Polysaccharide active ingredient, preparation thereof and application of polysaccharide active ingredient in preparation of anti-inflammatory drugs

Abstract

The invention belongs to the technical field of medicine development, and particularly discloses a brand new natural polysaccharide active component. According to a main chain structure, 4)-beta-D-Galp-(1-> 4)-alpha-D-Glcp-(1-> 3, 6)-beta-D-Galp-(1-> 3, 4)-alpha-D-Glcp-(1-> 4, 6)-alpha-D-Glcp-(1-> 3)-alpha-D-Glcp-(1-> 3)-alpha-L-Araf-(1-> 5)-alpha-L-Araf-(1-> 6) are sequentially connected with a main chain, and the main chain is formed by sequentially connecting three branched chains with the main chain. The invention also provides a preparation method of the compound and application of the compound in anti-inflammatory activity. The polysaccharide active component provided by the invention can effectively inhibit the inflammation level of macrophage RAW264.7, promote the improvement of the phagocytic ability of the macrophage RAW264.7 and inhibit the release of immune factors of the macrophage RAW264.7. And a feasible scheme is provided for discovering effective natural polysaccharide for treating inflammation.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to the field of anti-inflammatory drugs. Technical Background

[0002] Inflammation is a complex reaction process that occurs in biological organisms, showing the characteristics of resisting external stimuli, thereby achieving the purpose of protecting itself. The self-limiting and self-regulating properties of physiological inflammation are effective in promoting tissue healing and maintaining homeostasis. Acute inflammation may develop into chronic diseases over time, which will bring greater risks, such as tumors, cancer and diabetes. This is because inflammation causes the release of inflammatory factors and changes in vascular permeability. Therefore, finding drugs and ingredients that are effective in treating chronic inflammation has great application value.

[0003] Polysaccharides are widely present in nature as biological macromolecules. They are composed of different monosaccharides and have complex spatial structures and excellent biological activities. Studies on the anti-inflammatory activity of plant polysaccharides have found that polysaccharide components can enter cells or activate membrane receptors, affect intracellular gene replication and protein expression, affect the release of related inflammatory cytokines, and inhibit the continued occurrence of inflammation from the root. In addition, the preparation process of polysaccharide components is environmentally friendly and suitable for large-scale applications.

[0004] At present, existing technical inventions disclose polysaccharide components with anti-inflammatory activity. For example, patent No. CN116655820A discloses a polysaccharide of rattan tea acid that can be used to prepare anti-inflammatory cosmetics. Chinese patent literature with CN117343206A as publication number discloses a method for preparing fucoidan and its application in preparing anti-inflammatory drugs. CN117285658A discloses a codonopsis polysaccharide with significant anti-inflammatory effect and its extraction method and application.

[0005] According to existing patents, although many polysaccharide components with anti-inflammatory activity are disclosed, few of them have clear structures or their effects are not prominent. In addition, there is no report on the polysaccharide active components of the plants taken in the present invention, and there is also a lack of reports on the effects of anti-inflammatory activity. Summary of the invention

[0006] The first object of the present invention is to provide a novel polysaccharide active ingredient which can effectively promote macrophage proliferation, enhance macrophage phagocytosis ability and inhibit macrophage inflammation.

[0007] The second object of the present invention is to provide a method for preparing a polysaccharide active ingredient.

[0008] The third object of the present invention is to provide an anti-inflammatory drug comprising the polysaccharide active ingredient.

[0009] The polysaccharide active ingredient is a polysaccharide ingredient having the structure described in Formula 1:

[0010]

[0011] The present invention discloses for the first time in the industry a new polysaccharide having a structure of Formula 1, and through a series of research and exploration, it is found that the active ingredient of this polysaccharide has obvious biological activity in promoting macrophage proliferation, improving cell phagocytic activity, and affecting the release of inflammatory factors.

[0012] In the present invention, the polysaccharide component of formula 1 is a homogeneous acidic polysaccharide, and the monosaccharides constituting the acidic polysaccharide include: glucose, galactose, arabinose, rhamnose, galacturonic acid, mannose, glucuronic acid, and xylose, and the connection method is: →4)-β-D-Galp-(1→4)-α-D-Glcp-(1→3,6)-β-D-Galp-(1→3,4)-α-D-Glcp-(1→4, 6)-α-D-Glcp-(1→ is the main chain, and three side chains→5)-α-L-Araf-(1→ is connected to the main chain through O-3, α-D-Glcp-(1→3)-α-D-Glcp-(1→ is connected to the main chain through O-3, and α-L-Araf-(1→5)-α-L-Araf-(1→ is connected to the main chain through O-6 in sequence. The present invention provides a new polysaccharide, and it is found that it has a significant effect of inhibiting the development of inflammation.

[0013] The molecular weight of the polysaccharide active ingredient of Formula 1 of the present invention is 52000-55000, preferably 53000-54000.

[0014] The present invention provides a method for preparing the polysaccharide active component. The raw material of the trifoliate wood is extracted with water to obtain a crude extract; a sodium chloride aqueous solution is used as an eluent to perform cellulose exchange column chromatography elution on the crude extract, a target eluate with a concentration of 0.25 to 0.35 M sodium chloride aqueous solution is collected, and then the target eluate is eluted by dextran gel chromatography to obtain the polysaccharide active component.

[0015] The present invention shows that through the combination of water extraction, cellulose exchange column chromatography and dextran gel chromatography, and further coordinated with the joint control of the cellulose exchange column chromatography elution mechanism, the polysaccharide active ingredients with the novel structure and excellent anti-inflammatory activity can be unexpectedly separated.

[0016] In the present invention, the raw material of the truncatum truncatum is the rhizome part of the truncatum ....

[0017] In the present invention, before water is added, the raw material of Sanmei wood is pretreated and degreased, wherein the pretreatment includes the steps of washing, drying, crushing and screening in sequence.

[0018] In the present invention, the pretreated safflower raw material is dried and ground into powder, and then subjected to degreasing treatment. The solvent used in the degreasing stage is an alcohol-water solvent, and the alcohol content in the alcohol-water solvent can be above 50% (v / v), and can further be 60-90% (v / v). The temperature in the degreasing treatment stage is reflux, and the degreasing treatment time is above 1 hour, and can further be 1-3 hours.

[0019] In the present invention, the liquid-to-solid ratio in the water extraction stage is 10 to 25 mL / g.

[0020] In the present invention, in order to more completely utilize the components, the water extraction process can be repeated multiple times as needed.

[0021] The temperature of water extraction can be room temperature.

[0022] In the present invention, in order to improve the water extraction efficiency and effect, the water extraction described in the present invention can be carried out under the assistance of microwaves.

[0023] The microwave power in the water extraction stage is 250-650W.

[0024] The water extraction stage lasts for 10 to 30 minutes.

[0025] In the present invention, after the water extraction treatment, alcohol is added to the obtained water extract to perform alcohol precipitation treatment to obtain an alcohol precipitate, and then the alcohol precipitate is subjected to a deproteinization treatment to obtain the crude extract.

[0026] The alcohol is a C1-C4 mono- or polyol, preferably ethanol.

[0027] The volume ratio of the water extract to the alcohol is 1:1-10.

[0028] The alcohol precipitate is deproteinized using Sevage reagent to obtain crude polysaccharide;

[0029] The volume ratio of the alcohol precipitate to the Sevage reagent is 3 to 5:1.

[0030] The Sevage reagent is prepared in a volume ratio of chloroform to n-butanol of 3 to 5:1.

[0031] In the present invention, the cellulose exchange column is a DEAE-52 cellulose anion exchange column.

[0032] In the present invention, before elution begins, deionized water is used for pre-elution according to actual conditions; for example, the steps of the elution process may be: pre-elution with water, then elution with an aqueous solution of 0.05 to 0.15 M sodium chloride, then elution with a 0.25 to 0.35 M (further 0.29 to 0.31 M) sodium chloride aqueous solution, and collection of the target eluate containing the polysaccharide active ingredient.

[0033] In the present invention, the target eluate is desalted and then eluted by dextran gel column chromatography to obtain the polysaccharide active component.

[0034] In the present invention, the target eluate is subjected to a 3500Da dialysis desalination treatment.

[0035] In the present invention, the dextran gel column is a G-100 dextran gel column.

[0036] The present invention provides an application of the polysaccharide active ingredient in the preparation of anti-inflammatory drugs.

[0037] The polysaccharide active ingredient of the present invention can significantly promote macrophage proliferation, enhance cell phagocytic activity and affect the release of inflammatory factors.

[0038] The inflammatory factor is at least one of NO, ROS, IL-10, IL-6, IL-1β and TNF-α.

[0039] The present invention also provides an anti-inflammatory drug, which comprises a pharmaceutically effective amount of the polysaccharide active ingredient.

[0040] Preferably, the anti-inflammatory drug further comprises a pharmaceutically acceptable excipient;

[0041] Preferably, the anti-inflammatory drug is in any pharmaceutically acceptable dosage form.

[0042] Beneficial Effects

[0043] The polysaccharide component of a novel formula 1 structure provided by the present invention can more obviously inhibit inflammation and has a highly effective anti-inflammatory effect compared with crude polysaccharides and other homologous polysaccharides. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 One-dimensional NMR of the polysaccharide component of formula 1 1 H NMR (A) and 13 C NMR(B)

[0045] Figure 2 The two-dimensional nuclear magnetic resonance HH-COSY (A) HSQC (B) HMBC (C) NOSEY (D) images of the polysaccharide component of formula 1;

[0046] Figure 3 The main molecular structure diagram of the polysaccharide component of formula 1;

[0047] Figure 4 is the survival rate of macrophages after polysaccharide treatment in formula 1

[0048] Figure 5The changes of macrophage phagocytosis after treatment with polysaccharide component of formula 1

[0049] Figure 6 The changes of ROS after treatment with polysaccharide components of formula 1

[0050] Figure 7 The change of NO after polysaccharide treatment in formula 1

[0051] Figure 8 The content of TNF-α after polysaccharide treatment of formula 1

[0052] Fig. 9 The content of IL-1β after treatment with the polysaccharide component of formula 1

[0053] Fig.10 The content of IL-6 after polysaccharide treatment of formula 1

[0054] Fig.11 The content of IL-10 after polysaccharide treatment of formula 1

[0055] Fig.12 The changes of NF-κB protein in cells after treatment with polysaccharide of formula 1

[0056] Fig.13 Cell viability effects of polysaccharide of formula 1, comparison polysaccharide 2 and CLFP polysaccharide DETAILED DESCRIPTION

[0057] The following examples are intended to illustrate the present invention rather than to further limit the present invention.

[0058] The present invention discloses an optional method for preparing a polysaccharide active ingredient of formula 1, wherein the root and stem parts of the three-sister wood are washed, dried, crushed, and sieved, and then subjected to microwave-assisted water extraction, alcohol analysis, and deproteinization treatment to obtain a crude product; the crude product is subjected to column chromatography treatment, wherein water elution is used in advance, and then gradient elution is performed using a 0.1-0.7 mol / L salt solution, and an eluate of 0.25-0.35 M sodium chloride solution is collected as the target eluate, and desalination and concentration treatment are performed, and then purification treatment is performed on a dextran gel column to obtain the obtained product. The cellulose exchange column is a DEAE-52 cellulose ion exchange column, and the target eluate is desalted by dialysis, and the dextran gel column is a G-100 dextran gel column.

[0059] The following is a more specific implementation plan:

[0060] Example 1

[0061] Separation and purification experiment of polysaccharide of formula 1:

[0062] (1) Pretreatment: The raw materials of Sanmei wood are cleaned, dried, crushed with a grinder, sieved with a sieve, the sieved powder is peeled and weighed, sealed and dried for storage, and then used.

[0063] (2) Degreasing: Take 40g of Tripterygium wilfordii powder, add 80% ethanol by volume, solid-liquid ratio 1:15 (g / mL), reflux at 75°C, and react for 2h to degrease the sample. After the reaction is completed, use a Buchner funnel to filter under reduced pressure, retain the solid, and put it in a drying oven to dry overnight.

[0064] (3) Microwave-assisted extraction: Weigh an appropriate amount of defatted powder and add it to a conical flask. Add deionized water at a solid-liquid ratio of 1:15 (g / mL). Extract three times at a microwave power of 400 W, 30 min per group. Combine the three extracts and concentrate them by rotary evaporation to 100 mL.

[0065] (4) Alcohol precipitation: Slowly pour the concentrated extract into pre-cooled anhydrous ethanol at a ratio of 1:4, stirring vigorously. After precipitation at 4°C overnight, collect and dry the precipitate.

[0066] (5) Deproteinization: The obtained precipitate was redissolved in deionized water and deproteinized using Sevage reagent (chloroform: n-butanol, 4:1, v / v). 1 / 4 volume of Sevage reagent was added to the reconstituted solution, and the solution was shaken vigorously for 35 minutes, and then centrifuged at 7500 rpm for 8 minutes. The supernatant was taken and the protein layer was discarded. The above deproteinization operation was repeated 3-5 times until no protein layer was precipitated. The supernatant was concentrated, dialyzed, and freeze-dried to obtain crude polysaccharide, which was named CLFP.

[0067] (6) Column chromatography purification: Take the crude polysaccharide of Tripterygium wilfordii and dissolve it in a small amount of deionized water, and use it for column chromatography elution with DEAE-52 cellulose column filler. First, stir the filler thoroughly, wet-pack it into the column, let it stand for natural sedimentation, and then appropriately pressurize it until the column surface does not drop. At this time, the flow rate is about 7-10min / 15mL, and the DEAE-52 cellulose chromatography column is balanced with deionized water. Add the solution along the wall, and after the liquid surface is flush with the material, further elute with distilled water and NaCl solution (0.1, 0.3 and 0.5, 0.7mol / L) at a flow rate of 2.0mL / min. Collect 15mL of the eluate in each tube, and measure the absorbance value of the tube solution by the phenol-sulfuric acid method. Draw the elution curve with the number of tubes as the horizontal axis and the absorbance value as the vertical axis. At this time, two components are separated. The component obtained by eluting with 0.3 mol / L sodium chloride is named as polysaccharide (LFP-1) of formula 1, and the other component polysaccharide 2 is the component obtained by eluting with 0.5 mol / L sodium chloride.

[0068] The cellulose exchange column is a DEAE-52 cellulose ion exchange column.

[0069] The target eluate was desalted by dialysis.

[0070] The dextran gel column is a G-100 dextran gel column.

[0071] Example 2

[0072] Methylation experiment of polysaccharide of formula 1

[0073] (1) Methylation: Weigh the polysaccharide sample (3 mg) and place it in a glass reaction bottle, then add anhydrous DMSO (1 ml). Quickly add methylation reagent A solution, keep it in a closed state and dissolve it under ultrasonic action, and then add methylation reagent B solution. React in a magnetic stirring water bath at 30°C for 60 minutes. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction. Dialyze it in a 1000Da dialysis bag for 24 hours and then freeze-dry it. Accurately weigh 2 mg of sample and 200 mg of potassium bromide, press them into tablets, and the blank control is made of potassium bromide powder tablets. Place them in Fourier transform infrared spectrometer FT-IR650 (Tianjin Gangdong Technology Development Co., Ltd.) for scanning and recording to determine that the methylation reaction has been completed.

[0074] (2) Acetylation: Take the methylated polysaccharide, add 1 mL of 2M trifluoroacetic acid (TFA) and hydrolyze for 90 min, then evaporate to dryness. Add 2 mL of double distilled water to the residue, add 60 mg of sodium borohydride for reduction reaction for 8 hours, and then add glacial acetic acid to neutralize. Then, after rotary evaporation and drying, add 1 mL of acetic anhydride to acetylate at 100°C for 1 hour, and cool. Finally, add 10 mL of pure water to terminate the reaction.

[0075] The acetylated product was dissolved in 3 mL of dichloromethane and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, and the upper aqueous solution was removed. This process was repeated 4 times. The dichloromethane layer was dried with an appropriate amount of anhydrous sodium sulfate, concentrated to 1 mL, and placed in a liquid phase vial.

[0076] Thermo scientific, 1300-7000 gas chromatography-mass spectrometry was used to determine the acetylation product samples; GC-MS conditions: HP-INNOWAX column 30m*0.32mm*0.25um; program temperature conditions: starting temperature 140℃, heating to 230℃ at 1℃ / min; injection port temperature 250℃, detector temperature 250℃, carrier gas helium, flow rate 1mL / min.

[0077] The methylated sugar residues obtained in the experiment were compared with the mass spectra of standard PMAA (partially methylated sugar alcohol acetyl esters), and the identities and proportions of each sugar residue were determined based on retention time and mass-to-nuclear ratio. The analysis results of GC-MS data are shown in Table 1.

[0078] Table 1 GC-MS analysis results of polysaccharide of formula 1

[0079]

[0080]

[0081] Example 3

[0082] NMR experiment of polysaccharide of formula 1

[0083] 70 mg of LFP-1 was fully dissolved in 600 μL of heavy water and analyzed by one-dimensional and two-dimensional NMR using a Bruker Ascend 600 MHz instrument to obtain the sample 1 H, 13 C, HHCOSY, HMBC, HSQC, and NOESY spectra to analyze related connection structures.

[0084] Example 4

[0085] Analysis of the structure of the polysaccharide of formula 1

[0086] First, according to 1 H and 13 The number of anomeric hydrogen and anomeric carbon is determined in the anomeric region of the C spectrum, and the chemical shifts of the anomeric hydrogen and anomeric carbon are determined based on the chemical shifts of the anomeric hydrogen and anomeric carbon. Then, the chemical shifts of other hydrogens associated with the anomeric proton are found in the HH-COSY spectrum, and the chemical shifts of the carbons corresponding to the chemical shifts of the protons are found in the HSQC spectrum. 1 H. 13 C chemical shifts are shown in Table 2. After the carbon and hydrogen chemical shifts were assigned, the carbon-hydrogen related signal peaks were found through the HMBC spectrum, the connection mode and connection order of the sugar residues were determined, and the analysis of the NMR spectrum of the polysaccharide component of Formula 1 was completed.

[0087] Table 2 Different sugar residues 1 H. 13 C chemical shift

[0088]

[0089]

[0090] Example 5

[0091] Effects of polysaccharide of formula 1 on the biological activity of macrophage RAW264.7

[0092] The cells were cultured in DMEM medium containing 10% fetal bovine serum albumin and 1% penicillin-streptomycin solution. The suitable culture environment was a temperature of 37° C. and a carbon dioxide concentration of 5%.

[0093] When the cells reached the logarithmic growth phase, 1.5 × 10 4 Cells were inoculated into 96-well cell culture plates, and no cells were inoculated in the blank group. The cell growth status was observed at the same time. LFP-1 was dissolved in complete culture medium, filtered with a 0.22μm filter membrane, and gradiently diluted to different concentrations (12.5, 25, 50, 100, 200, 400μg / mL). After 24 hours of culture, the supernatant was removed, and 100μL of the corresponding sample solution or complete culture medium was added to each well. The LPS group added an equal amount of 1μg / ml LPS solution. Continue to incubate for 24 hours, aspirate the cell supernatant, wash with PBS 2-3 times, and add 100μL of ten-fold diluted CCK8 reagent to each well. Incubate in a 37℃ constant temperature incubator for about 40 minutes, measure the absorbance at 450nm under an enzyme marker, and calculate the effect of LFP-1 on macrophage viability.

[0094] The cell survival rate formula is: cell survival rate = (OD 实验组 -OD 空白组 ) / (OD 阴性对照组 -OD 空白组 )

[0095] Repeat the above cell culture and sample addition process. After 24 hours of culture, wash the cells three times with PBS. Add 100 μL of 0.1% neutral red dye to each well and culture at 37°C for 2 hours. Wash the dye between cells with PBS, add 100 μL of cell lysis solution (absolute ethanol: acetic acid = 1:1), incubate at 4°C for 3 hours, detect the absorbance at 540nm under an ELISA reader, and calculate the cell phagocytosis ability of LFP-1.

[0096] After 48 hours of incubation of cells in a 96-well plate, the supernatant of each well was transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, and 50 μL of the supernatant was added to the well, followed by the addition of 50 μL of Griess Reagent I and Griess Reagent II. The absorbance at 540 nm was measured on an ELISA reader to calculate the effect of LFP-1 culture on the release of nitric oxide in cells.

[0097] When the cells grow well, dilute the remaining cell suspension after subculturing and count them on a hemocytometer. 5Cells were inoculated into 6-well cell culture plates, and no cells were inoculated in the negative group, and the cell growth status was continuously observed. After 24 hours of incubator culture, the supernatant was aspirated and washed 3 times with PBS, and 2 mL of LFP-1 solution of different concentrations was added respectively, and culture was continued for 24 hours. The original culture medium was discarded, and after washing once with PBS, 1 mL of 1:1000 diluted DCFH-DA fluorescent probe was added to each well and incubated in a 37°C incubator for 30 minutes. The cells were washed three times with pure culture medium to remove intercellular DCFH-DA, 0.5 mL of PBS was added to scrape the cells, and centrifuged at 1500 rpm for 3 minutes, the supernatant was discarded, and 0.5 mL of PBS was added to resuspend the cells. The cell suspension was displayed using the FITC channel of flow cytometry, and the results were analyzed by Flowjo software.

[0098] When the cells grow well, dilute the remaining cell suspension after subculturing and count them on a hemocytometer. 4 Cells were inoculated into 96-well cell culture plates, and no cells were inoculated in the blank group to observe the cell growth status. After 24 hours of culture in the cell culture incubator, complete culture medium was used to prepare different concentrations of sample solutions (25, 50, 100, 200, 400 μg / mL), 100 μL of sample solution was added to the experimental group, 100 μL of complete culture medium was added to the control group, and 100 μL of 1 μg / mL LPS solution was added to the LPS group, and culture was continued for 24 hours. The content of inflammatory factors (IL-1β, IL-6, IL-10 and TNF-α) was determined using ELISA kits.

[0099] Cell culture, plating and sample loading were the same as above. After 24 h of culture, the supernatant culture medium was removed, and the cells were resuspended in 500 μL PBS and mixed thoroughly. After centrifugation, the supernatant was removed. Protein was extracted from the cells, measured with the BCA kit, and then the results were quantitatively analyzed using Image J software.

[0100] Comparative Example 1

[0101] Comparative efficacy test of polysaccharide fraction 2 (i.e., the fraction eluted with 0.5 mol / L sodium chloride solution described in Example 1) and crude polysaccharide CLFP of Example 1 on macrophage RAW264.7

[0102] Take macrophages in the logarithmic growth phase and dilute the cell density to 1×10 per ml using a cell counting plate. 5100 μL of cells were inoculated in each well of a 96-well plate, and no cells were inoculated in the blank group. The plates were placed in a cell culture incubator and cultured for 24 hours. The contrast polysaccharide components were prepared into solutions of 25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL using cell culture medium. 100 μL of cell culture medium was added to the negative control group, and 100 μL of the above-mentioned contrast polysaccharide component solutions of different concentrations were added to the experimental group, and the culture was continued for 24 hours. After washing away the original culture medium, 100 μL of CCK-8 reagent diluted 10 times with high-glucose DMEM medium was added to each well. After incubation for 1 hour, the OD value (absorbance) at 450 nm was measured with an enzyme reader, and the cell survival rate was calculated.

[0103] The cell survival rate formula is: cell survival rate = (OD 实验组 -OD 空白组 ) / (OD 阴性对照组 -OD 空白组 )

[0104] like Fig.13 As shown, the proliferation effect of the comparative polysaccharide component 2 on macrophages is not as good as that of the polysaccharide of formula 1, and the proliferation promoting effect of the crude polysaccharide CLFP is not as good as that of the polysaccharide purified by cellulose column. Therefore, it can be proved that the polysaccharide of formula 1 has priority in promoting cell activity and content.

Claims

1. A polysaccharide active ingredient, characterized in that: A polysaccharide component comprising the structure of Formula 1; 2. The polysaccharide active ingredient according to claim 1, characterized in that: The molecular weight of the polysaccharide active ingredient of Formula 1 is 52000-55000, preferably 53000-54000.

3. A method for preparing the polysaccharide active ingredient according to claim 1 or 2, characterized in that: The raw material of Tripterygium wilfordii is extracted with water to obtain a crude extract; a sodium chloride aqueous solution is used as an eluent to perform cellulose exchange column chromatography on the crude extract, and a target eluate having a concentration of 0.25 to 0.35 M sodium chloride aqueous solution is collected; and the target eluate is then eluted by dextran gel chromatography to obtain the polysaccharide active component.

4. The method for preparing the polysaccharide active ingredient according to claim 3, characterized in that: The raw material of the three-leaf twig is the rhizome of the three-leaf twig; Preferably, before water is added, the raw material of Sanmei wood is pretreated and degreased, wherein the pretreatment operation includes washing, drying, crushing and sieving in sequence, and after the powder is dried, 60-90% ethanol is used for reflux degreasing for more than 1 hour.

5. The method for preparing the polysaccharide active ingredient according to claim 3, characterized in that: The liquid-to-solid ratio in the water extraction stage is 10-25 mL / g; Preferably, the water extraction stage is carried out with microwave assistance; Preferably, the microwave power in the water extraction stage is 250-650W; Preferably, the water extraction stage lasts for 10 to 30 minutes.

6. The method for preparing the polysaccharide active ingredient according to claim 5, characterized in that: After the water extraction treatment, alcohol is added to the obtained water extract to perform alcohol precipitation treatment to obtain an alcohol precipitate, and then the alcohol precipitate is redissolved and deproteinized to obtain the crude extract; Preferably, the alcohol is a C1-C4 mono- or polyol, preferably ethanol; Preferably, the volume ratio of the water extract to the alcohol is 1:1 to 10; Preferably, the alcohol precipitate is deproteinized using Sevage reagent to obtain crude polysaccharide; Preferably, the volume ratio of the alcohol precipitate to the Sevage reagent is 3 to 5:

1.

7. The method for preparing the polysaccharide active ingredient according to claim 3, characterized in that: The cellulose exchange column is a DEAE-52 cellulose anion exchange column; Preferably, during the elution process, water is used for pre-elution, then an aqueous solution of 0.05-0.15 M sodium chloride is used for elution, and then an aqueous solution of 0.25-0.35 M sodium chloride is used for elution, and the target eluate containing the polysaccharide active ingredient is collected.

8. The method for preparing the polysaccharide active ingredient according to claim 3, characterized in that: The target eluate is dialyzed for desalting, and then eluted by dextran gel column chromatography to obtain the polysaccharide active ingredient; Preferably, the target eluate is desalted by 3500Da dialysis; Preferably, the dextran gel column is a G-100 dextran gel column.

9. Use of the polysaccharide active ingredient according to any one of claims 1 to 2 or the polysaccharide active ingredient prepared by the preparation method according to any one of claims 3 to 8 in the preparation of anti-inflammatory drugs.

10. An anti-inflammatory drug, characterized in that: A pharmaceutically effective amount of the polysaccharide active ingredient according to any one of claims 1 to 2 or the polysaccharide active ingredient prepared by the preparation method according to any one of claims 3 to 8; Preferably, the anti-inflammatory drug further comprises a pharmaceutically effective amount of an auxiliary material; Preferably, the anti-inflammatory drug also has a pharmaceutically acceptable dosage form.

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