A kind of wild silkworm pupa polysaccharide and its preparation method and application

Abstract

The present invention discloses a wild silkworm pupa polysaccharide and a preparation method and application thereof. The polysaccharide is extracted from wild silkworm pupa powder, and is a homopolysaccharide composed of α-D-glucose, comprising 1,4-linked, 1,4,6-linked and 1-linked α-D-glucose residues, and the molar ratio of the sugar residues is 4:1:1. The wild silkworm pupa polysaccharide has significant immunomodulatory activity, can be used to prepare medicines, and can also be used to prepare health products and foods for enhancing immunity, has good application prospects and commercial value, and can also improve the utilization value of silkworm pupae.

Description

Technical Field

[0001] The invention relates to the technical field of animal polysaccharide application, in particular to wild silkworm pupa polysaccharide and a preparation method and application thereof. Background Art

[0002] Polysaccharides are natural macromolecules formed by the polymerization of more than 10 monosaccharides through glycosidic bonds. They are widely found in plants, animals, and microorganisms, among which animal polysaccharides have been less studied. Polysaccharides are not only one of the four basic substances that constitute life, but also have significant biological activities, including immunomodulatory, anti-tumor, antioxidant, and antiviral activities. Due to its obvious therapeutic effects and almost no toxic side effects, it has received more and more attention in the biomedical field.

[0003] Wild silkworm (Bombyx mandarina M.) belongs to the phylum Arthropoda, class Insecta, order Lepidoptera, family Bombycidae, genus Bombyx. Silkworm pupae are the pupae of the silkworm, an insect of the family Bombycidae. They are the main by-product of the silkworm industry and are also traditional Chinese medicinal materials with high nutritional and medicinal value. Ancient medical works such as "Compendium of Materia Medica" record that silkworm pupae have the effects of promoting body fluid and quenching thirst, replenishing qi and nourishing blood, promoting digestion and regulating qi, and removing rheumatism. They can treat diseases such as malnutrition in children, ascariasis, various cardiovascular diseases, and chronic hepatitis. Modern research shows that silkworm pupae are rich in bioactive compounds, including proteins, oils, polysaccharides, chitin, vitamins, etc.

[0004] At present, there are no reports on the research and application of the fine structure and immune activity of wild silkworm pupa polysaccharides. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects of the prior art and provide a wild mulberry silkworm pupa polysaccharide and a preparation method and application thereof.

[0006] The specific technical solution is:

[0007] The invention provides a wild silkworm pupa polysaccharide, which is a homopolysaccharide composed of alpha-D-glucose.

[0008] Furthermore, the weight average molecular weight of the wild silkworm pupa polysaccharide is 200000-300000 Da (such as 200000, 205000, 210000, 215000, 220000, 225000, 230000, 234100, 235000, 240000, 245000, 250000, 255000, 260000, 265000, 270000, 275000, 280000, 285000, 290000, 295000, 300000 Da); in one embodiment of the present invention, the weight average molecular weight of the wild silkworm pupa polysaccharide is 234100 Da.

[0009] Furthermore, the chemical structure of the wild silkworm pupa polysaccharide contains 1,4-linked, 1,4,6-linked and 1-linked α-D-glucose residues in a molar ratio of 4:1:1.

[0010] Furthermore, in the chemical structure of the wild silkworm pupa polysaccharide, the main chain contains 1,4-α-D-glucose residues and 1,4,6-α-D-glucose residues, and the side chain contains 1-α-D-glucose residues.

[0011] Furthermore, the wild silkworm pupa polysaccharide comprises the following structure:

[0012]

[0013] wherein n is an integer from 205 to 308 (e.g., 205, 208, 210, 212, 215, 218, 220, 222, 225, 228, 230, 232, 235, 238, 240, 242, 245, 248, 250, 252, 255, 258, 260, 262, 265, 268, 270, 272, 275, 278, 280, 282, 285, 288, 290, 292, 295, 298, 300, 302, 305, 308).

[0014] The present invention also provides a method for preparing wild silkworm pupa polysaccharide, which comprises the step of extracting wild silkworm pupae.

[0015] Furthermore, the preparation method comprises the step of extracting crude polysaccharides by water extraction and alcohol precipitation.

[0016] Furthermore, the preparation method also includes the step of purifying the crude polysaccharide (such as by ion exchange column chromatography).

[0017] In one embodiment of the present invention, the preparation method comprises the following steps:

[0018] (1) taking wild silkworm pupa powder, extracting and defatting with petroleum ether to obtain defatted wild silkworm pupa powder;

[0019] (2) extracting the defatted wild silkworm pupa powder obtained in step (1) with water, and concentrating, ethanol precipitating, and removing protein from the obtained water extract to obtain crude polysaccharide;

[0020] (3) subjecting the crude polysaccharide obtained in step (2) to ion exchange column chromatography, eluting, and collecting the eluate;

[0021] (4) The eluate obtained in step (3) is dialyzed and concentrated using a dialysis bag.

[0022] Optionally, (5) freeze-drying the liquid in the dialysis bag after step (4) is completed.

[0023] Furthermore, in step (1), the solid-liquid ratio of wild silkworm pupa powder to petroleum ether is 1:1-10 (such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10); in one embodiment of the present invention, the solid-liquid ratio is 1:4.

[0024] Furthermore, in step (1), the extraction temperature is the boiling point of petroleum ether, i.e., 30-120° C. (e.g., 30, 40, 50, 60, 70, 80, 90, 100, 110, 120° C.).

[0025] Furthermore, in step (1), the number of extractions is 1-5 times (such as 1, 2, 3, 4, 5 times); in one embodiment of the present invention, the number of extractions is 3 times.

[0026] Furthermore, in step (1), each extraction time is 1-10 hours (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours); in one embodiment of the present invention, each extraction time is 5 hours.

[0027] In one embodiment of the present invention, step (1) comprises: taking wild silkworm pupa powder, adding petroleum ether, extracting at boiling temperature, collecting precipitate, and obtaining defatted wild silkworm pupa powder.

[0028] Furthermore, in step (2), the solid-liquid ratio of defatted wild silkworm pupa powder to water is 1:1-10 (such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10); in one embodiment of the present invention, the solid-liquid ratio is 1:3.

[0029] Furthermore, in step (2), the extraction temperature is 85-100°C (such as 85, 80, 95, 100°C); in one embodiment of the present invention, the extraction temperature is 97°C.

[0030] Furthermore, in step (2), the number of extractions is 1-5 times (such as 1, 2, 3, 4, 5 times); in one embodiment of the present invention, the number of extractions is 3 times.

[0031] Furthermore, in step (2), each extraction time is 1-10 hours (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours); in one embodiment of the present invention, each extraction time is 6 hours.

[0032] Furthermore, in step (2), in the alcohol precipitation step, the volume ratio of the aqueous extract concentrate to alcohol is 1:1-10 (such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10); in one embodiment of the present invention, the volume ratio is 1:4.

[0033] Furthermore, in step (2), in the alcohol precipitation step, the alcohol is ethanol.

[0034] Furthermore, in step (2), the protein is removed by the Sevage method.

[0035] In one embodiment of the present invention, step (2) comprises: extracting the defatted wild silkworm pupa powder obtained in step (1) with hot water, collecting the supernatant, concentrating it, adding anhydrous ethanol, collecting the precipitate, drying it, removing the protein therein, and obtaining crude polysaccharide.

[0036] Furthermore, in step (3), the ion exchange column is a cellulose column, and the filler of the cellulose column is DEAE-cellulose.

[0037] Furthermore, in step (3), the eluent used for elution is distilled water.

[0038] In one embodiment of the present invention, step (3) comprises: passing the aqueous solution of crude polysaccharide obtained in step (2) through a cellulose column in sequence, collecting the eluate, and concentrating it.

[0039] Furthermore, in step (4), the molecular weight cutoff of the dialysis bag is 5000-10000Da (such as 5000, 6000, 7000, 8000, 9000, 10000Da); in one embodiment of the present invention, the molecular weight cutoff is 7000Da.

[0040] In one embodiment of the present invention, step (4) comprises: placing the eluate obtained in step (3) in a dialysis bag for dialysis for 3 days, with the dialysis lasting for two days.

[0041] The present invention also provides a crude polysaccharide prepared by the method.

[0042] The present invention also provides an application of the wild silkworm pupa polysaccharide in the preparation of medicines for preventing and / or treating sepsis, beriberi, allergic diseases, gastrointestinal dysfunction, hyperlipidemia, hypertension, diabetes and cancer.

[0043] The present invention also provides an application of the wild silkworm pupa polysaccharide in preparing health products and foods for enhancing immunity.

[0044] In the above applications, the above polysaccharides can be used alone or in combination with other active ingredients.

[0045] The inventors of the present invention isolated and purified wild silkworm pupae polysaccharides from wild silkworm pupae, and conducted structural analysis and identification of its molecular weight, monosaccharide composition, chemical structure, etc., and determined its weight average molecular weight and structural composition. Cell experiments show that the polysaccharide has significant immunomodulatory activity. In particular, at a concentration of 2.5 μg / mL, the proliferation rate of T cells was the highest; at a concentration of 5 μg / mL, the proliferation rate of B cells and RAW264.7 cells was the highest. Based on this, the polysaccharide can be used to prepare immune-enhancing drugs, health products and foods, has good application prospects and commercial value, and can also provide the utilization value of silkworm pupae. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Shown is the infrared spectrum of wild silkworm pupa polysaccharide.

[0047] Figure 2 Shown is the HPLC spectrum of wild silkworm pupa polysaccharide.

[0048] Figure 3 Shown is the chemical structure of polysaccharide from Bombyx mori pupa.

[0049] Figure 4 The polysaccharide of wild silkworm pupa is shown 1 H NMR spectrum.

[0050] Figure 5 The polysaccharide of wild silkworm pupa is shown 13 C NMR spectrum.

[0051] Figure 6 The polysaccharide of wild silkworm pupa is shown 1 H- 1 H COSY spectrum.

[0052] Figure 7 Shown is the HMQC spectrum of Bombyx mandarinii pupa polysaccharide.

[0053] Figure 8 Shown is the HMBC spectrum of wild silkworm pupa polysaccharide.

[0054] Fig.9A Shown are the experimental results of the effect of wild silkworm pupa polysaccharide on T cell proliferation.

[0055] Fig. 9B Shown are the morphological images of T cells (A: blank group, B: LPS group, C: 1.25 μg / mL BMM-S, D: 2.5 μg / mL BMM-S, E: 5 μg / mL BMM-S).

[0056] Fig. 10A Shown are the experimental results of the effect of wild silkworm pupa polysaccharide on B cell proliferation.

[0057] Fig. 10BShown are the morphological images of B cells (A: blank group, B: LPS group, C: 1.25 μg / mL BMM-S, D: 2.5 μg / mL BMM-S, E: 5 μg / mL BMM-S).

[0058] Fig.11A Shown are the experimental results of the effect of wild silkworm pupa polysaccharide on macrophage proliferation.

[0059] Fig. 11B Shown are the morphological images of macrophages (A: blank group, B: LPS group, C: 1.25 μg / mL BMM-S, D: 2.5 μg / mL BMM-S, E: 5 μg / mL BMM-S). DETAILED DESCRIPTION

[0060] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0061] In the present invention, "wild silkworm (Bombyx mandarina M.)" refers to a wild insect belonging to the phylum Arthropoda, class Insecta, order Lepidoptera, family Bombycidae, genus Bombyx, which includes eggs, larvae, pupae and adults.

[0062] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0063] Example 1 Extraction, separation and purification of wild silkworm pupa polysaccharide BMM-S

[0064] 1.1 Extraction of crude polysaccharides from wild silkworm pupae by water extraction and alcohol precipitation

[0065] Weigh 200g of dried wild silkworm pupae (provided by the Sericulture Research Institute of Sichuan Academy of Agricultural Sciences), crush them, place the wild silkworm pupae powder and petroleum ether in a beaker at a solid-liquid ratio of 1:4, keep in a constant temperature water bath at boiling point temperature (boiling point 60-90°C) for 5h, collect the filter residue, and repeat 3 times. Place the defatted wild silkworm pupae powder and distilled water in a beaker at a solid-liquid ratio of 1:3, bathe in 97°C water for 6h, collect the supernatant and concentrate it after centrifugation, repeat 3 times, and finally concentrate all the supernatant to 200mL. Add anhydrous ethanol at a ratio of 1:4, let it stand to precipitate, collect the precipitate and dry it to remove the protein in the precipitate, so as to obtain crude polysaccharides from wild silkworm pupae.

[0066] 1.2 Separation and purification of crude polysaccharides from wild silkworm pupae by DEAE-cellulose column chromatography

[0067] Accurately weigh 50g of DEAE-52 cellulose and dissolve it in ultrapure water. Stir thoroughly and stop stirring if the cellulose is not in lumps and there are no obvious particles. Let it stand overnight, discard the supernatant and set aside. Prepare 0.5mol / L NaOH to soak the cellulose. After 6 hours, discard the supernatant and wash with ultrapure water until neutral. Discard the supernatant, add 0.5mol / L HCl to soak the cellulose. After 6 hours, discard the supernatant and wash with ultrapure water until neutral. Discard the supernatant again, add 0.5mol / L NaOH to soak the cellulose again for 6 hours, wash with ultrapure water until neutral, and obtain activated DEAE-52 cellulose. Let it stand for use.

[0068] The activated DEAE-52 cellulose was loaded into the column, and the crude polysaccharide was separated and purified after balancing the column with distilled water as the mobile phase for 3 hours. The diluted crude polysaccharide was centrifuged and the supernatant (6 mL) was evenly added to the activated DEAE cellulose column with distilled water as the mobile phase. The polysaccharide was determined by the phenol-sulfuric acid method. The eluate was collected and concentrated to 5 mL. Dialysis was performed using a dialysis bag (Mw ≥ 7000Da) for 48 hours. After centrifugation, the supernatant was collected and freeze-dried to obtain the wild mulberry silkworm pupa polysaccharide, named BMM-S.

[0069] Example 2 Structural Identification of Bombyx mori Polysaccharide BMM-S

[0070] 2.1 Determination of molecular weight

[0071] 10 mg of wild silkworm pupa polysaccharide BMM-S sample (obtained in Example 1, the same below) was accurately weighed, dissolved in 1 mL of ddH2O, subjected to ultrasonic treatment for 5 min, and analyzed by gel permeation chromatography.

[0072] 2.2 Infrared spectrum analysis

[0073] 5 mg of wild silkworm pupa polysaccharide BMM-S sample was accurately weighed, mixed with KBr, ground, and pressed into tablets. The sample was analyzed by Fourier transform infrared spectrometer (FT-IR) at 4000 cm -1 -400cm -1 Scan within range.

[0074] 2.3 Analysis of monosaccharide composition

[0075] Monosaccharide standards (glucose, mannose, ribose, rhamnose, galactose, xylose, arabinose, fucose, glucuronic acid, galacturonic acid) and 10 mg of wild silkworm pupa polysaccharide BMM-S sample after TFA acid hydrolysis were analyzed by high performance liquid chromatography (HPLC) using KH2PO4 and acetonitrile as mobile phases.

[0076] 2.4 Nuclear magnetic resonance analysis

[0077] Accurately weigh 50 mg of wild silkworm pupa polysaccharide BMM-S sample, dissolve it in 0.5 mL of heavy water (D2O), centrifuge it, put the supernatant into a nuclear magnetic resonance tube, and detect it on a nuclear magnetic resonance instrument (NMR).

[0078] 2.5 Methylation analysis

[0079] Accurately weigh 20 mg of wild silkworm pupa polysaccharide BMM-S sample, add 500 μL DMSO (dimethyl sulfoxide), mix well and add 10 mg of NaOH until it is insoluble. Ultrasonicate for 20 minutes, add 0.1 mL CH3I (methyl iodide), and ultrasonicate for 30 minutes. During the ultrasonication, control the temperature of the ultrasonic water bath to 18°C-20°C. Add water to terminate the reaction. Extract the product with chloroform, extract 4 times, combine the chloroform phases, and dry to obtain methylated polysaccharides. After the methylated polysaccharide is completely acid-hydrolyzed by TFA, add 0.1 mL methanol, dry and add 0.5 mL NaBD4, react at room temperature for 2 hours, add acetic acid to neutralize, add 0.1 mL methanol, blow dry, repeat 2 times, and obtain a methylated completely acid-hydrolyzed product.

[0080] After drying, add 0.5 mL of a freshly prepared mixed solution of pyridine and acetic anhydride (1:1) and react at 120°C for 30 min. Cool to room temperature, add 0.5 mL of methanol after drying, and then add 1 mL of dichloromethane, centrifuge at 12000 rpm / min for 20 min, take the supernatant, and place it in a sample bottle for GC-MS analysis.

[0081] 2.6 Test results

[0082] 2.6.1 Molecular weight of BMM-S polysaccharide

[0083] The molecular weight of wild silkworm pupa polysaccharide BMM-S is shown in Table 1, wherein the weight average molecular weight of wild silkworm pupa polysaccharide BMM-S is about 234100Da.

[0084] Table 1 Molecular weight of wild silkworm pupa polysaccharide (BMM-S)

[0085]

[0086] 2.6.2 IR spectroscopy analysis of wild silkworm pupa polysaccharide BMM-S

[0087] The Fourier transform infrared spectrum of wild silkworm pupa polysaccharide BMM-S is as follows Figure 1 As shown, at 3423.082cm -1 There is an obvious broadest absorption peak at 2923.602cm -1 Belongs to the stretching vibration of the CH of polysaccharide; signal 1635.366cm -1 Belongs to the stretching vibration of C=O; 1402.017 cm-1 The signal at 1083.815cm is attributed to the variable angle vibration of CH; -1 and 1039.460cm -1 The signal at 1200~1000cm -1 The signal is 580.478 cm-1, which is attributed to the stretching vibration of COC and is a characteristic peak of the pyranose ring. -1 This indicates that BMM-S exists in α configuration.

[0088] 2.6.3 Analysis of monosaccharide composition of wild silkworm pupa polysaccharide BMM-S

[0089] After the wild mulberry silkworm pupa polysaccharide BMM-S was hydrolyzed into monosaccharides, the monosaccharide composition was analyzed by high performance liquid chromatography. Figure 2 As shown in Figure 2, the main monosaccharide component of Bombyx mori polysaccharide BMM-S is glucose (98.31%), with a retention time of 27.044 min. Mannose, rhamnose, galactose, etc. are minor monosaccharide components.

[0090] 2.6.4 Gas chromatography and mass spectrometry analysis of BMM-S polysaccharide

[0091] The methylation results are shown in Table 2, indicating that the wild silkworm pupa polysaccharide BMM-S is mainly composed of 1,4-, 1,4,6- and 1-Glcp, and the corresponding ratio is about 4:1:1. Therefore, combined with the results of FT-IR, HPLC and GC-MS, it is speculated that the main chain of BMM-S is 1,4-α-D-Glcp, and the side chain is 1-α-D-Glcp connected to O-6 ( Figure 3 ).

[0092] Table 2 Results of methylation analysis of wild silkworm pupa polysaccharide (BMM-S)

[0093]

[0094] 2.6.5 NMR spectral analysis of Bombyx mori polysaccharide BMM-S

[0095] Polysaccharide BMM-S from wild silkworm pupa 1 H NMR spectrum ( Figure 4 )and 1 H- 1 H COSY( Figure 6) showed three signals at δ4.8-5.3ppm in the anomeric region. Combined with the literature, it was shown that only α-pyranose existed in the structure of wild silkworm pupa polysaccharide BMM-S, which was consistent with the results of FT-IR analysis. According to the methylation analysis results and the literature, the anomeric region signals δ5.27ppm, 5.23ppm and 4.84ppm corresponded to H-1 of residue A (1,4-α-D-Glcp), residue B (1,4,6-α-D-Glcp) and residue C (1-α-D-Glcp), respectively. The remaining signals at δ3.08-3.84ppm were attributed to the proton resonances on C-2 to C-6 in each monosaccharide. The hydrogen signal of water is δ4.70ppm. 13 C NMR spectrum ( Figure 5 ), δ99.80ppm, 99.95ppm and 98.66ppm in the anomeric carbon region are attributed to residue A, residue B and residue C, respectively. The signal between δ77-60ppm is attributed to the signal of C-2 to C-6 in the glucose residue. 1 H- 1 H COSY( Figure 6 ) and HMQC( Figure 7 ) Experimental relevance, 1 H (Table 3) and 13 The signals in the C NMR spectrum (Table 4) were assigned. The shifts of the signals δ3.29 / 76.48 and 3.37 / 74.80 ppm in the HMQC spectrum indicate that there is a substitution in C-4 of residues A and B. The signals δ3.66 / 71.10 and 3.92 / 71.10 ppm confirm the CH2-O-substitution of residue B. The signals at δ3.70 / 60.34 and 3.73 / 60.34 ppm indicate the presence of a sugar residue with unsubstituted CH2. In the HMBC spectrum of BMM-S ( Figure 8 ), the signal δ3.83 / 71.44 ppm is consistent with the correlation between H6 and C3 in residue A. In addition, the signal δ3.29 / 72.85 ppm is consistent with the correlation between H4 and C2 in residue A.

[0096] Table 3 Wild mulberry silkworm pupa polysaccharide (BMM-S) 1 Chemical shift of H

[0097]

[0098] Table 4 Wild mulberry silkworm pupa polysaccharide (BMM-S) 13 Chemical shift of C

[0099]

[0100]

[0101] Example 3 Study on the immunomodulatory activity of polysaccharides from wild silkworm pupae

[0102] 3.1 Reagents and instruments

[0103] RPMI-1640 culture medium, fetal bovine serum, double antibody, 0.5% trypsin, DMSO, lipopolysaccharide, CCK-8 kit, etc. are all commercially available products.

[0104] Microplate reader, cell culture incubator, inverted fluorescence microscope.

[0105] 3.2 Detection methods

[0106] The pharmacological evaluation of wild silkworm pupa polysaccharide BMM-S on the proliferation of T cells, B cells and RAW264.7 cells was detected by CCK-8 kit method. Cell culture medium (90% RPMI-1640 medium, 10% fetal bovine serum, 1% double antibody) was prepared. Cells in logarithmic growth phase (1×10 5 Cells / mL) were added to 96-well plates (100 μL / well) and incubated for 24 h (5% CO2, 37°C). Different concentrations of wild silkworm pupa polysaccharide BMM-S (final concentration 1.25, 2.5, 5 μg / mL) were added to 96-well plates (100 μL / well) and incubated for 24 h. Lipopolysaccharide LPS (final concentration 5 μg / mL) and cell culture fluid were used as positive control (LPS group) and negative control (blank group), respectively. Cell activity was detected according to the instructions of the CCK-8 kit. The optical density (OD) value was detected at 450 nm. An inverted fluorescence microscope was used to observe changes in cell morphology and cell number. The calculation method of cell viability is: cell proliferation rate (%) = [(As-Ac) / (Ac-Ab)] × 100%, where Ac is the absorbance of the negative control group (blank group), Ab is the absorbance of the cell culture fluid, and As is the absorbance of the experimental group (BMM-S) or the positive control group (LPS group).

[0107] 3.3 Statistics and Analysis

[0108] SPSS 26 software one-way analysis of variance combined with the least significant difference method was used to analyze the significant differences between the experimental group and the control group. All data presented are mean ± SD. *P < 0.05 indicates that there is a significant difference between the experimental group and the control group, and **P < 0.01 indicates that there is an extremely significant difference between the experimental group and the control group.

[0109] 3.4 Test results

[0110] 3.4.1 Effect of Bombyx mori polysaccharide BMM-S on T cell proliferation

[0111] Effect of wild silkworm pupa polysaccharide BMM-S on T cell proliferation Fig.9AAs shown. Compared with the blank group (cell proliferation rate is 0), wild mulberry silkworm pupa polysaccharide BMM-S and LPS can significantly promote T cell proliferation. When the final concentration of wild mulberry silkworm pupa polysaccharide BMM-S is 2.5μg / mL, the proliferation efficiency of T cells reaches the maximum value, increasing by 78.31%, which is equivalent to the efficiency of LPS (5μg / mL) in promoting T cell proliferation. It can be seen from the morphological diagram of T cells ( Fig. 9B ), T cells were spherical in the cell culture medium and in good condition; the cell clusters under BMM-S stimulation were larger than those in the blank group, and the cell clusters first increased and then decreased with the increase of BMM-S concentration; when the final concentration of BMM-S was 2.5 μg / mL, the cell clusters were the largest and had the largest number of cells; the cell clusters under LPS stimulation were larger than those in the blank group.

[0112] 3.4.2 Effect of Bombyx mori polysaccharide BMM-S on B cell proliferation

[0113] Effect of wild silkworm pupa polysaccharide BMM-S on B cell proliferation Fig. 10A As shown. Compared with the blank group (cell proliferation rate is 0), LPS (5μg / mL) can significantly promote B cell proliferation, with a proliferation rate of 64.81%. When the final concentration of wild silkworm pupa polysaccharide BMM-S is 2.5μg / mL and 5μg / mL, it can significantly promote B cell proliferation; when the final concentration of wild silkworm pupa polysaccharide BMM-S is 1.25μg / mL, it can significantly promote B cell proliferation. And when the final concentration of wild silkworm pupa polysaccharide BMM-S is 5μg / mL, the maximum proliferation rate is 83.60%, which is higher than the B cell proliferation rate stimulated by LPS (5μg / mL). It can be seen from the morphological diagram of B cells ( Fig. 10B ), B cells were spherical in the cell culture medium and in good condition; cells stimulated by BMM-S formed large clusters and increased in number, the cell clusters were larger than those in the blank group, and the cell clusters increased with the increase in BMM-S concentration; when the final concentration of BMM-S was 5 μg / mL, the cell clusters were the largest and had the largest number of cells; the cell clusters stimulated by LPS were larger than those in the blank group.

[0114] 3.4.3 Effect of Bombyx mori polysaccharide BMM-S on the proliferation of RAW264.7 cells

[0115] Effect of wild silkworm pupa polysaccharide BMM-S on the proliferation of RAW264.7 cells Fig.11A As shown. Compared with the blank group (cell proliferation rate is 0), LPS (5μg / mL) can significantly promote macrophage proliferation, with a proliferation rate of 89.14%. When the final concentration of wild silkworm pupa polysaccharide BMM-S is 1.25, 2.5, and 5μg / mL, it can significantly promote macrophage proliferation. At the same concentration (5μg / mL), the macrophage proliferation rate stimulated by wild silkworm pupa polysaccharide BMM-S (95.09%) is higher than that of LPS. It can be seen from the morphological diagram of macrophages ( Fig. 11B ), macrophages grew adherently in the cell culture medium, were round or oval in shape, and were in good condition; the number of cells under BMM-S stimulation was more than that in the blank group, and the number of cells increased with the increase of BMM-S concentration; the number of cells under LPS stimulation was more than that in the blank group.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A wild silkworm pupa polysaccharide, which is a homopolysaccharide composed of α-D-glucose. In the chemical structure of the wild silkworm pupa polysaccharide, the main chain contains 1,4-α-D-glucose residues and 1,4,6-α-D-glucose residues, and the side chain contains 1-α-D-glucose residues, and the molar ratio is 4:1:

1. The wild silkworm pupa polysaccharide contains the following structure: in, n is an integer from 205 to 308.

2. The wild silkworm pupa polysaccharide according to claim 1, characterized in that The weight average molecular weight of the wild silkworm pupa polysaccharide is 200000-300000Da.

3. The wild silkworm pupa polysaccharide according to claim 1 or 2, characterized in that The weight average molecular weight of the wild silkworm pupa polysaccharide is 234100Da.

4. A method for preparing the wild silkworm pupa polysaccharide according to any one of claims 1 to 3, the method comprising the following steps: (1) taking wild silkworm pupa powder, extracting and defatting with petroleum ether to obtain defatted wild silkworm pupa powder; (2) extracting the defatted wild silkworm pupa powder obtained in step (1) with water, and concentrating, ethanol precipitating, and removing protein from the obtained water extract to obtain crude polysaccharide; (3) subjecting the crude polysaccharide obtained in step (2) to ion exchange column chromatography, eluting, and collecting the eluate; (4) The eluate obtained in step (3) is dialyzed and concentrated using a dialysis bag.

5. The preparation method according to claim 4, characterized in that: In step (1), the material-liquid ratio of the wild silkworm pupa powder to petroleum ether is 1:1-10.

6. The preparation method according to claim 4 or 5, characterized in that: In step (1), the material-liquid ratio of the wild silkworm pupa powder to petroleum ether is 1:

4.

7. The preparation method according to claim 4, characterized in that: In step (1), the extraction temperature is the boiling point of petroleum ether, i.e., 30-120°C.

8. The preparation method according to claim 4, characterized in that: In step (1), the extraction times are 1 to 5 times.

9. The preparation method according to claim 4 or 8, characterized in that: In step (1), the extraction times are 3 times.

10. The preparation method according to claim 4, characterized in that: In step (1), the extraction time is 1-10 hours.

11. The preparation method according to claim 4 or 10, characterized in that: In step (1), the extraction time is 5 hours.

12. The preparation method according to claim 4, characterized in that: In step (2), the material-liquid ratio of the defatted wild silkworm pupa powder to water is 1:1-10.

13. The preparation method according to claim 4 or 12, characterized in that: In step (2), the solid-liquid ratio of the defatted wild silkworm pupa powder to water is 1:

3.

14. The preparation method according to claim 4, characterized in that: In step (2), the extraction temperature is 85-100°C.

15. The preparation method according to claim 4 or 14, characterized in that: In step (2), the extraction temperature is 97°C.

16. The preparation method according to claim 4, characterized in that: In step (2), the extraction times are 1 to 5 times.

17. The preparation method according to claim 4 or 16, characterized in that: In step (2), the extraction times are 3 times.

18. The preparation method according to claim 4, characterized in that: In step (2), the extraction time is 1-10 hours.

19. The preparation method according to claim 4 or 18, characterized in that: In step (2), the extraction time is 6 hours.

20. The preparation method according to claim 4, characterized in that: In step (2), the volume ratio of the water extract concentrate to the alcohol is 1:1-10.

21. The preparation method according to claim 4 or 20, characterized in that: In step (2), the volume ratio of the water extract concentrate to the alcohol is 1:

4.

22. The preparation method according to claim 4, characterized in that: In step (2), the alcohol is ethanol.

23. The preparation method according to claim 4, characterized in that: In step (3), the ion exchange column is a cellulose column, and its filler is DEAE-cellulose.

24. The preparation method according to claim 4, characterized in that: In step (3), the eluent used for elution is distilled water.

25. The preparation method according to claim 4, characterized in that: In step (4), the molecular weight cut-off of the dialysis bag is 5000-10000Da.

26. The preparation method according to claim 4 or 25, characterized in that: In step (4), the molecular weight cut-off of the dialysis bag is 7000Da.

27. Use of the polysaccharide according to any one of claims 1 to 3 in the preparation of a drug for enhancing immunity.

Images