Preparation of fucoidan degradation product with high uronic acid content and application thereof in reducing blood sugar

Abstract

The application discloses a preparation method of a high-glycuroninoglycan content fucoidan degradation product and application thereof. The specific preparation method is as follows: after fucoidan powder is treated by hot air assisted radio frequency drying, the fucoidan degradation product is obtained by acid hydrolysis combined with a two-step sedimentation method, the average molecular weight of the fucoidan degradation product is 7-9 kDa, the monosaccharide components are fucose, xylose, mannose, galactose, rhamnose, glucuronic acid and galacturonic acid, and the molar proportion of glycuroninoglycan is higher than 50%. The fucoidan degradation product provided by the application can effectively reduce the activities of alpha-amylase and alpha-glucosidase through reversible inhibition, and can effectively reduce the postprandial blood glucose level of mice in animal experiments. The fucoidan degradation product provided by the application has a good prospect in the field of developing blood glucose lowering food.

Description

Technical Field

[0001] This invention relates to the field of low molecular weight fucoidan preparation technology, specifically to the preparation of fucoidan degradation products with high uronic acid content and their application in lowering blood sugar. Background Technology

[0002] Type 2 diabetes, also known as non-insulin-dependent diabetes mellitus, is a group of metabolic diseases characterized by hyperglycemia caused by insulin resistance and abnormal insulin production. Blood glucose levels rise rapidly in patients with type 2 diabetes due to the hydrolysis of starch by pancreatic α-amylase and the absorption of glucose by α-glucosidase in the small intestine. Inhibiting these glycolytic enzymes can significantly reduce postprandial hyperglycemia after a mixed carbohydrate diet and is a key strategy for controlling diabetes. However, currently used hypoglycemic drugs, such as acarbose, may cause side effects such as bloating, flatulence, and vomiting. Therefore, it is necessary to find natural sources of ingredients to slow hyperglycemia and reduce side effects.

[0003] Fucoidan, extracted from brown algae or marine invertebrates, is mainly composed of fucose and sulfate groups, supplemented by trace amounts of xylose, mannose, arabinose, galactose, and glucuronic acid. Numerous studies have shown that fucoidan can regulate blood glucose levels through various mechanisms, which are closely related to its structure, with molecular weight being a crucial factor. Compared to high molecular weight polysaccharides, low molecular weight polysaccharides or oligosaccharides exhibit better solubility and bioavailability. However, the postprandial blood glucose-lowering effect of low molecular weight fucoidan is rarely reported. Literature reports that the monosaccharide composition of fucoidan also affects its hypoglycemic effect, with the content of fucose, uronic acid, and galactose potentially being important reasons for differences in activity; fucoidan with higher content of these monosaccharides exhibits better hypoglycemic activity. Therefore, preparing a low molecular weight fucoidan with high uronic acid content is of great significance for hypoglycemic research. Summary of the Invention

[0004] The purpose of this invention is to provide a fucoidan degradation product (FUDP) with high uronic acid content and its application in drugs that lower postprandial blood glucose.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing fucoidan degradation products with high uronic acid content, comprising the following steps:

[0006] Step 1: Fucoidan samples were treated by a two-step precipitation method combining acid hydrolysis with quaternary ammonium salt and alcohol precipitation to obtain fucoidan degradation products;

[0007] Step 2: Evaluate the in vitro hypoglycemic effect and analyze the mechanism of the fucoidan degradation products prepared in Step 1.

[0008] Furthermore, the fucoidan is a commercially available product derived from brown algae.

[0009] Furthermore, the fucoidan is pretreated before acid hydrolysis, specifically by using hot air-assisted radio frequency drying to dry the fucoidan powder at a temperature of 60°C until the moisture content reaches below 10%.

[0010] Furthermore, the acid solution is one or more of hydrochloric acid, trifluoroacetic acid, trichloroacetic acid, and phosphoric acid.

[0011] Furthermore, during the acidolysis treatment, the acidolysis temperature is controlled at 60-90℃, and the acidolysis time is 4-6 hours.

[0012] Furthermore, after acidolysis, volatile acids are removed by rotary evaporation under heating, and an alkaline solution is added to adjust the pH to neutral.

[0013] Furthermore, the quaternary ammonium salt is hexadecylpyridine chloride. 0.5-1.0% of the quaternary ammonium salt is added to the acid hydrolysate for sedimentation, and the sedimentation time is 12-24 hours. The precipitate is then obtained by centrifugation.

[0014] Furthermore, the alcohol precipitation reagent is anhydrous ethanol. The above precipitate is dissolved in sodium chloride solution and then mixed with anhydrous ethanol solution at a volume ratio of 1:3 to 5. The mixture is allowed to precipitate overnight, and the precipitate is obtained by centrifugation.

[0015] A high-uronic acid content fucoidan can be used to lower postprandial blood glucose and as an inhibitor of α-amylase and α-glucosidase, and can be used in the development of novel hypoglycemic drugs or functional foods.

[0016] This invention has at least the following beneficial effects:

[0017] This invention comprehensively investigated the hypoglycemic activity of FUDP through in vitro and in vivo experiments, focusing on its inhibitory effects on α-amylase and α-glucosidase activities, reaction kinetics, conformation, and physiological indicators of postprandial blood glucose in mice. The results showed that FUDP significantly inhibited the activities of both α-amylase and α-glucosidase, with half-maximal inhibitory concentrations (IC50) of 5.815 and 4.439 mg / mL, respectively. Reaction kinetics indicated that FUDP exhibited mixed inhibition of α-amylase and non-competitive inhibition of α-glucosidase. Circular dichroism and fluorescence spectroscopy analyses revealed that FUDP altered the polarity of the Trp and Tyr residues in both α-amylase and α-glucosidase, effectively modifying their secondary structures. In in vivo experiments, FUDP significantly inhibited the increase in postprandial blood glucose levels in normal mice and prediabetic mice.

[0018] Therefore, the FUDP described in this invention has a good hypoglycemic effect and, as an inhibitor of α-amylase and α-glucosidase, can be used in the development of novel hypoglycemic drugs or functional foods. Furthermore, the FUDP provided by this invention is an active ingredient derived from seaweed that can significantly reduce postprandial blood glucose, and is characterized by its green and safe nature. Attached Figure Description

[0019] Figure 1 Molecular weight distribution map of FUDP;

[0020] Figure 2 Graph showing the inhibitory effect of FUDP on α-amylase activity;

[0021] Figure 3 Graph showing the inhibitory effect of FUDP on α-glucosidase activity;

[0022] Figure 4 Inhibition kinetics of FUDP against α-amylase Figure 4 -A is the reaction kinetics diagram. Figure 4 -B is a Lineweaver-Burk plot;

[0023] Figure 5 Inhibitory kinetics of FUDP against α-glucosidase Figure 5 -A is the reaction kinetics diagram. Figure 5 -B is a Lineweaver-Burk plot;

[0024] Figure 6 Secondary structure changes of α-amylase and α-glucosidase Figure 6 -A is a circular dichroism chromatogram of the effects of different concentrations of FUDP on α-amylase. Figure 6 -B is a circular dichroism chromatogram of the effect of different concentrations of FUDP on α-glucosidase;

[0025] Figure 7 Fluorescence quenching spectra of α-amylase and α-glucosidase Figure 7 -A shows the fluorescence quenching spectra of different concentrations of FUDP on α-amylase. Figure 7 -B shows the fluorescence quenching spectra of different concentrations of FUDP on α-glucosidase;

[0026] Figure 8 FUDP lowers postprandial blood glucose. Figure 8 -A represents the effect of FUDP on reducing postprandial blood glucose in normal mice. Figure 8 -B represents the effect of FUDP on reducing postprandial blood glucose in prediabetic mice. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] Example 1: A method for preparing fucoidan degradation products with high uronic acid content, the specific process parameters are as follows:

[0029] Accurately weigh 8.0 g of fucoidan sample and dry it using hot air circulation in an RF device with an electrode gap of 100 mm and a temperature of 60℃. After drying, add 125 mL of 0.2 mol / L trifluoroacetic acid and hydrochloric acid solution respectively, dissolve completely, and react at 90℃ for 6 h with a shaking speed of 125 rpm. After the reaction, cool in ice water to terminate the reaction. Centrifuge the reaction solution at 8000 rpm for 15 min and collect the supernatant. Rotary evaporate the supernatant at 55℃ to remove volatile acids, and neutralize the solution with 0.2 mol / L NaOH. After deacidification, add 1% hexadecylpyridine chloride and allow to precipitate for 24 h. Centrifuge at 6000 rpm for 10 min to obtain the precipitate, and redissolve it with 20 mL of 3 mol / L sodium chloride solution. Add 4 volumes of anhydrous ethanol for overnight alcohol precipitation. Centrifuge at 8000 rpm for 10 min to obtain the precipitate, redissolve it again, desalt it through an ultrafiltration membrane, and freeze-dry to obtain the fraction, which is FUDP.

[0030] Example 2: Determination of FUDP Molecular Weight Distribution

[0031] The molecular weight distribution of FUDP in Example 1 was determined using high-performance liquid chromatography (HPLC). A W410 refractive index detector and a TSKgel G4000PWXL column (8 μm, 7.8 × 300 mm) were used. The system temperature was 35 °C, and the eluents were 0.2 mol / L NaNO3 and 0.01 mol / L NaH2PO4. 20 μL of FUDP solution (10 mg / mL) was injected into the column, and elution was performed at a flow rate of 0.5 mL / min. Standard curves were plotted using different molecular weights of dextran (80, 150, 270, 410, and 670 kDa).

[0032] Result: As Figure 1 As shown, FUDP is not a single polysaccharide component, and its average molecular weight is 8.06 kDa.

[0033] Example 3: Determination of FUDP monosaccharide composition

[0034] Weigh 8 mg of FUDP from Example 1 and dissolve it in 3 mL of 2 mol / L trifluoroacetic acid solution. Hydrolyze at 110 °C for 6 h. Remove TFA using a rotary vacuum evaporator. Adjust the pH of the solution to 7.0 with 6 mol / L NaOH. Add the monosaccharide standard and the acid hydrolysis product of SCGP to PMP solution (0.5 mL, 0.5 mol / L) and heat at 60 °C for 60 min. After cooling, adjust to neutral with NaOH solution, then extract five times with chloroform to remove excess PMP. Filter the supernatant through a 0.22 μm organic phase filter membrane. The chromatographic column was a ZORBAX 300XDB-C18 column with a wavelength of 245 nm. The mobile phase A was acetonitrile, and the mobile phase B was 0.05 mol / L phosphate buffer solution. Gradient elution was performed from 0 to 60 min. The initial setting was mobile phase A: mobile phase B = 17%: 83%, and the final elution ratio was mobile phase A: mobile phase B = 20%: 80%. The injection volume was 10 μL.

[0035] Results: As shown in Table 1, FUDP is composed of eight monosaccharides, and the composition and molar mass ratio of each monosaccharide are as follows: fucose: galactose: mannose: xylose: glucose: rhamnose: glucuronic acid: galacturonic acid = 15.62: 20.75: 0.50: 5.89: 5.30: 1.74: 40.04: 10.16.

[0036] Table 1. Composition of FUDP monosaccharides

[0037]

[0038] Example 4: Determination of FUDP uronic acid content

[0039] Prepare standard solutions of 12.5, 25.0, 37.5, 50.0, 62.5, 75.0, and 100 μg / mL in test tubes. Add 400 μL of the sample to each test tube. In an ice-water bath, add 2.4 mL of sodium tetraborate-sulfuric acid solution to each test tube. After the test tubes have completely cooled, remove them from the ice-water bath and vortex to mix. Immerse the test tubes in a boiling water bath for 20 min, cool with tap water, and let stand at room temperature for 5 min. Add 80 μL of 0.15% m-hydroxybiphenyl solution to each test tube, vortex rapidly to mix, let stand for 10 min, and measure the absorbance at 525 nm. A standard curve can be obtained based on the working standard solutions. Calculate the uronic acid content based on the standard curve.

[0040] Results: The total uronic acid content in FUDP was determined to be 23.20%.

[0041] Example 5: Determination of the inhibitory activity of FUDP against α-amylase

[0042] Porcine pancreatic α-amylase was dissolved at a concentration of 0.5 U / mL in 20 mM phosphate-buffered saline (PBS) (pH = 6.9). 125 μL of Example 1FUDP solution at different concentrations in 20 mM PBS was mixed with 125 μL of α-amylase solution. The mixture was incubated at 37°C for 30 min, followed by the addition of 350 μL of 1% soluble starch solution and a reaction time of 10 min. The reaction was terminated by adding 1 mL of DNS, and the mixture was boiled for 5 min. The absorbance was measured at 540 nm. The inhibition rate (%) was calculated using the following formula, and the half-inhibitory concentration (IC50) was calculated based on the inhibition curve. 50 )

[0043]

[0044] A3, A4, A1, and A2 are defined as the inhibition group (composed of FUDP and α-amylase), the inhibition control group (composed of FUDP but without enzyme), the blank control group (composed of α-amylase but without FUDP), and the blank control group (containing only PBS), respectively.

[0045] Result: Most of the dietary starch ingested by the human body is broken down into oligosaccharides or monosaccharides by α-amylase and α-glucosidase, and then absorbed by intestinal epithelial cells, leading to elevated blood glucose levels. Figure 2 As shown, the inhibition rates of FUDP and acarbose against α-amylase were positively correlated with increasing concentration within a specific range. Notably, the inhibition rate exceeded 80% when the FUDP concentration reached 10 mg / mL, achieving the optimal inhibitory effect of acarbose. The IC50 of FUDP was calculated to be... 50 The values ​​were 5.815 mg / mL.

[0046] Example 6: Determination of FUDP's inhibitory activity against α-glucosidase

[0047] 100 μL of FUDP solutions of different concentrations were mixed with an equal volume of α-glucosidase solution (0.5 U / mL, pH = 6.9) and incubated at 37 °C for 20 min. 100 μL of p-nitrophenyl-α-D-glucopyranoside (pNPG, 5 mM) was added and incubated at 37 °C for 30 min. The reaction was terminated by adding 1 mL of 1 mol / L Na2CO3, and the absorbance was measured at 405 nm. The inhibitory activity was calculated using equation (1).

[0048] Result: As Figure 3 As shown, the inhibitory effect of FUDP on α-glucosidase is dose-dependent. At a concentration of 10 mg / mL, FUDP achieves 100% inhibition of α-glucosidase. The calculated IC50 of FUDP... 50The concentration was 4.439 mg / mL. These results indicate that FUDP has a better inhibitory effect on α-glucosidase than on α-amylase.

[0049] Example 7: Inhibition kinetics and inhibition type determination of FUDP against α-amylase

[0050] The reaction kinetic parameters and maximum reaction rate (V) were determined using the Lineweaver-Burk plot of the Michaelis-Menten equation. max ) and Michaelis constant (K m To determine the concentrations of FUDP (5.0, 10.0, 15.0 mg / mL), α-amylase solutions of different concentrations (0.5, 1.0, 1.5, 2.0, and 2.5 U / mL) were incubated with inhibitor solutions at 37°C for 30 min, with the soluble starch concentration controlled at 1%. The reaction rate (mg / mL·min) was then measured. -1 The reaction rate was expressed as the amount of glucose produced per minute (mg). An enzyme kinetic graph was plotted with α-amylase concentration on the x-axis and reaction rate on the y-axis. With the α-amylase concentration fixed at 1.5 U / mL, the enzyme was incubated with inhibitor solutions at 37°C for 30 min. The reaction rates of different concentrations of FUDP (5, 10, 15 mg / mL) catalyzing soluble starch solutions of different concentrations (0.25%, 0.5%, 1.0%, 1.5%, and 2%) were then measured. Lineweaver-Burk curves were plotted with 1 / s on the x-axis and 1 / V on the y-axis to determine the inhibition type of the inhibitor.

[0051]

[0052] result: Figure 4 -A shows the inhibitory kinetics of FUDP on α-amylase. All hydrolysis rate curves intersected the origin with varying concentrations of FUDP and α-amylase, indicating that the inhibition of α-amylase by FUDP is reversible. Simultaneously, all slopes decreased with increasing inhibitor concentration, suggesting a positive correlation between the inhibitory effect of FUDP on α-amylase and its concentration. Reversible inhibition is generally classified into three types: competitive inhibition, non-competitive inhibition, and uncompetitive inhibition. Figure 4 As shown in Figure -B, with the increase of the inhibitor FUDP concentration, K m The concentration increased from 24.746 to 66.280 mg / mL, V max The decrease from 0.370 to 0.356 ΔOD / min indicates that the interaction between FUDP and α-amylase can be identified as a mixed competitive inhibition, including both competitive and non-competitive inhibition.

[0053] Example 8: Inhibition kinetics and inhibition type determination of FUDP against α-glucosidase

[0054] To determine the concentrations of FUDP (5.0, 7.5, and 10.0 mg / mL), α-glucosidase solutions of different concentrations (0.125, 0.250, 0.500, 1.000, and 1.500 U / mL) were incubated with inhibitor solutions at 37°C for 30 min. The pNPG concentration was controlled at 5 mM, and the reaction rate (mg / mL·min) was measured. -1 The reaction rate was expressed as the amount of substrate produced per minute (mg). An enzyme inhibition kinetic graph was plotted with α-glucosidase concentration on the x-axis and reaction rate on the y-axis. With the α-glucosidase concentration fixed at 0.5 U / mL, the substrate pNPG concentration was varied (1.25, 2.5, 5.0, 7.5, 10.0 mM), and the enzyme reaction rate was measured at different concentrations of FUDP (5.0, 7.5, 10.0 mg / mL). A Lineweaver-Burk double reciprocal curve was plotted with the reciprocal of the reaction rate (1 / V) on the y-axis and the reciprocal of the substrate concentration (1 / S) on the x-axis to determine the inhibition type of the inhibitor.

[0055] Results: In the reaction system, the concentrations of pNPG and inhibitor were kept constant. The effect of the inhibitor on the reaction rate of α-glucosidase and substrate was observed by changing the concentration of α-glucosidase. Figure 5 As shown in Figure -A, the enzymatic rate curves of α-glucosidase all pass through the origin, indicating that FUDP also has a reversible inhibitory effect on α-glucosidase, and the inhibitory effect is positively correlated with the inhibitor concentration. Figure 5 In -B, as the concentration of FUDP increases, the slope of the straight line representing V... max The value decreases, and almost all the straight lines intersect the x-axis at a single point, K. m The fact that the FUDP-mediated inhibition of α-glucosidase remains largely unchanged indicates that the FUDP-mediated inhibition follows a non-competitive inhibition mechanism. This suggests that the binding site of FUDP to α-glucosidase is in a non-competitive domain, rather than in the active site.

[0056] Example 9: Secondary structure analysis of α-amylase and α-glucosidase by FUDP

[0057] The CD spectra of α-amylase and α-glucosidase in the presence and absence of FUDP were determined using circular dichroism spectroscopy (190 nm–250 nm). α-amylase solutions (1.5 U / mL, 0.3 mL) were mixed with 0.3 mL of FUDP (5, 10, 15 mg / mL), and similarly, α-glucosidase (0.5 U / mL, 0.3 mL) and FUDP (5.0, 7.5, 10.0 mg / mL) were mixed. The mixtures were incubated at 37 °C for 15 min, and measurements were taken in the 190 nm–250 nm range by subtracting the background signal from the polysaccharide sample solution. The x-axis represents wavelength (nm), and the y-axis represents circular dichroism (mdeg). Chromatograms were plotted.

[0058] Results: Circular dichroism (CD) analysis is commonly used to evaluate changes in the secondary structure of proteins after interactions with other compounds. Figure 6 In -A, α-amylase showed two negative peaks at 209 nm and 228 nm, which are characteristic α-helical peaks caused by π-π* and n-π* transitions, respectively. The appearance of FUDP increased the CD spectrum of α-amylase, indicating that FUDP altered the secondary structure of α-amylase. Figure 6 As shown in Figure -B, α-glucosidase exhibits two characteristic negative peaks of the α-helix near 209 nm and 225 nm. Different concentrations of FUDP reduced the CD intensity near 209 nm and 225 nm, indicating that FUDP altered the secondary structure of α-glucosidase.

[0059] Example 10: Fluorescence spectral analysis of FUDP on α-amylase and α-glucosidase

[0060] Samples showing fluorescence quenching of α-amylase and α-glucosidase were analyzed using fluorescence spectroscopy. α-amylase (1.5 U / mL, 300 μL) and α-glucosidase (0.5 U / mL, 300 μL) were mixed with equal volumes of FUDP solutions of different concentrations and incubated at 37 °C for 15 min. The excitation wavelength was 280 nm, the emission wavelength was 300–500 nm, and the scan rate was 240 nm / min. The width of the two slits was set to 2.5 nm. Furthermore, fluorescence background correction was performed using sodium phosphate buffer (0.2 M, pH 6.9) as a blank.

[0061] Results: To further investigate the interaction between FUDP and digestive enzymes, fluorescence quenching analysis was performed to observe changes in the microenvironment of chromophores within the digestive enzymes. Proteins containing aromatic amino acid residues such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) fluoresce under excitation at 280 nm or 295 nm, with Trp exhibiting the highest fluorescence intensity and Phe showing the lowest. Figure 7As shown, the fluorescence intensity of α-amylase and α-glucosidase decreased upon the addition of FUDP, and this decrease was positively correlated with concentration, exhibiting typical fluorescence quenching. The slight shift in characteristic fluorescence peaks indicates that FUDP alters the enzyme microenvironment through hydrogen bonding and hydrophobic linkages. This binding leads to changes in the polarity of Trp and Tyr residues in the digestive enzymes, ultimately reducing their activity. Based on the results of CD, FUDP can alter the secondary structure of digestive enzymes and their surrounding microenvironment.

[0062] Example 11: Determination of postprandial blood glucose in mice

[0063] Six-week-old male ICR mice were housed in an air-conditioned room at 25±2℃ with a 12-hour light / dark cycle. After one week of acclimatization, the mice were divided into 8 groups (n=6). Four groups of male ICR mice were fasted for 12 hours and then administered 2.0 g / kg starch by gavage, followed by oral administration of 150 mg / kg FUDP 15 minutes later. Blood samples were collected from the tail after administration, and blood glucose levels were measured at 0, 30, 60, 90, and 120 minutes. Blood glucose levels were measured using a glucometer and compared with a control group that did not receive inhibitors. Additionally, a prediabetic model was established by feeding mice a high-sugar, high-fat diet for five weeks, and the above measurements were repeated.

[0064] Results: To confirm the in vivo relevance of the above in vitro results demonstrating the digestive enzyme inhibitory activity of FUDP, we conducted starch loading tests using ICR-normal mice and a prediabetic model. Figure 8 As shown in Figure A, compared with the control group, FUDP significantly reduced postprandial hyperglycemia induced 30 minutes after starch loading (P<0.01), with blood glucose decreasing from 13.7 mmol / L to 8.1 mmol / L. Furthermore, the peak blood glucose time in the FUDP group was delayed from 30 min to 60 min, a trend similar to that of acarbose. Figure 8 As shown in Figure B, compared with the normal group, the blood glucose level of mice in the prediabetic model group was significantly elevated 30 minutes after a meal. FUDP was able to restore the blood glucose level of prediabetic mice to normal levels 30 minutes after a meal, while acarbose caused the blood glucose level of prediabetic mice to be lower than normal. This indicates that FUDP can effectively improve the postprandial blood glucose level in prediabetic model mice. This result suggests that FUDP can reduce the postprandial glycemic response, and the different modes of action of inhibitors and digestive enzymes may be the main reason for their different hypoglycemic effects. The occurrence of type 2 diabetes is closely related to the elevation of postprandial blood glucose. Therefore, reducing the postprandial hyperglycemic response after a mixed carbohydrate diet is a key strategy for controlling diabetes.

[0065] This invention investigated the effects of FUDP on the biochemical properties of α-amylase and α-glucosidase by examining their reactivity, reaction kinetics, conformation, and fluorescence quenching. The results showed that FUDP is an ideal natural inhibitor of these enzymes. In vivo experiments demonstrated that FUDP significantly inhibited postprandial blood glucose levels in normal mice and prediabetic mice, proving that the hypoglycemic effect of oral FUDP is partly related to the activities of α-amylase and α-glucosidase. This invention not only provides valuable insights into the molecular interactions between FUDP and digestive enzymes in vivo but also greatly contributes to the development of natural and effective drugs or functional foods for the treatment and prevention of diabetes.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0067] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0068] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a fucoidan degradation product with high uronic acid content, characterized in that, Includes the following steps: Step 1: Fucoidan samples were treated by a two-step precipitation method combining acid hydrolysis with quaternary ammonium salt and alcohol precipitation to obtain fucoidan degradation products; Step 2: Evaluate the in vitro hypoglycemic effect and analyze the mechanism of the fucoidan degradation products prepared in Step 1; Pretreatment of fucoidan before acid hydrolysis includes: hot air-assisted radio frequency drying of fucoidan powder at a drying temperature of 60 ℃, and drying is stopped when the moisture content reaches below 10%. The acid is one or more selected from hydrochloric acid, trifluoroacetic acid, trichloroacetic acid, and phosphoric acid; During the acidolysis treatment, the acidolysis temperature is controlled at 60-90 ℃, and the acidolysis time is 4-6 h; The quaternary ammonium salt is hexadecylpyridine chloride. 0.5-1.0% of the quaternary ammonium salt is added to the acid hydrolysis solution for sedimentation. The sedimentation time is 12-24 h, and the precipitate is obtained by centrifugation. The fucoidan degradation product is not a single polysaccharide component, and its average molecular weight is 8.06 kDa. The fucoidan degradation product is composed of eight monosaccharides, and the composition and molar mass ratio of each monosaccharide are as follows: fucose: galactose: mannose: xylose: glucose: rhamnose: glucuronic acid: galacturonic acid = 15.62: 20.75: 0.50: 5.89: 5.30: 1.74: 40.04: 10.16; The total uronic acid content in the fucoidan degradation products was 23.20%.

2. The method for preparing a fucoidan degradation product with high uronic acid content according to claim 1, characterized in that: The fucoidan is a commercially available product derived from brown algae.

3. The method for preparing a fucoidan degradation product with high uronic acid content according to claim 1, characterized in that: After acidolysis, volatile acids are removed by rotary evaporation under heating, and an alkaline solution is added to adjust the pH to neutral.

4. The method for preparing a fucoidan degradation product with high uronic acid content according to claim 1, characterized in that: The alcohol precipitation reagent is anhydrous ethanol. The above precipitate is dissolved in sodium chloride solution and then mixed with anhydrous ethanol solution at a volume ratio of 1:3 to 5. The mixture is allowed to precipitate overnight, and the precipitate is obtained by centrifugation.

5. The high uronic acid content fucoidan degradation product prepared by the method according to claim 1, characterized in that: As an inhibitor of α-amylase and α-glucosidase, it can be used to prepare hypoglycemic drugs.

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