Rhododendron dauricum polysaccharide as well as preparation method and application thereof

Abstract

The invention relates to rhododendron dauricum polysaccharide as well as a preparation method and application thereof, 75 alcohol precipitation polysaccharide XNCDP-75 and four acidic homogeneous polysaccharides XNDHP-1, XNDHP-2, XNDHP-3 and XNDHP-4 are separated from rhododendron dauricum leaves, and experiments prove that the rhododendron dauricum polysaccharide has an obvious inhibition effect on complement activation, and can be used for preparing the rhododendron dauricum polysaccharide. The compound can significantly inhibit the inflammatory response of RAW264.7 cells induced by lipopolysaccharide LPS on the cellular level, and has a significant anti-inflammatory effect on mouse inflammation induced by LPS, influenza A virus H1N1 or influenza virus-drug-resistant bacterium co-infection in vivo. The compound can be further used as an active ingredient for preparing a novel complement inhibitor and a medicine for treating inflammation caused by bacteria, viruses or virus-bacteria co-infection.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicines, and in particular to a Rhododendron dasyphylla polysaccharide and a preparation method and application thereof. Background Art

[0002] The complement system is an important part of the human immune system. However, overactivation of this system can cause an overreaction of the human immune system, resulting in damage to the body's normal tissues. Overactivation of complement plays an important role in the pathogenesis of acute diseases such as rheumatoid arthritis, Alzheimer's disease, systemic lupus erythematosus (SLE), ischemia-reperfusion, acute myocardial infarction, acute respiratory distress syndrome (ARDS), and multiple organ failure syndrome. Currently, immunosuppressants such as glucocorticoids used in clinical practice are not specific complement inhibitors and are prone to a variety of complications and side effects. Therefore, new, efficient, low-toxic, and specific complement inhibitors are urgently needed in clinical practice.

[0003] Inflammation is a highly complex host pathophysiological reaction process and a natural defense reaction that aims to eliminate pathogens and reduce tissue damage. However, acute inflammation can cause large-scale neutrophil infiltration in a short period of time, forming local inflammatory centers and leading to varying degrees of tissue damage. Severe lung inflammation can cause acute lung injury (ALI), with symptoms including severe hypoxemia, respiratory distress, and even further deterioration to acute respiratory distress syndrome, ultimately leading to death of the patient (Long ME, Mallampalli RK, Horowitz JC. Clin Sci (Lond), 136 (10): 747-769). The complement system is closely related to pneumonia. Overactivation of the complement system is one of the important symptoms and causes of acute lung injury. Therefore, complement inhibitors can be used as potential drugs for the treatment of pneumonia and acute lung injury.

[0004] Since complement inhibition is one of the treatment strategies for infectious pneumonia, it is of great significance to develop highly effective and low-toxic complement inhibitors as anti-inflammatory drugs. Polysaccharides have the characteristics of high efficiency and low toxicity. It has been reported that anti-complement active polysaccharides with good anti-inflammatory activity have been isolated from medicinal plants (Chen MY, Li H, Lu XX, Ling LJ, Weng HB, Sun W, Chen DF, Zhang YY. Chin J Nat Med, 2019, 17: 187-197). Rhododendron dauricum L. is a semi-evergreen shrub of the genus Rhododendron. In traditional Chinese medicine, the leaves of Rhododendron dauricum are used as "Manshanhong" for medicine, which has the effect of clearing heat and removing phlegm, and is included in the "Chinese Pharmacopoeia" for the treatment of inflammation-related diseases such as pneumonia. At present, there are few reports on the research of Rhododendron dahurica polysaccharides. There is only one report on the extraction of 85% alcohol-precipitated crude polysaccharide RDPs from Rhododendron dahurica, and the separation of two uniform polysaccharides with antioxidant activity RDP-1 and RDP-2 from RDPs by macroporous resin adsorption method (Hu ZY, Yu RY, Sun JF, Duan YQ, Zhou HL, Zhou W, Li G. Process Biochem, 2022, 121: 113-125), and their derivatives AcRDP-1 and AcRDP-2 were obtained by acetylation modification (Hu Z, Sun J, Jin L, Zong T, Duan Y, Zhou H, Zhou W, Li G. Polymers (Basel). 2022, 14(15): 3130.). The derivatized uniform polysaccharides have certain anti-complement activity, while the natural polysaccharides RDPs, RDP-1, and RDP-2 themselves do not have anti-complement activity. At the same time, the five polysaccharides are all neutral uniform polysaccharides. Summary of the invention

[0005] The purpose of the present invention is to explore the polysaccharide with complement activity in Rhododendron dahurica and provide a Rhododendron dahurica polysaccharide and its preparation method and application.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] One of the technical solutions of the present invention is to provide a Rhododendron dahurica polysaccharide, which includes 75 alcohol precipitation polysaccharide XNDCP-75 and acidic uniform polysaccharides XNDHP-1, XNDHP-2, XNDHP-3, and XNDHP-4, and the structural characteristics thereof are respectively:

[0008] 75 alcohol precipitated polysaccharide XNDCP-75 is composed of monosaccharides in the following molar ratio: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 9.50: 4.51: 18.78: 39.65: 4.47: 9.40: 13.68; molecular weight range is 279.6-871.7 kDa; total sugar content is 57.34%; protein content is 2.21%; uronic acid content is 1.94%;

[0009] The acidic homogeneous polysaccharide XNDHP-1 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 4.69: 8.46: 4.49: 6.15: 1.93: 38.61: 35.67; the molecular weight is 371.5 kDa; the total sugar content is 91.15%; the protein content is 0.97%; the uronic acid content is 7.12%; the connection method includes terminal connection with arabinose sugars, terminally linked rhamnose, 1,5-linked arabinose, terminally linked glucuronic acid, terminally linked glucose, terminally linked galacturonic acid, 1,4-linked galactose, 1,3,6-linked galactose, 1,3,6-linked mannose, 1,3,4,6-linked galactose, with molar ratios of 19.87:6.12:13.16:2.11:3.17:4.78:33.02:3.96:6.21:7.60, respectively;

[0010] The acidic homogeneous polysaccharide XNDHP-2 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 7.41: 12.29: 2.40: 2.20: 5.97: 28.44: 41.29; the molecular weight is 385.4 kDa; the total sugar content is 89.96%; the protein content is 1.01%; the uronic acid content is 5.31%; the connection modes include terminal connection arabinose, terminal connection rhamnose, 1,3-connected arabinose, 1,5-connected arabinose, terminal connection glucuronic acid, terminal connection glucose , terminally linked to galacturonic acid, 1,3,5-linked to arabinose, 1,4-linked to galactose, 1,3-linked to glucose, 1,3,6-linked to galactose, 1,3,6-linked to glucose, 1,3,6-linked to mannose, 1,4,6-linked to mannose, 1,3,4-linked to rhamnose, 1,3,4,6-linked to galactose, with molar ratios of 18.96: 3.17: 5.79: 15.48: 2.78: 5.69: 1.96: 6.41: 15.26: 2.53: 1.87: 2.06: 2.07: 2.29: 6.71: 6.97, respectively;

[0011] The acidic homogeneous polysaccharide XNDHP-3 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 5.99: 3.19: 2.87: 1.79: 10.50: 36.14: 39.53; the molecular weight is 392.5 kDa; the total sugar content is 95.02%; the protein content is 1.27%; the uronic acid content is 3.21%; the connection modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked arabinose, 1,5-linked arabinose, terminal-linked The molar ratios of glucuronic acid, glucose, galacturonic acid, 1,3,5-arabinose, 1,4-galactose, 1,4-glucose, 1,3,6-mannose, 1,4,6-mannose, 1,4,6-galactose and 1,3,4,6-galactose are 17.34:4.39:2.21:11.45:1.79:3.68:2.74:9.81:18.62:3.64:4.45:2.97:5.97:11.18 respectively;

[0012] The acidic homogeneous polysaccharide XNDHP-4 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 6.27: 5.97: 2.79: 3.34: 9.58: 32.29: 39.76; the molecular weight is 425.4 kDa; the total sugar content is 94.23%; the protein content is 1.78%; the uronic acid content is 6.21%; the connection modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked arabinose, 1,5- The molar ratios of arabinose, 1,3-linked rhamnose, terminally linked glucuronic acid, terminally linked galacturonic acid, 1,3,5-linked arabinose, 1,4-linked glucose, 1,4-linked galactose, 1,3,6-linked mannose, 1,4,6-linked galactose, and 1,3,4,6-linked galactose are 14.01:2.94:14.10:4.21:3.01:2.84:1.99:6.41:3.67:33.11:5.13:1.87:6.74, respectively.

[0013] The second technical solution of the present invention is to provide a method for preparing Rhododendron dahurica polysaccharide as described in one of the above technical solutions, comprising the following steps:

[0014] S1. Extract the leaves of Rhododendron dahurica with ethanol, filter and dry them in turn. Extract the residue with hot water, filter, concentrate, stand and centrifuge. Centrifuge to obtain the precipitate. Add water to dissolve it and add trichloroacetic acid to centrifuge. Take the supernatant and adjust the pH to neutral. Then concentrate, dialyze and freeze-dry it in turn to obtain 75 alcohol-precipitated polysaccharide XNDCP-75.

[0015] S2, dissolving the 75 alcohol-precipitated polysaccharide XNDCP-75 obtained in step S1 in water, separating it by DEAE-cellulose column chromatography, eluting it with distilled water and gradient concentrations of NaCl solution in turn, collecting each fraction, and obtaining two acidic homogeneous polysaccharides XNDHP-2 and XNDHP-3 according to the ultraviolet detection results showing the reaction of the sugar content of each fraction and the uniformity of the high performance liquid chromatography test, and the remaining components are further purified by Sephacryl TM Two acidic homogeneous polysaccharides XNDHP-1 and XNDHP-4 were obtained by S400 gel chromatography.

[0016] Furthermore, in some specific embodiments, in step S1, the Rhododendron dahurica leaves are extracted by using 95% by mass ethanol.

[0017] Furthermore, in some specific embodiments, in step S1, 95% by mass ethanol is added before standing so that the ethanol concentration of the solution is 75% during standing.

[0018] Furthermore, in some specific embodiments, in step S1, trichloroacetic acid is added so that the concentration of trichloroacetic acid in the solution is 10%.

[0019] Furthermore, in some specific embodiments, in step S2, the gradient concentration range of the NaCl solution is 0.1-1.6 mol / L.

[0020] Furthermore, the gradient concentration of the NaCl solution is 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.8 mol / L, and 1.6 mol / L.

[0021] The third technical solution of the present invention is to provide an application of Rhododendron dahurica polysaccharide as described in one of the above technical solutions, wherein the Rhododendron dahurica polysaccharide is used for preparing anti-complement drugs.

[0022] A fourth technical solution of the present invention is to provide an application of the Rhododendron dahurica polysaccharide as described in one of the above technical solutions, wherein the Rhododendron dahurica polysaccharide is used for preparing anti-inflammatory drugs.

[0023] Furthermore, in some specific embodiments, the Rhododendron dahurica polysaccharide is used to prepare anti-inflammatory drugs for inflammation caused by bacterial infection, viral infection or viral-bacterial co-infection.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention separates 75 alcohol-precipitated polysaccharides XNDCP-75 and four acidic uniform polysaccharides XNDHP-1, XNDHP-2, XNDHP-3 and XNDHP-4 from the leaves of Dahurian rhododendron. Experiments confirm that the Dahurian rhododendron polysaccharides have a significant inhibitory effect on complement activation, can significantly inhibit the inflammatory response of RAW264.7 cells induced by lipopolysaccharide LPS at the cellular level, and have significant anti-inflammatory effects on inflammation in mice induced by LPS, influenza A virus H1N1 or influenza virus-resistant bacteria co-infection in vivo. The polysaccharides can be further used as active ingredients to prepare novel complement inhibitors and therapeutic drugs for inflammation caused by bacteria, viruses or virus-bacteria co-infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the separation flow chart of Rhododendron dahurica polysaccharide.

[0027] Figure 2 The chromatograms of HPGPC of XNDHP-1 (A, B), XNDHP-2 (C, D), XNDHP-3 (E, F), and XNDHP-4 (G, H) were obtained using SHODEX KS-802 (300×7.6 mm) and SHODEX KS-804 (300×7.6 mm) gel columns in series; detectors: evaporative light scattering detector (RID), differential refractometer detector (ELSD); eluent: distilled water; flow rate: 0.6 mL / min.

[0028] Figure 3 This is the effect of Rhododendron australis acidic homogeneous polysaccharides XNDHP-1~4 on the activity of RAW 264.7 cells.

[0029] Figure 4 Effects of XNDHP-1~4 homogeneous polysaccharides from Rhododendron dasyphylla on NO (in the form of NaNO) in RAW 264.7 cells induced by LPS 2 Compared with the LPS group, * P<0.05, ** P<0.01, *** P<0.001.

[0030] Figure 5 This study is to investigate the effects of XNDCP-75, an alcohol-precipitated polysaccharide from Rhododendron dasyphyllum 75, on systemic and pulmonary inflammation in mice induced by intraperitoneal injection of LPS. * P<0.05, ** P<0.01.

[0031] Figure 6 The effect of XNDCP-75, an alcohol-precipitated polysaccharide from Rhododendron dahurica 75, on pneumonia induced by LPS tracheal instillation in mice was investigated. Compared with the LPS group,* P<0.05, ** P<0.01.

[0032] Figure 7 The effect of alcohol-precipitated polysaccharide XNDCP-75 from Rhododendron dasyphylla 75 on pneumonia in mice induced by H1N1 nasal infection was investigated. Compared with the H1N1 group, * P<0.05, ** P<0.01.

[0033] Figure 8 The effect of XNDHP-2, an acidic homogeneous polysaccharide from Rhododendron dasyphyllum, on pneumonia in mice induced by H1N1 nasal infection was investigated. Compared with the H1N1 group, * P<0.05, ** P<0.01.

[0034] Fig. 9 The effect of XNDHP-2, an acidic homogeneous polysaccharide from Rhododendron dasyphyllum, on pneumonia in mice induced by H1N1-MRSA co-infection was shown in Figure 2. Compared with the H1N1-MRSA group, * P<0.05, ** P<0.01. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0036] In the following examples and comparative examples, unless otherwise specified, raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0037] Example 1: Preparation of Rhododendron dahurica polysaccharide

[0038] Figure 1The flow chart for preparing Rhododendron dahurica polysaccharide is as follows: 15 kg of dried Rhododendron dahurica leaves are crushed, extracted with 95% ethanol by mass, filtered, the residue is extracted with hot water for 3 times, concentrated, centrifuged, 95% ethanol by mass is added to the supernatant to 75% ethanol concentration, allowed to stand, centrifuged to remove the supernatant, the precipitate is re-dissolved with water, recovered under reduced pressure, and the ethanol is removed; re-dissolved, trichloroacetic acid is added to a trichloroacetic acid concentration of 10% to remove free protein, centrifuged, the supernatant is adjusted to neutral, dialyzed, concentrated, and freeze-dried to obtain crude polysaccharide XNDCP-75. 214 g of crude polysaccharide is dissolved in distilled water, centrifuged, and the supernatant is separated by DEAE-cellulose column chromatography. Elution was performed with distilled water, 0.1-1.6mol / L (0.1mol / L, 0.2mol / L, 0.4mol / L, 0.8mol / L, 1.6mol / L) NaCl solution, the elution volume was greater than 2 times the column volume (10L), the flow rate was 16mL / min, each fraction was collected, and the absorbance value at 490nm (after sulfuric acid-phenol method color development) was detected by a septum. According to the color reaction of the sugar content and the results of ultraviolet detection, the fractions were combined, concentrated, dialyzed and freeze-dried to obtain 6 secondary components: XND-1, XND-2, XND-3, XND-4, XND-5, and XND-6.

[0039] XND-3 and XND-4 were treated with Sephacryl TM The polysaccharides XNDHP-2 and XNDHP-3 were separated by S400 gel column chromatography and eluted with distilled water. The fractions were combined according to the color reaction of sugar content and the results of ultraviolet detection, and then concentrated, dialyzed and freeze-dried.

[0040] High performance gel permeation chromatography (HPGPC) assay showed that XND-2, XND-5, XNDHP-2, and XNDHP-3 were homogeneous polysaccharides, and XND2 and XND5 were named XNDHP-1 and XNDHP-4, respectively.

[0041] Example 2: Structural Characterization of Rhododendron dahurica Polysaccharide

[0042] (1) Determination of molecular weight

[0043] High performance gel permeation chromatography (HPGPC) is used to determine the relative molecular weight of polysaccharide samples. The basic principle is that uniform polysaccharides pass through gel permeation chromatography to form symmetrical chromatographic peaks. The peak time is related to the molecular weight. The calculation is based on the calibration curve obtained based on the known molecular weight. The results are as follows: Figure 2 As shown, Figure 2 AB is XNDHP-1, Figure 2 CD is XNDHP-2, Figure 2 EF is XNDHP-3, Figure 2GH is the chromatogram of HPGPC of XNDHP-4.

[0044] Chromatographic conditions: SHODEX KS-802 (300×7.6 mm) and SHODEX KS-804 (300×7.6 mm) gel columns were used for separation, with a flow rate of 0.6 mL / min, an injection volume of 20 μL, ultrapure water as the mobile phase, a column temperature of 37°C, and detectors such as differential refractive index detector (RID) and evaporative light scattering detector (ELSD).

[0045] Experimental method: Weigh 2.0 mg of homogeneous polysaccharide and Dextrans series standard products accurately, prepare a 2.0 mg / mL solution with ultrapure water, filter with a 0.45 μm microporous filter membrane before injection, record the retention time, draw a standard curve with the logarithm value (Lg) of the molecular weight of the standard polysaccharide as the ordinate and the retention time as the abscissa, obtain the corresponding linear regression equation, and calculate the relative molecular weight of the homogeneous polysaccharide. The relative molecular weights of XNDHP-1, XNDHP-2, XNDHP-3, and XNDHP-4 are 371.5 kDa, 385.4 kDa, 392.5 kDa, and 425.4 kDa, respectively.

[0046] (2) Determination of total sugar, uronic acid and protein content

[0047] The total sugar content of XNDCP-75 was 57.34% determined by sulfuric acid-phenol method; the total sugar content of XNDHP-1 was 91.15%; the total sugar content of XNDHP-2 was 89.96%; the total sugar content of XNDHP-3 was 95.02%; and the total sugar content of XNDHP-4 was 94.23%.

[0048] The uronic acid content was determined by the m-hydroxybiphenyl method. The uronic acid content of XNDCP-75 was 1.94%; the uronic acid content of XNDHP-1 was 7.12%; the uronic acid content of XNDHP-2 was 5.31%; the uronic acid content of XNDHP-3 was 3.21%; and the uronic acid content of XNDHP-4 was 6.21%.

[0049] The protein content was determined by Coomassie Brilliant Blue method: the protein content of XNDCP-75 was 2.21%; the protein content of XNDHP-1 was 0.97%; the protein content of XNDHP-2 was 1.01%; the protein content of XNDHP-3 was 1.27%; and the protein content of XNDHP-4 was 1.78%.

[0050] (3) Monosaccharide composition analysis

[0051] XNDCP-75, XNDHP-1, XNDHP-2, XNDHP-3 and XNDHP-4 were fully hydrolyzed with 2 mol / L trifluoroacetic acid (TFA) at 110 °C to obtain monosaccharide residue products, which were then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and analyzed by HPLC.

[0052] XNDCP-75 is a polysaccharide composed of seven monosaccharides, and the molar ratio of monosaccharides is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 9.50:4.51:18.78:39.65:4.47:9.40:13.68.

[0053] XNDHP-1 is a polysaccharide composed of seven monosaccharides, and the molar ratio of monosaccharides is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 4.69: 8.46: 4.49: 6.15: 1.93: 38.61: 35.67.

[0054] XNDHP-2 is a polysaccharide composed of seven monosaccharides, and the molar ratio of monosaccharides is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 7.41: 12.29: 2.40: 2.20: 5.97: 28.44: 41.29.

[0055] XNDHP-3 is a polysaccharide composed of seven monosaccharides, and the molar ratio of monosaccharides is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 5.99: 3.19: 2.87: 1.79: 10.50: 36.14: 39.53.

[0056] XNDHP-4 is a polysaccharide composed of seven monosaccharides, and the molar ratio of monosaccharides is mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 6.27: 5.97: 2.79: 3.34: 9.58: 32.29: 39.76.

[0057] (4) Methylation analysis

[0058] The acidic homogeneous polysaccharides of Rhododendron dasyphylla (XNDHP-1, XNDHP-2, XNDHP-3, and XNDHP-4) were subjected to uronic acid reduction using sodium borohydride. The homogeneous polysaccharides before and after reduction were subjected to methylation reaction using the modified Hakomori method. The methylated products were fully hydrolyzed with 2 mol / L TFA and NaBH 4 Reduction and acetic anhydride acetylation were used to prepare partially methylated alditol acetate derivatives, which were then analyzed by GC-MS. The methylation mass spectrometry results of the homogeneous polysaccharide before and after reduction were compared to determine the structures of different monosaccharide units in the polysaccharide.

[0059] The structure of XNDHP-1 contains: terminally linked arabinose, terminally linked rhamnose, 1,5-linked arabinose, terminally linked glucuronic acid, terminally linked glucose, terminally linked galacturonic acid, 1,4-linked galactose, 1,3,6-linked galactose, 1,3,6-linked mannose, and 1,3,4,6-linked galactose, with a molar ratio of 19.87:6.12:13.16:2.11:3.17:4.78:33.02:3.96:6.21:7.60.

[0060] The structure of XNDHP-2 contains: terminally linked arabinose, terminally linked rhamnose, 1,3-linked arabinose, 1,5-linked arabinose, terminally linked glucuronic acid, terminally linked glucose, terminally linked galacturonic acid, 1,3,5-linked arabinose, 1,4-linked galactose, 1,3-linked glucose, 1,3,6-linked galactose, 1,3,6-linked glucose, 1,3,6-linked mannose, 1,4,6-linked mannose, 1,3,4-linked rhamnose, 1,3,4,6-linked galactose, with a molar ratio of 18.96: 3.17: 5.79: 15.48: 2.78: 5.69: 1.96: 6.41: 15.26: 2.53: 1.87: 2.06: 2.07: 2.29: 6.71: 6.97.

[0061] The structure of XNDHP-3 contains: terminally linked arabinose, terminally linked rhamnose, 1,3-linked arabinose, 1,5-linked arabinose, terminally linked glucuronic acid, terminally linked glucose, terminally linked galacturonic acid, 1,3,5-linked arabinose, 1,4-linked galactose, 1,4-linked glucose, 1,3,6-linked mannose, 1,4,6-linked mannose, 1,4,6-linked galactose, 1,3,4,6-linked galactose, with a molar ratio of 17.34::4.39:2.21:11.45:1.79:3.68:2.74:9.81:18.62:3.64:4.45:2.97:5.97:11.18.

[0062] The structure of XNDHP-4 contains: terminally linked arabinose, terminally linked rhamnose, 1,3-linked arabinose, 1,5-linked arabinose, 1,3-linked rhamnose, terminally linked glucuronic acid, terminally linked galacturonic acid, 1,3,5-linked arabinose, 1,4-linked glucose, 1,4-linked galactose, 1,3,6-linked mannose, 1,4,6-linked galactose, and 1,3,4,6-linked galactose, with a molar ratio of 14.01:2.94:14.10:4.21:3.01:2.84:1.99:6.41:3.67:33.11:5.13:1.87:6.74.

[0063] Example 3: Classical pathway complement inhibition assay

[0064] Serum from 3-month-old guinea pigs was diluted to 1:100 with BBS buffer (barbital buffer, pH=7.4) as the source of complement for this classical pathway. Rabbit anti-sheep erythrocyte antibody was diluted to 1:1000 with BBS buffer as hemolysin; sheep erythrocytes (SRBC) were configured to 2% SRBC. 3 mg of Rhododendron dasyphyllum polysaccharide was accurately weighed, dissolved in BBS buffer, and diluted to 8 concentrations. 200 μL of Rhododendron dasyphyllum polysaccharide solution of different concentrations was pre-incubated with 200 μL of complement diluted to 1:100 at 37°C for 10 minutes, and then 100 μL of hemolysin (1:1000) and 100 μL of 2% SRBC were added in sequence. After being placed in a 37°C water bath for 30 minutes, the mixture was placed in a low-temperature high-speed centrifuge and centrifuged at 5000 rpm and 4°C for 10 minutes. 200 μL of supernatant was taken from each tube and placed in a 96-well plate, and the absorbance was measured at 405 nm. The experiment also set up a Rhododendron dahurica polysaccharide control group (200 μL of the corresponding concentration of Rhododendron dahurica polysaccharide plus 400 μL BBS buffer), a complement control group (200 μL BBS buffer instead of Rhododendron dahurica polysaccharide) and a full hemolysis group (100 μL 2% SRBC dissolved in 500 μL distilled water). The hemolysis inhibition rate was calculated by deducting the absorbance value of the Rhododendron dahurica polysaccharide group of each concentration from the absorbance value of the corresponding Rhododendron dahurica polysaccharide control group. The logarithm of the Rhododendron dahurica polysaccharide concentration was used as the X-axis and the hemolysis inhibition rate was used as the Y-axis to plot the obtained fitting curve to calculate the concentration of the test sample required for 50% inhibition of hemolysis (CH 50 Value). With heparin as the positive control drug, the results showed that 75 alcohol-precipitated polysaccharide and 4 homogeneous polysaccharides all had significant inhibitory activity on the activation of the classical complement pathway, that is, they had obvious anti-complement effects, CH 50 The values, i.e., the concentrations of the test substances required for 50% inhibition of hemolysis by the classical pathway, were 50±6μg / mL, 41±7μg / mL, 25±2μg / mL, 26±5μg / mL, and 76±4μg / mL, as shown in Table 1.

[0065] Table 1 Inhibitory effect of Rhododendron dahurica polysaccharide on complement activation

[0066]

[0067] CH 50 Values ​​are expressed as mean ± SD (n = 3); positive control heparin CH 50 :49±3μg / mL.

[0068] Example 4: Effects of Rhododendron australis acidic homogeneous polysaccharides XNDHP-1-4 on lipopolysaccharide (LPS)-induced RAW264.7 cells

[0069] RAW264.7 cells were cultured in DMEM medium mixed with 10% fetal bovine serum, 100 units / mL penicillin, and 100 mg / mL streptomycin. 5 The cells were inoculated at a concentration of 10 cells / well in a 96-well plate and cultured at 37°C in a 5% carbon dioxide atmosphere.

[0070] (1) Effects of Rhododendron australis acidic homogeneous polysaccharides XNDHP-1-4 on RAW264.7 cell viability

[0071] RAW264.7 cells seeded in 96-well plates were divided into 28 groups, each with 6 wells, including two normal control groups, two LPS groups (1 μg / mL), XNDHP-1~4 low-, medium-, and high-dose groups (62.5, 250, and 1000 μg / mL), and XNDHP-1~4 low-, medium-, and high-dose LPS groups (containing 62.5, 250, and 1000 μg / mL of acidic homogeneous polysaccharide, respectively, and 1 μg / mL of LPS). After the cells were inoculated and cultured for 24 hours, 100 μL of XTT reagent was added to each well and cultured for 2 hours. The absorbance was detected at 450 nm. The cell activity of the XNDHP-1~4 low, medium and high dose groups was obtained by comparing the absorbance values ​​of the XNDHP-1~4 low, medium and high dose groups with the absorbance values ​​of the normal group, and expressed as a percentage; the cell activity of the XNDHP-1~4 low, medium and high dose LPS groups was compared with the absorbance values ​​of the LPS group using the same method and expressed as a percentage.

[0072] like Figure 3 As shown, compared with the normal group, the activity of RAW264.7 cells in the low, medium and high dose groups of XNDHP-1-4 was higher than 90%; compared with the LPS group, the activity of RAW264.7 cells in the low, medium and high dose groups of XNDHP-1-4 was higher than 90%. The above results show that XNDHP-1-4 has no effect on the activity of RAW264.7 cells.

[0073] (2) Effects of Azalea dahurica acidic homogeneous polysaccharides XNDHP-1-4 on LPS-induced NO in RAW264.7 cell culture medium

[0074] RAW264.7 cells seeded in 96-well plates were divided into 15 groups, each with 6 wells, including normal control group, LPS group (1 μg / mL), positive control dexamethasone group (DXMS 60 μg / mL, LPS 1 μg / mL), XNDHP-1-4 low-, medium-, and high-dose LPS groups (containing acidic homogeneous polysaccharide 62.5, 250, and 1000 μg / mL, respectively, and LPS 1 μg / mL). After 24 hours of cell inoculation, the supernatant was taken and the NO content in the culture medium was determined by the Griess reaction method. The supernatant was determined according to the instructions of the NO kit, and NaNO 2 Concentration meter.

[0075] The results are as follows Figure 4 A-1, Figure 4 B-1, Figure 4 C-1, Figure 4 As shown in D-1, compared with the normal group, the NaNO 2 concentration was significantly increased (P<0.001); compared with the LPS group, the NaNO 2 concentrations were significantly decreased (P<0.001, P<0.001); compared with the LPS group, the NaNO 2 concentrations were significantly decreased (P<0.01, P<0.001, P<0.001); compared with the LPS group, the NaNO 2 concentration was significantly decreased (P<0.001); compared with the LPS group, the NaNO 2 The concentrations of both drugs were significantly decreased (P<0.01, P<0.001).

[0076] (3) Effects of Rhododendron australis acidic homogeneous polysaccharides XNDHP-1-4 on TNF-α, IL-6, and IL-1β in the culture medium of RAW264.7 cells induced by LPS

[0077] RAW264.7 cells seeded in 96-well plates were divided into 15 groups, each with 6 wells, including normal control group, LPS group (1 μg / mL), positive control dexamethasone group (DXMS 60 μg / mL, LPS 1 μg / mL), XNDHP-1-4 low-, medium-, and high-dose LPS groups (containing acidic homogeneous polysaccharide 62.5, 250, 1000 μg / mL, containing LPS 1 μg / mL, respectively). After 24 hours of cell inoculation, the supernatant was taken and the TNF-α, IL-6, and IL-1β contents in the culture medium were determined by Elisa method, and the supernatant was determined according to the instructions of TNF-α, IL-6, and IL-1β kits.

[0078] TNF-α content Figure 4 A-2, Figure 4 B-2, Figure 4 C-2, Figure 4 As shown in D-2, compared with the normal group, the TNF-α concentrations in the culture medium of the low, medium and high doses of LPS groups of XNDHP-1 to 4 were significantly increased (P<0.001); compared with the LPS group, the TNF-α concentrations in the culture medium of the medium and high doses of LPS groups of XNDHP-1 were significantly decreased (P<0.01, P<0.001); compared with the LPS group, the TNF-α concentrations in the culture medium of the low, medium and high doses of LPS groups of XNDHP-2 were significantly decreased (P<0.001, P<0.001, P<0.001); compared with the LPS group, the TNF-α concentration in the culture medium of the high dose of LPS group of XNDHP-3 was significantly decreased (P<0.01); compared with the LPS group, the TNF-α concentrations in the culture medium of the medium and high doses of LPS groups of XNDHP-4 were significantly decreased (P<0.01, P<0.001).

[0079] IL-6 content Figure 4 A-3, Figure 4 B-3, Figure 4 C-3, Figure 4 As shown in D-3, compared with the normal group, the IL-6 concentrations in the culture medium of the low, medium and high dose LPS groups of XNDHP-1~4 were significantly increased (P<0.001); compared with the LPS group, the IL-6 concentrations in the culture medium of the medium and high dose LPS groups of XNDHP-1 were significantly decreased (P<0.001, P<0.001); compared with the LPS group, the IL-6 concentrations in the culture medium of the low, medium and high dose LPS groups of XNDHP-2 were significantly decreased (P<0.01, P<0.001, P<0.001); compared with the LPS group, the IL-6 concentrations in the culture medium of the medium and high dose groups of XNDHP-3 were significantly decreased (P<0.01, P<0.001); compared with the LPS group, the IL-6 concentrations in the culture medium of the medium and high dose groups of XNDHP-4 were significantly decreased (P<0.05, P<0.001).

[0080] IL-1β content Figure 4 A-4, Figure 4 B-4, Figure 4 C-4, Figure 4 As shown in D-4, compared with the normal group, the IL-1β concentration in the culture medium of the low, medium and high doses of LPS groups of XNDHP-1 to 4 was significantly increased (P<0.001); compared with the LPS group, the IL-1β concentration in the culture medium of the high dose of LPS group of XNDHP-1 was significantly decreased (P<0.01); compared with the LPS group, the IL-1β concentration in the culture medium of the low, medium and high doses of LPS groups of XNDHP-2 was significantly decreased (P<0.05, P<0.05, P<0.001); compared with the LPS group, the IL-1β concentration in the culture medium of the high dose of LPS group of XNDHP-3 was significantly decreased (P<0.001); compared with the LPS group, the IL-1β concentration in the culture medium of the high dose of LPS group of XNDHP-4 was significantly decreased (P<0.05).

[0081] The above four Rhododendron australis acidic homogeneous polysaccharides XNDHP-1, XNDHP-2, XNDHP-3 and XNDHP-4 have a significant inhibitory effect on the secretion of inflammatory factors by RAW264.7 cells induced by LPS, and have significant anti-inflammatory activity.

[0082] Example 5: Effects of XNDCP-75, an alcohol-precipitated polysaccharide from Rhododendron dasyphyllum 75, on systemic and pulmonary inflammation in mice induced by intraperitoneal injection of LPS

[0083] This experiment included 5 groups, namely NC normal control group, LPS model group, XNDCP-75 (50mg / kg) group, XNDCP-75 (100mg / kg) group and positive drug dexamethasone DXMS group (3mg / kg). Balb / C male mice weighing 18-20g were gavaged according to body weight, and the normal control group and LPS model group were given double distilled water. On the second day of the experiment, mice in each group were also gavaged, and 10mg / kg LPS solution was injected intraperitoneally 1h after gavage to establish the model. The animals were killed 6h after modeling and the tissues were collected. First, mouse blood was obtained by removing the eyeballs and collecting blood in a centrifuge tube. After centrifugation at 3000rpm for 10min, mouse serum was obtained, which was aliquoted and frozen in a -80℃ refrigerator. The mice were killed by dislocating the cervical vertebra, and then the skin of the upper body of the mice was cut open to expose the trachea. 1mL PBS solution was used to lavage the lung tissue through the trachea, and the flushing was repeated 3 times. The lavage fluid was centrifuged at 3000 rpm for 10 min, and the obtained supernatant was aliquoted and stored in a -80°C refrigerator.

[0084] The levels of IL-6 and TNF-α in mouse serum and bronchoalveolar lavage fluid were determined by ELISA. The results showed that XNDCP-75 (100 mg / kg) significantly reduced the levels of IL-6 in mouse serum and bronchoalveolar lavage fluid (such as Figure 5 A, 5C) and serum TNF-α levels (such as Figure 5 B) (P<0.01), XNDCP-75 (50 mg / kg) significantly reduced the level of IL-6 in the bronchoalveolar lavage fluid of mice (e.g. Figure 5 C) and serum TNF-α levels (such as Figure 5 B) (P<0.05). The above results showed that XNDCP-75 significantly reduced LPS-induced systemic and pulmonary inflammation, had significant anti-inflammatory effects in vivo, and significantly reduced the levels of IL-6 and TNF-α in mouse serum and the level of IL-6 in bronchoalveolar lavage fluid.

[0085] Example 6: Effect of XNDCP-75, an alcohol-precipitated polysaccharide from Rhododendron dasyphyllum 75, on pneumonia in mice induced by tracheal instillation of LPS

[0086] This experiment included 4 groups, namely, sham operation group, LPS model group, XNDCP-75 (200mg / kg) group and positive drug dexamethasone DXMS group (4mg / kg). LPS solution was slowly injected into the body of 18-20g Balb / C male mice through a microsyringe. Immediately after suturing, the mice were rotated in four directions, front, back, left and right, so that the LPS solution was evenly distributed in the lungs. The dosage of LPS solution was 50μL / 20g body weight. After the operation was completed, the mice were immediately placed on a warm cotton pad to keep warm and wait for the mice to wake up. Drugs were administered 2h after modeling. The XNDCP-75 group was gavaged and the DXMS group was injected into the tail vein. After drug administration, the mice were given water and food normally and continued to be put back into the cage for feeding. The mice were sampled 24h after modeling. First, the mouse blood was obtained by removing the eyeball and collected in a centrifuge tube. After centrifugation at 3000rpm for 10min, the mouse serum was obtained and stored in a -80℃ refrigerator after aliquoting. The mice were killed by dislocating the cervical vertebrae, and then the skin of the upper body of the mice was cut open to expose the trachea and lungs. The right main bronchus of the mice was ligated to prevent the lavage fluid from flowing into the right lung during subsequent lavage. The left lung of the mouse was lavaged through the trachea, 0.4 mL each time, lavaged 4 times, and the lavage fluid was collected separately. The fresh upper lobe of the right lung of the mouse was stored in 10% formalin solution, and the middle lobe, lower lobe and lobule of the right lung were collected separately and stored in a -80℃ refrigerator.

[0087] The protein content in mouse bronchoalveolar lavage fluid was determined using a BCA kit. Figure 6As shown in A, the results showed that XNDCP-75 (200 mg / kg) significantly reduced the protein content in the alveolar lavage fluid of mice (P<0.05), indicating that XNDCP-75 significantly alleviated the inflammatory damage of the lung tissue of mice induced by LPS tracheal instillation.

[0088] The number of white blood cells in the bronchoalveolar lavage fluid of mice can be counted by staining the cell pellet in the bronchoalveolar lavage fluid with gentian violet stain and observing it under a microscope. Figure 6 As shown in B, the results showed that XNDCP-75 (200 mg / kg) significantly reduced the number of leukocytes in the alveolar lavage fluid of mice (P<0.01), indicating that XNDCP-75 significantly alleviated LPS tracheal instillation-induced lung inflammation in mice.

[0089] The levels of proinflammatory cytokines TNF-α and IL-6 in the bronchoalveolar lavage fluid of mice were tested using ELISA kits. Figure 6 As shown in CD, the results showed that XNDCP-75 (200 mg / kg) significantly reduced the levels of IL-6 and TNF-α (P<0.05) in bronchoalveolar lavage fluid, indicating that XNDCP-75 significantly alleviated LPS tracheal instillation-induced lung inflammation in mice.

[0090] XNDCP-75, an alcohol-precipitated polysaccharide from Rhododendron dahurica 75, has a significant anti-inflammatory effect in vivo, significantly reducing the protein content, white blood cell count and the levels of proinflammatory cytokines TNF-α and IL-6 in the bronchoalveolar lavage fluid of mice.

[0091] Example 7: Effects of Rhododendron dasyphylla polysaccharides XNDCP-75 and XNDHP-2 on pneumonia in mice induced by nasal infection with influenza A virus H1N1

[0092] After 1 day of feeding, the mice were anesthetized by inhalation of isoflurane. After the mice had no spontaneous activity, they were taken out of the anesthesia box, 30 μL of H1N1 virus solution was quickly administered by nasal drops, and the mice were immediately placed in a mouse cage to wait for awakening and normal access to water and food. The day when the mice were infected with the virus was counted as Day 0. The drug was administered from Day 0 and continued for 4 consecutive days, and the weight of the mice was recorded every day until Day 4. In this experiment, the drug administration method was gavage, and each drug group was given the corresponding drug by gavage. The normal control group and the H1N1 model group were given double distilled water. All mice were sampled on Day 4. Before sampling, the weight of the mice was first weighed and recorded. When sampling, the skin of the upper body and the chest cavity of the mice were cut open in turn to expose the lungs. Pinch the mouse trachea with forceps and remove the mouse lung tissue together with the heart. After removal, cut out the excess tissue such as the heart and trachea, and slightly wash the mouse lung tissue in ice PBS solution. After washing, the water was dried on filter paper, the weight of the whole lung was weighed and recorded, the upper lobe of the right lung was stored in formalin solution, and the rest of the lung tissue was stored in a -80°C refrigerator.

[0093] (1) Effect of XNDCP-75, an alcohol-precipitated polysaccharide from Rhododendron dasyphyllum 75, on pneumonia in mice induced by H1N1 nasal infection

[0094] Thirty Balb / C male mice weighing 14-16 g were randomly divided into five groups according to their body weight, namely, NC normal control group, H1N1 model group, XNDCP-75 (50 mg / kg) group, XNDCP-75 (100 mg / kg) group and positive drug oseltamivir (OST) group (20 mg / kg).

[0095] Compare the weight of each group of mice relative to their initial weight percentage, such as Figure 7 As shown in B, it was found that the body weight of mice in the H1N1 model group was the lowest, and XNDCP-75 (50, 100 mg / kg) increased the body weight of mice, indicating that XNDCP-75 protected the overall state of mice with H1N1-induced pneumonia.

[0096] The mouse lung index was calculated by lung weight (mg) / body weight (g). Figure 7 As shown in A, the results showed that XNDCP-75 (50, 100 mg / kg) significantly reduced the lung index of mice (P<0.05), indicating that XNDCP-75 significantly alleviated H1N1-induced inflammatory damage to the lung tissue of mice.

[0097] (2) Effect of XNDHP-2, an acidic homogeneous polysaccharide from Rhododendron dasyphyllum, on pneumonia in mice induced by H1N1 nasal infection

[0098] Thirty-six Balb / C male mice weighing 14-16 g were randomly divided into 6 groups according to their body weight, namely, NC normal control group, H1N1 model group, XNDHP-2 (5 mg / kg) group, XNDHP-2 (10 mg / kg) group, XNDHP-2 (20 mg / kg) group and positive drug oseltamivir (OST) group (20 mg / kg).

[0099] Compare the weight of each group of mice relative to their initial weight percentage, such as Figure 8 As shown in C, compared with the H1N1 model group, the body weight of mice in the XNDHP-2 (10, 20 mg / kg) groups increased, indicating that XNDHP-2 protected the overall status of mice with H1N1-induced pneumonia.

[0100] The mouse lung index was calculated by lung weight (mg) / body weight (g). Figure 8 As shown in B, the results showed that XNDHP-2 (10, 20 mg / kg) significantly reduced the lung index of mice (P<0.05), indicating that XNDHP-2 significantly alleviated H1N1-induced inflammatory damage to the lung tissue of mice.

[0101] The right upper lobe of the mouse lung was fixed in formalin solution, embedded, sliced, and stained with H&E, and the morphology of the lung tissue of each group of mice was observed under a microscope. Figure 8 As shown in A, the results showed that the lung tissues of mice in the H1N1 model group showed obvious collapse, edema, inflammatory cell infiltration and other pathological manifestations, while XNDHP-2 (10, 20 mg / kg) significantly reduced the above-mentioned inflammatory pathological damage to the lung tissues.

[0102] In addition, the levels of proinflammatory cytokines TNF-α and IL-6 in mouse lung tissue homogenates were measured by ELISA. Figure 8 As shown in DE, the results showed that XNDHP-2 (10, 20 mg / kg) significantly reduced the levels of TNF-α and IL-6 (P<0.05) in mouse lung homogenate, indicating that XNDHP-2 significantly alleviated H1N1-induced lung inflammation in mice.

[0103] The polysaccharides XNDCP-75 and XNDHP-2 from Rhododendron dasyphyllum have significant anti-inflammatory effects in vivo. XNDCP-75 and XNDHP-2 protect the overall state of mice with H1N1-induced pneumonia, reduce the weight loss of mice, and reduce the lung index. XNDHP-2 also significantly reduces H1N1-induced inflammatory damage in the lung tissue of mice and reduces the levels of proinflammatory cytokines IL-6 and TNF-α in the homogenate of mouse lung tissue.

[0104] Example 8: Effect of Rhododendron australis acidic homogeneous polysaccharide XNDHP-2 on pneumonia in mice induced by co-infection with H1N1-MRSA (influenza virus H1N1-methicillin-resistant Staphylococcus aureus MRSA)

[0105] Thirty-six 14-16g C57BL / 6 female mice were randomly divided into 6 groups according to their body weight, namely, NC normal control group, H1N1-MRSA model group, XNDHP-2 (5mg / kg) group, XNDHP-2 (10mg / kg) group, XNDHP-2 (20mg / kg) group and positive drug oseltamivir-linezolid (OST-LINE) group (20mg / kg). During modeling, the mice were anesthetized and quickly given 30μL H1N1 virus solution through nasal drops. The mice were placed in a mouse cage and waited to wake up and get water and food normally. The day when the mice were infected with the virus was counted as Day 0. On Day 3, 40μL MRSA bacterial solution was injected into the nose again. The drug was administered continuously for 5 days starting from Day 0, and the weight and death of the mice were recorded every day until Day 5. In this experiment, the drug administration method was oral gavage. Each drug administration group was given the corresponding drug by oral gavage, and the normal control group and the H1N1 model group were given double distilled water. All mice were sampled on Day 5. The weight of the mice was first weighed and recorded before sampling. When sampling, the skin of the upper body and the chest cavity of the mice were cut in turn to expose the lungs. Pinch the mouse trachea with tweezers and remove the mouse lung tissue together with the heart. After removal, cut out the excess tissue such as the heart and trachea, and slightly wash the mouse lung tissue in ice PBS solution. After washing, dry the water on filter paper, weigh the whole lung weight and record it, the upper lobe of the right lung was stored in formalin solution, and the rest of the lung tissue was stored in a -80℃ refrigerator.

[0106] Compare the weight of each group of mice relative to their initial weight percentage, such as Fig. 9 As shown in B, compared with the H1N1-MRSA model group, the body weight of mice in the XNDHP-2 (5, 10 mg / kg) groups increased, indicating that XNDHP-2 protected the overall status of mice with H1N1-MRSA-induced pneumonia.

[0107] The mouse lung index was calculated by lung weight (mg) / body weight (g). Fig. 9 As shown in A, the results showed that compared with the H1N1-MRSA model group, XNDHP-2 (10, 20 mg / kg) significantly reduced the lung index of mice (P<0.05), indicating that XNDHP-2 significantly alleviated H1N1-MRSA-induced inflammatory damage to mouse lung tissue.

[0108] The acidic homogeneous polysaccharide XNDHP-2 from Rhododendron dahurica has a significant anti-inflammatory effect in vivo, significantly reducing H1N1-MRSA-induced inflammatory damage in the lung tissue of mice, alleviating the weight loss of mice, and significantly reducing the lung index.

[0109] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. 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 work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A Rhododendron dasyphylla polysaccharide, characterized in that: Including 75 alcohol precipitated polysaccharide XNDCP-75 and acidic uniform polysaccharides XNDHP-1, XNDHP-2, XNDHP-3, XNDHP-4, their structural characteristics are: 75 alcohol precipitated polysaccharide XNDCP-75 is composed of monosaccharides in the following molar ratios: Composition: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 9.50: 4.51: 18.78: 39.65: 4.47: 9.40: 13.68; molecular weight range 279.6-871.7 kDa; total sugar content 57.34%; protein content 2.21%; uronic acid content 1.94%; The acidic homogeneous polysaccharide XNDHP-1 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 4.69: 8.46: 4.49: 6.15: 1.93: 38.61: 35.67; the molecular weight is 371.5 kDa; the total sugar content is 91.15%; the protein content is 0.97%; the uronic acid content is 7.12%; the connection method includes terminal connection with arabinose sugars, terminally linked rhamnose, 1,5-linked arabinose, terminally linked glucuronic acid, terminally linked glucose, terminally linked galacturonic acid, 1,4-linked galactose, 1,3,6-linked galactose, 1,3,6-linked mannose, 1,3,4,6-linked galactose, with molar ratios of 19.87:6.12:13.16:2.11:3.17:4.78:33.02:3.96:6.21:7.60, respectively; The acidic homogeneous polysaccharide XNDHP-2 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 7.41: 12.29: 2.40: 2.20: 5.97: 28.44: 41.29; the molecular weight is 385.4 kDa; the total sugar content is 89.96%; the protein content is 1.01%; the uronic acid content is 5.31%; the connection modes include terminal connection arabinose, terminal connection rhamnose, 1,3-connected arabinose, 1,5-connected arabinose, terminal connection glucuronic acid, terminal connection glucose , terminally linked to galacturonic acid, 1,3,5-linked to arabinose, 1,4-linked to galactose, 1,3-linked to glucose, 1,3,6-linked to galactose, 1,3,6-linked to glucose, 1,3,6-linked to mannose, 1,4,6-linked to mannose, 1,3,4-linked to rhamnose, 1,3,4,6-linked to galactose, with molar ratios of 18.96: 3.17: 5.79: 15.48: 2.78: 5.69: 1.96: 6.41: 15.26: 2.53: 1.87: 2.06: 2.07: 2.29: 6.71: 6.97, respectively; The acidic homogeneous polysaccharide XNDHP-3 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 5.99: 3.19: 2.87: 1.79: 10.50: 36.14: 39.53; the molecular weight is 392.5 kDa; the total sugar content is 95.02%; the protein content is 1.27%; the uronic acid content is 3.21%; the connection modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked arabinose, 1,5-linked arabinose, terminal-linked The molar ratios of glucuronic acid, glucose, galacturonic acid, 1,3,5-arabinose, 1,4-galactose, 1,4-glucose, 1,3,6-mannose, 1,4,6-mannose, 1,4,6-galactose and 1,3,4,6-galactose are 17.34:4.39:2.21:11.45:1.79:3.68:2.74:9.81:18.62:3.64:4.45:2.97:5.97:11.18 respectively; The acidic homogeneous polysaccharide XNDHP-4 is composed of monosaccharides in the following molar ratios: mannose: rhamnose: glucuronic acid: galacturonic acid: glucose: galactose: arabinose = 6.27: 5.97: 2.79: 3.34: 9.58: 32.29: 39.76; the molecular weight is 425.4 kDa; the total sugar content is 94.23%; the protein content is 1.78%; the uronic acid content is 6.21%; the connection modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked arabinose, 1,5- The molar ratios of arabinose, 1,3-linked rhamnose, terminally linked glucuronic acid, terminally linked galacturonic acid, 1,3,5-linked arabinose, 1,4-linked glucose, 1,4-linked galactose, 1,3,6-linked mannose, 1,4,6-linked galactose, and 1,3,4,6-linked galactose are 14.01:2.94:14.10:4.21:3.01:2.84:1.99:6.41:3.67:33.11:5.13:1.87:6.74, respectively.

2. A method for preparing Rhododendron dahurica polysaccharide according to claim 1, characterized in that: The steps include: S1. Extract the leaves of Rhododendron dahurica with ethanol, filter and dry them in turn. Extract the residue with hot water, filter, concentrate, stand and centrifuge. Centrifuge to obtain the precipitate. Add water to dissolve it and add trichloroacetic acid to centrifuge. Take the supernatant and adjust the pH to neutral. Then concentrate, dialyze and freeze-dry it in turn to obtain 75 alcohol-precipitated polysaccharide XNDCP-75. S2, dissolving the 75 alcohol-precipitated polysaccharide XNDCP-75 obtained in step S1 in water, separating it by DEAE-cellulose column chromatography, eluting it with distilled water and gradient concentrations of NaCl solution in turn, collecting each fraction, and obtaining two acidic homogeneous polysaccharides XNDHP-2 and XNDHP-3 according to the ultraviolet detection results showing the reaction of the sugar content of each fraction and the uniformity of the high performance liquid chromatography test, and the remaining components are further purified by Sephacryl TM Two acidic homogeneous polysaccharides XNDHP-1 and XNDHP-4 were obtained by S400 gel chromatography.

3. The method for preparing Rhododendron dahurica polysaccharide according to claim 2, characterized in that: In step S1, the Rhododendron dahurica leaves are extracted by using 95% ethanol.

4. The method for preparing Rhododendron dahurica polysaccharide according to claim 2, characterized in that: In step S1, 95% by mass ethanol is added before standing so that the ethanol concentration of the solution is 75% during standing.

5. The method for preparing Rhododendron dahurica polysaccharide according to claim 2, characterized in that: In step S1, trichloroacetic acid is added so that the concentration of trichloroacetic acid in the solution is 10%.

6. The method for preparing Rhododendron dahurica polysaccharide according to claim 2, characterized in that: In step S2, the gradient concentration range of the NaCl solution is 0.1-1.6 mol / L.

7. The method for preparing Rhododendron dahurica polysaccharide according to claim 6, characterized in that: The gradient concentration of NaCl solution is 0.1mol / L, 0.2mol / L, 0.4mol / L, 0.8mol / L, and 1.6mol / L.

8. A use of the Rhododendron dahurica polysaccharide as claimed in claim 1, characterized in that: The Rhododendron dahurica polysaccharide is used for preparing anti-complement drugs.

9. A use of the Rhododendron dahurica polysaccharide as claimed in claim 1, characterized in that: The Rhododendron dahurica polysaccharide is used for preparing anti-inflammatory drugs.

10. The use of Rhododendron dahurica polysaccharide according to claim 9, characterized in that: The Rhododendron dahurica polysaccharide is used for preparing anti-inflammatory drugs for inflammation caused by bacterial infection, viral infection or viral-bacterial co-infection.

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