Saxifraga stolonifera polysaccharide and application thereof in preparation of complement inhibitor or anti-inflammatory drug

Abstract

The invention relates to saxifraga stolonifera polysaccharide and application thereof in preparation of complement inhibitors or anti-inflammatory drugs. Based on activity guidance, it is found that a water extraction part of saxifraga stolonifera has good anti-complement activity when experimental screening is conducted on the anti-complement active part of saxifraga stolonifera, graded alcohol precipitation is conducted on the water extraction part, the activity of crude polysaccharide precipitated by 90 alcohol (90% ethyl alcohol) is higher than that of crude polysaccharide precipitated by 75 alcohol (75% ethyl alcohol), and the anti-complement activity of the crude polysaccharide precipitated by 90 alcohol is higher than that of a positive drug (heparin). On the basis, five neutral heteropolysaccharides with anticomplement activity, namely SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 or SSP-90-5, are separated from the 90 alcohol precipitation crude polysaccharide. Compared with the prior art, the structural characteristics of the saxifraga stolonifera polysaccharide are represented, and the preparation method of the saxifraga stolonifera polysaccharide and the application of the saxifraga stolonifera polysaccharide in preparation of complement inhibitors and anti-inflammatory drugs are provided. The invention verifies the anti-complement activity of the saxifraga stolonifera polysaccharide and the therapeutic effect of the saxifraga stolonifera polysaccharide on lipopolysaccharide (LPS)-induced acute lung injury and systemic inflammatory response.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and in particular relates to saxifraga polysaccharide and its use in the preparation of complement inhibitors or anti-inflammatory drugs. Background Art

[0002] Systemic inflammatory response syndrome (SIRS) is usually associated with multiple organ dysfunction syndrome caused by cytokine storm, pathogenic infection, over-activation of the complement system, massive infiltration of neutrophils, excessive oxidative stress, etc. The inflammatory response is a complex process, which protects the host from infections caused by pathogens, helps to clear pathogens, and reduces tissue damage. SIRS can lead to over-activation of the complement system and organ damage (such as acute lung injury, ALI). The complement system is an important part of the body's immune defense system, and normal complement activation is the key to maintaining the body's homeostasis and balance. Its functions include regulating the inflammatory response, enhancing the immune response, cell lysis, phagocytosis, and elimination of apoptotic cells, etc. However, over-activation or inappropriate inhibition of the complement system can lead to the occurrence of various diseases, including acute respiratory distress syndrome, rheumatoid arthritis, and systemic lupus erythematosus, etc. Inhibiting the over-activation of the complement system can be used as one of the strategies for treating inflammatory responses. However, clinically, chemically synthesized immunosuppressants such as glucocorticoids are mostly used as anti-inflammatory and anti-complement drugs, with poor selectivity, and long-term use will lead to a decline in immune function and other side effects. Relevant reports show that natural products of many heat-clearing traditional Chinese medicines have been proven to have anti-complement activity and anti-inflammatory activity. Therefore, it is of great significance to find anti-complement and anti-inflammatory components with mild toxic and side effects and definite curative effects from traditional Chinese medicines.

[0003] Saxifraga, a perennial herbaceous medicinal plant, is widely distributed in China with rich varieties. In inland provinces, the dried whole herb of Saxifraga stolonifera Curt of the genus Saxifraga in the family Saxifragaceae is mainly used. It is also known as tiger ear, stone lotus leaf, etc., and belongs to the lung, spleen, and large intestine meridians. A small amount of it can be burned and smoked to treat hemorrhoids and swelling toxins. Clinically, it can be decocted and taken to treat cough and asthma due to lung heat, and has significant curative effects when applied externally to treat skin urticaria, boils, etc.

[0004] Chinese Patent CN113786421A discloses a Saxifraga stolonifera extract with anti-inflammatory activity, its preparation method and application. The preparation method includes: S1: extracting fresh Saxifraga stolonifera and dried Saxifraga stolonifera respectively by different extraction methods to obtain extracts; S2: comparing the content differences of total flavonoids and total phenolic acids in the fresh Saxifraga stolonifera extract and dried Saxifraga stolonifera extract obtained by different extraction methods; S3: qualitatively analyzing the active ingredients in the fresh Saxifraga stolonifera extract and dried Saxifraga stolonifera extract; S4: establishing an inflammation model and adding the interacting fresh Saxifraga stolonifera extract and dried Saxifraga stolonifera extract respectively to verify the effects of the fresh Saxifraga stolonifera extract and dried Saxifraga stolonifera extract. However, in this application scheme, it is not clear which components in the Saxifraga stolonifera extract are the effective components that play a role. Summary of the Invention

[0005] Based on the current situation that there is no information in the prior art about which components in the Saxifraga stolonifera extract have anti-inflammatory effects, the present invention provides Saxifraga stolonifera polysaccharide and its use in the preparation of complement inhibitors or anti-inflammatory drugs.

[0006] Specifically, based on activity guidance, during the experimental screening of the anti-complement active site of Saxifraga stolonifera, it was found that its water extraction site has good anti-complement activity. The water extract was subjected to fractional alcohol precipitation, and the crude polysaccharide precipitated by 90% alcohol (90% ethanol) has stronger anti-complement activity than the crude polysaccharide precipitated by 75% alcohol (75% ethanol), and the anti-complement activity of the crude polysaccharide precipitated by 90% alcohol is stronger than that of the positive drug (heparin). Based on this, 5 neutral heteropolysaccharides with anti-complement activity were isolated from the crude polysaccharide precipitated by 90% alcohol.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] The present invention first provides Saxifraga stolonifera polysaccharide, which is selected from one or a combination of several of SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 or SSP-90-5:

[0009] Among them, SSP-90-1 is composed of five monosaccharides, with a molecular weight of 17.08 kDa; the total sugar content is 93.29 ± 1.71%; the protein content is 0.74 ± 0.24%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 6.3:16.3:14.3:42.3:20.8. The methylation result shows that this sugar is composed of arabinose linked at the end, rhamnose linked at the end, 1,5-linked arabinose, 1,2-linked rhamnose, 1,3-linked rhamnose, mannose linked at the end, glucose linked at the end, 1,3,5-linked arabinose, 1,4-linked galactose and 1,4,6-linked glucose, and the molar ratio is 10.1:3.4:1.8:9.0:3.8:5.9:3.1:9.3:42.3:11.3;

[0010] SSP-90-2 is composed of five monosaccharides, with a molecular weight of 21.46 kDa; the total sugar content is 92.83 ± 1.05%; the protein content is 2.04 ± 0.15%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 3.9:14.8:44.5:23.8:13.0. The methylation results show that this sugar is composed of arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, and 1,4,6-linked glucose, with a molar ratio of 8.8:5.8:8.1:3.6:20.7:3.0:25.1:15.5:9.3;

[0011] SSP-90-3 is composed of five monosaccharides, with a molecular weight of 23.37 kDa; the total sugar content is 94.76 ± 0.95%; the protein content is 1.79 ± 0.54%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 3.3:17.6:49.7:22.8:6.6. The methylation results show that this sugar is composed of arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked glucose, with a molar ratio of 5.0:12.2:6.1:4.2:26.0:5.4:19.6:9.5:5.1:6.9;

[0012] SSP-90-4 is composed of five monosaccharides, with a molecular weight of 24.06 kDa; the total sugar content is 92.42 ± 1.53%; the protein content is 0.93 ± 0.36%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 5.3:21.5:43.3:22.1:7.8. The methylation results show that this sugar is composed of arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked galactose, with a molar ratio of 4.7:11.5:9.5:5.5:24.1:4.5:12.7:17.8:2.6:7.1;

[0013] SSP-90-5 is composed of five monosaccharides, with a molecular weight of 24.77 kDa; the total sugar content is 93.56 ± 2.02%; the protein content is 1.61 ± 0.44%; it does not contain uronic acid, and the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 5.3:15.6:42.1:27.9:9.1. The methylation result shows that this sugar is composed of arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked galactose, with a molar ratio of 4.7:9.5:7.5:6.4:21.9:7.0:19.7:15.0:3.6:4.7.

[0014] The present invention further provides a preparation method of the saxifrage polysaccharide, comprising the following steps:

[0015] Saxifrage is defatted by extraction with 95% ethanol, filtered, and the residue is dried. Then, it is extracted with hot water. The extract is filtered, concentrated, 95% ethanol is added until the final ethanol concentration is 75%, left to stand, centrifuged to obtain the supernatant, the supernatant is concentrated, anhydrous ethanol is added until the final ethanol concentration is 90%, left to stand, centrifuged to discard the supernatant, the precipitate is dissolved in water, protein is removed with trichloroacetic acid, centrifuged, the supernatant is adjusted to neutrality, concentrated, dialyzed, and freeze-dried to obtain the 90% ethanol-precipitated crude polysaccharide SSP-90;

[0016] SSP-90 is dissolved in distilled water and preliminarily separated by DEAE-cellulose column chromatography, eluted with distilled water, 0.1, 0.2, 0.4, 0.8, and 1.6 mol / L NaCl solutions. Each fraction is collected, and the absorbance value at 490 nm (after color development by the sulfuric acid-phenol method) is detected every 250 mL. The fractions are combined, concentrated, dialyzed against running water, and freeze-dried to obtain 6 secondary fractions FW, F01, F02, F03, F04, and F05. Based on activity guidance, the FW fraction has no anti-complement activity, and the other 5 secondary fractions have anti-complement activity. Their homogeneity and molecular weight are verified by HPGPC-ELSD (high performance liquid gel permeation chromatography-evaporative light scattering detector) and HPGPC-RID (high performance liquid gel permeation chromatography-refractive index detector) to obtain the homogeneous polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4, and SSP-90-5.

[0017] The present invention further provides the use of the saxifrage polysaccharide in the preparation of complement inhibitors or anti-inflammatory drugs.

[0018] In one embodiment of the present invention, the saxifraga stolonifera polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4, and SSP-90-5 are used in the preparation of a complement inhibitor.

[0019] In one embodiment of the present invention, the saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 are used in the preparation of an anti-inflammatory drug or a drug for treating lung injury.

[0020] In one embodiment of the present invention, the saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 are used in the preparation of a drug for lipopolysaccharide (LPS)-induced acute lung injury.

[0021] In vitro experiments were conducted in the present invention, and the results confirmed that the saxifraga stolonifera polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4, and SSP-90-5 all had obvious inhibition on the hemolysis caused by the activation of the classical complement pathway, that is, they had obvious anti-complement effects. The CH 50 values (the concentration of the test sample required for 50% inhibition of hemolysis in the classical pathway) were 247.45 ± 4.32 μg / mL, 53.16 ± 3.23 μg / mL, 49.05 ± 1.79 μg / mL, 29.27 ± 2.06 μg / mL, and 59.13 ± 3.42 μg / mL, respectively.

[0022] Animal experiments in vivo proved that the saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 had significant improvement effects on the recruitment of inflammatory cells in the lungs of mice induced by LPS and the expression of inflammatory factors in the lungs and serum, and had significant therapeutic effects on LPS-induced lung injury and systemic inflammatory response, and could be used in the preparation of anti-inflammatory drugs.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] Five neutral heteropolysaccharides (SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4, and SSP-90-5) were isolated from the dried whole herb of the plant Saxifraga stolonifera Curt in the present invention, and the structural characteristics of the polysaccharides were characterized, and a preparation method of saxifraga stolonifera polysaccharides and their uses in the preparation of complement inhibitors and anti-inflammatory drugs were provided. The present invention confirmed the anti-complement activity of saxifraga stolonifera polysaccharides and their therapeutic effects on lipopolysaccharide (LPS)-induced acute lung injury and systemic inflammatory response. Description of the Drawings

[0025] Figure 1 , Flow chart for the isolation of homogeneous neutral heteropolysaccharides from Saxifraga stolonifera.

[0026] Figure 2 , HPGPC-ELSD chromatograms of Saxifraga stolonifera neutral heteropolysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 and SSP-90-5, where TSK-GEL GMPW XL gel column (300×7.6 mm); eluent: 20 mM ammonium acetate; flow rate: 0.6 mL / min.

[0027] Figure 3 , HPGPC-RID chromatograms of Saxifraga stolonifera neutral heteropolysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 and SSP-90-5, where TSK-GEL GMPW XL gel column (300×7.6 mm); eluent: 20 mM ammonium acetate; flow rate: 0.6 mL / min.

[0028] Figure 4 , Effects of Saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 on the total cell count in bronchoalveolar lavage fluid of LPS-induced acute lung injury in mice.

[0029] Figure 5 , Effects of Saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 on the expression of TNF-α and IL-6 in bronchoalveolar lavage fluid of LPS-induced acute lung injury in mice.

[0030] Figure 6 , Effects of Saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 on the expression of TNF-α and IL-6 in the serum of LPS-induced inflammatory mice. Detailed implementation mode

[0031] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1 Preparation of Saxifraga stolonifera polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 and SSP-90-5 Reference for the separation process of Saxifraga stolonifera homogeneous neutral heteropolysaccharides Figure 1 .

[0033] 15 kg of Saxifraga herb was crushed, extracted with 95% ethanol, filtered, the residue was dried, extracted with hot water for 3 times, the filtrate was combined, concentrated, 95% ethanol was added to the supernatant to a final ethanol concentration of 75%, allowed to stand, centrifuged to obtain the supernatant, the supernatant was concentrated, anhydrous ethanol was added to a final ethanol concentration of 90%, allowed to stand, centrifuged to discard the supernatant, the precipitate was re-dissolved with water, recovered under reduced pressure, residual ethanol was removed, re-dissolved with distilled water, free protein was removed with trichloroacetic acid, centrifuged, the supernatant was adjusted to neutral, dialyzed, concentrated, and freeze-dried to obtain crude polysaccharide SSP-90. 50 g of SSP-90 was dissolved in distilled water, centrifuged, and the supernatant was preliminarily separated by DEAE-cellulose column chromatography. The fractions were eluted with distilled water, 0.1, 0.2, 0.4, 0.8 and 1.6 mol / L NaCl solution in a gradient, with each gradient elution volume greater than 2 times the column volume (about 10 L) and a flow rate of 25 mL / min. The fractions were collected and the absorbance at 490 nm (after color development with sulfuric acid-phenol method) was detected every 250 mL. The fractions were combined according to the elution curve, concentrated, dialyzed and freeze-dried to obtain 6 secondary fractions (FW, F01, F02, F03, F04 and F05). Based on activity orientation, the FW fraction had no anti-complement activity, and the other 5 secondary fractions had It has anti-complement activity, and its homogeneity and molecular weight are verified by high performance gel chromatography-evaporative light scattering detector (HPGPC-ELSD) and high performance gel chromatography-differential refractive index detector (HPGPC-RID), all of which are single symmetrical peaks, indicating that it is a homogeneous component, and polysaccharides SSP-90-1 (721.57 mg), SSP-90-2 (354.03 mg), SSP-90-3 (503.37 mg), SSP-90-4 (382.19 mg) and SSP-90-5 (632.94 mg) are obtained. Among them, the HPGPC-ELSD chromatograms of Saxifraga neutral heteropolysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 and SSP-90-5 are as follows Figure 2 As shown, TSK-GEL GMPW XL Gel column (300×7.6 mm); eluent: 20 mM ammonium acetate; flow rate: 0.6 mL / min. The HPGPC-RID chromatograms of Saxifraga neutral heteropolysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 and SSP-90-5 are shown in Figure 3 As shown, TSK-GEL GMPW XL Gel column (300×7.6 mm); eluent: 20 mM ammonium acetate; flow rate: 0.6 mL / min.

[0034] Example 2 Structural Characterization of Saxifraga Polysaccharide

[0035] 1) Determination of molecular weight

[0036] The relative molecular weight of the polysaccharide sample was determined by high performance gel permeation chromatography (HPGPC). The basic principle is that homogeneous polysaccharides pass through gel permeation chromatography to form symmetric chromatographic peaks. The elution time is related to the molecular weight. Calculations are performed based on the calibration curve obtained from known molecular weights.

[0037] Chromatographic conditions: Separation was carried out using a TSK GMPW XL gel column (300×7.6 mm), with a flow rate of 0.6 mg / mL, an injection volume of 20 μL, a mobile phase of 20 mM ammonium acetate solution, a column temperature of 30 °C, and a differential refractive index detector (RID).

[0038] Experimental method: Accurately weigh 2 mg each of the homogeneous polysaccharides from Saxifraga stolonifera and Dextrans series standards (P800, P400, P200, P100, P50, P20, P10, P5), dissolve them in 20 mM ammonium acetate to form a 4 mg / mL solution, filter through a 0.22 μm microporous membrane and then detect. Record the retention time of the polysaccharide molecules. Plot a standard curve with the retention time as the abscissa and the logarithm (Log) of the standard polysaccharide molecular weight as the ordinate to obtain the corresponding linear regression equation. Substitute the retention time of the homogeneous polysaccharide to be measured into the regression equation to calculate the relative molecular weight of the homogeneous polysaccharide. The relative molecular weights of Saxifraga stolonifera polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4, and SSP-90-5 are 17.08 kDa, 21.46 kDa, 23.37 kDa, 24.06 kDa, and 24.77 kDa, respectively.

[0039] 2) Determination of the contents of total sugar, uronic acid and protein

[0040] The total sugar content of SSP-90-1 determined by the sulfuric acid-phenol method was 93.29 ± 1.71%; the total sugar content of SSP-90-2 was 92.83 ± 1.05%; the total sugar content of SSP-90-3 was 94.76 ± 0.95%; the total sugar content of SSP-90-4 was 92.42 ± 1.53%; the total sugar content of SSP-90-5 was 93.56 ± 2.02%.

[0041] The hydroxybiphenyl method was used to detect that polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4, and SSP-90-5 did not contain uronic acid.

[0042] The protein content was determined by Coomassie Brilliant Blue method. The protein content of SSP-90-1 was 0.74 ± 0.24%; the protein content of SSP-90-2 was 2.04 ± 0.15%; the protein content of SSP-90-3 was 1.79 ± 0.54%; the protein content of SSP-90-4 was 0.93 ± 0.36%; the protein content of SSP-90-5 was 1.61 ± 0.44%.

[0043] 3) Monosaccharide composition analysis

[0044] The products obtained by completely hydrolyzing Saxifraga stolonifera polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 and SSP-90-5 with 2 mol / L trifluoroacetic acid (TFA) at 110 °C were derivatized with PMP and then analyzed by high performance liquid chromatography.

[0045] SSP-90-1 is a polysaccharide composed of five monosaccharides, mannose:rhamnose:glucose:galactose:arabinose = 6.3:16.3:14.3:42.3:20.8.

[0046] SSP-90-2 is a polysaccharide composed of five monosaccharides, mannose:rhamnose:glucose:galactose:arabinose = 3.9:14.8:44.5:23.8:13.0.

[0047] SSP-90-3 is a polysaccharide composed of five monosaccharides, mannose:rhamnose:glucose:galactose:arabinose = 3.3:17.6:49.7:22.8:6.6.

[0048] SSP-90-4 is a polysaccharide composed of five monosaccharides, mannose:rhamnose:glucose:galactose:arabinose = 5.3:21.5:43.3:22.1:7.8.

[0049] SSP-90-5 is a polysaccharide composed of five monosaccharides, mannose:rhamnose:glucose:galactose:arabinose = 5.3:15.6:42.1:27.9:9.1.

[0050] 4) Methylation analysis

[0051] The Saxifraga stolonifera polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 and SSP-90-5 were methylated by the modified Hakomori method. The methylation products were completely hydrolyzed with 2 mol / L TFA, reduced with NaBH4 and acetylated with acetic anhydride to form partially methylated alditol acetate derivatives, and then analyzed by GC-MS.

[0052] The SSP-90-1 structure contains: arabinose linked at the end, rhamnose linked at the end, 1,5-linked arabinose, 1,2-linked rhamnose, 1,3-linked rhamnose, mannose linked at the end, glucose linked at the end, 1,3,5-linked arabinose, 1,4-linked galactose, and 1,4,6-linked glucose, with a molar ratio of 10.1:3.4:1.8:9.0:3.8:5.9:3.1:9.3:42.3:11.3.

[0053] The SSP-90-2 structure contains: arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, and 1,4,6-linked glucose, with a molar ratio of 8.8:5.8:8.1:3.6:20.7:3.0:25.1:15.5:9.3.

[0054] The SSP-90-3 structure contains: arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked glucose, with a molar ratio of 5.0:12.2:6.1:4.2:26.0:5.4:19.6:9.5:5.1:6.9.

[0055] The SSP-90-4 structure contains: arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked galactose, with a molar ratio of 4.7:11.5:9.5:5.5:24.1:4.5:12.7:17.8:2.6:7.1.

[0056] The SSP-90-5 structure contains: arabinose linked at the end, rhamnose linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked galactose, with a molar ratio of 4.7:9.5:7.5:6.4:21.9:7.0:19.7:15.0:3.6:4.7.

[0057] Classical pathway anti-complement activity in Example 3

[0058] Take the serum of 3-month-old guinea pigs, sensitize it with 2% sheep red blood cells, and dilute it to 1:80 with barbital buffer solution (BBS) as the complement source for the classical pathway in this experiment. Dilute the rabbit anti-sheep red blood cell antibody to 1:1000 with BBS as the hemolysin; prepare fresh sheep blood into 2% sheep blood cells (2% SRBC) with BBS. Weigh 1.5 mg of the polysaccharide sample precisely, dissolve it in BBS buffer solution, and serially dilute it into 8 concentrations. Mix 100 μL of polysaccharide solutions with different concentrations with 100 μL of complement diluted to 1:80, then successively add 50 μL of hemolysin (1:1000) and 50 μL of 2% SRBC. After incubating in a 37°C water bath for 30 min, place it in a low-temperature high-speed centrifuge and centrifuge at 4500 rpm for 5 min. Take 200 μL of the supernatant from each tube and transfer it to a 96-well plate, and measure the absorbance at 405 nm with an enzyme-linked immunosorbent assay (ELISA) reader. At the same time, set up a polysaccharide control group (100 μL of polysaccharide with the corresponding concentration plus 100 μL of BBS buffer solution, 50 μL of hemolysin (1:1000), and 50 μL of 2% SRBC), a zero-hemolysis group (250 μL of BBS buffer solution and 50 μL of 2% SRBC), and a complete-hemolysis group, namely the complement control group (100 μL of BBS buffer solution, 100 μL of 1:80 complement, 50 μL of hemolysin (1:1000), and 50 μL of 2% SRBC). After subtracting the absorbance value of the corresponding polysaccharide control group from the absorbance value of each polysaccharide group with different concentrations, calculate the CH 50 value (the concentration required for 50% inhibition of hemolysis in the classical pathway). Use heparin as a positive control drug, and the results show that 5 Saxifraga stolonifera neutral heteropolysaccharides all have significant inhibitory activities on the activation of the classical pathway of complement (see Table 1).

[0059] Table 1 Inhibitory effects of 5 Saxifraga stolonifera neutral heteropolysaccharides on complement activation

[0060]

[0061] CH 50 value is expressed as: mean ± SD (n = 3)

[0062] Example 4 Effects of Saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 on LPS-induced lung injury and systemic inflammatory response in mice

[0063] Fifty-four Balb / c mice (18 - 22 g) were randomly divided into 9 groups (A, B, C, D, E, F, G, H, I) according to body weight: Group A was the normal control group (Control), Group B was the LPS model group (LPS), Group C was the low-dose group of polysaccharide SSP-90-1 (SSP-90-1-L, 20 mg / kg), Group D was the medium-dose group of SSP-90-1 (SSP-90-1-M, 40 mg / kg), Group E was the high-dose group of SSP-90-1 (SSP-90-1-H, 80 mg / kg), Group F was the low-dose group of SSP-90-2 (SSP-90-2-L, 20 mg / kg), Group G was the medium-dose group of SSP-90-2 (SSP-90-2-M, 40 mg / kg), Group H was the high-dose group of SSP-90-2 (SSP-90-2-H, 80 mg / kg), and Group I was the positive control dexamethasone group (DEX, 4 mg / kg), with 6 mice in each group. The model was established by intraperitoneal injection at a dose of 10 mg / kg. Group A was injected with normal saline, and the other groups were injected with LPS. The administration method was intragastric administration at the above doses. Groups A and B were given normal saline of equal volume. Prophylactic administration was given once on the first day, and the drug was administered again 24 h later. One hour after drug administration, the model was established. Six hours after model establishment, blood was collected by eye enucleation. After the blood was left standing at 4°C for 1 - 2 h, the serum was taken after centrifugation for detecting the inflammatory factors TNF-α and IL-6. The mouse lung tissue was lavaged with normal saline to obtain bronchoalveolar lavage fluid (BALF). An appropriate amount was stained with 0.4% trypan blue solution, and the total cell count was detected with a cell counter. The remaining BALF was centrifuged to take the supernatant for detecting the inflammatory factors TNF-α and IL-6.

[0064] (1) Effects of Saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 on LPS-induced acute lung injury in mice

[0065] LPS-induced acute lung injury in mice leads to the recruitment of pulmonary inflammatory cells and the overexpression of inflammatory factors. By detecting the differences in the total cell count and the expression of inflammatory factors in the BALF of mice in each group, the pharmacological effects of polysaccharide drugs were evaluated. The results showed that the total cell count and the expression of inflammatory factors (TNF-α and IL-6) in the BALF of mice in the LPS group were significantly increased. The total cell count in the BALF of the SSP-90-1 (80 mg / kg) and SSP-90-2 (20 mg / kg, 40 mg / kg, and 80 mg / kg) administration groups was significantly lower than that in the LPS group (as Figure 4 shown), and the expression of TNF-α and IL-6 in the BALF of the SSP-90-1 (40 mg / kg and 80 mg / kg) and SSP-90-2 (20 mg / kg, 40 mg / kg, and 80 mg / kg) administration groups was significantly inhibited compared with that in the LPS group, and the effect of SSP-90-2 was stronger than that of SSP-90-1 (as Figure 5as shown

[0066] (2) Effects of Saxifraga stolonifera polysaccharides SSP-90-1 and SSP-90-2 on LPS-induced systemic inflammatory response in mice LPS-induced systemic inflammatory response in mice results in a large amount of expression of inflammatory factors in the blood. By detecting the expression of inflammatory factors in the serum of mice in each group, the anti-inflammatory effect of polysaccharides was evaluated. The results showed that the expressions of inflammatory factors (TNF-α and IL-6) in the serum of mice in the LPS group were significantly increased. The expressions of TNF-α and IL-6 in the serum of the administration groups of SSP-90-1 (40 mg / kg and 80 mg / kg) and SSP-90-2 (20 mg / kg, 40 mg / kg and 80 mg / kg) were significantly inhibited compared with those in the LPS group, and SSP-90-2 was stronger than SSP-90-1 (as Figure 6 shown

[0067] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A saxifraga stolonifera polysaccharide, characterized in that, It is SSP-90-1. SSP-90-1 is composed of five monosaccharides, with a molecular weight of 17.08 kDa; the total sugar content is 93.29 ± 1.71%; the protein content is 0.74 ± 0.24%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 6.3:16.3:14.3:42.3:20.

8. SSP-90-1 is composed of arabinose-linked at the end, rhamnose-linked at the end, 1,5-linked arabinose, 1,2-linked rhamnose, 1,3-linked rhamnose, mannose-linked at the end, glucose-linked at the end, 1,3,5-linked arabinose, 1,4-linked galactose, and 1,4,6-linked glucose, with a molar ratio of 10.1:3.4:1.8:9.0:3.8:5.9:3.1:9.3:42.3:11.

3.

2. A saxifraga polysaccharide, characterized in that, It is SSP-90-2. SSP-90-2 is composed of five monosaccharides, with a molecular weight of 21.46 kDa; the total sugar content is 92.83 ± 1.05%; the protein content is 2.04 ± 0.15%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 3.9:14.8:44.5:23.8:13.

0. SSP-90-2 is composed of arabinose-linked at the end, rhamnose-linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, and 1,4,6-linked glucose, with a molar ratio of 8.8:5.8:8.1:3.6:20.7:3.0:25.1:15.5:9.

3.

3. A saxifraga stolonifera polysaccharide, characterized in that, It is SSP-90-3. SSP-90-3 is composed of five monosaccharides, with a molecular weight of 23.37 kDa; the total sugar content is 94.76 ± 0.95%; the protein content is 1.79 ± 0.54%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 3.3:17.6:49.7:22.8:6.

6. SSP-90-3 is composed of arabinose-linked at the end, rhamnose-linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked glucose, with a molar ratio of 5.0:12.2:6.1:4.2:26.0:5.4:19.6:9.5:5.1:6.

9.

4. A saxifraga polysaccharide, characterized in that, It is SSP-90-4. SSP-90-4 is composed of five monosaccharides, with a molecular weight of 24.06 kDa; the total sugar content is 92.42 ± 1.53%; the protein content is 0.93 ± 0.36%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 5.3:21.5:43.3:22.1:7.

8. SSP-90-4 is composed of arabinose-linked at the end, rhamnose-linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked galactose, with a molar ratio of 4.7:11.5:9.5:5.5:24.1:4.5:12.7:17.8:2.6:7.

1.

5. A saxifraga polysaccharide, characterized in that, It is SSP-90-5. SSP-90-5 is composed of five monosaccharides, with a molecular weight of 24.77 kDa; the total sugar content is 93.56 ± 2.02%; the protein content is 1.61 ± 0.44%; it does not contain uronic acid, and the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 5.3:15.6:42.1:27.9:9.

1. SSP-90-5 is composed of arabinose-linked at the end, rhamnose-linked at the end, 1,2-linked rhamnose, 1,2-linked mannose, 1,2-linked glucose, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,6-linked glucose, and 1,4,6-linked galactose, with a molar ratio of 4.7:9.5:7.5:6.4:21.9:7.0:19.7:15.0:3.6:4.

7.

6. The preparation method of saxifraga stolonifera polysaccharide according to any one of claims 1-5, characterized in that, It includes the following steps: Saxifraga stolonifera is defatted by extraction with 95% ethanol, filtered, and the residue is dried. Then it is extracted with hot water. The extract is filtered, concentrated, and 95% ethanol is added until the final ethanol concentration is 75%. It is left to stand, and the supernatant is taken by centrifugation. The supernatant is concentrated, and anhydrous ethanol is added until the final ethanol concentration is 90%. It is left to stand, and the supernatant is discarded by centrifugation. The precipitate is redissolved in water, and the protein is removed with trichloroacetic acid. After centrifugation, the supernatant is adjusted to neutral, concentrated, dialyzed, and freeze-dried to obtain the 90% ethanol-precipitated crude polysaccharide SSP-90. SSP-90 is redissolved in distilled water and preliminarily separated by DEAE-cellulose column chromatography, eluted with distilled water, 0.1, 0.2, 0.4, 0.8, and 1.6 mol / L NaCl solutions. Each fraction is collected, and the absorbance value at 490 nm is detected every 250 mL. The fractions are combined, concentrated, dialyzed against running water, and freeze-dried to obtain 6 secondary fractions FW, F01, F02, F03, F04, and F05. Based on activity-guided separation, the FW fraction has no anti-complement activity, and the other 5 secondary fractions have anti-complement activity. Their homogeneity and molecular weights are verified by HPGPC-ELSD and HPGPC-RID to obtain the homogeneous polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4, or SSP-90-5.

7. Use of the saxifraga polysaccharide according to any one of claims 1-5 in the preparation of a complement inhibitor or an anti-inflammatory drug.

8. The preparation method of saxifraga stolonifera polysaccharide according to claim 7, characterized in that, Use of saxifraga polysaccharides SSP-90-1, SSP-90-2, SSP-90-3, SSP-90-4 or SSP-90-5 in the preparation of a complement inhibitor.

9. The preparation method of saxifraga stolonifera polysaccharide according to claim 7, characterized in that Use of saxifraga polysaccharides SSP-90-1 or SSP-90-2 in the preparation of an anti-inflammatory drug or a drug for treating lung injury.

10. The preparation method of saxifrage polysaccharide according to claim 9, characterized in that, Use of the saxifraga polysaccharides SSP-90-1, SSP-90-2 in the preparation of a drug for lipopolysaccharide-induced acute lung injury.

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