Traditional Chinese medicine composition homogeneous polysaccharide capable of diffusing lung and vanquishing toxin as well as preparation method and application of traditional Chinese medicine composition homogeneous polysaccharide

Abstract

The invention provides a homogeneous polysaccharide of a traditional Chinese medicine composition for diffusing lung and vanquishing toxin as well as a preparation method and application of the homogeneous polysaccharide, the homogeneous polysaccharide P2-2A and the homogeneous polysaccharide P2-2B are separated from active polysaccharide components of the traditional Chinese medicine composition for diffusing lung and vanquishing toxin by applying an extraction method, a water extraction and alcohol precipitation method, membrane dialysis, anion exchange resin chromatography and other methods, the total polysaccharide content of the P2-2A is 82.73%, and the total polysaccharide content of the P2-2B 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; the total polysaccharide content of the P2-2B is 64.75%, the uronic acid content is 29.23%, the protein content is 0.52%, and the molecular weight distribution ranges are respectively 8.367 * 10 < 4 > Da; the compound can be used for preparing medicines for treating inflammation, and another feasible scheme is provided for treatment of inflammation.

Description

Technical Field

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

[0002] The traditional Chinese medicine composition for Xuanfei Baidu is composed of 13 traditional Chinese medicines, 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 Xuanfei and dampness, clearing away heat and expelling evil, purging the lungs and detoxifying, and is suitable for the treatment of pneumonia infection caused by mild and common new coronavirus. It mainly contains flavonoids, phenylpropanoids, triterpenoid saponins, sesquiterpenes, cyclopentane, 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 connected by 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 concerned by researchers 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, long-term and excessive inflammation can cause great damage to the body. Once the inflammatory response is out of control, it can damage normal tissues and cause various acute and chronic inflammatory diseases. At present, 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, long-term 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 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 above-mentioned Xuanfei Baidu Chinese medicine composition.

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

[0008] A method for preparing a uniform polysaccharide of a Xuanfeibaidu Chinese medicine composition, the specific steps are as follows:

[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, knotweed, verbena, coix seed, reed root, scutellaria baicalensis, boiled bitter almond, red 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 (2) is detected as a precipitate layer component obtained after water extraction and alcohol precipitation, and the precipitate layer component is added with water and ultrasonicated to be fully dissolved or dispersed;

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

[0013] (5) concentrating the aqueous solution extracted 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 extra-membrane polysaccharide component and the intra-membrane polysaccharide component in step (5) and performing dialysis 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 ranges were 1.361×10 4 Da, IR spectrum at 2936.01cm -1 、1646.97cm -1 、933.88cm -1 and 847.09cm -1 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.

[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 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; 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 had a triple helix structure; scanning electron microscopy showed that P2-2A had an irregular shape and a rough surface porous structure.

[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 types of sugar residues of P2-2B were mainly →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, including galacturonic acid, galactose and arabinose, in the structure; the Congo red test showed that P2-2B had a triple helix structure; scanning electron microscopy showed that P2-2B had a smooth layered structure with a thickness of about 12.3 μm.

[0018] Preferably, in the method for preparing 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 the step (1) are as follows by weight: 6 parts of ephedra, 30 parts of gypsum, 10 parts of stir-fried atractylodes, 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 tangerine peel, and 10 parts of licorice.

[0019] Preferably, in the preparation method of the uniform polysaccharide of the above-mentioned 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 for uniform dispersion with ultrasonic assistance, and use n-butanol with an equal volume to water to extract three times, and combine the three extracts.

[0020] Preferably, in the method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition, the alcohol used in the water extraction and alcohol precipitation method in step (1) is 80% ethanol water solvent, and the solution 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 obtain the extract by ultrasonication for 30 minutes.

[0022] Preferably, in the method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition, the volume ratio of n-butanol to 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 water layer concentration 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 method for preparing the uniform polysaccharide of the above-mentioned Xuanfei Baidu Chinese medicine composition, the step (5) uses a 3.5 kDa 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 method for preparing 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 a 1 kDa dialysis membrane and a 50 kDa dialysis membrane for 3 days, respectively, collect the intramembrane and extramembrane solutions, recover them under reduced pressure and freeze-dry them 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 0.5 M NaCl solutions in sequence, collecting one tube for every 8 mL, determining the content of polysaccharides in the flow fractions, combining them, and dialyzing the obtained eluted components using a 3.5 kDa dialysis membrane for 3 days, recovering the samples under reduced pressure and freeze-drying them to obtain the uniform polysaccharides P2-2A and P2-2B of the Xuanfei Baidu Chinese medicine composition.

[0027] A uniform polysaccharide of Xuanfeibaidu Chinese medicine composition 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 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; 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 the four main sugar units, such as galactose and mannose, in the structure; the Congo red test showed that P2-2A had a triple helical structure; under the scanning electron microscope, P2-2A was observed to have a porous structure with irregular shape and rough surface;

[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 types of sugar residues of P2-2B were mainly →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, including galacturonic acid, galactose and arabinose, in the structure; the Congo red test showed that P2-2B had a triple helix structure; scanning electron microscopy showed that P2-2B had a smooth layered structure with a thickness of about 12.3 μm.

[0031] The uniform polysaccharide of the above-mentioned Xuanfei Baidu Chinese medicine composition was subjected to chemical analysis and homogeneity determination, and its total polysaccharide content, uronic acid content, protein content, and molecular weight distribution range were obtained; and its chemical structure information and characteristics were deeply elaborated by comprehensive methods such as IR, PMP-HPLC-DAD, NMR, methylation experiment, Congo red experiment and scanning electron microscopy. It is believed that P2-2A is a polysaccharide with a →4)-Glcp-(1→ as the main chain and a triple helical structure, and its surface is an irregular, rough porous structure; P2-2B is a galacturonic acid polysaccharide with a →4)-GalAp-(1→ as the main chain and a triple helical structure, and its surface is a smooth flaky 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 present invention is a flow chart for preparing components of the Xuanfeibaidu Chinese medicine composition;

[0036] Figure 2 Cell viability of macrophage RAW264.7 cells in samples with different concentrations of XFBD005;

[0037] Figure 3 The results of the determination of inflammatory indicators IL-1β, IL-6, and TNF-α in macrophage RAW264.7 cells after pretreatment with 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 The figure is a flow chart of the preparation and separation of polysaccharide components;

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

[0041] Figure 7 It is a flow chart of homogeneous polysaccharide purification;

[0042] Figure 8 UV scanning for uniform polysaccharide;

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

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

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

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

[0047] Fig.13 This is the GC-MS total ion chromatogram after methylation of uniform polysaccharides;

[0048] Fig.14 This is the Congo red test image of uniform polysaccharide;

[0049] Fig.15 This is a SEM electron microscope image of a uniform polysaccharide;

[0050] Fig.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. The process, conditions, reagents, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are common knowledge and common common sense in the art and there are no special restrictions.

[0052] Example 1

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

[0054] (I) Preparation and separation of components of Xuanfeibadu Chinese medicine composition

[0055] The preparation and separation of the Xuanfei Baidu Chinese medicine composition (prescription ratio is 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 stewed bitter almond, 15g of tangerine peel, and 10g of licorice) are carried out by solvent extraction, water extraction and alcohol precipitation and membrane dialysis. The specific steps are as follows: take the Xuanfei Baidu Chinese medicine composition 360g of the lyophilized powder (XFBD001) of the extract of the substance was added with 10 times the amount of water (3.6L) and dispersed evenly with ultrasound assistance. It was 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 the liquid was continuously stirred to finally configure the solution into an 80% ethanol solution, and 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) Screening of component samples and determination of NO content

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

[0059] As shown in Table 1, almost all component samples caused obvious cell death at 400 μg / mL. The XFBD005 component sample had a relatively good activity effect, less cell death, and a relatively low NO content. Although the NO concentration of the XFBD003 and XFBD004 component samples was low, the cells showed severe death. Therefore, the subsequent experiments mainly carried out further separation and purification of the XFBD005 component sample.

[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] The cells were plated at 3×104 / well 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 an enzyme reader.

[0065] The XFBD005 component sample significantly reduced the viability of RAW264.7 cells at a concentration of 50 μg / mL, which was highly toxic. Therefore, sample concentrations of 3.125, 6.25, 12.5, and 25 μg / mL were selected for subsequent experiments. Figure 2 shown.

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

[0067] According to the results of the MTT method, four dosing concentrations (the dosing concentrations of XFBD005 component samples were 3.125, 6.25, 12.5, and 25 μg / mL) were set for cell inflammatory factor detection. 3×105 cells / well were plated in a 6-well plate, and six experimental groups were set up, including a blank group, a model group, and four concentration dosing groups. After modeling and dosing, the cell supernatant was collected for the determination of IL-1β, IL-6, and TNF-α content (ELISA kit determination).

[0068] Compared with the blank group, LPS stimulation significantly increased the levels of IL-1β, IL-6, and TNF-α in macrophages. After pretreatment 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. 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] The cell plating was the same as step (2). After absorbing the supernatant culture medium, 1 mL of PBS was added to each well. The cells in the six-well plate were scraped off, blown evenly, centrifuged for 5 min, and the supernatant was aspirated to keep the cells. 500 μL of lysis buffer was added to lyse the cells in a 4°C refrigerator for 10 min. The enzyme activities of CAT, SOD, and GSH-Px and the content of MDA were determined using a kit, and the 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. After pretreatment with XFBD005 component samples, the MDA content decreased and the activities of CAT, SOD, and GSH-Px increased significantly, indicating that XFBD005 component samples can improve LPS-induced cell oxidative stress damage. The results of the determination of oxidative stress indicators CAT, SOD, GSH-Px enzyme activity 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 in Xuanfeibaidu traditional Chinese medicine composition

[0074] (I) Preparation and separation of polysaccharide components of Xuanfeibadu Chinese medicine composition

[0075] The preparation and separation of the Xuanfei Baidu Chinese medicine composition were carried out by solvent extraction and membrane dialysis. The specific steps were as follows: 110 g of the active component sample XFBD005 was added with 15 to 20 times of water, ultrasonicated for 30 minutes to dissolve or disperse it, and poured into a separatory funnel for use. After 100 mL of n-butanol and 500 mL of dichloromethane were stirred and mixed, the extract was fully shaken in a separatory funnel and then left to stand, and the extraction operation was performed 5 times in succession to remove the protein precipitate in the polysaccharide component. The solvent was further removed from the water layer and concentrated to a volume of about 1 L. The polysaccharide sample after protein removal was dialyzed for 3 days using a 3.5 kDa dialysis membrane, and the intramembrane and extramembrane solutions were collected. The solvent was recovered under reduced pressure and freeze-dried to obtain the extramembrane component (m < 3.5 kDa) P1 (43 g) and the intramembrane component (m > 3.5 kDa) P2 (34 g). Take the P1 component (6.4g), add 200mL of water to re-dissolve, 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 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), add 800mL of water to re-dissolve, 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 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 (50×110mm) and allowed to settle naturally. After the column chromatography was filled, 5 times the column volume of ultrapure water was used for balancing until there was no alcohol taste and it was set aside. Weigh 400 mg of the polysaccharide component P2-2 sample of the Xuanfei Baidu Chinese medicine composition, dissolve it in a 50 mL centrifuge tube with ultrapure water, centrifuge at 8000 rpm for 10 min, take the supernatant and load it on the top of the DEAE-52 chromatography column, and elute with 0, 0.1, 0.3 and 0.5 M NaCl solutions in sequence, collect one tube for every 8 mL, and determine the content of polysaccharides in the fraction 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 in the figure. 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 for 3 days using a 3.5 kDa dialysis membrane and freeze-dried. The specific operation process is as follows Figure 7 shown.

[0078] The obtained Xuanfeibaidu Chinese medicine composition homogeneous polysaccharides P2-2A and P2-2B were subjected to physicochemical and structural tests:

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

[0080] (1) Determination of total sugar content of Xuanfeibaidu 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] Take 20 mg of glucose standard, dissolve it in a 20 mL volumetric flask and dilute to the mark to complete the preparation of 1 mg / mL glucose standard stock solution. Take the stock solution and dilute it to different concentrations of 400, 200, 100, 75, 50, 25, 12.5 and 6.25 μg / mL glucose solutions for use. Take 0.5000 g of phenol, add 10 mL of water, ultrasonically dissolve, complete the preparation of 5% (w / v) phenol solution, and protect from light for use. Take 20 μL of glucose solutions of different concentrations, add 20 μL of the newly prepared 5% (w / v) phenol solution and 100 μL of concentrated sulfuric acid solution, and react in a 90°C water bath for 30 minutes. After the reaction is completed, take it out, cool it, and use a multifunctional microplate reader to measure its absorbance value A at 490 nm. Repeat the experimental operation 3 times for each concentration sample. With 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, a standard curve of glucose is drawn.

[0084] ②Calculation of total polysaccharide content

[0085] Take 1 mg of XFBD005 sample, add 10 mL of water, and prepare a 100 μg / mL sample solution. Take 20 μL of 100 μg / mL XFBD005 solution, follow the same steps as above, measure the absorbance value A1 after the reaction, substitute it into the standard curve to calculate the total sugar concentration C1 in the sample, and 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 is used to determine the uronic acid content in the sample to be tested. The specific steps are as follows:

[0090] ① Drawing of the standard curve Take 10.0 mg of galacturonic acid, dissolve it in a 10 mL volumetric flask and dilute it to the mark to complete the preparation of 1 mg / mL galacturonic acid mother solution, dilute the mother solution to 80 μg / mL, 60 μg / mL, 40 μg / mL, 20 μg / mL and 10 μg / mL galactose solutions of different concentrations for use. Take 15 mg of m-hydroxybiphenyl, dissolve it in a 10 mL volumetric flask with 5 mg / mL sodium hydroxide solution and dilute it to the mark to complete the preparation of 1.5 mg / mL m-hydroxybiphenyl-sodium hydroxide solution. Take 0.4780 g of sodium tetraborate solid, dissolve it in a 100 mL volumetric flask with concentrated sulfuric acid and dilute it to the mark to complete the preparation of 12.5 mM sodium tetraborate-concentrated sulfuric acid solution for use. Take 200 μL of galacturonic acid solution of different concentrations, slowly add 1.1 mL of sodium tetraborate-concentrated sulfuric acid solution, vortex and oscillate to mix thoroughly, then put it in a 90°C boiling water bath for 5 minutes, take it out, cool it, add 20 μL of 1.5 mg / mL m-hydroxybiphenyl-sodium hydroxide solution, mix it evenly, let it stand for 5 minutes, and take 100 μL of the reaction solution and use a multifunctional microplate reader to measure its absorbance value A at 525 nm. Repeat the experimental operation 3 times for each concentration sample. Use the galacturonic acid sample concentration as the horizontal axis and the absorbance value A of the reaction solution of different concentrations of samples as the vertical axis to draw a standard curve of galacturonic acid.

[0091] ②Calculation of uronic acid content Take 1 mg of XFBD005 sample, add 10 mL of water, and prepare a 100 μg / mL sample solution. Take 200 μL of 100 μg / mL XFBD005 solution, follow the same steps as above, measure the absorbance value A1 after the reaction, substitute it into the standard curve to calculate the uronic acid concentration C1 in the sample, and 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 2mL of 2mg / mL fetal bovine serum albumin, use a 50mL volumetric flask to dilute to the mark, dilute to 80μg / mL fetal bovine serum albumin solution as the mother solution, dilute the mother solution to 50μg / mL, 45μg / mL, 40μg / mL, 35μg / mL, 30μg / mL, 25μg / mL, 20μg / mL, 15μg / mL, 10μg / mL and 5μg / mL. Take 10mg of Coomassie Brilliant Blue G-250, add 5mL of 95% ethanol solution (v / v) and 10mL of 85% phosphoric acid solution (v / v) in a 100mL volumetric flask, use water to dilute to the mark, filter, and store in dark for later use. Take 40μL of bovine serum albumin solution of different concentrations, add 200μL of Coomassie Brilliant Blue solution, let stand at room temperature for 15min, and use a multifunctional microplate reader to measure its absorbance value A at 595nm. The experimental operation was repeated 3 times for each concentration sample. The standard curve of bovine fetal serum albumin was drawn with the concentration of bovine fetal serum albumin sample as the horizontal axis and the absorbance value A of the reaction solution of samples with different concentrations as the vertical axis.

[0098] ②Calculation of protein content

[0099] Take 1 mg of XFBD005 sample, add 10 mL of water, and prepare a 100 μg / mL sample solution. Take 40 μL of 100 μg / mL XFBD005 solution, and perform the same steps as above. Determine the absorbance value A1 after the reaction, substitute it into the standard curve to calculate the uronic acid concentration C1 in the sample, and 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 to be tested was determined by UV-visible spectrophotometry, and the specific steps are as follows:

[0104] ① Drawing of standard curve

[0105] Take 10.0 mg of rutin standard, add methanol to a 10 mL volumetric flask to dissolve and dilute to the mark to complete the preparation of 1 mg / mL rutin mother solution, and dilute the mother solution to 200 mg / mL, 100 mg / mL, 80 mg / mL, 60 mg / mL, 40 mg / mL and 20 mg / mL rutin solutions of different concentrations for use. Weigh 0.4000 g of sodium hydroxide solid, 0.5000 g of sodium nitrite solid and 1.000 g of aluminum nitrate solid, dissolve them in a 10 mL volumetric flask 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 different concentrations, add 100 μL of 5% sodium nitrite solution, vortex and mix, let it stand for 6 minutes, add 100 μL of 10% aluminum nitrate solution, vortex and mix, let it stand for 6 minutes, add 1 mL of 4% sodium hydroxide solution, add 1.25 mL of water, vortex and mix, let it stand for 15 minutes, take 200 μL of reaction solution and use a multifunctional microplate reader to measure its absorbance value A at 500 nm. Repeat the experimental operation 3 times for each concentration sample. Use the rutin sample concentration as the horizontal axis and the absorbance value A of the reaction solution of different concentrations of samples as the vertical axis to draw a standard curve of rutin.

[0106] ②Calculation of total flavonoid content

[0107] Take 1 mg of XFBD005 sample, add 10 mL of water, and 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. Determine the absorbance value A1 after the reaction, substitute it into the standard curve of rutin to calculate the uronic acid concentration C1 in the sample, and 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 Chinese medicine composition were measured, 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 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 fewer UV absorbing components.

[0117] 3. Infrared spectrum scanning of purified components

[0118] Weigh a small amount of polysaccharide sample and disperse it with methanol solution, add it dropwise onto the potassium bromide sheet and evaporate the solvent, repeat the operation until an appropriate amount of polysaccharide solid is precipitated on the surface of the potassium bromide sheet. Use Varian 640 FT-IR Fourier transform infrared spectrometer for scanning analysis. The instrument resolution is 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. Fig. 9 As shown. Among them, 3501.84~3286.69cm -1 The broad and strong absorption band at 2936.01 cm-1 is the stretching vibration peak of hydroxyl group. -1 The carbon-hydrogen stretching vibration peak of methyl or methylene is 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 2390 cm -1 The absorption peak at 1646.97 cm-1 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 existence of carboxyl groups, indicating that there are a small number of acidic sugar residues in P2-2A. 1149.41~1014.66cm -1 The absorption between 847.09cm indicates the presence of a pyranose ring. -1 The absorption peak at 933.88 cm-1 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 filter membrane, and seal in a sample injection 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 Waters Ultrahydrogel 2000 (12 μm, 300×7.8 mm), the flow rate was 1 mL / min, the mobile phase was water, the isocratic elution was 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] High performance gel exclusion chromatography-multi-angle laser scattering detector-differential refractive index detector (HPSEC-MALLS-RID) was used to determine the homogeneity and molecular weight range of polysaccharides of P2-2A and P2-2B. The chromatographic column was Waters Ultrahydrogel 2000 (8μm, 4.6×7.8mm), the mobile phase was 150mM ammonium formate, the isocratic elution was 30min, the column temperature was 30℃, the flow rate was 1mL / min, and the detectors were multi-angle laser scattering detector DAWN 8 HELEOS MALLS and differential refractive index detector RID-20A.

[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: Fig.10 and Fig.11 As shown in the figure, the chromatographic peaks of polysaccharide samples P2-2A and P2-2B under HPGPC-ELSD have good symmetry. After HPSEC-MALLS-RID detection, the molecular weights Mw of the two samples are 1.361×104 (±13.867%) Da and 8.367×104 (±81.616%) Da, respectively, and 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 uniform polysaccharides.

[0125] 5. Monosaccharide composition and distribution detection

[0126] The monosaccharide composition and distribution of the uniform polysaccharide separated from the Xuanfei Baidu Chinese medicine composition were analyzed by PMP-HPLC-DAD method, and the specific steps were 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 to mix and prepare a 1 mg / mL monosaccharide mixed standard solution.

[0128] ② Weigh 2.175g of 1-phenyl-3-methyl-5-pyrazolone (PMP) powder, add methanol to a 25mL volumetric flask and dissolve to the mark to complete the preparation of 0.5M PMP solution. Weigh 0.6g of sodium hydroxide solid, add water to a 25mL volumetric flask and dissolve to the mark to complete the preparation of 0.6M sodium hydroxide solution. Absorb 1.25mL of concentrated hydrochloric acid and 3.71mL of trichloroacetic acid, add water to a 25mL volumetric flask and dilute to the mark to complete the preparation of 0.6M hydrochloric acid solution and 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 water layer, centrifuge at 14000 rpm for 10 min, pass through a 0.22 μM microporous filter membrane, and seal in an injection vial for use.

[0130] ④ Weigh 2 mg of each homogeneous polysaccharide P2-2A and P2-2B, add 2.5 mL of 2M trichloroacetic acid solution, and react in a water bath at 100°C for 8 hours. After the reaction is completed, take it out, cool it, blow nitrogen to remove the solvent, add 1 mL of methanol to remove the residual reagent, blow nitrogen to dry, and repeat 3 times. After the acid hydrolysis is completed, add 200 μL of water to dissolve the dried sample, and perform the same operation as the above monosaccharide mixed standard solution PMP derivatization method.

[0131] ⑤Use Agilent 1260II high performance liquid chromatograph to analyze the monosaccharide composition and content of the PMP-derivatized samples. The chromatographic column is 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: Fig.12 As shown. By comparing the retention time of the chromatogram after derivatization with the monosaccharide mixed standard, it can be seen that sample P2-2A is mainly a polysaccharide composed of glucose and arabinose, and 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, and the specific monosaccharides included are mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose and fucose, and the content ratio is 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, add 1 mL of water to dissolve, add 100 mg / mL carbodiimide 1 mL to react for 2 hours. Add 1 mL each of 2M imidazole, 30 mg / mL NaBH4 and 30 mg / mL NaBD4, and react for 3 hours. Add 100 μL of glacial acetic acid to terminate the reaction. Dialyze the sample for 48 hours, freeze-dry the sample after dialysis is completed, and perform methylation treatment.

[0135] Take the freeze-dried samples of P2-2A and P2-2B after the reaction and add 500μL of DMSO solution to dissolve. Add 1mg of NaOH solid and incubate for 30min. Add 50μL of iodomethane solution and react for 1h. After the reaction is completed, add 1mL of water and 2mL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water washing 3 times. Take the lower dichloromethane phase and evaporate to dryness. Add 100μL of 2M trifluoroacetic acid and react at 121℃ for 90min. After the reaction is completed, evaporate to dryness at 30℃. Add 50μL of 2M ammonia and 1M NaBD4, mix well, and react at room temperature for 2.5h. After the reaction is completed, add 20μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250μL of methanol, and blow dry with nitrogen. Add 250μL of acetic anhydride, vortex to mix, and react at 100℃ for 2.5h. Add 1mL of water and let stand for 10min. Add 500 μL of dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat the water wash 3 times. Take the dichloromethane layer to prepare the analytical sample solution and set aside.

[0136] Agilent 7890A gas chromatography system was used to analyze the components of various methylated samples. The chromatographic column was an HP-5MS capillary column (30m×0.25mm×0.25μm), the carrier gas was high-purity helium (purity not less than 99.999%), the flow rate was 1.0mL / min, and the injection port temperature was 260°C. The injection volume was 1μL, split injection, split ratio 10:1, and solvent delay was 2.2min.

[0137] Program temperature rise: 50℃, hold for 1.0min, increase the temperature to 130℃ at 50℃ / min, increase the temperature to 230℃ at 3℃ / min, hold for 2min.

[0138] Mass spectrometry system: A quadrupole mass spectrometry detection system (Agilent 5977B) from Aiglent, USA, equipped with an electron impact ion source (EI) and a MassHunter workstation. Electron impact ion source (EI), injection port temperature 230°C, quadrupole temperature 150°C, electron energy 70eV. Scanning mode is full scan mode (SCAN), mass scanning range (m / z): 30-600.

[0139] The total ion chromatograms of the homogeneous polysaccharide samples P2-2A and P2-2B after methyl iodide methylation, hydrolysis, reduction and acetylation by GC-MS are shown in the figure. Fig.13 The results of the analysis are shown in Table 3. Compared with the standard mass spectrum library, it can be seen that the types of sugar residues in P2-2A are 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; the types of sugar residues in P2-2B are mainly →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 a 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 is reduced before methylation reduction, the terminal, 1-3 and 1-4 related methylation derivatives may be derived from the reduction of Gal and / or GalA existing itself.

[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 Analysis

[0147] Take 50 mg of P2-2A sample and dissolve it in 0.5 mL DMSO, and dissolve the same amount of P2-2B sample in 0.5 mL D2O for NMR measurement. 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] The terminal hydrogen region (δ H4.6~5.7ppm) signal indicates that P2-2A has both α- and β- configurations. According to HSQC spectrum analysis 1 H and 13 The chemical shifts of the terminal hydrogen and terminal carbon in the C signal revealed a total of four sugar units, namely, δ H / δ C The molecular weights of P2-2A and P2-2A were 5.23 / 91.99, 5.05 / 100.69, 4.94 / 106.87 and 4.78 / 98.95, which were α-Glcp-(1→, →4)-α-Glcp-(1→, →5)-α-Araf-(1→ and β-Xylp-(1→, respectively. According to the monosaccharide composition results, P2-2A was mainly composed of Man, Glc, Gal and Ara. The sugar units in the above NMR corresponded to t-Glc(p), 4-Glc(p), 5-Ara(f) and t-Xyl(p) in the methylation analysis results. According to the above monosaccharides, P2-2A was mainly glucan. The residues of P2-2A were analyzed in detail. 1 H and 13 The assignment of the chemical shifts of C is shown in Table 4.

[0150] Likewise, the terminal hydrogen region (δ H 4.5~5.8ppm) signal indicates that P2-2B has both α and β configurations. According to HSQC spectrum analysis 1 H and 13 The chemical shifts of the terminal hydrogen and terminal carbon in the C signal revealed a total of 7 sugar units, namely, 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, which are β-GlcAp-(1→, →2, 4-α-Rhap-(1→, →6)-β-Galp-(1→, →4)-α-GalAp-(1→, β-Ga lAp-(1→, α-GalAp-(1→ and →5)-α-Araf-(1→. According to its monosaccharide composition results, P2-2B is mainly composed of xylose, arabinose and fucose Man, Rha, GlcA, GalA, Glc, Gal, Xyl, Ara and Fuc. Based on the above information, P2-2B is mainly galacturonic acid polysaccharide. The residues of P2-2B were analyzed in detail. 1 H and 13 The assignment of the chemical shifts of C is shown in Table 5.

[0151] Table 4 NMR signals of sugar residues of P2-2A

[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 helix structure of the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition. The specific operation is as follows: take 2 mg of the uniform polysaccharide 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, take 100 μL of the reaction solution, and use a multifunctional microplate reader to measure its maximum absorption wavelength (λ max ). The same experimental operation was performed with aqueous solution instead of sample, and the experimental operation was repeated 3 times for each sample at different concentrations of sodium hydroxide. The hydrolysis curve of the polysaccharide triple helix structure was plotted with 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 Fig.14 As shown. Compared with the blank control group (Congo red solution), the results of the uniform polysaccharides P2-2A and P2-2B in the Xuanfei Baidu Chinese medicine composition are compared. As the NaOH concentration increases, the maximum absorption wavelength of the polysaccharide samples increases and red shifts; when the NaOH concentration reaches a certain level, the maximum absorption wavelength decreases and blue shifts. Based on the above phenomenon, it is shown that the uniform polysaccharides P2-2A and P2-2B in the Xuanfei Baidu Chinese medicine composition have a triple helix structure.

[0158] 9. Scanning electron microscopy analysis

[0159] The uniform polysaccharide samples P2-2A and P2-2B were freeze-dried, and an appropriate amount of solid was adhered to the black conductive glue. The floating sample on the conductive glue was blown off with an ear suction bulb, and gold was sprayed. The morphological characteristics of the uniform polysaccharide were observed using a scanning electron microscope (SEM), and representative fields of view were selected for image collection. The parameters used for SEM observation were: working voltage of 5 kV, and magnification of 50 to 1000 times.

[0160] After the homogeneous polysaccharide samples P2-2A and P2-2B were observed by scanning electron microscopy, the surrounding structures of the two samples were observed at different magnifications of 100 to 1000, such as Fig.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 irregular shape and rough surface, and P2-2B is a layered structure with smooth surface and a thickness of about 12.3 μm.

[0161] Example 3

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

[0163] 1. Experimental groups and research methods

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

[0165] The viability of RAW264.7 cells was determined by MTT method. RAW264.7 cells (105 cells / well) were seeded in 96-well plates and incubated in a 37°C and 5% CO2 incubator for 12 hours. Then, the cells were treated with different concentrations of XFBD005 (5, 10, 20 μg / mL), P2-2A (5, 10, 20 μg / mL) and P2-2B (5, 10, 20 μg / mL) samples for 12 hours, and the blank control wells were treated with DMSO in the same way. Subsequently, 50 μL of 2.5 mg / mL MTT solution was added to each well, and the supernatant was discarded after incubation in the incubator for 4 hours. 150 μL of DMSO was added to each well, mixed and allowed to stand for 5 minutes, and the OD value at 490 nm was measured using an enzyme marker (Bio-Tek) to calculate the cell survival rate.

[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. Cells were treated with different 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 detected at a wavelength of 490 nm using a microplate reader (Bio-Tek). RAW264.7 cells (3×105 / well) were seeded in 6-well plates and incubated for 12 hours. Subsequently, different concentrations of XFBD005 (5, 10, 20μg / mL), P2-2A (5, 10, 20μg / mL) and P2-2B (5, 10, 20μg / mL) were used for 12 hours, and the blank and positive controls were treated with DMSO and dexamethasone (50μg / mL), respectively. Then, the culture medium was aspirated and LPS (1μg / mL) was induced for 24 hours to establish an inflammatory model.

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

[0169] After establishing the RAW264.7 cell inflammation model and grouping and drug administration according to the above step 2, the NO content was determined by the Griess method. The operation was as follows: the RAW264.7 cell culture supernatant (50 μL / well) was taken in a 96-well plate, and equal volumes of Griess reagents I and II were mixed with the cell culture supernatant, and 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 inflammation-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 a concentration of 5-20 μg / mL. Based on this, 5, 10 and 20 μg / mL concentrations were selected to determine the content of inflammatory factors IL-1β, IL-6, and TNF-α. The specific steps are as follows: Also refer to 2 to establish the RAW264.7 cell inflammation model and group administration, collect the cell supernatant 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] After establishing the RAW264.7 cell inflammation model and grouping and dosing according to the above step 2, the supernatant culture medium was discarded and the cells of each group were collected. 500 μL of lysis solution was added to the cells and lysed in a 4°C refrigerator for 10 min. After complete lysis, the cells were centrifuged at 12000 rpm and 4°C for 10 min, and then the supernatant was collected and the total protein concentration was determined according to the Bradford kit. Subsequently, the CAT, SOD, GSH-Px enzyme activities and MDA content in the RAW264.7 cell lysate were detected according to the kit instructions.

[0174] 6. Data Analysis

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

[0176] (II) 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 Fig.16 As shown in the figure, compared with the CON group, XFBD005, P2-2A and P2-2B (5-20 μg / mL) samples had no significant effect on the viability of RAW264.7 cells (p>0.05), indicating that XFBD005, P2-2A and P2-2B samples at a concentration of 5-20 μg / mL had no cytotoxicity to RAW264.7 cells. Based on this, 5, 10 and 20 μg / mL concentrations 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), and after pre-administration of 5-20 μg / mL of XFBD005, P2-2A and P2-2B, the NO content in each administration 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 the P2-2A and P2-2B samples had a more obvious inhibitory effect on the NO content in LPS-induced 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 factors content of RAW264.7 cells induced by LPS by XFBD005, P2-2A and P2-2B

[0183]

[0184]

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

[0186] Compared with the XFBD-005 group (5 μg / 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 levels of inflammatory factors 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), proving that the P2-2A and P2-2B samples have a more obvious inhibitory effect on the expression level 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 CON group, #p<0.05, ##p<0.01. Compared with LPS group, *p<0.05, **p<0.01.

[0195] Compared with the XFBD-005 group (5 μg / 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 MDA content in the LPS group increased significantly, and the activities of CAT, SOD, and GSH-Px enzymes decreased significantly (p<0.01); after pre-administration protection with XFBD005, P2-2A, and P2-2B samples (5, 10, and 20 μg / mL), the MDA content decreased significantly (p<0.05 or p<0.01); the activities of CAT, SOD, and GSH-Px enzymes increased significantly (p<0.05 or p<0.01). In addition, the protective effect of P2-2A and P2-2B samples on CAT enzyme activity was better than that of each dose group of XFBD005. It is worth noting that when the administration concentration was 20 μg / mL, only the P2-2B sample significantly reduced the MDA content (p<0.05) and significantly increased the GSH-Px enzyme activity (p<0.05), proving that the components of the P2-2B sample have a more significant 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 design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for preparing a uniform polysaccharide of a Xuanfeibaidu Chinese medicine composition, 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, knotweed, verbena, coix seed, reed root, scutellaria baicalensis, boiled bitter almond, red 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 (2) is detected as a precipitate layer component obtained after water extraction and alcohol precipitation, and the precipitate layer 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) concentrating the aqueous solution extracted in step (4), and dialyzing it using a dialysis membrane to obtain two crude polysaccharide components P1 and P2 of the Xuanfei Baidu Chinese medicine composition outside the membrane and inside the membrane; (6) taking the extra-membrane polysaccharide component and the intra-membrane polysaccharide component in step (5) and performing dialysis purification respectively to obtain the purified polysaccharide components P1-1, P1-2 and P2-1, P2-2 of the Xuanfei Baidu Chinese medicine composition; (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 ranges were 1.361×10 4 Da, IR spectrum at 2936.01cm -1 、1646.97cm -1 、933.88cm -1 and 847.09cm -1 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 -1 There is an absorption peak.

2. The method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 1, 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, 15 parts of patchouli, 12 parts of artemisia annua, 20 parts of knotweed, 30 parts of verbena, 30 parts of coix seeds, 30 parts of reed rhizome, 15 parts of scutellaria baicalensis seeds, 15 parts of boiled bitter almonds, 15 parts of tangerine peel, and 10 parts of liquorice.

3. The method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 1, characterized in that: The extraction method in step (1) is to add 8 to 12 times the weight of the composition to be extracted with water to disperse it evenly with ultrasonic assistance, and use n-butanol with an equal volume to water to extract it 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.

4. The method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 1, characterized in that: 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 an extract; in step (4), the volume ratio of n-butanol to dichloromethane is n-butanol: dichloromethane = 1:5, and the number of extractions is 5 times.

5. The method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 1, characterized in that: In the step (5), a 3.5 kDa dialysis membrane is used to dialyze the polysaccharide component after the protein precipitate is removed in step (4) for 3 days, and the intramembrane and extramembrane solutions are collected. The samples are recovered under reduced pressure and freeze-dried to obtain the intramembrane polysaccharide component P2 and the extramembrane polysaccharide component P1, respectively.

6. The method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 1, characterized in that: The dialysis in step (6) is to repeatedly dialyze the intramembrane component and extramembrane component solutions in step (5) using 1 kDa dialysis membrane and 50 kDa dialysis membrane for 3 days, respectively, collect the intramembrane and extramembrane solutions, recover them under reduced pressure and freeze-dry them to obtain refined polysaccharide components P1-1, P1-2 and P2-1, P2-2.

7. The method for preparing the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 1, characterized in that: The method for further separation and purification by anion exchange column chromatography in step (7) is to subject the sample to DEAE-52 column chromatography, 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, combining them, and dialyzing the obtained eluted components using a 3.5 kDa dialysis membrane for 3 days, recovering the samples under reduced pressure and freeze-drying them to obtain the uniform polysaccharides P2-2A and P2-2B of the Xuanfei Baidu Chinese medicine composition.

8. A uniform polysaccharide in a Xuanfei Baidu Chinese medicine composition, prepared by the method according to any one of claims 1 to 7.

9. The homogeneous polysaccharide according to claim 8, characterized in that: It is a uniform polysaccharide 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 2936.01cm -1 、1646.97cm -1 、933.88cm -1 and 847.09cm -1 There is a strong absorption peak at the bottom; PMP-HPLC results show that the monosaccharide composition of P2-2A is mainly mannose, rhamnose, glucuronic acid, glucose, galactose and arabinose, with a content ratio of 26.1:1.6:18.6:29.2:24.4; methylation analysis results show that the types of sugar residues of P2-2A are 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; P2-2A has an irregularly shaped, rough-surfaced porous 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 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 -1 There is an absorption peak at ; PMP-HPLC results show that the main monosaccharide composition of P2-2B is 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; methylation analysis results show that the types of sugar residues of P2-2B are mainly →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 has a triple helix structure; Scanning electron microscopy showed that P2-2B has a smooth layered structure with a thickness of about 12.3μm.

10. Use of the uniform polysaccharide of the Xuanfei Baidu Chinese medicine composition according to claim 8 or 9 in the preparation of anti-inflammatory drugs.

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