A microalgae-derived glyceroglycoside preparation, and a preparation method and application thereof

Abstract

The present application provides a kind of microalgae source glycerol glucoside preparation and its preparation method and application, belong to medical technical field.The crude extract solution is obtained by low-osmotic extraction liquid extraction to salt stress algal mud, impurity is removed by filtration, and phycocyanin concentrated solution and glycerol glucoside containing permeate are obtained by membrane filtration separation, glycerol glucoside containing permeate is concentrated, bleaching and desalting, drying, and microalgae source glycerol glucoside is prepared, is mixed uniformly with gypenoside and bisabolol, and the mixture is prepared, after being embedded with oleic acid modified cholesterol and phospholipid to prepare modified liposome, mixed with chitosan solution and reacted, centrifuged, and the supernatant is discarded, and the solid is washed and dried to prepare microalgae source glycerol glucoside preparation.The present application realizes the technical effects of microalgae resource green low-carbon high-value utilization, product efficient purification, multi-target point synergistic anti-inflammatory and high transdermal absorption retention, and provides a kind of safe and effective, long-term use natural source new choice for treating atopic dermatitis externally.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a microalgae-derived glycerol glucoside preparation, its preparation method, and its application. Background Technology

[0002] Currently, the clinical treatment of atopic dermatitis (AD) mainly relies on anti-inflammatory drugs such as corticosteroids (e.g., hydrocortisone). While effective, long-term use may lead to side effects such as skin atrophy and immunosuppression. Furthermore, existing non-steroidal topical preparations (e.g., calcineurin inhibitors) also have issues with irritation or limited efficacy.

[0003] Traditional methods for producing glycerol glucosides primarily involve chemical and enzymatic processes. The research team has proposed a method using microalgae to produce glycerol glucosides, enabling direct conversion from carbon dioxide to the product within the same cell. This method boasts high conversion efficiency and low carbon emissions, making it a high-capacity production method. Microalgae, a treasure trove of marine biological resources, are rich in natural components such as polysaccharides, polyunsaturated fatty acids, and carotenoids. Their anti-inflammatory and antioxidant activities have been confirmed by extensive basic research, giving them a unique advantage in developing novel drugs. Glycerol glucoside (GG) is a natural functional molecule synthesized and accumulated by prokaryotic microalgae in response to salt stress. It possesses excellent moisturizing properties and is widely used as a high-end moisturizing ingredient in the daily chemical industry. Recent studies have found that it also exhibits excellent anti-inflammatory activity, significantly alleviating inflammation in mouse skin inflammation models and effectively inhibiting the expression of key inflammatory factors such as IL-6, IL-1β, and TNF-α. It holds promise as a novel candidate drug for the treatment of atopic dermatitis, filling the market gap for naturally derived AD treatments and propelling microalgae resources from basic research to industrial application.

[0004] The inventors in patent CN110804078B provided a more efficient method for pigment removal, including preliminary decolorization using a membrane treatment device and deep decolorization using a silicon-carbon resin adsorbent. This disclosure demonstrates that using the aforementioned resin material to adsorb pigments can effectively improve the adsorption effect on small molecule pigments. However, this method suffers from drawbacks such as poor regeneration performance of the silicon-carbon resin, difficulty in separation, and low purification efficiency.

[0005] Therefore, it is of great significance to develop a safe, effective, and long-term usable natural-derived topical preparation. Summary of the Invention

[0006] The purpose of this invention is to propose a microalgal-derived glycerol glucoside preparation, its preparation method, and its application. Addressing the issues of significant side effects, poor decolorization of microalgal-derived glycerol glucoside, and difficulties in regeneration and separation associated with existing atopic dermatitis treatments, this invention achieves the following technical effects: salt-stressed low-osmotic extraction of microalgae, membrane separation, and decolorization purification using magnetically modified microspheres to obtain glycerol glucoside. This glycerol glucoside is then compounded with asiaticoside and bisabolol and delivered via oleic acid-modified cholesterol liposomes and chitosan encapsulation. This process achieves green, low-carbon, and high-value utilization of microalgal resources, highly efficient and selective purification of the product, multi-target synergistic anti-inflammatory effects, and high transdermal absorption and retention. It provides a safe, effective, and long-term usable new option for topical treatment of atopic dermatitis from a natural source.

[0007] The technical solution of this invention is implemented as follows: This invention provides a method for preparing a microalgal-derived glycerol glucoside preparation. Salt-stressed algal sludge is extracted with a hypotonic extract to obtain a crude extract. After filtration to remove impurities, membrane filtration is used to separate a phycocyanin concentrate and a permeate containing glycerol glucoside. The permeate containing glycerol glucoside is concentrated, decolorized, desalted, and dried to obtain microalgal-derived glycerol glucoside. This mixture is then uniformly mixed with asiaticoside and bisabolol to obtain a mixture. Modified liposomes are prepared by embedding oleic acid-modified cholesterol and phospholipids. These liposomes are then mixed with a chitosan solution, centrifuged, and the supernatant is discarded. The solid is washed and dried to obtain the microalgal-derived glycerol glucoside preparation.

[0008] As a further improvement to the present invention, the following steps are included: S1. Obtaining salt-stressed algal sludge; S2. Mix the salt-stressed algal mud with the hypotonic extract, swell it in the dark, and separate the solid and liquid to obtain the crude extract; S3. The crude extract is filtered through multiple stages to remove impurities, resulting in a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration to obtain a phycocyanin concentrate and a permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside was concentrated, decolorized by modified microspheres, and desalted by cation exchange resin and anion exchange resin to obtain a purified glycerol glucoside solution, which was then dried to obtain microalgae-derived glycerol glucoside. S6. Mix microalgae-derived glycerol glucoside, asiaticoside, and bisabolol evenly to obtain a mixture; S7. Oleic acid, cholesterol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in dimethylformamide, and the mixture was stirred at room temperature under an inert atmosphere. The mixture was then dialyzed, centrifuged, and the supernatant was dried to obtain oleic acid-modified cholesterol. S8. Dissolve the mixture in ethanol, add oleic acid-modified cholesterol and phospholipids, add dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add PBS solution, heat and stir, sonicate, cool, filter, mix the filtrate with chitosan solution, stir to react, centrifuge, discard the supernatant, wash the solid, dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0009] As a further improvement of the present invention, the hypotonic extraction solution in step S2 is a 0.5-1 g / L calcium chloride, potassium chloride, or sodium chloride solution, the volume ratio of the salt-stressed algal sludge to the hypotonic extraction solution is 1:20-50, and the light-shielding swelling time is 24-48 h; the multi-stage filtration in step S3 includes at least two stages of filter membranes with different pore sizes, the pore size decreasing step by step, the first stage filter membrane having a pore size of 0.1 μm-5 μm, and the last stage filter membrane having a pore size of 1500D-0.1 μm; the membrane filtration in step S4 has a membrane pore size of 1500D-0.1 μm.

[0010] As a further improvement of the present invention, the membrane pore size selected for concentration in step S5 is no greater than 300D, and the preparation method of the modified microspheres is as follows: T1. Dissolve sodium phosphate, ammonia, and urea in water to obtain an alkaline solution; dissolve tetrabutyl titanate in ethanol to obtain a tetrabutyl titanate solution; add Fe3O4NPs and hexadecyltrimethylammonium bromide to ethanol and disperse evenly, while simultaneously adding the alkaline solution and tetrabutyl titanate solution dropwise, stirring and mixing evenly, stirring at room temperature, aging, separating with a magnet, washing, drying, and calcining to obtain porous doped TiO2@Fe3O4 microspheres; T2. Porous doped TiO2@Fe3O4 microspheres were added to Tris-HCl solution, dopamine hydrochloride was added, the mixture was heated and stirred, separated by magnet, washed, and dried to obtain PDA@porous doped TiO2@Fe3O4 microspheres; T3. PDA@porous doped TiO2@Fe3O4 microspheres and emulsifier were added to liquid paraffin, followed by the addition of gelatin solution and chitosan solution. The mixture was emulsified, heated and stirred to react, and glutaraldehyde solution was added dropwise. The mixture was then cross-linked and cured, separated by a magnet, washed, and dried to obtain the modified microspheres.

[0011] As a further improvement of the present invention, in step T1, the mass ratio of sodium phosphate, ammonia, urea, tetrabutyl titanate, Fe3O4NPs, and hexadecyltrimethylammonium bromide is 0.5-1.5:2-4:2-6:80-100:30-50:8-12; the stirring reaction at room temperature lasts for 2-4 hours; the aging time is 10-15 hours; and the calcination temperature is 400-500℃ for 2-4 hours. In step T2, the pH value of the Tris-HCl solution is 8.5-9.5. The mass ratio of porous doped TiO2@Fe3O4 microspheres to dopamine hydrochloride is 10:3-6, and the heating and stirring reaction temperature is 45-55℃ for 3-5 hours. In step T3, the emulsifier is a Span-type emulsifier, and the mass ratio of PDA@porous doped TiO2@Fe3O4 microspheres, emulsifier, gelatin, and chitosan is 5-7:0.5-1:20-40:10. The heating and stirring reaction temperature is 45-55℃ for 15-25 minutes, and the crosslinking and curing time is 2-4 hours.

[0012] As a further improvement of the present invention, the mass ratio of microalgae-derived glycerol glucoside, asiaticoside and bisabolol in step S6 is 10:2-5:1-3.

[0013] As a further improvement of the present invention, the mass ratio of oleic acid, cholesterol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in step S7 is 2.8-3:3.8-4:4.9-5.1:0.2-0.3, and the stirring reaction time at room temperature is 10-14 h.

[0014] As a further improvement of the present invention, the mass ratio of the mixture, oleic acid modified cholesterol, phospholipid and chitosan in step S8 is 2-4:4-8:16-32:4-8, the temperature of the heating, stirring and ultrasonication is 40-50℃, the time is 30-50min, the power of the ultrasonication is 200-300W, and the time is 5-15min.

[0015] This invention further protects a microalgal-derived glycerol glucoside preparation obtained by the above-described preparation method.

[0016] This invention further protects the use of the above-mentioned microalgal-derived glycerol glucoside preparation in the preparation of a medicament for the prevention and / or treatment of atopic dermatitis.

[0017] The present invention has the following beneficial effects: 1. This invention uses salt-stressed microalgae as raw material and employs a low-osmotic extraction process to simultaneously obtain phycocyanin concentrate and glycerol glucoside in the same process, achieving the graded extraction and comprehensive utilization of multiple high-value active components from microalgae. Compared to traditional chemical synthesis or enzymatic catalysis methods, this invention utilizes glycerol glucoside generated by the microalgae themselves for extraction, eliminating the need for complex substrate feeding and enzyme catalysts. It boasts advantages such as high conversion efficiency, low carbon emissions, and low production costs, aligning with the development direction of green biomanufacturing.

[0018] 2. This invention innovatively uses gelatin / chitosan@PDA@porous doped TiO2@Fe3O4 magnetic microspheres as a decolorizing material. This microsphere, with Fe3O4 as its magnetic core, endows the material with excellent magnetic responsiveness, enabling rapid separation and recovery via an external magnetic field. It can also be regenerated using solvents, overcoming the shortcomings of traditional silicon-carbon resins, such as difficulty in separation and poor regeneration performance after decolorization. The polydopamine (PDA) layer, through its abundant catechol groups, enhances the adsorption capacity for pigment molecules and exhibits good interaction with chitosan and gelatin, thus forming a gelatin / chitosan shell on the surface of the prepared PDA@porous doped TiO2@Fe3O4 microspheres, achieving in-situ synthesis of the microspheres. The porous doped TiO2 provides a large specific surface area and abundant active sites, and under the synergistic doping effect of P and N, it significantly improves the utilization rate of visible light, thereby effectively decomposing pigment molecules. Simultaneously, the abundant pores also contribute to the adsorption of pigments. The gelatin / chitosan shell further improves biocompatibility and mechanical stability, while also enhancing the adsorption effect on pigment molecules and increasing the decolorization rate. This modified microsphere is reusable, significantly reducing purification costs and improving the purification efficiency and quality of glycerol glucoside.

[0019] 3. This invention scientifically combines microalgae-derived glycerol glucoside with asiaticoside and bisabolol, which work synergistically to exert anti-inflammatory, repairing, and soothing effects. Glycerol glucoside blocks inflammatory signaling pathways by inhibiting the expression of key inflammatory factors such as IL-6, IL-1β, and TNF-α; asiaticoside promotes skin barrier repair and accelerates the healing of damaged tissue; and bisabolol exerts a soothing and calming effect, reducing skin stinging and burning sensations. The synergistic effect of these three components can comprehensively intervene in the pathological process of atopic dermatitis from three dimensions: inflammation suppression, barrier repair, and symptom relief. The efficacy is superior to that of a single component, and it avoids the side effects such as skin atrophy and immunosuppression caused by long-term use of glucocorticoid drugs.

[0020] 4. This invention uses oleic acid to hydrophobically modify cholesterol, and utilizes the transdermal promoting properties of oleic acid to enhance the affinity between liposomes and the stratum corneum of the skin, thereby improving the deformability and transdermal penetration efficiency of liposomes. At the same time, chitosan is used to positively charge the surface of liposomes, which bind tightly to the negatively charged skin surface through electrostatic interaction, prolonging the retention time of active ingredients at the target site on the skin, and achieving sustained release and long-lasting effects of the drug.

[0021] 5. In the preparation method of the present invention, the hypotonic extraction is carried out under normal temperature and light-proof conditions, and the key steps such as membrane filtration, magnetic separation, and liposome preparation are all operated under mild conditions. There is no need for high temperature and high pressure or large-scale use of toxic solvents. The process is highly safe, the equipment requirements are low, and it is easy to achieve large-scale continuous production, which has good industrialization prospects.

[0022] 6. The formulation of this invention uses glycerol glucoside of natural microalgae as the core active ingredient, combined with plant-derived asiaticoside and bisabolol. The entire formula is safe and mild, with no risk of hormone dependence, and can be used for a long time. It fills the market gap for topical drugs for the treatment of atopic dermatitis of natural origin and provides a new candidate drug for clinical use that combines efficacy and safety. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Hysteresis curves of porous doped TiO2@Fe3O4 microspheres prepared in Example 1.

[0025] Figure 2 SEM image of the modified microspheres prepared in Example 4.

[0026] Figure 3 The infrared spectrum of the modified microspheres prepared in Example 4.

[0027] Figure 4 These are comparative photos of mice in each OVA-sensitized group before and after administration in Test Example 5.

[0028] Figure 5 This is a comparison of HE staining in mice from different OVA-sensitized groups after administration in test case 5.

[0029] Figure 6 This is a comparison of TB staining in mice from different OVA-sensitized groups after administration in test case 5. Detailed Implementation

[0030] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Fe3O4NPs are Fe3O4 nanoparticles with an average particle size of 50 nm.

[0032] Preparation Example 1: Porous Doped TiO2@Fe3O4 Microspheres The preparation method is as follows: Dissolve 0.05g sodium phosphate, 0.2g ammonia, and 0.2g urea in 10mL of water to obtain an alkaline solution; dissolve 8g tetrabutyl titanate in 100mL of ethanol to obtain a tetrabutyl titanate solution; add 3g Fe3O4NPs and 0.8g hexadecyltrimethylammonium bromide to 100mL of ethanol and disperse evenly, while simultaneously adding the alkaline solution and the tetrabutyl titanate solution, stirring and mixing evenly, stirring at room temperature for 2h, aging for 10h, separating with a magnet, washing, drying, and calcining at 400℃ under a nitrogen atmosphere for 4h to obtain porous doped TiO2@Fe3O4 microspheres. Figure 1 The figure shows the hysteresis curve of the porous doped TiO2@Fe3O4 microspheres. As can be seen from the figure, the saturation magnetization of the porous doped TiO2@Fe3O4 microspheres is 27 emu / g.

[0033] Preparation Example 2: Porous Doped TiO2@Fe3O4 Microspheres The preparation method is as follows: Dissolve 0.15g sodium phosphate, 0.4g ammonia, and 0.6g urea in 10mL of water to obtain an alkaline solution; dissolve 10g tetrabutyl titanate in 100mL of ethanol to obtain a tetrabutyl titanate solution; add 5g Fe3O4NPs and 1.2g hexadecyltrimethylammonium bromide to 100mL of ethanol and disperse evenly, while simultaneously adding the alkaline solution and the tetrabutyl titanate solution, stirring and mixing evenly, stirring at room temperature for 4h, aging for 15h, separating with a magnet, washing, drying, and calcining at 500℃ under a nitrogen atmosphere for 2h to obtain porous doped TiO2@Fe3O4 microspheres.

[0034] Preparation Example 3: Porous Doped TiO2@Fe3O4 Microspheres The preparation method is as follows: Dissolve 0.1g sodium phosphate, 0.3g ammonia, and 0.4g urea in 10mL of water to obtain an alkaline solution; dissolve 9g tetrabutyl titanate in 100mL of ethanol to obtain a tetrabutyl titanate solution; add 4g Fe3O4NPs and 1g hexadecyltrimethylammonium bromide to 100mL of ethanol and disperse evenly, while simultaneously adding the alkaline solution and the tetrabutyl titanate solution, stirring and mixing evenly, stirring at room temperature for 3h, aging for 12h, separating with a magnet, washing, drying, and calcining at 450℃ under a nitrogen atmosphere for 3h to obtain porous doped TiO2@Fe3O4 microspheres.

[0035] Comparative Preparation Example 1 The only difference from Preparation Example 3 is that sodium phosphate was not added.

[0036] The preparation method is as follows: Dissolve 0.3g ammonia and 0.5g urea in 10mL of water to obtain an alkaline solution; dissolve 9g tetrabutyl titanate in 100mL of ethanol to obtain a tetrabutyl titanate solution; add 4g Fe3O4NPs and 1g hexadecyltrimethylammonium bromide to 100mL of ethanol and disperse evenly, while simultaneously adding the alkaline solution and tetrabutyl titanate solution dropwise, stirring and mixing evenly, stirring at room temperature for 3h, aging for 12h, separating with a magnet, washing, drying, and calcining at 450℃ for 3h to obtain porous doped TiO2@Fe3O4 microspheres.

[0037] Comparative Preparation Example 2 The only difference from Preparation Example 3 is that urea was not added.

[0038] The preparation method is as follows: Dissolve 0.5g sodium phosphate and 0.3g ammonia in 10mL of water to obtain an alkaline solution; dissolve 9g tetrabutyl titanate in 100mL of ethanol to obtain a tetrabutyl titanate solution; add 4g Fe3O4NPs and 1g hexadecyltrimethylammonium bromide to 100mL of ethanol and disperse evenly, while simultaneously adding the alkaline solution and tetrabutyl titanate solution dropwise, stirring and mixing evenly, stirring at room temperature for 3h, aging for 12h, separating with a magnet, washing, drying, and calcining at 450℃ for 3h to obtain porous doped TiO2@Fe3O4 microspheres.

[0039] Comparative preparation example 3 The only difference from Preparation Example 3 is that sodium phosphate and urea were not added.

[0040] The preparation method is as follows: Dissolve 0.3g of ammonia in 10mL of water to obtain an alkaline solution; dissolve 9g of tetrabutyl titanate in 100mL of ethanol to obtain a tetrabutyl titanate solution; add 4g of Fe3O4NPs and 1g of hexadecyltrimethylammonium bromide to 100mL of ethanol and disperse evenly, while simultaneously adding the alkaline solution and the tetrabutyl titanate solution, stirring and mixing evenly, stirring at room temperature for 3h, aging for 12h, separating with a magnet, washing, drying, and calcining at 450℃ for 3h to obtain porous TiO2@Fe3O4 microspheres.

[0041] Comparative preparation example 4 The only difference from Preparation Example 3 is that hexadecyltrimethylammonium bromide was not added.

[0042] The preparation method is as follows: Dissolve 0.1g sodium phosphate, 0.3g ammonia and 0.4g urea in 10mL of water to obtain an alkaline solution; dissolve 9g tetrabutyl titanate in 100mL of ethanol to obtain a tetrabutyl titanate solution; add 4g Fe3O4NPs to 100mL of ethanol and disperse evenly, while simultaneously adding the alkaline solution and the tetrabutyl titanate solution, stirring and mixing evenly, stirring at room temperature for 3h, aging for 12h, separating with a magnet, washing, drying, and calcining at 450℃ for 3h to obtain doped TiO2@Fe3O4 microspheres.

[0043] Test Example 1 The specific surface area and average pore size of the doped TiO2@Fe3O4 microspheres prepared in Examples 1-3 and Comparative Examples 1-4 were determined by nitrogen adsorption-desorption testing (Quantachrome Instruments NOVA 2000). The results are shown in Table 1.

[0044] Table 1

[0045] As can be seen from the table above, the doped TiO2@Fe3O4 microspheres prepared in Examples 1-3 of this invention have a large specific surface area and average pore size.

[0046] Test Example 2 20 mg of doped TiO2@Fe3O4 microspheres prepared in Preparation Examples 1-3 and Comparative Preparation Examples 1-4 and 200 mL of a 10 mg / L Rhodamine B aqueous solution were added to a 250 mL cylindrical reactor. A quartz cooling well with a built-in 500 W xenon lamp was inserted into the reactor, and magnetic stirring was started. After stirring in the dark for 30 min to reach adsorption-desorption equilibrium, the xenon lamp was turned on to begin photocatalytic testing. After 60 min, 5 mL of the solution was collected, centrifuged, and the supernatant was used to measure the absorption spectrum of the Rhodamine B aqueous solution using a spectrophotometer. The intensity of the maximum absorption peak was used as the basis for calculation. The results are shown in Table 2.

[0047] The degradation rate of Rhodamine B was calculated as follows: Rhodamine B degradation rate = (1 - A / A0) × 100% In the formula: A0 is the maximum absorption peak intensity of the initial absorption spectrum, and A is the maximum absorption peak intensity of the absorption spectrum at time t. Table 2

[0048] As can be seen from the table above, the doped TiO2@Fe3O4 microspheres prepared in Examples 1-3 of this invention have a good effect on photocatalytic degradation of organic matter.

[0049] Preparation Example 4: Modified Microspheres The preparation method is as follows: T1. 10g of porous doped TiO2@Fe3O4 microspheres prepared in Preparation Example 1 were added to 300mL of Tris-HCl solution with pH=8.5-9.5, 3g of dopamine hydrochloride was added, the mixture was heated to 55℃, stirred for 3h, separated by magnet, washed, and dried to obtain PDA@porous doped TiO2@Fe3O4 microspheres; T2. Add 0.5g PDA@porous doped TiO2@Fe3O4 microspheres and 0.05g Span 85 to 150mL of liquid paraffin, add 40mL of aqueous solution containing 2g gelatin and 30mL of solution containing 1g chitosan (solvent is 2wt% acetic acid), emulsify at 10000r / min for 15min, heat to 55℃, stir for 15min, add 1mL of 5wt% glutaraldehyde solution, crosslink and cure for 2h, separate with a magnet, wash, dry, and obtain modified microspheres. Figure 2 The image shows a SEM image of the modified microspheres. As can be seen from the image, the microspheres have a porous structure. Figure 3 The infrared spectrum of the prepared modified microspheres.

[0050] Preparation Example 5: Modified Microspheres The preparation method is as follows: T1. 10g of porous doped TiO2@Fe3O4 microspheres prepared in Preparation Example 2 were added to 300mL of Tris-HCl solution with pH=8.5-9.5, 6g of dopamine hydrochloride was added, the mixture was heated to 45℃, stirred for 5h, separated by magnet, washed, and dried to obtain PDA@porous doped TiO2@Fe3O4 microspheres; T2. Add 0.7g PDA@porous doped TiO2@Fe3O4 microspheres and 0.1g Span 85 to 150mL liquid paraffin, add 40mL of aqueous solution containing 4g gelatin and 30mL of solution containing 1g chitosan (solvent is 2wt% acetic acid), emulsify at 10000r / min for 15min, heat to 45℃, stir for 25min, add 1mL 5wt% glutaraldehyde solution, crosslink and cure for 4h, separate with a magnet, wash, dry, and obtain modified microspheres.

[0051] Preparation Example 6: Modified Microspheres The preparation method is as follows: T1. 10g of porous doped TiO2@Fe3O4 microspheres prepared in Preparation Example 3 were added to 300mL of Tris-HCl solution with pH=8.5-9.5, 4.5g of dopamine hydrochloride was added, the mixture was heated to 50℃, stirred for 4h, separated by magnet, washed, and dried to obtain PDA@porous doped TiO2@Fe3O4 microspheres; T2. Add 0.6g of PDA@porous doped TiO2@Fe3O4 microspheres and 0.08g of Span 85 to 150mL of liquid paraffin, add 40mL of aqueous solution containing 3g of gelatin and 30mL of solution containing 1g of chitosan (solvent is 2wt% acetic acid), emulsify at 10000r / min for 15min, heat to 50℃, stir for 20min, add 1mL of 5wt% glutaraldehyde solution, crosslink and cure for 3h, separate with a magnet, wash, dry, and obtain modified microspheres.

[0052] Comparative preparation examples 5-8 The only difference from Preparation Example 6 is that the porous doped TiO2@Fe3O4 microspheres were prepared by Comparative Preparation Examples 1-4.

[0053] Comparative preparation example 9 The only difference from preparation example 6 is that step T1 was not performed.

[0054] The preparation method is as follows: T1. 10g of the porous doped TiO2@Fe3O4 microspheres prepared in Preparation Example 3 were added to 300mL of Tris-HCl solution with pH=8.5-9.5, and 4.5g of dopamine hydrochloride were added. The mixture was heated to 50℃ and stirred for 4h. The mixture was separated by a magnet, washed, and dried to obtain PDA@porous doped TiO2@Fe3O4 microspheres, which are the modified microspheres.

[0055] Comparative preparation example 10 The only difference from preparation example 6 is that step T2 was not performed.

[0056] The preparation method is as follows: 0.6 g of porous doped TiO2@Fe3O4 microspheres prepared in Preparation Example 3 and 0.08 g of Span 85 were added to 150 mL of liquid paraffin. 40 mL of an aqueous solution containing 3 g of gelatin and 30 mL of a solution containing 1 g of chitosan (solvent: 2 wt% acetic acid) were added. The mixture was emulsified at 10000 r / min for 15 min, heated to 50 °C, and stirred for 20 min. 1 mL of 5 wt% glutaraldehyde solution was added dropwise, and the mixture was crosslinked and cured for 3 h. The microspheres were separated by magnetism, washed, and dried to obtain the modified microspheres.

[0057] Test Example 3 Modified microspheres prepared in Preparation Examples 4-6 and Comparative Preparation Examples 5-10 (with a mass of 0.5 g / L) were added to 50 mL of concentrated solution containing glycerol glucoside. The mixture was stirred and oscillated for 60 min for adsorption. The decolorization rate and the recovery rate of glycerol glucoside were calculated. The results are shown in Table 3.

[0058] Table 3

[0059] As shown in the table above, the modified microspheres prepared in Examples 4-6 of this invention have a good decolorization effect on the concentrated solution containing glycerol glucoside, and the recovery rate of glycerol glucoside is high.

[0060] Test Example 4 The modified microspheres prepared in Preparation Example 6 (0.5 g / L) were added to 50 mL of concentrated solution containing glycerol glucoside. The mixture was stirred and oscillated for 60 min to adsorb the microspheres. After magnetic separation, the microspheres were added to a 60 wt% ethanol solution and sonicated at 200 W for 30 min. After magnetic separation, washing, and drying, the decolorization process was repeated five times, and the decolorization rate was calculated for each step. The results are shown in Table 4.

[0061] Table 4

[0062] As can be seen from the table above, the method for regenerating the modified microspheres prepared in Example 6 of this invention is simple, and the regenerated microspheres still have a high decolorization effect.

[0063] Example 1 This embodiment provides a method for preparing a microalgae-derived glycerol glucoside preparation, including the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 500 mL of 0.5 g / L calcium chloride solution, allow it to swell in the dark for 24 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract is filtered through two stages: the first stage has a pore size of 0.1 μm and the last stage has a pore size of 1500D to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 1500D membrane pore size, 30℃ temperature and 1MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 4 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Mix 1g of microalgae-derived glycerol glucoside, 0.2g of asiaticoside, and 0.1g of bisabolol evenly to obtain a mixture; S7. Dissolve 0.28g oleic acid, 0.38g cholesterol, 0.49g dicyclohexylcarbodiimide and 0.02g 4-dimethylaminopyridine in 100mL dimethylformamide, stir at room temperature for 10h under nitrogen protection, dialyze for 48h, centrifuge, freeze dry the supernatant to obtain oleic acid modified cholesterol. S8. Dissolve 20 mg of the mixture in 50 mL of ethanol, add 40 mg of oleic acid-modified cholesterol and 160 mg of phospholipid, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 40 °C, stir for 30 min, sonicate at 200 W for 15 min, cool to 4 °C, filter through a 0.45 μm filter membrane, mix the filtrate with 40 mL of 1 mg / mL chitosan solution (solvent is 2 wt% acetic acid), stir and react for 3 h, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0064] Example 2 This embodiment provides a method for preparing a microalgae-derived glycerol glucoside preparation, including the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 200 mL of 1 g / L calcium chloride solution, allow it to swell in the dark for 48 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract is filtered through two stages: the first stage has a pore size of 5 μm and the last stage has a pore size of 0.1 μm to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 0.1 μm membrane pore size, 40℃ temperature and 2 MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 5 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Mix 1g of microalgae-derived glycerol glucoside, 0.5g of asiaticoside, and 0.3g of bisabolol evenly to obtain a mixture; S7. Dissolve 0.3g oleic acid, 0.4g cholesterol, 0.51g dicyclohexylcarbodiimide and 0.03g 4-dimethylaminopyridine in 100mL dimethylformamide, stir at room temperature for 14h under nitrogen protection, dialyze for 48h, centrifuge, freeze dry the supernatant to obtain oleic acid modified cholesterol. S8. Dissolve 40 mg of the mixture in 50 mL of ethanol, add 80 mg of oleic acid-modified cholesterol and 320 mg of phospholipid, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 50 °C, stir for 50 min, sonicate at 300 W for 5 min, cool to 4 °C, filter through a 0.45 μm filter membrane, mix the filtrate with 40 mL of 2 mg / mL chitosan solution (solvent is 2 wt% acetic acid), stir and react for 3 h, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0065] Example 3 This embodiment provides a method for preparing a microalgae-derived glycerol glucoside preparation, including the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 350 mL of 0.8 g / L calcium chloride solution, allow it to swell in the dark for 36 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract was filtered through two stages: the first stage had a pore size of 0.2 μm and the last stage had a pore size of 3000 D to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 3000D membrane pore size, 35℃ temperature and 1.5MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 6 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Mix 1g of microalgae-derived glycerol glucoside, 0.35g of asiaticoside, and 0.2g of bisabolol evenly to obtain a mixture; S7. Dissolve 0.29g oleic acid, 0.39g cholesterol, 0.5g dicyclohexylcarbodiimide and 0.024g 4-dimethylaminopyridine in 100mL dimethylformamide, stir at room temperature for 12h under nitrogen protection, dialyze for 48h, centrifuge, freeze dry the supernatant to obtain oleic acid modified cholesterol. S8. Dissolve 30 mg of the mixture in 50 mL of ethanol, add 60 mg of oleic acid-modified cholesterol and 280 mg of phospholipid, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 45 °C, stir for 40 min, sonicate at 250 W for 10 min, cool to 4 °C, filter through a 0.45 μm filter membrane, mix the filtrate with 40 mL of 1.5 mg / mL chitosan solution (solvent is 2 wt% acetic acid), stir and react for 3 h, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0066] Comparative Example 1 The only difference from the example is that asiaticoside was not added in step S6.

[0067] Includes the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 350 mL of 3 g / L calcium chloride solution, allow it to swell in the dark for 36 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract was filtered through two stages: the first stage had a pore size of 0.2 μm and the last stage had a pore size of 3000 D to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 3000D membrane pore size, 35℃ temperature and 1.5MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 6 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Mix 1g of microalgae-derived glycerol glucoside and 0.55g of bisabolol evenly to obtain a mixture; S7. Dissolve 0.29g oleic acid, 0.39g cholesterol, 0.5g dicyclohexylcarbodiimide and 0.024g 4-dimethylaminopyridine in 100mL dimethylformamide, stir at room temperature for 48h under nitrogen protection, dialyze for 48h, centrifuge, freeze dry the supernatant to obtain oleic acid modified cholesterol. S8. Dissolve 30 mg of the mixture in 50 mL of ethanol, add 60 mg of oleic acid-modified cholesterol and 280 mg of phospholipid, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 45 °C, stir for 40 min, sonicate at 250 W for 10 min, cool to 4 °C, filter through a 0.45 μm filter membrane, mix the filtrate with 40 mL of 1.5 mg / mL chitosan solution (solvent is 2 wt% acetic acid), stir and react for 3 h, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0068] Comparative Example 2 The only difference from the example is that bisabolol was not added in step S6.

[0069] Includes the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 350 mL of 3 g / L calcium chloride solution, allow it to swell in the dark for 36 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract was filtered through two stages: the first stage had a pore size of 0.2 μm and the last stage had a pore size of 3000 D to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 3000D membrane pore size, 35℃ temperature and 1.5MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 6 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Mix 1g of microalgae-derived glycerol glucoside and 0.55g of asiaticoside evenly to obtain a mixture; S7. Dissolve 0.29g oleic acid, 0.39g cholesterol, 0.5g dicyclohexylcarbodiimide and 0.024g 4-dimethylaminopyridine in 100mL dimethylformamide, stir at room temperature for 48h under nitrogen protection, dialyze for 48h, centrifuge, freeze dry the supernatant to obtain oleic acid modified cholesterol. S8. Dissolve 30 mg of the mixture in 50 mL of ethanol, add 60 mg of oleic acid-modified cholesterol and 280 mg of phospholipid, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 45 °C, stir for 40 min, sonicate at 250 W for 10 min, cool to 4 °C, filter through a 0.45 μm filter membrane, mix the filtrate with 40 mL of 1.5 mg / mL chitosan solution (solvent is 2 wt% acetic acid), stir and react for 3 h, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0070] Comparative Example 3 The only difference from the example is that asiaticoside and bisabolol were not added in step S6.

[0071] Includes the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 350 mL of 3 g / L calcium chloride solution, allow it to swell in the dark for 36 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract was filtered through two stages: the first stage had a pore size of 0.2 μm and the last stage had a pore size of 3000 D to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 3000D membrane pore size, 35℃ temperature and 1.5MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 6 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Dissolve 0.29g oleic acid, 0.39g cholesterol, 0.5g dicyclohexylcarbodiimide and 0.024g 4-dimethylaminopyridine in 100mL dimethylformamide, stir at room temperature for 48h under nitrogen protection, dialyze for 48h, centrifuge, freeze dry the supernatant to obtain oleic acid modified cholesterol. S7. Dissolve 30 mg of microalgal glycerol glucoside in 50 mL of ethanol, add 60 mg of oleic acid-modified cholesterol and 280 mg of phospholipid, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 45 °C, stir for 40 min, sonicate at 250 W for 10 min, cool to 4 °C, filter through a 0.45 μm filter membrane, mix the filtrate with 40 mL of 1.5 mg / mL chitosan solution (solvent is 2 wt% acetic acid), stir and react for 3 h, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgal glycerol glucoside preparation.

[0072] Comparative Example 4 The only difference from the embodiment is that step S7 is not performed.

[0073] Includes the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 350 mL of 3 g / L calcium chloride solution, allow it to swell in the dark for 36 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract was filtered through two stages: the first stage had a pore size of 0.2 μm and the last stage had a pore size of 3000 D to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 3000D membrane pore size, 35℃ temperature and 1.5MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 6 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Mix 1g of microalgae-derived glycerol glucoside, 0.35g of asiaticoside, and 0.2g of bisabolol evenly to obtain a mixture; S7. Dissolve 30 mg of the mixture in 50 mL of ethanol, add 60 mg of cholesterol and 280 mg of phospholipids, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 45 °C, stir for 40 min, sonicate at 250 W for 10 min, cool to 4 °C, filter through a 0.45 μm filter membrane, mix the filtrate with 40 mL of 1.5 mg / mL chitosan solution (solvent is 2 wt% acetic acid), stir and react for 3 h, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0074] Comparative Example 5 The only difference from the example is that chitosan was not added in step S8.

[0075] Includes the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix 10 mL of salt-stressed algal mud with 350 mL of 3 g / L calcium chloride solution, allow it to swell in the dark for 36 h, and centrifuge to separate the solid and liquid phases to obtain the crude extract; S3. The crude extract was filtered through two stages: the first stage had a pore size of 0.2 μm and the last stage had a pore size of 3000 D to remove impurities and obtain a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration. Ultrafiltration was carried out under the conditions of 3000D membrane pore size, 35℃ temperature and 1.5MPa pressure to obtain phycocyanin concentrate and permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside is concentrated, the membrane pore size is not greater than 300D, the modified microspheres prepared in Preparation Example 6 are decolorized, the modified microspheres are regenerated and reused by magnetic separation, and then the liquid is desalted by passing it through cation exchange resin and anion exchange resin in sequence to obtain glycerol glucoside purified solution, which is dried to obtain microalgae-derived glycerol glucoside. S6. Mix 1g of microalgae-derived glycerol glucoside, 0.35g of asiaticoside, and 0.2g of bisabolol evenly to obtain a mixture; S7. Dissolve 0.29g oleic acid, 0.39g cholesterol, 0.5g dicyclohexylcarbodiimide and 0.024g 4-dimethylaminopyridine in 100mL dimethylformamide, stir at room temperature for 48h under nitrogen protection, dialyze for 48h, centrifuge, freeze dry the supernatant to obtain oleic acid modified cholesterol. S8. Dissolve 30 mg of the mixture in 50 mL of ethanol, add 60 mg of oleic acid-modified cholesterol and 280 mg of phospholipids, add 5 mL of dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add 30 mL of PBS solution with pH=6.8, heat to 45 °C, stir for 40 min, sonicate at 250 W for 10 min, cool to 4 °C, filter through a 0.45 μm filter membrane, centrifuge, discard the supernatant, wash the solid, freeze dry, and obtain the microalgae-derived glycerol glucoside preparation.

[0076] Test Example 5 Eight-week-old female SPF-grade BALB / c mice, weighing 18-25g, were selected for the experiment. BALB / c mice exhibited more pronounced Th2-type inflammatory responses and showed higher sensitivity to allergens.

[0077] Ovalbumin (OVA) sensitization model: After hair removal and skin preparation in the same area on the back of mice, OVA hydrogel was applied to the epidermis for three consecutive weeks for sensitization and challenge. After the sensitization was completed, the serum OVA-specific IgE level of mice was significantly increased to more than 1 times that before sensitization by ELISA. Based on the results, the mice were randomly divided into the model group, the example groups 1-3, the comparative examples 1-5, and the positive drug group, with 10 mice in each group.

[0078] After grouping, the sample group received a 10wt% aqueous solution of the prepared microalgal glycerol glucoside formulation, applied topically twice daily at intervals >10 hours, with 0.5 mL administered each time. The model group and negative control group received an equal volume of distilled water. The positive drug group received hydrocortisone cream applied topically every three days.

[0079] At the end of modeling and 30 minutes after drug administration on day 7 of the experiment, the skin on the back of the mice was photographed, collected, and stained with hematoxylin and eosin (HE) and TB. Results are shown below. Figure 4 , 5 6.

[0080] Depend on Figure 4 It can be seen that after applying the microalgae-derived glycerol glucoside preparations obtained in Examples 1-3 of this invention to the epidermis, the skin inflammation in mice was significantly improved compared with the model group, and it had a significant ameliorative effect on OVA-induced dermatitis. Figure 5 It can be seen that the skin tissue morphology and structure of mice in Examples 1-3 and the positive drug group were normal, with intact skin appendages, visible hair follicles in the dermis, normal gland distribution and morphology, no obvious inflammatory cell infiltration, and normal arrangement and distribution of collagen fibers in the dermis. In contrast, the model group mice showed significant thickening of the epidermal spinous layer and dermis, disordered and significantly increased collagen fiber distribution, reduced hair follicles, atrophy of glands, fat layer, and muscle layer, surrounding inflammatory cell infiltration, and unclear boundary between the dermis and subcutaneous tissue. Therefore, the degree of inflammation in Examples 1-3 was significantly improved compared to the model group. Figure 6 It can be seen that there are very few mast cells in the skin of mice in the Example 1-3 groups and the positive drug group, while the number of mast cells in the skin of mice in the model group is significantly increased; the number of mast cells in the Example 1-3 groups is significantly lower than that in the model group.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a microalgae-derived glycerol glucoside preparation, characterized in that, Salt-stressed algal sludge was extracted with a hypotonic extract to obtain a crude extract. After filtration to remove impurities, membrane filtration was used to separate a phycocyanin concentrate and a permeate containing glycerol glucoside. The permeate containing glycerol glucoside was concentrated, decolorized, desalted, and dried to obtain microalgal-derived glycerol glucoside. This was then mixed evenly with asiaticoside and bisabolol to obtain a mixture. Modified liposomes were prepared by encapsulating cholesterol and phospholipids with oleic acid. The mixture was then reacted with chitosan solution, centrifuged, the supernatant was discarded, the solid was washed, and dried to obtain the microalgal-derived glycerol glucoside preparation.

2. The preparation method according to claim 1, characterized in that, Includes the following steps: S1. Obtaining salt-stressed algal sludge; S2. Mix the salt-stressed algal mud with the hypotonic extract, swell it in the dark, and separate the solid and liquid to obtain the crude extract; S3. The crude extract is filtered through multiple stages to remove impurities, resulting in a permeate containing phycocyanin and glycerol glucoside. S4. Phycocyanin and glycerol glucoside were separated by membrane filtration to obtain a phycocyanin concentrate and a permeate containing glycerol glucoside, respectively. S5. The permeate containing glycerol glucoside was concentrated, decolorized by modified microspheres, and desalted by cation exchange resin and anion exchange resin to obtain a purified glycerol glucoside solution, which was then dried to obtain microalgae-derived glycerol glucoside. S6. Mix microalgae-derived glycerol glucoside, asiaticoside, and bisabolol evenly to obtain a mixture; S7. Oleic acid, cholesterol, dicyclohexylcarbodiimide and 4-dimethylaminopyridine were dissolved in dimethylformamide, and the mixture was stirred at room temperature under an inert atmosphere. The mixture was then dialyzed, centrifuged, and the supernatant was dried to obtain oleic acid-modified cholesterol. S8. Dissolve the mixture in ethanol, add oleic acid-modified cholesterol and phospholipids, add dichloromethane to promote dissolution, remove the solvent under reduced pressure to form a lipid film, add PBS solution, heat and stir, sonicate, cool, filter, mix the filtrate with chitosan solution, stir to react, centrifuge, discard the supernatant, wash the solid, dry, and obtain the microalgae-derived glycerol glucoside preparation.

3. The preparation method according to claim 2, characterized in that, In step S2, the hypotonic extract is a 0.5-1 g / L solution of calcium chloride, potassium chloride, or sodium chloride. The volume ratio of the salt-stressed algal sludge to the hypotonic extract is 1:20-50. The time for light-shielding swelling is 24-48 h. In step S3, the multi-stage filtration includes at least two stages of filter membranes with different pore sizes, wherein the pore size decreases progressively. The first-stage filter membrane has a pore size of 0.1 μm-5 μm, and the last-stage filter membrane has a pore size of 1500D-0.1 μm. In step S4, the membrane filtration has a pore size of 1500D-0.1 μm.

4. The preparation method according to claim 2, characterized in that, The membrane pore size selected for concentration in step S5 is no greater than 300D, and the preparation method of the modified microspheres is as follows: T1. Dissolve sodium phosphate, ammonia, and urea in water to obtain an alkaline solution; dissolve tetrabutyl titanate in ethanol to obtain a tetrabutyl titanate solution; add Fe3O4 NPs and hexadecyltrimethylammonium bromide to ethanol and disperse evenly, while simultaneously adding the alkaline solution and tetrabutyl titanate solution dropwise, stirring and mixing evenly, stirring at room temperature, aging, separating with a magnet, washing, drying, and calcining to obtain porous doped TiO2@Fe3O4 microspheres; T2. Porous doped TiO2@Fe3O4 microspheres were added to Tris-HCl solution, dopamine hydrochloride was added, the mixture was heated and stirred, separated by magnet, washed, and dried to obtain PDA@porous doped TiO2@Fe3O4 microspheres; T3. PDA@porous doped TiO2@Fe3O4 microspheres and emulsifier were added to liquid paraffin, followed by the addition of gelatin solution and chitosan solution. The mixture was emulsified, heated and stirred to react, and glutaraldehyde solution was added dropwise. The mixture was then cross-linked and cured, separated by a magnet, washed, and dried to obtain the modified microspheres.

5. The preparation method according to claim 4, characterized in that, In step T1, the mass ratio of sodium phosphate, ammonia, urea, tetrabutyl titanate, Fe3O4 NPs, and hexadecyltrimethylammonium bromide is 0.5-1.5:2-4:2-6:80-100:30-50:8-12. The stirring reaction at room temperature lasts for 2-4 hours, the aging time is 10-15 hours, and the calcination temperature is 400-500℃ for 2-4 hours. In step T2, the pH value of the Tris-HCl solution is 8.5-9.

5. The porous doped TiO2@Fe3O4 microspheres and hydrochloric acid... The mass ratio of dopamine is 10:3-6, the heating and stirring reaction temperature is 45-55℃, and the time is 3-5h; the emulsifier in step T3 is a Span-type emulsifier, the mass ratio of PDA@porous doped TiO2@Fe3O4 microspheres, emulsifier, gelatin and chitosan is 5-7:0.5-1:20-40:10, the heating and stirring reaction temperature is 45-55℃, the time is 15-25min, and the crosslinking and curing time is 2-4h.

6. The preparation method according to claim 2, characterized in that, The mass ratio of microalgal glycerol glucoside, asiaticoside, and bisabolol in step S6 is 10:2-5:1-3.

7. The preparation method according to claim 2, characterized in that, In step S7, the mass ratio of oleic acid, cholesterol, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine is 2.8-3:3.8-4:4.9-5.1:0.2-0.3, and the stirring reaction at room temperature is carried out for 10-14 hours.

8. The preparation method according to claim 2, characterized in that, In step S8, the mass ratio of the mixture, oleic acid modified cholesterol, phospholipid, and chitosan is 2-4:4-8:16-32:4-8. The heating, stirring, and ultrasonication are performed at a temperature of 40-50°C for 30-50 minutes, with an ultrasonic power of 200-300W for 5-15 minutes.

9. A microalgae-derived glycerol glucoside preparation obtained by the preparation method according to any one of claims 1-8.

10. The use of a microalgae-derived glycerol glucoside preparation as described in claim 9 in the preparation of a medicament for the prevention and / or treatment of atopic dermatitis.

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