A method for preparing active fragments of laminarin with low molecular weight and high purity

Abstract

The invention discloses a method for preparing a low-molecular-weight and high-purity active fragment of kelp polysaccharide, and belongs to the technical field of polysaccharide degradation. The operation steps are as follows: (1) the uniform component of kelp polysaccharide is dissolved in acetic acid-sodium acetate buffer and ultrasonically stretched, the chain structure of the polysaccharide is stretched, β-mannanase is added for enzymolysis, and fixed-point cutting is implemented to obtain a low-molecular-weight kelp polysaccharide mixture; (2) after separation and purification by dextran gel, a uniform low-molecular-weight active fragment of kelp polysaccharide is obtained, and the active fragment of kelp polysaccharide is powdered, with a molecular weight of 1371 Daltons, and mainly contains glucose, mannose and galactose. The present invention has the advantages of simple operation, no pollution, and stable reaction, and the degraded polysaccharide has obvious activity of inhibiting macrophage foaming.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide degradation, and particularly relates to a method for degrading laminarin polysaccharide by combining ultrasound with enzyme. Background Art

[0002] Laminaria japonica (scientific name Laminaria japonica ) is a plant of the Laminariaceae family. Its holdfast is in the form of a rhizoid, with a short and thick stalk that serves as a connection. Its leaves are wide and long, and are usually sold in the form of dried products. Kelp is rich in polysaccharides, proteins, amino acids, dietary fiber, and minerals. It has a high yield, abundant resources, and is easy to cook. It is an ideal natural food.

[0003] The main active ingredient of kelp is polysaccharide. Studies have shown that kelp polysaccharide has a variety of biological activities including anti-tumor, anti-virus, anti-atherosclerosis, hypoglycemia, and hypolipidemic activities. The research team previously extracted a uniform kelp polysaccharide from kelp. Studies have found that this uniform kelp polysaccharide can significantly inhibit macrophage foaming. However, its molecular weight is as high as 1.96×10 6 Dalton, too large molecular weight leads to high viscosity of polysaccharides, poor water solubility and low absorption rate, which seriously hinders the further research and development of kelp polysaccharides in the fields of food and medicine. In recent years, many studies have proved that there are one or more low molecular weight fragments in polysaccharide molecules as "active centers". When polysaccharides enter the body, these fragments can bind to cell membrane surface receptors and exert biological activity. Therefore, the degradation of polysaccharides and further separation and purification to obtain uniform low molecular weight active fragments is a hot topic in current polysaccharide research.

[0004] Traditional polysaccharide degradation includes physical degradation, chemical degradation and biological degradation. Among them, physical degradation includes microwave degradation, ultrasonic degradation and radiation degradation. These degradation methods are simple to operate and widely applicable, but they are inefficient and difficult to destroy the glycosidic bonds in the polysaccharide molecules. They are usually used as pretreatments for other degradation methods. Chemical degradation includes acid hydrolysis and free radical degradation. Free radical degradation is also inefficient. Acid hydrolysis is efficient, but the reaction is violent, the degradation products are messy and easily produce harmful substances. The biological degradation method is mainly enzymatic degradation, including specific enzymes and non-specific enzymes. The enzymatic degradation reaction is mild. By selecting different enzymes, the polysaccharide can be cut at a specific point. However, for different polysaccharides, different enzymes need to be selected, and multiple experiments are required to optimize the reaction conditions. Summary of the invention

[0005] In order to realize simple operation and high efficiency in preparing purified degradation products of laminarin with low molecular weight and high purity, the present invention provides a method for preparing active fragments of laminarin with low molecular weight and high purity.

[0006] The preparation steps of a low molecular weight and high purity active fragment of kelp polysaccharide are as follows:

[0007] (1) Degradation of kelp polysaccharides

[0008] The uniform components of laminarin polysaccharide are dissolved in acetic acid-sodium acetate buffer, subjected to ultrasound, and β-mannanase is added for enzymolysis; the enzymolysis product is boiled and inactivated, centrifuged to remove enzymes, dialyzed, and freeze-dried to obtain a low molecular weight laminarin polysaccharide mixture;

[0009] The kelp polysaccharide is uniformly distributed in powder form with a molecular weight of 1.96×10 6 Dalton, composed of mannose, glucose and galactose in a molar ratio of 1:1.85:2.92;

[0010] (2) Separation and purification of low molecular weight kelp polysaccharide mixture

[0011] Separating and purifying the low molecular weight laminarin mixture through a dextran gel column to obtain uniform low molecular weight laminarin active fragments;

[0012] The active fragment of laminarin is in powder form and has a molecular weight of 1371 Daltons.

[0013] The further technical solution is as follows:

[0014] In step (1), the repeating unit of the structure of the homogeneous component of kelp polysaccharide mainly comprises →3,6)-α-D-Man p -(1→、→4)-α-D-Man p -(1→,→4)-2-O-acetyl-β-D-Glc p -(1→、→4)-β-D-Glc p -(1→,→6)-4-O-SO3-β-D-Gal p -(1→、→6)-β-D-Gal p -(1→、→3)-β-D-Gal p -(1→ and terminal sugar residue α-D-Glc p -(1→.

[0015] In step (1), 100 mg of laminarin component was dissolved in 10 mL of acetic acid-sodium acetate buffer with a concentration of 50 mmol / L and a pH of 4.5, and ultrasonicated at a frequency of 40 kHz for 1.5 h; 10 mg of β-mannanase (10 u / mg) was added, and the reaction was carried out at 50° C. for 3 h; the mixture was in a water bath at 100° C. for 10 min, and the supernatant was collected by centrifugation to obtain a low molecular weight laminarin mixture.

[0016] In step (2), 10 mg of the low molecular weight kelp polysaccharide mixture is dissolved in 1 mL of distilled water, added to a pre-treated G-25 polysaccharide gel column, eluted with double distilled water at a flow rate of 0.15 mL / min, and a tube of eluate is collected every 10 minutes by a drop counting automatic collector. The polysaccharide content is determined by the phenol-sulfuric acid method, an elution curve is drawn, and the eluates of the same elution peak are combined. The homogeneity and molecular weight are detected by high performance liquid chromatography; and freeze-dried to obtain a uniform low molecular weight kelp polysaccharide active fragment.

[0017] The application of the low-molecular-weight and high-purity active fragment of kelp polysaccharide in the preparation of a product for inhibiting macrophage foaming.

[0018] The beneficial technical effects of the present invention are embodied in the following aspects:

[0019] 1. The molecular weight of the active fragment of kelp polysaccharide prepared by the preparation method of the present invention is low, ranging from 1.96×10 6 Dalton is reduced to 1371 Daltons, see Figure 1 , and in the form of a single symmetrical peak, indicating that it is a highly uniform component. The active fragment of kelp polysaccharide has high purity and can effectively improve the autophagic flow damage caused by oxidized low-density lipoprotein, reduce lipid aggregation in macrophages, and inhibit the transformation of macrophages into foam cells.

[0020] 2. The present invention uses ultrasound combined with β-degradation of laminarin to degrade, and further separates and purifies the degradation products through G-25 polysaccharide gel, ultimately obtaining a polysaccharide with low molecular weight and high purity. The degradation process does not involve the use of any organic reagents, thereby greatly reducing the pollution to the environment. In addition, cell experiments have shown that the active fragments of laminarin prepared in this study can significantly inhibit macrophage foaming, indicating that the active fragments of laminarin prepared in the present invention are the structural basis for laminarin to exert its activity in inhibiting macrophage foaming. The present invention will provide support for the "active center" theory and provide a theoretical basis for the research and promotion of laminarin.

[0021] The present invention uses ultrasound combined with beta-deoxyglucanase to degrade the homogeneous component of laminarin polysaccharide, and further separates and purifies the degradation product through G-25 dextran gel, and finally obtains a polysaccharide with low molecular weight and high purity. At the same time, it is found through experiments that neither ultrasound nor enzyme treatment can directly reduce the molecular weight of the homogeneous component of laminarin polysaccharide, so we speculate that ultrasonic pretreatment can destroy the dense and orderly structure of the homogeneous component of laminarin polysaccharide, so that the chain structure of the polysaccharide is stretched, which is conducive to the penetration of the enzyme in the polysaccharide structure, so that the polysaccharide is effectively degraded. The low molecular weight laminarin polysaccharide mixture after degradation is separated in sequence according to the molecular weight through G-25 dextran gel, and the eluent of the same elution peak is combined after the polysaccharide content is determined by phenol sulfuric acid method, and finally analyzed by high performance liquid chromatography to obtain a uniform low molecular weight laminarin polysaccharide active fragment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart for the preparation of active fragments of kelp polysaccharides.

[0023] Figure 2 It is the molecular weight distribution diagram of the homogeneous components of laminarin polysaccharide and the active fragments of laminarin polysaccharide.

[0024] Figure 3 This is a UV spectrum scan of the active fragments of kelp polysaccharides.

[0025] Figure 4 This is an infrared spectrum scan of the active fragments of kelp polysaccharide.

[0026] Figure 5 This is a monosaccharide composition analysis diagram of the active fragments of kelp polysaccharides.

[0027] Figure 6 This is the methylation analysis diagram of the active fragments of kelp polysaccharide.

[0028] Figure 7 This is the NMR image of the active fragment of kelp polysaccharide;

[0029] Figure 7 A in: one dimension 1 H spectrum; B: one-dimensional 13 C spectrum; C: two-dimensional 1 H- 1 H COSY spectrum can reflect the signals between adjacent hydrogen atoms in the same sugar residue; D: 2D HSQC spectrum can attribute the chemical shift between carbon and hydrogen in the same sugar residue; E: 2D HMBC spectrum can confirm the connection order between different sugar residues. (All coordinate axis units are displacement ppm).

[0030] Figure 8 It is the effect of active fragments of laminarin polysaccharide on inhibiting the formation of foam cells induced by oxidized low-density lipoprotein;

[0031] Figure 8 A: Total cholesterol content and free cholesterol content in RAW264.7 cells with different treatments; B: Cholesterol ester ratio in RAW264.7 cells with different treatments. Each statistical graph was obtained from 3 independent experiments, and the statistical difference was expressed in the form of standard error. # or * (p < 0.05) indicates a significant difference, and ## or ** (p < 0.01) indicates an extremely significant difference.

[0032] Fig. 9 It is the effect of active fragments of laminarin on inhibiting lipid deposition induced by oxidized low-density lipoprotein;

[0033] Fig. 9 A: Oil red staining of RAW264.7 cells with different treatments, arrows indicate lipid aggregation points; B: Statistical graph of oil red staining, the ordinate is the relative lipid content. Each statistical graph was obtained from 3 independent experiments, and the statistical difference is expressed in the form of standard error, # (p < 0.05) indicates a significant difference, and ## or ** (p < 0.01) indicates an extremely significant difference.

[0034] Fig.10 The effect of different contents of active fragments of laminarin and oxidized low-density lipoprotein on the expression of proteins in macrophages;

[0035] Fig.10 A: Western blot test; B: Expression of LC3-Ⅱ in macrophages with different treatments; C: Expression of p62 in macrophages with different treatments; D: Expression of ABCG1 in macrophages with different treatments.

[0036] Fig.11 It is the effect of active fragments of kelp polysaccharides on the autophagic flow damage of macrophages caused by oxidized low-density lipoprotein. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with the embodiments and drawings.

[0038] The reagents and equipment used in the following Example 1 without mentioning the manufacturer are all commercially available products. If the conditions are not specified in the examples, they are all carried out according to the conditions recommended by the manufacturer or conventional conditions.

[0039] Example 1

[0040] See also Figure 1 The steps for preparing the active fragment of laminarin with low molecular weight and high purity of the present invention are as follows:

[0041] (1) Preparation of kelp powder

[0042] Select fresh kelp leaves, rinse them and place them in clean water for 3 days to remove salt, during which the water is replaced every 3 hours. After the removal of salt, cut them into small pieces, dry and grind them, and pass them through a 60-mesh sieve to obtain kelp powder.

[0043] (2) Preparation of crude polysaccharide powder

[0044] Weigh 80 g of kelp powder, add 4 L of distilled water, heat in a water bath at 90° C. with stirring for 3 h, filter, adjust the pH value of the filtrate to 1.0 with concentrated hydrochloric acid, centrifuge and take the supernatant, adjust the pH value to 7.0 with a 10 mol / L sodium hydroxide solution, and evaporate and concentrate to 1 / 20 volume at 65° C. under reduced pressure; add 4 times the volume of anhydrous ethanol and stir evenly; let stand at 20° C. for 24 h, filter and take the precipitate, add 100 mL of distilled water to dissolve it, remove the protein by mixing and centrifuging the mixed solution, dialyze under tap water for 48 h using a 4000 Dalton dialysis bag, and freeze-dry to obtain a crude polysaccharide powder.

[0045] (3) Purification of crude kelp polysaccharide

[0046] Weigh 100 mg of crude polysaccharide powder, dissolve it in 10 mL of distilled water, slowly add it to the pressed anion exchange column, use distilled water at a flow rate of 2 mL / min for elution, collect the eluate, slowly add the eluate to the pre-treated propylene dextran S-500 gel chromatography column, use double distilled water at a flow rate of 0.15 mL for elution again, and collect one tube of eluate every 10 minutes by a drop counting automatic collector. Determine the polysaccharide content by the phenol-sulfuric acid method, draw the elution curve, combine the eluates of the same elution peak, use a 4000 Dalton dialysis bag for dialyzed in distilled water for 48 hours, and concentrate and freeze-dry to obtain a uniform kelp polysaccharide component.

[0047] (4) Degradation of kelp polysaccharides

[0048] Weigh 100 mg of kelp polysaccharide component, dissolve it in 10 mL of acetic acid-sodium acetate buffer with a concentration of 50 mmol / L and a pH of 4.5, and ultrasonicate at a frequency of 40 kHz for 1.5 hours; add 10 mg of β-mannanase (10u / mg), react at 50°C for 3 hours, water bath at 100°C for 10 minutes, centrifuge and take the supernatant to obtain a low molecular weight kelp polysaccharide mixture.

[0049] (5) Separation and purification of low molecular weight kelp polysaccharide mixture

[0050] Weigh 10 mg of a low molecular weight kelp polysaccharide mixture, dissolve it in 1 mL of distilled water, add it to a pre-treated G-25 polysaccharide gel column, use double distilled water at a flow rate of 0.15 mL / min for elution, collect a tube of eluate every 10 minutes using an automatic drop collector, determine the polysaccharide content by the phenol-sulfuric acid method, draw an elution curve, combine the eluates of the same elution peak, detect the homogeneity and molecular weight by high performance liquid chromatography, and freeze-dry to obtain a uniform low molecular weight kelp polysaccharide active fragment.

[0051] The active fragment of laminarin in Example 1 is in powder form and has a molecular weight of 1371 Daltons.

[0052] Effect Example 1

[0053] Determination of chemical indicators

[0054] 1. Polysaccharide Purification and Molecular Weight Determination

[0055] The uniform laminarin component prepared above and the laminarin active fragment finally prepared were respectively taken and their molecular weights were determined by high performance liquid chromatography. The detailed steps are as follows:

[0056] The sample was prepared into a 1 mg / mL solution using double distilled water. After passing through a 0.22 μm filter membrane, 20 μL was taken and injected into a high performance liquid chromatography column. The column model was TSKG5000 PWx1. The chromatographic conditions were: double distilled water as the mobile phase, a flow rate of 0.5 mL / min, and a column temperature of 30°C. At the same time, the molecular weight logarithm of the Detran series standard glucose and its retention time were used to draw a standard curve. The molecular weight of the polysaccharide sample was calculated based on the standard curve. The results are as follows: Figure 2 shown.

[0057] After the kelp polysaccharide is degraded by the method of the present invention, the molecular weight is reduced from 1.96×10 6 The Dalton was reduced to 1371 Daltons and was in the form of a single symmetrical peak, indicating that it was a highly homogeneous component.

[0058] 2. UV detection of polysaccharides

[0059] An appropriate amount of active fragments of laminarin was weighed and prepared into a 2 mg / mL solution. The solution was scanned at a wavelength of 190-400 nm using a UV-visible spectrophotometer to detect whether there were absorption peaks of nucleic acid and protein conjugates at 260 nm and 280 nm.

[0060] The results of UV detection are as follows Figure 3 As shown, there are no absorption peaks at 260nm and 280nm, indicating that the active fragments of laminarin do not contain nucleic acids and proteins.

[0061] 3. Infrared detection of polysaccharides

[0062] Take 2 mg of fully dried active fragments of kelp polysaccharide, add potassium bromide, mix and press into tablets, place on a Fourier infrared spectrometer scanner, and scan at 4000-500 cm -1 Scan within the range.

[0063] The results of infrared detection are as follows Figure 4 As shown in the figure, the active fragment of kelp polysaccharide has obvious polysaccharide characteristic absorption peak. -1 The absorption peak at 2988 cm corresponds to the stretching vibration of -OH. -1 The absorption peak at 1635 cm corresponds to the stretching vibration of CH. -1 The absorption peak at 1380cm is the stretching vibration peak of C=O. -1 The absorption peak at 1225cm is the angle-variation vibration peak of CH. -1 The absorption peak at 1027 cm -1 The vibration peak at 899 cm corresponds to the CO stretching vibration peak in COC, -1 The absorption peak at indicates that LJP61A-1 contains β-glycosidic bonds.

[0064] 4. Analysis of monosaccharide composition of polysaccharides

[0065] The active fragment of kelp polysaccharide prepared in Example 1 was taken and, after acid hydrolysis and derivatization, was compared with the monosaccharide standard that had undergone the same derivatization to determine its monosaccharide composition. The detailed steps are as follows:

[0066] Take 3 mg of sample, dissolve it in 1 mL of distilled water, add 0.5 mL of 3 mol / L HCl solution and acid hydrolyze it at 110 °C in an oxygen-free environment for 1 hour, and adjust the pH to neutral with 3 mol / L sodium hydroxide solution. Take 0.4 mL, add 0.4 mL of 1-phenyl-3-methyl-5-pyrazolone (PMP)-methanol solution (0.5 mol / L) and 0.4 mL of sodium hydroxide solution (0.3 mol / L), seal the tube and place it in a water bath at 70 °C for 100 min. After cooling, add 0.5 ml of dilute hydrochloric acid (0.3 mol / L), add 2 mL of chloroform solution and shake to wash, remove the lower chloroform layer, repeat 3 times, centrifuge and take the supernatant to obtain the derivatized sample solution, and use the same derivatization method to treat 2 mg / mL monosaccharide standard solution and 6 mg / mL glucosamine hydrochloride internal standard solution.

[0067] After the above solution passed through a 0.22qm filter membrane, 20 μL was taken and injected into a high performance liquid chromatography equipped with a UV detector, using octadecylsilane bonded silica gel as a filler, and the chromatographic conditions were: acetonitrile-0.02 mol / L ammonium acetate solution (20:80) as the mobile phase, a flow rate of 0.5 mL / min, and a detection wavelength of 250 nm.

[0068] The calculation method of monosaccharide molar ratio is:

[0069]

[0070] The monosaccharide composition results are as follows Figure 5 As shown, the active fragment of laminarin prepared by the present invention is composed of glucose, mannose and galactose, and the molar ratio is 4.57:1.08:1.00.

[0071] 5. Methylation Analysis of Polysaccharides

[0072] Table 1: Summary of mass spectrometry analysis of active fragments of methylated kelp polysaccharides

[0073]

[0074] The active fragments of laminarin were methylated and replaced by methyl iodide, and the degree of methylation was detected by infrared spectroscopy. The powder of the fully methylated active fragments of laminarin was acid-degraded, reduced by sodium borohydride, and acetylated. The resulting sugar alcohol acetate products were analyzed by gas chromatography-mass spectrometry to determine the type of sugar residues and the connection mode. Figure 6 As shown, the active fragments of kelp polysaccharide mainly include four kinds of sugar alcohol acetates. After identifying their mass-to-charge ratios, the sugar residues to which they belong are assigned. The results are shown in Table 1.

[0075] 6. NMR Analysis of Polysaccharides

[0076] The results are as follows Figure 7 As shown, through 1 H-NMR( Figure 7 A) Assign chemical shifts to the anomeric hydrogen to determine the configuration of the polysaccharide; 13 C-NMR( Figure 7 B) can be assigned to the chemical shift of the anomeric carbon and determine the substitution site of the polysaccharide; δ96.8ppm, δ101.3ppm, δ106.5ppm, δ104.0ppm, δ103.5ppm in the figure correspond to the sugar residue a(1-linkedGlc p )、b(1,3,6-linked Man p )、c(1,6-linked Gal p )、d(1,4-linked Glc p ), the signal peak at δ178ppm is the signal peak of the carbonyl carbon of the acetyl group, indicating that there are acetylated sugar residues in the active fragment of kelp polysaccharide. Compared with the unacetylated sugar residues, the chemical shift of hydrogen moves to the low field, while the chemical shift of carbon moves to the high field, so it is determined that 1,4-linked Glc p The 2-position of is replaced by an acetyl group.

[0077] Table 2: Chemical shifts of sugar residues in active fragments of kelp polysaccharides

[0078]

[0079] 1 H- 1 H COSY( Figure 7 C) can reflect the signals between adjacent hydrogen atoms in the sugar residue. As shown in the figure, taking sugar residue a as an example, H1 / H2 (5.13ppm / 3.75ppm), H2 / H3 (3.75ppm / 3.94ppm), H3 / H4 (3.94ppm / 3.97ppm), H4 / H5 (3.97ppm / 4.13ppm), and H5 / H6 (4.13ppm / 3.93ppm) can be found in sequence. The results are shown in the table. HSQC ( Figure 7 D) can be assigned to chemical shifts between carbon and hydrogen in the same sugar residue, such as Figure 7 As shown in D in, the cross peaks between anomeric carbon and anomeric hydrogen are δ96.8 / 5.13, δ101.3 / 5.31, δ106.5 / 4.40, δ104.0 / 4.35, δ103.5 / 4.62, and all chemical shifts are shown in Table 2. HMBC ( Figure 7 E) can be used to determine the order in which sugar residues are linked, such as Figure 7 As shown in E, the cross peak aH1 / eC4 indicates that the 1st position of a is connected to the 4th position of e, eH1 / dC4 indicates that the 1st position of e is connected to the 4th position of d, dH1 / bC3 indicates that the 1st position of d is connected to the 3rd position of b, bH1 / cC6 indicates that the 1st position of b is connected to the 6th position of c, and aH1 / bC6 indicates that the 1st position of a is connected to the 6th position of c. The structure of the active fragment of kelp polysaccharide obtained in the end is:

[0080]

[0081] Effect example 2:

[0082] 1. Effect of active fragments of kelp polysaccharides on inhibiting macrophage foaming

[0083] RAW264.7 cells were cultured in an incubator at 37°C and 5% carbon dioxide in a DMEM high-glucose medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells in the logarithmic phase were cultured at 1×10 6Cells were seeded in a 24-well plate at a concentration of 100 μg / mL, with 470 μL per well. After the cells adhered to the wall, oxidized low-density lipoprotein (Ox-LDL) was added to each well at a final concentration of 50 μg / mL. The polysaccharide intervention group was added with active fragments of kelp polysaccharides at final concentrations of 5 μg / mL and 25 μg / mL, respectively. The normal group was added with an equal volume of DMEM medium without serum. The cells were cultured in an incubator for 24 h, and the cells were digested and collected. The total cholesterol content and free cholesterol content were determined using a total cholesterol (TC) content kit and a free cholesterol (FC) content kit. The cholesterol ester (CE) content was obtained by subtracting the free cholesterol content from the total cholesterol content. The cholesterol content was statistically shown in the following table. Figure 8 As shown in A, cholesterol esters such as Figure 8 The cells were treated in the same manner, stained with Oil Red O staining kit, and counterstained with hematoxylin. After staining, the cells were observed and photographed under a microscope. After photographing, an equal volume of anhydrous ethanol was added, and the absorbance was measured at 500 nm. The staining results are shown in Fig. 9 As shown in A, the statistical results are as follows Fig. 9 As shown in B.

[0084] like Figure 8 As shown in A, compared with the normal group, the addition of active fragments of kelp polysaccharides alone did not significantly change the total cholesterol content and free cholesterol content in the cells, while the treatment of oxidized low-density lipoprotein significantly increased the total cholesterol content in the cells. Figure 8 B in the formula increased the proportion of cholesterol esters. After being treated with active fragments of kelp polysaccharides, the total cholesterol content was significantly reduced, the proportion of cholesterol esters was also reduced accordingly, and the improvement effect of the high-dose group was better than that of the low-dose treatment group. Fig. 9 In A, the cells added with oxidized low-density lipoprotein absorbed a large amount of oxidized low-density lipoprotein, resulting in lipid aggregation. Red particles were clearly observed in the cells. Compared with the model group, the red particles in the kelp polysaccharide active fragment intervention group were significantly reduced, and the improvement effect of the high-dose group was better than that of the low-dose treatment group. Fig. 9 The statistical results of B in also demonstrated the improvement effect of the active fragments of kelp polysaccharides.

[0085] 2. Effects of active fragments of kelp polysaccharides on the expression of LC3-Ⅱ, p62 and ABCG1 in macrophages

[0086] RAW264.7 cells were treated in the same manner as in Example 2.1, and different concentrations of oxidized low-density lipoprotein and active fragments of laminarin were added. After cell culture, the cells were washed with sterile PBS, and then fully lysed on ice for half an hour with RIPA lysis buffer containing phosphatase inhibitors and protease inhibitors, and then centrifuged at 10000rpm for 20min, the supernatant was aspirated, 1 / 4 volume of 5× loading buffer was added, and denatured at 100℃ for 10min, and the resulting protein was stored at -80℃. The protein blot test was performed, and the specific steps were as follows: 10 microliters of each denatured protein was taken, and another 10 microliters of pre-stained rainbow protein was taken and added to the prepared concentrated gel loading well, the lower layer was the separation gel, the electrolytic tank was filled with electrolyte, and the constant voltage 80V electrophoresis was performed until the protein entered the separation gel, and the voltage was adjusted to 120V and continued electrophoresis for 1h. After the electrophoresis, the gel block was soaked in transfer buffer, and the sandwich was assembled in the order of sponge-filter paper-gel-PVDF membrane-filter paper-sponge. The membrane was transferred in an ice-water bath for 1.5 hours, and the transfer current was 200 mA. After the transfer, it was washed 3 times with 20 mL TBS buffer on a shaker for 10 minutes each time, and then the PVDF membrane was blocked with 5% skim milk powder for 2 hours. After the end, it was washed again 3 times with 20 mL TBS buffer on a shaker for 10 minutes each time, and the membrane was immersed in the primary antibody solution diluted with TBS buffer 1000:1, and shaken at 4°C for 12 hours. After taking it out, it was washed 3 times with TBS buffer, and then soaked and shaken for 1 hour with the secondary antibody solution diluted with TBS buffer 5000:1. The membrane was washed 3 times with TBS after being taken out, and 10 mL of luminescent liquid was added, and imaged and observed in a chemiluminometer.

[0087] Specific results such as Fig.10 As shown in A, the expression of β-actin in different groups is consistent, indicating that the loading amount and protein concentration are consistent. Image-J software was used to analyze the gray value of the immune trace band to obtain the ratio of the gray value of different groups to the corresponding β-actin gray value. The expression level was statistically analyzed with the value of the normal group as the control. Fig.10 B in the figure is the statistics of LC3 expression. Fig.10 The C in the figure is the statistics of p62 expression. Fig.10 D in the figure is a statistical analysis of the expression of ABCG1. It can be seen that only in cells treated with oxidized low-density lipoprotein, the expression of LC3-Ⅱ and p62 increased, indicating that oxidized low-density lipoprotein inhibited the autophagy process of macrophages; after treatment with active fragments of laminarin polysaccharides, the expression of LC3-Ⅱ increased further, while p62 decreased, indicating that autophagy was activated; at the same time, the expression of ABCG1 in macrophages treated with active fragments of laminarin polysaccharides was significantly higher than that in macrophages treated with oxidized low-density lipoprotein, indicating that the treatment with active fragments of laminarin polysaccharides enhanced the cholesterol efflux of macrophages.

[0088] 3. Effects of active fragments of laminarin on autophagic flux in foamy macrophages

[0089] RAW264.7 cells in the logarithmic growth phase were seeded in a 3.5 cm laser confocal culture dish at a concentration of 0.2×10 6 / mL, 2mL per culture dish, after culturing for 24 hours, the culture medium was removed, and 1mL of new culture medium containing 5mL mGFP-RFP-LC3 double-labeled adenovirus was added again. After culturing for 2 hours, 1mL of fresh culture medium was added to each well. After continuing to culture for 24 hours, the old culture medium with the virus was removed, and each culture dish was washed with 1mL PBS. Fresh culture medium containing different concentrations of polysaccharides, oxidized low-density lipoprotein and autophagy inhibitors were added to different components respectively, and cultured in the incubator again for 24 hours. After taking out, the culture medium was removed, and each culture dish was washed with 1mL PBS, fixed with 4% paraformaldehyde for 15 minutes and washed again, stained with a nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) at a concentration of 5 μg / mL for 20 minutes, the dye was removed and washed, and the number of autophagosomes and autophagolysosomes was observed and counted under a laser confocal microscope.

[0090] The specific statistical results are as follows: Fig.11 As shown in the figure, green fluorescent protein (GFP) and red fluorescent protein (RFP) emit yellow signals in autophagosomes at neutral pH. When autophagosomes and lysosomes combine to form autophagolysosomes, the signal of pH-sensitive green fluorescent protein is weakened, while the signal of red fluorescent protein is retained. Therefore, the intensity of green fluorescence and red fluorescence can be used as an evaluation index of cell autophagic flow. Compared with the normal group, cells treated with only laminarin active fragments did not change significantly, while the green signal of cells in the oxidized low-density lipoprotein treatment group was retained in large quantities, indicating that the intracellular autophagic flow was significantly damaged; the treatment of laminarin active fragments significantly reduced the signal of green fluorescent protein, and the improvement effect was more obvious with the increase of polysaccharide concentration; CQ is an autophagy inhibitor, and the addition of CQ significantly reduced the repair effect of laminarin active fragments on cell autophagic flow, indicating that laminarin active fragments can repair the autophagic flow disorder induced by oxidized low-density lipoprotein.

[0091] The above results show that the active fragments of kelp polysaccharides can effectively improve the autophagic flow damage caused by oxidized low-density lipoprotein, reduce lipid aggregation in macrophages, and then inhibit the transformation of macrophages into foam cells.

[0092] In summary, the degradation method of the present invention can effectively degrade laminarin, and the use of dextran gel can separate and obtain low molecular weight uniform polysaccharides from the degradation products, and the obtained polysaccharides can effectively inhibit macrophage foaming.

[0093] Finally, it should be understood that the above embodiment 1 does not limit the present invention. For ordinary technical personnel in the relevant field, other different forms of changes or modifications can be made without departing from the basic principles of the present invention, and these changes or modifications are still within the protection scope of the present invention.

Claims

1. A method for preparing a low molecular weight and high purity active fragment of laminarin, characterized in that: The steps are as follows: (1) Degradation of kelp polysaccharides The uniform components of laminarin polysaccharide are dissolved in acetic acid-sodium acetate buffer, subjected to ultrasound, and β-mannanase is added for enzymolysis; the enzymolysis product is boiled and inactivated, centrifuged to remove enzymes, dialyzed, and freeze-dried to obtain a low molecular weight laminarin polysaccharide mixture; The kelp polysaccharide is uniformly divided into powders with a molecular weight of 1.96×10 6 Dalton, composed of mannose, glucose and galactose in a molar ratio of 1:1.85:2.92; The specific operations are as follows: 100 mg of laminarin component was dissolved in 10 mL of acetic acid-sodium acetate buffer with a concentration of 50 mmol / L and a pH of 4.5, and ultrasonicated at a frequency of 40 kHz for 1.5 h; 10 mg of β-mannanase 10 u / mg was added, and the reaction was carried out at 50°C for 3 h; the mixture was placed in a water bath at 100°C for 10 min, and the supernatant was collected by centrifugation to obtain a low molecular weight laminarin mixture; (2) Separation and purification of low molecular weight kelp polysaccharide mixture Separating and purifying the low molecular weight laminarin mixture through a dextran gel column to obtain uniform low molecular weight laminarin active fragments; The specific operations are as follows: Dissolve 10 mg of low-molecular-weight kelp polysaccharide mixture in 1 mL of distilled water, add to a pre-treated G-25 polysaccharide gel column, elute with double distilled water at a flow rate of 0.15 mL / min, collect a tube of eluate every 10 minutes by a drop counting automatic collector, determine the polysaccharide content by phenol sulfuric acid method, draw an elution curve, combine the eluates of the same elution peak, and detect the homogeneity and molecular weight by high performance liquid chromatography; freeze-dry to obtain a uniform low-molecular-weight kelp polysaccharide active fragment; The active fragment of laminarin is in powder form and has a molecular weight of 1371 Daltons.

2. The method for preparing a low molecular weight and high purity active fragment of laminarin according to claim 1, characterized in that: In step (1), the repeating unit of the structure of the homogeneous component of kelp polysaccharide mainly comprises →3,6)-α-D-Man p -(1→、→4)-α-D-Man p -(1→,→4)-2-O-acetyl-β-D-Glc p -(1→、→4)-β-D-Glc p -(1→,→6)-4-O-SO3-β-D-Gal p -(1→、→6)-β-D-Gal p -(1→、→3)-β-D-Gal p -(1→ and terminal sugar residue α-D-Glc p -(1→.

3. The use of a low molecular weight and high purity active fragment of laminarin according to claim 1, characterized in that: The application of the low-molecular-weight and high-purity active fragment of laminarin in the preparation of a product for inhibiting macrophage foaming.

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