Preparation method of fructus xanthii polysaccharide with immunocompetence

Abstract

The invention discloses a preparation method of fructus xanthii polysaccharide with immunocompetence, and belongs to the technical field of polysaccharide. According to the invention, a water extraction and alcohol precipitation method is combined with ion exchange column chromatography, anti-inflammatory activity screening and sephadex column chromatography, the preparation process is mild in condition, simple to operate and environment-friendly, and the prepared fructus xanthii polysaccharide has the characteristics of anti-inflammatory activity, slightly acidic pH value, definite monosaccharide composition, definite molecular weight, uniform polysaccharide component, clear glycosidic bond linking mode and the like. The specific structure of the fructus xanthii polysaccharide with immune anti-inflammatory activity is analyzed, and a new research thought is provided for further structural research of the fructus xanthii polysaccharide.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharides, and particularly relates to a method for preparing Xanthium sibiricum polysaccharide with immune activity. Background Art

[0002] Xanthium sibiricum is the mature fruit with involucre of Xanthium sibiricum Patr., a plant of the Asteraceae family. It is also known as Xanthium sibiricum, old Xanthium sibiricum, and cattle lice. Xanthium sibiricum polysaccharide is a natural biomacromolecule extracted from Xanthium sibiricum, which is rich in nutrients. Xanthium sibiricum polysaccharide has multiple pharmacological activities, including antiviral, antioxidant, and antibacterial effects.

[0003] However, there are few reports on the structure of Xanthium sibiricum polysaccharides, and due to the difficulty of NMR characterization and analysis of polysaccharide structures, the drug basis for the immune and anti-inflammatory activity of Xanthium sibiricum polysaccharides has not yet been clarified. These studies provide a theoretical basis for further research and application of Xanthium sibiricum polysaccharides: Lu Jing (Anti-inflammatory effect and mechanism of different solvent extracts of Xanthium sibiricum) et al. studied the anti-inflammatory effect and mechanism of different solvent extracts of Xanthium sibiricum on LPS-induced RAW264.7 cells, and found that water extract (STW), ethanol extract (CTW) and ether extract (MTW) ​​can significantly reduce the content of inflammatory factors TNF-α, IL-6 and NO, and inhibit inflammatory response by regulating the STAT3 signaling pathway. Among them, water extract and ethanol extract have stronger anti-inflammatory activity, while water extract has toxic effect on cells at high concentrations. Shi Yang (Study on the Physicochemical Properties and Biological Activities of Xanthium sibiricum Polysaccharides) et al. extracted four polysaccharide components from Xanthium sibiricum by using hot water, chelating agents, and NaOH solvents of different concentrations. They found that they differed in monosaccharide composition and confirmed their polysaccharide structures by Fourier transform infrared spectroscopy (FTIR) analysis. Antioxidant activity tests showed that AXP exhibited the best antioxidant effect in vitro, and all polysaccharide components could exhibit anti-inflammatory activity by inhibiting LPS-induced NO secretion by RAW264.7 macrophages. The results of the study showed that different extraction methods had a significant effect on the physicochemical properties and biological activities of Xanthium sibiricum polysaccharides, providing theoretical support for the development and utilization of Xanthium sibiricum polysaccharides.

[0004] At present, the main deficiencies are as follows: 1. Common structural analysis is only targeted at Xanthium sibiricum polysaccharides with smaller molecular weights, and there is a lack of research on the diversity of Xanthium sibiricum polysaccharide structures; 2. Research on the material basis for the efficacy of Xanthium sibiricum polysaccharides is still insufficient. Summary of the invention

[0005] In order to solve the above technical problems, the present invention aims to provide a method for preparing Xanthium sibiricum polysaccharide with immunological activity, which is achieved by the following technical scheme:

[0006] A Xanthium sibiricum polysaccharide, wherein the monosaccharide composition of the Xanthium sibiricum polysaccharide is arabinose, galacturonic acid and galactose, and the molar ratio of each monosaccharide is 12:7:1; the main glycosidic bond connection mode of the main chain in the Xanthium sibiricum polysaccharide molecule is: →4)-α-L-Arap-(1→4)-β-D-GalAp-(1→.

[0007] Furthermore, the molecular weight of the Xanthium sibiricum polysaccharide is 158.5 kDa.

[0008] A method for preparing Xanthium sibiricum polysaccharide comprises the following steps:

[0009] The Xanthium sibiricum was subjected to hot water extraction, defatting, deproteinization, alcohol precipitation, centrifugation and freeze drying to obtain the Xanthium sibiricum crude polysaccharide.

[0010] Ion exchange column chromatography is used to separate and purify the crude polysaccharide of Xanthium sibiricum, and NaCl solutions of different concentrations are used as eluents for elution at a flow rate of 0.5-2.0 mL / min. The eluents are collected to obtain a refined extract of Xanthium sibiricum polysaccharide;

[0011] The Xanthium sibiricum polysaccharide extract is separated and purified by dextran gel column chromatography and freeze-dried to obtain the Xanthium sibiricum polysaccharide.

[0012] Furthermore, the hot water extraction comprises decocting Xanthium sibiricum and 100°C hot water in a volume ratio of 1:4-10, repeating the process multiple times (e.g. 3-5 times), decocting for 45-60 minutes each time, collecting the extracts from each decoction together and concentrating them to obtain a concentrated extract; the Xanthium sibiricum adopts dried Xanthium sibiricum fruit.

[0013] Furthermore, the degreasing is performed by adding 2-5 times of petroleum ether to the concentrated extract for extraction, and this step is repeated 3 times before collecting the degreasing extract.

[0014] Furthermore, the deproteinization is performed by mixing a sevage reagent with the defatted extract and then standing for 1 hour for separation to remove the protein. This step is repeated 3 times and the deproteinized extract is collected.

[0015] Furthermore, the volume ratio of the sevage reagent to the defatted extract is 1:4.

[0016] Furthermore, the sevage reagent is a solution of chloroform: n-butanol=5:1 by volume.

[0017] Furthermore, the alcohol precipitation centrifugation is to concentrate the deproteinized extract and mix it with anhydrous ethanol in a volume ratio of 1:4-6, precipitate at 4-10°C for 24 hours, centrifuge at 4500-5000rpm for 10-15min, and filter the precipitate; the concentration is to concentrate the deproteinized extract to 1 / 8-1 / 10 of the original volume.

[0018] Furthermore, the precipitate obtained by alcohol precipitation and centrifugation is completely dissolved in pure water and then placed in a freeze dryer for freeze drying to obtain Xanthium sibiricum crude polysaccharide.

[0019] Furthermore, the filler of the ion exchange column chromatography is DEAE-52 cellulose, and the specification is 3.5×30 cm.

[0020] In a specific embodiment, the crude polysaccharide of Xanthium sibiricum was separated and purified by DEAE-52 anion exchange column chromatography with a specification of 3.5×30 cm, and 0 mol / L, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions were used as eluents for elution at a flow rate of 2.0 mL / min to obtain 4 groups of Xanthium sibiricum polysaccharide extracts.

[0021] Furthermore, the dextran gel column chromatography uses a Sephadex G-200 dextran column with a specification of 16×1000 mm.

[0022] In a specific embodiment, a Sephadex G-200 glucan column with a specification of 16×1000 mm is used to separate and purify the Xanthium sibiricum polysaccharide extract, and a NaCl solution (0 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L) with a concentration corresponding to that during elution by ion exchange column chromatography is used to elute the Xanthium sibiricum polysaccharide extract, and the elution flow rate is 0.2-0.5 mL / min. The eluate components are dialyzed through a dialysis bag and then freeze-dried to obtain Xanthium sibiricum polysaccharide.

[0023] In a preferred embodiment of the present invention, a method for preparing Xanthium sibiricum polysaccharide having immunological and anti-inflammatory activity is also provided, comprising the following steps:

[0024] Obtain multiple batches of Xanthium sibiricum from different regions, and subject the multiple batches of Xanthium sibiricum to hot water extraction, defatting, deproteinization, alcohol precipitation, centrifugation, and freeze-drying to obtain multiple batches of Xanthium sibiricum crude polysaccharides;

[0025] Multiple batches of crude Xanthium sibiricum polysaccharides were screened for the first anti-inflammatory activity to obtain the batch of crude Xanthium sibiricum polysaccharides with the best anti-inflammatory activity;

[0026] The optimal batch of crude Xanthium sibiricum polysaccharides was separated and purified by ion exchange column chromatography, and NaCl solutions of different concentrations were used as eluents for elution at an elution flow rate of 0.5-2.0 mL / min. The polysaccharide content in the eluent was detected by phenol-sulfuric acid colorimetry, and the eluent was collected to obtain multiple groups of Xanthium sibiricum polysaccharide extracts;

[0027] A second anti-inflammatory activity screening was performed on multiple groups of Xanthium sibiricum polysaccharide extracts to obtain the Xanthium sibiricum polysaccharide extract with the best anti-inflammatory activity;

[0028] The Xanthium sibiricum polysaccharide extract of the optimal group is separated, purified and freeze-dried by dextran gel column chromatography to obtain the Xanthium sibiricum polysaccharide.

[0029] Wherein, the first anti-inflammatory activity screening and the second anti-inflammatory activity screening use MH7A cells.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention combines a water extraction and alcohol precipitation method with ion exchange column chromatography, anti-inflammatory activity screening, and dextran gel column chromatography to prepare Xanthium sibiricum polysaccharide having the characteristics of anti-inflammatory activity, acidic pH value, clear monosaccharide composition, determined molecular weight, uniform polysaccharide components, clear glycosidic bond linking mode, etc. The preparation process conditions are mild, the operation is simple, and the environment is friendly. The present invention combines one-dimensional and two-dimensional nuclear magnetic resonance technologies to analyze the specific structure of Xanthium sibiricum polysaccharide with immune anti-inflammatory activity, and provides a new research idea for further structural research on Xanthium sibiricum polysaccharide.

[0032] Other features and advantages of the present invention will be described in the following description, and partly become obvious from the description, or be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the specific embodiments pointed out in the description. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The flowchart of the preparation process of Xanthium sibiricum polysaccharide S10a-III-a in Example 1 of the present invention is shown;

[0034] Figure 2 The inflammatory cell activity screening diagram of Xanthium sibiricum polysaccharides S1a-S10a in Example 1 of the present invention is shown;

[0035] Figure 3 The DEAE-52 anion exchange column chromatography elution diagram of Xanthium sibiricum polysaccharide S10a in Example 1 of the present invention is shown;

[0036] Figure 4 The inflammatory cell activity screening diagram of Xanthium sibiricum polysaccharides S10a-I-S10a-IV in Example 1 of the present invention is shown;

[0037] Figure 5 The dextran column chromatography elution diagram of Xanthium sibiricum polysaccharide S10a-III in Example 1 of the present invention is shown;

[0038] Figure 6 The total sugar determination results of Xanthium sibiricum polysaccharides S1-S10 in Example 2 of the present invention are shown; wherein A: total sugar standard curve; B: total sugar content;

[0039] Figure 7 (A) shows the uronic acid determination results of Xanthium sibiricum polysaccharide S10a-III-a in Example 2 of the present invention; wherein A: uronic acid content standard curve; B: uronic acid content;

[0040] Figure 8 The ultraviolet spectrum of Xanthium sibiricum polysaccharide S10a-III-a in Example 2 of the present invention is shown;

[0041] Fig. 9 The HPGPC diagram of Xanthium sibiricum polysaccharide S10a-III-a in Example 2 of the present invention is shown;

[0042] Fig.10 The triple helical structure analysis diagram of Xanthium sibiricum polysaccharide S10a-III-a in Example 2 of the present invention is shown;

[0043] Fig.11 The particle size distribution diagram of Xanthium sibiricum polysaccharide S10a-III-a in Example 2 of the present invention is shown;

[0044] Fig.12 The potential distribution diagram of Xanthium sibiricum polysaccharide S10a-III-a in Example 2 of the present invention is shown;

[0045] Fig.13 The infrared spectrum of Xanthium sibiricum polysaccharide S10a-III-a in Example 2 of the present invention is shown;

[0046] Fig.14 The monosaccharide composition analysis diagram of Xanthium sibiricum polysaccharide S10a-III-a in Example 3 of the present invention is shown, wherein 1: galacturonic acid; 2: PMP; 3: galactose; 4: arabinose;

[0047] Fig.15 The Xanthium sibiricum polysaccharide S10a-III-a in Example 3 of the present invention is shown. 1 H NMR spectra (A) and 13 C NMR spectrum (B);

[0048] Fig.16 The Xanthium sibiricum polysaccharide S10a-III-a in Example 3 of the present invention is shown. 1 H- 1H COSY spectrum (A), HSQC spectrum (B) and HMBC spectrum (C);

[0049] Fig.17 The structural diagram of Xanthium sibiricum polysaccharide S10a-III-a in Example 3 of the present invention is shown. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the present invention clearer, the implementation scheme of the present application will be described in detail below in conjunction with specific examples. The technical terms and scientific terms used in the present invention have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs, without other definitions. Without departing from the concept of the present invention, those skilled in the art may make various improvements and changes to the specific implementation methods of the present invention specification, which all belong to the protection scope of the present invention. The raw materials used in the present invention are all commercially available unless otherwise specified.

[0051] Example 1

[0052] See also Figure 1 , a method for preparing Xanthium sibiricum polysaccharide, comprising the following steps:

[0053] (1) In this example, 10 batches of Xanthium sibiricum S1-S10 collected from different places were subjected to hot water extraction, defatting, deproteinization, alcohol precipitation and centrifugation, and freeze-drying to obtain Xanthium sibiricum crude polysaccharides S1a-S10a.

[0054] Hot water extraction: according to the solid-liquid ratio of 1:4, accurately weigh 100 g of dried Xanthium sibiricum fruit and add 400 mL of distilled water, put it into a pot with hot water at 100°C and boil for 1 hour, collect the decoction, repeat this operation 3 times, boil for 45 minutes for the last time, collect the extract, collect the three extracts together, concentrate by rotary evaporation, and obtain 50 mL of concentrated extract for use.

[0055] Degreasing: Add 5 times petroleum ether to the concentrated extract for extraction, collect the aqueous phase, repeat this step 3 times and collect the defatted extract.

[0056] Deproteinization: Sevage reagent (chloroform: n-butanol = 5:1 (v:v)) was mixed with the extract at a ratio of 1:4 (v:v). After complete mixing, the mixture was allowed to stand for 1 hour for separation to remove protein. This step was repeated 3 times and the deproteinized extract was collected.

[0057] Alcohol precipitation and centrifugation: The deproteinized extract was concentrated and mixed with anhydrous ethanol in a volume ratio of 1:4. The extract was precipitated at 4°C for 24 hours, centrifuged at 4750 rpm for 15 minutes, and the precipitate was filtered.

[0058] Freeze-drying: After the precipitate is completely dissolved in pure water, it is placed in a freeze dryer for freeze-drying to obtain the Xanthium sibiricum crude polysaccharide freeze-dried powder.

[0059] (2) First anti-inflammatory activity screening: The anti-inflammatory activity of crude Xanthium sibiricum polysaccharides S1a-S10a was screened using MH7A cells to obtain the best batch of crude Xanthium sibiricum polysaccharides S10a with anti-inflammatory activity. Figure 2 In the figure, Normal is the normal group and Control is the control group. It can be seen from the figure that S10a has the highest inhibitory activity on inflammatory cells.

[0060] (3) A DEAE-52 anion exchange column (3.5×30 cm) was used for chromatography. The eluent was 0, 0.1, 0.2, and 0.3 mol / L NaCl solution at a flow rate of 2.0 mL / min. The polysaccharide content in the eluent was detected by phenol-sulfuric acid colorimetry at 490 nm. The polysaccharide-containing eluent was collected to obtain a Xanthium sibiricum polysaccharide extract. Four components were obtained, namely S10a-I eluted with 0 mol / L NaCl solution, S10a-II eluted with 0.1 mol / L NaCl solution, S10a-III eluted with 0.2 mol / L NaCl solution, and S10a-IV eluted with 0.3 mol / L NaCl solution, see Figure 3 The horizontal axis is the number of elution tubes, and the vertical axis is the absorbance value and the concentration of NaCl solution. From the figure, we can see the predicted polysaccharides of four different components.

[0061] (4) Second anti-inflammatory activity screening: The anti-inflammatory activity of the four components was screened using MH7A cells, and the best anti-inflammatory activity group of Xanthium sibiricum polysaccharide extract S10a-Ⅲ was obtained. Figure 4 , S10a-Ⅰ eluted with 0mol / L NaCl solution, S10a-Ⅱ eluted with 0.1mol / L NaCl solution, S10a-Ⅲ eluted with 0.2mol / L NaCl solution, S10a-Ⅳ eluted with 0.3mol / L NaCl solution, and Control.

[0062] (5) See Figure 5 Elution diagram of Xanthium sibiricum polysaccharide S10a-Ⅲ by dextran column chromatography. The optimal group of Xanthium sibiricum polysaccharide extract S10a-Ⅲ was separated and purified by Sephadex G-200 dextran gel column chromatography with a specification of 16×1000mm. 10 mg / mL of Xanthium sibiricum polysaccharide extract S10a-Ⅲ was eluted with 0.2 mol / L NaCl solution at an elution flow rate of 0.2 mL / min. The obtained eluate components were dialyzed through a dialysis bag and then freeze-dried to obtain Xanthium sibiricum polysaccharide, which was named S10a-Ⅲ-a.

[0063] Example 2

[0064] The physicochemical properties of Xanthium sibiricum polysaccharide are analyzed as follows:

[0065] (1) The total sugar content of the Xanthium sibiricum polysaccharides (S1a-S10a) obtained in Example 1 was determined by the phenol-sulfuric acid method.

[0066] Weigh 10 mg of D-anhydrous glucose accurately, place it in a 100 mL volumetric flask, add distilled water to dissolve and dilute to obtain a glucose reference solution with a concentration of 0.1 mg / mL. Then, take 0.00 mL, 0.20 mL, 0.40 mL, 0.60 mL, 0.80 mL, and 1.0 mL of the prepared standard solution and place them in 25 mL stoppered glass test tubes, add distilled water to make up to 2.0 mL, add 1.0 mL of 5% phenol solution and 5.0 mL of concentrated sulfuric acid solution, mix well, place in a boiling water (80°C) water bath and heat for 15 min, then take it out, cool to room temperature, use water as a blank control, measure the absorbance value at a wavelength of 490 nm, and measure it three times in parallel. Use glucose concentration (Concentration) as the horizontal axis and absorbance (Absorbance) as the vertical axis to draw a standard curve, obtain the regression equation and calculate the correlation coefficient (R).

[0067] Accurately weigh 5 mg of each of the 10 batches of crude Xanthium sibiricum polysaccharides S1a-S10a prepared in Example 1, add distilled water to make up to 50 mL in a volumetric flask, accurately pipette 1 mL of the sample and dissolve it in a stoppered glass test tube, add water to make up to 2 mL, and determine the absorbance of the sample at 490 nm according to the drawing and determination method of the standard curve.

[0068] Establish the standard curve of total sugar in Xanthium sibiricum Figure 6 (A) shows that y = 0.0862x + 0.1426, R 2 =0.9994. Substituting the absorbance values ​​of 10 groups of samples measured at 490nm into the above standard curve equation, the following is calculated: Figure 6 (B) Total sugar content shown.

[0069] (2) The m-hydroxybiphenyl method was used to determine the uronic acid content of the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1.

[0070] In this example, the m-hydroxybiphenyl method is used to determine the uronic acid content in the Xanthium sibiricum polysaccharide. This method is simple, easy to operate, highly specific, and suitable for the determination of the uronic acid content in the Xanthium sibiricum polysaccharide.

[0071] Accurately weigh 5 mg of anhydrous galacturonic acid in a 10 mL volumetric flask, add distilled water to dissolve and dilute to volume to obtain a galacturonic acid standard solution with a concentration of 0.5 mg / mL; accurately weigh 477 mg of sodium tetraborate in a 50 mL volumetric flask, add concentrated sulfuric acid to dissolve and dilute to the scale line; accurately weigh 50 mg of solid sodium hydroxide in a 10 mL volumetric flask, add distilled water to dissolve and dilute to volume to obtain a sodium hydroxide aqueous solution; weigh 15 mg of m-hydroxybiphenyl in a 10 mL volumetric flask, dissolve and dilute to volume with the freshly prepared sodium hydroxide solution. Accurately measure 0 μL, 50 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL and 600 μL of 0.5 mg / mL galacturonic acid standard solution into a 20 mL stoppered test tube, add water to 1 mL, add 5 mL of sodium tetraborate solution, shake well, heat in boiling water for 20 min, then take out, cool to room temperature, add 50 μL of m-hydroxybiphenyl solution, shake well, let stand at room temperature for 30 min, measure the absorbance of the standard at 525 nm, and draw a curve. Accurately weigh 5 mg of S10aⅢ-a sample, place it in a 10 mL volumetric flask, add distilled water to the scale, then pipette 400 μL into a 20 mL stoppered test tube, make up to 1 mL, add 5 mL of sodium tetraborate solution, shake well, heat in boiling water for 20 min, then cool to room temperature, add 100 μL of m-hydroxybiphenyl solution, mix well, and leave at room temperature for 30 min. Measure the absorbance of the sample at 525 nm and record it.

[0072] Establish a standard curve for the content of sugar acid in Xanthium sibiricum Figure 7 (A) shows that y = 0.0127x + 0.259, R 2 =0.9997. Substituting the absorbance value of S10a-Ⅲ-a measured at 525nm into the above standard curve equation, the following is obtained by calculation: Figure 7 The uronic acid content shown in (B) is 70.84%, which means that S10a-Ⅲ-a may be an acidic polysaccharide, which supports the subsequent structural analysis. The instrument used is SHIMADZU UV-2550 UV-visible spectrophotometer.

[0073] (3) The ultraviolet absorption of the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 was measured by UV scanning.

[0074] See also Figure 8 After testing, the UV scanning spectrum of the obtained Xanthium sibiricum polysaccharide S10a-Ⅲ-a showed no absorption peaks of pigment, protein and nucleic acid, indicating that the purity of S10a-Ⅲ-a was high. The instrument used was SHIMADZU UV-2550 UV-visible spectrophotometer with a scanning range of 200-800nm.

[0075] (4) The homogeneity and relative molecular weight of the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 were determined by high performance gel permeation chromatography (HPGPC).

[0076] Test conditions: Agilent 1260 Infinity system; 0.05 mol / L phosphate (PH=6.7) buffer: acetonitrile = 79:21, column temperature 35°C; detection wavelength: 245 nm; injection volume 10 μL; flow rate 1.0 mL / min.

[0077] See also Fig. 9 After testing, the Xanthium sibiricum polysaccharide S10a-Ⅲ-a obtained in Example 1 is a uniform polysaccharide with a relative molecular mass of 158.5 kDa.

[0078] (5) The triple helical structure of the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 was determined by UV scanning.

[0079] A 1 mg / mL Xanthium sibiricum polysaccharide S10a-Ⅲ-a solution was prepared and mixed with a Congo red solution (100 μM) at a ratio of 1:1 (v:v) to form a complex for later use. Subsequently, NaOH solutions with concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 and 0.5 mol / L were added, and the reaction solution was scanned at an ultraviolet wavelength of 400-600 nm.

[0080] The structure of S10a-Ⅲ-a was characterized by the Congo red experiment. The principle of the Congo red experiment is: under alkaline conditions, if the polysaccharide has a triple helix structure, it can be complexed with the Congo red reagent, and the maximum absorption wavelength of the complex will be red-shifted compared to the Congo red solution. As the alkaline concentration increases, the maximum absorption wavelength of the polysaccharide-Congo red complex will drop sharply due to the destruction of the triple helix structure. Experimental characterization such as Fig.10 As shown, the maximum absorption wavelength of the complex (S10a-Ⅲ-a+Congo Red) is 509 nm. Compared with 500 nm of Congo red solution (Congo Red), the maximum absorption wave shows only a slight red shift trend and tends to be stable with the increase of alkaline concentration, indicating that S10a-Ⅲ-a does not have a triple helical structure.

[0081] (6) The particle size distribution of the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 was measured using a nanoparticle analyzer. Fig.11 and potential analysis see Fig.12Sample 1-3 are samples of Xanthium sibiricum polysaccharide S10a-Ⅲ-a. The sample concentrations are Sample 1 (5 mg / mL), Sample 2 (50 mg / mL), and Sample 3 (0.5 mg / mL). The stability of the polysaccharide solution can be evaluated by the particle size distribution diagram and the potential analysis diagram. The particle size distribution diagram reflects the dispersion state and particle size of the polysaccharide in the solution. Fig.11 The particle size distribution of Xanthium sibiricum polysaccharide S10a-Ⅲ-a is 700-800nm; the potential analysis diagram can reflect the stability of particles in liquid. The higher the absolute value of the potential, the greater the electrostatic repulsion between particles, and the more stable the particles. Fig.12 The absolute value of the midpoint potential is 17-37mV.

[0082] (7) The characteristic groups of the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 were analyzed using an infrared spectrometer.

[0083] Weigh 2.0 mg of dried S10a-Ⅲ-a polysaccharide sample, mix with 200 mg of KBr powder, press into tablets, and then scan and analyze on L1600400Spectrum TWO FT-IR Fourier transform infrared spectrometer with a scanning range of 4000 cm -1 -400cm -1 .

[0084] See also Fig.13 After testing, the Xanthium sibiricum polysaccharide S10a-Ⅲ-a obtained in Example 1 has a characteristic peak of polysaccharide, 3400cm -1 The broad peak at 2935 cm -1 There is a narrow peak at 1620 cm-1, which belongs to the asymmetric stretching vibration of CH. -1 and 1410cm -1 The peak at 1240 cm represents the -COOH bending vibration, which indicates that S10aⅢ-a should contain uronic acid molecules. -1 、1142cm -1 and 1020cm -1 Three absorption peaks appeared at 1020 cm-1, indicating the presence of COC bonds. Based on these findings, S10aⅢ-a should be a polysaccharide. -1 and 1620cm -1 The two peaks at represent the presence of CO stretching vibration and CH bending vibration, respectively.

[0085] Example 3

[0086] The chemical structure of Xanthium sibiricum polysaccharide is identified as follows:

[0087] (1) The monosaccharide composition of the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 was analyzed by HPLC pre-column derivatization with PMP reagent, see Fig.14 , Fig.14 Among them, 1 is galacturonic acid, 2 is PMP (1-phenyl-3-methyl-5-pyrazolone), 3 is galactose, and 4 is arabinose.

[0088] After testing, it was found that the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 was composed of arabinose, galacturonic acid and galactose.

[0089] (2) The chemical structure characteristics of the Xanthium sibiricum polysaccharide S10a-Ⅲ-a obtained in Example 1 were determined by nuclear magnetic resonance spectroscopy (NMR).

[0090] Take 30 mg of polysaccharide and completely dissolve it in 99.9% D2O, put it into a nuclear magnetic tube, and perform NMR measurement.

[0091] Detection conditions: VNMRS600 superconducting nuclear magnetic resonance spectrometer; One NMR probe (5mm); 1 H spectrum operating frequency 599.81MHz; 13 C spectrum working frequency 150.84MHz; measured at 25℃ 1 H spectrum, measured at 25°C 13 C, COSY, HSQC, and HMBC spectra.

[0092] See also Figure 15-16 After testing, the Xanthium sibiricum polysaccharide S10a-III-a obtained in Example 1 is composed of →3,4)-α-L-Arap-(1→,1,4)-α-D-GalAp-(6→,α-D-Galp-(1→ and β-L-Arap-(1→, wherein Fig.15 A is the hydrogen spectrum ( 1 H NMR spectrum), Fig.15 B is the carbon spectrum ( 13 C NMR spectrum), Fig.16 A is the homonuclear chemical shift correlation spectrum ( 1 H- 1 H COSY diagram), Fig.16 B in the figure is the heteronuclear single quantum relationship spectrum (HSQC diagram), Fig.16 C in the middle is a multi-bond carbon-hydrogen relationship spectrum (HMBC diagram). The chemical shift analysis of the four sugar residues is shown in Table 1. The specific NMR spectrum structure analysis is as follows:

[0093] Table 1. NMR analysis of Xanthium sibiricum polysaccharide S10a-Ⅲ-a

[0094]

[0095] Based on the above analysis, the following results can be obtained: the Xanthium sibiricum polysaccharide S10a-Ⅲ-a has a uniform composition and a relative molecular mass of 158.5 kDa. The monosaccharide composition analysis results show that the Xanthium sibiricum polysaccharide S10a-Ⅲ-a in the present invention contains furanose arabinose (Arap), galactopyranose galacturonic acid (GalAp) and galactopyranose (Galp). The molar ratio of the monosaccharide composition of the Xanthium sibiricum polysaccharide S10a-Ⅲ-a is furanose arabinose: galactopyranose galacturonic acid: galactopyranose = 12:7:1; The results of nuclear magnetic resonance analysis are shown in Table 1. The main configuration of sugar residues is α-type. The primary structure of polysaccharides mainly refers to the composition of sugar residues, the connection order and connection mode of sugar residues, the conformation of sugar rings and the configuration of glycosidic bonds. This study mainly focuses on the characterization of the primary structure of polysaccharides. The primary structural unit of Xanthium sibiricum polysaccharide S10a-Ⅲ-a contains four sugar residues: (1→3,4)-α-L-Arap-(1→, 1,4)-α-D-GalAp-(6→, α-D-Galp-(1→ and β-L-Arap-(1→). The connection modes of the four sugar residues are as follows: Fig.17 As shown, the Xanthium sibiricum polysaccharide S10a-III-a with a molecular weight of 158.5 kDa is composed of multiple such primary structural units.

[0096] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A Xanthium sibiricum polysaccharide, characterized in that: The monosaccharide composition of the Xanthium sibiricum polysaccharide is arabinose, galacturonic acid and galactose, and the molar ratio of each monosaccharide is 12:7:1; The main glycosidic bond connection mode of the main chain in the Xanthium sibiricum polysaccharide molecule is: →4)-α-L-Arap-(1→4)-β-D-GalAp-(1→.

2. The Xanthium sibiricum polysaccharide according to claim 1, characterized in that The molecular weight of the Xanthium sibiricum polysaccharide is 158.5 kDa.

3. A method for preparing Xanthium sibiricum polysaccharide according to any one of claims 1 to 2, characterized in that: The steps include: The Xanthium sibiricum was subjected to hot water extraction, defatting, deproteinization, alcohol precipitation, centrifugation and freeze drying to obtain the Xanthium sibiricum crude polysaccharide. Ion exchange column chromatography is used to separate and purify the crude polysaccharide of Xanthium sibiricum, and NaCl solutions of different concentrations are used as eluents for elution at a flow rate of 0.5-2.0 mL / min. The eluents are collected to obtain a refined extract of Xanthium sibiricum polysaccharide; The Xanthium sibiricum polysaccharide extract is separated and purified by dextran gel column chromatography and freeze-dried to obtain the Xanthium sibiricum polysaccharide.

4. The method for preparing Xanthium sibiricum polysaccharide according to claim 3, characterized in that: The hot water extraction comprises decocting Xanthium sibiricum and 100° C. hot water in a volume ratio of 1:4-10, repeating the process for multiple times, decocting for 45-60 minutes each time, collecting the extracts from each decoction together and concentrating them to obtain a concentrated extract.

5. The method for preparing Xanthium sibiricum polysaccharide according to claim 3, characterized in that: The degreasing is performed by adding 2-5 times of petroleum ether to the concentrated extract for extraction.

6. The method for preparing Xanthium sibiricum polysaccharide according to claim 3, characterized in that: The deproteinization comprises mixing a sevage reagent with the defatted extract and then standing the mixture to separate the liquids, removing the protein, and collecting the deproteinized extract; The volume ratio of the sevage reagent to the defatted extract is 1:

4.

7. The method for preparing Xanthium sibiricum polysaccharide according to claim 6, characterized in that: The sevage reagent is a solution of chloroform: n-butanol=5:1 by volume.

8. The method for preparing Xanthium sibiricum polysaccharide according to claim 3, characterized in that: The alcohol precipitation centrifugation is to concentrate the deproteinized extract and mix it with anhydrous ethanol in a volume ratio of 1:4-6, precipitate at 4-10°C for 24 hours, centrifuge at 4500-5000rpm for 10-15min, and filter and collect the precipitate; the concentration is to concentrate the deproteinized extract to 1 / 8-1 / 10 of the original volume.

9. The method for preparing Xanthium sibiricum polysaccharide according to claim 3, characterized in that: The filler of the ion exchange column chromatography is DEAE-52 cellulose.

10. The method for preparing Xanthium sibiricum polysaccharide according to claim 3, characterized in that: The dextran gel column chromatography uses a Sephadex G-200 dextran column.

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