Homogeneous polysaccharide with immunomodulatory activity as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to a homogeneous polysaccharide with immunoregulatory activity and a preparation method and application thereof. The homogeneous polysaccharide FPS1-2 with immunoregulation activity is extracted and can recover the body weight of immunocompromised mice, increase spleen indexes and thymus indexes, improve spleen lesion conditions, promote proliferation of CD4 + T cells and release of related cell factors, recover metabolites such as short-chain fatty acids in intestinal tracts and improve the immune regulation activity of the mice. The flora type in the intestinal microbiota is improved. Therefore, the homogeneous polysaccharide has a good application prospect in preparation of functional food for enhancing immunity or medicines for treating immunosuppression.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to a uniform polysaccharide with immunomodulatory activity and a preparation method and use thereof. Background Art

[0002] With the accelerated pace of modern life and the doubling of pressure, people generally have low immunity, and sub-health status has gradually been taken seriously by people. About 75% of the world's population is in a sub-health state. Immune system regulation is mainly manifested in the interaction between immune cells and immune molecules. The body regulates the occurrence of immune responses and maintains the key to the stability of the immune environment in the body through the synergistic interaction between multiple systems, multiple cells and multiple molecules. When the immune regulation function is abnormal, the immune system stress will produce a strong immune attack on itself, causing cell damage and loss of function, thereby causing autoimmune diseases such as rheumatoid arthritis and lupus erythematosus. On the other hand, the immune system cannot produce a moderate response to infection by external pathogenic microorganisms, or a low response may lead to serious infection, and an excessive response may cause an allergic reaction and cause damage to the body. With the increasing demand for the treatment of autoimmune diseases, the research and development and market application demand for related immune regulation drugs continue to expand. Therefore, the development of safer, more effective and non-toxic immune regulation drugs is one of the main directions of scientific research at present.

[0003] The Chinese herbal medicine Aconite is the root of the plant Aconitum of the Ranunculaceae family, which has a wide range of pharmacological effects, such as anti-arrhythmic, regulating energy metabolism, anti-inflammatory and anti-tumor activities. More and more studies have shown that the improvement of immune function by Aconite is mainly attributed to Aconite polysaccharides.

[0004] However, although these research results show the immunomodulatory effect of aconite polysaccharides, the specific pharmacological material basis of aconite polysaccharides in regulating the immune system is still unclear due to the low proportion of active polysaccharides in aconite, the lack of separation and purification methods, and the difficulty in characterizing the polysaccharide structure. This makes the existing technology of using aconite and its polysaccharides for immunomodulation not efficient and accurate enough. Summary of the invention

[0005] In view of the defects of the prior art, the present invention provides a homogeneous polysaccharide with immunomodulatory activity and a preparation method and use thereof. The purpose is to prepare a neutral homogeneous polysaccharide from the traditional Chinese medicine aconite, characterize its structure, provide a polysaccharide with immunomodulatory activity formed by condensation of monosaccharide molecules, and study the immunomodulatory activity of the obtained homogeneous polysaccharide.

[0006] A homogeneous polysaccharide with immunomodulatory activity, which is composed of glucose;

[0007] The infrared characteristic spectrum of the homogeneous polysaccharide is at 3310.2 cm -1There is an absorption peak at 2930.4cm -1 and 1419.5cm -1 There is an absorption peak of CH at 1644.7 cm -1 There is an absorption peak of complex carbohydrate nature at 1016.2 cm -1 and 992.3cm -1 There is an absorption peak of COC at

[0008] The homogeneous polysaccharide 1 In the H NMR signal, there are eight anomeric proton chemical shifts at δ 5.32, 5.30, 5.28, 5.25, 5.14, 4.90, 4.88, and 4.57 ppm;

[0009] The homogeneous polysaccharide 13 In the C NMR signal, there are eight terminal carbon chemical shifts at δ 99.90, 99.70, 99.65, 99.54, 98.68, 98.55, 95.77 and 91.87.

[0010] The absorption peak of the infrared characteristic spectrum, 1 H NMR signals and 13 The error range of C NMR signals is ±2%

[0011] A homogeneous polysaccharide with immunomodulatory activity is prepared by a method comprising the following steps:

[0012] Step 1, preparing water extract from Radix Aconiti Lateralis Preparata;

[0013] Step 2, deproteinizing the water extract and obtaining crude polysaccharide FPS after dialysis;

[0014] Step 3, purifying the crude polysaccharide FPS through a chromatographic column and eluting with water to obtain FPS1; the FPS1 is separated by gel, eluted with H2O at a flow rate of 0.45-0.55 mL / min, and the components with a retention volume of 264-348 mL are collected.

[0015] Preferably, the specific process of step 1 is:

[0016] Step 1.1, extracting the aconite root with water at a mass-to-volume ratio of 1:36-44, at an extraction temperature of 90-100° C., for an extraction time of 3.6-4.4 hours, collecting the filtrate, and concentrating to obtain a concentrate;

[0017] Step 1.2, adding 3.6-4.4 times the volume of ethanol to the concentrate of step 1.1 for precipitation, to obtain;

[0018] Preferably, the specific process of step 2 is:

[0019] Step 2.1, deproteinizing the water extract of step 1 using a mixed solution of chloroform and n-butanol in a volume ratio of 3.6-4.4:1, and dialyzing with water for 28-34 hours to obtain a dialysis solution;

[0020] Step 2.2, freeze-drying the dialysis solution in step 2.1 to obtain;

[0021] Preferably, the specific process of step 3 is:

[0022] Step 3.1, further purifying the crude polysaccharide FPS described in step 2 by passing through a DEAE-52 column, eluting with H2O at a flow rate of 1.8-2.2 mL / min, collecting the components eluted with H2O, and obtaining polysaccharide FPS1;

[0023] Step 3.2, further separate the polysaccharide FPS1 described in step 3.1 using Sephadex G-100, elute with H2O at a flow rate of 0.45-0.55 mL / min, and collect the components with a retention volume of 264-348 mL.

[0024] Preferably, the number average molecular weight M of the homogeneous polysaccharide is n The weight average molecular weight is 16.56±2.786% kDa, and the weight average molecular weight is M w 19.23 ± 3.316% kDa;

[0025] Preferably, the glycosidic bond connection composition and ratio of the homogeneous polysaccharide are T-Glcp 15.41%, 1,3-Glcp 4.06%, 1,6-Glcp 2.85%, 1,4-Glcp 60.47%, 1,3,4-Glcp 5.42% and 1,4,6-Glcp 11.79%;

[0026] Preferably, the main chain structure of the homogeneous polysaccharide is →4)-α-D-Glcp-(1→; α-D-Glcp-(1→3)-α-D-Glcp and α-D-Glcp-(1→6)-α-D-Glcp, respectively.

[0027] The O-3 position of -D-Glcp-(1→) and the O-6 position of →4,6)-α-D-Glcp-(1→) are connected to the main chain.

[0028] A homogeneous polysaccharide with immunomodulatory activity, which is composed of glucose; the number average molecular weight M of the homogeneous polysaccharide is n The weight average molecular weight is 16.56±2.786% kDa, and the weight average molecular weight is M w 19.23 ± 3.316% kDa;

[0029] The glycosidic bond composition and ratio of the homogeneous polysaccharide are T-Glcp 15.41%, 1,3-Glcp

[0030] 4.06%, 1,6-Glcp 2.85%, 1,4-Glcp 60.47%, 1,3,4-Glcp 5.42% and 1,4,6-Glcp11.79%;

[0032] The main chain structure of the homogeneous polysaccharide is →4)-α-D-Glcp-(1→; α-D-Glcp-(1→3)-α-D-Glcp and α-D-Glcp-(1→6)-α-D-Glcp are connected to the main chain through the O-3 position of →3,4)-α-D-Glcp-(1→ and the O-6 position of →4,6)-α-D-Glcp-(1→, respectively.

[0033] The present invention also provides a method for preparing the above-mentioned uniform polysaccharide, which comprises the following steps:

[0034] Step 1, preparing water extract from Radix Aconiti Lateralis Preparata;

[0035] Step 2, deproteinizing the water extract and obtaining crude polysaccharide FPS after dialysis;

[0036] Step 3, purifying the crude polysaccharide FPS through a chromatographic column and eluting with water to obtain FPS1; the FPS1 is separated by gel, eluted with H2O at a flow rate of 0.45-0.55 mL / min, and the components with a retention volume of 264-348 mL are collected.

[0037] The present invention provides a use of a uniform polysaccharide with immunomodulatory activity, characterized in that the polysaccharide is used in the preparation of products for regulating immunity, including but not limited to functional foods, health foods, foods for special medical purposes or new drugs.

[0038] Preferably, the regulation of immunity is the regulation of immunity induced by chemotherapeutic drugs.

[0039] The present invention also provides a health product or medicine for improving immunity, which is obtained by using any of the above-mentioned uniform polysaccharides as an active substance and adding pharmaceutically acceptable excipients or auxiliary components.

[0040] The present invention extracts a homogeneous polysaccharide FPS1-2 with immunomodulatory activity from aconite by designing the process flow of extraction and purification and optimizing the process parameters. The homogeneous polysaccharide FPS1-2 can restore the weight of immunosuppressed mice, increase the spleen index and thymus index, improve spleen lesions, promote the proliferation of CD4+T cells and the release of related cytokines, restore metabolites such as short-chain fatty acids in the intestine, and improve the flora type in the intestinal microbiota. This shows that the homogeneous polysaccharide has a good application prospect in the preparation of functional foods for enhancing immunity or drugs for treating immunosuppression.

[0041] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0042] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Flow chart of separating and purifying homogeneous polysaccharide FPS1-2 from Radix Aconiti Lateralis Preparata medicinal materials in the present invention;

[0044] Figure 2 A is the elution curve of FPS on DEAE-52, and B is the elution curve of FPS1 on Sephadex G-100.

[0045] Figure 3 The analytical spectra are obtained by size exclusion chromatography coupled with a differential detector and a multi-angle laser light scattering instrument (GPC-MALLs-RI). A is the analytical spectra of FPS1-1, and B is the analytical spectra of FPS1-2.

[0046] Figure 4 A is the standard sample spectrum, and B is the monosaccharide composition diagram of FPS1-2.

[0047] Figure 5 This is the Fourier transform infrared spectrum of FPS1-2.

[0048] Figure 6 This is the methylation analysis spectrum of FPS1-2.

[0049] Figure 7 This is the NMR spectrum of FPS1-2.

[0050] Figure 8The effects of FPS1-2 on body weight, thymus organ index, spleen organ index, number of leukocytes, lymphocytes and erythrocytes in cyclophosphamide (CTX)-induced immunosuppressive mice.

[0051] Fig. 9 This is a pathological staining image of spleen tissue.

[0052] Fig.10 This is the effect of FPS1-2 on INF-γ, IL-4, IL-17A and TNF-α in mouse serum.

[0053] Fig.11 Immunohistochemical detection of the effect of FPS1-2 on CD4+ expression. A is the staining picture of CD4+ protein, and B is the content of CD4+ protein.

[0054] Fig.12 The effect of FPS1-2-H on the contents of acetate, propionate, butyrate and valerate in colon contents.

[0055] Fig.13 The effect of FPS1-2-H on the composition and structure of intestinal microbiota. A is the distribution diagram at the microbial phylum level, B is the relative abundance of Firmicutes, Bacteroidetes and Ascomycetes, C is the distribution diagram at the microbial genus level, D is the relative abundance of Lactobacillus and Akkermansia, E is the linear discriminant analysis (LDA) score diagram, and F is the linear discriminant analysis effect size (LEfSe) diagram of lineage distribution. DETAILED DESCRIPTION

[0056] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0057] Example 1 Homogeneous polysaccharide FPS1-2 with immunomodulatory activity and its preparation method Figure 1 As shown, the uniform polysaccharide FPS1-2 of Aconite root in this example was prepared by the following method:

[0058] (1) 500 g of dried Aconite powder was extracted with 40 volumes of water at 100° C. for 4 hours. The filtrate was collected, concentrated, and precipitated with 4 volumes of ethanol.

[0059] (2) Dissolve the precipitate and remove the protein from the solution using chloroform: n-butanol (volume ratio 4:1). Repeat this process until there is no flocculent material in the middle of the solution, indicating that the protein has been completely removed. Dialyze the solution with distilled water for 31 hours.

[0060] (3) The obtained solution is freeze-dried to obtain crude polysaccharide (hereinafter referred to as FPS).

[0061] (4) FPS was further purified by DEAE-52 column eluting with H2O, 0.2M and 0.5M NaCl solutions at a flow rate of 2.0 mL / min. Figure 2 As shown, the polysaccharide fraction eluted with H2O was collected and freeze-dried to obtain polysaccharide (hereinafter referred to as FPS1).

[0062] (5) FPS1 was further separated using Sephadex G-100 and eluted with H2O at a flow rate of 0.5 mL / min, collecting 6 mL in each tube. Figure 2 As shown, the fractions from tubes 32 to 42 were collected, combined, concentrated and freeze-dried to obtain white powder polysaccharides named FPS1-1, and the fractions from tubes 44 to 58 were collected, combined, concentrated and freeze-dried to obtain white powder polysaccharides named FPS1-2.

[0063] The technical solution of the present invention is further described below through experiments. The samples FPS1-2 tested in the following experimental examples are all prepared by the method of Example 1.

[0064] Experimental Example 1 Molecular Weight Determination

[0065] 1. Experimental Methods

[0066] The molecular weight of polysaccharides was accurately determined by high performance size exclusion chromatography-multi-angle laser light scattering detector-differential refractive index detector (HPSEC-MALLS-RID) with the following specific operations:

[0067] Weigh the polysaccharide samples and prepare 5.0 mg / mL sample solutions. All solutions were filtered through a 0.22 μm microporous membrane and then analyzed by HPSEC-MALLS-RID. Chromatographic columns: OHpak SB-806HQ 300×8mm, OHpak SB-804HQ 300×8mm (Shodex, Japan) in series; mobile phase: 0.2M NaCl; flow rate: 1.0 mL / min; detector: (RID-G7162A) differential detector and DAWN detector (Wyatt Technology Co., USA); Determination of weight average molecular weight and number average molecular weight (M w and M n ) and polydispersity index (M w / M n ). ASTRA 8 software was used for data collection and calculation.

[0068] 2. Experimental Results

[0069] The results are shown in Table 1 and Figure 3 Shown: The number average molecular weight M of the homogeneous polysaccharide FPS1-2 n The weight average molecular weight is 16.56±2.786% kDa, and the weight average molecular weight is M w The molecular weight of the polysaccharide was 19.23±3.316% kDa and the dispersion index PDI was 1.16, indicating that the polysaccharide was a broadly distributed polymer.

[0070] The molecular weight and radius of the separated polysaccharides are shown in Table 1:

[0071] Table 1 Average molecular weight and radius

[0072]

[0073]

[0074] Note: Mw is weight average molecular weight, Mn is number average molecular weight, Mp is peak molecular weight, Rz is c is the root mean square radius.

[0075] Experimental Example 2 Monosaccharide composition analysis

[0076] 1. Experimental Methods

[0077] 1. Experimental reagents

[0078] Standard monosaccharides (arabinose, ribose, xylose, glucose, fructose, mannose, galactose, fucose, rhamnose, galacturonic acid, guluronic acid, glucuronic acid and mannonic acid) were used and the solvents for HPLC were of chromatographic purity.

[0079] 2. Experimental conditions

[0080] The monosaccharide composition of FPS1-2 was determined by high performance liquid ion chromatography, and the specific operation was as follows:

[0081] FPS1-2 sample (5.0 mg) was hydrolyzed in 5.0 mL trifluoroacetic acid (TFA, 2 M) at 120°C for 2 h. The chromatographic system used was a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), and the monosaccharide components were analyzed and detected using an electrochemical detector. Dionex TM CarboPac TMPA20 (150*3.0mm, 10μm) liquid chromatography column; injection volume is 5μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1M NaOH, 0.2M NaAc), flow rate 0.5mL / min; column temperature 30℃; elution gradient: 0min A phase / B phase / C phase (95:5:0, V / V), 26min A phase / B phase / C phase (85﹕5﹕10, V / V), 42min A phase / B phase / C phase (85﹕5﹕10, V / V), 42.1min A phase / B phase / C phase (60:0:40, V / V), 52min A phase / B phase / C phase (60﹕40﹕0, V / V), 52.1min A phase / B phase / C phase (95﹕5﹕0, V / V), 60min Phase A / Phase B / Phase C (95﹕5﹕0, V / V).

[0082] 2. Experimental Results

[0083] The results are as follows Figure 4 Shown: FPS1-2 is composed only of glucose, indicating that it is a glucan.

[0084] Experimental Example 3 Fourier Transform Infrared Spectroscopy Analysis

[0085] 1. Experimental Methods

[0086] Fourier transform infrared spectroscopy was used to reveal the characteristic functional groups of polysaccharides. The specific operation was as follows:

[0087] 1.5 mg of polysaccharide samples (FPS1-1 and FPS1-2) were weighed and mixed with 200 mg of KBr. The samples were analyzed by infrared spectroscopy (V70 Advance Bruker, Germany) at 4000-400 cm -1 Data detection and analysis are carried out within the scope.

[0088] 2. Experimental Results

[0089] The results are as follows Figure 5 Shown: 3310.2cm -1 The broad absorption peak near 2930.4 cm is caused by the stretching vibration of -OH. -1 and 1419.5cm -1 The absorption peak at 1644.7cm is caused by CH stretching vibration. -1 The absorption peak at 1016.2cm indicates the presence of complex carbohydrate characteristics. -1 and 992.3cm -1 The strong absorption peak near 954cm is caused by the stretching vibration of COC in the pyranose ring. -1 and 574.3cm -1The characteristic absorption peak at is caused by the α / β-glycosidic bond.

[0090] Experimental Example 4 Methylation Analysis

[0091] 1. Experimental Methods

[0092] The type and percentage of glycosidic linkages in FPS1-2 were determined by methylation analysis as follows:

[0093] (1) 10.0 mg of polysaccharide sample was dissolved in 2.0 mL of thoroughly dried anhydrous DMSO, and 30 mg of NaOH powder was added and heated and stirred to allow it to fully dissolve. In an ice-water bath and under N2 protection, CH3I was slowly added, and the mixture was protected from light and ultrasonicated for 2 h. 1.0 mL of distilled water was added to quench the reaction. Finally, 2.5 mL of chloroform and water (1:1, v / v) were used to extract three times, and the organic phase was dried with magnesium sulfate to obtain methylated polysaccharide. The above experiment was repeated twice. (2) The obtained methylated polysaccharide was hydrolyzed, reduced and acetylated. The acetylated product was dissolved with 3.0 mL of CH2Cl2, transferred to a 10 mL separatory funnel, and 2.0 mL of ultrapure water was added. The mixture was shaken, and after static stratification, the upper aqueous solution was removed. This was repeated three times. The CH2Cl2 solutions were combined, and after removing the possible water contained in the funnel filled with anhydrous Na2SO4, the volume was adjusted to 10.0 mL in a volumetric flask, and the final product was analyzed by GC-MS. (3) Detection conditions: Chromatographic column: RXI-5SIL MS chromatographic column 30×0.25 mm; injection mode: split; split ratio: 50.0; injection volume: 1.0 μL. The program temperature conditions are: starting temperature 120°C, 3°C·min -1 Heating to 250℃·min -1 ; Maintain for 5 min; Inlet temperature 250°C, detector temperature 250°C, flow rate 1.0 mL min -1 , the carrier gas is helium.

[0094] 2. Experimental Results

[0095] The results are shown in Table 2. The types and proportions of glycosidic bonds in FPS1-2 are inferred from mass spectrometry fragmentation data as T-Glcp (15.41%), 1,3-Glcp (4.06%), 1,6-Glcp (2.85%), 1,4-Glcp (60.47%), 1,3,4-Glcp (5.42%) and 1,4,6-Glcp (11.79%). The above data indicate that the main glycosidic bond type of FPS1-2 is 1,4-Glcp (60.47%). In addition, the presence of 1,4,6-Glcp indicates that some 1,4-Glcp units have branches at O-6.

[0096] Table 2 Methylation analysis and glycosidic bond connection of FPS1-2

[0097]

[0098]

[0099] Experimental Example 5 Nuclear Magnetic Resonance Spectrum Analysis

[0100] 1. Experimental Methods

[0101] NMR analysis is used to obtain the main information of the isomeric configuration and type of polysaccharide branches. The specific operation is as follows:

[0102] 50 mg of dried FPS1-2 was dissolved in D2O (0.6 mL) and exchanged three times before NMR analysis. NMR spectra were recorded on a Bruker Advance III 600-MHz NMR spectrometer.

[0103] 2. Experimental Results

[0104] One-dimensional ( 1 H and 13 C) NMR spectrum results are as follows Figure 7 A, 7B: FPS1-2 1 The H NMR signals are mainly concentrated between δ3.2 and 5.5 ppm. The signals at δ5.32, 5.30, 5.28, 5.25, 5.14, 4.90, 4.88 and 4.57 ppm belong to the terminal protons. Therefore, the polysaccharide residues can be labeled as a, b, c, d, e, f, g and h, respectively. The signals in the range of δ3.2-4.2 ppm correspond to the protons H-2 to H-6 of the residues. 13 The C NMR signals are mainly concentrated between δ60 and 110, and the terminal carbon region of the glycosidic bond is mainly located between δ95 and 102. The main terminal carbon signal peaks are observed at δ99.90, 99.70, 99.65, 99.54, 98.68, 98.55, 95.77 and 91.87. The chemical shifts of C2 to C6 of the sugar ring are mainly located between δ60 and 85.

[0105] The results of 2D NMR spectroscopy are as follows Figure 7 As shown in C-7F: A characteristic terminal hydrogen signal appears at δ5.32 in the heteronuclear single quantum coherence (HSQC) spectrum, and the corresponding signal is δ99.54. From the hydrogen-hydrogen correlation ( 1 H- 1H-1 / H-2 / H-3 / H-4 / H-5 signals can be observed in the H COSY spectrum. Therefore, the proton signals of H-1, H-2, H-3, H-4 and H-5 are inferred to be δ5.32, 3.55, 3.87, 3.58 and 3.75, respectively. In the nuclear Overhauser effect (NOESY) spectrum, there are correlation peaks between δ5.32, δ3.55 and δ3.76, indicating that the chemical shift of H-6 is δ3.76. According to the HSQC and heteronuclear multiple bond correlation (HMBC) spectra, the chemical shifts of C-1, C-2, C-3, C-4, C-5 and C-6 are δ99.54, 71.47, 73.31, 76.61, 71.15 and 60.44, respectively. Therefore, these signals should be attributed to the glycosidic bond of (→4)-α-Glcp-(1→). All signals within the residue were determined in a similar manner and are summarized in Table 3.

[0106] The structural identification results showed that the main chain structure of the polysaccharide was →4)-α-D-Glcp-(1→.α-D-Glcp-(1→3)-α-D-Glcp and α-D-Glcp-(1→6)-α-D-Glcp were connected to the main chain via the O-3 position of →3,4)-α-D-Glcp-(1→ and the O-6 position of →4,6)-α-D-Glcp-(1→, respectively.

[0107] Table 3 FPS1-2 1 H and 13 C NMR signal assignment (ppm)

[0108]

[0109] Experimental Example 6 Regulatory Effects on Immunodeficient Mice

[0110] 1. Experimental Methods

[0111] 1. Experimental Animals

[0112] Genus: SPF KM mice, weight: 18-22g, 56, male.

[0113] 2. Rearing conditions

[0114] Temperature range: 23℃±1℃; humidity range: 40%~70%; ventilation frequency: no less than 15 times of fresh air / hour; 12 hours light and 12 hours dark alternation.

[0115] 3. Construction of mouse immunodeficiency model

[0116] Fifty-six male SPF KM mice (18-22 g) were adaptively housed at 23±1℃ with free access to water and food for 7 days. Subsequently, they were randomly assigned to seven experimental groups (8 mice in each group): normal control group (NC), model group (M), positive control group (PC), low-dose FPS1-1 group (FPS1-1-L), high-dose FPS1-1 group (FPS1-1-H), low-dose FPS1-2 group (FPS1-2-L), and high-dose FPS1-2 group (FPS1-2-H). Mice in the NC group were injected with normal saline, and mice in the other groups were injected with cyclophosphamide (CTX) at a dose of 80 mg / kg for 3 consecutive days to complete the induction of the immune injury model. Then, the PC group was orally administered with Lentinan (5 mg / kg) every day. The experimental groups were orally administered with FPS1-1 and FPS1-2 once a day (200 mg / kg in the low-dose group and 400 mg / kg in the high-dose group). After 10 days of treatment, all mice were killed by injection of pentobarbital. The spleen, thymus, eye blood, colon contents and feces of each mouse were collected, and the experimental results were statistically processed.

[0117] 4. Determination of mouse body weight

[0118] During the experiment, the body weights of the seven groups of mice were weighed and recorded every day, and a curve was drawn based on the changes in body weight.

[0119] 5. Calculation of organ index

[0120] The thymus and spleen were dissected and accurately weighed with an electronic analytical balance. The thymus index and spleen index were calculated according to the following formula.

[0121] Thymus index = average thymus mass (mg) / average mouse weight (g);

[0122] Spleen index = average spleen mass (mg) / average mouse body weight (g).

[0123] 6. Counting of immune cells

[0124] Blood samples were collected from the eyeball using heparin tubes, and the number of immune cells in the blood was measured using a fully automatic blood analyzer (BC-5120, Myriad) within 24 hours.

[0125] 7. Histomorphological analysis

[0126] The spleen was fixed with 10% formalin solution, routinely paraffin-embedded, sectioned, dehydrated and sealed after hematoxylin-eosin staining, and the pathological morphology of the tissue was observed under an optical microscope.

[0127] 8. Determination of serum cytokine levels

[0128] After the last administration, blood was collected from the eyeballs of the animals in each group the next morning, and serum was obtained after centrifugation at 3000r / min for 15min and stored in a refrigerator at 4°C. The NO content in the serum of mice was then determined using Griess reagent. The TNF-α, INF-γ, IL-4 and IL-17A contents in the serum were determined according to the operating instructions of the ELISA kit.

[0129] 9. Determination of CD4+ protein level

[0130] Spleen sections were deparaffinized with xylene and then gradually hydrated. Paraffin-embedded tissue sections were treated with 0.3% H2O2 for 15 minutes. Then, the samples were rinsed with PBS to remove excess reagents and immersed in 10% goat serum for 15 minutes to block nonspecific binding sites. In addition, the sections were incubated with anti-CD4+ mouse primary antibody (1:200) at 4°C overnight. Unbound primary antibody was rinsed with PBS, and tissue sections were then incubated with secondary antibody for 30 minutes. Then, the sections were stained with diaminobenzidine (DAB) and re-stained with hematoxylin. Finally, the total amount of CD4+ protein was calculated using Image Pro Plus 6.0 software.

[0131] 10. Determination of short-chain fatty acid content

[0132] The colon contents (0.5 g) were dissolved in a mixed solution of 1 mL of water and 300 μL of acetone. The resulting solution was homogenized for 3 minutes and then centrifuged at 12,000 rpm. The supernatant was evaluated for its specific SCFAs content by an Agilent 7820 gas chromatograph equipped with a DB-FFAP capillary column (30.00 m × 0.25 mm × 0.25 μm). The column temperature was initially maintained at 70 °C for 5.0 minutes and then increased to 100 °C at a rate of 6 °C / min.

[0133] 11. Determination of intestinal microbiota composition

[0134] Genomic DNA was extracted from mouse fecal samples. The purity of the extracted DNA was assessed by 1.0% agarose gel electrophoresis and amplified by PCR using primers amplifying relevant fragments of the 16S rDNA V3-V4 region. α-Diversity was measured by calculating Chao1 and Simpson_e indices. β-Diversity was analyzed by calculating PCoA and NMDS. All analyses were performed by R language software. Linear discriminant analysis (LDA) and linear discriminant analysis effect size (LEfSe) methods were used to compare and visualize the microorganisms of different groups.

[0135] 2. Experimental Results

[0136] 1. Effects on thymus index, spleen index and immune cell count

[0137] CTX is an anticancer drug with antimitotic and apoptosis-inducing effects, which can lead to a decrease in body weight, spleen, and thymus weight in mice. Therefore, the effects of FPS1-1 and FPS1-2 on body weight and spleen and thymus organ indexes in CTX-induced immunosuppressive mice were studied. Figure 8 As shown, the body weight, spleen index, and thymus index of CTX-treated mice were significantly decreased compared with the NC group. However, the body weight, spleen index, and thymus index of the FPS1-1 and FPS1-2 treatment groups (200 and 400 mg / kg) showed an upward trend. Among them, the effect of the FPS1-2-H group was comparable to that of the positive drug group. The results showed that FPS1-2 can effectively restore the body weight, spleen index, and thymus index of immunosuppressed mice.

[0138] Lymphocytes are crucial immune cells in the human immune system and are an important defense barrier against pathogen invasion. Therefore, the effects of FPS1-1 and FPS1-2 on immune cell counts were evaluated. Figure 8 D-8F shows that compared with the NC group, the white blood cell and lymphocyte counts in the M group were decreased (P<0.05), and the white blood cell count in the FPS1-2-H group increased significantly, reaching 2.06×10 9 / L. Both low-dose and high-dose groups of FPS1-1 and FPS1-2 promoted lymphocyte proliferation, and the effect of the FPS1-2-H group was comparable to that of the PC group. In addition, the red blood cell levels of mice were not affected by CTX or drug treatment. The results show that FPS1-2 can significantly increase the number of immune cells.

[0139] 2. Histopathological analysis

[0140] The results are as follows Fig. 9 As shown: In the NC group, the boundary between the red pulp and white pulp in the spleen was obvious, and after CTX treatment, the boundary between the red pulp and white pulp became blurred, and the number and size of splenic nodules were significantly reduced. However, these adverse reactions were significantly reversed by the FPS1-2 group and the PC group. Compared with the M group, the boundary between the red pulp and white pulp in the FPS1-2 group gradually returned to normal. At the same time, the cell density in the FPS1-2 group increased, and the cells were arranged tightly and orderly. The results showed that FPS1-2 can effectively improve the pathological conditions of the spleen in immunosuppressed mice.

[0141] 3. Effects on serum cytokine levels

[0142] In response to antigen stimulation, CD4+T cell subsets secrete various cytokines, which play a vital role in immune response. For example, Th1, Th2, and Th17 helper CD4+T cell subsets can secrete cytokines such as TNF-α, INF-γ, IL-4, and IL-17A. Therefore, the immunomodulatory effect of FPS1-2 was determined by detecting CD4+T cell-related cytokines. The results are shown in Fig.10 As shown: The levels of INF-γ, IL-4, IL-17A and TNF-α in the M group were significantly lower than those in the NC group (P<0.05). After FPS1-2 treatment, the secretion levels of INF-γ, IL-4, IL-17A and TNF-α were significantly increased compared with the M group. The results showed that FPS1-2 can significantly promote the secretion of cytokines related to CD4+T cell subsets.

[0143] 4. Immunohistochemistry to evaluate its effect on CD4+ expression

[0144] The spleen is a vital immune organ in the immune system and an important site for CD4+T cells to execute immune responses. Therefore, the number of CD4+T cells in the spleen of each group of mice was determined by observing the expression of CD4+ antibodies through immunohistochemistry. Fig.11 As shown: CD4+ antibody is brown. Compared with the NC group, the CD4+ protein level in the M group was significantly reduced (P<0.01), indicating that CTX can inhibit the proliferation of CD4+ T cells, leading to immunosuppression. Compared with the M group, the CD4+ protein content in the FPS1-1-H, FPS1-2-L, and FPS1-2-H groups was significantly upregulated (P<0.01 or 0.05 compared with the M group). The results show that FPS1-2 can promote the proliferation of CD4+ T cells in the spleen.

[0145] 5. Effects on short-chain fatty acids in colon contents

[0146] Short-chain fatty acids (SCFA) are metabolites produced by intestinal microbiota during polysaccharide digestion and are closely related to immune cell proliferation and dysfunction. Therefore, the SCFA levels of the NC, M, and FPS1-2-H groups were measured to investigate the effect of FPS1-2-H on short-chain fatty acids produced by intestinal microbiota. Fig.12 As shown: Compared with the NC group, the contents of the three major SCFAs (acetic acid, propionic acid, and butyric acid) were reduced in the M group. After FPS1-2-H treatment, the contents of the three short-chain fatty acids increased significantly, and immunosuppression was reversed. However, there was no significant difference in the content of valeric acid. The results showed that FPS1-2-H can enhance the production of short-chain fatty acids in immunosuppressed mice.

[0147] 6. Effects on the composition and structure of the intestinal microbiota

[0148] The intestinal microbiota is composed of a vast community of trillions of bacteria that interact with the host's immune system in various ways. In recent years, various polysaccharides have been shown to have the ability to regulate the ecological balance of the intestinal microbiota. Therefore, the present invention selected the NC, M, and FPS1-2-H (F2H) groups for further 16S rRNA sequencing to study the composition of the intestinal microbiota. The results are shown in Figure 2. Fig.13 As stated: At the microbial phylum level, Firmicutes and Bacteroidetes were the dominant phyla in the intestinal community, accounting for more than 90% of the total. In the M group, the abundance of Firmicutes increased and the abundance of Bacteroidetes decreased. In contrast, the F2H group reduced the ratio of Firmicutes to Bacteroidetes. The relative abundance of harmful bacteria such as Actinobacteria decreased slightly. At the microbial genus level, the relative abundance of Lachnospiraceae_NK4A136, Lactobacillus, Bacteroides, and Akkermansia in the M group was significantly reduced compared with the NC group, while the relative abundance of uncultured bacterium, Ruminococacaceae_UCG-014, and Mucispirillum increased significantly. However, the F2H group can regulate the relative abundance of Lactobacillus and Akkermansia. LEfSe results showed that the F2H group displayed 7 enriched taxa (LDA>3.0, P<0.05), and Ruminococacaceae, RuminococceaeUCG_014 and Rikenellaceae were identified as the main enriched taxa. It is worth noting that Ruminococcocacae and RuminococacaeUCG_014 are key beneficial bacteria widely distributed in the intestinal microbiota of healthy mice. In the M group, the main enriched taxa were Desulfovibrionaceae and Gutmetagenome. These results indicate that the main microbiota composition of the F2H group was different compared with that of the M group. The branch diagram of the F2H group showed that the three marker species (Rikenellaceae, Ruminococacae and

[0149] The results showed that FPS1-2 altered the types of key bacteria in the gut microbiota of immunodeficient mice and promoted the proliferation of specific bacteria.

[0150] The above results indicate that the homogeneous polysaccharide FPS1-2 has a significant immunomodulatory effect on immunodeficient mice, and its effect is significantly better than that of another homogeneous polysaccharide FPS1-1 isolated from Aconitum.

[0151] Through the above embodiments and experimental examples, it can be seen that the present invention extracts a homogeneous polysaccharide FPS1-2 with immunomodulatory activity, which can restore the weight of immunosuppressed mice, increase spleen index and thymus index, improve spleen lesions, promote the proliferation of CD4+T cells and the release of related cytokines, and restore metabolites such as short-chain fatty acids in the intestine, and improve the flora type in the intestinal microbiota. This shows that the homogeneous polysaccharide has a good application prospect in the preparation of functional foods for enhancing immunity or drugs for treating immunosuppression.

Claims

1. A homogeneous polysaccharide having immunomodulatory activity, characterized in that: The homogeneous polysaccharide consists of glucose; The infrared characteristic spectrum of the homogeneous polysaccharide is at 3310.2 cm -1 There is an absorption peak at 2930.4cm -1 and 1419.5cm -1 There is an absorption peak of CH at 1644.7 cm -1 There is an absorption peak of complex carbohydrate nature at 1016.2 cm -1 and 992.3cm -1 There is an absorption peak of COC at The homogeneous polysaccharide 1 In the H NMR signal, there are eight anomeric proton chemical shifts at δ 5.32, 5.30, 5.28, 5.25, 5.14, 4.90, 4.88, and 4.57 ppm; The homogeneous polysaccharide 13 In the C NMR signal, there are eight terminal carbon chemical shifts at δ 99.90, 99.70, 99.65, 99.54, 98.68, 98.55, 95.77, and 91.87; The absorption peak of the infrared characteristic spectrum, 1 H NMR signals and 13 The error range of C NMR signals is ±2%.

2. A homogeneous polysaccharide having immunomodulatory activity, characterized in that: It is prepared by a method comprising the following steps: Step 1, preparing water extract from Radix Aconiti Lateralis Preparata; Step 2, deproteinizing the water extract and obtaining crude polysaccharide FPS after dialysis; Step 3, purifying the crude polysaccharide FPS through a chromatographic column and eluting with water to obtain FPS1; the FPS1 is separated by gel, eluted with H2O at a flow rate of 0.45-0.55 mL / min, and the components with a retention volume of 264-348 mL are collected.

3. The homogeneous polysaccharide according to claim 2, characterized in that: The specific process of step 1 is as follows: Step 1.1, extracting the aconite root with water at a mass-to-volume ratio of 1:36-44, at an extraction temperature of 90-100° C., for an extraction time of 3.6-4.4 hours, collecting the filtrate, and concentrating to obtain a concentrate; Step 1.2, adding 3.6-4.4 times the volume of ethanol to the concentrate of step 1.1 for precipitation, to obtain; And / or, the specific process of step 2 is: Step 2.1, deproteinizing the water extract of step 1 using a mixed solution of chloroform and n-butanol in a volume ratio of 3.6-4.4:1, and dialyzing with water for 28-34 hours to obtain a dialysis solution; Step 2.2, freeze-drying the dialysis solution described in step 2.1 to obtain.

4. The homogeneous polysaccharide according to claim 2, characterized in that: The specific process of step 3 is as follows: Step 3.1, further purifying the crude polysaccharide FPS described in step 2 by passing through a DEAE-52 column, eluting with H2O at a flow rate of 1.8-2.2 mL / min, collecting the components eluted with H2O, and obtaining polysaccharide FPS1; Step 3.2, further separate the polysaccharide FPS1 described in step 3.1 using Sephadex G-100, elute with H2O at a flow rate of 0.45-0.55 mL / min, and collect the components with a retention volume of 264-348 mL.

5. The homogeneous polysaccharide according to any one of claims 1 to 4, characterized in that: The number average molecular weight M of the homogeneous polysaccharide n The weight average molecular weight is 16.56±2.786% kDa, and the weight average molecular weight is M w 19.23 ± 3.316% kDa; and / or, the glycosidic bond connection composition and ratio of the homogeneous polysaccharide is T-Glcp 15.41%, 1,3-Glcp 4.06%, 1,6-Glcp 2.85%, 1,4-Glcp 60.47%, 1,3,4-Glcp 5.42% and 1,4,6-Glcp 11.79%; And / or, the main chain structure of the homogeneous polysaccharide is →4)-α-D-Glcp-(1→; α-D-Glcp-(1→3)-α-D-Glcp and α-D-Glcp-(1→6)-α-D-Glcp respectively through the connection with →3,4)-α The O-3 position of -D-Glcp-(1→) and the O-6 position of →4,6)-α-D-Glcp-(1→) are connected to the main chain.

6. A homogeneous polysaccharide having immunomodulatory activity, characterized in that: Composed of glucose; the number average molecular weight M of the homogeneous polysaccharide n The weight average molecular weight is 16.56±2.786% kDa, and the weight average molecular weight is M w 19.23 ± 3.316% kDa; The glycosidic bond composition and ratio of the homogeneous polysaccharide are T-Glcp 15.41%, 1,3-Glcp 4.06%, 1,6-Glcp 2.85%, 1,4-Glcp 60.47%, 1,3,4-Glcp 5.42% and 1,4,6-Glcp11.79%; The main chain structure of the homogeneous polysaccharide is →4)-α-D-Glcp-(1→; α-D-Glcp-(1→3)-α-D-Glcp and α-D-Glcp-(1→6)-α-D-Glcp are connected to the main chain through the O-3 position of →3,4)-α-D-Glcp-(1→ and the O-6 position of →4,6)-α-D-Glcp-(1→, respectively.

7. The method for preparing a uniform polysaccharide according to any one of claims 1 to 6, characterized in that: The steps include: Step 1, preparing water extract from Radix Aconiti Lateralis Preparata; Step 2, deproteinizing the water extract and obtaining crude polysaccharide FPS after dialysis; Step 3, purifying the crude polysaccharide FPS through a chromatographic column and eluting with water to obtain FPS1; the FPS1 is separated by gel, and the components with a retention volume of 264-348 mL are collected to obtain.

8. Use of a homogeneous polysaccharide having immunomodulatory activity, characterized in that: Use in the preparation of products for regulating immunity, including but not limited to functional foods, health foods, foods for special medical purposes or new drugs.

9. The use according to claim 8, characterized in that: The regulation of immunity is the regulation of immunity induced by chemotherapy drugs.

10. A health product or medicine for improving immunity, characterized in that: The invention is obtained by taking the uniform polysaccharide described in any one of claims 1 to 6 as an active substance and adding pharmaceutically acceptable auxiliary materials or auxiliary components.

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