Corn bran polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses corn bran polysaccharide and a preparation method and application thereof, and belongs to the technical field of corn deep processing. In order to solve the technical problems that in the prior art, the utilization rate of corn byproducts is low, economic benefits are poor, chemical residues exist in a functional sugar preparation method, and the function is single, the invention provides the preparation method of the corn bran polysaccharide. The corn bran polysaccharide is prepared sequentially through degreasing, water extraction, alcohol precipitation, protease enzymolysis, glycosidase XynB enzymolysis and separation and purification of ion exchange chromatography and gel column chromatography. In-vivo and in-vitro experiments show that the corn bran polysaccharide prepared by the method disclosed by the invention has the functions of reducing blood sugar and relieving intestinal injury caused by diabetes mellitus.

Description

Technical Field

[0001] The invention belongs to the technical field of corn deep processing, and particularly relates to corn bran polysaccharide and a preparation method and application thereof. Background Art

[0002] Corn is the largest grain crop in my country by planting area and total output. The agricultural corn production process generates a variety of wastes, which are also a renewable resource. The amount of waste generated during corn processing, such as corn silk, corn husks, and corn cobs, is considerable. However, these wastes generated during corn processing are usually treated as feed or crushed and returned to the field, which greatly reduces the added value of corn. Therefore, there is an urgent need to find ways to efficiently utilize these wastes to achieve deep processing of corn and thereby improve subsequent economic benefits.

[0003] Functional sugars are a class of carbohydrates with special benefits, primarily including functional oligosaccharides and functional sugar alcohols. Corn processing byproducts contain a high concentration of macromolecular polysaccharide compounds, which serve as ideal raw materials for functional sugars. Currently, functional sugars are mostly produced using chemical methods. While chemical preparation yields high-purity functional sugars, it also carries environmental risks and cannot avoid the problem of residual chemical reagents. Furthermore, chemically produced functional sugars are primarily oligosaccharides with a single function, which limits the development of diverse functional sugars. Therefore, the safe and environmentally friendly production of diverse functional sugars is a key issue currently under investigation in the deep processing of corn byproducts. Summary of the Invention

[0004] In order to solve the technical problems in the prior art of low utilization rate of corn by-products, poor economic benefits, chemical residues in the preparation method of functional sugars, and single functions, the present invention provides a preparation method of corn bran polysaccharide. The method uses corn bran as raw material and sequentially undergoes defatting, water extraction, alcohol precipitation, protease hydrolysis, glycosidase XynB hydrolysis, and separation and purification by ion exchange chromatography and gel column chromatography to prepare a corn bran polysaccharide with the function of lowering blood sugar and alleviating intestinal damage caused by diabetes.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide a method for preparing corn bran polysaccharide, the preparation method comprising the following steps:

[0007] S1. Raw material pretreatment: corn bran is washed, aired, crushed, and sieved to obtain corn bran dry powder, which is then defatted with petroleum ether to obtain defatted corn bran;

[0008] S2, water extraction: The defatted corn bran obtained in S1 was mixed with water at a solid-liquid ratio of 1 g:15 mL, and water-extracted at 90°C for 3 h. The filtrate was collected by filtration. This was performed three times in total, and the filtrates obtained from each extraction were combined.

[0009] S3, purification: the filtrate obtained in S2 was subjected to rotary evaporation, and after rotary evaporation, anhydrous ethanol was added for alcohol precipitation, the precipitate obtained by the alcohol precipitation was redissolved in water, and residual ethanol was removed by concentration under reduced pressure, and papain was added in an amount of 300 U / mL, and enzymatic hydrolysis was carried out at 60° C. for 6 h. Sevage reagent was added to the obtained enzymatic hydrolyzate, and the upper aqueous phase was collected by centrifugation after thorough mixing. The steps of thorough mixing with Sevage reagent and centrifugation were repeated 3 times, and residual Sevage reagent was removed by concentration under reduced pressure. The crude corn bran polysaccharide was dialyzed against tap water, deionized water, and ultrapure water for one day each, and freeze-dried to obtain the crude corn bran polysaccharide.

[0010] S4, enzymatic hydrolysis: The crude corn bran polysaccharide obtained in S3 was prepared into an aqueous solution with a concentration of 0.2 g / mL, 2200 U / g glycosidase XynB was added, and the mixture was incubated on a shaker at 37°C for 6 h. After the enzyme was inactivated, the supernatant was collected by centrifugation, dialyzed, and freeze-dried to obtain enzymatically hydrolyzed corn bran polysaccharide;

[0011] S5. Separation and purification: The enzymatically hydrolyzed corn bran polysaccharide obtained in S4 was separated and purified using a DEAE celluLose-52 ion exchange column and a Sephadex G-200 gel column in sequence to obtain corn bran polysaccharide.

[0012] In one embodiment of the present invention, the solid-liquid ratio of the corn bran dry powder and petroleum ether in S1 is 1 g:2 mL, and the degreasing treatment time is 24 h.

[0013] In one embodiment of the present invention, the alcohol precipitation in S3 is performed by adding anhydrous ethanol until the volume fraction of ethanol is 75%, and standing at 4° C. overnight; the volume ratio of the enzymatic hydrolysate to the Sevage reagent is 1:5.

[0014] In one embodiment of the present invention, the purification method of the ion exchange column described in S5 is as follows: prepare a polysaccharide sample with a concentration of 25 mg / mL, centrifuge and discard the precipitate; load the sample, and the sample volume does not exceed 3% of the column volume; elute with 0 mol / L, 0.1 mol / L, and 0.3 mol / L NaCl solutions in sequence, control the flow rate to 1 mL / min, collect with an automatic collector, collect 10 mL into 1 tube, and track the sample using the phenol-sulfuric acid method; finally, draw an elution curve with the number of eluent tubes as the horizontal axis and the A490 nm absorbance value as the vertical axis; collect and combine the same components, dialyze for 3 days with deionized water in a dialysis bag with a cutoff flow of 3500 Da, and then freeze-dry to obtain the corresponding components.

[0015] In one embodiment of the present invention, the purification method of the gel column described in S5 is as follows: the polysaccharide sample is configured into a 15 mg / mL polysaccharide solution, and after centrifugation, it is filtered through a 0.45 μm water filter membrane; the sample is loaded, and the loading volume does not exceed 3% of the column volume, and eluted with deionized water. The flow rate is controlled to 0.3 mL / min, and collected by an automatic collector, one tube is collected for every 3 mL, and a differential refractometer is used to track the sample; the same components are collected and combined, and after dialyzing for 3 days through a dialysis bag with deionized water with a cut-off flow rate of 3500 Da, the purified polysaccharide sample is obtained by freeze-drying.

[0016] The second object of the present invention is to provide corn bran polysaccharide obtained by the above preparation method.

[0017] The third object of the present invention is to provide the use of the corn bran polysaccharide in the preparation of a drug for lowering blood sugar.

[0018] The fourth object of the present invention is to provide the use of the above-mentioned corn bran polysaccharide in the preparation of a medicament for preventing and / or treating diabetes.

[0019] The fifth object of the present invention is to provide the use of the corn bran polysaccharide in the preparation of a medicament for alleviating intestinal damage caused by diabetes.

[0020] Beneficial effects of the present invention:

[0021] The present invention uses corn bran as raw material, and sequentially undergoes defatting, water extraction, alcohol precipitation, protease hydrolysis, glycosidase XynB hydrolysis, and separation and purification by ion exchange chromatography and gel column chromatography to prepare a corn bran polysaccharide. In the preparation process of corn bran polysaccharide, the present invention optimizes the conditions of water extraction, and with the hypoglycemic activity of the hydrolyzed product in mice as a guide, optimizes the selection of glycosidase and the hydrolysis time, and obtains enzymatically hydrolyzed corn bran polysaccharide (XynB-CBP-6h) with hypoglycemic activity. The enzymatically hydrolyzed corn bran polysaccharide is separated and purified by ion exchange chromatography and gel column chromatography to obtain three components: ECBP-1, ECBP-2, and ECBP-3. After hypoglycemic activity screening, it is found that ECBP-3 has good hypoglycemic activity and can be used to alleviate intestinal damage caused by diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figure is a flow chart of the extraction process of CBP and the preparation process of ECBP; wherein, Figure 1 A in the figure is the extraction flow chart of CBP. Figure 1 B in FIG. 1 is a flow chart for the preparation of ECBP;

[0023] Figure 2 This is the single factor optimization result diagram of CBP extraction conditions; Figure 2A in the figure is the effect of material-liquid ratio on polysaccharide yield. Figure 2 B in the figure is the effect of extraction time on polysaccharide yield. Figure 2 C in the figure is the result graph of the effect of extraction times on polysaccharide yield. Figure 2 D in the figure is the effect of extraction temperature on polysaccharide yield;

[0024] Figure 3 The figure shows the evaluation results of the hypoglycemic activity of CBP and its five enzymatic hydrolysates; * indicates p < 0.05, *** indicates p < 0.001;

[0025] Figure 4 The graph shows the effect of different hydrolysis times of CBP by XynB on the hypoglycemic activity of the hydrolysate; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001;

[0026] Figure 5 The figure shows the effects of different doses of XynB-CBP-6h on postprandial blood glucose and glucose tolerance in mice; Figure 5 A in the figure shows the effect of different doses of XynB-CBP-6h on postprandial blood glucose in mice. Figure 5 Figure B shows the effect of different doses of XynB-CBP-6h on oral glucose tolerance in mice; * indicates p < 0.05, ** indicates p < 0.01;

[0027] Figure 6 This is the result of the effect of XynB hydrolysis time on the infrared spectrum of the hydrolysis product;

[0028] Figure 7 This is a graph showing the relationship between apparent viscosity and shear rate of the hydrolyzed products obtained by XynB hydrolysis of CBP at different times;

[0029] Figure 8 This is the elution curve of CBP and ECBP obtained by DEAE celluLose-52 purification; Figure 8 A in the figure is the elution curve obtained by purification of CBP by DEAE celluLose-52. Figure 8 Figure B is the elution curve obtained by purification of ECBP by DEAEcelluLose-52;

[0030] Figure 9 is the differential detector spectra of CBP-3 and ECBP-3;

[0031] Figure 10 This is the result of screening the active components of ECBP for hypoglycemic activity; Figure 10 A in the figure is the test result of postprandial blood glucose during the 28-day treatment period of each active component. Figure 10 B in the figure is a graph showing the changes in fasting blood sugar before and after administration of each active ingredient; Figure 10 C in the figure is the OGTT diagram after the administration of each active component. Figure 10 D in the table is the area under the curve (AUC) of each active component; ** indicates p < 0.01, *** indicates p < 0.001;

[0032] Figure 11 is a multi-detector gel permeation chromatogram of CBP-3 and ECBP-3; wherein, Figure 11 A in FIG is a multi-detector gel permeation chromatogram of CBP-3. Figure 11 B in FIG is a multi-detector gel permeation chromatogram of ECBP-3;

[0033] Figure 12 HPAEC-PAD chromatograms of CBP-3 and ECBP-3;

[0034] Figure 13 This is a statistical graph showing the inhibition rates of α-amylase by CBP-3 and ECBP-3 at different concentrations;

[0035] Figure 14 This is a statistical graph showing the inhibition rates of glucosidase by CBP-3 and ECBP-3 at different concentrations;

[0036] Figure 15 The figure shows the statistical results of DPPH free radical scavenging rate of CBP-3 and ECBP-3 at different concentrations;

[0037] Figure 16 is the total ion current of CBP-3 and ECBP-3 after methylation; Figure 16 A in the figure is the total ion current after CBP-3 methylation. Figure 16 B in the figure is the total ion current after ECBP-3 methylation;

[0038] Figure 17 is the one-dimensional nuclear magnetic resonance spectrum of CBP-3 and ECBP-3; Figure 17 A in the figure is the one-dimensional NMR spectrum of CBP-3. Figure 17 B in the figure is the one-dimensional NMR spectrum of ECBP-3;

[0039] Figure 18 is the two-dimensional nuclear magnetic resonance spectrum of ECBP-3; Figure 18 A in is the HSQC spectrum, Figure 18 B in is the HMBC spectrum, Figure 18 C in 1 H / 1 Hcosy spectrum;

[0040] Figure 19 is the predicted structural formula of ECBP-3;

[0041] Figure 20 The intestinal morphology and H&E staining results of the front, middle, and back of the small intestine and colon of each group of mice are shown in the figure. Figure 20 A in the figure is the intestinal morphology observation diagram of each group of mice. Figure 20 B is the H&E staining result of the front end of the small intestine of each group of mice. Figure 20 C in the figure is the H&E staining result of the middle part of the small intestine of mice in each group. Figure 20 D in the figure is the H&E staining result of the posterior end of the small intestine of each group of mice. Figure 20 E in the figure is the H&E staining result observation picture of the colon of mice in each group;

[0042] Figure 21 Statistical results of the expression levels of related genes in each group of mice; Figure 21 A in the figure is the statistical result of IL-6 gene expression level in the intestine of each group of mice. Figure 21 Figure B is the statistical result of the intestinal IL-1β gene expression level of each group of mice. Figure 21 C in the figure is the statistical result of the expression level of GLP-1 gene in the intestine of each group of mice. Figure 21 D in the figure is the statistical result of GLUT-2 gene expression level in the liver of mice in each group; * indicates p < 0.05, ** indicates p < 0.01, and ns indicates no statistical difference. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. Except for the contents specifically mentioned below, the processes, conditions, reagents, experimental methods, etc. for implementing the present invention are common knowledge and common common sense in the art and are not particularly limited in the present invention.

[0044] The purchase sources of some reagents, medicines and kits involved in the present invention are shown in Table 1.

[0045] Table 1 Purchase sources of some reagents, drugs and kits involved in the present invention

[0046]

[0047]

[0048] The detection method involved in the present invention is as follows:

[0049] 1. Determination of total sugar content:

[0050] The total sugar content was determined using the phenol-sulfuric acid method, and the polysaccharide content was calculated using a glucose standard curve using an ultraviolet spectrophotometer at a wavelength of 490 nm. The specific method is as follows:

[0051] To prepare a glucose standard curve, take 1 mL of a serially diluted glucose standard solution (0.1, 0.08, 0.06, 0.04, 0.02, 0 mg / mL) and add distilled water to 2.0 mL. Then, add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid, mix thoroughly, and cool to room temperature. Measure the absorbance at 490 nm using a UV spectrophotometer.

[0052] Determination of total sugar content: Prepare and take 1 mL of CBP (0.1 mg / mL) and add distilled water to make up to 2 mL. Then measure the absorbance value three times according to the total sugar content detection method of the above glucose standard solution.

[0053] 2. Determination of reducing sugar content:

[0054] In addition to polysaccharides, crude polysaccharides also contain reducing sugars. This part needs to be removed from the total sugar when calculating the polysaccharide content. The dinitrosalicylic acid method is usually used to determine the reducing sugar content. The specific method is as follows:

[0055] Preparation of glucose standard curve: Take 1 mL of glucose standard solution (0.1, 0.08, 0.06, 0.04, 0.02, 0 mg / mL) prepared by gradient dilution, make up to 2.0 mL with distilled water, add 1.5 mL of DNS, mix thoroughly, boil in a water bath for 5 min, and then cool to room temperature. Use water as a control and measure the absorbance at a wavelength of 540 nm using a UV spectrophotometer.

[0056] Determination of reducing sugar content:

[0057] Prepare and take 1 mL of CBP (5 mg / mL), add distilled water to 2 mL, and then measure the absorbance value three times according to the reducing sugar content detection method of the above-mentioned glucose standard solution.

[0058] 3. Determination of uronic acid content:

[0059] Preparation of standard curve: Take 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mL of standard solution (100 μg / mL), add water to 1 mL (corresponding to concentration 0-100 μg / mL), slowly add 5 mL of sodium tetraborate-sulfuric acid solution in an ice-water bath, vortex mix, heat in a boiling water bath for 20 min, immediately cool to room temperature, add 0.2 mL of carbazole solution, shake well, and place at room temperature in the dark for 2 h. Use blank (distilled water) as reference, measure the absorbance at 530 nm, and draw a standard curve;

[0060] Sample preparation: Accurately weigh 10 mg of the polysaccharide sample to be tested, dissolve it in distilled water and dilute to 10 mL (concentration 1 mg / mL), take 1 mL of the sample solution, and operate according to the standard curve steps, repeating 3 times in parallel;

[0061] Absorbance determination and calculation: According to the sample absorbance value, the corresponding galacturonic acid concentration is obtained from the standard curve.

[0062] 4. Protein content determination:

[0063] Preparation of Coomassie Brilliant Blue Reagent: Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 mL of 95% ethanol and stir until completely dissolved. Add 100 mL of 85% phosphoric acid and continue stirring to mix thoroughly. Add ultrapure water to 1 L, filter to remove the precipitate, and store at 4°C in the dark.

[0064] Preparation of standard curve: dilute BSA stock solution (1 mg / mL) with PBS to a gradient concentration (0, 0.125, 0.25, 0.5, 1.0 mg / mL), take 5 μL of each concentration standard, add 1 mL of Coomassie Brilliant Blue reagent, incubate at room temperature for 5-10 min, read the absorbance at 595 nm, and fit the standard curve with protein concentration as the horizontal axis and the absorbance at 595 nm as the vertical axis;

[0065] Calculation of protein content: Dilute the sample to be tested to the estimated concentration range, take 5-20 μL of sample, add the reagent according to the above standard steps and measure the absorbance, and calculate the sample protein concentration according to the standard curve.

[0066] 5. Ion exchange column purification:

[0067] Column Packing: First, wash the DEAE celluLose-52 with deionized water to remove the alcohol smell. Swell the DEAE celluLose-52 with ultrasonically treated deionized water for activation. Add 1 / 4 column volume of deionized water to the column. Next, slowly drain the fully swollen DEAE celluLose-52 through a glass rod for wet packing (Φ5 cm × 60 cm). Avoid creating fractures or bubbles during the packing process. Finally, equilibrate the packed ion exchange chromatography column with ultrasonically treated deionized water (1 mL / min) and elute for 4-6 column volumes.

[0068] Elution: Prepare a polysaccharide sample (25 mg / mL), centrifuge at 4000g for 15 min, and discard the precipitate; load the sample with a sample volume not exceeding 3% of the column volume; elute with 0 mol / L, 0.1 mol / L, and 0.3 mol / L NaCl solutions in sequence at a flow rate of 1 mL / min, collect in an automatic collector, and collect 10 mL into one tube. Use the phenol-sulfuric acid method to track the sample; finally, draw an elution curve with the number of eluent tubes as the horizontal axis and the A490 nm absorbance as the vertical axis; collect and combine the same fractions, dialyze against deionized water in a dialysis bag (cut-off capacity 3500 Da) for 3 days, and then freeze-dry to obtain the corresponding fractions.

[0069] 6. Gel column purification

[0070] Column Packing: Activate Sephadex G-200 by fully swelling it with ultrasonically treated deionized water. First, add 1 / 4 column volume of deionized water to the column. Slowly drain the fully swollen Sephadex G-200 through a glass rod for wet packing (Φ2.5 cm × 100 cm). Avoid generating fractures and bubbles during the packing process. Equilibrate the packed column with ultrasonically treated deionized water (0.30 mL / min) and elute for 4-6 column volumes.

[0071] Elution: The polysaccharide sample was prepared into a 15 mg / mL polysaccharide solution; centrifuged at 4000 g for 15 min and then filtered through a 0.45 μm water filter; the sample was loaded with a sample volume not exceeding 3% of the column volume and eluted with deionized water at a flow rate of 0.3 mL / min. The solution was collected in an automatic collector, with one tube collecting every 3 mL. The sample was tracked using a differential refractive index detector (RID); the same fractions were collected and combined, dialyzed against deionized water in a dialysis bag (cut-off flow rate 3500 Da) for three days, and freeze-dried to obtain the purified polysaccharide sample.

[0072] 7. Fourier transform infrared spectroscopy:

[0073] Fourier transform infrared spectroscopy (FT-IR) is a technique used to analyze the molecular structure and chemical composition of substances. 3 mg of polysaccharide sample was weighed, mixed with a small amount of KBr, finely ground, and pressed into granules. FT-IR spectrometer (Nicolet is50, USA) was used to analyze the molecular structure and chemical composition of substances at 400-4000 cm -1 The infrared spectrum was recorded in the range of 100 nm and scanned 32 times.

[0074] 8. Rheological analysis:

[0075] The modular rotational rheometer applies controllable shear stress or strain to the material by replacing different fixtures (such as cone and plate, parallel plates or coaxial cylinders) and control systems, and measures the strain or stress response generated, thereby calculating rheological parameters such as viscosity and elastic modulus. The apparent viscosity was determined using a modular rotational rheometer (HAAKE MARS60, USA) with a cone and plate geometry (diameter 60 mm, gap 1 mm). Specifically, the samples (80 mg / mL, 2 mL) were transferred to the plates respectively (the NG group was the same volume of deionized water), and the shear rate was reduced from 10s to 20s at a temperature of 25±0.1°C. -1 Scan up to 1000 s-1. Flow curves were recorded by performing rheological tests at different shear rates.

[0076] 9. Monosaccharide composition detection:

[0077] 3 mg of polysaccharide sample was accurately weighed and hydrolyzed at 110°C for 4 h with 1 mL of 3M TFA. An appropriate amount of methanol solution was then added and the mixture was concentrated to dryness at 50°C under reduced pressure three times to remove residual TFA. Dissolved in 1 mL of pure water, the mixture was centrifuged (14,000 rpm, 10 min), and the supernatant was filtered through a 0.22 μm aqueous filter for analysis by HPAEC-PAD. Separation was performed using a Dionex CarboPac™ PA20 guard column (3 × 30 mm) and an analytical column (3 × 150 mm, 10 μm). The column temperature was set at 25°C and the flow rate was 0.5 mL / min during injection. Mobile phases A consisted of pure water, B consisted of 100 mM NaOH, and C consisted of 100 mM CH₃COONa. The elution program was as follows: 0-12 min, 8.5%-97.4% A, 1.5%-2.6% B; 12-15 min, 97.4%-97% A, 2.6%-3% B; 15.01-20 min, 94%-91% A, 6%-9% B; 20.01-30 min, 32%-8% A, 8%-2% B, 60%-90% C.

[0078] 10. Molecular weight analysis:

[0079] High-performance size-exclusion chromatography coupled with multi-angle laser light scattering and differential refractive index detection (HPSEC-MALLS-RID) can accurately determine the molecular weight, conformation, and other structural information of polysaccharides. 5 mg of polysaccharide sample was precisely weighed and dissolved in 1 mL of 50 mM ammonium formate solution. After thorough vortexing, the sample was centrifuged (15,000 rpm for 10 min) to remove insoluble matter. The supernatant was filtered through a 0.22 μm aqueous filter and injected. The instrument consisted of a Waters 2695, a Waters 2414 RID, and a Wyatt DAWN HELEOS-II MALLS laser detector. The chromatographic column was a TSK-Gel G-4000PWXL (300 mm × 7.8 mm, TOSOH Bioscience, Tokyo, Japan). The mobile phase was 50 mM ammonium formate, the injection volume was 50 μL, the flow rate was 0.5 mL / min, and the column temperature was 35°C.

[0080] 11. Methylation analysis:

[0081] Methylation: Weigh 2 mg of sample in a 2 mL reactor and dry it in P2O5 overnight; add 1 mL of anhydrous DMSO to the reactor, seal the bottle, and stir at room temperature for 12 hours until completely dissolved. Do not allow contact with water during the dissolution process; add about 2 g of NaOH to a mortar (do not weigh), quickly grind to a fine powder, take about 50 mg and add the suspension (do not weigh), and stir for 2 hours; put the reactor in ice until the suspension freezes (DMSO freezing point 18 ° C), wipe the outer surface of the reactor dry, and add 300 μL of iodomethane three times, 100 μL each time, and react for 15 minutes for the first two times. The reaction was performed for 30 min at a time; 1 mL of H2O was added to ice water; 0.5 mL was added to terminate the reaction, and the suspension was transferred to a hydrolysis tube. 0.5 mL was then added to clean the reactor, and the cleaning liquid was transferred to the hydrolysis tube; N2 was blown to make the solution clear from turbidity (to remove excess iodomethane); an equal volume of chloroform was added for extraction, vortexed for 60 s, and then centrifuged (3 min, 2400 rpm), and the upper layer of water was discarded; an equal volume of water was added to wash the chloroform layer, vortexed for 60 s, and then centrifuged (3 min, 2400 rpm), and discarded; the washing was repeated 4 times, and the upper layer of water and the middle white emulsion layer were discarded for the fourth time; N2 was blown dry.

[0082] Hydrolysis and reductive acetylation: Add 1 mL of 2M TFA to the dried sample and seal the bottle cap; heat in a metal bath or oven for hydrolysis at 120°C for 2 h; dry in a water bath <40°C with N2, or concentrate under reduced pressure; add approximately 3 mg of NaBD4 and dropwise add 1–1.5 mL of 1M NH3·H2O, and reduce at 40°C for 60 min or at room temperature for 18 h; add 10% acetic acid / methanol dropwise in an ice-water bath; transfer to a pear-shaped flask and rinse the reaction tube with methanol; spin dry at 40–50°C, then add 10% acetic acid / methanol once (1.5–2 mL), spin dry, and then add methanol to the pear-shaped flask 5–6 times to remove the acetic acid.

[0083] Acetylation: Add 1 mL of acetic anhydride and 0.1 mL of 1-methylimidazole; seal the reactor and acetylate at room temperature for 30 min; add 1 mL of pure water to ice to terminate the reaction, hydrolyze the acetic anhydride, and transfer the product to a hydrolysis tube; add an equal volume of chloroform for extraction, vortex and oscillate for 60 s, then centrifuge (3 min, 2400 rpm), and discard the upper layer of water; add an equal volume of water to wash the chloroform layer, vortex and oscillate for 60 s, then centrifuge (3 min, 2400 rpm), discard, repeat the washing four times, and discard the upper layer of water; pass the chloroform layer through a membrane (nylon 0.45 μm) and transfer to a gas phase bottle.

[0084] On-line monitoring: Total ion monitoring, scan range: 50-650 m / z. Ion source and quadrupole temperatures: 230°C and 150°C, respectively. Inlet: 250°C. Split ratio: 1:10. A DB-5 column was used. Temperature program: 110°C to 230°C at 5°C / min, then to 300°C at 10°C / min, with a 5-min solvent delay.

[0085] 12. Nuclear magnetic resonance analysis:

[0086] 20 mg of sample was accurately dissolved in 0.5 mL of D2O. The measurement was performed using a fully digital superconducting NMR spectrometer (AvanceNEO600M, USA). The measured spectrum included 1 H and 13 C, heteronuclear single quantum coherence spectroscopy (HSQC), heteronuclear multiple bond correlation spectroscopy (HMBC), hydrogen-hydrogen correlation spectroscopy ( 1 H / 1 H COSY) and Nuclear Overhauser Effect Spectroscopy (NOESY).

[0087] 13. In vitro activity detection

[0088] α-glucosidase inhibition rate:

[0089] Thoroughly mix 40 μL of 5 mmol / L pNPG (prepared in neutral sodium phosphate buffer) and 20 μL of polysaccharide sample solutions of varying concentrations. Incubate at 37°C for 5 minutes. Add 10 μL of 1 U / mL α-glucosidase solution prepared in neutral sodium phosphate buffer, mix thoroughly, and react for 10 minutes (37°C). Terminate the reaction by adding 140 μL of 2 mol / L NaCO3 solution to obtain the sample solution to be tested. Replace the sample solution with 40% ethanol and the enzyme solution with buffer, and follow the above steps. These are recorded as blank and background, respectively. Acarbose is used as the positive control. Measure the absorbance (405 nm) of the reaction solution using the following formula:

[0090] α-glucosidase inhibition rate (%) = 1-(A 样品 -A 背景 ) / A 空白 ×100%

[0091] α-amylase inhibition rate:

[0092] Weigh a certain amount of polysaccharide sample and prepare polysaccharide sample solutions of different concentrations with 20mmol / L sodium phosphate buffer (pH=6.9). Mix 200μL of α-amylase solution (1U / mL, dissolved in sodium phosphate buffer at pH=6.9) and 200μL of polysaccharide solution and record it as the sample; replace the polysaccharide solution with a buffer containing NaCl as the negative control, use the buffer instead of the enzyme solution as the background, and use acarbose as the positive control. Incubate at 37°C for 10min, add 400μL of 1% starch solution, and water bath (37°C) for 10min. Add 1mL DNS color developer to terminate the reaction, add 10mL of distilled water, and measure the absorbance of the background, negative control, and sample at 540nm. The calculation formula is as follows:

[0093] α-amylase inhibition rate (%) = 1-(A 样品 -A 背景 ) / A 阴性 ×100%

[0094] DPPH free radical scavenging rate determination:

[0095] Weigh a certain amount of polysaccharide sample and prepare 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg / mL samples and VC solution with distilled water. Take 1 mL of each concentration sample solution in a test tube. Prepare 300 mL of 0.2 mM DPPH solution, weigh 23.6592 mg of DPPH powder and dissolve it in 300 mL of anhydrous ethanol. Add 2 mL of DPPH solution to the test tube with the sample, mix the sample and react in the dark for 30 minutes. Measure the absorbance at λ = 517 nm. Use VC instead of polysaccharide as the positive control group, water instead of polysaccharide as the blank group, and anhydrous ethanol instead of DPPH as the background group. The calculation formula is as follows:

[0096] DPPH free radical scavenging rate = [A0-(A S -A1)] / A0·100%

[0097] A S --Experimental group = 2 mL sample + 2 mL DPPH-ethanol

[0098] A1--Background = 2mL sample + 2mL anhydrous ethanol

[0099] A0--Blank control = 2mL distilled water + 2mL DPPH-ethanol

[0100] 14. Detection of mRNA expression levels of mouse-related genes

[0101] Total RNA extraction: In this experiment, the Trizol method was used to extract total RNA from mouse spleen tissue. The specific operation method can be found in the "Molecular Cloning Experimental Guide, Fourth Edition".

[0102] cDNA synthesis: The extracted RNA was used as a template and the cDNA was synthesized using Vazyme Extract using the IIQ RT SuperMix for qPCR (+DNAwiper) Kit. For detailed steps, refer to the instructions. Aliquot and freeze after the reaction.

[0103] Detection of related genes by qPCR: Synthesized cDNA was used as a template using the Vazyme ChamQ Universal SYBR qPCR Master Mix kit. Reaction conditions are shown in Table 2. Fluorescence quantitative PCR primers are shown in Table 3.

[0104] Table 2 qPCR reaction system

[0105]

[0106]

[0107] Table 3 qPCR primer information

[0108]

[0109] 15. Statistical analysis methods:

[0110] Each value is expressed as the mean ± standard deviation. Each experiment was performed with at least three biological replicates and three technical replicates. All statistical analyses were performed using GraphiPad Prism 10.0. Differences between groups were assessed using SPSS 23.0 software. Data are presented as the mean ± standard deviation (n = 3). All analyses were statistically significant (P < 0.05).

[0111] Example 1: Preparation method of enzymatically hydrolyzed corn bran polysaccharide

[0112] 1. Extraction of Corn Bran Polysaccharide (CBP)

[0113] The extraction method of corn bran polysaccharide (CBP) is based on Figure 1 The details of A are as follows:

[0114] 1. Processing of raw materials

[0115] After cleaning the corn bran raw material, air it in a cool, sealed place for 3 days, grind it using a grinder, and pass it through a 60-mesh sieve. The resulting corn bran dry powder was then thoroughly mixed with 30-60 boiling range petroleum ether at a material-liquid ratio of 1g:2mL, sealed, and defatted at room temperature for 24 hours. After completion, the supernatant was discarded, dried, and used in subsequent experiments.

[0116] 2. CBP extraction optimization

[0117] Defatted corn bran was extracted using water extraction, and the extraction conditions were optimized using a single-factor plus orthogonal design. Based on a solid-liquid ratio of 1 g:15 mL, extraction temperature of 80°C, extraction time of 2 h, and extraction times of 2, the effects of solid-liquid ratios of 1:5, 1:10, 1:15, 1:20, and 1:25 g / mL, extraction temperatures of 60, 70, 80, 90, and 100°C, extraction times of 1, 2, 3, and 4 h, and extraction times of 1, 2, 3, and 4 times on polysaccharide yield were investigated.

[0118] On the basis of the single-factor experiment, an orthogonal experimental design was conducted for L9(34), and the polysaccharide yield was used as the evaluation index to optimize the extraction process. The orthogonal experimental design factors and levels are shown in Table 4.

[0119] Table 4 Orthogonal test factors and levels

[0120]

[0121]

[0122] The purity of polysaccharides has a significant impact on subsequent experiments. The higher the purity, the easier it is to produce uniform polysaccharides after column chromatography separation and purification. Low purity will lead to heterogeneity in column chromatography results, and impurities will also have a certain impact on the chromatography filler. In addition, the activity testing of polysaccharides also requires high-purity crude polysaccharides. For example, certain proteins and small molecules can affect the activity of polysaccharides, affecting the judgment of the activity of the polysaccharide itself. Therefore, optimizing polysaccharide extraction conditions to achieve the optimal extraction yield and total sugar content is the basis for subsequent experiments.

[0123] The single factor optimization results of CBP extraction conditions are as follows Figure 2 As shown by Figure 2 As shown in A, the extraction rate increases with the increase of the solid-liquid ratio. However, after 1:20, the increase in the extraction rate tends to slow down. Based on cost and time considerations, the solid-liquid ratios of 1:5, 1:10, and 1:15 (g / mL) were selected for subsequent experiments. For the same reason, the extraction time and number of extractions were finally selected as 1, 2, and 3 hours and 1, 2, and 3 times (see Figure 2 B and C in ). By Figure 2 It can be seen from D that at 60-90℃, the extraction rate increased significantly with the increase of temperature, but when it reached 100℃, the extraction rate began to decrease. This may be due to the destruction of some polysaccharide components in corn bran that are not resistant to high temperatures. Based on this, 80, 90, and 100℃ were finally selected for subsequent experiments.

[0124] The results of the orthogonal test for extraction process optimization are shown in Table 5. It can be seen from Table 5 that when polysaccharide content is used as the evaluation index, the order of influence of each factor on the polysaccharide extraction rate is A>D>B>C, that is, solid-liquid ratio>number of extractions>extraction temperature>extraction time, and the best combination scheme is A3B2C3D3.

[0125] Table 5 Orthogonal test results

[0126]

[0127] Verification tests were conducted on the two optimal combinations (A3B2C3D3 and A3B2C1D3) obtained from the orthogonal experiment. The polysaccharide extraction rate of A3B2C3D3 was 3.89%, higher than the 3.57% of A3B2C1D3. Therefore, the optimal process conditions for corn bran polysaccharide extraction were determined to be a solid-liquid ratio of 1:15 (g / mL), an extraction temperature of 90°C, an extraction time of 3 hours, and three extractions.

[0128] The defatted corn bran is extracted using the optimal extraction method of corn bran polysaccharide to obtain crude polysaccharide.

[0129] 3. Preliminary purification of CBP extraction

[0130] The crude polysaccharide extract obtained by the optimal extraction method was rotary evaporated to an appropriate volume. Anhydrous ethanol was then added to a final ethanol concentration of 75%. The extract was allowed to stand overnight at 4°C and centrifuged. The precipitate was reconstituted with water and concentrated under reduced pressure to remove residual ethanol. Papain was then added at 300 U / mL and enzymatically digested at 60°C for 6 hours. The supernatant was thoroughly mixed with Sevage reagent (chloroform to n-butanol, volume ratio = 1:5) and centrifuged. The upper aqueous phase was separated into three layers and repeated four times. The aqueous phase was concentrated under reduced pressure to remove residual Sevage reagent. The extract was dialyzed against tap water, deionized water, and ultrapure water for one day each, and then freeze-dried to obtain CBP.

[0131] The identification results of total sugar, reducing sugar, uronic acid and protein content are shown in Table 6. The total sugar content was 87.5% and the protein content was 2.1%, indicating that the early extraction and purification were in line with expectations.

[0132] The crude polysaccharide extracted by hot water extraction is not entirely polysaccharide, but also contains some reducing sugars. Therefore, it is necessary to determine the reducing sugar content to calculate the polysaccharide content. As shown in Table 6, the polysaccharide content of CBP is 82.4%, and it contains 2.3% uronic acid.

[0133] Table 6 Content of each component in CBP

[0134]

[0135] Monosaccharide composition analysis (Table 7) revealed that CBP contains Fuc (fucose), Ara (arabinose), Rha (rhamnose), Gal (galactose), Glc (glucose), Xyl (xylose), Man (mannose), GalA (galacturonic acid), and GlcA (glucuronic acid), with a relative molar ratio of 0.15:19.67:1.27:6.40:56.75:12.08:1.36:1.14:1.18. This was the basis for the selection of various glycosidases in subsequent enzymatic hydrolysis experiments.

[0136] Table 7 Monosaccharide composition analysis of CBP

[0137]

[0138] 2. Preparation of Enzymatic Corn Bran Polysaccharide (ECBP)

[0139] The preparation method of ECBP refers to Figure 1B: First, select a suitable glycosidase based on the monosaccharide composition of CBP. Prepare CBP into a 0.2 g / mL solution, add glycosidase at a dosage of 2200 U / g, mix thoroughly, and incubate on a shaker at 37°C. After incubation, inactivate in a 100°C water bath for 15 minutes, then centrifuge at 4000 g for 15 minutes, collect the supernatant, dialyze, and then lyophilize.

[0140] The study of CBP enzymatic conditions was activity-oriented. The enzymatic conditions for corn bran polysaccharide hydrolysis were investigated by selecting various glycoside hydrolases (FaeA, AbfA, AraA, XlnA, and XynB) and varying hydrolysis times (3, 6, and 12 hours). Detailed screening through animal experiments identified the optimal enzymatic conditions. The CBP hydrolyzate prepared using this optimal method was named ECBP and used in subsequent experiments.

[0141] In vivo hypoglycemic activity assay:

[0142] 1. Establishment of diabetic mouse model

[0143] The method for establishing a type 2 diabetes mouse model is as follows: All mice were housed in a controlled environment (room temperature 23±2°C, 12 / 12h light / dark cycle) and provided with a normal diet. After one week of adaptation, streptozotocin (STZ, 40 mg / kg) was injected daily. During the modeling period, the mice were fed a high-fat, high-sugar diet. Mice in the control group (NG) were injected with an equal volume of normal saline and fed a standard diet. Five days after the injection, tail blood samples were collected to measure blood glucose levels. Postprandial blood glucose levels greater than 16.7 mmol / L and fasting blood glucose levels greater than 11.1 mmol / L indicated that the model was successfully established. Otherwise, the modeling will continue until it is successfully established. Each experimental group in this study consisted of 12 mice (n=12).

[0144] 2. Screening of enzymatic hydrolysis conditions:

[0145] For the enzymatic hydrolysis screening experiment, mice were administered via oral gavage for 14 days at a daily dose of 100 mg / kg. The model group (MG) was injected with an equal volume of saline. Following the experiment, the mice underwent an oral glucose tolerance test (OGTT) and the area under the curve (AUC) was calculated. The optimal enzymatic hydrolysis conditions were determined by analyzing changes in blood glucose levels before and after administration.

[0146] OGTT is a classic experimental method for assessing glucose metabolism and insulin sensitivity. Before the experiment, ensure that the mice are in good health and fast for 12-16 hours to reduce interference from gastric contents. The bedding needs to be changed during the fasting period to prevent the mice from consuming residual food. After weighing on the day of the experiment, fasting blood glucose is measured by taking blood from the tail: the first drop of blood is discarded and the second drop of blood is used for testing (the blood glucose meter needs to be calibrated to avoid interference from alcohol residue). In the OGTT, glucose solution is gavaged according to body weight (2g / kg). Immediately after gavage, the time is started and blood glucose is continuously monitored at time points such as 0, 30, 60, 90, and 120 minutes. The ambient temperature must be kept constant during the experiment to reduce stress. AUC is calculated using GraphpadPrism10.

[0147] 3. Active component screening

[0148] For the active ingredient screening experiment, mice were administered via oral gavage for 28 days at a daily dose of 100 mg / kg. The model group (MG) was injected with an equal volume of normal saline. Blood glucose was measured every seven days. After the experiment, the mice underwent an oral glucose tolerance test (OGTT) according to the above method, and the area under the curve (AUC) was calculated. The active ingredient was identified by the changes in blood glucose after administration.

[0149] Effects of various glycosidases on the hypoglycemic effect of CBP:

[0150] The hypoglycemic activities of CBP and five enzymatic hydrolysates were preliminarily evaluated in a diabetic mouse model. Figure 3 As shown, the hypoglycemic activity of CBP polysaccharides after different enzymatic hydrolysis treatments was significantly altered compared to the untreated state. Compared to unhydrolyzed CBP, the enzymatic hydrolyzates AbfA-CBP and XynB-CBP demonstrated varying degrees of enhanced hypoglycemic activity in diabetic mice. XynB-CBP exhibited a more significant hypoglycemic effect, with a 26.7% reduction (p < 0.01). Therefore, XynB-CBP received particular attention, and further studies were conducted to determine the optimal enzymatic hydrolysis conditions.

[0151] Effects of different enzymatic hydrolysis times on the blood sugar-lowering effect of CBP:

[0152] The biological evaluation of polysaccharides with different enzymatic hydrolysis times was carried out in a diabetic mouse model. Figure 4 As shown, different hydrolysis times exhibited different activity levels. As the hydrolysis time increased from 0 h to 6 h, the blood glucose reduction rate increased from 4.1% to 21.1%. Notably, as the hydrolysis time was further extended to 12 h, the blood glucose reduction rate showed a downward trend (18.3%). Therefore, considering the actual effect, 6 h was determined to be the most effective hydrolysis time for XynB to hydrolyze CBP.

[0153] Dose dependence and OGTT:

[0154] Dose dependence refers to the phenomenon that the effect of a drug or biologically active substance changes regularly with the dose. Figure 5 As shown in Figure A, with increasing doses (25-50-100 mg / kg), the blood glucose lowering rate increased from 6% to 27.7%, showing an obvious dose-dependent effect.

[0155] OGTT can reflect the degree of dependence of the sample on insulin. Figure 5 As shown in Figure 3B, blood glucose levels peaked at 30 minutes in all groups. The 100 mg / kg group showed a 9.1% decrease in blood glucose compared to the MG group. The most significant hypoglycemic effect was observed at 120 minutes, with a 36.1% decrease compared to the MG group.

[0156] Overall, XynB-CBP-6h was the hydrolyzate with the most significant increase in glucose-lowering activity, with a clear dose-dependent effect. Therefore, XynB-CBP-6h was selected for subsequent experiments and named ECBP.

[0157] 3. Analysis of changes in physical and chemical properties of CBP after enzymatic hydrolysis:

[0158] 1. Infrared spectrum analysis:

[0159] Infrared spectroscopy detects the vibration characteristics of functional groups such as hydroxyl (-OH), CH bond, carbonyl (C=O) in polysaccharide molecules, as well as specific absorption peaks (such as 890cm -1 and 830cm -1 Corresponding to β- and α-glycosidic bonds, respectively), it can quickly identify the functional group composition, sugar ring type and glycosidic bond configuration of polysaccharides, and is an indispensable preliminary analytical method for analyzing the structural characteristics of polysaccharides. Figure 6 As shown in Figure 2, as the hydrolysis time increases, the signal peaks in the infrared spectrum gradually become clearer. In particular, the increase in hydrolysis time significantly increases the -CH2 / -CH3 group at 2850 cm -1 and 2950cm -1 The relative content of the double peak region between 1000cm -1 The vibrations of nearby CO and glycosidic bonds are also significantly enhanced. This phenomenon suggests that enzymatic hydrolysis targets specific chemical bonds within the polysaccharide chain, leading to their cleavage and degradation. This degradation reduces the size of the polysaccharide molecules, loosens their structure, and exposes more functional groups, resulting in distinct absorption peaks in the infrared spectrum. Due to the cleavage and degradation of the polysaccharide chain, previously masked or overlapping absorption peaks are released, resulting in shifts in their corresponding absorption peaks.

[0160] 2. Rheological properties analysis:

[0161] The viscosity characteristics of polysaccharides are closely related to their release and absorption in the body. Therefore, this study tested the viscosity curves of products after different enzymatic hydrolysis times. Figure 7 The relationship between viscosity and shear rate is shown. -1 ), the viscosity of CBP decreases significantly with increasing shear rate, showing typical shear-thinning fluid behavior. This indicates that as the shear rate increases, the fluidity of the solution increases, the mobility of the molecular chains accelerates, and the viscosity decreases. The flow characteristics of this solution are consistent with the typical characteristics of non-Newtonian fluids. In contrast, XynB-CBP-3h has a relatively high viscosity within 10-100s. -1 The viscosity of XynB-CBP-3h was 100-1000s. -1 range, and 10-1000s for XynB-CBP-6h and XynB-CBP-12h -1 Within the range, the viscosity of all three products did not change significantly with increasing shear rate, and the non-Newtonian fluid behavior of CBP was lost. This indicates that XynB successfully modified the rheological properties of CBP.

[0162] 4. Isolation and Purification of ECBP

[0163] From the above experiments, we can see that ECBP has good hypoglycemic activity. In order to analyze the main components of ECBP that exert hypoglycemic activity, ECBP will be separated and purified to explore its active ingredients.

[0164] 1. Ion exchange chromatography results:

[0165] After CBP and ECBP were purified by DEAE celluLose-52, the absorbance of the solutions eluted with 0 mol / L, 0.1 mol / L, and 0.3 mol / L NaCl gradients was measured at 490 nm using the phenol-sulfuric acid method. The elution curves were plotted as follows: Figure 8 The horizontal axis represents the number of elution tubes, and the vertical axis represents the absorbance (490 nm). Six purified fractions were obtained from the three gradient elutions and named p-CBP-1 / p-ECBP-1, p-CBP-2 / p-ECBP-2, and p-CBP-3 / p-ECBP-3.

[0166] 2. Gel chromatography results:

[0167] p-CBP-3 and p-ECBP-3 were purified separately using Sephadex G-200, eluted with deionized water, and tracked using a differential refractive index detector (RID). Figure 9 As shown, p-CBP-1 eluted as a single major component, designated CBP-1, while p-ECBP-3 eluted as two major components, designated ECBP-3 and ECBP-3-1. RID signals during the separation process revealed that p-ECBP-3 exhibited a lower molecular weight minor component, ECBP-3-1, compared to p-CBP-3. This component is likely an oligosaccharide product of enzymatic hydrolysis. Due to the low abundance of ECBP-3-1, only the structural features of CBP-3 and ECBP-3 are reported in this study.

[0168] The total sugar, protein and uronic acid contents were determined by colorimetry. The results are shown in Table 8. No protein components were found and the total sugar content was high, thus proving that the purification effect was good.

[0169] Table 8 Content detection results of each component of CBP-3 and ECBP-3

[0170]

[0171] 5. Screening of ECBP for its hypoglycemic activity

[0172] Postprandial blood sugar and fasting blood sugar are key criteria for diagnosing diabetes. Figure 10 A and Figure 10 As shown in Figure B, after the start of the experiment, the blood glucose levels of the NG group remained stable within a certain range after meal and fasting. In contrast, the blood glucose levels of the MG group continued to rise during the treatment, which is a characteristic of diabetic model mice. Figure 10 As shown in Figure A, after 28 days of treatment, the postprandial blood glucose level in the ECBP-3 group decreased by 24.2% compared with the level before treatment (p<0.01). Figure 10 As shown in Figure B, the fasting blood glucose level in the ECBP-3 group was significantly reduced, down 19.4% compared to the level before treatment (P<0.01). The fasting blood glucose levels in all other groups (except the NG group) showed varying degrees of increase.

[0173] Oral glucose tolerance test (OGTT) and area under the curve (AUC) are commonly used clinical methods for diagnosing diabetes, which can determine the degree of dependence of the sample on insulin. Figure 10 As shown in Figure C, except for the ECBP-3 group (which peaked at 30 minutes), all groups had peak blood glucose levels at 60 minutes. Among them, the MG group had the highest blood glucose level, exceeding 33mmol / L per mouse. Over time, the blood glucose levels of all groups gradually decreased to varying degrees. Throughout the experiment, the ECBP-3 group showed faster blood glucose regulation, with blood glucose levels significantly lower than those of the MG group (P<0.05). Figure 10As shown in Figure D, the ECBP-3 group had the smallest AUC, indicating reduced insulin dependence and reduced progression of diabetes. These results indicate that ECBP-3 is the most effective component isolated from ECBP.

[0174] VI. Changes in physicochemical properties and in vitro activity before and after enzymatic hydrolysis

[0175] 1. Molecular weight analysis

[0176] High-performance liquid chromatography-multi-angle light scattering detection-refractive index detection-ultraviolet-visible detection (HPSEC-MALLS-RID-Vis) was used to analyze the molecular weight and conformation of CBP-3 and ECBP-3. First, HPSEC-RID of CBP-3 and ECBP-3 showed a single and symmetrical peak, indicating that they are homogeneous polysaccharides ( Figure 11 ). In addition, the M of CBP-3 W is 9.658×10 4 g / mol, while ECBP-3's M W is 4.252×10 4 g / mol, showing a significant decrease, indicating that the enzymatic treatment caused the molecular weight of corn bran polysaccharides to change, which corresponds to the minor component ECBP-3-1 in the separation process ( Figure 9 The polydispersity indices (Mw / Mn, PDI) of CBP-3 and ECBP-3 were 1.229 and 1.326, respectively, indicating that CBP-3 and ECBP-3 had good purity with narrow Mw distribution.

[0177] At the same time, SEC-MALS-RI-Vis can directly measure the V value of the polymer, which can be used to preliminarily determine the molecular conformation. Generally, a V value of less than 0.33, 0.50-0.60, or greater than 1.0 indicates that the polymer molecular conformation is spherical, flexible, or rigid. The measurement results show that CBP-3 has a V value of 0.60, which indicates a flexible coil, while CBP-3 has a V value of 0.33, which indicates a spherical shape. This indicates that the molecular conformation has undergone significant changes after enzymatic hydrolysis. The reduction in molecular weight and the change in molecular conformation may be one of the reasons for its enhanced hypoglycemic activity.

[0178] 2. Monosaccharide composition analysis

[0179] HPAEC-PAD was used to analyze the monosaccharide composition of CBP-3 and ECBP-3. HPAEC-PAD detection showed that both CBP-3 and ECBP-3 are composed of Fuc, Rha, Ara, Gal, Glc, Man, Xyl, GalA and GlcA (see Figure 12However, considering the incomplete hydrolysis caused by the resistance of GalA during polysaccharide hydrolysis, as well as the overhydrolysis of Xyl, Rha, and Ara, the monosaccharide composition profile is more qualitative, and the results are summarized in Table 9. Overall, both CBP-3 and ECBP-3 are complex acidic heteropolysaccharides, and the co-presence of Rha and GalA suggests the possibility of RG-I type pectin.

[0180] Table 9 Monosaccharide composition results of CBP and ECBP-3

[0181]

[0182]

[0183] 3. α-amylase inhibition rate

[0184] The inhibition rate of polysaccharides on α-amylase reflects their ability to regulate postprandial blood glucose levels by delaying starch digestion and reducing glucose production, which is of great significance for diabetes management and the development of natural hypoglycemic drugs. Figure 13 As shown. Overall, the inhibition rate of α-amylase after enzymatic hydrolysis (ECBP-3) was significantly improved. For ECBP-3, the inhibition rate increased rapidly with the increase of concentration in the range of 0.5-2 mg / mL. The maximum inhibition rate (2.18 times) was reached at 2 mg / mL compared to before enzymatic hydrolysis. However, when the concentration exceeded 2 mg / mL, the inhibitory effect of CBP-3 and ECBP-3 on α-amylase tended to stabilize as the concentration continued to increase. At the maximum inhibitory concentration, ECBP-3 showed a 1.72-fold increase in α-amylase inhibition compared to CBP-3. Overall, the inhibition of α-amylase was significantly improved after enzymatic hydrolysis compared to before enzymatic hydrolysis, which may be one of the reasons why the inhibitory effect on mouse blood glucose after enzymatic hydrolysis was better than before enzymatic hydrolysis in in vivo experiments.

[0185] 4. Glucosidase activity

[0186] The inhibition rate of polysaccharides on α-glucosidase reflects its ability to reduce postprandial blood glucose levels by slowing down the rate at which carbohydrates are broken down into glucose, which is of great significance for the dietary regulation and blood glucose management of diabetes (especially type 2 diabetes). Figure 14 It can be seen that, overall, the inhibition rate of glucosidase after enzymatic hydrolysis did not show a significant advantage over that of α-amylase. When the maximum concentration (26.7%) was reached, the inhibition rate after enzymatic hydrolysis increased by 1.69 times compared to that before enzymatic hydrolysis.

[0187] 5. DPPH free radical scavenging rate

[0188] The scavenging rate of polysaccharides for DPPH free radicals reflects their ability to act as electron or hydrogen donors and is an important indicator for evaluating the antioxidant activity of polysaccharides. The higher the scavenging rate, the stronger the effect of terminating free radical chain reactions through reduction reactions, which can be used as a basis for the development of natural antioxidants. Figure 15 It can be seen that, on the whole, the scavenging rate of DPPH free radicals (ECBP-3) was significantly improved after enzymatic hydrolysis. For ECBP-3, the inhibition rate increased rapidly with the increase of concentration in the range of 0.05-0.1 mg / mL. The maximum advantageous inhibition rate (1.72 times) was reached at 2 mg / mL compared to before enzymatic hydrolysis. However, when the concentration exceeded 2 mg / mL, as the concentration continued to increase, the scavenging rate of DPPH free radicals by CBP-3 and ECBP-3 tended to stabilize. At the maximum inhibitory concentration, ECBP-3 showed that the scavenging rate of DPPH free radicals increased by 1.52 times compared with CBP-3. Overall, the scavenging rate of DPPH free radicals was significantly improved after enzymatic hydrolysis compared to before enzymatic hydrolysis.

[0189] 6. Methylation analysis

[0190] The methylation analysis results of CBP-3 and ECBP-3 are shown in Tables 10 and 11. The total ion currents of CBP-3 and ECBP-3 after methylation are shown in Tables 10 and 11. Figure 16 shown.

[0191] Table 10 CBP-3 methylation analysis results

[0192]

[0193]

[0194] Table 11ECBP-3 methylation analysis results

[0195]

[0196]

[0197] 7. Nuclear Magnetic Resonance Spectroscopy and Structural Formula Prediction

[0198] Nuclear magnetic resonance spectroscopy is an important method for polysaccharide structural analysis. One-dimensional and two-dimensional NMR spectra of polysaccharides can be used to identify the configuration and linkage of glycosidic bonds within them. This study combined the results of one-dimensional and two-dimensional NMR spectra of ECBP-3 with methylation analysis and analyzed the unit sequence and linkage pattern of ECBP-3 in conjunction with previously reported literature.

[0199] exist 1 In the anomeric proton region (4.3-5.8 ppm) of the H NMR spectrum (see Figure 17), found several groups of H1 signals of the anomeric protons of residual sugars, and determined the main peak positions of 5.00, 5.06, 5.13, 5.17, 4.58, 4.60, 4.57, 5.04, 4.94, 5.16, 5.21, 5.12, 4.57, 4.60, 4.43, 4.57 and 4.83δ, confirming that the residual sugars in the polysaccharide have both α and β configurations. The signals at 2.09 and 1.20 ppm may be attributed to the acetylated group and the C6 methyl group of Rha, respectively. The signal at 3.20 ppm may be attributed to H4 of Rha. The weak signal at 3.30 to 3.45 ppm may be attributed to H2 of GalA. Finally, the signal at 5.0 to 5.5 ppm may be attributed to the H1 signals of GalA, Rha and Ara.

[0200] exist 13 C NMR spectrum (see Figure 17 ), a clear carbonyl signal was observed at 165-180 ppm in the low field, which is a typical -COOR signal of uronic acid, thus confirming that the polysaccharide is acidic. This result is consistent with the monosaccharide composition analysis. 13 In the CNMR spectrum, 107.8, 107.4, 106.4, 107.0, 99.7, 100.1, 99.7, 98.0, 98.1, 97.7, 100.5, 100.6, 99.7 and 103.1 anomeric carbons indicate that the polysaccharide is composed of multiple residues. 13 In the C NMR spectrum, the signals at 106 to 108 ppm may be attributed to the C1 position of Ara, and the signals at 20 to 30 ppm may be attributed to the acetylated group and the C6 methyl group of Rha, respectively.

[0201] 2D-NMR( 1 H / 1 HCOSY, HSQC, HMBC and NOESY spectra were used to analyze the structure of the polysaccharide. 17 major H1 / C1 related signals were identified in the HSQC spectra (see Figure 18 A in ). They are designated as residues AQ respectively. 1 H- 1 HCOS Y spectrum ( Figure 18 C) Find the coupling peak of HH, and then 1 H– 13 C HSQC spectrum (see Figure 18 A) and find the HC coupling peak, and then analyze the methylation results and reported literature. 1 H and 13 The main chemical shifts of C are shown in Table 12.

[0202] Based on the magnitude of the main chemical shift intensities and the peak signals in the research literature, a comprehensive analysis of the main abnormal residues was performed. The residues AO were Araf-(1→、→3)-Araf-(1→、→3,5)Araf-(1→、→5)-Araf-(1→、→4)-β-Galp-(1→、→4,6)-β-Galp-(1→、Glcp-(1→、→4)-Glcp-(1→、→4,6)-Glcp-(1→、→2)-α-Rhap-(1→、→2,4)-α-Rhap-(1→、β-Xylp-(1→、→4)-β-Xylp-(1→、β-GlcpA-(1→、 and →4)-α-GalA-(1→[89-00].

[0203] The correlation of sugar residues was further analyzed using HMBC. Figure 18 B in can observe D H1 -L C4 and D H1 -L H4 The signal of →5)-Araf-(1→2,4)-α-Rhap-(1→ is detected by C H1 -D C5 and C H1 -D H5 It can be determined that there is a structure of →3,5)-Araf-(1→5)-Araf-(1→. H1 -C C3 and B H1 -C H3 It can be determined that there is a structure of →3)-Araf-(1→3,5)-Araf-(1→. H1 -B C3 、A H1 -D C5 and A H1 -C C3 It can be determined that there are structures of Araf-(1→3)-Araf-(1→, Araf-(1→5)-Araf-(1→ and Araf-(1→3,5)-Araf-(1→. H1 -L C4 、F H1 -E C4 、D H1 -I H4 and G H1 -E C4It can be determined that 4)-β-Galp-(1→2,4)-α-Rhap-(1→, →4,6)-β-Galp-(1→4)-β-Galp-(1→ and β-Galp-(1→4)-β-Galp-(1→. By I H1 -L H4 and H H1 -I H3 It can be determined that the structures of →4)-Glcp-(1→2,4)-α-Rhap-(1→ and Glcp-(1→4)-Glcp-(1→ exist.

[0204] In summary, it is speculated that the structural formula of ECBP-3 is as follows Figure 19 shown.

[0205] Table 12 ECBP-3 1 H and 13 Summary of the main chemical shifts of C

[0206]

[0207]

[0208] VII. Changes in in vitro activity before and after enzymatic hydrolysis

[0209] 1. ECBP-3 can alleviate intestinal damage:

[0210] At the end of the experiment, three mice were randomly selected from each group for intestinal morphological observation. Figure 20 As shown in Figure A, the intestinal structure of the MG group was significantly atrophied, the intestinal wall was fragile, the permeability increased, and it was easily damaged. The colon length was also significantly shortened, which may be related to inflammatory infiltration or intestinal obstruction.

[0211] After H&E staining of the proximal, middle and distal small intestine and colon, Figure 20 As shown in Figures B, C, D, and E, compared with the NG group, the MG group exhibited disorganized crypt architecture, with a significant decrease in the number of goblet and absorptive cells within the crypts, abnormal epithelial cell morphology, uneven nuclear distribution, and blurred cell borders. The colonic glandular architecture also showed signs of disorganization. In contrast, the ECBP-3 group demonstrated significant restoration of intestinal architecture, with overall normalization and improved morphological and functional characteristics. These results indicate that ECBP-3 treatment partially reversed the intestinal damage induced by the MG group and improved intestinal morphology.

[0212] 2. Analysis of mRNA expression levels of genes related to mouse transcription level:

[0213] In diabetic mice, intestinal IL-6 mRNA expression increased significantly, which may be related to chronic low-grade inflammation and intestinal barrier damage caused by hyperglycemia. The intestinal IL-1β mRNA level of diabetic mice also increased significantly, which may be related to impaired intestinal barrier integrity. The expression of IL-6 mRNA and IL-1β mRNA in the model group (MG) was significantly upregulated compared with the normal group (NG), while the expression in the ECBP-3 group was significantly restored compared with the model group. This indicates that the chronic intestinal inflammation caused by hyperglycemia has been suppressed, and the integrity of the intestinal barrier has been restored (see Figure 21 A and Figure 21 B) in.

[0214] In diabetic mice, the level of intestinal GLP-1 mRNA concentration decreased significantly. The possible reasons are as follows: first, the function of enteroendocrine cells (L cells) is impaired: hyperglycemia and lipotoxicity inhibit the proliferation and hormone secretion of L cells; second, intestinal flora disorder: under the diabetic state, the abundance of short-chain fatty acid (SCFAs) producing bacteria (such as Clostridium butyricum) decreases, resulting in the weakening of the SCFAs-GLP-1 axis function. The expression of this gene was significantly downregulated in the model group (MG) compared with the normal group (NG), while the expression of this gene was significantly restored in the ECBP-3 group after drug treatment (see Figure 21 C).

[0215] In the liver tissue of diabetic mice, the mRNA expression level of GLUT-2 was significantly higher than that of the normal control group. This upregulation may be due to the adaptive regulation of glucose metabolism by hepatocytes under hyperglycemic conditions, which promotes glucose uptake and metabolism by increasing the expression of GLUT-2. The expression of this gene was significantly upregulated in the model group (MG) compared with the normal group (NG), while the expression levels of ECBP-2 and ECBP-3 groups were significantly restored compared with the model group (see Figure 21 D) in.

Claims

1. A method for preparing corn bran polysaccharide, characterized in that: The steps include: S1. Raw material pretreatment: corn bran is washed, aired, crushed, and sieved to obtain corn bran dry powder, which is then defatted with petroleum ether to obtain defatted corn bran; S2, water extraction: The defatted corn bran obtained in S1 was mixed with water at a solid-liquid ratio of 1 g:15 mL, and water-extracted at 90°C for 3 h. The filtrate was collected by filtration. This was performed three times in total, and the filtrates obtained from each extraction were combined. S3, purification: the filtrate obtained in S2 was subjected to rotary evaporation, and after rotary evaporation, anhydrous ethanol was added for alcohol precipitation, the precipitate obtained by the alcohol precipitation was redissolved in water, and residual ethanol was removed by concentration under reduced pressure, and papain was added in an amount of 300 U / mL, and enzymatic hydrolysis was carried out at 60° C. for 6 h. Sevage reagent was added to the obtained enzymatic hydrolyzate, and the upper aqueous phase was collected by centrifugation after thorough mixing. The steps of thorough mixing with Sevage reagent and centrifugation were repeated 3 times, and residual Sevage reagent was removed by concentration under reduced pressure. The crude corn bran polysaccharide was dialyzed against tap water, deionized water, and ultrapure water for one day each, and freeze-dried to obtain the crude corn bran polysaccharide. S4, enzymatic hydrolysis: The crude corn bran polysaccharide obtained in S3 was prepared into an aqueous solution with a concentration of 0.2 g / mL, 2200 U / g glycosidase XynB was added, and the mixture was incubated on a shaker at 37°C for 6 h. After the enzyme was inactivated, the supernatant was collected by centrifugation, dialyzed, and freeze-dried to obtain enzymatically hydrolyzed corn bran polysaccharide; S5. Separation and purification: The enzymatically hydrolyzed corn bran polysaccharide obtained in S4 was separated and purified using a DEAE celluLose-52 ion exchange column and a Sephadex G-200 gel column in sequence to obtain corn bran polysaccharide.

2. The preparation method according to claim 1, characterized in that The solid-liquid ratio of the corn bran dry powder to petroleum ether in S1 is 1 g:2 mL, and the degreasing treatment time is 24 h.

3. The preparation method according to claim 1, characterized in that The alcohol precipitation in S3 was performed by adding anhydrous ethanol until the volume fraction of ethanol was 75%, and standing at 4° C. overnight; the volume ratio of the enzymatic hydrolysate to the Sevage reagent was 1:

5.

4. The preparation method according to claim 1, characterized in that The purification method of the ion exchange column described in S5 is as follows: prepare a polysaccharide sample with a concentration of 25 mg / mL, centrifuge and discard the precipitate; load the sample with a sample volume not exceeding 3% of the column volume; elute with 0 mol / L, 0.1 mol / L, and 0.3 mol / L NaCl solutions in sequence, with a flow rate controlled at 1 mL / min, collect the solution using an automatic collector, and collect 10 mL into one tube; track the sample using the phenol-sulfuric acid method; finally, draw an elution curve with the number of eluent tubes as the horizontal axis and the A490 nm absorbance as the vertical axis; The same fractions were collected and combined, dialyzed with deionized water in a dialysis bag with a cutoff flow rate of 3500 Da for 3 days, and then freeze-dried to obtain the corresponding fractions.

5. The preparation method according to claim 1, characterized in that The purification method of the gel column described in S5 is as follows: a polysaccharide sample is prepared into a 15 mg / mL polysaccharide solution, which is centrifuged and filtered through a 0.45 μm aqueous filter membrane; the sample is loaded, with the loading volume not exceeding 3% of the column volume, and eluted with deionized water at a flow rate of 0.3 mL / min. The sample is collected in an automatic collector, with each 3 mL collected in one tube, and the sample is tracked using a differential refractive index detector; The same fractions were collected and combined, dialyzed with deionized water in a dialysis bag with a cut-off flow rate of 3500Da for 3 days, and then freeze-dried to obtain the purified polysaccharide sample.

6. Corn bran polysaccharide obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the corn bran polysaccharide according to claim 6 in the preparation of a drug for lowering blood sugar.

8. Use of the corn bran polysaccharide according to claim 6 in the preparation of a medicament for preventing and / or treating diabetes.

9. Use of the corn bran polysaccharide according to claim 6 in the preparation of a medicament for alleviating intestinal damage caused by diabetes.

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