Preparation of fucoidin degradation product with high uronic acid content and application of fucoidin degradation product in blood sugar reduction

Abstract

The invention discloses a preparation method and application of a high-uronic-acid-content fucoidin degradation product, and the specific preparation method is as follows: fucoidin powder subjected to hot air assisted radio frequency drying treatment is subjected to acid hydrolysis and two-step sedimentation to obtain the fucoidin degradation product, the average molecular weight is 7-9kDa, monosaccharide components are fucose, xylose and mannose, and the fucoidin degradation product has the advantages of high uronic-acid-content fucoidin degradation product and high-uronic-acid-content fucoidin degradation product and high-uronic-acid-content fucoidin degradation product. Galactose, rhamnose, glucuronic acid and galacturonic acid, and the molar ratio of the glucuronic acid is higher than 50%. The fucoidin degradation product provided by the invention can effectively reduce the activity of alpha-amylase and alpha-glucosidase through reversible inhibition, can effectively reduce the postprandial blood sugar level of mice in animal experiments, and has a good prospect in the field of development of blood sugar reducing foods.

Description

Technical Field

[0001] The present invention relates to the technical field of low molecular weight fucoidan preparation, and specifically to the preparation of a fucoidan degradation product with a high uronic acid content and its application in reducing blood sugar. Background Art

[0002] Non-insulin-dependent diabetes mellitus, or type 2 diabetes, is a group of metabolic diseases characterized by hyperglycemia caused by insulin resistance and abnormal insulin production. Due to the hydrolysis of starch by pancreatic α-amylase and the absorption of glucose by α-glucosidase in the small intestine, blood glucose levels in patients with type 2 diabetes rise rapidly. Inhibition of these glycolytic enzymes can significantly reduce postprandial hyperglycemia after a mixed carbohydrate diet and is a key strategy for controlling diabetes. However, current drugs used to lower blood sugar, such as acarbose, may cause side effects such as bloating, flatulence, and vomiting, so it is necessary to find natural ingredients to delay hyperglycemia and reduce side effects.

[0003] Fucoidan is extracted from brown algae or marine invertebrates, and is mainly composed of fucose and sulfate groups, supplemented by trace amounts of xylose, mannose, arabinose, galactose and glucuronic acid. A large number of studies have shown that fucoidan can regulate blood sugar levels through a variety of mechanisms, which are closely related to its structure, and molecular weight is one of the important factors. Compared with high molecular weight polysaccharides, low molecular weight polysaccharides or oligosaccharides have better solubility and bioavailability. However, there are few reports on the effect of low molecular weight fucoidan on lowering postprandial blood sugar. It has been reported in the literature that the monosaccharide composition of fucoidan also affects its hypoglycemic effect, among which the content of fucose, uronic acid and galactose may be an important reason for the difference in activity. Fucoidan with high monosaccharide content has better hypoglycemic activity. Therefore, the preparation of a low molecular weight fucoidan with a high uronic acid content is of great significance for hypoglycemic research. Summary of the invention

[0004] The purpose of the present invention is to provide a fucoidan degradation product (FUDP) with high uronic acid content and its application in a drug for reducing postprandial blood sugar.

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

[0006] Step 1: Treat the fucoidan sample by acid hydrolysis combined with quaternary ammonium salt and alcohol precipitation to obtain the fucoidan degradation product;

[0007] Step 2: Conduct in vitro blood sugar-lowering evaluation and mechanism analysis on 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 comprising: treating the fucoidan powder with hot air-assisted radio frequency drying, the drying temperature is 60° C., and the drying is stopped when 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 acid hydrolysis treatment, the acid hydrolysis temperature is controlled to be 60-90°C and the acid hydrolysis treatment time is 4-6h.

[0012] Furthermore, after the acid hydrolysis is completed, the volatile acid is removed by heating and rotary evaporation, and an alkaline solution is added to adjust to neutrality.

[0013] Furthermore, the quaternary ammonium salt is cetylpyridinium chloride, and 0.5-1.0% of the quaternary ammonium salt is added to the acid hydrolysis solution for sedimentation, the sedimentation time is 12-24 hours, and the precipitate is 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 in a volume ratio of 1:3-5, and precipitated overnight by alcohol, and the precipitate is obtained by centrifugation.

[0015] A fucoidan with high uronic acid content is used for reducing postprandial blood sugar. As an alpha-amylase and alpha-glucosidase inhibitor, it can be used for the development of new blood sugar-lowering drugs or functional foods.

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

[0017] The present invention has comprehensively studied the hypoglycemic activity of FUDP from aspects such as inhibiting the activity of alpha-amylase and alpha-glucosidase, reaction kinetics, conformation and physiological indicators of postprandial blood glucose in mice through in vitro and in vivo experiments. The results show that FUDP has a significant inhibitory effect on the activity of alpha-amylase and alpha-glucosidase, and the half inhibition concentration (IC50) is 5.815 and 4.439mg / mL respectively. The reaction kinetics show that FUDP is a mixed inhibition to alpha-amylase, and is a non-competitive inhibition to alpha-glucosidase. It can be seen from circular dichroism and fluorescence spectroscopy that FUDP changes the polarity of Trp and Tyr residues of alpha-amylase and alpha-glucosidase, and effectively changes the secondary structure of alpha-amylase and alpha-glucosidase. In the in vivo experiment, FUDP significantly inhibited the rise of postprandial blood glucose level in normal mice and prediabetic mice.

[0018] Therefore, the FUDP of the present invention has a good hypoglycemic effect, and as an α-amylase and α-glucosidase inhibitor, can be used in the development of new hypoglycemic drugs or functional foods. At the same time, the FUDP provided by the present invention is an active ingredient derived from seaweed that can significantly reduce postprandial blood sugar, and has the characteristics of being green and safe. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0020] Figure 2 Figure 3 Inhibitory effect of FUDP on α-amylase activity;

[0021] Figure 3 Figure 3 Inhibitory effect of FUDP on α-glucosidase activity;

[0022] Figure 4 The inhibition kinetics of FUDP on α-amylase, Figure 4 -A is the reaction kinetics diagram, Figure 4 -B is Lineweaver-Burk diagram;

[0023] Figure 5 Inhibition kinetics of α-glucosidase by FUDP, Figure 5 -A is the reaction kinetics diagram, Figure 5 -B is Lineweaver-Burk diagram;

[0024] Figure 6 Secondary structure changes of α-amylase and α-glucosidase, Figure 6 -A is the circular dichroism spectrum of different concentrations of FUDP on α-amylase, Figure 6 -B is the circular dichroism spectra 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 is the fluorescence quenching spectrum of different concentrations of FUDP on α-amylase, Figure 7 -B is the fluorescence quenching spectra of different concentrations of FUDP on α-glucosidase;

[0026] Figure 8 FUDP reduces postprandial blood sugar. Figure 8 -A is the effect of FUDP on lowering blood sugar after a starch meal in normal mice, Figure 8 -B is the effect of FUDP on reducing blood glucose after a starch meal in prediabetic mice. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0029] Accurately weigh 8.0g of fucoidan sample and dry it by hot air circulation in the radio frequency equipment, with an electrode gap of 100mm and a temperature of 60℃. After drying, add 125mL of 0.2mol / L trifluoroacetic acid and hydrochloric acid solution respectively, fully dissolve, react at 90℃ for 6h, oscillate at 125rpm, and cool in ice water after the reaction to terminate the reaction. The reaction solution was centrifuged at 8000rpm for 15min, and the supernatant was collected. The supernatant was evaporated at 55℃ to remove volatile acids, and 0.2mol / L NaOH was added to the solution for neutralization. After deacidification, 1% hexadecylpyridinium chloride was added and precipitated for 24h, centrifuged at 6000rpm for 10min to obtain a precipitate, and 20mL of 3mol / L sodium chloride solution was added for re-dissolution. Add 4 times the volume of anhydrous ethanol for overnight alcohol precipitation. Centrifuge at 8000rpm for 10min to obtain a precipitate, re-dissolve and desalt through an ultrafiltration membrane, and the component obtained after freeze-drying is FUDP.

[0030] Example 2 FUDP molecular weight distribution determination

[0031] The molecular weight distribution of FUDP in Example 1 was determined using high performance liquid chromatography (HPLC). Equipped with a W410 refractive index detector and a TSKgel G4000PWXL chromatographic column (8μm, 7.8×300mm). The system temperature was 35°C, and the eluents were 0.2mol / LNaNO3 and 0.01mol / L NaH2PO4. 20μL FUDP solution (10mg / mL) was injected into the column and eluted at a flow rate of 0.5mL / min. A standard curve was drawn using different molecular weights of dextran (80, 150, 270, 410 and 670kDa).

[0032] Results: 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] 8 mg of FUDP in Example 1 was weighed and dissolved in 3 mL of 2 mol / L trifluoroacetic acid solution, hydrolyzed at 110°C for 6 h, TFA was removed by rotary vacuum evaporator, and the pH of the solution was adjusted to 7.0 with 6 mol / L NaOH. The monosaccharide standard and the acid hydrolyzate of SCGP were added to PMP solution (0.5 mL, 0.5 mol / L) and heated at 60°C for 60 min. After cooling, the solution was adjusted to neutral with NaOH solution, and then extracted with chloroform five times to remove excess PMP, and the supernatant was filtered through a 0.22 μm organic phase filter. The chromatographic column is a ZORBAX300XDB-C18 column with a wavelength of 245 nm; the detection mobile phase A is acetonitrile, the mobile phase B is 0.05 mol / L phosphate buffer solution, the gradient elution is 0-60 min, the initial setting is mobile phase A: mobile phase B = 17%: 83%, the final elution ratio is mobile phase A: mobile phase B = 20%: 80%, and the injection volume is 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 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 FUDP monosaccharide composition

[0037]

[0038] Example 4 Determination of FUDP uronic acid content

[0039] Prepare 12.5, 25.0, 37.5, 50.0, 62.5, 75.0, 100 μg / mL standard solutions in test tubes respectively, take 400 μL of the sample to be tested in the test tube, add 2.4 mL of sodium tetraborate concentrated sulfuric acid solution to each test tube in an ice water bath, take out the test tube from the ice water bath after it is completely cooled, and vortex mix. Immerse the test tube in a boiling water bath and heat for 20 minutes, cool with tap water, and leave at room temperature for 5 minutes. Add 80 μL of 0.15% m-hydroxybiphenyl solution to each test tube, vortex mix quickly, let stand for 10 minutes, and measure the absorbance at 525 nm. A standard curve can be obtained based on the working standard solution, and the uronic acid content can be calculated based on the standard curve.

[0040] Results: The results showed that the total uronic acid content in FUDP was 23.20%.

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

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

[0043]

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

[0045] Results: Most of the dietary starch ingested by the human body is broken down into oligosaccharides or monosaccharides by α-amylase and α-glucosidase and absorbed by intestinal epithelial cells, leading to increased blood sugar levels. Figure 2 As shown in the figure, the inhibition rate of FUDP and acarbose on α-amylase is positively correlated with the increase of concentration within a specific range. It is worth noting that when the FUDP concentration reaches 10 mg / mL, the inhibition rate exceeds 80%, achieving the best inhibition effect of acarbose. After calculation, the IC 50 The values ​​were 5.815 mg / mL respectively.

[0046] Example 6 Determination of the inhibitory activity of FUDP on α-glucosidase

[0047] 100 μL of FUDP solution of different concentrations was 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-pyranoglucoside (pNPG, 5 mM) was added and incubated at 37°C for 30 min. 1 mL of 1 mol / L Na2CO3 was added to terminate the reaction, the absorbance was measured at 405 nm, and the inhibitory activity was calculated using formula (1).

[0048] Results: Figure 3 As shown in the figure, the inhibitory effect of FUDP on α-glucosidase is dose-dependent. When the concentration is 10 mg / mL, the inhibition rate of FUDP on α-glucosidase can reach 100%. After calculation, the IC 50It is 4.439 mg / mL. The above results show that the inhibitory effect of FUDP on α-glucosidase is better than that on α-amylase.

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

[0050] The Lineweaver-Burk plot of the Michaelis-menten equation was used to determine the reaction kinetic parameters, the maximum reaction rate (V max ) and Michaelis constant (K m The concentration of FUDP (5.0, 10.0, 15.0 mg / mL) was determined, and different concentrations of α-amylase solutions (0.5, 1.0, 1.5, 2.0 and 2.5 U / mL) were incubated with the inhibitor solution at 37°C for 30 min, and the soluble starch concentration was controlled to be 1%. The reaction rate (mg / mL·min -1 ), the reaction rate is measured in terms of the amount of glucose generated per minute (mg). The horizontal axis is the α-amylase concentration, and the vertical axis is the reaction rate to draw the enzymatic reaction kinetics. The concentration of the α-amylase solution was fixed at 1.5U / mL. After incubation with the inhibitor solution at 37°C for 30 minutes, the reaction rates of different concentrations of FUDP (5, 10, 15 mg / mL) catalyzing 0.25%, 0.5%, 1.0%, 1.5%, and 2% soluble starch solutions were measured. The Lineweaver-Burk curve was drawn with 1 / S as the horizontal axis and 1 / V as the vertical axis to determine the inhibition type of the inhibitor.

[0051]

[0052] result: Figure 4 -A shows the inhibition kinetics of FUDP on α-amylase. With the different concentrations of FUDP and α-amylase, all hydrolysis rate curves intersect with the origin, indicating that the inhibitory effect of FUDP on α-amylase is reversible. At the same time, all slopes decrease with the increase of inhibitor concentration, indicating that the inhibitory effect of FUDP on α-amylase is positively correlated with its concentration. Reversible inhibition is generally divided into three types: competitive inhibition, non-competitive inhibition, and anti-competitive inhibition. Figure 4 As shown in Figure 2B, with the increase of the concentration of inhibitor FUDP, K m From 24.746 to 66.280 mg / mL, V max The decrease from 0.370 to 0.356ΔOD / min indicated that the interaction between FUDP and α-amylase could be identified as mixed competitive inhibition, including competitive inhibition and non-competitive inhibition.

[0053] Example 8 Inhibition kinetics and inhibition type judgment of FUDP on α-glucosidase

[0054] The concentration of FUDP (5.0, 7.5, 10.0 mg / mL) was determined. The α-glucosidase solutions of different concentrations (0.125, 0.250, 0.500, 1.000 and 1.500 U / mL) were incubated with the inhibitor solution at 37°C for 30 min. The pNPG concentration was controlled to be 5 mM. The reaction rate (mg / mL·min -1 ), the reaction rate is measured in terms of substrate generated per minute (mg). The horizontal axis is the concentration of α-glucosidase, and the vertical axis is the reaction rate to draw the enzyme inhibition reaction kinetic image. The concentration of α-glucosidase is fixed at 0.5U / mL, the concentration of substrate pNPG is changed (1.25, 2.5, 5.0, 7.5, 10.0mM), and the enzymatic reaction rate under different concentrations of FUDP (5.0, 7.5, 10.0mg / mL) is measured. With the reciprocal of the reaction rate (1 / V) as the vertical axis and the reciprocal of the substrate concentration (1 / S) as the horizontal axis, a Lineweaver-Burk double reciprocal curve is drawn to determine the inhibition type of the inhibitor.

[0055] Results: In the reaction system, the base pNPG concentration and the inhibitor concentration were controlled as constants, and the effect of the inhibitor on the reaction rate of α-glucosidase and substrate was observed by changing the α-glucosidase concentration. Figure 5 -A shows that 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 -B, as the concentration of FUDP increases, the V max decreases, and all straight lines almost intersect the x-axis at one point, K m The results showed that the FUDP-mediated inhibition of α-glucosidase followed a non-competitive inhibition mechanism. This indicates that the binding site of FUDP to α-glucosidase is in the non-competitive domain, not 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 (190nm-250nm). α-amylase solution (1.5U / mL, 0.3mL) was mixed with 0.3mL FUDP (5, 10, 15mg / mL), and α-glucosidase (0.5U / mL, 0.3mL) and FUDP (5.0, 7.5, 10.0mg / mL) were mixed in the same way, incubated at 37°C for 15min, and measured in the range of 190nm-250nm by subtracting the background signal of the polysaccharide sample solution. The horizontal axis is the wavelength (nm), the vertical axis is the circular dichroism (mdeg), and the chromatogram is drawn.

[0058] Results: Circular dichroism (CD) analysis is often used to evaluate the changes in the secondary structure of proteins after interaction with other compounds. Figure 6 -A, α-amylase showed two negative peaks at 209nm and 228nm, which were the α-helix characteristic peaks caused by π-π* and n-π* transitions. The appearance of FUDP increased the α-amylase CD spectrum, indicating that FUDP changed the secondary structure of α-amylase. Figure 6 As shown in Figure 1-B, α-glucosidase has two characteristic negative peaks of α-helix near 209nm and 225nm. Different concentrations of FUDP reduce the CD intensity near 209nm and 225nm, indicating that FUDP changes the secondary structure of α-glucosidase.

[0059] Example 10 Fluorescence spectroscopy analysis of α-amylase and α-glucosidase by FUDP

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

[0061] Results: To further investigate the interaction between FUDP and digestive enzymes, fluorescence quenching analysis was performed to observe the microenvironmental changes of the chromophores in digestive enzymes. Proteins containing aromatic amino acid residues such as tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe) will emit fluorescence under excitation light at 280nm or 295nm. Trp has the highest fluorescence intensity, while Phe has a lower fluorescence intensity. Figure 7As shown in the figure, the fluorescence intensity of α-amylase and α-glucosidase decreased after the addition of FUDP and was positively correlated with the concentration, showing a typical fluorescence quenching phenomenon. The slight shift of the characteristic fluorescence peak indicates that FUDP changes the microenvironment of the enzyme through the combination of hydrogen bonds and hydrophobic connections. This combination leads to changes in the polarity of Trp and Tyr residues in the digestive enzymes, ultimately reducing their activity. Combined with the results of CD, FUDP can change the secondary structure of the digestive enzymes and the surrounding microenvironment.

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

[0063] Six-week-old ICR male mice were selected and raised in an air-conditioned room with a temperature of 25±2℃ and a light / dark cycle of 12h. After one week of adaptive feeding, the mice were divided into 8 groups (n=6). Among them, 4 groups of male ICR mice were fasted for 12h, and each mouse was gavaged with 2.0g / kg starch, and 150mg / kg FUDP was orally administered after an interval of 15min. Blood samples were drawn from the tail after administration, and blood glucose levels were measured at 0, 30, 60, 90 and 120 minutes. Blood glucose levels were measured with a blood glucose meter and compared with the control group that did not take the inhibitor. In addition, mice fed with high sugar and high fat for five weeks were used to construct a prediabetes model, and the above measurements were repeated.

[0064] Results: To confirm the in vivo relevance of the in vitro findings that FUDP has digestive enzyme inhibitory activity, we performed starch loading tests using ICR normal mice and a prediabetic model. Figure 8 -A shows that compared with the control group, FUDP can significantly reduce postprandial hyperglycemia caused by starch loading half an hour later (P<0.01), and blood glucose dropped from 13.7mmol / L to 8.1mmol / L. In addition, the peak time of blood glucose in the FUDP group was delayed from 30min to 60min, and the change trend was similar to that of acarbose. Figure 8 -B shows that compared with the normal group, the blood glucose of mice in the prediabetes model group increased significantly 30 minutes after the meal. FUDP can restore the blood glucose of prediabetes mice to normal levels 30 minutes after the meal, while acarbose can make the blood glucose of prediabetes mice lower than normal levels. This shows that FUDP can effectively improve the postprandial blood glucose level of mice in the prediabetes model group. This result shows that FUDP can reduce the postprandial blood glucose 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 increase in postprandial blood glucose. Therefore, reducing the postprandial hyperglycemia response after a mixed carbohydrate diet is a key strategy for controlling diabetes.

[0065] The present invention studies the influence of FUDP on the biochemical properties of α-amylase and α-glucosidase from the aspects of enzyme reaction activity, reaction kinetics, conformation and fluorescence quenching, and the results show that FUDP is an ideal natural enzyme inhibitor. In vivo experiments, FUDP can significantly inhibit the postprandial blood glucose level of normal mice and prediabetic mice, proving that the hypoglycemic effect of oral FUDP is partly related to the activity of α-amylase and α-glucosidase. The content of the present invention not only provides some valuable inspiration for understanding the molecular interaction between FUDP and digestive enzymes in vivo, but also is of great help in developing natural and effective diabetes treatment and prevention drugs or functional foods.

[0066] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

[0067] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

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

Claims

1. A method for preparing a degradation product of fucoidan with high uronic acid content, characterized in that: The following steps are involved: Step 1: Treat the fucoidan sample by acid hydrolysis combined with quaternary ammonium salt and alcohol precipitation to obtain the fucoidan degradation product; Step 2: Conduct in vitro blood sugar-lowering evaluation and mechanism analysis on the fucoidan degradation products prepared in step 1.

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

3. The method for preparing a degradation product of fucoidan with high uronic acid content according to claim 1, characterized in that: The fucoidan is pretreated before acid hydrolysis, specifically including: treating the fucoidan powder with hot air-assisted radio frequency drying, the drying temperature is 60° C., and the drying is stopped when the moisture content reaches below 10%.

4. The method for preparing a degradation product of fucoidan with high uronic acid content according to claim 3, characterized in that: The acid solution is one or more of hydrochloric acid, trifluoroacetic acid, trichloroacetic acid and phosphoric acid.

5. The method for preparing a degradation product of fucoidan with high uronic acid content according to claim 3, characterized in that: During the acid hydrolysis treatment, the acid hydrolysis temperature is controlled at 60-90°C and the acid hydrolysis treatment time is 4-6h.

6. The method for preparing a degradation product of fucoidan with high uronic acid content according to claim 3, characterized in that: After the acid hydrolysis is completed, the volatile acid is removed by heating and rotary evaporation, and an alkaline solution is added to adjust to neutrality.

7. The method for preparing a degradation product of fucoidan with high uronic acid content according to claim 3, characterized in that: The quaternary ammonium salt is cetylpyridinium 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 hours, and the precipitate is obtained by centrifugation.

8. The method for preparing a degradation product of fucoidan with high uronic acid content according to claim 7, 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 in a volume ratio of 1:3-5, and precipitated overnight by alcohol, and the precipitate is obtained by centrifugation.

9. The use of a high uronic acid content fucoidan as claimed in claim 1 for reducing postprandial blood sugar, characterized in that: As α-amylase and α-glucosidase inhibitors, they can be used in the development of new hypoglycemic drugs or functional foods.

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