Tupistra chinensis polysaccharide TCP-1 as well as preparation method, identification method and application thereof

Abstract

The invention discloses tupistra chinensis polysaccharide TCP-1 as well as a preparation method, an identification method and application thereof, and relates to the technical field of medicines and health foods, the tupistra chinensis polysaccharide TCP-1 is composed of fructose and glucose, and the molecular weight range of the tupistra chinensis polysaccharide TCP-1 is 1000-100000Da. The preparation method is simple, reaction conditions are mild, and large-scale production can be achieved; the chemical structure of the obtained high-purity tupistra chinensis polysaccharide is identified, the structure of the tupistra chinensis polysaccharide is defined, and a structural basis is provided for exploring the pharmacological activity mechanism of the tupistra chinensis polysaccharide. Meanwhile, the obtained tupistra chinensis polysaccharide TCP-1 lays a foundation for tupistra chinensis polysaccharide medicines, health-care foods and functional foods, quality control of the tupistra chinensis polysaccharide medicines, health-care foods and functional foods, and deep research of structure-function relationships and action mechanisms of the tupistra chinensis polysaccharide medicines, health

Description

Technical Field

[0001] The invention relates to the technical field of medicine and health food, and in particular to a polysaccharide TCP-1 and a preparation method, an identification method and an application thereof. Background Art

[0002] Ulcerative colitis (UC), also known as idiopathic colorectal inflammation and chronic nonspecific ulcerative colitis, is an inflammatory bowel disease of unknown etiology that occurs specifically in the rectum and colon. In recent years, the global incidence and prevalence of UC have continued to rise, with the peak age of onset being between 30 and 40 years old. It is prone to recurrence and is difficult to cure, and has become a global health problem that seriously affects the quality of life of patients. The main clinical symptoms of UC are diarrhea, abdominal pain, mucus, pus and blood in the stool, etc., which generally lead to weight loss, anemia, water and electrolyte disorders and hypoproteinemia. If not well controlled, it is prone to cancer and is accompanied by a variety of extraintestinal complications. At present, the etiology and pathogenesis of UC are not fully understood, so the treatment of UC remains a clinical treatment challenge worldwide.

[0003] Polysaccharides, also known as polysaccharides, are a class of bioinformatic macromolecules widely found in animals, plants and microorganisms. Natural polysaccharides play an important role in a variety of diseases, including tumors, anti-inflammatory and cardiovascular diseases, due to their outstanding antioxidant and immunomodulatory activities. More and more evidence shows that plant polysaccharides can exert multiple effects such as hypoglycemic, immunomodulatory and anti-inflammatory by affecting intestinal homeostasis. Therefore, searching for potential therapeutic drugs from natural polysaccharides is expected to become an effective strategy for the prevention and treatment of ulcerative colitis.

[0004] Tupistra chinensis Baker is a plant of the Lily family, and its rhizome is its medicinal part. It tastes bitter and pungent, is cold in nature, and enters the lung, stomach, and liver meridians. It clears away heat and detoxifies, dispels wind and dampness, and relieves blood stasis and relieves pain. It is clinically used for sore throat, rheumatic pain, traumatic injury, stomachache, carbuncle, ulcer, snake bite, rabies bite, etc. The chemical components of Tupistra chinensis Baker mainly include steroids, flavonoids, proteins, polysaccharides and other components. Previous studies on the active ingredients of Tupistra chinensis Baker have mainly focused on steroid compounds. As one of the main active ingredients of Tupistra chinensis Baker, its pharmacological effects have not been widely studied. At present, there are few reports on the homogeneous polysaccharide structure and anti-inflammatory effect of Tupistra chinensis polysaccharide. Therefore, it is necessary to provide a method for extracting and purifying Tupistra chinensis polysaccharide to lay a foundation for the quality control of Tupistra chinensis polysaccharide and in-depth research on the activity of Tupistra chinensis Baker in treating ulcerative colitis. Summary of the invention

[0005] The first technical problem to be solved by the present invention is: in view of the deficiencies in the prior art, a polysaccharide TCP-1 is provided, which has a significant therapeutic effect on ulcerative colitis.

[0006] To solve the above-mentioned first technical problem, the technical solution of the present invention is:

[0007] The invention discloses an open-mouthed arrow polysaccharide TCP-1, which is composed of fructose and glucose and has a molecular weight range of 1000-100000Da.

[0008] Preferably, the main chain of the open arrow polysaccharide TCP-1 is composed of →6)-α-D-Glcp-(1→, →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→; the side chain is composed of →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→.

[0009] The second technical problem to be solved by the invention is: in view of the shortcomings of the existing technology, a method for preparing TCP-1 polysaccharide is provided, and the obtained TCP-1 polysaccharide has a significant therapeutic effect on ulcerative colitis.

[0010] To solve the above second technical problem, the technical solution of the present invention is:

[0011] A method for preparing TCP-1 polysaccharide comprises the following steps:

[0012] S1. Shear

[0013] Slice the dried rhizomes of the open arrowhead, clean them with water, and air-dry them to obtain open arrowhead slices, wherein the size of the open arrowhead slices is 3 to 5 cm in length and 0.5 to 2.5 cm in diameter;

[0014] S2. Water extraction

[0015] Add the open arrow slices obtained in step S1 into water, heat and extract, and filter to obtain an extract;

[0016] S3, alcohol precipitation

[0017] The extract obtained in step S2 is concentrated under a vacuum degree of ≤-0.1 MPa to obtain a concentrated solution; anhydrous ethanol is added to the concentrated solution until the ethanol volume concentration is a%, and the solution is allowed to stand and separated to obtain a precipitated crude polysaccharide TC, wherein 10≤a<100;

[0018] S4. Purification

[0019] S4-01, primary purification

[0020] The crude polysaccharide TC obtained in step S3 is subjected to protein removal, dialyzation, and freeze-drying to obtain the open arrow polysaccharide TC;

[0021] S4-02, Secondary Purification

[0022] The purified TC polysaccharide was subjected to ion exchange column chromatography, gradient elution was performed with 0-2M NaCl solution, the elution curve was tracked by phenol-sulfuric acid method, and the sugar parts were collected according to the elution curve, concentrated, and freeze-dried to obtain TCP polysaccharide;

[0023] The open-mouthed arrow polysaccharide TCP is dissolved with water and centrifuged, the supernatant is taken and the precipitate is discarded, the supernatant is subjected to molecular sieve gel column chromatography, eluted with water, the elution curve is detected by phenol-sulfuric acid method, the sugar part is collected according to the elution curve, concentrated, and freeze-dried to obtain the open-mouthed arrow polysaccharide TCP-1.

[0024] The present invention combines water extraction with alcohol precipitation, and high-concentration ethanol can separate polysaccharides with high polarity and good water solubility from polysaccharides with low polarity and poor water solubility, thus solving the problem that the traditional water boiling method for extracting polysaccharides leads to complicated and difficult subsequent separation.

[0025] Preferably, the amount of water added in step S2 is 6-10 times the weight of the open arrow.

[0026] Preferably, the heating temperature in step S2 is 60-80° C., the heating extraction time is 1-3 h, and the number of heating extractions is 2-4 times.

[0027] Preferably, in step S3, the concentration temperature is 40-70° C. and the standing time is 10-15 h.

[0028] The third technical problem to be solved by the invention is: in view of the shortcomings of the existing technology, a method for identifying the open-mouthed arrow polysaccharide TCP-1 is provided, and the structural information of the open-mouthed arrow polysaccharide TCP-1 is successfully obtained.

[0029] To solve the third technical problem, the technical solution of the present invention is:

[0030] A method for identifying the open-mouthed arrow polysaccharide TCP-1 comprises the following steps:

[0031] (1) Taking the open arrow polysaccharide TCP-1, hydrolyzing it with TFA acid, and detecting the hydrolyzate by high performance liquid chromatography using amino column RID;

[0032] (2) Taking the open arrow polysaccharide TCP-1, drying it, pressing it into tablets, and performing infrared spectroscopy detection;

[0033] (3) Taking the open arrow polysaccharide TCP-1, methylating, hydrolyzing, reducing, acetylating, and performing GC-MS detection;

[0034] (4) Dissolving the open arrow polysaccharide TCP-1 in D2O and performing nuclear magnetic resonance analysis;

[0035] (5) The molecular conformation of TCP-1 polysaccharide was analyzed by Congo red method;

[0036] (6) The surface characteristics of TCP-1 polysaccharide were analyzed using a scanning electron microscope.

[0037] The invention uses dried rhizomes of ophiopogon japonicus as raw materials, adopts a water extraction and alcohol precipitation method to separate and obtain crude polysaccharides, deproteinizes the extracted crude polysaccharides, and then purifies the ophiopogon japonicus crude polysaccharides by ion exchange chromatography and gel molecular sieve column chromatography methods, and prepares a pure ophiopogon japonicus polysaccharide for the first time. The physicochemical properties, molecular weight, monosaccharide composition and the like of the pure polysaccharide are systematically analyzed and identified, and the structural information of the ophiopogon japonicus polysaccharide is successfully obtained, wherein the ophiopogon japonicus polysaccharide TCP-1 is a fructan, the main chain of which is composed of →6)-α-D-Glcp-(1→, →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→); and the side chains are composed of →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→.

[0038] Preferably, the TCP-1 methylated to the open arrow polysaccharide in step (3) is added to anhydrous DMSO in a reaction bottle, and then dry sodium hydroxide is added, and ultrasonic treatment is performed for 20 to 40 minutes. Under ice bath conditions, methyl iodide is added three times in the dark, and each ice bath ultrasonic treatment is performed for 20 to 40 minutes. After the reaction is completed, distilled water is added to decompose the residual methyl iodide, and chloroform is added for extraction. The chloroform layer is centrifuged to obtain the methylated open arrow polysaccharide TCP-1;

[0039] Hydrolysis and reduction: taking the methylated open arrow polysaccharide TCP-1 and placing it in a stoppered test tube, then adding TFA, hydrolyzing it in a constant temperature oil bath at 45-60°C for 25-35 minutes, evaporating it to dryness under reduced pressure, repeatedly adding methanol and spinning it to a neutral pH, and then reducing the hydrolysis product with NaBH4 at 35-45°C for 25-35 minutes;

[0040] The acetylation is to add glacial acetic acid to the reduced hydrolyzate to terminate the reaction and spin-dry the product, and then add acetic anhydride and pyridine to obtain the acetylated product.

[0041] The fourth technical problem to be solved by the invention is: in view of the deficiencies in the prior art, an application of TCP-1 polysaccharide in the preparation of drugs or health products for treating colitis is provided.

[0042] Due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0043] 1. The present invention adopts water extraction and alcohol precipitation method to perform preliminary separation of the open arrow polysaccharide, which has significant effect. In addition, the preparation method is simple, the reaction conditions are mild, and it can be produced on a large scale.

[0044] 2. The present invention uses column chromatography to perform secondary separation and purification on the crude polysaccharide of the open-mouthed arrow, with significant results, and prepares a pure product of the open-mouthed arrow polysaccharide TCP-1 for the first time.

[0045] 3. The present invention identified the structure of the purified open arrow polysaccharide TCP-1 pure product, clarified the physicochemical properties and structure of each polysaccharide component, and provided a structural basis for exploring its pharmacological activity mechanism.

[0046] 4. The pure TCP-1 polysaccharide obtained by the present invention has well-preserved components, clear structure, and controllable quality. It can restore the length of the fruit fly intestine and the number of intestinal epithelial cells and inhibit the abnormal proliferation of intestinal stem cells to play the role of treating ulcerative colitis, providing a basis for the application of TCP-1 polysaccharide in the fields of medicine, health care products, etc.

[0047] 5. The present invention lays a foundation for the quality control of the polysaccharide drug and the in-depth study of its structure-activity relationship and mechanism of action. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is the HPLC chromatogram of monosaccharide composition detected by RID of open arrow polysaccharide TCP-1 amino column;

[0049] Figure 2 This is the infrared spectrum of the open arrow polysaccharide TCP-1;

[0050] Figure 3 TCP-1 1 H NMR spectrum;

[0051] Figure 4 TCP-1 13 C NMR spectrum;

[0052] Figure 5 TCP-1 1 H- 1 H COSY spectrum;

[0053] Figure 6 This is the HSQC spectrum of the open arrow polysaccharide TCP-1;

[0054] Figure 7 This is the HMBC spectrum of the open-pointed arrow polysaccharide TCP-1;

[0055] Figure 8 This is the Congo red result of the open arrow polysaccharide TCP-1;

[0056] Fig. 9 This is the surface characteristic image of the open arrow polysaccharide TCP-1;

[0057] Fig.10This is the primary structure diagram of the open arrow polysaccharide TCP-1;

[0058] Fig.11 This is a diagram showing the effect of the open arrow polysaccharide TCP-1 on the length of the Drosophila intestine;

[0059] Fig.12 This is a diagram showing the effect of the open-mouthed arrow polysaccharide TCP-1 on the number of intestinal stem cells in Drosophila;

[0060] Fig.13 This is a diagram showing the effect of open arrow polysaccharide TCP-1 on the number of intestinal epithelial cells in Drosophila. DETAILED DESCRIPTION

[0061] The present invention will be further described below in conjunction with the embodiments.

[0062] Example 1

[0063] A method for preparing TCP-1 polysaccharide comprises the following steps:

[0064] S1. Shear

[0065] Slice 1 kg of dried rhizomes of open arrowhead, wash quickly with cold water, and air-dry to obtain open arrowhead slices 3 to 5 cm long and 0.5 to 2.5 cm in diameter;

[0066] S2. Water extraction

[0067] Add 8 times the weight of water to the open arrow slices obtained in step S1, heat to 70°C for extraction, extract for 1 hour, collect the extract, and air-dry the residue to obtain the extract and the residue;

[0068] S3, alcohol precipitation

[0069] The extract obtained in step S2 was concentrated under reduced pressure at 60°C and a vacuum degree of ≤-0.1MPa, and ethanol was added to a volume concentration of 80%. After standing at room temperature for 24 hours, the extract was centrifuged and the precipitate was collected to obtain crude polysaccharide TC;

[0070] S4. Purification

[0071] S4-01, primary purification

[0072] The TC crude polysaccharide obtained in step S3 is respectively deproteinized by the Sevag method. After the deproteinization, the crude polysaccharide is dialyzed with a dialysis bag (molecular weight cutoff of 1000Da) and freeze-dried to obtain the open arrow polysaccharide TC;

[0073] S4-02, Secondary Purification

[0074] 150 mg of open arrow polysaccharide TC was taken, dissolved in 5 mL of deionized water, loaded on a DEAE-FF column, and gradient eluted with 0-2 M NaCl solution, and an elution peak appeared, wherein the elution peak was the 0.05 M NaCl elution part (the elution curve was tracked by the phenol-sulfuric acid method during the elution process, and the sugar parts were collected according to the elution curve). The obtained eluate was concentrated and freeze-dried to obtain an open arrow polysaccharide TCP;

[0075] The above-mentioned freeze-dried open arrow polysaccharide TCP was dissolved in water, centrifuged, the supernatant was taken, and applied to a Sephacryl S-100 column, eluted with water, and the elution curve was tracked using the phenol-sulfuric acid method. A single symmetrical peak appeared, and the main peak was collected, concentrated, and freeze-dried to obtain open arrow polysaccharide TCP-1.

[0076] Example 2

[0077] Structural Analysis of the Polysaccharide TCP-1 from the Radix Astragali

[0078] (I) Test materials: TCP-1 polysaccharide in Example 1.

[0079] (II) Test methods:

[0080] 1. Monosaccharide composition analysis

[0081] Sample processing:

[0082] 4 mg of open arrow polysaccharide TCP-1 was accurately weighed and hydrolyzed with 1.8 mL of 0.2 M TFA at 70°C for 30 min. After the reaction, the hydrolyzate of open arrow polysaccharide TCP-1 was concentrated three times with anhydrous methanol to completely remove the remaining TFA. After being spin-dried, 1 mL of 70% acetonitrile was added to dissolve it, and the organic phase was filtered with a 0.22 μm filter membrane for HPLC injection analysis.

[0083] Chromatographic column:

[0084] Welch Ultimate XB-NH2 column (250mm×4.6mm, 5μm); mobile phase: acetonitrile-water solution (75:25, v / v); detector: RID; flow rate: 1.0mL / min; injection volume: 20μL; column oven temperature: 30℃.

[0085] 2. Infrared spectrum detection

[0086] 2.0 mg of the dried open arrow polysaccharide TCP-1 test material was ground with KBr and pressed into tablets. The IR Affinity-1 was used to analyze the -1 Scan within the range.

[0087] 3. Methylation / GC-MS Analysis

[0088] Weigh 8.0 mg of dry open arrow polysaccharide TCP-1 test material into a reaction bottle, add 8 mL of anhydrous DMSO, then add 800 mg of dry sodium hydroxide, ultrasonicate for 30 min, add 3.0 mL of iodomethane in an ice bath in the dark, add it three times, and ultrasonicate for 30 min each time in an ice bath. After the reaction is completed, add 2 mL of distilled water to decompose the residual iodomethane, add 1 mL of chloroform to extract, and centrifuge to obtain the chloroform layer to obtain the methylated open arrow polysaccharide TCP-1.

[0089] The methylated open arrow polysaccharide TCP-1 sample was placed in a stoppered test tube and hydrolyzed with 2 mol / L TFA in a constant temperature oil bath at 50°C for 30 min, evaporated to dryness under reduced pressure, and then repeatedly added methanol and dried to a neutral pH, and then 20 mg of NaBH4 was used to reduce the hydrolysis product at 40°C for 30 min. The reaction was terminated with 100 μL of glacial acetic acid, the sample was dried under low pressure, and then 2 mL of acetic anhydride and pyridine were added for acetylation to obtain the acetylated product.

[0090] The acetylated product was kept at 95°C with magnetic stirring for 2 h, and then methanol was added repeatedly for 3 times, spin-dried, dissolved in 1 mL of chloroform, and washed 3 times with an equal volume of distilled water. The water layer was removed, and finally the chloroform layer was dried over anhydrous sodium sulfate, and then the sodium sulfate solid was removed by filtration, and the product was concentrated and evaporated to dryness under reduced pressure for GC-MS analysis.

[0091] 4. Nuclear Magnetic Resonance Analysis

[0092] After repeated freeze-drying of each open arrow polysaccharide TCP-1, 60 mg was dissolved in 0.6 mL D2O and placed in a nuclear magnetic resonance tube. The results were recorded using a 400 MHz nuclear magnetic resonance spectrometer Bruker AV-600. 1 H NMR, 13 C NMR, HSQ C, HMBC, etc. spectra.

[0093] 5. Congo Red Analysis

[0094] After dissolving TCP-1 polysaccharide (0.5 mg / mL), the mixture was fully mixed with Congo red solution (50 μmol / L) and NaOH solution (0.00-0.60 M) in a concentration gradient. Each mixed solution was then scanned in the range of 400-600 nm using a UV-visible spectrophotometer. The maximum absorption wavelength (λ max ) and the maximum absorption wavelength (λ max ) for comparison.

[0095] 6. Scanning Electron Microscope Analysis

[0096] A small amount of open arrow polysaccharide TCP-1 was fixed on an aluminum tube using carbon tape, and the sample was scanned and recorded using a field emission scanning electron microscope with different magnifications.

[0097] (III) Test results:

[0098] 1. Structural analysis of the polysaccharide TCP-1 from the open-mouthed arrow

[0099] (1) Monosaccharide composition analysis

[0100] like Figure 1 As shown in the HPLC spectrum, TCP-1 contains fructose and glucose. (Chromatographic peak sequence: 1: fructose, 2: glucose)

[0101] (2) Infrared spectroscopy analysis

[0102] like Figure 2 As shown, from the infrared spectrum of the open-arrow polysaccharide TCP-1, it can be seen that the open-arrow TCP-1 contains the infrared characteristic absorption peak of the polysaccharide.

[0103] (3) Methylation / GC-MS analysis

[0104] The methylation analysis of the open-mouthed arrow polysaccharide TCP-1 was carried out after hydrolysis, reductive acetylation and GC-MS detection. The GC-MS spectrum showed that the open-mouthed arrow polysaccharide TCP-1 contained sugar residues such as →6)-α-D-Glcp-(1→, →1)-β-D-Fr uf-(2→, →1,6)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→, β-D-Fruf-(2→ and →6)-β-D-Fruf-(2→).

[0105] (4) Nuclear Magnetic Resonance Analysis

[0106] This test passed 1 H NMR, 13 C NMR, 1 H- 1 The chemical shifts of carbon and hydrogen atoms of the sugar residues of TCP-1 were assigned by H COSY and HSQC, and the connection order was confirmed by HMBC. Figure 3-7 TCP-1 1 H NMR, 13 C NMR, 1 H- 1 H COSY, HSQC and HMBC spectra.

[0107] according to Figure 3-7 The NMR spectrum and carbon-hydrogen assignment of open-ended arrow polysaccharide TCP-1 are shown in Table 1.

[0108] Table 1 NMR analysis results of open-mouthed arrow polysaccharide TCP-1

[0109]

[0110] In summary: TCP-1 is a fructan composed of fructose and glucose. Methylation analysis shows that it contains →6)-α-D-Glcp-(1→, →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→, β-D-Fruf-(2→ and →6)-β-D-Fruf-(2→). The connection sequence between different sugar residues was obtained by two-dimensional nuclear magnetic resonance HMBC spectrum analysis. The structure of open arrow polysaccharide TCP-1 is as follows: Fig.10 shown.

[0111] (5) Congo red analysis

[0112] like Figure 8 The Congo red results showed that the open arrow polysaccharide TCP-1 did not have a three-dimensional helical structure.

[0113] (6) Scanning electron microscopy analysis

[0114] like Fig. 9 As shown, the surface of open arrow polysaccharide TCP-1 is rough and has an irregular pore structure.

[0115] Example 3

[0116] Study on the effect of pure TCP-1 polysaccharide from Poly ...

[0117] (I) Test materials: TCP-1 polysaccharide in Example 1.

[0118] (II) Experimental subjects: Drosophila (provided by the School of Pharmacy, Guangdong Pharmaceutical University).

[0119] (III) Test methods:

[0120] 1. Drosophila culture:

[0121] They were raised on standard corn agar medium at a uniform temperature of 25 °C and a humidity level of 60-70%, adhering to a 12 h light / dark regime.

[0122] 2. Drug administration

[0123] W 1118Wild-type and esg-Gal4 UAS-GFP / Cyo transgenic flies were divided into control (NC), dextran sodium sulfate (DSS)-treated and open arrow polysaccharide TCP-1 + DSS-treated groups, 30 flies in each group (half male and half female). Before the experiment, flies in each group were starved for 2 h and then transferred to filter paper bottles filled with new medium, and the filter paper was changed every day.

[0124] 3. Lifespan Experiment

[0125] W 1118 Seven days after wild-type drug treatment, the number of surviving and dead flies was recorded.

[0126] 4. Intestinal Length Experiment

[0127] W 1118 96 h after wild-type drug treatment, an appropriate amount of isoflurane was added to the bottle to anesthetize the fruit flies. Afterwards, the intestines of the fruit flies were stripped and sealed with 78% glycerol, and then imaged with a microscope. The intestinal length was quantified by ImageJ software for detailed evaluation.

[0128] 5. The Smurf Experiment

[0129] W 1118 At the end of the 96 h of wild-type dosing treatment, each group of flies was anesthetized with isoflurane and imaged using a stereomicroscope. After imaging, phosphate buffer was added, the flies were homogenized, and the absorbance was measured at 632 nm to assess the extent of blue dye infiltration into the flies.

[0130] 6. Intestinal stem cell population experiment

[0131] After 96 hours of treatment with Esg-Gal4 UAS-GFP / Cyo transgenic fruit flies, the intestines of the fruit flies were collected and washed three times with PBS solution. After washing, the obtained intestines were attached to 4% paraformaldehyde and stained with 50 μL DAPI solution for 10 min under dark conditions. The stained intestines were washed twice with PBS to remove unreacted DAPI, sealed with 78% glycerol, and then observed and photographed under a fluorescence microscope, and the fluorescence intensity was quantified using ImageJ software.

[0132] 7. Intestinal epithelial cell number experiment

[0133] W 1118After 96h of wild-type drug treatment, incubate with 50μL 7-AAD solution (5μg / mL) for 30min in dark conditions. Then, the stained intestine was washed three times in PBS to eliminate excess 7-AA D and then fixed with 4% paraformaldehyde. After fixation, each sample was rinsed three times with PBS and then incubated with 50μL DAPI solution for 10min in the dark. After staining, the samples were washed with PBS twice to remove unreacted DAPI solution. Finally, the samples were preserved with 78% glycerol and then observed by fluorescence microscopy. Fluorescence intensity was measured using ImageJ software for subsequent data analysis.

[0134] (IV) Experimental results

[0135] like Figure 11-13 As shown, TCP-1, a polysaccharide from the open arrow, can protect the intestinal barrier damaged by DSS, restore the intestinal length and the number of intestinal epithelial cells, and inhibit the abnormal proliferation of intestinal stem cells to exert its activity in treating ulcerative colitis.

[0136] In summary, the pure TCP-1 polysaccharide prepared by the present invention can restore the intestinal barrier, length and number of intestinal epithelial cells, inhibit the abnormal proliferation of intestinal stem cells, and thus exert the ability to treat ulcerative colitis.

[0137] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

Claims

1. A polysaccharide TCP-1, characterized in that: The open arrow polysaccharide TCP-1 is composed of fructose and glucose, and its molecular weight ranges from 1000 to 100000Da.

2. The open arrow polysaccharide TCP-1 according to claim 1, characterized in that: The main chain of the open arrow polysaccharide TCP-1 is composed of →6)-α-D-Glcp-(1→, →1)-β-D-Fruf-(2→, →1,6)-β-D-Fruf-(2→, →6)-β-D-Fruf-(2→ and β-D-Fruf-(2→; the side chain is composed of →6)-β-D-Fr uf-(2→ and β-D-Fruf-(2→.

3. A method for preparing the open arrow polysaccharide TCP-1 according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1. Shear Slice the dried rhizomes of the open arrowhead, clean them with water, and air-dry them to obtain open arrowhead slices, wherein the size of the open arrowhead slices is 3 to 5 cm in length and 0.5 to 2.5 cm in diameter; S2. Water extraction Add the open arrow slices obtained in step S1 into water, heat and extract, and filter to obtain an extract; S3, alcohol precipitation The extract obtained in step S2 is concentrated under a vacuum degree of ≤-0.1 MPa to obtain a concentrated solution; anhydrous ethanol is added to the concentrated solution until the ethanol volume concentration is a%, and the solution is allowed to stand and separated to obtain a precipitated crude polysaccharide TC, wherein 10≤a<100; S4. Purification S4-01, primary purification The crude polysaccharide TC obtained in step S3 is subjected to protein removal, dialyzation, and freeze-drying to obtain the open arrow polysaccharide TC; S4-02, Secondary Purification The purified TC polysaccharide was subjected to ion exchange column chromatography, gradient elution was performed with 0-2M NaCl solution, the elution curve was tracked by phenol-sulfuric acid method, and the sugar parts were collected according to the elution curve, concentrated, and freeze-dried to obtain TCP polysaccharide; The open-mouthed arrow polysaccharide TCP is dissolved with water and centrifuged, the supernatant is taken and the precipitate is discarded, the supernatant is subjected to molecular sieve gel column chromatography, eluted with water, the elution curve is detected by phenol-sulfuric acid method, the sugar part is collected according to the elution curve, concentrated, and freeze-dried to obtain the open-mouthed arrow polysaccharide TCP-1.

4. The method for preparing the open arrow polysaccharide TCP-1 according to claim 3, characterized in that: The amount of water added in step S2 is 6-10 times the weight of the open arrow sheet.

5. The method for preparing the open arrow polysaccharide TCP-1 according to claim 3, characterized in that: The heating temperature in step S2 is 60-80° C., the heating extraction time is 1-3 hours, and the heating extraction times are 2-4 times.

6. The method for preparing the open arrow polysaccharide TCP-1 according to claim 3, characterized in that: In step S3, the concentration temperature is 40-70° C. and the standing time is 10-15 h.

7. The method for identifying the open-mouthed arrow polysaccharide TCP-1 according to any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Taking the open arrow polysaccharide TCP-1, hydrolyzing it with TFA acid, and detecting the hydrolyzate by high performance liquid chromatography using amino column RID; (2) Taking the open arrow polysaccharide TCP-1, drying it, pressing it into tablets, and performing infrared spectroscopy detection; (3) Taking the open arrow polysaccharide TCP-1, methylating, hydrolyzing, reducing, acetylating, and performing GC-MS detection; (4) Dissolving the open arrow polysaccharide TCP-1 in D2O and performing nuclear magnetic resonance analysis; (5) The molecular conformation of TCP-1 polysaccharide was analyzed by Congo red method; (6) The surface characteristics of TCP-1 polysaccharide were analyzed using a scanning electron microscope.

8. The method for identifying the open arrow polysaccharide TCP-1 according to claim 7, characterized in that: The TCP-1 methylated in step (3) is added to anhydrous DMSO in a reaction bottle, and then dry sodium hydroxide is added, and ultrasonic treatment is performed for 20 to 40 minutes. Under ice bath conditions, methyl iodide is added three times in the dark, and each ice bath ultrasonic treatment is performed for 20 to 40 minutes. After the reaction is completed, distilled water is added to decompose the residual methyl iodide, and chloroform is added for extraction. The chloroform layer is centrifuged to obtain the methylated TCP-1 methylated polysaccharide. Hydrolysis and reduction: taking the methylated open arrow polysaccharide TCP-1 and placing it in a stoppered test tube, then adding TFA, hydrolyzing it in a constant temperature oil bath at 45-60°C for 25-35 minutes, evaporating it to dryness under reduced pressure, repeatedly adding methanol and spinning it to a neutral pH, and then reducing the hydrolysis product with NaBH4 at 35-45°C for 25-35 minutes; The acetylation is to add glacial acetic acid to the reduced hydrolyzate to terminate the reaction and spin-dry the product, and then add acetic anhydride and pyridine to obtain the acetylated product.

9. Use of the open-mouthed arrow polysaccharide TCP-1 according to any one of claims 1 to 6 in the preparation of drugs or health products for treating colitis.

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