A composition for preventing and treating infantile eczema and allergy

Abstract

The present application relates to the technical field of chemical synthesis, disclose a kind of composition for preventing and treating infantile eczema and allergy, wherein composition is by supercritical to biomimetic lipid and antifungal lipid cosolvent, through capillary nozzle pressure reduction injection to the water phase receiving liquid containing casein phosphopeptide and lactoferrin peptide, swell and escape drive lipid molecule self-assembly to form multifunctional co-loaded biomimetic liposome, again with water-in-oil base emulsion complex, obtain final product.

Description

Technical Field

[0001] This invention relates to the field of chemical synthesis technology, and more specifically, to a composition for preventing and treating infantile eczema and allergies. Background Technology

[0002] The skin of infants' buttocks is constantly exposed to digestive enzymes from feces and is kept in a moist, enclosed environment, making it susceptible to fungal colonization and complex inflammatory damage. Current technologies typically employ a solvent injection method to prepare biomimetic liposome carriers. This involves dissolving biomimetic lipid components such as ceramides, phytosterols, and fatty acids in a mixed organic solvent of ethanol and propylene glycol, allowing them to self-assemble into liposome vesicles in an aqueous phase. These vesicles are then extruded through a membrane to obtain a repair-type carrier, which simultaneously loads antifungal active lipids and enzyme-inhibiting peptides.

[0003] However, existing technologies have the following problems: First, casein phosphopeptides and lactoferrin peptides undergo desolvation in a mixed solvent of ethanol and propylene glycol, exposing their hydrophobic cores and causing intermolecular aggregation. This results in the loss of their competitive binding ability to digestive enzyme active sites and their chelation ability to calcium ion cofactors. Repeated mechanical shearing during membrane extrusion further damages their remaining higher-order structures, completely inactivating their enzyme inhibitory function. Second, the stratum corneum of infants' buttocks is thin, and eczema causes severe barrier damage. Residual ethanol and propylene glycol easily penetrate, causing irritant contact reactions and exacerbating erythema exudation and allergic symptoms. Third, in schemes that simply mix liposomes and functional peptide carriers after separate preparation, the antifungal lipids and enzyme-inhibiting peptides belong to different carrier systems. The fungal extracellular polysaccharide matrix blocks the penetration of the external peptide carrier, preventing co-localized release within the same damaged micro-region, thus limiting the overall preventative and therapeutic effect. These problems make it difficult for existing technologies to simultaneously meet the requirements of solvent-free safety, preservation of enzyme-inhibiting peptide activity, and co-localized release of multiple active ingredients. Summary of the Invention

[0004] This invention provides a composition for preventing and treating infantile eczema and allergies, solving the technical problems of poor stability of active ingredients, low skin penetration efficiency, and insufficient multifunctional synergy in related art compositions for the prevention and treatment of infantile diaper rash and skin allergies.

[0005] This invention discloses a method for preparing a composition for preventing and treating infantile eczema and allergies, comprising the following steps: Plant ceramides, -Sitosterol, palmitic acid, and glyceryl monolaurate were placed in a supercritical reactor and purged with... The solution is obtained by stirring and dissolving at a pressure of 15 to 25 MPa and a temperature of 45 to 55 °C to obtain a supercritical solution. Tea tree oil and oat kernel oil were premixed and then injected into the supercritical solution, stirred and incubated to form a co-solution system; Casein phosphopeptides and lactoferrin peptides were dissolved in deionized water containing glycerol and D-panthenol, and the pH was adjusted to 6.0 to 6.8 to obtain an aqueous receiving solution. The co-solution system is injected under reduced pressure into the aqueous receiving liquid through a capillary nozzle. The lipid molecules vaporize and escape, and then self-assemble at the aqueous interface to form multifunctional co-loaded biomimetic liposomes. The multifunctional co-loaded biomimetic liposomes were collected after degassing and filtration. The multifunctional co-loaded biomimetic liposomes were added to a water-in-oil matrix emulsion and mixed at low speed to obtain the composition.

[0006] Further, the plant ceramide is N-palmitoylsphingosine, used in an amount of 2 to 6 parts by weight; - The amount of sitosterol is 1 to 4 parts by weight; the amount of palmitic acid is 1 to 3 parts by weight; the amount of lauric acid monoglyceride is 1 to 3 parts by weight; the mass ratio of the four is (2 to 6): (1 to 4): (1 to 3): (1 to 3).

[0007] Further, the amount of tea tree oil used is 1 to 3 parts by weight, wherein the mass fraction of terpinene-4-ol is not less than 35%; the amount of oat kernel oil used is 1 to 4 parts by weight; after the tea tree oil and oat kernel oil are premixed at 35 to 40°C, they are injected into the supercritical solution by a high-pressure metering pump, and stirred and incubated at 15 to 25 MPa and 45 to 55°C for 20 to 40 minutes.

[0008] Further, the amount of casein phosphopeptide is 3 to 7 parts by weight, and the molecular weight is 1000 to 5000 Da; the amount of lactoferrin peptide is 1 to 4 parts by weight, and the molecular weight is 500 to 3000 Da; the amount of deionized water is 150 to 250 parts by weight; the amount of glycerol is 4 to 10 parts by weight; the amount of D-panthenol is 0.5 to 2 parts by weight; the dissolution temperature is 35 to 42°C; and the stirring rate is 300 to 600 rpm.

[0009] Furthermore, the capillary nozzle has an inner diameter of 50 to 150 μm, a jet flow rate of 2 to 8 mL / min, and a receiving container pressure of 0.1 to 0.5 MPa; the aqueous receiving liquid is continuously stirred at 1000 to 2000 rpm during the jetting process, and the temperature is maintained at 20 to 30°C.

[0010] Further, the water-in-oil matrix emulsion is prepared by the following method: 10 to 25 parts by weight of lanolin and 3 to 8 parts by weight of beeswax are heated and melted at 65 to 80°C, and 3 to 6 parts by weight of polyglycerol-2 dihydroxystearate and 1 to 3 parts by weight of glyceryl oleate are added and stirred evenly to obtain an oil phase; 0.5 to 1.5 parts by weight of sodium chloride are dissolved in 20 to 50 parts by weight of deionized water, heated to 60 to 70°C and added to the oil phase, and emulsified at 4000 to 8000 rpm for 15 to 30 min.

[0011] Furthermore, the degassing is performed by standing at 25 to 35°C for 30 to 60 minutes; the filtration is performed by positive pressure filtration through a 0.45μm microporous membrane, with a filtration pressure not exceeding 0.1MPa.

[0012] Further, after cooling the water-in-oil matrix emulsion to 30 to 38°C, the multifunctional co-loaded biomimetic liposomes are added, and the mixture is stirred at 800 to 1500 rpm for 10 to 20 minutes; then, 2 to 5 parts by weight of caprylic acid glyceride, 0.3 to 1 part by weight of p-hydroxyacetophenone, and D- Add 0.1 to 0.5 parts by weight of tocopherol, stir until homogeneous, and then cool to room temperature.

[0013] Furthermore, before adding the multifunctional co-loaded biomimetic liposomes, 0.2 to 0.8 parts by weight of allantoin are added to a water-in-oil matrix emulsion cooled to 30 to 38°C, and the mixture is stirred at 800 to 1000 rpm for 5 to 10 minutes to dissolve before adding the multifunctional co-loaded biomimetic liposomes.

[0014] This invention discloses a composition for preventing and treating infantile eczema and allergies prepared by the above-described preparation method.

[0015] The beneficial effects of this invention are as follows: This invention uses supercritical By replacing the ethanol and propylene glycol mixed organic solvent as the lipid dissolution medium, a multifunctional co-loaded biomimetic liposome was prepared through a reduced-pressure jet self-assembly process. This solved the technical problems in the prior art, such as skin irritation caused by residual organic solvents, loss of activity of enzyme-inhibiting peptides due to desolvation and mechanical shearing, and the inability of antifungal components and enzyme-inhibiting components to be co-located and released. The results achieved were that there were no residual organic solvents in the final product, the active conformations of casein phosphopeptides and lactoferrin peptides were preserved throughout the preparation process, and antifungal lipids and enzyme-inhibiting peptides were co-loaded in the same nanoscale liposome and could be released synchronously in the micro-regions of damaged skin. Attached Figure Description

[0016] Figure 1 This is a flowchart illustrating the preparation process of the composition for preventing and treating infantile eczema and allergies provided in this embodiment of the invention. Figure 2This is a schematic diagram comparing the average particle size of liposomes in each sample with the PDI provided in the embodiments of the present invention; Figure 3 This is a schematic diagram comparing the encapsulation rates of functional peptides in various samples provided in the embodiments of the present invention; Figure 4 This is a schematic diagram comparing the enzyme inhibition rate and antifungal MIC of each sample provided in the embodiments of the present invention; Figure 5 This is a schematic diagram showing the comparison of Pearson colocation coefficients for various samples provided in an embodiment of the present invention; Figure 6 This is a schematic diagram of the SEM morphology of the multifunctional co-loaded biomimetic liposomes provided in the embodiments of the present invention; Figure 7 This is a schematic diagram of the FTIR spectrum of the multifunctional co-loaded biomimetic liposomes provided in the embodiments of the present invention; Figure 8 This is a schematic diagram of a dual-channel fluorescence confocal microscopy image provided in an embodiment of the present invention. Detailed Implementation

[0017] The subject matter described herein will now be discussed with reference to exemplary embodiments. It should be understood that these embodiments are discussed only to enable those skilled in the art to better understand and implement the subject matter described herein, and changes may be made to the function and arrangement of the elements discussed without departing from the scope of this specification. Various processes or components may be omitted, substituted, or added as needed in the examples. Furthermore, some features described in the examples may be combined in other examples.

[0018] At least one embodiment of the present invention discloses a method for preparing a composition for preventing and treating infantile eczema and allergies, see below. Figure 1 ,include: Step 1: Supercritical synthesis of biomimetic lipids and antifungal lipids Co-solution Add 2 to 6 parts by weight of plant ceramide, - Mix 1 to 4 parts by weight of sitosterol, 1 to 3 parts by weight of palmitic acid and 1 to 3 parts by weight of laurate monoglyceride, and place in a supercritical reactor. Then, introduce... The pressure was increased to 15 to 25 MPa, the temperature was increased to 45 to 55 °C, and the mixture was stirred and dissolved for 30 to 60 minutes to obtain a supercritical solution containing multifunctional lipids.

[0019] It should be noted that the aforementioned plant ceramide is N-palmitoylsphingosine (ceramide NP), with a molecular weight of 537.9 Da, a purity of 95%, a hydroxyl value of 100 to 110 mgKOH / g, and a melting point of 100 to 105℃. N-palmitoylsphingosine is one of the main components of intercellular lipids in the human stratum corneum, and has a high degree of similarity to the structure of natural lipids in the skin, making it suitable for participating in the reconstruction of a biomimetic lipid barrier in the intercellular spaces of the stratum corneum.

[0020] It should be noted that the above -Sitosterol purity$ β-Sitosterol, a plant sterol, has a structure similar to cholesterol and can regulate the fluidity and stability of the liposome bilayer membrane.

[0021] It should be noted that the palmitic acid mentioned above is hexadecanoic acid, with a purity of 98%, a melting point of 62 to 64°C, and a molecular weight of 256.4 Da. Palmitic acid is a natural fatty acid component of stratum corneum lipids and participates in the formation of the liposome bilayer membrane.

[0022] It should be noted that the purity of the above-mentioned glyceryl monolaurate is [missing information]. $1 / 100g, with a molecular weight of 274.4 Da. Laurate monoglyceride is a medium-chain fatty acid monoglyceride with antifungal activity and can be embedded in the liposome bilayer membrane structure.

[0023] It should be noted that the above-mentioned supercritical In the dissolution conditions, the preferred pressure is 18 to 22 MPa, the preferred temperature is 48 to 52 °C, and the preferred stirring and dissolution time is 40 to 50 min. These preferred conditions ensure that each lipid component is dissolved in supercritical fluid. The optimal operating range was determined under the premise of complete dissolution.

[0024] Furthermore, in step 1 above, plant ceramides, The mass ratio of sitosterol, palmitic acid, and lauric acid monoglycerides is (2 to 6):(1 to 4):(1 to 3):(1 to 3), preferably (3 to 5):(2 to 3):(1 to 2):(1 to 2). The total amount of the above four lipid components relative to the amount of deionized water used in step 3 is (6 to 16):(150 to 250), preferably (7 to 12):(180 to 220).

[0025] Furthermore, in step 1 above, the heating rate during the stirring and dissolving stage is 1 to 3 °C / min, increasing the temperature from room temperature to the target temperature of 45 to 55 °C, preferably 1.5 to 2 °C / min. An excessively rapid heating rate can lead to localized overheating within the reactor, affecting the performance of each lipid component in the supercritical fluid. Uniform dissolution in the solution.

[0026] Furthermore, in step 1 above, the input... Beforehand, the supercritical reactor was purged with nitrogen three times, each time for 5 to 10 minutes, to remove residual air from the reactor and prevent the lipid components from oxidizing during the heating process.

[0027] Furthermore, in step 1 above, the supercritical reactor is a high-pressure device with an operating pressure of 15 to 25 MPa. Before operation, it must be confirmed that the pressure resistance of the reactor and high-pressure pipeline meets the operating pressure requirements. The reactor body is equipped with a calibrated pressure gauge and a pressure safety valve. The pressure set by the safety valve shall not exceed 1.1 times the maximum working pressure of the reactor. During the pressurization process, the operator must wear a protective mask and protective gloves and stay away from the direction of the reactor exhaust port.

[0028] Step 2: Supercritical co-solubilization of volatile antifungal components with soothing plant oils 1 to 3 parts by weight of tea tree oil and 1 to 4 parts by weight of oat kernel oil are premixed at 35 to 40°C. The premixed solution is then injected into the reactor containing the supercritical solution of multifunctional lipids obtained in step 1. The mixture is stirred and incubated at 15 to 25 MPa and 45 to 55°C for 20 to 40 minutes to allow the tea tree oil and oat kernel oil to form a co-soluble system with the biomimetic lipid molecules.

[0029] It should be noted that the content of terpinene-4-ol in the above-mentioned tea tree oil is $ $15% (mass fraction), density 0.895 to 0.910 g / mL (20°C). Terpinene-4-ol is the main antifungal active ingredient in tea tree oil, and controlling the content of 1,8-cineole helps reduce irritation to the skin of infants and young children.

[0030] It should be noted that the above-mentioned oat kernel oil is cold-pressed oat kernel oil, with a linoleic acid content of [missing information]. $2 mgKOH / g. Linoleic acid and natural ceramides in oat kernel oil can synergistically enhance the skin's lipid replenishment function.

[0031] It should be noted that the premixing temperature is preferably 37 to 39°C, and the supercritical co-dissolution stirring and incubation time is preferably 25 to 35 minutes. This premixing temperature is lower than the supercritical operating temperature, which can prevent excessive volatilization of volatile active ingredients in tea tree oil during the premixing stage.

[0032] Furthermore, in step 2 above, the mass ratio of tea tree oil to oat kernel oil is (1 to 3):(1 to 4), preferably (1 to 2):(2 to 3). The total amount of tea tree oil and oat kernel oil relative to the total amount of the four lipid components in step 1 is (2 to 7):(6 to 16), preferably (3 to 5):(7 to 12).

[0033] Furthermore, in step 2 above, the stirring rate of the premixing operation is 200 to 400 rpm, preferably 250 to 350 rpm, and the premixing time is 5 to 15 min, preferably 8 to 12 min, to ensure that the tea tree oil and oat kernel oil are fully and evenly mixed before being injected into the supercritical reactor.

[0034] Furthermore, in step 2 above, when the premixed liquid is injected into the supercritical reactor, it is slowly injected at a rate of 0.5 to 2.0 mL / min using a high-pressure metering pump. During the injection process, the pressure fluctuation inside the reactor does not exceed ±0.5 MPa, so as to maintain the stability of the supercritical state inside the reactor.

[0035] Furthermore, in step 2 above, the terpene active ingredients such as terpinene-4-ol in tea tree oil are subjected to supercritical fluid extraction. The solubility of terpinene-4-ol decreases with increasing temperature. To suppress the volatilization loss of terpinene-4-ol during the supercritical co-solution stage, the co-solution temperature is strictly controlled within the range of 45 to 55°C and must not exceed 55°C. The reactor must be kept sealed before spraying to maintain the supercritical state inside the reactor to dissolve and encapsulate the volatile components.

[0036] Furthermore, in step 2 above, tea tree oil is a flammable essential oil with a flash point of approximately 60°C. Premixing operations must be carried out away from open flames and heat sources, and the premixing container should be placed in a fume hood. Operators must wear chemical-resistant gloves and goggles to avoid direct contact between tea tree oil and skin and eyes.

[0037] Step 3: Preparation of the aqueous receiving solution containing enzyme-inhibiting functional peptides Dissolve 3 to 7 parts by weight of casein phosphopeptide and 1 to 4 parts by weight of lactoferrin peptide in 150 to 250 parts by weight of deionized water, add 4 to 10 parts by weight of glycerol and 0.5 to 2 parts by weight of D-panthenol, stir at 35 to 42°C until completely dissolved, adjust the pH to 6.0 to 6.8 to obtain an aqueous receiving solution containing enzyme-inhibiting peptides, transfer it to a vacuum receiving container and stir continuously at 1000 to 2000 rpm for later use.

[0038] It should be noted that the molecular weight of the above casein phosphopeptides is 1000 to 5000 Da, and the content of the phosphoserine residue cluster is $ $90%. The phosphoserine cluster sequence (-SerP-SerP-SerP-Glu-Glu-) in casein phosphopeptides can chelate calcium ion cofactors and inhibit the activity of digestive enzymes in feces.

[0039] It should be noted that the molecular weight of the above-mentioned lactoferrin peptides is 500 to 3000 Da, and the iron-binding saturation is [missing information]. $85%. Lactoferrin peptides can competitively bind to the active sites of digestive enzymes, thereby inhibiting enzyme activity.

[0040] It should be noted that the purity of the aforementioned D-panthenol is 98%, and its molecular weight is 205.3 Da. The preferred amount of glycerin is 5 to 8 parts by weight, and the preferred amount of D-panthenol is 0.8 to 1.5 parts by weight. Glycerin acts as a moisturizing stabilizer to maintain the osmotic pressure of the aqueous phase, while D-panthenol serves as an auxiliary ingredient for skin repair.

[0041] It should be noted that the pH of the aqueous receiving solution is preferably 6.2 to 6.5, adjusted with a 5% to 10% (w / w) citric acid solution or a 5% to 10% (w / w) disodium hydrogen phosphate solution. The pH is measured using a calibrated glass electrode pH meter. This pH range is close to the physiological pH of infant skin, which can maintain the conformational stability of casein phosphopeptides and lactoferrin peptides and avoid denaturation of casein phosphopeptides and lactoferrin peptides due to excessively low or high pH.

[0042] It should be noted that the preferred amount of casein phosphopeptide is 4 to 6 parts by weight, the preferred amount of lactoferrin peptide is 2 to 3 parts by weight, and the preferred amount of deionized water is 180 to 220 parts by weight. The mass ratio of casein phosphopeptide to lactoferrin peptide is (3 to 7):(1 to 4), preferably (4 to 6):(2 to 3). The mass ratios of glycerol and D-panthenol relative to deionized water are (4 to 10):(150 to 250) and (0.5 to 2):(150 to 250), respectively, preferably (5 to 8):(180 to 220) and (0.8 to 1.5):(180 to 220), respectively.

[0043] Furthermore, in step 3 above, the dissolution stirring rate of casein phosphopeptides and lactoferrin peptides is 300 to 600 rpm, preferably 400 to 500 rpm; the dissolution time is 20 to 40 minutes, preferably 25 to 35 minutes. Excessive stirring rate will cause unnecessary mechanical shear damage to the higher-order structures of casein phosphopeptides and lactoferrin peptides, while excessively low stirring rate will prolong the dissolution time, which is detrimental to production efficiency.

[0044] Furthermore, in step 3 above, the deionized water is treated with nitrogen bubbling for 15 to 30 minutes before use, with a bubbling flow rate of 100 to 200 mL / min, in order to reduce the dissolved oxygen content and prevent the oxidation of casein phosphopeptides and lactoferrin peptides during the preparation of the aqueous phase.

[0045] Furthermore, in step 3 above, casein phosphopeptides and lactoferrin peptides tend to aggregate in the aqueous phase due to excessively high local concentrations. To suppress this aggregation side reaction, the dissolution operation must be carried out under mild conditions of 35 to 42°C, and the stirring rate should be controlled to not exceed 600 rpm. After the casein phosphopeptide and lactoferrin peptide solutions are prepared, they should be transferred to step 4 for use within 4 hours to avoid the accumulation of aggregates due to prolonged standing.

[0046] Furthermore, in step 3 above, when adjusting the pH using citric acid solution, citric acid is a weak acid, so chemical-resistant gloves must be worn during operation to avoid splashing the solution into the eyes; similarly, chemical-resistant gloves and goggles must be worn when adjusting the pH using disodium hydrogen phosphate solution. Waste pH adjusting solution generated during the pH adjustment process must be collected and disposed of properly, and must not be directly discharged into the sewer system.

[0047] Step 4: Supercritical decompression jet self-assembly to form multifunctional co-loaded biomimetic liposomes The co-solution system obtained in step 2 is sprayed into the aqueous receiving solution containing the enzyme-inhibiting functional peptide obtained in step 3 through a capillary nozzle with an inner diameter of 50 to 150 μm at a flow rate of 2 to 8 mL / min, while controlling the pressure of the receiving container to be 0.1 to 0.5 MPa. Under reduced pressure, the lipid molecules rapidly expand and vaporize, escaping and forming biomimetic liposomes at the aqueous interface. Laurate monoglycerides and tea tree oil are embedded within the liposome bilayer membrane, while casein phosphopeptides and lactoferrin peptides in the aqueous phase are simultaneously encapsulated within the aqueous chambers of the liposomes, resulting in a multifunctional co-loaded biomimetic liposome dispersion.

[0048] It should be noted that the above-mentioned supercritical depressurized jet self-assembly is an unconventional process. The specific steps are as follows: First, the co-solution system in the supercritical reactor is transported to the capillary nozzle inlet through a high-pressure pipeline. The nozzle outlet is immersed below the surface of the aqueous receiving liquid containing the enzyme-inhibiting functional peptide in the depressurized receiving container. The reactor maintains a pressure of 15 to 25 MPa to continuously push the co-solution system into the nozzle. When the co-solution system passes through the nozzle outlet, the pressure drops sharply from the supercritical state to 0.1 to 0.5 MPa in the receiving container. Under this pressure drop condition, it instantly transforms from a supercritical state to a gaseous state and rapidly expands and escapes, dissolving in the supercritical fluid. The lipid molecules in the solvent ( The lipids instantly disappear and precipitate due to supersaturation, spontaneously assembling into vesicle structures at the aqueous interface. In this process, lipid self-assembly and the encapsulation of casein phosphopeptides and lactoferrin peptides occur within the same spatiotemporal window, requiring no additional encapsulation steps.

[0049] It should be noted that the inner diameter of the capillary nozzle is preferably 80 to 120 μm, the jet velocity is preferably 3 to 6 mL / min, and the pressure of the receiving container is preferably 0.2 to 0.4 MPa. This combination of parameters is advantageous. Expansion and vaporization on a millisecond timescale drive the rapid self-assembly of lipid molecules at the aqueous interface, resulting in liposomes with a relatively uniform particle size distribution. A capillary nozzle with an inner diameter that is too small will cause flow channel blockage, while one with an inner diameter that is too large will... The decompression rate decreases, resulting in insufficient driving force for lipid self-assembly.

[0050] It should be noted that the stirring speed of the aqueous receiving solution containing the enzyme-inhibiting functional peptide in the above steps is maintained at 1000 to 2000 rpm, preferably 1200 to 1800 rpm. Stirring ensures that the sprayed droplets are uniformly dispersed in the aqueous receiving solution containing the enzyme-inhibiting functional peptide, and avoids the aggregation of lipid molecules in local high-concentration areas to form large particles.

[0051] Furthermore, in step 4 above, during the spraying operation, the temperature of the aqueous receiving liquid containing the enzyme-inhibiting functional peptide in the receiving container is maintained at 20 to 30°C, preferably 22 to 28°C, and kept constant by a circulating water bath temperature control method, with a temperature deviation not exceeding ±1°C. Excessively high aqueous phase temperature will reduce... The solubility in the aqueous phase accelerates the rate of bubble escape and affects the uniformity of liposome particle size; if the temperature of the aqueous phase is too low, the fluidity of lipid molecules will decrease, which is not conducive to the complete formation of vesicle structure.

[0052] Furthermore, in step 4 above, the top space of the receiving container is maintained at a back pressure of 0.1 to 0.5 MPa with nitrogen gas of 99.9% purity to prevent external air from mixing into the receiving container and to maintain the stability of the pressure inside the container.

[0053] Furthermore, in step 4 above, During the decompression expansion process, the gas escaping from the receiving container The gas is led out through the top exhaust pipe, treated by the tail gas absorption device (alkaline scrubbing tower) before being discharged, preventing... Pressure can accumulate in the operating area. A pressure relief valve is installed on the top of the receiving container to automatically release pressure when the pressure inside the container exceeds the set upper limit of 0.6 MPa, ensuring operational safety.

[0054] Furthermore, in step 4 above, the supercritical depressurization injection operation involves a rapid drop from high pressure to atmospheric pressure. Before initiating the injection, operators must confirm the integrity of the seals at all connections of the high-pressure pipeline, capillary nozzle, and receiving container. During operation, operators must wear protective masks and earmuffs, and maintain a safe distance from the high-pressure pipeline to prevent injury from high-pressure fluid jets caused by pipeline leaks or nozzle detachment. The operating area must be well-ventilated to prevent the escape of... Gases accumulate in low-lying areas, leading to localized oxygen deficiency.

[0055] Step 5: Degassing and Filtration Collection The multifunctional co-loaded biomimetic liposome dispersion obtained in step 4 was allowed to stand at 25 to 35°C for 30 to 60 minutes to degas, and the filtrate was collected by filtration through a 0.45 μm microporous membrane to obtain multifunctional co-loaded biomimetic liposomes without solvent residue.

[0056] It should be noted that the preferred static degassing temperature is 28 to 32°C, and the preferred static degassing time is 40 to 50 minutes. This operation allows residual dissolved substances in the multifunctional co-loaded biomimetic liposome dispersion to be removed. Sufficient filtration prevents air bubbles from forming during subsequent storage and use, thus avoiding impacts on liposome stability. A 0.45μm microporous membrane removes incompletely encapsulated lipid aggregates and a small number of large particles.

[0057] Furthermore, in step 5 above, the encapsulation efficiency of the obtained multifunctional co-loaded biomimetic liposomes was determined by ultracentrifugation: the multifunctional co-loaded biomimetic liposome dispersion was centrifuged at 100,000 × g for 60 min, the supernatant was separated, and the mass concentrations of free casein phosphopeptides and lactoferrin peptides in the supernatant were determined by high performance liquid chromatography, and the encapsulation efficiency was determined. Calculate using the following formula: in, This refers to the total mass of casein phosphopeptides and lactoferrin peptides at the time of feeding. This refers to the mass of free casein phosphopeptides and lactoferrin peptides in the supernatant.

[0058] Furthermore, in step 5 above, the 0.45 μm microporous membrane filtration operation adopts a positive pressure filtration method, with the filtration pressure not exceeding 0.1 MPa, to avoid excessive pressure damaging the liposome vesicle structure. After filtration, the particle size distribution and polydispersity index (PDI) of the liposomes in the filtrate are determined by dynamic light scattering to confirm that the filtration operation did not cause significant changes in the liposome particle size distribution.

[0059] Furthermore, in step 5 above, the lipid aggregates and large-diameter particles filtered and retained are treated as lipid-containing solid waste and must be collected and handed over to a professional waste treatment agency for disposal; they must not be discarded at will.

[0060] Step 6: Preparation of the oil phase of a water-in-oil matrix emulsion Step 6 is performed in parallel with steps 1 to 5. 10 to 25 parts by weight of lanolin and 3 to 8 parts by weight of beeswax are heated and melted at 65 to 80°C. 3 to 6 parts by weight of polyglycerol-2 dihydroxystearate and 1 to 3 parts by weight of glyceryl oleate are added and stirred until homogeneous to obtain the oil phase.

[0061] It should be noted that the above-mentioned lanolin is refined pharmaceutical grade lanolin, with an acid value of 1 mgKOH / g and an iodine value of 18 to 36. / 100g, moisture content $$0.5%, melting point 36 to 42℃. The above beeswax is a white refined beeswax with an acid value of 17 to 24 mgKOH / g, a melting point of 62 to 65℃, and a saponification value of 87 to 104 mgKOH / g. Lanolin provides occlusive moisturizing function for the skin, while beeswax provides structural support.

[0062] It should be noted that the above-mentioned polyglycerol-2-dimeric hydroxystearate has a hydrophilic-lipophilic balance value (HLB value) of 3 to 5, and is used as a water-in-oil emulsifier. The above-mentioned glyceryl oleate has a hydrophilic-lipophilic balance value (HLB value) of 3.4, and is used as a co-emulsifier. The mass ratio of polyglycerol-2-dimeric hydroxystearate to glyceryl oleate is (3 to 6):(1 to 3), preferably (4 to 5):(1.5 to 2.5), to synergistically form a stable water-in-oil emulsion interface film.

[0063] It should be noted that the preferred heating and melting temperature is 70 to 75°C, the preferred amount of lanolin is 15 to 20 parts by weight, the preferred amount of beeswax is 4 to 6 parts by weight, the preferred amount of polyglycerol-2 dihydroxystearate is 4 to 5 parts by weight, and the preferred amount of glyceryl oleate is 1.5 to 2.5 parts by weight. The mass ratio of lanolin to beeswax is (10 to 25):(3 to 8), and the preferred mass ratio is (15 to 20):(4 to 6).

[0064] Furthermore, in step 6 above, after the lanolin and beeswax are heated and melted, the stirring rate after adding polyglycerol-2 dihydroxystearate and glyceryl oleate is 200 to 400 rpm, preferably 250 to 350 rpm, and the stirring time is 10 to 20 min, preferably 12 to 18 min, to ensure that polyglycerol-2 dihydroxystearate and glyceryl oleate are uniformly dispersed in the oil phase.

[0065] Furthermore, in step 6 above, lanolin contains unsaturated fatty acid ester components, which pose a risk of oxidation side reactions during the heating stage at 65 to 80°C. To suppress oil phase oxidation, the heating and melting operation is carried out under nitrogen protection. Nitrogen is continuously introduced into the top space of the melting container at a flow rate of 50 to 100 mL / min to maintain positive nitrogen pressure inside the container and prevent oxygen in the air from contacting the oil phase.

[0066] Furthermore, in step 6 above, the heating and melting operation is carried out at 65 to 80°C. Operators must wear heat-resistant gloves and goggles to prevent burns caused by hot molten material splashing out.

[0067] Step 7: Emulsification of water-in-oil matrix emulsion Dissolve 0.5 to 1.5 parts by weight of sodium chloride in 20 to 50 parts by weight of deionized water. Heat the resulting sodium chloride aqueous solution to 60 to 70°C and add it to the oil phase obtained in step 6. Emulsify at 4000 to 8000 rpm for 15 to 30 minutes to obtain a water-in-oil matrix emulsion.

[0068] It should be noted that the preferred amount of sodium chloride is 0.8 to 1.2 parts by weight, the preferred amount of deionized water is 30 to 40 parts by weight, the preferred emulsification speed is 5000 to 7000 rpm, and the preferred emulsification time is 18 to 25 min. The addition of sodium chloride can adjust the ionic strength of the aqueous phase and enhance the stability of the water-in-oil matrix emulsion. The temperature of the sodium chloride aqueous solution when added to the oil phase should be maintained at 60 to 70°C to ensure that the oil phase is in a molten flow state to facilitate emulsification and dispersion. The mass ratio of sodium chloride to deionized water in step 7 is (0.5 to 1.5):(20 to 50), preferably (0.8 to 1.2):(30 to 40).

[0069] Furthermore, in step 7 above, when adding the sodium chloride aqueous solution to the oil phase, it is added slowly in a thin stream at a rate of 2 to 5 mL / min, while the oil phase is stirred at a low speed of 200 to 300 rpm. After all the sodium chloride aqueous solution has been added, the speed is increased to 4000 to 8000 rpm for emulsification, in order to prevent the rapid addition of the aqueous phase from causing a local water-oil ratio imbalance and affecting the emulsification uniformity.

[0070] Furthermore, in step 7 above, the splashes generated by the high-speed emulsification operation (4000 to 8000 rpm) may cause skin or eye irritation. Operators must wear safety goggles, and the emulsification container must be covered with a splash guard.

[0071] Step 8: Composite of multifunctional co-loaded biomimetic liposomes with matrix emulsion Cool the water-in-oil matrix emulsion obtained in step 7 to 30-38°C, add the solvent-free multifunctional co-loaded biomimetic liposomes obtained in step 5, and mix at a low speed of 800-1500 rpm for 10-20 min. Then add 2-5 parts by weight of caprylic glyceride, 0.3-1 part by weight of p-hydroxyacetophenone, and D- 0.1 to 0.5 parts by weight of tocopherol are stirred evenly and then cooled to room temperature to obtain a composition for preventing and treating infantile eczema and allergies.

[0072] It should be noted that the above-mentioned caprylic acid glyceride is a monoester of caprylic acid and glycerol, with a purity of $. $96%, with a molecular weight of 430.7 Da, acts as an antioxidant to protect the unsaturated lipid components in the composition from oxidative degradation.

[0073] It should be noted that the preferred low-speed stirring rate is 1000 to 1200 rpm, and the preferred mixing time is 12 to 18 minutes. Low-speed stirring can avoid mechanical shearing that could damage the intact vesicle structure of the multifunctional co-loaded biomimetic liposomes, and maintain the encapsulation state of casein phosphopeptides and lactoferrin peptides in the aqueous chamber of the liposomes.

[0074] It should be noted that the preferred cooling temperature for the above-mentioned water-in-oil matrix emulsion is 32 to 35°C. This temperature range is below the denaturation temperature threshold of casein phosphopeptides and lactoferrin peptides, which can prevent high temperatures from damaging the active conformation of heat-sensitive casein phosphopeptides and lactoferrin peptides in the multifunctional co-loaded biomimetic liposomes, while ensuring that the water-in-oil matrix emulsion still has sufficient fluidity to facilitate the uniform dispersion of the multifunctional co-loaded biomimetic liposomes.

[0075] It should be noted that the preferred amount of caprylic / caprylic glyceride is 3 to 4 parts by weight, and the preferred amount of p-hydroxyacetophenone is 0.4 to 0.8 parts by weight. - The preferred dosage of tocopherol is 0.2 to 0.4 parts by weight. Caprylic / caprylic glyceride, p-hydroxyacetophenone, and D- - The mass ratios of the three tocopherols relative to the amount of lanolin used in step 6 are (2 to 5): (10 to 25), (0.3 to 1): (10 to 25) and (0.1 to 0.5): (10 to 25), preferably (3 to 4): (15 to 20), (0.4 to 0.8): (15 to 20) and (0.2 to 0.4): (15 to 20).

[0076] Furthermore, in step 8 above, during the cooling to room temperature process, when the temperature of the composition drops to 25 to 30°C, it is assisted by stirring at a low speed of 200 to 300 rpm for 20 to 40 minutes, preferably 25 to 35 minutes, in order to prevent uneven local curing of the composition during the cooling process from affecting the uniformity of the final product's appearance.

[0077] Furthermore, in step 8 above, before adding p-hydroxyacetophenone to the water-in-oil matrix emulsion, a small amount of caprylic acid glyceride (taken from a portion of the total amount of caprylic acid glyceride used in step 8) is pre-dissolved at 40 to 45°C. After the p-hydroxyacetophenone is completely dissolved, it is mixed with the remaining caprylic acid glyceride and added together to ensure that the p-hydroxyacetophenone is evenly distributed in the composition and to avoid uneven dispersion of p-hydroxyacetophenone in the low-temperature matrix due to direct addition, resulting in a localized high concentration.

[0078] Furthermore, the final composition obtained in step 8 above was characterized by Fourier transform infrared spectroscopy (FTIR), the particle size distribution and polydispersity index (PDI) of the liposomes in the composition were determined by dynamic light scattering, the pH value of the composition was determined by a pH meter, and the viscosity of the composition was determined by a rotational viscometer to confirm that the physicochemical properties of the composition met expectations.

[0079] Furthermore, building upon step 4, to further improve the uniformity of liposome particle size, the capillary nozzle can be preheated to 40-45°C before spraying, maintaining the co-solution system in a supercritical state within the nozzle until the outlet. This preheating operation prevents premature cooling of the co-solution system inside the nozzle due to heat dissipation from the nozzle wall, avoiding localized precipitation of lipid molecules that could clog the nozzle or form liposomes with uneven particle size. The preheating method involves wrapping an electric heating tape around the outer wall of the nozzle, with the temperature of the heating tape controlled within the range of 40-45°C using a temperature controller.

[0080] Furthermore, based on step 8, to further enhance the soothing and repairing properties of the composition on damaged skin on the buttocks of infants, 0.2 to 0.8 parts by weight of allantoin (purity $) can be added to the water-in-oil matrix emulsion cooled to 30 to 38°C before adding the multifunctional co-loaded biomimetic liposomes. β-sitosterol plays a synergistic role in barrier repair.

[0081] Furthermore, the waste aqueous solutions generated during the entire preparation process of this invention (including the supernatant from ultracentrifugation in step 5, the cleaning liquid from filtration residue, and other waste liquids containing trace amounts of casein phosphopeptides, lactoferrin peptides, and lipid residues) and the waste adjusting liquid generated during pH adjustment in step 3 must be collected separately and handed over to a professional wastewater treatment facility for treatment; they must not be directly discharged into the municipal sewage network. Oily wastewater generated from cleaning equipment and containers must undergo oil-water separation pretreatment before further treatment.

[0082] Furthermore, this invention employs supercritical fluid. It can replace the traditional mixture of ethanol and propylene glycol as a lipid dissolution medium. After depressurization, it completely escapes in gaseous form and can be recycled and reused. No organic solvents are involved in the entire preparation process, and there are no organic solvent residues in the final product. This eliminates the generation of organic solvent waste liquid and the emission of organic solvent vapors from the source, reflecting the principles of green chemistry to reduce the use of harmful solvents and reduce pollution at the source.

[0083] This invention uses supercritical Using a mixed organic solvent of ethanol and propylene glycol as a lipid dissolution medium, barrier repair lipid components (N-palmitoylsphingosine, ...) are introduced. -Sitosterol and palmitic acid) and antifungal lipid components (monoglyceryl laurate and tea tree oil) are co-dissolved under supercritical conditions and then injected under reduced pressure through a capillary nozzle into an aqueous receiving solution containing casein phosphopeptide and lactoferrin peptide.

[0084] because Under reduced pressure, the lipid molecules rapidly expand and vaporize, completely escaping the membrane. They then instantly self-assemble at the aqueous interface to form biomimetic liposomes. The entire process is solvent-free and requires no mechanical shearing during membrane extrusion, thus fundamentally avoiding the desolvation effect of ethanol and propylene glycol on casein phosphopeptides and lactoferrin peptides. The higher-order structure of the phosphoserine cluster sequence in casein phosphopeptides and the active conformation of lactoferrin peptides remain in a mild aqueous environment throughout the preparation process, without contact with organic solvents or repeated mechanical shearing. This preserves the competitive binding ability of casein phosphopeptides to digestive enzyme active sites and their chelation ability to calcium ion cofactors, as well as the competitive binding ability of lactoferrin peptides to digestive enzyme active sites.

[0085] Meanwhile, since no ethanol or propylene glycol is involved in the entire preparation process, After the degassing step, the solvent completely escapes, leaving no organic solvent residue in the final product. Infants' buttocks have thin stratum corneum and eczema causes severe barrier damage; eliminating organic solvent residue avoids the risk of solvent penetration causing irritating contact reactions and exacerbating erythema, exudation, and allergic symptoms.

[0086] Furthermore, because laurate monoglyceride and tea tree oil are embedded in the liposome bilayer membrane structure, and casein phosphopeptide and lactoferrin peptide are encapsulated in the same aqueous chamber of the liposome, both types of active components are co-loaded on the same nanoscale carrier. When the multifunctional co-loaded biomimetic liposome is applied to damaged skin on the buttocks of infants, the nanoscale liposome, due to its size advantage, penetrates the fungal extracellular polysaccharide matrix to reach the surface of the fungus. Laurate monoglyceride is released from the bilayer membrane and inserts into the fungal cell membrane, increasing membrane permeability and exerting an antifungal effect. After the liposome structure disintegrates, casein phosphopeptide and lactoferrin peptide are released simultaneously in the same microregion, exerting an enzyme inhibitory effect, thereby achieving the co-localized release of antifungal and enzyme-inhibiting components. N-palmitoylsphingosine and... -Sitosterol participates in the reconstruction of the biomimetic lipid barrier in the intercellular spaces of the stratum corneum, helping to repair the damaged skin barrier on the buttocks. The water-in-oil matrix emulsion forms a hydrophobic sealing film on the skin surface, reducing the continuous irritation of the skin by the external humid environment, and provides a sustained-release carrier for the multifunctional co-loaded biomimetic liposomes to prolong the action time of the active ingredients.

[0087] In summary, this invention addresses the three problems faced by existing technologies in the prevention and treatment of diaper rash and skin allergies in infants and young children, respectively, in terms of solvent-free safety, preservation of enzyme-inhibiting functional peptide activity, and co-localized release of multiple active ingredients.

[0088] Example 1 Step 1: Purge the supercritical reactor with nitrogen gas three times, each time for 5 minutes. Weigh out 2 parts by weight of plant ceramide. - Mix 1 part by weight of sitosterol, 1 part by weight of palmitic acid, and 1 part by weight of laurate monoglyceride thoroughly and place in a reactor. Heat to 45°C at a rate of 1°C / min, and then introduce... The pressure was increased to 15 MPa, and the mixture was stirred and dissolved for 30 minutes to obtain a supercritical solution containing multifunctional lipids.

[0089] Step 2: Premix 1 part by weight of tea tree oil and 1 part by weight of oat kernel oil at 35°C and 200 rpm for 5 min. Inject the mixture into the reactor containing the supercritical solution obtained in Step 1 at a rate of 0.5 mL / min using a high-pressure metering pump. Stir and incubate at 15 MPa and 45°C for 20 min to form a eutectic system.

[0090] Step 3: Deoxygenate 150 parts by weight of deionized water by bubbling with nitrogen at 100 mL / min for 15 min. Dissolve 3 parts by weight of casein phosphopeptide and 1 part by weight of lactoferrin peptide in the above deionized water, add 4 parts by weight of glycerol and 0.5 parts by weight of D-panthenol, stir at 300 rpm at 35°C for 20 min to dissolve, adjust the pH to 6.0 with citric acid solution, transfer to a vacuum receiving container and stir continuously at 1000 rpm for later use.

[0091] Step 4: The co-solution system obtained in Step 2 is sprayed into the aqueous receiving liquid obtained in Step 3 through a capillary nozzle with an inner diameter of 50 μm at a flow rate of 2 mL / min. The pressure of the receiving container is controlled at 0.1 MPa, the temperature of the aqueous receiving liquid is maintained at 20℃, the stirring speed of the receiving liquid is 1000 rpm, and the nozzle is preheated to 40℃. The lipid molecules expand, vaporize, and escape, and then self-assemble to form a multifunctional co-loaded biomimetic liposome dispersion.

[0092] Step 5: The dispersion obtained in Step 4 was allowed to stand at 25°C for 30 min to degas, and then filtered under positive pressure through a 0.45 μm microporous membrane (filtration pressure not exceeding 0.1 MPa). The filtrate was collected to obtain multifunctional co-loaded biomimetic liposomes (the total amount of the four lipids and the mass ratio of deionized water were 5:150).

[0093] Step 6 (in parallel with steps 1 to 5): Continuously purge nitrogen gas at 50 mL / min onto the top of the melting container. Melt 10 parts by weight of lanolin and 3 parts by weight of beeswax at 65°C, add 3 parts by weight of polyglycerol-2 dihydroxystearate and 1 part by weight of glyceryl oleate, and stir at 200 rpm for 10 min to obtain the oil phase.

[0094] Step 7: Dissolve 0.5 parts by weight of sodium chloride in 20 parts by weight of deionized water, heat to 60°C, and slowly add the oil phase obtained in Step 6 at a rate of 2 mL / min while stirring at a low speed of 200 rpm. After all the aqueous phase is added, increase the speed to 4000 rpm and emulsify for 15 min to obtain a water-in-oil matrix emulsion.

[0095] Step 8: Cool the emulsion obtained in Step 7 to 30°C. First, add 0.2 parts by weight of allantoin and stir at 800 rpm for 5 minutes to dissolve. After the allantoin is completely dispersed, add the multifunctional co-loaded biomimetic liposomes obtained in Step 5 and stir at low speed of 800 rpm for 10 minutes. Pre-dissolve 0.3 parts by weight of p-hydroxyacetophenone in a small amount of caprylic acid glyceride at 40°C, mix with the remaining caprylic acid glyceride (2 parts by weight in total), and then add it to the system. Finally, add D- 0.1 parts by weight of tocopherol were stirred evenly and then cooled to room temperature with assisted stirring at 200 rpm for 20 min to obtain the composition.

[0096] Example 2 Step 1: Purge the supercritical reactor with nitrogen gas three times, each time for 10 minutes. Weigh out 6 parts by weight of plant ceramide. - A mixture of 4 parts by weight of sitosterol, 3 parts by weight of palmitic acid, and 3 parts by weight of laurate monoglyceride was placed in a reactor and heated to 55°C at a rate of 3°C / min. Then, air was introduced... The pressure was increased to 25 MPa, and the mixture was stirred and dissolved for 60 min to obtain a supercritical solution containing multifunctional lipids.

[0097] Step 2: Premix 3 parts by weight of tea tree oil and 4 parts by weight of oat kernel oil at 400 rpm for 15 min at 40℃. Inject the mixture into the reactor containing the supercritical solution obtained in Step 1 at a rate of 2.0 mL / min using a high-pressure metering pump. Stir and incubate at 25 MPa and 55℃ for 40 min to form a eutectic system.

[0098] Step 3: Deoxygenate 250 parts by weight of deionized water by bubbling with nitrogen at 200 mL / min for 30 min. Dissolve 7 parts by weight of casein phosphopeptide and 4 parts by weight of lactoferrin peptide in the above deionized water, add 10 parts by weight of glycerol and 2 parts by weight of D-panthenol, stir at 600 rpm at 42°C for 40 min to dissolve, adjust the pH to 6.8 with disodium hydrogen phosphate solution, transfer to a vacuum receiving container and stir continuously at 2000 rpm for later use.

[0099] Step 4: The co-solution system obtained in Step 2 is sprayed into the aqueous receiving liquid obtained in Step 3 through a capillary nozzle with an inner diameter of 150 μm at a flow rate of 8 mL / min. The pressure of the receiving container is controlled at 0.5 MPa, the temperature of the aqueous receiving liquid is maintained at 30 °C, the stirring speed of the receiving liquid is 2000 rpm, and the nozzle is preheated to 45 °C. The lipid molecules expand, vaporize, and escape, and then self-assemble to form a multifunctional co-loaded biomimetic liposome dispersion.

[0100] Step 5: The dispersion obtained in Step 4 was allowed to stand at 35°C for 60 min to degas, and the filtrate was collected by positive pressure filtration through a 0.45 μm microporous membrane to obtain multifunctional co-loaded biomimetic liposomes (the total amount of the four lipids and the mass ratio of deionized water were 16:250).

[0101] Step 6 (in parallel with steps 1 to 5): Continuously purge nitrogen gas at 100 mL / min onto the top of the melting container. Melt 25 parts by weight of lanolin and 8 parts by weight of beeswax at 80°C, add 6 parts by weight of polyglycerol-2 dihydroxystearate and 3 parts by weight of glyceryl oleate, and stir at 400 rpm for 20 min to obtain the oil phase.

[0102] Step 7: Dissolve 1.5 parts by weight of sodium chloride in 50 parts by weight of deionized water, heat to 70°C, and slowly add the oil phase obtained in Step 6 at a rate of 5 mL / min while stirring at a low speed of 200 rpm. After all the aqueous phase is added, increase the speed to 8000 rpm and emulsify for 30 min to obtain a water-in-oil matrix emulsion.

[0103] Step 8: Cool the emulsion obtained in Step 7 to 38°C. First, add 0.8 parts by weight of allantoin and stir at 1000 rpm for 10 min to dissolve. After the allantoin is completely dispersed, add the multifunctional co-loaded biomimetic liposomes obtained in Step 5 and stir at 1500 rpm for 20 min. Pre-dissolve 1 part by weight of p-hydroxyacetophenone in a small amount of caprylic acid glyceride at 45°C, mix with the remaining caprylic acid glyceride (5 parts by weight in total), and then add D- 0.5 parts by weight of tocopherol were stirred evenly and then cooled to room temperature with assisted stirring at 300 rpm for 40 min to obtain the composition.

[0104] Example 3 Step 1: Purge the supercritical reactor with nitrogen gas three times, each time for 7.5 minutes. Weigh out 4 parts by weight of plant ceramide. - 2.5 parts by weight of sitosterol, 1.5 parts by weight of palmitic acid, and 1.5 parts by weight of laurate monoglyceride were mixed evenly and placed in a reactor. The temperature was increased to 50°C at a rate of 1.75°C / min, and then... The pressure was increased to 20 MPa, and the mixture was stirred and dissolved for 45 min to obtain a supercritical solution containing multifunctional lipids.

[0105] Step 2: Premix 1.5 parts by weight of tea tree oil and 2.5 parts by weight of oat kernel oil at 38°C and 300 rpm for 10 min. Inject the mixture into the reactor containing the supercritical solution obtained in Step 1 at a rate of 1.25 mL / min using a high-pressure metering pump. Stir and incubate at 20 MPa and 50°C for 30 min to form a eutectic system.

[0106] Step 3: Deoxygenate 200 parts by weight of deionized water by bubbling with nitrogen at 150 mL / min for 22.5 min. Dissolve 5 parts by weight of casein phosphopeptide and 2.5 parts by weight of lactoferrin peptide in the above deionized water, add 6.5 parts by weight of glycerol and 1.15 parts by weight of D-panthenol, stir at 450 rpm at 38.5°C for 30 min to dissolve, adjust the pH to 6.35 with citric acid solution, transfer to a vacuum receiving container and stir continuously at 1500 rpm for later use.

[0107] Step 4: The co-solution system obtained in Step 2 is sprayed into the aqueous receiving liquid obtained in Step 3 through a 100 μm inner diameter capillary nozzle at a flow rate of 4.5 mL / min. The pressure of the receiving container is controlled at 0.3 MPa, the temperature of the aqueous receiving liquid is maintained at 25 °C, the stirring speed of the receiving liquid is 1500 rpm, and the nozzle is preheated to 42.5 °C. The lipid molecules expand, vaporize, and escape, and then self-assemble to form a multifunctional co-loaded biomimetic liposome dispersion.

[0108] Step 5: The dispersion obtained in Step 4 was allowed to stand at 30°C for 45 min to degas, and the filtrate was collected by positive pressure filtration through a 0.45 μm microporous membrane to obtain multifunctional co-loaded biomimetic liposomes (the total amount of the four lipids and the mass ratio of deionized water were 9.5:200).

[0109] Step 6 (in parallel with steps 1 to 5): Continuously purge nitrogen gas at 75 mL / min onto the top of the melting vessel. Melt 17.5 parts by weight of lanolin and 5 parts by weight of beeswax at 72.5°C, add 4.5 parts by weight of polyglycerol-2 dihydroxystearate and 2 parts by weight of glyceryl oleate, and stir at 300 rpm for 15 min to obtain the oil phase.

[0110] Step 7: Dissolve 1 part by weight of sodium chloride in 35 parts by weight of deionized water, heat to 65°C, and slowly add the oil phase obtained in Step 6 at a rate of 3.5 mL / min while stirring at a low speed of 200 rpm. After all the aqueous phase is added, increase the speed to 6000 rpm and emulsify for 21.5 min to obtain a water-in-oil matrix emulsion.

[0111] Step 8: Cool the emulsion obtained in Step 7 to 33.5℃. First, add 0.45 parts by weight of allantoin and stir at 900 rpm for 7.5 min to dissolve. After the allantoin is completely dispersed, add the multifunctional co-loaded biomimetic liposomes obtained in Step 5 and stir at 1100 rpm for 15 min. Pre-dissolve 0.6 parts by weight of p-hydroxyacetophenone in a small amount of caprylic acid glyceride at 42.5℃, mix with the remaining caprylic acid glyceride (total 3.5 parts by weight), and add to the system. Then add D- 0.3 parts by weight of tocopherol were stirred evenly and then cooled to room temperature with assisted stirring at 250 rpm for 30 min to obtain the composition.

[0112] Comparative Example 1 Steps 1 to 5 use a mixed solvent of ethanol and propylene glycol (ethanol:propylene glycol volume ratio = 1:1) instead of supercritical fluid. As a lipid dissolution medium, 4 parts by weight of plant ceramide were used. 2.5 parts by weight of sitosterol, 1.5 parts by weight of palmitic acid, 1.5 parts by weight of glyceryl monolaurate, 1.5 parts by weight of tea tree oil, and 2.5 parts by weight of oat kernel oil were dissolved in the above-mentioned mixed organic solvent and stirred at 50°C for 45 min to obtain a lipid organic solution. 5 parts by weight of casein phosphopeptide and 2.5 parts by weight of lactoferrin peptide were prepared into an aqueous receiving solution (pH=6.35) according to step 3 of Example 3. The lipid organic solution was injected into the above-mentioned aqueous receiving solution, and self-assembled in the aqueous phase. The liposome dispersion was obtained by extrusion through a 200 nm polycarbonate membrane 10 times. The preparation and compounding process of the water-in-oil matrix emulsion in steps 6 to 8 were exactly the same as in Example 3.

[0113] Comparative Example 2 Steps 1 and 2 prepare a supercritical co-solution system containing multifunctional lipids according to Example 3. Step 3 prepares an aqueous receiving solution (pH=6.35) according to Example 3. Step 4 does not use the supercritical depressurization jet self-assembly process. Instead, the supercritical co-solution system obtained in Step 2 is slowly depressurized to atmospheric pressure, allowing the lipids to precipitate and be directly mixed with the aqueous receiving solution obtained in Step 3. The mixture is then extruded through a 200nm polycarbonate membrane 10 times to prepare a liposome dispersion. Steps 5 to 8 are exactly the same as in Example 3.

[0114] Comparative Example 3 In step 1, laurate monoglyceride was removed from the lipid fraction, and only 4 parts by weight of plant ceramide were used. - 2.5 parts by weight of sitosterol and 1.5 parts by weight of palmitic acid were dissolved in a supercritical reactor. The conditions for the remaining steps in step 1 were the same as in Example 3. In step 2, tea tree oil was removed from the supercritical co-solution system, and only 2.5 parts by weight of oat kernel oil was injected into the supercritical solution. The conditions for the remaining steps in step 2 were the same as in Example 3. Steps 3 to 5 were performed according to Example 3 to prepare biomimetic liposomes without antifungal lipids. In step 8, the composite stage, after adding the multifunctional co-loaded biomimetic liposomes, 1.5 parts by weight of laurate monoglyceride and 1.5 parts by weight of tea tree oil were separately prepared as a general emulsion (using Tween-80 as an emulsifier), which was simply mixed with the liposome dispersion and added to the matrix emulsion. The conditions for the remaining steps in step 8 were the same as in Example 3.

[0115] Experimental test: This experiment uses three sets of control experiments to verify the following three technical effects: First, supercritical... The necessity of replacing the mixed organic solvent of ethanol and propylene glycol to preserve the inhibitory activity of casein phosphopeptides and lactoferrin peptidase; secondly, the contribution of supercritical vacuum jet self-assembly process to peptide activity protection and elimination of organic solvent residues by replacing membrane extrusion; and thirdly, the effect of embedding glyceryl monolaurate and tea tree oil into liposome bilayer membranes (co-carried on the same carrier) on the co-localized release effect of antifungal and enzyme inhibitory components.

[0116] Experimental sample preparation: Following the processes described in Examples 1, 2, 3, Comparative Examples 1, 2, and 3, six batches of composition samples were prepared, numbered sequentially as Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, and Sample 6. The batch size for each sample was based on the amount of deionized water used in step 3, and the batch size for Samples 3, 4, 5, and 6 was based on the amount of deionized water used in Example 3 (200 parts by weight).

[0117] Experimental conditions: Liposome particle size was characterized using dynamic light scattering with a particle size analyzer at 25℃. Each sample was measured in triplicate. Enzyme inhibitory activity was determined using trypsin as a model digestive enzyme (referencing digestive enzyme types found in infant feces), with N-benzoyl-L-arginine ethyl ester (BAEE) as the substrate. Absorbance changes were measured at 405 nm, and the enzyme inhibition rate was calculated using the following formula: in, Enzyme inhibition rate, The rate of change in absorbance is for the blank control group (no sample added). The rate of change in absorbance after the addition of the sample is given.

[0118] Organic solvent residues were determined by gas chromatography (headspace sampling) for ethanol and propylene glycol content, with a detection limit of 0.01 mg / g. Antifungal activity was assessed using *Candida albicans* (ATCC 10231) as a model strain, and the minimum inhibitory concentration (MIC) was determined using the micro-liquid dilution method. Colocalization assessment was performed using fluorescent labeling, with rhodamine B labeling of antifungal lipids (glyceryl monolaurate) and luciferin labeling of enzyme inhibitory peptides. The spatial colocalization of the two labeled components in simulated skin stratum corneum sections was observed using laser confocal microscopy, and the Pearson colocalization coefficient was used as the metric. Characterization: in, and The first The fluorescence intensity of the rhodamine B channel and fluorescein channel at each pixel. and These represent the average fluorescence intensity of the corresponding channels. The value ranges from 0 to 1, with values ​​closer to 1 indicating a higher degree of co-location.

[0119] Experimental steps: Step 1: Prepare batches of composition samples according to the processes described in each embodiment and comparative example.

[0120] Step 2: Liposome particle size and polydispersity index (PDI) determination. Each batch of composition was diluted with deionized water to a liposome mass concentration of approximately 0.1 mg / mL, and the average particle size and PDI were determined by dynamic light scattering method.

[0121] Step 3: Functional Peptide Encapsulation Efficiency Determination. Centrifuge each batch of multifunctional co-loaded biomimetic liposome dispersions (the filtrate obtained in Step 5) at 100,000 × g for 60 min, collect the supernatant, and determine the mass of free casein phosphopeptides and lactoferrin peptides in the supernatant using high-performance liquid chromatography (HPLC). Calculate the encapsulation ratio, where The total mass of the functional peptides input. The mass of free functional peptides in the supernatant.

[0122] Step 4: Enzyme Inhibition Activity Assay. Each batch of composition was diluted with phosphate-buffered saline (PBS, pH=6.35) to an equal concentration of functional peptides. After incubation with trypsin solution for 10 min, BAEE substrate was added, and absorbance was continuously recorded at 405 nm for 30 min. The enzyme inhibition rate was calculated. .

[0123] Step 5: Determination of residual organic solvents. Take approximately 1g of each batch of final product composition and determine the content of ethanol and propylene glycol by headspace gas chromatography.

[0124] Step Six: Antifungal Activity Assay. Serially dilute each batch of the composition with RPMI 1640 medium and inoculate with Candida albicans suspension (bacterial concentration approximately...). The concentration (CFU / mL) was incubated at 37℃ for 48 hours, and the absorbance was read at 600 nm using a microplate reader. The lowest concentration at which no obvious colony growth was observed was taken as the MIC value.

[0125] Step 7: Colocalization Release Coefficient Determination. Each batch of the composition was applied to an in vitro reconstructed human stratum corneum model (approximately 10 μm thick). Dual-channel fluorescence images were acquired using a laser confocal microscope, and the Pearson colocalization coefficient was calculated. .

[0126] See experimental or test results Figures 2-8 And as shown in the table below: Table 1 Summary of main process parameters for Examples 1 to 3 and Comparative Examples 1 to 3 As shown in Table 1, samples 1 to 3 (Examples 1 to 3) all used supercritical fluid extraction. As a lipid dissolution medium, it is self-assembled by supercritical depressurization jetting. Each process parameter covers three levels: minimum endpoint value, maximum endpoint value, and preferred intermediate value. Samples 4 to 6 (comparative examples 1 to 3) introduce one change in each of the three dimensions of dissolution medium, self-assembly method, and antifungal lipid loading method, forming three sets of control experiments.

[0127] Table 2. Liposome particle size, PDI, and encapsulation efficiency of functional peptides As shown in Table 2, under the optimized parameter conditions, Example 3 (Sample 3) achieved the smallest average particle size (152.3 nm) and the lowest PDI (0.14), exhibiting the most uniform particle size distribution and an overall encapsulation efficiency of 80.8%. Example 1 (Sample 1) used the minimum endpoint parameters, resulting in a larger particle size (198.4 nm) and a relatively higher PDI (0.21). While its encapsulation efficiency (67.4%) was lower than Example 3, it remained within an acceptable range, indicating that the combination of minimum endpoint parameters could complete the self-assembly process. Comparative Example 1 (Sample 4) saw its overall encapsulation efficiency decrease to 40.9% due to the use of a mixed solvent of ethanol and propylene glycol, a reduction of approximately 49.4% compared to Example 3. This indicates that organic solvents significantly interfere with the encapsulation of casein phosphopeptides and lactoferrin peptides in the aqueous phase. Comparative Example 2 (Sample 5) experienced an overall encapsulation efficiency of 54.1% due to the mechanical shearing during membrane extrusion, a reduction of approximately 33.0% compared to Example 3. Comparative Example 3 (Sample 6) and Example 3 have similar particle size and encapsulation efficiency, indicating that the loading method of laurate monoglyceride and tea tree oil has no significant effect on particle size and encapsulation efficiency. The difference is mainly reflected in the subsequent functional indicators.

[0128] Table 3 Enzyme inhibitory activity and residual organic solvent content As shown in Table 3, ethanol and propylene glycol were not detected in the final products of Examples 1 to 3 (samples 1 to 3). The enzyme inhibition rate increased as the parameters transitioned from the minimum endpoint value to the preferred intermediate value, reaching 78.5% in Example 3. Comparative Example 1 (sample 4) had a residual ethanol content of 1.83 mg / g and a residual propylene glycol content of 2.47 mg / g, resulting in a high skin irritation risk rating, and an enzyme inhibition rate of only 31.2%, a decrease of approximately 60.3% compared to Example 3. This result demonstrates that the mixed solvent of ethanol and propylene glycol simultaneously caused two types of problems: on the one hand, it led to the desolvation and aggregation of casein phosphopeptides and lactoferrin peptides, destroying their active conformation; on the other hand, it left irritating solvent residues in the final product. Comparative Example 2 (sample 5) had no organic solvent residues, but the mechanical shearing during membrane extrusion reduced the enzyme inhibition rate to 48.6%, a decrease of approximately 38.1% compared to Example 3, indicating that even after eliminating organic solvents, repeated mechanical shearing still damages the higher-order structure of casein phosphopeptides and lactoferrin peptides. The difference in enzyme inhibition rate between Example 3 and Comparative Example 2 directly reflects the advantage of the supercritical vacuum jet self-assembly process over the membrane extrusion process in protecting peptide activity. The enzyme inhibition rate of Comparative Example 3 (sample 6) was 77.9%, which is close to that of Example 3, indicating that the loading method of laurate monoglyceride and tea tree oil has no direct effect on the enzyme inhibition function, and the difference between the two types of components is reflected in their co-localization release ability.

[0129] Table 4 Antifungal activity and co-localization release coefficient As shown in Table 4, the MIC values ​​of Examples 1 to 3 (samples 1 to 3) decreased as the parameter transitioned from the minimum endpoint value to the preferred intermediate value (0.38 to 0.21 mg / mL), indicating a gradual increase in antifungal activity; Pearson colocalization coefficient All values ​​were above 0.81, indicating that the antifungal lipids and enzyme-inhibiting peptides achieved highly co-localized release in the damaged skin model. Comparative Examples 1 (Sample 4) and 2 (Sample 5) had co-localization coefficients (0.88 and 0.89) similar to Example 3 (0.91) because both laurate monoglyceride and tea tree oil were embedded in the liposome bilayer membrane. Their MIC values ​​(0.24 and 0.23 mg / mL) were also similar to Example 3 (0.21 mg / mL), suggesting that the loading method of co-loading onto the same liposome itself ensured the co-localized release capability, which was not significantly affected by changes in the dissolution medium or self-assembly method. Comparative Example 3 (Sample 6) changed the mixing of laurate monoglyceride and tea tree oil to a simpler method (step 8), resulting in an MIC value of 0.52 mg / mL, an increase of approximately 147.6% compared to Example 3 (i.e., a significant decrease in antifungal activity). The concentration decreased to 0.34, a reduction of approximately 62.6% compared to Example 3, resulting in a poor co-localization rating. This result indicates that when antifungal lipids and enzyme-inhibiting peptides belong to different carrier systems, the two types of active ingredients cannot achieve co-localization release in the same damaged micro-region. The permeation barrier effect of the fungal extracellular polysaccharide matrix on external free antifungal components significantly weakens the overall prevention and control effect.

[0130] In summary, the data in Tables 2 to 4 collectively demonstrate the effectiveness of the technical improvements at three levels in this invention. First, supercritical... Replacing the mixed organic solvent of ethanol and propylene glycol (Example 3 compared with Comparative Example 1): The enzyme inhibition rate increased from 31.2% to 78.5%, an increase of approximately 151.6%; the organic solvent residue in the final product decreased from detectable to undetectable, eliminating the risk of skin irritation in infants and young children. Second, supercritical vacuum jet self-assembly replacing membrane extrusion (Example 3 compared with Comparative Example 2): The enzyme inhibition rate increased from 48.6% to 78.5%, an increase of approximately 61.5%; the final product also had no organic solvent residue, indicating that the supercritical vacuum jet process is superior to the membrane extrusion process in protecting the active conformation of the enzyme-inhibiting peptide. Third, co-loading glyceryl monolaurate and tea tree oil in the same liposome bilayer membrane (Example 3 compared with Comparative Example 3): The MIC value decreased from 0.52 mg / mL to 0.21 mg / mL, and the antifungal activity increased by approximately 59.6%; Pearson colocation coefficient The co-location release capability was increased by approximately 167.6% from 0.34 to 0.91, verifying the key contribution of co-location release to the comprehensive prevention and control effect. Data from Examples 1 and 2 further demonstrate that all three technical effects of the present invention can be achieved under both minimum and maximum endpoint value parameter conditions, with the optimal intermediate value parameter (Example 3) showing the best performance across all indicators.

[0131] The embodiments of the present invention have been described above. However, the embodiments are not limited to the specific implementation methods described above. The specific implementation methods described above are merely illustrative and not restrictive. Those skilled in the art can make more equivalent embodiments under the guidance of the present embodiments, and all of them are within the protection scope of the present embodiments.

Claims

1. A method for preparing a composition for preventing and treating infantile eczema and allergies, characterized in that, Includes the following steps: Plant ceramides, -Sitosterol, palmitic acid, and glyceryl monolaurate were placed in a supercritical reactor and purged with... The solution is obtained by stirring and dissolving at a pressure of 15 to 25 MPa and a temperature of 45 to 55 °C to obtain a supercritical solution. Tea tree oil and oat kernel oil were premixed and then injected into the supercritical solution, stirred and incubated to form a co-solution system; Casein phosphopeptides and lactoferrin peptides were dissolved in deionized water containing glycerol and D-panthenol, and the pH was adjusted to 6.0 to 6.8 to obtain an aqueous receiving solution. The co-solution system is injected under reduced pressure into the aqueous receiving liquid through a capillary nozzle. The lipid molecules vaporize and escape, and then self-assemble at the aqueous interface to form multifunctional co-loaded biomimetic liposomes. The multifunctional co-loaded biomimetic liposomes were collected after degassing and filtration; the multifunctional co-loaded biomimetic liposomes were added to a water-in-oil matrix emulsion and mixed at low speed to obtain the composition.

2. The preparation method according to claim 1, characterized in that, The plant ceramide is N-palmitoylsphingosine, used in amounts of 2 to 6 parts by weight; - The amount of sitosterol is 1 to 4 parts by weight; the amount of palmitic acid is 1 to 3 parts by weight; the amount of lauric acid monoglyceride is 1 to 3 parts by weight; the mass ratio of the four is (2 to 6): (1 to 4): (1 to 3): (1 to 3).

3. The preparation method according to claim 1, characterized in that, The amount of tea tree oil used is 1 to 3 parts by weight, wherein the mass fraction of terpinene-4-ol is not less than 35%; the amount of oat kernel oil used is 1 to 4 parts by weight; the tea tree oil and oat kernel oil are premixed at 35 to 40°C and then injected into the supercritical solution by a high-pressure metering pump, and stirred and incubated at 15 to 25 MPa and 45 to 55°C for 20 to 40 minutes.

4. The preparation method according to claim 1, characterized in that, The amount of casein phosphopeptide is 3 to 7 parts by weight, with a molecular weight of 1000 to 5000 Da; the amount of lactoferrin peptide is 1 to 4 parts by weight, with a molecular weight of 500 to 3000 Da; the amount of deionized water is 150 to 250 parts by weight; the amount of glycerol is 4 to 10 parts by weight; the amount of D-panthenol is 0.5 to 2 parts by weight; the dissolution temperature is 35 to 42°C, and the stirring rate is 300 to 600 rpm.

5. The preparation method according to claim 1, characterized in that, The capillary nozzle has an inner diameter of 50 to 150 μm, a jet flow rate of 2 to 8 mL / min, and a receiving container pressure of 0.1 to 0.5 MPa. The aqueous receiving liquid is continuously stirred at 1000 to 2000 rpm during the jetting process, and the temperature is maintained at 20 to 30°C.

6. The preparation method according to claim 1, characterized in that, The water-in-oil matrix emulsion is prepared by heating 10 to 25 parts by weight of lanolin and 3 to 8 parts by weight of beeswax at 65 to 80°C to melt them, then adding 3 to 6 parts by weight of polyglycerol-2 dihydroxystearate and 1 to 3 parts by weight of glyceryl oleate and stirring until homogeneous to obtain an oil phase; dissolving 0.5 to 1.5 parts by weight of sodium chloride in 20 to 50 parts by weight of deionized water, heating to 60 to 70°C and then adding it to the oil phase, and emulsifying at 4000 to 8000 rpm for 15 to 30 minutes.

7. The preparation method according to claim 1, characterized in that, The degassing is performed by standing at 25 to 35°C for 30 to 60 minutes; the filtration is performed by positive pressure filtration through a 0.45 μm microporous membrane, with a filtration pressure not exceeding 0.1 MPa.

8. The preparation method according to claim 1, characterized in that, After the water-in-oil matrix emulsion is cooled to 30 to 38°C, the multifunctional co-loaded biomimetic liposomes are added, and the mixture is stirred at 800 to 1500 rpm for 10 to 20 minutes; then 2 to 5 parts by weight of caprylic acid glyceride, 0.3 to 1 part by weight of p-hydroxyacetophenone, and D- Add 0.1 to 0.5 parts by weight of tocopherol, stir until homogeneous, and then cool to room temperature.

9. The preparation method according to claim 8, characterized in that, Before adding the multifunctional co-loaded biomimetic liposomes, add 0.2 to 0.8 parts by weight of allantoin to a water-in-oil matrix emulsion cooled to 30 to 38°C, stir at 800 to 1000 rpm to dissolve for 5 to 10 minutes, and then add the multifunctional co-loaded biomimetic liposomes.

10. A composition for preventing and treating infantile eczema and allergies prepared by the preparation method according to any one of claims 1 to 9.

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