Traditional Chinese medicine polysaccharide composition with immune regulation function as well as preparation method and application of traditional Chinese medicine polysaccharide composition

Abstract

The invention discloses a preparation method of a traditional Chinese medicine polysaccharide composition with an immunoregulation function, high-purity traditional Chinese medicine polysaccharides can be prepared through specific process steps such as extraction, concentration, alcohol precipitation, purification and the like, the traditional Chinese medicine polysaccharides are combined according to a specific proportion, and the composition has a better immunoregulation effect.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of traditional Chinese medicine polysaccharide compositions, and in particular to a traditional Chinese medicine polysaccharide composition with immunomodulatory function, a preparation method and application thereof. Background Art

[0002] At present, the extraction, purification and functional research of Chinese medicine polysaccharides are concentrated on the research of single medicinal polysaccharides. The chemical composition of Chinese medicine is complex, containing many types of chemical components such as flavonoids, saponins, polysaccharides, saponins, inorganic salts, etc. Modern research has shown that polysaccharide components have good immunoregulatory, hypoglycemic, anti-tumor and other effects. At present, there is little research on the immunoregulatory function of polysaccharide combinations.

[0003] Traditional Chinese medicine polysaccharides have good immunomodulatory function, but polysaccharides are a large class of substances, and traditional Chinese medicine contains many other ingredients, so it is difficult to study the extraction and purification of polysaccharides. The present invention provides a method for extracting and purifying traditional Chinese medicine polysaccharides and preparing a polysaccharide composition, which has not been reported in the literature or disclosed in the patent. Summary of the invention

[0004] The purpose of the present invention is to provide a Chinese medicine polysaccharide composition and a preparation method and application thereof. The polysaccharides in Codonopsis pilosula, Angelica sinensis, Rehmannia glutinosa and Achyranthes bidentata are extracted and purified, and the purified polysaccharides are combined in proportion to determine the immunoregulatory function of the composition, thereby providing a new direction for the extraction, purification and immunoregulatory function research of Chinese medicine polysaccharides.

[0005] The technical solution of the present invention is:

[0006] A method for preparing a Chinese medicinal polysaccharide composition having an immunomodulatory function comprises the following steps:

[0007] (1) Extraction: Take appropriate amounts of Codonopsis pilosula, Angelica sinensis, Achyranthes bidentata, and Rehmannia glutinosa, add 10 to 15 times the amount of 30% to 40% ethanol, heat and reflux at 75° C. to 85° C. for 2 to 3 times, each time for 1 to 2 hours, and filter to obtain a polysaccharide composition extract;

[0008] (2) Concentration: The filtered extracts are concentrated under reduced pressure to 80-120 mL at a temperature of 55-65° C.

[0009] (3) Alcohol precipitation: add 90% to 100% ethanol to the concentrated solution to precipitate the solution until the ethanol concentration is 70% to 80%, collect the precipitate by centrifugation, and repeat the operation 2 to 3 times;

[0010] (4) Macroporous resin purification:

[0011] Four kinds of precipitates, namely, Codonopsis pilosula, Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa, were purified by macroporous resin respectively, and the eluents were collected to obtain the crude polysaccharide extracts, which were then concentrated and dried under vacuum.

[0012] (5) Gel resin purification:

[0013] Purify the crude extracts of Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa obtained in step (4) by gel resin, respectively, and concentrate and dry under vacuum to obtain the corresponding polysaccharide extracts;

[0014] (6) Combination ratio:

[0015] The codonopsis polysaccharide obtained in step (4) and the angelica, rehmannia and achyranthes polysaccharides obtained in step (5) are combined in a mass ratio of 55-65:15-25:9-11:9-11 and mixed evenly.

[0016] Preferably, the purification step of Codonopsis pilosula in step (4) comprises: dissolving the precipitate in water, passing through D101 macroporous resin, eluting with 15-25% ethanol 10-15 times, collecting the eluate to obtain a crude extract of the polysaccharide composition, and concentrating and drying in vacuum at a temperature of 55-65°C.

[0017] Preferably, the purification step of Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa in step (4) comprises: dissolving the precipitate in water, passing through D101 macroporous resin, eluting with 10 to 15 times of water, collecting the eluate to obtain a crude extract of the polysaccharide composition, and concentrating and drying in vacuum at a temperature of 55 to 65° C.;

[0018] Preferably, in step (5), the gel resin purification step of Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa comprises: dissolving the crude polysaccharide extract in water, passing through DEAE Seplife A-25 resin, eluting with 0.3-0.5 M NaCl, collecting the eluate, dialyzing with purified water for 24-48 hours, concentrating and drying under vacuum pressure at a temperature of 55-65° C., to obtain a polysaccharide extract.

[0019] As another object of the present invention, the present invention also provides a Chinese medicinal polysaccharide composition prepared by the above method.

[0020] As another object of the present invention, the present invention also provides the use of the above-mentioned traditional Chinese medicine polysaccharide composition in the preparation of a drug with immunomodulatory function, wherein the effective concentration of the polysaccharide composition is 50-100 ug / mL.

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

[0022] 1. The extraction and purification methods of four polysaccharides, namely, codonopsis pilosula, angelica sinensis, achyranthes bidentata and rehmannia glutinosa, are clarified: polysaccharides are a type of macromolecular substance with immunomodulatory, hypoglycemic and anti-tumor effects. The extraction and purification processes of polysaccharides from different medicinal materials are different. The present invention starts with the polysaccharide extraction process and uses different extraction processes to extract and purify the polysaccharides from the four medicinal materials. After purification, the polysaccharide contents of codonopsis pilosula polysaccharide, angelica sinensis polysaccharide, achyranthes bidentata polysaccharide and rehmannia glutinosa polysaccharide are 88.36%, 69.70%, 59.06% and 55.11%, respectively, and the weight average molecular weights are 2.838*10 4 ,3.490*10 5 ,1.831*10 4 ,2.536*10 4 .

[0023] 2. Clear efficacy: The immunomodulatory function of the polysaccharide mixture obtained by the present invention was studied, and the results showed that the polysaccharide composition ratio set in the experiment had obvious immunomodulatory efficacy compared with polysaccharide extracts with other ratios. DETAILED DESCRIPTION

[0024] The present application is further explained below in conjunction with specific implementation methods, but the embodiments do not limit the present application in any form.

[0025] In the following examples, the experimental conditions used are:

[0026] 1. Content determination and molecular weight analysis of Codonopsis pilosula polysaccharide

[0027] UV-visible spectrophotometry was used, with D-anhydrous glucose as the reference substance, and the absorbance of Codonopsis pilosula polysaccharide was determined at 490 nm after color development with sulfuric acid phenol method, and the content of Codonopsis pilosula polysaccharide was calculated. Gel permeation chromatography was used, Shodex OHpakSB-803HQ and Shodex OHpak SB-805HQ columns were connected in series, the mobile phase was 0.15M sodium nitrate solution, and the flow rate was 0.6mL·min -1 , injection volume 200μL, elution time 60min.

[0028] 2. Monosaccharide composition analysis

[0029] Weigh approximately 100 mg of each of the four polysaccharides accurately and use an appropriate amount of 2 mol·L -1 Dissolve in trifluoroacetic acid and dilute to 10 mL in a volumetric flask. Tighten the cap and seal with a sealing film. Place in a 100°C water bath for hydrolysis for 8 hours. Take out and cool. Use 200 g·L -1 Adjust the pH of the sodium hydroxide solution to about 7.0, transfer to a 50 mL volumetric flask, dissolve to volume; 3000 r·min -1 Centrifuge for 15 minutes and keep the supernatant for use in PMP derivatization.

[0030] Weigh D-mannose, rhamnose, D-galacturonic acid, D-anhydrous glucose, galactose, fructose, and L(+)-arabinose as 7 monosaccharide reference substances, dissolve them in water and make up to 10 mL. Take 1 mL of each of the prepared 7 monosaccharide standards, monosaccharide mixed solution, and 4 polysaccharide hydrolyzate samples in stoppered tubes, and add 1 mL of 0.5 mol·L -1 PMP methanol solution and 0.3 mol·L -1 NaOH solution, mix well; react in a 70℃ water bath for 30 min, take out, cool for 10 min, and use 1mL0.3 mol·L -1 Neutralize with HCl; add 1 mL of chloroform to extract, shake thoroughly, discard the lower layer, repeat 3 times. The upper layer is the aqueous phase, mix well, filter, and use it as the test solution.

[0031] Shimadzu chromatography system was used: detection wavelength was 250 nm, Waters T3 column, column temperature was 30 °C, acetonitrile and 0.05 mol·L -1 Potassium dihydrogen phosphate aqueous solution (pH 6.90 adjusted with sodium hydroxide) was used as the mobile phase for gradient elution, and the flow rate of the mobile phase was 1.0 mL·L -1 min, sample injection volume 20μL, running time 90min. Elution gradient is shown in Table 1:

[0032] Table 1 Mobile phase gradient setting table

[0033]

[0034] The retention time is used for qualitative analysis and the corresponding chromatographic peak area is used for quantitative analysis. A curve is established using the concentrations of monosaccharide mixed standard solutions of different concentrations and the corresponding chromatographic peak areas. The chromatographic peak area of ​​the sample solution is substituted into the curve to calculate the content of each sugar substance in the sample solution.

[0035] 3. Research on immunomodulatory activity

[0036] 3.1 Sample preparation

[0037] Take appropriate amount of four polysaccharide extracts respectively and prepare them into 1 mg·mL with purified water -1 The mother liquor is prepared and used immediately.

[0038] 3.2 Maximum tolerable concentration (MTC) determination

[0039] Wild-type AB zebrafish 3 days post-fertilization (3 dpf) were randomly selected and placed in a 6-well plate, with 30 fish per well. Four polysaccharide mother solutions with different concentrations (1000, 500, 250, 125, 62.5, 31.25 μg mL -1), 3 mL of diluted polysaccharide solution was added to each well of the polysaccharide groups with different concentrations, and 3 mL of standard dilution water was added to each well of the normal control group and the model control group. Except for the normal control group, all other experimental groups were intravenously injected with a mixture of vinorelbine tartrate and fluorescent microspheres to establish a zebrafish immunodeficiency model; the death of zebrafish was observed with the naked eye every day, and the toxic phenotype of each organ of zebrafish was observed under a microscope. The maximum concentration that would not cause the death of zebrafish and was not toxic to the organs of zebrafish larvae was determined as the MTC of the four polysaccharides for the model zebrafish.

[0040] 3.3 Evaluation of the efficacy of promoting macrophage phagocytosis

[0041] Wild-type AB zebrafish 3 days after fertilization (3 dpf) were randomly selected and intravenously injected with fluorescent microspheres to establish a zebrafish macrophage phagocytosis model. The injected zebrafish were randomly distributed into 6-well plates, with 30 fish per well. The four polysaccharide stock solutions were diluted to different concentrations with standard dilution water, and the low concentration (25 μg mL -1 ), medium (50 μg·mL -1 ), high (100 μg·mL -1 ) dose group, 3 mL of diluted polysaccharide solution was added to each well of each polysaccharide low, medium and high dose groups, and a positive control group and a model control group were set up, and 3 mL of standard diluted water was added to each well. After treatment at 28°C for 48 hours, the fish were washed three times with standard diluted water and re-administered once. After treatment for 96 hours, the zebrafish in each group were placed under a fluorescence microscope and photographed, and the pictures were saved and the number of remaining fluorescent particles in the zebrafish was analyzed using Image-J image processing software.

[0042] 3.4 Statistical methods

[0043] SPSS26.0 was used for statistical analysis, and the results were expressed as mean ± standard deviation. If the results were in accordance with normal distribution and homogeneity of variance, the t test was used to compare the two groups, and the Turkey or Dunnet test of one-way analysis of variance was used to compare multiple groups; if the results were in accordance with normal distribution but the variance was unequal, the t′ test was used to compare the two groups, and the nonparametric Kruskal-Wallis test was used to compare multiple groups; if the results were not in accordance with normal distribution, regardless of homogeneity of variance, the Mann-Whitney test was used to compare the two groups, and the nonparametric Kruskal-Wallis test was used to compare multiple groups. P < 0.05 indicated that the difference was statistically significant.

[0044] Example 1

[0045] This embodiment provides a method for preparing a Chinese medicine polysaccharide composition with immunomodulatory function, comprising the following steps:

[0046] (1) Extraction: Take 100 g of each of Codonopsis pilosula, Angelica sinensis, Achyranthes bidentata, and Rehmannia glutinosa, respectively, and extract with 30% ethanol for 3 times, adding 1000 mL each time, extract at 80°C for 1.5 hours, and filter and collect the combined filtrate;

[0047] (2) Concentration: Concentrate the above extracts to 100 mL under reduced pressure and vacuum at 55°C;

[0048] (3) Alcohol precipitation: Add 90% ethanol to the concentrated solutions of the four medicinal materials to precipitate to 70% ethanol concentration, collect the precipitate by centrifugation, and repeat the operation three times;

[0049] (4) Macroporous resin purification:

[0050] ① Codonopsis pilosula: Dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 10 times 15% ethanol, collect the eluate to obtain a crude extract of polysaccharide composition, concentrate and dry it in vacuum at 55°C to obtain 2.11g of Codonopsis pilosula polysaccharide extract;

[0051] ② Angelica sinensis: Dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 10 times of water, collect the eluate, concentrate and dry it in vacuum at 55°C, and obtain 2.62g of Angelica sinensis crude polysaccharide extract;

[0052] ③ Achyranthes bidentata: Dissolve the precipitate in water, pass through D101 macroporous resin, elute with 10 times of water, collect the eluate, concentrate and dry in vacuum at 55°C, and obtain 2.43g of Achyranthes bidentata crude polysaccharide extract;

[0053] ④ Rehmannia glutinosa: dissolve the precipitate in water, pass through D101 macroporous resin, elute with 10 times water, collect the eluate, vacuum concentrate and dry at 55°C to obtain 2.31g of crude polysaccharide extract of Rehmannia glutinosa polysaccharide;

[0054] (5) Gel resin purification:

[0055] ① Codonopsis pilosula: No need for gel resin purification.

[0056] ② Angelica: Take 1 g of the crude polysaccharide extract of Angelica, dissolve it in water, pass it through DEAE Seplife A-25 resin, use 0.3 M NaCl to elute, collect the eluate, dialyze it with purified water for 24 hours, and vacuum concentrate and dry it at 55°C to obtain 0.35 g of the polysaccharide composition extract.

[0057] ③ Achyranthes bidentata: The crude extract of Achyranthes bidentata polysaccharide was dissolved in water, passed through DEAE Seplife A-25 resin, eluted with 0.3M NaCl, the eluate was collected, dialyzed with purified water for 24h, concentrated and dried under vacuum pressure at 55°C, and 0.28g of Achyranthes bidentata polysaccharide extract was obtained.

[0058] ④ Rehmannia glutinosa: The crude extract of Rehmannia glutinosa polysaccharide was dissolved in water, passed through DEAE Seplife A-25 resin, eluted with 0.3M NaCl, the eluate was collected, dialyzed with purified water for 24 hours, concentrated and dried under vacuum pressure at 55°C, and 0.36g of Rehmannia glutinosa polysaccharide extract was obtained.

[0059] After testing, the polysaccharide contents of purified Codonopsis pilosula polysaccharide, Angelica sinensis polysaccharide, Achyranthes bidentata polysaccharide and Rehmannia glutinosa polysaccharide were 88.36%, 69.70%, 59.06% and 55.11%, respectively, and the weight average molecular weight was 2.838*10 4 ,3.490*10 5 ,1.831*10 4 ,2.536*10 4 .

[0060] (6) Combination ratio: The above-obtained Codonopsis pilosula, Angelica sinensis, Rehmannia root and Achyranthes bidentata polysaccharides were combined in a ratio of 55:25:9:11 and mixed evenly.

[0061] The zebrafish macrophage model was used to study the immune regulation function. Compared with the model control group, the number of remaining fluorescent microspheres in the zebrafish of the normal control group and the positive control group was significantly reduced, indicating that the phagocytic function of macrophages was increased and the model was successfully established. The polysaccharide combination group was at 50 μg·mL -1 After 96 hours of treatment, the number of remaining fluorescent microspheres in zebrafish was significantly less than that in the model control group (P < 0.05). -1 After treatment, the number of remaining fluorescent microspheres in zebrafish was not significantly different from that in the model control group, indicating that the effective concentration of the polysaccharide composition in promoting the phagocytosis of zebrafish macrophages was 50 μg mL -1 about.

[0062] Example 2

[0063] This embodiment provides a method for preparing a Chinese medicine polysaccharide composition with immunomodulatory function, comprising the following steps:

[0064] (1) Extraction: Take 100 g of each of Codonopsis pilosula, Angelica sinensis, Achyranthes bidentata, and Rehmannia glutinosa, extract with 35% ethanol for 3 times, adding 1500 mL each time, extract at 75°C for 2 hours, and filter and collect the combined filtrate.

[0065] (2) Concentration: The extracts were concentrated to 80 mL under reduced pressure and vacuum at 60°C;

[0066] (3) Alcohol precipitation: Add 95% ethanol to the four medicinal material concentrates to precipitate to 75% ethanol concentration, collect the precipitate by centrifugation, and repeat the operation twice;

[0067] (4) Macroporous resin purification:

[0068] ① Codonopsis pilosula: dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 15 times 20% ethanol, collect the eluate to obtain a crude extract of polysaccharide composition, concentrate and dry it in vacuum at 60°C to obtain 2.15g of Codonopsis pilosula polysaccharide extract;

[0069] ② Angelica sinensis: Dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 15 times of water, collect the eluate, concentrate and dry it in vacuum at 60°C, and obtain 2.43g of Angelica sinensis crude polysaccharide extract;

[0070] ③ Achyranthes bidentata: Dissolve the precipitate in water, pass through D101 macroporous resin, elute with 15 times of water, collect the eluate, concentrate and dry in vacuum at 60°C to obtain 2.40 g of Achyranthes bidentata crude polysaccharide extract;

[0071] ④ Rehmannia glutinosa: dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 15 times of water, collect the eluate, concentrate and dry it in vacuum at 60°C, and obtain 2.24g of crude polysaccharide extract of Rehmannia glutinosa polysaccharide;

[0072] (5) Gel resin purification:

[0073] ① Codonopsis pilosula: No need for gel resin purification.

[0074] ② Angelica: Take 1 g of the crude polysaccharide extract of Angelica, dissolve it in water, pass it through DEAE Seplife A-25 resin, use 0.4 M NaCl to elute, collect the eluate, dialyze it with purified water for 36 hours, and vacuum concentrate and dry it at 60°C to obtain 0.30 g of the polysaccharide composition extract.

[0075] ③ Achyranthes bidentata: The crude extract of Achyranthes bidentata polysaccharide was dissolved in water, passed through DEAE Seplife A-25 resin, eluted with 0.4 M NaCl, the eluate was collected, dialyzed with purified water for 36 h, concentrated and dried under vacuum pressure at 60°C, and 0.26 g of Achyranthes bidentata polysaccharide extract was obtained.

[0076] ④ Rehmannia glutinosa: The crude extract of Rehmannia glutinosa polysaccharide was dissolved in water, passed through DEAE Seplife A-25 resin, eluted with 0.4 M NaCl, the eluate was collected, dialyzed with purified water for 36 h, concentrated and dried under vacuum pressure at 60°C, and 0.33 g of Rehmannia glutinosa polysaccharide extract was obtained.

[0077] (6) Combination ratio:

[0078] The above-obtained Codonopsis pilosula, Angelica sinensis, Rehmannia root and Achyranthes bidentata polysaccharides were combined in a ratio of 60:20:10:10 and mixed evenly.

[0079] The zebrafish macrophage model was used to study the immune regulation function. Compared with the model control group, the number of remaining fluorescent microspheres in the zebrafish of the normal control group and the positive control group was significantly reduced, indicating that the phagocytic function of macrophages was increased and the model was successfully established. The polysaccharide combination group was at 100 μg·mL -1 After 96 hours of treatment, the number of remaining fluorescent microspheres in zebrafish was significantly less than that in the model control group (P < 0.05). -1 After treatment, the number of remaining fluorescent microspheres in zebrafish was not significantly different from that in the model control group, indicating that the effective concentration of the polysaccharide composition in promoting the phagocytosis of zebrafish macrophages was 100 μg mL -1 about.

[0080] Example 3

[0081] This embodiment provides a method for preparing a Chinese medicine polysaccharide composition with immunomodulatory function, comprising the following steps:

[0082] (1) Extraction: Take 100 g of each of Codonopsis pilosula, Angelica sinensis, Achyranthes bidentata, and Rehmannia glutinosa, extract with 40% ethanol for 3 times, adding 1500 mL each time, extract at 80°C for 1 hour, and filter and collect the combined filtrate.

[0083] (2) Concentration: The extracts were concentrated to 120 mL under reduced pressure and vacuum at 65°C;

[0084] (3) Alcohol precipitation: add anhydrous ethanol to the four medicinal material concentrates to precipitate to 80% ethanol concentration, collect the precipitate by centrifugation, and repeat the operation three times;

[0085] (4) Macroporous resin purification:

[0086] ① Codonopsis pilosula: dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 12 times 25% ethanol, collect the eluate to obtain a crude extract of polysaccharide composition, concentrate and dry it in vacuum at 65°C to obtain 2.23g of Codonopsis pilosula polysaccharide extract;

[0087] ② Angelica sinensis: Dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 12 times of water, collect the eluate, concentrate and dry it in vacuum at 65°C, and obtain 2.55g of Angelica sinensis crude polysaccharide extract;

[0088] ③ Achyranthes bidentata: Dissolve the precipitate in water, pass through D101 macroporous resin, elute with 12 times of water, collect the eluate, concentrate and dry in vacuum at 65°C to obtain 2.38g of Achyranthes bidentata crude polysaccharide extract;

[0089] ④ Rehmannia glutinosa: dissolve the precipitate in water, pass it through D101 macroporous resin, elute with 12 times of water, collect the eluate, concentrate and dry it in vacuum at 65°C, and obtain 2.19g of crude polysaccharide extract of Rehmannia glutinosa polysaccharide;

[0090] (5) Gel resin purification:

[0091] ① Codonopsis pilosula: No need for gel resin purification.

[0092] ② Angelica: Take 1 g of the crude polysaccharide extract of Angelica, dissolve it in water, pass it through DEAE Seplife A-25 resin, use 0.5 M NaCl to elute, collect the eluate, dialyze it with purified water for 48 hours, and vacuum concentrate and dry it at 65°C to obtain 0.32 g of the polysaccharide composition extract.

[0093] ③ Achyranthes bidentata: The crude extract of Achyranthes bidentata polysaccharide was dissolved in water, passed through DEAE Seplife A-25 resin, eluted with 0.5M NaCl, the eluate was collected, dialyzed with purified water for 48h, concentrated and dried under vacuum pressure at 65°C, and 0.28g of Achyranthes bidentata polysaccharide extract was obtained.

[0094] ④ Rehmannia glutinosa: The crude extract of Rehmannia glutinosa polysaccharide was dissolved in water, passed through DEAE Seplife A-25 resin, eluted with 0.5 M NaCl, the eluate was collected, dialyzed with purified water for 48 h, concentrated and dried under vacuum pressure at 65°C, and 0.31 g of Rehmannia glutinosa polysaccharide extract was obtained.

[0095] (7) Combination ratio:

[0096] The above-obtained Codonopsis pilosula, Angelica sinensis, Rehmannia root and Achyranthes bidentata polysaccharides were combined in a ratio of 65:15:9:11 and mixed evenly.

[0097] The zebrafish macrophage model was used to study the immune regulation function. Compared with the model control group, the number of remaining fluorescent microspheres in the zebrafish of the normal control group and the positive control group was significantly reduced, indicating that the phagocytic function of macrophages was increased and the model was successfully established. The polysaccharide combination group was at 75 μg·mL -1 After 96 hours of treatment, the number of remaining fluorescent microspheres in zebrafish was significantly less than that in the model control group (P < 0.05). -1 After treatment, the number of remaining fluorescent microspheres in zebrafish was not significantly different from that in the model control group, indicating that the effective concentration of the polysaccharide composition in promoting the phagocytosis of zebrafish macrophages was 75 μg mL -1 about.

[0098] Comparative Example 1

[0099] Compared with Example 1, the only difference is that in step (6), the ratio of Codonopsis pilosula polysaccharide: Angelica sinensis polysaccharide: Achyranthes bidentata polysaccharide: Rehmannia glutinosa polysaccharide is 10:10:25:55.

[0100] The zebrafish macrophage model was used to study the immune regulation function. Compared with the model control group, the number of remaining fluorescent microspheres in the zebrafish of the normal control group and the positive control group was significantly reduced, indicating that the phagocytic function of macrophages was increased and the model was successfully established. The polysaccharide combination group was at 100 μg·mL -1 After 96 hours of treatment, the number of remaining fluorescent microspheres in the zebrafish had no significant difference compared with the model control group (P>0.05).

[0101] Comparative Example 2

[0102] Compared with Example 1, the only difference is that in step (6), the ratio of Codonopsis pilosula polysaccharide: Angelica sinensis polysaccharide: Achyranthes bidentata polysaccharide: Rehmannia glutinosa polysaccharide is 10:30:25:35.

[0103] The zebrafish macrophage model was used to study the immune regulation function. Compared with the model control group, the number of remaining fluorescent microspheres in the zebrafish of the normal control group and the positive control group was significantly reduced, indicating that the phagocytic function of macrophages was increased and the model was successfully established. The polysaccharide combination group was at 100 μg·mL -1 After 96 hours of treatment, the number of remaining fluorescent microspheres in the zebrafish had no significant difference compared with the model control group (P>0.05).

[0104] Comparative Example 3

[0105] Compared with Example 1, the only difference is that in step (6), the ratio of Codonopsis pilosula polysaccharide: Angelica sinensis polysaccharide: Achyranthes bidentata polysaccharide: Rehmannia glutinosa polysaccharide is 30:10:20:40.

[0106] The zebrafish macrophage model was used to study the immune regulation function. Compared with the model control group, the number of remaining fluorescent microspheres in the zebrafish of the normal control group and the positive control group was significantly reduced, indicating that the phagocytic function of macrophages was increased and the model was successfully established. The polysaccharide combination group was at 100 μg·mL -1 After 96 hours of treatment, the number of remaining fluorescent microspheres in the zebrafish had no significant difference compared with the model control group (P>0.05).

[0107] Obviously, the embodiments listed in this application are only examples for more clearly illustrating the present invention, and are not intended to limit the implementation methods of this application. For those skilled in the art, this application may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this application shall be included in the protection scope of this application.

Claims

1. A method for preparing a Chinese medicine polysaccharide composition having immunomodulatory function, characterized in that: The following steps are involved: (1) Extraction: Take appropriate amounts of Codonopsis pilosula, Angelica sinensis, Achyranthes bidentata, and Rehmannia glutinosa, add 10 to 15 times the amount of 30% to 40% ethanol, heat and reflux at 75° C. to 85° C. for 2 to 3 times, each time for 1 to 2 hours, and filter to obtain a polysaccharide composition extract; (2) Concentration: The filtered extracts are concentrated under reduced pressure and vacuum to 80-120 mL at a temperature of 55-65° C.; (3) Alcohol precipitation: add 90% to 100% ethanol to the concentrated solution to precipitate the solution until the ethanol concentration is 70% to 80%, collect the precipitate by centrifugation, and repeat the operation 2 to 3 times; (4) Macroporous resin purification: Four kinds of precipitates, namely, Codonopsis pilosula, Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa, were purified by macroporous resin respectively, and the eluents were collected to obtain the crude polysaccharide extracts, which were then concentrated and dried under vacuum. (5) Gel resin purification: Purify the crude extracts of Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa obtained in step (4) by gel resin, respectively, and concentrate and dry under vacuum to obtain the corresponding polysaccharide extracts; (6) Combination ratio: The codonopsis polysaccharide obtained in step (4) and the angelica, rehmannia and achyranthes polysaccharides obtained in step (5) are combined in a mass ratio of 55-65:15-25:9-11:9-11 and mixed evenly.

2. The method according to claim 1, characterized in that The purification step of Codonopsis pilosula in step (4) includes: dissolving the precipitate in water, passing through D101 macroporous resin, eluting with 15-25% ethanol 10-15 times, collecting the eluate to obtain a crude extract of the polysaccharide composition, and concentrating and drying in vacuum at a temperature of 55-65°C.

3. The method according to claim 1, characterized in that The purification step of Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa in step (4) comprises: dissolving the precipitate in water, passing through D101 macroporous resin, eluting with 10 to 15 times of water, collecting the eluate to obtain a crude extract of the polysaccharide composition, and concentrating and drying in vacuum at a temperature of 55 to 65°C.

4. The method according to claim 1, characterized in that: In step (5), the gel resin purification step of Angelica sinensis, Achyranthes bidentata and Rehmannia glutinosa comprises: dissolving the crude polysaccharide extract in water, passing through DEAE Seplife A-25 resin, eluting with 0.3-0.5M NaCl, collecting the eluate, dialyzing with purified water for 24-48h, concentrating and drying under vacuum pressure at a temperature of 55-65°C to obtain a polysaccharide extract.

5. A Chinese medicinal polysaccharide composition prepared by the method according to any one of claims 1 to 4.

6. Use of the Chinese medicinal polysaccharide composition according to claim 5 in the preparation of a drug having an immunomodulatory function, characterized in that: The effective concentration of the polysaccharide composition is 50-100 ug / mL.

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