Open arrowhead polysaccharide TCP-1, and preparation method, identification method and application thereof

Abstract

The application discloses a TCP-1 of gomphrena celosiae polysaccharide, a preparation method and application thereof, relates to the technical field of medicine and health food, and the TCP-1 of gomphrena celosiae polysaccharide is composed of fructose and glucose, and the molecular weight ranges from 1000 to 100000 Da. The preparation method is simple, the reaction condition is mild, and large-scale production is realized; the chemical structure of the obtained high-purity gomphrena celosiae polysaccharide is identified, the structure is clear, and structural basis is provided for exploring the pharmacological activity mechanism. Meanwhile, the TCP-1 of gomphrena celosiae polysaccharide obtained by the application is a gomphrena celosiae polysaccharide medicine, health food and functional food, and lays a foundation for quality control, further research on structure-activity relationship and action mechanism.

Description

Technical Field

[0001] The present 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 colorectalitis 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 recurring attacks and is difficult to cure. It 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 imbalance and hypoproteinemia. If not properly 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. Therefore, the treatment of UC remains a clinical treatment challenge worldwide.

[0003] Polysaccharides, also known as polysaccharides, are a class of bioinformatic macromolecules found widely in plants, animals, and microorganisms. Natural polysaccharides, due to their outstanding antioxidant and immunomodulatory activities, play a vital role in a variety of diseases, including tumors, anti-inflammatory, and cardiovascular diseases. Increasing evidence suggests that plant polysaccharides can exert multiple effects, including hypoglycemic, immunomodulatory, and anti-inflammatory, by influencing intestinal homeostasis. Therefore, identifying potential therapeutic agents 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 Liliaceae family, the Convallaria family, whose rhizome is its medicinal part. It has a bitter and pungent taste and a cold nature, and enters the lung, stomach, and liver meridians. It clears heat and detoxifies, dispels wind and dampness, and relieves blood stasis and pain. It is clinically used for sore throat, rheumatic pain, traumatic injuries, stomachache, carbuncles, and snake and rabies bites. The chemical components of Tupistra chinensis Baker mainly include steroids, flavonoids, proteins, polysaccharides, and other components. Previous research on the active ingredients of Tupistra chinensis Baker has mainly focused on steroid compounds. As one of the main active ingredients of Tupistra chinensis, the pharmacological effects of polysaccharides have not been widely studied. Currently, there are few reports on the homogeneous polysaccharide structure and anti-inflammatory effects of Tupistra chinensis polysaccharides. Therefore, it is necessary to provide a method for extracting and purifying Tupistra chinensis polysaccharides to lay the foundation for quality control of Tupistra chinensis polysaccharides and in-depth research on their activity in treating ulcerative colitis. Summary of the Invention

[0005] The first technical problem to be solved by the present invention is: to address the deficiencies in the existing technology and provide a polysaccharide TCP-1 with significant therapeutic effect on ulcerative colitis.

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

[0007] The invention discloses an open 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 from Trichoderma lucidum is provided, and the obtained TCP-1 polysaccharide from Trichoderma lucidum has a significant therapeutic effect on ulcerative colitis.

[0010] In order 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 rhizome of the open arrow, wash it with water, and air-dry it to obtain open arrow slices, wherein the size of the open arrow slices is 3 to 5 cm long and 0.5 to 2.5 cm in diameter;

[0014] S2. Water extraction

[0015] Add the open arrow slices obtained in step S1 to 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 deproteinization, dialysis, and freeze-drying to obtain open arrow polysaccharide TC;

[0021] S4-02, secondary purification

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

[0023] The open 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 the phenol-sulfuric acid method, the sugar part is collected according to the elution curve, concentrated, and freeze-dried to obtain the open 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 causes 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 hours, and the number of heating extractions is 2-4 times.

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

[0028] The third technical problem to be solved by the invention is: to address the deficiencies in the existing technology, to provide an identification method for the open-mouthed arrow polysaccharide TCP-1, and to successfully obtain the structural information of the open-mouthed arrow polysaccharide TCP-1.

[0029] In order to solve the third technical problem mentioned above, the technical solution of the present invention is:

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

[0031] (1) TCP-1 polysaccharide was hydrolyzed with TFA, and the hydrolyzate was detected 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 detecting it by infrared spectroscopy;

[0033] (3) TCP-1 polysaccharide was taken, methylated, hydrolyzed, reduced, acetylated, and then analyzed by GC-MS;

[0034] (4) TCP-1 polysaccharide was dissolved in D2O and analyzed by nuclear magnetic resonance;

[0035] (5) The molecular conformation of TCP-1 polysaccharide was analyzed using the 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 the common angelica 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 common angelica crude polysaccharides by ion exchange chromatography and gel molecular sieve column chromatography. A pure common angelica polysaccharide is prepared for the first time, and 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 common angelica polysaccharide is successfully obtained. The common angelica polysaccharide TCP-1 is a fructan, the main chain of which consists 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 chains consist of (→6)-β-D-Fruf-(2→) and β-D-Fruf-(2→).

[0038] Preferably, the TCP-1 methylated in step (3) is added to anhydrous DMSO in a reaction flask, and then dry sodium hydroxide is added, ultrasonicated for 20 to 40 minutes, and iodomethane is added three times under ice bath conditions and in the dark, each time ultrasonicated for 20 to 40 minutes. After the reaction is completed, distilled water is added to decompose the residual iodomethane, and chloroform is added for extraction, and the chloroform layer is centrifuged to obtain the methylated TCP-1.

[0039] Hydrolysis and reduction: Take the methylated open arrow polysaccharide TCP-1 and place it in a stoppered test tube, then add TFA, hydrolyze it in a constant temperature oil bath at 45-60°C for 25-35 minutes, evaporate to dryness under reduced pressure, repeatedly add methanol and spin dry until the pH is neutral, and then reduce the hydrolysis product with NaBH4 at 35-45°C for 25-35 minutes;

[0040] The acetylation is carried out by adding glacial acetic acid to the reduced hydrolyzed product to terminate the reaction and spin-drying the product, and then adding 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 shortcomings of the existing technology, a method is provided for the use of TCP-1 polysaccharide in the preparation of drugs or health products for treating colitis.

[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 carry out 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 arrow, with significant results, and prepares a pure product of the open arrow polysaccharide TCP-1 for the first time.

[0045] 3. The present invention identified the structure of the purified TCP-1 polysaccharide, 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, a 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 an active role in treating ulcerative colitis, providing a basis for the application of TCP-1 polysaccharide in the fields of medicine, health care products, etc.

[0047] 5. This invention lays the foundation for the quality control of Kaikoujian polysaccharide drugs and in-depth research on their 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 arrow polysaccharide TCP-1;

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

[0056] Figure 9 This is the surface feature map of the open arrow polysaccharide TCP-1;

[0057] Figure 10The primary structure diagram of the open arrow polysaccharide TCP-1;

[0058] Figure 11 This is a diagram showing the effect of TCP-1, a polysaccharide of open arrows, on the length of the Drosophila intestine;

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

[0060] Figure 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 with reference to 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, quickly wash 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 ≤-0.1 MPa, 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 crude TC polysaccharide obtained in step S3 was deproteinized using the Sevag method. After deproteinization, the crude polysaccharide was dialyzed using a dialysis bag (molecular weight cut-off of 1000 Da) and freeze-dried to obtain open arrow polysaccharide TC;

[0073] S4-02, secondary purification

[0074] 150 mg of open arrow polysaccharide TC was dissolved in 5 mL of deionized water and loaded onto a DEAE-FF column. A gradient elution was performed using a 0-2 M NaCl solution. An elution peak appeared, wherein the elution peak was the 0.05 M NaCl elution portion (the elution curve was tracked using the phenol-sulfuric acid method during the elution process, and the sugar portion was collected according to the elution curve). The resulting eluate was concentrated and freeze-dried to obtain open arrow polysaccharide TCP.

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

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

[0079] (2) Test methods:

[0080] 1. Monosaccharide composition analysis

[0081] Sample processing:

[0082] Accurately weigh 4 mg of TCP-1 and hydrolyze it with 1.8 mL of 0.2 M TFA at 70°C for 30 min. After the reaction, concentrate the TCP-1 hydrolyzate three times with anhydrous methanol to completely remove any remaining TFA. After spin drying, dissolve it in 1 mL of 70% acetonitrile. Filter the organic phase through a 0.22 μm filter and prepare for HPLC analysis.

[0083] Chromatographic column:

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

[0085] 2. Infrared spectroscopy detection

[0086] 2.0 mg of the dried open arrow polysaccharide TCP-1 test material was ground with KBr and pressed into a tablet. The tablet was then analyzed by IR Affinity-1 at 4000-400 cm -1 Scan within the range.

[0087] 3. Methylation / GC-MS Analysis

[0088] Weigh 8.0 mg of dried 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 minutes, add 3.0 mL of iodomethane in an ice bath in the dark, add it three times, and ultrasonicate in an ice bath for 30 minutes each time. After the reaction is completed, add 2 mL of distilled water to decompose the residual iodomethane, and add 1 mL of chloroform for extraction. Centrifuge and take the chloroform layer to obtain methylated open arrow polysaccharide TCP-1.

[0089] A methylated TCP-1 polysaccharide sample was hydrolyzed in a stoppered test tube with 2 mol / L TFA in a 50°C oil bath for 30 minutes. The sample was then evaporated to dryness under reduced pressure, and methanol was added repeatedly until the pH was neutral. The hydrolysis product was then reduced with 20 mg of NaBH₄ at 40°C for 30 minutes. The reaction was terminated with 100 μL of glacial acetic acid, and the sample was dried under reduced pressure. Acetylation was then performed with 2 mL of acetic anhydride and pyridine 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 3 times, spin-dried, dissolved in 1 mL of chloroform, and washed 3 times with an equal volume of distilled water. The aqueous layer was removed, and the chloroform layer was finally dried over anhydrous sodium sulfate, filtered to remove the sodium sulfate solid, and concentrated under reduced pressure to dryness for GC-MS analysis.

[0091] 4. Nuclear Magnetic Resonance Analysis

[0092] After repeated freeze-drying, 60 mg of each open arrow polysaccharide TCP-1 was dissolved in 0.6 mL of 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 and other spectra.

[0093] 5. Congo Red Analysis

[0094] After dissolving TCP-1 (0.5 mg / mL) of open arrow polysaccharide, the mixture was thoroughly 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] (3) Test results:

[0098] 1. Structural Analysis of the Polysaccharide TCP-1 from the Open Arrow

[0099] (1) Monosaccharide composition analysis

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

[0101] (2) Infrared spectroscopy analysis

[0102] like Figure 2 As shown in FIG, the infrared spectrum of the open-arrow polysaccharide TCP-1 shows 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 arrow polysaccharide TCP-1 was carried out after hydrolysis, reductive acetylation and GC-MS detection. The GC-MS spectrum showed that the open 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 linkage order was confirmed by HMBC. Figures 3-7 TCP-1, a polysaccharide of open arrow 1 H NMR, 13 C NMR, 1 H- 1 H COSY, HSQC and HMBC spectra.

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

[0108] Table 1 Nuclear magnetic resonance 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 order between different sugar residues was obtained by two-dimensional nuclear magnetic resonance HMBC spectrum analysis. The structure of the open arrow polysaccharide TCP-1 is as follows Figure 10 shown.

[0111] (5) Congo red analysis

[0112] like Figure 8 Congo red results showed that TCP-1 polysaccharide had no three-dimensional helical structure.

[0113] (6) Scanning electron microscopy analysis

[0114] like Figure 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 Kaempferia scoparia in treating ulcerative colitis in Drosophila

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

[0118] (2) Experimental subjects: Drosophila (provided by the School of Pharmacy, Guangdong Pharmaceutical University).

[0119] (3) 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. Medication

[0123] W 1118Wild-type and esg-Gal4 UAS-GFP / Cyo transgenic flies were divided into control (NC), dextran sodium sulfate (DSS)-treated (DSS)-treated (NC) and open arrow polysaccharide TCP-1 + DSS-treated (TCP-1 + DSS) groups, with 30 flies in each group (half male and half female). Before the experiment, each group of flies was starved for 2 hours and then transferred to a filter paper bottle filled with fresh medium, with the filter paper 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 hours after wild-type treatment, an appropriate amount of isoflurane was added to the vial to anesthetize the flies. The flies' intestines were then dissected and sealed with 78% glycerol before imaging using a microscope. Intestinal length was quantified using ImageJ software for detailed evaluation.

[0128] 5. The Smurf Experiment

[0129] W 1118 After 96 hours of wild-type drug 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 at 632 nm was measured 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 flies, the intestines were harvested and washed three times with PBS. After washing, the intestines were fixed in 4% paraformaldehyde and stained with 50 μL of DAPI solution for 10 minutes in the dark. The stained intestines were washed twice with PBS to remove unreacted DAPI, sealed with 78% glycerol, and observed and photographed under a fluorescence microscope. Fluorescence intensity was quantified using ImageJ software.

[0132] 7. Intestinal epithelial cell number experiment

[0133] W 1118After 96 hours of wild-type drug treatment, the cells were incubated with 50 μL 7-AAD solution (5 μg / mL) for 30 minutes in the dark. The stained intestines were then washed three times in PBS to eliminate excess 7-AAD and then fixed with 4% paraformaldehyde. After fixation, each sample was rinsed three times with PBS and then incubated with 50 μL DAPI solution for 10 minutes 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 observed by fluorescence microscopy. Fluorescence intensity was measured using ImageJ software for subsequent data analysis.

[0134] (IV) Experimental results

[0135] like Figures 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 intended 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 use of TCP-1 polysaccharide in the preparation of a drug for treating colitis, characterized by: The open arrow polysaccharide TCP-1 is composed of fructose and glucose, and its molecular weight ranges from 1000 to 100000 Da; 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→; The preparation method of the open-mouthed arrow polysaccharide TCP-1 comprises the following steps: S1, shear Slice the dried rhizome of the open arrow, wash it with water, and air-dry it to obtain open arrow slices, wherein the size of the open arrow slices is 3 to 5 cm long and 0.5 to 2.5 cm in diameter; S2. Water extraction Add the open arrow slices obtained in step S1 to water, heat and extract, and filter to obtain an extract; S3, alcohol precipitation The extract obtained in step S2 was concentrated under a vacuum degree of ≤-0.1 MPa to obtain a concentrated solution; anhydrous ethanol was added to the concentrated solution to a volume concentration of 80% ethanol, and the solution was allowed to stand and separated to obtain a precipitated crude polysaccharide TC; S4. Purification S4-01, primary purification The crude polysaccharide TC obtained in step S3 is subjected to deproteinization, dialysis, and freeze-drying to obtain open arrow polysaccharide TC; S4-02, secondary purification The purified TC was subjected to ion exchange column chromatography, and gradient elution was performed with 0-2M NaCl solution. The elution curve was tracked using the phenol-sulfuric acid method. The 0.05M NaCl elution fraction was collected according to the elution curve, concentrated, and freeze-dried to obtain TCP. The open 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 the phenol-sulfuric acid method, the sugar part is collected according to the elution curve, concentrated, and freeze-dried to obtain the open arrow polysaccharide TCP-1.

2. The use of the open arrow polysaccharide TCP-1 according to claim 1 in the preparation of a drug for treating colitis, characterized in that: The amount of water added in step S2 is 6-10 times the weight of the open arrow slice.

3. The use of the open arrow polysaccharide TCP-1 according to claim 1 in the preparation of a drug for treating colitis, 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.

4. The use of the open arrow polysaccharide TCP-1 according to claim 1 in the preparation of a drug for treating colitis, characterized in that: In step S3, the concentration temperature is 40-70° C., and the standing time is 10-15 hours.

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