Sodium alginate multifunctional composite microsphere as well as preparation method and application thereof

Abstract

The invention relates to the technical field of microbial probiotic preparations, in particular to a sodium alginate multifunctional composite microsphere as well as a preparation method and application thereof. The sodium alginate multifunctional composite microspheres are obtained by dissolving the carbohydrate vesicles, the traditional Chinese medicine nanoparticles and the probiotics in a sodium alginate solution and then dropwise adding the solution into a calcium chloride solution through an ionic cross-linking effect. According to the invention, the amphiphilic xylooligosaccharide-cystamine derivative with H2S response is synthesized through a click reaction on the basis of prebiotic xylooligosaccharide. And the amphipathic saccharide vesicles are obtained through self-assembly by virtue of hydrophilic and hydrophobic effects. Sanguinarine iodide prepared by purification, separation and reflux and baicalin are self-assembled in an aqueous solution through a non-covalent bond, and the traditional Chinese medicine nanoparticles are prepared through ultrasonic treatment. An in-vitro simulated release experiment proves that the sodium alginate multifunctional composite microsphere is relatively low in release rate under an acidic condition, so that substances are favorably delivered to intestinal tracts; and the release of encapsulated substances can be better realized in neutral and alkaline environments.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial probiotic preparations, and specifically relates to sodium alginate multifunctional composite microspheres and a preparation method and application thereof. Background Art

[0002] In the modern animal husbandry industry, diarrhea, as a common disease, not only significantly increases the economic cost of farming, but also poses a serious constraint on the sustainable benefits of the farming industry. Traditional large-scale farming methods often rely on the widespread use of antibiotics to control the occurrence of diseases such as diarrhea. However, this irrational antibiotic application strategy has led to the widespread spread of bacterial resistance and the problem of antibiotic residues in animals, posing a potential threat to food safety and human health. Therefore, exploring and designing green, safe, and efficient antibiotic alternatives to effectively prevent and treat diarrhea in animals has become a key issue that needs to be urgently addressed in the current farming industry.

[0003] In recent years, alternative antibiotics have attracted much attention due to their unique advantages and significant therapeutic effects. These alternative antibiotics, mainly including prebiotics, probiotics, traditional Chinese medicine and plant extracts, act on the intestines of animals through different mechanisms, showing effects comparable to or even superior to antibiotics. Prebiotics and probiotics can regulate the intestinal function and immune system of livestock and poultry, effectively avoid and treat bacterial diarrhea, and thus improve the overall health of animals. They have become an important tool for regulating modern intestinal microecology and preventing intestinal diseases. On the other hand, traditional Chinese medicine and plant extracts have become the leaders among antibiotic alternatives due to their natural sources, efficient antibacterial and anti-inflammatory effects, and low side effects. In particular, the risk of these natural extracts inducing bacterial resistance is extremely low, providing safety guarantees for long-term use.

[0004] Among the many traditional Chinese medicines and plant extracts, sanguinarine and baicalin have attracted much attention due to their broad-spectrum anti-inflammatory and antibacterial effects. They can not only effectively inhibit the growth of pathogens, but also regulate the inflammatory response of the intestine, and have a significant effect on the prevention and treatment of diarrhea.

[0005] Based on this, the present invention provides a sodium alginate multifunctional composite microsphere for co-loading prebiotics, traditional Chinese medicine nanoparticles, and probiotics. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a sodium alginate multifunctional composite microsphere and a preparation method and application thereof, which can be used for co-loading of prebiotics, traditional Chinese medicine nanoparticles and probiotics.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The invention provides a sodium alginate multifunctional composite microsphere. The sodium alginate multifunctional composite microsphere is obtained by dissolving sugar vesicles, traditional Chinese medicine nanoparticles and probiotics in a sodium alginate solution and then dropping the solution into a calcium chloride solution through ion crosslinking.

[0009] The present invention also provides a method for preparing the sodium alginate multifunctional composite microspheres, comprising the following steps:

[0010] (1) using a monoalkynyl cystamine derivative containing a disulfide bond and a prebiotic oligoxylose derivative to prepare an amphiphilic molecule oligoxylose-cystamine derivative, then mixing the amphiphilic molecule oligoxylose-cystamine derivative with a methanol solution, evaporating the solvent in a vacuum to form a dry lipid film on the bottle wall, then adding ultrapure water and placing the mixture in an ultrasonic cleaning machine for ultrasonic treatment and standing to obtain sugar vesicles;

[0011] (2) mixing the ISA solution and the BA solution, placing the mixture in an ultrasonic cleaning machine for ultrasonic treatment and allowing the mixture to stand, thereby obtaining Chinese medicine nanoparticles;

[0012] (3) The sugar vesicles prepared in step (1) are mixed with the traditional Chinese medicine nanoparticles and probiotic freeze-dried powder prepared in step (2) and sodium alginate colloid, and stirred to obtain a sodium alginate composite solution, and then the sodium alginate composite solution is added dropwise to the CaCl2 solution to obtain sodium alginate multifunctional composite microspheres.

[0013] Furthermore, in step (1), the method for preparing the disulfide bond-containing monoalkynyl cystamine derivative comprises the following steps:

[0014] (1.1) Cystamine dihydrochloride and NaOH are mixed and stirred at a molar ratio of 1:2 to 2.4, and distilled water is evaporated in vacuo, and the crude product is dissolved in a large amount of dichloromethane and subjected to ultrasonic extraction; the collected organic phase is dried, filtered, and evaporated in vacuo to remove the solvent to obtain cystamine;

[0015] (1.2) adding cystamine and EDCI obtained in step (1.1) to a flask, adding a dichloromethane solution, stirring, dissolving n-octanoic acid in dichloromethane and adding it to the flask, so that the molar ratio of cystamine to n-octanoic acid is 2.5-3:1, and the mass volume ratio of n-octanoic acid to dichloromethane is 1 g:5-10 mL, mixing to obtain a mixture, stirring the mixture at room temperature, then pouring it into a separatory funnel, washing with a saturated sodium chloride solution, drying the collected organic phase, filtering, evaporating the solvent in a vacuum, and then separating and purifying it by column chromatography to obtain compound 2;

[0016] (1.3) Add the purified product of step (1.2) and EDCI to a flask, add dichloromethane solution, the mass volume ratio of the purified product of step (1.2) to dichloromethane is 1 g: 10-15 mL, stir, dissolve 5-hexynoic acid in dichloromethane and add it to the flask, so that the mass volume ratio of 5-hexynoic acid to dichloromethane is 1 g: 5-10 mL, and the molar ratio of the purified product of step (1.2) to 5-hexynoic acid is 2.5-3: 1, to obtain a mixture, stir the mixture at room temperature, then pour it into a separatory funnel, wash it with saturated sodium chloride solution, dry the collected organic phase, filter and evaporate the solvent in a vacuum, and then separate and purify it by column chromatography to obtain a monoalkynyl cystamine derivative containing a disulfide bond.

[0017] Furthermore, in step (1), the method for preparing the prebiotic xylo-oligosaccharide derivative comprises the following steps:

[0018] (2.1) Tetraethylene glycol and dichloromethane solution are mixed in a mass volume ratio of 1 g: 10-15 mL, stirred at room temperature, and then p-toluene yellow acid chloride and triethylamine are dissolved in dichloromethane and added to a flask, so that the mass volume ratio of p-toluene yellow acid chloride and dichloromethane solution is 1 g: 5-10 mL, and the mass volume ratio of triethylamine and dichloromethane solution is 1 g: 5-10 mL. The mixture is stirred at room temperature, then poured into a separatory funnel, washed with water, and the collected organic phase is dried, filtered, and the solvent is evaporated in vacuo, and then separated and purified by column chromatography to obtain the product;

[0019] (2.2) Add xylooligosaccharides and iodine in a mass ratio of 300 mg:25-30 mg to a flask, add acetic anhydride under ice bath to make the mass volume ratio of xylooligosaccharides and acetic anhydride 0.3 g:5-8 mL to obtain a mixture, stir the mixture at room temperature overnight, add saturated Na2S2O3 to the organic phase to adjust it to weak alkalinity, transfer the organic phase to a beaker with dichloromethane, add saturated sodium bicarbonate solution, stir well with a glass rod, pour into a separatory funnel for extraction, extract with water and saturated sodium chloride solution in turn, dry the collected organic phase, filter and evaporate the solvent in vacuo to obtain compound 4;

[0020] (2.3) Under N2 conditions, the purified products of step (2.1) and step (2.2) are mixed in a mass ratio of 400-410 mg:270 mg, anhydrous dichloromethane is added to dissolve, so that the mass volume ratio of the purified product to dichloromethane is 1 g:5-10 mL, boron trifluoride ether is added dropwise to the flask, so that the mass volume ratio of the purified product in step (2.2) to boron trifluoride ether is 270 mg:135-140 μL, stirred at room temperature, and then poured into a separatory funnel, dichloromethane is added, and extraction is carried out with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence, and the collected organic phase is dialyzed with a dialysis bag, the dialyzed solution is filtered with a funnel and placed in an oven for drying, the product on the filter paper is dissolved and collected with dichloromethane, filtered and evaporated in vacuo to remove the solvent to obtain compound 5;

[0021] (2.4) Under N2 conditions, compound 5 and sodium azide were mixed in a flask at a mass ratio of 200 mg:20-25 mg, and N,N-anhydrous dimethylformamide solution was added to dissolve the mixture to obtain a mixture. The mixture was stirred for reaction. After the reaction was completed, the mixture was poured into a separatory funnel, and dichloromethane was added. After extraction with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence, the collected organic phase was filtered and the solvent was evaporated in vacuo to obtain a prebiotic oligoxylose derivative.

[0022] Furthermore, in step (1), the preparation method of the amphiphilic molecule oligoxylose-cystamine derivative comprises the following steps:

[0023] (3.1) Under N2 conditions, the monoalkynyl cystamine derivative containing a disulfide bond, the prebiotic oligoxylose derivative, CuSO4·5H2O and ascorbic acid are dissolved in N,N-anhydrous dimethylformamide and added to a flask to obtain a mixture, wherein the mass volume ratio of the mixture is 158.9 mg:445.5 mg:33.6 mg:45.9 mg:5-6 mL, the mixture is stirred for reaction, and after the reaction is completed, it is dialyzed, the dialyzed solution is filtered with a funnel and placed in an oven for drying, the solid on the filter paper is dissolved with dichloromethane and collected, filtered, the solvent is evaporated in vacuo, and then separated and purified by column chromatography to obtain compound 7;

[0024] (3.2) Under N2 conditions, compound 7 and sodium methoxide and methanol solution were mixed in a mass volume ratio of 286.8 mg:33-35 mg:5-6 mL and stirred at room temperature. After the reaction was completed, ion exchange resin was added and stirred at room temperature again. The mixture was filtered with methanol solution, and the organic phase was collected and evaporated in vacuo to remove the solvent to obtain an amphiphilic molecule oligoxylose-cystamine derivative.

[0025] Furthermore, in step (2), the method for preparing the ISA solution comprises the following steps:

[0026] (4.1) Under light-proof conditions, a crude sample of sanguinarine hydrochloride and anhydrous ethanol solution were added to a flask at a mass volume ratio of 1.5 g:15-20 mL. 0.1 mol / L NaOH was added gradually during stirring to adjust the pH to a weakly alkaline state. The filtrate was collected by suction filtration, sodium borohydride was added to the filtrate until the raw material was completely consumed, the filtrate was collected and the solvent was evaporated in a vacuum, ultrasonically extracted with dichloromethane, filtered, and then poured into a separatory funnel and washed with water. The collected organic phase was dried, filtered, and the solvent was evaporated in a vacuum, and then purified by column chromatography to obtain compound ISA2;

[0027] (4.2) At room temperature, compound ISA2 and iodine were dissolved in 5 mL of anhydrous ethanol, with the mass volume ratio of iodine to anhydrous ethanol being 116 mg:5-6 mL. After reflux heating for a period of time, saturated Na2S2O3 was added to change the solution from brown-black to light red. The solution was filtered and dried, and the orange-red solid on the filter paper was collected to obtain compound ISA1.

[0028] (4.3) Compound ISA1 is mixed evenly with ultrapure water to obtain ISA solution.

[0029] Furthermore, in step (3), the mass volume ratio of the probiotic freeze-dried powder, the traditional Chinese medicine nanoparticles, the sugar vesicles and the sodium alginate colloid is 40 mg: 1 mL: 1 mL: 2 mL.

[0030] Furthermore, in step (3), the concentration of the sugar vesicles is 2 mg / mL, the concentration of the traditional Chinese medicine nanoparticle solution is 0.1 mM, the mass concentration of the sodium alginate colloid is 1%, and the mass concentration of the CaCl2 solution is 0.5%.

[0031] Furthermore, in step (1), the ultrasonic treatment time is 20 minutes and the standing time is 1 hour; in step (2), the ultrasonic treatment time is 15 minutes and the standing time is 1 hour; in step (3), the specific method of dripping is to use a 2μL flat-head liquid phase needle to absorb the sodium alginate composite solution and place it at a position 3 cm vertically above the liquid surface, connect the rubber tube mouth of argon gas to a 5mL syringe, align the nipple with the flat-head liquid phase needle needle, and fix it with an iron stand, and push the syringe piston handle at a uniform speed.

[0032] The present invention also provides an application of the sodium alginate multifunctional composite microspheres in the preparation of sustained-release drugs. The sodium alginate multifunctional composite microspheres can be used for co-loading of prebiotics, traditional Chinese medicine nanoparticles and probiotics.

[0033] The present invention discloses the following technical effects:

[0034] (1) The present invention synthesizes a prebiotic oligoxylose derivative and a monoalkynyl cystamine derivative containing a disulfide bond, and connects the two by a click reaction to synthesize an amphiphilic sugar molecule containing a disulfide bond and having a H2S response; iodine sanguinarine and baicalin are ultrasonicated in an aqueous solution to prepare Chinese medicine nanoparticles through non-covalent self-assembly, which can reduce the cytotoxicity of the two when used alone.

[0035] (2) The present invention provides a sodium alginate multifunctional composite microsphere for co-loading prebiotics, Chinese medicine nanoparticles, and probiotics. The pH responsiveness of the sodium alginate microspheres and the H2S responsiveness of the disulfide bonds in the sugar vesicles can be used to achieve the gradual release of sugar vesicles, probiotics, and Chinese medicine nanoparticles under specific conditions, thereby avoiding premature degradation of active ingredients and providing a new method for the co-delivery of composite microecological preparations.

[0036] (3) In vitro simulated release experiments have shown that the release rate of sodium alginate multifunctional composite microspheres is low under acidic conditions, which is conducive to the delivery of substances to the intestine; the release of encapsulated substances can be better achieved in neutral and alkaline environments. At 37°C, the release rates of sodium alginate microspheres in PBS (pH = 7.4), pH = 6.8, and pH = 1.2 aqueous solutions after 12 hours were 65.9%, 29.5%, and 11.8%, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 The synthetic reaction flow chart of sodium alginate multifunctional composite microspheres in the present invention;

[0039] Figure 2 The synthetic reaction flow chart of the amphiphilic molecule xylooligosaccharide-cystamine derivative in the present invention;

[0040] Figure 3 The synthetic reaction flow chart of sanguinarine iodide in the present invention is as follows;

[0041] Figure 4 is the nuclear magnetic hydrogen spectrum of the monoalkynyl cystamine derivative containing a disulfide bond in the present invention;

[0042] Figure 5 is a Fourier infrared spectrum of the prebiotic oligoxylose derivative of the present invention;

[0043] Figure 6 is a mass spectrum of the amphiphilic molecule oligoxylose-cystamine derivative of the present invention;

[0044] Figure 7 It is the nuclear magnetic hydrogen spectrum of sanguinarine iodide in the present invention.

[0045] Figure 8 The characterization diagrams of sodium alginate multifunctional composite microspheres in Experimental Example 1 of the present invention are shown in Figure 1; wherein (a) is an optical microscope characterization diagram; (b) is a SEM characterization diagram;

[0046] Fig. 9 This is a diagram showing the release effect of the sodium alginate multifunctional composite microspheres in Experimental Example 1 of the present invention. DETAILED DESCRIPTION

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0049] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0050] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0051] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0052] The test methods used in the following implementations, unless otherwise specified, are all commonly used experimental methods in this field;

[0053] Unless otherwise specified, the experimental materials used in the following examples are all commonly used experimental materials in this field.

[0054] The sodium alginate multifunctional composite microspheres in the present invention are based on prebiotic oligoxylose, and amphiphilic oligoxylose-cystamine derivatives with H2S response are synthesized through click reaction, and then amphiphilic sugar vesicles are obtained by self-assembly by hydrophilic and hydrophobic effects; at the same time, the purified, separated, and refluxed sanguinarine iodide and baicalin are self-assembled by non-covalent bonds in an aqueous solution to obtain Chinese medicine nanoparticles by ultrasound; then the sugar vesicles, Chinese medicine nanoparticles, and probiotics are dissolved in a sodium alginate solution, and then dripped into a calcium chloride solution to obtain the result through ion crosslinking. The synthesis process is as follows: Figure 1 As shown, the specific preparation method is as follows:

[0055] (1) Cystamine dihydrochloride and NaOH are mixed at a molar ratio of 1:2 to 2.4, stirred with distilled water for 1 hour, and the distilled water is removed by evaporation in a vacuum (50° C.), and the crude product is dissolved in a large amount of dichloromethane and subjected to ultrasonic extraction; the collected organic phase is dried, filtered, and evaporated in a vacuum to remove the solvent to obtain cystamine (Compound 1);

[0056] (2) Add the cystamine and EDCI obtained in step (1) to a flask, add a dichloromethane solution, and stir for 0.5 h. Dissolve n-octanoic acid in dichloromethane and add it to the flask so that the molar ratio of cystamine to n-octanoic acid is 2.5 to 3:1, and the mass volume ratio of n-octanoic acid to dichloromethane is 1 g: 5 to 10 mL. Mix to obtain a mixture, stir the mixture at room temperature for 24 h, then pour it into a separatory funnel, wash it with a saturated sodium chloride solution, dry the collected organic phase, filter it, evaporate the solvent in a vacuum, and then separate and purify it by column chromatography to obtain compound 2.

[0057] (3) The purified product obtained in step (2) and EDCI are added to a flask, and a dichloromethane solution is added, wherein the mass volume ratio of the purified product in step (1.2) to dichloromethane is 1 g: 10-15 mL, and the mixture is stirred for 0.5 h. 5-hexynoic acid is dissolved in dichloromethane and added to the flask, wherein the mass volume ratio of 5-hexynoic acid to dichloromethane is 1 g: 5-10 mL, and the molar ratio of the purified product in step (1.2) to 5-hexynoic acid is 2.5-3: 1, to obtain a mixture, and the mixture is stirred at room temperature for 24 h, then poured into a separatory funnel, washed with a saturated sodium chloride solution, and the collected organic phase is dried, filtered, and the solvent is evaporated in a vacuum, and then separated and purified by column chromatography to obtain compound 3.

[0058] (4) Tetraethylene glycol and dichloromethane solution are mixed in a mass volume ratio of 1 g: 10-15 mL, and stirred at room temperature for 0.5 h. At 0°C, p-toluene yellow acid chloride and triethylamine are dissolved in dichloromethane and added to a flask so that the mass volume ratio of p-toluene yellow acid chloride and dichloromethane solution is 1 g: 5-10 mL, and the mass volume ratio of triethylamine and dichloromethane solution is 1 g: 5-10 mL. The mixture is stirred at room temperature for 12 h, then poured into a separatory funnel, washed with water, and the collected organic phase is dried, filtered, and the solvent is evaporated in vacuo, and then separated and purified by column chromatography to obtain the product.

[0059] (5) Add xylooligosaccharides and iodine in a mass ratio of 300 mg:25-30 mg to a flask, add acetic anhydride under ice bath to make the mass volume ratio of xylooligosaccharides and acetic anhydride 0.3 g:5-8 mL to obtain a mixture, stir the mixture at room temperature overnight, add saturated Na2S2O3 to the organic phase to adjust it to weak alkalinity, transfer the organic phase to a beaker with dichloromethane, add saturated sodium bicarbonate solution, stir well with a glass rod, pour into a separatory funnel, extract three times, extract three times with water and saturated sodium chloride solution in turn, dry the collected organic phase, filter and evaporate the solvent in a vacuum to obtain compound 4.

[0060] (6) Under N2 conditions, the purified products of step (4) and step (5) were mixed in a mass ratio of 400-410 mg:270 mg, and anhydrous dichloromethane was added to dissolve the mixture so that the mass volume ratio of the purified product to dichloromethane was 1 g:5-10 mL. Boron trifluoride etherate was added dropwise into the flask so that the mass volume ratio of the purified product in step (5) to boron trifluoride etherate was 270 mg:135-140 μL. The mixture was stirred at room temperature for 12 h, then poured into a separatory funnel, dichloromethane was added, and the mixture was extracted with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence. The collected organic phase was dialyzed with a dialysis bag for 48 h. The dialyzed solution was filtered with a funnel and dried in an oven. The product on the filter paper was dissolved with dichloromethane and collected. The mixture was filtered and the solvent was evaporated in a vacuum to obtain compound 5.

[0061] (7) Under N2 conditions, the purified product in step (6) and sodium azide were mixed in a flask at a mass ratio of 200 mg: 20-25 mg, and a dry N,N-dimethylformamide solution was added to dissolve the mixture to obtain a mixture, and the mixture was stirred at 85°C for 24 hours. The mass volume ratio of the purified product in step (6) and the dry N,N-dimethylformamide solution was 0.2 g: 3-5 mL. After the reaction was completed, it was poured into a separatory funnel, dichloromethane was added, and it was extracted with a saturated sodium bicarbonate solution, water and a saturated sodium chloride solution in sequence. The collected organic phase was filtered and the solvent was evaporated in a vacuum to obtain compound 6.

[0062] (8) Under N2 conditions, the purified products of step (3) and step (7), CuSO4·5H2O, and ascorbic acid were dissolved in N,N-anhydrous dimethylformamide and added to a flask to obtain a mixture, wherein the mass volume ratio of the above mixture was 158.9 mg:445.5 mg:33.6 mg:45.9 mg:5-6 mL. The mixture was stirred at 80°C for 24 h. After the reaction was completed, it was dialyzed for 48 h. The dialyzed solution was filtered with a funnel and placed in an oven for drying. The solid on the filter paper was dissolved with dichloromethane and collected. The solid was filtered and the solvent was evaporated in vacuo, and then separated and purified by column chromatography to obtain compound 7.

[0063] (9) Under N2 conditions, the purified product in step (8) and sodium methoxide and methanol solution were mixed in a mass volume ratio of 286.8 mg: 33-35 mg: 5-6 mL, and stirred at room temperature for 12 h. After the reaction was completed, an ion exchange resin was added, and the mixture was stirred at room temperature for another 2 h. The mixture was filtered with a methanol solution, and the organic phase was collected and evaporated in vacuo to remove the solvent to obtain an amphiphilic molecule oligoxylose-cystamine derivative (Compound 8).

[0064] (10) Under light-shielding conditions, a crude sample of sanguinarine hydrochloride (Compound ISA1) and anhydrous ethanol solution were added to a flask at a mass volume ratio of 1.5 g:15-20 mL, and 0.1 mol / L NaOH was added gradually during stirring to adjust the pH to weak alkalinity. The filtrate was collected by suction filtration, and sodium borohydride was added to the filtrate until the raw material was completely consumed. The filtrate was collected and the solvent was evaporated in a vacuum. The product was extracted with dichloromethane by ultrasonication, filtered, and then poured into a separatory funnel and washed with water. The collected organic phase was dried, filtered, and the solvent was evaporated in a vacuum. The product was separated and purified by column chromatography to obtain Compound ISA2.

[0065] (11) At room temperature, ISA2 and iodine were mixed in a molar ratio of 1:2.4-2.6; the mass volume ratio of ISA2 and anhydrous ethanol was 61 mg:5-6 mL, and refluxed at 78°C for 2.5 h. Saturated Na2S2O3 was added to change the solution from brown-black to light red. The solution was filtered and dried, and the orange-red solid on the filter paper was collected to obtain sanguinarine iodide (compound ISA1).

[0066] (12) Compound 8 obtained in step (9), methanol and water were added to a round-bottom flask in a mass volume ratio of 3 mg:3-5 mL:3 mL, and the solvent was evaporated in vacuo to form a dry lipid film on the flask wall. Ultrapure water was added and the mixture was placed in an ultrasonic cleaner for ultrasonic treatment and allowed to stand for 1 h.

[0067] (13) The compound ISA1 obtained in step (11) was mixed with ultrapure water at a mass volume ratio of 4.6 mg:100 mL to obtain a sanguinarine iodide solution.

[0068] (14) Baicalin was mixed with ultrapure water at a mass volume ratio of 4.6 mg:100 mL and a NaOH solution to dissolve the mixture, thereby obtaining a baicalin solution.

[0069] (15) The products obtained in step (13) and step (14) are mixed in a volume ratio of 1:1 to 1.2 mL, placed in an ultrasonic cleaning machine for ultrasonic treatment, and allowed to stand to obtain traditional Chinese medicine nanoparticles.

[0070] (16) Sodium alginate and ultrapure water were mixed at a volume ratio of 100 mg:10 mL to obtain a sodium alginate solution.

[0071] (17) Mix CaCl2 and ultrapure water at a ratio of 1 g:200 mL and sterilize by high pressure to obtain a CaCl2 solution, which is stored for future use.

[0072] (18) The product obtained in step (12), step (15) and step (16) and the lyophilized probiotic powder were mixed uniformly in a mass volume ratio of 1 mL:1 mL:2 mL:40 mg to obtain a mixed solution.

[0073] (19) The CaCl2 solution obtained in step (17) is placed on a magnetic stirring stirrer and stirred at a constant speed. A flat-head liquid phase needle is used to draw the mixed solution obtained in step (18) and placed in a position perpendicular to the liquid surface. The rubber tube of argon gas is connected to the syringe, the nipple is aligned with the needle of the flat-head liquid phase needle, and fixed with an iron stand. The syringe piston handle is pushed in at a uniform speed for dropwise addition. After the dropwise addition is completed, the solution is collected with a filter and stored at 4°C to obtain sodium alginate multifunctional composite microspheres.

[0074] The synthetic reaction flow chart of the amphiphilic molecule oligoxylose-cystamine derivative in the above steps is as follows: Figure 2 As shown, the synthetic reaction flow chart of sanguinarine iodide is as follows Figure 3 shown.

[0075] The present invention will be further described below by way of specific embodiments.

[0076] Example 1

[0077] This embodiment follows Figure 2 The chemical reaction equation shown in the figure is used to synthesize the amphiphilic molecule oligoxylose-cystamine derivative, which specifically includes the following steps:

[0078] (1) Synthesis of monoalkynyl cystamine derivatives containing disulfide bonds

[0079] At 0℃ and N2, accurately weigh Figure 1Compound 2 (1.2 g, 4.31 mmol) and EDCI (1.03 g, 5.39 mmol) shown in were added to a 50 mL flask. Then 40 mL of dichloromethane solution was added and stirred for 0.5 h. 5-Hexynoic acid (0.38 mL, 3.59 mmol) was dissolved in 5 mL of dichloromethane and added to the flask. The mixture was stirred at room temperature for 24 h, then poured into a separatory funnel and washed with saturated sodium chloride solution (3×80 mL). The collected organic phase was dried over anhydrous sodium sulfate, filtered and evaporated in vacuo to remove the solvent. The crude product was separated by column chromatography using dichloromethane and methanol (75:1) as eluent to obtain compound 3 (see NMR hydrogen spectrum). Figure 4 ) Column separation yield 57%;

[0080] (2) Synthesis of prebiotic oligoxylose derivatives

[0081] Under N2 conditions, accurately weigh Figure 1 Compound 5 (0.20 g) and sodium azide (20.53 mg, 0.32 mmol) shown in the figure were placed in a 10 mL flask, and 3 mL of N,N-anhydrous dimethylformamide (DMF) solution was added to dissolve them. The mixture was stirred at 85°C for 24 h. After the reaction was completed, it was poured into a separatory funnel, 20 mL of dichloromethane was added, and it was extracted with saturated sodium bicarbonate solution (3×40 mL), water (3×40 mL) and saturated sodium chloride (3×40 mL) solution in sequence. The collected organic phase was filtered and the solvent was evaporated in vacuo to obtain compound 6 (see H NMR spectrum). Figure 5 )

[0082] (3) Synthesis of amphiphilic oligoxylose-cystamine derivatives

[0083] Under N2 conditions, compound 3 (158.9 mg), compound 6 (445.5 mg, 1.2 mmol), and CuSO4·5H2O (33.61 mg, 0.34 mmol) were weighed and dissolved in 4 mL of N,N-anhydrous dimethylformamide (DMF) solution and added to a 25 mL flask. Ascorbic acid (45.9 mg, 0.66 mmol) was dissolved in 1 mL of DMF and added to the flask. The mixture was stirred at 80°C for 24 h, and after the reaction was completed, it was dialyzed for 48 h. The dialyzed solution was filtered with a funnel and placed in an oven for drying. The solid on the filter paper was dissolved and collected with dichloromethane, filtered, and the solvent was evaporated in a vacuum. Compound 7 was obtained by column chromatography with a column separation yield of 56.5%;

[0084] Under N2 conditions, compound 7 (286.8 mg) and sodium methoxide (32.4 mg, 0.6 mmol) were added to a 10 mL flask. 5 mL of methanol solution was added and stirred at room temperature for 12 h. After the reaction was completed, ion exchange resin was added to neutralize the pH to 7 and stirred at room temperature for another 2 h. The mixture was filtered with methanol solution, and the organic phase was collected and evaporated in vacuo to remove the solvent. A yellow solid compound 8 was obtained (see H NMR spectrum). Figure 6 ) Column separation yield 85%;

[0085] Example 2

[0086] This embodiment follows Figure 3 The chemical reaction equation shown in Figure 1 is used to synthesize sanguinarine iodide (ISA), which specifically includes the following steps:

[0087] (1) Under light-proof conditions, add 1.5 g of crude sanguinarine hydrochloride sample and anhydrous ethanol (20 mL) solution to a 100 mL flask, add 0.1 mol / L NaOH gradually during stirring to adjust the pH to weak alkalinity, and collect the filtrate by suction filtration. At room temperature, add 0.2 g of sodium borohydride to the filtrate every 15 minutes, and monitor the reaction progress by TLC (petroleum ether and ethyl acetate 7:1) until the raw material is completely consumed. Add cotton to the filter paper for filtering, collect the filtrate and evaporate the solvent in a vacuum. The concentrate is ultrasonically extracted with a large amount of dichloromethane for multiple times, add cotton to the filter paper for filtering, then pour into a separatory funnel and wash with water. The collected organic phase is dried over anhydrous sodium sulfate, filtered and evaporated in a vacuum to remove the solvent. The crude product obtained is separated by column chromatography using petroleum ether and ethyl acetate (25:1) as eluent to obtain ISA2, with a column separation yield of 12%;

[0088] (2) At room temperature, ISA2 (61 mg, 0.183 mmol) and iodine (115.9 mg, 0.46 mmol) were dissolved in 5 mL of anhydrous ethanol and added to a 10 mL flask. After reflux heating at 78°C for 2.5 h, the reaction solution was naturally cooled to room temperature, and saturated Na2S2O3 was added dropwise to change the solution from brown-black to light red. The solution was filtered, and the filter paper was placed in an oven and dried at 35°C. The orange-red solid on the filter paper was collected to obtain compound ISA (see H NMR spectrum). Figure 7 ), column separation yield 93.3%.

[0089] Test Example 1

[0090] Test Example 1 of the present invention uses the amphiphilic molecule oligoxylose-cystamine derivative (Compound 8) prepared in Example 1 to form sugar vesicles under hydrophilic and hydrophobic interactions. Sanguinarine iodide (Compound ISA) prepared in Example 2 and baicalin are self-assembled by non-covalent bonds in an aqueous solution to prepare Chinese medicine nanoparticles by ultrasound. Sugar vesicles, Chinese medicine nanoparticles, and probiotics are dissolved in a sodium alginate solution, and then dripped into a calcium chloride solution to prepare sodium alginate multifunctional composite microspheres through ionic crosslinking, and the loading and release effects thereof are studied. The specific method is as follows:

[0091] (1) 3 mg of amphiphilic oligoxylose-cystamine derivative (HXLC) and 2 mL of methanol solution were added to a 5 mL round-bottom flask, and the solvent was evaporated in a vacuum to form a dry lipid film on the flask wall. After adding 3 mL of ultrapure water, the flask was placed in an ultrasonic cleaner for ultrasonic treatment for 20 min and allowed to stand for 1 h to obtain HXLC sugar vesicles.

[0092] (2) Accurately weigh 4.6 mg of ISA powder and pour it into a 250 mL beaker, then add 100 mL of ultrapure water, and stir it evenly with a magnetic stirrer to obtain a 0.1 mM ISA solution. Accurately weigh 4.5 mg of baicalin (BA) powder and pour it into a 250 mL beaker, then add 100 mL of ultrapure water. Slowly add 10 mM NaOH to the stirring solution to dissolve it. Stir it evenly with a magnetic stirrer to obtain a 0.1 mM BA solution. Add 2 mL of 0.1 mM ISA solution and BA solution to a glass test tube respectively, mix them, put them in an ultrasonic cleaner, ultrasonicate them for 15 min, and let them stand for 1 h to obtain ISABA nanoparticles.

[0093] (3) Accurately weigh 100 mg of sodium alginate and pour it into a 25 mL beaker, add 10 mL of ultrapure water. Stir evenly with a magnetic stirrer to obtain a sodium alginate solution. Accurately weigh 1 g of dry CaCl2 solid and pour it into a 250 mL beaker, add 200 mL of ultrapure water. Stir evenly with a magnetic stirrer to obtain a CaCl2 solution, sterilize it under high pressure and store it for later use.

[0094] Ⅰ) Accurately weigh 40 mg of probiotic freeze-dried powder, absorb 1 mL (0.1 mM) of traditional Chinese medicine nanoparticle solution, 1 mL (2 mg / mL) of sugar vesicles, and 2 mL of sodium alginate colloid (1%) into a 25 mL beaker, and stir evenly with a magnetic stirrer to obtain a sodium alginate composite solution (A).

[0095] II) Pour 1 mL (0.1 mM) ISA and 1 mL 1% sodium alginate colloid into a 25 mL beaker and stir evenly with a magnetic stirrer to obtain a sodium alginate composite solution (B).

[0096] Place the prepared CaCl2 solution on a magnetic stirring stirrer and stir at a constant speed. Use a 2μL flat-head liquid phase needle to draw sodium alginate composite solution (A) and (B) respectively and place it at a position 3cm vertical to the liquid surface. Connect the rubber tube of argon gas to a 5mL syringe, align the nipple with the flat-head liquid phase needle needle, and fix it with an iron stand, and push the syringe piston handle at a uniform speed.

[0097] The sodium alginate multifunctional composite microspheres (A) were collected by filtration and characterized by optical microscopy and SEM (e.g. Figure 8 shown).

[0098] Sodium alginate multifunctional composite microspheres (B) were collected with a filter, washed with CaCl2 solution, dried to constant weight, added to PBS buffer solution, and then anhydrous ethanol was added for ultrasonic mixing. After centrifuging the solution, the absorbance of the supernatant was measured, and the encapsulation rate of sodium alginate was calculated to be 77.3% by quantitative analysis. In order to further explore the release rate of ISA in sodium alginate microspheres under different conditions and time. The release ability of sodium alginate multifunctional composite microspheres for ISA was studied by in vitro simulated release experiments. Sodium alginate multifunctional composite microspheres were continuously stirred in PBS (pH = 7.4), pH = 6.8, and pH = 1.2 solutions at 37°C. 100 μL of dialysate was drawn into a 96-well plate every 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours, and its absorbance was measured with the help of an enzyme reader. After each measurement, 100 μL of dialysate was added, and the release curve of sodium alginate multifunctional composite microspheres was calculated after quantitative analysis. The results are shown in Fig. 9 .Depend on Fig. 9 It can be seen that sodium alginate multifunctional composite microspheres showed good release effect.

[0099] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A sodium alginate multifunctional composite microsphere, characterized in that: The sodium alginate multifunctional composite microspheres are obtained by dissolving sugar vesicles, traditional Chinese medicine nanoparticles and probiotics in a sodium alginate solution and then dropping the solution into a calcium chloride solution to undergo ion crosslinking.

2. The method for preparing sodium alginate multifunctional composite microspheres according to claim 1, characterized in that: The following steps are involved: (1) using a monoalkynyl cystamine derivative containing a disulfide bond and a prebiotic oligoxylose derivative to prepare an amphiphilic molecule oligoxylose-cystamine derivative, then mixing the amphiphilic molecule oligoxylose-cystamine derivative with a methanol solution, evaporating the solvent in a vacuum to form a dry lipid film on the bottle wall, then adding ultrapure water and placing the mixture in an ultrasonic cleaning machine for ultrasonic treatment and standing to obtain sugar vesicles; (2) mixing the ISA solution and the BA solution, placing the mixture in an ultrasonic cleaning machine for ultrasonic treatment and allowing the mixture to stand, thereby obtaining Chinese medicine nanoparticles; (3) The sugar vesicles prepared in step (1) are mixed with the traditional Chinese medicine nanoparticles and probiotic freeze-dried powder prepared in step (2) and sodium alginate colloid, and stirred to obtain a sodium alginate composite solution, and then the sodium alginate composite solution is added dropwise to the CaCl2 solution to obtain sodium alginate multifunctional composite microspheres.

3. The method for preparing sodium alginate multifunctional composite microspheres according to claim 2, characterized in that: In step (1), the method for preparing the disulfide bond-containing monoalkynyl cystamine derivative comprises the following steps: (1.1) Cystamine dihydrochloride and NaOH are mixed and stirred at a molar ratio of 1:2 to 2.4, and distilled water is evaporated in vacuo, and the crude product is dissolved in a large amount of dichloromethane and subjected to ultrasonic extraction; the collected organic phase is dried, filtered, and evaporated in vacuo to remove the solvent to obtain cystamine; (1.2) adding cystamine and EDCI obtained in step (1.1) to a flask, adding a dichloromethane solution, stirring, dissolving n-octanoic acid in dichloromethane and adding it to the flask, so that the molar ratio of cystamine to n-octanoic acid is 2.5-3:1, and the mass volume ratio of n-octanoic acid to dichloromethane is 1 g:5-10 mL, mixing to obtain a mixture, stirring the mixture at room temperature, then pouring it into a separatory funnel, washing with a saturated sodium chloride solution, drying the collected organic phase, filtering, evaporating the solvent in a vacuum, and then separating and purifying it by column chromatography to obtain compound 2; (1.3) Add the purified product of step (1.2) and EDCI to a flask, add dichloromethane solution, the mass volume ratio of the purified product of step (1.2) to dichloromethane is 1 g: 10-15 mL, stir, dissolve 5-hexynoic acid in dichloromethane and add it to the flask, so that the mass volume ratio of 5-hexynoic acid to dichloromethane is 1 g: 5-10 mL, and the molar ratio of the purified product of step (1.2) to 5-hexynoic acid is 2.5-3: 1, to obtain a mixture, stir the mixture at room temperature, then pour it into a separatory funnel, wash it with saturated sodium chloride solution, dry the collected organic phase, filter and evaporate the solvent in a vacuum, and then separate and purify it by column chromatography to obtain a monoalkynyl cystamine derivative containing a disulfide bond.

4. The method for preparing sodium alginate multifunctional composite microspheres according to claim 2, characterized in that: In step (1), the method for preparing the prebiotic oligoxylose derivative comprises the following steps: (2.1) Tetraethylene glycol and dichloromethane solution are mixed in a mass volume ratio of 1 g: 10-15 mL, stirred at room temperature, and then p-toluene yellow acid chloride and triethylamine are dissolved in dichloromethane and added to a flask, so that the mass volume ratio of p-toluene yellow acid chloride and dichloromethane solution is 1 g: 5-10 mL, and the mass volume ratio of triethylamine and dichloromethane solution is 1 g: 5-10 mL. The mixture is stirred at room temperature, then poured into a separatory funnel, washed with water, and the collected organic phase is dried, filtered, and the solvent is evaporated in vacuo, and then separated and purified by column chromatography to obtain the product; (2.2) Add xylooligosaccharides and iodine in a mass ratio of 300 mg:25-30 mg to a flask, add acetic anhydride under ice bath to make the mass volume ratio of xylooligosaccharides and acetic anhydride 0.3 g:5-8 mL to obtain a mixture, stir the mixture at room temperature overnight, add saturated Na2S2O3 to the organic phase to adjust it to weak alkalinity, transfer the organic phase to a beaker with dichloromethane, add saturated sodium bicarbonate solution, stir well with a glass rod, pour into a separatory funnel for extraction, extract with water and saturated sodium chloride solution in turn, dry the collected organic phase, filter and evaporate the solvent in vacuo to obtain compound 4; (2.3) Under N2 conditions, the purified products of step (2.1) and step (2.2) are mixed in a mass ratio of 400-410 mg:270 mg, anhydrous dichloromethane is added to dissolve, so that the mass volume ratio of the purified product to dichloromethane is 1 g:5-10 mL, boron trifluoride ether is added dropwise to the flask, so that the mass volume ratio of the purified product in step (2.2) to boron trifluoride ether is 270 mg:135-140 μL, stirred at room temperature, and then poured into a separatory funnel, dichloromethane is added, and extraction is carried out with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence, and the collected organic phase is dialyzed with a dialysis bag, the dialyzed solution is filtered with a funnel and placed in an oven for drying, the product on the filter paper is dissolved and collected with dichloromethane, filtered and evaporated in vacuo to remove the solvent to obtain compound 5; (2.4) Under N2 conditions, compound 5 and sodium azide were mixed in a flask at a mass ratio of 200 mg:20-25 mg, and N,N-anhydrous dimethylformamide solution was added to dissolve the mixture to obtain a mixture. The mixture was stirred for reaction. After the reaction was completed, the mixture was poured into a separatory funnel, and dichloromethane was added. After extraction with saturated sodium bicarbonate solution, water and saturated sodium chloride solution in sequence, the collected organic phase was filtered and the solvent was evaporated in vacuo to obtain a prebiotic oligoxylose derivative.

5. The method for preparing sodium alginate multifunctional composite microspheres according to claim 2, characterized in that: In step (1), the method for preparing the amphiphilic molecule oligoxylose-cystamine derivative comprises the following steps: (3.1) Under N2 conditions, the monoalkynyl cystamine derivative containing a disulfide bond, the prebiotic oligoxylose derivative, CuSO4·5H2O and ascorbic acid are dissolved in N,N-anhydrous dimethylformamide and added to a flask to obtain a mixture, wherein the mass volume ratio of the mixture is 158.9 mg:445.5 mg:33.6 mg:45.9 mg:5-6 mL, the mixture is stirred for reaction, and after the reaction is completed, it is dialyzed, the dialyzed solution is filtered with a funnel and placed in an oven for drying, the solid on the filter paper is dissolved with dichloromethane and collected, filtered, the solvent is evaporated in vacuo, and then separated and purified by column chromatography to obtain compound 7; (3.2) Under N2 conditions, compound 7 and sodium methoxide and methanol solution were mixed in a mass volume ratio of 286.8 mg:33-35 mg:5-6 mL and stirred at room temperature. After the reaction was completed, ion exchange resin was added and stirred at room temperature again. The mixture was filtered with methanol solution, and the organic phase was collected and evaporated in vacuo to remove the solvent to obtain an amphiphilic molecule oligoxylose-cystamine derivative.

6. The method for preparing sodium alginate multifunctional composite microspheres according to claim 2, characterized in that: In step (2), the method for preparing the ISA solution comprises the following steps: (4.1) Under light-proof conditions, a crude sample of sanguinarine hydrochloride and anhydrous ethanol solution were added to a flask at a mass volume ratio of 1.5 g:15-20 mL. 0.1 mol / L NaOH was added gradually during stirring to adjust the pH to a weakly alkaline state. The filtrate was collected by suction filtration, sodium borohydride was added to the filtrate until the raw material was completely consumed, the filtrate was collected and the solvent was evaporated in a vacuum, ultrasonically extracted with dichloromethane, filtered, and then poured into a separatory funnel and washed with water. The collected organic phase was dried, filtered, and the solvent was evaporated in a vacuum, and then purified by column chromatography to obtain compound ISA2; (4.2) At room temperature, compound ISA2 and iodine were dissolved in 5 mL of anhydrous ethanol, the molar ratio of ISA2 to iodine was 1:2.4-2.6; the mass volume ratio of ISA2 to anhydrous ethanol was 61 mg:5-6 mL. After reflux heating for a period of time, saturated Na2S2O3 was added to change the solution from brown-black to light red. The solution was filtered and dried, and the orange-red solid on the filter paper was collected to obtain compound ISA1; (4.3) Compound ISA1 is mixed evenly with ultrapure water to obtain ISA solution.

7. The method for preparing sodium alginate multifunctional composite microspheres according to claim 2, characterized in that: In step (3), the mass volume ratio of the probiotic freeze-dried powder, the traditional Chinese medicine nanoparticles, the sugar vesicles and the sodium alginate colloid is 40 mg: 1 mL: 1 mL: 2 mL.

8. The method for preparing sodium alginate multifunctional composite microspheres according to claim 2, characterized in that: In step (3), the concentration of the sugar vesicles is 2 mg / mL, the concentration of the traditional Chinese medicine nanoparticle solution is 0.1 mM, the mass concentration of the sodium alginate colloid is 1%, and the mass concentration of the CaCl2 solution is 0.5%.

9. The method for preparing sodium alginate multifunctional composite microspheres according to claim 2, characterized in that: In step (1), the ultrasonic treatment time is 20 minutes and the standing time is 1 hour; in step (2), the ultrasonic treatment time is 15 minutes and the standing time is 1 hour; in step (3), the specific method of dripping is to use a 2μL flat-head liquid phase needle to absorb the sodium alginate composite solution and place it at a position 3 cm vertically above the liquid surface, connect the rubber tube mouth of argon gas to a 5mL syringe, align the nipple with the flat-head liquid phase needle needle, and fix it with an iron stand, and push the syringe piston handle at a uniform speed.

10. Use of the sodium alginate multifunctional composite microspheres according to claim 1 in the preparation of sustained-release drugs, characterized in that: The sodium alginate multifunctional composite microspheres can be used for co-loading of prebiotics, traditional Chinese medicine nanoparticles and probiotics.

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