Polysaccharides from Rhododendron principis Bur. var. omeiense (Balf. f. et W. W. Smith) Tagg and Its Preparation Method and Application

Abstract

The present invention relates to the polysaccharides of Rhododendron principis Bur. and their preparation methods and applications. The polysaccharides of Rhododendron principis Bur. include the 90% ethanol precipitation polysaccharide ZNDCP-90 of Rhododendron principis Bur. and four homogeneous polysaccharides ZNDHP-1, ZNDHP-2, ZNDHP-3, and ZNDHP-4. It has been experimentally confirmed that the polysaccharides of Rhododendron principis Bur. all have significant inhibitory effects on complement activation and can be further used as active ingredients to prepare novel complement inhibitors; it has been confirmed by cell model experiments that ZNDCP-90, ZNDHP-2, ZNDHP-3, and ZNDHP-4 can significantly inhibit the inflammatory response of LPS-induced RAW264.7 cells, have anti-inflammatory activity, and can be further used as active ingredients to prepare anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, relates to polysaccharides, and in particular to polysaccharides from Rhododendron principis Bur. and their preparation methods and applications. Background Art

[0002] The complement system is an important part of the human immune system. However, when the complement system is over-activated, it will cause an over-reaction of the human immune system, resulting in damage to the body's own normal tissues. For example, in the pathogenesis of rheumatoid arthritis, Alzheimer's disease, systemic lupus erythematosus (SLE), as well as acute diseases such as ischemic reperfusion, acute myocardial infarction, acute respiratory distress syndrome (ARDS), and multiple organ failure syndrome, the over-activation of complement plays an important role. Currently, immunosuppressants such as glucocorticoids used clinically are not specific complement inhibitors and are prone to produce various complications and side effects. Therefore, there is an urgent need for a new type of highly efficient, low-toxic, and specific complement inhibitor clinically.

[0003] The inflammatory reaction is a highly complex host pathophysiological reaction process, which is a natural defense reaction aimed at clearing pathogens and thus reducing tissue damage. However, the acute inflammatory reaction will trigger a large area of neutrophil infiltration in a short time, forming a local inflammation center, resulting in varying degrees of tissue damage. Systemic inflammatory response syndrome (SIRS) is a systemic inflammatory reaction induced by factors such as infection, trauma, and ischemic reperfusion. It is mostly manifested as neutrophil inflammatory infiltration caused by factors such as over-activation of the complement system and excessive oxidative stress, forming a cytokine storm. The excessive release of inflammatory cytokines acting on endothelial cells will affect the microcirculation in organs, further leading to multiple organ dysfunction syndrome, and ultimately resulting in the death of the patient (Bone RC. Jama, 1992; 268: 3452-3455). The complement system is closely related to the inflammatory reaction. The over-activation of the complement system is one of the important symptoms and inducements of systemic inflammatory response syndrome. Therefore, complement inhibitors can be used as potential drugs for treating inflammation.

[0004] Since complement inhibition is one of the potential therapeutic targets for inflammation, it is of great significance to develop highly effective and low-toxic complement inhibitors into 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). Due to factors such as altitude and location, a unique Tibetan medicine system has been formed in the Tibet region. Many Tibetan medicines have obvious regulatory effects on the immune system and are valuable resources for searching for complement inhibitors and anti-inflammatory drug precursors.

[0005] Rhododendron principis Bur. et Franch. is an evergreen small tree of the genus Rhododendron in the family Ericaceae and is widely distributed in eastern and southern Tibet. In traditional Tibetan medicine, the branches and leaves of Rhododendron principis are used as "Dama", which are recorded in "Jingzhu Materia Medica" and have the effect of drying body pus and blood, and are used to treat pneumonia and other inflammation-related diseases. However, at present, there are no reports on the structure, anti-complement activity and anti-inflammatory activity of the polysaccharides from Rhododendron principis. Summary of the Invention

[0006] Based on the current situation of the existing technology, the object of the present invention is to provide components with anti-complement activity and anti-inflammatory activity, specifically related to the polysaccharides from Rhododendron principis and their preparation methods and applications, especially the 90% ethanol-precipitated polysaccharides from Rhododendron principis (ZNDCP-90) and four homogeneous polysaccharides from Rhododendron principis (ZNDHP-1, ZNDHP-2, ZNDHP-3, ZNDHP-4) and their preparation methods and applications in the preparation of complement inhibitor drugs and anti-inflammatory drugs.

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

[0008] One of the technical solutions of the present invention is to provide the polysaccharides from Rhododendron principis, including the 90% ethanol-precipitated polysaccharides from Rhododendron principis ZNDCP-90 and four homogeneous polysaccharides from Rhododendron principis ZNDHP-1, ZNDHP-2, ZNDHP-3, ZNDHP-4, which have the following structural characteristics respectively:

[0009] The 90% ethanol-precipitated polysaccharides from Rhododendron principis ZNDCP-90: a polysaccharide composed of seven monosaccharides, with a molecular weight range of 353.2-2208.5 kDa; the total sugar content is 64.31%, the protein content is 1.94%, and the uronic acid content is 1.31%; the molar ratio of monosaccharides mannose: rhamnose: glucose: galacturonic acid: galactose: xylose: arabinose = 11.22: 2.20: 16.22: 24.33: 9.66: 24.59: 11.78;

[0010] Homogeneous polysaccharide ZNDHP-1 from Rhododendron principis Bur. var. farrerae (Tagg) Chamberlain: A polysaccharide composed of five monosaccharides, with a molecular weight of 633.8 kDa; the total sugar content is 91.69%, the protein content is 1.01%, and the uronic acid content is 1.68%; the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 9.85:9.05:56.02:15.99:9.10; the linkage modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked rhamnose, terminal-linked glucose, terminal-linked galactose, 1,5-linked arabinose, 1,3-linked glucose, 1,4-linked glucose, 1,4-linked galactose, 1,4-linked mannose, 1,3,5-linked arabinose, 1,2,6-linked glucose, 1,2,4-linked glucose, 1,4,6-linked galactose, 1,4,6-linked mannose, 1,3,6-linked glucose, 1,3,6-linked mannose, and the molar ratios are 2.21:6.03:5.02:17.59:2.58:2.21:2.04:25.74:1.98:2.97:5.69:3.75:3.36:8.78:2.87:2.54:4.64 respectively;

[0011] Homogeneous polysaccharide ZNDHP-2 from Rhododendron principis Bur. var. farrerae (Tagg) Chamberlain: A polysaccharide composed of five monosaccharides, with a molecular weight of 599.7 kDa; the total sugar content is 94.31%, the protein content is 1.31%, and the uronic acid content is 1.01%; the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 2.21:5.96:48.34:16.98:26.51; the linkage modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked arabinose, 1,3-linked rhamnose, terminal-linked glucose, 1,5-linked arabinose, 1,2-linked glucose, 1,4-linked glucose, 1,3,5-linked arabinose, 1,2,6-linked glucose, 1,2,6-linked galactose, 1,3,6-linked galactose, 1,3,6-linked glucose, 1,4,6-linked mannose, and the molar ratios are 14.01:2.79:2.21:1.98:11.04:6.90:25.98:1.88:9.45:5.79:10.06:1.79:3.15:2.97 respectively;

[0012] Uniform polysaccharide ZNDHP-3 from Rhododendron principis Bur. var. chienii (Wils.) Rehd.: A polysaccharide composed of five monosaccharides, with a molecular weight of 651.5 kDa; the total sugar content is 92.14%, the protein content is 1.98%, and the uronic acid content is 1.08%; the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 1.67:1.94:71.85:11.30:13.24; the linkage modes include terminal-linked arabinose, 1,3-linked rhamnose, 1,5-linked arabinose, terminal-linked glucose, 1,3,5-linked arabinose, 1,4-linked glucose, 1,4-linked galactose, 1,4,6-linked galactose, 1,3,6-linked mannose, and the molar ratios are 3.24:2.21:2.29:25.93:7.14:46.99:2.33:7.16:2.71 respectively;

[0013] Uniform polysaccharide ZNDHP-4 from Rhododendron principis Bur. var. chienii (Wils.) Rehd.: A polysaccharide composed of three monosaccharides, with a molecular weight of 691.9 kDa; the total sugar content is 90.87%, the protein content is 1.65%, and the uronic acid content is 0.87%; the molar ratio of monosaccharides is glucose:galactose:arabinose = 65.18:10.11:24.71; the linkage modes include terminal-linked arabinose, 1,5-linked arabinose, terminal-linked glucose, 1,3,5-linked arabinose, 1,4-linked glucose, 1,4-linked galactose, 1,4,6-linked glucose, 1,3,6-linked glucose, and the molar ratios are 7.17:4.89:11.87:7.24:46.69:7.82:7.43:6.89 respectively.

[0014] The second technical solution of the present invention is to provide a preparation method of Rhododendron principis Bur. var. chienii (Wils.) Rehd. polysaccharide, which includes the following steps:

[0015] S1. Take the leaves of Rhododendron principis Bur. var. chienii (Wils.) Rehd., and successively carry out ethanol extraction, filtration, and drying. The dried residue is extracted with hot water, filtered, concentrated, left standing for the first time, centrifuged. The supernatant after centrifugation is concentrated, left standing for the second time, centrifuged. The precipitate after centrifugation is redissolved in water, trichloroacetic acid is added and centrifuged. The supernatant after centrifugation is adjusted to neutral pH, and then concentrated, dialyzed, and freeze-dried in sequence to obtain Rhododendron principis Bur. var. chienii (Wils.) Rehd. 90% ethanol-precipitated polysaccharide ZNDCP-90;

[0016] S2. Dissolve the Rhododendron principis Bur. & Franch. 90% ethanol-precipitated polysaccharide ZNDCP-90 prepared in step S1 in water, and separate it by DEAE-cellulose column chromatography in batches. Elute it successively with distilled water and NaCl solutions of gradient concentrations, collect each fraction, and according to the ultraviolet detection results of the color reaction of the sugar content in each fraction, combine the fractions and concentrate, dialyze, and freeze-dry them successively. After detecting the homogeneity by high-performance liquid chromatography and the anti-complement activity, obtain the Rhododendron principis Bur. & Franch. homogeneous polysaccharide ZNDHP-1, the Rhododendron principis Bur. & Franch. homogeneous polysaccharide ZNDHP-2, the Rhododendron principis Bur. & Franch. homogeneous polysaccharide ZNDHP-3, and the Rhododendron principis Bur. & Franch. homogeneous polysaccharide ZNDHP-4.

[0017] Furthermore, in step S1, extract the leaves of Rhododendron principis Bur. & Franch. with ethanol of 95% mass fraction.

[0018] Furthermore, in step S1, add ethanol of 95% mass fraction before the first static settling so that the ethanol concentration in the extraction solution during the first static settling is 75%.

[0019] Furthermore, in step S1, add anhydrous ethanol before the second static settling so that the ethanol concentration in the supernatant during the second static settling is 90%.

[0020] Furthermore, in step S1, after adding trichloroacetic acid, the concentration of trichloroacetic acid in the solution is 10%.

[0021] Furthermore, in step S2, the gradient concentrations of the NaCl solution are 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.8 mol / L, and 1.6 mol / L in sequence.

[0022] The third technical solution of the present invention is to provide an application of Rhododendron principis Bur. & Franch. polysaccharide, and the Rhododendron principis Bur. & Franch. polysaccharide is used for preparing an anti-complement drug.

[0023] Furthermore, the anti-complement drug is a drug that inhibits the hemolysis of cells caused by the activation of the classical complement pathway.

[0024] The fourth technical solution of the present invention is to provide an application of Rhododendron principis Bur. & Franch. polysaccharide, and the Rhododendron principis Bur. & Franch. polysaccharide is used for preparing an anti-inflammatory drug.

[0025] Furthermore, the anti-inflammatory drug is a drug that inhibits the secretion of inflammatory factors induced by lipopolysaccharide.

[0026] Even further, the anti-inflammatory drug is a drug that inhibits lipopolysaccharide-induced pneumonia.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] Five Rhododendron principis polysaccharides were isolated from the leaves of Rhododendron principis, namely one 90% ethanol precipitation polysaccharide ZNDCP-90 of Rhododendron principis and four homogeneous polysaccharides ZNDHP-1, ZNDHP-2, ZNDHP-3, and ZNDHP-4 of Rhododendron principis. It was confirmed by in vitro experiments that the polysaccharides of Rhododendron principis all have significant complement inhibitory activity and can be further used as active ingredients for the preparation of anti-complement drugs; it was confirmed by in vivo experiments that ZNDCP-90, ZNDHP-2, ZNDHP-3, and ZNDHP-4 have significant inhibitory effects on inflammatory responses and can be further used as active ingredients for the preparation of anti-inflammatory drugs. Description of the Drawings

[0029] Figure 1 It is a flowchart for the isolation of homogeneous polysaccharides of Rhododendron principis.

[0030] Figure 2 It is a chromatogram of high performance gel permeation chromatography (HPGPC) of homogeneous polysaccharides of Rhododendron principis. Among them, the gel column uses a tandem gel column of SHODEX KS-802 (300×7.6 mm) and SHODEX KS-804 (300×7.6 mm); the detector is a refractive index detector (RID) and an evaporative light scattering detector (ELSD); the eluent is distilled water; the flow rate is 0.6 mL / min.

[0031] Figure 3 It is the result of the effect of 90% ethanol precipitation polysaccharide ZNDCP-90 of Rhododendron principis on TNF-α, IL-6 in the serum of mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI) and IL-6 in bronchoalveolar lavage fluid (BALF). Among them, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, with the Model Control group as the control.

[0032] Figure 4 It is the result of the effect of polysaccharides ZNDHP-2, ZNDHP-3, and ZNDHP-4 of Rhododendron principis on the activity of RAW 264.7 cells.

[0033] Figure 5 It is the result of the effect of different concentrations of polysaccharides ZNDHP-2, ZNDHP-3, and ZNDHP-4 of Rhododendron principis on the NO release of LPS-induced RAW264.7 cells. The results are calculated based on the concentration of NaNO 2 Among them, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, with the LPS group as the control.

[0034] Figure 6Effects of Rhododendron principis Bur. polysaccharides ZNDHP-2, ZNDHP-3, and ZNDHP-4 at different concentrations on the release of cytokine TNF-α in LPS-induced RAW264.7 cells. Among them, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, with the LPS column as the control.

[0035] Figure 7 Effects of Rhododendron principis Bur. polysaccharides ZNDHP-2, ZNDHP-3, and ZNDHP-4 at different concentrations on the release of cytokine IL-6 in LPS-induced RAW264.7 cells. Among them, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, with the LPS column as the control.

[0036] Figure 8 Effects of Rhododendron principis Bur. polysaccharides ZNDHP-2, ZNDHP-3, and ZNDHP-4 at different concentrations on the release of cytokine IL-1β in LPS-induced RAW264.7 cells. Among them, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, with the LPS column as the control. Detailed implementation manners

[0037] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0038] In the following examples and comparative examples, unless otherwise specified, the raw materials or processing techniques are conventional commercially available raw material products or conventional processing techniques in the art.

[0039] Example 1: Preparation of Rhododendron principis Bur. polysaccharide

[0040] Take 10 kg of the branches and leaves of Rhododendron principis Bur., crush them, extract with ethanol with a mass fraction of 95%, filter, extract the residue with water 3 times, concentrate and centrifuge. Add ethanol with a mass fraction of 95% to the supernatant after centrifugation until the ethanol concentration in the supernatant is 75%, let it stand, centrifuge and take the supernatant. Concentrate the supernatant and add anhydrous ethanol until the ethanol concentration is 90%, let it stand, centrifuge and remove the supernatant. Redissolve the precipitate with water, recover under reduced pressure to remove ethanol; redissolve with water, add trichloroacetic acid until the trichloroacetic acid concentration is 10% to remove free protein, centrifuge and take the supernatant. Adjust the supernatant to neutral, dialyze, concentrate, and freeze-dry to obtain the crude polysaccharide Rhododendron principis Bur. 90% ethanol-precipitated polysaccharide ZNDCP-90.

[0041] Take 75 g of the 90% ethanol-precipitated polysaccharide ZNDCP-90 from Rhododendron principis Bur. et Franch., dissolve it in distilled water, centrifuge and take the supernatant. The supernatant was fractionated and separated by DEAE-cellulose column chromatography. It was eluted successively with distilled water and 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.8 mol / L, and 1.6 mol / L NaCl solutions. The elution volume was greater than 2 times the column volume (10 L), the flow rate was 16 mL / min, and each fraction was collected. After color development by the sulfuric acid-phenol method, the absorbance value at 490 nm was detected every other tube. According to the ultraviolet detection results of the color reaction of sugar content, the fractions were combined, concentrated, dialyzed, and freeze-dried to obtain 6 sub-components: ZND-1, ZND-2, ZND-3, ZND-4, ZND-5, and ZND-6. By HPGPC detection, ZND-2, ZND-3, ZND-4, and ZND-5 were homogeneous polysaccharides, as Figure 2 shown, and were named ZNDHP-1, ZNDHP-2, ZNDHP-3, and ZNDHP-4, respectively.

[0042] Example 2: Structural Characterization of Polysaccharides from Rhododendron principis Bur. et Franch.

[0043] (1) Determination of Molecular Weight

[0044] The relative molecular weight of the polysaccharide sample from Rhododendron principis Bur. et Franch. was determined by 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. Calculation is performed based on the calibration curve obtained from known molecular weights.

[0045] Chromatographic conditions: Separation was carried out using a tandem gel column of SHODEX KS-802 (300×7.6 mm) and SHODEX KS-804 (300×7.6 mm). The flow rate was 0.6 mL / min, the injection volume was 20 μL, ultrapure water was used as the mobile phase, the column temperature was 37 °C, and the detectors were a differential refractive index detector (RID) and an evaporative light scattering detector (ELSD).

[0046] Experimental method: Accurately weigh 2.0 mg each of the homogeneous polysaccharide from Rhododendron principis Bur. et Franch. and Dextrans series standard products, dissolve them in ultrapure water to prepare a 2.0 mg / mL solution, filter through a 0.45 μm microporous filter membrane before injection and then detect, record the retention time. Plot a standard curve with the logarithm of the molecular weight of the standard polysaccharide (Lg) 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 ZNDCP-90, ZNDHP-1, ZNDHP-2, ZNDHP-3, and ZNDHP-4 were 353.2 - 2208.5 kDa, 633.8 kDa, 599.7 kDa, 651.5 kDa, and 691.9 kDa, respectively.

[0047] (2) Determination of total sugar, uronic acid, and protein content

[0048] The total sugar content of ZNDCP-90 determined by the sulfuric acid-phenol method was 64.31%; the total sugar content of ZNDHP-1 was 91.69%; the total sugar content of ZNDHP-2 was 94.31%; the total sugar content of ZNDHP-3 was 92.14%; the total sugar content of ZNDHP-4 was 90.87%.

[0049] The uronic acid content was detected by the m-hydroxybiphenyl method. The uronic acid content of ZNDCP-90 was 1.31%; the uronic acid content of ZNDHP-1 was 1.68%; the uronic acid content of ZNDHP-2 was 1.01%; the uronic acid content of ZNDHP-3 was 1.08%; the uronic acid content of ZNDHP-4 was 0.87%.

[0050] The protein content was determined by the Coomassie brilliant blue method: the protein content of ZNDCP-90 was 1.94%; the protein content of ZNDHP-1 was 1.01%; the protein content of ZNDHP-2 was 1.31%; the protein content of ZNDHP-3 was 1.98%; the protein content of ZNDHP-4 was 1.65%.

[0051] (3) Monosaccharide composition analysis

[0052] ZNDCP-90, ZNDHP-1, ZNDHP-2, ZNDHP-3, and ZNDHP-4 were each completely 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 high-performance liquid chromatography.

[0053] ZNDCP-90 is a polysaccharide composed of seven monosaccharides, and the molar ratio of monosaccharides is mannose:rhamnose:glucose:galacturonic acid:galactose:xylose:arabinose = 11.22:2.20:16.22:24.33:9.66:24.59:11.78.

[0054] ZNDHP-1 is a polysaccharide composed of five monosaccharides, and the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 9.85:9.05:56.02:15.99:9.10.

[0055] ZNDHP-2 is a polysaccharide composed of five monosaccharides, and the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 2.21:5.96:48.34:16.98:26.51.

[0056] ZNDHP-3 is a polysaccharide composed of five monosaccharides, and the molar ratio of the monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 1.67:1.94:71.85:11.30:13.24.

[0057] ZNDHP-4 is a polysaccharide composed of three monosaccharides, and the molar ratio of the monosaccharides is glucose:galactose:arabinose = 65.18:10.11:24.71.

[0058] (4) Methylation analysis

[0059] ZNDHP-1, ZNDHP-2, ZNDHP-3, and ZNDHP-4 were methylated respectively using the modified Hakomori method. The methylated products were completely hydrolyzed with 2 mol / L TFA, reduced with NaBH 4 and acetylated with acetic anhydride to form partially methylated alditol acetate derivatives, and then subjected to GC-MS analysis.

[0060] The structure of ZNDHP-1 contains: arabinose linked at the end, rhamnose linked at the end, 1,3-linked rhamnose, glucose linked at the end, galactose linked at the end, 1,5-linked arabinose, 1,3-linked glucose, 1,4-linked glucose, 1,4-linked galactose, 1,4-linked mannose, 1,3,5-linked arabinose, 1,2,6-linked glucose, 1,2,4-linked glucose, 1,4,6-linked galactose, 1,4,6-linked mannose, 1,3,6-linked glucose, 1,3,6-linked mannose, and the molar ratio is 2.21:6.03:5.02:17.59:2.58:2.21:2.04:25.74:1.98:2.97:5.69:3.75:3.36:8.78:2.87:2.54:4.64.

[0061] The structure of ZNDHP-2 contains: arabinose linked at the end, rhamnose linked at the end, 1,3-linked arabinose, 1,3-linked rhamnose, glucose linked at the end, 1,5-linked arabinose, 1,2-linked glucose, 1,4-linked glucose, 1,3,5-linked arabinose, 1,2,6-linked glucose, 1,2,6-linked galactose, 1,3,6-linked galactose, 1,3,6-linked glucose, 1,4,6-linked mannose, and the molar ratio is 14.01:2.79:2.21:1.98:11.04:6.90:25.98:1.88:9.45:5.79:10.06:1.79:3.15:2.97.

[0062] The ZNDHP-3 structure contains: arabinose linked at the end, rhamnose linked at 1,3, arabinose linked at 1,5, glucose linked at the end, arabinose linked at 1,3,5, glucose linked at 1,4, galactose linked at 1,4, galactose linked at 1,4,6, mannose linked at 1,3,6, with a molar ratio of 3.24:2.21:2.29:25.93:7.14:46.99:2.33:7.16:2.71.

[0063] The ZNDHP-4 structure contains: arabinose linked at the end, arabinose linked at 1,5, glucose linked at the end, arabinose linked at 1,3,5, glucose linked at 1,4, galactose linked at 1,4, glucose linked at 1,4,6, glucose linked at 1,3,6, with a molar ratio of 7.17:4.89:11.87:7.24:46.69:7.82:7.43:6.89.

[0064] Example 3: Classical pathway complement inhibition assay

[0065] Take the serum of 3-month-old guinea pigs and dilute it 1:100 with BBS buffer (barbiturate buffer, pH = 7.4) as the complement source for this classical pathway. Dilute the rabbit anti-sheep red blood cell antibody 1:1000 with BBS buffer as the hemolysin; prepare 2% SRBC with sheep red blood cells (SRBC). Weigh 3 mg of Rhododendron principis polysaccharide precisely, dissolve it in BBS buffer, and serially dilute it to 8 concentrations. After pre-incubating 200 μL of the polysaccharide solution at different concentrations with 200 μL of the complement diluted to 1:100 at 37 °C for 10 min, successively add 100 μL of hemolysin (1:1000) and 100 μL of 2% SRBC, incubate in a 37 °C water bath for 30 min, then place it in a low-temperature high-speed centrifuge and centrifuge at 5000 rpm and 4 °C for 10 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. At the same time, set up a polysaccharide control group (200 μL of the corresponding concentration of Rhododendron principis polysaccharide plus 400 μL of BBS buffer), a complement control group (replace Rhododendron principis polysaccharide with 200 μL of BBS buffer), and a complete hemolysis group (100 μL of 2% SRBC dissolved in 500 μL of distilled water). After subtracting the absorbance value of the corresponding Rhododendron principis polysaccharide control group from the absorbance value of each concentration of Rhododendron principis polysaccharide group, calculate the hemolysis inhibition rate. Use the logarithm of the Rhododendron principis polysaccharide concentration as the X-axis and the hemolysis inhibition rate as the Y-axis to plot a graph, and calculate the concentration (CH 50 value) of the test sample required to inhibit 50% hemolysis from the fitted curve obtained. Using heparin as a positive control drug, the results show that both the 90% ethanol-precipitated polysaccharide of Rhododendron principis and 4 homogeneous polysaccharides of Rhododendron principis have significant inhibitory activities on the activation of the classical pathway of complement, as shown in Table 1.

[0066] Table 1 Inhibitory effect of Rhododendron principis Bur. polysaccharide on complement activation

[0067]

[0068] Among them, CH 50 value is expressed as: mean ± SD (n = 3); positive control heparin CH 50 : 49 ± 3 μg / mL.

[0069] Example 4: Effects of ZNDCP-90 on cytokines in serum and bronchoalveolar lavage fluid of LPS-induced acute lung injury mice

[0070] Thirty Balb / c mice (18 - 22 g) were randomly divided into 5 groups (A, B, C, D, E) according to body weight: Group A was the normal control group, Group B was the LPS-induced acute lung injury model group, Group C was the positive control dexamethasone group (DXMS, 4 mg / kg), Group D was the low-dose ZNDCP-90 group (50 mg / kg), and Group E was the high-dose ZNDCP-90 group (100 mg / kg), with 6 mice in each group. Groups C, D, and E were given intragastric administration of 0.5% carboxymethylcellulose sodium (CMC-Na) as the positive control group and experimental groups, and administered continuously for two days. After two consecutive days of administration, 1 h after the last intragastric administration, Groups B, C, D, and E were given subcutaneous injection of LPS (10 mg / kg). 6 h after the injection of LPS, blood was taken from the eyeballs, and mouse serum was obtained by centrifugation. 1 mL of normal saline was repeatedly injected into the lungs of mice with a syringe to obtain bronchoalveolar lavage fluid, and the serum and bronchoalveolar lavage fluid were used to detect the indexes of inflammatory factors in pneumonia.

[0071] (1) Effects of ZNDCP-90 on TNF-α and IL-6 in serum of LPS-induced acute lung injury mice

[0072] The serum was centrifuged, and the supernatant was measured according to the TNF-α and IL-6 kit instructions by Elisa method. The results are as Figure 3 shown. Compared with the normal group, the levels of TNF-α and IL-6 in the serum of the model group were significantly increased (P < 0.001, P < 0.001); after administration, compared with the model group, the levels of TNF-α and IL-6 in the serum of the low-dose ZNDCP-90 group had no significant change, and the levels of TNF-α and IL-6 in the serum of the high-dose ZNDCP-90 group were significantly decreased (P < 0.01, P < 0.05).

[0073] (2) Effects of ZNDCP-90 on IL-6 in bronchoalveolar lavage fluid of LPS-induced acute lung injury mice

[0074] The bronchoalveolar lavage fluid was centrifuged, and the supernatant was measured according to the IL-6 kit instructions by Elisa method. The results are asFigure 3 As shown, compared with the normal group, the level of IL-6 in the bronchoalveolar lavage fluid of the model group was significantly increased (P<0.001); after administration, compared with the model group, there was no significant change in the level of IL-6 in the bronchoalveolar lavage fluid of the low-dose ZNDCP-90 group, and the level of IL-6 in the bronchoalveolar lavage fluid of the high-dose ZNDCP-90 group was significantly decreased (P<0.01).

[0075] Example 5: Effects of ZNDHP-2, ZNDHP-3, and ZNDHP-4 on LPS-induced RAW264.7 cells

[0076] RAW264.7 cells were cultured in DMEM medium mixed with 10% fetal bovine serum, 100 unit / mL penicillin, and 100 mg / mL streptomycin, and seeded in 96-well plates at a concentration of 10 5 cells / well and cultured in an environment of 37°C and 5% carbon dioxide gas.

[0077] (1) Effects of ZNDHP-2, ZNDHP-3, and ZNDHP-4 on the viability of RAW264.7 cells

[0078] The RAW264.7 cells inoculated in 96-well plates were divided into 22 groups, with 6 wells in each group (A-V): Groups A and B were normal control groups, groups C and D were LPS groups (1 μg / mL), group E was the low-dose ZNDHP-2 group (50 mg / mL), group F was the medium-dose ZNDHP-2 group (100 mg / mL), group G was the high-dose ZNDHP-2 group (200 mg / mL), group H was the low-dose ZNDHP-2 + LPS group (50 mg / mL, LPS 1 μg / mL), group I was the medium-dose ZNDHP-2 + LPS group (100 mg / mL, LPS 1 μg / mL), group J was the high-dose ZNDHP-2 + LPS group (200 mg / mL, LPS 1 μg / mL), group K was the low-dose ZNDHP-3 group (50 mg / mL), group L was the medium-dose ZNDHP-3 group (100 mg / mL), group M was the high-dose ZNDHP-3 group (200 mg / mL), group N was the low-dose ZNDHP-3 + LPS group (50 mg / mL, LPS 1 μg / mL), group O was the medium-dose ZNDHP-3 + LPS group (100 mg / mL, LPS 1 μg / mL), group P was the high-dose ZNDHP-3 + LPS group (200 mg / mL, LPS 1 μg / mL), group Q was the low-dose ZNDHP-4 group (50 mg / mL), group R was the medium-dose ZNDHP-4 group (100 mg / mL), group S was the high-dose ZNDHP-4 group (200 mg / mL), group T was the low-dose ZNDHP-4 + LPS group (50 mg / mL, LPS 1 μg / mL), group U was the medium-dose ZNDHP-4 + LPS group (100 mg / mL, LPS 1 μg / mL), and group V was the high-dose ZNDHP-4 + LPS group (200 mg / mL, LPS 1 μg / mL). After the RAW264.7 cells were inoculated and cultured for 24 h, 100 μL of XTT reagent was added to each well and cultured for 2 h, and the absorbance was measured at 450 nm. As Figure 4 shown, compared with the normal control group and the LPS group respectively, the low-, medium-, and high-dose ZNDHP-2 groups, the low-, medium-, and high-dose ZNDHP-3 groups, and the low-, medium-, and high-dose ZNDHP-4 groups had no effect on the viability of RAW264.7 cells cultured with or without LPS.

[0079] (2) Effects of ZNDHP-2, ZNDHP-3, and ZNDHP-4 on NO in the medium of LPS-induced RAW264.7 cells

[0080] The RAW264.7 cells inoculated in 96-well plates were divided into 12 groups, with 6 wells in each group (A - L): Group A was the normal control group, Group B was the LPS group (1 μg / mL), Group C was the positive control dexamethasone group (DXMS 60 μg / mL, LPS 1 μg / mL), Group D was the low-dose ZNDHP-2 group (50 mg / mL, LPS 1 μg / mL), Group E was the medium-dose ZNDHP-2 group (100 mg / mL, LPS 1 μg / mL), Group F was the high-dose ZNDHP-2 group (200 mg / mL, LPS 1 μg / mL), Group G was the low-dose ZNDHP-3 group (50 mg / mL, LPS 1 μg / mL), Group H was the medium-dose ZNDHP-3 group (100 mg / mL, LPS 1 μg / mL), Group I was the high-dose ZNDHP-3 group (200 mg / mL, LPS 1 μg / mL), Group J was the low-dose ZNDHP-4 group (50 mg / mL, LPS 1 μg / mL), Group K was the medium-dose ZNDHP-4 group (100 mg / mL, LPS 1 μg / mL), and Group L was the high-dose ZNDHP-4 group (200 mg / mL, LPS 1 μg / mL). After the RAW264.7 cells were inoculated and cultured for 24 h, the supernatant was taken, and the Griess reaction method was used to measure the NO content in the culture medium. The supernatant was measured according to the NO kit instructions, with NaNO 2 concentration meter. The results are as Figure 5 shown. Compared with the normal group, the NaNO 2 concentration in the culture medium of the LPS group was significantly increased (P < 0.001); compared with the LPS group, the NaNO 2 concentrations in the culture media of the low-, medium-, and high-dose ZNDHP-2 groups were all significantly decreased (P < 0.001, P < 0.001, P < 0.001); compared with the LPS group, the NaNO 2 concentrations in the culture media of the low-, medium-, and high-dose ZNDHP-3 groups were all significantly decreased (P < 0.001, P < 0.001, P < 0.001); compared with the LPS group, the NaNO 2 concentrations in the culture media of the low-, medium-, and high-dose ZNDHP-4 groups were all significantly decreased (P < 0.001, P < 0.001, P < 0.001).

[0081] (3) Effects of ZNDHP-2, ZNDHP-3, and ZNDHP-4 on TNF-α, IL-6, and IL-1β in the culture medium of LPS-induced RAW264.7 cells

[0082] The RAW264.7 cells inoculated in 96-well plates were divided into 12 groups, with 6 wells in each group (A-L): A was the normal control group, B was the LPS group (1 μg / mL), C was the positive control dexamethasone group (DXMS 60 μg / mL, LPS 1 μg / mL), D was the low-dose ZNDHP-2 group (50 mg / mL, LPS 1 μg / mL), E was the medium-dose ZNDHP-2 group (100 mg / mL, LPS 1 μg / mL), F was the high-dose ZNDHP-2 group (200 mg / mL, LPS 1 μg / mL), G was the low-dose ZNDHP-3 group (50 mg / mL, LPS 1 μg / mL), H was the medium-dose ZNDHP-3 group (100 mg / mL, LPS 1 μg / mL), I was the high-dose ZNDHP-3 group (200 mg / mL, LPS 1 μg / mL), J was the low-dose ZNDHP-4 group (50 mg / mL, LPS 1 μg / mL), K was the medium-dose ZNDHP-4 group (100 mg / mL, LPS 1 μg / mL), and L was the high-dose ZNDHP-4 group (200 mg / mL, LPS 1 μg / mL). After the RAW264.7 cells were inoculated and cultured for 24 h, the supernatant was taken, and the contents of TNF-α, IL-6, and IL-1β in the culture medium were measured by the Elisa method. The supernatant was measured according to the kit instructions for TNF-α, IL-6, and IL-1β. As for the content of TNF-α Figure 6 shown, compared with the normal group, the concentration of TNF-α in the culture medium of the LPS group was significantly increased (P < 0.001); compared with the LPS group, the concentrations of TNF-α in the culture medium of the low-, medium-, and high-dose ZNDHP-2 groups were all significantly decreased (P < 0.001, P < 0.001, P < 0.001); compared with the LPS group, the concentrations of TNF-α in the culture medium of the low-, medium-, and high-dose ZNDHP-3 groups were all significantly decreased (P < 0.01, P < 0.01, P < 0.001); compared with the LPS group, the concentrations of TNF-α in the culture medium of the low-, medium-, and high-dose ZNDHP-4 groups were all significantly decreased (P < 0.05, P < 0.01, P < 0.001). As for the content of IL-6 Figure 7 shown, compared with the normal group, the concentration of IL-6 in the culture medium of the LPS group was significantly increased (P < 0.001); compared with the LPS group, the concentrations of IL-6 in the culture medium of the low-, medium-, and high-dose ZNDHP-2 groups were all significantly decreased (P < 0.001, P < 0.001, P < 0.001); compared with the LPS group, the concentrations of IL-6 in the culture medium of the low-, medium-, and high-dose ZNDHP-3 groups were all significantly decreased (P < 0.01, P < 0.01, P < 0.001); compared with the LPS group, the concentrations of IL-6 in the culture medium of the low-, medium-, and high-dose ZNDHP-4 groups were all significantly decreased (P < 0.01, P < 0.01, P < 0.001). As for the content of IL-1βFigure 8 As shown, compared with the normal group, the concentration of IL-1β in the culture medium of the LPS group increased significantly (P<0.001); compared with the LPS group, the concentrations of IL-1β in the culture media of the low, medium, and high dose groups of ZNDHP-2 decreased significantly (P<0.001, P<0.001, P<0.001); compared with the LPS group, the concentrations of IL-1β in the culture media of the low, medium, and high dose groups of ZNDHP-3 decreased significantly (P<0.001, P<0.001, P<0.001); compared with the LPS group, the concentrations of IL-1β in the culture media of the low, medium, and high dose groups of ZNDHP-4 decreased significantly (P<0.001, P<0.001, P<0.001).

[0083] 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. Rhododendron principis polysaccharide, characterized in that, it includes Rhododendron principis 90% ethanol precipitation polysaccharide ZNDCP-90 and four Rhododendron principis homogeneous polysaccharides ZNDHP-1, ZNDHP-2, ZNDHP-3, ZNDHP-4, which respectively have the following structural characteristics: Rhododendron principis 90% ethanol precipitation polysaccharide ZNDCP-90: a polysaccharide composed of seven monosaccharides, with a molecular weight range of 353.2 - 2208.5 kDa; the total sugar content is 64.31%, the protein content is 1.94%, and the uronic acid content is 1.31%; the molar ratio of monosaccharides mannose:rhamnose:glucose:galacturonic acid:galactose:xylose:arabinose = 11.22:2.20:16.22:24.33:9.66:24.59:11.78; Rhododendron principis homogeneous polysaccharide ZNDHP-1: a polysaccharide composed of five monosaccharides, with a molecular weight of 633.8 kDa; the total sugar content is 91.69%, the protein content is 1.01%, and the uronic acid content is 1.68%; the molar ratio of monosaccharides mannose:rhamnose:glucose:galactose:arabinose = 9.85:9.05:56.02:15.99:9.10; the linkage modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked rhamnose, terminal-linked glucose, terminal-linked galactose, 1,5-linked arabinose, 1,3-linked glucose, 1,4-linked glucose, 1,4-linked galactose, 1,4-linked mannose, 1,3,5-linked arabinose, 1,2,6-linked glucose, 1,2,4-linked glucose, 1,4,6-linked galactose, 1,4,6-linked mannose, 1,3,6-linked glucose, 1,3,6-linked mannose, and the molar ratios are 2.21:6.03:5.02:17.59:2.58:2.21:2.04:25.74:1.98:2.97:5.69:3.75:3.36:8.78:2.87:2.54:4.64 respectively; Homogeneous polysaccharide ZNDHP-2 from Rhododendron principis Bur. var. chienianum (Cowan) Chamberlain: A polysaccharide composed of five monosaccharides with a molecular weight of 599.7 kDa; the total sugar content is 94.31%, the protein content is 1.31%, and the uronic acid content is 1.01%; the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 2.21:5.96:48.34:16.98:26.51; the linkage modes include terminal-linked arabinose, terminal-linked rhamnose, 1,3-linked arabinose, 1,3-linked rhamnose, terminal-linked glucose, 1,5-linked arabinose, 1,2-linked glucose, 1,4-linked glucose, 1,3,5-linked arabinose, 1,2,6-linked glucose, 1,2,6-linked galactose, 1,3,6-linked galactose, 1,3,6-linked glucose, 1,4,6-linked mannose, and the molar ratios are 14.01:2.79:2.21:1.98:11.04:6.90:25.98:1.88:9.45:5.79:10.06:1.79:3.15:2.97 respectively; Homogeneous polysaccharide ZNDHP-3 from Rhododendron principis Bur. var. chienianum (Cowan) Chamberlain: A polysaccharide composed of five monosaccharides with a molecular weight of 651.5 kDa; the total sugar content is 92.14%, the protein content is 1.98%, and the uronic acid content is 1.08%; the molar ratio of monosaccharides is mannose:rhamnose:glucose:galactose:arabinose = 1.67:1.94:71.85:11.30:13.24; the linkage modes include terminal-linked arabinose, 1,3-linked rhamnose, 1,5-linked arabinose, terminal-linked glucose, 1,3,5-linked arabinose, 1,4-linked glucose, 1,4-linked galactose, 1,4,6-linked galactose, 1,3,6-linked mannose, and the molar ratios are 3.24:2.21:2.29:25.93:7.14:46.99:2.33:7.16:2.71 respectively; Homogeneous polysaccharide ZNDHP-4 from Rhododendron principis Bur. var. chienianum (Cowan) Chamberlain: A polysaccharide composed of three monosaccharides with a molecular weight of 691.9 kDa; the total sugar content is 90.87%, the protein content is 1.65%, and the uronic acid content is 0.87%; the molar ratio of monosaccharides is glucose:galactose:arabinose = 65.18:10.11:24.71; the linkage modes include terminal-linked arabinose, 1,5-linked arabinose, terminal-linked glucose, 1,3,5-linked arabinose, 1,4-linked glucose, 1,4-linked galactose, 1,4,6-linked glucose, 1,3,6-linked glucose, and the molar ratios are 7.17:4.89:11.87:7.24:46.69:7.82:7.43:6.89 respectively.

2. A preparation method of the Rhododendron principis Bur. var. chienianum (Cowan) Chamberlain polysaccharide as described in claim 1, characterized in that, it comprises the following steps: S1. Take the leaves of Rhododendron principis Bur. and carry out ethanol extraction, filtration, and drying in sequence. The dried residue is extracted with hot water, filtered, concentrated, statically settled for the first time, and centrifuged. The supernatant after centrifugation is concentrated, statically settled for the second time, and centrifuged. The precipitate after centrifugation is redissolved in water, trichloroacetic acid is added and centrifuged. The supernatant after centrifugation is adjusted to neutral pH, and then concentrated, dialyzed, and freeze-dried in sequence to obtain the 90% ethanol-precipitated polysaccharide ZNDCP-90 of Rhododendron principis Bur.; S2. Dissolve the 90% ethanol-precipitated polysaccharide ZNDCP-90 of Rhododendron principis Bur. prepared in step S1 in water, and separate it by DEAE-cellulose column chromatography in batches. It is eluted with distilled water and NaCl solutions with gradient concentrations in sequence. Each fraction is collected. According to the ultraviolet detection results of the color reaction of the sugar content in each fraction, the fractions are combined and concentrated, dialyzed, and freeze-dried in sequence. After being inspected by high-performance liquid chromatography for homogeneity and anti-complement activity detection, the homogeneous polysaccharide ZNDHP-1 of Rhododendron principis Bur., the homogeneous polysaccharide ZNDHP-2 of Rhododendron principis Bur., the homogeneous polysaccharide ZNDHP-3 of Rhododendron principis Bur., and the homogeneous polysaccharide ZNDHP-4 of Rhododendron principis Bur. are obtained. Among them, in step S1, anhydrous ethanol is added before the second static settlement so that the ethanol concentration in the supernatant during the second static settlement is 90%.

3. The preparation method of the Rhododendron principis Bur. polysaccharide according to claim 2, characterized in that, in step S1, the leaves of Rhododendron principis Bur. are extracted with ethanol with a mass fraction of 95%.

4. The preparation method of the Rhododendron principis Bur. polysaccharide according to claim 2, characterized in that, in step S1, anhydrous ethanol with a mass fraction of 95% is added before the first static settlement so that the ethanol concentration in the extraction solution during the first static settlement is 75%.

5. The preparation method of the Rhododendron principis Bur. polysaccharide according to claim 2, characterized in that, in step S1, after adding trichloroacetic acid, the concentration of trichloroacetic acid in the solution is 10%.

6. The preparation method of the Rhododendron principis Bur. polysaccharide according to claim 2, characterized in that, in step S2, the gradient concentrations of the NaCl solution are 0.1 mol / L, 0.2 mol / L, 0.4 mol / L, 0.8 mol / L, and 1.6 mol / L in sequence.

7. An application of the Rhododendron principis Bur. polysaccharide as claimed in claim 1, characterized in that, the Rhododendron principis Bur. polysaccharide is used for preparing anti-complement drugs.

8. An application of the Rhododendron principis Bur. polysaccharide as claimed in claim 1, characterized in that, the Rhododendron principis Bur. polysaccharide is used for preparing anti-inflammatory drugs.

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