Diosmim oligosaccharides, methods for preparing the same, and use thereof in the preparation of products for preventing or treating ulcerative colitis

Abstract

The present application relates to the technical field of oligosaccharide, in particular to a Dioscorea panthaica Prain et Buck oligosaccharide, a preparation method thereof and application of the Dioscorea panthaica Prain et Buck oligosaccharide in preparing a product for preventing or treating ulcerative colitis. In the present application, Dioscorea panthaica Prain et Buck polysaccharide and a ferrous ion solution are mixed, pH is adjusted to 2.0-6.0, a hydrogen peroxide solution is added, and ultrasonic treatment is carried out in an ultrasonic cell crusher to obtain a Dioscorea panthaica Prain et Buck polysaccharide degradation solution; the Dioscorea panthaica Prain et Buck polysaccharide degradation solution and a sodium hydroxide solution are mixed, solid substances and hydrogen peroxide are removed, and after purification and drying, the Dioscorea panthaica Prain et Buck oligosaccharide is obtained. The Dioscorea panthaica Prain et Buck oligosaccharide can be used for preparing a product related to preventing or treating ulcerative colitis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of oligosaccharides, in particular to a Dioscorea panthaica oligosaccharide, a preparation method thereof and application of the Dioscorea panthaica oligosaccharide in preparing a product for preventing or treating ulcerative colitis. BACKGROUND

[0002] Oligosaccharide, also known as oligosaccharide, is a carbohydrate containing a small amount of monosaccharide residues. According to different biological functions, oligosaccharides can be divided into ordinary oligosaccharides and functional oligosaccharides. There is no enzyme system for hydrolyzing functional oligosaccharides (except isomaltulose) in the human gastrointestinal tract, so they are not digested and absorbed directly into the large intestine, thereby being preferentially utilized by intestinal flora. As an excellent functional food base, functional oligosaccharides can be used as dietary fiber and prebiotics to enrich the diet and improve the intestinal microecology, and have the characteristics of anti-tumor, anti-inflammatory, anti-oxidation and blood lipid-lowering.

[0003] Inflammatory bowel disease (IBD) generally refers to inflammatory lesions of the colon caused by genetic susceptibility, intestinal microbiome changes, congenital and adaptive immune system defects, and different environmental exposures, and mainly includes ulcerative colitis (UC) and Crohn's disease (CD). The first-line drugs for treating ulcerative colitis in clinical practice mainly include western medicines (such as salicylic acid, glucocorticoids, immunosuppressants, etc.) and biological agents. These drugs have significant drawbacks: salicylic acid drugs can relieve symptoms, but have limited effect; glucocorticoids have significant side effects; immunosuppressants treat ulcerative colitis by suppressing cellular immunity and humoral immunity, and are prone to cause adverse reactions; biological agents are prone to drug resistance when used for a long time. SUMMARY

[0004] Therefore, the present application aims to provide a Dioscorea panthaica oligosaccharide, a preparation method thereof and application of the Dioscorea panthaica oligosaccharide in preparing a product for preventing or treating ulcerative colitis, which at least solves one problem in the prior art.

[0005] In a first aspect, the present application provides a Dioscorea panthaica oligosaccharide, which has a molecular weight of 2 kDa, and a monosaccharide composition comprising glucose (Glc), galacturonic acid (Gal-UA), galactose (Gal) and rhamnose (Rha), and a molar ratio of glucose, galacturonic acid, galactose and rhamnose being 59.3:2.6:2.0:1.0.

[0006] In a second aspect, the present application provides a preparation method of the Dioscorea panthaica oligosaccharide, which comprises the following steps:

[0007] The Pachyrhizus polysaccharide and ferrous ion solution are mixed, the pH is adjusted to 2.0-6.0, a hydrogen peroxide solution is added, and the mixture is subjected to ultrasonic treatment in an ultrasonic cell crusher to obtain a Pachyrhizus polysaccharide degradation solution.

[0008] The Pachyrhizus polysaccharide degradation solution and a sodium hydroxide solution are mixed, and solid substances and hydrogen peroxide are removed, and after purification and drying, Pachyrhizus oligosaccharides are obtained.

[0009] Ultrasonic treatment is a "green" technology for degrading various forms of polymers including polysaccharides. Fenton oxidation reaction has the characteristics of high efficiency, low toxicity, mild reaction conditions, environmental protection and low cost, and is an effective advanced oxidation process technology. In the present application, ultrasonic treatment and Fenton oxidation reaction are combined (referred to as ultrasonic-Fenton combined technology), which can combine the advantages of the two methods and work synergistically. The ultrasonic-Fenton combined technology uses ultrasonic to accelerate the decomposition of hydrogen peroxide, thereby accelerating the reaction rate and efficiency of polysaccharide degradation.

[0010] In some alternative embodiments, the Pachyrhizus polysaccharide has a loose sheet structure, a relative weight average molecular weight of 26.5 kDa, a total sugar content of 94.3%, and a uronic acid content of 5.5%.

[0011] In some alternative embodiments, the concentration of ferrous ions in the ferrous ion solution is 1 mM-5 mM. Preferably, the concentration of ferrous ions in the ferrous ion solution is 2 mM-4 mM. The amount of ferrous ions required to prepare 1 kg of Pachyrhizus oligosaccharides is 112 g-222 g.

[0012] In some alternative embodiments, the pH is adjusted to 2.0-6.0. Preferably, the pH is adjusted to 3.0.

[0013] In some alternative embodiments, the concentration of the hydrogen peroxide solution is 10 mM-40 mM. Preferably, the concentration of the hydrogen peroxide solution is 15 mM-30 mM. The amount of hydrogen peroxide solution required to prepare 1 kg of Pachyrhizus oligosaccharides is 306 mL-612 mL.

[0014] In some alternative embodiments, the ultrasonic intensity is 14.0 W / mL-28.0 W / mL. Preferably, the ultrasonic intensity is 17.5 W / mL-21.0 W / mL.

[0015] In some alternative embodiments, the ultrasonic treatment time is 10 min-60 min. Preferably, the ultrasonic treatment time is 20 min-40 min.

[0016] In some alternative embodiments, the concentration of the sodium hydroxide solution is 0.1 M to 1.0 M. The amount of the sodium hydroxide solution is equal to the volume of the Pachyrhizus polysaccharide degradation solution in terms of volume, that is, the Pachyrhizus polysaccharide degradation solution and the sodium hydroxide solution are mixed at a volume ratio of 1:1.

[0017] In some alternative embodiments, removing the solid substances and hydrogen peroxide includes removing the solid substances by centrifugation and removing the hydrogen peroxide by heating.

[0018] In some alternative embodiments, the purification refers to dialysis and the drying refers to freeze-drying. Preferably, the molecular weight cut-off of the dialysis is less than 1 kDa.

[0019] In a third aspect, the present application provides use of the Pachyrhizus oligosaccharide in the preparation of a product for preventing or treating ulcerative colitis.

[0020] In some alternative embodiments, the product for preventing or treating ulcerative colitis is a product for reducing weight loss, reducing disease activity index score.

[0021] In some alternative embodiments, the product for preventing or treating ulcerative colitis is a product for reducing colon shortening, reducing colon bleeding.

[0022] In some alternative embodiments, the product for preventing or treating ulcerative colitis is a product for reducing colon structure disorder, reducing inflammatory cell infiltration.

[0023] Due to the adoption of the above technical solutions, the embodiments of the present application at least have the following beneficial effects: a Pachyrhizus oligosaccharide with novel structural characteristics and a preparation method thereof are provided, the Pachyrhizus oligosaccharide has a molecular weight of 2 kDa, has the effects of reducing disease activity index, reducing colon bleeding, repairing colon structure, and reducing inflammatory cell infiltration, is safe and effective, can be taken for a long time without drug resistance, makes up for the defects of western medicine preparations such as salicylic acid, glucocorticoids, immunosuppressants, and biological preparations, and can be used for preparing a product for preventing or treating ulcerative colitis; the preparation method of the Pachyrhizus oligosaccharide, that is, the ultrasonic-Fenton combined technology, has a simple process flow, low requirements for equipment, low cost, and a high yield of oligosaccharide of more than 50%, and is suitable for large-scale industrial promotion. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 is the HP GPC chart of the Pachyrhizus oligosaccharide in Example 1 of the present application.

[0025] Figure 2 is the monosaccharide composition HPLC chart of the Pachyrhizus oligosaccharide in Example 1 of the present application.

[0026] Figure 3is the weight coefficient change curve of mice in Example 2 of the present application.

[0027] Figure 4 is the DAI score change curve of mice in Example 2 of the present application.

[0028] Figure 5 is the colon length change of mice in Example 2 of the present application.

[0029] Figure 6 is the hematoxylin-eosin (HE) staining of mouse colon tissue pathological analysis result in Example 2 of the present application. DETAILED DESCRIPTION

[0030] The concept and technical effects of the present application will be described clearly and completely below to fully illustrate the purpose, scheme and effects of the present application.

[0031] In the present application, soybean oligosaccharides are prepared by degrading soybean polysaccharides through ultrasonic-Fenton combined technology. The polysaccharide degradation to prepare oligosaccharides has the advantages of simplicity, low cost and easy reproduction on an industrial scale. The ultrasonic-Fenton combined technology uses ultrasonic to accelerate the decomposition of Fenton reagent and promote the oxidation of organic matter, thereby accelerating the degradation rate and efficiency of polymers such as polysaccharides to prepare functional oligosaccharides. According to animal experiments, it is found that the prepared soybean oligosaccharides have the effect of preventing or treating ulcerative colitis, which provides a reference for the development of new products for preventing or treating ulcerative colitis.

[0032] Reagents and samples used in the following examples or comparative examples:

[0033] Anhydrous ethanol, ferrous sulfate heptahydrate, concentrated hydrochloric acid, hydrogen peroxide, sodium hydroxide, sodium nitrate, dextran series standard, monosaccharide and uronic acid standard (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, mannuronic acid), phenol, concentrated sulfuric acid, trifluoroacetic acid (TFA), dinitrosalicylic acid (DNS), carbazole, bovine serum albumin, coomassie brilliant blue, heavy water, dextran sulfate sodium (DSS), paraformaldehyde fixing solution, dry ice, liquid nitrogen, SPF level 8-week-old male C57BL / 6J mice (weight 22±2 g, Henan Skb Bioscience Co., Ltd.).

[0034] Instruments and equipment used in the following examples or comparative examples:

[0035] Electronic balance, adjustable pipette, vortex, magnetic heating stirrer, rotary evaporator, freeze dryer, pH meter, ultrasonic cell crusher, table low-temperature high-speed centrifuge, high-performance liquid chromatograph, multifunctional enzyme marker, ultraviolet spectrophotometer, ion chromatograph, -80°C ultra-low temperature refrigerator, -20°C refrigerator, and full-automatic sample rapid grinder.

[0036] Example 1

[0037] The present example provides a preparation method of Dioscorea panthaica oligosaccharide, which comprises the following steps:

[0038] 100 mg of Dioscorea panthaica polysaccharide was accurately weighed, dissolved in 10 mL of ferrous sulfate solution (ferrous ion concentration of 3 mM), the pH was adjusted to 3.0, 15.3 μL of hydrogen peroxide solution (concentration of 15 mM) was added, and ultrasonic treatment was performed in an ultrasonic cell crusher, the ultrasonic power was 17.5 W / mL, the ultrasonic time was 30 min, immediately after ultrasonic treatment, an equal volume of sodium hydroxide solution (concentration of 0.5 M) was added to terminate the reaction, the solid material was removed by centrifugation, the hydrogen peroxide was removed by heating, dialysis (molecular weight cut-off <1 kDa), freeze-drying, and Dioscorea panthaica oligosaccharide was obtained.

[0039] The total sugar content of the Dioscorea panthaica oligosaccharide sample prepared by the method of the present example was determined by the phenol-sulfuric acid method with glucose as the standard. Specifically, 5 mg of the standard was weighed into a 50 mL volumetric flask (the weight was recorded), as the standard solution. The gradient of the standard was 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL, and 1.2 mL of the diluted standard, and the water was added to 2 mL. 5 mg of the sample was weighed into a 50 mL volumetric flask (the weight was recorded), 1 mL of the sample solution was taken into a test tube, and the water was added to 2 mL. 1.0 mL of 3% phenol solution was added to the standard and the sample, respectively, 5 mL of concentrated sulfuric acid was slowly added, and the solution was shaken and shaken constantly while adding the concentrated sulfuric acid to make the solution uniform and fully reacted. After standing for 30 min, the absorbance was measured at 490 nm. The glucose solution concentration-absorbance was used as the horizontal and vertical coordinates to draw the standard curve. The total sugar content of the sample was calculated by substituting the absorbance of the sample into the standard curve. The total sugar content of the Dioscorea panthaica oligosaccharide prepared by the method of the present example was 70.9%.

[0040] The reducing sugar content in the oligosaccharide sample prepared by the method of the embodiment is determined by the DNS method with glucose as the standard. Specifically, a 1 mg / mL glucose standard solution is prepared. 0, 0.1, 0.15, 0.20, 0.25, 0.3, 0.35, and 0.40 mL of the glucose standard solution (1 mg / mL) are respectively taken in a glass test tube, and water is added to 0.5 mL. A 1 mg / mL sample solution is prepared, and 0.5 mL is taken. The standard and the sample are respectively added with 0.5 mL of DNS, boiled in a water bath for 5 min, cooled, then added with 4 mL of distilled water, and immediately measured for absorbance at 540 nm. The glucose solution concentration-absorbance is taken as the horizontal and vertical coordinates to draw a standard curve. The absorbance of the sample is substituted into the standard curve to calculate the reducing sugar content. The reducing sugar content of the oligosaccharide prepared by the method of the embodiment is 12.5% by detection.

[0041] The uronic acid content in the oligosaccharide sample prepared by the method of the embodiment is determined by the improved sulfuric acid-carbazole method with galacturonic acid as the standard. Specifically, 5 mg of the galacturonic acid standard is weighed into a 50 mL volumetric flask (the weight is recorded), as the standard solution. The gradient of the standard is 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the diluted standard, and water is added to 1 mL. 5 mg of the sample is weighed into a 50 mL volumetric flask (the weight is recorded), and 1 mL of the sample solution is taken in a test tube. The standard and the sample are respectively added with 6 mL of concentrated sulfuric acid in an ice bath, then boiled in a water bath at 85°C for 20 min, and then added with 0.2 mL of 0.1% carbazole-ethanol, mixed, and then placed for 2 h, and the absorbance is measured at 530 nm. The galacturonic acid solution concentration-absorbance is taken as the horizontal and vertical coordinates to draw a standard curve. The absorbance of the sample is substituted into the standard curve to calculate the uronic acid content. The uronic acid content of the oligosaccharide prepared by the method of the embodiment is 21.2% by detection.

[0042] The protein content of the Dioscorea alata oligosaccharide sample prepared by the method of the embodiment was determined by the Coomassie brilliant blue method with bovine serum albumin as the standard. Specifically, the protein standard was completely dissolved, 10 μL of which was diluted with pure water to 250 μL to make the final concentration 0.2 mg / mL. 1 mL of 5×G250 staining solution was taken, 4 mL of double distilled water was added, and the mixture was uniformly mixed to form 1×G250 staining solution. 0 μL, 2 μL, 4 μL, 6 μL, 8 μL, 12 μL, 16 μL, and 20 μL of the standard solution were respectively taken into a 96-well plate, and PBS diluent was added to make up to 20 μL. After the sample was appropriately diluted, 20 μL of the sample was added to the sample well of the 96-well plate. Due to the error of the pipette when taking a small amount, the points in front of the standard line may not be very accurate, so the sample points are as far as possible to fall behind 1 / 2 of the standard line. 200 μL of the diluted 1×G250 staining solution was added to each well, and the plate was placed at room temperature for 3-5 min. The absorbance at 595 nm or other wavelengths between 560 nm and 610 nm was determined by an enzyme-labeled instrument. The standard curve was plotted with the concentration of bovine serum albumin solution as the horizontal coordinate and the absorbance as the vertical coordinate. The protein content of the sample was calculated by substituting the absorbance of the sample into the standard curve. It was detected that no protein was detected in the Dioscorea alata oligosaccharide prepared by the method of the embodiment, proving that the prepared oligosaccharide has high purity.

[0043] The molecular weight and purity were determined by high-performance gel permeation chromatography, the effects of various factors on the degradation of Dioscorea alata polysaccharide in the process of ultrasonic combined with Fenton reaction were studied, and the specific detection conditions were as follows: high-performance liquid chromatography, differential detector, Ultrahydrogel TM Linear gel column (7.8×300 mm); mobile phase: 0.1 M NaNO3 solution, flow rate: 0.6 mL / min; sample size: 20 μL; column temperature: 35℃; data acquisition time: 30 min. As shown in Figure 1 The molecular weight of the Dioscorea alata oligosaccharide prepared by the method of the embodiment was calculated to be 2 kDa.

[0044] The monosaccharide composition of the Dioscorea alata oligosaccharide was detected by high-performance anion exchange chromatography pulse amperometry. Specifically, an appropriate amount of Dioscorea alata oligosaccharide was weighed, 1 mL of 2 M TFA solution was added, and heating was performed at 121℃ for 2 hours. Nitrogen was passed, and the sample was blown dry. 99.99% methanol was added for washing, and then the sample was blown dry again. The methanol washing was repeated for 2-3 times. Sterile water was added for dissolution, and the sample was transferred into a chromatography bottle for detection. The instrument parameters were as follows: the chromatography system was a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), an electrochemical detector was used for analysis and detection of the monosaccharide composition; Dionex TM CarboPac TMPA20 (150*3.0mm, 10 μm) liquid chromatography column; the injection amount was 5 μL; the mobile phase A (H2O), the mobile phase B (0.1 M NaOH), the mobile phase C (0.1 M NaOH, 0.2 M NaAc), the flow rate was 0.5 mL / min; the column temperature was 30°C. As shown in Figure 2 It was calculated that the Pueraria polysaccharide was mainly composed of glucose, galacturonic acid, galactose and rhamnose, and the molar ratio was 59.3:2.6:2.0:1.0.

[0045] Comparative Example 1

[0046] The present comparative example provides a method for degrading Pueraria polysaccharide, and the steps are as follows:

[0047] Accurately take 100 mg of Pueraria polysaccharide, dissolve in 10 mL of water, adjust the pH to 3.0, add 30.6 μL of hydrogen peroxide solution (concentration of 30 mM), and place in an ultrasonic cell crusher for ultrasonic treatment, the ultrasonic power is 24.5 W / mL, and the ultrasonic time is 30 min. Immediately after ultrasonic treatment, add an equal volume of sodium hydroxide solution (concentration of 0.5 M) to terminate the reaction, remove the solid material by centrifugation, remove the hydrogen peroxide by heating, dialysis (molecular weight cut-off <1 kDa), freeze-drying, and obtain the Pueraria degradation product.

[0048] In the present comparative example, no ferrous ions were added, and the molecular weight of the Pueraria degradation product obtained was 20.8 kDa, which was 10 times the molecular weight of the Pueraria oligosaccharide prepared in Example 1. From the comparison of Comparative Example 1 and Example 1, it can be seen that under acidic conditions, metal ions can catalyze the decomposition of hydrogen peroxide to produce highly active hydroxyl radicals, initiate radical chain reactions, and promote the oxidation of polysaccharides, thereby promoting degradation. If no ferrous ions are added, hydrogen peroxide cannot be decomposed.

[0049] Comparative Example 2

[0050] The present comparative example provides a method for degrading Pueraria polysaccharide, and the steps are as follows:

[0051] Accurately take 100 mg of Pueraria polysaccharide, dissolve in 10 mL of water, adjust the pH to 3.0, add 30.6 μL of hydrogen peroxide solution (concentration of 30 mM), and place in an ultrasonic cell crusher for ultrasonic treatment, the ultrasonic power is 24.5 W / mL, and the ultrasonic time is 30 min. Immediately after ultrasonic treatment, add an equal volume of sodium hydroxide solution (concentration of 0.5 M) to terminate the reaction, remove the solid material by centrifugation, remove the hydrogen peroxide by heating, dialysis (molecular weight cut-off <1 kDa), freeze-drying, and obtain the Pueraria degradation product.

[0052] In the present comparative example, no hydrogen peroxide was added, and the molecular weight of the obtained Pachyrhizus oligosaccharide was 18.8 kDa, which was 9 times the molecular weight of the Pachyrhizus oligosaccharide prepared in Example 1. From the comparison between Comparative Example 2 and Example 1, it can be seen that under acidic conditions, metal ions can catalyze the decomposition of hydrogen peroxide to produce highly active hydroxyl radicals, initiate a radical chain reaction, and promote the oxidation of polysaccharides to promote degradation. If no hydrogen peroxide is added, highly active hydroxyl radicals cannot be produced.

[0053] Comparative Example 3

[0054] The present comparative example provides a method for degrading Pachyrhizus polysaccharides, which comprises the following steps:

[0055] Accurately weigh 100 mg of Pachyrhizus polysaccharides, dissolve in 10 mL of ferrous sulfate solution (ferrous ion concentration 2 mM), adjust the pH to 3.0, add 30.6 μL of hydrogen peroxide solution (concentration 30 mM), add an equal volume of sodium hydroxide solution (concentration 0.5 M) to terminate the reaction, centrifuge to remove solid materials, heat to remove hydrogen peroxide, dialysis (molecular weight cut-off <1 kDa), lyophilization, and obtain Pachyrhizus degradation products.

[0056] In the present comparative example, no ultrasonic treatment was performed, and the molecular weight of the obtained Pachyrhizus degradation products was 9.9 kDa, which was 5 times the molecular weight of the Pachyrhizus oligosaccharide prepared in Example 1. From the comparison between Comparative Example 3 and Example 1, it can be seen that when ultrasonic waves act on a medium, mechanical and cavitation effects can be produced, which can promote the degradation of polymers such as polysaccharides. If no ultrasonic treatment is performed, mechanical and cavitation effects cannot be produced.

[0057] Example 2

[0058] In the present example, the therapeutic effect of Pachyrhizus oligosaccharides on DSS-induced ulcerative colitis in mice was investigated through animal experiments. The present animal experiment complies with the Guidelines for the Care and Use of Laboratory Animals, and was approved by the Animal Experiment Management Committee of Nanchang University (Approval No. 0064257).

[0059] (1) Experimental animals and feeding conditions

[0060] The experimental animals were 40 SPF level 8-week-old (22±2 g) male C57BL / 6J mice. Before the formal experiment began, the animals were placed in a constant temperature (25±0.5℃) and constant humidity (50-60%) environment, and were allowed to freely drink and eat, and were fully adapted for 7 days (the day the mice arrived was day 0), with a light-dark cycle of 12 h. The bedding was changed every three days. The body weight and food and water intake of the mice were recorded every day.

[0061] (2) Animal grouping and intervention

[0062] After 7 days of adaptive feeding, the mice were randomly divided into the following 4 groups (n=10 / group) and given different interventions for 3 weeks:

[0063] ① Control group: the mice were given normal drinking water;

[0064] ② Model group: the mice were given normal drinking water for the first 2 weeks and 3% DSS (m / v) solution for drinking water for the third week;

[0065] ③ Dioscorea panthaica polysaccharide group: the mice were given normal drinking water for the first 2 weeks and 3% DSS (m / v) solution for drinking water for the third week, and were given 100 mg / kg / day of Dioscorea panthaica polysaccharide by gavage for 3 weeks;

[0066] ④ Dioscorea panthaica oligosaccharide group: the mice were given normal drinking water for the first 2 weeks and 3% DSS (m / v) solution for drinking water for the third week, and were given 100 mg / kg / day of Dioscorea panthaica oligosaccharide by gavage for 3 weeks.

[0067] During the experiment, the body weight and food and water intake of the mice were recorded every day. As shown in Table 2, the body weight of the mice decreased significantly after modeling. Figure 3

[0068] The Dioscorea panthaica oligosaccharide used in this experiment was prepared in Example 1.

[0069] (3) Disease activity index score (DAI score)

[0070] To evaluate the severity of DSS-induced UC (ulcerative colitis), the body weight loss, fecal consistency, and fecal occult blood of the mice were recorded every day during the DSS exposure, and were scored according to the criteria shown in Table 1. Then the three scores were added to obtain the DAI score.

[0071] Table 1: DAI score criteria

[0072]

[0073] As shown in Table 3, by statistically analyzing the disease activity index (DAI), colon length, and the morphology of the colon section of the control group, the model group, the Dioscorea panthaica polysaccharide group, and the Dioscorea panthaica oligosaccharide group, it can be determined that the DAI index of the mice increased significantly after modeling; after the mice were given Dioscorea panthaica polysaccharide and Dioscorea panthaica oligosaccharide, the DAI index decreased significantly, and the decrease in the DAI index was more obvious in the Dioscorea panthaica oligosaccharide group than in the Dioscorea panthaica polysaccharide group. Figure 4 (4) Animal tissue sample collection

[0074]

[0075] ​​After 3 weeks of continuous intervention, the mice were taken blood from the eyeball, immediately centrifuged at 4℃, 3000 rpm for 20 min, and the serum was separated and collected. The mice were decapitated, and the spleen, liver, kidney, colon, colon contents and cecum contents were collected. After measuring the length of the colon, the distal colon 0.5 cm was placed in 4% paraformaldehyde fixing solution, and the spleen, liver and kidney were weighed, respectively. Then all the collected samples were aliquoted and frozen with liquid nitrogen, and stored at -80℃ for further analysis.

[0076] As shown in Figure 5 , after modeling, the colon of the mice was significantly shortened, and the average colon length of the mice gavaged with DSS and DSS oligosaccharides was significantly increased, and DSS oligosaccharides increased more than DSS, indicating that both DSS and DSS oligosaccharides could alleviate the colonitis of mice, and the alleviating effect of DSS oligosaccharides was better.

[0077] (5) Colon HE staining

[0078] ①Preparation of paraffin sections: after the colon tissues fixed by 4% paraformaldehyde were dehydrated, transparent, immersed in wax, and embedded in each link, the sections with a thickness of 4 μm were prepared;

[0079] ②Dewaxing of paraffin sections to water: sequentially place the sections in xylene I for 20 min-xylene II for 20 min-anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-75% alcohol for 5 min, and rinse with tap water;

[0080] ③Staining with hematoxylin: stain with hematoxylin staining solution for 3-5 min, differentiate with differentiation solution, and finally use blue return solution to return blue, and rinse the sections with running water after each operation is completed;

[0081] ④Eosin staining: sequentially place the sections in 85% and 95% gradient alcohol for 5 min each, and stain with eosin staining solution for 5 min;

[0082] ⑤Dehydration and mounting: sequentially place the sections in anhydrous ethanol I for 5 min-anhydrous ethanol II for 5 min-anhydrous ethanol III for 5 min-xylene I for 5 min-xylene II for 5 min, and complete the mounting with neutral gum;

[0083] ⑥Microscope examination, image acquisition and analysis.

[0084] The results of HE staining of the colon of mice are shown in Figure 6 , the colon structure of the control group mice was complete, the crypts were arranged in order, and the epithelial cells were arranged closely; while the colon structure of the model group mice was severely damaged, the crypt structure was damaged, almost no goblet cells were found, and a large area of inflammatory infiltration was observed; compared with the model group, the crypt structure of the DSS and DSS oligosaccharide groups was complete, the number of goblet cells was increased, and the degree of inflammatory cell infiltration was reduced.

[0085] The above merely illustrates the preferred embodiments of the present application, and the present application is not limited to the above-mentioned embodiments. Any modification and change of the technical solutions and / or embodiments within the scope of the present application shall fall within the protection scope of the present application.

Claims

1. A kind of soybean-jaw oligosaccharide, characterized in that, The molecular weight is 2 kDa, and the total sugar content is 70.9%. The monosaccharide composition includes glucose, galacturonic acid, galactose, and rhamnose, and the molar ratio of glucose, galacturonic acid, galactose, and rhamnose is 59.3:2.6:2.0:1.

0. The preparation method of the soybean-pickled oligosaccharide includes the following steps: Dioscorea bean polysaccharide and ferrous ion solution were mixed, the pH was adjusted to 2.0-6.0, hydrogen peroxide solution was added, and the mixture was placed in an ultrasonic cell disruptor for ultrasonic treatment to obtain dioscorea bean polysaccharide degradation solution. The soybean polysaccharide degradation solution was mixed with sodium hydroxide solution to remove solid matter and hydrogen peroxide. After purification and drying, soybean oligosaccharide was obtained. The concentration of ferrous ions in the ferrous ion solution is 1 mM to 5 mM, the concentration of the hydrogen peroxide solution is 10 mM to 40 mM, and the concentration of the sodium hydroxide solution is 0.1 M to 1.0 M; the ultrasonic intensity is 14.0 W / mL to 28.0 W / mL, and the ultrasonic treatment time is 10 min to 60 min.

2. The use of the soybean-pickled oligosaccharide according to claim 1 in the preparation of products for the prevention or treatment of ulcerative colitis.

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