A uniform polysaccharide of Xuanfeibaidu Chinese medicine composition and its preparation method and application

Abstract

The present invention provides a uniform polysaccharide in a Xuanfei Baidu traditional Chinese medicine composition, and a preparation method and application thereof. The uniform polysaccharides P2-2A and P2-2B are separated from the active polysaccharide components of the Xuanfei Baidu traditional Chinese medicine composition by using extraction, water extraction and alcohol precipitation, membrane dialysis, and anion exchange resin chromatography. The total polysaccharide content of P2-2A is 82.73%, the uronic acid content is 13.11%, the protein content is 0.57%, and the molecular weight distribution range is 1.361×10 4 The total polysaccharide content of P2-2B is 64.75%, the uronic acid content is 29.23%, the protein content is 0.52%, and the molecular weight distribution range is 8.367×10 4 Da; can be used to prepare drugs for treating inflammation, providing another feasible solution for the treatment of inflammation.

Description

Technical Field

[0001] The present invention relates to the technical field of separation and purification of macromolecular polysaccharides in traditional Chinese medicine compounds, in particular to a uniform polysaccharide in a Xuanfei Baidu traditional Chinese medicine composition and a preparation method and application thereof. Background Art

[0002] The Xuanfei Baidu Chinese medicine composition is composed of thirteen Chinese herbs, including ephedra, atractylodes, licorice, knotweed, verbena, reed root, patchouli, tangerine peel, bitter almond, artemisia annua, scutellaria baicalensis, coix seed, and gypsum. It has the effects of promoting lung function and eliminating dampness, clearing heat and expelling pathogens, and purging the lungs and detoxifying. It is suitable for the treatment of mild and common pneumonia caused by the new coronavirus. It mainly contains flavonoids, phenylpropanoids, triterpenoid saponins, sesquiterpenes, iridoids, alkaloids and other ingredients, and has anti-inflammatory, immunomodulatory, antiviral and antioxidant effects. Polysaccharides are commonly found in the cells of animals, plants, and microorganisms. They are polymers formed by connecting monosaccharides. They are widely available and have low cytotoxicity. They participate in and mediate the regulation of various life phenomena and physiological processes of cells. In the study of polysaccharides, the most research-focused ones are traditional Chinese medicine polysaccharides.

[0003] Inflammation is a normal defense reaction of the body and a protective response produced by the body when it is injured. Under normal circumstances, inflammation is beneficial to the body, as it can eliminate harmful stimuli and initiate the healing process. However, prolonged and excessive inflammation can cause great damage to the body. Once the inflammatory response gets out of control, it can damage normal tissues and cause various acute and chronic inflammatory diseases. Currently, there are two main types of anti-inflammatory drugs used in clinical practice, including steroidal anti-inflammatory drugs and non-steroidal anti-inflammatory drugs. Although both drugs have good anti-inflammatory effects, prolonged use or excessive use can cause damage to the body. Therefore, it is particularly important to develop new, highly effective and low-toxic anti-inflammatory drugs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a uniform polysaccharide in a traditional Chinese medicine composition for purifying the lungs and defeating toxicity.

[0005] Another technical problem to be solved by the present invention is to provide a method for preparing the uniform polysaccharide of the Xuanfei Baidu traditional Chinese medicine composition.

[0006] Another technical problem to be solved by the present invention is to provide an application of the uniform polysaccharide in the Xuanfei Baidu Chinese medicine composition.

[0007] The technical solution adopted in the present invention is:

[0008] A method for preparing a uniform polysaccharide in a Xuanfei Baidu traditional Chinese medicine composition, comprising the following specific steps:

[0009] (1) using an extraction method and a water extraction and alcohol precipitation method to treat a Xuanfei Baidu Chinese medicine composition to obtain an extract of the Xuanfei Baidu Chinese medicine composition, wherein the Xuanfei Baidu Chinese medicine composition is composed of ephedra, gypsum, bran-fried atractylodes, patchouli, artemisia annua, polygonum cuspidatum, verbena, coix seed, reed root, scutellaria baicalensis, braised bitter almond, dried tangerine peel, and liquorice;

[0010] (2) using a cell experiment to detect the biological activity of the extract of the Xuanfei Baidu Chinese medicine composition obtained in step (1);

[0011] (3) the active polysaccharide component of the Xuanfei Baidu Chinese medicine composition with anti-inflammatory activity obtained in step (1) is detected as a precipitate component obtained after water extraction and alcohol precipitation, and the precipitate component is added with water and ultrasonicated to be fully dissolved or dispersed;

[0012] (4) extracting the aqueous solution obtained in step (3) with a mixture of n-butanol and dichloromethane to remove substances such as proteins in the polysaccharide component;

[0013] (5) concentrating the aqueous solution after extraction in step (4), and dialyzing it using a dialysis membrane to obtain two crude polysaccharide components of the Xuanfei Baidu Chinese medicine composition, P1 (extra-membrane polysaccharide component) and P2 (intra-membrane polysaccharide component);

[0014] (6) taking the extracellular polysaccharide component and the intracellular polysaccharide component in step (5) and performing dialysis and purification respectively to obtain the purified polysaccharide components P1-1, P1-2 and P2-1, P2-2 of the Xuanfei Baidu Chinese medicine composition;

[0015] (7) The purified polysaccharide component P2-2 of the Xuanfei Baidu Chinese medicine composition in step (6) was further separated and purified using an anion exchange column to obtain uniform polysaccharides P2-2A and P2-2B, wherein the total polysaccharide content of P2-2A was 82.73%, the uronic acid content was 13.11%, the protein content was 0.57%, and the molecular weight distribution range was 1.361×10 4 Da, IR spectrum at 2936.01 cm -1 、1646.97cm -1 、933.88cm -1 and 847.09cm -1 There is a strong absorption peak at the bottom; the total polysaccharide content of P2-2B is 64.75%, the uronic acid content is 29.23%, the protein content is 0.52%, and the molecular weight distribution range is 8.367×10 4 Da, IR spectrum at 2943.29 cm -1 、2891.73cm -1 、2390cm -1 、1568.05cm -1 、898.28cm-1 and 833.06cm -1 There is an absorption peak at.

[0016] The total polysaccharide content of P2-2A is 82.73%, the uronic acid content is 13.11%, the protein content is 0.57%, and the molecular weight distribution range is 1.361×10 4 Da; IR spectrum at 2936.01cm -1 、1646.97cm -1 、933.88cm -1 and 847.09cm -1 The results of PMP-HPLC showed that the monosaccharide composition of P2-2A mainly consisted of mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose, with a content ratio of 26.1:1.6:18.6:29.2:24.4; the results of methylation analysis showed that the types of sugar residues in P2-2A were mainly →4)-Glcp-(1→、Glcp-(1→、→4,6)-Glcp-(1→、→2)-Glcp-(1→、→5)-Ara(f)-(1→) and Ara(f)-(1→, with a molar ratio of 41.651:16.495:9.587:6.220:5.282:5.161; in the H NMR spectrum, C NMR spectrum and HSQC spectrum, the terminal hydrogen region (δ H The signal of 4.6~5.7ppm indicates that there are both α- and β-type sugars in the P2-2A structure. H / δ C The correlation signals of 5.23 / 91.99, 5.05 / 100.69, 4.94 / 106.87 and 4.78 / 98.95 confirmed the connection of four main sugar units such as galactose and mannose in the structure; the Congo red test showed that P2-2A has a triple helical structure; scanning electron microscopy showed that P2-2A has a porous structure with irregular shape and rough surface.

[0017] The total polysaccharide content of P2-2B is 64.75%, the uronic acid content is 29.23%, the protein content is 0.52%, and the molecular weight distribution range is 8.367×10 4 Da; IR spectrum at 2943.29cm -1 、2891.73cm -1 、2390cm -1 、1568.05cm -1 、898.28cm -1 and 833.06cm -1The results of PMP-HPLC showed that the main monosaccharide components of P2-2B were mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose, with a content ratio of 5.5:8.4:4.1:23.5:5.7:29.2:1.4:19.1:3.2; the results of methylation analysis showed that the main types of sugar residues in P2-2B were →4)-GalAp-(1→、→6)-Galp-(1→、→2,4-Rha(p)-(1→、GalAp-(1→) and GlcAp-(1→, with a molar ratio of 38.944:8.508:7.152:6.071:5.012; in the H NMR spectrum, C NMR spectrum and HSQC spectrum, the terminal hydrogen region (δ H The signal of 4.5~5.8ppm indicates that there are both α- and β-type sugars in P2-2B. H / δ C The correlation signals of 4.54 / 102.57, 5.12 / 99.03, 5.08 / 98.97, 5.80 / 106.79, 4.61 / 96.16, 5.33 / 96.19 and 4.65 / 103.36 confirmed the presence of seven sugar units in the structure, including galacturonic acid, galactose, and arabinose; the Congo red test showed that P2-2B has a triple helical structure; scanning electron microscopy showed that P2-2B has a smooth layered structure with a thickness of about 12.3 μm.

[0018] Preferably, in the preparation method of the uniform polysaccharide of the above-mentioned Xuanfei Baidu Chinese medicine composition, the amounts of the components of the Xuanfei Baidu Chinese medicine composition in step (1) are as follows by weight: 6 parts of ephedra, 30 parts of gypsum, 10 parts of fried atractylodes lancea, 15 parts of patchouli, 12 parts of artemisia annua, 20 parts of knotweed, 30 parts of verbena, 30 parts of coix seed, 30 parts of reed root, 15 parts of scutellaria baicalensis, 15 parts of boiled bitter almonds, 15 parts of dried tangerine peel, and 10 parts of licorice.

[0019] Preferably, in the preparation method of the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition, the extraction method in step (1) is to add 8 to 12 times the weight of water to be extracted, ultrasonically assist in uniform dispersion, and use n-butanol with an equal volume to water to extract three times, and combine the three extracts.

[0020] Preferably, in the preparation method of the uniform polysaccharide of the Xuanfei Baidu traditional Chinese medicine composition, the alcohol used in the water extraction and alcohol precipitation method in step (1) is 80% ethanol water solvent, and the mixture is placed in a 4°C environment for alcohol precipitation for 24 hours.

[0021] Preferably, in the preparation method of the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition, the extraction method in step (3) is to mix the active polysaccharide component of the Xuanfei Baidu Chinese medicine composition with distilled water at a solid-liquid ratio of 1 g:15-20 mL, and ultrasonicate for 30 minutes to obtain the extract.

[0022] Preferably, in the method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition, the volume ratio of n-butanol and dichloromethane in step (4) is n-butanol:dichloromethane=1:5, and the number of extractions is 5 times.

[0023] Preferably, in the method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition, the concentration of the aqueous layer in step (5) is to concentrate the extract after extraction in step (4) to 30% to 50% of the original volume.

[0024] Preferably, in the preparation method of the uniform polysaccharide of the above-mentioned Xuanfei Baidu Chinese medicine composition, the step (5) uses a 3.5kDa dialysis membrane to dialyze the polysaccharide component after removing the protein precipitate in step (4) for 3 days, collects the intramembrane and extramembrane solutions, recovers the samples under reduced pressure and freeze-dries them to obtain the intramembrane polysaccharide component P2 and the extramembrane polysaccharide component P1, respectively.

[0025] Preferably, in the preparation method of the uniform polysaccharide of the above-mentioned Xuanfei Baidu Chinese medicine composition, the dialysis in step (6) is to repeatedly dialyze the intramembrane component and extramembrane component solutions in step (5) using 1kDa dialysis membrane and 50kDa dialysis membrane for 3 days, respectively, collect the intramembrane and extramembrane solutions, recover them under reduced pressure and freeze-dry to obtain refined polysaccharide components P1-1, P1-2 and P2-1, P2-2.

[0026] Preferably, in the preparation method of the uniform polysaccharide of the above-mentioned Xuanfei Baidu Chinese medicine composition, the anion exchange column chromatography in the step (7) is further separated and purified by DEAE-52 column chromatography, eluting with 0, 0.1, 0.3 and 0.5M NaCl solutions in sequence, collecting one tube for every 8 mL, determining the content of polysaccharides in the flow fractions, combining, and dialyzing the obtained eluted fractions using a 3.5kDa dialysis membrane for 3 days, recovering the samples under reduced pressure and freeze-drying to obtain the uniform polysaccharides P2-2A and P2-2B of the Xuanfei Baidu Chinese medicine composition.

[0027] A uniform polysaccharide of a traditional Chinese medicine composition for purifying the lungs and defeating toxicity is prepared by the above method.

[0028] Preferably, the purified homogeneous polysaccharide of the Xuanfei Baidu Chinese medicine composition is homogeneous polysaccharide P2-2A and P2-2B, wherein:

[0029] The total polysaccharide content of P2-2A was 82.73%, the uronic acid content was 13.11%, the protein content was 0.57%, and the molecular weight distribution range was 1.361×10 4 Da; IR spectrum at 2936.01cm -1 、1646.97cm -1 、933.88cm -1 and 847.09cm -1 The results of PMP-HPLC showed that the monosaccharide composition of P2-2A mainly consisted of mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose, with a content ratio of 26.1:1.6:18.6:29.2:24.4; the results of methylation analysis showed that the types of sugar residues in P2-2A were mainly →4)-Glcp-(1→、Glcp-(1→、→4,6)-Glcp-(1→、→2)-Glcp-(1→、→5)-Ara(f)-(1→) and Ara(f)-(1→, with a molar ratio of 41.651:16.495:9.587:6.220:5.282:5.161; in the H NMR spectrum, C NMR spectrum and HSQC spectrum, the terminal hydrogen region (δ H The signal of 4.6~5.7ppm indicates that there are both α- and β-type sugars in the P2-2A structure. H / δ C Correlation signals of 5.23 / 91.99, 5.05 / 100.69, 4.94 / 106.87, and 4.78 / 98.95 confirmed the connection of four major sugar units in the structure, including galactose and mannose. Congo red assays revealed that P2-2A has a triple helical structure. Scanning electron microscopy revealed that P2-2A has an irregularly shaped, rough-surfaced porous structure.

[0030] The total polysaccharide content of P2-2B was 64.75%, the uronic acid content was 29.23%, the protein content was 0.52%, and the molecular weight distribution range was 8.367×10 4 Da; IR spectrum at 2943.29cm -1 、2891.73cm -1 、2390cm -1 、1568.05cm -1 、898.28cm -1 and 833.06cm -1The results of PMP-HPLC showed that the main monosaccharide components of P2-2B were mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose, with a content ratio of 5.5:8.4:4.1:23.5:5.7:29.2:1.4:19.1:3.2; the results of methylation analysis showed that the main types of sugar residues in P2-2B were →4)-GalAp-(1→、→6)-Galp-(1→、→2,4-Rha(p)-(1→、GalAp-(1→) and GlcAp-(1→, with a molar ratio of 38.944:8.508:7.152:6.071:5.012; in the H NMR spectrum, C NMR spectrum and HSQC spectrum, the terminal hydrogen region (δ H The signal of 4.5~5.8ppm indicates that there are both α- and β-type sugars in P2-2B. H / δ C The correlation signals of 4.54 / 102.57, 5.12 / 99.03, 5.08 / 98.97, 5.80 / 106.79, 4.61 / 96.16, 5.33 / 96.19 and 4.65 / 103.36 confirmed the presence of seven sugar units in the structure, including galacturonic acid, galactose, and arabinose; the Congo red test showed that P2-2B has a triple helical structure; scanning electron microscopy showed that P2-2B has a smooth layered structure with a thickness of about 12.3 μm.

[0031] The uniform polysaccharide physicochemical analysis and homogeneity determination of the above-mentioned Xuanfei Baidu Chinese medicine composition were carried out to obtain its total polysaccharide content, uronic acid content, protein content, and molecular weight distribution range. IR, PMP-HPLC-DAD, NMR, methylation experiment, Congo red experiment, and scanning electron microscopy were used to further elaborate its chemical structure information and characteristics. It was found that P2-2A is a glucan with a triple helical structure based on →4)-Glcp-(1→ as the main chain, and its surface is an irregular, rough porous structure; P2-2B is a galacturonic acid polysaccharide with a triple helical structure based on →4)-GalAp-(1→ as the main chain, and its surface is a smooth lamellar structure.

[0032] Application of the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition in the preparation of anti-inflammatory drugs.

[0033] The beneficial effects of the present invention are:

[0034] The above-mentioned uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition is obtained by separating the active polysaccharide components of the Xuanfei Baidu Chinese medicine composition through extraction, water extraction and alcohol precipitation, membrane dialysis and anion exchange resin chromatography. It can be used to prepare drugs for treating inflammation, providing another feasible solution for the treatment of inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The figure is a flow chart for the preparation of components of the Xuanfei Baidu Chinese medicine composition;

[0036] Figure 2 The cell viability of macrophage RAW264.7 cells in the presence of different concentrations of XFBD005 samples;

[0037] Figure 3 The results of the measurement of inflammatory indicators IL-1β, IL-6, and TNF-α in macrophage RAW264.7 cells after pretreatment with the component XFBD005;

[0038] Figure 4 The results of the determination of oxidative stress indicators CAT, SOD, GSH-Px enzyme activities and MDA content in macrophage RAW264.7 cells after pretreatment with component XFBD005;

[0039] Figure 5 This is a flow chart for the preparation and separation of polysaccharide components;

[0040] Figure 6 This is the elution curve for homogeneous polysaccharide purification;

[0041] Figure 7 This is a flow chart for the purification of homogeneous polysaccharides;

[0042] Figure 8 UV scanning for uniform polysaccharide;

[0043] Figure 9 It is the infrared absorption spectrum of uniform polysaccharide;

[0044] Figure 10 It is the HPGPC-ELSD chromatogram of homogeneous polysaccharide;

[0045] Figure 11 It is the HPSEC-MALLS-RID chromatogram of homogeneous polysaccharide;

[0046] Figure 12 It is the homogeneous polysaccharide PMP-HPLC-DAD chromatogram (250nm);

[0047] Figure 13 This is the GC-MS total ion chromatogram of uniform polysaccharide after methylation;

[0048] Figure 14 This is a Congo red test image of uniform polysaccharide;

[0049] Figure 15 It is a SEM electron microscope image of uniform polysaccharide;

[0050] Figure 16This is a bar graph showing the effects of samples XFBD005, P2-2A, and P2-2B at concentrations of 5, 10, and 20 μg / mL on the viability of RAW264.7 cells. DETAILED DESCRIPTION

[0051] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. Except for the contents specifically mentioned below, the processes, conditions, reagents, experimental methods, etc. for implementing the present invention are common knowledge and common common sense in the art and are not particularly limited.

[0052] Example 1

[0053] Preparation of components of Xuanfei Baidu Chinese medicine composition and study on its anti-inflammatory activity

[0054] (1) Preparation and separation of components of Xuanfeibaidu Chinese medicine composition

[0055] The preparation and separation of the Xuanfei Baidu Chinese medicine composition (prescription ratio: 6g of ephedra, 30g of gypsum, 10g of bran-fried atractylodes, 15g of patchouli, 12g of artemisia annua, 20g of polygonum cuspidatum, 30g of verbena, 30g of coix seed, 30g of reed root, 15g of scutellaria baicalensis, 15g of braised bitter almond, 15g of tangerine peel, and 10g of licorice) were carried out by solvent extraction, water extraction and alcohol precipitation, and membrane dialysis. The specific steps are as follows: taking the Xuanfei Baidu Chinese medicine composition 360g of the freeze-dried powder (XFBD001) of the extract of the substance was added with 10 times the amount of water (3.6L) and ultrasonically dispersed evenly, and extracted three times with an equal volume (3.6L) of n-butanol. The three extracts were combined and the solvent was recovered under reduced pressure to obtain 277.0g of the extracted water layer component (XFBD002), 64.1g of the n-butanol component (XFBD003) and 17.0g of the insoluble component (XFBD004). 250.0g of the XFBD002 component was taken and re-dissolved with 800mL of water, and 3.2L of anhydrous ethanol was slowly added while constantly stirring the liquid to finally configure the solution into an 80% ethanol solution. It was placed in an environment of 4°C for alcohol precipitation for 24h, filtered and the solvent was recovered to obtain 160.7g of the alcohol precipitation component (XFBD005) and 88.3g of the supernatant component (XFBD006). The specific separation process of the components is as follows. Figure 1 shown.

[0056] (II) Cell experiment to detect the pharmacological activity of the components of the Xuanfei Baidu Chinese medicine composition

[0057] (1) Component sample screening and NO content determination

[0058] RAW264.7 cells were cultured and passaged, and cells in the logarithmic growth phase were selected for subsequent experiments. A blank control group, LPS model group, groups treated with different concentrations of dexamethasone, and a dexamethasone group (50 μg / mL) were set up. The concentrations of the different concentrations of dexamethasone were set at 400 μg / mL and 200 μg / mL, and the component samples were dissolved in DMSO. RAW264.7 cells were plated at 1×105 / well in a 24-well plate and cultured in a constant temperature incubator at 37°C with 5% CO2. After modeling and drug administration, NO content was measured using the Griess method.

[0059] As shown in Table 1, nearly all fraction samples caused significant cell death at 400 μg / mL. Fraction XFBD005 exhibited relatively good activity, exhibited minimal cell death, and exhibited relatively low NO levels. Fractions XFBD003 and XFBD004, despite exhibiting lower NO concentrations, nonetheless exhibited significant cell death. Therefore, subsequent experiments focused on further separation and purification of fraction XFBD005.

[0060] Table 1 Inhibition of cell viability and NO release by XBG components

[0061]

[0062]

[0063] (2) MTT assay to detect the viability of macrophage RAW264.7 cells

[0064] 3×104 cells / well were plated in a 96-well plate, and a blank group and different concentration drug groups were set up. The drug groups were set up with 9 concentrations (1.6, 3.125, 6.25, 12.5, 25, 50, 100, 200, 400 μg / mL), and 6 replicate wells were set up in each group. After culturing in an incubator for 12 hours, 50 μL of MTT was added to each well and the plates were placed in an incubator. After 4 hours, the supernatant was aspirated and 150 μL of DMSO was added. The plates were allowed to stand for 5 minutes, and the absorbance was measured at 450 nm using a microplate reader.

[0065] The XFBD005 component sample at a concentration of 50 μg / mL significantly reduced the viability of RAW264.7 cells and showed great toxicity. Therefore, sample concentrations of 3.125, 6.25, 12.5, and 25 μg / mL were selected for subsequent experiments. The cell viability of macrophage RAW264.7 cells in the presence of different concentrations of XFBD005 samples is shown in Figure 2. Figure 2 shown.

[0066] (3) Determination of inflammatory indicators IL-1β, IL-6, and TNF-α in macrophage RAW264.7 cells

[0067] Based on the results of the MTT assay, four dosing concentrations (3.125, 6.25, 12.5, and 25 μg / mL for XFBD005 component samples) were set for cellular inflammatory factor detection. Cells were plated in 6-well plates at 3×105 / well and six experimental groups were set up: a blank group, a model group, and four dosing concentration groups. After modeling and dosing, cell supernatants were collected for the determination of IL-1β, IL-6, and TNF-α levels (assayed with ELISA kits).

[0068] Compared with the blank group, LPS stimulation significantly increased the levels of IL-1β, IL-6, and TNF-α in macrophages. After pre-treatment with XFBD005 component samples, the levels of IL-1β, IL-6, and TNF-α were significantly reduced, indicating that XFBD005 component samples have good anti-inflammatory effects. The results of the determination of inflammatory indicators IL-1β, IL-6, and TNF-α in macrophage RAW264.7 are as follows: Figure 3 shown.

[0069] (4) Determination of oxidative stress indicators CAT, SOD, GSH-Px enzyme activities and MDA content in macrophage RAW264.7 cells

[0070] Cell plating was performed in the same manner as in step (2). After aspirating the supernatant culture medium, 1 mL of PBS was added to each well. The cells in the six-well plate were scraped and evenly mixed. The plate was centrifuged for 5 min, and the supernatant was aspirated, retaining the cells. 500 μL of lysis buffer was added and the cells were lysed in a 4°C refrigerator for 10 min. The enzyme activities of CAT, SOD, and GSH-Px, as well as the content of MDA, were determined using a kit. Protein concentration was determined using a Bradford kit.

[0071] Compared with the blank group, LPS induction significantly increased the MDA content in macrophages and significantly reduced the activities of CAT, SOD, and GSH-Px enzymes. After pretreatment with the XFBD005 component sample, the MDA content decreased and the activities of CAT, SOD, and GSH-Px enzymes increased significantly, indicating that the XFBD005 component sample can improve LPS-induced cell oxidative stress damage. The results of the determination of oxidative stress indicators CAT, SOD, GSH-Px enzyme activities and MDA content in macrophage RAW264.7 are shown in Figure 2. Figure 4 shown.

[0072] Example 2

[0073] Preparation and structural identification of uniform polysaccharide from Xuanfei Baidu traditional Chinese medicine composition

[0074] (1) Preparation and separation of polysaccharide components of Xuanfei Baidu Chinese medicine composition

[0075] The Xuanfei Baidu Chinese medicine composition was prepared and isolated using solvent extraction and membrane dialysis. The specific steps were as follows: 110 g of the active ingredient sample, XFBD005, was added to 15-20 times the volume of water, sonicated for 30 minutes to dissolve or disperse it, and then poured into a separatory funnel for later use. After stirring and mixing 100 mL of n-butanol and 500 mL of dichloromethane, the mixture was thoroughly shaken in a separatory funnel with the extract and allowed to stand for five consecutive extractions to remove the protein precipitate from the polysaccharide fraction. The aqueous layer was further stripped of solvent and concentrated to approximately 1 L. The deproteinized polysaccharide sample was dialyzed using a 3.5 kDa dialysis membrane for three days. The intramembrane and extramembrane solutions were collected, the solvent was recovered under reduced pressure, and the mixture was freeze-dried to yield the extramembrane fraction (m < 3.5 kDa) P1 (43 g) and the intramembrane fraction (m > 3.5 kDa) P2 (34 g). Take the P1 component (6.4g) and add it to 200mL of water to re-dissolve it, use a 1kDa dialysis membrane to perform repeated dialysis for 3 days, collect the intra-membrane and extra-membrane solutions, recover the solvent under reduced pressure and freeze-dry it to obtain the extra-membrane component (m<1kDa) P1-1 (5.70g) and the intra-membrane component (1kDa<m<3.5kDa) P1-2 (0.44g). Take the P2 component (27g) and add it to 800mL of water to re-dissolve it, use a 50kDa dialysis membrane to perform repeated dialysis for 3 days, collect the intra-membrane and extra-membrane solutions, recover the solvent under reduced pressure and freeze-dry it to obtain the extra-membrane component (3.5kDa<m<50kDa) P2-1 (17.5g) and the intra-membrane component (50kDa<m) P2-2 (8.85g). The specific operation process is as follows. Figure 5 shown.

[0076] (II) Purification of uniform polysaccharide in Xuanfeibaidu Chinese medicine composition

[0077] The uniform polysaccharide of Xuanfei Baidu Chinese medicine composition was purified by anion exchange column chromatography. The specific steps are as follows: the pre-activated DEAE-52 cellulose filler was wet loaded onto a column (50×110 mm) and allowed to settle naturally. After the column chromatography was completed, it was balanced with 5 times the column volume of ultrapure water until there was no alcohol taste and set aside. 400 mg of the polysaccharide component P2-2 sample of the Xuanfei Baidu Chinese medicine composition was weighed, dissolved in a 50 mL centrifuge tube with ultrapure water, centrifuged at 8000 rpm for 10 min, the supernatant was taken and loaded onto the top of the DEAE-52 chromatography column, and eluted with 0, 0.1, 0.3 and 0.5 M NaCl solutions in sequence. One tube was collected for every 8 mL, and the polysaccharide content in the fraction was determined by the phenol-sulfuric acid method. The number of tubes of the received fraction was used as the horizontal coordinate, and the absorbance value A of the sample determined by the phenol-sulfuric acid method was used as the vertical coordinate to depict the elution curve of the polysaccharide fraction, as shown below: Figure 6 The samples of the same elution peak were combined to obtain two elution fractions, namely P2-2A and P2-2B. The solvent was recovered and dialyzed using a 3.5 kDa dialysis membrane for 3 days and freeze-dried. The specific operation process is as follows: Figure 7 shown.

[0078] The obtained Xuanfei Baidu Chinese medicine composition homogeneous polysaccharides P2-2A and P2-2B were subjected to physical, chemical and structural tests:

[0079] 1. Determination of the physicochemical properties of Xuanfei Baidu homogeneous polysaccharide

[0080] (1) Determination of total sugar content of Xuanfei Baidu homogeneous polysaccharide

[0081] The content of Xuanfeibaidu homogeneous polysaccharide was determined by sulfuric acid-phenol method. The specific steps are as follows:

[0082] ① Drawing of standard curve

[0083] Dissolve 20 mg of glucose standard in a 20 mL volumetric flask and dilute to the mark to prepare a 1 mg / mL glucose standard stock solution. Dilute the stock solution to different concentrations of 400, 200, 100, 75, 50, 25, 12.5, and 6.25 μg / mL glucose solutions for later use. Dissolve 0.5000 g of phenol in 10 mL of water and ultrasonically dissolve to prepare a 5% (w / v) phenol solution. Protect from light and set aside. Add 20 μL of glucose solutions of varying concentrations to 20 μL of the newly prepared 5% (w / v) phenol solution and 100 μL of concentrated sulfuric acid solution. Incubate in a 90°C water bath for 30 minutes. After the reaction is complete, remove the solution, cool it, and aspirate 100 μL of the reaction solution. Measure the absorbance (A) at 490 nm using a multifunctional microplate reader. Repeat the experiment three times for each concentration. Draw a standard curve for glucose using the glucose sample concentration as the horizontal axis and the absorbance value A of the reaction solution of samples with different concentrations as the vertical axis.

[0084] ②Calculation of total polysaccharide content

[0085] Take 1 mg of XFBD005 sample and add 10 mL of water to prepare a 100 μg / mL sample solution. Take 20 μL of the 100 μg / mL XFBD005 solution and perform the same steps as above. Measure the absorbance value A1 after the reaction and substitute it into the standard curve to calculate the total sugar concentration C1 in the sample. Calculate the total sugar content in the sample according to the following formula:

[0086]

[0087] Where V is the volume of the sample solution to be tested; M is the weighed weight of the sample to be tested.

[0088] (2) Determination of uronic acid content

[0089] The m-hydroxybiphenyl method was used to determine the uronic acid content in the sample. The specific steps are as follows:

[0090] ① Preparation of the Standard Curve: Dissolve 10.0 mg of galacturonic acid in a 10 mL volumetric flask and dilute to the mark to prepare a 1 mg / mL galacturonic acid stock solution. Dilute this stock solution to 80 μg / mL, 60 μg / mL, 40 μg / mL, 20 μg / mL, and 10 μg / mL galactose solutions for later use. Dissolve 15 mg of m-hydroxybiphenyl in a 5 mg / mL sodium hydroxide solution in a 10 mL volumetric flask and dilute to the mark to prepare a 1.5 mg / mL m-hydroxybiphenyl-sodium hydroxide solution. Dissolve 0.4780 g of solid sodium tetraborate in a 100 mL volumetric flask with concentrated sulfuric acid and dilute to the mark to prepare a 12.5 mM sodium tetraborate-concentrated sulfuric acid solution for later use. Take 200 μL of galacturonic acid solution of varying concentrations and slowly add 1.1 mL of sodium tetraborate-concentrated sulfuric acid solution. Vortex and mix thoroughly. Place in a 90°C boiling water bath for 5 minutes. Remove, cool, and add 20 μL of 1.5 mg / mL m-hydroxybiphenyl-sodium hydroxide solution. Mix thoroughly and let stand for 5 minutes. Aspirate 100 μL of the reaction solution and measure its absorbance (A) at 525 nm using a multifunctional microplate reader. Repeat the experiment three times for each sample concentration. Draw a standard curve for galacturonic acid, with the galacturonic acid sample concentration as the horizontal axis and the absorbance (A) of the reaction solutions of the different sample concentrations as the vertical axis.

[0091] ②Calculation of uronic acid content Take 1 mg of XFBD005 sample and add 10 mL of water to prepare a 100 μg / mL sample solution. Take 200 μL of the 100 μg / mL XFBD005 solution and perform the same steps as above. Measure the absorbance value A1 after the reaction and substitute it into the standard curve to calculate the uronic acid concentration C1 in the sample. Calculate the uronic acid content in the sample according to the following formula:

[0092]

[0093] Where V is the volume of the sample solution to be tested; M is the weighed weight of the sample to be tested.

[0094] (3) Protein content determination

[0095] The protein content in the sample was determined by the Coomassie brilliant blue method. The specific steps are as follows:

[0096] ① Drawing of standard curve

[0097] Take 2 mL of 2 mg / mL fetal bovine serum albumin (FBS) and dilute to the mark in a 50 mL volumetric flask. Dilute to 80 μg / mL FBS solution as a stock solution. Dilute the stock solution to 50 μg / mL, 45 μg / mL, 40 μg / mL, 35 μg / mL, 30 μg / mL, 25 μg / mL, 20 μg / mL, 15 μg / mL, 10 μg / mL, and 5 μg / mL. Take 10 mg of Coomassie Brilliant Blue G-250 and add 5 mL of 95% ethanol solution (v / v) and 10 mL of 85% phosphoric acid solution (v / v) to a 100 mL volumetric flask. Dilute to the mark with water, filter, and store in dark until ready to use. Take 40 μL of FBS solutions of varying concentrations and add 200 μL of Coomassie Brilliant Blue solution. Let the solution stand at room temperature for 15 minutes. Measure the absorbance (A) at 595 nm using a multifunctional microplate reader. The experimental operation was repeated three times for each concentration of sample. The standard curve of bovine fetal serum albumin was drawn with the concentration of bovine fetal serum albumin as the horizontal axis and the absorbance value A of the reaction solution of different concentrations of samples as the vertical axis.

[0098] ②Calculation of protein content

[0099] Take 1 mg of XFBD005 sample and add 10 mL of water to prepare a 100 μg / mL sample solution. Take 40 μL of the 100 μg / mL XFBD005 solution and perform the same steps as above. Measure the absorbance value A1 after the reaction and substitute it into the standard curve to calculate the uronic acid concentration C1 in the sample. Calculate the protein content in the sample according to the following formula:

[0100]

[0101] Where V is the volume of the sample solution to be tested; M is the weight of the sample to be tested.

[0102] (4) Determination of total flavonoid content

[0103] The total flavonoids content in the sample was determined by UV-visible spectrophotometry. The specific steps are as follows:

[0104] ① Drawing of standard curve

[0105] Take 10.0 mg of rutin standard, dissolve it in methanol in a 10 mL volumetric flask and dilute to the mark to complete the preparation of 1 mg / mL rutin stock solution. Dilute the stock solution to 200 mg / mL, 100 mg / mL, 80 mg / mL, 60 mg / mL, 40 mg / mL and 20 mg / mL rutin solutions for later use. Weigh 0.4000 g of solid sodium hydroxide, 0.5000 g of solid sodium nitrite and 1.000 g of solid aluminum nitrate, dissolve them in 10 mL volumetric flasks and dilute to the mark to complete the preparation of 4% (w / v) sodium hydroxide solution, 5% (w / v) sodium nitrite solution and 10% (w / v) aluminum nitrate solution. Take 50 μL of rutin solution of varying concentrations, add 100 μL of 5% sodium nitrite solution, vortex to mix, let stand for 6 minutes, then add 100 μL of 10% aluminum nitrate solution, vortex to mix, let stand for 6 minutes, add 1 mL of 4% sodium hydroxide solution, add 1.25 mL of water, vortex to mix, let stand for 15 minutes, aspirate 200 μL of the reaction solution and measure its absorbance (A) at 500 nm using a multifunctional microplate reader. Repeat the experiment three times for each sample concentration. Draw a standard curve for rutin, with the rutin sample concentration as the horizontal axis and the absorbance (A) of the reaction solution of the different sample concentrations as the vertical axis.

[0106] ②Calculation of total flavonoid content

[0107] Take 1 mg of XFBD005 sample and add 10 mL of water to prepare a 100 μg / mL sample solution. Take 40 μL of the 100 μg / mL XFBD005 sample solution and perform the same steps as above. Measure the absorbance value A1 after the reaction and substitute it into the rutin standard curve to calculate the uronic acid concentration C1 in the sample. Calculate the rutin content in the sample according to the following formula:

[0108]

[0109] Where V is the volume of the sample solution to be tested; M is the weighed weight of the sample to be tested.

[0110] The total sugar content, uronic acid content, and protein content of the polysaccharide components P2-2A and P2-2B of the Xuanfei Baidu traditional Chinese medicine composition were determined, and the results are shown in Table 2.

[0111] Table 2 Physical and chemical determination of homogeneous polysaccharides

[0112]

[0113] 2. UV full wavelength scanning of purified components

[0114] The specific steps of ultraviolet full wavelength scanning of the purified components of the Xuanfei Baidu Chinese medicine composition are as follows:

[0115] An appropriate amount of polysaccharide sample was weighed and dissolved in water to prepare a 0.1 mg / mL polysaccharide solution, which was then subjected to UV scanning in the range of 200 to 400 nm using a multifunctional microplate reader, with water used as a blank.

[0116] The results are as follows Figure 8 As shown, the UV absorption spectra of the two homogeneous polysaccharides are basically consistent with that of blank water, indicating that they contain relatively few UV absorbing components.

[0117] 3. Infrared spectrum scanning of purified components

[0118] A small amount of polysaccharide sample was weighed and dispersed in methanol solution, then added dropwise to a potassium bromide sheet and the solvent evaporated. This process was repeated until an appropriate amount of polysaccharide solid was precipitated on the surface of the potassium bromide sheet. Scanning analysis was performed using a Varian 640 FT-IR Fourier transform infrared spectrometer. The instrument resolution was 4.00 cm -1 , scanning range is 4000~400cm -1 , the number of scans is 32 times.

[0119] The homogeneous polysaccharide samples P2-2A and P2-2B were measured by Fourier transform infrared spectroscopy. Figure 9 As shown. Among them, 3501.84~3286.69cm -1 The broad and strong absorption band at 2936.01 cm is the stretching vibration peak of hydroxyl group. -1 The carbon-hydrogen stretching vibration peak of methyl or methylene is located at 2943.29 and 2891.73 cm-1, respectively. -1 , and at 1568.05cm -1 The characteristic absorption peak of GalA esterification is generated at and above, and it is speculated that P2-2B contains galacturonic acid. P2-2B has a peak at 2390 cm -1 The absorption peak at 1646.97 cm is inferred to be a small amount of unsaturated impurities in P2-2B. -1 The absorption peak at 1411.40 cm -1 The nearby absorption bands also prove the presence of carboxyl groups, indicating that there are a small number of acidic sugar residues in P2-2A. -1 The absorption between 847.09 cm indicates the presence of a pyranose ring. -1 The absorption peak at 933.88 cm indicates the presence of an α-configuration glycosidic bond. -1 and 847.09cm -1 The absorption peak at 898.28 cm indicates that there are both α-glycosidic bonds and β-glycosidic bonds in its structure; similarly, the absorption peak of P2-2B at 898.28 cm-1 and 833.06cm -1 The absorption peak at 761.13~770.82cm indicates that there are both α-glycosidic bonds and β-glycosidic bonds in its structure. -1 The absorption peaks at are C–O–C asymmetric stretching vibration and C–O stretching vibration.

[0120] 4. Determination of molecular weight and homogeneity of purified components

[0121] Take 2 mg of each P2-2A and P2-2B sample, add 1 mL of ultrapure water to dissolve, centrifuge at 14000 rpm for 10 min, filter through a 0.22 μM microporous membrane, and seal in a sample vial for later use.

[0122] The polysaccharide homogeneity of P2-2A and P2-2B was determined using an Agilent 1260II high-performance liquid chromatograph. The chromatographic column was a Waters Ultrahydrogel 2000 (12 μm, 300 × 7.8 mm), the flow rate was 1 mL / min, the mobile phase was water, the elution was isocratic for 30 min, the column temperature was 30°C, the injection volume was 10 μL, and the detector was an evaporative light scattering detector (ELSD).

[0123] The polysaccharide homogeneity and molecular weight range of P2-2A and P2-2B were determined using high-performance gel exclusion chromatography coupled with a multi-angle laser light scattering detector and a differential refractive index detector (HPSEC-MALLS-RID). The column was a Waters Ultrahydrogel 2000 (8 μm, 4.6 × 7.8 mm), the mobile phase was 150 mM ammonium formate, and the elution was isocratic for 30 min at a column temperature of 30°C and a flow rate of 1 mL / min. The detectors were a DAWN 8 HELEOS MALLS multi-angle laser light scattering detector and a RID-20A differential refractive index detector.

[0124] Chromatographic homogeneity analysis was performed on polysaccharide samples P2-2A and P2-2B of the Xuanfei Baidu Chinese medicine composition. The chromatograms detected by high performance gel exclusion chromatography (HPGPC-ELSD) and HPSEC-MALLS-RID were as follows: Figure 10 and Figure 11 As shown in the figure, the chromatographic peaks of polysaccharide samples P2-2A and P2-2B under HPGPC-ELSD are well symmetrical. After HPSEC-MALLS-RID detection, the corresponding molecular weights Mw of the two samples are 1.361×104 (±13.867%) Da and 8.367×104 (±81.616%) Da, respectively. The symmetry-related values ​​Mw / Mn are 1.561 (±21.433%) and 1.698 (±113.09%), respectively, indicating that the samples have good symmetry and the two polysaccharide samples can be considered to be homogeneous polysaccharides.

[0125] 5. Monosaccharide composition and distribution detection

[0126] The monosaccharide composition and distribution of the uniform polysaccharide isolated from the Xuanfei Baidu Chinese medicine composition were analyzed using the PMP-HPLC-DAD method. The specific steps are as follows:

[0127] ① Weigh 1 mg each of ribose, rhamnose, glucosamine, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose, dissolve them in 1 mL of ultrapure water to prepare a 1 mg / mL standard monosaccharide solution, and pipette 100 μL of each solution to prepare a 1 mg / mL monosaccharide mixed standard solution.

[0128] ② Weigh 2.175g of 1-phenyl-3-methyl-5-pyrazolone (PMP) powder and dissolve to the mark in methanol in a 25mL volumetric flask to prepare a 0.5M PMP solution. Weigh 0.6g of sodium hydroxide solid and dissolve to the mark in water in a 25mL volumetric flask to prepare a 0.6M sodium hydroxide solution. Pipette 1.25mL of concentrated hydrochloric acid and 3.71mL of trichloroacetic acid into a 25mL volumetric flask and dilute to the mark with water to prepare a 0.6M hydrochloric acid solution and a 2M trichloroacetic acid solution.

[0129] ③ Take 200 μL of the monosaccharide mixed standard solution in a glass container, add 200 μL of 0.6 M sodium hydroxide solution and 400 μL of 0.5 M PMP solution, shake well, react in a 70°C water bath for 3 h, take out and cool, add 200 μL of 0.6 M hydrochloric acid solution, and then add 1 mL of chloroform to extract, repeat 3 times, retain the aqueous layer, centrifuge at 14000 rpm for 10 min, filter through a 0.22 μM microporous filter membrane, and seal in a sample vial for later use.

[0130] ④ Weigh 2 mg each of the homogeneous polysaccharides P2-2A and P2-2B, add 2.5 mL of 2M trichloroacetic acid solution, and react in a 100°C water bath for 8 h. After completion of the reaction, remove the sample, cool it, remove the solvent with nitrogen purge, add 1 mL of methanol to remove residual reagents, and purge dry with nitrogen purge. Repeat this three times. After acid hydrolysis, dissolve the dried sample in 200 μL of water and perform the same PMP derivatization procedure as described above for the monosaccharide standard solution.

[0131] ⑤ The monosaccharide composition and content of the PMP-derived samples were analyzed using an Agilent 1260II high performance liquid chromatograph. The chromatographic column was Cosmosil 5C 18-MS-Ⅱ (1.7μm, 150mm×4.6mm), the mobile phase was 0.05mM ammonium acetate solution and acetonitrile, the elution program was 17% acetonitrile isocratic elution for 30min, the column temperature was 30℃, the flow rate was 1mL / min, the sample volume was 10μL, and the detection wavelength was 250nm.

[0132] The chromatograms of the homogeneous polysaccharide samples P2-2A and P2-2B on HPLC-DAD after acid hydrolysis and PMP derivatization are as follows: Figure 12 As shown. By comparing the retention times of the chromatograms after derivatization with a mixed monosaccharide standard, it can be seen that sample P2-2A is mainly a polysaccharide composed of glucose and arabinose. The specific monosaccharides included are mannose, rhamnose, glucuronic acid, glucose, galactose, and arabinose, with a content ratio of 26.1:1.6:18.6:29.2:24.4. Sample P2-2B is mainly a polysaccharide composed of galacturonic acid, galactose, and arabinose. The specific monosaccharides included are mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose, with a content ratio of 5.5:8.4:4.1:23.5:5.7:29.2:1.4:19.1:3.2.

[0133] 6. Methylation analysis

[0134] Weigh 10 mg of P2-2B sample and dissolve it in 1 mL of water. Add 1 mL of 100 mg / mL carbodiimide and react for 2 hours. Add 1 mL each of 2 M imidazole, 30 mg / mL NaBH₄, and 30 mg / mL NaBD₄ and react for 3 hours. Terminate the reaction by adding 100 μL of glacial acetic acid. Dialyze the sample for 48 hours. After dialysis, freeze-dry the sample and proceed to methylation.

[0135] Dissolve the freeze-dried samples of P2-2A and P2-2B in 500 μL of DMSO. Add 1 mg of solid NaOH and incubate for 30 minutes. Add 50 μL of iodomethane solution and react for 1 hour. After the reaction is complete, add 1 mL of water and 2 mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Aspirate the lower dichloromethane phase and evaporate to dryness. Add 100 μL of 2M trifluoroacetic acid and react at 121°C for 90 minutes. After the reaction is complete, evaporate to dryness at 30°C. Add 50 μL each of 2M ammonia and 1M NaBD4, mix well, and react at room temperature for 2.5 hours. After the reaction is complete, terminate the reaction with 20 μL of acetic acid, blow dry with nitrogen, wash twice with 250 μL of methanol, and blow dry with nitrogen. Add 250 μL of acetic anhydride, vortex to mix, and react at 100°C for 2.5 hours. Add 1 mL of water and let stand for 10 minutes. Add 500 μL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash three times. Prepare the dichloromethane layer for analytical sample solution and set aside.

[0136] The composition of various methylated samples was analyzed using an Agilent 7890A gas chromatograph. The chromatographic column was an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm), and the carrier gas was high-purity helium (≥99.999%) at a flow rate of 1.0 mL / min. The inlet temperature was 260°C. The injection volume was 1 μL, and a split injection with a split ratio of 10:1 and a solvent delay of 2.2 min was used.

[0137] Temperature program: 50°C, hold for 1.0 min, increase the temperature at 50°C / min to 130°C, increase the temperature at 3°C / min to 230°C, and hold for 2 min.

[0138] Mass spectrometry system: Agilent 5977B quadrupole mass spectrometer (Agilent, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. The EI source had an inlet temperature of 230°C, a quadrupole temperature of 150°C, and an electron energy of 70 eV. The scan mode was full scan (SCAN), with a mass scan range (m / z) of 30–600.

[0139] The total ion current of the homogeneous polysaccharide samples P2-2A and P2-2B after methyl iodide methylation, hydrolysis, reduction and acetylation after GC-MS detection is as follows Figure 13 The analysis results are shown in Table 3. Compared with the standard mass spectrum library, it can be seen that the main types of sugar residues in P2-2A are →4)-Glcp-(1→, Glcp-(1→, →4,6)-Glcp-(1→, →2)-Glcp-(1→, →5)-Ara(f)-(1→ and Ara(f)-(1→), with a molar ratio of 41.651:16.495:9.587:6.220:5.282:5.161; the main types of sugar residues in P2-2B are →4)-GalAp-(1→, →6)-Galp-(1→, →2,4-Rha(p)-(1→, GalAp-(1→) and GlcAp-(1→), with a molar ratio of 38.944:8.508:7.152:6.071:5.012.

[0140] It can be preliminarily inferred that the main chain structure of P2-2A is neutral glucan connected by →4)-Glcp-(1→, Glcp-(1→, →4,6)-Glcp-(1→, →2)-Glcp-(1→, and the side chain structure includes →5)-Ara(f)-(1→, Ara(f)-(1→, →2)-Manp-(1→, →4)-Galp-(1→ and →3,5)-Ara(f)-(1→. The main chain structure of P2-2B is →4)-GalAp-(1→, and the side chain structure Including →6)-Galp-(1→、→2,4-Rha(p)-(1→、GalAp-(1→、GlcAp-(1→、Galp-(1→、→2)-Rhap-(1→、→5)-Ara(f)-(1→、Ara(f)-(1→ and →4)-Galp-(1→. Since GalA was reduced before methylation reduction, the terminal, 1-3 and 1-4 related methylation derivatives may be derived from the existing Gal and / or GalA reduction.

[0141] Methylation analysis was performed on the homogeneous polysaccharide of the Xuanfei Baidu Chinese medicine composition, and the results are shown in Table 3.

[0142] Table 3 Methylation results of homogeneous polysaccharides

[0143]

[0144]

[0145]

[0146] 7. Nuclear magnetic resonance spectroscopy

[0147] 50 mg of P2-2A sample was dissolved in 0.5 mL of DMSO, and an equal amount of P2-2B sample was dissolved in 0.5 mL of D2O for NMR analysis. 1 H NMR, 1 H- 1 H COSY and TOCSY NMR spectra were measured on a Bruke AV III 500 MHz operating at 500 MHz. 13 C NMR, 1 H- 13 C HSQC, TOCSY, HMBC, NOESY and other nuclear magnetic resonance spectra.

[0148] The NMR spectra provided the primary structural information of the homogeneous polysaccharides P2-2A and P2-2B.

[0149] Terminal hydrogen region (δ HThe signals (4.6 - 5.7 ppm) indicate the simultaneous presence of both α- and β-configurations in P2-2A. According to the HSQC spectrum analysis 1 H and 13 C signal chemical shifts of the anomeric hydrogen and anomeric carbon at the termini, a total of 4 sugar units were found, which are δ H / δ C 5.23 / 91.99, 5.05 / 100.69, 4.94 / 106.87, and 4.78 / 98.95, corresponding to α-Glcp-(1→, →4)-α-Glcp-(1→, →5)-α-Araf-(1→, and β-Xylp-(1→ respectively. According to the results of its monosaccharide composition, P2-2A is mainly composed of Man, Glc, Gal, and Ara. The above sugar units in the NMR correspond to t-Glc(p), 4-Glc(p), 5-Ara(f), and t-Xyl(p) in the methylation analysis results. Based on the above monosaccharides, it shows that P2-2A is mainly glucan. A detailed analysis of the residues of P2-2A was carried out, and its 1 H and 13 C chemical shift assignments are shown in Table 4.

[0150] Similarly, the signals in the anomeric hydrogen region (δ H 4.5 - 5.8 ppm) indicate the simultaneous presence of both α- and β-configurations in P2-2B. According to the HSQC spectrum analysis 1 H and 13 C signal chemical shifts of the anomeric hydrogen and anomeric carbon at the termini, a total of 7 sugar units were found, which are δ H / δ C 4.54 / 102.57, 5.12 / 99.03, 5.08 / 98.97, 5.80 / 106.79, 4.61 / 96.16, 5.33 / 96.19, and 4.65 / 103.36, corresponding to β-GlcAp-(1→, →2,4-α-Rhap-(1→, →6)-β-Galp-(1→, →4)-α-GalAp-(1→, β-GalAp-(1→, α-GalAp-(1→, and →5)-α-Araf-(1→ respectively. According to the results of its monosaccharide composition, P2-2B is mainly composed of xylose, arabinose, fucose, Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara, and Fuc. Based on the above information, it shows that P2-2B is mainly galacturonan. A detailed analysis of the residues of P2-2B was carried out, and its 1 H and 13 C chemical shift assignments are shown in Table 5.

[0151] Table 4 NMR Signals of P2-2A Sugar Residues

[0152]

[0153] Table 5 NMR signals of sugar residues of P2-2B

[0154]

[0155] 8. Congo Red Test

[0156] The Congo red experiment was used to analyze the triple helical structure of the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition. The specific operation is as follows: take 2 mg each of the uniform polysaccharides P2-2A and P2-2B of the Xuanfei Baidu Chinese medicine composition, add ultrapure water to make it into a 2 mg / mL aqueous solution, take 20 μM of each sample solution, add 20 μM of 80 mM Congo red solution and 100 μM of sodium hydroxide solution with different concentration gradients (0-1.0 M), vortex and oscillate to mix thoroughly, and react at room temperature for 10 minutes, draw 100 μL of the reaction solution, and use a multifunctional microplate reader to measure its maximum absorption wavelength (λ max The same experimental procedures were repeated three times for each sample using aqueous solutions instead of samples at different sodium hydroxide concentrations. The hydrolysis curve of the polysaccharide triple helix structure was plotted using the added sodium hydroxide concentration as the horizontal axis and the measured maximum absorption wavelength of the sample as the vertical axis.

[0157] The results of the triple helix determination of the homogeneous polysaccharide samples P2-2A and P2-2B by Congo red test are as follows Figure 14 As shown in Figure 2, the results for the homogeneous polysaccharides P2-2A and P2-2B in the Xuanfei Baidu Chinese medicine composition were compared with the blank control (Congo red solution). As the NaOH concentration increased, the maximum absorption wavelength of the polysaccharide samples increased, resulting in a red shift. When the NaOH concentration reached a certain level, the maximum absorption wavelength decreased, resulting in a blue shift. These observations indicate that the homogeneous polysaccharides P2-2A and P2-2B in the Xuanfei Baidu Chinese medicine composition possess a triple helical structure.

[0158] 9. Scanning electron microscopy analysis

[0159] Homogeneous polysaccharide samples P2-2A and P2-2B were freeze-dried, and an appropriate amount of the solid was applied to a black conductive adhesive. The suspended solid was removed using an earbud, and the samples were gold-sprayed. The morphological characteristics of the homogeneous polysaccharides were observed using a scanning electron microscope (SEM), and images were collected from representative fields. The SEM observation parameters used were an operating voltage of 5 kV and a magnification of 50-1000x.

[0160] After the homogeneous polysaccharide samples P2-2A and P2-2B were observed by scanning electron microscopy, the periscopic structures of the two samples were observed at different magnifications of 100 to 1000, as shown in Figure 2. Figure 15It can be clearly seen from the figure that the uniform polysaccharide P2-2A of the Xuanfei Baidu Chinese medicine composition is a porous structure with an irregular shape and a rough surface, while P2-2B is a layered structure with a smooth surface and a thickness of about 12.3 μm.

[0161] Example 3

[0162] Study on the anti-inflammatory activity of uniform polysaccharide from Xuanfei Baidu traditional Chinese medicine composition

[0163] (1) Experimental groups and research methods

[0164] 1. RAW264.7 cell viability assay, the specific steps are as follows:

[0165] RAW264.7 cell viability was determined using the MTT assay. RAW264.7 cells (105 cells / well) were seeded into 96-well plates and incubated at 37°C in a 5% CO2 incubator for 12 hours. The cells were then treated with various concentrations of XFBD005 (5, 10, and 20 μg / mL), P2-2A (5, 10, and 20 μg / mL), and P2-2B (5, 10, and 20 μg / mL) for 12 hours. Blank control wells were treated with DMSO. Subsequently, 50 μL of a 2.5 mg / mL MTT solution was added to each well. After incubation for 4 hours, the supernatant was discarded, and 150 μL of DMSO was added to each well. The mixture was mixed and allowed to stand for 5 minutes. OD values ​​at 490 nm were measured using a microplate reader (Bio-Tek) to calculate cell viability.

[0166] 2. Establishment of LPS-induced RAW264.7 cell inflammation model and grouped drug administration

[0167] RAW 264.7 cells were seeded in 96-well plates for 12 hours. The cells were then treated with various doses of P2-2A, P2-2B, and dexamethasone for 12 hours. Subsequently, MTT solution was added for 4 hours. The absorbance of each well was measured at 490 nm using a microplate reader (Bio-Tek). RAW 264.7 cells (3×105 cells / well) were seeded in 6-well plates and incubated for 12 hours. Subsequently, the cells were treated with various concentrations of XFBD005 (5, 10, and 20 μg / mL), P2-2A (5, 10, and 20 μg / mL), and P2-2B (5, 10, and 20 μg / mL) for 12 hours. Blank and positive controls were treated with DMSO and dexamethasone (50 μg / mL), respectively. Subsequently, the culture medium was aspirated, and the cells were induced with LPS (1 μg / mL) for 24 hours to establish an inflammatory model.

[0168] 3. Determination of NO content in LPS-induced RAW264.7 cells

[0169] Following the establishment of a RAW264.7 cell inflammatory model and group-administered medication as described in step 2 above, NO levels were determined using the Griess method. The procedure was as follows: 50 μL of RAW264.7 cell culture supernatant (per well) was placed in a 96-well plate. Equal volumes of Griess reagents I and II were mixed with the cell culture supernatant. The OD value was measured at 540 nm using a microplate reader (Bio-Tek) to calculate the NO content in the cells.

[0170] 4. Determination of the content of inflammatory-related factors in LPS-induced RAW264.7 cells

[0171] According to previous studies, when the concentrations of samples XFBD005, P2-2A, and P2-2B were 5, 10, and 20 μg / mL, there was no significant effect on the viability of RAW264.7 cells, indicating that the samples were not cytotoxic to RAW264.7 cells at concentrations of 5 to 20 μg / mL. Based on this, concentrations of 5, 10, and 20 μg / mL were selected for the determination of the content of inflammatory factors IL-1β, IL-6, and TNF-α. The specific operation steps are as follows: Similarly, refer to 2 to establish a RAW264.7 cell inflammation model and group administration, collect cell supernatants, and detect the content of IL-1β, IL-6, and TNF-α in the cells according to the ELISA kit instructions.

[0172] 5. Determination of LPS-induced oxidative stress levels in RAW264.7 cells

[0173] Following the establishment of the RAW264.7 cell inflammatory model and group dosing as described in Step 2 above, the supernatant culture medium was discarded and cells from each group were collected. 500 μL of lysis buffer was added to the cells and lysed in a refrigerator at 4°C for 10 minutes. After complete lysis, the cells were centrifuged at 12,000 rpm and 4°C for 10 minutes. The supernatant was then collected and the total protein concentration was determined using the Bradford assay. Subsequently, the RAW264.7 cell lysate was assayed for CAT, SOD, GSH-Px enzyme activities, and MDA content according to the assay kit instructions.

[0174] 6. Data Analysis

[0175] Experimental data were analyzed using GraphPad Prism software and expressed as mean ± SD (n = 3). Differences between groups were determined using one-way ANOVA followed by Tukey's post hoc test, and p < 0.05 was considered statistically significant.

[0176] (2) Activity study results and discussion

[0177] 1. Effects of XFBD005, P2-2A, and P2-2B on RAW264.7 cell viability

[0178] The experimental results are as follows Figure 16 As shown in the figure, compared with the CON group, XFBD005, P2-2A, and P2-2B samples (5-20 μg / mL) had no significant effect on RAW264.7 cell viability (p>0.05), indicating that XFBD005, P2-2A, and P2-2B samples at concentrations of 5-20 μg / mL were not cytotoxic to RAW264.7 cells. Based on this, concentrations of 5, 10, and 20 μg / mL were selected for subsequent related experiments.

[0179] 2. Effects of XFBD005, P2-2A, and P2-2B on NO Release in RAW264.7 Cells

[0180] The experimental results showed that compared with the CON group, the NO content in LPS-induced RAW264.7 macrophages was significantly increased (p<0.01). However, after pre-administration of 5-20 μg / mL of XFBD005, P2-2A, and P2-2B, the NO content in each treatment group was significantly reduced (p<0.05 or p<0.01). In addition, compared with the XFBD-005 group, the NO content in RAW264.7 macrophages in the P2-2A and P2-2B groups was significantly reduced (p<0.05 or p<0.01), indicating that P2-2A and P2-2B samples have a more significant inhibitory effect on LPS-induced NO content in RAW264.7 macrophages.

[0181] 3. Determination of the content of inflammatory factors in LPS-induced RAW264.7 cells by XFBD005, P2-2A and P2-2B.

[0182] Table 6 Determination of inflammatory cytokine levels in LPS-induced RAW264.7 cells by XFBD005, P2-2A, and P2-2B

[0183]

[0184]

[0185] Compared with the CON group, #p<0.05, ##p<0.01. Compared with the LPS group, *p<0.05, **p<0.01.

[0186] Compared with the XFBD-005 group (5μg / mL), the P2-2A group: ☆ p<0.05, ☆☆ p<0.01. P2-2B group: ★ p<0.05, ★★ p<0.01.

[0187] Compared with the XFBD-005 group (10 μg / mL), the P2-2A group: $ p<0.05, $$ p<0.01. P2-2B group: & p<0.05, && p<0.01.

[0188] Compared with the XFBD-005 group (20 μg / mL), the P2-2A group: ￥ p<0.05, ￥￥ p<0.01. P2-2B group: γ p<0.05, γγ p<0.01.

[0189] The results in Table 6 show that LPS can stimulate the release of IL-1β, IL-6, and TNF-α in RAW264.7 cells. Compared with the CON group, the IL-1β, IL-6, and TNF-α contents in the LPS group were significantly increased (p<0.01). After pre-administration protection of each sample (5, 10, and 20 μg / mL), the IL-1β, IL-6, and TNF-α contents were significantly decreased (p<0.05 or p<0.01). However, when the concentration of XFBD-005 was 5 μg / mL, the inhibitory effect on TNF-α was not obvious. In addition, compared with the XFBD-005 (5, 10, 20 μg / mL) group, the inflammatory factor levels in the P2-2A (5, 10, 20 μg / mL) and P2-2B (5, 10, 20 μg / mL) groups were significantly reduced, with significant differences (p<0.05 or p<0.01), demonstrating that the P2-2A and P2-2B samples had a more significant inhibitory effect on the expression levels of LPS-induced macrophage inflammatory factors.

[0190] 4. Effects of XFBD005, P2-2A, and P2-2B on LPS-induced oxidative stress in RAW264.7 cells

[0191] Table 7 Effects of XFBD005, P2-2A and P2-2B on LPS-induced oxidative stress in RAW264.7 cells

[0192]

[0193]

[0194] Compared with the CON group, #p<0.05, ##p<0.01. Compared with the LPS group, *p<0.05, **p<0.01.

[0195] Compared with the XFBD-005 group (5μg / mL), the P2-2A group: ☆ p<0.05,☆☆ p<0.01. P2-2B group: ★ p<0.05, ★★ p<0.01.

[0196] Compared with the XFBD-005 group (10 μg / mL), the P2-2A group: $ p<0.05, $$ p<0.01. P2-2B group: & p<0.05, && p<0.01.

[0197] Compared with the XFBD-005 group (20 μg / mL), the P2-2A group: ￥ p<0.05, ￥￥ p<0.01. P2-2B group: γ p<0.05, γγ p<0.01.

[0198] The results in Table 7 show that compared with the CON group, the LPS group showed a significant increase in MDA content and a significant decrease in CAT, SOD, and GSH-Px enzyme activities (p < 0.01). Pre-administration of XFBD005, P2-2A, and P2-2B samples (5, 10, and 20 μg / mL) significantly reduced MDA content (p < 0.05 or p < 0.01), while significantly increasing CAT, SOD, and GSH-Px enzyme activities (p < 0.05 or p < 0.01). Furthermore, the protective effects of P2-2A and P2-2B on CAT enzyme activity were superior to those of all XFBD005 dose groups. Notably, at a concentration of 20 μg / mL, only P2-2B significantly decreased MDA content (p < 0.05) and increased GSH-Px enzyme activity (p < 0.05), demonstrating that the components of the P2-2B sample possess a more pronounced antioxidant effect.

[0199] The above-described embodiments are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements to the technical solutions of the present invention made by ordinary engineers and technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A homogeneous polysaccharide composition of a traditional Chinese medicine for Xuanfei Baidu, characterized by: It is a homogeneous polysaccharide P2-2A or P2-2B, wherein The total polysaccharide content of P2-2A was 82.73%, the uronic acid content was 13.11%, the protein content was 0.57%, and the molecular weight Mw was 1.361×10 4 Da; IR spectrum at 2936.01cm -1 、1646.97cm -1 、933.88cm -1 and 847.09cm -1 There is a strong absorption peak at the end of the experiment; the results of PMP-HPLC showed that the monosaccharide composition of P2-2A mainly included mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose, with a content ratio of 26.1:1.6:18.6:29.2:24.4; the sugar residue types of P2-2A were mainly →4)-Glcp-(1→, Glcp-(1→, →4,6)-Glcp-(1→, →2)-Glcp-(1→, →5)-Ara(f)-(1→ and Ara(f)-(1→, with a molar ratio of 41.651:16.495:9.587:6.220:5.282:5.161; P2-2A has a triple helical structure; The total polysaccharide content of P2-2B was 64.75%, the uronic acid content was 29.23%, the protein content was 0.52%, and the molecular weight Mw was 8.367×10 4 Da; IR spectrum at 2943.29cm -1 、2891.73cm -1 、2390cm -1 、1568.05cm -1 、898.28cm -1 and 833.06cm -1 The results of PMP-HPLC showed that the main monosaccharide components of P2-2B were mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose, with a content ratio of 5.5:8.4:4.1:23.5:5.7:29.2:1.4:19.1:3.2; the main sugar residue types of P2-2B were →4)-GalAp-(1→, →6)-Galp-(1→, →2,4-Rha(p)-(1→, GalAp-(1→) and GlcAp-(1→, with a molar ratio of 38.944:8.508:7.152:6.071:5.012; P2-2B had a triple helical structure.

2. The homogeneous polysaccharide according to claim 1, characterized in that: Scanning electron microscopy showed that P2-2A had a porous structure with irregular shape and rough surface; P2-2B had a layered structure with smooth surface and a thickness of about 12.3 μm.

3. The method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 1, characterized in that: The specific steps are as follows: (1) using an extraction method and a water extraction and alcohol precipitation method to treat a Xuanfei Baidu Chinese medicine composition to obtain an extract of the Xuanfei Baidu Chinese medicine composition, wherein the Xuanfei Baidu Chinese medicine composition is composed of ephedra, gypsum, bran-fried atractylodes, patchouli, artemisia annua, polygonum cuspidatum, verbena, coix seed, reed root, scutellaria baicalensis, braised bitter almond, dried tangerine peel, and liquorice; (2) using a cell experiment to detect the biological activity of the extract of the Xuanfei Baidu Chinese medicine composition obtained in step (1); (3) the active polysaccharide component of the Xuanfei Baidu Chinese medicine composition with anti-inflammatory activity obtained in step (1) is detected as a precipitate component obtained after water extraction and alcohol precipitation, and the precipitate component is added with water and ultrasonicated to be fully dissolved or dispersed; (4) extracting the aqueous solution obtained in step (3) using a mixture of n-butanol and dichloromethane; (5) The aqueous layer solution after extraction in step (4) was concentrated, and the polysaccharide component after removing the protein precipitate in step (4) was dialyzed for 3 days using a 3.5 kDa dialysis membrane, and the intramembrane and extramembrane solutions were collected. The samples were recovered under reduced pressure and freeze-dried to obtain the extramembrane polysaccharide component P1 and the intramembrane polysaccharide component P2, respectively; (6) The extramembrane polysaccharide fraction P1 and the intramembrane polysaccharide fraction P2 solutions in step (5) were dialyzed repeatedly for 3 days using a 1 kDa dialysis membrane and a 50 kDa dialysis membrane, respectively. The intramembrane and extramembrane solutions were collected, recovered under reduced pressure, and freeze-dried to obtain the extramembrane purified polysaccharide fraction P1-1 and the intramembrane fraction P1-2 of P1, and the extramembrane purified polysaccharide fraction P2-1 and the intramembrane fraction P2-2 of P2; (7) The refined polysaccharide component P2-2 of the Xuanfei Baidu Chinese medicine composition in step (6) was further separated and purified using an anion exchange column to obtain uniform polysaccharides P2-2A and P2-2B.

4. The preparation method according to claim 3, characterized in that The amounts of the components of the Xuanfei Baidu Chinese medicine composition in step (1) are as follows by weight: 6 parts of ephedra, 30 parts of gypsum, 10 parts of fried atractylodes lancea, 15 parts of patchouli, 12 parts of artemisia annua, 20 parts of knotweed, 30 parts of verbena, 30 parts of coix seed, 30 parts of reed root, 15 parts of scutellaria baicalensis, 15 parts of boiled bitter almonds, 15 parts of dried tangerine peel, and 10 parts of licorice.

5. The preparation method according to claim 3, wherein: The extraction method in step (1) is to add 8 to 12 times the weight of the composition to be extracted with water and ultrasonically assist in uniform dispersion, and use n-butanol with an equal volume to water to extract three times, and combine the three extracts; the alcohol used in the water extraction and alcohol precipitation method in step (1) is 80% ethanol water solvent, and the mixture is placed in a 4°C environment for alcohol precipitation for 24 hours.

6. The preparation method according to claim 3, wherein: The extraction method in step (3) is to mix the active polysaccharide component of the Xuanfei Baidu Chinese medicine composition with distilled water according to a material-liquid ratio of 1g:15-20mL, and ultrasonicate for 30 minutes to obtain an extract; in the step (4), the volume ratio of n-butanol and dichloromethane is n-butanol:dichloromethane=1:5, and the number of extractions is 5 times.

7. The preparation method according to claim 3, wherein: The further separation and purification method by anion exchange column chromatography in step (7) is to perform DEAE-52 column chromatography on the sample, eluting with 0, 0.1, 0.3 and 0.5 M NaCl solutions in sequence, collecting one tube for every 8 mL, determining the content of polysaccharides in the fractions, and combining to obtain two elution fractions. The obtained elution fractions are dialyzed using a 3.5 kDa dialysis membrane for 3 days, and the sample is recovered under reduced pressure and freeze-dried to obtain the uniform polysaccharides P2-2A and P2-2B of the Xuanfei Baidu Chinese medicine composition.

8. Use of the uniform polysaccharide of the Xuanfei Baidu traditional Chinese medicine composition according to claim 1 or 2 in the preparation of anti-inflammatory drugs.

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