Active polysaccharide from Pteris chinensis, preparation method and application thereof

Abstract

The present invention provides an active polysaccharide from the genus Pteris serrata, a preparation method, and an application thereof. The polysaccharide comprises mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose, and fucose in a molar ratio of (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). The active polysaccharide prepared by the present invention can increase the phagocytic activity of RAW264.7 cells, increase the serum IgM level of immunosuppressed mice, improve the spleen tissue morphology of immunosuppressed mice, increase the organ index of immunosuppressed mice, and increase the proportion of T lymphocytes in the spleen of immunosuppressed mice, thereby significantly improving immunity.

Description

Technical Field

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

[0002] Polysaccharides are complex polymers composed of multiple monosaccharide molecules linked by glycosidic bonds. They possess diverse biological activities, including immunomodulatory, anti-tumor, antioxidant, and anti-aging properties. Immunomodulatory activity is a hot topic in polysaccharide pharmacological research. Currently, over 100 traditional Chinese medicine polysaccharides have been found to possess immunomodulatory properties. These include lentinan, ginseng polysaccharide, and cordyceps polysaccharide, which have been developed and marketed for clinical use in the treatment of infectious diseases, certain autoimmune diseases, and tumors. Polysaccharides are widely available, abundant in resources, and have minimal toxic side effects. As natural immunomodulators, they hold enormous potential for development and broad application prospects.

[0003] The fern, also known as the multi-toothed fern, belongs to the fern family Pteridaceae. It has the properties of clearing heat and detoxifying, killing insects and alleviating pain. It is used to treat exogenous wind-heat, fever, aversion to wind, sore throat, dry mouth, and rashes, making it an important traditional Chinese medicine. The fern is often boiled in water and taken internally, so its polysaccharide may be its main active ingredient.

[0004] In the previous experiments, the applicant used Pteris serrata to extract crude polysaccharides and further purified them to prepare fractionated polysaccharides. However, the immune activity of the fractionated polysaccharides was low. Summary of the Invention

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

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0007] The invention discloses an active polysaccharide from Pteris cerana. The monosaccharide composition of the active polysaccharide from Pteris cerana is as follows: 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).

[0008] The molecular weight of the active polysaccharide from the Pteris chinensis is 4170-4175 Da, preferably 4172 Da.

[0009] 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.

[0010] The preparation method comprises preparing crude polysaccharide of Pteris cerana, preparing target graded polysaccharide of Pteris cerana, and purifying the polysaccharide. The purification method comprises dissolving the target graded polysaccharide of Pteris cerana, eluting the polysaccharide with a 0.09-0.12 mol / L NaCl aqueous solution, collecting the eluted components, concentrating the components, and then dialysing the components with a 3500Da dialysis bag, first with tap water and then with distilled water, and freeze-drying the components to obtain the active polysaccharide of Pteris cerana.

[0011] The dialysis time of the tap water is 47-49 hours, and the dialysis time of the distilled water is 23-25 ​​hours.

[0012] The monosaccharide composition of the target graded polysaccharide of the monkey leg ferns is 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.

[0013] 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 carried out with 0, 0.1, 0.2, 0.3 and 0.4 mol / L NaCl aqueous solutions in sequence, the eluted components of 0.2 mol / L NaCl aqueous solution are collected, and after concentration, the eluted components are dialyzed with tap water and then with distilled water using a 3500Da dialysis bag, and the target graded polysaccharide of Pteris cerana is obtained after freeze-drying.

[0014] The dialysis time of the tap water is 47-49 hours, and the dialysis time of the distilled water is 23-25 ​​hours.

[0015] The preparation method of the crude polysaccharide of Pteris cerana is as follows: the dried rhizome of Pteris cerana is crushed, and then refluxed in methanol; the obtained medicinal residue is refluxed in distilled water, and then filtered; the concentrated liquid is precipitated with ethanol, washed with ethanol, and protein is removed, and then freeze-dried to obtain the crude polysaccharide of Pteris cerana.

[0016] The application of the active polysaccharide of Pteris argentea in preparing health-care products or medicines for improving immunity.

[0017] Compared with the prior art, the present invention achieves the following beneficial effects:

[0018] (1) The active polysaccharide of the present invention has a high yield of 27.8%.

[0019] (2) The active polysaccharide of the monkey leg ferns 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 T lymphocytes in the spleen of immunosuppressed mice, which has a significant effect on improving immunity.

[0020] (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

[0021] Figure 1 This is the FT-IR spectrum of the active polysaccharide of Pteris chinensis prepared in Example 1 of the present invention;

[0022] Figure 2 The active polysaccharide of Pteris chinensis prepared in Example 1 of the present invention 1 H NMR spectrum;

[0023] Figure 3 The active polysaccharide of Pteris chinensis prepared in Example 1 of the present invention 13 C NMR spectrum;

[0024] Figure 4 The molecular weight chromatograms of the polysaccharides prepared in Example 1 of the present invention and Comparative Example 1 are shown;

[0025] 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;

[0026] Figure 5 This is a bar graph showing the effect of the active polysaccharide of Pteris argentea prepared by the present invention on the serum IgM level of immunosuppressive mice;

[0027] Figure 6 This is a microscopic picture (20 times) showing the effect of the active polysaccharide of Pteris chinensis prepared in the present invention on the spleen tissue morphology of immunosuppressed mice;

[0028] 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.

[0029] Figure 7 This is a flow cytometric 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 immunosuppressive mice;

[0030] 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.

[0031] Figure 8 This is a bar graph showing the effect of the active polysaccharide of Pteris argentea prepared by the present invention on the proportion of T lymphocytes in the spleen of immunosuppressive mice;

[0032] Figure 9 This is a line graph showing the effect of the active polysaccharide of Pteris argentea prepared by the present invention on the body weight of normal mice. DETAILED DESCRIPTION

[0033] Example 1 A method for preparing active polysaccharides from Pteris chinensis

[0034] The following steps are involved:

[0035] (1) Preparation of Crude Polysaccharide from Pteris chinensis

[0036] 2 kg of dried rhizomes of Pteris cerana were crushed to a particle size of 4 mm, 8 times the volume of methanol was added, refluxed twice, each time for 1.5 h, filtered to remove methanol, and the residue was dried in the shade. 6 times the volume of distilled water was added, refluxed three times, each time for 1.5 h, filtered while hot, concentrated under reduced pressure to 200 mL, and 3 times the volume of anhydrous ethanol was added. The resulting precipitate was collected, rinsed with a small amount of anhydrous ethanol, dried at 50 ° C for 3 h, and redissolved in distilled water (1 g: 18 mL). After deproteinization using Sevage reagent (chloroform: n-butanol = 4:1, v / v), the supernatant was freeze-dried to obtain brown Pteris cerana crude polysaccharide.

[0037] (2) Preparation of target graded polysaccharides from Pteris chinensis

[0038] 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 gradient 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 fractions and color development with phenol-sulfuric acid test solution, the absorbance at 490 nm was 0, and the elution was stopped. The elution fractions of the same concentration were combined, and each elution fraction was concentrated to 100 mL. Then, the fractions were dialyzed with tap water for 48 h and distilled water for 24 h using a 3500 Da dialysis bag. After the dialysis, the fractions were freeze-dried to obtain five types of graded polysaccharides from Pteris cerana. The above five graded polysaccharides were taken for in vitro immune activity screening. The results showed that the graded polysaccharide of Pteris cerana obtained by elution with 0.2 mol / L NaCl aqueous solution had the best in vitro immune activity. The graded polysaccharide of Pteris cerana obtained by elution with 0.2 mol / L NaCl aqueous solution was marked as the target graded polysaccharide.

[0039] 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.

[0040] (3) Preparation of active polysaccharides from Pteris chinensis

[0041] 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 mixture was dialyzed with tap water for 48 h and distilled water for 24 h using a 3500 Da dialysis bag, and then freeze-dried to obtain a light yellow-brown active polysaccharide from the monkey leg horn fern.

[0042] Based on 8 g of target graded polysaccharide, the yield of the active polysaccharide from Pteris chinensis prepared by the method of Example 1 was 27.8%.

[0043] The characterization spectrum of the active polysaccharide of Pteris chinensis prepared in Example 1 is shown in Figures 1 to 3 .

[0044] 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).

[0045] Table 1 Molecular weight and monosaccharide composition of the active polysaccharide of Pteris chinensis prepared by the present invention

[0046]

[0047] Figure 1 It can be seen that 3396cm −1 、602cm −1 The characteristic absorption peaks of polysaccharides are shown at 2937cm, which belong to the stretching vibration and swing vibration of OH. −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 5.2-5.4 ppm is the terminal hydrogen signal of the α-configuration sugar residue, 4.5-4.9 ppm is the terminal hydrogen signal of the β-configuration sugar residue, and 3.0-4.4 ppm is the signal peak of the H2-H6 sugar residue. Figure 3 101-103 ppm is the terminal carbon signal of the β-configuration sugar residue, 100.5-95 ppm is the terminal carbon signal of the α-configuration sugar residue, and 45-85 ppm is the C2-C6 signal peak of the sugar residue.

[0048] Comparative Example 1

[0049] Based on Example 1, the only change 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 in Example 1.

[0050] The molecular weight of the polysaccharide prepared in Comparative Example 1 is 3520Da (see 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.

[0051] Test Example 1 In vitro phagocytic activity assay

[0052] RAW264.7 cells were collected at 1×10 4 Cells were seeded at a density of 100 μL / well in a 96-well plate and incubated in a 37°C incubator for 48 hours. The drug wells were pretreated for 24 hours with 100 μL of various concentrations of the polysaccharide sample prepared in Comparative Example 1, the active polysaccharide sample prepared in Example 1, or the target graded polysaccharide sample prepared in Step 2 of Example 1 (20 μg / mL, 40 μg / mL, and 80 μg / mL). After treatment, the cells were washed twice with PBS, and 100 μL of a 0.05% neutral red solution (m / v) was added to each well. The cells were 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. Phagocytic activity was determined by measuring absorbance at 540 nm using a multifunctional microplate reader. The results are shown in Table 2.

[0053] Table 2 Results of the determination of phagocytic activity of RAW264.7 cells

[0054]

[0055] Phagocytic activity is an important indicator for evaluating the immune activity of a drug. The stronger the effect of a drug molecule on cellular phagocytic activity, the stronger the drug's immune activity. As shown in Table 2, the active polysaccharide from Pteris chinensis prepared in Example 1 of the present invention significantly improved the phagocytic activity of RAW264.7 cells, compared to the polysaccharide prepared in Comparative Example 1 and the target fractionated polysaccharide prepared in Step 2 of Example 1.

[0056] The active polysaccharide of Pteris chinensis prepared in Example 1 was subjected to the following in vivo immunomodulatory activity experiment.

[0057] Experimental Example 2 Effect of the active polysaccharide of Pteris chinensis on the serum immunoglobulin IgM level in immunosuppressive mice

[0058] The ELISA method was used to determine the effect of active polysaccharides from Pteris chinensis on the serum IgM level of immunosuppressive 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).

[0059] The specific dosage is as follows:

[0060] Normal group (NG): gavage with equal volume of normal saline for 30 days, and starting from the 26th day, intraperitoneal injection of equal volume of normal saline for 5 days;

[0061] Model group (CG): gavage with an equal volume of normal saline for 30 days, and starting from the 26th day, intraperitoneal injection of cyclophosphamide (CXT) for 5 days, 60 mg / kg / d;

[0062] Positive control group (AG-C): Oral administration of Astragalus polysaccharide (AG) for 30 days (200 mg / kg / d), and concurrent intraperitoneal injection of CXT for 5 days (60 mg / kg / d) starting from day 26;

[0063] 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);

[0064] 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);

[0065] 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);

[0066] After the drug treatment, the mice were weighed and their weights were recorded. The eyeballs were then removed to collect blood, which was centrifuged and serum was collected. The IgM levels were determined according to the instructions. The results were as follows: Figure 5 shown.

[0067] 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 the immunosuppressive 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 increasing the IgM level is higher than that of the positive control group (AG-C), indicating that the active polysaccharide of the present invention prepared from the fern can significantly increase the level of immunoglobulin IgM in the serum of immunosuppressive mice and has immunoregulatory activity.

[0068] Experimental Example 3 Effect of the active polysaccharide of Pteris chinensis on the spleen tissue morphology of immunosuppressed mice

[0069] HE staining was used to observe the effect of the active polysaccharide of Pteris cerana on the spleen tissue morphology of CXT-induced immunosuppressive mice. The specific experimental method was as follows: after blood was taken from the mice in Experimental Example 2, they were killed by dislocation, and spleen tissues of each group of mice (3 mice per 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. The effect of the active polysaccharide of Pteris cerana on the spleen tissue morphology of immunosuppressive mice of the present invention is shown in FIG. Figure 6shown.

[0070] 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 between the white pulp and red pulp more compact and regular ( Figure 6 D, 6E, 6F). At a dose of 200 mg / kg / day, the active polysaccharide from Pteris serrata was more effective than astragalus polysaccharide (200 mg / kg / day) in improving spleen tissue morphology in immunosuppressed mice. The active polysaccharide from Pteris serrata of the present invention can significantly improve CXT-induced changes in spleen tissue morphology in mice and exhibits significant immunomodulatory activity.

[0071] Experimental Example 4 Effects of Active Polysaccharides from Pteris chinensis on Organ Indexes in Immunosuppressed Mice

[0072] Spleen and lymph node tissues were collected from mice and their organ indices were measured. The specific experimental method was as follows: After completing the sampling procedure for Experimental Example 2, spleen and lymph node tissues were collected from each group of mice, weighed, and organ mass recorded to calculate the organ index (organ index = organ mass (mg) / mouse body weight (g)). The results of the effects of the active polysaccharide from the present invention on the organ indices of immunosuppressed mice are shown in Table 3.

[0073] Table 3 Effects of the active polysaccharide of Pteris chinensis prepared by the present invention on organ indexes of immunosuppressive mice (n = 8)

[0074]

[0075] Compared with the normal group:* p <0.05,** p <0.01; compared with the model group: Δ p <0.05, ΔΔ p <0.01

[0076] 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.

[0077] Experimental Example 5 Effect of the active polysaccharide of Pteris chinensis on the proportion of T lymphocytes in the spleen of immunosuppressed mice

[0078] The effect of active polysaccharides from Pteris chinensis on the proportion of T lymphocytes in mouse spleen was determined by flow cytometry. The specific experimental method was as follows: after the organ index was determined in Experiment 4, the mouse spleen was rinsed 3 times with PBS, ground with a syringe in a clean bench, and filtered through a 74 μm nylon filter to prepare a monolayer of spleen cells. After the red blood cells were removed with red blood cell lysis buffer, the cells were washed 3 times with PBS, and RPMI 1640 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 minute, incubated in the dark for 15 minutes, centrifuged at 1500 rpm for 5 minutes, washed 3 times with PBS, discarded the supernatant, and resuspended in 300μL PBS. The T cell percentage was analyzed on a flow cytometer. The results are shown in Figure 7 and Figure 8 .

[0079] from Figure 7 and Figure 8 As can be seen, compared with the model group, the active polysaccharide from Pteris argentea at a dose of 50-200 mg / kg / d increased the proportion of T cells in the spleen cells of immunosuppressed mice, with a positive vector-effect relationship. Furthermore, the effect of increasing the proportion of T cells in the spleen of immunosuppressed mice was superior to that of Astragalus polysaccharide (200 mg / kg / d). The active polysaccharide from Pteris argentea prepared by this invention can increase the proportion of T lymphocytes in the spleen tissue of immunosuppressed mice and exhibits good immunomodulatory activity.

[0080] Example 6 Safety Investigation of Active Polysaccharides from Pteris chinensis

[0081] 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: normal group (NG), low-dose administration group (LG), and 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 this period, the mice were allowed to drink water and take food freely. 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 shown in the figure. Figure 9 The changes of organ indexes are shown in Table 4.

[0082] Table 4 Effects of the active polysaccharide of Pteris chinensis prepared by the present invention on the organ indexes of normal mice (n = 8)

[0083]

[0084] Compared with the normal group:* p <0.05

[0085] The long-term toxicity results showed that the mice in each group were in good spirits, with smooth fur, nocturnal activity, and a normal diet during the administration period. 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 administration group. As can be seen from Table 4, after 90 days of administration of active polysaccharides to mice, there was no abnormal increase in the liver index and no abnormal decrease in the kidney index compared with the normal group. Figure 9 It can be seen that the active polysaccharide of the present invention can slowly increase the weight of mice at doses of 200 mg / kg / d and 400 mg / kg / d, and the weight gain trend is similar to that of the normal group. Long-term administration of the active polysaccharide of the present invention at doses of 200 mg / kg / d and 400 mg / kg / d has no obvious toxic side effects on normal mice and is safe and reliable.

[0086] 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 principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An active polysaccharide from Pteris chinensis, characterized by: The monosaccharide composition of the active polysaccharide of Pteris chinensis is: mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and fucose, with a molar ratio of (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); The molecular weight of the active polysaccharide of Pteris chinensis is 4170-4175Da.

2. The active polysaccharide from 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.

3. The method for preparing the active polysaccharide of Pteris chinensis according to any one of claims 1 to 2, characterized in that: The preparation method comprises preparing crude polysaccharide of Pteris cerana, preparing target graded polysaccharide of Pteris cerana, and purifying the polysaccharide. The purification method comprises dissolving the target graded polysaccharide of Pteris cerana, eluting the polysaccharide with a 0.09-0.12 mol / L NaCl aqueous solution, collecting the eluted components, concentrating the components, and then dialysing the components with a 3500Da dialysis bag, first with tap water and then with distilled water, and freeze-drying the components to obtain the active polysaccharide of Pteris cerana.

4. The method according to claim 3, wherein: 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 carried out with 0, 0.1, 0.2, 0.3 and 0.4 mol / L NaCl aqueous solutions in sequence, the eluted components of 0.2 mol / L NaCl aqueous solution are collected, and after concentration, the eluted components are dialyzed with tap water and then with distilled water using a 3500Da dialysis bag, and the target graded polysaccharide of Pteris cerana is obtained after freeze-drying.

5. The method according to claim 3, wherein: The preparation method of the crude polysaccharide of Pteris cerana is as follows: the dried rhizome of Pteris cerana is crushed, and then refluxed in methanol; the obtained medicinal residue is refluxed in distilled water, and then filtered; the concentrated liquid is precipitated with ethanol, washed with ethanol, and protein is removed, and then freeze-dried to obtain the crude polysaccharide of Pteris cerana.

6. Use of the active polysaccharide of Pteris chinensis according to any one of claims 1 to 2 in the preparation of health products or medicines for improving immunity.

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