Preparation method and application of polygala japonica acidic polysaccharide

Abstract

The application provides a preparation method and application of a polygonatum cyrtonema acid polysaccharide, and belongs to the technical field of polygonatum cyrtonema polysaccharide extraction. The application adopts a separation and purification method of anion exchange chromatography combined with gel chromatography, and finally obtains a polygonatum cyrtonema acid uniform polysaccharide (PCPA) with high purity. In-vivo pharmacodynamic experiments show that the prepared PCPA can significantly relieve intestinal mucosa injury and has high intestinal immune regulation activity. The preparation method provided by the application is stable in process, the prepared polygonatum cyrtonema acid polysaccharide is high in purity, is a uniform polysaccharide, and has an intestinal immune regulation effect.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction technology of Polygonatum multiflorum, and particularly relates to a method for preparing acidic polysaccharides of Polygonatum multiflorum and their application. Background Technology

[0002] Polygonatum multiflorum, a traditional Chinese medicine used in both food and medicine, possesses the effects of tonifying the spleen and replenishing qi, nourishing the kidneys and moistening the lungs. It contains various bioactive components, such as polysaccharides, steroidal saponins, and flavonoids. Polysaccharides, as one of the main active ingredients in Polygonatum multiflorum, have various pharmacological effects, including lowering blood lipids and blood sugar, enhancing immunity, anti-oxidation, and anti-tumor activity. Polygonatum multiflorum originates from nature, is inexpensive and readily available, and has few side effects on the human body, giving it significant advantages and making it an excellent natural immunomodulator.

[0003] Currently, most studies focus on water-washed neutral polysaccharides from Polygonatum odoratum, while there are few reports on the isolation, purification, and activity of acidic polysaccharides from Polygonatum odoratum. Furthermore, the yield of acidic polysaccharides from Polygonatum odoratum is low, and the separation methods are complex. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for preparing acidic polysaccharides of Polygonatum odoratum. The method provided by this invention is simple and stable, and the prepared acidic polysaccharides of Polygonatum odoratum have high purity and high intestinal immunomodulatory activity.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] A method for preparing acidic polysaccharides from Polygonatum multiflorum includes the following steps:

[0007] The roots of Polygonatum multiflorum were successively defatted with ethanol and extracted with water to obtain an aqueous extract.

[0008] The aqueous extract was mixed with α-amylase for enzymatic hydrolysis and ethanol precipitation to obtain the ethanol precipitate.

[0009] The alcohol precipitate was redissolved to obtain a reconstituted solution. The reconstituted solution was then deproteinized and dialyzed to retain components with a molecular weight greater than 3500 Da, thus obtaining crude polysaccharide of Sophora flavescens.

[0010] The crude polysaccharide of Polygonatum odoratum was initially separated by an anion exchange column and eluted with NaCl solution to obtain polygonatum odoratum salt-washed polysaccharide.

[0011] The polysaccharide of Polygonatum odoratum was purified by agarose gel column elution and eluted with ultrapure water to obtain an eluent containing acidic polysaccharide of Polygonatum odoratum.

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

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

[0014] Preferably, the aqueous 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-volume ratio of (2000-5000) g: 1 mL for enzymatic hydrolysis. The enzymatic hydrolysis temperature is 60-70℃ and the time is 0.5-3 h.

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

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

[0017] Preferably, the polysaccharide obtained by washing polysaccharide with 0.2M NaCl is a component obtained by elution.

[0018] Another object of the present invention is to provide an acidic polysaccharide of Polygonatum odoratum prepared by the preparation method described above, wherein the molecular weight of the acidic polysaccharide of Polygonatum odoratum is 18.594 to 25.755 kDa.

[0019] Preferably, the acidic polysaccharide of Polygonatum odoratum includes galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose, and mannose, and 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 preparation method described herein or the application of the acidic polysaccharide of Polygonatum odoratum in the preparation of intestinal immunomodulatory drugs.

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

[0022] This invention provides a method for preparing acidic polysaccharides from Polygonatum odoratum. The method involves first defatting the dried rhizomes of Polygonatum odoratum with ethanol solution, then obtaining an aqueous extract containing the polysaccharides through water extraction. The starch in the aqueous extract is removed using α-amylase, followed by alcohol precipitation to obtain an alcohol precipitate containing protein and crude Polygonatum odoratum polysaccharides. The protein in the precipitate is then removed. Finally, the extract is purified by anion exchange column elution and agarose gel column chromatography to obtain a high-purity, homogeneous acidic polysaccharide from Polygonatum odoratum. The polysaccharide is mainly composed of galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose, and mannose, with a molar ratio of 41.9:16.55:11.01:11.7:8.51:8.21:2.11. The preparation method provided by this invention has the advantages of simple and stable process, and the prepared acidic polysaccharide from Polygonatum odoratum is of high purity, homogeneous, and has intestinal immune-regulating effects. Attached Figure Description

[0023] Figure 1 DEAE elution curve of Polygonatum multiflorum polysaccharide;

[0024] Figure 2 The 6FF elution curve of acidic polysaccharides from Polygonatum multiflorum is shown.

[0025] Figure 3 The ultraviolet spectrum of acidic homogeneous polysaccharide from Polygonatum multiflorum;

[0026] Figure 4 Infrared spectrum of acidic homogeneous polysaccharide from Polygonatum multiflorum;

[0027] Figure 5 This is a graph showing the absolute molecular weight distribution of acidic homogeneous polysaccharides from Polygonatum multiflorum.

[0028] Figure 6 Spatial conformational analysis diagram of acidic homogeneous polysaccharide from Polygonatum multiflorum;

[0029] Figure 7 To evaluate the in vitro intestinal mucosal immunomodulatory activity of different eluted fractions of Polygonatum odoratum polysaccharide; *p<0.05, **p<0.01 compared with the control group;

[0030] Figure 8 The results show the effects of acidic homogeneous polysaccharide from Polygonatum odoratum on body weight and food intake in immunosuppressed mice; where A represents changes in mouse body weight; B represents changes in food intake; #p<0.05, ##p<0.01 compared with the Ctrl group; *p<0.05 compared with the Cy group;

[0031] Figure 9The results show the effects of acidic homogeneous polysaccharide from Polygonatum odoratum on immune organ indices in immunosuppressed mice; where A represents the spleen index and B represents the thymus index; #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 HE staining images of sections of the duodenum, jejunum, ileum, and colon. Detailed Implementation

[0033] This invention provides a method for preparing acidic polysaccharides from Polygonatum odoratum, comprising the following steps: Polygonatum odoratum roots are sequentially defatted with ethanol and extracted with water to obtain an aqueous extract; the aqueous extract is mixed with α-amylase for enzymatic hydrolysis and ethanol precipitation to obtain an ethanol precipitate; the ethanol precipitate is redissolved to obtain a reconstituted solution; the reconstituted solution is deproteinized and dialyzed to retain components with a molecular weight greater than 3500 Da, yielding crude polysaccharides from Polygonatum odoratum; the crude polysaccharides are preliminarily separated using an anion exchange column and eluted with NaCl solution to obtain salt-washed polysaccharides from Polygonatum odoratum; the salt-washed polysaccharides are purified using an agarose gel column and eluted with ultrapure water to obtain an eluent containing acidic polysaccharides from Polygonatum odoratum.

[0034] In this invention, the root of Polygonatum multiflorum is preferably dried and then pulverized, and the pulverization is preferably passed through a 45-mesh sieve to obtain Polygonatum multiflorum root powder.

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

[0036] In this invention, the water extraction includes mixing defatted Polygonatum odoratum powder with water and then performing a hot extraction to obtain an aqueous extract. The mass-to-volume ratio of the defatted Polygonatum odoratum powder to water in this invention is 1g:(15-30)mL, preferably 1g:(20-30)mL, more preferably 1g:30mL; the water extraction temperature is preferably 60-100℃, further preferably 70-80℃, more preferably 80℃; the number of water extractions is preferably 1-4 times, more preferably 2 times; and the extraction time for each extraction is preferably 1-3 hours, more preferably 2 hours.

[0037] After obtaining the water extract, the water extract is concentrated to obtain a concentrated solution. The concentration of the concentrated solution is 0.2-2 g / mL, preferably 1.7 g / mL.

[0038] After obtaining the concentrated solution, the present invention mixes the concentrated solution with α-amylase for enzymatic hydrolysis. Following hydrolysis, the enzyme is inactivated by boiling in a water bath. An appropriate amount of anhydrous ethanol is added for alcohol precipitation to obtain the precipitate. In this invention, the concentrated solution and α-amylase are mixed at a mass-to-volume ratio of (2000–5000) g:1 mL, preferably at 3000 g:1 mL; the α-amylase activity is preferably 46 U / mL; the hydrolysis temperature is 60–70°C, preferably 65°C; the hydrolysis time is preferably 0.5–3 h, more preferably 1 h; the enzyme inactivation time by boiling in a 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%, more 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 this invention, after obtaining the alcohol precipitate, pure water is added to the alcohol precipitate and the mixture is reconstituted by stirring in a water bath to obtain a reconstituted solution. In this 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 1g:(30–70)mL, more preferably 1g:(40–60)mL, and more preferably 1g:50mL.

[0040] In this invention, after obtaining the reconstituted solution, the solution is deproteinized using the Sevag method and then dialyzed to retain components with a molecular weight cutoff greater than 3500 Da, yielding crude polysaccharides from Polygonatum odoratum roots. In this invention, the reconstituted solution, chloroform, and n-butanol are mixed at a volume ratio of 30:5:1. After mixing and stirring for 2 hours, centrifugation (at 4500 rpm for 15 minutes) is performed to remove the protein. This deproteinization process is repeated 5-8 times until no obvious protein layer is visible. The solution is then concentrated under reduced pressure at 60°C to remove residual organic solvents. After dialyzing with running water using a dialysis bag with a molecular weight cutoff of 3500 Da for 72 hours, the solution is concentrated under reduced pressure, and the concentrate is freeze-dried under vacuum to obtain crude polysaccharides from Polygonatum odoratum roots.

[0041] In this invention, after obtaining the crude polysaccharide from the roots of *Polygonatum cyrtonema*, the crude polysaccharide is initially separated using an anion exchange column and eluted with NaCl solution to obtain *Polygonatum cyrtonema* salt-washed polysaccharide. The concentration of the NaCl solution used in this invention is 0.1–0.5 mol / mL, preferably using ultrapure water, 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, 0.4 M NaCl, and 0.5 M NaCl solutions for elution. The eluents from each tube are collected, and the absorbance values ​​of each tube are detected using the phenol-sulfuric acid method to plot elution curves. Based on the elution curves, the eluents containing polysaccharides are combined and collected, and then concentrated under reduced pressure. In this invention, four polysaccharide fractions were obtained by elution with NaCl solution: PCP-W (eluted with ultrapure water), PCP-A1 (eluted with 0.1M NaCl), PCP-A2 (eluted with 0.2M NaCl), and PCPA-3 (eluted with 0.3M NaCl). The polysaccharide obtained by eluting with *Polygonatum sibiricum* salt was the fraction obtained from the 0.2M NaCl elution solution. In this invention, the anion exchange column preferably includes a DEAE-52 anion exchange column, with a column height 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 eluent is collected at 10 mL / tube to obtain the DEAE eluted fraction (PCP-A2) of *Polygonatum sibiricum* polysaccharide with high intestinal immunomodulatory activity.

[0042] In this invention, the collected 0.2M NaCl eluents are combined and concentrated under reduced pressure. After dialysis with running water using a dialysis bag with a molecular weight cutoff of 3500 Da, the solution is then dialyzed with deionized water. The concentrate is then freeze-dried under vacuum to obtain a polysaccharide component of Polygonatum odoratum salt with high intestinal immunomodulatory activity. The running water dialysis time is 48 hours, and the deionized water dialysis time is 24 hours.

[0043] The polysaccharide prepared by eluting crude sugar from the root part of Polygonatum odoratum using NaCl solution has a larger molecular weight and a different monosaccharide composition compared with the neutral water-washed sugar prepared by elution with ultrapure water. It also has higher intestinal immune-regulating activity compared with the neutral water-washed sugar.

[0044] After obtaining the polysaccharide washed from Polygonatum cyrtonema, this invention purifies the polysaccharide using an agarose gel column, eluting with ultrapure water. The eluent is collected at 2 mL / tube, and the absorbance of each tube is measured using the phenol-sulfuric acid method to plot the elution curve. Based on the elution curve, the eluents containing polysaccharides are combined to obtain an eluent containing acidic polysaccharides from Polygonatum cyrtonema. The eluent containing acidic polysaccharides from Polygonatum cyrtonema is concentrated under reduced pressure and then freeze-dried to obtain homogeneous acidic polysaccharide from Polygonatum cyrtonema (PCPA). In this invention, the agarose gel column preferably includes a Sepharose 6FF column, with a column height preferably 50–90 cm, more preferably 75–90 cm, and more preferably 85 cm; the flow rate of the Sepharose 6FF column is preferably 0.1–0.5 mL / min, more preferably 0.2 mL / min.

[0045] This invention employs anion exchange chromatography combined with gel chromatography for separation and purification, ultimately yielding a homogeneous acidic polysaccharide from Polygonatum odoratum exhibiting high intestinal immunomodulatory activity. In vivo pharmacodynamic experiments demonstrate that the prepared PCPA significantly alleviates intestinal mucosal damage and possesses high intestinal immunomodulatory activity. The preparation method provided by this invention is stable and can obtain high-purity, homogeneous acidic polysaccharide from Polygonatum odoratum with intestinal immunomodulatory activity.

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

[0047] This invention also provides the preparation method described above or the application of the acidic polysaccharide of Polygonatum odoratum in the preparation of intestinal immunomodulatory 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 scope of protection of the present invention.

[0049] Example 1

[0050] An acidic polysaccharide from Polygonatum multiflorum is prepared by the following steps:

[0051] 1. Take 100g of dried Polygonatum odoratum rhizome powder, mix it with an 85% ethanol solution and stir thoroughly; the mass-volume ratio of the acid of Polygonatum odoratum to the ethanol solution is 1g:10mL; the extraction temperature is room temperature, the extraction time is 24h, the extraction is performed once, the ethanol is removed by filtration, and after evaporation at room temperature until there is no alcohol odor, the defatted Polygonatum odoratum powder is obtained.

[0052] 2. 86.24 g of defatted Polygonatum powder was extracted twice with 30 times the amount of water and heated (80℃) for 2 h each time. The extracts were combined. The volume was reduced to 600 mL under reduced pressure at 60℃. 200 μL of α-amylase (46 U / mL) (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number A109181-100g) was added. The mixture was stirred in a water bath at 65℃ for 1 h, then inactivated at 100℃ for 15 min to remove starch. Anhydrous ethanol was added until the final ethanol concentration was 80%. The mixture was precipitated for 24 h. The supernatant was discarded by centrifugation to obtain the Polygonatum polyphylla ethanol precipitate.

[0053] 3. After evaporating the polysaccharide precipitate at room temperature until it is completely odorless, add distilled water and heat in a 65°C water bath until it is completely dissolved to obtain the precipitate solution.

[0054] 4. The alcohol precipitate solution, chloroform and n-butanol were mixed and stirred for 2 hours, and then centrifuged at 4500 rpm for 15 minutes. The volume ratio of the alcohol precipitate solution, chloroform and n-butanol was 30:5:1. After centrifugation, the protein was removed 8 times, and the residual organic solvent was removed by concentration under reduced pressure at 60°C. The solution was dialyzed with running water for 48 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and then dialyzed with deionized water for 24 hours. The concentrate was freeze-dried under vacuum to obtain 12.08 g of crude polysaccharide from Polygonatum odoratum.

[0055] 5. Dissolve 200 mg of crude polysaccharide from Polygonatum odoratum in 5 mL of ultrapure water, filter through a 0.45 μm filter membrane, and load the filtrate onto a equilibrated DEAE-52 anion exchange column (50 cm high). Elute sequentially with ultrapure water, 0.1 M NaCl, 0.2 M NaCl, 0.3 M NaCl, 0.4 M NaCl, and 0.5 M NaCl solutions at a rate of 5 mL / min. Collect 10 mL of eluent per tube: PCP-W (ultrapure water elution), PCP-A1 (0.1 M NaCl elution), PCP-A2 (0.2 M NaCl elution), and PCPA-3 (0.3 M NaCl elution). Collect the eluent from each tube, and determine the absorbance of each tube using the phenol-sulfuric acid method. Plot the elution curve. Based on the elution curve... Figure 1 The eluent was collected at 10 mL / tube, concentrated under reduced pressure, dialyzed with running water for 48 h using a dialysis bag with a molecular weight cutoff of 3500 Da, and then dialyzed with deionized water for 24 h. The concentrate was then freeze-dried under vacuum to obtain the polysaccharide fraction of Polygonatum odoratum (PCP-A2).

[0056] 6. Dissolve 50 mg of the polysaccharide fraction washed from Polygonatum odoratum in 5 mL of distilled water and filter through a 0.22 μm filter membrane. Load the filtrate onto a pre-equilibrated Sepharose 6FF column (85 cm high). Elute with ultrapure water at a rate of 0.2 mL / min, collecting 2 mL of eluent per tube. Detect the absorbance of each tube using the phenol-sulfuric acid method and plot the 6FF elution curve. Based on the elution curve (…),… Figure 2 The two tubes at and near the point of highest absorbance were combined to collect the eluent containing polysaccharides. The eluent was concentrated under reduced pressure, and the concentrate was freeze-dried under vacuum to obtain polysaccharide (PCPA) of Polygonatum odoratum with an elution yield of 89.32±0.49%.

[0057] Test Example 1

[0058] The structure and activity of the PCPA prepared in Example 1 were determined as follows:

[0059] 1. PCPA structural determination

[0060] 1.1 Determination of the physicochemical properties of PCPA

[0061] 1.1.1 Determination of total carbohydrate, protein and uronic acid content

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

[0063] 1.1.2 Ultraviolet and Infrared Spectroscopy Measurement

[0064] PCPA was prepared into a 0.1 mg / mL solution and scanned in the wavelength range of 200–800 nm.

[0065] After thoroughly drying PCPA, weigh out 2 mg and analyze it using an infrared spectrometer at 4000–4000 cm⁻¹. -1 The area is scanned.

[0066] 1.1.3 Determination of absolute molecular weight

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

[0068] 1.1.4 Congo Red Experiment

[0069] PCPA (2.5 mg / mL) and Congo red solution (80 μmol / L) were thoroughly mixed at a 1:1 (v / v) ratio. The mixture was gradually adjusted 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). The maximum absorption wavelength of the Congo red polysaccharide solutions at different concentrations was recorded by ultraviolet scanning (200–600 nm).

[0070] 1.2 PCPA Monosaccharide Composition

[0071] Take a clean chromatographic vial, weigh an appropriate amount of PCPA sample, add 1 mL of 2M TFA acid solution, and heat at 60℃ for 1 hour. Purge with nitrogen and dry. Wash with 99.99% methanol, then dry again, repeating the methanol washing 2-3 times. Dissolve in an appropriate amount of sterile water, transfer to a chromatographic vial, and proceed with the analysis.

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

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

[0074] 2.1 Preparation of conditioned medium

[0075] Preparation of conditioned medium: Mice were euthanized by vertebral dislocation. After disinfection with 75% alcohol, the lower abdominal skin was carefully incised, and the small intestine was aseptically removed. The intestine was washed with cold HBSS solution, and Peyer's knots were carefully removed from the intestinal wall and placed in cold complete culture medium (RPMI-1640-FBS) containing 5% FBS and 1% penicillin antibiotics. The Peyer's knots were ground up using a syringe plunger through four layers of 200-mesh gauze. The cells were washed twice with HBSS containing 5% FBS (HBSS-FBS), centrifuged at 4°C for 10 min each time (1500 rpm), and finally resuspended in complete culture medium. Viable cell counts were performed by trypan blue staining, and the concentration of the Peyer's knot cell suspension was adjusted to 2 × 10⁻⁶ cells / mL. 6 Cells / mL. Add 180 μL of Peyer's knot cell suspension to each well of a 96-well plate. Add 20 μL of different concentrations of Polygonatum sibiricum polysaccharide eluent to each well (final concentrations of 0, 25, 50, 100, and 200 μg / mL), with 5 replicates per group. Incubate the 96-well plate in a 5% CO2 incubator at 37°C for 5 days. After incubation, centrifuge and collect the supernatant (conditioned medium) for later use.

[0076] 2.2 Preparation of bone marrow cell suspension

[0077] Mice were euthanized by vertebral dislocation. After disinfection with 75% alcohol, the femur and tibia were aseptically harvested, muscle tissue was removed, and the bones were placed in a petri dish containing cold HBSS solution. The femur was washed with complete culture medium until it turned white. The resulting suspension was centrifuged at 4°C (1500 rpm × 10 min) to obtain bone marrow cells. After erythrocyte lysis, the cells were washed twice with HBSS-FBS, centrifuged at 4°C for 10 min each time (1500 rpm / min). Trypan blue staining was performed to count viable cells, and the concentration of the bone marrow cell suspension was adjusted to 2.5 × 10⁻⁶ cells / min. 5 per mL.

[0078] 2.3 Evaluation of intestinal mucosal immune activity

[0079] Bone marrow cell suspension was added to 96-well plates at a density of 100 μL per well, along with 50 μL of RPMI-1640-FBS and 50 μL of conditioned medium. The plates were then incubated in a 5% CO2 incubator at 37°C for 6 days. Five hours before the end of the incubation period, 20 μL of Lamar Blue reagent was added to each well, and the plates were incubated until the end of the incubation period. Fluorescence intensity was measured at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

[0080] 3. In vivo pharmacodynamic studies of PCPA

[0081] 3.1 Animal grouping, model establishment, and drug administration regimen

[0082] Sixty 6-8 week old C57 / BL6 mice were acclimatized for one week with free access to water and food. After the acclimatization period, they were randomly divided into four groups: a normal control group (Ctrl), a model group (Cy group), a positive control 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 control group, the mice in the model group and each PCPA dose group were intraperitoneally injected with cyclophosphamide (Cy) (80 mg / kg) for 3 days to establish an immunosuppressive mouse model. After modeling, the LH group was administered levamisole hydrochloride (40 mg / kg) by gavage, while the low-dose and high-dose groups were administered 50 mg / kg and 200 mg / kg PCPA solution by gavage, respectively, once daily for 7 consecutive days. The mice were weighed daily, and their mental state, activity level, and coat luster were observed routinely. On day 8, mice were euthanized after anesthesia, their body weight was measured, and all mouse serum, spleen, duodenum, jejunum, ileum, and colon tissues were collected and stored at -80°C, or some tissues were fixed in 4% neutral formalin for further research.

[0083] 3.2 Immune organ index

[0084] After euthanizing the mice, the spleen was rapidly and aseptically separated. Adhering fat and fascia were removed, and the spleen was quickly washed in physiological saline to remove blood. The spleen was then blotted dry on filter paper, weighed immediately, and the weight recorded. The formula for calculating 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 assess the severity of intestinal injury, portions of the duodenum, jejunum, ileum, and colon were washed with pre-cooled PBS, immediately fixed in 10% paraformaldehyde solution for 24 hours, dehydrated and embedded in paraffin, and then sectioned into 5 mm sections using a tissue sectioner. Morphological changes in the duodenum, jejunum, ileum, and colon were examined using hematoxylin and eosin (H&E) staining. HE-stained sections of each intestinal segment are shown below. Figure 10 As shown.

[0088] 4. Statistical methods

[0089] GraphPad 9.5 software was used for plotting and one-way ANOVA. 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 content

[0093] The physicochemical properties of PCPA are shown in Table 1: PCPA contains more than 90% carbohydrates, contains a certain amount of uronic acid, and has a low protein content.

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

[0095] Acidic homogeneous polysaccharide of Polygonatum multiflorum content carbohydrate 90.32±1.26％ protein 1.56±0.18％ Glucuronic acid 36.91±0.56％

[0096] 5.1.2 Ultraviolet and Infrared Spectroscopic Analysis

[0097] like Figure 3 As shown, PCPA has no obvious absorption peaks at 260nm and 280nm, indicating that it does not contain nucleic acids and proteins. Figure 4 Indicates: 3389cm -1 The peak value is the stretching vibration of -OH, 2934 cm⁻¹. -1 The peak value is the stretching vibration of CH, 1737 cm. -1 The peak value is the stretching vibration of C=O on the carboxylic acid (or acetoxy group), at 1425 cm⁻¹.-1 The peak value is due to the bending vibration of pyranose, at 1141 cm⁻¹. -1 The peak value is due to the CO vibration of the O-acetyl group, at 1100 cm⁻¹. -1 The nearby peaks indicate the presence of furan ring structures in the polysaccharide, 500–900 cm⁻¹ -1 The peak value indicates the presence of a pyranose ring backbone in the polysaccharide.

[0098] 5.1.3 Absolute molecular weight analysis

[0099] LS represents multi-angle laser light scattering signal, and RI represents differential signal. Figure 5 Plotting the retention time (Time, min) on the x-axis and the relative scale on the y-axis, the weight-average molecular weight (MW) of PCPA was found to be 25.755 kDa, the number-average molecular weight (Mn) was 21.807 kDa, and the polydispersity coefficient Mw / Mn was 1.181.

[0100] 5.1.4 Spatial Conformation Analysis

[0101] like Figure 6 As shown, the maximum absorption wavelength of PCPA solution in 0–0.5 mol NaOH solution did not undergo a significant red shift, indicating that it does not have 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%, respectively, and a molar ratio of 41.9:16.55:11.01:11.7:8.51:8.21:2.11.

[0104] Table 2 Monosaccharide composition analysis of PCPA

[0105] Monosaccharide composition percentage Moor ratio Galacturonic acid 38.92％ 41.9 fructose 17.03％ 16.55 Rhamnose 10.32％ 11.01 Arabic sugar 10.07％ 11.7 Galactose 9.11％ 8.51 glucose 8.76％ 8.21 Mannose 2.11％ 2.11

[0106] 5.3 Evaluation and screening of in vitro intestinal mucosal activity of Polygonatum multiflorum polysaccharides from different elution fractions

[0107] like Figure 7As shown, in vitro intestinal mucosal immunomodulatory activity evaluation analysis indicated that PCP-W, PCP-A2, and PCP-A3 all significantly promoted bone marrow cell proliferation by stimulating Peyer's knot cells to produce conditioned medium, and this effect was dose-dependent. When the polysaccharide sample concentration was 200 μg / mL, all three polysaccharide components promoted the proliferation of bone marrow cells to its maximum extent via Peyer's knot cells. Among them, PCP-A2 exhibited stronger intestinal mucosal immunomodulatory activity than the other components.

[0108] 5.4 In vivo intestinal immune-modulating drug efficacy experiment

[0109] 5.4.1 Effects of PCPA on body weight and physiological state in immunosuppressed mice

[0110] like Figure 8 As shown in (A), after Cy injection, the body weight of mice in all Cy-treated groups showed a decreasing trend, which stopped on day 6. Compared with the Cy group, both LH and high-dose PCPA treatment slowed down the recovery of body weight (p<0.05). The food intake of mice was as follows... Figure 8 As shown in (B), after three consecutive days of intraperitoneal injection of Cy, the food intake of mice decreased sharply. After the Cy injection was stopped, the food intake of mice in each group gradually recovered on the 6th day.

[0111] 5.4.2 Effects of PCPA on Immune Organ Indices in Immunosuppressed Mice

[0112] like Figure 9 As shown, compared with the control group, the spleen index of Cy group mice was abnormally high and the thymus index was significantly low (p<0.05). After treatment with LH and different doses of PCPA, compared with the Cy group, the abnormal increase in spleen index and the abnormal decrease in thymus index of LH, PCPA-L and PCPA-H groups were significantly improved (p<0.05). The results show that PCPA has a significant immunomodulatory effect.

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

[0114] like Figure 10 As shown, H&E staining was used to analyze the effects of PCPA on the morphology of the duodenum, jejunum, ileum, and colon in Cy-treated mice. In the control group, the villi were neat, dense, and intact, but after Cy treatment, the villi of the duodenum, jejunum, and ileum 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 mucosae in the small and large intestine regions was significantly increased. These results indicate that PCPA can improve intestinal nutrient absorption and enhance the physical barrier function of the intestinal mucosa by repairing Cy-induced intestinal mucosal damage.

[0115] In summary, this invention employs a separation and purification method combining anion exchange chromatography and gel chromatography to ultimately obtain a homogeneous acidic polysaccharide from Polygonatum sibiricum with intestinal immunomodulatory activity. In vivo and in vitro intestinal immunomodulatory activity tests show that the prepared PCPA possesses high intestinal immunomodulatory activity. The preparation method provided by this invention is stable and can yield a homogeneous polysaccharide with intestinal immunomodulatory activity.

[0116] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing acidic polysaccharides from Polygonatum odoratum, characterized in that, Includes the following steps: The roots of Polygonatum odoratum were sequentially defatted with ethanol and then extracted with water to obtain an aqueous extract. The volume concentration of the ethanol solution used in the defatting process was 70%–90%, and the mass-to-volume ratio of the Polygonatum odoratum root to the ethanol solution was 1 g:(5–15) mL. The water extraction involved mixing the defatted product with water at a ratio of 1 g:(15–30) mL and then extracting. The temperature of the water extraction was 60–100°C, and the extraction time was 1–3 h. The aqueous extract and α-amylase were mixed and enzymatically hydrolyzed, followed by alcohol precipitation to obtain an alcohol precipitate. The aqueous extract was concentrated to 0.2–2 g / mL to obtain a concentrated solution. The concentrated solution and α-amylase were mixed at a mass-volume ratio of (2000–5000) g: 1 mL and enzymatically hydrolyzed at a temperature of 60–70 °C for 0.5–3 h. The alcohol precipitate was redissolved to obtain a reconstituted solution. The reconstituted solution was then deproteinized and dialyzed to retain components with a molecular weight greater than 3500 Da, thus obtaining crude polysaccharide of Sophora flavescens. The crude polysaccharide of Polygonatum odoratum was initially separated by an anion exchange column and eluted with NaCl solution to obtain polygonatum odoratum salt-washed polysaccharide; the concentration of the NaCl solution was 0.1-0.5 mol / mL; the polygonatum odoratum salt-washed polysaccharide was the component obtained by 0.2M NaCl elution solution; The polysaccharide of Polygonatum odoratum was purified by agarose gel column elution and eluted with ultrapure water to obtain an eluent containing acidic polysaccharide of Polygonatum odoratum.

2. 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%.

3. The acidic polysaccharide of Polygonatum cyrtonema prepared by the preparation method according to claim 1 or 2, characterized in that, The molecular weight of the polysaccharide from Polygonatum odoratum is 18.594–25.755 kDa.

4. The acidic polysaccharide of Polygonatum odoratum according to claim 3, characterized in that, The polysaccharide of Polygonatum odoratum comprises galacturonic acid, fructose, rhamnose, arabinose, galactose, glucose, and mannose, wherein 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.

5. The preparation method according to claim 1 or 2, or the application of the polysaccharide of Polygonatum odoratum according to claim 3 or 4, in the preparation of intestinal immunomodulatory drugs.

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