Rosa roxburghii tratt residue pectin polysaccharide as well as preparation method and application thereof

Abstract

The invention provides the roxburgh rose residue pectin polysaccharide. The roxburgh rose residue pectin polysaccharide prepared by the method is high in purity and relatively uniform in molecular weight distribution. The invention further provides a preparation method and application of the roxburgh rose residue pectin polysaccharide. The roxburgh rose residue pectin polysaccharide is structurally modified through a low-temperature controllable alkaline degreasing technology, a targeted enzyme digestion technology and a controllable partial acid hydrolysis technology, the structural characteristic change of the roxburgh rose residue pectin polysaccharide before and after modification is compared, and the effect of the roxburgh rose residue pectin polysaccharide and the modified product thereof on improving mouse ulcerative colitis is provided. The modified rosa roxburghii tratt fruit residue pectin polysaccharide has a better improvement effect on the mouse ulcerative colitis, and especially has the best improvement effect on the rosa roxburghii tratt fruit residue pectin polysaccharide prepared by a targeted enzyme digestion technology.

Description

Technical Field

[0001] The invention relates to a roxburghii pomace pectin polysaccharide, a preparation method and application thereof. Background Art

[0002] Rosa roxburghii, also known as silk-reeling flower, is a plant of the genus Rosa in the Rosaceae family. It is widely distributed in the mountainous areas of southwestern China, particularly in Guizhou Province. Rosa roxburghii is a spreading shrub, growing from 1 to 2.5 meters tall. Its leaves are odd-pinnately compound with prickly petioles. The flowers are pale red or pink, and the fruit is densely covered with thorns, hence the name "thorny pineapple." Rosa roxburghii fruit is rich in functional nutrients such as polysaccharides, flavonoids, vitamin C, organic acids, and minerals. The "Compendium of Materia Medica" records that Rosa roxburghii fruit has stomachic, digestive, and antidiarrheal properties, and is primarily used to treat food stagnation, bloating, and enteritis-induced diarrhea. Polysaccharides are one of the main active ingredients in Rosa roxburghii fruit. Numerous studies have demonstrated that Rosa roxburghii fruit polysaccharides exhibit significant in vitro antioxidant, hypoglycemic, antitumor, and anti-inflammatory activities, as well as intestinal microbial regulation. Notably, Rosa roxburghii fruit polysaccharides can repair intestinal barrier damage and alleviate inflammation by inhibiting the endotoxin-Toll-like receptor 4-nuclear factor κB signaling pathway, suggesting potential therapeutic benefits for inflammatory bowel disease and other inflammatory-related diseases. Sea buckthorn residue is the main processing by-product produced in the production of sea buckthorn juice, and these sea buckthorn residue processing by-products account for about 40% to 50% of the total fruit mass. Due to the rough texture and poor taste of these sea buckthorn residue processing by-products, they are often discarded directly, which will lead to a large amount of resource waste and a potential burden on the environment. Research results show that compared with sea buckthorn fruit, sea buckthorn residue also has extremely high nutritional and health value. Among raw materials of equal quality, the content of active polysaccharides contained in sea buckthorn residue is even higher than that of sea buckthorn fruit. Application number 202211191416.7, invention name: Method and system for simultaneous extraction of sea buckthorn polysaccharides, sea buckthorn polyphenols, sea buckthorn SOD and Vc from sea buckthorn residue. This patent provides a method and system for simultaneous extraction of sea buckthorn polysaccharides, sea buckthorn polyphenols, sea buckthorn SOD and Vc from sea buckthorn residue. The present invention only measures the yield of sea buckthorn crude polysaccharides during the extraction process. The literature (Zhu Jiaxi, Chen Zhenghao, Li Guangjiang, Qiu Shuyi, Luo You, Song Angxin, Study on Ultrasonic-Assisted Extraction of Seabuckthorn Pear Residue Polysaccharides and Their Antioxidant Activity, Journal of Food Safety and Quality, 2024, 15(12):81-88) reported the effect of ultrasonic-assisted extraction technology on the structure and in vitro antioxidant activity of crude polysaccharides from seabuckthorn pomace (total polysaccharide content is 63.99% to 77.18%, and the molecular weight distribution diagram shows multiple different molecular weight components). However, there is currently no research on high-purity seabuckthorn pomace pectin polysaccharides and their related structural modifications, and there is no relevant report on the in vivo health effects of seabuckthorn pomace pectin polysaccharides. Therefore, there is an urgent need to clarify the structural characteristics and in vivo health effects of seabuckthorn pomace pectin polysaccharides, so as to promote the application of seabuckthorn pomace pectin polysaccharides in the field of medicine and health, and enhance the high-value utilization of seabuckthorn pomace by-products.

[0003] Ulcerative colitis (UC) is a bowel disease characterized by weight loss, bloody stools, diarrhea, intestinal mucosal damage, colonic ulceration, and severe inflammation. Its pathogenesis is multifactorial, involving unhealthy dietary habits, genetic factors, epithelial barrier impairment, immune dysregulation, and intestinal microbial imbalance. While the disease is primarily chronic and generally less aggressive, its increasing incidence places a significant economic burden on patients and healthcare systems. Currently, drug therapy is the primary intervention for UC, with clinicians selecting appropriate medications based on disease severity. Commonly used medications in clinical treatment can be categorized into the following categories: aminosalicylic acid compounds, corticosteroids, immunosuppressants, biologics, probiotics, and traditional Chinese medicines. These drugs play a crucial role in disease management. While drug therapy can effectively alleviate symptoms, it is associated with high relapse rates and adverse drug reactions. Therefore, the development of natural, safe, and effective treatment options, such as the use of natural product polysaccharides, is crucial. Studies have shown that natural product polysaccharides have advantages in the treatment of UC, such as good safety, definite efficacy, and few adverse reactions. Their mechanisms of action involve multiple aspects such as immune cell regulation, signal transduction intervention, regulation of inflammatory factor secretion, intestinal flora balance, and mucosal barrier protection. At the same time, the combined use of natural product polysaccharides with other drugs has become an important direction of current research. Natural product polysaccharides are mainly decomposed in the intestine by polysaccharide-degrading enzymes secreted by intestinal flora, showing potential prebiotic activity in both non-disease and disease states, especially in the treatment or prevention of intestinal diseases and the promotion of intestinal health. Studies have shown that the molecular weight, monosaccharide composition, uronic acid content, glycosidic bond type, etc. of natural product polysaccharides will affect their anti-inflammatory activity. Studies have shown that rosa roxburghii fruit polysaccharides have potential therapeutic effects on inflammatory bowel disease (Wang Lei, Zhang Pan, Li Chao, Xu Fei, Chen Jie, A polysaccharide from Rosa roxburghii Trattfruit attenuates high-fat diet-induced intestinal barrier dysfunction and inflammation in mice by modulating the gut microbiota, Food & Function, 2022, 13: 530-547). However, there are currently no reports on the effect of rosa roxburghii pectin polysaccharides in improving ulcerative colitis, and their structure-activity relationship is still unclear, which leads to the extremely low utilization rate of rosa roxburghii pectin polysaccharides in related products in the field of medicine and health. Summary of the Invention

[0004] The invention provides a roxburghii pear residue pectin polysaccharide and also provides a preparation method and application of the roxburghii pear residue pectin polysaccharide.

[0005] The present invention provides a pectin polysaccharide from roxburghii pear residue. The pectin polysaccharide prepared by the present invention has high purity and relatively uniform molecular weight distribution. Each 100 mg of pectin polysaccharide from roxburghii pear residue contains: 90.19 mg ± 1.67 mg–91.74 mg ± 0.18 mg of total polysaccharide; 24.23 mg ± 1.82 mg–73.53 mg ± 2.11 mg of total uronic acid; and 1.73 mg GAE ± 0.01 mg GAE–3.66 mg GAE ± 0.09 mg GAE (gallic acid equivalent). The degree of esterification of the pectin polysaccharide from roxburghii pear residue is 17.96% ± 0.55%–56.38% ± 0.88%, and the molecular weight is (0.811 ± 0.026) × 10 4 Da–(4.641±0.043)×10 4 The molar proportion of Da, galacturonan (HG) is 20.83mol%-68.39mol%, the molar proportion of type I rhamnogalacturonan (RG-I) is 24.00mol%-69.52mol%, and the side chain length of RG-I is 1.00-11.22.

[0006] The molar percentages of each monosaccharide in the pectin polysaccharides from sea buckthorn residue are as follows: galacturonic acid (GalA) 28.99 mol%-76.40 mol%, galactose (Gal) 7.99 mol%-25.96 mol%, arabinose (Ara) 0.00 mol%-27.25 mol%, rhamnose (Rha) 4.24 mol%-8.15 mol%, glucose (Glc) 3.02 mol%-5.38 mol%, mannose (Man) 1.64 mol%-3.17 mol%, glucuronic acid (GlcA) 1.36 mol%-2.46 mol%, and xylose (Xyl) 0.00 mol%-0.94 mol%.

[0007] The roxburgh pear residue pectin polysaccharide of the present invention is prepared by adopting a low eutectic solvent-assisted extraction technology, and then the roxburgh pear residue pectin polysaccharide is modified by a low-temperature controllable alkaline deesterification technology, a targeted enzyme cleavage technology or a controllable partial acid hydrolysis technology to obtain the roxburgh pear residue modified pectin polysaccharide.

[0008] The present invention also provides a method for preparing the roxburghii pear residue pectin polysaccharide, which comprises the following steps:

[0009] a. Preparation of roxburghii pectin polysaccharide RPDP by deep eutectic solvent-assisted extraction technology;

[0010] b. Take the roxburghii pear residue pectin polysaccharide RPDP prepared in step a and prepare the roxburghii pear residue modified pectin polysaccharide RPDP-E by using targeted enzymatic cleavage technology;

[0011] or,

[0012] c. Taking the pear residue pectin polysaccharide RPDP prepared in step a, a low-temperature controllable alkaline deesterification technology was used to prepare a pear residue modified pectin polysaccharide PRDP-A;

[0013] or,

[0014] d. Take the roxburghii residue pectin polysaccharide RPDP prepared in step a and use controlled partial acid hydrolysis technology to prepare roxburghii residue-modified pectin polysaccharide RPDP-T.

[0015] Wherein, the preparation method of the deep eutectic solvent-assisted extraction technology in step a is:

[0016] The prickly pear residue powder was mixed with 80% ethanol at a ratio of 1:10 (w / v), and ultrasonically treated in an ultrasonic cleaning tank at 480W and 25°C; after ultrasonic treatment, the mixture was centrifuged at 5000×g for 10 min, and the precipitate was mixed with a low eutectic solvent at a ratio of 1:40 (w / v), wherein the low eutectic solvent consisted of choline chloride, ethylene glycol and ultrapure water, and the molar ratio of choline chloride to ethylene glycol was 1:3, and the water content of the low eutectic solvent was 55% (v / v). The polysaccharide was extracted at 90°C for 3h; after extraction, the mixture was centrifuged at 4000×g for 15min, and the above mixture was separated. The supernatant was concentrated using a rotary evaporator, and the concentrate was sequentially added with heat-stable α-amylase (5U / mL), saccharifying enzyme (5U / mL), and pancreatin (5U / mL) to remove starch, dextrin, and protein. The supernatant was then inactivated at 95°C for 30 minutes, centrifuged (4000×g, 15 minutes), and 4 volumes of 95% ethanol (v / v) were added. The supernatant was precipitated overnight at 4°C. The precipitate was reconstituted with ultrapure water and separated by 3kDa and 100kDa ultrafiltration centrifuge tubes, respectively. A polysaccharide fraction with a molecular weight between 3kDa and 100kDa was finally obtained. The purified polysaccharide fraction was then freeze-dried in a vacuum at -80°C for 48 hours to obtain a high-purity pectin polysaccharide from sea pear residue, named RPDP.

[0017] The targeted enzyme cleavage technology described in step b is specifically:

[0018] The pectin polysaccharide RPDP solution from roxburghii pear residue was mixed with a pectinase solution and then reacted at 40°C for 9 hours. After the reaction, the enzyme was inactivated at 95°C for 30 minutes. The upper layer was then collected by ultrafiltration using a 3kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain a pectin polysaccharide modified with roxburghii pear residue, named RPDP-E.

[0019] The low-temperature controllable alkaline deesterification technology described in step c is specifically:

[0020] The RPDP solution of pectin polysaccharide from roxburghii pear residue was mixed with a NaOH solution (the final pH value of the mixed solution was 11), and then stirred at 4°C for 30 minutes. After the reaction, HCl solution (1M) was added dropwise to the mixed solution until the mixed solution was neutral (pH = 7). Finally, the upper layer was collected by ultrafiltration using a 3kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain the pectin polysaccharide modified from roxburghii pear residue, named PRDP-A.

[0021] The controllable partial acid hydrolysis technology described in step d is specifically:

[0022] The RPDP solution of pectin polysaccharide from roxburghii pear residue was mixed with a TFA solution (final TFA concentration of 0.25 M) and then reacted at 95°C for 180 minutes. After the reaction, a 1M NaOH solution was added dropwise to the mixed solution until the mixed solution was neutral (pH = 7). Finally, the upper layer was collected by ultrafiltration using a 3kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain a pectin polysaccharide modified from roxburghii pear residue, named RPDP-T.

[0023] The invention provides a pharmaceutical composition for treating or preventing ulcerative colitis. The pharmaceutical composition contains the roxburghii pear residue pectin polysaccharide as an active ingredient and is added with pharmaceutically acceptable auxiliary ingredients to prepare a commonly used pharmaceutical preparation.

[0024] Wherein, the preparation is an oral preparation or a colon administration preparation.

[0025] The present invention structurally modifies roxburgh pear residue pectin polysaccharides through a low-temperature controllable alkaline deesterification technology, a targeted enzyme cleavage technology, and a controllable partial acid hydrolysis technology. The prepared pectin polysaccharides have a uniform molecular weight distribution. The structural characteristics of the roxburgh pear residue pectin polysaccharides before and after modification are compared. The effects of the roxburgh pear residue pectin polysaccharides and their modified products on improving ulcerative colitis in mice are compared, indicating that the activity of the roxburgh pear residue-modified pectin polysaccharides in improving ulcerative colitis is significantly enhanced, and in particular, the roxburgh pear residue-modified pectin polysaccharides prepared by the targeted enzyme cleavage technology have the best improvement effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 High-performance size exclusion chromatograms of RPDP, RPDP-T, RPDP-E, and RPDP-A (Note: RPDP, RPDP-E, RPDP-A, and RPDP-T are the products of deep eutectic solvent-assisted extraction of pectin polysaccharides from roxburghii pear residue, the product modified by targeted enzymatic cleavage, the product modified by low-temperature controlled alkaline deesterification, and the product modified by controlled partial acid hydrolysis, respectively);

[0027] Figure 2HPLC chromatograms of monosaccharide compositions of RPDP, RPDP-T, RPDP-E, and RPDP-A (Note: RPDP, RPDP-E, RPDP-A, and RPDP-T are the products of deep eutectic solvent-assisted extraction of pectin polysaccharides from roxburghii pear residue, the products modified by targeted enzymatic cleavage, the products modified by low-temperature controlled alkaline deesterification, and the products modified by controlled partial acid hydrolysis, respectively);

[0028] Figure 3 Fourier transform infrared spectra of RPDP, RPDP-E, RPDP-A, and RPDP-T (Note: RPDP, RPDP-E, RPDP-A, and RPDP-T are the products of deep eutectic solvent-assisted extraction of pectin polysaccharides from roxburghii pear residue, the products modified by targeted enzymatic cleavage technology, the products modified by low-temperature controllable alkaline deesterification technology, and the products modified by controlled partial acid hydrolysis technology, respectively);

[0029] Figure 4 RPDP, RPDP-E, RPDP-A and RPDP-T 1 H NMR (left) and 13 C NMR spectra (right) (Note) RPDP, RPDP-E, RPDP-A, and RPDP-T represent the products of deep eutectic solvent-assisted extraction of pectin polysaccharides from roxburghii pear residue, the products modified by targeted enzymatic cleavage, the products modified by low-temperature controlled alkaline deesterification, and the products modified by controlled partial acid hydrolysis, respectively.

[0030] Figure 5 Modeling process of DSS-induced ulcerative colitis mice;

[0031] Figure 6 Body weight changes (A) and DAI scores (B) of mice (Note) Experimental results are expressed as mean values. Significance analysis, *p < 0.05, **p < 0.01;

[0032] Figure 7 Representative images of mouse colon (A) and statistical histogram of colon length (B) (Note) af indicates significant differences among the Control group, DSS group, ASA group, RPDP group, RPDP-T group, RPDP-E group, and RPDP-A group (p < 0.05);

[0033] Figure 8 H&E staining, PAS staining, PSR staining (A), H&E staining histological score (B), PAS positive area statistical histogram (C), PSR fibrosis area statistical histogram (D) (Note) ae indicates significant differences among the Control group, DSS group, ASA group, RPDP group, RPDP-T group, RPDP-E group, and RPDP-A group (p < 0.05).

[0034] Figure 9 Immunofluorescence analysis of ZO-1 and occludin (A), statistical histogram of ZO-1 fluorescence area (B), statistical histogram of occludin fluorescence area (C) (Note) af indicates significant differences among the Control group, DSS group, ASA group, RPDP group, RPDP-T group, RPDP-E group, and RPDP-A group (p < 0.05);

[0035] Figure 10 The expression levels of LPS (A) and cytokines IL-10 (B), IL-6 (C), IL-1β (D), and TNF-α (E) (Note) ag indicates significant differences among the Control group, DSS group, ASA group, RPDP group, RPDP-T group, RPDP-E group, and RPDP-A group (p < 0.05).

[0036] Figure 11 Concentrations of acetic acid (A), propionic acid (B), butyric acid (C), and total short-chain fatty acids (D) (Note) af indicates significant differences among the Control group, DSS group, ASA group, RPDP group, RPDP-T group, RPDP-E group, and RPDP-A group (p < 0.05).

[0037] Figure 12 PCoA (A) and cluster (B) analysis of mouse intestinal flora;

[0038] Figure 13 Abundance histograms of the mouse intestinal flora at the phylum level (A) and genus level (B), as well as histograms of individual phyla (C) and individual bacteria (D) (Note) 1a-g indicate significant differences among the control group, DSS group, ASA group, RPDP group, RPDP-T group, RPDP-E group, and RPDP-A group (p < 0.05).

[0039] Figure 14 Correlation analysis heat map (Note: Significant differences in the correlation between indicators are indicated by *p < 0.05 and **p < 0.01). DETAILED DESCRIPTION

[0040] Example 1 Preparation and structural characterization of roxburghii residue pectin polysaccharides and their modified products

[0041] 1 Experimental Materials and Reagents

[0042] 1.1 Experimental Materials

[0043] The roxburghii residue used in this experiment is a processing byproduct (roxburghii residue) produced by roxburghii juice production enterprises during the processing of roxburghii fruit. The roxburghii residue was freeze-dried, powdered, and sieved (60 mesh) to prepare roxburghii residue powder.

[0044] 2 Experimental methods

[0045] 2.1 Preparation of pectin polysaccharides from roxburghii residue using deep eutectic solvent-assisted extraction technology

[0046] 10 g of roxburghii pear residue powder was mixed with 80% ethanol at a 1:10 (w / v) ratio and sonicated in an ultrasonic bath at 480W and 25°C for 30 minutes to remove alcohol-soluble components. After sonication, the mixture was centrifuged at 5000×g for 10 minutes. The precipitate was then mixed with a 1:40 (w / v) deep eutectic solvent (choline chloride, ethylene glycol, and ultrapure water, with a molar ratio of choline chloride to ethylene glycol of 1:3) with a 55% water content. Polysaccharides were extracted at 90°C for 3 hours. After extraction, the mixture was centrifuged at 4000×g for 15 minutes, and the supernatant was rotary evaporated to approximately 100 mL. A thermostable α-amylase (5 U / mL), a saccharifying enzyme (5 U / mL), and a pancreatin (5 U / mL) were added to the concentrate to remove starch, dextrin, and protein. The supernatant was then inactivated at 95°C for 30 minutes and centrifuged (4000×g for 15 minutes). Four volumes of 95% ethanol (v / v) were added and the mixture was precipitated overnight at 4°C. The precipitate was reconstituted with ultrapure water and separated through 3kDa and 100kDa ultrafiltration centrifuge tubes, respectively, to obtain polysaccharide fractions with molecular weights between 3kDa and 100kDa. The purified polysaccharide fractions were then freeze-dried at -80°C for 48 hours to obtain a high-purity pectin-based polysaccharide from the prickly pear residue, designated RPDP.

[0047] 2.2 Preparation of pectin-modified polysaccharides from roxburghii residue using targeted enzymatic digestion technology

[0048] A 5.0 mg / mL solution of pectin polysaccharide from roxburghii pear residue was mixed with a 0.3 U / mL pectinase solution and then reacted at 40°C for 9 hours. After the reaction, the enzyme was inactivated at 95°C for 30 minutes. The supernatant was then collected by ultrafiltration using a 3 kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain a modified pectin polysaccharide from roxburghii pear residue, named RPDP-E.

[0049] 2.3 Preparation of pectin-modified polysaccharides from roxburghii residue using low-temperature controlled alkaline deesterification technology

[0050] A pectin polysaccharide solution (5 mg / mL) from roxburghii pear residue was mixed with a NaOH solution (the final pH of the mixed solution was 11), and then stirred at 4°C for 30 minutes. After the reaction, a 1M HCl solution was added dropwise to the mixed solution until the mixed solution became neutral (pH = 7). Finally, the upper layer was collected by ultrafiltration using a 3kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain a pectin polysaccharide modified from roxburghii pear residue, named PRDP-A.

[0051] 2.4 Preparation of pectin-modified polysaccharides from roxburghii residue using controlled partial acid hydrolysis technology

[0052] A 5.0 mg / mL solution of pectin polysaccharide from roxburghii pear residue was mixed with a 0.5 M TFA solution (final TFA concentration of 0.25 M) and then reacted at 95°C for 180 minutes. After the reaction, a 1 M NaOH solution was added dropwise to the mixed solution until the mixed solution was neutral (pH = 7). Finally, the upper layer was collected by ultrafiltration using a 3 kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain a pectin polysaccharide modified from roxburghii pear residue, named RPDP-T.

[0053] 2.5 Chemical composition and structural analysis of pectin polysaccharides from roxburghii residue and their modified products

[0054] (1) Chemical composition analysis

[0055] The total polysaccharide content was determined by the phenol-sulfuric acid method. The standard curve for the determination of total polysaccharide content was prepared using a mixed standard solution of galacturonic acid and galactose. The sample to be tested was diluted to an appropriate concentration with ultrapure water. 400 μL of the sample to be tested was added with 200 μL of 6% phenol, shaken well, and then 1 mL of concentrated sulfuric acid was added. The mixture was incubated at 90°C for 10 minutes. After cooling, the absorbance was measured at 490 nm.

[0056] The total uronic acid content was determined by the m-hydroxybiphenyl method. A standard curve was prepared using galacturonic acid as the standard. 100 μL of the sample solution to be tested was mixed with 1 mL of sodium tetraborate sulfuric acid solution (12.5 mM). The mixture was incubated in a 95°C water bath for 10 min. After the reaction was complete, the mixture was rapidly cooled to room temperature. 20 μL of 3-phenylphenol solution (0.15%, w / v) was added and mixed. The mixture was reacted at room temperature for 15 min, and the absorbance was measured at 520 nm.

[0057] Total protein content was determined using the Coomassie Brilliant Blue method. Using bovine serum albumin (BSA) as the standard, the sample was prepared at 2 mg / mL. 200 μL of the sample was added to 1 mL of Coomassie Brilliant Blue solution, shaken, and allowed to stand for 10 minutes. The absorbance was then measured at 595 nm.

[0058] The total bound phenol content was determined using the folin-phenol method, with gallic acid as the standard to generate a standard curve. 50 μL of the sample solution was mixed with 250 μL of a 0.2 M folin-phenol solution and allowed to react for 3 minutes. Then, 250 μL of a 20% w / v sodium carbonate solution was added and the mixture was incubated in the dark for 30 minutes. The absorbance was measured at 760 nm.

[0059] (2) Molecular weight determination

[0060] The molecular weights (M) of pectin polysaccharides from roxburghii pear residue and their modified products were determined by high performance size exclusion chromatography coupled with multi-angle laser scattering and differential detection (HPSEC-MALLS-RID). w ) and polydispersity (M w / M n The sample was prepared at a concentration of 1.0 mg / mL, and the injection volume was 100 μL. The sample was separated using a Shodex OHpak SB-806M HQ column (300 mm × 8.9 mm, ID) with a 0.9% NaCl aqueous solution as the mobile phase at a flow rate of 0.5 mL / min. A dn / dc value of 0.15 mL / g was selected, and data were acquired and analyzed using ASTRA Version 7.1.3.

[0061] (3) Determination of sugar composition

[0062] High-performance liquid chromatography (HPLC) combined with pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) was used to determine the compositional sugar types of pectin polysaccharides from roxburghii pear residue and their modified products. First, 5.0 mg of the sample was accurately weighed and hydrolyzed with 2.0 mL of 4.0 M trifluoroacetic acid at 95°C for 8 h. The hydrolyzed sample was then evaporated to dryness in a rotary evaporator under vacuum and washed three times with methanol to remove residual trifluoroacetic acid. The dried hydrolyzate was then dissolved in 1 mL of water and derivatized. Then, 100 μL of the hydrolyzate was mixed with 100 μL of 0.6 M sodium hydroxide solution and 100 μL of 0.5 M PMP methanol solution. The mixture was vortexed and reacted at 70°C for 100 min. After the reaction is completed, 100 μL of 0.3 M hydrochloric acid solution is used to further neutralize the mixture and diluted to 1 mL with ultrapure water. 1 mL of chloroform is added to the mixture, shaken vigorously, and after standing to separate the layers, the organic layer is removed. This process is repeated 3 times. Finally, the mixture is passed through a 0.22 μm organic filter membrane and further analyzed by high-performance liquid chromatography. Among them, eight standard substances including glucuronic acid, galacturonic acid, glucose, mannose, galactose, arabinose, rhamnose, and xylose are mixed in proportion to prepare a mixed standard. 20 μL of PMP derivative was injected into the high performance liquid chromatography system. The chromatographic column was an Agilent ZORBAX Eclipse XDB-C18 column (250×4.6 mm, id.5 μm), 0.1 M phosphate buffer (pH=6.7) and acetonitrile (82:18, v / v) were used as the eluent. The operating temperature was 30°C, the flow rate was 1.0 mL / min, and the detection wavelength of DAD was 245 nm.

[0063] (4) Infrared spectroscopy

[0064] The infrared spectral characteristics of pectin polysaccharides from roxburghii pear residue and their modified products were detected by Fourier transform infrared spectroscopy (FT-IR). The sample to be tested was mixed with dry potassium bromide in a mass ratio of 1:80, ground and pressed into tablets, and the infrared spectrometer at 4000-400 cm -1 The absorption value of the frequency range is 1700~1750cm -1 (esterified aldehyde) and 1600-1630cm -1 The degree of esterification of the sample to be tested was calculated based on the free aldehyde acid.

[0065] The formula for calculating the degree of esterification is as follows:

[0066]

[0067] (5) Nuclear magnetic resonance spectroscopy analysis

[0068] 40 mg of the dried sample to be tested was dissolved in 1 mL of D2O, and the proton and carbon frequencies were set to 600.13 and 150.90 Hz, respectively, and the spectrometer was operated on a Bruker AVIII-600 NMR spectrometer at 25 °C. 1 H NMR and 13 C NMR spectra were collected. Finally, the data were analyzed using MestReNova-6.1.1.

[0069] 3 Results and Analysis

[0070] 3.1 Chemical composition of pectin polysaccharides from roxburghii residue and their modified products

[0071] Table 1 summarizes the chemical compositions of pectin polysaccharides from roxburghii pear residue and their modified products. The results showed that the chemical compositions of their modified products (RPDP-E, RPDP-A, and RPDP-T) varied somewhat compared to the original pectin polysaccharide RPDP. Specifically, the total polysaccharide contents of pectin polysaccharides from roxburghii pear residue and their modified products were relatively stable, with the total polysaccharide contents of RPDP, RPDP-E, RPDP-A, and RPDP-T being 91.08 ± 0.9 mg / 100 mg, 91.74 ± 0.18 mg / 100 mg, 90.96 ± 0.64 mg / 100 mg, and 90.19 ± 1.67 mg / 100 mg, respectively. This indicates that pectin polysaccharides from roxburghii pear residue and their modified products possess extremely high polysaccharide purity. However, compared with RPDP, the uronic acid content of RPDP-E and RPDP-A decreased from 37.89±1.86 mg / 100 mg to 35.73±1.54 mg / 100 mg and 24.23±1.82 mg / 100 mg, respectively. This indicates that degradation by pectinase reduced the uronic acid content. Furthermore, the β-elimination reaction during alkaline deesterification also destroyed the HG backbone, leading to a decrease in uronic acid content. Compared with RPDP, the uronic acid content of RPDP-T increased significantly to 73.53±2.11 mg / 100 mg. This suggests that the protons provided during acid hydrolysis can attack and break the glycosidic bonds, and that low acid concentrations can preferentially hydrolyze the neutral sugar side chains (RG-I side chains) of the pectin polysaccharides in the roxburghii pomace, thereby exposing the backbone and ultimately leading to an increase in uronic acid content. In addition, pectin polysaccharides from roxburghii pear residue and their modified products contain a small amount of protein, ranging from 1.32±0.2 mg / 100 mg to 2.64±0.14 mg / 100 mg. The total bound phenolic content of pectin polysaccharides from roxburghii pear residue modified by different techniques decreased to some extent. Specifically, compared with RPDP, the total bound phenolic content of RPDP-E, RPDP-A, and RPDP-T decreased from 3.66±0.09 mg GAE / 100 mg to 1.91±0.06 mg GAE / 100 mg, 2.79±0.03 mg GAE / 100 mg, and 1.73±0.01 mg GAE / 100 mg, respectively.

[0072] Table 1 Chemical composition, molecular weight and monosaccharide composition of pectin polysaccharides from roxburghii residue and their modified products

[0073]

[0074] Notes: 1) RPDP, RPDP-E, RPDP-A, and RPDP-T represent the extraction of pectin polysaccharides from roxburghii pear residue using a deep eutectic solvent, the product modified by targeted enzymatic cleavage, the product modified by low-temperature controlled alkaline deesterification, and the product modified by controlled partial acid hydrolysis, respectively. 2) mg GAE / 100 mg, gallic acid equivalent per 100 mg of polysaccharide. 3) Superscript (ad) indicates significant differences among groups (p < 0.05). 4) Calculation formula: HG (mol%) = GalA (mol%) - Rha (mol%); RG-I (mol%) = GalA (mol%) - HG (mol%) + Rha (mol%) + Gal (mol%) + Ara (mol%); RG-I side chain length = (Ara (mol%) + Gal (mol%)) / Rha (mol%).

[0075] 3.2 Molecular weight and distribution of pectin polysaccharides from roxburghii residue and their modified products

[0076] Figure 1 The high-performance size exclusion chromatography (HSEC) spectra of pectin polysaccharides from roxburghii pear residue and their modified products were presented. The results showed that the molecular weight distribution of pectin polysaccharides from roxburghii pear residue and their modified products was relatively uniform, showing high chromatographic purity. In addition, compared with RPDP, the elution curves of the different modified products (RPDP-E, RPDP-A, and RPDP-T) all shifted to the right to a certain extent, indicating that different modification techniques reduced the molecular weight of pectin polysaccharides from roxburghii pear residue to varying degrees. As shown in Table 1, after modification by different techniques, the molecular weight of pectin polysaccharide RPDP from roxburghii pear residue increased from (4.641±0.043)×10 4 Da decreased to (0.811±0.026)×10 4 Da(RPDP-E), (3.556±0.033)×10 4 Da(RPDP-A), (0.842±0.007)×10 4 Da(RPDP-T). Compared with RPDP, the molecular weight of RPDP-A is only 4.641×10 4 Da decreased slightly to 3.556×10 4 The decrease in its molecular weight may be due to the β-elimination reaction during the low-temperature alkaline deesterification process, which triggers the breakage of the pectin main chain. Compared with RPDP, the molecular weight of RPDP-T is significantly reduced to 0.842×10 4 Da, the molecular weight distribution also decreased from 1.771 to 1.363, which shows that acidic conditions can promote the cleavage of glycosidic bonds, thereby reducing the molecular weight. Finally, the most obvious decrease in molecular weight is RPDP-E, whose molecular weight decreased to 0.811×10 4Da, while the molecular weight distribution increased to 1.866. This is because pectinase degrades the HG backbone, and HG is degraded into fragments of different molecular weights. This process not only reduces the molecular weight of pectin polysaccharides from roxburghii pear residue, but also broadens its molecular weight distribution range. In summary, different modification technologies will reduce the molecular weight of pectin polysaccharides from roxburghii pear residue to varying degrees. Among them, low-temperature alkaline deesterification treatment has a relatively small effect on its molecular weight.

[0077] 3.3 Monosaccharide composition of pectin polysaccharides from roxburghii residue

[0078] Figure 2 The following is a liquid chromatography-mass spectrometry analysis of the sugar compositions of pectin polysaccharides from roxburghii pear residue and their modified products. The results show that RPDP, RPDP-E, RPDP-A, and RPDP-T are primarily composed of eight monosaccharides: galacturonic acid (GalA), galactose (Gal), arabinose (Ara), rhamnose (Rha), glucose (Glc), xylose (Xyl), mannose (Man), and glucuronic acid (GlcA), with the predominant monosaccharides being GalA, Gal, Ara, and Rha. Generally, the major monosaccharide units in the RG-I domain are GalA, Rha, Gal, and Ara, while the major monosaccharide unit in the HG domain is GalA. Therefore, the sugar composition analysis indicates that pectin polysaccharides from roxburghii pear residue and their modified products are primarily composed of the HG and RG-I domains. The experimental results show that compared with RPDP, the GalA molar ratio of RPDP-E decreased from 42.24 mol% to 39.22 mol%, while the Gal and Ara molar ratios increased from 22.52 mol% and 19.59 mol% to 23.22 mol% and 24.38 mol%. Furthermore, the molar ratio of the HG domain decreased from 36.35 mol% to 34.98 mol%, while the RG-I ratio and RG-I side chain length increased from 53.91 mol% and 7.15 to 56.08 mol% and 11.22. Compared with RPDP, the most significant change in RPDP-A was the decrease in GalA, from 42.24 mol% to 28.99 mol%, a trend that was consistent with the change in total uronic acid determined by colorimetry. Compared with RPDP, the molar ratio of GalA to Rha and the proportion of HG domains in RPDP-T were significantly increased, reaching 76.40 mol% and 68.39 mol%, respectively, while the molar percentages of Gal and Ara were significantly decreased to 7.99 mol% and 0 mol%. Compared with Gal, Ara belongs to the furanose ring glycosidic bond and is more susceptible to acid hydrolysis.

[0079] 3.4 Infrared spectral characteristics of pectin polysaccharides from roxburghii residue and their modified products

[0080] Figure 3The following are Fourier transform infrared spectra of pectin polysaccharides from roxburghii pear residue and their modified products. The results show that the infrared spectra of pectin polysaccharides from roxburghii pear residue treated with different modification techniques are similar, indicating that pectin polysaccharides from roxburghii pear residue and their modified products have similar chemical groups. Specifically, the typical adsorption bands of RPDP, RPDP-E, RPDP-A, and RPDP-T are 3408.26 cm -1 、2921.34cm -1 、1738.77cm -1 、1623.76cm -1 、1419.61cm -1 、1240.01cm -1 、1099.75cm -1 and 1018.25cm -1 These adsorption bands correspond to complex pectin polysaccharides, indicating that their chemical groups remain stable after being treated with different modification techniques. -1 The absorption band near 1738.77 cm corresponds to the asymmetric stretching vibration of the carbonyl double bond (C=O), which can be used to evaluate the degree of esterification (DE) of pectin polysaccharides. -1 and 1623.76cm -1 The absorption bands at represent the vibration of esterified carboxyl group (COO-R) and free carboxyl group (COO-), respectively. Based on the peak areas of these two places, the esterification degree of pectin polysaccharides from roxburghii pear residue and its modified products can be calculated. Figure 3 It can also be observed at 1738.77cm -1 Compared with RPDP, the absorption peak signals of RPDP-T1, RPDP-T2 and RPDP-A are significantly weakened; the corresponding absorption peak signals at 1623.76 cm -1 At 1738.77 cm, their absorption peak signals are significantly enhanced. -1 and 1623.76cm -1 The esterification degree of the pectin polysaccharides from roxburghii pear residue and their modified products can be calculated from the ratio of the adsorption peaks. Compared with RPDP, the esterification degree of RPDP-A decreased from 56.38% ± 0.88% to 22.21% ± 0.62%, indicating a significant decrease in esterification after low-temperature alkaline deesterification, successfully preparing pectin polysaccharides modified with low esterification degrees from roxburghii pear residue. Compared with RPDP, the esterification degree of RPDP-E decreased slightly to 53.07% ± 0.59%. Compared with RPDP, the esterification degree of RPDP-T decreased significantly to 17.96% ± 0.55%, likely due to acid hydrolysis of the esterified carboxyl groups to free carboxyl groups.

[0081] 3.5 Nuclear magnetic resonance spectral characteristics of pectin polysaccharides from roxburghii residue and their modified products

[0082] Figure 4 Rosa roxburghii pectin polysaccharide and its modified products 1 H and 13 C NMR spectrum. The results showed that 1 H NMR signals representing α-D-GalAp, α-L-Rhap, and α-L-Araf residues were observed at 4.46 ppm to 4.64 ppm 1 H NMR signals represent β-D-Galp residues. Observed at 99.53 ppm to 109.11 ppm 13 C NMR signals represent α-D-GalAp, β-D-Galp and α-L-Araf residues at 3.80 ppm ( 1 H) and 52.73 ppm ( 13 C) The typical esterification signals of GalA-OCH3 were observed at 2.06 ppm and 2.17 ppm ( 1 The signal at H) indicates the presence of O-3 and O-6 acetyl groups in the GalAp residues, and typical one-dimensional NMR signals of the HG and RG-I domains can be observed ( Figure 4 Compared with RPDP, the main change of RPDP-A is at 3.80ppm ( 1 H) and 52.73 ppm ( 13 The GalA-OCH3 signal at C) is significantly weakened, which indicates that the controlled low-temperature alkaline deesterification treatment significantly reduces the methyl esterification of pectin polysaccharides in roxburghii pear residue, but the low-temperature alkaline deesterification technology has a limited impact on the primary chemical structure of RPDP. Compared with RPDP, the main changes of RPDP-E are at 1.25ppm ( 1 H) and 1.31 ppm ( 1 The Rhap residue signal at 5.31 ppm ( 1 The signal at H) was preliminarily judged to be the H-1 signal of 1,2-α-L-Rhap residue, which is similar to the previous research results that pectinase can degrade the HG domain and thus increase the proportion of RG-Ⅰ domain. It is worth noting that compared with RPDP, RPDP-T at 3.80ppm ( 1 H) and 52.73 ppm ( 1 H) and the GalA-OCH3 signal at 2.06 ppm ( 1 H) and 2.17 ppm ( 1 The O-acetyl signal at H) was significantly weakened, which is consistent with the decreasing trend of its esterification degree. Compared with RPDP, RPDP-T at 5.09ppm ( 1 H), 5.15ppm(1 H), 107.36ppm( 13 C) and 109.11ppm( 13 The Araf residue signal at C) changes significantly. In RPDP-T, the Araf residue signal basically disappears, and its signal is between 4.46 ppm and 4.64 ppm ( 1 H) and 104.22 ppm ( 13 The Galp residue signals in C) were significantly weakened or disappeared. These results indicate that acid hydrolysis can degrade the RG-Ⅰ side chain of the pectin polysaccharide from the roxburghii residue, thereby exposing the main chain.

[0083] 4. Summary of this section

[0084] The present invention uses low-temperature controlled alkaline deesterification technology, targeted enzymatic cleavage technology, and controlled partial acid hydrolysis technology to structurally modify the pectin polysaccharide from roxburghii pear residue. The results show that the targeted enzymatic cleavage technology can reduce the HG ratio of pectin polysaccharide from roxburghii pear residue (from 36.35% to 34.98%) and significantly reduce its molecular weight (from 4.641×10 4 Da is reduced to 0.811×10 4 In addition, the low-temperature controlled alkaline deesterification technology can significantly reduce the esterification degree of pectin polysaccharides from roxburghii pear residue (from 56.38% to 22.21%), significantly reduce its HG ratio (from 36.35% to 20.83%), and reduce its molecular weight (from 4.641×10 4 Da is reduced to 3.556×10 4 Da). Finally, controlled partial acid hydrolysis technology can remove the RG-I arabinose side chains of the pectin polysaccharide and significantly reduce the galactose side chains. The arabinose molar ratio is reduced from 19.59 mol% to 0, and the galactose molar ratio is significantly reduced from 22.52 mol% to 7.99 mol%, and the molecular weight is also reduced from 4.641×10 4 Da was significantly reduced to 0.842×10 4 In summary, the present invention successfully prepared roxburghii residue-modified pectin polysaccharides with different structural characteristics by using targeted enzymatic digestion technology, low-temperature controllable alkaline deesterification technology, and controllable partial acid hydrolysis technology.

[0085] The beneficial effects of the present invention are demonstrated by specific pharmacodynamic tests below.

[0086] Example 1 Effects of pectin polysaccharides from roxburghii residue and their modified products on improving ulcerative colitis and their structure-activity relationship

[0087] 1 Materials and Reagents

[0088] 1.1 Experimental Materials

[0089] The present invention uses roxburghii pear residue pectin polysaccharides (RPDP) extracted with the assistance of a deep eutectic solvent, roxburghii pear residue-modified pectin polysaccharides (RPDP-E) prepared by targeted enzymatic cleavage technology, roxburghii pear residue-modified pectin polysaccharides (RPDP-A) prepared by controllable low-temperature alkaline deesterification technology, and roxburghii pear residue-modified pectin polysaccharides (RPDP-T) prepared by controllable partial acid hydrolysis technology for experiments.

[0090] 2 Experimental methods

[0091] 2.1 Establishment of a dextran sulfate sodium-induced ulcerative colitis mouse model

[0092] Eight-week-old, 22g, C57BL / 6 male mice from Spafford were used in this experiment. After adaptive feeding, the mice were divided into seven groups, each containing six mice, based on the absence of significant differences in mean body weight. These groups included a blank group (Control), a model group (dextran sulfate sodium, DSS), a positive drug group (5-aminosalicylic acid, ASA), a roxburghii pectin polysaccharide group (RPDP), a modified pectin polysaccharide prepared by targeted enzymatic cleavage technology (RPDP-E), a modified pectin polysaccharide prepared by controlled low-temperature alkaline deesterification technology (RPDP-A), and a modified pectin polysaccharide prepared by controlled partial acid hydrolysis technology (RPDP-T). The control, DSS, RPDP, RPDP-E, RPDP-A, and RPDP-T groups were given 2.5% DSS ad libitum for 10 days. The RPDP, RPDP-E, RPDP-A, and RPDP-T groups were then gavaged with 150 mg / kg of DSS on days 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16 (a total of 10 times). The positive drug group was gavaged with 100 mg / kg of 5-aminosalicylic acid (5-ASA). The blank and model groups were gavaged with the same volume of normal saline. Body weight and disease activity index were recorded daily during feeding. Finally, on the 17th day, mice were anesthetized and killed after fasting for 12 hours. Blood was collected from the mice. Fresh feces were collected 24 hours before the mouse dissection for intestinal flora detection. Colon tissues of the mice were collected and the length was measured. 1-2 cm of colon tissue was fixed in 4% paraformaldehyde solution. A portion of the colon tissue was homogenized and stored at -80°C for subsequent cytokine determination (see Figure 5 ).

[0093] 2.2 Recording of mouse body weight and assessment of disease activity index (DAI)

[0094] During the modeling process, the body weight, fecal status, and blood in stool of the mice were detected and recorded daily to assess the severity of colitis in the mice (Table 2).

[0095] Table 2 DAI scoring criteria

[0096]

[0097] 2.3 Analysis of mouse colon length and colon histopathology

[0098] On the 17th day, the mice were anesthetized and killed for dissection. During the dissection, the colon was carefully removed from 1 cm above the anus to the upper end of the cecum. After measuring the full length of the colon, rinse with clean water. The colon tissue of about 0.5 cm at the distal end was selected for the preparation of colon pathological sections of the experimental mice, and preserved with paraformaldehyde fixative, and then stained. The specific steps of H&E staining are as follows: the paraformaldehyde-fixed colon tissue is dehydrated and embedded in paraffin, sliced ​​using a pathological slicer, and then dewaxed, pretreated with high-definition constant staining (1 min), stained with hematoxylin (3-5 min), differentiated, and blued, then rinsed with running water, placed in 95% alcohol for dehydration for 1 min, and finally stained in eosin stain for about 15 s. After dehydration and sealing, the sections were observed under a microscope, and H&E staining was scored according to Table 3. The PAS staining procedure involves embedding and sectioning fixed tissues, then staining them in PAS staining solution B for 10-15 minutes, then in solution A for 25-30 minutes, and finally in solution C for 30 seconds. The tissues are then differentiated with hydrochloric acid, blued with ammonia, mounted, and observed under a microscope. The PSR staining procedure involves embedding and sectioning embedded tissues, staining with picrosirius red for 8 minutes, dehydrating, mounting, and observing under a microscope. The stained areas of PAS and PSR staining were quantified using Image J software.

[0099] Table 3 H&E staining scoring table

[0100]

[0101] 2.4. Immunofluorescence detection of tight junction protein levels in mouse colon

[0102] Immunofluorescence was used to assess the intestinal barrier repair effects of pectin polysaccharides derived from roxburghii pear residue and their modified products. Paraffin sections were first dewaxed to an aqueous phase and then subjected to antigen retrieval in EDTA (pH 8.0) antigen retrieval buffer. Sections were blocked with BSA for 30 minutes, then diluted primary antibodies were added dropwise and incubated overnight at 4°C in a humidified chamber. Following incubation, sections were washed three times with PBS and then incubated with diluted secondary antibodies for 50 minutes. Subsequently, DAPI staining solution was added dropwise and incubated for 10 minutes at room temperature in the dark. An autofluorescence quencher was then added for 5 minutes, followed by rinsing with running water for 10 minutes. Finally, sections were mounted with anti-fluorescence quenching mounting media, and images were acquired using a scanner. Under UV light excitation, DAPI-stained cell nuclei appear blue, while positive expression is detected as red or green fluorescence from the corresponding fluorescent markers. Finally, the fluorescence area was quantified using Image J software.

[0103] 2.5 Analysis of serum LPS levels and colon tissue inflammatory factor levels

[0104] Blood was collected from mice and allowed to stand at room temperature for a period of time before centrifugation (3000r, 15min). The supernatant was collected and stored at -80°C for use in determining LPS levels. Part of the mouse colon was removed and rinsed with pre-chilled PBS (0.01M, pH=7.4) to remove residual feces and blood. After weighing, PBS was added at a ratio of 1:9 (mass:volume). The colon tissue was broken up using a tissue homogenizer and then centrifuged at 5000×g for 10min at 4°C, and the supernatant was collected. According to the operating procedures of the enzyme-linked immunosorbent assay (ELISA) kit instructions, the levels of cytokines (including TNF-α, IL-1β, IL-10, and IL-6) in the colon tissue of each group of mice and LPS in the serum were detected.

[0105] 2.6 Determination of short-chain fatty acids in mouse feces

[0106] 4-Methylvaleric acid was used as the internal standard, and an internal standard solution (solvent: methyl tert-butyl ether) was prepared at a concentration of 50 μg / mL. Acetic acid, propionic acid, and butyric acid were weighed separately, and nine standard solutions with different concentration gradients were prepared using the internal standard solution. 100 mg of fecal sample from each group was added with 100 μL of 10% H₃PO₄ and 500 μL of the internal standard solution. The samples were homogenized by shaking for 1 minute, then sonicated for 10 minutes. The supernatant was collected and filtered through a 0.22 μm organic filter membrane for gas chromatography-mass spectrometry (GC-MS) analysis. GC-MS analysis was performed using an HP-INNOWax capillary column (30 m × 0.25 mm × 0.25 mm) with a split ratio of 10:1 and an injection volume of 1 μL. The inlet, ion source, and transfer line temperatures were set at 250°C, 230°C, and 250°C, respectively. Nitrogen was used as the carrier gas at a flow rate of 1.0 mL / min. The column temperature program was as follows: initial temperature of 80°C for 3 minutes, then ramped to 180°C at a rate of 10°C / min, and finally ramped to 250°C at a rate of 25°C / min and held for 3 minutes. For concentration calculations, standard curves for acetic acid, propionic acid, and butyric acid were plotted, with the standard concentration as the horizontal axis and the standard to internal standard peak area ratio as the vertical axis. The short-chain fatty acid content in the sample was calculated using these standard curves.

[0107] 2.7 Intestinal flora analysis

[0108] Mouse feces were collected and subsequently subjected to 16S rRNA sequencing analysis. Genomic DNA was extracted using the CTBA extraction method, and its purity and concentration were tested. The genomic DNA was diluted to 1 ng / mL and amplified by PCR (using specific primers with barcodes and NEB). Ultra-fidelity PCR premix and high-efficiency high-fidelity enzyme); PCR products were detected and purified by agarose gel electrophoresis; Ultra TMLibraries were prepared using the QIIME2 DNA library preparation kit and quantified by Qubit and Q-PCR. Qualified libraries were sequenced using the NovaSeq 6000. 16S rRNA gene sequencing data were analyzed using the Jizhi Cloud platform of Tianjin Jizhi Gene Technology Co., Ltd. The raw data were first spliced ​​using FLASH software, followed by quality control using fastp software. Chimeras were removed using vsearch software, and noise was reduced using the DADA2 method, ultimately generating high-quality sequences (ASVs). The ASVs were then annotated to species using the classify-sklearn algorithm in QIIME2, and the species information for each ASV was obtained by comparison with the database to provide a foundation for subsequent analysis. PCoA and LEfSe analyses were performed on the Jizhi Cloud platform, and intergroup correlation analysis was performed using the ChiPlot platform.

[0109] 3 Results and Analysis

[0110] 3.1 Effects of pectin polysaccharides from roxburghii residue and their modified products on body weight and disease index in mice

[0111] Mouse weight is one of the key indicators for evaluating ulcerative colitis. Mice with ulcerative colitis usually experience a gradual weight loss, and the DAI score can accurately assess the pathological state of UC mice. In this experiment, the weight and disease index of mice were recorded daily during the ten days of modeling. The experimental results showed that compared with the blank group, the weight of mice in the other groups decreased from the 10th day, with the positive group showing the lowest weight loss and the DSS model group showing the most severe weight loss ( Figure 6 In addition, the blank group mice had smooth fur, black and shiny, normal luster, normal diet and drinking water, a steady increase in weight, good activity, firm feces, brown-yellow color, and no diarrhea or bloody stools. Compared with the blank group, the DSS model group mice had a significant weight loss from the 10th day, an increase in DAI score, and obvious diarrhea and bloody stools from the 13th day, accompanied by decreased appetite, fatigue, laziness, and dull fur color, indicating that the model was successfully established ( Figure 6). After gavage with pectin polysaccharides from sea buckthorn residue and their modified products, the weight loss and rising trend of DAI scores in UC mice were slowed down, but the mitigating effects of the four polysaccharides were different. Among them, the modified polysaccharides were better than the original polysaccharides, and the RPDP-E group showed better effects among the modified polysaccharides. Compared with the RPDP group, the rising trend of DAI scores in the RPDP-E group slowed down from the 13th day, close to that of the positive drug group, and could significantly and effectively slow down the weight loss and rising DAI scores in UC mice; the improvement effect of RPDP-T was second, and the last was RPDP-A; compared with all modified samples (RPDP-E, RPDP-T, RPDP-A), the original pectin polysaccharide RPDP from sea buckthorn residue had the weakest mitigating effect. In summary, pectin polysaccharides from sea buckthorn residue and their modified products can alleviate the weight loss and rising DAI scores of mice caused by DSS, and the pectin polysaccharides modified from sea buckthorn residue have better effects.

[0112] 3.2 Effects of pectin polysaccharides from roxburghii pear residue and their modified products on colon length in mice with ulcerative colitis

[0113] Under normal circumstances, the colon of mice will atrophy due to the effects of ulcerative colitis. Ten days after modeling, the mice were fasted for 12 hours and anesthetized and killed. The length of the colon was measured after dissection. The results are as follows Figure 7 As shown, compared with the blank group, the colon length of mice in the DSS model group was significantly shortened (p<0.01), indicating that DSS induced colon shortening in mice, and the colon became darker and showed obvious congestion. Rosa roxburghii pectin polysaccharides and their modified products can alleviate the DSS-induced colon shortening in mice. Specifically, compared with the RPDP group, the RPDP-E sample group and the RPDP-T sample group had a more significant effect on restoring the colon length of mice, and the cecal tissue was more plump. Both PRDP-E and PRDP-T had a good alleviating effect.

[0114] 3.3 Effects of pectin polysaccharides from roxburghii pear residue and their modified products on colon pathological tissue sections of mice with ulcerative colitis

[0115] like Figure 8As shown in the figure, H&E, PAS, and PSR staining of the mouse colon cross-sections clearly revealed the structural characteristics of each layer of colonic tissue. All three results indicate that the colonic mucosal tissue of mice in the DSS model group was severely damaged, the crypt structure was destroyed, and there was a certain amount of edema, accompanied by the overflow of inflammatory factors and the presence of cell infiltration. H&E staining results showed that compared with the control group, the DSS model group showed significant overflow of inflammatory factors and edema, as well as damaged epithelial structure and loss of crypt structure. PAS staining results showed that the DSS model group had a large number of goblet cells missing, with the number significantly lower than that of the other groups. PSR staining results showed that the colonic fibrosis in the DSS model group was the most severe, with a noticeable redness, indicating damage to the colonic tissue. After intervention with pectin polysaccharides from roxburghii pomace and positive drugs, symptoms such as intestinal mucosal tissue damage, crypt atrophy, cell infiltration, fibrosis, and edema in the colitis mice were improved. Compared with RPDP, the modified RPDP-E and RPDP-T showed the most significant improvement. In particular, the RPDP-E group showed clear crypt structure and relatively intact epithelial structure, approaching that of the control group. However, the original RPDP sample showed a significant loss of crypt structure and a relatively low number of goblet cells.

[0116] 3.4 Effects of pectin polysaccharides from roxburghii pear residue and their modified products on tight junction protein expression in mice with ulcerative colitis

[0117] like Figure 9As shown, the expression levels of ZO-1 and occludin in colonic tissue were measured using immunofluorescence. The intestinal mucosal barrier, composed of intestinal epithelial cells and tight junction proteins, plays an important role in maintaining a stable intestinal environment and protecting against intestinal infections. ZO-1 and occludin are key components of tight junction proteins and play an essential role in maintaining intestinal barrier function. When intestinal barrier function is impaired, intestinal permeability increases, allowing harmful bacteria or toxins to enter the tissue through the bloodstream, leading to intestinal diseases. Immunofluorescence staining revealed that tight junction proteins in the periphery of colonic epithelial cells in the control group exhibited intact, continuous ring-like structures. In contrast, the ZO-1 and occludin proteins in the model group were significantly disrupted, with fluorescence intensities significantly lower than those in the other groups. Compared with the model group, the expression levels of ZO-1 and occludin were significantly enhanced in all four polysaccharide-treated samples. Furthermore, analysis of the staining area of ​​ZO-1 and occludin using Image J software further confirmed the impaired expression of these two proteins in the model group. The results showed that pectin polysaccharides from roxburghii pear residue and their modified products could upregulate the expression of ZO-1 and occludin, thereby effectively repairing intestinal barrier damage. Compared with the RPDP group, the staining areas of ZO-1 and occludin in the RPDP-T, RPDP-E, and RPDP-A groups were significantly increased, especially in the RPDP-E group, where the levels were comparable to those in the positive drug group.

[0118] 3.5 Effects of pectin polysaccharides from roxburghii residue and their modified products on inflammatory factors and serum LPS in mice with ulcerative colitis

[0119] According to previous studies, the lipopolysaccharide (LPS) content in serum can indirectly reflect the degree of inflammation in mice. LPS is a unique component of the cell wall of Gram-negative bacteria, also known as endotoxin, which can activate the host's immune system and trigger an inflammatory response. When the intestinal barrier of mice is damaged, endotoxins will enter the blood circulation, activate a series of signaling pathways, and induce the release of proinflammatory cytokines (such as TNF-α, IL-6, etc.), leading to systemic inflammation. In the DSS-induced inflammatory model, the increase in serum LPS levels is closely related to the intensity of the inflammatory response. The imbalance of cytokines is closely related to the pathogenesis of UC. IL-1β and IL-6 are two proinflammatory cytokines that are positively correlated with colonic inflammation. Among them, IL-1β mainly mediates the initial stage of the inflammatory response and maintains its persistence, while IL-6 mainly affects intestinal mucosal lesions. In addition, TNF-α has been shown to play an indispensable role in the pathogenesis of UC. At the same time, IL-10, as an important anti-inflammatory cytokine, can reduce the inflammatory response and maintain intestinal immune homeostasis, and has been shown to have a protective effect on UC. As Figure 10As shown in the figure, this experiment used ELISA to measure the expression levels of inflammatory factors TNF-α, IL-6, IL-1β, and IL-10 in the colon tissue of mice, as well as the content of LPS in the mouse serum. The results showed that compared with the control group, the LPS content in the model group mice was significantly increased, indicating that there was a significant inflammatory response in the model group mice. In addition, the expression levels of TNF-α, IL-6, and IL-1β in the colon tissue of the model group mice were significantly increased, while the expression level of IL-10 was significantly decreased, indicating that DSS caused severe inflammation in the mice. After intervention with pectin polysaccharides from sea buckthorn residue and their modified products, the levels of pro-inflammatory factors such as TNF-α, IL-6, IL-1β and the content of serum lipopolysaccharide (LPS) were significantly reduced, and the reduction effect of RPDP-E was the most obvious, followed by RPDP-T, while the level of anti-inflammatory factor IL-10 was significantly increased. This indicates that pectin acidic polysaccharides from sea buckthorn residue and their modified products can inhibit the pathological inflammation of colitis by downregulating the levels of TNF-α, IL-6, IL-1β, reducing the content of serum LPS, and upregulating the level of IL-10, and the improvement effect of the modified pectin polysaccharides from sea buckthorn residue is significantly better than that of the original pear residue polysaccharide, especially RPDP-E.

[0120] 3.6 Effects of pectin polysaccharides from roxburghii pear residue and their modified products on the content of short-chain fatty acids in feces of mice with ulcerative colitis

[0121] Short-chain fatty acids are the main metabolites produced by intestinal flora in the process of metabolizing natural product polysaccharides, and are therefore closely related to intestinal flora. Common short-chain fatty acids include acetic acid, propionic acid, and butyric acid. Studies have found that short-chain fatty acids can promote the proliferation of beneficial bacteria, thereby helping to restore intestinal flora imbalance, and the restoration of intestinal flora homeostasis can further increase the production of short-chain fatty acids. In addition, short-chain fatty acids can also upregulate the expression of tight junction proteins, maintain intestinal barrier function, and regulate inflammatory responses. This experiment measured the concentration of short-chain fatty acids in the cecal contents of mice. Figure 11As shown, compared with the control group, the DSS model group significantly reduced the concentrations of total short-chain fatty acids (SCFAs) and acetic, propionic, and butyric acid. After treatment with roxburghii pear pectin polysaccharides and their modified products, the concentrations of SCFAs, acetic, propionic, and butyric acid increased in all four polysaccharide sample groups, with the modified roxburghii pear pectin polysaccharides having a more pronounced restorative effect, particularly in the RPDP-E group. These results suggest that roxburghii pectin polysaccharides and their modified products can improve DSS-induced colitis by increasing the production of SCFAs. Both roxburghii pectin polysaccharides and their modified products are pectin polysaccharides, which are readily fermented by intestinal microflora to produce butyric and acetic acid. Butyric acid may inhibit the progression of colitis by regulating the excessive proliferation of intestinal epithelial cells to maintain their integrity. The increase in SCFAs is closely related to the metabolic activity of the intestinal microbiota, particularly the proliferation of beneficial bacteria. Short-chain fatty acids inhibit the secretion of these inflammatory factors by activating G protein-coupled receptors (GPR43), and by promoting the expression of tight junction proteins (ZO-1, Occludin), they enhance the tight junctions between intestinal epithelial cells, reduce intestinal permeability, and thus alleviate inflammation.

[0122] 3.7 Effects of pectin polysaccharides from roxburghii pear residue and their modified products on the intestinal flora of mice with ulcerative colitis

[0123] The intestinal flora is a collection of symbiotic microorganisms in the host, which is widely involved in the regulation of the host's metabolism, immunity, endocrine and other physiological functions, and is of great significance in maintaining health and preventing diseases. A large number of studies have pointed out that the intestinal flora plays a key role in promoting the development of the intestinal mucosal immune system, resisting the invasion of pathogenic microorganisms, and maintaining the balance of intestinal microecology and the stability of the internal environment. Therefore, effective regulation of intestinal microbial flora is considered to be an effective way to improve health, prevent and treat diseases. The present invention uses 16S rRNA gene amplicon sequencing technology to deeply analyze the effects of pectin polysaccharides from sea buckthorn residue and their modified products on the intestinal flora of mice. Figure 12 As shown in the figure, PCoA analysis results showed that the pectin polysaccharide RPDP from roxburghii pear residue and its modified products RPDP-T and RPDP-A showed a clear clustering trend, while RPDP-E was at a certain distance from them. In addition, the distance between the DSS group and the control group was the greatest, indicating that the intestinal microbiota structure in the model group had undergone significant changes. However, the intestinal microbiota structure was restored to a certain extent after polysaccharide intervention. Cluster analysis further revealed the relationship between different pectin polysaccharides from roxburghii pear residue. The results showed that the microbiota structure between RPDP and RPDP-A and RPDP-T was relatively close, while RPDP-E showed some differences. In addition, there was a certain distance between the polysaccharide sample group and both the model group and the blank group, further confirming the significant changes in the intestinal microbiota structure after polysaccharide intervention.

[0124] In order to further explore the specific effects of pectin polysaccharides from roxburghii pear residue and its modified products on the intestinal microorganisms of mice, the composition of the intestinal flora was further analyzed in detail at the phylum and genus levels. Figure 13 As shown, at the phylum level, the gut microbiota in the blank control group (Control group) was primarily composed of Bacteroidetes and Firmicutes, with Bacteroidetes comprising approximately 70% of the total microbiota. Studies have shown that Bacteroidetes plays a crucial role in the development of ulcerative colitis (UC), with reduced abundance being one of the primary characteristics of intestinal dysbiosis in UC patients. Bacteroidetes can ameliorate intestinal inflammation and promote the production of short-chain fatty acids. Firmicutes are also major producers of short-chain fatty acids, while butyrate is a major energy source for colonic epithelial cells and has anti-inflammatory effects. Compared with the Control group, the DSS model group showed a significant decrease in the abundance of Bacteroidetes and a significant increase in the abundance of Proteobacteria and Deferribacterota. Increased abundance of Proteobacteria and Deferribacterota may lead to excessive production of proinflammatory cytokines, thus triggering colitis. Increased abundance of Proteobacteria, in particular, is closely associated with the development of inflammation. Studies have shown that reducing the ratio of Firmicutes to Bacteroidetes (F / B) may reduce intestinal inflammation and lower the risk of chronic inflammatory diseases such as inflammatory bowel disease (IBD). Compared to the DSS model group, the positive drug group only slightly increased the abundance of Bacteroidetes. 5-ASA primarily exerts its anti-inflammatory effects by inhibiting the cyclooxygenase (COX) and lipoxygenase (LOX) pathways, reducing the production of prostaglandins and leukotrienes, and its direct regulatory effects on the microbiome itself are relatively weak. Treatment with pectin-based polysaccharides from roxburghii pear residue and its modified products restored the DSS-induced intestinal microbiome structure of mice to varying degrees. Compared to the DSS model group, the abundance of Bacteroidetes increased significantly in all four polysaccharide-treated groups, while the abundance of Firmicutes decreased significantly. The abundance of Proteobacteria and Deferribacteria also decreased significantly after polysaccharide treatment. In the RPDP-E group, the abundance of Bacteroidetes returned to levels close to those of the control group, while Firmicutes also decreased significantly compared to the DSS group. The RPDP-T group showed a relatively weaker recovery of the microbiome than the RPDP-E group, but no significant difference was observed. The relative abundance of Bacteroidetes and Firmicutes in the RPDP and RPDP-A groups did not differ significantly, but overall, both groups were superior to the positive drug group. These results suggest that pectin-based polysaccharides from roxburghii pear residue and their modified products have a significant regulatory effect on intestinal microbiota.

[0125] Further analysis was conducted at the genus level, and the top 40 bacterial genera with the highest relative abundance were selected for in-depth analysis. The results showed that compared with the control group, the relative abundance of Muribaculaceae in the DSS model group decreased from approximately 60% to approximately 20%. After intervention with RPDP, RPDP-T, RPDP-E, and RPDP-A, the relative abundance of Muribaculaceae increased significantly, with RPDP-E in particular restoring its relative abundance to approximately 40%. Muribaculaceae produces SCFAs, especially acetic acid and propionic acid, through the fermentation of dietary fiber. These metabolites can regulate the host immune system, inhibit inflammatory responses, regulate NF-κB and natural killer cell signaling pathways, and enhance the intestinal barrier. Under the intervention of RPDP-E, the relative abundance of Bacteroides also increased. Bacteroides can enhance intestinal barrier function and reduce inflammatory responses by producing short-chain fatty acids (SCFAs), thereby alleviating colitis. Compared with the DSS group, RPDP-E, RPDP-A, and RPDP significantly upregulated the abundance of Clostridia_UCG-014. Studies have shown that Clostridia_UCG-014 can promote the differentiation and proliferation of regulatory T cells (Tregs), enhance anti-inflammatory responses, and thus alleviate colitis symptoms in mice. It also metabolizes dietary fiber to produce short-chain fatty acids such as butyrate, inhibiting the release of proinflammatory cytokines and strengthening tight junctions in intestinal epithelial cells, thereby alleviating the inflammatory response in colitis mice. RPDP-E also significantly upregulated the abundance of Turicibacter and Christensenellaceae_R-7_group. Turicibacter bacteria are generally considered commensal bacteria in the intestine and possess certain probiotic properties. They can regulate host glucose and lipid metabolism through polysaccharide metabolism and serve as a targeted microbial group for butyrate production, positively impacting intestinal health. Christensenellaceae_R-7_group is closely associated with host health status, with its relative abundance negatively correlated with body mass index (BMI), indicating that it is more abundant in healthy individuals. In addition, it participates in carbohydrate metabolism and produces short-chain fatty acids (primarily butyrate) to provide energy for intestinal epithelial cells, promoting cell repair and maintaining intestinal barrier function. Rosa roxburghii pectin polysaccharides and their modified products increased the relative abundance of Prevotellaceae_UCG-001, Eubacterium_coprostanoligenes_group, Ruminococcus, and Lactobacillus to some extent, but the effect was not significant.The abundance of Escherichia-Shigella, Colidextribacter, and Parasutterella increased significantly in the DSS model group. Under the intervention of pectin polysaccharides from sea buckthorn residue and their modified products, the abundance of Escherichia-Shigella, Colidextribacter, and Parasutterella in the mouse intestine was significantly reduced, with no significant difference between samples. Escherichia-Shigella is a representative genus of Proteobacteria, closely related to metabolic endotoxemia and intestinal diseases, and is commonly found in patients with ulcerative colitis. Colidextribacter may promote tumor development by increasing the expression of oncogenes (such as Bcl-2). This potential tumor-promoting effect makes it considered a potentially harmful bacterium, which may be related to intestinal barrier dysfunction. Disruption of intestinal barrier function may lead to an imbalance in the intestinal microbial community, thereby triggering inflammation or other health problems. Multiple studies have found that the abundance of Parasutterella is positively correlated with BMI (body mass index) in patients with obesity and type 2 diabetes, and that its abundance is significantly increased in patients with irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD). These results further confirm the regulatory effects of pectin polysaccharides from roxburghii pear residue and their modified products on intestinal microbiota and their potential therapeutic effects.

[0126] Polysaccharides cannot be directly absorbed by the human body and must rely on intestinal microbes for their degradation and utilization. Intestinal microbes often utilize their own carbohydrate-activated enzymes (CAZymes) to degrade polysaccharides. Compared to RPDP, RPDP-A exhibits superior intestinal microbiota-modulating abilities, likely due to its lower degree of esterification. Studies have shown that highly esterified pectin polysaccharides contain more methylated groups, which intestinal microbes preferentially degrade when degrading highly esterified pectins. The degradation of these groups requires a greater variety of CAZymes encoded by intestinal microbes. Compared to RPDP and RPDP-T, RPDP-E exhibits the best intestinal microbiota-modulating abilities, likely related to its lowest molecular weight and longer RG-I side chain length. Low-molecular-weight polysaccharides, with their smaller molecular size and simpler linear structure, are more easily recognized and broken down into monosaccharides or oligosaccharides by carbohydrate-activated enzymes secreted by intestinal microbes. High-molecular-weight polysaccharides, on the other hand, are complex structures that require specific enzymes secreted by intestinal microbes to break them down into smaller molecules. Furthermore, studies have shown that arabinan side chains may help the gut microbiota utilize the entire pectin polysaccharide domain to produce more SCFAs during colitis. Arabinose-rich pectin polysaccharides can increase the abundance of Bacteroidetes, Bifidobacterium, and acetate and butyrate production. Arabinose can increase the production of SCFAs and organic acids in the gut and promote the growth of the anti-inflammatory bacteria Actinobacteria in mice with DSS-induced colitis. In summary, RPDP-E has the lowest molecular weight, followed by RPDP-T, while RPDP-A has a molecular weight closer to that of the original sample RPDP. Their ability to restore the intestinal Bacteroidetes flora is negatively correlated with molecular weight, indicating that lower molecular weight is associated with a stronger restoration effect. Further analysis revealed that RPDP-E has a molecular weight similar to RPDP-T, but its side chain length is significantly longer than that of RPDP-T. Therefore, it is speculated that molecular weight and RG-I side chain length may be key effector structures in the ameliorative effects of roxburghii pectin polysaccharides on ulcerative colitis.

[0127] 3.8 Analysis of potential pathways of action of pectin polysaccharides from roxburghii pomace and their modified products in improving ulcerative colitis in mice

[0128] like Figure 14As shown, in order to clarify the correlation between the various indicators, the present invention conducted a correlation analysis on the relationship between intestinal flora, short-chain fatty acid content, inflammatory factors, tissue pathological damage and tight junction protein-related parameters. The results showed that Bacteroidetes and Muribaculaceae were significantly positively correlated with acetic acid, propionic acid, n-butyric acid and total short-chain fatty acid content. Firmicutes were significantly negatively correlated with total short-chain fatty acids and n-butyric acid content, and Proteobacteria were significantly negatively correlated with acetic acid and propionic acid. Colidextribacter and Parasutterella were significantly negatively correlated with total short-chain fatty acids, acetic acid and propionic acid. Escherichia-Shigella was significantly negatively correlated with acetic acid and propionic acid. As a harmful bacterium, it may promote the reduction of anti-inflammatory cytokines and the increase of pro-inflammatory cytokines, leading to the occurrence of colitis. The above results indicate that beneficial bacteria can promote the production of SCFAs, while harmful bacteria inhibit the production of SCFAs. Furthermore, Muribaculaceae and Bacteroidetes showed positive correlations not only with short-chain fatty acids but also with the anti-inflammatory cytokine IL-10, PAS score (goblet cell number), and expression of immunofluorescent proteins. Meanwhile, they showed significant negative correlations with proinflammatory cytokines such as TNF-α, IL-6, IL-1β, lipopolysaccharide (LPS), and H&E score. Muribaculaceae exerts its biological effects by promoting cytokine production and the synthesis of short-chain fatty acids. Short-chain fatty acids can regulate the activity of innate immune cells such as neutrophils, macrophages, and natural killer cells. Correlation analysis further revealed that short-chain fatty acids were significantly negatively correlated with colonic inflammatory factors, H&E score, and PSR score, and significantly positively correlated with colonic anti-inflammatory factors, tight junction proteins, and PAS score. SCFAs inhibit the secretion of these inflammatory factors, promote the expression of tight junction proteins (ZO-1 and Occludin), strengthen tight junctions between intestinal epithelial cells, and reduce intestinal permeability, thereby reducing the inflammatory response induced by LPS.

[0129] In summary, roxburghii pectin polysaccharides and their modified products can alleviate ulcerative colitis by reshaping the balance of intestinal microbiota. Specifically, roxburghii pectin polysaccharides and their modified products may inhibit the growth of harmful bacteria (such as Escherichia-Shigella and Parasutterella) while promoting the proliferation of beneficial bacteria (such as Muribaculaceae and Clostridia_UCG-01), thereby increasing the production of short-chain fatty acids. Short-chain fatty acids play a role in improving ulcerative colitis by regulating the levels of inflammatory factors, repairing intestinal barrier function, and upregulating the expression of tight junction proteins.

[0130] 4. Summary of this section

[0131] The present invention establishes a mouse model of ulcerative colitis by using DSS to explore the effects and potential structure-activity relationships of pectin polysaccharides from roxburghii pear residue and their modified products in improving ulcerative colitis. The experimental results show that oral administration of pectin polysaccharides from roxburghii pear residue (RPDP) and its modified products (RPDP-T, RPDP-E, and RPDP-A) significantly improves DSS-induced colitis in mice. Compared with RPDP, the improvement effects of RPDP-T, RPDP-E, and RPDP-A are significantly enhanced, especially RPDP-E prepared using targeted enzymatic cleavage technology. By analyzing their structural characteristics, we found that RPDP-E has the lowest molecular weight, followed by RPDP-T, while the molecular weight of RPDP-A is closer to that of the original sample RPDP. Their improvement effects on colitis may be negatively correlated with molecular weight, indicating that the lower the molecular weight, the more obvious the improvement effect. Further analysis found that the molecular weight of RPDP-E was close to that of RPDP-T, but the side chain length of RPDP-E was significantly longer than that of RPDP-T. Therefore, it is speculated that the molecular weight and RG-Ⅰ side chain length may be the key effector structures of sea buckthorn pomace pectin polysaccharides in improving ulcerative colitis.

Claims

1. A pectin polysaccharide from roxburghii residue, characterized in that: The invention is prepared by extracting roxburghii residue, a by-product of roxburghii fruit processing, as raw material. Each 100 mg contains 90.19 mg ± 1.67 mg–91.74 mg ± 0.18 mg of total polysaccharides, 24.23 mg ± 1.82 mg–73.53 mg ± 2.11 mg of total uronic acid, 1.32 mg ± 0.2 mg–2.64 mg ± 0.14 mg of total protein, and 1.73 mg GAE ± 0.01 mg GAE–3.66 mg GAE ± 0.09 mg GAE (gallic acid equivalent). The degree of esterification is 17.96% ± 0.55%–56.38% ± 0.88% and the molecular weight is (0.811 ± 0.026) × 10 4 Da–(4.641±0.043)×10 4 Da.

2. The roxburghii residue pectin polysaccharide according to claim 1, wherein: Its monosaccharide composition and molar percentage are: galacturonic acid (GalA) 28.99mol%-76.40mol%, galactose (Gal) 7.99mol%-25.96mol%, arabinose (Ara) 0.00mol%-27.25mol%, rhamnose (Rha) 4.24mol%-8.15mol%, glucose (Glc) 3.02mol%-5.38mol%, mannose (M an) 1.64mol%-3.17mol%, glucuronic acid (GlcA) 1.36mol%-2.46mol%, xylose (Xyl) 0.00mol%-0.94mol%; the molar proportion of its galacturonan (HG) is 20.83mol%-68.39mol%, and the molar proportion of type I rhamnogalacturonan (RG-Ⅰ) is 24.00mol%-69.52mol%.

3. The roxburghii pear residue pectin polysaccharide according to claim 1 or 2, characterized in that: The RPDP is a pectin polysaccharide from roxburghii pear residue prepared by deep eutectic solvent-assisted extraction technology. Per 100 mg, it contains 91.08 mg ± 0.9 mg of total polysaccharides, 37.89 mg ± 1.86 mg of total uronic acid, 1.91 mg ± 0.13 mg of total protein, and 3.66 mg GAE ± 0.09 mg GAE of total bound phenols. Its degree of esterification is 56.38% ± 0.88%, and its molecular weight is (4.641 ± 0.043) × 10 4 Da, polydispersity coefficient M w / M n is 1.771; its monosaccharide composition and molar percentage are: galacturonic acid (GalA) 42.24mol%, galactose (Gal) 22.52mol%, arabinose (Ara) 19.59mol%, rhamnose (Rha) 5.89mol%, glucose (Glc) 5.38mol%, mannose (Man) 1.64mol%, glucuronic acid (GlcA) 1.86mol%, xylose (Xyl) 0.88mol%; the molar proportion of HG is 36.35mol%, the molar proportion of RG-I is 53.91mol%, and the side chain length of RG-I is 7.

15.

4. The roxburghii residue pectin polysaccharide according to claim 3, wherein: The invention relates to a method for preparing roxburghii residue pectin polysaccharide RPDP by low eutectic solvent assisted extraction technology, and modifying it by targeted enzyme cutting technology to obtain roxburghii residue modified pectin polysaccharide RPDP-E; modifying it by low temperature controllable alkaline deesterification technology to obtain roxburghii residue modified pectin polysaccharide PRDP-A; and modifying it by controlled partial acid hydrolysis technology to obtain roxburghii residue modified pectin polysaccharide RPDP-T. Each 100 mg of RPDP-E contains 91.74 mg ± 0.18 mg of total polysaccharides, 35.73 mg ± 1.54 mg of total uronic acid, 1.32 mg ± 0.2 mg of total protein, and 1.91 mg GAE ± 0.06 mg GAE of total bound phenols. Its degree of esterification is 53.07% ± 0.59%, and its molecular weight is (0.811 ± 0.026) × 10 4 Da, polydispersity coefficient M w / M n is 1.866; its monosaccharide composition and molar percentage are: galacturonic acid (GalA) 39.22mol%, galactose (Gal) 23.22mol%, arabinose (Ara) 24.38mol%, rhamnose (Rha) 4.24mol%, glucose (Glc) 4.04mol%, mannose (Man) 2.60mol%, glucuronic acid (GlcA) 1.36mol%, xylose (Xyl) 0.94mol%; its HG molar proportion is 34.98mol%, RG-I molar proportion is 56.08mol%, and RG-I side chain length is 11.22; Each 100 mg of PRDP-A contains 90.96 mg ± 0.64 mg of total polysaccharide, 24.23 mg ± 1.82 mg of total uronic acid, 2.64 mg ± 0.14 mg of total protein, and 2.79 mg GAE ± 0.03 mg GAE of total bound phenol. Its degree of esterification is 22.21% ± 0.62%, and its molecular weight is (3.556 ± 0.033) × 10 4 Da, polydispersity coefficient M w / M n is 1.425; its monosaccharide composition and molar percentage are: galacturonic acid (GalA) 28.99mol%, galactose (Gal) 25.96mol%, arabinose (Ara) 27.25mol%, rhamnose (Rha) 8.15mol%, glucose (Glc) 4.04mol%, mannose (Man) 2.49mol%, glucuronic acid (GlcA) 2.46mol%, xylose (Xyl) 0.66mol%; its HG molar proportion is 20.83mol%, RG-I molar proportion is 69.52mol%, and RG-I side chain length is 6.53; Each 100 mg of RPDP-T contains 90.19 mg ± 1.67 mg of total polysaccharide, 73.53 mg ± 2.11 mg of total uronic acid, 2.35 mg ± 0.08 mg of total protein, and 1.73 mg GAE ± 0.01 mg GAE of total bound phenols. Its degree of esterification is 17.96% ± 0.55%, and its molecular weight is (0.842 ± 0.007) × 10 4 Da, polydispersity coefficient M w / M n is 1.363; its monosaccharide composition and molar percentage are: galacturonic acid (GalA) 76.40mol%, galactose (Gal) 7.99mol%, arabinose (Ara) 0.00mol%, rhamnose (Rha) 8.00mol%, glucose (Glc) 3.02mol%, mannose (Man) 3.17mol%, glucuronic acid (GlcA) 1.42mol%, xylose (Xyl) 0.00%; its HG molar proportion is 68.39mol%, RG-I molar proportion is 24.00mol%, and RG-I side chain length is 1.

00.

5. The method for preparing the roxburghii residue pectin polysaccharide according to any one of claims 1 to 4, characterized in that: It includes the following steps: a. Preparation of roxburghii pectin polysaccharide RPDP by deep eutectic solvent-assisted extraction technology; b. Take the roxburghii pear residue pectin polysaccharide RPDP prepared in step a and prepare the roxburghii pear residue modified pectin polysaccharide RPDP-E by using targeted enzymatic cleavage technology; or, c. Taking the pear residue pectin polysaccharide RPDP prepared in step a, a low-temperature controllable alkaline deesterification technology was used to prepare a pear residue modified pectin polysaccharide PRDP-A; or, d. Take the roxburghii residue pectin polysaccharide RPDP prepared in step a and use controlled partial acid hydrolysis technology to prepare roxburghii residue-modified pectin polysaccharide RPDP-T.

6. The method for preparing the roxburghii pear residue pectin polysaccharide according to claim 5, wherein: The preparation method of the deep eutectic solvent-assisted extraction technology in step a is: The prickly pear residue powder was mixed with 80% ethanol at a ratio of 1:10 (w / v) and ultrasonically treated in an ultrasonic cleaning tank at 480W and 25°C; after ultrasonic treatment, the mixture was centrifuged at 5000×g for 10 min, and the precipitate was mixed with a low eutectic solvent at a ratio of 1:40 (w / v), wherein the low eutectic solvent consisted of choline chloride, ethylene glycol and ultrapure water, and the molar ratio of choline chloride to ethylene glycol was 1:3, and the water content of the low eutectic solvent was 55% (v / v). The polysaccharide was extracted at 90°C for 3h; after extraction, the mixture was centrifuged at 4000×g for 15min, and the supernatant was concentrated by rotary evaporation, and the concentrate was added to the heat-stable α -amylase (5U / mL), saccharifying enzyme (5U / mL) and pancreatin (5U / mL) to remove starch, dextrin and protein; then inactivate at 95°C for 30 minutes, centrifuge (4000×g, 15 minutes), take the supernatant, add 4 volumes of 95% ethanol (v / v), and precipitate overnight at 4°C; the precipitate is reconstituted with ultrapure water and separated by 3kDa and 100kDa ultrafiltration centrifuge tubes respectively; finally, a polysaccharide fraction with a molecular weight between 3kDa and 100kDa is obtained; then, the purified polysaccharide fraction is vacuum freeze-dried at -80°C for 48 hours to obtain a high-purity pectin polysaccharide from roxburghii pear residue, named RPDP; The targeted enzyme cleavage technology described in step b is specifically: The pectin polysaccharide RPDP solution of roxburghii residue was mixed with the pectinase solution and then reacted at 40°C for 9 hours. After the reaction, the enzyme was inactivated at 95°C for 30 minutes. The upper layer was then collected by ultrafiltration using a 3kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain the pectin polysaccharide modified with roxburghii residue, named RPDP-E. The low-temperature controllable alkaline deesterification technology described in step c is specifically: The RPDP solution of pectin polysaccharide from roxburghii residue was mixed with a NaOH solution (the final pH value of the mixed solution was 11), and then stirred at 4°C for 30 minutes. After the reaction, HCl solution (1M) was added dropwise to the mixed solution until the mixed solution was neutral (pH = 7). Finally, the upper layer was collected by ultrafiltration using a 3kDa ultrafiltration centrifuge tube and freeze-dried at -80°C for 48 hours to obtain the pectin polysaccharide modified from roxburghii residue, which was named PRDP-A. The controllable partial acid hydrolysis technology described in step d is specifically: The RPDP solution of pectin polysaccharide from sea buckthorn residue was mixed with TFA solution (the final concentration of TFA was 0.25 M), and then placed at 95°C for reaction for 180 minutes; after the reaction, NaOH solution (1 M) was added dropwise to the mixed solution until the mixed solution was neutral (pH = 7), and finally the upper layer was collected by ultrafiltration using a 3kDa ultrafiltration centrifuge tube, and freeze-dried at -80°C for 48 hours to obtain the pectin polysaccharide modified from sea buckthorn residue, named RPDP-T.

7. Use of the roxburghii pome pectin polysaccharide according to any one of claims 1 to 4 in the preparation of a medicament for preventing or treating ulcerative colitis.

8. A pharmaceutical composition for treating or preventing ulcerative colitis, characterized in that: The invention contains the pectin polysaccharide of roxburghii residue according to any one of claims 1 to 4 as an active ingredient, and is added with pharmaceutically acceptable auxiliary ingredients to prepare a commonly used pharmaceutical preparation.

9. The pharmaceutical composition for treating or preventing ulcerative colitis according to claim 8, characterized in that: The preparation is an oral preparation or a colon administration preparation.

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