Polygonum cuspidatum homogeneous polysaccharide as well as preparation method and application thereof

Abstract

The invention provides a common clubmoss herb homogeneous polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of traditional Chinese medicines. The uniform polysaccharide of the Bidens pilosa is obtained through water extraction and alcohol precipitation, impurity removal, ion exchange column purification and gel filtration chromatography purification, is composed of galactose and glucose, is novel in structure, has an excellent immune regulation function, can remarkably promote the proliferation, phagocytosis and NO generation capacity of macrophages RAW264.7, and can be used for preparing the polysaccharide of the Bidens pilosa, so that the polysaccharide of the Bidens pilosa can be used for preparing the polysaccharide of the Bidens pilosa and the polysaccharide of the Bidens pilosa, and the polysaccharide of the Bidens pilosa can be used for preparing the polysaccharide of the Bidens pilosa. The cytokines (proinflammatory factors TNF-alpha and IL-6) generated by the macrophages RAW264.7 are obviously increased, and the inflammatory inhibition factors (IL-10) generated by the macrophages RAW264.7 are obviously reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines and relates to a uniform polysaccharide of Herba Lycopodii, and particularly relates to a uniform polysaccharide of Herba Lycopodii and a preparation method and application thereof. Background Art

[0002] Immune-mediated diseases (IMD) are chronic diseases characterized by excessive, uncontrolled inflammation and thrombosis, which can easily lead to organ damage, cancer, atherosclerosis, inflammation and other diseases, and even cause death. Currently, a variety of therapeutic drugs and regimens have been approved for the treatment of IMD, including broad-spectrum immunomodulators such as glucocorticoids and small molecule chemical drugs; and highly specific drugs, including immunostimulants such as B lymphocyte stimulators and granulocyte-macrophage colony stimulating factor, immunomodulators such as human immunoglobulin and interferon, and cytokine inhibitors such as TNF, IL, and IAK inhibitors. However, the therapeutic effects of current therapeutic drugs on IMD patients vary from person to person, and only 30-40% of patients have achieved ideal therapeutic effects, which fails to meet clinical needs. Therefore, finding and developing safe, effective and low-cost immunomodulators has very important theoretical, economic and social significance.

[0003] As natural plants and their extracts or active ingredients continue to deepen in the research of immune regulation, anti-virus, anti-tumor, anti-inflammatory, etc., they have aroused widespread research interest due to their advantages such as low toxicity and side effects, precise efficacy, and low resistance to drugs. The polysaccharide components in natural plants are a very important class of bioactive macromolecules, which can achieve disease prevention and control through the dual regulatory effects of immune activation and inhibition. In recent years, researchers have made great progress in the study of the chemical structure and mechanism of action of immunoactive polysaccharides. Polysaccharides can affect the body's immune regulation in many ways, such as activating macrophages and complement systems, stimulating the production of related cytokines to activate T / B lymphocytes, and improving the immune system's ability to recognize antigens and immune tolerance. Therefore, finding plant polysaccharides with immunomodulatory activity is a current research hotspot.

[0004] Sarcopyramis nepalensis Wall, S. nepalensis Wa1., is a dried whole herb of Sarcopyramis nepalensis Wall, a plant of the Melastomataceae family. It is a widely used and valuable Chinese herbal medicine in southern Fujian. It has the effects of clearing lung heat and removing liver fire, and is mainly used to treat rheumatic pain, tinnitus, deafness, and photophobia. The chemical components that have been separated and identified from Sarcopyramis nepalensis Wall mainly include flavonoids, polysaccharides, organic acids, etc. As one of the main active ingredients of Sarcopyramis nepalensis Wall, the content of polysaccharides is as high as 15.46-16.21%, and it shows obvious biological activity in immunomodulation, anti-inflammatory, anti-tumor and other aspects, and has good development prospects. The invention patent with the publication number CN101664418A discloses the use of a polysaccharide of Rhamnose chinensis in the preparation of drugs against hepatitis B virus. The polysaccharide is composed of three monosaccharides, namely rhamnose, glucose and galactose, with a molecular weight of 84818Da, and has the potential of anti-oxidation, liver protection and anti-hepatitis B virus. However, for the same medicinal plant polysaccharide, polysaccharides of different relative molecular weights can be produced by separation and purification, and their physical and chemical properties and efficacy are significantly different; the monosaccharide composition, side chain position and higher-order structure of the polysaccharide have a significant impact on the pharmacological activity.

[0005] At present, there are the following problems: the extraction rate of polysaccharides from Psoralea corylifolia is low, and it is difficult to separate and purify them, so most pharmacological studies use crude polysaccharides; the structural research of polysaccharides from Psoralea corylifolia focuses on monosaccharide composition, side chain position, etc., and there are fewer studies on higher-order structures and structure-activity relationships, while the higher-order structure of polysaccharides has a significant impact on pharmacological activity; the composition and molecular weight distribution of polysaccharides obtained by different extraction methods are very different, and its chemical structure needs to be further clarified to explore the mechanism of action of the structure and function of polysaccharides from Psoralea corylifolia. In recent years, the chemical composition and pharmacological activity of Psoralea corylifolia have been widely studied, but the structural analysis and immunomodulatory activity of polysaccharides from Psoralea corylifolia have not been studied in depth. Therefore, it is necessary to improve the extraction rate of polysaccharides from Psoralea corylifolia, strengthen the separation and purification of polysaccharides from Psoralea corylifolia, and strengthen the research on structure-activity relationship of polysaccharides from Psoralea corylifolia, so as to provide theoretical guidance for the product development of polysaccharides from Psoralea corylifolia in functional foods and medicines. Summary of the invention

[0006] The present invention aims at the problems existing in the prior art and provides a uniform polysaccharide of Herba Eupatorii and a preparation method and application thereof. The uniform polysaccharide of Herba Eupatorii is obtained by water extraction and alcohol precipitation, impurity removal, ion exchange column purification and gel filtration chromatography purification. The uniform polysaccharide of Herba Eupatorii is composed of galactose and glucose, has a novel structure, and has a more excellent immunomodulatory function. It can significantly promote the proliferation, phagocytosis and NO production of macrophages RAW264.7, significantly increase the production of cytokines (pro-inflammatory factors TNF-α, IL-6) of macrophages RAW264.7, and significantly reduce the production of anti-inflammatory factors (IL-10) of macrophages RAW264.7.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for preparing a uniform polysaccharide of Herba Lycopodii, comprising the following steps:

[0009] 1) Preparing crude polysaccharide of Herba Lycopodii by water extraction and alcohol precipitation;

[0010] 2) removing impurities from the crude polysaccharide of Herba Lycopodii obtained in step 1);

[0011] 3) The crude polysaccharide of the herbaceous wormwood obtained in step 2) after impurities removal is sequentially purified by ion exchange column and gel filtration chromatography to obtain a uniform polysaccharide of the herbaceous wormwood; the uniform polysaccharide of the herbaceous wormwood consists of galactose and glucose.

[0012] Specifically, the water extraction and alcohol precipitation method of step 1) is as follows: reflux extraction of the herb and water according to a solid-liquid ratio of 1kg:8-10L, the number of extractions is 2-3 times, each extraction time is 1-2h, the extract is concentrated, and alcohol precipitation is performed using a 75-85% ethanol aqueous solution; preferably, the precipitate obtained by the alcohol precipitation is washed, preferably using a 90-99% volume fraction ethanol aqueous solution for washing. In one embodiment, the herb and water are mixed at a solid-liquid ratio of 1kg:8L, reflux extraction is performed twice, each time for 2h; the extracts are combined, concentrated, and 80% volume fraction ethanol aqueous solution is used, and the precipitate is placed overnight to obtain the precipitate, the supernatant is removed, and the precipitate is washed with a 95% volume fraction ethanol aqueous solution, evaporated until there is no alcohol taste, and dried to obtain the crude polysaccharide of the herb.

[0013] In step 2), the impurity removal includes: protein removal, resin adsorption and dialysis. Preferably, the impurity removal is specifically:

[0014] S1) protein removal: dissolving crude polysaccharide of Herba Lycopodii in water, adding papain for enzymolysis to obtain enzymolysis solution; removing protein by Sevage method, and collecting the aqueous phase;

[0015] S2) Resin adsorption: adding macroporous resin X-5 to the aqueous phase for adsorption;

[0016] S3) Dialysis: collect the liquid, dialyze and dry it to obtain the crude polysaccharide sample of Herba Lycopodii after the impurities are removed.

[0017] In one embodiment, the impurity removal is specifically:

[0018] S1) removing protein: dissolving crude polysaccharide of Herba Eupatorii in water, adding papain for enzymolysis to obtain an enzymolysis solution; adding equal volumes of Sevage reagent and petroleum ether to the enzymolysis solution in sequence for extraction, wherein the Sevage reagent is a chloroform-n-butanol mixture with a volume ratio of 4:1, and collecting the aqueous phase;

[0019] S2) Resin adsorption: Add macroporous resin X-5 to the aqueous phase, mix thoroughly, and adsorb overnight;

[0020] S3) Dialysis: collect the liquid, dialyze it with a dialysis bag (3000Da) for 48 hours to remove small molecular components, and freeze-dry it to obtain the crude polysaccharide sample of the Herba Lycopodii after impurities removal.

[0021] In the step 3), the ion exchange column purification is specifically as follows: the impurity-removed sedge coleus crude polysaccharide obtained in step 2) is dissolved in water, and purified by ion exchange column DEAE Seplife FF26 / 300, and gradient elution is performed using pure water and 0.1-0.3M NaCl solution in sequence, preferably pure water, 0.1M, 0.2M, and 0.3M NaCl solution in sequence, and more preferably 0.1M NaCl solution is used as the eluent to obtain the eluted sedge coleus crude polysaccharide. In one embodiment, the impurity-removed sedge coleus crude polysaccharide is dissolved in water to obtain a sedge coleus crude polysaccharide mother liquor; separated by DEAE Seplife FF26 / 300 ion exchange column, gradient elution is performed using pure water, 0.1M, 0.2M, and 0.3M NaCl solution in sequence, and the eluent is collected, an ion purification elution curve is drawn, and the eluents corresponding to the same elution peak are combined, and the ion exchange column-purified sedge coleus crude polysaccharide is obtained by concentration, desalting, and drying.

[0022] After the crude polysaccharide of the herb is purified by ion exchange column, it is purified by gel filtration chromatography. The gel purification is specifically as follows: TM 26 / 60Sephacryl TM In one embodiment, the crude polysaccharide of Herba Lycopodii obtained by eluting with 0.1M NaCl solution as the eluent is purified by gel filtration chromatography, and the gel chromatography column is HiPrep TM 26 / 60Sephacryl TM , the eluent is water, and the uniform polysaccharide of Herba Lycopersicum is obtained. In one embodiment, the crude polysaccharide of Herba Lycopersicum purified by ion exchange column is dissolved in water to obtain the crude polysaccharide mother liquor of Herba Lycopersicum, and the mother liquor is purified by HiPrep TM 26 / 60Sephacryl TMSeparate and purify, use water for elution, collect the eluate, use the phenol-sulfuric acid method to determine the polysaccharide content and draw an elution curve, and combine the eluates corresponding to the same elution peak, concentrate and dry, so as to obtain the uniform polysaccharide of Fengguidoucao.

[0023] In a second aspect, the present invention also provides a uniform polysaccharide of Herba Lycopodii obtained by the preparation method described in the above technical scheme, wherein the uniform polysaccharide of Herba Lycopodii is composed of galactose and glucose in a molar ratio of 42.03:54.14.

[0024] The purity of the homogeneous polysaccharide of the herb is 92.7%, the weight average molecular weight is 10-20 kDa, preferably 19.320 kDa, and the structure is as follows:

[0025]

[0026] Among them, m is 8 and n is 7.

[0027] In a third aspect, the present invention also provides a method of preparing the uniform polysaccharide of Herba Lycopodii using the preparation method described in the above technical solution or using the uniform polysaccharide of Herba Lycopodii described in the above technical solution in an immunomodulator.

[0028] Preferably, the immunomodulator is a drug that regulates macrophages, and the regulation of macrophages includes one or more of promoting macrophage proliferation, enhancing macrophage phagocytic activity, promoting macrophage NO release, and regulating macrophage cytokine release. The cytokines include one or more of pro-inflammatory factors and anti-inflammatory factors, the pro-inflammatory factors include one or more of TNF-α and IL-6, and the anti-inflammatory factors include IL-10.

[0029] In a fourth aspect, the present invention further provides an immunomodulator, comprising the uniform polysaccharide of Herba Lycopodii obtained by the preparation method described in the above technical scheme or the uniform polysaccharide of Herba Lycopodii described in the above technical scheme.

[0030] Compared with the prior art, the present invention has the following beneficial effects: the present invention extracts and precipitates the herb with water, removes impurities, and purifies it with DEAE ion exchange and gel filtration chromatography to prepare the herb uniform polysaccharide. The herb uniform polysaccharide obtained by the present invention has a high purity of 92.7%, a weight average molecular weight Mw of 19.320 kDa, and is composed of galactose and glucose in a molar ratio of 42.03:54.14. Combined with methylation and nuclear magnetic resonance analysis, its chemical structure is analyzed, and it is determined that the herb uniform polysaccharide (HLS-D1N1) has a novel structure. The monosaccharide composition, side chain position and higher-order structure of the polysaccharide have a significant effect on the pharmacological activity. The uniform polysaccharide of Herba Lycopodii obtained by the present invention has a more excellent immunoregulatory function. In the concentration range of 6.25-100 μg / mL, it can significantly promote the proliferation, phagocytosis and NO production ability of macrophages RAW264.7, significantly increase the production of cytokines (pro-inflammatory factors TNF-α, IL-6) by macrophages RAW264.7, and significantly reduce the production of anti-inflammatory factors (IL-10) by macrophages RAW264.7. It can be used to prepare immunoregulatory drugs, providing a theoretical basis for the application of the uniform polysaccharide of Herba Lycopodii in products such as functional foods, health products and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the elution curve of the uniform polysaccharide ion purification of Herba Lycopodii;

[0032] Figure 2 This is the elution curve of the homogeneous polysaccharide HLS-D1 from Herba Lycopodii by gel filtration chromatography.

[0033] Figure 3 This is the absolute molecular weight analysis chart of the homogeneous polysaccharide HLS-D1N1 of Herba Lycopodii;

[0034] Figure 4 This is the molecular configuration analysis diagram of the uniform polysaccharide HLS-D11N1 of Herba Lycopodii;

[0035] Figure 5 is the ion chromatogram of the mixed standard;

[0036] Figure 6 This is the HLS-D1N1 ion chromatogram of the homogeneous polysaccharide of Herba Lycopodii;

[0037] Figure 7 This is the GC-MS total ion chromatogram of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii;

[0038] Figure 8 This is the NMR one-dimensional 1H spectrum of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii;

[0039] Fig. 9This is the one-dimensional 13C NMR spectrum of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii;

[0040] Fig.10 This is the NMR two-dimensional COSY spectrum of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii, where + represents the cross peak at the position (f2, f1);

[0041] Fig.11 This is the NMR two-dimensional NOESY spectrum of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii, where + represents the cross peak at the position (f2, f1);

[0042] Fig.12 This is the NMR two-dimensional HSQC spectrum of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii, where + represents the cross peak at the position (f2, f1);

[0043] Fig.13 This is the NMR two-dimensional HMBC spectrum of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii, where + represents the cross peak at the position (f2, f1);

[0044] Fig.14 This is the result of the effect of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii on the proliferation of RAW264.7 cells (compared with the Con. group, * indicates statistical difference, P < 0.05; ** indicates significant difference, P < 0.01; *** indicates extremely significant difference, P < 0.001);

[0045] Fig.15 This is the result of the effect of the homogeneous polysaccharide HLS-D1N1 of Herba Lycopodii on the phagocytic ability of RAW264.7 cells (compared with the Con. group, * indicates statistical difference, P < 0.05; ** indicates significant difference, P < 0.01; *** indicates extremely significant difference, P < 0.001);

[0046] Fig.16 This is a graph showing the effect of the homogeneous polysaccharide HLS-D1N1 from Herba Lycopodii on the secretion of NO in RAW264.7 cells (compared with the Con. group, * indicates statistical difference, P < 0.05; ** indicates significant difference, P < 0.01; *** indicates extremely significant difference, P < 0.001);

[0047] Fig.17 This is a graph showing the effect of the homogeneous polysaccharide HLS-D1N1 from Herba Lycopodii on the secretion of TNF-α in RAW264.7 cells (*** indicates extremely significant difference, P < 0.001);

[0048] Fig.18 This is a graph showing the effect of the homogeneous polysaccharide HLS-D1N1 from Herba Lycopodii on the secretion of IL-6 in RAW264.7 cells (*** indicates extremely significant difference, P < 0.001);

[0049] Fig.19 This is a graph showing the effect of HLS-D1N1, a homogeneous polysaccharide from Herba Lycopodii, on the secretion of IL-10 in RAW264.7 cells (** indicates a significant difference, P < 0.01; *** indicates an extremely significant difference, P < 0.001). DETAILED DESCRIPTION

[0050] It is worth noting that, unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0051] The following sources of raw materials are provided for illustrative purposes:

[0052] Fengguidoucao preparation: Fengguidoucao was purchased from Shanzhen Zhongcaotang (Zhangzhou, Fujian), and was identified by Professor Wang Xiaoping of Zhangzhou Health Vocational College as the dried whole herb of Sarcopyramis nepalensis Wall, a plant of the Melastomataceae family.

[0053] Example 1

[0054] Preparation of Uniform Polysaccharide HLS-D1N1 from Herba Lycopodii

[0055] 1. Extraction of crude polysaccharides from Herba Lycopodii:

[0056] Take 2 kg of processed Herba Lycopodii, add it into water at a solid-liquid ratio (kg: L) of 1:8, and reflux extract twice, each time for 2 hours; combine the extracts, concentrate to 4 L, add ethanol to make the ethanol content reach 80%, leave it to precipitate overnight, remove the supernatant, wash the precipitate with 8 L of 95% ethanol, evaporate until there is no alcohol taste, and dry to obtain the crude Herba Lycopodii polysaccharide.

[0057] 2. Removal of impurities from crude polysaccharide from Fengguidou grass:

[0058] (1) Take 30 g of crude polysaccharide from Psoralea corylifolia, add it into 3 L of pure water to fully dissolve the crude polysaccharide, add 1.5% papain (45 g), and perform enzymolysis overnight to obtain an enzymolysis solution;

[0059] (2) Add 750 mL of chloroform and n-butanol to the enzymatic hydrolyzate (aqueous phase), mix thoroughly, and collect the upper aqueous phase;

[0060] (3) Continue to add 1.5 L of petroleum ether to the aqueous phase, mix thoroughly, and collect the lower aqueous phase;

[0061] (4) Add 400 g of macroporous resin X-5 to the aqueous phase, mix thoroughly, and adsorb overnight;

[0062] (5) The liquid was collected and dialyzed with a dialysis bag (3000Da) for 48 hours to remove small molecular components. The polysaccharide liquid was freeze-dried to obtain 8.7 g of a crude polysaccharide sample of Psoralea corylifolia after impurities removal with a purity of 86.0%.

[0063] 3. Ion exchange purification and gel filtration chromatography purification

[0064] 1. Ion exchange purification:

[0065] (1) taking the impurity-removed crude polysaccharide sample of Herba Lycopodii and dissolving it in pure water to prepare a 20 mg / mL crude polysaccharide mother solution of Herba Lycopodii;

[0066] (2) The crude polysaccharide mother liquor of Psoralea corylifolia was centrifuged at 10000 g for 10 min, and the supernatant was purified by ion exchange column DEAE Seplife FF26 / 300 at a flow rate of 4 mL / min; pure water, 0.1 M, 0.2 M and 0.3 M NaCl solution were used for gradient elution in sequence, and one tube was collected for every 15 mL, and all the eluate was collected;

[0067] (3) Accurately measure an appropriate amount of the eluent in each collection tube, add sulfuric acid-phenol reagent, react in the dark for 10 minutes, measure the absorbance at 490 nm, and use the sulfuric acid-phenol method to determine the total sugar content; use the number of eluent tubes as the horizontal axis and the total sugar content and NaCl concentration in the eluent as the vertical axis to draw the ion purification elution curve of the sample, see Figure 1 , Figure 1 This is the elution curve of the uniform polysaccharide ion purification of Herba Lycopodii;

[0068] Depend on Figure 1 It can be seen that this ion purification mainly obtained two elution peak components, ion elution peak 1: tubes 7-19, recorded as HLS-D1; ion elution peak 2: tubes 28-40, recorded as HLS-D2;

[0069] (4) combining the eluates from each collection tube corresponding to the same elution peak;

[0070] (5) Concentrate the eluate from each collection tube by rotary evaporation to 1 / 5 of the original volume;

[0071] (6) Desalination was performed by dialyzing for 48 h using a 3000 Da dialysis bag, and the desalted solution was freeze-dried.

[0072] 2. Gel filtration chromatography purification:

[0073] (1) taking 1.0 g of the homogeneous polysaccharide HLS-D1 of the herb with the highest peak concentration after ion purification, adding pure water to prepare a 2 mg / mL mother solution of the homogeneous polysaccharide HLS-D1 of the herb;

[0074] (2) The mother liquor of the homogeneous polysaccharide HLS-D1 of the herb was centrifuged at 10000 g for 10 min, and the supernatant was passed through a gel chromatography column HiPrep TM 26 / 60Sephacryl TM Separation and purification, flow rate 1mL / min, use pure water to elute 1.5 times column volume, collect one tube for every 12mL, and collect all the eluate;

[0075] (3) The total sugar content of the eluate in each collection tube was determined by the sulfuric acid-phenol method, and the gel purification elution curve of the sample was plotted with the number of eluate tubes as the abscissa and the total sugar content as the ordinate, see Figure 2 , Figure 2 This is the elution curve of the homogeneous polysaccharide HLS-D1 from Herba Lycopodii by gel filtration chromatography.

[0076] Depend on Figure 2 It can be seen that this gel filtration chromatography purification mainly obtained one elution peak component, gel elution peak 1: tubes 35-49, recorded as HLS-D1N1;

[0077] (4) combining the eluates from each collection tube corresponding to the elution peak and concentrating them by rotary evaporation to 1 / 5 of the original volume;

[0078] (5) freeze-drying, and identifying the content and purity of the polysaccharide after gel purification by sulfuric acid-phenol method, to obtain 1.99 g of the uniform polysaccharide HLS-D1N1 from the herbaceous holmium wilfordii, with a comprehensive yield of 39.8% and a polysaccharide purity of 92.7%.

[0079] Example 2

[0080] Structural Identification of the Homogeneous Polysaccharide HLS-D2N1 from Herba Lycopodii

[0081] (1) The molecular weight of the homogeneous polysaccharide HLS-D1N1 of Herba Lycopodii obtained in Example 1 was determined by high performance gel chromatography:

[0082] Chromatographic conditions: Gel chromatography-differential-multi-angle laser light scattering system was used: the liquid phase system was U3000 (Thermo, USA), the differential detector was Optilab T-rex (Wyatt technology, CA, USA), and the laser light scattering detector was DAWN HELEOSⅡ (Wyatt technology, CA, USA); gel exclusion chromatography columns Ohpak SB-805HQ (300×8mm) and Ohpak SB-803HQ (300×8mm) were connected in series; column temperature: 45°C; injection volume: 100μL; mobile phase A: 0.02wt% NaN3, 0.1M NaNO3; flow rate: 0.6mL / min; 100% A phase was isocratic eluted for 75min.

[0083] Sample preparation: The sample was dissolved in 0.1 M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, filtered through a filter with a pore size of 0.45 μm and then tested on an instrument.

[0084] Determination results: The absolute molecular weight analysis of the uniform polysaccharide HLS-D1N1 of the herb is shown in the figure below: Figure 3 As shown in the figure, Molar Mass represents the molar mass, LS represents the multi-angle laser light scattering signal (the scattered light intensity is proportional to the molecular size and molecular weight of the substance), and RI represents the difference signal (the response value depends on the change in the refractive index of the post-column effluent, which is related to the type, concentration and molecular weight of the substance). Figure 4 As shown; according to the Mark-Houwink Equation, the weight average molecular weight Mw of the uniform polysaccharide HLS-D1N1 of the Herba Lycopodii was calculated to be 19.320 kDa.

[0085] (2) The composition of the homogeneous polysaccharide HLS-D1N1 of Herba Lycopodii was analyzed by ion chromatography:

[0086] Chromatographic conditions:

[0087] Chromatographic system: Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA) uses an electrochemical detector to analyze and detect monosaccharide components; liquid chromatography column Dionex TM CarboPac TM PA20 (150*3.0mm, 10μm); injection volume: 5μL; mobile phase: mobile phase A is H2O, mobile phase B is 0.1M NaOH, mobile phase C is 0.1M NaOH (containing 0.2M NaAc); flow rate: 0.5 mL / min; column temperature: 30°C; elution gradient: 0 min: phase A / phase B / phase C (95:5:0, V / V / V); 26 min: phase A / phase B / phase C (85:5:10, V / V / V); 42 min: phase A / phase B / phase C (85:5:10, V / V / V); 42.1 min: phase A / phase B / phase C (60:0:40, V / V / V); 52 min: phase A / phase B / phase C (60:40:0, V / V / V); 52.1 min: phase A / phase B / phase C (95:5:0, V / V / V); 60 min: phase A / phase B / phase C (95:5:0, V / V / V).

[0088] Preparation and calculation method of standard solution:

[0089] Take 13 kinds of monosaccharide standards: fucose (Fuc), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), glucuronic acid (Glc-UA), mannuronic acid (Man-UA), guluronic acid (Gul-UA)), and prepare about 10 mg / mL standard stock solution; take appropriate amount of standard stock solution and mix to prepare the highest index concentration of 60 μg / mL, 50 μg / mL or 40 μg / mL standard mixed standard, and prepare the series of standards required for the instrument according to the concentration gradient in Table 1;

[0090] Table 1 Monosaccharide mixed standard series concentration table

[0091]

[0092]

[0093] Acid hydrolysis: Weigh 5.0 mg of the homogeneous polysaccharide HLS-D1N1 sample of Herba Lycopodii, add 1 mL of 2M TFA (trifluoroacetic acid) acid solution, heat at 121°C for 2 hours, blow dry with nitrogen, add 99.99% methanol to wash, blow dry again, repeat the methanol washing 3 times, add redistilled water to dissolve, and perform ion chromatography detection.

[0094] Measurement results:

[0095] The ion chromatograms of the mixed standard and the homogeneous polysaccharide of Herba Lycopodii are shown in Figure 5 and Figure 6 .

[0096] The content of each component in the sample (μg / mg) = C*V*F / M;

[0097] Where: C is the concentration, in μg / mL; V is the volume of the sample extract, in mL; F is the dilution factor; M is the total amount of sample weighed, in mg.

[0098] After calculation and comparison with the monosaccharide standard, it was found that the uniform polysaccharide HLS-D1N1 of Herba Lycopodii was composed of galactose and glucose. The molar ratio of each monosaccharide in HLS-D1N1 was galactose: glucose was 42.03:54.14.

[0099] (3) The bonding mode of the glycosidic bonds of the homogeneous polysaccharide HLS-D1N1 of the Herba Lycopodii was analyzed by methylation and gas chromatography-mass spectrometry (GC-MS):

[0100] Analytical conditions: Agilent Technologies Inc. CA, UAS 6890A-5977B gas spectrometer, autosampler model G4567A; chromatographic column: BPX70 (30m×0.25mm×0.25μm, SGE, Australia); injection volume: 1μL; split ratio: 10:1; carrier gas: high-purity helium, flow rate 1.5ml / min; the initial temperature of the column oven was 140°C for 2.0min, then programmed to 230°C at 3°C / min and maintained for 3min; mass spectrometry conditions: electron impact ion source (EI) and MassHunter workstation, analytes were detected in full scan (SCAN) mode, mass scan range (m / z): 50-350.

[0101] Sample treatment: Take 2 mg of the uniform polysaccharide HLS-D1N1 sample of Herba Lycopodii, add 500 μL DMSO to dissolve, add 1 mg NaOH, and incubate for 30 min; add 50 μL iodomethane solution to react for 1 h, then add 1 mL water and 2 mL dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase; absorb the lower dichloromethane phase and blow it dry with nitrogen; add 100 μL 2M TFA, react at 121°C for 90 min, and evaporate to dryness at 30°C; add 50 μL 2M ammonia, 50 μL 1M NaBD4, mix well, react at room temperature for 2.5 hours, add 20 μL acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL methanol, blow dry with nitrogen; add 250 μL acetic anhydride, vortex mix well, react at 100 ° C for 2.5 hours, add 1 mL of water and let stand for 10 minutes, then add 500 μL dichloromethane, vortex mix well, centrifuge, discard the aqueous phase, take the lower dichloromethane phase, and detect on GC-MS.

[0102] Test results:

[0103] The GC-MS total ion current results of the uniform polysaccharide HLS-D1N1 of Psoralea corylifolia are shown in Figure 7 and Table 2;

[0104] Table 2 Bonding structure analysis results of HLS-D1N1 polysaccharide sample of Fengguidoucao uniform polysaccharide

[0105]

[0106] (4) Analyze the NMR signals of sugar residues by NMR spectroscopy:

[0107] Instrument conditions: NMR spectrometer: Bruker (AVANCE NEO 500M, Germany) 500 MHz; scanning temperature 25 °C; QXI 1 H / 31 P / 13 C / 15N 5mm quadruple resonance reverse detection probe (Z-gradient, ATM Acc), technical parameters: signal-to-noise ratio (1H): 888, resolution (Hz): 0.32 (rotating); BBFO 1 H- 19 F, 31 P- 15 N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradien, ATM), technical parameters: signal-to-noise ratio ( 1 H):798, resolution (Hz): 0.26 (rotating); signal-to-noise ratio ( 13 C): 32, resolution (Hz): 0.1.

[0108] Sample treatment: Take an appropriate amount of HLS-D1N1 polysaccharide from Herba Lycopodii and fully dissolve it in D2O to prepare a solution with a concentration of 40 mg / mL; transfer the dissolved solution to a nuclear magnetic resonance tube and add 0.5 mL.

[0109] Test results: Place the NMR tube into the NMR spectrometer to scan one dimension 1 H spectrum, 13 C spectrum, 2D COSY, 2D NOESY, 2D HSQC, 2D HMBC spectra, the results are shown in order Figure 8-13 .

[0110] The specific results are as follows:

[0111] Sugar residue A: Anomeric signal δ = 5.33 / 99.54 ppm (H1 / C1) indicates that residue A is an α-configuration glucose residue. 1 The H1 chemical shift of sugar residue A determined by H NMR is δ=5.33ppm. In the COSY spectrum, the H2 chemical shift of residue A is determined to be δ=3.56ppm based on the cross peak δ5.33 / 3.56ppm. According to the same method, the H3-H6 signals can be determined in sequence through the COSY spectrum, that is, the H3-H6 chemical shifts of sugar residue A are assigned to δ=3.88ppm, δ=3.6ppm, δ=3.77ppm and δ=3.75(3.86)ppm respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the chemical shifts of C2-C6 on the sugar ring can be assigned by HSQC signals, which are δ=71.79ppm, 73.33ppm, 76.62ppm, 71.21ppm and 60.72ppm respectively. The chemical shifts of C1 and C4 shifted to the downfield, indicating that the residue was substituted at the O-1 and O-4 positions of the sugar ring. Combining the methylation analysis results and literature reports, it was inferred that the sugar residue A might be →4)-α-D-Glcp-(1→;

[0112] Sugar residue B: Anomeric signal δ = 4.57 / 104.34 ppm (H1 / C1) indicates that residue B may be a β-galactose residue. 1 The chemical shift of H1 of sugar residue B determined by H NMR is δ=4.57ppm. The H2-H5 signals can be determined in sequence through the cross peaks of the COSY spectrum (the method is the same as that of sugar residue A, the same below). The chemical shifts of H2-H6 of sugar residue B are assigned to δ=3.61ppm, 3.89ppm, 4.10ppm, 3.70ppm and 3.59(3.49)ppm respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the chemical shifts of C2-C6 on the sugar ring can be assigned by HSQC signals, which are δ=71.50ppm, 71.35ppm, 77.64ppm, 73.18ppm and 62.42ppm respectively. Among them, the chemical shifts of C1 and C4 shift to the low field, indicating that the residue has been substituted at the O-1 and O-4 positions of the sugar ring. Combining the results of methylation analysis and literature reports, it is inferred that sugar residue B may be →4)-β-D-Galp-(1→;

[0113] Sugar residue C: Anomeric signal δ = 5.17 / 92.3 ppm (H1 / C1) indicates that residue C is a glucose residue in α-configuration. 1 The H1 chemical shift of sugar residue C determined by H NMR is δ=5.17ppm. The H2-H6 signals can be determined in sequence through the COSY spectrum cross peaks. The H2-H6 chemical shifts of sugar residue C are assigned to δ=3.49ppm, 3.69ppm, 3.77ppm, 3.71ppm and 3.76ppm, respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the chemical shifts of C2-C6 on the sugar ring can be assigned by HSQC signals, which are δ=71.14ppm, 73.29ppm, 76.52ppm, 71.77ppm and 60.39ppm, respectively. Among them, the chemical shift of C4 shifts to the low field, indicating that the residue has been substituted at the O-4 position of the sugar ring. Combined with the methylation analysis results and literature reports, it is inferred that sugar residue C may be →4)-α-D-Glcp;

[0114] Sugar residue D: Anomeric signal δ = 4.53 / 104.46 ppm (H1 / C1) indicates that residue D is a galactose residue in β-configuration. 1The chemical shift of H1 of sugar residue D determined by H NMR is δ=4.53ppm. The H2-H6 signals can be determined in sequence through the cross peaks of the COSY spectrum. The chemical shifts of H2-H6 of sugar residue D are assigned to δ=3.51ppm, 3.63ppm, 3.83ppm, 3.75ppm and 3.58ppm respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the chemical shifts of C2-C6 on the sugar ring can be assigned by HSQC signals, which are δ=71.45ppm, 74.46ppm, 68.85ppm, 71.90ppm and 62.47ppm respectively. Among them, the chemical shift of C1 shifts to the low field, indicating that the residue has been substituted at the O-1 position of the sugar ring. Combined with the results of methylation analysis and literature reports, it is inferred that sugar residue D may be β-D-Galp-(1→;

[0115] Sugar residue E: Anomeric signal δ = 4.89 / 98.53 ppm (H1 / C1) indicates that residue E is a glucose residue of α-configuration. 1 The chemical shift of H1 of sugar residue E determined by H NMR is δ=4.89ppm. The H2-H6 signals can be determined in sequence through the cross peaks of the COSY spectrum. The chemical shifts of H2-H6 of sugar residue E are assigned to δ=3.48ppm, 3.78ppm, 3.58ppm, 3.75ppm and 3.79(3.84)ppm respectively. After assigning the chemical shifts of hydrogen on the sugar ring, the chemical shifts of C2-C6 on the sugar ring can be assigned by HSQC signals, which are δ=71.46ppm, 77.50ppm, 76.62ppm, 70.48ppm and 68.72ppm respectively. Among them, the chemical shifts of C1, C4 and C6 shift to the low field, indicating that the residue has been substituted at the O-1, O-4 and O-6 positions of the sugar ring. Combining the results of methylation analysis and literature reports, it is inferred that sugar residue E may be →4,6)-α-D-Glcp-(1→

[0116] Combined with one-dimensional NMR ( Figure 8-9 ) and two-dimensional NMR ( Figure 10-13 ) spectrum, and the sugar residue signals of the samples were assigned. The results are shown in Table 3. Figure 10-13 In the figure, + indicates the cross peak at the position (f2, f1).

[0117] Table 3 Sugar residues 1 H and 13 Chemical shift of C

[0118]

[0119] In summary, according to the sugar residues in the sample 13 C and 1The chemical shift of H is combined with HMBC and NOESY spectra to analyze the structure and connection mode of the polysaccharide. Combined with the HMBC spectrum, the connection order of each residue in the polysaccharide is inferred:

[0120] There is a cross peak δ=5.33 / 76.62 ppm between sugar residue A-H1 and sugar residue A-C4, there is a cross peak δ=99.54 / 3.6 ppm between sugar residue A-C1 and sugar residue A-H4, there is a cross peak δ=104.34 / 4.1 ppm between sugar residue B-C1 and sugar residue B-H4. Combined with the NOESY spectrum, the connection order of each residue in the polysaccharide was inferred: there was a cross peak δ=5.33 / 3.6ppm between sugar residue A-H1 and sugar residue A-H4, there were cross peaks δ=5.33 / 3.79ppm and δ5.33 / 3.84ppm between sugar residue A-H1 and sugar residue E-H6, there was a cross peak δ=4.57 / 3.6ppm between sugar residue B-H1 and sugar residue A-H4, there was a cross peak δ=4.57 / 4.1ppm between sugar residue B-H1 and sugar residue B-H4, there was a cross peak δ=4.53 / 3.58ppm between sugar residue D-H1 and sugar residue E-H4, there was a cross peak δ=4.89 / 3.77ppm between sugar residue E-H1 and sugar residue C-H4.

[0121] Based on the analysis of one-dimensional NMR, two-dimensional NMR information and methylation results, it is inferred that the polysaccharide is mainly composed of →4)-β-D-Galp-(1→, →4)-α-D-Glcp-(1→, →4,6)-α-D-Glcp-(1→ and →4)-α-D-Glcp, etc., which are connected to each other to form the main chain, and the side chain is mainly composed of β-D-Galp-(1→ connected to the O-4 position of the sugar residue →4,6)-α-D-Glcp-(1→, etc. Therefore, the structure of the Fengguidoucao uniform polysaccharide HLS-D1N1 (chain) is as follows:

[0122]

[0123] Test Example 1

[0124] Regulatory effects of homogeneous polysaccharide HLS-D1N1 from Herba Lycopodii on immune cells

[0125] (1) RAW264.7 cell culture:

[0126] RAW264.7 cells were cultured in DMEM medium (containing 10% FBS, 100 U / mL penicillin and 100 μg / mL streptomycin) in a 37° C., 5% CO 2 incubator, and the cell culture medium was replaced every 3 days.

[0127] (2) Effects of HLS-D1N1 and LPS from Herba Lycopodii on the proliferation of RAW264.7 cells:

[0128] Select cells in the logarithmic growth phase and count them at 5×10 4 / mL, 100μL / well were inoculated in a 96-well plate, and after incubation in a CO2 incubator overnight, 100μL of the HLS-D1N1 solution of the uniform polysaccharide of the herb obtained in Example 1 (6.25, 12.5, 25, 50, 100μg / mL) was added. The blank control group (Con.) was not added with drugs, and an equal volume of the drug solvent was added. A background control well was set up separately, and 6 duplicate wells were set up in each group. The culture was carried out at 37°C for 24h. 10μL of CCK-8 solution was added to each well 1h before the end, and the absorbance was measured at 450nm. The cell survival rate was calculated according to the following formula:

[0129] Cell survival rate = (OD experimental well - OD background well) / (OD blank well - OD background well) × 100%.

[0130] The results of the effect of HLS-D1N1, a homogeneous polysaccharide from Herba Lycopodii, on the proliferation of RAW264.7 cells are shown in Fig.14 As shown in the figure, 12.5-100μg / mL HLS-D1N1, a uniform polysaccharide from the herb H. truncatum, has a significant proliferative effect on RAW264.7 cells in a dose-dependent manner, and the cell viability is the highest at a concentration of 100μg / mL. Based on the fact that HLS-D1N1, a uniform polysaccharide from the herb H. truncatum, has no cytotoxic effect on RAW264.7 cells under treatment with various concentrations, a sample concentration of 6.25-100μg / mL was used for subsequent experiments.

[0131] (3) Determination of the phagocytic ability of the homogeneous polysaccharide HLS-D1N1 of Psoralea corylifolia on RAW264.7 cells

[0132] Cells in the logarithmic growth phase were selected and cultured at 2.5×10 4 / mL, 100μL / well were inoculated in a 96-well plate and incubated in a CO2 incubator overnight, and then 100μL of different concentrations of the uniform polysaccharide HLS-D1N1 solution of Herba Lycopodii obtained in Example 1 (6.25, 12.5, 25, 50, 100μg / mL) were added and cultured for 24h. DMEM medium was used as the blank control group, and LPS (1μg / mL) was used as the positive control group. After the culture was completed, the supernatant was discarded, and the cells were washed twice with pre-cooled PBS, and 1% neutral red dye was added and incubated in the incubator for 1h. The neutral red was then aspirated, washed twice with PBS, and then cell lysis solution (50% ethanol, 1% acetic acid) was added to extract the neutral red, and the absorbance was detected at 540nm. The results of the effect of the uniform polysaccharide HLS-D1N1 of Herba Lycopodii on the phagocytic ability of RAW264.7 cells are shown in Fig.15 .

[0133] The results show that Fig.14The proliferation-promoting effect presented by the uniform polysaccharide HLS-D1N1 of Herba Lycopodii was similar. Within the concentration range of 6.25-100 μg / mL, the uniform polysaccharide HLS-D1N1 of Herba Lycopodii increased the phagocytic ability and activity of RAW264.7 cells to varying degrees, and was concentration-dependent.

[0134] (4) Effects of HLS-D1N1 polysaccharide from Herba Lycopodii on the release of NO and cytokines from RAW264.7 cells:

[0135] Select cells in the logarithmic growth phase and calculate 5×10 5 / mL, 100μL / well were inoculated in a 96-well plate and placed in a CO2 incubator for overnight culture, the culture medium was discarded, and a serum-free DMEM solution was added. The cells were starved for 2h to keep them at the same level. The experimental group was added with different concentrations of the HLS-D1N1 solution of the uniform polysaccharide of the herb obtained in Example 1 (6.25, 12.5, 25, 50, 100μg / mL), the DMEM culture medium was the blank control group, and LPS (1μg / mL) was the positive control group. After 24h of culture, the cell supernatant was collected for the determination of the production of NO and cytokines. NO was determined using Griess reagent, and the cytokine production was determined using an ELASA kit. The results of the effects of the uniform polysaccharide of the herb HLS-D1N1 on the secretion of NO, TNF-α, IL-6, and IL-10 by RAW264.7 cells are shown in Figures 16-19, respectively.

[0136] from Figure 16-19 It can be seen that compared with the blank control group, the amount of NO, IL-6 and TNF-α released by RAW264.7 cells treated with the experimental concentration of HLS-D1N1 polysaccharide from Herba Lycopodii increased (P<0.05, 0.01), and the release of the three cytokines was the largest at a concentration of 100 μg / mL. This shows that HLS-D1N1 polysaccharide from Herba Lycopodii has a promoting effect on the secretion of NO, IL-6, TNF-α and IL-10 by RAW264.7 cells within the concentration range of 6.25-100 μg / mL, and it shows a dose-dependent relationship, indicating that HLS-D1N1 polysaccharide from Herba Lycopodii has certain immune activation properties.

[0137] Depend on Fig.19It can be seen that compared with the blank control group, the secretion of the anti-inflammatory factor IL-10 in the supernatant of RAW264.7 cells treated with the experimental concentration of HLS-D1N1 polysaccharide from H. sylvestris was significantly reduced (P<0.05, 0.01). At a concentration of 12.5-100 μg / mL, with the increase of the concentration of HLS-D1N1 polysaccharide from H. sylvestris, the secretion of IL-10 showed a dose-effect dependence to a certain extent. At a concentration of 100 μg / mL, the secretion of IL-10 was equivalent to that of the positive control group (LPS, 11 μg / mL), indicating that HLS-D1N1 polysaccharide from H. sylvestris can effectively inhibit the secretion of the anti-inflammatory factor IL-10.

[0138] In summary, within the experimental concentration range (6.25-100 μg / mL), HLS-D1N1, a homogeneous polysaccharide from Herba Lycopodii, can significantly promote the proliferation and differentiation of RAW264.7 cells and enhance their phagocytic ability. It can also play an immunomodulatory role by promoting the release of cytokines such as NO, TNF-α and IL-6 and inhibiting the secretion of the anti-inflammatory factor IL-10.

[0139] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing uniform polysaccharide from Herba Lycopodii, It is characterized in that The following steps are involved: 1) Preparing crude polysaccharide of Herba Lycopodii by water extraction and alcohol precipitation; 2) removing impurities from the crude polysaccharide of Herba Lycopodii obtained in step 1); 3) The crude polysaccharide of the herbaceous wormwood obtained in step 2) after impurities removal is sequentially purified by ion exchange column and gel filtration chromatography to obtain a uniform polysaccharide of the herbaceous wormwood; the uniform polysaccharide of the herbaceous wormwood consists of galactose and glucose.

2. The preparation method according to claim 1, It is characterized in that The water extraction and alcohol precipitation method in step 1) is specifically as follows: subjecting the herb to reflux extraction with water at a solid-liquid ratio of 1kg:8-10L, the extraction times are 2-3 times, each extraction time is 1-2h, the extract is concentrated, and alcohol precipitation is performed using an ethanol aqueous solution with a volume fraction of 75-85%.

3. According to the preparation method described in claim 1, It is characterized in that In the step 2), impurity removal includes: protein removal, resin adsorption and dialysis.

4. According to the preparation method described in claim 3, It is characterized in that The impurity removal in step 2) is specifically as follows: S1) protein removal: dissolving crude polysaccharide of Herba Lycopodii in water, adding papain for enzymolysis to obtain enzymolysis solution; removing protein by Sevage method, and collecting the aqueous phase; S2) Resin adsorption: adding macroporous resin X-5 to the aqueous phase for adsorption; S3) Dialysis: collecting the liquid, dialyzing and drying, and obtaining the crude polysaccharide sample of Herba Lycopodii after impurities removal; The ion exchange purification in step 3) is specifically as follows: dissolving the impurity-removed crude polysaccharide of Herba Lycopersici in water to obtain a crude polysaccharide mother liquor of Herba Lycopersici; purifying through an ion exchange column DEAE Seplife FF26 / 300, using pure water and 0.1-0.3M NaCl solution for gradient elution in sequence; collecting the eluate, drawing an ion purification elution curve and combining the eluates corresponding to the same elution peak, and concentrating, desalting and drying to obtain the Herba Lycopersici polysaccharide purified by the ion exchange column; In step 3), the gel filtration chromatography purification is specifically as follows: the crude polysaccharide of Herba Lycopodii purified by ion exchange column is dissolved in water to obtain a mother liquor of Herba Lycopodii polysaccharide, and then the mother liquor is purified by gel filtration chromatography column HiPrep TM 26 / 60Sephacryl TM Separate and purify, use water for elution, collect the eluate, draw a gel filtration chromatography purification elution curve and combine the eluates corresponding to the same elution peak, concentrate and dry to obtain the uniform polysaccharide of Herba Lysimachiae.

5. A uniform polysaccharide of Herba Lycopodii, It is characterized in that The polysaccharide is prepared by the preparation method described in any one of claims 1 to 4, and is composed of galactose and glucose in a molar ratio of 42.03:54.

14.

6. According to claim 5, the homogeneous polysaccharide of Herba Lycopodii, It is characterized in that The uniform polysaccharide structure of the Herba Lycopodii is as follows: Among them, m is 8 and n is 7.

7. The homogeneous polysaccharide of Herba Lycopodii according to claim 6, It is characterized in that The purity of the uniform polysaccharide of Herba Lycopodii is 92.7% and the weight average molecular weight is 19.320 kDa.

8. Use of the homogeneous polysaccharide of Herba Lycopodii obtained by the preparation method according to any one of claims 1 to 4 or the homogeneous polysaccharide of Herba Lycopodii according to any one of claims 5 to 7 in immunomodulators.

9. The use according to claim 8, Features: The immunomodulator is a drug that regulates macrophages, and the regulation of macrophages includes one or more of promoting macrophage proliferation, enhancing macrophage phagocytic activity, promoting macrophage release of NO, and regulating macrophage release of cytokines; the cytokines include one or more of pro-inflammatory factors and anti-inflammatory factors, the pro-inflammatory factors include one or more of TNF-α and IL-6, and the anti-inflammatory factors include IL-10.

10. An immunomodulator, It is characterized in that It includes the uniform polysaccharide of Herba Lycopodii prepared by the preparation method described in any one of claims 1-4 or the uniform polysaccharide of Herba Lycopodii described in any one of claims 5-7.

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