Actinidia kolomikta active polysaccharide as well as preparation method and application thereof

Abstract

The invention provides an athyrium multidentatum active polysaccharide as well as a preparation method and application thereof, and is characterized in that the monosaccharide of the athyrium multidentatum active polysaccharide is prepared from mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and fucose according to the molar ratio of (9.6 to 9.8) to (8.5 to 8.7) to (3.1 to 3.3) to (37.0 to 37.2) to (21.3 to 21.5) to (4.4 to 4.6) to (7.2 to 7.4) to (5.8 to 6.0). The athyrium multidentatum active polysaccharide prepared by the invention can improve the phagocytic activity of RAW264.7 cells, can improve the IgM level in serum of an immunosuppressive mouse, can improve the spleen tissue morphology of the immunosuppressive mouse, can increase the organ index of the immunosuppressive mouse, can improve the spleen T lymphocyte proportion of the immunosuppressive mouse, and has a remarkable immunity improving effect.

Description

Technical Field

[0001] The invention relates to an active polysaccharide of Pteris cerana, a preparation method and application thereof, and belongs to the technical field of polysaccharides. Background Art

[0002] Polysaccharides are a class of complex high-molecular compounds formed by multiple monosaccharide molecules connected by glycosidic bonds. They have multiple biological activities such as immunomodulation, anti-tumor, anti-oxidation, and anti-aging. Among them, immunomodulatory activity is a hot topic in the study of polysaccharide pharmacological effects. At present, more than 100 traditional Chinese medicine polysaccharides have been found to have immunomodulatory effects, such as Lentinan, ginseng polysaccharide, and cordyceps polysaccharide, which have been developed and marketed one after another and are used clinically to treat infectious diseases, certain autoimmune diseases, and tumors. Polysaccharides have a wide range of sources, rich resources, and few toxic and side effects. As natural immunomodulators, they have great development potential and broad application prospects.

[0003] Monkey leg fern, formerly known as multi-toothed fern, is a fern plant of the family Pteridaceae. It has the effects of clearing away heat and detoxifying, killing insects and relieving pain. It can be used for exogenous wind-heat, fever, aversion to wind, sore throat, dry mouth, rash and other symptoms. It is an important Chinese medicinal material. Monkey leg fern is often boiled in water for oral administration, so polysaccharides may be its main active ingredient.

[0004] In the previous experiments, the applicant used Pteris fasciata to extract crude polysaccharides, and further purified them to prepare graded polysaccharides, but the immune activity of the graded polysaccharides was low. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides an active polysaccharide of Pteris cretica, a preparation method and an application thereof, to achieve the following invention objectives: to improve the immunomodulatory activity of the active polysaccharide of Pteris cretica.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: A kind of active polysaccharide of Pteris cerana. The monosaccharide composition of the active polysaccharide of Pteris cerana is: mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and fucose, and the molar ratio is (9.6-9.8): (8.5-8.7): (3.1-3.3): (37.0-37.2): (21.3-21.5): (4.4-4.6): (7.2-7.4): (5.8-6.0).

[0007] The molecular weight of the active polysaccharide from Pteris chinensis is 4170-4175 Da, preferably 4172 Da.

[0008] The molar ratio of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and fucose is 9.69: 8.63: 3.21: 37.13: 21.37: 4.53: 7.29: 5.95.

[0009] The preparation method comprises preparing crude polysaccharide of Pteris cerana, preparing target graded polysaccharide of Pteris cerana and purifying; the purification method comprises dissolving the target graded polysaccharide of Pteris cerana, eluting with a 0.09-0.12 mol / L NaCl aqueous solution, collecting the eluted components, concentrating, using a 3500Da dialysis bag, first dialyzing with tap water, then dialyzing with distilled water, and freeze-drying to obtain the active polysaccharide of Pteris cerana.

[0010] The tap water dialysis time is 47-49 hours, and the distilled water dialysis time is 23-25 ​​hours.

[0011] The target graded polysaccharide of the monkey leg ferns is composed of monosaccharides of mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose, fucose, xylose, ribose, galacturonic acid and mannuronic acid, and the molar ratio is (0.134-0.138): (0.069-0.073): (0.030-0.034): (0.343-0.347): (0.139-0.143): (0.07 7-0.081): (0.019-0.023): (0.132-0.136): (0.002-0.004): (0.031-0.035): (0.004-0.006); preferably 0.136: 0.071: 0.032: 0.345: 0.141: 0.079: 0.021: 0.134: 0.003: 0.033: 0.005.

[0012] The preparation method of the target graded polysaccharide of Pteris cerana is as follows: after dissolving the crude polysaccharide of Pteris cerana, gradient elution is performed with 0, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl aqueous solutions in sequence, elution components of 0.2 mol / L NaCl aqueous solution are collected, and after concentration, the elution components are dialyzed with tap water and then with distilled water using a 3500Da dialysis bag, and freeze-dried to obtain the target graded polysaccharide of Pteris cerana.

[0013] The tap water dialysis time is 47-49 hours, and the distilled water dialysis time is 23-25 ​​hours.

[0014] The preparation method of the crude polysaccharide of Pteris cerana is as follows: the dried rhizomes of Pteris cerana are crushed, and then refluxed in methanol, the obtained medicinal residues are refluxed in distilled water, and then filtered, and the concentrated liquid is precipitated with ethanol, rinsed with ethanol, and protein is removed, and then freeze-dried to obtain the crude polysaccharide of Pteris cerana.

[0015] The application of the active polysaccharide of Pteris chinensis in preparing health products or medicines for improving immunity.

[0016] Compared with the prior art, the present invention achieves the following beneficial effects: (1) The active polysaccharide of the Pteris chinensis of the present invention has a high yield of 27.8%.

[0017] (2) The active polysaccharide prepared by the present invention can improve the phagocytic activity of RAW264.7 cells; it can increase the IgM level in the serum of immunosuppressed mice, improve the spleen tissue morphology of immunosuppressed mice, increase the organ index of immunosuppressed mice, and increase the proportion of spleen T lymphocytes in immunosuppressed mice, which has a significant effect on improving immunity.

[0018] (3) The active polysaccharide of Pteris chinensis prepared by the present invention has no obvious toxic side effects on normal mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 FT-IR spectrum of the active polysaccharide of Pteris chinensis prepared in Example 1 of the present invention; Figure 2 The active polysaccharide of Pteris chinensis prepared in Example 1 of the present invention 1 H NMR spectrum; Figure 3 The active polysaccharide of Pteris chinensis prepared in Example 1 of the present invention 13 C NMR spectrum; Figure 4 The molecular weight chromatograms of the polysaccharides prepared in Example 1 of the present invention and Comparative Example 1; Wherein A is a chromatogram of the molecular weight of the active polysaccharide of Pteris chinensis prepared by the present invention, and B is a chromatogram of the molecular weight of the polysaccharide prepared in Comparative Example 1; Figure 5 A bar graph showing the effect of the active polysaccharide of Pteris chinensis prepared by the present invention on the serum IgM level of immunosuppressed mice; Figure 6 This is a microscope picture (20 times) showing the effect of the active polysaccharide of Pteris hymenoptera prepared by the present invention on the spleen tissue morphology of immunosuppressed mice; A is a microscopic image of the spleen tissue morphology of mice in the normal group; B is a microscopic image of the spleen tissue morphology of mice in the model group; C is a microscopic image of the spleen tissue morphology of mice in the positive control group; D is a microscopic image of the spleen tissue morphology of mice in the low-dose active polysaccharide group; E is a microscopic image of the spleen tissue morphology of mice in the medium-dose active polysaccharide group; F is a microscopic image of the spleen tissue morphology of mice in the high-dose active polysaccharide group; Figure 7The flow cytometric graph shows the effect of the active polysaccharide of Pteris chinensis prepared by the present invention on the proportion of spleen T lymphocytes in immunosuppressed mice; A is the flow cytometry of spleen cells of mice in the normal group; B is the flow cytometry of spleen cells of mice in the model group; C is the flow cytometry of spleen cells of mice in the positive control group; D is the flow cytometry of spleen cells of mice in the low-dose active polysaccharide group; E is the flow cytometry of spleen cells of mice in the medium-dose active polysaccharide group; F is the flow cytometry of spleen cells of mice in the high-dose active polysaccharide group; Figure 8 A bar graph showing the effect of the active polysaccharide of Pteris chinensis prepared by the present invention on the proportion of T lymphocytes in the spleen of immunosuppressed mice; Fig. 9 It is a line graph showing the effect of the active polysaccharide of Pteris hystrix prepared by the present invention on the body weight of normal mice. DETAILED DESCRIPTION

[0020] Example 1 A method for preparing active polysaccharides from Pteris chinensis The following steps are involved: (1) Preparation of crude polysaccharide from Pteris chinensis 2 kg of dried rhizomes of Pteris fasciata were crushed to a particle size of 4 mm, and 8 times the volume of methanol was added. The mixture was refluxed twice for 1.5 h each time. The mixture was filtered to remove the methanol. After the residue was dried in the shade, 6 times the volume of distilled water was added. The mixture was refluxed three times for 1.5 h each time. The mixture was filtered while hot and concentrated under reduced pressure to 200 mL. 3 times the volume of anhydrous ethanol was added, and the resulting precipitate was collected, rinsed with a small amount of anhydrous ethanol, dried at 50°C for 3 h, and then redissolved in distilled water (1 g:18 mL). After deproteinization with Sevage reagent (chloroform: n-butanol = 4:1, v / v), the supernatant was freeze-dried to obtain brown Pteris fasciata crude polysaccharide.

[0021] (2) Preparation of target graded polysaccharides from Pteris chinensis 30 g of crude polysaccharides from Pteris cerana were dissolved in 240 mL of distilled water and centrifuged. The supernatant was applied to a DEAE-52 cellulose column (12 cm × 90 cm) and eluted with 0, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl aqueous solutions in sequence. The phenol-sulfuric acid method was used for tracking detection. After elution to the same concentration of NaCl elution components and phenol-sulfuric acid test solution for color development, the absorbance at 490 nm was 0, and the elution was stopped. The elution components of the same concentration were combined, and each elution component was concentrated to 100 mL, and then dialyzed with tap water for 48 h and distilled water for 24 h using a 3500Da dialysis bag. After the dialysis, it was freeze-dried to obtain 5 types of graded polysaccharides from Pteris cerana. The above five graded polysaccharides were screened for in vitro immune activity. The results showed that the graded polysaccharides of Pteris crenata obtained by eluting with 0.2 mol / L NaCl aqueous solution had the best in vitro immune activity. The graded polysaccharides of Pteris crenata obtained by eluting with 0.2 mol / L NaCl aqueous solution were marked as target graded polysaccharides.

[0022] The molecular weight of the target graded polysaccharide is 17412 Da, and the monosaccharide composition and molar ratio are mannose: rhamnose: glucuronic acid: glucose: galactose: arabinose: fucose: xylose: ribose: galacturonic acid: mannuronic acid = 0.136: 0.071: 0.032: 0.345: 0.141: 0.079: 0.021: 0.134: 0.003: 0.033: 0.005.

[0023] (3) Preparation of active polysaccharides from Pteris chinensis 8 g of target graded polysaccharide was dissolved in 50 mL of distilled water and centrifuged. The supernatant was applied to a Sephadex G 100 column (2 cm × 100 cm) and eluted with a 0.1 mol / L NaCl aqueous solution. The phenol-sulfuric acid method was used for tracking detection. The elution curve was drawn according to the elution volume and the absorbance at 490 nm after the eluate reacted with the phenol-sulfuric acid test solution. The symmetrical components of the elution peak were combined and concentrated to 50 mL. The polysaccharide was dialyzed with tap water for 48 h and distilled water for 24 h using a 3500Da dialysis bag. The polysaccharide was then freeze-dried to obtain a light yellow-brown active polysaccharide from the monkey leg ferns.

[0024] Based on 8 g of target graded polysaccharide, the yield of the active polysaccharide of Pteris crenata prepared by the method of Example 1 was 27.8%.

[0025] The characterization spectrum of the active polysaccharide of Pteris chinensis prepared in Example 1 is shown in Figures 1 to 3 .

[0026] The molecular weight of the active polysaccharide from Pteris chinensis prepared in Example 1 is 4172 Da (see the molecular weight chromatogram). Figure 4A), the monosaccharide composition and molar ratio are mannose (Man): rhamnose (Rha): glucuronic acid (GlcA): glucose (Glc): galactose (Gal): xylose (Xyl): arabinose (Ara): fucose (Fuc) = 9.69: 8.63: 3.21: 37.13: 21.37: 4.53: 7.29: 5.95 (see Table 1).

[0027] Table 1 Molecular weight and monosaccharide composition of the active polysaccharide of Pteris chinensis prepared by the present invention

[0028] Figure 1 It can be seen that 3396cm −1 、602cm −1 The characteristic absorption peaks of polysaccharides are shown at 2937cm −1 The signal at 1616 cm is caused by the stretching vibration of CH in the methylene group. −1 and 1418cm −1 The signal peaks at 1077 cm-1 belong to the asymmetric and symmetric vibrations of -COOH, respectively. −1 The nearby absorption peak is caused by the COC stretching vibration of the pyranose ring. Figure 2 5.2-5.4ppm is the terminal hydrogen signal of the α-configuration sugar residue, 4.5-4.9ppm is the terminal hydrogen signal of the β-configuration sugar residue, and 3.0-4.4ppm is the signal peak of the H2-H6 of the sugar residue. Figure 3 101-103ppm is the terminal carbon signal of the β-configuration sugar residue, 100.5-95ppm is the terminal carbon signal of the α-configuration sugar residue, and 45-85ppm is the C2-C6 signal peak of the sugar residue.

[0029] Comparative Example 1 The only difference from Example 1 is that in step (3), 0 mol / L NaCl aqueous solution is used for elution, and the rest of the operations are the same as those in Example 1.

[0030] The molecular weight of the polysaccharide prepared in Comparative Example 1 is 3520Da (see the molecular weight chromatogram Figure 4 B), whose monosaccharide composition is rhamnose, arabinose, galactose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, with a molar ratio of 0.026:0.025:0.033:0.507:0.192:0.189:0.024:0.004.

[0031] Test Example 1 In vitro phagocytic activity assay RAW264.7 cells were collected at 1×10 4The cells were inoculated in a 96-well plate at a density of 100 μL / well and incubated in a constant temperature incubator at 37°C for 48 hours. The drug wells were added with 100 μL of polysaccharide samples prepared in Comparative Example 1, active polysaccharide samples prepared in Example 1, or target graded polysaccharide samples prepared in Step 2 of Example 1 (20 μg / mL, 40 μg / mL, 80 μg / mL) of different concentrations, respectively, and pretreated for 24 hours. After the treatment, the cells were washed twice with PBS, and 100 μL of neutral red solution (0.05%, m / v) was added to each well and incubated for 30 minutes. After washing three times with PBS, 100 μL of glacial acetic acid-anhydrous ethanol (1:1, v / v) was added to each well and incubated at 37°C for 1 hour. The absorbance value was detected at 540 nm using a multifunctional microplate reader to determine the phagocytic activity. The experimental results are shown in Table 2.

[0032] Table 2 Results of the determination of phagocytic activity of RAW264.7 cells

[0033] Phagocytic activity is an important indicator for evaluating the immune activity of drugs. The stronger the effect of drug molecules on cell phagocytic activity, the stronger the immune activity of the drug. As can be seen from Table 2, the active polysaccharide of Pteris cerana prepared in Example 1 of the present invention can significantly improve the phagocytic activity of RAW264.7 cells compared with the polysaccharide prepared in Comparative Example 1 and the target graded polysaccharide prepared in Step 2 of Example 1.

[0034] The active polysaccharide of Pteris chinensis prepared in the above Example 1 was subjected to the following in vivo immunomodulatory activity experiment.

[0035] Experimental Example 2 Effect of active polysaccharide from Pteris chinensis on serum immunoglobulin IgM level in immunosuppressed mice The ELISA method was used to determine the effect of active polysaccharides from Pteris chinensis on the serum IgM level of immunosuppressed mice. The specific experimental method was as follows: 72 SPF-grade 36-40 g Kunming male mice were randomly divided into 6 groups, including normal group (NG), model group (CG), positive control group (AG-C), active polysaccharide low-dose group (LG-C), active polysaccharide medium-dose group (MG-C), and active polysaccharide high-dose group (GG-C).

[0036] The specific dosage is as follows: Normal group (NG): gavage with an equal volume of saline for 30 days, and from the 26th day, intraperitoneal injection of an equal volume of saline for 5 days; Model group (CG): gavage with an equal volume of normal saline for 30 days, and from the 26th day, intraperitoneal injection of cyclophosphamide (CXT) for 5 days, 60 mg / kg / d; Positive control group (AG-C): gavage of astragalus polysaccharide (AG) for 30 days (200 mg / kg / d), and from the 26th day, intraperitoneal injection of CXT for 5 days (60 mg / kg / d); Active polysaccharide low-dose group (LG-C): oral administration of active polysaccharide for 30 days (50 mg / kg / d), and starting from the 26th day, intraperitoneal injection of CXT for 5 days (60 mg / kg / d); Active polysaccharide medium dose group (MG-C): oral administration of active polysaccharide for 30 days (100 mg / kg / d), and starting from the 26th day, intraperitoneal injection of CXT for 5 days (60 mg / kg / d); Active polysaccharide high-dose group (GG-C): oral administration of active polysaccharide for 30 days (200 mg / kg / d), and starting from the 26th day, intraperitoneal injection of CXT for 5 days (60 mg / kg / d); After the drug treatment, the mice were weighed and their weights were recorded. Then, the eyeballs were removed to collect blood, which was centrifuged and serum was collected. The IgM level was determined according to the instructions. The results were as follows: Figure 5 shown.

[0037] from Figure 5 It can be seen that when the active polysaccharide of the present invention is administered at a dosage of 50 mg / kg / d-200 mg / kg / d, the level of IgM in the serum of immunosuppressed mice is significantly increased compared with the model group. When the active polysaccharide of the present invention is administered at a dosage of 50 mg / kg / d and 100 mg / kg / d, the effect of improving the IgM level is higher than that of the positive control group (AG-C), indicating that the active polysaccharide of Pteris fasciata prepared by the present invention can significantly increase the level of immunoglobulin IgM in the serum of immunosuppressed mice and has immunoregulatory activity.

[0038] Experimental Example 3 Effect of the active polysaccharide of Pteris chinensis on the spleen tissue morphology of immunosuppressed mice HE staining was used to observe the effect of active polysaccharides from Pteris cerana on the morphology of spleen tissue in CXT-induced immunosuppressive mice. The specific test method was as follows: after blood was taken from the mice in Test Example 2, they were killed by dislocation, and spleen tissues of mice in each group (3 mice in each group) were taken, fixed with 4% paraformaldehyde, dehydrated, waxed, embedded and sliced, and then HE staining was performed to observe the changes in tissue morphology under a microscope. Figure 6 shown.

[0039] from Figure 6 It can be seen that compared with the model group ( Figure 6 B), the active polysaccharide of the present invention can significantly increase the white pulp area and the number of red pulp spleen cells in the spleen tissue of immunosuppressed mice, making the spleen cords clearer and the spleen cells at the boundary of the white pulp and red pulp more compact and regular ( Figure 6 D, 6E, 6F). When the active polysaccharide of Pteris cerana was administered at a dose of 200 mg / kg / d, the effect of improving the spleen tissue morphology of immunosuppressed mice was better than that of Astragalus polysaccharide (200 mg / kg / d). The active polysaccharide of Pteris cerana of the present invention can significantly improve the changes in the spleen tissue morphology of mice caused by CXT and has significant immunomodulatory activity.

[0040] Experimental Example 4 Effects of the active polysaccharide of Pteris chinensis on organ indexes of immunosuppressed mice The spleen and lymph node tissues of mice were taken and the organ index was determined. The specific test method was as follows: After completing the sampling of the mice in Test Example 2 in Test Example 3, the spleen and lymph node tissues of mice in each group were taken, weighed, the organ mass was recorded, and the organ index was calculated (organ index = organ mass (mg) / mouse weight (g)). The results of the effect of the active polysaccharide of the present invention on the organ index of immunosuppressed mice are shown in Table 3.

[0041] Table 3 Effects of the active polysaccharide of Pteris chinensis prepared by the present invention on organ indexes of immunosuppressed mice (n = 8)

[0042] Compared with the normal group:* p <0.05,** p <0.01; compared with the model group: Δ p <0.05, ΔΔ p <0.01 As can be seen from Table 3, the active polysaccharide of the present invention can improve the spleen and lymph node indexes of immunosuppressed mice and has a significant immunoregulatory effect.

[0043] Experimental Example 5 Effect of the active polysaccharide of Pteris chinensis on the proportion of spleen T lymphocytes in immunosuppressed mice The effect of active polysaccharides from Pteris cerana on the percentage of T lymphocytes in mouse spleen was determined by flow cytometry. The specific experimental method was as follows: the mouse spleen after the organ index was determined in Experiment 4 was rinsed with PBS three times, ground with a syringe in a clean bench, and filtered through a 74 μm nylon filter to prepare a single layer of spleen cells. After the red blood cells were removed by red blood cell lysis solution, the cells were washed with PBS three times, and RPMI 1640 culture medium was added to adjust the cell concentration of each group to 5×10 6 Each group of cells was added with 5μL CD3 and 5μL CD4 antibodies, vortexed for 1 min, incubated in the dark for 15 min, centrifuged at 1500 rpm for 5 min, washed with PBS 3 times, discarded the supernatant, and resuspended with 300μL PBS. The percentage of T cells was analyzed on a flow cytometer. The results are shown in Figure 7 and Figure 8 .

[0044] from Figure 7 and Figure 8 It can be seen that compared with the model group, the active polysaccharide of Pteris cerana increased the proportion of T cells in the spleen cells of immunosuppressed mice at 50mg / kg / d-200mg / kg / d, and showed a positive vector-effect relationship. At the same time, the effect of improving the proportion of T cells in the spleen of immunosuppressed mice was better than that of Astragalus polysaccharide (200mg / kg / d). The active polysaccharide of Pteris cerana prepared by the present invention can increase the proportion of T lymphocytes in the spleen tissue of immunosuppressed mice and has good immunoregulatory activity.

[0045] Example 6 Safety investigation of active polysaccharides from Pteris chinensis A long-term toxicity experiment was used to investigate the toxic and side effects of the active polysaccharide of Pteris cerana. The specific experimental method was as follows: 24 SPF-grade 36-40g Kunming male mice were adaptively raised for 3 days and then randomly divided into 3 groups: a normal group (NG), a low-dose administration group (LG), and a high-dose administration group (GG). The normal group was gavaged with an equal volume of normal saline for 90 days, the low-dose administration group (LG) was gavaged with a low dose of Pteris cerana active polysaccharide for 90 days (200 mg / kg / d), and the high-dose administration group (GG) was gavaged with a high dose of Pteris cerana active polysaccharide for 90 days (400 mg / kg / d), during which they were free to drink water and take food. The morphology, living habits, and mental state of the mice were observed, and the daily weight of the mice was recorded. At the end of the experiment, the weight changes and survival rates of the mice were counted, and the organ index was calculated. The effect of the active polysaccharide of Pteris cerana on the weight of mice of the present invention is as follows: Fig. 9 The changes of organ indexes are shown in Table 4.

[0046] Table 4 Effects of the active polysaccharide of Pteris chinensis prepared by the present invention on the organ indexes of normal mice (n = 8)

[0047] Compared with the normal group:* p <0.05 The long-term toxicity results showed that the mice in each group were in good mental state, with smooth fur, sleeping during the day and coming out at night, and eating normally. No mice died during the experiment. Compared with the normal group, there was no significant difference in the living habits and mental state of the mice in the drug-treated group. As can be seen from Table 4, after 90 days of administration of active polysaccharides to mice, compared with the normal group, the liver index of the mice did not increase abnormally, and the kidney index did not decrease abnormally. Fig. 9It can be seen that the active polysaccharide of the present invention can slowly increase the weight of mice at a dose of 200 mg / kg / d and 400 mg / kg / d, and the weight gain trend is similar to that of the normal group. The active polysaccharide of the present invention has no obvious toxic side effects on normal mice when taken for a long time at a dose of 200 mg / kg / d and 400 mg / kg / d, and is safe and reliable.

[0048] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An active polysaccharide from Pteris chinensis, characterized in that: The monosaccharide composition of the active polysaccharide of the ferns is: mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and fucose, and the molar ratio is (9.6-9.8): (8.5-8.7): (3.1-3.3): (37.0-37.2): (21.3-21.5): (4.4-4.6): (7.2-7.4): (5.8-6.0).

2. The active polysaccharide of Pteris chinensis according to claim 1, characterized in that: The molecular weight of the active polysaccharide from Pteris chinensis is 4170-4175Da.

3. The active polysaccharide of Pteris chinensis according to claim 1, characterized in that: The molar ratio of mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and fucose is 9.69: 8.63: 3.21: 37.13: 21.37: 4.53: 7.29: 5.

95.

4. The method for preparing the active polysaccharide of Pteris chinensis according to any one of claims 1 to 3, characterized in that: The preparation method comprises preparing crude polysaccharide of Pteris cerana, preparing target graded polysaccharide of Pteris cerana and purifying; the purification method comprises dissolving the target graded polysaccharide of Pteris cerana, eluting with a 0.09-0.12 mol / L NaCl aqueous solution, collecting the eluted components, concentrating, using a 3500Da dialysis bag, first dialyzing with tap water, then dialyzing with distilled water, and freeze-drying to obtain the active polysaccharide of Pteris cerana.

5. The method according to claim 4, characterized in that: The target graded polysaccharide of the monkey leg ferns is composed of mannose, rhamnose, glucuronic acid, glucose, galactose, arabinose, fucose, xylose, ribose, galacturonic acid, and mannuronic acid, and the molar ratio is (0.134-0.138): (0.069-0.073): (0.030-0.034): (0.343-0.347): (0.139-0.143): (0.077-0.081): (0.019-0.023): (0.132-0.136): (0.002-0.004): (0.031-0.035): (0.004-0.006).

6. The method according to claim 4, characterized in that: The preparation method of the target graded polysaccharide of Pteris cerana is as follows: after dissolving the crude polysaccharide of Pteris cerana, gradient elution is performed with 0, 0.1, 0.2, 0.3, and 0.4 mol / L NaCl aqueous solutions in sequence, elution components of 0.2 mol / L NaCl aqueous solution are collected, and after concentration, the elution components are dialyzed with tap water and then with distilled water using a 3500Da dialysis bag, and freeze-dried to obtain the target graded polysaccharide of Pteris cerana.

7. The method according to claim 4, characterized in that: The preparation method of the crude polysaccharide of Pteris cerana is as follows: the dried rhizomes of Pteris cerana are crushed, and then refluxed in methanol, the obtained medicinal residues are refluxed in distilled water, and then filtered, and the concentrated liquid is precipitated with ethanol, rinsed with ethanol, and protein is removed, and then freeze-dried to obtain the crude polysaccharide of Pteris cerana.

8. Use of the active polysaccharide of Pteris chinensis according to any one of claims 1 to 3 in the preparation of health products or medicines for improving immunity.

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