Preparation and application of a kind of nano micelles of brown algae polysaccharide derivatives

Abstract

The invention discloses a preparation and application of a fucoidan derivative nano-micelle, and relates to the field of marine biomedicine technology. The fucoidan derivative nano-micelle, its raw material fucoidan derivative; the fucoidan derivative is a fucoidan sulfonyl derivative, obtained by chemically connecting the hydroxyl group in the fucoidan structure with the sulfonyl chloride in the structure of 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester; the fucoidan is extracted from kelp. The fucoidan derivative nano-micelle prepared by the present invention has stronger amylin aggregation inhibition activity, and the anticoagulant ability is also improved; and it can be used as a good carrier material, with more excellent drug loading capacity and encapsulation rate.

Description

Technical Field

[0001] The invention belongs to the technical field of marine biomedicine, and specifically relates to the preparation and application of nano micelles of brown algae polysaccharide derivatives. Background Art

[0002] Polysaccharides in marine organisms have antiviral, antitumor, anti-radiation, anti-mutation, antioxidant and immune-enhancing effects. Factors affecting the activity of polysaccharides include the main chain properties, branch chain properties and higher-order structures of polysaccharide molecules. In order to improve the activity of polysaccharides, molecular modification and structural modification of polysaccharides are of great significance. At present, chemical methods are mainly used to modify the hydroxyl, carboxyl, amino and other groups on the sugar residues, including sulfation, carboxylmethylation, selenization, methylation, phosphation, and digroup derivatization. Brown algae polysaccharides can be extracted from many marine organisms, with a wide range of sources and large storage capacity. In recent years, researchers have confirmed that it has multiple biological activities such as antitumor, antiviral, and anticoagulant. However, some of its biological activities are not ideal. For example, its antiviral effect is not ideal, and it only works in the stage of avian leukosis virus adsorbing host cells, and has no antiviral effect in the stage of virus reproduction. Therefore, improving its biological activity will have great research significance and application value.

[0003] In the field of nanotechnology, nanoparticles refer to particles with a particle size between 1 and 100 nm; while nanoparticles (nano drugs or nano drug delivery systems) in pharmacy generally refer to nano carriers with a particle size between 10 and 1000 nm for delivering drugs or biomolecules, mainly including nanospheres, nanocapsules, nano micelles, nano liposomes, solid lipid nanoparticles, etc. Compared with traditional drug dosage forms, nano drug delivery systems have the following advantages: they can penetrate capillaries to avoid the rapid clearance of phagocytes and prolong their retention time in the blood; they can penetrate cells and tissue gaps to reach target organs such as the liver, spleen, lungs, spinal cord and lymph; the biodegradability, pH, ion and / or temperature sensitivity of the material itself determine the controlled release characteristics of the loaded drug, etc. At present, nano drug delivery systems have been widely used in the fields of small molecule drugs, peptides, proteins (vaccines), nucleic acids, etc., and have shown great application potential in the fields of biology, medicine and pharmaceuticals. Therefore, marine polysaccharides are ideal raw materials for preparing nanomaterials for drug (or bioactive substances) delivery. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of brown algae polysaccharide derivative nano-micelles, which have stronger amylin aggregation inhibition activity and improved anticoagulant ability; and can be used as a good carrier material with better drug loading capacity and encapsulation rate.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0006] A fucoidan derivative, which is a sulfonated fucoidan derivative, obtained by connecting a hydroxyl group in the fucoidan structure with a sulfonyl chloride in the structure of tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate through a chemical bond;

[0007] The above-mentioned brown algae polysaccharide is extracted from kelp. The brown algae polysaccharide involved in the present invention is extracted from natural kelp, and its raw material resources are abundant and naturally non-toxic; the brown algae polysaccharide derivatives chemically modified with 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester have better biological activity, significantly enhanced anticoagulant ability, improved amylin aggregation inhibition activity, and significantly improved anti-diabetic effects. The brown algae polysaccharide derivatives prepared by the present invention can be well applied to the preparation of nano-micelles, and while maintaining more excellent biological activity, can also be used as a carrier to encapsulate active substances, and have higher encapsulation rate and drug loading capacity.

[0008] Furthermore, the monosaccharide composition and molar ratio of brown algae polysaccharide are mannose: rhamnose: glucuronic acid: glucose: xylose: fucose = 1: 0.742: 0.907: 0.348: 1.325: 1.589.

[0009] Furthermore, the monosaccharide composition and molar ratio of brown algae polysaccharide are mannose: rhamnose: glucuronic acid: glucose: galactose: xylose: fucose = 1: 0.429: 0.304: 0.742: 1.143: 0.587: 0.947.

[0010] The invention also discloses a method for preparing the brown algae polysaccharide, which adopts a composite enzymatic hydrolysis method, wherein the composite enzyme comprises pectinase, cellulase and papain.

[0011] Specifically, the method for preparing the brown algae polysaccharide comprises:

[0012] Step 1: Material pretreatment:

[0013] Wash the kelp, crush and sieve it, dry it to constant weight, and then use Soxhlet extraction method to heat and reflux with acetone, petroleum ether and anhydrous ethanol as solvents to obtain kelp powder;

[0014] Step 2: Compound enzyme extraction:

[0015] Take kelp powder and phosphate buffer solution, add compound enzyme, mix well and perform enzymolysis, and inactivate the enzyme in boiling water to obtain enzymolysis solution;

[0016] Step 3: Concentrated alcohol precipitation:

[0017] The enzymatic hydrolysate obtained in step 2 is centrifuged and concentrated under reduced pressure, ethanol is added to a concentration of 20-30%, the precipitate is allowed to stand, centrifuged to remove the precipitate, and then ethanol is added to a final concentration of 60-70%, and the precipitate is freeze-dried to obtain crude brown algae polysaccharide;

[0018] Step 4, separation and purification: The crude polysaccharide of brown algae is subjected to enzymatic hydrolysis combined with Savage method deproteinization, DEAE-52 ion exchange column treatment, and Sephadex G-200 gel column chromatography separation and purification to obtain brown algae polysaccharide.

[0019] Furthermore, the solid-liquid ratio of kelp powder and phosphate buffer solution (pH 6.0-6.5) is 1:35-45.

[0020] Furthermore, the enzymatic hydrolysis conditions include: water bath temperature 45~55°C, and enzymatic hydrolysis time 4~6h.

[0021] Furthermore, the mass ratio of pectinase, cellulase and papain is 1:0.6~0.8:0.2~0.4.

[0022] Preferably, alfalfa lactone is also added during the composite enzymatic extraction process; the amount of alfalfa lactone added is 0.2-2wt%. In the present invention, alfalfa lactone is also added during the composite enzymatic extraction process of brown algae polysaccharide, and the yield of the obtained crude polysaccharide is significantly improved, and the protein content in the crude polysaccharide is significantly reduced. It is speculated that the presence of alfalfa lactone may have a beneficial effect on the enzyme cells, improve their enzyme activity, act on the kelp substrate, and prepare a single component of brown algae polysaccharide, whose monosaccharide composition and molar ratio are mannose: rhamnose: glucuronic acid: glucose: xylose: fucose = 1: 0.742: 0.907: 0.348: 1.325: 1.589, which has more excellent amylin aggregation inhibition activity, better therapeutic effect on diabetes; and its anticoagulant activity is also significantly improved.

[0023] The method for preparing the above-mentioned fucoidan derivative comprises: the fucoidan derivative is prepared by a sulfonylation reaction.

[0024] Furthermore, the preparation method of the above-mentioned brown algae polysaccharide derivative specifically comprises:

[0025] Take N,N-dimethylformamide, slowly add 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester under ice bath conditions, and stand for 30-40 minutes to obtain solution A; take the above-mentioned brown algae polysaccharide, add formamide, stir at 50-60°C for 20-40 minutes, then add the above-mentioned solution A dropwise, and react for 2-4 hours after the addition is complete; then pour out the reaction solution, add 70-80% ethanol to precipitate in the presence of 0.1-0.12% KCl, wash with 90-95% ethanol 2-4 times, dry and then dissolve in distilled water, add 2M sodium hydroxide solution to adjust the pH to 7-8, then dialyze, dialyze with tap water for 1-2 days, dialyze with distilled water for 1-2 days; finally, rotary evaporate and vacuum freeze-dry to obtain the brown algae polysaccharide derivative.

[0026] Furthermore, the solid-liquid ratio of tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate to N,N-dimethylformamide is 0.2~0.3 g:1 mL; the solid-liquid ratio of brown algae polysaccharide to formamide is 0.018~0.026 g:1 mL; and the volume ratio of solution A to formamide is 0.3~0.4:1.

[0027] A brown algae polysaccharide derivative nano-micelle, the raw materials of which include the above-mentioned brown algae polysaccharide derivative. The present invention uses inert organic reagent formamide as a solvent, and obtains nano-micelles by self-assembly of the prepared brown algae polysaccharide derivative through dialysis method, which is smooth spherical, evenly dispersed, and has uniform particle size; it can interact with lipid membranes and the like through the lipophilic part of the micelle, thereby effectively enhancing its biological activity; in vitro cell experiments have proved that the brown algae polysaccharide derivative nano-micelles prepared by the present invention have better anticoagulant effect, and have high application value in the field of preparing drugs related to the treatment of vascular diseases. In addition, the brown algae polysaccharide derivative nano-micelles prepared by the present invention can also be used as a carrier material to expand its application range; wherein, the brown algae polysaccharide is chemically modified with 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester to obtain its derivatives, and then the nano-micelles are prepared, which can significantly improve the drug loading capacity and encapsulation effect of the nano-micelles; at the same time, the brown algae polysaccharide extracted with alfalfa lactone is added during the composite enzymatic hydrolysis process, and the nano-micelles are prepared after derivatization, which can also improve the encapsulation effect of the nano-micelles to a certain extent.

[0028] The method for preparing the above-mentioned brown algae polysaccharide derivative nano micelles comprises:

[0029] The brown algae polysaccharide derivative prepared in Example 3 was dissolved in formamide to obtain a 1-1.5 mg / mL solution, and then dialyzed in water using a 500-1000 Da dialysis bag for 48-52 h to allow the brown algae polysaccharide derivative to obtain nano micelles through self-assembly under the action of the solvent, and finally freeze-dried to obtain the brown algae polysaccharide derivative nano micelles.

[0030] Another object of the present invention is to provide the use of the above-mentioned fucoidan derivatives in the preparation of nanomaterials for delivering drugs or bioactive substances.

[0031] Another object of the present invention is to provide the use of the above-mentioned fucoidan derivative nano-micelles in the preparation of anti-diabetic and anticoagulant drugs.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention adopts a composite enzymatic hydrolysis method to extract brown algae polysaccharide from kelp, and its crude polysaccharide yield is significantly improved, and the protein content is significantly reduced. After separation and purification, a single component of brown algae polysaccharide is obtained, which has more excellent amylin aggregation inhibition activity and anticoagulant activity. Then, 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester is used to chemically modify the brown algae polysaccharide derivative, and the anticoagulant ability and the activity of inhibiting amylin aggregation are significantly enhanced; nano micelles are obtained by self-assembly through dialysis, which effectively enhances its biological activity; at the same time, it can also be used as a carrier to encapsulate active substances, and has a higher encapsulation rate and drug loading capacity. Among them, the brown algae polysaccharide extracted by adding alfalfa lactone in the composite enzymatic hydrolysis process is used, and the nano micelles are prepared after derivatization, which also improves the encapsulation effect of the nano micelles to a certain extent.

[0034] Therefore, the present invention provides a preparation method and application of brown algae polysaccharide derivative nano-micelles, wherein the brown algae polysaccharide derivative nano-micelles have stronger amylin aggregation inhibition activity and improved anticoagulant ability; and can be used as a good carrier material with more excellent drug loading capacity and encapsulation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the high performance liquid chromatography test result of brown algae polysaccharide in Example 1 of the present invention;

[0036] Figure 2 The HPLC test result of the mixed monosaccharide standard sample solution in Example 1 of the present invention;

[0037] Figure 3 The infrared spectrum test results of Experimental Example 1 of the present invention are as follows;

[0038] Figure 4 This is the SEM test result in Experimental Example 1 of the present invention.

[0039] Reference numerals:

[0040] a-PMP, b-mannose, c-rhamnose, d-glucuronic acid, e-glucose, f-galactose, g-xylose, h-fucose, i-galacturonic acid. DETAILED DESCRIPTION

[0041] The technical solution of the present invention is further described in detail below in conjunction with specific implementations and drawings:

[0042] Embodiment 1:

[0043] A preparation method of brown algae polysaccharide:

[0044] Step 1: Material pretreatment:

[0045] Wash the kelp, crush it, pass it through a 60-mesh sieve, and dry it at 70°C to constant weight; then use the Soxhlet extraction method, use acetone, petroleum ether, and anhydrous ethanol as solvents in sequence, heat and reflux for 2 hours to remove lipids and pigments that are easily soluble in organic solvents, and obtain kelp powder;

[0046] Step 2: Compound enzyme extraction:

[0047] Take kelp powder and phosphate buffer solution (pH 6.0) at a solid-liquid ratio of 1:40, add complex enzyme (the mass ratio of pectinase, cellulase and papain is 1:0.72:0.31) and mix well, then add 0.9wt% of alfalfa lactone, hydrolyze for 5.5h in a water bath at 52℃, then inactivate the enzyme in a boiling water bath for 10min to obtain the enzymatic solution;

[0048] Step 3: Concentrated alcohol precipitation:

[0049] The enzymatic hydrolyzate obtained in step 2 is centrifuged, the supernatant is concentrated under reduced pressure, ethanol is added to a concentration of 24%, the precipitate is allowed to stand, centrifuged to remove the precipitate, and then ethanol is added to a final concentration of 66%, and the precipitate is freeze-dried to obtain crude brown algae polysaccharide;

[0050] Step 4: separation and purification:

[0051] Enzymatic hydrolysis combined with Savage method for protein removal: take crude brown algae polysaccharide and add distilled water to a concentration of 0.025 g / mL, add papain at a mass volume ratio of 3.1%, mix well and perform constant temperature enzymolysis for 1 hour in a water bath at 37°C; then add 1 / 5 volume of Savage reagent and slowly stir to react for 1 hour; then centrifuge, the middle layer of the solution is flocculent denatured protein, repeat the above operation until no flocculent protein precipitates; reduce pressure concentration and freeze-dry to obtain the deproteinized crude brown algae polysaccharide;

[0052] DEAE-52 ion exchange column treatment, take the deproteinized brown algae crude polysaccharide and dissolve it in deionized water to obtain a solution with a concentration of 0.05g / mL, pass it through a 0.45μm membrane to remove impurities and then load it onto a column (DEAE-52 ion exchange column specification 2.6×60cm), flow rate 1.2mL / min, and use 0, 0.25, 0.5, 1, 2M NaCl concentrations for elution in sequence, and use phenol-sulfuric acid method for tracking detection, each gradient elution until no elution peak appears, collect the components and merge the main peak (large peak with good repeatability) according to the color reaction results, dialyze, concentrate, freeze-dry, and proceed to the next step;

[0053] Sephadex G-200 gel column chromatography, take the sample treated with DEAE-52 ion exchange column, dissolve it in deionized water, the concentration is 12 mg / mL, pass it through a 0.45μm filter membrane to remove impurities, then load the sample (Sephadex G-200 gel column specification is 2.6×60cm), elute it with deionized water, the flow rate is 0.6mL / min, and the phenol-sulfuric acid method is used for tracking detection. The main peak parts are combined for dialyzation, concentration, and freeze-drying to obtain brown algae polysaccharide.

[0054] Analysis of monosaccharide composition of brown algae polysaccharide:

[0055] HPLC method was used for analysis. Sample treatment: brown algae polysaccharide was added to 2M sulfuric acid in a solid-liquid ratio of 1mg:1mL, and hydrolyzed under vacuum and 100℃ for 9h. Then, the pH was adjusted to neutral with 0.3M sodium hydroxide solution. After centrifugation, the supernatant was taken to obtain the complete hydrolyzate of polysaccharide. PMP derivatization, monosaccharide and mixed monosaccharide standard solutions required for the experiment were prepared, 200μL was placed in a 2.0mL EP tube, 100μL of 0.5M PMP methanol solution and 100μL of 0.3M sodium hydroxide solution were added in sequence, mixed, reacted in a 70℃ water bath for 60min, taken out, cooled to room temperature, 100μL of 0.3M hydrochloric acid was added, 1mL of chloroform was added for extraction, and the chloroform phase was removed by centrifugation. Repeat 4 times, and then the aqueous phase was filtered through a 0.22μm filter for HPLC analysis. The chromatographic conditions included: chromatographic column Agilent HC-C18 (4.6×250cm, 5μm); injection volume 10μL; mobile phase 0.05M phosphate buffer solution (pH 6.5) + acetonitrile (v / v, 4:1); flow rate 1.2mL / min; column temperature 30℃; detection wavelength 245nm.

[0056] Results showed that the HPLC analysis of brown algae polysaccharide and its mixed monosaccharide standard samples was as follows: Figure 1-2 The brown algae polysaccharide prepared in this embodiment was analyzed by high performance liquid chromatography, and 6 peaks were separated. By comparing with the high performance liquid chromatography of standard monosaccharides, it can be obtained that the monosaccharide composition and molar ratio of brown algae polysaccharide are mannose: rhamnose: glucuronic acid: glucose: xylose: fucose = 1: 0.742: 0.907: 0.348: 1.325: 1.589.

[0057] Determination of molecular weight of brown algae polysaccharide:

[0058] The test was carried out using the HPGPC method, and the molecular weight of the polygonatum sibiricum polysaccharide prepared in this example was calculated to be 8.12 kDa by comparing with the regression curve of the molecular weight of the polysaccharide standard sample.

[0059] Embodiment 2:

[0060] The difference between the preparation of brown algae polysaccharide and Example 1 is that no alfalfa lactone is added during the composite enzymatic extraction process.

[0061] The relative molecular weight was determined in the same manner as in Example 1, and the relative molecular weight of the obtained polygonatum polysaccharide was 9.01 kDa. The monosaccharide composition analysis was the same as in Example 1, and the results showed that the monosaccharide composition and molar ratio of the extracted brown algae polysaccharide were mannose: rhamnose: glucuronic acid: glucose: galactose: xylose: fucose = 1: 0.429: 0.304: 0.742: 1.143: 0.587: 0.947.

[0062] Embodiment 3:

[0063] Preparation of a brown algae polysaccharide derivative:

[0064] Take N,N-dimethylformamide, slowly add tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate (solid-to-liquid ratio with N,N-dimethylformamide is 0.26g:1mL) under ice bath condition, and stand for 36 minutes to obtain solution A; take the brown algae polysaccharide prepared in Example 1 (solid-to-liquid ratio with formamide is 0.21g:1mL), add formamide, stir at 56°C for 35 minutes, and then dropwise add the above solution A (volume ratio with formamide is 0.34:1), and react for 3.5 hours after the dropwise addition is completed; then pour out the reaction solution, add 80% ethanol to precipitate in the presence of 0.12% KCl, wash with 95% ethanol 4 times, dry and then dissolve in distilled water, add 2M sodium hydroxide solution to adjust the pH to 7.4, then dialyze, dialyze with tap water for 2d and with distilled water for 1d; finally, rotary evaporate and vacuum freeze-dry to obtain the brown algae polysaccharide derivative.

[0065] Embodiment 4:

[0066] The difference between the preparation of a brown algae polysaccharide derivative and Example 3 is that the brown algae polysaccharide used is prepared in Example 2.

[0067] Embodiment 5:

[0068] Preparation of a kind of brown algae polysaccharide derivative nano micelles:

[0069] The brown algae polysaccharide derivative prepared in Example 3 was dissolved in formamide to obtain a 1.3 mg / mL solution, and then dialyzed in water for 48 h using a 500-1000 Da dialysis bag to allow the brown algae polysaccharide derivative to obtain nano micelles through self-assembly under the action of the solvent, and finally freeze-dried to obtain the brown algae polysaccharide derivative nano micelles.

[0070] Embodiment 6:

[0071] The difference between the preparation of a brown algae polysaccharide derivative nano-micelle and Example 5 is that the brown algae polysaccharide derivative used is prepared in Example 4.

[0072] Embodiment 7:

[0073] The difference between the preparation of the brown algae polysaccharide derivative and that of Example 3 is that chlorosulfonic acid is used instead of tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate.

[0074] The difference between the preparation of a brown algae polysaccharide nano-derivative micelle and Example 5 is that the brown algae polysaccharide derivative is prepared in this example.

[0075] Embodiment 8:

[0076] The difference between the preparation of the brown algae polysaccharide derivative and Example 4 is that chlorosulfonic acid is used instead of tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate.

[0077] The difference between the preparation of a brown algae polysaccharide nano-derivative micelle and Example 5 is that the brown algae polysaccharide derivative is prepared in this example.

[0078] Test Example 1:

[0079] Infrared spectroscopy characterization

[0080] The sample was fully ground with KBr under the irradiation of infrared lamp, pressed into tablets and then analyzed by NICOLET 380 infrared spectrometer. Test parameters: wavelength range 4000~500cm -1 .

[0081] The above test was performed on the fucoidan prepared in Example 1 and the fucoidan derivative prepared in Example 3. The results are as follows: Figure 3 As shown in the figure, it can be seen that in the infrared spectrum of the brown algae polysaccharide prepared in Example 1, the -1 The stretching vibration peak of OH appears in the range of 3000~2800cm -1 The characteristic absorption peaks of methyl and methylene appear in the range of 1610cm -1 The characteristic absorption peak of C=O in -CHO appears near 1250cm -1 The characteristic absorption peak of S=O appears near 1250~1000cm -1 There are characteristic absorption peaks of COH and COC in the range. Compared with the infrared test results of the brown algae polysaccharide prepared in Example 1, the infrared spectrum of the brown algae polysaccharide derivative prepared in Example 3 shows a peak at 1682 cm -1 The characteristic absorption peak of C=O in the ester group appears near 1600~1500cm -1The benzene ring skeleton vibration absorption peak appears in the range of 1319cm -1 The characteristic absorption peak of CN bond appears near 1250cm -1 The absorption intensity of the characteristic peak of S=O nearby is enhanced; the above results indicate that the brown algae polysaccharide derivative in Example 3 was successfully prepared.

[0082] SEM characterization

[0083] Scanning electron microscopy (SEM) was used to analyze the particle size and morphology of the nanomicelle samples.

[0084] The above test was performed on the nano micelles prepared in Example 5, and the results are as follows: Figure 4 As shown in the figure, it can be seen that the nano micelles of the brown algae polysaccharide derivative prepared in Example 5 of the present invention are spherical in appearance, evenly dispersed, and uniform in shape, proving that the micelles were successfully prepared.

[0085] Crude polysaccharide yield and protein content determination

[0086] Brown algae crude polysaccharide yield % = Brown algae crude polysaccharide mass / kelp powder mass × 100%

[0087] Protein content determination:

[0088] Weigh bovine serum albumin and dissolve it in distilled water to prepare a 0.1 mg / mL bovine serum albumin standard solution. Prepare the protein standard curve according to the conventional operation, and the linear regression equation is y=2.47x+0.0207, r 2 =0.992. Prepare a crude polysaccharide sample solution of appropriate concentration, take 1.0 mL and operate according to the standard curve determination method, and calculate the protein content according to the protein standard curve.

[0089] The above test was performed on the crude brown algae polysaccharides prepared in Example 1 and Example 2, and the results are shown in Table 1:

[0090] Table 1 Test results of crude polysaccharide yield and protein content

[0091] sample Crude polysaccharide yield (%) Protein content / % Example 1 5.76 1.84 Example 2 4.47 2.52

[0092] From the data analysis in Table 1, it can be seen that the yield of crude brown algae polysaccharides prepared by the method provided in Example 1 is significantly higher than that in Example 2, and its protein content is lower than that in Example 2, indicating that the addition of alfalfa lactone in the composite enzymatic hydrolysis process is speculated to have a beneficial effect on the enzyme, thereby promoting the enzymatic hydrolysis process, so that the yield of crude brown algae polysaccharides is significantly improved while effectively reducing the protein content.

[0093] Test Example 2:

[0094] Antidiabetic activity assay

[0095] Test method: Thioflavin fluorescence method, using Th-T to specifically label amylin fibers. The specific operation process includes: taking amylin (5μg / mL) and the sample to be tested (90μg / mL) and incubating them together at 37°C, and after 24 hours, using amylin incubated alone as a control to detect the Th-T fluorescence intensity, that is, taking 180μL of Th-T solution and adding it to a 96-well plate, and then adding 20μL of the sample to be tested; setting up a blank control group and adding an equal amount of PBS (pH 7.4); then placing it on a multifunctional microplate reader to read the value (excitation wavelength 450nm, emission wavelength 480nm), and subtracting the fluorescence value of the blank control group from the measured fluorescence value.

[0096] The above test was performed on the fucoidan prepared in Example 1-2 and the fucoidan derivative prepared in Example 3-4. The results are shown in Table 2:

[0097] Table 2 Test results of inhibitory activity of amylin aggregation

[0098] sample Fluorescence reduction percentage (%) Example 1 81.2 Example 2 62.7 Example 3 92.5 Example 4 71.6

[0099] From the data in Table 2, it can be concluded that the inhibition percentage of amylin aggregation by the fucoidan prepared in Example 1 of the present invention is significantly higher than that in Example 2, indicating that the fucoidan obtained by adding alfalfa lactone extraction during the composite enzymatic hydrolysis process has a more excellent anti-diabetic activity. The effect of Example 3 is better than that of Example 1, and the effect of Example 4 is better than that of Example 2, indicating that chemical modification of fucoidan with 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester to obtain its derivatives can significantly enhance the anti-diabetic activity of the derivatives.

[0100] Anticoagulant activity assay

[0101] Take 5 mL of fresh rabbit blood and add 0.5 mL of the sample to be tested and sodium citrate solution respectively, and record the coagulation time of rabbit blood. In order to compare the anticoagulant activity of the sample to be tested and sodium citrate, the test is based on the sulfate equivalent (the amount of carboxyl groups in sodium citrate is converted into sulfate groups with equal charge, i.e. 1 mol / L COO - =0.5 mol / L SO4 2- ) as the solute concentration. The solute concentration in this test is 0.5 mg / mL. Recalcify the blood by adding CaCl2 solution to the beaker containing the sample solution to be tested without coagulation. Observe and record the coagulation time.

[0102] The above test was performed on the fucoidan prepared in Example 1-2, the fucoidan derivative prepared in Example 3-4, and the fucoidan derivative nano-micelles prepared in Example 5-8. The results are shown in Table 3:

[0103] Table 3 Anticoagulant activity test results

[0104] sample Coagulation time (min) Sodium Citrate — Example 1 25 Example 2 14 Example 3 39 Example 4 28 Example 5 50 Example 6 37 Example 7 36 Example 8 24

[0105] Note: “—” means that after blood was added, the coagulation time was too short to be measured under the experimental conditions.

[0106] From the data in Table 3, it can be concluded that the clotting time of the brown algae polysaccharide prepared in Example 1 of the present invention is significantly higher than that of Example 1, indicating that the brown algae polysaccharide obtained by adding alfalfa lactone extraction during the composite enzymatic hydrolysis process has a better anticoagulant ability. The effect of Example 3 is better than that of Example 1, and the effect of Example 4 is better than that of Example 2, indicating that the use of 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester to chemically modify brown algae polysaccharide to obtain its derivatives can significantly enhance the anticoagulant activity of the derivatives. The clotting time of the brown algae polysaccharide derivative nano-micelles prepared in Example 5 of the present invention is significantly higher than that of Example 3 and Example 7, and the effect of Example 6 is better than that of Example 4 and Example 8, indicating that the preparation of brown algae polysaccharide derivatives into nano-micelles can further enhance the biological activity of the nano-micelles, and the coagulation activity is significantly enhanced; and the anticoagulant activity of the derivatives obtained by chemically modifying brown algae polysaccharide using 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester is better than that of the brown algae polysaccharide derivatives modified with chlorosulfonic acid.

[0107] Test Example 3:

[0108] Determination of drug loading and encapsulation efficiency of nanomicelles

[0109] Drug loading determination: Take the nanomicelle sample and dissolve it in DMSO to prepare a solution with a concentration of 1 mg / mL. Take 5 mL and add 0.01, 0.1, 0.5, 1.0, 2.0, and 4.0 mL of 0.2 mg / mL β-carotene solution (solvent is DMSO) respectively. Mix well and dialyze in a large amount of water for 3 days, changing the water every 6 hours. Then make the solution volume uniform to 25 mL, filter with a 0.45 μm microporous membrane to remove the unencapsulated β-carotene, and measure the ultraviolet absorbance of the solution (A 278 ), and the content was calculated based on the standard curve of β-carotene.

[0110] Calculation of drug loading and encapsulation efficiency:

[0111] Take two 5 mL DMSO and add 0.1 mg / mL and 0.2 mg / mL DMSO solutions containing β-carotene as control groups, ultrasonicate for 30 min, and measure the UV absorbance at 278 nm. Calculate the drug loading and encapsulation efficiency according to the following formula:

[0112] Drug loading = drug amount contained in micelles / total amount of micelles added × 100%

[0113] Encapsulation efficiency = amount of drug contained in micelles / total amount of drug added × 100%

[0114] The above test was performed on the fucoidan derivative nano-micelles prepared in Examples 5-6 and the fucoidan nano-micelles prepared in Examples 7-8. The results are shown in Table 4:

[0115] Table 4 Drug loading and encapsulation efficiency test results

[0116] sample Drug loading (%) Encapsulation rate (%) Example 5 16.3 55.1 Example 6 14.4 43.0 Example 7 7.7 42.8 Example 8 7.2 34.6

[0117] From the data in Table 4, it can be concluded that the drug loading and encapsulation efficiency of the brown algae polysaccharide derivative nano-micelles prepared in Example 5 of the present invention are significantly higher than those in Example 7, and the effect of Example 6 is better than that of Example 8, indicating that the use of 2-(chlorosulfonyl)-1H-indole-1-carboxylic acid tert-butyl ester to chemically modify brown algae polysaccharide to obtain its derivatives and then prepare nano-micelles can significantly improve the drug loading capacity and encapsulation effect of nano-micelles. In addition, the effect of Example 5 is better than that of Example 6, and the effect of Example 7 is better than that of Example 8, indicating that adding brown algae polysaccharide extracted from alfalfa lactone during the composite enzymatic hydrolysis process and preparing nano-micelles after derivatization can effectively improve the encapsulation effect of nano-micelles.

[0118] The conventional techniques in the above embodiments are prior arts known to those skilled in the art, and thus will not be described in detail here.

[0119] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A fucoidan derivative, which is a sulfonated fucoidan derivative, obtained by chemically connecting the hydroxyl group in the fucoidan structure with the sulfonyl chloride in the structure of tert-butyl 2-(chlorosulfonyl)-1H-indole-1-carboxylate; The brown algae polysaccharide is extracted from kelp; the monosaccharide composition and molar ratio of the brown algae polysaccharide are mannose: rhamnose: glucuronic acid: glucose: xylose: fucose = 1:0.742:0.907:0.348:1.325:1.

589.

2. The brown algae polysaccharide derivative according to claim 1, characterized in that: The preparation method of brown algae polysaccharide adopts a composite enzymatic hydrolysis method, and the composite enzyme comprises pectinase, cellulase and papain.

3. The brown algae polysaccharide derivative according to claim 2, characterized in that: In the process of preparing brown algae polysaccharide by the composite enzymatic hydrolysis method, alfalfa lactone is also added.

4. The preparation method according to claim 2, characterized in that: The mass ratio of the pectinase, the cellulase and the papain is 1:0.6-0.8:0.2-0.

4.

5. The method for preparing the brown algae polysaccharide derivative according to claim 1, comprising: The fucoidan derivative is prepared by sulfonylation reaction.

6. A fucoidan derivative nano-micelle, wherein the raw material of the nano-micelle comprises the fucoidan derivative according to any one of claims 1 to 5.

7. Use of the fucoidan derivative according to claim 1 in preparing nanomaterials for delivering drugs or bioactive substances.

8. Use of the brown algae polysaccharide derivative nano-micelles according to claim 6 in the preparation of anti-diabetic and anticoagulant drugs.

Images