Laminarin and preparation method and application thereof

Abstract

This invention discloses a kelp fucoidan, its preparation method, and its application. The kelp fucoidan includes LF3-1 and / or LF4-1. The main chain of LF3-1 is composed of alternating →3)-α-L-Fucp-(1→ and →3)-β-D-Galp-(1→). The fucose residues on the main chain of LF3-1 are sulfated at the C-4 position or connected to a first branch, which is composed of →4)-α-L-Fucp-(1→. The galactose residues on the main chain of LF3-1 are sulfated at the C-6 position or connected to a second branch, which is composed of →2,3)-β-D-Manp-(1→ and β-D-Galp-(1→). The sulfate content of LF3-1 is 26.3%, and the weight-average molecular weight of LF3-1 is 305. LF4-1 has a molecular weight of 32.7% and a weight-average molecular weight of 182.1 kDa. Its main chain consists of alternating links of →3)-α-L-Fucp-(1→ and →6)-β-D-Galp-(1→). The fucose in the main chain of LF4-1 is sulfated at C-4 or has a third branch, which is composed of →4)-α-L-Fucp-(1→. The galactose in the main chain of LF4-1 is sulfated at C-3 or has a fourth branch, which is composed of →2,4)-β-D-Manp-(1→ and β-D-Galp-(1→). LF4-1 effectively improves blood lipids and insulin resistance, promotes lipid oxidation and glucose metabolism gene expression, and thus plays a role in weight loss and lipid reduction.

Description

Technical Field

[0001] This invention belongs to the field of bio-extraction technology, specifically relating to a kelp fucoidan, its preparation method, and its application. Background Technology

[0002] Fucoidan is the main active substance extracted from Sargassum fusiforme, named for its primary monosaccharide component, fucose. It is also a water-soluble polyanionic homopolysaccharide, rich in sulfate groups, hence also called fucose sulfate. Besides the main L-fucoose, the monosaccharide composition of fucoidan includes small amounts of rhamnose, arabinose, and galactose, with the monosaccharides primarily linked by α-1,2, α-1,3, or α-1,4 glycosidic bonds. Numerous studies have shown that fucoidan has broad health benefits and therapeutic effects, exhibiting biological activities such as lowering blood lipids, lowering blood sugar, antioxidation, anticancer activity, and immune regulation. Its biological activity is closely related to the molecular weight, sulfate group content, and chemical structure of the polysaccharide; therefore, a deeper understanding of the structure-activity relationship of fucoidan has become a key focus of future research. Summary of the Invention

[0003] The purpose of this invention is to overcome the defects of the prior art and provide a kelp fucoidan.

[0004] Another object of the present invention is to provide a method for preparing the above-mentioned kelp fucoidan.

[0005] Another object of the present invention is to provide the application of the above-mentioned kelp fucoidan.

[0006] The technical solution of the present invention is as follows:

[0007] A type of kelp fucoidan, comprising LF3-1 and / or LF4-1;

[0008] The structure of LF3-1 is as follows: R1 is -SO3 or the first branch, which is composed of →4)-α-L-Fucp-(1→; R2 is -SO3 or the second branch, which is composed of →2,3)-β-D-Manp-(1→ and β-D-Galp-(1→ alternately; the sulfate content in LF3-1 is 26.3%; the weight-average molecular weight of LF3-1 is 305.3 kDa.

[0009] The structure of LF4-1 is as follows R3 is -SO3 or the third branch, which is composed of →4)-α-L-Fucp-(1→. R4 is -SO3 or the fourth branch, which is composed of →2,4)-β-D-Manp-(1→ and β-D-Galp-(1→ alternately. The sulfate content in LF4-1 is 32.7%, and the weight-average molecular weight of LF4-1 is 182.1 kDa.

[0010] In some possible implementations, the molar mass ratio of mannose residues, galactose residues, and fucose residues in LF3-1 is 3.59:11.97:14.25;

[0011] The molar mass ratio of mannose residues, galactose residues and fucose residues in LF4-1 is 2.21:13.11:7.13.

[0012] In some possible implementations, LF3-1 consists of mannose residues, glucosamine residues, glucuronic acid residues, galactose residues and fucose residues in a molar mass ratio of 3.59:1.00:2.41:11.97:14.25;

[0013] LF4-1 is composed of mannose residues, glucosamine residues, glucuronic acid residues, rhamnose residues, galactose residues and fucose residues in a molar mass ratio of 2.21:1.00:1.00:1.25:13.11:7.13.

[0014] In some possible implementations, kelp fucoidan comprises LF3-1 and LF4-1 in a mass ratio of 0.8-1.2:0.8-1.2.

[0015] In some possible implementations, the mass ratio of LF3-1 to LF4-1 is 1:1.

[0016] A method for preparing the above-mentioned kelp fucoidan includes the following steps:

[0017] (1) Mix kelp powder and an aqueous solution containing 0.2-0.6wt% sodium dodecyl sulfate at 60-80℃ at a mass-to-volume ratio of 1:20-40, let stand for 2-4 hours, then centrifuge, collect the supernatant and concentrate in sequence to obtain the first concentrated solution;

[0018] (2) Mix the first concentrate and 10wt% calcium chloride at a volume ratio of 3-4:1, then filter, collect and concentrate the filtrate to obtain the second concentrate;

[0019] (3) Mix the second concentrate with anhydrous ethanol to obtain the first mixed solution, wherein the amount of anhydrous ethanol added is 20-30% of the total volume of the first mixed solution. Stir until a white granular precipitate appears and the color of the first mixed solution becomes lighter. Let it stand, collect the supernatant, add anhydrous ethanol to the supernatant to obtain the second mixed solution, wherein the amount of anhydrous ethanol added is 50-60% of the total volume of the second mixed solution. Stir until a white granular precipitate appears and the color of the second mixed solution becomes lighter. Let it stand, collect the precipitate, wash the precipitate with anhydrous ethanol, add water to dissolve the precipitate, and then dialyze and freeze dry to obtain crude kelp fucoidan. The molecular weight cutoff for dialysis is 8-14 kDa.

[0020] (4) The crude kelp fucoidan was eluted isocratically in a DEAE-52 cellulose column with 0.5 mol / L, 0.7 mol / L and 1.0 mol / L NaCl solutions, and the fractions eluted with 0.7 mol / L and / or 1.0 mol / L NaCl solutions were collected to obtain LF3 and / or LF4.

[0021] (5) Elute LF3 and / or LF4 with 0.5 mol / L NaCl solution in a Sephadex G-200 chromatography column to obtain LF3-1 and / or LF4-1.

[0022] In some possible implementations, in step (3) of mixing the second concentrate with anhydrous ethanol, the amount of anhydrous ethanol added is 25% of the total volume of the second concentrate and anhydrous ethanol.

[0023] More preferably, in step (3), when collecting the supernatant and adding anhydrous ethanol, the amount of anhydrous ethanol added is 56% of the total volume of the supernatant and anhydrous ethanol.

[0024] More preferably, the kelp powder is obtained by defatting crude kelp powder. The defatting process is as follows: the crude kelp powder is refluxed with 90%-98% ethanol at 82-90℃ for 3.5-4.5 hours, and then dried to obtain kelp powder.

[0025] A weight loss and lipid-lowering composition, the effective ingredient of which is the above-mentioned kelp fucoidan.

[0026] The above-mentioned application of kelp fucoidan in the preparation of weight loss and lipid-lowering compositions.

[0027] The present invention has at least the following beneficial effects:

[0028] 1. The kelp fucoidan isolated and extracted by this invention can effectively improve blood lipid levels and insulin resistance levels, promote the expression of lipid oxidation genes (PPAR-α, PPAR-γ, CPT-1FAS, LXR and SREBP-1c) and glucose metabolism genes (IRS1 and Glut4), thereby playing a role in treating and improving obesity and related diseases.

[0029] 2. The method for preparing kelp fucoidan provided by the present invention firstly involves hot water extraction of kelp powder and precipitation with CaCl2 to remove alginate impurities, followed by fractional precipitation with ethanol of different concentrations to obtain the target compound. The method is simple to operate and suitable for industrial production.

[0030] 3. High-purity kelp fucoidan components with different structures can be rapidly separated and obtained by using a DEAE-52 cellulose column and different concentrations of NaCl. Attached Figure Description

[0031] Figure 1 Elution curves of crude kelp fucoidan in a DEAE-52 cellulose column with different NaCl concentrations;

[0032] Figure 2 Elution curves of LF3 and LF4 obtained by Sephadex G-200 chromatography column; where (a) represents LF3-1 and (b) represents LF4-1.

[0033] Figure 3 High-performance gel permeation chromatograms of LF3-1 and LF4-1 are shown; where (a) represents LF3-1 and (b) represents LF4-1.

[0034] Figure 4 The ultraviolet spectra of LF3-1 and LF4-1 are shown.

[0035] Figure 5 Infrared spectra of LF3-1 and LF4-1;

[0036] Figure 6 The graph shows the monosaccharide composition of LF3-1 and LF4-1, where HB refers to the monosaccharide standard.

[0037] Figure 7 The NMR spectra of LF3-1 are shown; among them, (a) 1 H NMR, (b) 13 C NMR, (c) 1 H / 13 C HSQC、(d) 1 H / 1 H COSY, (e) 1 H / 13 C HMBC, (f)1 H / 1 H NOESY;

[0038] Figure 8 The images show the nuclear magnetic resonance (NMR) spectra of LF4-1; among them, (a) 1 H NMR, (b) 13 C NMR, (c) 1 H / 13 C HSQC、(d) 1 H / 1 H COSY, (e) 1 H / 13 C HMBC, (f) 1 H / 1 H NOESY;

[0039] Figure 9 The graph shows the effects of LF3-1 and LF4-1 on mouse body weight; where ND refers to a normal diet and HFD refers to a high-fat diet.

[0040] Figure 10 The graph shows the effects of LF3-1 and LF4-1 on serum lipids in mice; where ND refers to a normal diet and HFD refers to a high-fat diet.

[0041] Figure 11 The figure shows the effects of LF3-1 and LF4-1 in a 1:1 mass ratio on the body weight and other parameters of mice on a high-fat diet; (a) mouse body weight, (b) mouse body weight gain, and (c) mouse food intake.

[0042] Figure 12 The figure shows the effects of 1:1 mass ratio of LF3-1 and LF4-1 fucoidan on oral glucose tolerance in mice on a high-fat diet. Among them, (a) oral glucose tolerance (OGTT), (b) area under the curve (AUC), (c) insulin level, and (d) insulin resistance index (HOMA-IR).

[0043] Figure 13 The figure shows the effects of LF3-1 and LF4-1 in a 1:1 mass ratio on serum triglycerides and other parameters in mice on a high-fat diet. Among them, (a) the effects of triglycerides (TG), (b) total cholesterol (TC), (c) low-density lipoprotein (LDL-C), and (d) high-density lipoprotein (HDL-C) are shown.

[0044] Figure 14The figure shows the effect of 1:1 mass ratio of LF3-1 and LF4-1 fucoidan on the relative mRNA expression levels of lipid metabolism in mouse liver; (a) PPAR-α, (b) PPAR-γ, (c) CPT-1, (d) FAS, (e) LXR, (f) SREBP-1c.

[0045] Figure 15 The figure shows the effect of 1:1 mass ratio of LF3-1 and LF4-1 fucoidan on the relative expression levels of mRNA in the liver of mice with glucose metabolism; (a) IRS1, (b) Glut4. Detailed Implementation

[0046] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0047] In this invention, all reagents and materials are commercially available products or can be obtained through conventional technical means in the field. The DEAE-52 cellulose column was purchased from Solarbio Science & Technology Co., Ltd. Unless otherwise specified, content refers to mass percentage.

[0048] Example 1: Preparation of kelp fucoidan

[0049] (1) The crude kelp powder was refluxed with 95% ethanol at 85°C for 4 hours and then dried at 50°C to obtain kelp powder. The purpose of this step is to remove lipids from the crude kelp powder.

[0050] (2) Kelp powder and hot water containing 0.4wt% sodium dodecyl sulfate at 70℃ are mixed at a mass-to-volume ratio of 1:30. After standing for 3 hours, the mixture is centrifuged, the supernatant is collected and concentrated to obtain the first concentrated liquid. The purpose of this step is to extract kelp fucoidan and some impurities from the kelp powder.

[0051] (3) Mix the first concentrate and 10wt% calcium chloride solution at a volume ratio of 4:1, then filter, collect the filtrate and concentrate to obtain the second concentrate. This step removes alginate impurities through calcium chloride.

[0052] (4) While stirring, slowly pour anhydrous ethanol into the second concentrate to mix the two solutions, obtaining a first mixed solution. The amount of anhydrous ethanol added is 25% of the total volume of the first mixed solution. Continue stirring until a white granular precipitate appears in the first mixed solution and the color of the first mixed solution becomes lighter. Let it stand at 4°C for 12 hours, collect the supernatant, and add anhydrous ethanol to the supernatant while stirring to obtain a second mixed solution. The amount of anhydrous ethanol added is 56% of the total volume of the second mixed solution. Stir until a white granular precipitate appears in the second mixed solution and the color of the second mixed solution becomes lighter. Let it stand at 4°C for 12 hours and collect the precipitate. Wash the precipitate three times with a small amount of anhydrous ethanol, add an appropriate amount of distilled water to dissolve the precipitate, and then dialyze it using a dialysis bag with a molecular weight cutoff of 8-14 kDa. After that, freeze-dry to obtain crude kelp fucoidan. The yield of crude kelp fucoidan is 5.73% by weight.

[0053] (5) The crude kelp fucoidan was separated by DEAE-52 cellulose column chromatography, and isocratic elution was performed with deionized water and 0.1 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.7 mol / L, 1.0 mol / L, 1.5 mol / L and 2.0 mol / L NaCl solutions, respectively. Figure 1 It can be seen that the elution components of 0.4, 0.5, 0.7, 1.0 and 1.5 mol / L NaCl solutions have obvious single peaks. The above elution components were collected to obtain LF1, LF2, LF3, LF4 and LF5. However, the yields of LF1, LF2 and LF5 were low and difficult to further purify, so only LF3 and LF4 were retained.

[0054] (6) LF3 and LF4 were eluted separately in a Sephadex G-200 chromatography column with 0.5 mol / L NaCl solution, as follows: Figure 2 The LF3-1 and LF4-1 shown.

[0055] In Example 1, crude kelp fucoidan was isocratically eluted with deionized water and 0.1, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, or 2.0 mol / L NaCl solutions to obtain LF1, LF2, LF3, LF4, and LF5 for subsequent analysis. If only the kelp fucoidan provided in this application is to be prepared, 0.5 mol / L NaCl can be used first to remove the lower yields of LF1 and LF2, followed by elution with 0.7 mol / L and / or 1.0 mol / L NaCl solutions as needed. Specifically, if only 0.7 mol / L is used for elution, LF3 is obtained; if only 1.0 mol / L NaCl solution is used for elution, a mixture of LF3 and LF4 is obtained; if 0.7 mol / L and 1.0 mol / L NaCl solutions are used sequentially, separated LF3 and LF4 can be obtained. LF3 and LF4 were purified by Sephadex G-200 chromatography column to obtain LF3-1 and LF4-1, respectively.

[0056] Comparative Example 1: Preparation of fucoidan

[0057] (1) The crude kelp powder was refluxed with 95% ethanol at 85°C for 4 hours, and then dried at 50°C to obtain kelp powder.

[0058] (2) Mix kelp powder and 70℃ hot water at a mass-to-volume ratio of 1:30, let stand for 3 hours, then centrifuge, collect the supernatant and concentrate to obtain the first concentrated liquid.

[0059] (3) Mix the first concentrate and 10wt% calcium chloride solution at a volume ratio of 4:1, then filter, collect the filtrate and concentrate to obtain the second concentrate.

[0060] (4) While stirring, slowly pour anhydrous ethanol into the second concentrate to mix the second concentrate and anhydrous ethanol. The amount of anhydrous ethanol added is 70% of the total volume of the mixture. Stir until white granular precipitate appears in the solution and the color of the solution becomes lighter. Let it stand at 4℃ for 12 hours, collect the precipitate, add an appropriate amount of distilled water until the precipitate is completely dissolved, and then add anhydrous ethanol to obtain the first mixed solution. The amount of anhydrous ethanol added is 30% of the total volume of the first mixed solution. Stir until white granular precipitate appears in the first mixed solution and the color of the first mixed solution becomes lighter. Let it stand at 4℃ for 12 hours, collect the supernatant, add anhydrous ethanol to the supernatant to obtain the second mixed solution. The amount of anhydrous ethanol added is 70% of the total volume of the second mixed solution. Stir until white granular precipitate appears in the second mixed solution and the color of the second mixed solution becomes lighter. Let it stand at 4℃ for 12 hours and collect the precipitate. The precipitate was washed three times with a small amount of anhydrous ethanol, dissolved in an appropriate amount of distilled water, and then dialyzed using a dialysis bag with a molecular weight cutoff of 8-14 kDa. Afterward, it was lyophilized to obtain crude fucoidan for comparison. The yield of crude fucoidan for comparison was 4.86% by weight.

[0061] (5) The crude kelp fucoidan was separated by DEAE-52 cellulose column chromatography. It was eluted isocratically with deionized water and 0.1 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L, 0.7 mol / L, 1.0 mol / L, 1.5 mol / L and 2.0 mol / L NaCl solutions, respectively. The elution fractions of 0.7 mol / L and 1.0 mol / L NaCl solutions were collected as control LF3 and control LF4, respectively.

[0062] (6) Comparative LF3 and comparative LF4 were eluted with 0.5 mol / L NaCl solution in a Sephadex G-200 chromatography column, collected and detected, but LF3-1 or LF4-1 in Example 1 could not be detected.

[0063] In Comparative Example 1, step (3) alcohol precipitation was performed using existing methods. After obtaining the second concentrate, it was mixed with anhydrous ethanol and the precipitate was collected. In Example 1, the supernatant was collected. The yield of crude fucoidan in Comparative Example 1 was lower than that of crude kelp fucoidan in Example 1, and LF3-1 or LF4-1 could not be effectively collected and detected after purification.

[0064] Example 2: Determination of the physicochemical properties of kelp fucoidan

[0065] 1. Purity and molecular weight determination

[0066] The molecular weights of LF3-1 and LF4-1 were determined by high-performance gel permeation chromatography.

[0067] The high-performance liquid chromatography (HPLC) system was an Agilent 1260Ⅱ, the column was a Shodex Sugar KS-804 (8 mm × 300 mm, 7 μm) column, the detector was a differential refractive index detector, the mobile phase was ultrapure water, the flow rate was 0.4 mL / min, the column temperature was 80 °C, and the injection volume was 20 μL. Figure 3 It can be seen that LF3-1 and LF4-1 are high-purity homogeneous polysaccharides with weight-average molecular weights of 305.3 kDa and 182.1 kDa, respectively.

[0068] 2. Determination of total sugar content

[0069] The total sugar content of LF3-1 and LF4-1 was determined by the phenol-sulfuric acid method.

[0070] Prepare a 0.1 mg / mL sample solution. Take 0 mL and 0.1 mL of the 0.1 mg / mL sample solution, respectively, and make up to 1 mL with deionized water. Add 0.5 mL of 6% phenol solution and 2.5 mL of concentrated sulfuric acid to each tube, and let them stand at room temperature for 30 min. Measure the OD value at 490 nm. Plot a glucose standard curve with glucose concentration on the x-axis and absorbance at 490 nm on the y-axis (y = 4.8229x - 0.0017, R0). 2 =0.9995), the total sugar content of LF3-1 and LF4-1 was found to be 65.8% and 73.9%, respectively.

[0071] 3. Determination of L-(-)-fucose content

[0072] The L-(-)-fucose content of LF3-1 and LF4-1 was determined by the Dische method.

[0073] Prepare a 200 μg / mL sample solution. Take 1 mL of the solution into a 25 mL stoppered glass test tube. Add 4.5 mL of 87% (v / v) H₂SO₄ solution to the tube in an ice-water bath. Shake well and place in an ice bath for 1 min. Heat in a boiling water bath for 10 min. Add 0.2 mL of 3% L-cysteine ​​hydrochloride solution to the tube at room temperature. Shake at 160 r / min and 40 °C for 40 min. Measure the absorbance A at 396 nm and 427 nm, respectively. 396 and A 427 A standard curve was plotted using the concentration and absorbance (A1-A2) of L-(-)-fucose standard (y = 0.0151x + 0.0345, R0). 2 =0.9989), the L-(-)-fucose contents of LF3-1 and LF4-1 were 31.9% and 27.8%, respectively.

[0074] 4. Determination of sulfate content

[0075] The sulfate content was determined using a modified barium chloride-gelatin method.

[0076] Weigh 10g of sample and dissolve it in 5mL of 1mol / L HCl solution. Incubate in a boiling water bath for 5 hours. Take 0.2mL of the solution and add 3.8mL of 3% trichloroacetic acid and 1mL of barium chloride-gelatin solution. Shake well and let stand at room temperature for 15 minutes. Measure the absorbance at 360nm (A1). Repeat the experiment using 1mL of 0.5% gelatin solution instead of barium chloride-gelatin, measuring the absorbance (A2). Determine the absorbance difference (A1-A2). The standard is potassium sulfate standard solution, and the blank is distilled water. Plot a standard curve (y = 0.0026x - 0.0156, R0) based on sulfate concentration and absorbance (A1-A2). 2 =0.9944), the sulfate contents of LF3-1 and LF4-1 were 26.3% and 32.7%, respectively.

[0077] 5. Nucleic acid and protein detection

[0078] Impurity analysis of LF3-1 and LF4-1 was performed using ultraviolet spectrophotometry.

[0079] Prepare 0.5 mg / mL solutions of LF3-1 and LF4-1, and perform a full-wavelength UV scan from 200 to 400 nm to detect any impurities such as nucleic acids in LF3-1 and LF4-1. Figure 4 As shown, 260nm and 280nm are the characteristic absorption wavelengths of nucleic acids and proteins. LF3-1 and LF4-1 components showed no obvious absorption peaks, indicating that impurities such as nucleic acids and proteins have been basically removed.

[0080] Quantitative analysis of proteins was performed using the Folin-phenol method. 0.5 mg / mL fractions of LF3-1 and LF4-1 were prepared. 1 mL of each fraction was placed in a test tube, 5 mL of reagent A was added, followed by 0.5 mL of reagent B (Folin-phenol reagent). The solution was incubated at room temperature for 30 min, and the absorbance was measured at 700 nm. A standard curve was plotted using bovine serum albumin as the standard (y = 0.0025x + 0.0101, R²). 2 =0.9956), the protein contents of LF3-1 and LF4-1 were 2.9% and 3.2%, respectively.

[0081] Example 3: Structural Identification of Laminaria Fucoidan

[0082] 1. Infrared spectroscopy analysis

[0083] Dry LF3-1 and LF4-1 were separately mixed with potassium bromide, thoroughly ground in an agate mortar, and then compressed into tablets. A NICOLET iS50 infrared spectrometer was used to scan the wavelength range of 4000-400 cm⁻¹. -1The number of scans was 32, and the resolution was 4cm. -1 .like Figure 5 As shown, 3490cm was obtained. -1 The peak at 2954 cm⁻¹ represents the vibrational absorption peaks of OH and NH in sugar residues. -1 The weak absorption peak is related to the stretching vibration of the CH group of the methyl group in fucose; 1651 cm⁻¹ -1 The strong absorption peak at 1394 cm⁻¹ is caused by the C=O vibration of the acetylamino group. -1 The weak absorption peak at 1267 cm⁻¹ is a CO stretching vibration absorption peak, proving the presence of uronic acid; -1 The sharp absorption peak at 1054 cm⁻¹ is attributed to the S=O stretching vibration of the sulfate group, indicating the presence of a sulfate ester group; -1 The peak at 850 cm⁻¹ is a vibrational absorption peak resulting from the overlap of the COC and COH sugar rings. -1 The absorption peaks indicate the presence of α-pyranose in the polysaccharide structure.

[0084] 2. Monosaccharide composition analysis

[0085] The monosaccharide composition of LF3-1 and LF4-1 was determined by derivatization combined with high performance liquid chromatography.

[0086] Weigh 5 mg of sample into a hydrolysis tube, add 1 mL of 2 mol / L trifluoroacetic acid solution, hydrolyze at 110 °C for 2 h, evaporate to dryness under reduced pressure at 60 °C, and repeat the operation 3 times with 1 mL of methanol to ensure complete removal of excess trifluoroacetic acid. Then dissolve in distilled water. Take 300 μL of the hydrolyzed solution into a hydrolysis tube, add 100 μL of 0.5 mol / L PMP methanol solution, then add 300 μL of 0.3 mol / L NaOH solution, seal the tube with nitrogen, and hydrolyze at 70 °C for 1 h. After cooling to room temperature, neutralize with 300 μL of 0.3 mol / L HCl solution, then add 1 mL of chloroform for extraction. Collect the supernatant, repeat the operation several times, filter through a 0.45 μm microporous membrane, and inject for high performance liquid chromatography analysis. The high-performance liquid chromatography (HPLC) detection conditions were as follows: an ion chromatograph (Shimadzu LC-16, Shimadzu Corporation, USA) was used for detection, employing a ShimNex CS C18 column (5 μm, 4.6 x 250 mm) and a UV detector; column temperature: 30℃; flow rate: 0.5 mL / min; injection volume: 10 μL; mobile phase: 80% 0.1 M phosphate buffer and 20% acetonitrile. Various monosaccharide standards (rhamnose, xylose, glucose, galactose, mannose, fucose, arabinose, glucuronic acid, and glucosamine) were weighed, dissolved separately in distilled water, and analyzed according to the method. Based on the retention time of the standards, the monosaccharide composition in the polysaccharide sample could be determined, and the molar percentage of monosaccharides in the sample could be calculated based on the peak area and molar mass of each monosaccharide. The results showed that LF3-1 was composed of mannose, glucosamine, glucuronic acid, galactose and fucose, with a molar ratio of 3.59:1.00:2.41:11.97:14.25; LF4-1 was composed of mannose, glucosamine, glucuronic acid, rhamnose, galactose and fucose, with a molar ratio of 2.21:1.00:1.00:1.25:13.11:7.13.

[0087] 3. Methylation analysis

[0088] The glycosidic bond linkage of LF3-1 and LF4-1 was determined using a methylation assay. 2.5 mg of dried sample was weighed and placed in a hydrolysis tube, which was dried at 60 °C for 12 h. 2 mL of dried DMSO was added and allowed to dissolve completely. 30 mg of dried NaOH powder was added, the tube was sealed, and stirred until completely dissolved. The hydrolysis tube was placed in an ice bath, and under light-protected conditions, 1 mL of iodomethane was slowly added dropwise. The reaction was carried out at low temperature for 2.5 h, followed by the addition of 0.5 mL of water to terminate the reaction. 2 mL of dichloromethane was added to the sample. The aqueous and organic layers were separated by low-speed centrifugation. The aqueous layer was removed, and 2 mL of water was added to wash until the aqueous layer was colorless. The bottom organic layer was then transferred to a new tube, dried under nitrogen, and analyzed by infrared spectroscopy for the presence of methyl groups.

[0089] Add 1 mL of 4M TFA (3.1 mL TFA + 6.9 mL water) to the dried reactants, hydrolyze under nitrogen in separate tubes at 110 °C for 6 h, evaporate the liquid to dryness, wash twice with 1 mL methanol to remove trifluoroacetic acid, and evaporate to dryness again. Reduction: Add 1 mL of distilled water to the obtained sample, dissolve the sample, add 20 mg of sodium borohydride, and react overnight at room temperature. Then add 5 drops of glacial acetic acid to neutralize the sodium borohydride, then add 500 mL of a methanol-acetic acid mixture (methanol:acetic acid = 9:1), dry under nitrogen, and repeat twice; then add 500 mL of methanol, dry under nitrogen, and repeat twice. Add 0.5 mL of acetic anhydride to the dried sample, separate the tubes under nitrogen, react at 120 °C for 1 h, dry under nitrogen, add 0.5 mL of water to terminate the reaction, add 1 mL of dichloromethane, extract, remove the aqueous layer, add 1 mL of water again, extract to remove the aqueous layer, dry under nitrogen, and add 0.5 mL of dichloromethane (chromatographic grade) to reconstitute.

[0090] Finally, GC-MS analysis was performed: the column was an Rtx-5MS (60 m x 0.32 mm x 0.25 μm), and the temperature program was as follows: (a) initial temperature 120 °C, held for 5 min; (b) increased to 150 °C at a rate of 5 °C / min and held for 2 min; (c) increased to 220 °C at a rate of 3 °C / min and held for 2 min; (d) increased to 280 °C at a rate of 5 °C / min and held for 2 min; (e) held at 280 °C for 5 min. Mass spectra were obtained in the range of 30–500 m / z. Figure 6 As shown, LF3-1 is composed of (3→4)-Fucp, (1→4)-Fucp, (1→3)-Fucp(4SO4), (1→3)-Fucp, 1-Galp, (1→3)-Galp(6SO4), (3→6)-Galp, (2→4)-Manp and (2→3)-Manp; LF4-1 is composed of (3→4)-Fucp, (1→4)-Fucp, (1→3)-Fucp(4SO4), (1→3)-Fucp, 1-Galp, (1→3)-Galp, (3→6)-Galp, (1→6)-Galp(3SO4), (2→4)-Manp and (2→3)-Manp.

[0091] 4. NMR Analysis

[0092] like Figure 7 and Figure 8As shown, the signals at δ1.31 and δ1.32 are terminal hydrogen signals of fucose, the signals at δ15.44 and δ15.83 are terminal carbon signals of fucose, the signals at δ5.25-5.45 are anodic hydrogen signals of fucose, and the signals at δ98-100 are anodic carbon signals of fucose. The signal peak appearing at δ98.91-99.94 ppm belongs to the anodic carbon C-1 signal peak of fucose, and the signal peaks appearing at δ1.31 and δ1.32 ppm belong to the anodic carbon H6 signal peak of fucose. In LF3-1, H1 (δ5.44ppm) of sugar residue A is coupled with C3 (δ77.33ppm) of sugar residue G, and H1 (δ4.42ppm) of sugar residue G is coupled with C3 (δ77.01ppm) of sugar residue A. In LF4-1, H1 (δ5.43ppm) of sugar residue A is coupled with C6 (δ67.14ppm) of sugar residue H, and H1 (δ5.36ppm) of sugar residues B and C is coupled with C6 (δ67.14ppm) of sugar residue G. Therefore, the main chain of LF3-1 is composed of →3)-α-L-Fucp-(1→ and →3)-β-D-Galp-(1→), with the fucose in the main chain sulfated at the C-4 position or having a first branch, which is composed of →4)-α-L-Fucp-(1→; the galactose in the main chain is sulfated at the C-6 position or has a second branch, which is composed of →2,3)-β-D-Manp-(1→ and β-D-Galp-(1→). The main chain of LF4-1 is composed of →3)-α-L-Fucp-(1→ and →6)-β-D-Galp-(1→). The fucose in the main chain is sulfated at the C-4 position or has a third branch, which is composed of →4)-α-L-Fucp-(1→. The galactose in the main chain is sulfated at the C-3 position or has a fourth branch, which is composed of →2,4)-β-D-Manp-(1→ and β-D-Galp-(1→).

[0093] Example 4: Study on the weight-loss activity of kelp fucoidan

[0094] 1. Effects of LF3-1 and LF4-1 on mouse body weight and serum lipids, respectively.

[0095] LF3-1 and LF4-1 were prepared into solutions for animal experiments. Male C57BL / 6J mice (6 weeks old) were selected and administered the following treatments by gavage: the LF3-1 group received 100 mg / kg / day of LF3-1 solution by gavage; the LF4-1 group received 100 mg / kg / day of LF4-1 solution by gavage; and the ND group (normal diet) and HFD group (high-fat diet) received the same volume of 0.9% saline by gavage. The administration time was fixed daily for 6 consecutive weeks, and body weight was measured weekly. Figure 9 It was found that by week 6, the body weight of mice in the LF3-1 group and LF4-1 group was significantly different from that in the HFD group, decreasing by 9.65% and 9.43% respectively, while there was no significant difference compared with the ND group (p>0.05).

[0096] The concentrations of TG and TC in serum were determined using an enzyme-linked immunosorbent assay (ELISA) according to the kit instructions. Figure 10 It was found that, compared with the HFD group, the levels of TC and TG in the LF3-1 and LF4-1 intervention groups were significantly decreased (p < 0.05). Therefore, LF3-1 and LF4-1 can effectively improve the blood lipid levels in obese mice.

[0097] 2. Effects of kelp fucoidan on body weight and diet in mice

[0098] Because LF3-1 and LF4-1 are structurally similar in terms of monosaccharide composition, fucose sulfate substitution type, and glycosidic bond type, and both can affect the body weight and blood lipids of mice on a high-fat diet, these two components were mixed in a 1:1 mass ratio to prepare a kelp fucoidan solution for subsequent animal experiments. Male C57BL / 6J mice (6 weeks old) were selected and administered the following treatments by gavage: the AL group received 60 mg / kg / day orlistat solution by gavage; the FUC100 group received 100 mg / kg / day of kelp fucoidan solution by gavage; the FUC300 group received 300 mg / kg / day of kelp fucoidan solution by gavage; and the ND and HFD groups received the same volume of 0.9% saline by gavage. The administration time by gavage was fixed daily for 24 consecutive weeks. Body weight-related indicators were then measured.

[0099] Depend on Figure 11 It was found that by week 24, the body weights of the AL, FUC100, and FUC300 groups were significantly lower than those of the HFD group, decreasing by 30.07%, 28.79%, and 31.41%, respectively, with no significant difference compared to the ND group (p > 0.05). Compared to the HFD group, the weight gain in the AL, FUC100, and FUC300 groups was significantly reduced (p < 0.01), decreasing by 56.95%, 52.59%, and 59.54%, respectively. Regarding daily food intake, there were no significant differences among the experimental groups (p > 0.05), indicating that the change in mouse body weight was not related to food intake. Therefore, intervention with kelp fucoidan (a 1:1 mass ratio of LF3-1 and LF4-1) can effectively inhibit the weight gain of mice on a high-fat diet without changing food intake.

[0100] 3. Effects of kelp fucoidan on organ indices in mice

[0101] After weighing the mouse organs, the organ index is calculated using the following formula:

[0102]

[0103] The results showed that compared with the HFD group, the liver and fat coefficients of mice in the AL, FUC100, and FUC300 groups were significantly reduced (p < 0.01). Specifically, the liver coefficient decreased by 12.24%, 18.37%, and 20.41%, respectively; the epididymal fat coefficient decreased by 58.82%, 60.29%, and 61.76%, respectively; and the perirenal fat coefficient decreased by 80.00%, 60.00%, and 73.33%, respectively. Therefore, kelp fucoidan (a composition of LF3-1 and LF4-1 in a 1:1 mass ratio) can effectively reduce the liver, epididymal, and perirenal fat coefficients in mice fed a high-fat diet, and can effectively inhibit fat accumulation in the body.

[0104] 4. Effects of kelp fucoidan on serum blood glucose in mice

[0105] Mice were fed for 24 weeks. On the last day at 20:00, they were fasted but allowed free water. At 8:00 the following day, mouse weight was measured, and then they were administered a 1g / kg glucose solution by gavage. Blood glucose concentrations were measured by collecting blood from the tail vein at 0, 15, 30, 60, 90, and 120 minutes. A graph was plotted on blood glucose concentration over time to observe the effect of kelp fucoidan on glucose metabolism in high-fat diet-induced obese mice. The insulin resistance index was calculated using the following formula: like Figure 12 As shown, the blood glucose and AUC of mice in the kelp fucoidan intervention group were significantly lower than those in the HFD group at all time points (p < 0.01), indicating that the treatment group could effectively enhance glucose tolerance and improve blood glucose in mice. After 24 weeks of kelp fucoidan intervention, the insulin level in the FUC 100 group decreased by 15.9% compared with the HFD group, but the difference was not statistically significant, while the HOMA-IR index showed a significant difference (p < 0.01); the insulin level in the FUC 300 group was significantly lower than that in the HFD group (p < 0.01), and the HOMA-IR index was close to that in the ND group, indicating that kelp fucoidan (a combination of LF3-1 and LF4-1 in a 1:1 mass ratio) can dose-dependently inhibit the increase of serum insulin levels in mice and improve insulin resistance.

[0106] 5. Effects of kelp fucoidan on serum lipids in mice

[0107] The concentrations of TG, TC, LDL-C, and HDL-C in serum were determined using an enzyme-linked immunosorbent assay (ELISA) according to the kit instructions. Figure 13It was found that, compared with the HFD group, the levels of TC, TG, and LDL-C in mice treated with kelp fucoidan were significantly decreased (p < 0.01), while the level of HDL-C was significantly increased (p < 0.01). Therefore, kelp fucoidan (a combination of LF3-1 and LF4-1 in a 1:1 mass ratio) can effectively improve the blood lipid levels in obese mice.

[0108] 6. Effects of kelp fucoidan on lipid and glucose metabolism in mouse liver

[0109] Weigh an appropriate amount of tissue for total RNA extraction using BeyoRT. TM Total RNA was reverse transcribed using the II cDNA first-strand synthesis kit and BeyoFast. TM The SYBR Green qPCR Mix (2X) kit, after adding reagents according to the specified ratio, allows for real-time quantitative PCR analysis of sample cDNA (20 μL reaction volume). For example... Figure 14 and Figure 15 As shown, kelp fucoidan can promote the expression levels of lipid oxidation genes (PPAR-α, PPAR-γ, and CPT-1) and glucose metabolism genes (IRS1 and Glut4) in mice fed a high-fat diet, while inhibiting the expression levels of fatty acid synthesis genes (FAS, LXR, and SREBP-1c). This indicates that kelp fucoidan (a 1:1 mass ratio of LF3-1 and LF4-1) exerts its weight-loss and lipid-lowering effects by promoting fatty acid oxidation and glucose metabolism and inhibiting fatty acid synthesis-related genes.

[0110] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A type of kelp fucoidan, characterized in that, Including LF3-1 and / or LF4-1; The structure of LF3-1 is as follows: R1 is -SO3 or a first branch, the first branch being composed of →4)-α-L-Fucp-(1→, R2 is -SO3 or a second branch, the second branch being composed of →2,3)-β-D-Manp-(1→ and β-D-Galp-(1→ alternately, the sulfate content in LF3-1 is 26.3%, and the weight-average molecular weight of LF3-1 is 305.3 kDa; The structure of the LF4-1 is as follows: R3 is -SO3 or a third branch, the third branch being composed of →4)-α-L-Fucp-(1→, R4 is -SO3 or a fourth branch, the fourth branch being composed of →2,4)-β-D-Manp-(1→ and β-D-Galp-(1→ alternately, the sulfate content in LF4-1 is 32.7%, and the weight-average molecular weight of LF4-1 is 182.1 kDa; The LF3-1 is composed of mannose residues, glucosamine residues, glucuronic acid residues, galactose residues and fucose residues in a molar mass ratio of 3.59:1.00:2.41:11.97:14.

25. The LF4-1 is composed of mannose residues, glucosamine residues, glucuronic acid residues, rhamnose residues, galactose residues and fucose residues in a molar mass ratio of 2.21:1.00:1.00:1.25:13.11:7.

13.

2. The kelp fucoidan as described in claim 1, characterized in that, This includes LF3-1 and LF4-1 with a mass ratio of 0.8-1.2:0.8-1.

2.

3. The kelp fucoidan as described in claim 2, characterized in that, The mass ratio of LF3-1 to LF4-1 is 1:

1.

4. A method for preparing kelp fucoidan as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Mix kelp powder and an aqueous solution containing 0.2-0.6wt% sodium dodecyl sulfate at 60-80℃ at a mass-to-volume ratio of 1:20-40, let stand for 2-4 h, and then centrifuge, collect the supernatant and concentrate in sequence to obtain the first concentrated solution; (2) Mix the first concentrate and 10wt% calcium chloride at a volume ratio of 3-4:1, then filter, collect and concentrate the filtrate to obtain the second concentrate; (3) The second concentrate is mixed with anhydrous ethanol to obtain a first mixed solution, wherein the amount of anhydrous ethanol added is 20-30% of the total volume of the first mixed solution. Stir until a white granular precipitate appears and the color of the first mixed solution becomes lighter. Let it stand, collect the supernatant, add anhydrous ethanol to the supernatant to obtain a second mixed solution, wherein the amount of anhydrous ethanol added is 50-60% of the total volume of the second mixed solution. Stir until a white granular precipitate appears and the color of the second mixed solution becomes lighter. Let it stand, collect the precipitate, wash the precipitate with anhydrous ethanol, dissolve the precipitate with water, and then obtain crude kelp fucoidan by dialysis and freeze drying. The molecular weight cutoff of the dialysis is 8-14 kDa. (4) The crude kelp fucoidan was separated using a DEAE-52 cellulose column and eluted isocratically with 0.5 mol / L, 0.7 mol / L and 1.0 mol / L NaCl solutions in sequence. The fractions obtained by elution with 0.7 mol / L and / or 1.0 mol / L NaCl solutions were collected to obtain LF3 and / or LF4. (5) Elute the LF3 and / or the LF4 in a Sephadex G-200 chromatography column with 0.5 mol / L NaCl solution to obtain the LF3-1 and / or the LF4-1.

5. The preparation method according to claim 4, characterized in that, In step (3), the amount of anhydrous ethanol added is 25% of the total volume of the second concentrate and anhydrous ethanol. In the process of adding anhydrous ethanol, the amount of anhydrous ethanol added is 56% of the total volume of the supernatant and anhydrous ethanol.

6. The preparation method according to claim 4, characterized in that, The kelp powder is obtained by defatting crude kelp powder. The defatting process is as follows: the crude kelp powder is refluxed with 90%-98% ethanol at 82-90℃ for 3.5-4.5 hours, and then dried to obtain the kelp powder.

7. A weight-loss and lipid-lowering composition, characterized in that, Its active ingredient includes the kelp fucoidan described in any one of claims 1-3.

8. The use of kelp fucoidan according to any one of claims 1-3 in the preparation of a weight-loss and lipid-lowering composition.

Images