Application of Laiyang pear residue polysaccharide in preparing medicine for treating and / or preventing ulcerative colitis

Abstract

The present invention relates to the field of biomedicine technology, and specifically to the use of Laiyang pear residue polysaccharide in the preparation of drugs for treating and / or preventing ulcerative colitis. The in vitro digestion characteristics of Laiyang pear residue polysaccharide, its mechanism of action in improving ulcerative colitis and related signal pathways were studied. Laiyang pear residue polysaccharide was partially degraded under simulated salivary-gastrointestinal digestion conditions, resulting in a significant increase in reducing sugar content and a change in molecular weight. By constructing a dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model, it was proved that Laiyang pear residue polysaccharide has an improving effect on UC; at the same time, it provides a basis for the research and development of Laiyang pear residue polysaccharide drugs for the treatment of ulcerative colitis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of Laiyang pear residue polysaccharide in preparing medicine for treating and / or preventing ulcerative colitis. Background Art

[0002] Polysaccharides from natural sources need to be digested and absorbed by the human body before they can play a role after reaching the colon. After being digested by gastric juice and intestinal juice, polysaccharides reach the colon in a complete or incomplete form and interact with the intestinal flora to exert their functional activity. Therefore, to study the functional activity of polysaccharides, it is necessary to study their digestion characteristics. Undigested polysaccharides can be used by intestinal flora to promote the production of beneficial metabolites such as short-chain fatty acids, and regulate the structure and composition of the microbiome, making them potential prebiotics.

[0003] Ulcerative colitis (UC) is a chronic idiopathic inflammatory bowel disease (IBD) that seriously affects the quality of life of patients, and its incidence is on the rise in Asia and around the world. Clinically, UC patients often present with symptoms such as blood in stool, abdominal pain, and low-grade fever, accompanied by a series of complications, and may even increase the risk of colorectal cancer. The symptoms of UC mainly affect the colonic mucosa. Intestinal mucosal damage or changes in intestinal flora may be triggers for UC. Studies have shown that intestinal flora and the progression of UC are interrelated through regulating immune responses, inflammation, and epithelial barrier function. Therefore, the intestinal microbiota may be a new target for the prevention and alleviation of UC, because regulating the intestinal microbiota can improve chronic inflammation and slow the progression of UC. However, many existing treatment strategies have adverse effects on patient health, and expensive drugs and long-term use may lead to immunosuppression and increased susceptibility to infection. Therefore, it is necessary to develop alternative treatment strategies without side effects. Therefore, it is of great significance to find anti-inflammatory polysaccharides from natural plants. China is rich in pear resources. Laiyang pear is the fresh fruit of the cultivated variety of Pyrus dasyphylla, a plant of the Rosaceae family. It is one of the traditional precious varieties of pears in Shandong. Laiyang pear residue is rich in dietary fiber (mainly polysaccharides) and has high nutritional value. However, Laiyang pear residue is often regarded as waste and has low value for comprehensive utilization.

[0004] Chinese patent CN 115025112A discloses the use of Angelica dahurica polysaccharide in preparing drugs for preventing and treating ulcerative colitis, and Chinese patent CN 113491706A discloses the use of chickpea polysaccharide in preparing drugs for treating and / or preventing ulcerative colitis. However, Angelica dahurica and chickpea in this application have medicinal value; while the Laiyang pear residue in this application itself is waste. Summary of the invention

[0005] In view of the problem of low utilization of Laiyang pear residue at this stage, the present invention provides a new use of Laiyang pear residue polysaccharide, studies the in vitro digestion characteristics of Laiyang pear residue polysaccharide and its mechanism of action and related signal pathways in improving ulcerative colitis, and provides a basis for the development of Laiyang pear residue polysaccharide drugs for the treatment of ulcerative colitis.

[0006] The technical solution of this application is as follows:

[0007] Application of Laiyang pear residue polysaccharide in preparing medicine for treating and / or preventing ulcerative colitis.

[0008] Furthermore, the Laiyang pear residue polysaccharide is prepared by the following method:

[0009] (1) Laiyang pear residue is dried, crushed, and extracted with hot water;

[0010] (2) centrifuging the extract from step (1) and concentrating the supernatant;

[0011] (3) The concentrated solution of step (2) is subjected to alcohol precipitation; centrifugation, and drying;

[0012] (4) After the precipitate is re-dissolved, it is centrifuged to remove impurities, dialyzed with running deionized water, and freeze-dried.

[0013] Preferably, the hot water extraction process in step (1) is: 80-100°C, the solid-liquid ratio is 1:20-40, and the time is 3-5h.

[0014] Preferably, the supernatant in step (2) is concentrated to 1 / 3 or less of the original volume.

[0015] Preferably, the conditions for the alcohol precipitation in step (3) are: using 2-4 volumes of 85-95% ethanol for alcohol precipitation.

[0016] Preferably, the conditions for the flowing deionized water dialysis in step (4) are 3000-4000 kDa, 50-90 h.

[0017] Furthermore, the Laiyang pear residue polysaccharide is combined with one or more pharmaceutically acceptable excipients to prepare a preparation.

[0018] Preferably, the auxiliary materials include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers or lubricants in the pharmaceutical field.

[0019] Furthermore, the preparation is a tablet, a pill, a capsule, a suspension or an emulsion.

[0020] Furthermore, during the digestion stage in the stomach and intestines, the glycosidic bonds of the Laiyang pear residue polysaccharide are destroyed, the reducing sugar content is increased, the mannose Man content is increased, the molecular weight is changed, and the antioxidant activity is enhanced.

[0021] The study found that: Monosaccharide composition and molecular weight analysis showed that Laiyang pear residue polysaccharide had no significant changes in the oral digestion stage. In the gastric and intestinal digestion stages, the glycosidic bonds of Laiyang pear residue polysaccharide were destroyed, the reducing sugar content increased significantly, and the molecular weight changed. Laiyang pear residue polysaccharide alleviates intestinal pathological damage by enhancing intestinal barrier integrity and upregulating the expression of tight junction proteins. Laiyang pear residue polysaccharide relieves intestinal inflammation by inhibiting the NF-κB signaling pathway. Laiyang pear residue polysaccharide reduces the ratio of Firmicutes / Bacteroidetes, increases the relative abundance of Lactobacillus, and changes the diversity and composition of the intestinal microbiota, thereby improving colitis.

[0022] Beneficial Effects of the Invention

[0023] For the first time, an in vitro digestion model was used to study the digestion characteristics of Laiyang pear residue polysaccharide during in vitro digestion. The results showed that Laiyang pear residue polysaccharide was partially degraded under simulated salivary-gastrointestinal digestion conditions, resulting in a significant increase in reducing sugar content and a change in molecular weight. By constructing a dextran sulfate sodium (DSS)-induced ulcerative colitis mouse model, it was demonstrated that Laiyang pear residue polysaccharide has an improvement effect on UC. Therefore, Laiyang pear residue polysaccharide has the potential to become a functional food that improves the intestinal flora environment, enhances health and prevents diseases, such as a prebiotic. These findings provide theoretical guidance and practical experience for further research on the therapeutic effects of Laiyang pear residue polysaccharide and its products, and have important theoretical and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flow chart of extraction and in vitro digestion of Laiyang pear residue polysaccharides;

[0025] Figure 2 Effects of different factors on the yield of polysaccharides from Laiyang pear residue: (A) solid-liquid ratio; (B) extraction temperature; (C) extraction time; (D) extraction times;

[0026] Figure 3 Changes in structural characteristics of Laiyang pear residue polysaccharides during in vitro digestion: (A) monosaccharide composition; (B) Fourier transform infrared spectrum; (C) Congo red staining curve; (D) thermogravimetric curve; (E) ABTS free radical scavenging activity; (F) DPPH free radical scavenging activity;

[0027] Figure 4Effects of Laiyang pear residue polysaccharide on body weight and colon length in colitis mice: (A) Experimental design process; (B) Mouse body weight; (C) Disease activity index (DAI); (D) Colon image; (E) Average colon length; (F) Spleen index;

[0028] Figure 5 Effects of Laiyang pear residue polysaccharide on inflammatory response in colitis mice: (AC) Expression levels of IL-1β, IL-6, and TNF-α in colon; (D) Representative WB immunoblot images; (E) Relative expression density ratios of p-NF-κB / NF-κB, iNOS / β-Actin, and COX-2 / β-Actin;

[0029] Figure 6 Effects of Laiyang pear residue polysaccharide on colonic histopathological changes in colitis mice: (A) Colonic H&E staining, Alcian blue staining and ZO-1 immunohistochemical analysis; (B) Histological score; (C) Goblet cells / crypts; (D) Positive density;

[0030] Figure 7 Effects of Laiyang pear residue polysaccharide on intestinal barrier integrity in colitis mice: (AC) ZO-1, Occludin, Claudin-1 mRNA expression; (D) WB representative immunoblot images; (E) Relative expression density ratio of Occludin / β-Actin and Claudin-1 / β-Actin;

[0031] Figure 8 Effects of Laiyang pear residue polysaccharide on alpha diversity of intestinal flora in colitis mice: (A) Alpha diversity analysis; (B) Venn diagram;

[0032] Fig. 9 Effects of Laiyang pear residue polysaccharide on the β-diversity of intestinal flora in colitis mice: (A) PCA analysis; (B) PCoA analysis; (C) NMDS analysis;

[0033] Fig.10 Effects of Laiyang pear residue polysaccharide on the intestinal flora composition of colitis mice: (A) Relative abundance at the phylum level; (B) Relative abundance at the genus level; (C) Heat map at the phylum level; (D) Heat map at the genus level. DETAILED DESCRIPTION

[0034] Example 1 Extraction and in vitro digestion of Laiyang pear residue polysaccharides

[0035] 1.1 Extraction of Laiyang pear residue polysaccharides

[0036] Raw material pretreatment: From the perspective of rational utilization of resources, Laiyang pear residue was purchased from Yantai Aitaozhang Food Co., Ltd. as raw material, dried in an oven at 60 ℃, crushed with a high-speed universal grinder, passed through a 60-mesh sieve, and pear residue dry powder was obtained and sealed in a sealed bag for later use.

[0037] Determination of extraction method: In the preliminary experimental study, we compared the effects of hot acid extraction, hot alkali extraction, ultrasound-assisted extraction and traditional hot water extraction on the yield of Laiyang pear residue polysaccharides, and finally chose the hot water extraction method based on the comprehensive yield and the uniformity and integrity of the biological properties of polysaccharides. At the same time, in order to take into account the extraction rate and extraction energy consumption, the solid-liquid ratio was determined to be 1:20-40, the extraction temperature was 80-100 ℃, and the extraction time was 3-5 h. After the extraction, the supernatant was collected by centrifugation at 5000 r / min for 15 min, and the precipitate was repeated twice by hot water extraction. The supernatant was combined and concentrated to one-third of the original volume. Three times the volume of 95% ethanol was added to the concentrate for alcohol precipitation (3 times alcohol precipitation), and the alcohol precipitation was centrifuged (5000 r / min, 15min), and the precipitate was dried at 60℃. The obtained precipitate was re-dissolved, centrifuged to remove impurities, dialyzed with flowing deionized water (3500kDa) for 72 h, and then freeze-dried, named LPP, and sealed for storage.

[0038] 1.2 In vitro digestion of Laiyang pear residue polysaccharides

[0039] 1.2.1 In vitro digestion

[0040] The simulated saliva formula was a mixture of KSCN (14.54 g), NaCl (127.49 g), KCl (65.16 g), NaHCO3 (61.60 g), Na2SO4 (41.45 g), NaH2PO4 (64.58 g), urea (18.18 g), α-amylase (210.90 mg), and distilled water (1.0 L), pH 6.8. 50 mL of TFP solution (6.67 mg / mL) was mixed with 50 mL of simulated saliva and incubated in a 37°C water bath with shaking. The digestion samples were collected after 0.25, 0.5, and 1.0 h, and the enzyme was inactivated in a boiling water bath for 10 min. 0.45 g pepsin was dissolved in HCl (0.1 M) and added to 50 mL saliva digestion sample to start gastric digestion, and 0.5 mol / L HCl solution was continuously added to adjust the solution pH to 2.0 within 1 h. After incubation at 37 °C for 0.5, 1.0, 2.0, 4.0, and 6.0 h, the partially digested samples were collected again and the enzyme was inactivated in a boiling water bath for 10 min. Finally, the pH of the sample solution after saliva gastric digestion was adjusted to pH 6.8 by adding NaHCO3 solution, and a mixture of pancreatic enzyme (3.5 mg / mL) and bile salt (22 mg / mL) was added to the remaining digestion solution at a ratio of 3:10 (v / v). After incubation at 37 °C for 0.5, 1.0, 2.0, 4.0, and 6.0 h, the sample digestion solution was collected again and the enzyme was inactivated for subsequent detection. The samples collected from each digestion process were centrifuged and the supernatant was taken, precipitated with alcohol, redissolved and dialyzed. The dialyzed solution was collected, concentrated and freeze-dried to obtain digestion samples at different stages, named LPP-S, LPP-G and LPP-I.

[0041] 1.2.2 Determination of basic indicators

[0042] 1.2.2.1 Determination of reducing sugar content

[0043] Preparation of DNS reagent: Accurately weigh 0.63 g of 3,5-dinitrosalicylic acid, 26.2 mL of 2 mol / L sodium hydroxide, 0.5 g of crystalline phenol, and 0.5 g of sodium sulfite, dissolve in 50 mL of 0.36 g / mL hot aqueous solution of potassium sodium tartrate, make up to 100 mL with deionized water, store in a dark place, and let stand at 4°C for one week before use.

[0044] Drawing of standard curve: Dry anhydrous glucose to constant weight and prepare 2 mg / mL glucose standard solution for use. Accurately pipette 0, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mL into a 15 mL stoppered test tube, add water to 1 mL, add 1.5 mL of DNS solution, mix well, heat in a boiling water bath for 5 min, cool quickly, add water to 15 ml, and measure the absorbance at 540 nm. The linear regression equation Y=1.1914X-0.0074, R 2 =0.9995.

[0045] Ensure that the pH value of the sample is around 7.0, dilute the digestion solution in an appropriate concentration gradient, and perform the measurement according to the above steps. Substitute the absorbance value into the standard curve to measure the reducing sugar content.

[0046] 1.2.2.2 Component Analysis

[0047] The total sugar content, uronic acid content and total protein content of LPP and the products of different digestion stages, LPP-S, LPP-S and LPP-I, were determined respectively.

[0048] 1.2.2.3 Monosaccharide composition analysis

[0049] Weigh 5 mg of sample into a stoppered test tube, add 5.0 mL of 2 M TFA, acid hydrolyze at 110 ° C for 8 h, evaporate TFA with nitrogen, and reconstitute with 5.0 mL of ultrapure water. Use Thermo Fisher U3000 instrument and Xtimate C18 column for detection, column temperature: 30 ° C, flow rate 1.0 mL / min, detection wavelength 250 nm, injection volume 20 μL, mobile phase 0.05 M potassium dihydrogen phosphate solution and acetonitrile.

[0050] 1.2.2.4 Infrared spectroscopy analysis

[0051] 1 mg polysaccharide powder and 190 mg KBr were mixed and ground evenly, and pressed into tablets. The test conditions were wave number 400~4000cm -1 , resolution 4cm -1 , the cumulative number of scans is 32 times.

[0052] 1.2.2.5 Thermogravimetric analysis

[0053] Accurately weigh 3.0 mg of sample into a crucible and use a thermogravimeter under N2 atmosphere to measure the temperature in the temperature range of 25-600℃ at a heating rate of 10℃ / min to obtain a thermogravimetric curve (TG).

[0054] 1.2.2.6 Congo red staining experiment

[0055] The polysaccharide samples LPP, LPP-S, LPP-G, and LPP-I were respectively prepared into 0.5 mg / mL solutions, and 80 μmol / L Congo red reagent and 1 mol / L sodium hydroxide solution were prepared for use. Take 2 mL of polysaccharide sample, add 2 mL of Congo red reagent, and then add 2 mL of sodium hydroxide solution to increase the concentration of sodium hydroxide in the system from 0 mol / mL to 0.5 mol / mL, and measure the maximum absorption wavelength within 200-400 nm by UV-visible spectrometer.

[0056] Experimental results: During the entire digestion process, the total sugar content changed significantly, and the uronic acid level and protein content decreased significantly. In addition, LPP is composed of mannose (Man), ribose (Rib), rhamnose (Rha), glucose (Glc), caramel (Fuc), glucuronic acid (GlcA), xylose (Xyl), galactose (Gal), galacturonic acid (GalA) and arabinose (Ara). After digestion, the Man content increased, indicating that in vitro digestion can affect the monosaccharide composition of polysaccharides. Polysaccharides with a relatively high Man content may have a stronger effect in alleviating colitis. In addition, LPP is also a polysaccharide with a triple helix structure.

[0057] Table 1 Summary of data for LPP, LPP-S, LPP-G, and LPP-I

[0058]

[0059] Note: ad in the table indicates significant difference with p < 0.05.

[0060] Table 2 Reducing sugars of LPP (C R ) Content changes

[0061]

[0062] Note: ad in the table indicates significant difference with p < 0.05.

[0063] Example 2 Improvement of Laiyang pear residue polysaccharide on DSS-induced UC mice

[0064] 2.1 Modeling and treatment

[0065] 40 male mice, 6 weeks old, were selected and housed in an environment with a constant temperature of 25°C, relative humidity of 35%-55%, light for 12 h, and free access to food. After the mice adapted for 1 week, they were randomly divided into 5 groups. Figure 3 As shown in A. At the end of the experiment, the mice were fasted for 12 hours and anesthetized with 4% chloral hydrate (0.1 mL / 10 g .p) and then killed.

[0066] 2.2 Biochemical analysis

[0067] After killing the mice, blood was collected from the ophthalmic venous plexus and centrifuged (12 000 rpm, 4°C, 10 min) to obtain serum. Colon tissue was added with physiological saline and ground into a homogenate, centrifuged at 1700g and 4°C for 10 min, and the supernatant was collected. ELISA kits were used to detect the levels of inflammation-related factors (TNF-α, IL-6, IL-1β) and oxidative stress-related indicators (SOD, GSH, CAT, and MDA).

[0068] 2.3 Histopathological analysis

[0069] Colonic histopathology was evaluated by H&E staining and Alcian blue staining. Briefly, colonic tissue samples were fixed in 4% paraformaldehyde (PFA) overnight (4 °C), dehydrated in graded ethanol, and then embedded in paraffin, sectioned at 4 μm, stained with hematoxylin-eosin dye or Alcian blue dye, and then histopathologically observed under an Olympus BX-FM light microscope.

[0070] 2.4 PCR

[0071] qPCR was used to detect the expression levels of ZO-1, Occludin, and Claudin-1 mRNA. Briefly, RNA from mouse colon tissue was extracted using the Tissue Total RNA Isolation Kit, and its concentration was determined by nucleic acid quantification, diluted, reverse transcribed, and analyzed. All reactions were performed on the ABI 7500 Real-Time PCR System according to standard protocols. The threshold cycle (Ct) values ​​were compared using 2 −ΔΔCT Methods Calculation and analysis were performed with U6 as internal control.

[0072] 2.5 WB

[0073] Western blot was used to detect the expression levels of NF-κB and tight junction proteins (ZO-1, Occludin, and Claudin-1) in colon tissue. Proteins were extracted from mouse colon tissue and then separated by SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were incubated with primary antibodies at 4°C overnight. After incubation with horseradish peroxidase (HRP), the bands were determined.

[0074] 2.6 Immunohistochemical analysis

[0075] ZO-1 levels were detected by immunohistochemistry. Briefly, tissue sections were incubated with the corresponding primary antibodies overnight and titrated with the corresponding secondary antibodies for 30 min. Sections were developed by adding DAB colorimetric reagent, observed on a light microscope, and analyzed using Image ProPlus 6.0.

[0076] 2.7 16S rRNA sequencing

[0077] The mice were placed on sterile filter paper, and 3-5 pieces of fresh feces were collected in sterile EP tubes, immediately frozen with liquid nitrogen for 15 minutes, and stored at -80°C. DNA was extracted from mouse fecal samples using the e.zn.a.® soil DNA kit. Primers 338F and 806R were used to determine the V3 and V4 regions of the bacterial 16S rRNA gene sequences in feces. Purification and quantification were performed, and sequencing libraries were constructed for sequencing analysis. Data were analyzed on the Majorbio cloud platform.

[0078] Experimental results: Laiyang pear residue polysaccharide delayed the degree of weight loss in UC mice and improved symptoms such as colon shortening and bloody stools. Laiyang pear residue polysaccharide significantly inhibited the loss of goblet cells, alleviated the damage to colon tissue, and reduced intestinal pathological damage. At the same time, it upregulated the expression of tight junction proteins and protected the integrity of the intestinal barrier. Laiyang pear residue polysaccharide significantly reduced the expression of proinflammatory cytokines (IL-1β, IL-6 and TNF-α) and inhibited colon inflammation. In addition, Laiyang pear residue polysaccharide reduced the ratio of Firmicutes / Bacteroidetes, increased the relative abundance of Lactobacillus, and changed the diversity and composition of the intestinal microbiota, thereby improving colitis.

Claims

1. Application of Laiyang pear residue polysaccharide in the preparation of a drug for treating and / or preventing ulcerative colitis; characterized in that: The Laiyang pear residue polysaccharide is prepared by the following method: (1) Laiyang pear residue is dried, crushed, and extracted with hot water; (2) centrifuging the extract of step (1) and concentrating the supernatant; (3) The concentrated solution of step (2) is subjected to alcohol precipitation; centrifugation, and drying; (4) After the precipitate is re-dissolved, it is centrifuged to remove impurities, dialyzed with running deionized water, and then freeze-dried.

2. The use according to claim 1, characterized in that: The hot water extraction process in step (1) is: 80-100°C, a solid-liquid ratio of 1:20-40, and a time of 3-5 hours.

3. The use according to claim 1, characterized in that: The supernatant in step (2) is concentrated to 1 / 3 or less of the original volume.

4. The use according to claim 1, characterized in that: The conditions for the alcohol precipitation in step (3) are: using 2-4 times the volume of 85-95% ethanol for alcohol precipitation.

5. The use according to claim 1, characterized in that: The conditions for the flowing deionized water dialysis in step (4) are 3000-4000 kDa, 50-90 h.

6. The use according to any one of claims 1 to 5, characterized in that: The Laiyang pear residue polysaccharide is combined with one or more pharmaceutically acceptable excipients to prepare a preparation.

7. The use according to claim 6, characterized in that: The auxiliary materials include conventional diluents, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants in the pharmaceutical field.

8. The use according to claim 6, characterized in that: The preparation is in the form of tablets, pills, capsules, suspensions or emulsions.

9. The use according to any one of claims 1 to 5, characterized in that: During the digestion stage in the stomach and intestine, the glycosidic bonds of the Laiyang pear residue polysaccharide are destroyed, the reducing sugar content is increased, the mannose Man content is increased, the molecular weight is changed, and the antioxidant activity is enhanced.

Images