Herba siegesbeckiae polysaccharide, and preparation method and application thereof

Abstract

The present application relates to the preparation method and application of the polysaccharide of Herba Siegesbeckiae, which includes total polysaccharide SOP and two uniform polysaccharides SOPC2-3 and SOPC3. The experiments prove that the polysaccharide of Herba Siegesbeckiae has a significant inhibitory effect on complement activation, and can be further used as an active ingredient to prepare a new complement inhibitor. The cell model experiments prove that the polysaccharide of Herba Siegesbeckiae can significantly inhibit the inflammatory response of LPS-induced RAW264.7 cells, and has anti-inflammatory activity, and can be further used as an active ingredient to prepare an anti-inflammatory drug. The animal experiments prove that the polysaccharide of Herba Siegesbeckiae (SOP and SOPC3) can effectively relieve LPS-induced acute lung injury in mice, and can be further used as an active ingredient to prepare a drug for preventing and treating acute lung injury.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and relates to polysaccharides, especially to Siegesbeckia orientalis polysaccharide and its preparation method and application. Background Technology

[0002] The complement system, a crucial immune defense system and inflammatory mediator in the human body, is essential for clearing pathogens, repairing damage, and maintaining homeostasis when properly activated. However, overactivation can lead to tissue damage and local or systemic edema. Severe inflammatory diseases such as acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and rheumatoid arthritis (RA) are all closely related to the overactivation of the complement system. Currently used immunosuppressants (such as glucocorticoids) are not specific complement inhibitors and are prone to causing various complications and side effects. Therefore, developing novel, highly effective, low-toxicity, and highly selective complement inhibitors is an urgent clinical need.

[0003] Acute lung injury (ALI) is a critical clinical condition with high morbidity and mortality, characterized by severe lung inflammation and edema caused by diffuse alveolar-capillary membrane damage. Its etiology is complex, commonly involving bacterial or viral infections, hemorrhagic shock, and trauma. In recent years, ALI caused by bacterial or viral infections, or bacterial-viral co-infections, has drawn increasing attention to lung diseases. In severe cases, the "cytokine storm" that erupts in the body can ultimately lead to acute respiratory distress syndrome (ARDS), with hypoxemia and multiple organ failure being the leading causes of death. Multiple studies have found that excessive complement activation is considered an important marker of ALI and ARDS associated with severe sepsis, cytokine storms, and multiple organ failure. Autopsy studies have further confirmed that plasma complement depletion exacerbates alveolar-capillary wall damage, increases vascular permeability, and promotes the release of inflammatory mediators, thereby worsening lung tissue damage. Therefore, complement inhibitors may be considered potential drugs for the treatment of acute lung injury.

[0004] Currently, the clinical treatment of acute lung injury mainly relies on anti-infection, anti-inflammatory, and respiratory and circulatory support therapies, but the efficacy is limited, and commonly used glucocorticoid anti-inflammatory drugs have significant side effects. Given that complement inhibition has become one of the important potential therapeutic targets for bacterial or viral acute lung injury, the development of highly effective and low-toxicity complement inhibitors is of great significance. Since many traditional Chinese medicine components have significant regulatory effects on the immune system, they provide valuable resources for finding novel complement inhibitors, anti-inflammatory drugs, and prodrugs for treating acute lung injury.

[0005] Siegesbeckia orientalis was first recorded in the Tang Dynasty's "Newly Revised Materia Medica". The 2020 edition of the Chinese Pharmacopoeia lists it as Siegesbeckia orientalis, a plant in the Asteraceae family. Siegesbeckia orientalis L., Siegesbeckia orientalis S. pubescens Makino or hairy-stalked Siegesbeckia or Siegesbeckia orientalis S. glabrescens The dried aerial parts of Makino are cold in nature, pungent and bitter in taste, and enter the liver and kidney meridians. They have the effects of clearing heat and detoxifying, dispelling wind and dampness, and relieving pain. Clinically, they are often used to treat rheumatic pain, rashes and sores, boils and carbuncles. The "Collection of Folk Prescriptions of Guizhou" records that it has the effect of "moistening the lungs and relieving cough". Studies have shown that the water extract of Siegesbeckia orientalis can effectively alleviate acute lung injury induced by influenza virus infection in mice, and can significantly reduce alveolar inflammatory exudate and reduce the expression of inflammatory factors (Wang Huan, Fan Kaifang, Chen Yuan, Wang Tengyue, Zhao Mingyue. Chinese Journal of Traditional Chinese Medicine, 2024, 39(06):3089-3093; Fan Kaifang, Wang Huan, Wang Tengyue, Zhao Mingyue, Chen Yuan. Chinese Journal of Traditional Chinese Medicine, 2024, 39(06):2879-2882). However, there are currently no studies on the structure, anticomplement activity, anti-inflammatory activity, and prevention and treatment effects of Siegesbeckia orientalis polysaccharides. Summary of the Invention

[0006] The purpose of this invention is to provide active ingredients with anti-complement, anti-inflammatory, and anti-acute lung injury properties based on the current state of the technology. Specifically, it relates to Siegesbeckia orientalis polysaccharides, their preparation methods, and applications, especially Siegesbeckia orientalis total polysaccharides (SOP) and two Siegesbeckia orientalis homogeneous polysaccharides (SOPC2-3 and SOPC3), their preparation methods, and their applications in the preparation of complement inhibitor drugs, anti-inflammatory drugs, and drugs for the prevention and treatment of acute lung injury.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention discloses Siegesbeckia orientalis polysaccharides, including total Siegesbeckia orientalis polysaccharide SOP and two homogeneous Siegesbeckia orientalis polysaccharides SOPC2-3 and SOPC3, which have the following structural features:

[0009] SOP (Standard Operating Procedure) of total polysaccharides from Siegesbeckia orientalis: a polysaccharide composed of six monosaccharides with a molecular weight range of 8.1-960.7 kDa; total sugar content of 67.3%, protein content of 31.1%, and uronic acid content of 21.3%; the monosaccharide molar ratio is mannose:rhamnose:galacturonic acid:glucose:galactose:arabinose = 6.7:7.7:28.9:11.6:29.0:16.1.

[0010] Siegesbeckia orientalis homogeneous polysaccharide SOPC2-3: a polysaccharide composed of four monosaccharides with a molecular weight of 121.2 kDa; total sugar content of 98.6%, no protein, uronic acid content of 42.4%; monosaccharide molar ratio of rhamnose: galacturonic acid: galactose: arabinose = 16.1: 43.6: 27.8: 12.5;

[0011] SOPC3, a homogeneous polysaccharide from Siegesbeckia orientalis, is a polysaccharide composed of four monosaccharides with a molecular weight of 16.6 kDa. It contains 81.6% total sugar, 10.9% protein, and 47.4% uronic acid. The monosaccharide molar ratio is rhamnose:galacturonic acid:galactose:arabinose = 11.5:54.9:20.7:12.9. The protein is mainly composed of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, arginine, and proline, with a relative mass ratio of 4.513:1.877:2.125:4.803:2.262:2.204:2.040:4.847.

[0012] Accordingly, a method for preparing the polysaccharide from Siegesbeckia orientalis includes the following steps:

[0013] S1. Take the herbal material of Siegesbeckia orientalis and process it in sequence by crushing, hot water extraction, filtration, concentration and centrifugation. After centrifugation, precipitate the supernatant with alcohol, redissolve the precipitate with water, add trichloroacetic acid, centrifuge, adjust the pH of the supernatant to neutral, and then concentrate, dialyze and freeze dry to obtain Siegesbeckia orientalis total polysaccharide SOP.

[0014] S2. Dissolve the total polysaccharide SOP obtained in step S1 in water, separate it using a DEAE-52 anion exchange column, and elute sequentially with distilled water and gradient concentration NaCl solutions. Collect each fraction, combine the target fractions based on the UV detection results of the sugar content color reaction, and perform concentration, dialysis, and freeze-drying sequentially. Further purify the dried sample using a Sepharose CL-6B gel column, eluting with 0.15 M NaCl solution. Collect and combine the fractions based on the UV detection (sugar content color reaction) results, and perform concentration, dialysis, and freeze-drying sequentially. Verify homogeneity by high performance liquid chromatography. For non-homogeneous polysaccharide components, separate them again using a Sepharose CL-6B gel column with 0.15 M NaCl solution. After elution with NaCl solution, the fractions were combined based on the UV detection results, and the concentration, dialysis, and freeze-drying steps were repeated. The homogeneity and anticomplement activity were tested by high performance liquid chromatography to obtain homogeneous polysaccharides SOPC2-3 and SOPC3 from Siegesbeckia orientalis.

[0015] In step S1, the hot water extraction method is as follows: Siegesbeckia orientalis is extracted with water at a material-to-liquid ratio of 1:15, the extraction temperature is 95-100 ℃, the extraction time is 3-4 h, and the extraction is repeated 3-4 times before the extracts are combined.

[0016] Preferably, in step S1, 4 times the volume of 95% ethanol is added before alcohol precipitation so that the final concentration of ethanol in the extraction solution is 76%.

[0017] Preferably, in step S1, the addition of trichloroacetic acid makes the concentration of trichloroacetic acid in the solution 10%.

[0018] Preferably, in step S2, the gradient concentrations of the NaCl solution used for separation on the DEAE-52 anion exchange column are 0.1 mol / L, 0.5 mol / L, and 1.0 mol / L, respectively.

[0019] Accordingly, the application of *Siegesbeckia orientalis* polysaccharide prepared by the aforementioned method in the preparation of an anticomplement drug. The anticomplement drug is a drug that inhibits cell hemolysis caused by activation of the classical complement pathway.

[0020] Accordingly, the application of *Siegesbeckia orientalis* polysaccharide prepared by the aforementioned method in the preparation of anti-inflammatory drugs. The anti-inflammatory drug is a drug that inhibits the secretion of inflammatory factors induced by lipopolysaccharide.

[0021] Accordingly, the application of *Siegesbeckia orientalis* polysaccharide prepared by the aforementioned method in the preparation of a drug for preventing and treating acute lung injury. The drug for preventing and treating acute lung injury is a drug that inhibits lipopolysaccharide-induced acute lung injury.

[0022] The present invention has the following beneficial effects:

[0023] This invention isolates three polysaccharides from Siegesbeckia orientalis: one total polysaccharide SOP and two homogeneous polysaccharides SOPC2-3 and SOPC3. In vitro experiments confirmed that these polysaccharides possess anti-complement activity and can be further used as active ingredients in the preparation of anti-complement drugs. Cell model experiments confirmed that these polysaccharides have significant anti-inflammatory effects and can be further used as active ingredients in the preparation of anti-inflammatory drugs. Animal experiments confirmed that Siegesbeckia orientalis polysaccharides (SOP and SOPC3) can effectively alleviate LPS-induced acute lung injury in mice and can be further used as active ingredients in the preparation of drugs for the prevention and treatment of acute lung injury. Attached Figure Description

[0024] Figure 1 Flowchart for the separation of homogeneous polysaccharides from Siegesbeckia orientalis;

[0025] Figure 2 The chromatogram of homogeneous polysaccharide from Siegesbeckia orientalis by high-performance gel permeation chromatography (HPGPC);

[0026] Figure 3 The results show the effects of total polysaccharide SOP and homogeneous polysaccharides SOPC2-3 and SOPC3 from Siegesbeckia orientalis on the viability of RAW 264.7 cells;

[0027] Figure 4The results show the effects of total polysaccharide SOP and homogeneous polysaccharides SOPC2-3 and SOPC3 on lipopolysaccharide (LPS)-induced nitric oxide (NO) release in RAW264.7 cells;

[0028] Figure 5 The results show the effects of total polysaccharide SOP from Siegesbeckia orientalis on the total cell count, TNF-α, IL-6, and IL-1β in bronchoalveolar lavage fluid (BALF) of mice with LPS-induced acute lung injury.

[0029] Figure 6 The results show the effects of total polysaccharide SOP from Siegesbeckia orientalis on serum TNF-α and IL-6 in mice with LPS-induced acute lung injury.

[0030] Figure 7 This study presents the effects of SOPC3, a homogeneous polysaccharide from Siegesbeckia orientalis, on TNF-α, IL-6, and IL-1β in lung homogenates from LPS-induced acute lung injury in mice. Detailed Implementation

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0033] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0034] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0035] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0036] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0037] Example 1: Preparation of Siegesbeckia orientalis polysaccharide

[0038] The medicinal herb *Siegesbeckia orientalis* was pulverized and extracted with water at a ratio of 1:15 (material to liquid) using hot water at 100 ℃ for 3 h. This extraction was repeated four times. The extracts were then combined, filtered, concentrated, and centrifuged. Four times the volume of 95% ethanol was added to the supernatant after centrifugation until the ethanol concentration in the supernatant was 76%. The mixture was allowed to stand, centrifuged again, and the precipitate was redissolved in water. Trichloroacetic acid was added until the trichloroacetic acid concentration was 10% to remove free proteins. The supernatant was then collected by centrifugation, adjusted to neutral, and then dialyzed, concentrated, and freeze-dried to obtain the total polysaccharide SOP of *Siegesbeckia orientalis*.

[0039] 72.3 g of total polysaccharide SOP from Siegesbeckia orientalis was dissolved in ultrapure water, centrifuged, and the supernatant was collected. The supernatant was then separated fractionally using a DEAE-52 anion exchange column. Elution was performed sequentially with distilled water and 0.1 mol / L, 0.5 mol / L, and 1.0 mol / L NaCl solutions, with an elution volume greater than twice the column volume and a flow rate of 1.5 mL / min. Each fraction was collected, and the absorbance at 490 nm was measured after colorimetric analysis using the sulfuric acid-phenol method. Based on the UV detection results of the sugar content colorimetric reaction, the fractions were combined, concentrated, dialyzed, and freeze-dried to obtain four secondary components: SOPA, SOPB, SOPC, and SOPD.

[0040] 12.53 g of the polysaccharide fraction SOPC from *Siegesbeckia orientalis* was dissolved in ultrapure water, centrifuged, and the supernatant was collected. The supernatant was then separated using Sepharose CL-6B gel column chromatography, eluted with 0.15 mol / L NaCl solution at a flow rate of 0.3 mL / min. Each fraction was collected, and the absorbance at 490 nm was measured after color development using the sulfuric acid-phenol method. Based on the UV detection results of the sugar content color reaction, the fractions were combined, concentrated, dialyzed, and freeze-dried to obtain three secondary fractions: SOPC1, SOPC2, and SOPC3. SOPC2 was further separated using Sepharose CL-6B gel column chromatography to obtain three secondary fractions: SOPC2-1, SOPC2-2, and SOPC2-3. HPGPC analysis showed that SOPC2-3 and SOPC3 were homogeneous polysaccharides. Figure 1 and 2 As shown.

[0041] Example 2: Structural characterization of Siegesbeckia orientalis polysaccharides

[0042] (1) Determination of molecular weight

[0043] HPGPC was used to determine the relative molecular weight of polysaccharide samples from Siegesbeckia orientalis. The basic principle is that homogeneous polysaccharides form symmetrical chromatographic peaks through gel permeation chromatography, and the elution time is related to the molecular weight. The molecular weight is calculated based on the calibration curve obtained from the known molecular weight.

[0044] Chromatographic conditions: Separation was performed using SHODEX KS-802 (300 × 7.6 mm) and KS-804 (300 × 7.6 mm) tandem gel columns at a flow rate of 0.6 mL / min, an injection volume of 20 μL, a mobile phase of 0.02 mol / L ammonium acetate, a column temperature of 40 ℃, and a refractive index detector (RID).

[0045] Experimental Methods: Accurately weigh 2.0 mg each of total polysaccharide, homogeneous polysaccharide, and dextran standards from *Siegesbeckia orientalis*. Prepare a 2.0 mg / mL solution using 0.02 mol / L ammonium acetate. Filter the solution through a 0.22 μm microporous membrane and record the retention time. Plot a standard curve with the logarithm of the standard molecular weight (Lg) on ​​the ordinate and the retention time on the abscissa. Obtain the corresponding linear regression equation and calculate the relative molecular weight of *Siegesbeckia orientalis* polysaccharides. The molecular weight range of SOP is 8.1-960.7 kDa; the relative molecular weights of SOPC2-3 and SOPC3 are 121.2 kDa and 16.6 kDa, respectively.

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

[0047] The total sugar content of SOP determined by the sulfuric acid-phenol method was 67.3%; the total sugar content of SOPC2-3 was 98.6%; and the total sugar content of SOPC3 was 81.6%.

[0048] The uronic acid content was determined by the m-hydroxybiphenyl method: the uronic acid content of SOP was 21.3%; the uronic acid content of SOPC2-3 was 42.4%; and the uronic acid content of SOPC2-3 was 47.4%.

[0049] Protein content was determined by BCA method: SOP had a protein content of 31.1%; SOPC2-3 contained no protein; SOPC3 had a protein content of 10.9%. An automated amino acid analyzer revealed that the protein in SOPC3 mainly consisted of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, arginine, and proline, with a relative mass ratio of 4.513:1.877:2.125:4.803:2.262:2.204:2.040:4.847.

[0050] (3) Monosaccharide composition analysis

[0051] SOP, SOPC2-3, and SOPC3 were 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.

[0052] SOP is a polysaccharide composed of six monosaccharides, with a monosaccharide molar ratio of mannose:rhamnose:galacturonic acid:glucose:galactose:arabinose = 6.7:7.7:28.9:11.6:29.0:16.1.

[0053] SOPC2-3 is a polysaccharide composed of four monosaccharides, with a molar ratio of rhamnose:galacturonic acid:galactose:arabinose = 16.1:43.6:27.8:12.5.

[0054] SOPC3 is a polysaccharide composed of four monosaccharides, with a molar ratio of rhamnose: galacturonic acid: galactose: arabinose = 11.5: 54.9: 20.7: 12.9.

[0055] Example 3: Classical pathway complement inhibition test

[0056] Serum from 3-month-old guinea pigs was diluted 1:100 with barbiturate buffer (BBS, pH 7.4) to serve as a complement source for classical complement pathway activation. Rabbit anti-sheep erythrocyte antibody was diluted 1:1000 with BBS buffer to serve as hemolysin, and sheep erythrocytes (SRBCs) were prepared as a 2% suspension (v / v). 3 mg of *Siegesbeckia orientalis* polysaccharide was accurately weighed, dissolved in BBS buffer, and serially diluted to obtain eight concentration gradients. 200 μL of each polysaccharide solution was mixed with 200 μL of 1:100 diluted complement and pre-incubated at 37 °C for 10 min. Then, 100 μL of hemolysin (1:1000) and 100 μL of 2% SRBC suspension were added sequentially, and incubation continued at 37 °C for 30 min. After incubation, the reaction tubes were placed in a pre-cooled centrifuge at 4 °C and centrifuged at 5000 rpm for 10 min. 200 μL of supernatant was transferred from each tube to a 96-well plate, and the absorbance (OD value) was measured at 405 nm. The following control groups were also included in the experiment: (1) Polysaccharide control group: 200 μL of polysaccharide solution of the corresponding concentration + 400 μL BBS buffer (used to subtract the background of the polysaccharide itself); (2) Complement control group (100% hemolysis control): 200 μL BBS buffer (replacing polysaccharide) + 200 μL 1:100 complement + 100 μL hemolysin + 100 μL 2% SRBC; (3) Total hemolysis group (100% hemolysis reference): 100 μL 2% SRBC + 500 μL distilled water. The hemolysis inhibition rate was calculated by subtracting the absorbance value of the corresponding Siegesbeckia orientalis polysaccharide control group from the absorbance values ​​of each concentration of Siegesbeckia orientalis polysaccharide group. The formula for calculating the hemolysis inhibition rate is: Hemolysis inhibition rate (%) = [1 - (OD value of the corrected sample group / OD value of the complement control group)] × 100%. A dose-response curve was plotted with the logarithm of the polysaccharide concentration on the x-axis and the hemolysis inhibition rate on the y-axis. The polysaccharide concentration (CH) required to achieve a 50% hemolysis inhibition rate was calculated through curve fitting. 50 (Value). Using heparin as a positive control, the results showed that both total polysaccharides from Siegesbeckia orientalis and two homogeneous polysaccharides from Siegesbeckia orientalis had significant inhibitory activity against classical complement pathway activation, as shown in Table 1.

[0057] Table 1. Inhibitory effect of Siegesbeckia orientalis polysaccharides on complement activation

[0058] Siegesbeckia orientalis polysaccharide SOP SOPC2-3 SOPC3 heparin <![CDATA[CH 50 (μg / mL)]]> 38.4±2.6 749.2±26.6 203.1±0.6 112.1±4.6

[0059] CH 50 The value is expressed as: mean ± SD (n=3).

[0060] Example 4: Effect of Siegesbeckia orientalis polysaccharide on LPS-induced RAW264.7 cell viability

[0061] Mouse macrophages (RAW267.4) were resuspended in DMEM medium at 3 × 10⁻⁶. 4 Cells were seeded at a concentration of 100 μL / well in 96-well plates. They were cultured for 24 h in a 5% CO2, 37 ℃ incubator. After the cells had covered the bottom of the wells, the following drugs were administered: A control group and a group containing Siegesbeckia orientalis polysaccharides (SOP, SOPC2-3, and SOPC3); the drug concentrations were 15.625, 31.25, 62.5, 125, 250, and 500 μg / mL, respectively. Each group had three replicates. After drug administration, the cells were cultured for another 24 h. The next day, 10 μL of MTT reagent (5 mg / mL) was added to each well, and the cells were cultured for another 4 h. The supernatant was discarded, and 100 μL of formazan was added to each well. After shaking for 10 min, the absorbance was measured at 570 nm. The results are as follows: Figure 3 As shown, the polysaccharides SOP, SOPC2-3, and SOPC3 of Siegesbeckia orientalis showed no toxicity to RAW264.7 cells in the range of 15.625-500 μg / mL, and had a certain proliferative effect.

[0062] Example 5: Effect of Siegesbeckia orientalis polysaccharide on LPS-induced NO release in RAW264.7 cells

[0063] Mouse macrophages (RAW267.4) were used at a rate of 3 × 10⁻⁶. 4 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured adherently for 12 h in a cell culture incubator (37 ℃, 5% CO2). The following experimental groups were set up: blank control group, inflammation model group (LPS, 2 μg / mL), Siegesbeckia orientalis polysaccharide group (concentration gradients of 15.625, 31.25, 62.5, 125, and 250 μg / mL), and positive control drug dexamethasone group (DEX, 60 μg / mL). Each group had three replicates, and the corresponding drug or control sample was added and cultured for another 24 h. Except for the blank control group, all other groups were treated with LPS and cultured for another 24 h. The supernatant from each group was collected, and the release of nitric oxide (NO) was detected using a Beyotime reagent kit. Results are as follows: Figure 4 As shown, compared with the normal control group, the NO concentration in RAW267.4 cells in the LPS group was significantly increased ( p <0.001 indicates that LPS stimulation of RAW264.7 cells produced an inflammatory response and excessive cytokine expression; compared with the LPS group, the SOP and SOPC2-3 groups significantly inhibited NO release from RAW267.4 cells in the range of 15.625-250 μg / mL. p <0.01); SOPC3 group significantly inhibited NO release from RAW267.4 cells in the range of 62.5-250 μg / mL ( p(<0.05) In summary, Siegesbeckia orientalis polysaccharides SOP, SOPC2-3, and SOPC3 all exhibit significant anti-inflammatory activity.

[0064] Example 6: Effects of Siegesbeckia orientalis polysaccharide SOP on LPS-induced acute lung injury in mice

[0065] Thirty male BALB / c mice (18-22 g) were randomly assigned to five groups according to body weight: normal group, LPS group, low-dose total polysaccharide group of Siegesbeckia orientalis, high-dose total polysaccharide group of Siegesbeckia orientalis, and dexamethasone positive control group. The administration of drugs to different groups was as follows: the normal group (Normal) and the model group (LPS) were given sodium carboxymethyl cellulose (0.5% CMC-Na); the low-dose total polysaccharide group of Siegesbeckia orientalis (SOP 50) and the high-dose total polysaccharide group of Siegesbeckia orientalis (SOP 100) were given 50 mg / kg and 100 mg / kg SOP, respectively; and the positive control group (DEX) was given 4 mg / kg dexamethasone. Mice were administered the drugs by gavage once daily for two consecutive days. One hour after administration on the second day, except for the normal group mice which were injected intraperitoneally with saline, the other mice were injected intraperitoneally with 10 mg / kg LPS to induce acute lung injury. Six hours after LPS treatment, blood was collected from the eyes of all mice. The blood was centrifuged to obtain mouse serum. 1 mL of physiological saline was repeatedly injected into the lungs of the mice using a syringe to obtain bronchoalveolar lavage fluid. The serum was used to detect inflammatory factor markers, and the bronchoalveolar lavage fluid was used to detect the total number of lung cells and inflammatory factor markers.

[0066] (1) Effect of SOP on the total cell count in bronchoalveolar lavage fluid of mice with LPS-induced acute lung injury

[0067] Mouse bronchoalveolar lavage fluid (BALF) was centrifuged at 3500 rpm for 10 min at 4 ℃, and the supernatant was collected. Cells were centrifuged again to obtain a cell pellet, and then resuspended. 20 μL of the cell suspension was added to 0.4% trypan blue solution and mixed thoroughly. Then, 10 μL was added through the gap between the coverslip and the counting chamber of a hemocytometer, and the total cell count was determined using a cell counter. Results are as follows: Figure 5 As shown in Figure A, both low and high doses of total polysaccharide SOP from Siegesbeckia orientalis significantly inhibited the significantly elevated total cell count in the lungs of mice with LPS-induced acute lung injury. p <0.001), thereby alleviating LPS-induced increase in lung permeability in mice.

[0068] (2) Effects of SOP on TNF-α, IL-6 and IL-β in bronchoalveolar lavage fluid of mice with LPS-induced acute lung injury

[0069] The supernatant from BALF sample centrifugation was used to prepare blank and sample wells according to the instructions of each inflammatory factor detection kit. Each sample was diluted to an appropriate concentration and then added to the kit along with the reagents. The levels of inflammatory factors (TNF-α, IL-6, IL-1β) in each sample were calculated based on the measured absorbance values. Results are as follows: Figure 5 As shown in BD, both low and high doses of total polysaccharide SOP from Siegesbeckia orientalis significantly reduced the expression of inflammatory factors (TNF-α, IL-6, IL-1β, ...) in the bronchoalveolar lavage fluid (BALF) of mice with LPS-induced acute lung injury. p < 0.01).

[0070] (3) Effects of SOP on serum TNF-α and IL-6 in mice with LPS-induced acute lung injury

[0071] Serum samples were centrifuged at 4500 rpm for 10 min at 4 ℃, and the supernatant was collected. The levels of inflammatory factors (TNF-α and IL-6) in each sample were calculated according to the absorbance values ​​measured in the instructions of each inflammatory factor detection kit. The results are as follows: Figure 6 As shown, both low and high doses of total polysaccharide SOP from Siegesbeckia orientalis significantly reduced serum inflammatory factors (TNF-α and IL-6) in mice with LPS-induced acute lung injury. p The expression < 0.01).

[0072] Example 7: Effects of Siegesbeckia orientalis polysaccharide SOPC3 on TNF-α, IL-6 and IL-β in lung tissue of mice with LPS-induced acute lung injury

[0073] Thirty-six male BALB / c mice (18-22 g) were randomly assigned to six groups according to body weight: normal group, LPS group, low-dose Siegesbeckia orientalis polysaccharide group, medium-dose Siegesbeckia orientalis polysaccharide group, high-dose Siegesbeckia orientalis polysaccharide group, and dexamethasone positive control group. The administration of drugs to different groups was as follows: the normal group and the model group (LPS) were given sodium carboxymethyl cellulose (0.5% CMC-Na); the low-dose Siegesbeckia orientalis polysaccharide group (SOPC3 50), the medium-dose Siegesbeckia orientalis polysaccharide group (SOPC3 100), and the high-dose Siegesbeckia orientalis polysaccharide group (SOPC3 200) were given 50 mg / kg, 100 mg / kg, and 200 mg / kg of the homogeneous polysaccharide SOPC3, respectively; and the positive control group (DEX) was given 4 mg / kg of dexamethasone. The mice were administered the drugs by gavage once daily for two consecutive days. One hour after administration on day 2, except for the normal control group mice which were injected intraperitoneally with saline, the other mice were injected intraperitoneally with 10 mg / kg LPS to induce acute lung injury. Six hours after LPS treatment, blood was collected from the eyes of all mice and whole lung tissue was collected. The lung tissue was used to detect inflammatory markers.

[0074] Lung tissue was added to lysis buffer at a ratio of 1:9 (mg / μL), homogenized, and lysed at 4 ℃ for 30 min. After centrifugation at 7000 rpm for 10 min, the supernatant was collected and stored at -80 ℃. Protein content was quantified using a protein assay kit. After boiling at 100 ℃ for 10 min, electrophoresis was performed, followed by membrane transfer. After blocking for 30 min, the membrane was incubated with primary antibody overnight at 4 ℃. The next day, the primary antibody was washed off, and the membrane was incubated with secondary antibody for 1 h. ECL chemiluminescence buffer was added for development, bands were recorded, and semi-quantitative acquisition and statistical analysis were performed using ImageJ software. Results are as follows: Figure 7 As shown, the low-dose and medium-dose SOPC3 groups of Siegesbeckia orientalis polysaccharide significantly reduced the expression level of IL-1β in the lungs of mice with LPS-induced acute lung injury after administration. p <0.001), but had no significant effect on the expression of TNF-α and IL-6; high-dose SOPC3 treatment significantly reduced the expression of inflammatory factors (TNF-α, IL-6, IL-1β) in the lungs of mice with acute lung injury. p <0.05).

Claims

1. Siegesbeckia orientalis polysaccharide, characterized in that, These include total polysaccharide SOP from Siegesbeckia orientalis, homogeneous polysaccharide SOPC2-3 from Siegesbeckia orientalis glycoprotein SOPC3 from Siegesbeckia orientalis, each possessing the following structural characteristics: SOP for total polysaccharides from Siegesbeckia orientalis: Siegesbeckia orientalis was pulverized and extracted with water at a material-to-liquid ratio of 1:15 at 100℃ for 3 hours. This extraction was repeated 4 times. The extracts were then combined, filtered, concentrated, and centrifuged. Four times the volume of 95% ethanol was added to the supernatant after centrifugation until the ethanol concentration in the supernatant was 76%. The mixture was allowed to stand, centrifuged again, and the precipitate was reconstituted with water. Trichloroacetic acid was added until the trichloroacetic acid concentration was 10% to remove free proteins. The supernatant was then centrifuged and adjusted to neutral. The supernatant was then dialyzed, concentrated, and freeze-dried to obtain the SOP for total polysaccharides from Siegesbeckia orientalis. SOPC2-3, a homogeneous polysaccharide from Siegesbeckia orientalis, is a polysaccharide composed of four monosaccharides with a molecular weight of 121.2 kDa. It contains 98.6% total sugar, has a protein content below the BCA detection limit, and a uronic acid content of 42.4%. The monosaccharide molar ratio is rhamnose:galacturonic acid:galactose:arabinose = 16.1:43.6:27.8:12.

5. Siegesbeckia orientalis glycoprotein SOPC3: a polysaccharide composed of four monosaccharides with a molecular weight of 16.6 kDa; total sugar content is 81.6%, protein content is 10.9%, and uronic acid content is 47.4%; the monosaccharide molar ratio is rhamnose: galacturonic acid: galactose: arabinose = 11.5: 54.9: 20.7: 12.9; the protein is mainly composed of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, arginine, and proline, with a relative mass ratio of 4.513: 1.877: 2.125: 4.803: 2.262: 2.204: 2.040: 4.

847.

2. The method for preparing Siegesbeckia orientalis polysaccharide according to claim 1, characterized in that, Includes the following steps: S1. Take the medicinal material Siegesbeckia orientalis, crush it, add water at a material-to-liquid ratio of 1:15 for hot water extraction at 100 ℃ for 3 h. Repeat the extraction 4 times and combine the extracts. Filter, concentrate and centrifuge. Add 4 times the volume of 95% ethanol to the supernatant after centrifugation until the ethanol concentration in the supernatant is 76%. Let stand, centrifuge, redissolve the precipitate after centrifugation with water, add trichloroacetic acid to the trichloroacetic acid concentration to 10% to remove free protein, centrifuge and take the supernatant. Adjust the supernatant to neutral, dialyze, concentrate and freeze dry to obtain Siegesbeckia orientalis total polysaccharide SOP. S2. Dissolve the total polysaccharide SOP obtained from Siegesbeckia orientalis in water, separate it using a DEAE-52 anion exchange column, and elute sequentially with distilled water and NaCl solutions of varying concentrations. Collect each fraction, combine the target fractions based on the UV detection results of the sugar content color reaction, and perform concentration, dialysis, and freeze-drying sequentially. Further purify the dried sample using a Sepharose CL-6B gel column, elute with 0.15 M NaCl solution, collect and combine the fractions based on the UV detection results of the sugar content color reaction, and perform concentration, dialysis, and freeze-drying sequentially. Verify homogeneity by high performance liquid chromatography. For non-homogeneous polysaccharide components, separate them again using a Sepharose CL-6B gel column, elute with 0.15 M NaCl solution, combine the fractions based on the UV detection results, and repeat the concentration, dialysis, and freeze-drying steps. The homogeneity and anticomplement activity of Siegesbeckia orientalis were determined by high performance liquid chromatography, and the homogeneous polysaccharide SOPC2-3 and Siegesbeckia orientalis glycoprotein SOPC3 were obtained.

3. The method for preparing Siegesbeckia orientalis polysaccharide according to claim 2, characterized in that, In step S2, the gradient concentrations of the NaCl solution used for separation on the DEAE-52 anion exchange column are 0.1 mol / L, 0.5 mol / L, and 1.0 mol / L, respectively.

4. The use of total polysaccharide SOP from Siegesbeckia orientalis as described in claim 1 in the preparation of an anticomplement drug.

5. The use of the total polysaccharide SOP, homogeneous polysaccharide SOPC2-3, or glycoprotein SOPC3 of Siegesbeckia orientalis as described in claim 1 in the preparation of anti-inflammatory drugs.

6. The use of the total polysaccharide SOP or SOPC3 of Siegesbeckia or Siegesbeckia orientalis polysaccharide as described in claim 1 in the preparation of a drug for the prevention and treatment of acute lung injury.

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