CN112076207A

CN112076207A - High molecular weight cordyceps militaris polysaccharide, preparation method thereof and application of high molecular weight cordyceps militaris polysaccharide in preparation of anticomplement medicines

Info

Publication number: CN112076207A
Application number: CN202011035760.8A
Authority: CN
Inventors: 孙金凤; 胡政宇; 李镐; 周微; 王佳鸣; 金龙
Original assignee: Yanbian University
Current assignee: Yanbian University
Priority date: 2020-09-27
Filing date: 2020-09-27
Publication date: 2020-12-15
Anticipated expiration: 2040-09-27
Also published as: CN112076207B

Abstract

The invention belongs to the technical field of polysaccharide medicines, and particularly relates to three uniform high molecular weight polysaccharides in cordyceps militaris, a preparation method thereof and application thereof in preparation of anticomplement medicines. The invention separates three homogeneous high molecular weight polysaccharides CMP-1, CMP-2 and CMP-3 from Cordyceps militaris, and experiments prove that the homogeneous high molecular weight polysaccharides have obvious inhibition effect on complement activation, and can be further used as active ingredients to prepare novel anticomplement drugs.

Description

High molecular weight cordyceps militaris polysaccharide, preparation method thereof and application of high molecular weight cordyceps militaris polysaccharide in preparation of anticomplement medicines

Technical Field

The invention belongs to the technical field of polysaccharide medicines, and particularly relates to three uniform high molecular weight polysaccharides in cordyceps militaris, a preparation method thereof and application thereof in preparation of anticomplement medicines.

Background

The prior art discloses that a complement system is the first defense line of body natural immune defense, plays important roles of monitoring, defense and elimination in eliminating exogenous pathogenic microorganisms and apoptotic cell metabolism, and has a profound influence on activation and correct guidance of adaptive immunity. The complement system is a 'double-edged sword', and the excessive activation can cause the damage of the normal tissues of the human body, such as rheumatoid arthritis, senile dementia, systemic lupus erythematosus, rejection reaction after organ transplantation and the like; studies have shown that over-activation of complement also plays an important role in multiple organ failure syndromes, such as acute diseases like ischemic reperfusion, acute myocardial infarction, Acute Respiratory Distress Syndrome (ARDS), which is one of the major causes of death in patients infected with the 2019 novel coronavirus (covi-19). At present, the immunosuppressive agents such as glucocorticoid, cyclophosphamide, methylamine pterine and the like which are widely used clinically have a certain treatment effect on certain diseases related to excessive complement activation, but because the drugs are not specific complement inhibitors, the long-term application of the drugs can reduce the body defense function, cause the reduction of the anti-infection capability, facilitate secondary infection, spread potential focuses, generate various complications, side effects and other defects, and thus, a novel complement inhibitor with high efficiency, low toxicity and specificity is urgently needed clinically. Active ingredients with anticomplementary action are widely present in nature. The compounds such as natural polysaccharide, flavone, protein, polypeptide and alkaloid have better complement inhibition effect. Wherein the natural polysaccharide is the main anti-complement active component, is safe and reliable, has low toxicity and has a unique immune regulation mechanism. The inventors of the present application therefore sought to enhance the study of the anticomplementary active components of the natural polysaccharides, providing a material basis for the treatment of diseases associated with excessive complement system activation and ARDS patients infected with COVID-19.

Cordyceps militaris (L.ex Fr.) Link, a model species of Clavicipitaceae and Cordyceps, also called Cordyceps militaris, is a unique and precious medicinal fungus mainly produced in Jilin, Liaoning and inner Mongolia of China. According to the records of the modern Chinese medicine Ju Ji (China medicine sea): north Chinese Caterpillar fungus, sweet in nature and taste, enters lung and kidney meridians. It can be used for treating deficiency of lung and kidney, chronic cough, asthma, and chronic cough with phlegm and blood. The cordyceps sinensis is expensive and extremely deficient in wild resources, and the cordyceps militaris has similar chemical components and medicinal characteristics to the cordyceps sinensis, so that the cordyceps sinensis is considered to be the most ideal substitute of the cordyceps sinensis. Cordyceps militaris polysaccharide is one of the main active ingredients, and has multiple biological activities. However, no report on the separation and preparation of homogeneous high molecular weight polysaccharides with anticomplementary activity from Cordyceps militaris has been found so far.

Disclosure of Invention

The invention aims to provide active ingredients with anticomplementary effect in natural medicines, relates to high molecular weight cordyceps militaris polysaccharide, a preparation method thereof and application thereof in pharmacy, and particularly relates to three uniform high molecular weight cordyceps militaris polysaccharides (CMP-1, CMP-2 and CMP-3), a preparation method thereof and application thereof in preparation of complement inhibition medicines.

The invention separates the water extract of the cordyceps militaris to obtain three homogeneous polysaccharides (CMP-1, CMP-2 and CMP-3), and in vitro experiments prove that the three polysaccharides have obvious complement inhibition activity and can be developed and researched as an anticomplement medicament.

In the invention, the Cordyceps militaris (L.ex Fr.) Link is a complex consisting of two parts, namely a stroma and a sclerotium, belonging to the Clavicipitaceae and Cordyceps genus.

The structural characteristics of the cordyceps militaris polysaccharides CMP-1, CMP-2 and CMP-3 are described as follows:

(1) CMP-1 is a polysaccharide consisting of eleven monosaccharides, with a weight average molecular weight of about 929.41 kDa; the total sugar content is 91.78 +/-2.11%; the uronic acid content is 3.40 + -0.35%; the monosaccharide composition of the polysaccharide shows that it consists of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose in a molar ratio of 39.35:4.03:3.98:2.56:1.62:1.52:70.52:26.90:1.00:3.23: 4.23;

(2) CMP-2 is a polysaccharide consisting of ten monosaccharides, with a weight average molecular weight of about 907.99 kDa; the total sugar content is 92.95 plus or minus 1.17 percent; the uronic acid content is 4.83 + -0.23%; the monosaccharide composition of the polysaccharide shows that it consists of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and fucose in a molar ratio of 13.62:86.70:7.80:6.22:1.47:2.99:17.80:9.26:1.00: 3.02;

(3) CMP-3 is a polysaccharide consisting of ten monosaccharides, with a weight average molecular weight of about 1258.9 kDa; the total sugar content is 94.18 + -2.18%; the uronic acid content is 5.93 + -0.28%; the monosaccharide composition of the polysaccharide shows that the polysaccharide consists of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and fucose, and the molar ratio is 33.61:44.67:27.34:31.84:7.32:8.39:102.23:38.27:1.00: 5.79;

the cordyceps militaris polysaccharide (CMP-1, CMP-2 and CMP-3) is prepared by the following method:

(1) extracting cordyceps militaris with 95% ethanol for 1-3 times, filtering, taking dregs of a decoction, and drying to constant weight to obtain dried cordyceps militaris dregs of a decoction;

(2) weighing the dried cordyceps militaris dregs obtained in the step (1), extracting for 1-3 h at an extraction temperature of 85-95 ℃ according to a liquid-material ratio of 30: 1-40: 1(mL/g), filtering to obtain a filtrate, concentrating the filtrate to 1/5 of the original volume, adding 95% ethanol until the final concentration is 85%, standing for 24h at 4 ℃, centrifuging to remove a supernatant, taking a precipitate, and drying to constant weight to obtain cordyceps militaris crude polysaccharide;

(3) and (3) adding deionized water into the cordyceps militaris crude polysaccharide obtained in the step (2) for re-dissolution, carrying out column chromatography separation by using DEAE-52 cellulose, sequentially eluting by using deionized water, 0.1mol/L and 0.2mol/L NaCl solutions at the flow rate of 1mL/min, merging the same polysaccharide components, respectively putting into dialysis bags for dialysis separation, dialyzing for 24-48 h, freeze-drying, and detecting the anticomplementary activity to obtain the polysaccharide with the anticomplementary activity and uniform high molecular weight.

In the preparation method, in the step (3), the molecular weight cut-off of the dialysis bag is 8kDa to 14 kDa.

In the above production method, the polysaccharide having a uniform high molecular weight is produced in step (3) as CMP-1, CMP-2 or CMP-3.

In vitro tests prove that the cordyceps militaris polysaccharide CMP-1, CMP-2 or CMP-3 has obvious inhibition on cell hemolysis caused by activation of classical and alternative complement pathways, namely has obvious anticomplementary effect.

CH of CMP-1, CMP-2 and CMP-3₅₀The values (concentration of sample required for 50% inhibition of hemolysis by the classical pathway) were 0.43. + -. 0.07mg/mL, 0.41. + -. 0.10mg/mL and 0.27. + -. 0.04mg/mL, respectively. AP (Access Point)₅₀The values (concentration of sample required for 50% inhibition of hemolysis by the alternative pathway) were 0.42. + -. 0.12mg/mL, 0.38. + -. 0.09mg/mL and 0.33. + -. 0.07mg/mL, respectively. CMP-1 can interact with target C2, C4, C9, Factor B, Factor D or Factor P to inhibit complement activation; CMP-2 can interact with target C2, C4, C3, C5, C9, Factor B, Factor d, or Factor P to inhibit complement activation; CMP-3 can interact with target C2, C3, C5, C9, Factor B, Factor D, or Factor P to inhibit complement activation.

The invention has the advantages of

The invention separates the water extract of the cordyceps militaris to obtain three uniform cordyceps militaris polysaccharides CMP-1, CMP-2 and CMP-3 with high molecular weight, and in vitro experiments prove that the three cordyceps militaris polysaccharides have obvious complement inhibition activity and can be further used for preparing anticomplement medicines.

Drawings

FIG. 1 shows the separation and elution curve of Cordyceps militaris polysaccharide.

FIG. 2 shows HPGPC chromatograms of Cordyceps militaris polysaccharides, wherein A-C are HPGPC chromatograms of Cordyceps militaris polysaccharides CMP-1, CMP-2, and CMP-3, respectively.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Unless otherwise specified, the experimental methods adopted by the invention are all conventional methods, and experimental equipment, materials, reagents and the like used in the experimental method can be purchased from chemical companies.

Materials used in the following examples:

cordyceps militaris: artificially cultivated cordyceps militaris (cultivated c. militaris) is obtained from the scientific research institute of Yanbian forestry.

Example 1 preparation of Cordyceps militaris polysaccharides CMP-1, CMP-2 and CMP-3

2.5kg of cordyceps militaris medicinal materials are taken and crushed, extracted for three times by 95 percent ethanol, filtered to obtain medicine residues, and dried to constant weight. Weighing dry Cordyceps residue, extracting at 90 deg.C for 2 hr with liquid-material ratio of 35:1(mL/g), and filtering to obtain filtrate. Concentrating the filtrate to 1/5, adding 95% ethanol to final concentration of 85%, standing at 4 deg.C for 24 hr, centrifuging to remove supernatant, and drying the precipitate to constant weight to obtain Cordyceps militaris crude polysaccharide. Loading the pretreated DEAE-52 cellulose into a chromatographic column by adopting a wet method, dissolving the cordyceps militaris crude polysaccharide in deionized water, loading the sample, eluting by using deionized water, 0.1mol/L NaCl solution and 0.2mol/L NaCl solution at the flow rate of 1mL/min and the column volume of 3 times in sequence, collecting by separating bottles, drawing an elution curve according to a phenol-sulfuric acid method chromogenic reaction, combining the same polysaccharide components to obtain three polysaccharide components, wherein the elution curve is shown in figure 1. The three polysaccharide components are respectively put into dialysis bags (the cut-off molecular weight is 8 kDa-14 kDa), dialyzed for 36 hours under flowing water, and freeze-dried to obtain three uniform high molecular weight polysaccharides, namely CMP-1, CMP-2 and CMP-3.

Example 2 structural characterization of Cordyceps militaris polysaccharides (CMP-1, CMP-2, and CMP-3)

(1) Weight average molecular weight measurement

The uniformity and molecular weight of Cordyceps militaris polysaccharide are determined by high performance gel permeation chromatography combined with refractive index detector (HPGPC-RID). 20 μ L of Cordyceps militaris polysaccharide solution (2.0 mg/mL) was filtered through a 0.45 μm microporous filter and then injected into a Shodex sugar KS-804 column (8.0 mm. times.300 mm). The chromatographic conditions are as follows: ultrapure water is used as a mobile phase, the flow rate is 1.0mL/min, the column temperature is 50 ℃, and the temperature of the differential refractive index detector is 35 ℃. Data processing was recorded on an N2000 GPC chromatography workstation. The molecular weight of the polysaccharide was calculated using a series of dextrans of different weight average molecular weight as standards for calibration curves.

The results of the experiments showed that the weight average molecular weights of CMP-1, CMP-2 and CMP-3 were 929.41kDa, 907.99kDa and 1258.9kDa, respectively. As shown in FIG. 2, the HPGPC chromatogram of the Cordyceps militaris polysaccharide is homogeneous and has a weight average molecular weight of greater than 900 kDa. Therefore, CMP-1, CMP-2 and CMP-3 are three homogeneous high molecular weight Cordyceps militaris polysaccharides.

(2) Total sugar and uronic acid content determination

Determination of total sugar content by sulfuric acid-phenol method: the total sugar content of CMP-1, CMP-2 and CMP-3 was 91.78 + -2.11%, 92.95 + -1.17% and 94.18 + -2.18%, respectively.

The content of uronic acid is determined and detected by an m-hydroxy biphenyl method: the uronic acid saccharide contents of CMP-1, CMP-2 and CMP-3 were 3.40 + -0.35%, 4.83 + -0.23% and 5.93 + -0.28%, respectively.

(3) Monosaccharide composition determination

The monosaccharide component is determined by a 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method. Precisely weighing 2mg of cordyceps militaris polysaccharide, mixing the cordyceps militaris polysaccharide with 1mL of 2mol/L trifluoroacetic acid (TFA), sealing, reacting and hydrolyzing for 2 h. Adding 200 mu L of 0.5mol/L PMP methanol solution and 0.3mol/L NaOH solution into the hydrolysate in sequence, carrying out water bath at 70 ℃ for 1h, adding 200 mu L of 0.3mol/L HCl, extracting with chloroform for three times, and collecting a water layer. Derivatization procedures were as above with mannose (Man), glucosamine (GluN), ribose (Rib), rhamnose (Rha), glucuronic acid (GluA), galacturonic acid (GalA), glucose (Glu), galactose (Gal), xylose (Xyl), arabinose (Arab) and fucose (Fuc) as standard controls. And (3) passing the pretreated sample and the standard substance through a 0.45-micron microporous filter membrane, and injecting a sample. Chromatographic conditions are as follows: supersil ODS2 column (5mm, 4.6X 250 mm)²) The column temperature is 30 ℃, the detector wavelength is 245nm, the flow rate is 0.8mL/min, the sample injection amount is 20 mu L, and the mobile phase is acetonitrile: 0.1mol/mL phosphate buffer solution (ph6.8) ═ 82:18 (v/v). As a result, as shown in Table 1, CMP-1 was composed of eleven monosaccharides, and CMP-2 and CMP-3 were composed of ten monosaccharides.

TABLE 1 molar ratio of monosaccharide components of three Cordyceps militaris polysaccharides

Remarking: "-" is no monosaccharide detected.

Example 3 classical pathway complement inhibition assay

GVB-Ca is taken from 6% sheep red blood cells²⁺/Mg²⁺(Beijing Ku Lai Bo technology Limited, Cat: DZSL0356-500ML) buffer to 2X 10⁹cells/mL, and hemolysin (SBJ-RXS 10, product number: 1) in a ratio of 1:1, performing water bath at 37 ℃ for 30min, and centrifuging to remove unbound hemolysin to obtain sensitized sheep red blood cells (EAs) for later use. Normal healthy adult human serum (NHSP) serves as the complement source for this classical pathway. Precisely weighing Cordyceps militaris polysaccharide with GVB-Ca²⁺/Mg²⁺Preparing buffer solution into different concentration gradients, adding 1:80NHSP, pre-incubating in 37 deg.C water bath for 30min, adding sensitized sheep red blood cell, water bath at 37 deg.C for 30min, centrifuging, collecting supernatant, placing in 96-well plate, measuring absorbance at 540nm with enzyme labeling instrument, and setting blank group, total hemolysis group, NHSP group, negative drug control group (glucose) and positive drug control group (heparin) respectively. Calculating hemolysis inhibition rate of Cordyceps militaris polysaccharide under different concentration gradient, and calculating Concentration (CH) of 50% polysaccharide required for inhibiting hemolysis of each component with GraphPad prism 6.0₅₀Value). The results show that CH for CMP-1, CMP-2 and CMP-3₅₀The values are 0.43 + -0.07 mg/mL, 0.41 + -0.10 mg/mL and 0.27 + -0.04 mg/mL respectively, and all three homogeneous high molecular weight polysaccharides have significant inhibitory activity on complement classical pathway activation (as shown in Table 2).

The components and the addition amount in each group are as follows:

blank group: 40 μ L of EAs +260 μ L of GVB-Ca²⁺/Mg²⁺；

Total hemolysis group: 40 μ L EAs +260 μ L DDW;

NHSP group: 150 μ L NHSP +40 μ L EAs +110 μ L GVB-Ca²⁺/Mg²⁺；

Negative control group: 30 μ L glucose +150 μ L NHSP +40 μ L EAs +80 μ L GVB-Ca²⁺/Mg²⁺；

Positive control group: 12 μ L heparin +150 μ L NHSP +40 μ L EAs +98 μ L GVB-Ca²⁺/Mg²⁺；

Administration group: 6 μ L samples at different concentrations +150 μ L NHSP +104 μ L GVB-Ca²⁺/Mg²⁺+40μL EAs。

Example 4 alternative pathway complement inhibition assay

Normal healthy human serum passes GVB-Mg²⁺EGTA buffer incubation for 15min on ice was used as the complement source for this alternative pathway. GVB-Mg for 2% rabbit erythrocyte²⁺/EGTA buffer to 5 × 10⁸Adding NHSP and diluted cordyceps militaris polysaccharide with different concentrations after cell/mL, pre-incubating for 15min, adding rabbit red blood cells (Erab), incubating in a water bath at 37 ℃ for 30min, cooling on ice, terminating the reaction, and centrifuging. And (3) respectively taking the supernatant of each tube to dilute in a 96-well plate, measuring the absorbance value at 412nm, and simultaneously respectively setting a blank group, a full hemolysis group, an NHSP group, a negative medicine control group (glucose) and a positive medicine control group (heparin) in the experiment. Calculating the hemolysis inhibition rate of the cordyceps militaris polysaccharide under different concentration gradients. And the concentration of 50% of the polysaccharide required to inhibit hemolysis (AP) for each fraction was calculated using GraphPad prism 6.0₅₀Value). The results show that CH for CMP-1, CMP-2 and CMP-3₅₀Values of 0.42 + -0.12 mg/mL, 0.38 + -0.09 mg/mL and 0.33 + -0.07 mg/mL, respectively, all three homogeneous high molecular weight polysaccharides had significant inhibitory activity against activation of the alternative complement pathway (as shown in Table 2).

The components and the addition amount in each group are as follows:

blank group: 25 μ L Erab +75 μ L GVB-Mg²⁺/EGTA；

Total hemolysis group: 25 μ L Erab +75 μ L DDW;

NHSP group: 10 μ L NHSP +25 μ L Erab +65 μ L GVB-Mg²⁺/EGTA；

Negative control group: 1 μ L glucose +10 μ L NHSP +25 μ L Erab +64 μ L GVB-Mg²⁺/EGTA；

Positive control group: 11 μ L heparin +54 μ L NHSP +25 μ L Erab +54 μ L GVB-Mg²⁺/EGTA；

Administration group: 1 μ L samples at different concentrations +10 μ L NHSP +25 μ L Erab +64 μ L GVB-Mg²⁺/EGTA。

TABLE 2 inhibition of complement activation by Cordyceps militaris polysaccharides

Remarking: all data are expressed as mean ± SD (n ═ 3);

CH₅₀and AP₅₀Represents the 50% hemolysis inhibitory concentration by the classical and alternative pathways, respectively;

heparin was the positive control and glucose was the negative control.

Example 5 complement System target assay

The classical pathway: selecting the minimum concentration of polysaccharide required for inhibiting hemolysis to be close to 100% in the classical pathway as the critical concentration of anticomplement action target, and sequentially adding deletion serum (C2, C3, C4, C5, C9) and sensitized sheep red blood cell (2.0 × 10)⁹cells/mL), mixing, incubating in a water bath at 37 ℃ for 30min, and centrifuging at 4 ℃ for 5min by using a centrifuge of 2000 Xg. Three groups are made, and each group is respectively provided with a blank group, a positive drug control group, a serum control group and a whole blood dissolving group. Taking 0.2mL of supernatant of each tube to a 96-well plate, measuring the absorbance value at 540nm, subtracting the absorbance value of the corresponding control group, and calculating the hemolysis rate.

The alternative pathway: selecting the lowest concentration of polysaccharide required for inhibiting hemolysis to be close to 100% under the alternative pathway as the critical concentration of anticomplementary action target, and sequentially adding deletion serum (C3, C5, C9, Factor B, Factor D, Factor P) and rabbit erythrocyte (5.0 × 10)⁸cells/mL) for 30min, incubated on ice for 5min to stop the reaction, and centrifuged at 1000Xg for 3min at 4 ℃. Three groups are made, and each group is respectively provided with a blank group, a positive drug control group, a serum control group and a whole blood dissolving group. Transfer 30. mu.L of supernatant to a 96-well plate, add 270. mu.L of GVB-Mg to each well²⁺Diluting with EGTA buffer solution, measuring absorbance at 412nm, and calculating hemolysis rate by subtracting absorbance of corresponding control group.

The experimental result shows that the CMP-1 can interact with the targets C2, C4, C9, Factor B, Factor D and Factor P to inhibit the complement activation; CMP-2 can interact with targets C2, C4, C3, C5, C9, Factor B, Factor d, and Factor P to inhibit complement activation; CMP-3 can interact with targets C2, C3, C5, C9, Factor B, Factor D, and Factor P to inhibit complement activation.

The embodiments described above are presented to enable those skilled in the art to make and use the invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the embodiments described herein, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Claims

1. The application of the cordyceps militaris polysaccharide in preparing complement inhibition drugs, wherein the cordyceps militaris polysaccharide is CMP-1, CMP-2 or CMP-3, and has the following structural characteristics:

(3) CMP-3 is a polysaccharide consisting of ten monosaccharides, with a weight average molecular weight of about 1258.9 kDa; the total sugar content is 94.18 + -2.18%; the uronic acid content is 5.93 + -0.28%; the monosaccharide composition of the polysaccharide shows that the polysaccharide is composed of mannose, glucosamine, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and fucose, and the molar ratio is 33.61:44.67:27.34:31.84:7.32:8.39:102.23:38.27:1: 5.79.

2. The use according to claim 1, wherein CMP-1, CMP-2 or CMP-3 inhibits hemolysis of a cell caused by activation of the classical or alternative complement pathway.

3. The use according to claim 1, wherein said CMP-1 inhibits complement activation, its CH₅₀0.43. + -. 0.07mg/mL, AP₅₀At 0.42. + -. 0.12mg/mL, interaction of CMP-1 with target C2, C4, C9, Factor B, Factor D, or Factor P inhibits complement activation.

4. The use according to claim 1, wherein said CMP-2 inhibits complement activation, its CH₅₀0.41 + -0.10 mg/mL, AP₅₀At 0.38. + -. 0.09mg/mL, interaction of CMP-2 with target C2, C4, C3, C5, C9, Factor B, Factor D, or Factor P inhibits complement activation.

5. The use according to claim 1, wherein said CMP-3 inhibits complement activation, its CH₅₀0.27. + -. 0.04mg/mL, AP₅₀0.33. + -. 0.07mg/mL, interaction of CMP-3 with target C2, C3, C5, C9, Factor B, Factor D, or Factor P inhibits complement activation.

6. The use of claim 1, wherein the cordyceps militaris polysaccharide is prepared by the following steps:

7. The use of claim 6, wherein in step (3), the dialysis bag has a molecular weight cut-off of 8kDa to 14 kDa.

8. Use according to claim 6, characterized in that the homogeneous high molecular weight polysaccharide with anticomplementary activity is CMP-1, CMP-2 or CMP-3.