Akebia fruit polysaccharide as well as preparation method and application thereof to autoimmune diseases

Abstract

The invention belongs to the technical field of polysaccharide extraction, and particularly relates to fructus akebiae polysaccharide, a preparation method thereof and application of the fructus akebiae polysaccharide to autoimmune diseases. The fructus akebiae polysaccharide comprises structural units provided by the following sugar residues: galacturonic acid residues, rhamnose residues, glucose residues, galactose residues and xylose residues, wherein the molar ratio of the galacturonic acid residues to the rhamnose residues to the glucose residues to the galactose residues to the xylose residues is (2-3): (1-2): (7-8): (1-2): (1-2). The akebia fruit polysaccharide is an acidic homogeneous heteropolysaccharide which is separated from Anhui Jinzhai akebia fruit for the first time, is clear in monosaccharide composition, definite in molecular weight and clear in glycosidic bond linking mode, has immunocompetence and can be applied to development of 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 and a preparation method thereof and an application thereof to autoimmune diseases. Background Art

[0002] The fruit of the three-leaf akebia is a medicinal and edible fruit, also known as "Akebia" and "Akebia". The fruit of the three-leaf akebia matures in August and cracks in the peel, so it is also called "August Explosion". The flesh is sweet and rich in various amino acids. It is one of the new health fruits. There are many records in ancient books such as "Kaibao Materia Medica" and "Dietary Materia Medica" that prove that it has a long history as a traditional Chinese medicine. Modern scientific research has found that the main chemical substances of the akebia are triterpenoid saponins, phenolic alcohols and their glycosides, steroids, polysaccharides, etc. The polysaccharide components have been proven to have diuretic, liver regeneration, neuroprotection, hypolipidemic, analgesic, anti-inflammatory and weight loss effects, as well as effects on ethanol metabolism.

[0003] At present, the research on the seed of the Chinese genus is still focused on the small molecule stage, and the triterpenoid saponin α-hedera saponin is often used as a reference substance for research; however, the specific pharmacological material basis of the biological activity of the important macromolecular substances such as polysaccharides in the medicinal materials and in line with the theory of Chinese medicine decoction has not been studied in depth, and the specific pharmacological material basis of the biological activity of the polysaccharide components of the seed of the Chinese genus is still unclear. In addition, due to the structural complexity, variety, and heterogeneity of the components of the seed of the Chinese genus polysaccharide, the structural analysis of the polysaccharide has always been difficult and challenging. At present, there are mainly the following two shortcomings: 1. There is a lack of pharmacological activity analysis corresponding to the polysaccharide components of the seed of the Chinese genus; 2. Conventional structural characterization mainly analyzes the total sugar content in the seed of the Chinese genus polysaccharide, and lacks the analysis of the monosaccharide composition and connection mode in the polysaccharide molecule, which makes the characteristics of the reported seed of the Chinese genus polysaccharide appear rough and inaccurate. More accurate structural characterization is more valuable in revealing the pharmacological activity of the seed of the Chinese genus polysaccharide, and also provides safety for the quality control of the medicinal materials. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a polysaccharide of the seed of the genus Aesculus and its preparation method and use for autoimmune diseases. The present invention clarifies the material basis of the efficacy of the polysaccharide of the seed of the genus Aesculus guided by the "immune active component", and combines the two-dimensional nuclear magnetic resonance technology to analyze the specific structure of the polysaccharide of the seed of the genus Aesculus that exerts immune activity, providing a safety guarantee for the further quality control of the polysaccharide of the seed of the genus Aesculus.

[0005] In the first aspect, the present invention provides a polysaccharide of the seed of the thorn, 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 precocious seed polysaccharide has the structure shown below:

[0007]

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

[0009] m is an integer of 1-30, preferably 3-28, more preferably 5-25, 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 sucrose polysaccharide is 30 to 50 kDa.

[0012] In a second aspect, the present invention provides a method for preparing the aforementioned polysaccharide of the Chinese thorn fruit, comprising: extracting the dried fruit of the Chinese thorn fruit 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 polysaccharide of the Chinese thorn fruit; re-dissolving the crude polysaccharide of the Chinese thorn fruit 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, and dialyzing and freeze-drying the eluate to obtain the polysaccharide of the Chinese thorn fruit.

[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 temperature of the alcohol precipitation is 3-5°C, preferably 4°C, and the time of the alcohol precipitation 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 more, 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 time of the alcohol precipitation 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 specification 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 h, preferably a 2500 Da dialysis bag for 48 h.

[0018] In a third aspect, the present invention provides a composition comprising the aforementioned schizonepeta polysaccharide, and other 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 schizophyllum 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 cyperus polysaccharide provided by the present invention is an acidic homogeneous heteropolysaccharide separated from the cyperus rotundus in Jinzhai, Anhui Province for the first time, with a clear monosaccharide composition, a determined molecular weight, and a clear glycosidic bond linking mode. It has immune activity and can be used in the development of drugs for the treatment of 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 of ethanol at different concentrations and chromatography on a dextran gel column, 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 the environment is friendly, and the method is suitable for large-scale production and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solution in the embodiment of the present method, the following is a brief introduction 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 paying 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 process flow chart of the preparation of the AFP-ⅡA polysaccharide of the present invention in Example 1 is shown;

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

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

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

[0031] Figure 5 The monosaccharide composition analysis diagram of the polysaccharide AFP-ⅡA of the formosan seed in 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 diagram of the predicted polysaccharide AFP-ⅡA 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 the effect of the AFP-ⅡA polysaccharide of Example 1 of the present invention on the proliferation of MH7A cells are shown. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1 Preparation of AFP-ⅡA polysaccharide

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

[0038] Step 1: Extraction of total polysaccharides from koreana: Figure 1 180 g of the dried fruit of Anhui Jinzhai Precocious Fruit as shown in A was repeatedly boiled with hot water / ethanol (volume ratio 1:1) for 3 times, the volume ratio of Precocious Fruit to hot water / ethanol was 1:4, the hot water temperature was 100°C, filtered, the filtrates from the 3 times were combined and concentrated, anhydrous ethanol was added for alcohol precipitation, the volume ratio of the filtrate to anhydrous ethanol was 1:4, the alcohol precipitation temperature was 4°C, and the alcohol precipitation time was 12h. Centrifuged at 5000rpm / min for 15min, the precipitate was collected, and the total polysaccharide of Precocious Fruit was obtained.

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

[0040] Step 3, secondary purification: Dissolve the crude polysaccharide AFP of the seed of the Chinese wolfberry in pure water and precipitate it with alcohol again, add 95% v / v ethanol to the ethanol concentration of the system to 20% v / v, 40% v / v, 60% v / v, 80% v / v, respectively, the alcohol precipitation temperature is 4°C, the alcohol precipitation time is 12h, collect the precipitate, and obtain the seed of the Chinese wolfberry polysaccharide AFP-Ⅱ corresponding to the different alcohol concentration systems, and dissolve it in pure water to obtain a seed of the Chinese wolfberry polysaccharide AFP-Ⅱ solution with a concentration of 0.2-1.0 mg / mL. Then take 2.0 mL and react with the newly prepared reaction mixture DPPH (0.2mM methanol solution, 2.0 mL) at room temperature (25°C) in the dark for 30 minutes. Then measure the absorbance value of the mixture at 517nm to screen out the best component. The results of the DPPH antioxidant experiment show that the seed of the Chinese wolfberry polysaccharide AFP-Ⅱ corresponding to the 40% v / v alcohol concentration system has the best antioxidant effect.

[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 form a 5 mg / mL cyperus polysaccharide AFP-Ⅱ solution, and passed through a Sephadex G-200 polyacrylamide gel chromatography column (1.6×100 cm), and the eluent was pure water, 0.1 mol / L NaCl solution, and 0.2 mol / L NaCl solution, with a flow rate of 0.5 mL / min. The polysaccharide content in the eluate was detected by phenol-sulfuric acid colorimetry, and the polysaccharide-containing eluate was collected to obtain a cyperus polysaccharide refined extract, and 3 components were obtained, such as Figure 3As shown in A, the AFP-ⅡA polysaccharide eluted with pure water, the AFP-ⅡB polysaccharide eluted with 0.1mol / L NaCl solution, and the AFP-ⅡC polysaccharide eluted with 0.2mol / L NaCl solution were dialyzed for 48 hours in a 2500Da dialysis bag, and then freeze-dried to obtain freeze-dried powders of each component. The screening results of the anti-rheumatoid arthritis cell experiment showed that the AFP-ⅡA component was the best, with an extraction rate 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 to 6 shown.

[0043] Example 2 Analysis of the physicochemical properties of the polysaccharide AFP-ⅡA

[0044] (1) Determination of polysaccharide content of AFP-ⅡA polysaccharide

[0045] Accurately weigh 10 mg of D-anhydrous glucose, place it in a 100 mL volumetric flask, add distilled water to dissolve and make up to volume to obtain a glucose reference solution with a concentration of 0.1 mg / mL; Drawing of the standard curve: accurately aspirate 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 and place it in a 25 mL stoppered glass test tube, 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 it in a water bath (80°C) and heat for 15 min, then take it out, cool to room temperature, use an equal amount of pure water as a blank control, use SHIMADZUUV-2550 UV-visible spectrophotometer to measure the absorbance at a wavelength of 490 nm, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa. Weigh 10 mg of lyophilized powder of AFP-ⅡA polysaccharide from the plant, dissolve it in pure water to form a 0.1 mg / mL polysaccharide solution, take 0.4 mL of the solution and place it in a 10 mL test tube. Determine the absorbance of the sample solution and calculate the polysaccharide content.

[0046] It was determined that the polysaccharide content in the lyophilized powder of AFP-ⅡA obtained in Example 1 was 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 it 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 glucose standard solution into 10mL test tubes, 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, shake and mix well, place it in a boiling water bath for 20min, take it out and cool it to room temperature. Add 100μL of m-hydroxybiphenyl solution to each tube, mix well and let it stand for 30min. Weigh 10 mg of polysaccharide freeze-dried powder accurately, dissolve it in pure water to form a 0.5 mg / mL polysaccharide solution, take 0.4 mL of the solution and place it in a 10 mL test tube. Use a SHIMADZU UV-2550 ultraviolet-visible spectrophotometer to measure the absorbance value at a wavelength of 525 nm.

[0049] It was determined 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 B, after testing, 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 SHIMADZU UV-2550 UV-visible spectrophotometer with a scanning range of 200-800nm.

[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 refractometer.

[0054] like Figure 3 As shown in Figure C, after testing, the AFP-ⅡA polysaccharide obtained in Example 1 is a uniform 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 tablets, and then scan and analyze it on a L1600400Spectrum TWO FT-IR Fourier transform infrared 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 3430cm -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; 902cm -1 Probably due to the β-D-glucose residues.

[0058] Example 3 Chemical structure identification of the polysaccharide of the genus

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

[0060] Weigh 60 mg of AFP-ⅡA lyophilized powder from the fruit of the Chinese wolfberry and dissolve it in 3 mL of ultrapure water. Add it to a stoppered test tube and stir it with a magnetic stirrer. Then add 3 mL of 4 mol / L trifluoroacetic acid and place it in an oil bath at 110°C for 5 hours. Transfer the sample solution obtained after acid hydrolysis to a spinner flask and add 10 mL of methanol solution to evaporate it to dryness. Repeat this operation 3 times to remove the acid. Dissolve it in 2 mL of ultrapure water, filter it, and take the solution for derivatization.

[0061] Derivatization: Pipette 1mL of hydrolyzate into a test tube, then take 1mL of 0.3mol / L NaOH solution and 0.5mol / L PMP methanol solution, mix well, place in a water bath at 70℃ for 1 hour, and cool naturally for 10min. Add 1mL of 0.3mol / L HCl, then add 1mL of dichloromethane to extract 3 times. The upper layer is the water layer. Take the water layer and add 1mL of ultrapure water to mix and dilute. Filter with a 0.22μm microporous membrane, dilute to 10mL volumetric flask, and take 1mL for injection.

[0062] Test conditions: Agilent 1260 Infinity system; column No. 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 by methylation analysis

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

[0066] Take the completely methylated sample and place it in a 10mL ampoule, add 3mL of 4mol / L trifluoroacetic acid, seal the tube, hydrolyze at 120℃ for 6h, cool at room temperature, transfer the hydrolyzate to a rotary evaporator and evaporate to dryness, then add appropriate amount of methanol to evaporate to dryness several times to completely remove the residual trifluoroacetic acid, then add 1.5mL 0.05MNaOH solution and 45mg sodium borohydride, react at room temperature for 12h, neutralize with 100μL of acetic acid to neutrality, and place the reaction solution on a rotary evaporator to evaporate to dryness; add 1mL of acetic anhydride and 1mL of pyridine to the reaction solution in sequence, seal it and react at 90℃ for 2h; after the reaction, extract the acetylation product with dichloromethane, remove residual water from the extract with anhydrous sodium sulfate, and perform GC-MS determination. Detection conditions: BR-17 chromatographic column (0.25μm×250μm×30m), carrier gas: high-purity helium (purity 99.999%) and high-purity nitrogen (purity 99.999%): Nanjing Shangyuan Industrial Gas Plant; column flow rate: 1.0mL / min; split injection, inlet temperature 250℃. The temperature program was set as follows: initial temperature 50℃, 25℃ / min to 100℃, maintained for 3min, and then 4℃ / min to 180℃. Mass spectrometry conditions: EI ion source, electron energy 70eV, ion source temperature 220℃, transmission line temperature 280℃, quadrupole temperature 40℃. Full scan mode, scan range m / z 40~600, mass spectrometry standard library: 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 Analysis of methylation of AFP-ⅡA in polysaccharide of cyperus rotundus

[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, it was found that the predicted polysaccharide AFP-ⅡA obtained in Example 1 was 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 can be obtained: the composition of the safflower polysaccharide AFP-ⅡA is uniform, and the relative molecular mass is 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 thereof 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 Psoralea corylifolia

[0072] (1) Cell culture

[0073] DMEM cell culture medium containing 10% FBS and 1% double antibody was cultured in a constant temperature incubator at 37°C, 5% CO2, and saturated humidity. When MH7A cells grew to cover 70%-80% of the bottom of the culture flask, PBS solution was aspirated with a 3mL pipette and washed three times, and the cells were digested with 0.25% trypsin digestion solution, and the cell digestion was terminated with DMEM complete culture medium containing serum, and the cells were passaged at a ratio of 1:2 to complete the relevant experiments.

[0074] (2) Cell proliferation activity assay

[0075] MH7A cells growing normally in the logarithmic phase were taken, digested, centrifuged and collected, and counted under a counting plate to make 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 and 5% CO2. After the cells adhered to the wall 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 further incubation for 1 hour. The absorbance (OD) value of each well at 450 nm was measured with an ELISA instrument. The experiment was repeated 3 times, the average 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, compared with the normal group, the absorbance value after treatment with the cyperus rotundus 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 cyperus rotundus polysaccharide, indicating that the cyperus rotundus 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, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A polysaccharide of cyperus rotundus, characterized in that The polysaccharide of the seed of the genus contains structural units provided by the following sugar residues: galacturonic acid residue, rhamnose residue, glucose residue, galactose residue, xylose residue, and the molar ratio thereof is 2-3:1-2:7-8:1-2:1-2.

2. The polysaccharide of claim 1, characterized in that The precocious seed polysaccharide has the structure shown below: Wherein, m is an integer of 1 to 30; n is an integer of 1 to 30; m:n=1:

1.

3. The polysaccharide of claim 1, characterized in that The weight average molecular weight of the cyperus polysaccharide is 30-50 kDa.

4. A method for preparing the polysaccharide of the fruit of claim 1, characterized in that: The preparation method comprises: The dried fruit of the genus Aesculus was extracted with an ethanol aqueous solution, and the extract was concentrated and then precipitated with alcohol to obtain a precipitate a; The precipitate a is re-dissolved in water, defatted, deproteinized, concentrated and freeze-dried to obtain crude polysaccharide of the seed of the Chinese wolfberry; After the crude polysaccharide of the seed of the genus Preparata was redissolved in water, it was 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.

5. The preparation method according to claim 4, 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 solid-liquid ratio is 1 g:3-5 mL.

6. The preparation method according to claim 4, 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 temperature of the alcohol precipitation is 3-5°C, and the time of the alcohol precipitation is 10-14h.

7. The preparation method according to claim 4, 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 more, the alcohol solvent is added to the system until the alcohol concentration is 20-80% v / v, the temperature of the alcohol precipitation is 3-5°C, and the time of the alcohol precipitation is 10-14h.

8. The preparation method according to claim 4, characterized in that: A Sephadex G-200 polysaccharide gel chromatography column is used and eluted with pure water; and / or, a 2000-3000 Da dialysis bag is used for dialysis for 40-50 hours.

9. A composition, characterized in that The composition comprises the schizonepeta polysaccharide according to any one of claims 1 to 3.

10. Use of the polysaccharide of any one of claims 1 to 3 or the composition of claim 9 in the preparation of a medicament for treating autoimmune diseases.

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