecular weight cut-off of 3500 Da for 72 h under running water, it is concentrated under reduced pressure, and the concentrated solution is freeze-dried under vacuum to obtain crude polysaccharide from the roots of Polygonatum cyrtonema Hua.

[0041] After obtaining the crude polysaccharide from the roots of Polygonatum cyrtonema Hua in the present invention, the crude polysaccharide from the roots of Polygonatum cyrtonema Hua is preliminarily separated by an anion exchange column and eluted with an NaCl solution to obtain the NaCl-eluted polysaccharide of Polygonatum cyrtonema Hua. The concentration of the NaCl solution in the present invention is 0.1 - 0.5 moL / mL. Preferably, ultra-pure water, 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, 0.4 M NaCl, and 0.5 M NaCl solutions are used for elution. The eluates of each tube are collected, and the absorbance values of each tube are detected by the phenol-sulfuric acid method to plot an elution curve. According to the elution curve, the eluates containing polysaccharides are combined and collected and concentrated under reduced pressure. After elution with an NaCl solution in the present invention, 4 polysaccharide components are obtained: PCP-W (eluted with ultra-pure water), PCP-A1 (eluted with 0.1 M NaCl), PCP-A2 (eluted with 0.2 M NaCl), and PCPA-3 (eluted with 0.3 M NaCl); the NaCl-eluted polysaccharide of Polygonatum cyrtonema Hua is the component obtained from the 0.2 M NaCl eluate. In the present invention, the anion exchange column preferably includes a DEAE-52 anion exchange column, the column height is preferably 30 - 60 cm, more preferably 50 cm; the elution rate of the DEAE-52 anion exchange column is preferably 2 - 6 mL / min, more preferably 5 mL / min; the eluates are collected at 10 mL / tube to obtain the DEAE elution fraction (PCP-A2) of the polysaccharide of Polygonatum cyrtonema Hua with high intestinal immune regulation activity.

[0042] In the present invention, the collected 0.2 M NaCl eluate is combined and concentrated under reduced pressure, dialyzed against running water with a dialysis bag having a molecular weight cut-off of 3500 Da, and then dialyzed against deionized water. The concentrated solution is freeze-dried under vacuum to obtain the NaCl-eluted polysaccharide fraction of Polygonatum cyrtonema Hua with high intestinal immune regulation activity; the running water dialysis time is 48 h, and the deionized water dialysis time is 24 h.

[0043] The NaCl-eluted polysaccharide of Polygonatum cyrtonema Hua prepared by eluting the crude sugar from the roots of Polygonatum cyrtonema Hua with an NaCl solution in the present invention has a larger molecular weight and different monosaccharide compositions compared with the neutral water-washed sugar prepared by eluting with ultra-pure water, and has higher intestinal immune regulation activity compared with the neutral water-washed sugar.

[0044] After obtaining the refined polysaccharide of Polygonatum cyrtonema Hua, the refined polysaccharide of Polygonatum cyrtonema Hua is purified by an agarose gel column, eluted with ultrapure water, the eluate is collected at 2 mL / tube, the absorbance value of each tube is detected by the phenol-sulfuric acid method, and an elution curve is plotted. According to the elution curve, the eluates containing polysaccharides are combined to obtain an eluate containing acidic polysaccharide of Polygonatum cyrtonema Hua. The eluate of the acidic polysaccharide of Polygonatum cyrtonema Hua is concentrated under reduced pressure and then freeze-dried to obtain acidic homogeneous polysaccharide of Polygonatum cyrtonema Hua (PCPA). In the present invention, the agarose gel column preferably comprises a Sepharose 6FF chromatographic column, the column height is preferably 50-90 cm, more preferably 75-90 cm, and still more preferably 85 cm; the flow rate of the Sepharose 6FF chromatographic column is preferably 0.1-0.5 mL / min, and more preferably 0.2 mL / min.

[0045] The present invention adopts an anion exchange chromatography combined with gel chromatography separation and purification method, and finally obtains an acidic homogeneous polysaccharide of Polygonatum cyrtonema Hua with high intestinal immune regulation activity. In vivo pharmacodynamic experiments show that the prepared PCPA can significantly relieve intestinal mucosal damage and has high intestinal immune regulation activity. The preparation method provided by the present invention has stable process and can obtain acidic homogeneous polysaccharide of Polygonatum cyrtonema Hua with high purity and intestinal immune regulation activity.

[0046] The present invention also provides the acidic polysaccharide of Polygonatum cyrtonema Hua prepared by the above preparation method. The molecular weight of the acidic polysaccharide of Polygonatum cyrtonema Hua is 18.594-25.755 kDa. Preferably, the acidic polysaccharide of Polygonatum cyrtonema Hua is mainly composed of galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose and mannose, and the molar ratio is 41.9:16.55:11.01:11.7:8.51:8.21:2.11.

[0047] The present invention also provides the application of the above preparation method or the acidic polysaccharide of Polygonatum cyrtonema Hua in the preparation of intestinal immune regulation drugs.

[0048] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0049] Example 1

[0050] An acidic polysaccharide of Polygonatum cyrtonema Hua is prepared by the following steps:

[0051] 1. Take 100 g of dry Polygonatum cyrtonema Hua rhizome powder, mix it with an ethanol solution with a volume concentration of 85%, and stir well; the mass-volume ratio of the acidic Polygonatum cyrtonema Hua to the ethanol solution is 1 g:10 mL; the extraction temperature is room temperature, the extraction time is 24 h, extract once, filter off the ethanol, and volatilize at room temperature until there is no alcohol smell to obtain defatted powder of Polygonatum cyrtonema Hua;

[0052] 2. Take 86.24 g of defatted polygonatum sibiricum powder, add 30 times the amount of water and heat (80 °C) for extraction twice, 2 h each time, and combine the extracts; recover under reduced pressure at 60 °C to 600 mL, add 200 μL of α-amylase (46 U / mL) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., product number A109181-100 g), water bath at 65 °C, stir for 1 h, inactivate at 100 °C for 15 min, remove starch, add absolute ethanol until the final ethanol concentration is 80%, precipitate with alcohol for 24 h, centrifuge and discard the supernatant to obtain polygonatum cyrtonema alcohol precipitate;

[0053] 3. After volatilizing the polygonatum cyrtonema alcohol precipitate at room temperature until there is no alcohol smell completely, add distilled water and water bath at 65 °C until completely dissolved to obtain an alcohol precipitate solution;

[0054] 4. Mix and stir the alcohol precipitate solution, chloroform and n-butanol for 2 h, then centrifuge at a centrifugal speed of 4500 rpm for 15 min. The volume ratio of the alcohol precipitate solution, chloroform and n-butanol is 30:5:1; after centrifugation, deproteinize 8 times, concentrate under reduced pressure at 60 °C to remove the residual organic solvents, dialyze with a dialysis bag with a molecular weight cut-off of 3500 Da against running water for 48 h, then continue to dialyze with deionized water for 24 h, and freeze-dry the concentrated solution under vacuum to obtain 12.08 g of crude polygonatum cyrtonema polysaccharide;

[0055] 5. Take 200 mg of crude polygonatum cyrtonema polysaccharide, dissolve it in 5 mL of ultrapure water, filter through a 0.45 μm filter membrane, load the filtrate onto a pre-equilibrated DEAE-52 anion exchange column with a column height of 50 cm, and sequentially elute with ultrapure water, 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, 0.4 M NaCl, 0.5 M NaCl solutions in segments at an elution speed of 5 mL / min, collect the eluate at 10 mL / tube: PCP-W (eluted with ultrapure water), PCP-A1 (eluted with 0.1 M NaCl), PCP-A2 (eluted with 0.2 M NaCl), PCP-A3 (eluted with 0.3 M NaCl), collect the eluate of each tube, detect the absorbance value of each tube by the phenol-sulfuric acid method, and draw an elution curve. According to the elution curve ( Figure 1 ) collect the eluate at 10 mL / tube, concentrate under reduced pressure, dialyze with a dialysis bag with a molecular weight cut-off of 3500 Da against running water for 48 h, then continue to dialyze with deionized water for 24 h, and freeze-dry the concentrated solution under vacuum to obtain the polygonatum cyrtonema salt-washed polysaccharide fraction (PCP-A2);

[0056] 6. Dissolve 50 mg of the refined polysaccharide component of Polygonatum cyrtonema Hua in 5 mL of distilled water, filter through a 0.22-μm filter membrane, load the filtrate onto a pre-equilibrated Sepharose 6FF chromatographic column with a column height of 85 cm, elute with ultrapure water at an elution rate of 0.2 mL / min, collect the eluate in 2-mL fractions, detect the absorbance value of each fraction by the phenol-sulfuric acid method, and plot the 6FF elution curve. According to the elution curve ( Figure 2 ), collect the two fractions on the left and right near the highest absorbance value and combine them to obtain the eluate containing polysaccharides. Concentrate under reduced pressure, and lyophilize the concentrated solution under vacuum to obtain the acidic homogeneous polysaccharide of Polygonatum cyrtonema Hua (PCPA) with an elution yield of 89.32 ± 0.49%.

[0057] Test Example 1

[0058] Perform structure and activity determination on the PCPA prepared in Example 1 as follows:

[0059] 1. PCPA Structure Determination

[0060] 1.1 PCPA Physicochemical Property Determination

[0061] 1.1.1 Determination of Total Carbohydrate, Protein, and Uronic Acid Contents

[0062] Determine the contents of total carbohydrate, protein, and uronic acid by the phenol-sulfuric acid method, Coomassie brilliant blue method, and m-hydroxybiphenyl colorimetric method, respectively.

[0063] 1.1.2 Ultraviolet and Infrared Spectroscopy Determination

[0064] Prepare a 0.1-mg / mL solution of PCPA and scan it in the wavelength range of 200 - 800 nm.

[0065] Fully dry PCPA, weigh 2 mg, and scan it with an infrared spectrometer in the range of 4000 - 400 cm -1 region.

[0066] 1.1.3 Absolute Molecular Weight Determination

[0067] The chromatographic system used is a gel chromatography - differential - multi-angle laser light scattering system, the liquid phase system is U3000 (Thermo, USA), the differential detector is Optilab T-rEX (Wyatttechnology, CA, USA), and the laser light scattering detector is DAWN HELEOSⅡ (Wyatttechnology, CA, USA).

[0068] 1.1.4 Congo Red Experiment

[0069] Prepare a mixture by fully mixing PCPA (2.5 mg / mL) and Congo red solution (80 μmol / L) in a ratio of 1:1 (v / v). Gradually adjust the mixture to different NaOH concentrations (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, and 0.5 mol / L) by adding NaOH solution (4 M). Record the maximum absorption wavelength of Congo red polysaccharide solutions at different concentrations by ultraviolet scanning (200 - 600 nm).

[0070] 1.2 Monosaccharide composition of PCPA

[0071] Take a clean chromatographic vial, weigh an appropriate amount of PCPA sample, add 1 mL of 2 M TFA acid solution, and heat at 60 °C for 1 hour. Pass nitrogen and blow dry. Add 99.99% methanol for cleaning and then blow dry again. Repeat the methanol cleaning 2 - 3 times. Add an appropriate amount of sterile water to dissolve and transfer it to the chromatographic vial for testing.

[0072] The chromatographic system used is the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and an electrochemical detector is used to analyze and detect monosaccharide components.

[0073] 2. Evaluation of in vitro intestinal mucosal immune activity of PCPA

[0074] 2.1 Preparation of conditioned medium

[0075] Preparation of conditioned medium: Dispose of mice by cervical dislocation. After disinfection by soaking in 75% alcohol, carefully cut open the abdominal skin, aseptically remove the small intestine, wash it with cold HBSS solution, carefully remove Peyer's patches from the intestinal wall and place them in cold complete culture medium (RPMI - 1640 - FBS) containing 5% FBS and 1% double antibody. Grind Peyer's patches with the piston of a syringe through four layers of 200 - mesh gauze, wash the cells twice with HBSS containing 5% FBS (HBSS - FBS), centrifuge at 4 °C for 10 min (1500 r / min) each time, and finally resuspend the Peyer's patch cells with the complete culture medium. Perform trypan blue staining for viable cell counting and adjust the concentration of the Peyer's patch cell suspension to 2×10 6 cells / mL. Add the Peyer's patch cell suspension to a 96 - well culture plate, 180 μL per well, and add 20 μL of elution fractions of Polygonatum cyrtonema Hua polysaccharide at different concentrations (final concentrations are 0, 25, 50, 100, 200 μg / mL) to each well. Set 5 replicates for each group. Place the 96 - well plate in a CO2 incubator at 5% CO2 and 37 °C for 5 days. After the culture is completed, centrifuge and take the supernatant (conditioned medium) for standby.

[0076] 2.2 Preparation of bone marrow cell suspension

[0077] The mice were sacrificed by cervical dislocation. After being disinfected by immersion in 75% alcohol, the femurs and tibias of the mice were aseptically taken, the muscle tissues were removed, and they were placed in a petri dish containing cold HBSS solution. The leg bones were rinsed with complete culture medium until they turned white. The washed suspension was centrifuged at 4°C (1500 rpm × 10 min) to obtain bone marrow cells. After lysis of red blood cells, it was washed twice with HBSS-FBS, and centrifuged at 4°C for 10 min (1500 r / min) each time. Viable cell counting was performed by trypan blue staining, and the concentration of the bone marrow cell suspension was adjusted to 2.5×10 5 cells / mL.

[0078] 2.3 Evaluation of intestinal mucosal immune activity

[0079] The bone marrow cell suspension was added to a 96-well plate, 100 μL per well. At the same time, 50 μL of RPMI-1640-FBS and 50 μL of conditioned medium were added. The 96-well plate was cultured in an incubator at 5% CO2 and 37°C for 6 d. 5 h before the end of the culture, 20 μL of AlamarBlue reagent was added to each well, and the culture was continued until the end. The fluorescence intensity was measured under the conditions of excitation wavelength 544 nm and emission wavelength 590 nm.

[0080] 3. In vivo pharmacodynamic experiment of PCPA

[0081] 3.1 Animal grouping, model establishment and dosing regimen

[0082] Sixty 6- to 8-week-old C57 / BL6 mice were acclimated for one week, during which they had free access to water and food. After the acclimation period, they were randomly divided into a normal group (Ctrl), a model group (Cy group), a positive drug levamisole hydrochloride group (LH group), a low-dose PCPA group (PCPA-L), and a high-dose PCPA group (PCPA-H), with 12 mice in each group. Except for the normal group, the mice in the model group and each dose group of PCPA were intraperitoneally injected with cyclophosphamide (Cy) drug (80 mg / kg) for 3 days to establish an immunosuppressed mouse model. After the model establishment, the LH group was given levamisole hydrochloride by gavage (40 mg / kg), and the low-dose and high-dose dosing groups were given 50 mg / kg and 200 mg / kg PCPA solution by gavage, once a day for 7 consecutive days. And the body weights of each group of mice were weighed and the mental state, activity status, hair glossiness and other routine conditions were clinically observed on time every day. On the 8th day, they were sacrificed after anesthesia, the body weights of the mice were weighed, the sera, spleens, duodenums, jejuns, ileums and colon tissues of all mice were collected and stored at -80°C, or some tissues were fixed in 4% neutral formalin for the next step of research.

[0083] 3.2 Immune organ index

[0084] After sacrificing the mice, the spleens were aseptically isolated quickly, the adherent fat and fascia on the surface were removed, and they were washed rapidly in physiological saline. After removing the blood stains, they were blotted dry on filter paper and weighed immediately, and the weight was recorded. The calculation formula for the immune organ index is as follows:

[0085] Organ index (mg / g) = Organ weight (mg) / Body weight (g)

[0086] 3.3 Histological examination of each intestinal segment

[0087] To evaluate the severity of intestinal injury, the duodenum, jejunum, ileum, and colon parts were washed with pre-cooled PBS and then fixed immediately in 10% paraformaldehyde solution for 24 h. After dehydration and paraffin embedding, they were cut into 5-mm-thick slices with a microtome. The hematoxylin and eosin staining (H&E) method was used to detect the morphological changes of the duodenum, jejunum, ileum, and colon tissues. The HE staining sections of each intestinal segment are shown as Figure 10 shown.

[0088] 4. Statistical methods

[0089] GraphPad 9.5 software was used for drawing and one-way analysis of variance. The results are expressed as mean ± SD; a P value less than 0.05 was considered statistically significant.

[0090] 5. Experimental results

[0091] 5.1 Determination of the physicochemical properties of PCPA

[0092] 5.1.1 Analysis of total carbohydrate and protein contents

[0093] The results of the determination of the physicochemical properties of PCPA are shown in Table 1: The carbohydrate content in PCPA reaches more than 90%, contains a certain amount of uronic acid, and the protein content is low.

[0094] Table 1 Determination of the physicochemical properties of PCPA

[0095] Polygonatum cyrtonema Hua acidic homogeneous polysaccharide Content Carbohydrate 90.32±1.26％ Protein 1.56±0.18％ Uronic acid 36.91±0.56％

[0096] 5.1.2 Ultraviolet and infrared spectroscopy analysis

[0097] As Figure 3 shown: At 260 nm and 280 nm, PCPA has no obvious absorption peak, indicating that it does not contain nucleic acid and protein. Figure 4 It shows that the peak at 3389 cm -1 is the stretching vibration of -OH, the peak at 2934 cm -1 is the stretching vibration of C-H, the peak at 1737 cm -1 is the stretching vibration of C=O on the carboxylic acid (or acetoxy group), and the peak at 1425 cm-1 The peak is the bending vibration of pyranose, 1141 cm -1 The peak is the C-O vibration of O-acetyl, 1100 cm -1 The peak near indicates the presence of a furan ring structure in the polysaccharide, 500 - 900 cm -1 The peak indicates the presence of a pyranose ring skeleton in the polysaccharide.

[0098] 5.1.3 Absolute molecular weight analysis

[0099] LS represents the multi-angle laser light scattering signal, and RI represents the differential signal. Figure 5 Taking the detected retention time (Time, min) as the abscissa and the relative scale Relative Scale as the ordinate, the weight-average molecular weight (MW) of PCPA is 25.755 kDa, the number-average molecular weight (Mn) is 21.807 kDa, and the polydispersity coefficient Mw / Mn is 1.181.

[0100] 5.1.4 Spatial conformation analysis

[0101] As Figure 6 shown, in the NaOH solution of 0 - 0.5 moL, the maximum absorption wavelength of the PCPA solution did not show an obvious red shift, indicating the absence of a triple helix structure.

[0102] 5.2 Monosaccharide composition

[0103] As shown in Table 2, PCPA is mainly composed of galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose, and mannose, with percentages of 38.92%, 17.03%, 10.32%, 10.07%, 9.11%, 8.76%, and 2.11%, and a molar ratio of 41.9:16.55:11.01:11.7:8.51:8.21:2.11.

[0104] Table 2 Analysis of the monosaccharide composition of PCPA

[0105] Monosaccharide composition Percentage Molar ratio Galacturonic acid 38.92％ 41.9 Fructose 17.03％ 16.55 Rhamnose 10.32％ 11.01 Arabinose 10.07％ 11.7 Galactose 9.11％ 8.51 Glucose 8.76％ 8.21 Mannose 2.11％ 2.11

[0106] 5.3 In vitro intestinal mucosa activity evaluation and screening of different elution parts of Polygonatum cyrtonema polysaccharide

[0107] As Figure 7As shown, in vitro evaluation and analysis of intestinal mucosal immune activity indicated that PCP-W, PCP-A2, and PCP-A3 could all promote the proliferation of bone marrow cells significantly in a dose-dependent manner by stimulating Peyer's patch cells to produce conditioned medium. When the concentration of the polysaccharide sample was 200 μg / mL, the three polysaccharide components promoted the proliferation of bone marrow cells to the maximum through Peyer's patch cells. Among them, the PCP-A2 component showed stronger intestinal mucosal immune regulatory activity than the other components.

[0108] 5.4 In vivo intestinal immune regulation pharmacodynamic experiment

[0109] 5.4.1 Effects of PCPA on the body weight and physiological status of immunosuppressed mice

[0110] As Figure 8 shown in (A) below, after injecting Cy, the body weights of mice in each Cy administration group showed a downward trend and stopped decreasing on the 6th day. Compared with the Cy group, treatment with LH and high-dose PCPA both slowed down the weight recovery (p < 0.05). The food intake of the mice was as Figure 8 shown in (B) below. After intraperitoneal injection of Cy for three consecutive days, the food intake of the mice decreased sharply. After stopping the injection of Cy, the food intake of each group of mice began to gradually recover on the 6th day.

[0111] 5.4.2 Effects of PCPA on the immune organ indices of immunosuppressed mice

[0112] As Figure 9 shown below, compared with the control group, the spleen index of mice in the Cy group was abnormally increased, and the thymus index was significantly decreased (p < 0.05). After treatment with LH and different doses of PCPA, compared with the Cy group, the abnormal increase in the spleen index and abnormal decrease in the thymus index of mice in the LH, PCPA-L, and PCPA-H groups were significantly improved (p < 0.05). The results showed that PCPA had a significant immune regulatory effect.

[0113] 5.4.3 Effects of PCPA on the tissues of each intestinal segment of immunosuppressed mice

[0114] As Figure 10 shown below, H&E staining was used to analyze the effects of PCPA on the morphology of the duodenum, jejunum, ileum, and colon of Cy-treated mice. The villi of the control group were neat, dense, and intact. However, after Cy treatment, the villi of the duodenum, jejunum, and ileum of the mice became short, sparse, and broken. After treatment with LH and different doses of PCPA, the intestinal villi of Cy-treated mice recovered to a shape close to that of the control group, and the thickness of the intestinal mucosa and muscularis mucosa in the small intestine and large intestine regions increased significantly. These results indicated that PCPA could improve intestinal nutrient absorption and enhance the physical barrier function of the intestinal mucosa by repairing the intestinal mucosal damage caused by Cy.

[0115] In summary, the present invention adopts a separation and purification method combining anion exchange chromatography and gel chromatography, and finally obtains a homogeneous acidic polysaccharide from Polygonatum cyrtonema Hua with intestinal immunomodulatory activity. The in vivo and in vitro intestinal immunomodulatory activities indicate that the prepared PCPA has high intestinal immunomodulatory activity. The preparation method provided by the present invention has stable process and can obtain a homogeneous polysaccharide with intestinal immunomodulatory activity.

[0116] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing polysaccharide from Polygonatum cyrtonema, characterized in that: The steps include: Degreasing and water extracting the root of Polygonatum cyrtonema in turn to obtain a water extract; The water extract is mixed with α-amylase, and subjected to enzymatic hydrolysis and alcohol precipitation to obtain an alcohol precipitate; The alcohol precipitate is redissolved to obtain a reconstituted solution, the reconstituted solution is deproteinized and then dialyzed to retain components with a molecular weight greater than 3500 Da to obtain a crude Polygonatum cyrtonema polysaccharide; Preliminarily separating the crude Polygonatum cyrtonema polysaccharide through an anion exchange column, and eluting with a NaCl solution to obtain the Polygonatum cyrtonema salt-washed polysaccharide; The salt-washed Polygonatum cyrtonema polysaccharide is purified by an agarose gel column and eluted with ultrapure water to obtain an eluate containing Polygonatum cyrtonema acidic polysaccharide.

2. The preparation method according to claim 1, characterized in that: The volume concentration of the ethanol solution used in the ethanol degreasing is 70% to 90%, and the mass volume ratio of the Polygonatum cyrtonema root to the ethanol solution is 1g: (5 to 15) mL.

3. The preparation method according to claim 1, characterized in that: The water extraction comprises mixing the ethanol defatted product with water at a ratio of 1 g: (15-30) mL and then extracting the mixture. The water extraction temperature is 60-100° C. and the water extraction time is 1-3 hours.

4. The preparation method according to claim 1, characterized in that: The water extract is concentrated to 0.2-2 g / mL to obtain a concentrated solution, and the concentrated solution and α-amylase are mixed at a mass volume ratio of (2000-5000) g:1 mL for enzymolysis. The enzymolysis temperature is 60-70° C. and the time is 0.5-3 h.

5. The preparation method according to claim 1, characterized in that: The final concentration of the ethanol solution in the alcohol precipitation is 60% to 90%.

6. The preparation method according to claim 1, characterized in that: The concentration of the NaCl solution is 0.1-0.5 mol / mL.

7. The preparation method according to claim 1, characterized in that: The salt-washed Polygonatum cyrtonema polysaccharide is a component obtained from a 0.2M NaCl eluent.

8. The polygonatum cyrtonema acidic polysaccharide prepared by the preparation method according to any one of claims 1 to 7, characterized in that: The molecular weight of the polygonatum sibiricum acidic polysaccharide is 18.594-25.755 kDa.

9. The Polygonatum cyrtonema acidic polysaccharide according to claim 8, characterized in that The polygonatum sibiricum acidic polysaccharide comprises galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose and mannose, and the molar ratio of the galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose and mannose is 41.9:16.55:11.01:11.7:8.51:8.21:2.

11.

10. Use of the preparation method according to any one of claims 1 to 7 or the Polygonatum cyrtonema acidic polysaccharide according to claim 8 or 9 in the preparation of intestinal immunomodulatory drugs.

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