CN117024616A - Preparation and application of brown algae polysaccharide derivative nano micelle - Google Patents
Preparation and application of brown algae polysaccharide derivative nano micelle Download PDFInfo
- Publication number
- CN117024616A CN117024616A CN202310834747.6A CN202310834747A CN117024616A CN 117024616 A CN117024616 A CN 117024616A CN 202310834747 A CN202310834747 A CN 202310834747A CN 117024616 A CN117024616 A CN 117024616A
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- CN
- China
- Prior art keywords
- brown algae
- fucoidan
- algae polysaccharide
- derivative
- polysaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses a preparation method and application of a brown algae polysaccharide derivative nano micelle, and relates to the technical field of marine biological medicines. The brown algae polysaccharide derivative nano micelle is prepared from brown algae polysaccharide derivatives; the brown algae polysaccharide derivative is a brown algae polysaccharide sulfonylation derivative, and is obtained by connecting hydroxyl in a brown algae polysaccharide structure with sulfonyl chloride in a 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester structure through a chemical bond; the brown algae polysaccharide is extracted from herba Zosterae Marinae. The brown algae polysaccharide derivative nano micelle prepared by the invention has stronger amylin aggregation inhibition activity, and the anticoagulation capability is also improved; and can be used as a good carrier material, and has more excellent drug carrying capacity and encapsulation efficiency.
Description
Technical Field
The invention belongs to the technical field of marine biological medicines, and particularly relates to preparation and application of a brown algae polysaccharide derivative nano micelle.
Background
The polysaccharides in marine organisms have antiviral, antitumor, radioprotective, antimutagenic, antioxidant and immunity enhancing effects. Factors influencing polysaccharide activity include the backbone properties, the branching properties, and the higher order structure of the polysaccharide molecule. In order to improve the activity of the polysaccharide, the molecular modification and structural transformation of the polysaccharide are of great significance. Currently, hydroxyl, carboxyl, amino and other groups on sugar residues are mainly used for modification by a chemical method, and the modification includes sulfation, carboxymethylation, selenization, methylation, phosphorylation, double-group derivatization modification and the like. The brown algae polysaccharide can be extracted from a plurality of marine organisms, has wide sources and large storage capacity, and has various biological activities such as anti-tumor, antiviral, anticoagulation and the like proved by researchers in recent years. However, some of the bioactive effects are not ideal, for example, the antiviral effects are not ideal, and the effect is only exerted during the stage of adsorption of avian leukosis virus to host cells, and the antiviral effects are not exerted during the virus propagation stage. Therefore, the improvement of the biological activity of the recombinant strain has great research significance and application value.
In the field of nanotechnology, nanoparticles refer to particles with a particle size of 1-100 nm; the nanoparticles (nano-drug or nano-drug delivery system) in the pharmacy generally refer to nano-carriers with the particle size of 10-1000 nm for delivering drugs or biomolecules, and mainly comprise nanospheres, nanocapsules, nano-micelles, nano-liposomes, solid lipid nanoparticles and the like. Compared with the traditional drug dosage form, the nano drug delivery system has the following advantages: the capillary vessel can be penetrated, so that the rapid clearance of phagocytes is avoided, and the residence time of the phagocytes in blood is prolonged; can penetrate through the cell and tissue gap to reach target organs such as liver, spleen, lung, spinal cord, lymph and the like; the biodegradability, pH, ionic and/or temperature sensitivity of the material itself determine the controlled release characteristics of the loaded drug, etc. At present, the nano drug delivery system is widely applied to the fields of small molecule drugs, polypeptides, proteins (vaccines), nucleic acids and the like, and shows great application potential in the fields of biology, medicine, pharmacy and the like. Thus, marine polysaccharides are ideal raw materials for preparing drug (or bioactive substance) delivery nanomaterials.
Disclosure of Invention
The invention aims to provide a preparation method and application of brown algae polysaccharide derivative nano-micelle, wherein the brown algae polysaccharide derivative nano-micelle has stronger amylin aggregation inhibition activity and improved anticoagulation capability; and can be used as a good carrier material, and has more excellent drug carrying capacity and encapsulation efficiency.
The technical scheme adopted by the invention for achieving the purpose is as follows:
a fucoidan derivative is a fucoidan sulfonylation derivative, which is prepared by connecting hydroxyl in fucoidan structure with sulfonyl chloride in 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester structure through chemical bond;
the brown algae polysaccharide is extracted from herba Zosterae Marinae. The brown algae polysaccharide is extracted from natural kelp, and the brown algae polysaccharide has rich raw material resources and is natural and nontoxic; the brown algae polysaccharide derivative is chemically modified by adopting the 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester, so that the brown algae polysaccharide derivative has better biological activity, the anticoagulation capability is obviously enhanced, the amylin aggregation inhibition activity is improved, and the anti-diabetes effect is obviously improved. The brown algae polysaccharide derivative prepared by the invention can be well applied to the preparation of nano-micelles, can be used as a carrier for encapsulating active substances while maintaining more excellent bioactivity, and has higher encapsulation efficiency and drug carrying capacity.
Further, the brown algae polysaccharide comprises mannose in the following monosaccharide composition and molar ratio: rhamnose: glucuronic acid: glucose: xylose: fucose=1:0.742:0.907:0.348: 1.325:1.589.
Further, the brown algae polysaccharide comprises mannose in the following monosaccharide composition and molar ratio: rhamnose: glucuronic acid: glucose: galactose: xylose: fucose=1:0.429:0.304:0.742:1.143:0.587:0.947.
The invention also discloses a preparation method of the brown algae polysaccharide, which adopts a complex enzymolysis method, wherein the complex enzyme comprises pectase, cellulase and papain.
Specifically, the preparation method of the brown algae polysaccharide comprises the following steps:
step one, material pretreatment:
cleaning herba Zosterae Marinae, pulverizing, sieving, drying to constant weight, and sequentially heating and refluxing with acetone, petroleum ether, and anhydrous ethanol as solvents to obtain herba Zosterae Marinae powder;
step two, extracting by a complex enzyme method:
adding compound enzyme into kelp powder and phosphate buffer solution, uniformly mixing and carrying out enzymolysis, and inactivating enzyme in boiling water bath to obtain enzymolysis liquid;
step three, concentrating and precipitating with alcohol:
centrifuging the enzymolysis liquid obtained in the second step, concentrating under reduced pressure, adding ethanol until the concentration is 20-30%, standing for precipitation, centrifuging to remove the precipitate, adding ethanol until the final concentration is 60-70%, and freeze-drying the precipitate to obtain brown algae crude polysaccharide;
step four, separating and purifying: and (3) taking brown algae crude polysaccharide, carrying out enzymolysis, deproteinization by a Savage method, treatment by a DEAE-52 ion exchange column, and separation and purification by a Sephadex G-200 gel column chromatography to obtain brown algae polysaccharide.
Further, the feed liquid ratio of the kelp powder to the phosphate buffer solution (pH is 6.0-6.5) is 1:35-45.
Further, the enzymolysis conditions include: the water bath temperature is 45-55 ℃, and the enzymolysis time is 4-6 hours.
Further, the mass ratio of pectase, cellulase and papain is 1:0.6 to 0.8:0.2 to 0.4.
Preferably, alfalfa lactone is also added in the extraction process of the composite enzymolysis method; the addition amount of the alfalfa lactone is 0.2-2wt%. In the invention, alfalfa lactone is also added in the process of extracting the brown algae polysaccharide by a composite enzymolysis method, the yield of the obtained crude polysaccharide is obviously improved, the protein content in the crude polysaccharide is obviously reduced, the existence of the alfalfa lactone possibly has beneficial effects on enzyme cells, the enzyme activity of the alfalfa lactone is improved, the kelp substrate is acted, and the single-component brown algae polysaccharide is prepared by the single-component brown algae polysaccharide, wherein the monosaccharide composition and the molar ratio are mannose: rhamnose: glucuronic acid: glucose: xylose: fucose=1:0.742:0.907:0.348: 1.325:1.589, has more excellent amylin aggregation inhibition activity and better treatment effect on diabetes; and the anticoagulant activity of the composition is also obviously improved.
The preparation method of the brown algae polysaccharide derivative comprises the following steps: the brown algae polysaccharide derivative is prepared through a sulfonylation reaction.
Further, the preparation method of the brown algae polysaccharide derivative specifically comprises the following steps:
slowly adding 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester into N, N-dimethylformamide under ice bath condition, and standing for 30-40 min to obtain a solution A; taking the brown algae polysaccharide, adding formamide, stirring for 20-40 min at 50-60 ℃, then dripping the solution A, and reacting for 2-4 h after dripping; pouring out the reaction liquid, adding 70-80% ethanol for precipitation in the presence of 0.1-0.12% KCl, washing 2-4 times with 90-95% ethanol, drying, dissolving in distilled water, adding 2M concentration sodium hydroxide solution for regulating pH to 7-8, dialyzing with tap water for 1-2 d, and dialyzing with distilled water for 1-2 d; finally, rotary evaporation and vacuum freeze-drying are carried out to obtain the brown algae polysaccharide derivative.
Further, the solid-to-liquid ratio of the tert-butyl 2- (chlorosulfonyl) -1H-indole-1-carboxylate to the N, N-dimethylformamide is 0.2-0.3 g:1mL; the solid-to-liquid ratio of the brown seaweed polysaccharide to the formamide is 0.018-0.026 g:1mL; the volume ratio of the solution A to the formamide is 0.3-0.4:1.
A brown algae polysaccharide derivative nano micelle comprises the brown algae polysaccharide derivative. The invention takes inert organic reagent formamide as a solvent, and the prepared brown algae polysaccharide derivative is subjected to self-assembly by a dialysis method to obtain nano-micelle which is smooth spherical, uniformly dispersed and uniform in particle size; can generate interaction with lipid membrane through micelle lipophilic part, etc., thereby effectively enhancing the bioactivity; in vitro cell experiments prove that the brown algae polysaccharide derivative nano micelle prepared by the invention has better anticoagulation effect and has high application value in the field of medicaments related to vascular diseases. In addition, the brown algae polysaccharide derivative nano micelle prepared by the invention can also be used as a carrier material, so that the application range of the brown algae polysaccharide derivative nano micelle is widened; wherein, 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester is adopted to chemically modify brown seaweed polysaccharide to obtain derivatives thereof, and then nano micelle is prepared, so that the drug carrying capacity and encapsulation effect of the nano micelle can be obviously improved; meanwhile, brown algae polysaccharide extracted from alfalfa lactone is added in the composite enzymolysis process, and the nano micelle is prepared after derivatization, so that the encapsulation effect of the nano micelle can be improved to a certain extent.
The preparation method of the brown algae polysaccharide derivative nano micelle comprises the following steps:
dissolving the brown alga polysaccharide derivative prepared in the embodiment 3 in formamide to obtain 1-1.5 mg/mL solution, dialyzing the solution in water for 48-52 h by using a 500-1000Da dialysis bag to enable the brown alga polysaccharide derivative to obtain nano-micelle through self-assembly under the action of a solvent, and finally freeze-drying to obtain the brown alga polysaccharide derivative nano-micelle.
Still another object of the present invention is to provide the use of the fucoidan derivative described above for preparing a drug or bioactive substance delivery nanomaterial.
The invention also aims at providing the application of the brown algae polysaccharide derivative nano micelle in preparing antidiabetic and anticoagulation medicines.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the compound enzymolysis method to extract fucoidin from kelp, the yield of crude polysaccharide is obviously improved, the protein content is obviously reduced, and single-component fucoidin is obtained after separation and purification, and the fucoidin has more excellent amylin aggregation inhibition activity and anticoagulation activity. Then adopting 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester to chemically modify brown algae polysaccharide derivative, and obviously enhancing anticoagulant capability and amylin aggregation inhibition activity; the nano micelle is obtained through the self-assembly effect of a dialysis method, so that the biological activity of the nano micelle is effectively enhanced; meanwhile, the active substance can be used as a carrier for encapsulating active substances, and has higher encapsulation efficiency and drug carrying capacity. Wherein, brown algae polysaccharide extracted from alfalfa lactone is added in the composite enzymolysis process, and the nano micelle is prepared after derivatization, so that the encapsulation effect of the nano micelle is improved to a certain extent.
Therefore, the invention provides the preparation and the application of the brown algae polysaccharide derivative nano micelle, and the brown algae polysaccharide derivative nano micelle has stronger amylin aggregation inhibition activity and improves the anticoagulation capability; and can be used as a good carrier material, and has more excellent drug carrying capacity and encapsulation efficiency.
Drawings
FIG. 1 shows the result of HPLC test of fucoidan in example 1 of the present invention;
FIG. 2 shows the results of a HPLC test of a standard sample solution of mixed monosaccharides in example 1 of the present invention;
FIG. 3 shows the results of the IR spectrum test in test example 1 according to the invention;
FIG. 4 shows SEM test results of test example 1 according to the present invention.
Reference numerals:
a-PMP, b-mannose, c-rhamnose, d-glucuronic acid, e-glucose, f-galactose, g-xylose, h-fucose, i-galacturonic acid.
Detailed Description
The technical scheme of the invention is further described in detail below with reference to the specific embodiments and the attached drawings:
example 1:
a preparation method of brown algae polysaccharide comprises the following steps:
step one, material pretreatment:
cleaning herba Zosterae Marinae, pulverizing, sieving with 60 mesh sieve, and drying at 70deg.C to constant weight; then adopting a Soxhlet extraction method, sequentially using acetone, petroleum ether and absolute ethyl alcohol as solvents, heating and refluxing for 2 hours, and removing lipid and pigment which is easy to dissolve in organic solvents to obtain kelp powder;
step two, extracting by a complex enzyme method:
taking kelp powder and phosphate buffer solution (pH is 6.0) according to the ratio of 1:40, adding compound enzyme (pectase, cellulase and papain with the mass ratio of 1:0.72:0.31), uniformly mixing, adding 0.9wt% of alfalfa lactone, carrying out enzymolysis for 5.5 hours under the water bath condition of 52 ℃, and then carrying out boiling water bath enzyme deactivation for 10 minutes to obtain enzymolysis liquid;
step three, concentrating and precipitating with alcohol:
centrifuging the enzymolysis liquid obtained in the second step, concentrating the supernatant under reduced pressure, adding ethanol until the concentration is 24%, standing for precipitation, centrifuging to remove the precipitate, then adding ethanol until the final concentration is 66%, and freeze-drying the precipitate to obtain brown algae crude polysaccharide;
step four, separating and purifying:
the enzymolysis is combined with the Savage method for deproteinization, the brown algae crude polysaccharide is taken, distilled water with the concentration of 0.025g/mL is added, papain is added according to the mass volume ratio of 3.1%, and after being uniformly mixed, the mixture is subjected to constant temperature enzymolysis for 1h under the water bath condition of 37 ℃; then adding 1/5 volume of Savage reagent, and slowly stirring to react for 1h; centrifuging, wherein the middle layer of the solution is flocculent denatured protein, and repeating the operation until no flocculent protein is precipitated; concentrating under reduced pressure, and lyophilizing to obtain deproteinized brown algae crude polysaccharide;
treating with DEAE-52 ion exchange column, dissolving deproteinized brown algae crude polysaccharide in deionized water to obtain 0.05g/mL solution, purifying with 0.45 μm membrane, loading into column (DEAE-52 ion exchange column with specification of 2.6X10 cm) with flow rate of 1.2mL/min, eluting with NaCl of 0, 0.25, 0.5,1, 2M concentration, detecting by phenol-sulfuric acid method, eluting with each gradient until no eluting peak appears, collecting components, mixing main peak (with large outlet peak and good repeatability) according to color reaction result, dialyzing, concentrating, lyophilizing, and performing next step;
and (3) carrying out Sephadex G-200 gel column chromatography, dissolving a sample treated by a DEAE-52 ion exchange column with deionized water at a concentration of 12mg/mL, filtering with a 0.45 mu m filter membrane to remove impurities, loading the sample (the specification of the Sephadex G-200 gel column is 2.6X60 cm), eluting with deionized water at a flow rate of 0.6mL/min, adopting a phenol-sulfuric acid method for tracking detection, combining main peak parts for dialysis, concentrating and freeze-drying to obtain brown algae polysaccharide.
Monosaccharide composition analysis of brown algae polysaccharide:
analysis was performed by HPLC. Sample treatment: the solid-to-liquid ratio is 1mg:1mL of brown algae polysaccharide is taken according to the proportion, 2M sulfuric acid is added for mixing, the mixture is hydrolyzed for 9 hours under the conditions of vacuum and 100 ℃, then 0.3M sodium hydroxide solution is used for adjusting the pH value to be neutral, and supernatant fluid is taken after centrifugation, so that the complete hydrolysate of the polysaccharide is obtained. Preparing standard solutions of monosaccharides and mixed monosaccharides required by experiments, placing 200 mu L of standard solutions into a 2.0mL EP tube, sequentially adding 100 mu L of 0.5M PMP methanol solution and 100 mu L of 0.3M sodium hydroxide solution, uniformly mixing, reacting for 60min at 70 ℃, taking out, cooling to room temperature, adding 100 mu L of 0.3M hydrochloric acid, adding 1mL of chloroform for extraction, centrifuging to remove chloroform phase, repeating for 4 times, and then filtering the water phase by a 0.22 mu M filter head for HPLC analysis. The chromatographic conditions include: column chromatography Agilent HC-C18 (4.6X105 cm,5 μm); the sample injection amount is 10 mu L; the mobile phase was 0.05M phosphate buffer (pH 6.5) +acetonitrile (v/v, 4:1); the flow rate is 1.2mL/min; column temperature is 30 ℃; the detection wavelength is 245nm.
And (3) analyzing results, wherein the high performance liquid chromatography of the brown algae polysaccharide and the mixed monosaccharide standard sample is shown in figures 1-2. The fucoidan prepared in this example is analyzed by high performance liquid chromatography, and separated to obtain 6 peaks, and compared with the high performance liquid chromatography of standard monosaccharides, the fucoidan can be obtained with a monosaccharide composition and molar ratio of mannose: rhamnose: glucuronic acid: glucose: xylose: fucose=1:0.742:0.907:0.348: 1.325:1.589.
Determination of brown algae polysaccharide molecular weight:
the test is carried out by adopting an HPGPC method, and the molecular weight of the polygonatum polysaccharide prepared in the embodiment is calculated to be 8.12kDa by contrasting with the regression curve of the molecular weight of the polysaccharide standard sample.
Example 2:
the brown algae polysaccharide is prepared in comparison with example 1 in that: the alfalfa lactone is not added in the extraction process of the composite enzymolysis method.
The relative molecular weight of the prepared Polygonatum sibiricum polysaccharide was 9.01kDa by the same method as in example 1; the monosaccharide composition analysis method is the same as in example 1, and the result shows that the monosaccharide composition and the molar ratio of the extracted fucoidin are mannose: rhamnose: glucuronic acid: glucose: galactose: xylose: fucose=1:0.429:0.304:0.742:1.143:0.587:0.947.
Example 3:
preparation of brown algae polysaccharide derivatives:
slowly adding 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester (the solid-liquid ratio of the N, N-dimethylformamide is 0.26g:1 mL) into N, N-dimethylformamide under ice bath condition, and standing for 36min to obtain solution A; taking brown algae polysaccharide prepared in example 1 (solid-to-liquid ratio of the brown algae polysaccharide to the formamide is 0.21g:1 mL), adding the formamide, stirring for 35min at 56 ℃, then dropwise adding the solution A (volume ratio of the brown algae polysaccharide to the formamide is 0.34:1), and reacting for 3.5h after the dropwise adding is finished; pouring out the reaction solution, adding 80% ethanol for precipitation in the presence of 0.12% KCl, washing for 4 times with 95% ethanol, drying, dissolving in distilled water, adding 2M sodium hydroxide solution for regulating pH to 7.4, dialyzing with tap water for 2d, and dialyzing with distilled water for 1d; finally, rotary evaporation and vacuum freeze-drying are carried out to obtain the brown algae polysaccharide derivative.
Example 4:
the preparation of fucoidan derivatives differs from example 3: the brown seaweed polysaccharide used was prepared in example 2.
Example 5:
the preparation method of the brown algae polysaccharide derivative nano micelle comprises the following steps:
dissolving the brown algae polysaccharide derivative prepared in the embodiment 3 in formamide to obtain a solution of 1.3mg/mL, dialyzing in water for 48h by using a 500-1000Da dialysis bag, enabling the brown algae polysaccharide derivative to obtain nano-micelle through self-assembly under the action of a solvent, and finally lyophilizing to obtain the brown algae polysaccharide derivative nano-micelle.
Example 6:
the preparation of brown algae polysaccharide derivative nano micelle is different from that of example 5: the fucoidan derivative used was prepared in example 4.
Example 7:
the brown algae polysaccharide derivative is prepared in a manner different from that of example 3 in that: chlorosulfonic acid is used instead of tert-butyl 2- (chlorosulfonyl) -1H-indole-1-carboxylate.
The preparation of fucoidan polysaccharide nano derivative micelle is different from that of example 5: the fucoidan derivative was prepared in this example.
Example 8:
the brown algae polysaccharide derivative is prepared in a manner different from that of example 4 in that: chlorosulfonic acid is used instead of tert-butyl 2- (chlorosulfonyl) -1H-indole-1-carboxylate.
The preparation of fucoidan polysaccharide nano derivative micelle is different from that of example 5: the fucoidan derivative was prepared in this example.
Test example 1:
characterization by Infrared Spectroscopy
And (3) fully grinding the sample and KBr under the irradiation of an infrared lamp, pressing the sample and KBr into tablets, and carrying out structural analysis by using a NICOLET 380 type infrared spectrometer. Test parameters: wavelength range 4000-500 cm -1 。
The fucoidan prepared in example 1 and the fucoidan derivative prepared in example 3 were subjected to the above-described test, and the results are shown in fig. 3. As can be seen from the analysis of the figure, in the infrared spectrum of the fucoidan prepared in example 1, the infrared spectrum is 3500-3300 cm -1 The stretching vibration peak of O-H appears in the range of 3000-2800 cm -1 The characteristic absorption peaks of methyl and methylene appear in the range of 1610cm -1 Characteristic absorption peak of C=O in-CHO appears nearby, 1250cm -1 Characteristic absorption peak with S=O appears nearby, 1250-1000 cm -1 Characteristic absorption peaks for C-O-H and C-O bonds in C-O-C are present in the range. Compared with the infrared test result of fucoidan prepared in example 1, the infrared spectrum of fucoidan derivative prepared in example 3 was as high as 1682cm -1 Characteristic absorption peak of C=O in ester group appears in the vicinity of 1600-1500 cm -1 The vibration absorption peak of the benzene ring framework appears in the range of 1319cm -1 Characteristic absorption peak of C-N bond appears nearby, 1250cm -1 Characteristic peak absorption intensity of s=o in the vicinity is enhanced; the above results indicate that fucoidan derivatives were successfully prepared in example 3.
SEM characterization
The particle size and morphology of the nano-micelle samples were analyzed using Scanning Electron Microscopy (SEM).
The above test was performed on the nano micelle prepared in example 5, and the result is shown in fig. 4. From the analysis of the figure, the brown algae polysaccharide derivative nano micelle prepared in the embodiment 5 of the invention has the appearance of sphere, uniform dispersion and uniform shape, and proves that the micelle preparation is successful.
Crude polysaccharide yield and protein content determination
Brown algae crude polysaccharide yield% = brown algae crude polysaccharide mass/kelp powder mass x 100%
Protein content determination:
weighing bovine serum albumin, dissolving in distilled water to obtain 0.1mg/mL bovine serum albumin standardPreparing a protein standard curve from the quasi-solution according to conventional operation, and obtaining a linear regression equation of y=2.47x+0.0207, r 2 =0.992. Preparing a crude polysaccharide sample solution with proper concentration, taking 1.0mL to operate according to a standard curve measurement method, and calculating the protein content according to a protein standard curve.
The brown algae crude polysaccharide prepared in example 1 and example 2 was subjected to the above test, and the results are shown in table 1:
TABLE 1 crude polysaccharide yield and protein content test results
| Sample of | Crude polysaccharide yield (%) | Protein content/% |
| Example 1 | 5.76 | 1.84 |
| Example 2 | 4.47 | 2.52 |
As can be seen from the data analysis in Table 1, the brown algae crude polysaccharide prepared by the method provided in example 1 has a significantly higher yield than that of example 2, and has a lower protein content than that of example 2, which indicates that the addition of alfalfa lactone in the complex enzymolysis process can be expected to exert a beneficial effect on enzymes, thereby promoting the enzymolysis process, so that the brown algae crude polysaccharide yield is significantly improved, and the protein content is effectively reduced.
Test example 2:
determination of antidiabetic Activity
The testing method comprises the following steps: thioflavin fluorescence method, using Th-T specificity labeling of amylin fiber. The specific operation process comprises the following steps: taking amylin (5 mug/mL) and a sample to be detected (90 mug/mL) to be incubated at 37 ℃ together, taking the independently incubated amylin as a control after 24 hours, and detecting the fluorescence intensity of Th-T, namely taking 180 mug of Th-T solution to be added into a 96-well plate, and then adding 20 mug of sample to be detected; a blank control group was set and equal amount of PBS (pH 7.4) was added; then the sample is placed on a multifunctional enzyme labeling instrument to read the value (excitation wavelength is 450nm, emission wavelength is 480 nm), and the measured fluorescence value is subtracted from the fluorescence value of the blank control group.
The fucoidan prepared in examples 1-2 and fucoidan derivatives prepared in examples 3-4 were subjected to the above test, and the results are shown in Table 2:
TABLE 2 results of test for the Activity of inhibiting the aggregation of amylin
| Sample of | Percent fluorescence reduction (%) |
| Example 1 | 81.2 |
| Example 2 | 62.7 |
| Example 3 | 92.5 |
| Example 4 | 71.6 |
As can be seen from the data in Table 2, the inhibition percentage of the fucoidan prepared in example 1 of the present invention to amylin aggregation is significantly higher than that of example 2, which indicates that fucoidan extracted by adding alfalfa lactone in the complex enzymatic hydrolysis process has more excellent antidiabetic activity. Example 3 is better than example 1, and example 4 is better than example 2, indicating that chemical modification of fucoidan with tert-butyl 2- (chlorosulfonyl) -1H-indole-1-carboxylate to give derivatives can significantly enhance the antidiabetic activity of the derivatives.
Measurement of anticoagulant Activity
Fresh rabbit blood 5mL is taken, 0.5 mL sample to be tested and sodium citrate solution are respectively added, and the rabbit blood clotting time is recorded. To facilitate comparison of the anticoagulation activity of the sample to be tested and sodium citrate, the test was conducted with the equivalent amount of sulfuric acid groups (the amount of sodium citrate carboxyl groups was converted into the amount of sulfuric acid groups of the same charge, i.e., 1 mol/L COO - =0.5 mol/L SO 4 2- ) As a solute concentration, the concentration of the solute in the test was 0.5mg/mL. Re-calcilytic, re-adding CaCl into a non-coagulated beaker added with the sample solution to be tested 2 Solution, observe and record clotting time.
The brown seaweed polysaccharide prepared in examples 1-2, brown seaweed polysaccharide derivatives prepared in examples 3-4, and brown seaweed polysaccharide derivative nano-micelles prepared in examples 5-8 were subjected to the above test, and the results are shown in Table 3:
TABLE 3 anticoagulant Activity test results
| Sample of | 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 |
Annotation: "-" indicates that the clotting time is too short after blood addition to be measured under experimental conditions.
As can be seen from the data in Table 3, the clotting time of the fucoidan prepared in example 1 of the present invention is significantly higher than that of example 1, indicating that fucoidan extracted by adding alfalfa lactone during the complex enzymatic hydrolysis process has better anticoagulation ability. Example 3 is better than example 1, and example 4 is better than example 2, which shows that the chemical modification of fucoidan with 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester to obtain its derivative can significantly enhance the anticoagulant activity of the derivative. The clotting time of the brown seaweed polysaccharide derivative nano-micelle prepared in the embodiment 5 of the invention is obviously higher than that of the embodiment 3 and the embodiment 7, and the effect of the embodiment 6 is better than that of the embodiment 4 and the embodiment 8, which shows that the biological activity of the nano-micelle can be further enhanced and the clotting activity is obviously enhanced by preparing the brown seaweed polysaccharide derivative into the nano-micelle; and the anticoagulant activity of the derivative obtained by chemically modifying the fucoidin with 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester is better than that of the fucoidin derivative modified by chlorosulfonic acid.
Test example 3:
determination of drug loading and encapsulation efficiency of nano micelle
Drug loading measurement: dissolving a nano micelle sample in DMSO to prepare a solution with the concentration of 1mg/mL, taking 5mL, respectively adding 0.01,0.1,0.5,1.0,2.0 and 4.0mL of beta-carotene solution with the volume of 0.2mg/mL (the solvent is DMSO), uniformly mixing, dialyzing in a large amount of water for 3d, and changing the water once for 6 h; then the solution was uniformly sized to 25mL, filtered through a 0.45 μm microporous membrane to remove the unencapsulated beta-carotene, and the ultraviolet absorbance of the solution was measured (A 278 ) The content was calculated in combination with the standard curve of beta-carotene.
Calculation of drug loading and encapsulation efficiency:
two 5mL DMSO portions were added with 0.1mg/mL and 0.2mg/mL of a beta-carotene-containing DMSO solution as a control group, and the ultrasonic treatment was performed for 30min to determine the ultraviolet absorbance at 278 nm. Drug loading and encapsulation efficiency were calculated according to the following formulas:
drug loading = amount of drug entrapped in micelle/total amount of micelle added x 100%
Encapsulation efficiency = amount of drug entrapped in micelle/total amount of drug added x 100%
The brown seaweed polysaccharide derivative nano-micelle prepared in examples 5 to 6 and the brown seaweed polysaccharide nano-micelle prepared in examples 7 to 8 were subjected to the above test, and the results are shown in Table 4:
table 4 drug loading and encapsulation efficiency test results
| Sample of | Drug loading (%) | Encapsulation efficiency (%) |
| Example 5 | 16.3 | 55.1 |
| Example 6 | 14.4 | 43.0 |
| Example 7 | 7.7 | 42.8 |
| Example 8 | 7.2 | 34.6 |
From the data in table 4, it can be seen that the drug loading rate and encapsulation efficiency of the brown seaweed polysaccharide derivative nano micelle prepared in example 5 of the present invention are obviously higher than those of example 7, and the effect of example 6 is better than that of example 8, which shows that the chemical modification of the brown seaweed polysaccharide by using the tert-butyl 2- (chlorosulfonyl) -1H-indole-1-carboxylate to obtain the derivative thereof, and the nano micelle prepared by the present invention can significantly improve the drug loading capacity and encapsulation effect of the nano micelle. In addition, the effect of example 5 is better than that of example 6, and the effect of examples 7 is better than that of example 8, which shows that the encapsulation effect of the nano-micelle can be effectively improved by adding brown seaweed polysaccharide extracted from alfalfa lactone in the composite enzymolysis process and preparing the nano-micelle after derivatization.
The conventional technology in the above embodiments is known to those skilled in the art, and thus is not described in detail herein.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A fucoidan derivative is a fucoidan sulfonylation derivative, which is prepared by connecting hydroxyl in fucoidan structure with sulfonyl chloride in 2- (chlorosulfonyl) -1H-indole-1-carboxylic acid tert-butyl ester structure through chemical bond;
the brown algae polysaccharide is extracted from kelp.
2. The fucoidan derivative according to claim 1, wherein: the composition and the molar ratio of the monosaccharide of the brown algae polysaccharide are mannose: rhamnose: glucuronic acid: glucose: xylose: fucose=1:0.742:0.907:0.348: 1.325:1.589.
3. The fucoidan derivative according to claim 1, wherein: the composition and the molar ratio of the monosaccharide of the 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.
4. A fucoidan derivative according to claim 2 or 3, wherein: the preparation method of the brown algae polysaccharide adopts a complex enzymolysis method, wherein the complex enzyme comprises pectase, cellulase and papain.
5. The fucoidan derivative of claim 4, wherein: alfalfa lactone is also added in the process of preparing brown algae polysaccharide by the composite enzymolysis method.
6. The method of manufacturing according to claim 4, wherein: the mass ratio of the pectase to the cellulase to the papain is 1:0.6 to 0.8:0.2 to 0.4.
7. The method for preparing fucoidan derivatives of claim 1, comprising: the brown algae polysaccharide derivative is prepared through a sulfonylation reaction.
8. A fucoidan derivative nano-micelle, the raw material of which comprises the fucoidan derivative of any one of claims 1-7.
9. Use of fucoidan derivatives according to claim 1 for the preparation of drugs or bioactive substance delivery nanomaterials.
10. Use of the fucoidan derivative nano-micelle of claim 9 in the preparation of antidiabetic and anticoagulant drugs.
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