Application of swimming bladder polysaccharide in preparation of product for relieving colitis

Abstract

The invention discloses application of swimming bladder polysaccharide in preparation of a product for relieving colitis, and belongs to the technical field of biological medicine preparation. The swim bladder polysaccharide provided by the invention has no toxic or side effect on animals. By adopting the swim bladder polysaccharide provided by the invention, the DAI index of a mouse with DSS-induced colitis can be remarkably reduced, and weight loss and colon shortening can be relieved. The swim bladder polysaccharide provided by the invention can be used for relieving crypt atrophy, epithelial cell destruction, goblet cell reduction and massive inrush of inflammatory cells caused by DSS, and obviously reducing histological scores. Compared with mice in a DSS group, proinflammatory factors IL-1beta, IL-6 and TNF-alpha in colons of mice in a swim bladder polysaccharide intervention group are remarkably reduced, and an anti-inflammatory factor IL-10 is remarkably increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical preparation, and particularly relates to the application of fish swim bladder polysaccharide in the preparation of products for relieving colitis. Background Art

[0002] Inflammatory bowel disease (IBD) has become an increasingly prominent global health problem with a rising incidence year by year. This rapidly growing disease severely affects the daily lives of patients and greatly disrupts their normal functions and well-being. Ulcerative colitis (UC) is the primary subtype of IBD, mainly characterized by persistent inflammation of the mucosa and epithelium of the colon and rectum. This inflammation can trigger a variety of symptoms, from diarrhea and bloody stools to weight loss and intestinal microbiota disorders. UC is also a lifelong and recurrent disease, which can lead to partial colon resection or colorectal cancer in severe cases. Although traditional therapies (including 5-ASA, corticosteroids, and immunosuppressants) have made progress, current treatment methods for UC are often associated with a series of adverse reactions and symptom recurrence.

[0003] The fish swim bladder is the buoyancy regulation organ of fish, containing rich proteins, polysaccharides, and trace elements. Emerging scientific evidence indicates that fish swim bladder polysaccharide has broad therapeutic potential. Previous studies have found that fish swim bladder polysaccharide effectively prevents and inhibits the development and spread of colorectal cancer. In addition, fish swim bladder polysaccharide can also relieve charcoal-induced constipation in mice and prevent rifampicin-induced gastric ulcer damage, indicating its potential role in regulating intestinal health. However, there is no report on the use of fish swim bladder polysaccharide in relieving colitis. Summary of the Invention

[0004] [Technical Problem]

[0005] The technical problem to be solved by the present invention is that traditional therapies for treating colitis will cause adverse reactions and recurrence.

[0006] [Technical Solution]

[0007] In order to overcome the deficiencies of the prior art, the present invention provides an application of fish swim bladder polysaccharide in the preparation of products for relieving colitis. This fish swim bladder polysaccharide can effectively improve the severe symptoms of colitis, including weight loss, diarrhea, bloody stools, and colon shortening, has no toxic side effects, and has broad application prospects. The relieving effect of fish swim bladder polysaccharide on colitis is mainly achieved by inhibiting inflammatory factors, increasing anti-inflammatory factors, and enhancing intestinal barrier function.

[0008] The present invention provides an application of fish swim bladder polysaccharide in the preparation of products for relieving colitis.

[0009] In one embodiment of the present invention, the product is a drug or a food.

[0010] In one embodiment of the present invention, the colitis is ulcerative colitis.

[0011] In one embodiment of the present invention, the total sugar content of the fish swim bladder polysaccharide is 63.53 ± 1.59%, the protein content is 4.63 ± 0.14%, and the contents of uronic acid and sulfate groups are 6.04 ± 0.19% and 13.05 ± 0.75% respectively.

[0012] In one embodiment of the present invention, the dosage of the fish swim bladder polysaccharide is 300 mg / kg.

[0013] In one embodiment of the present invention, the fish swim bladder polysaccharide is prepared by an enzymatic hydrolysis and alcohol precipitation method.

[0014] In one embodiment of the present invention, the preparation method of the fish swim bladder polysaccharide comprises the following steps:

[0015] Cut the fish swim bladder into pieces, soak it in acetone for defatting, dry and grind it into powder. Mix the fish swim bladder powder with deionized water, add pepsin, and adjust the pH to 4.5; after the reaction, adjust the pH to 8.0, add trypsin, and after the reaction, adjust the pH to neutral and heat to inactivate the enzyme to obtain an enzymatic hydrolysis mixture; finally, centrifuge, take the supernatant, concentrate it, add ethanol for alcohol precipitation, take the precipitate, redissolve it, remove protein, dialyze, and freeze-dry to obtain the fish swim bladder polysaccharide.

[0016] In one embodiment of the present invention, the fish swim bladder powder is mixed with deionized water at a ratio of 1:30 (w / v, g / mL).

[0017] In one embodiment of the present invention, the addition amounts of pepsin and trypsin are both 3-4% of the mass of the fish swim bladder.

[0018] In one embodiment of the present invention, the fish swim bladder polysaccharide relieves colitis mainly by inhibiting inflammatory factors, increasing anti-inflammatory factors, and improving intestinal barrier function.

[0019] In one embodiment of the present invention, the fish swim bladder polysaccharide can reduce the disease activity index (DAI), and relieve weight loss and colon shortening.

[0020] In one embodiment of the present invention, the fish swim bladder polysaccharide can relieve the damage of colon tissue.

[0021] Advantages of the present invention:

[0022] (1) The fish swim bladder polysaccharide provided by the present invention has no toxic and side effects on animals.

[0023] (2) Using the fish swim bladder polysaccharide provided by the present invention can significantly reduce the DAI index of mice with DSS-induced colitis, and relieve weight loss and colon shortening.

[0024] (3) The use of the isinglass polysaccharide provided by the present invention can alleviate crypt atrophy, epithelial cell damage, goblet cell reduction, and massive influx of inflammatory cells caused by DSS, and significantly reduce the histological score.

[0025] (4) Compared with the mice in the DSS group, the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α in the colon of the mice in the isinglass polysaccharide intervention group were significantly reduced, and the anti-inflammatory factor IL-10 was significantly increased.

[0026] (5) Compared with using sea cucumber polysaccharide to relieve colitis in Patent CN119119321A, the DSS dose used in the present invention is 3.5%, which is higher than the dose in that patent and causes greater damage to the colon tissue. However, the inhibitory effect of the isinglass polysaccharide of the present invention on inflammatory factors is more stable and effective. In addition, the present invention can not only inhibit related inflammatory factors, but also significantly improve the symptoms of weight loss, colon shortening, and tissue damage caused by colitis. Description of the Drawings

[0027] Figure 1 It is the DAI graph (A), body weight graph (B), colon morphology (C), and colon length graph (D) of mice between different groups;

[0028] Figure 2 It is the HE staining graph (A) of the colon tissue of mice between different groups and the intestinal tissue damage score graph (B);

[0029] Figure 3 It is the graph of the content of each cytokine of mice between different groups. Detailed Embodiments

[0030] The present invention will be further described in detail below in conjunction with specific embodiments for the understanding of those skilled in the art.

[0031] Pepsin and trypsin: Pepsin (Sigma 9001-75-6 ≥ 500 U / mg); Trypsin (Sigma 9002-07-7 1000-2000 BAEE U / mg).

[0032] The ELISA kits for TNF-α (ml002095V), IL-10 (ml037873-2), IL-6 (ml063159), and IL-1β (ml063132) were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0033] The total sugar content of the isinglass polysaccharide was determined by the phenol-sulfuric acid method. The specific determination method is as follows:

[0034] Weigh 20 mg of the standard glucose with constant weight after drying into a 500 mL volumetric flask, make up the volume, respectively pipette 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6, 2.0 mL, each make up to 2.0 mL with distilled water, then add 1.0 mL of 5% phenol, and finally slowly add 5.0 mL of concentrated sulfuric acid, shake well and cool, develop color in a boiling water bath for 10 min and then cool to room temperature, measure the absorbance at 490 nm, use 1.0 mL of deionized water as the blank, with the abscissa being the polysaccharide concentration (mg / mL) and the ordinate being the absorbance value to obtain the standard curve; then calculate the polysaccharide content in the sample based on the absorbance of the measured sample.

[0035] The uronic acid content was determined by the m-hydroxybiphenyl method. The specific determination method is as follows:

[0036] (1) First, prepare 0.0125 mol / L sodium tetraborate-sulfuric acid solution, 0.15% m-hydroxybiphenyl solution and 60 μg / mL glucuronic acid standard stock solution;

[0037] (2) Pipette 0.00, 0.05, 0.10, 0.15, 0.20, 0.25 mL of 60 μg / mL glucuronic acid stock solution into glass test tubes and make up the volume to 0.25 mL with water;

[0038] (3) Place the glass test tubes in an ice-water bath, add 1.50 mL of 0.0125 mol / L sodium tetraborate-sulfuric acid solution in equal amounts, mix well, and then heat in a boiling water bath for 5 min;

[0039] (4) After cooling, add 25 μL of 0.15% m-hydroxybiphenyl solution in equal amounts, mix well, measure the absorbance at 520 nm, and all experiments are in triplicate. Use water as the blank control, with the uronic acid content as the abscissa and the absorbance value at 520 nm as the ordinate to draw the standard curve;

[0040] (5) Weigh 10 mg of the fish swim bladder polysaccharide sample and dissolve it in 10 mL of deionized water. Using the above experimental method, measure the absorbance at 520 nm, and substitute the measured absorbance value into the standard curve for calculation.

[0041] The content of sulfate groups was quantified by the barium chloride-gelatin turbidimetry method. The specific determination method is as follows:

[0042] (1) Place K2SO4 in an oven at 105 °C and dry to constant weight. Weigh 108.75 mg of the dried K2SO4, make up the volume to 100 mL with 1.0 M hydrochloric acid, shake well to obtain a standard solution with a sulfate group concentration of 0.6 mg / mL;

[0043] (2) Weigh accurately 0.5 g of gelatin and 1.0 g of BaCl₂, add them to 100 mL of deionized water, heat to dissolve, centrifuge and take the supernatant to obtain a BaCl₂-gelatin turbid solution;

[0044] (3) Pipette 0.00, 0.04, 0.08, 0.12, 0.16, and 0.20 mL of the standard solution into 10-mL test tubes respectively, add 1.0 M hydrochloric acid to make up to 0.2 mL, and in addition, add 3.8 mL of trichloroacetic acid and 1.0 mL of BaCl₂-gelatin turbid solution to each tube, mix well, and let stand for 15 min;

[0045] (4) Measure the absorbance at 360 nm, repeat three times, use the mass of sulfate group as the abscissa and the absorbance value as the ordinate to make a standard curve, measure the absorbance value of the sample according to the above operation method, and calculate the sulfate group content in the sample based on the standard curve.

[0046] The protein content was determined by the Coomassie brilliant blue method. The specific determination method is as follows:

[0047] Use the total protein (TP) assay kit (A045-2-2) from Nanjing Jiancheng Bioengineering Institute to detect the protein content in the fish swim bladder polysaccharide sample.

[0048] Example 1

[0049] First, cut 30 g of fish swim bladder into small pieces to increase its surface area, then immerse it in 100% acetone and completely cover it, degrease overnight at room temperature with stirring, dry the degreased fish swim bladder and grind it into powder for subsequent treatment. Then, mix the fish swim bladder powder with deionized water at a ratio of 1:30 (w / v), add 4% (w / v) pepsin, adjust the pH to 4.5, and react in a 37°C constant temperature water bath for 2 h for preliminary hydrolysis. Then, adjust the pH to 8.0, add 4% (w / v) trypsin, and react in a 37°C constant temperature water bath for 2 h for further hydrolysis. Subsequently, adjust the pH to neutral and heat to 80°C for 20 min to inactivate the enzyme. After centrifuging the enzymatic hydrolysis mixture at 8000 g for 30 min, collect the supernatant. After concentrating the supernatant, add 4 volumes of ethanol to a final concentration of 80% (v / v) to precipitate the polysaccharide, and let stand at 4°C for 24 h. Centrifuge to collect the crude polysaccharide precipitate, dissolve it in distilled water, add one-fourth volume of Sevage reagent (chloroform:n-butanol = 4:1), stir in a magnetic stirrer for 30 min, then centrifuge at 5000 r / min at room temperature for 5 min, repeat this operation until there is no protein residue in the middle layer. Finally, combine the supernatant, concentrate by rotary evaporation, and dialyze with a 3500 Da dialysis bag against running water for three days, and then lyophilize to obtain purified fish swim bladder polysaccharide.

[0050] The total sugar content of the fish swim bladder polysaccharide was 63.53 ± 1.59%, the protein content was 4.63 ± 0.14%, and in addition, the contents of uronic acid and sulfate groups were 6.04 ± 0.19% and 13.05 ± 0.75%, respectively.

[0051] Example 2

[0052] Improvement effect of fish swim bladder polysaccharide on colitis

[0053] Forty male C57BL / 6 mice were acclimated to the new environment for one week. Subsequently, the mice were randomly assigned to four different groups, with each group consisting of 10 mice, namely the control group (Con), DSS group, CIP group, and CIP + DSS group;

[0054] The mice in the CIP and CIP + DSS groups were orally gavaged with 300 mg / kg 小鼠 of fish swim bladder polysaccharide, while the control group and DSS group received 200 μL of normal saline, and gavage was performed for 21 days; on the 14th day, colitis was induced in the DSS group and DSS + CIP group, and a solution containing 3.5% DSS was continuously added to the drinking water for seven days to induce colitis in the DSS group and DSS + CIP group.

[0055] During the entire study process, the body weights of the mice were recorded daily, the characteristics of the feces and the presence of occult blood were observed, and scoring was performed according to the scoring criteria of the disease activity index in Table 1. The total score of the three obtained results was divided by 3 to obtain the disease activity index (DAI). All animal experiments were approved by the Animal Ethics Committee of Dalian Polytechnic University, and the experiments were conducted in accordance with the guidelines of the National Institute of Animal Experimentation.

[0056] Table 1 Scoring criteria for the disease activity index

[0057] Score Body weight Fecal characteristics Occult blood condition 0 No weight loss Normal formed feces No bloody stool 1 1 - 5% weight loss Slightly soft, unformed Small amount of bloody stool (occult blood) 2 5 - 10% weight loss Loose stool Obvious bloody stool 3 10 - 20% weight loss Diarrhea Large amount of bloody stool 4 >20% weight loss Severe diarrhea or bloody stool Large amount of bloody stool with anal bleeding

[0058] After the experiment, the mice were sacrificed by cervical dislocation after carbon dioxide inhalation anesthesia, the colon tissues were collected and the colon length was measured. Distal colon tissues of each group were taken and immersed in 4% paraformaldehyde solution for preservation. After preservation, these samples were embedded in paraffin and cut into 5-μm-thick sections. In order to observe the tissue structure characteristics, these sections were stained by hematoxylin-eosin (H&E) staining method. After staining, the morphological characteristics of the colon tissue sections were carefully observed and photographed using a Nikon Eclipse TI-S microscope in Japan.

[0059] After the experiment, the mice were sacrificed, and the colon tissues were used to measure the levels of cytokines (TNF-α, IL-6, IL-1β, IL-10) in the mouse colon tissues using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions.

[0060] Result measurement and analysis

[0061] 1. Relief of symptoms of colitis in mice by isinglass polysaccharide

[0062] DSS can induce severe diarrhea and bloody stools in mice, and at the same time, there is a significant decrease in body weight, which are the characteristics of ulcerative colitis. Treatment with isinglass polysaccharide can significantly improve these symptoms. The DAI score is measured based on the weight loss, diarrhea, and bloody stools of mice. Treatment with isinglass polysaccharide significantly reduces the DAI value (as shown in Figure 1 A and B).

[0063] Another characteristic of DSS colitis is the shortening of the colon length. After euthanasia, the colon length of each experimental group was measured. Compared with the control group, isinglass polysaccharide intervention significantly reduced the shortening of the colon length caused by DSS.

[0064] The histological score reflects the degree of damage to the colon tissue by quantitatively evaluating key histological features such as the degree of inflammatory cell infiltration, the degree of epithelial cell damage, the degree of edema, and the formation of ulcers in the colon tissue. Scoring is carried out according to the histological scoring criteria in Table 2, and the total score obtained from the four items is the histological score. As shown in Figure 2 A, DSS treatment led to obvious inflammatory reactions and severe histological damage, manifested as crypt atrophy, epithelial cell destruction, and a large influx of inflammatory cells. On the contrary, the crypt structure in the CIP + DSS group was better preserved and the inflammatory cell infiltration decreased, indicating that isinglass polysaccharide has a protective effect on colitis. In addition, isinglass polysaccharide treatment significantly reduced the histological score, further confirming its protective effect ( Figure 2 B).

[0065] Table 2 Histological scoring criteria

[0066]

[0067] Therefore, the isinglass polysaccharide of the present invention can relieve the symptoms of ulcerative colitis and reduce the damage to the colon tissue.

[0068] 2. Anti-inflammatory effect of isinglass polysaccharide

[0069] As shown in Figure 3 , compared with the normal group, DSS treatment led to a significant increase in the levels of pro-inflammatory cytokines IL-1β, IL-6, and TNF-α, while the level of the anti-inflammatory cytokine IL-10 was significantly decreased. On the contrary, the levels of IL-1β, IL-6, and TNF-α in the DSS + CIP group were significantly decreased, and the level of IL-10 was significantly increased. Therefore, isinglass polysaccharide significantly reduces pro-inflammatory factors and increases the content of anti-inflammatory factors, proving that isinglass polysaccharide has a good anti-inflammatory effect.

[0070] The embodiments provided above are not intended to limit the scope covered by the present invention, nor are the described steps intended to limit the order of their execution. Obvious improvements made by those skilled in the art to the present invention in combination with the existing well-known general knowledge also fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of fish maw polysaccharide in the preparation of products for relieving colitis.

2. The use according to claim 1, characterized in that: The product is a medicine or a food.

3. The use according to claim 1, characterized in that: The colitis is ulcerative colitis.

4. The use according to claim 1, characterized in that: Fish maw polysaccharides mainly relieve colitis by inhibiting inflammatory factors, increasing anti-inflammatory factors and improving intestinal barrier function.

5. The use according to claim 1, characterized in that: Fish maw polysaccharides can reduce disease activity index, reduce weight loss and colon shortening.

6. The use according to claim 1, characterized in that: Fish bladder polysaccharides can alleviate colon tissue damage.

7. The use according to claim 1, characterized in that: Fish maw polysaccharide is prepared by enzymatic hydrolysis and alcohol precipitation.

8. The use according to claim 1, characterized in that: The preparation method of fish maw polysaccharide comprises the following steps: The fish maw is cut into pieces and immersed in acetone for defatting, dried and ground into powder, the fish maw powder is mixed with deionized water, pepsin is added, and the pH is adjusted to 4.5; after the reaction is completed, the pH is adjusted to 8.0, trypsin is added, and after the reaction is completed, the pH is adjusted to neutral and heated to inactivate the enzyme to obtain an enzymatic hydrolysis mixture; finally, centrifugation is performed, the supernatant is concentrated, ethanol is added for alcohol precipitation, the precipitate is re-dissolved, deproteinized, dialyzed, and freeze-dried to obtain fish maw polysaccharide.

9. The use according to claim 1, characterized in that: The fish maw powder was mixed with deionized water at a ratio of 1:30, w / v, g / mL.

10. The use according to claim 1, characterized in that: The added amount of pepsin and trypsin is 3-4% of the mass of the fish maw.

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