A sanguisorba officinalis polysaccharide, its preparation method and uses

Abstract

The present invention relates to a sanguisorba officinalis polysaccharide, its preparation method and uses. In the sanguisorba officinalis polysaccharide of the present invention, by weight percentage, the neutral sugar content is 79% - 85%, the uronic acid content is 13% - 19%, and the protein content is 1% - 4%. The composition of the polysaccharide includes mannose, rhamnose, galacturonic acid, galactose, xylose and arabinose. In vitro and in vivo experiments have proved that the sanguisorba officinalis polysaccharide can significantly inhibit the expression of inflammation-related factors in the enteritis cell model and relieve the symptoms of dextran sulfate sodium-induced acute colitis in mice, including the weight loss of mice, the increase of spleen weight index, the reduction of colon length and the intestinal structure damage. And it has no effect on the growth viability of normal intestinal epithelial cells in vitro and has no toxic effect on the important organs of mice in vivo, with high safety. Therefore, the polysaccharide has the potential to treat gastrointestinal inflammatory diseases and is expected to become a candidate carbohydrate drug for treating gastrointestinal inflammatory diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of polysaccharides, and particularly relates to an acidic sanguisorba polysaccharide, a preparation method thereof, and its use in the preparation of a drug for preventing and / or treating gastrointestinal inflammatory diseases. Background Art

[0002] Inflammatory bowel diseases (IBD) are chronic, progressive, immune-mediated diseases, and their incidence rates are increasing year by year worldwide. With the accumulation of inflammation, it often leads to irreversible intestinal damage and may cause complication syndromes including intestinal stricture, intestinal obstruction, intestinal fistula, loss of colorectal function, and even colorectal cancer. In addition, IBD patients are prone to infections of joints, skin, eyes and other organs, seriously reducing the quality of life of patients and bringing great pain and economic burden to patients and their families. However, the pathological process of IBD is not yet clear at present, and possible pathogenic factors include genes, environment, intestinal flora, immune system imbalance, etc., and there is no clinical drug that can cure IBD. Therefore, finding a drug that can effectively treat IBD has important clinical significance.

[0003] Sanguisorba officinalis L. is a perennial herb of the genus Sanguisorba in the Rosaceae family. According to the "Compendium of Materia Medica", Sanguisorba officinalis L. has the effects of cooling blood, hemostasis, purging fire and astringency. The active ingredients in Sanguisorba officinalis L. mainly include tannins, triterpenoid saponins, flavonoids and polysaccharide compounds. Modern research has found that Sanguisorba officinalis L. has activities such as detumescence, hemostasis, anti-tumor, antioxidant and antibacterial. As one of the main active substances in Chinese herbal medicines, polysaccharides play an important role in the exertion of their biological activities and have also attracted more and more researchers' attention. However, there are few reports on natural polysaccharides with anti-colitis activity in Chinese herbal medicines. Summary of the Invention

[0004] The present invention adopts a simple and effective extraction process and method for sanguisorba polysaccharide, and obtains an acidic polysaccharide (named 147-02-S2 in the text) using Sanguisorba officinalis L. as the raw material. In vitro experiments show that 147-02-S2 can significantly reduce the expression of inflammation-related factors in the enteritis cell model; and it is found in in vivo experiments of mice that this polysaccharide can improve the intestinal damage situation and reduce the inflammation level in mice with dextran sulfate sodium-induced acute colitis model, and is expected to be developed into a saccharide drug for anti-inflammatory bowel diseases.

[0005] In view of this, one of the purposes of the present invention is to provide a sanguisorba polysaccharide.

[0006] Another purpose of the present invention is to provide a preparation method of the sanguisorba polysaccharide.

[0007] A third object of the present invention is to provide a pharmaceutical composition comprising the sanguisorba officinalis polysaccharide.

[0008] A fourth object of the present invention is to provide the use of the sanguisorba officinalis polysaccharide or a pharmaceutical composition comprising the sanguisorba officinalis polysaccharide in the preparation of a drug for preventing and / or treating gastrointestinal inflammatory diseases.

[0009] In order to achieve the above objects of the present invention, the following technical solutions are specifically adopted:

[0010] On the one hand, the present invention provides a sanguisorba officinalis polysaccharide (147 - 02 - S2), comprising neutral sugars with a weight percentage of 79% - 85% (such as 80%, 81%, 82%, 83%, 84%), uronic acids with a weight percentage of 13% - 19% (such as 14%, 15%, 16%, 17%, 18%), and proteins with a weight percentage of 1% - 4% (such as 1.5%, 2%, 2.5%, 3%, 3.5%) (where the weight percentage is calculated based on the weight of the sanguisorba officinalis polysaccharide). Among them, the polysaccharide is mainly composed of mannose, rhamnose, galacturonic acid, galactose, xylose, and arabinose.

[0011] In some embodiments, the structural unit of the polysaccharide is mainly composed of α - galacturonic acid connected by 1→4 and α - rhamnose connected by 1→2 as the main chain. Branches exist at the C - 4 position of the α - pyran rhamnose and the C - 3 position of the α - pyran galacturonic acid. The branches are composed of mannose (terminal β - pyran mannose), galactose (1,6 - β - pyran galactose, 1,3,6 - β - pyran galactose, 1,4 - β - pyran galactose, and terminal β - pyran galactose), xylose (terminal β - pyran xylose), and arabinose (1,5 - α - furan arabinose, 1,3,5 - α - furan arabinose, and terminal - α - furan arabinose) residues.

[0012] In some embodiments, the range of the weight - average molecular weight of the sanguisorba officinalis polysaccharide is 10 - 1000 kDa, preferably 20 - 800 kDa, more preferably 40 - 600 kDa, and further preferably 60 - 100 kDa.

[0013] In some embodiments, the molar ratio of mannose, rhamnose, galacturonic acid, galactose, xylose, and arabinose is 4 - 8:12 - 20:12 - 20:16 - 24:2.5 - 6.5:32 - 40, preferably 6.18:16.65:15.63:20.41:4.66:36.47.

[0014] In some embodiments, the Sanguisorba officinalis polysaccharide 147-02-S2 contains 81.9% neutral sugar, 15.63% uronic acid, and 2.3% protein, and its weight average molecular weight is 88.37 kDa.

[0015] In some embodiments, the Sanguisorba officinalis polysaccharide has substantially Figure 1 High performance gel permeation chromatography (HPGPC) diagram shown.

[0016] In some embodiments, the Sanguisorba officinalis polysaccharide has substantially the same Figure 2 In the infrared spectrum of the Sanguisorba officinalis polysaccharide 147-02-S2, 3332.96 cm -1 The absorption peak comes from OH stretching vibration, 2933.45 cm -1 The absorption peak is CH, which represents stretching vibration, 1409.69 cm -1 The angle-variable vibration absorption peak from CH, 1012.62 cm -1 The absorption peaks near the ring are attributed to the stretching vibration of the exocyclic C-(COH) and the intracyclic CO(COC). -1 The stretching vibration of carboxyl C=O indicates that the polysaccharide contains uronic acid and is an acidic polysaccharide.

[0017] In some embodiments, the Sanguisorba officinalis polysaccharide has substantially the same Figure 3 The main signal in 13 C NMR spectrum. 13 In the C NMR spectrum, the signal peaks of δ110.46 / δ107.61, δ108.56, and δ108.23 were assigned to the C-1 of terminal α-arabinofuranose, 1,5-α-arabinofuranose, and 1,3,5-α-arabinofuranose; the signal peaks of δ105.54-δ104.60 were assigned to the terminal β-xylopyranose, the terminal β-mannopyranose, 1,6-β-galactopyranose, 1,3,6-β-galactopyranose, and 1,4-β- C-1 of galactopyranose and terminal β-galactopyranose; the signal peaks at δ100.83-δ98.70 are attributed to C-1 of 1,2,4-α-rhamnose, 1,2-α-rhamnose, 1,4-α-galactopyranosyl acid and 1,3,4-α-galactopyranosyl acid; the signal peaks at δ175.99 and δ176.57 are attributed to the carboxyl carbon of galacturonic acid; δ17.81 and δ18.06 are attributed to the signal peak of rhamnose C6.

[0018] In some embodiments, the Sanguisorba officinalis polysaccharide has substantially the same Figure 4 The main signal in 1 H NMR spectrum.1 In the 1H NMR spectrum, the signals of H-1 of terminal α-arabinofuranose, 1,5-α-arabinofuranose and 1,3,5-α-arabinofuranose are attributed to δ5.31 / δ5.36, δ5.23 / δ5.25 and δ5.14 respectively; the signals of H-1 of 1,2,4-α-rhamnopyranose and 1,2-α-rhamnopyranose overlap at δ5.31; the signals of H-1 of 1,4-α-galactopyranuronic acid, 1,3,4-α-galactopyranuronic acid and terminal α-galactopyranuronic acid are attributed to δ5.12 - δ5.07; the signal of H-1 of β-mannopyranose is attributed to δ4.82; the signals of H-1 of terminal β-galactopyranose, 1,4-β-galactopyranose, 1,6-β-galactopyranose, 1,3,6-β-galactopyranose and terminal β-xylopyranose are attributed to δ4.75 - δ4.55 respectively; in addition, the signals of H-6 of 1,2,4-α-rhamnopyranose and 1,2-α-rhamnopyranose are attributed to δ1.38 and δ1.36 respectively.

[0019] In some embodiments, the sanguisorba officinalis polysaccharide of the present invention is extracted and isolated from the roots of sanguisorba officinalis produced in Zhejiang.

[0020] On the other hand, the present invention provides a method for preparing the sanguisorba officinalis polysaccharide (147 - 02 - S2), comprising the following steps:

[0021] a. Extraction of sanguisorba officinalis polysaccharide:

[0022] The tuberous roots of sanguisorba officinalis are extracted with boiling water, and the obtained extract is concentrated, dialyzed, re - concentrated, centrifuged to remove the precipitate. The obtained supernatant is precipitated with alcohol, centrifuged and separated. The obtained precipitate is washed and dried to obtain the crude water - extracted sanguisorba officinalis polysaccharide.

[0023] Preferably, step a includes: before boiling water extraction, the tuberous roots of sanguisorba officinalis are soaked in ethanol for 3 - 10 days (such as 7 days) and then air - dried; the dried tuberous roots of sanguisorba officinalis are taken, added with about 10 - 30 times (such as 15 - 20 times) the weight of water, the temperature is raised to 100 °C for boiling water extraction for 3 - 5 h, and the extraction is carried out 3 - 9 times (such as 7 - 9 times). The extraction solutions are combined and concentrated, dialyzed, re - concentrated, centrifuged to obtain the supernatant, added with about 3 - 10 times (such as 5 - 10 times) the volume of ethanol of the supernatant, centrifuged to obtain the precipitate. The precipitate is washed 2 - 6 times (such as 3 - 5 times) with absolute ethanol and acetone, and obtained the crude water - extracted sanguisorba officinalis polysaccharide by vacuum drying or freeze - drying.

[0024] More preferably, step a includes: taking dried Sanguisorba officinalis, adding about 20 times the weight of deionized water, raising the temperature to 100 °C for boiling water extraction for 4 h, extracting 8 times in total, combining and concentrating the extraction solution, dialyzing, concentrating the internal dialysis solution, centrifuging, obtaining the supernatant, adding about 5 times the volume of ethanol to the supernatant, centrifuging to obtain a precipitate, washing the precipitate alternately with absolute ethanol and acetone 3 times, drying in an oven under vacuum, redissolving, and then freeze-drying to obtain crude water-extracted Sanguisorba officinalis polysaccharide;

[0025] b. Purification of Sanguisorba officinalis polysaccharide:

[0026] b1. Dissolve the crude water-extracted Sanguisorba officinalis polysaccharide prepared in step a in water, centrifuge, and subject the supernatant to preliminary fractionation and purification on an anion exchange column, eluting successively with water and 0.05 - 0.4 M NaCl aqueous solution, collecting the elution fraction of about 0.2 M NaCl aqueous solution to obtain Sanguisorba officinalis polysaccharide 147-02;

[0027] Preferably, step b1 includes: taking the crude water-extracted Sanguisorba officinalis polysaccharide prepared in step a, adding it to dissolve in about 10 - 20 times the weight of water, centrifuge, subject the supernatant to separation on an anion exchange column, eluting successively with deionized water, 0.05 M, 0.1 M, 0.2 M, and 0.4 M NaCl aqueous solutions, detecting with sulfuric acid-phenol, collecting and combining the eluate of about 0.2 M NaCl aqueous solution according to the elution curve, concentrating, centrifuging, dialyzing the supernatant, and freeze-drying to obtain preliminarily purified Sanguisorba officinalis polysaccharide 147-02;

[0028] More preferably, step b1 includes: taking the crude water-extracted Sanguisorba officinalis polysaccharide prepared in step a, adding it to dissolve in about 16.7 times the weight of water, centrifuge, subject the supernatant to separation on a DEAE Sepharose Fast Flow anion exchange column, eluting successively with deionized water, 0.05 M, 0.1 M, 0.2 M, and 0.4 M NaCl aqueous solutions, detecting with sulfuric acid-phenol, collecting and combining the eluate of 0.2 M NaCl solution according to the elution curve, concentrating, centrifuging, dialyzing the supernatant, and freeze-drying to obtain preliminarily purified Sanguisorba officinalis polysaccharide 147-02;

[0029] b2. Dissolve the Sanguisorba officinalis polysaccharide 147-02 prepared in step b1 in about 10 - 30 times the weight of 0.2 M NaCl aqueous solution, centrifuge, subject the supernatant to purification on a gel chromatography column, eluting with 0.2 M NaCl aqueous solution, collecting and combining the main component of Sanguisorba officinalis polysaccharide 147-02-S2 according to the elution curve, concentrating, dialyzing, and freeze-drying to obtain the Sanguisorba officinalis polysaccharide 147-02-S2;

[0030] Preferably, step b2 includes: dissolving the sanguisorba officinalis polysaccharide 147-02 prepared in step b1 in an aqueous solution of 0.2 M NaCl at about 20 times its weight, centrifuging, purifying the supernatant through a Sephacryl HR S-300 gel chromatography column, eluting with a 0.2 M NaCl solution, detecting by the sulfuric acid-phenol method, collecting and combining the main component, sanguisorba officinalis polysaccharide 147-02-S2, according to the elution curve, concentrating, dialyzing, and freeze-drying to obtain the sanguisorba officinalis polysaccharide 147-02-S2.

[0031] In some embodiments, in step a, the ethanol may be an aqueous ethanol solution of about 70% (v / v) or more, preferably an aqueous ethanol solution of about 85% (v / v) or more, and particularly an aqueous ethanol solution of about 95% (v / v) or more.

[0032] In another aspect of the present invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of the sanguisorba officinalis polysaccharide as an active ingredient, and the composition may further include pharmaceutically acceptable pharmaceutical excipients, such as carriers, excipients, adjuvants, and / or diluents, etc.

[0033] In yet another aspect of the present invention, there is provided the use of the sanguisorba officinalis polysaccharide or the pharmaceutical composition in the preparation of a drug for treating and / or preventing gastrointestinal inflammatory diseases.

[0034] In the present invention, the gastrointestinal inflammatory diseases include inflammatory bowel disease and acute gastritis, etc.

[0035] In yet another aspect of the present invention, there is also provided a method for improving gastrointestinal inflammatory diseases, particularly inflammatory bowel disease, and for preventing and / or treating gastrointestinal inflammatory diseases, particularly inflammatory bowel disease, the method comprising administering a therapeutically effective amount of the sanguisorba officinalis polysaccharide or the pharmaceutical composition to a subject in need of such treatment.

[0036] In the present invention, the inflammatory bowel disease is particularly a chronic non-specific intestinal inflammatory disease, such as ulcerative colitis (UC) and Crohn's disease (CD).

[0037] The technical solution of the present invention has at least the following technical effects:

[0038] The present invention prepares a homogeneous sanguisorba officinalis polysaccharide 147-02-S2 component (with a molecular weight within a certain range (showing a normal distribution), having a definite sugar residue linkage mode and sugar composition) through specific extraction and separation methods. In vivo experiments have proved that this polysaccharide 147-02-S2 can reduce dextran sulfate sodium-induced colitis, including increasing the colon length, restoring the intestinal microscopic morphology, and reducing the expression of inflammation-related factors, and at the same time has no toxic effect on other important organs. And this polysaccharide 147-02-S2 has no effect on the growth viability of intestinal epithelial cells in vitro, can significantly inhibit the expression of inflammation-related factors in the enteritis cell model, and has great application prospects in the candidate drugs for preventing and / or treating gastrointestinal and digestive tract inflammatory diseases, especially inflammatory bowel disease.

[0039] The present invention has been described in detail above, but the above embodiments are essentially illustrative only and are not intended to limit the present invention. In addition, the present invention is not limited by any theory described in the foregoing prior art or the invention content or the following examples. Brief Description of the Drawings

[0040] Figure 1 Characteristic high performance gel permeation chromatogram of the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0041] Figure 2 Characteristic infrared spectrum of the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0042] Figure 3 Characteristic 13 13C NMR spectrum of the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0043] Figure 4 Characteristic 1 1H NMR spectrum of the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0044] Figure 5 Cytotoxicity test results of the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0045] Figure 6 In vitro cell Elisa test results of the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0046] Figure 7 In vivo safety test results of the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0047] Figure 8 Improvement results of the colon length of model mice by the sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1.

[0048] Figure 9 Improvement results of sanguisorba officinalis polysaccharide 147-02-S2 prepared in Example 1 on the colonic intestinal structure of model mice. Detailed implementation manners

[0049] In this article, all features or conditions defined in the form of numerical ranges or percentage ranges are only for the sake of brevity and convenience. Accordingly, the description of a numerical range or percentage range should be regarded as having covered and specifically disclosed all possible sub-ranges and individual numerical values within the range, especially integer numerical values. For example, the description of the range "1 to 8" should be regarded as having specifically disclosed all sub-ranges such as 1 to 7, 2 to 8, 2 to 6, 3 to 6, 4 to 8, 3 to 8, etc., especially sub-ranges defined by all integer numerical values, and should be regarded as having specifically disclosed individual numerical values such as 1, 2, 3, 4, 5, 6, 7, 8 within the range. Unless otherwise specified, the foregoing interpretation method applies to all contents of the present invention throughout the text, regardless of the breadth of the range.

[0050] If a quantity or other numerical value or parameter is expressed in the form of a range, a preferred range or a series of upper and lower limits, it should be understood that all ranges formed by any pair of the upper limit or preferred value of the range and the lower limit or preferred value of the range have been specifically disclosed herein, whether or not these ranges are separately disclosed. In addition, when a numerical range is mentioned in this article, unless otherwise stated, the range should include its endpoints and all integers and fractions within the range.

[0051] In this article, on the premise of achieving the purpose of the invention, the numerical value should be understood as having the precision of the significant digits of the numerical value. For example, the number 40.0 should be understood as covering the range from 39.50 to 40.49.

[0052] The following further illustrates the present invention in conjunction with embodiments. It should be noted that the following embodiments are provided only for the purpose of illustration and do not constitute a limitation on the scope of protection required by the present invention.

[0053] Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods.

[0054] Unless otherwise specified, the materials, reagents, etc. used in the embodiments can all be obtained from commercial channels.

[0055] Materials:

[0056] The dried sanguisorba officinalis root tubers were purchased from Bozhou Chinese Herbal Pieces Company, and the origin was Zhejiang, China.

[0057] Equipment:

[0058] High performance gel permeation chromatography used a tandem column of Shodex SUGAR KS-804 (8.0 mm × 300 mm, Agilent, USA) and Shodex SUGAR KS-802 (8.0 mm × 300 mm, Agilent, USA) to prepare a standard curve with T-series standard dextrans of different molecular weights;

[0059] High performance liquid chromatography (HPLC) was determined using an Agilent 1260Seri high performance liquid system (Agilent, USA);

[0060] Infrared analysis was determined using a Perkin-Elmer 599B infrared spectrophotometer (Perkin-Elmer, USA);

[0061] Nuclear magnetic resonance analysis was determined using a Brucker AM-500 nuclear magnetic resonance spectrometer (Brucker, Germany).

[0062] Preparation Example 1: Preparation of Sanguisorba officinalis polysaccharide 147-02-S2

[0063] a. Extraction of Sanguisorba officinalis polysaccharide:

[0064] Before boiling water extraction, the Sanguisorba officinalis medicinal materials were soaked in ethanol for 3 - 10 days and then air-dried. Take 5 kg of dry Sanguisorba officinalis root tubers, add 20 L × 5 of deionized water, and perform boiling water extraction at 100 °C for 4 h each time, for a total of 8 times. Combine and concentrate the extraction solution, and dialyze with running water for 3 days. Heat-concentrate the inner dialysate to 3 L, centrifuge to discard the precipitate, add 5 times the volume (15 L) of 95% v / v ethanol to the supernatant under stirring, let it stand for overnight alcohol precipitation, centrifuge and separate. The obtained precipitate was washed alternately with absolute ethanol and acetone 3 times, the precipitate was centrifuged and taken, vacuum-dried at 50 °C, redissolved in 2 L of deionized water, and freeze-dried to obtain 172 g of crude water-extracted Sanguisorba officinalis polysaccharide (yield 3.4%).

[0065] b. Purification of Sanguisorba officinalis polysaccharide:

[0066] Take 6 g of the crude water-extracted Sanguisorba officinalis polysaccharide prepared above, dissolve it in 100 mL of deionized water, centrifuge at 4000 r / min for 10 min to remove insoluble substances. The supernatant was separated by a DEAE Sepharose Fast Flow anion exchange column, and gradient elution was carried out successively with deionized water, 0.05 M, 0.1 M, 0.2 M, and 0.4 M NaCl aqueous solutions, detected by sulfuric acid-phenol, and an elution curve was plotted. According to the elution curve, collect and combine the 0.2 M NaCl eluate, concentrate, centrifuge, dialyze the supernatant, and freeze-dry to obtain about 452 mg of preliminarily purified Sanguisorba officinalis polysaccharide 147-02 (yield 7.53%).

[0067] Take 200mg 147-02 and dissolve it in 4mL of 0.2M NaCl solution, centrifuge at 4000r / min for 10min, and pass the supernatant through a Sephacryl HR S-300 gel chromatography column, elute with 0.2M NaCl solution and control the flow rate to 5mL / 15min. Detect by sulfuric acid-phenol method and draw the elution curve, collect and combine the main component of Sanguisorba officinalis polysaccharide 147-02-S2 according to the elution curve, concentrate, dialyze, and freeze-dry to obtain about 42.6mg of Sanguisorba officinalis polysaccharide 147-02-S2 (yield 21.3%). The yield of the obtained polysaccharide is calculated based on the crude polysaccharide in the previous step.

[0068] c. Structural identification and analysis of Sanguisorba officinalis polysaccharide 147-02-S2:

[0069] (1) The molecular weight and purity of Sanguisorba officinalis polysaccharide 147-02-S2 were determined by high performance gel permeation chromatography (HPGPC). It showed a single symmetrical peak in the HPGPC spectrum and was normally distributed, as shown in Figure 2. Figure 1 As shown (the small peak at about 35.5min in the figure is the solvent peak). According to the dextran standard curve, the relative molecular mass of 147-02-S2 was calculated to be 88.37kDa. Using glucose as the standard, the sugar content was determined by the phenol-sulfuric acid method, indicating that the sugar content of Sanguisorba officinalis polysaccharide 147-02-S2 was 81.9%; in addition, the protein content was detected by the BCA Protein Assay kit (the kit was purchased from Biyuntian Biotechnology) method, which was 2.3%.

[0070] The complete hydrolysis sample of sanguisorba officinalis polysaccharide 147 - 02 - S2 was measured for its monosaccharide composition by PMP derivatization method. It was determined that sanguisorba officinalis polysaccharide 147 - 02 - S2 mainly contains mannose (Man), rhamnose (Rha), galacturonic acid (GalA), galactose (Gal), xylose (Xyl) and arabinose (Ara), and their molar ratio is 6.18:16.65:15.63:20.41:4.66:36.47. Methylation analysis was carried out on 147 - 02 - S2 and its reduced polysaccharide. It was found that the sugar residues of 147 - 02 - S2 mainly include terminal arabinofuranose, 1,5 - arabinofuranose, 1,3,5 - arabinofuranose, terminal xylopyranose, terminal galactopyranose, 1,4 - galactopyranose, 1,3,6 - galactopyranose and 1,4 - galactopyranose, terminal mannosepyranose, 1,2 - rhamnopyranose, 1,2,4 - rhamnopyranose, 1,4 - galactopyranuronic acid, 1,3,4 - galactopyranuronic acid and terminal galactopyranuronic acid. In addition, based on the comparative analysis of the structures of sanguisorba officinalis polysaccharide 147 - 02 - S2 and its partial acid hydrolysis product 147 - 02 - S205a, the backbone information of 147 - 02 - S2 was determined. The monosaccharide composition of the partial acid hydrolysis product 147 - 02 - S205a showed that it consists of Rha, GalA, Gal and Xyl, and their relative molar ratio is 28.34:45.33:19.54:6.77. By comparing and analyzing with the monosaccharide composition results of 147 - 02 - S2, it was found that the contents of Rha and GalA in 147 - 02 - S205a increased significantly, indicating that these two sugar residues are located on the main chain, while Man and Ara have completely disappeared, and the content of Gal decreased, indicating that they may exist on the branches. Further methylation was carried out on 147 - 02 - S205a and its reduced product. It was determined that 147 - 02 - S205a mainly consists of terminal xylopyranose, 1,4 - galactopyranose, 1,6 - galactopyranose, 1,2 - rhamnopyranose, 1,2,4 - rhamnopyranose, 1,4 - galactopyranuronic acid, 1,3,4 - galactopyranuronic acid and terminal galactopyranuronic acid. This result further indicates that the main chain of 147 - 02 - S2 is 1,4 - galactopyranuronic acid, 1,3,4 - galactopyranuronic acid, 1,2 - rhamnopyranose and 1,2,4 - rhamnopyranose, and the branches exist at the C - 3 position of 1,3,4 - galactopyranuronic acid and the C - 4 position of 1,2,4 - rhamnopyranose. The branches are composed of terminally - linked arabinofuranose, 1,5 - arabinofuranose, 1,3,5 - arabinofuranose, terminally - linked xylopyranose, terminally - linked galactopyranose, 1,4 - galactopyranose, 1,3,6 - galactopyranose and 1,6 - galactopyranose, terminally - linked mannosepyranose and terminally - linked galactopyranuronic acid.

[0071] (2) Infrared spectrum (such as Figure 2),3332.96 cm -1 The absorption peak is derived from the O-H stretching vibration, 2933.45 cm -1 The absorption peak is derived from the C-H stretching vibration, 1409.69 cm -1 The absorption peak at 1012.62 cm is derived from the bending vibration of C-H -1 The absorption peak near is attributed to the stretching vibrations of exocyclic C-O (C-O-H) and endocyclic C-O (C-O-C). In addition, the absorption peak at 1706.08 cm -1 is attributed to the stretching vibration of the carboxyl C=O, indicating that the polysaccharide contains uronic acid and belongs to acidic polysaccharide.

[0072] (3) NMR analysis:

[0073] Take 30 mg of sanguisorba polysaccharide 147-02-S2, dissolve it in 500 μL of D2O, add 2.5 μL of acetone as an internal standard (δH = 2.29 ppm, δC = 31.5 ppm), and measure the one-dimensional and two-dimensional nuclear magnetic resonance spectra at 25 °C on a Bruker AVANCE III 500M nuclear magnetic resonance spectrometer. The structure of sanguisorba polysaccharide 147-02-S2 was confirmed by referring to the nuclear magnetic resonance spectra. The 13 C NMR and 1 H NMR results are as Figure 3 and Figure 4 .

[0074] In the 13 C NMR spectrum of 147-02-S2 ( Figure 3 ), the signal peaks at δ110.46 / δ107.61, δ108.56, and δ108.23 are attributed to the C-1 of terminal α-L-arabinofuranose, 1,5-α-L-arabinofuranose, and 1,3,5-α-L-arabinofuranose; the signal peaks at δ105.54 - δ104.60 are attributed to the C-1 of terminal β-D-xylopyranose, terminal β-D-mannopyranose, 1,6-β-D-galactopyranose, 1,3,6-β-D-galactopyranose, 1,4-β-D-galactopyranose, and terminal β-D-galactopyranose; the signal peaks at δ100.83 - δ98.70 are attributed to the C-1 of 1,2,4-α-L-rhamnopyranose, 1,2-α-L-rhamnopyranose, 1,4-α-L-galactopyranuronic acid, and 1,3,4-α-L-galactopyranuronic acid. The signal peaks at δ175.99 and δ176.57 are attributed to the carboxyl carbon of galacturonic acid. The signal peaks at δ17.81 and δ18.06 are attributed to the C6 of rhamnose.

[0075] In the 1 H NMR spectrum of 147-02-S2 ( Figure 4) The H-1 signals of terminal α-arabinofuranose, 1,5-α-arabinofuranose, and 1,3,5-α-arabinofuranose are attributed to δ5.31 / δ5.36, δ5.23 / δ5.25, and δ5.14 respectively; the H-1 signals of 1,2,4-α-rhamnopyranose and 1,2-α-rhamnopyranose overlap at δ5.31; the H-1 signals of 1,4-α-galactopyranuronic acid, 1,3,4-α-galactopyranuronic acid, and terminal α-galactopyranuronic acid are attributed to δ5.12 - δ5.07; the H-1 signal of β-mannopyranose is attributed to δ4.82; the H-1 signals of terminal β-galactopyranose, 1,4-β-galactopyranose, 1,6-β-galactopyranose, 1,3,6-β-galactopyranose, and terminal β-xylopyranose are attributed to δ4.75 - δ4.55 respectively. In addition, the H-6 signals of 1,2,4-α-rhamnopyranose and 1,2-α-rhamnopyranose are attributed to δ1.38 and δ1.36 respectively.

[0076] Test Example 1: Activity of Sanguisorba officinalis polysaccharide 147-02-S2 against colitis

[0077] a. Sanguisorba officinalis polysaccharide 147-02-S2 has no cytotoxicity to normal colonic epithelial cells NCM460

[0078] NCM460 cells were cultured adherently for 12 h, the original medium was removed and co-incubated with a medium containing 147-02-S2 polysaccharide at different concentrations (0.1 mg / mL, 0.5 mg / mL, and 1.0 mg / mL), and then the cell growth viability was detected. The MTT results showed ( Figure 5 ) that after co-incubation with the polysaccharide for 12 h, 24 h, and 48 h, the cell viability was greater than 90%, indicating that 147-02-S2 had no obvious inhibitory effect on cell growth viability and no obvious cytotoxicity.

[0079] b. Sanguisorba officinalis polysaccharide 147-02-S2 can significantly inhibit the expression of inflammatory factors in the colitis cell model

[0080] NCM460 cells were cultured adherently for 12 h. After treating the cells with lipopolysaccharide (LPS, 1 μg / mL) for 12 h, different concentrations of 147-02-S2 polysaccharide (L group: 0.1 mg / mL, M group: 0.5 mg / mL, H group: 1.0 mg / mL) or a medium containing a positive control drug (PO group, 5-aminosalicylic acid, 5-ASA, 20 μg / mL) were added and the cells were cultured for another 12 h. The cell supernatant was collected to detect the secretion levels of inflammatory factors TNF-α and IL-1β. The Elisa results showed ( Figure 6 ) that the H group could effectively inhibit the protein secretion levels of inflammatory factors TNF-α and IL-1β.

[0081] c. Mouse model and the treatment protocol of sanguisorba officinalis polysaccharide 147-02-S2

[0082] The mice were allowed to drink and eat freely. After one week of adaptation, they were randomly divided into 7 groups (7 mice in each group), namely the normal control group (Control), the model group (Model), the positive drug control group (5-ASA), the low-dose administration group (Low), the medium-dose administration group (Middle), the high-dose administration group (High), and the safety evaluation group (147-02-S2). The Control group was not given any treatment. The mice in the safety evaluation group ate normally and were intragastrically administered 80 mg / kg of 147-02-S2 polysaccharide every day for 7 consecutive days. The positive drug control group and the 147-02-S2 polysaccharide treatment groups ate normally and drank 2.5% dextran sulfate sodium solution. On this basis, the 5-ASA group was intragastrically administered 200 mg / kg of 5-ASA, and the Low, Middle, and High groups were intragastrically administered 5, 20, and 80 mg / kg of 147-02-S2 polysaccharide respectively, all for 7 consecutive days. At the end of the experiment, the mice were sacrificed and samples were taken from the heart, liver, spleen, lung, kidney, and colon.

[0083] Result 1: Sanguisorba officinalis polysaccharide 147-02-S2 has no obvious toxicity to the important organs of mice

[0084] At the end of the experiment, the mice were sacrificed. After preserving the heart, liver, spleen, lung, and kidney tissues in 4% paraformaldehyde solution, H&E staining was performed. The results showed ( Figure 7 ), compared with the normal control group Control, there were no obvious changes in the important organs of the 147-02-S2 group.

[0085] Result 2: Sanguisorba officinalis polysaccharide 147-02-S2 can effectively improve the intestinal damage in model mice

[0086] (1) Changes in the length of the mouse colon ( Figure 8 ): The colon tissues of the mice in the Control group and the 147-02-S2 group were normal and pinkish, with good toughness. However, the colon tissues of the mice in the Model group were red, swollen, and inflamed, easy to break during sampling, with obvious redness and blood streaks visible to the naked eye on the intestinal wall, and the colon length was significantly shortened. The three concentration groups of 147-02-S2 administration reduced the degree of colon redness and shortening to a certain extent, which was basically equivalent to that of the positive drug 5-ASA administration group, indicating that the treatment group could significantly improve the phenomenon of colon shortening in mice, and the improvement degree of the Middle group was higher than that of the 5-ASA group.

[0087] (2) Mouse colon tissue ( Figure 9)After H&E staining, it was observed under a light microscope. In the colon of mice in the Control group, no obvious lesions were observed. The structure of the colonic epithelial cells was intact, and goblet cells and crypt structures were clearly visible. The glands were arranged neatly, and no edema was seen in the submucosa. In contrast, the colon in the Model group showed obvious inflammatory symptoms, with the epithelial cell layer damaged and disordered, a reduction in goblet cells, damage to the glandular tubular structure, and a large number of inflammatory cells infiltrating the mucosa and submucosa. Compared with the mice in the Model group, the mucosal tissue damage in the three 147-02-S2 treatment groups at different concentrations was alleviated to varying degrees. Among them, in the Middle group with a dose of 20 mg / kg, the colonic tissue structure was relatively intact, the lesion area was reduced, the goblet cell structure was relatively intact, the epithelial cells were arranged neatly, the crypts were relatively intact, only a small part of the glandular structure was damaged, and there was only a little infiltration of inflammatory cells, which was comparable to that of the positive drug 5-ASA control group.

[0088] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: within the scope of the spirit and essence defined by the claims of the present invention, the technical solutions described in the foregoing embodiments can be modified, or some or all of the technical features can be equivalently replaced; and these modifications or replacements are still within the scope defined by the claims of the present invention.

Claims

1. A preparation method of sanguisorba officinalis polysaccharide for preventing and / or treating inflammatory bowel disease, characterized in that, The preparation method includes the following steps: a. Extraction of sanguisorba officinalis polysaccharide: The roots of sanguisorba officinalis are extracted with boiling water. The obtained extract is concentrated, dialyzed, re-concentrated, and centrifuged to remove the precipitate. The supernatant is precipitated with alcohol and centrifuged for separation. The obtained precipitate is washed and dried to obtain the crude water-extracted sanguisorba officinalis polysaccharide; b. Purification of sanguisorba officinalis polysaccharide: b1. Dissolve the crude water-extracted sanguisorba officinalis polysaccharide prepared in step a in water, centrifuge, and the supernatant is preliminarily fractionated and purified by an anion exchange column, eluting successively with water and 0.05 - 0.4 M NaCl aqueous solution. Collect the elution fraction of 0.2 M NaCl aqueous solution to obtain sanguisorba officinalis polysaccharide 147-02; b2. Dissolve the sanguisorba officinalis polysaccharide 147-02 prepared in step b1 in 10 - 30 times the weight of 0.2 M NaCl aqueous solution, centrifuge, and the supernatant is purified by a gel chromatography column, eluting with 0.2 M NaCl aqueous solution. Collect and combine the sanguisorba officinalis polysaccharide fractions, wherein, by weight percentage, in the sanguisorba officinalis polysaccharide, the content of neutral sugar is 79% - 85%, the content of uronic acid is 13% - 19%, the content of protein is 1% - 4%, and the polysaccharide includes mannose, rhamnose, galacturonic acid, galactose, xylose, and arabinose. Among them, the molar ratio of mannose, rhamnose, galacturonic acid, galactose, xylose, and arabinose is 4 - 8: 12 - 20: 12 - 20: 16 - 24: 2.5 - 6.5: 32 - 40, and the structural unit of the sanguisorba officinalis polysaccharide is mainly composed of α-galactopyranosyluronic acid connected by 1→4 and α-rhamnopyranose connected by 1→2 as the main chain. There are branches at the C-4 position of α-rhamnopyranose and the C-3 position of α-galactopyranosyluronic acid. The branches are composed of 1, 3, 6-β-galactopyranose, 1, 6-β-galactopyranose, 1, 4-β-galactopyranose, terminal-linked β-galactopyranose, terminal-linked β-mannopyranose, terminal-linked β-xylopyranose, 1, 5-α-furan arabinose, 1, 3, 5-α-furan arabinose, terminal-linked α-furan arabinose, and terminal-linked α-galactopyranosyluronic acid.

2. The preparation method according to claim 1, wherein step a includes: before boiling water extraction, soak the sanguisorba officinalis tubers in ethanol for 3 - 10 days and air-dry; take the dried roots of sanguisorba officinalis, add 10 - 30 times the weight of water, carry out boiling water extraction for 3 - 5 hours, extract 3 - 9 times, combine the extracts, concentrate, dialyze, re-concentrate, centrifuge to obtain the supernatant, add 3 - 10 times the volume of ethanol to the supernatant, centrifuge to obtain the precipitate, wash the precipitate with absolute ethanol and acetone for 2 - 6 times, and dry to obtain the crude water-extracted sanguisorba officinalis polysaccharide; and / or step b includes: b1. Take the crude sanguisorba officinalis polysaccharide prepared in step a, dissolve it in water with a weight ratio of 10 - 20 times, and centrifuge; the supernatant is separated by an anion exchange column and eluted successively with water, 0.05 M, 0.1 M, 0.2 M, and 0.4 M NaCl aqueous solutions. Collect and combine the eluate of 0.2 M NaCl aqueous solution, concentrate, centrifuge, dialyze the supernatant, and freeze-dry to obtain sanguisorba officinalis polysaccharide 147-02; b2. Dissolve the sanguisorba officinalis polysaccharide 147-02 prepared in step b1 in 0.2 M NaCl aqueous solution with a weight ratio of 10 - 30 times, centrifuge, purify the supernatant through a gel chromatography column, elute with 0.2 M NaCl aqueous solution, detect with sulfuric acid-phenol, collect and combine this component of sanguisorba officinalis polysaccharide according to the elution curve, concentrate, dialyze, and freeze-dry to obtain the said sanguisorba officinalis polysaccharide.

3. The preparation method according to claim 2, characterized in that, In step a, the ethanol is an ethanol aqueous solution with a concentration of 70% v / v or more.

4. The preparation method according to claim 2, characterized in that, In step a, the ethanol is an ethanol aqueous solution with a concentration of 85% v / v or more.

5. The preparation method according to claim 2, wherein In step a, the ethanol is an ethanol aqueous solution with a concentration of 95% v / v or more.

6. A sanguisorba officinalis polysaccharide for preventing and / or treating inflammatory bowel disease, which is prepared by the preparation method described in any one of claims 1-5.

7. The sanguisorba officinalis polysaccharide according to claim 6, characterized in that, The range of the weight-average molecular weight of the said sanguisorba officinalis polysaccharide is 10-1000 kDa.

8. The sanguisorba officinalis polysaccharide according to claim 6, characterized in that The range of the weight-average molecular weight of the said sanguisorba officinalis polysaccharide is 20-800 kDa.

9. The sanguisorba officinalis polysaccharide according to claim 6, characterized in that The range of the weight-average molecular weight of the said sanguisorba officinalis polysaccharide is 40-600 kDa.

10. The sanguisorba officinalis polysaccharide according to claim 6, wherein, The range of the weight-average molecular weight of the said sanguisorba officinalis polysaccharide is 60-100 kDa.

11. The sanguisorba officinalis polysaccharide according to claim 6, wherein By weight percentage, in the said sanguisorba officinalis polysaccharide, the content of neutral sugar is 81.9%, the content of uronic acid is 15.63%, and the content of protein is 2.3%.

12. According to the sanguisorba officinalis polysaccharide described in claim 6, characterized in that The molar ratio of mannose, rhamnose, galacturonic acid, galactose, xylose, and arabinose is 6.18: 16.65:15.63:20.41:4.66: 36.

47.

13. A pharmaceutical composition for preventing and / or treating inflammatory bowel disease, which consists of the sanguisorba officinalis polysaccharide prepared by the preparation method described in any one of claims 1-5 or the sanguisorba officinalis polysaccharide described in any one of claims 6-12, and optionally pharmaceutically acceptable excipients.

14. Use of the sanguisorba officinalis polysaccharide prepared by the preparation method described in any one of claims 1-5, or the sanguisorba officinalis polysaccharide described in any one of claims 6-12, or the pharmaceutical composition described in claim 13 in the preparation of a drug for preventing and / or treating inflammatory bowel disease.

15. The use according to claim 14, characterized in that, The said inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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