A kind of euphorbia polysaccharide and its preparation method and use in autoimmune diseases

Abstract

The present invention belongs to the technical field of polysaccharide extraction, and particularly relates to a cyperus polysaccharide, a preparation method thereof, and an application for treating autoimmune diseases. The cyperus polysaccharide of the present invention comprises structural units provided by the following sugar residues: galacturonic acid residues, rhamnose residues, glucose residues, galactose residues, and xylose residues, with a molar ratio of 2-3:1-2:7-8:1-2:1-2. The cyperus polysaccharide of the present invention is an acidic homogeneous heteropolysaccharide with a clear monosaccharide composition, a determined molecular weight, and a clear glycosidic bond linkage mode, which is isolated and obtained for the first time from the cyperus in Jinzhai, Anhui Province. It has immune activity and can be used to develop drugs for treating autoimmune diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and particularly relates to a cyperus polysaccharide, a preparation method thereof, and an application thereof in autoimmune diseases. Background Art

[0002] The fruit of the three-leafed akebia tree, also known as "mutong" and "yuzhizi," ripens in August due to the cracking of the peel, earning it the nickname "August Explosion." Its flesh is sweet and rich in various amino acids, making it a new type of health fruit. Ancient texts such as the Kaibao Materia Medica and the Dietary Materia Medica attest to its long history as a traditional Chinese medicine. Modern scientific research has revealed that yuzhizi primarily contains triterpenoid saponins, phenolic alcohols and their glycosides, steroids, and polysaccharides. These polysaccharides have been shown to have diuretic, liver regenerative, neuroprotective, lipid-lowering, analgesic, anti-inflammatory, and weight-loss properties, as well as to influence ethanol metabolism.

[0003] Current research on the herb remains focused on small molecules, often using the triterpenoid saponin α-hedera saponin as a reference. However, the specific pharmacological mechanisms underlying the biological activities of the herb's polysaccharides, important macromolecular components that adhere to the principles of traditional Chinese medicine decoction, have yet to be fully explored. Furthermore, due to the structural complexity, diversity, and heterogeneity of herb polysaccharides, structural elucidation of the polysaccharides remains difficult and challenging. Currently, there are two major limitations: 1. Analyses of the pharmacological activities of the herb's polysaccharides are lacking; 2. Conventional structural characterization focuses primarily on the total sugar content of herb polysaccharides, lacking detailed analysis of the monosaccharide composition and linkage within the polysaccharide molecules. This results in a crude and inaccurate characterization of the herb's polysaccharides. More accurate structural characterization would be more valuable in revealing the pharmacological activities of herb polysaccharides and providing a safety guarantee for herb quality control. Summary of the Invention

[0004] In light of the shortcomings of existing technologies, the present invention provides a polysaccharide derived from a plant called a cyperus rotundus, a method for its preparation, and its use in treating autoimmune diseases. This invention clarifies the material basis for the efficacy of the polysaccharide derived from a plant called a cyperus rotundus, guided by its "immune active component," and combines two-dimensional nuclear magnetic resonance (NMR) technology to reveal the specific structure of the polysaccharide that exerts immune activity, providing a safety guarantee for further quality control of the polysaccharide derived from a plant called a cyperus rotundus.

[0005] In a first aspect, the present invention provides a polysaccharide of the fruit of the Chinese wolfberry tree, which comprises structural units provided by the following sugar residues: galacturonic acid residues, rhamnose residues, glucose residues, galactose residues, and xylose residues, and the molar ratio thereof is 2-3:1-2:7-8:1-2:1-2, preferably 2.75:1.14:7.56:1.06:1.62.

[0006] In some embodiments, the sucrose polysaccharide has the structure shown below:

[0007]

[0008] Wherein, m is an integer from 1 to 30; n is an integer from 1 to 30; m:n=1:1.

[0009] m is an integer of 1 to 30, preferably 3 to 28, more preferably 5 to 25, and most preferably 20.

[0010] n is an integer of 1 to 30, preferably 3 to 28, more preferably 5 to 25, and most preferably 20.

[0011] In some embodiments, the weight average molecular weight of the seed polysaccharide is 30 to 50 kDa.

[0012] In a second aspect, the present invention provides a method for preparing the aforementioned korean polysaccharide, comprising: extracting the dried fruit of the korean with an ethanol-water solution, precipitating the extract with alcohol to obtain a precipitate a; re-dissolving the precipitate a with water, defatting, deproteinizing, concentrating and freeze-drying to obtain a crude korean polysaccharide; re-dissolving the crude korean polysaccharide with water, and re-precipitating with alcohol to obtain a precipitate b; re-dissolving the precipitate b with water, passing it through a gel chromatography column and eluting it, dialyzing the eluate and freeze-drying it to obtain the korean polysaccharide.

[0013] In some embodiments, in the ethanol-water extraction: the concentration of the ethanol-water is 40-60% v / v, the extraction temperature is 100° C., the solid-liquid ratio is 1 g:3-5 mL, preferably 1 g:4 mL; the number of extractions is 1 to 3 times, preferably 3 times.

[0014] In some embodiments, in the alcohol precipitation for preparing precipitate a: the alcohol solvent is anhydrous ethanol, the volume ratio of the extract to anhydrous ethanol is 1:3-5, preferably 1:4; the alcohol precipitation temperature is 3-5°C, preferably 4°C, and the alcohol precipitation time is 10-14h, preferably 12h.

[0015] In some embodiments, in the alcohol precipitation for preparing precipitate b: the alcohol solvent is an ethanol solution with a concentration of 95% v / v or above, preferably an ethanol solution with a concentration of 95% v / v, the alcohol concentration of the system added with the alcohol solvent is 20-80% v / v, for example, it can be an alcohol concentration system of 20% v / v, 40% v / v, 60% v / v, 80% v / v, preferably an alcohol concentration system of 40% v / v, the temperature of the alcohol precipitation is 3-5°C, preferably 4°C, and the alcohol precipitation time is 10-14h, preferably 12h.

[0016] In some embodiments, a Sephadex G-200 dextran gel chromatography column is used and eluted with pure water. Preferably, the precipitate b is redissolved in water to a concentration of 8 to 12 mg / mL, preferably 10 mg / mL; the size of the dextran gel chromatography column is 1.5×100 cm to 1.7×100 cm, preferably 1.6×100 cm; the flow rate is 0.4 to 0.6 mL / min, preferably 0.5 mL / min;

[0017] In some embodiments, dialysis is performed using a 2000-3000 Da dialysis bag for 40-50 hours, preferably a 2500 Da dialysis bag for 48 hours.

[0018] In a third aspect, the present invention provides a composition comprising the aforementioned cyperus polysaccharide, and the remaining components are not specifically limited, for example, they may be pharmaceutically acceptable auxiliary ingredients.

[0019] In a fourth aspect, the present invention provides the use of the aforementioned cyperus polysaccharide or composition in the preparation of a medicament for treating autoimmune diseases, wherein the autoimmune diseases include but are not limited to rheumatoid arthritis, osteoarthritis, gouty arthritis, psoriasis, inflammatory bowel disease, multiple sclerosis, systemic lupus erythematosus, aplastic anemia, Sjögren's syndrome, urticaria, allergic rhinitis or asthma, psoriasis, ankylosing spondylitis, preferably rheumatoid arthritis, osteoarthritis, and gouty arthritis.

[0020] The beneficial effects of the present invention are:

[0021] The polysaccharide provided by the present invention is an acidic homogeneous heteropolysaccharide isolated for the first time from the euphorbia pulex in Jinzhai, Anhui Province, with a clear monosaccharide composition, a determined molecular weight, and a clear glycosidic bond linkage mode. It has immune activity and can be used to develop drugs for treating autoimmune diseases.

[0022] The preparation method provided by the present invention is obtained by combining a water extraction and alcohol precipitation method with graded precipitation with ethanol of different concentrations and dextran gel chromatography column chromatography, and is optimized through a DPPH free radical experiment and an immune-related MH7A cell experiment. The preparation process conditions are mild, the operation is simple, and it is environmentally friendly, and is suitable for large-scale production applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present method, a brief introduction will be given below to the drawings required for use in the description of the implementation cases. Obviously, the drawings described below are some implementation cases of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0024] Figure 1 A shows the appearance of the predictor in Example 1 of the present invention;

[0025] Figure 1 B shows the appearance of the AFP-ⅡA polysaccharide in Example 1 of the present invention;

[0026] Figure 2 The flowchart of the preparation process of the polysaccharide AFP-ⅡA of the genus AFP in Example 1 of the present invention is shown;

[0027] Figure 3 A shows the different fractions obtained by eluting the AFP-ⅡA polysaccharide from a DEAE-52 dextran gel column in step 4 of Example 1 of the present invention;

[0028] Figure 3 B shows the ultraviolet spectrum of the polysaccharide AFP-ⅡA of the seed of the present invention in Example 1;

[0029] Figure 3 C shows the HPGPC diagram of the polysaccharide AFP-ⅡA of the seed of Example 1 of the present invention;

[0030] Figure 4 The infrared spectrum of the polysaccharide AFP-ⅡA of the schizonepeta tenuifolia of Example 1 of the present invention is shown;

[0031] Figure 5 The monosaccharide composition analysis diagram of the polysaccharide AFP-ⅡA of the fruit of Example 1 of the present invention is shown, wherein from left to right are: galacturonic acid, rhamnose, glucose, galactose, and xylose;

[0032] Figure 6 The structure of the AFP-ⅡA polysaccharide of Example 1 of the present invention is shown, wherein: m=20; n=20; m:n=1:1;

[0033] Figure 7 The experimental results of the effect of the AFP-ⅡA-C polysaccharide on the proliferation of MH7A cells in Example 1 of the present invention are shown;

[0034] Figure 8 The experimental results of Example 1 of the present invention showing the effect of the AFP-ⅡA polysaccharide of the genus AFP on the proliferation of MH7A cells. DETAILED DESCRIPTION

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0036] Example 1 Preparation of AFP-ⅡA Polysaccharide from Apocynum serrata

[0037] Preparation process flow chart Figure 2 As shown, the specific steps include:

[0038] Step 1: Extraction of total polysaccharides from koji seeds: Figure 1 180 g of dried fruit of the Anhui Jinzhai euphorbia pulex (A) was decocted three times with hot water / ethanol (volume ratio of 1:1), with the volume ratio of euphorbia pulex to hot water / ethanol being 1:4, and the hot water temperature being 100°C. The mixture was filtered, the filtrates from the three decoctations were combined and concentrated, and then precipitated with anhydrous ethanol, with the volume ratio of filtrate to anhydrous ethanol being 1:4, the precipitation temperature being 4°C, and the precipitation time being 12 hours. The mixture was centrifuged at 5000 rpm / min for 15 minutes, and the precipitate was collected to obtain euphorbia pulex total polysaccharide.

[0039] Step 2: Primary purification: The total polysaccharide of the angelica dahurica was dissolved in pure water, defatted with petroleum ether, deproteinized with a Seveg reagent, and then concentrated and freeze-dried to obtain the crude polysaccharide AFP of the angelica dahurica.

[0040] Step 3, Secondary Purification: The crude AFP polysaccharide from the iris was dissolved in pure water and precipitated again with alcohol. 95% v / v ethanol was added until the ethanol concentrations reached 20%, 40%, 60%, and 80% v / v, respectively. The precipitation temperature was 4°C and the precipitation time was 12 hours. The precipitates were collected to obtain AFP-II polysaccharides corresponding to different alcohol concentrations. These AFP-II solutions were dissolved in pure water to concentrations of 0.2 to 1.0 mg / mL. 2.0 mL of each solution was then reacted with a freshly prepared DPPH reaction mixture (2.0 mL of a 0.2 mM methanol solution) at room temperature (25°C) in the dark for 30 minutes. The absorbance of the mixture was measured at 517 nm to identify the optimal fraction. DPPH antioxidant assay results showed that the AFP-II polysaccharide from the iris polysaccharide obtained with a 40% v / v alcohol concentration exhibited the best antioxidant activity.

[0041] Step 4, three purifications: The 40% v / v alcohol concentration system corresponding to the cyperus polysaccharide AFP-Ⅱ was dissolved in pure water to a 5 mg / mL cyperus polysaccharide AFP-Ⅱ solution, and passed through a Sephadex G-200 polyacrylamide gel chromatography column (1.6×100 cm). The eluent used pure water, 0.1 mol / L NaCl solution, and 0.2 mol / L NaCl solution at a flow rate of 0.5 mL / min. The polysaccharide content in the eluate was detected by phenol-sulfuric acid colorimetry. The polysaccharide-containing eluate was collected to obtain the cyperus polysaccharide extract, and 3 components were obtained, such as Figure 3As shown in Figure A, the AFP-ⅡA polysaccharide from the iris eluted with pure water, the AFP-ⅡB polysaccharide from the iris eluted with a 0.1 mol / L NaCl solution, and the AFP-ⅡC polysaccharide from the iris eluted with a 0.2 mol / L NaCl solution were dialyzed for 48 hours using a 2500 Da dialysis bag and then freeze-dried to obtain freeze-dried powders of each component. Screening results from an anti-rheumatoid arthritis cell experiment showed that the AFP-ⅡA component was the most optimal, with an extraction yield of 0.19%.

[0042] Step 5, structural characterization: the appearance, UV spectrum, HPGPC, infrared spectrum, monosaccharide composition analysis and structure of the AFP-ⅡA polysaccharide are shown in the following figure: Figure 1 B、3B、 Figure 3 C. Figures 4-6 shown.

[0043] Example 2 Analysis of the physicochemical properties of AFP-ⅡA polysaccharide from the fruit of the Chinese wolfberry

[0044] (1) Determination of polysaccharide content of AFP-ⅡA polysaccharide from the fruit of the Chinese wolfberry

[0045] Accurately weigh 10 mg of D-anhydrous glucose and place it in a 100 mL volumetric flask. Dissolve it in distilled water and make up to volume to obtain a glucose reference solution with a concentration of 0.1 mg / mL. For the drawing of the standard curve, accurately pipette 0.00 mL, 0.10 mL, 0.20 mL, 0.30 mL, 0.40 mL, 0.50 mL, 0.60 mL, 0.80 mL, and 1.0 mL of the prepared standard solution into 25 mL stoppered glass test tubes, add distilled water to make up to 2.0 mL respectively, add 1.0 mL of 5% phenol solution and 5.0 mL of concentrated sulfuric acid solution, mix well, place in a water bath (80°C) and heat for 15 min, then take out and cool to room temperature. Use an equal amount of pure water as a blank control, and use a SHIMADZU UV-2550 UV-visible spectrophotometer to measure the absorbance at a wavelength of 490 nm. Plot the standard curve with absorbance as the ordinate and concentration as the abscissa. Weigh 10 mg of lyophilized AFP-ⅡA polysaccharide powder from the plant and dissolve it in pure water to form a 0.1 mg / mL polysaccharide solution. Place 0.4 mL of the solution in a 10 mL test tube. Measure the absorbance of the sample solution and calculate the polysaccharide content.

[0046] The polysaccharide content in the AFP-ⅡA freeze-dried powder of the schizonepeta polysaccharide obtained in Example 1 was determined to be 87%.

[0047] (2) Determination of the uronic acid content of the AFP-ⅡA polysaccharide obtained in Example 1 by the m-hydroxybiphenyl method

[0048] Weigh 0.955g of sodium tetraborate and dissolve it in 100mL of concentrated sulfuric acid. Weigh 0.015g of m-hydroxybiphenyl and dissolve it in 0.5% NaOH solution and dilute to 10mL. Accurately weigh 5mg of anhydrous galacturonic acid, dissolve it in pure water and dilute to volume with a 10mL volumetric flask to obtain a 0.5mg / mL galacturonic acid standard solution. Accurately pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0mL of the above-mentioned glucose standard solution into 10mL test tubes respectively, and fill each tube with double distilled water to 1.0mL. Place the test tube in an ice bath and add 5mL of cooled borate solution dropwise. After shaking and mixing, place it in a boiling water bath for 20min, take it out and cool to room temperature. Add 100μL of m-hydroxybiphenyl solution to each tube, mix well and let it stand for 30min. Accurately weigh 10 mg of lyophilized polysaccharide powder and dissolve it in pure water to form a 0.5 mg / mL polysaccharide solution. Place 0.4 mL of the solution in a 10 mL test tube. Measure the absorbance at 525 nm using a SHIMADZU UV-2550 UV-visible spectrophotometer.

[0049] The results showed that the uronic acid content in the AFP-ⅡA polysaccharide obtained in Example 1 was 18.96%.

[0050] (3) UV scanning was used to measure the ultraviolet absorption of the AFP-ⅡA polysaccharide obtained in Example 1.

[0051] like Figure 3 As shown in Figure B, the UV scanning spectrum of the obtained lyophilized powder of AFP-ⅡA showed no absorption peaks of pigments, proteins and nucleic acids. The instrument used was a SHIMADZU UV-2550 UV-visible spectrophotometer with a scanning range of 200 to 800 nm.

[0052] (4) The homogeneity and relative molecular weight of the AFP-ⅡA polysaccharide obtained in Example 1 were determined by HPGPC method.

[0053] Test conditions: Agilent 1260 Infinity system; SRT SEC-150 (7.8×300 mm) chromatographic column; mobile phase: double-distilled water; injection volume: 4 μL; flow rate: 1.0 mL / min; column temperature: 35°C; differential refractive index detector.

[0054] like Figure 3 As shown in Figure C, the detection showed that the AFP-ⅡA polysaccharide obtained in Example 1 was a homogeneous polysaccharide with a relative molecular mass of 41.9 kDa.

[0055] (5) Analyze the characteristic groups of the AFP-ⅡA polysaccharide obtained in Example 1 using an infrared spectrometer

[0056] Weigh about 5.0 mg of the dried polysaccharide freeze-dried powder sample, mix it with 200 mg of KBr powder, press it into a tablet, and then scan and analyze it on a L1600400Spectrum TWO FT-IR spectrometer with a scanning range of 4000 cm -1 ~400cm -1 .

[0057] See also Figure 4 After testing, the AFP-ⅡA polysaccharide obtained in Example 1 has a characteristic peak of polysaccharide at 3430 cm -1 The broad peak at 2850 cm-1 is attributed to the -OH group. -1 The peak at 1640 cm is attributed to CH stretching vibration. -1 The peak at 1590 cm is attributed to C=O stretching vibration. -1 The peak at 1350cm is attributed to the stretching vibration of CH (C=O on C). -1 The peak at 1040 cm is attributed to the CO stretching vibration of the carboxylic acid. -1 The peak at 1350 cm-1 is attributed to the CO stretching vibration of the pyranose ring. -1 、1040cm -1 , 1150cm -1 The three absorption peaks that appeared indicated the existence of COC bonds; 902 cm -1 Probably due to the β-D-glucose residues.

[0058] Example 3 Chemical Structure Identification of Precocious Seed Polysaccharide

[0059] (1) The monosaccharide composition of the AFP-ⅡA polysaccharide obtained in Example 1 was determined by PMP pre-column derivatization method.

[0060] Weigh 60 mg of lyophilized AFP-ⅡA polysaccharide powder and dissolve it in 3 mL of ultrapure water. Add the solution to a stoppered test tube and stir with a magnetic stirrer. Then, add 3 mL of 4 mol / L trifluoroacetic acid and incubate in an oil bath at 110°C for 5 h. Transfer the resulting sample solution to a spinner flask and add 10 mL of methanol to evaporate it to dryness. Repeat this process three times to remove the acid. Dissolve the sample in 2 mL of ultrapure water, filter it, and use the solution for derivatization.

[0061] Derivatization: Pipette 1 mL of the hydrolyzate into a test tube, then add 1 mL of 0.3 mol / L NaOH solution and 0.5 mol / L PMP methanol solution, mix well, incubate in a 70°C water bath for 1 hour, and allow to cool naturally for 10 minutes. Add 1 mL of 0.3 mol / L HCl, and extract three times with 1 mL of dichloromethane. The upper layer is the aqueous layer. Dilute the aqueous layer with 1 mL of ultrapure water, filter through a 0.22 μm microporous membrane, dilute to a 10 mL volumetric flask, and inject 1 mL.

[0062] Test conditions: Agilent 1260 Infinity system; column 1 (0.25 μm × 250 μm × 30 m); mobile phase: 0.05 mol / L phosphate (pH = 6.7) buffer: acetonitrile = 79:21, column temperature: 35°C; detection wavelength: 245 nm; flow rate: 1 mL / min; injection volume: 10 μL.

[0063] See also Figure 5 After testing, the AFP-ⅡA polysaccharide obtained in Example 1 is composed of galacturonic acid: rhamnose: glucose: galactose: xylose, and the molar ratio of galacturonic acid: rhamnose: glucose: galactose: xylose is 2.75:1.14:7.56:1.06:1.62.

[0064] (2) Glycosidic bond type of the AFP-ⅡA polysaccharide obtained in Example 1 was analyzed by methylation

[0065] Weigh 15 mg of fully dried polysaccharide, add 2 mL of anhydrous dimethyl sulfoxide (DMSO) to fully dissolve it, then add 90 mg of NaOH powder, stir and react for 2 hours, then add 2 mL of anhydrous iodomethane, react in a sealed and light-proof place at 35°C for 12 hours, add an appropriate amount of distilled water to terminate the reaction, and obtain a completely methylated sample.

[0066] A fully methylated sample was placed in a 10 mL ampoule, 3 mL of 4 mol / L trifluoroacetic acid was added, the tube was sealed, and hydrolysis was carried out at 120°C for 6 h. The sample was cooled to room temperature, and the hydrolyzate was transferred to a rotary evaporator and evaporated to dryness. Appropriate amounts of methanol were added several times and evaporated to dryness to completely remove residual trifluoroacetic acid. 1.5 mL of 0.05 M NaOH solution and 45 mg of sodium borohydride were then added. The sample was reacted at room temperature for 12 h, neutralized with 100 μL of acetic acid until neutral, and the reaction solution was evaporated to dryness on a rotary evaporator. 1 mL of acetic anhydride and 1 mL of pyridine were added to the reaction solution in sequence. The solution was sealed and acetylated at 90°C for 2 h. After the reaction, the acetylated product was extracted with dichloromethane, and the extract was used to remove residual water with anhydrous sodium sulfate, and then analyzed by GC-MS. Detection conditions: A BR-17 column (0.25 μm × 250 μm × 30 m) was used. Carrier gases: high-purity helium (99.999%) and high-purity nitrogen (99.999%), purchased from Nanjing Shangyuan Industrial Gas Plant; column flow rate: 1.0 mL / min; split-flow injection was used, with an inlet temperature of 250°C. The temperature program was set as follows: initial temperature of 50°C, ramped at 25°C / min to 100°C, held for 3 min, then ramped at 4°C / min to 180°C. Mass spectrometry conditions: EI ion source, electron energy 70 eV, ion source temperature 220°C, transmission line temperature 280°C, and quadrupole temperature 40°C. Full scan mode was used, with a scan range of m / z 40 to 600. The mass spectral standard library was NIST 11.L.

[0067] After testing, the methylated product of the AFP-ⅡA polysaccharide obtained in Example 1 is shown in Table 1. The specific methylated structure analysis is as follows:

[0068] Table 1 Methylation analysis of AFP-ⅡA polysaccharide from the fruit of the Chinese wolfberry

[0069] Glycosidic bond type molar ratio 1,4-β-D-GalAp 2.7 3,4)-α-L-Rhap- 1.1 1,2,4-β-D-Xylp 1.6 1,4-β-D-Galp 1.0 1,4-α-D-Glcp 6.3 1,2,4-α-D-Glcp 1.2

[0070] After testing, the predicted seed polysaccharide AFP-ⅡA obtained in Example 1 is composed of 1,4-β-D-GalAp, 3,4)-α-L-Rhap, 1,2,4-β-D-Xylp, 1,4-β-D-Galp, 1,4-α-D-Glcp, and 1,2,4-α-D-Glcp sugar residues. Based on the above analysis, the following results were obtained: the composition of the safflower polysaccharide AFP-ⅡA is uniform, with a relative molecular mass of 41.9 kDa. The monosaccharide composition analysis and methylation results show that the safflower polysaccharide AFP-ⅡA in the present invention contains pyranose glucose (Glcp), pyranose galactose (Galp), furanose rhamnose (Rhap), furanose xylose (Xylp) and pyranose galacturonic acid (GalAp), and the molar ratio is 7.56:1.06:1.14:1.62:2.75; its primary structural unit contains six sugar residues: 1,4-β-D-GalAp, 3,4)-α-L-Rhap-, 1,2,4-β-D-Xylp, 1,4-β-D-Galp, 1,4-α-D-Glcp and 1,2,4-α-D-Glcp. The possible connection modes of the six sugar residues are as follows: Figure 6 shown.

[0071] Example 4 Experimental study on the anti-rheumatoid arthritis effect of polysaccharide from Rhizoma Coptidis

[0072] (1) Cell culture

[0073] Culture in DMEM medium supplemented with 10% FBS and 1% double-antibody in a 37°C, 5% CO2, saturated humidity incubator. When MH7A cells reach 70%-80% confluence at the bottom of the flask, wash three times with PBS using a 3 mL pipette. Disintegrate the cells with 0.25% trypsin solution and terminate digestion with serum-suppled DMEM complete medium. Subculture the cells at a 1:2 ratio to complete the experiment.

[0074] (2) Cell proliferation activity assay

[0075] MH7A cells in normal growth in logarithmic phase were taken, digested, centrifuged and collected, and counted under a counting plate to prepare 1×10 5 A single cell suspension of 100 μL per well was inoculated into a 96-well plate and cultured overnight at 37°C with 5% CO2. After the cells adhered and grew for 24 hours, they were stimulated with TNF-α (10 ng / mL) and treated with different concentrations (50, 100, 200 μg / mL) of the AFP-ⅡA-C prepared in Example 1 for 24 hours. 10 μL of CCK-8 solution was added to each well and the plates were placed in an incubator for another 1 hour. The absorbance (OD) value of each well at 450 nm was measured using a microplate reader. The experiment was repeated 3 times, the average value was taken, and the cell survival rate was calculated. The results are shown in FIG. Figure 7 shown.

[0076] The results are as follows Figure 8 As shown in the results, compared with the normal group, the absorbance value after treatment with the polysaccharide prepared in Example 1 of the present invention was significantly reduced, and the degree of reduction was positively correlated with the concentration of the polysaccharide, indicating that the polysaccharide prepared in the present invention can effectively inhibit the growth of arthritis fibroblasts MH7A cells, showing an excellent anti-rheumatoid arthritis effect.

[0077] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A polysaccharide of koreana, characterized in that The polysaccharide of the fruit of the Chinese wolfberry contains structural units provided by the following sugar residues: galacturonic acid residues, rhamnose residues, glucose residues, galactose residues, and xylose residues, and the molar ratio thereof is 2-3:1-2:7-8:1-2:1-2; The polysaccharide has the structure shown below: Wherein, m is an integer from 1 to 30; n is an integer from 1 to 30; m:n=1:1; The weight average molecular weight of the schizonepeta polysaccharide is 30-50 kDa.

2. A method for preparing the polysaccharide of the fruit of the Chinese wolfberry according to claim 1, characterized in that: The preparation method comprises: The dried fruit of the genus Precocious is extracted with an ethanol-water solution, the extract is concentrated and then precipitated with alcohol to obtain precipitate a; The precipitate a is redissolved in water, defatted, deproteinized, concentrated and freeze-dried to obtain crude polysaccharide of the fruit of the Chinese wolfberry; After the crude polysaccharide of the seed of the Chinese wolfberry is redissolved in water, it is precipitated with alcohol again to obtain precipitate b; The precipitate b is redissolved in water, passed through a gel chromatography column and eluted, and the eluate is dialyzed and freeze-dried to obtain the cyperus polysaccharide.

3. The preparation method according to claim 2, characterized in that In the ethanol-water extraction, the concentration of the ethanol-water is 40-60% v / v, the extraction temperature is 100° C., and the material-liquid ratio is 1 g:3-5 mL.

4. The preparation method according to claim 2, characterized in that In the alcohol precipitation for preparing precipitate a: the alcohol solvent is anhydrous ethanol, the volume ratio of the extract to anhydrous ethanol is 1:3~5, the alcohol precipitation temperature is 3~5°C, and the alcohol precipitation time is 10~14h.

5. The preparation method according to claim 2, characterized in that In the alcohol precipitation for preparing precipitate b: the alcohol solvent is an ethanol solution with a concentration of 95% v / v or above, and the alcohol solvent is added until the alcohol concentration of the system is 20~80% v / v. The temperature of the alcohol precipitation is 3~5°C, and the alcohol precipitation time is 10~14h.

6. The preparation method according to claim 2, characterized in that Use Sephadex G-200 dextran gel chromatography column and elute with pure water; and / or, dialyze using a 2000~3000Da dialysis bag for 40~50 hours.

7. A composition, characterized in that The composition comprises the schizonepeta polysaccharide according to claim 1.

8. Use of the polysaccharide of Rhizoma Coptidis according to claim 1 or the composition according to claim 7 in the preparation of a medicament for treating rheumatoid arthritis.

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