A polysaccharide for preventing, treating or ameliorating chronic constipation, and a preparation method and application thereof

Abstract

The present application discloses a polysaccharide for preventing, treating or improving chronic constipation, and a preparation method and application thereof. The polysaccharide can improve or restore intestinal barrier integrity, increase the expression amount of colon tight junction proteins, regulate the proportion of intestinal microbe beneficial flora, increase the content of short-chain fatty acids, improve gastrointestinal motility, regulate intestinal oxidative stress, increase the fecal water content and intestinal transport rate of constipation subjects, reduce intestinal tissue damage, and the like, thereby being capable of being used for preventing, treating or improving chronic constipation. In addition, the polysaccharide has the advantages of high safety and good curative effect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a polysaccharide for the prevention, treatment or improvement of chronic constipation, its preparation method and application. Background Technology

[0002] Chronic constipation is a gastrointestinal disease characterized by its widespread incidence and high recurrence rate. Typical clinical manifestations include reduced bowel movement frequency, abnormal intestinal motility, and symptoms of anorectal obstruction. Data analysis shows that the global prevalence of constipation is approximately 15%, making it a global health concern. This is due to factors such as lack of exercise, poor coordination between mucosal transport and the defecation reflex, adverse reactions to opioids, and irritable bowel syndrome. Current research indicates that the pathogenic mechanism of constipation involves impaired sensorimotor function of the colon and pelvic floor.

[0003] Current treatment options mainly relieve constipation through osmotic laxatives and stimulant laxatives, but these therapies can cause side effects such as abdominal cramps and diarrhea, and may also lead to risks such as cardiovascular disease.

[0004] Therefore, there is an urgent need to develop a safer and more effective drug that can prevent, treat or improve chronic constipation. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, the present invention provides a polysaccharide for the prevention, treatment, or improvement of chronic constipation, a method for its preparation, and its applications. Specifically, the present invention includes the following:

[0006] In a first aspect, the present invention provides a polysaccharide for the prevention, treatment or improvement of chronic constipation, the main chain of which has a structure such as →6)-α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ glycosidic bond, terminal β-D-Glc p -(1→through→4,6)-α-D-Glc p -(1→ is connected to the main chain, and the polysaccharide has the structure shown in Formula I: Formula I; Where n is selected from natural numbers from 2 to 20.

[0007] In some embodiments, the polysaccharide for preventing, treating, or improving chronic constipation according to the present invention includes at least one of the following: (1) Improve or restore the integrity of the intestinal barrier; (2) Increase the expression level of colonic tight junction protein; (3) Regulate the proportion of beneficial gut microbiota; (4) Increase the content of short-chain fatty acids; (5) Improves gastrointestinal motility; (6) Regulate intestinal oxidative stress.

[0008] In a second aspect, the invention provides a pharmaceutical composition for the prevention, treatment or improvement of chronic constipation, comprising the polysaccharide described herein.

[0009] In some embodiments, the pharmaceutical composition according to the present invention further comprises a pharmaceutically acceptable carrier.

[0010] A third aspect of the present invention provides a method for preparing a polysaccharide for the prevention, treatment, or improvement of chronic constipation according to the present invention, comprising the following steps: (1) Extract fruit pulp with an aqueous solvent at 60-100℃ for 30-120 min to obtain an extract. Add a precipitant to precipitate the extract and remove the protein from the precipitate to obtain crude polysaccharide. (2) The crude polysaccharide is separated and purified to obtain the polysaccharide.

[0011] In some embodiments, according to the method for preparing polysaccharides for the prevention, treatment or improvement of chronic constipation according to the present invention, the ratio of the fruit pulp to the aqueous solvent is 1:(2-10)g / mL.

[0012] In some embodiments, according to the method for preparing polysaccharides for the prevention, treatment or improvement of chronic constipation according to the present invention, proteins in the precipitate are removed by the Sevag method.

[0013] In some embodiments, the method for preparing polysaccharides for the prevention, treatment or improvement of chronic constipation according to the present invention includes separation and purification comprising ion exchange column chromatography and non-ionic gel chromatography.

[0014] In some embodiments, the method for preparing polysaccharides for the prevention, treatment, or improvement of chronic constipation according to the present invention, wherein the separation and purification includes: The crude polysaccharide was separated by ion exchange column chromatography, and the elution fraction corresponding to the first peak was collected. Using water as the mobile phase, the elution fraction corresponding to the first peak is passed through a non-ionic gel filter column to obtain the polysaccharide used for the prevention, treatment, or improvement of chronic constipation.

[0015] A fourth aspect of the invention provides the use of the polysaccharide according to the invention in the preparation of a medicament for the prevention, treatment or improvement of chronic constipation.

[0016] The polysaccharide of this invention can enhance the permeability of the intestinal barrier by upregulating intestinal closure protein and sealing protein-1, restore gastrointestinal motility balance, alleviate intestinal dysfunction and tissue damage, effectively restore the abundance, diversity and structural composition of the microbial community in the cecal contents of constipated mice, significantly improve intestinal microbial dysbiosis in constipated mice, increase the content of short-chain fatty acids, enhance intestinal immunity and thus maintain the intestinal barrier, thereby ultimately achieving the effect of effectively relieving constipation. Attached Figure Description

[0017] Figure 1 The elution curve for DEAE-52.

[0018] Figure 2 The results of the activity screening for different polysaccharide components in relieving chronic constipation are shown. In the table, A represents fecal particle count, B represents fecal weight, C represents fecal water content, and D represents small intestinal transit rate.* p <0.05,** p <0.01, *** p <0.001, **** p <0.0001 VS model group.

[0019] Figure 3 Elution curves for Sephadex G-50.

[0020] Figure 4 The results show the molecular weight determination of the polysaccharides of this invention.

[0021] Figure 5 The results show the monosaccharide composition determination of the polysaccharides of this invention.

[0022] Figure 6 The results are shown in the ultraviolet spectral scan of the polysaccharide of this invention.

[0023] Figure 7 The results are shown in the infrared spectral analysis of the polysaccharide of this invention.

[0024] Figure 8 The polysaccharide of this invention 13 Results of C NMR spectral analysis.

[0025] Figure 9 The polysaccharide of this invention 1 H- 1 HCl COSY spectral analysis results.

[0026] Figure 10 The results are shown in the HSQC spectral analysis of the polysaccharide of this invention.

[0027] Figure 11 The results of HMBC spectral analysis of the polysaccharide of this invention are shown.

[0028] Figure 12 The results of TOCSY spectral analysis of the polysaccharide of this invention are shown.

[0029] Figure 13 The results of the polysaccharide HPUP of this invention in relieving chronic constipation induced by loperamide hydrochloride are shown in the figure. In the figure, A is the small intestinal transit rate, B is the number of fecal particles, C is the fecal weight, D is the fecal water content, EI is the serum MTL, SP, 5-HT, GAS and VIP content, and JL is the colonic SOD, GSH-px and MDA content.

[0030] Figure 14 This image shows the improvement of the intestinal tissue of mice with chronic constipation by the polysaccharide HPUP of this invention. In the image, A is the H&E staining map of the duodenum, jejunum, and jejunum; B is the H&E staining map of the colon; C is the Alcian blue map of the colon; DF are the statistical results of the villus length of the duodenum, jejunum, and ileum, respectively; G is the result of the colonic crypt thickness; H is the result of the colonic muscle layer length; and I is the statistical graph of the area of ​​colonic goblet cells.

[0031] Figure 15 The results show the enhancing effect of the polysaccharide HPUP of this invention on tight junction proteins in the colonic tissue of mice with chronic constipation. Among them, A is the immunoblot band image of mouse colonic closure protein and sealing protein-1, B is the quantitative map of expression of mouse colonic closure protein and sealing protein-1, C is the mRNA expression analysis map of mouse colonic closure protein and sealing protein-1, D and E are the immunofluorescence maps of mouse colonic closure protein and sealing protein-1, respectively, and F and G are the fluorescence intensity statistics of mouse colonic closure protein and sealing protein-1.

[0032] Figure 16 The results of diversity analysis of mouse cecal contents are shown in the figure. A is the abundance rank plot, B is the sparse curve plot, C is the Chao1 plot, D is the Simpson plot, E is the Shannon plot, F is the Observed_species plot, G is the PCoA plot, H is the PCA plot, I is the NMDS plot, and J is the Venn plot.

[0033] Figure 17 The results show the composition, differential analysis, and changes in short-chain fatty acid content in the cecal contents of mice. In this paper, A represents the changes in bacterial abundance at the phylum level, B represents the changes in bacterial abundance at the genus level, and C represents the content of short-chain fatty acids in feces. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention.

[0037] In this invention, the term "prevention, treatment, or improvement" includes improving the condition of a disease or functional disorder before or after its onset. This improvement or prevention, measured by any standard technique, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% compared to an untreated control group under equivalent conditions. Beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and reduction (whether partial or complete). The prevention, treatment, or improvement includes, but is not limited to, the following conditions associated with chronic constipation: improved or restored intestinal barrier integrity, increased expression of colonic tight junction proteins, regulation of the proportion of beneficial gut microbiota, increased short-chain fatty acid content in feces, enhanced gastrointestinal motility, regulation of intestinal oxidative stress, reduction of intestinal tissue damage, and increased fecal weight, fecal water content, and intestinal transit rate in constipated subjects.

[0038] polysaccharides In one aspect, the present invention provides a polysaccharide for the prevention, treatment, or improvement of chronic constipation, the main chain of which has a structure such as →6)-α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ glycosidic bond, terminal β-D-Glc p -(1→through→4,6)-α-D-Glc p -(1→ is connected to the main chain, and the polysaccharide has the structure shown in Formula I: Formula I; Wherein, n is selected from natural numbers from 2 to 20, preferably from 3 to 19, even more preferably from 4 to 18, further preferably from 5 to 17, more preferably from 6 to 16, and even more preferably from 7 to 15, for example 7, 8, 9, 10, 11, 12, 13, 14, 15. In a specific embodiment, n is 12.

[0039] The present invention provides structural analysis and characterization of the obtained polysaccharide HPUP. In a specific embodiment, high-performance gel permeation chromatography (HPGPC) was used to determine the molecular weight, and the relative molecular weight of the polysaccharide HPUP of the present invention was 2.99 kDa.

[0040] In the specific implementation scheme, the polysaccharide HPUP is detected by infrared spectroscopy analysis using IR mode, with a scanning wavenumber range of 4000 cm⁻¹. -1 -400 cm -1 The polysaccharide HPUP of this invention is at 3389 cm⁻¹ -1 The stretching vibration peak of the OH bond in the carbohydrate molecule is located at 2927 cm⁻¹. -1 The stretching vibration peak of the CH bond in carbohydrate molecules is located at 1421 cm⁻¹. -1 The bending vibration peak with CH bonds at 1047 cm⁻¹; -1 The peak of deformation vibration of the COH bond with the pyranose ring is located at this point.

[0041] In a specific implementation scheme, the specific chemical structure of the polysaccharide HPUP was determined by nuclear magnetic resonance analysis. The results showed that β-D-Glc exists in the polysaccharide HPUP. p -(1→and→4,6)-α-D-Glc p The connection method of -(1→ exists, and -α-D-Glc exists.) p -(1→6)-α-D-Glc p -(1→ repeating unit, exists →6)-α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ Connection method at C6 bit.)

[0042] Pharmaceutical Composition In one aspect, the present invention provides a pharmaceutical composition for the prevention, treatment or improvement of chronic constipation, comprising the polysaccharides described herein.

[0043] In a preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier that participates in the delivery or transport of the drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable," meaning it is compatible with other components of the formulation (e.g., the polysaccharides of this invention) and does not harm the patient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, emulsifiers, colorants, diluents, fillers, wetting agents, binders, lubricants, sweeteners, antioxidants, etc. Examples of buffers include, but are not limited to, citrate, histidine, succinate, etc.; examples of emulsifiers include, but are not limited to, polysorbate, etc.; examples of colorants include, but are not limited to, sodium copper chlorophyllin, betaine, curcumin, β-carotene, anthocyanins, etc.; examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, dispersion media, etc.; fillers include, but are not limited to, sucrose, trehalose, xylitol, etc.; wetting agents include, but are not limited to, water, etc.; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, etc.; lubricants include, but are not limited to, magnesium stearate, micronized silica gel, etc.; sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, aspartame, etc.; antioxidants include, but are not limited to, ascorbic acid, sodium ascorbate, tea polyphenols, etc.

[0044] Preparation method In one aspect, the present invention provides a method for preparing polysaccharides for the prevention, treatment or improvement of chronic constipation, comprising steps (1) and (2), which are described in detail below.

[0045] Step (1) of the present invention is to extract fruit pulp with an aqueous solvent at 60-100℃ for 30-120 min to obtain an extract, add a precipitant to precipitate and obtain a precipitate, remove the protein in the precipitate and obtain crude polysaccharide.

[0046] In a preferred embodiment, step (1) of the present invention includes extracting fruit pulp using an aqueous solvent at 60-100°C (preferably 65-100°C, more preferably 70-100°C, even more preferably 75-100°C, and even more preferably 80-100°C, such as 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100°C) for 30-120 min (preferably 30-110 min, more preferably 30-100 min, even more preferably 30-90 min, and even more preferably 30-80 min, such as 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 min) to obtain an extract, and then rubbing the extract at 6000-10000 rpm (preferably 6200-9800 rpm, even more preferably 6400-9600 rpm, and even more preferably 6600-9400 rpm). Centrifuge at rpm, more preferably 6800-9200 rpm, even more preferably 7000-9000 rpm, such as 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000 rpm, for 5-30 min (preferably 5-25 min, even more preferably 5-20 min, more preferably 5-15 min, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min), add a precipitant (e.g., ethanol with a final concentration of 70-90%, preferably 72-88%, even more preferably 74-86%, more preferably 76-84%, such as 76, 77, 78, 79, 80, 81, 82, 83, 84%) and allow to precipitate for 30-60 h (preferably 32-58 h, even more preferably 34-56 h). The precipitate is obtained by precipitating for 36-54 hours (h, more preferably 36-54 hours, e.g., 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 hours). Proteins in the precipitate are removed by the Sevag method to obtain crude polysaccharide. The ratio of fruit pulp to aqueous solvent is 1:(2-10) g / mL, preferably 1:(3-9) g / mL, and even more preferably 1:(3-8) g / mL, e.g., 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 g / mL. It is understood that the extraction process can be repeated several times to better extract the active ingredients, and the extract can be concentrated or the crude polysaccharide can be freeze-dried; all of these are within the scope of this invention.

[0047] In this invention, the aqueous solvent includes, but is not limited to, distilled water, deionized water, reverse osmosis water, ultrapure water, etc.; the fruit includes, but is not limited to, dragon fruit, bird's nest fruit, custard apple, snake fruit, jaboticaba, pineapple, apple, banana, sweet orange, pineapple, grape, strawberry, etc.; and the precipitant includes, but is not limited to, ethanol, propanol, isopropanol, acetone, etc.

[0048] Step (2) of the present invention is to separate and purify the crude polysaccharide to obtain the polysaccharide HPUP.

[0049] In a preferred embodiment, step (2) of the present invention includes loading the crude polysaccharide aqueous solution onto an ion exchange chromatography column (e.g., but not limited to, a DEAE-52 anion exchange resin column), and performing gradient elution sequentially using 0 M NaCl, 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, and 0.7 M NaCl solutions as eluents at a rate of 0.1-5 mL / min (preferably 0.2-4 mL / min, more preferably 0.3-3 mL / min, further preferably 0.4-2 mL / min, and more preferably 0.5-1.5 mL / min, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, and 1.5 mL / min), collecting the elution fraction corresponding to the 0 M NaCl; and loading the elution fraction corresponding to the 0 M NaCl onto a non-ionic gel filtration column (e.g., but not limited to, Sephadex). The gel was eluted using a G-50 gel column with water as the eluent at a rate of 0.1-2 mL / min (preferably 0.1-1.8 mL / min, more preferably 0.1-1.6 mL / min, even more preferably 0.1-1.4 mL / min, more preferably 0.1-1.2 mL / min, and even more preferably 0.1-1 mL / min, for example 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 mL / min). The gel was then freeze-dried to obtain the polysaccharide HPUP.

[0050] application In one aspect, the present invention provides the use of the polysaccharide described herein in the preparation of a medicament for the prevention, treatment or improvement of chronic constipation.

[0051] In this invention, the prevention, treatment, or improvement is achieved by administering a therapeutically effective amount of the drug to a subject. Subjects include, but are not limited to, mammals, including but not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, and pigs.

[0052] The therapeutically effective dose described in this invention refers to a pharmaceutically recognized effective dosage, meaning the amount of the active compound (i.e., the polysaccharide of this invention) is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the drug is typically 0.01-1000 mg / kg, preferably 1-500 mg / kg, even more preferably 10-400 mg / kg, further preferably 30-300 mg / kg, and more preferably 50-200 mg / kg, for example, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 mg / kg. It can be administered as a single dose once daily, multiple times daily, or at intervals.

[0053] In this invention, there are no particular limitations on the method of administration of the drug. Representative methods of administration include, but are not limited to, oral, rectal, and parenteral (intravenous, intramuscular, or subcutaneous) administration. Accordingly, the pharmaceutical compositions of this invention can be formulated into various clinically acceptable dosage forms, examples of which include, but are not limited to, decoctions, powders, pills, ointments, tablets, capsules, granules, and oral liquids.

[0054] This invention also provides a method for in vitro regulation of cell function, comprising the step of contacting cells in vitro with the polysaccharide or pharmaceutical composition described in this invention. The regulation includes, but is not limited to, providing the expression levels of tight junction proteins. In some embodiments, the method may be a therapeutic method, i.e., for therapeutic purposes. In other embodiments, the method is a non-therapeutic method, such as for efficacy evaluation, drug screening, disease mechanism research, etc.

[0055] In a preferred embodiment, the method for in vitro regulation of cell function according to the present invention includes: (1) culturing cells in vitro (e.g., but not limited to Caco-2 cells, HT-29 cells, MODE-K cells, primary colonic epithelial cells, etc.); (2) contacting the cells with the polysaccharide or pharmaceutical composition of the present invention in vitro; and (3) detecting the expression level of tight junction proteins, etc. The detection can be performed using methods and apparatus known in the art, and is not particularly limited thereto.

[0056] Example 1 The following illustrates the preparation and characterization of the polysaccharide of the present invention for the prevention, treatment or improvement of chronic constipation.

[0057] 1. Preparation method 1.1 Extraction of crude polysaccharides Dragon fruit was washed, peeled, and sliced, then homogenized and mixed with deionized water at a solid-liquid ratio of 1:5 (g / mL). Extraction was carried out at 100℃ for 45 min. The extracted mixture was centrifuged at 8000 rpm for 10 min at 4℃, and the process was repeated once. The supernatant was collected and concentrated. The concentrate was then precipitated with ethanol (to achieve an alcohol content of 80%) for 48 h. The precipitate was redissolved in deionized water and treated with a mixture of n-butanol and chloroform at a ratio of 1:4 (v / v), repeated three times to remove proteins. The polysaccharide solution was freeze-dried to obtain crude polysaccharide.

[0058] 1.2 Separation and Purification 1.2.1 Ion exchange column chromatography Take an appropriate amount of crude polysaccharide sample, dissolve it thoroughly in distilled water, and then filter it through a 0.45 μm filter membrane to remove insoluble impurities. Separate the solution using a DEAE-52 anion exchange resin column. First, pack an appropriate amount of activated resin into a glass chromatography column (2.6 cm × 40 cm), and equilibrate it with distilled water. After equilibration, load an appropriate amount of the crude polysaccharide solution that passed through the filter membrane into the chromatography column. Then, perform gradient elution using 0 M NaCl, 0.1 M NaCl, 0.3 M NaCl, 0.5 M NaCl, and 0.7 M NaCl solutions in sequence, at an elution rate of 1 mL / min. The collection volume per tube is approximately 10 mL. The elution curve is shown below. Figure 1 As shown. After collection, the polysaccharide content of the eluent in each tube was determined using the phenol-sulfuric acid method, and identical components were combined for subsequent separation.

[0059] 1.2.2 Screening of polysaccharides with constipation-relieving activity To screen for the most active polysaccharide fractions, the alleviating effects of five different DEAE-52 eluent polysaccharide fractions on loperamide hydrochloride-induced constipation in mice were investigated. Fifty-six Balb / c mice were acclimatized for 7 days and then randomly divided into seven groups of eight each: a blank control group (Ctrl), a loperamide hydrochloride group (model group), and dragon fruit polysaccharide groups (HPUP-0, HPUP-1, HPUP-2, HPUP-3, HPUP-4). Except for the blank control group, the other groups were administered 10 mg / kg loperamide hydrochloride via gavage to induce a mouse constipation model for 14 consecutive days. Simultaneously, the dragon fruit polysaccharide groups were administered 100 mg / kg of the corresponding polysaccharide extracts daily via gavage, while the blank control group and model group were administered the same volume of purified water daily via gavage. The general physiological condition and fecal consistency of the mice were monitored daily. On the last day of the experiment, six mice were randomly selected and administered 0.2 mL of activated charcoal solution via gavage. Afterward, the mice were placed in clean, individual cages with free access to food and water. The quantity and weight of black feces were recorded within 6 hours after gavage. The collected feces were dried at 60°C for 48 hours, and the moisture content was calculated. After statistical analysis, the mice were fasted for 12 hours with free access to water, and then administered 0.2 mL of activated charcoal solution by gavage. The mice were sacrificed 30 minutes later, and the small intestine was harvested to measure the transport distance of the activated charcoal solution.

[0060] The results are as follows Figure 2 As shown, compared with the blank control group, the basic fecal indicators of the model group mice were significantly reduced (p<0.05), while the HPUP-0 group significantly restored the related indicators such as small intestinal passage rate, fecal weight, quantity, and water content (p<0.01). Therefore, HPUP-0 has the best ability to relieve constipation in mice, and HPUP-0 was selected for subsequent research.

[0061] 1.2.3 Gel column chromatography HPUP-0 was further separated and purified using Sephadex G-50 gel. The procedures were the same as described above: column packing, equilibration, and sample loading. Distilled water was used as the eluent at a rate of 0.5 mL / min. After confirming the target component, the collected eluent containing the same component was concentrated to a suitable volume, freeze-dried, and finally yielded a white solid polysaccharide, named HPUP.

[0062] The results are as follows Figure 3 As shown, the elution curve of HPUP is a single peak, indicating that the eluted component is a single polysaccharide component with a uniform molecular weight distribution.

[0063] 2. Compositional analysis, physicochemical properties, and structural characterization 2.1 Composition Analysis The total sugar content in polysaccharides was determined using the phenol-sulfuric acid method: A standard glucose solution (0-0.1 mg / mL) was prepared. 1 mg of HPUP was accurately weighed and dissolved in 1 mL of distilled water. 100 µL of each solution was placed in a 2 mL EP tube. 100 µL of 5% phenol and 500 µL of sulfuric acid were added to both the standard and the sample. The mixture was stirred and boiled at 100 °C for 10 min. After the reaction, 200 µL of the sample was placed in a 96-well plate, and the absorbance was immediately measured at 490 nm. A standard curve was plotted, and the total sugar content in HPUP was calculated.

[0064] The Bradford assay was used to determine the polysaccharide-protein content: Prepare bovine serum albumin (BSA) standard solution (0-1 mg / mL) and take 4 µL of each solution into a 96-well plate. Accurately weigh 1 mg of HPUP and dissolve it in 1 mL of distilled water, and take 4 µL of each solution. Add 200 µL of Bradford reagent to the standard and the sample, and mix well. Immediately measure the absorbance at 595 nm, and calculate the protein concentration in the sample based on the standard curve.

[0065] The uronic acid content in polysaccharides was determined using the sulfuric acid-m-hydroxyphenol method: A standard galacturonic acid solution (0-0.1 mg / mL) was prepared. 1 mg of HPUP was accurately weighed and dissolved in 1 mL of distilled water. After complete dissolution, 400 μL of the sample was pipetted into a 5 mL EP tube and 2.4 mL of sodium tetraborate-sulfuric acid solution (12.5 mM) was added. The mixture was stirred and reacted in a boiling water bath for 5 min. The mixture was then immediately cooled to room temperature in an ice bath. 40 μL of 0.15% m-hydroxybiphenyl (dissolved in 0.5% sodium hydroxide) was added, stirred, and the spectrophotometry was immediately measured at 520 nm. The uronic acid content (calculated as galacturonic acid) in the sample was calculated based on the standard curve.

[0066] The results showed that HPUP had a total sugar content of more than 98%, contained trace amounts of uronic acid (less than 1%), and contained no protein.

[0067] 2.2 Homogeneity and Molecular Weight Analysis High-performance gel permeation chromatography (HPGPC) was used to identify the purity and determine the molecular weight of HPUP samples. Pullulan was used as a standard to construct a standard curve, and the molecular weight of HPUP was calculated. The chromatographic conditions were: column temperature 30℃, mobile phase 0.1 M NaNO3, and flow rate 0.7 mL / min.

[0068] The results are as follows Figure 4 As shown, a distinct narrow peak appears on both sides, and the peak shape is symmetrical, indicating that the HPUP molecular weight is uniformly distributed. The relative molecular weight of HPUP is 2.99 kDa, which can be used for further structural analysis.

[0069] 2.3 Monosaccharide Composition Analysis High-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD) was used to determine the monosaccharide composition of hydrolysis products in HPUP samples. The instrument was a Thermo ICS 5000 ion chromatograph with a Thermo Scientific™ Dionex™ CarboPac™ PA100 column. Mobile phase A consisted of 20 mM NaOH, and mobile phase B consisted of 100 mM NaOH + 500 mM NaAc. The column temperature was 30 °C, and the flow rate was 0.4 mL / min.

[0070] The results are as follows Figure 5 As shown, HPUP's monosaccharide composition is mainly glucose, with small amounts of galactose, fructose, mannose, and arabinose. Glucose is the dominant monosaccharide (accounting for over 97%), indicating that HPUP is a glucan.

[0071] 2.4 Ultraviolet Spectroscopy Analysis Weigh an appropriate amount of HPUP sample and dissolve it completely in ultrapure water. Scan the sample using a UV spectrophotometer with the following settings: wavelength range: 200-800 nm; time interval: 1 nm; blank control: ultrapure water. Plot the HPUP absorption spectrum curve.

[0072] The results are as follows Figure 6 As shown, the ultraviolet scanning data of HPUP indicates that HPUP has no characteristic absorption in the wavelength range of 260 nm to 280 nm, thus inferring that there are no protein or nucleic acid components remaining in the sample.

[0073] 2.5 Fourier Transform Infrared Spectroscopy Analysis The characteristic absorption peaks of HPUP samples were detected using Fourier transform infrared spectroscopy (FTIR). HPUP and KBr were mixed at a mass ratio of 1:100 and ground into powder in an agate mortar. The sample was then compressed into tablets using a tablet press, and the IR mode was used for detection, with a scanning wavenumber range of 4000 cm⁻¹. -1 up to 400 cm -1 .

[0074] The results are as follows Figure 7 As shown, at 3389 cm -1 The strong absorption peak at 2927 cm⁻¹ is caused by the stretching vibration of the OH bonds in the polysaccharide backbone; -1 The absorption peak at 1421 cm⁻¹ corresponds to the stretching vibration of CH in the polysaccharide functional group; -1 The absorption peak at 1047 cm⁻¹ is related to the bending vibration of CH₄; -1 The strong absorption peak at the point represents the deformation vibration of COH in the pyranose ring structure, indicating that pyranose, as the main monosaccharide, constitutes the dragon fruit pulp polysaccharide.

[0075] 2.6 Methylation Analysis HPUP was methylated, acid-hydrolyzed, reduced, and acetylated to obtain derivatives, which were then analyzed by gas chromatography-mass spectrometry (GC-MS). Capillary column: HP-5MS (0.25 μm, 30 m × 0.25 mm). Initial temperature: 80 °C, held for 2 min. Then, the temperature was increased to 320 °C at a rate of 15 °C / min and held for 2 min. High-purity helium was used as the carrier gas at a flow rate of 1 mL / min.

[0076] By comparing retention time and fragment ion mass spectrometry with the database of the Complex Carbohydrate Research Center, the glycosidic bond linkage mode was determined. Based on the analysis of the glycosidic bonds, the results are shown in Table 1. HPUP is a type of carbohydrate mainly composed of →6)-Glc p The glucan is composed of -(1→, and also contains β-Glc p -(1→and→4,6)-Glc p -(1→residue), the molar ratios of these bonds are 6.09:86.96:6.85.

[0077] Table 1 Glycosidic bond configuration 2.7 Nuclear Magnetic Resonance Analysis One-dimensional and two-dimensional spectra of HPUP in D2O were analyzed using liquid-state nuclear magnetic resonance spectroscopy. Chemical shifts are expressed in parts per million (ppm). 1 The chemical reference shift for H was set to 4.78 ppm (D₂O). The spectra were analyzed using Top Spin software.

[0078] By comprehensive application 13 C 1 H- 1 The fine structure of HPUP was systematically characterized using HCOSY, HSQC, HMBC, and TOCSY nuclear magnetic resonance techniques. Combined with monosaccharide composition and methylation analysis results, the structure of each sugar residue was further analyzed. 1 H and 13 The C chemical shifts were assigned in detail, and the results are shown in Table 2. 13 C-NMR spectrum ( Figure 8 The results showed that the anomeric carbon signals, ranked from highest to lowest chemical shift, were 95.9 ppm (residue A), 93.2 ppm (residue C), and 92.1 ppm (residue B). These signals were observed in the HSQC spectrum (…). Figure 10 The corresponding cross peaks in the COSY spectrum were 4.56 / 95.9 ppm, 5.15 / 93.2 ppm, and 5.14 / 92.1 ppm, respectively. Subsequently, combined with the COSY spectrum... Figure 9The H1-H2 related peaks (4.56 / 3.18 ppm) and TOCSY ( Figure 12 Based on the chromatogram, the H-2 chemical shift of residue A is estimated to be 3.18 ppm, and the H3-H6 / C3-C6 signals are 3.40 / 75.6 ppm, 3.15 / 73.3 ppm, 3.38 / 75.7 ppm, and 3.65 / 60.8 ppm, respectively. Therefore, residue A is assigned as β-D-Glc. p -(1→).

[0079] Table 2. Sugar residues 1 H and 13 C chemical shift assignment Analysis of the COSY and TOCSY results revealed H2-H6 chemical shifts of residue B to be 3.45 ppm, 3.62 ppm, 3.32 ppm, 3.76 ppm, and 3.33 ppm, respectively. Combined with HSQC spectroscopy, the corresponding C2-C6 chemical shifts were 71.0 ppm, 72.3 ppm, 69.4 ppm, 71.6 ppm, and 69.7 ppm, respectively. The C6 signal showed a significant low-field shift (approximately 10 ppm), indicating substitution at this position. Therefore, residue B is assigned to →6)-α-D-Glc p Similarly, based on the combined analysis of COSY, TOCSY, and HSQC spectra, the H2 / C2-H6 / C6 chemical shifts of residue C are 3.45 / 71.8 ppm, 3.74 / 71.0 ppm, 3.60 / 77.6 ppm, 3.76 / 71.3 ppm, and 3.32 / 70.2 ppm, respectively. Compared with unsubstituted Glc residues, both its C4 and C6 signals are shifted to the lower field by approximately 8-10 ppm, indicating that both C4 and C6 positions of residue C are substituted. Therefore, residue C is assigned as →4,6)-α-D-Glc. p -(1→. Based on HMBC spectrum( Figure 11 The correlation peaks observed in the signal further confirmed the linkage order of residues A and C. The signal AH1-CC4 (4.56 / 77.6 ppm) indicates β-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→connected at C4; signal BH1-BC6 (5.14 / 69.7 ppm) indicates the presence of →)-α-D-Glc p -(1→6)-α-D-Glc p -(1→repeating unit; the related peaks BH1-CC6 (5.14 / 70.2 ppm) and CH1-BC6 (5.15 / 69.7 ppm) indicate →6)-α-D-Glcp -(1→and→4,6)-α-D-Glc p -(1→ interconnected at C6 bit.

[0080] Example 2 The following illustrates the application of the polysaccharides of the present invention for the prevention, treatment or improvement of chronic constipation.

[0081] 1. Experimental Methods 1.1 Establishing a mouse model of chronic constipation Forty male Balb / c mice were acclimatized for 7 days and then randomly divided into five groups of eight each: a blank control group, a loperamide hydrochloride group (model group), a positive control group (Mos 2 mg / kg), a low-dose HPUP group (HPUP 100 mg / kg), and a high-dose HPUP group (HPUP 200 mg / kg). Except for the blank control group, the other groups were administered 10 mg / kg loperamide hydrochloride by gavage to induce constipation for 14 consecutive days. Simultaneously, the HPUP group received HPUP 100 mg / kg or 200 mg / kg daily, the positive control group received mosapride 2 mg / kg, and the control and model groups received the same volume of purified water by gavage. The general physiological condition and fecal consistency of the mice were monitored daily. After the experiment, the mice were euthanized by sodium pentobarbital anesthesia, and samples were collected for further analysis.

[0082] 1.2 Measurement of constipation-related indicators On the last day of the experiment, each mouse was administered 0.2 mL of activated charcoal solution by gavage. Afterward, the mice were placed in clean, individual cages with free access to food and water, and the experimental procedures were performed as described in Example 1.

[0083] 1.3 ELISA method for determination Blood was collected from mice via the orbital sinus and serum was collected by centrifugation (3000 rpm, 10 min, 4℃). The concentrations of substance P (SP), motilin (MTL), gastrin (GAS), vasoactive intestinal peptide (VIP), and serotonin (5-HT) in serum were detected using a double-antibody sandwich assay. Colon tissue was collected, and the expression levels of superoxide dismutase (SOD), glutathione (GSH-px), and malondialdehyde (MDA) were detected according to the manufacturer's instructions.

[0084] 1.4 Histopathological Analysis The duodenum, jejunum, ileum, and colon of mice were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4 μm. Hematoxylin and eosin (H&E) staining was used to assess the degree of lesions. The colon was stained with Alcian blue to observe mucus secretion. After staining, the sections were fixed with neutral resin and air-dried. Quantitative analysis was performed using software.

[0085] 1.5 Immunoblot detection of colon tissue proteins Mouse colon tissue samples were collected, and protein samples were extracted using RIPA lysis. Protein concentration was detected using the BCA method. Protein samples were separated and electrophoresed, and protein bands were transferred to an NC membrane. The membrane was incubated overnight at 4°C with diluted primary antibody on a shaker. After incubation with secondary antibody at room temperature on a shaker for 1 h, protein signals were detected using an enhanced chemiluminescence visualization (ECL) system. The intensity of the protein bands was analyzed using software.

[0086] 1.6 Expression of tight junction proteins in colonic immunofluorescence Mouse colon tissue was fixed in pre-chilled acetone (15 min) and infiltrated with 3% Triton × 100. Sections were blocked with 1% bovine serum albumin at room temperature (30 min). Sections were washed repeatedly with PBS and incubated overnight at 4°C. Primary antibodies included ocludin (1:1000) and claudin-1 (1:1000). The next day, secondary antibodies were added, and the sections were incubated at room temperature for 2 h, followed by staining with 4',6-diamino-2-phenylindole (DAPI) reagent. Imaging was performed using a fluorescence microscope. Images of target protein expression in colon sections were acquired using an optical microscope. Positive regions were analyzed using software.

[0087] 1.7 16S rRNA gene sequencing analysis of cecal contents Nucleic acid was extracted from the contents of the mouse cecum using a kit. Specific primers 338F and 806R for the V3-V4 region of bacterial 16S rRNA were added. The 16S rRNA was sequenced using a sequencing platform.

[0088] 1.8 Determination of Short-Chain Fatty Acid Content in Feces 20 mg of mouse fecal sample was added to 800 μL of a methanol:acetonitrile mixture (1:1), ground, and centrifuged (4000 rpm, 4℃, 10 min). 40 μL of the supernatant was collected, and 20 μL of 200 mM 3-nitrophenylhydrazine (3NPH) was added and mixed. Then, 20 μL of 120 mM (EDC)•HCl-6% pyridine solution was added, and the mixture was shaken at 1200 rpm for 30 min, followed by centrifugation (12000 rpm, 10 min). 150 μL of formic acid-water mixture was added to 50 μL of the supernatant, and the mixture was centrifuged at 12000 rpm for 10 min. After separation, the upper organic phase was collected for subsequent GC-MS analysis. Gas chromatography-mass spectrometry (GC-MS) was performed using an Agilent DB-WAX capillary column (30 m × 0.25 mm ID × 0.25 μm) and a flame ionization detector. The injection volume was 1 μL, and the injection temperature was 250℃.

[0089] 1.9 Data Analysis All data are expressed as mean ± standard deviation (SD). Data analysis and plotting were performed using GraphPad Prism 10.0. Statistical comparisons were performed using one-way ANOVA and Dunnett's post-test; p < 0.05 was considered statistically significant.

[0090] 2. Experimental Results 2.1 HPUP has a relieving effect on constipation in mice. Slow transit constipation clinically manifests as reduced fecal water content and slowed intestinal motility. The incidence of constipation in the model mice was significantly higher than in normal mice, demonstrating the successful establishment of the mouse constipation model. Results are as follows... Figure 13 As shown in the AD diagram, compared with the control group, the baseline fecal parameters of the model group mice were significantly reduced (p < 0.05), while low- and high-dose HPUP and Mos significantly restored related parameters such as small intestinal passage rate, fecal weight, quantity, and water content (p < 0.01). Therefore, HPUP can effectively relieve constipation.

[0091] To elucidate the mechanism by which HPUP promotes gastrointestinal motility and to examine its effect on relieving constipation in mice, this study used mouse serum to measure the levels of key peptides related to intestinal regulation. MTL, GAS, SP, and 5-HT are key gastrointestinal hormones that precisely regulate gastrointestinal motility, digestive fluid secretion, and intestinal barrier function, while VIP is an inhibitory neurotransmitter; its decreased levels can lead to intestinal motility and are one of the important mechanisms inducing constipation. Results are as follows: Figure 13 As shown in the EI, HPUP significantly increased the levels of MTL, SP, 5-HT and GAS in mouse serum, while significantly reducing the VIP level in mice with loperamide hydrochloride-induced constipation (p < 0.05).

[0092] Due to the disruption of the colonic mucosal barrier and the infiltration of inflammatory cells, excessive ROS is produced in the intestine, leading to changes in SOD, GSH-px, and MDA, which impairs the antioxidant mechanism and further aggravates colonic damage. The results are as follows: Figure 13 As shown in the JL, a significant increase in the lipid peroxidation product MDA was observed in the colon of constipated mice, indicating colonic damage. After administration, both the Mos group and the HPUP group significantly reduced the MDA level in the colon of constipated mice, reaching levels almost equivalent to those in the control group. The activities of SOD and GSH-px in constipated mice were significantly inhibited, while the high- and low-dose HPUP groups significantly restored the activities of SOD and GSH-px in the colon (p < 0.05). The results indicate that HPUP can alleviate the degree of colonic mucosal damage by restoring the activity of antioxidant enzymes and reducing oxidative stress. Therefore, HPUP has a significant ameliorative effect on loperamide hydrochloride-induced constipation in mice.

[0093] 2.2 Effects of HPUP on histopathological changes in constipated mice H&E staining histological analysis results are as follows: Figure 14 As shown in A and B, constipation causes damage to intestinal tissues, characterized by inflammatory cell infiltration, villus disorder, and crypt structure destruction. Figure 14 DG analysis showed a significant decrease in small intestinal villi and colonic mucosa in the model group (p < 0.01), but HPUP mitigated this trend. Furthermore, Figure 14 The H-values ​​showed that the therapeutic effect significantly improved with increasing HPUP dosage. HPUP at a dose of 200 mg / kg significantly restored muscle layer thickness (p < 0.0001), significantly reducing colonic muscle thickness in constipated mice. This restoration of muscle thickness improved constipation symptoms in the mice. Goblet cells are a special type of epithelial cell, mainly distributed in the inner lining of the intestine. They secrete a gel-like substance—mucus—forming a natural protective layer on the intestinal surface, playing a crucial role in protecting the intestinal tissue from external stimuli and maintaining intestinal homeostasis. The area of ​​goblet cells in the colon was observed using AB staining, and the results are as follows: Figure 14 As shown in C, the quantitative results are as follows: Figure 14 As shown in Figure I, the relative proportion of goblet cells in the model group mice was significantly decreased, while the relative proportion of goblet cells in the HPUP group mice was significantly increased. The results indicate that HPUP has a significant restorative effect on pathological tissue damage in the small intestine and colon of mice with loperamide hydrochloride-induced constipation.

[0094] 2.3 Enhancement of tight junction proteins in the colonic barrier by HPUP in constipated mice Tight junction proteins, as important components of the intestinal barrier, can form specialized complexes between adjacent epithelial cells to construct and maintain a barrier structure with selective permeability. This structure can prevent intestinal inflammation and infection, strengthen the intestinal barrier, and thus alleviate gastrointestinal damage caused by constipation. First, the expression levels of tight junction proteins were analyzed, and the results are as follows: Figure 15 As shown in the AC results, the expression levels of closure protein and sealing protein-1 in the colon tissue of model group mice were significantly decreased in WB and RT-qPCR (p < 0.05), while HPUP could restore their expression levels. Immunofluorescence staining results ( Figure 15 The results were further confirmed by the DG, indicating that HPUP can enhance the intestinal barrier and thus alleviate intestinal tissue damage induced by loperamide hydrochloride in mice with constipation.

[0095] 2.4 Effects of HPUP on intestinal microbial diversity, composition, and short-chain fatty acids in constipated mice To further investigate the regulatory effect of HPUP on the gut microbiota imbalance induced by loperamide hydrochloride-induced constipation in mice, 16S rRNA sequencing was used to determine gut microbial diversity and study changes in gut microbiota after HPUP treatment. First, the sparse curves and rank-abundance curves of each sample group were analyzed. The sparse curves confirmed sufficient sequencing depth and coverage, while the rank-abundance curves confirmed that most microbial diversity was captured (e.g., ...). Figure 16 As shown in A and B), the number of species and the size of the sequencing samples in each gut microbiota group are sufficient to support further analysis. Further analysis of the alpha diversity of the samples included the Chao1 index, Simpson index, Shannon index, and Observed_species index. Figure 16 The results of CF analysis showed that phenobarbital hydrochloride (HPUP) reduced the abundance and diversity of the gut microbiota, while HPUP treatment significantly increased α-diversity of the gut microbiota (p < 0.05), indicating that HPUP has a significant protective effect on microbial diversity. Further analysis using PCoA, PCA, and NMDS revealed... Figure 16 The GI results showed that the gut microbiota composition of the control and HPUP groups was significantly different from that of the model group, indicating that the microbial community structure of constipated mice had changed. Notably, HPUP treatment reversed these changes, restoring them to normal. Analysis of the samples using Venn diagrams yielded the following results: Figure 16 As shown in Figure J, there were unique or shared gut microbiota among the sample groups. A total of 536 operating units (OTUs) were found across the three groups. The control group had 1489 unique OTUs, the model group had 1017 unique OTUs, and the HPUP group had 1413 unique OTUs. This indicates that loperamide hydrochloride altered the composition of OTUs, while HPUP was able to restore them to near-normal control levels.

[0096] Further in-depth analysis was conducted on the changing trends of the abundance of gut microbiota in mice. Figure 17 The results showed that, at the phylum level, community abundance analysis revealed that the model group had an increased relative abundance of Firmicutes while a decreased relative abundance of Bacteroidetes, and HPUP treatment reversed the effects of loperamide hydrochloride on these species. Correspondingly, the increased Firmicutes / Bacteroidetes (F / B) ratio was also alleviated by HPUP treatment. Figure 17B showed that examining the abundance of gut microbiota at the genus level, the model group increased the abundance of harmful genera such as *Desulfovibrio*, disrupting the dynamic homeostasis of the intestinal mucosa. Simultaneously, it reduced beneficial taxa, such as *Lactobacillus*, *Lawsonia*, and *Prevotella*, which are associated with SCFAs and capable of immunomodulation and intestinal barrier maintenance. HPUP intervention effectively reversed this dysbiosis trend. Short-chain fatty acids (SCFAs) are the main metabolites produced by gut probiotics fermenting indigestible carbohydrates. They play a crucial role in reducing inflammation and maintaining intestinal barrier integrity. The results of SCFA detection in feces are as follows: Figure 17 As shown in Figure C, HPUP treatment effectively reversed the defects induced by loperamide hydrochloride. Compared with the model group, the concentrations of acetic acid (p < 0.001), propionic acid (p < 0.01), butyric acid (p < 0.0001), isobutyric acid (p < 0.01), valeric acid (p < 0.001), and isovaleric acid (p < 0.01) in the feces of the HPUP group were significantly increased, approaching those of the control group. These results indicate that HPUP may enhance gastrointestinal motility in constipated mice by promoting the synthesis of acetic acid, valeric acid, and butyric acid. In conclusion, HPUP treatment can significantly restore the damage to the intestinal microbiota induced by loperamide hydrochloride in mice, and may help alleviate intestinal inflammation and protect the intestinal barrier by restoring the diversity of intestinal microbiota, thereby achieving the therapeutic effect on constipated mice and supporting the diversity of microbiota and the energy supply of intestinal microbiota.

[0097] 2.5 Relationship between characteristic bacteria and constipation detection indicators Spearman correlation analysis was used to analyze the relationship between gut microbiota and constipation-related indicators. The results showed that the relative abundance of *Prevotella*, *Dunaliella*, and *Lactobacillus* was positively correlated with various beneficial metabolites such as acetic acid, propionic acid, butyric acid, and isobutyric acid, while negatively correlated with VIP and MDA. In this study, HPUP intervention increased the levels of SCFAs in mouse feces, particularly acetic acid, propionic acid, and butyric acid, which was associated with higher abundance of *Prevotella*, *Dunaliella*, and *Lactobacillus*. Therefore, gut microbiota interactions can alter their related metabolites. Correlation analysis also showed that the relative abundance of *Lactobacillus* and *Prevotella* was positively correlated with closure protein, sealing protein-1, gastrointestinal hormones MTL, SP, GAS, and 5-HT, and oxidative stress indicators SOD and GSH-px. These findings indicate that HPUP intervention effectively restored loperamide hydrochloride-induced constipation in mice, mainly by improving the composition of the gut microbiota. In addition, HPUP can maintain the intestinal barrier and resist oxidative stress, thus having a dual regulatory effect.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of polysaccharides in the preparation of remedies for the prevention, treatment, or improvement of chronic constipation, characterized in that, The polysaccharide backbone has →6)-α-D-Glc p -(1→and→4,6)-α-D-Glc p -(1→ glycosidic bond, terminal β-D-Glc p -(1→through→4,6)-α-D-Glc p -(1→ is connected to the main chain, and the monosaccharide composition of the polysaccharide contains more than 97% glucose and less than 3% galactose, fructose, mannose and arabinose, and the polysaccharide has the structure shown in Formula I: Equation I; Where n is selected from natural numbers from 2 to 20; The polysaccharide is prepared by the following method: (1) Extract dragon fruit pulp with an aqueous solvent at 60-100℃ for 30-120 min to obtain an extract. Add a precipitant to precipitate the extract and remove the protein from the precipitate to obtain crude polysaccharide. (2) The crude polysaccharide is separated and purified to obtain the polysaccharide.

2. The use of the polysaccharide according to claim 1 in the preparation of a medicament for the prevention, treatment, or improvement of chronic constipation, characterized in that, The application includes at least one of the following scenarios: (1) Improve or restore the integrity of the intestinal barrier; (2) Increase the expression level of colonic tight junction protein; (3) Regulate the proportion of beneficial gut microbiota; (4) Increase the content of short-chain fatty acids; (5) Improves gastrointestinal motility; (6) Regulate intestinal oxidative stress.

3. The use of the polysaccharide according to claim 1 in the preparation of a medicament for the prevention, treatment or improvement of chronic constipation, wherein the ratio of the dragon fruit pulp to the aqueous solvent is 1:(2-10) g / mL.

4. The use of the polysaccharide according to claim 1 in the preparation of a medicament for the prevention, treatment, or improvement of chronic constipation, characterized in that, Proteins in the precipitate were removed using the Sevag method.

5. The use of the polysaccharide according to claim 1 in the preparation of a medicament for the prevention, treatment, or improvement of chronic constipation, characterized in that, The separation and purification include ion exchange column chromatography and non-ionic gel chromatography.

6. The use of the polysaccharide according to claim 1 in the preparation of a medicament for the prevention, treatment, or improvement of chronic constipation, characterized in that, The separation and purification process includes: The crude polysaccharide was separated by ion exchange column chromatography, and the elution fraction corresponding to the first peak was collected. Using water as the mobile phase, the elution fraction corresponding to the first peak is passed through a non-ionic gel filtration column to obtain the polysaccharide.

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