A glabridin-hydroxyl asiaticoside double-cabin liposome and a preparation method and application thereof

Abstract

The present application relates to the technical field of cosmetics, in particular to a glabridin-hydroxyl asiaticoside double-cabin liposome and a preparation method and application thereof, according to mass percentage, the preparation raw material comprises the following components: soybean lecithin 6.5-9.5%, co-emulsifier 0.8-2.0%, glycerol 18-25%, glabridin 0.4-1.0%, hydroxyl asiaticoside 0.5-1.5%, 1,3-butanediol 6-10%, preservative 0.5-1.0%, and the balance is deionized water; the co-emulsifier comprises sucrose stearate and steareth-2 with a mass ratio of 1.5-4:1; the double-cabin liposome is prepared by a glycerol dilution method, so that the provided product can simultaneously and efficiently co-load polar contrast double active substances, and the stability and transdermal rate are effectively improved.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a glycyrrhizin-hydroxyasiaticoside dual-compartment liposome, its preparation method, and its application. Background Technology

[0002] Pigmentation is a complex physiological process regulated by melanin synthesis, transport, inflammatory cascades, and epidermal barrier function. Inflammatory pigmentation (PIH) accounts for more than 60% of clinical pigmentation types and is the core cause of dullness in sensitive skin. Glycyrrhizin, a flavonoid active compound extracted from the root of *Glycyrrhiza glabra*, can precisely bind to the active site of tyrosinase, blocking the conversion of dopa to dopaquinone. It is currently recognized as one of the strongest natural tyrosinase inhibitors. However, using glycyrrhizin alone has two major problems: first, its phenolic hydroxyl structure easily causes skin irritation and sensitization, making it unsuitable for sensitive skin; second, it can only intervene in the downstream stages of melanin production and cannot block the activation of melanocytes by inflammatory signals, making it almost ineffective against PIH.

[0003] Hydroxyacine is a pentacyclic triterpenoid saponin extracted from Centella asiatica. It can promote the synthesis of type I / III collagen by regulating the TGF-β / Smad pathway, while downregulating pro-inflammatory factors, thus blocking the PIH pathway at its source. Theoretically, both can simultaneously address the two major problems of pigmentation and inflammation induction. However, their physicochemical properties are extremely contrasting: glycyrrhizin is highly lipophilic (logP≈4.85) and only 0.10 mg / L water-soluble, easily crystallizing and highly sensitive to photothermal and oxygen; hydroxyasiacine is a strongly hydrophilic macromolecule (molecular weight 975 Da), which can hardly penetrate the lipophilic stratum corneum of the skin. This polarity difference makes it difficult for the two to coexist stably in the same formulation, becoming the core technical bottleneck restricting their synergistic application.

[0004] Existing formulation technologies cannot effectively solve the above problems: nanoemulsions have high surfactant content and are prone to instability due to Oswald ripening; cyclodextrin encapsulation and hydrogels can only improve the solubility of glycyrrhizin, but cannot solve the transdermal problem of asiaticoside; for example, Chinese patent application CN120960061A discloses a liposome of Babao raw powder, which uses ethanol as an organic solvent and improves the water dispersibility of a single solid Chinese herbal raw powder by combining a dispersant with phospholipids. Although traditional liposomes can simultaneously encapsulate lipid-soluble and water-soluble components, they are highly dependent on volatile organic solvents such as chloroform and ethanol, and solvent residues can easily damage the skin barrier; on the other hand, the encapsulation rate for polarity-contrasting dual active ingredients is low, usually <70% for lipid-soluble components and <40% for water-soluble components; and long-term storage can easily lead to problems such as increased particle size and precipitation of active ingredients. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a glycyrrhizin-hydroxyasiaticoside dual-compartment liposome. By optimizing the formulation of raw materials and controlling the process, the product can simultaneously and efficiently co-load two polarity-contrasting active ingredients, effectively improving stability and transdermal permeability. It is free of volatile organic solvents and cholesterol, better meeting practical application requirements.

[0006] This invention provides a glycyrrhizin-asiaticoside dual-compartment liposome, the raw materials of which, by mass percentage, include the following components: 6.5%-9.5% soybean lecithin, 0.8%-2.0% co-emulsifier, 18%-25% glycerol, 0.4%-1.0% glycyrrhizin, 0.5%-1.5% asiaticoside, 6%-10% 1,3-butanediol, 0.5%-1.0% preservative, and the balance being deionized water; The co-emulsifier comprises sucrose stearate and stearyl alcohol polyether-2 in a mass ratio of 1.5-4:1; The dual-compartment liposomes were prepared using the glycerol dilution method. In the dual-compartment liposomes, glycyrrhizin was embedded in the liposome's lipophilic bilayer in an amorphous state, while asiaticoside was completely encapsulated in the hydrophilic inner cavity of the liposome.

[0007] Existing liposome products often only achieve inefficient loading of a single active ingredient or simply combine active ingredients with similar polarities. They fail to effectively address the coexistence problem caused by the extreme contrast in physicochemical properties between glycyrrhizin and asiaticoside, generally exhibiting low encapsulation efficiency, poor stability, and weak transdermal absorption. Furthermore, traditional preparation processes rely on volatile organic solvents, which can easily cause skin irritation and fail to meet the whitening needs of sensitive skin. This application utilizes 6.5%-9.5% soybean lecithin as the main emulsifier, combined with sucrose stearate and stearyl alcohol polyether-2 in a mass ratio of 1.5-4:1 as co-emulsifiers, and 18%-25% glycerol as a gel phase forming agent. A glycerol dilution method is used to prepare bilayer liposomes, achieving precise, partitioned loading of glycyrrhizin in an amorphous state within the liposome's lipophilic bilayer, while asiaticoside is completely encapsulated within the liposome's hydrophilic lumen. It completely solves the problems of easy precipitation and inactivation of highly crystalline, lipid-soluble glycyrrhizin and poor transdermal permeability of highly hydrophilic macromolecular hydroxyasiaticoside. It also achieves efficient co-loading and simultaneous targeted delivery of dual active components, while leaving no volatile organic solvent residues throughout the process. It also has barrier repair effects and is suitable for sensitive skin.

[0008] The rationale behind this invention is likely as follows: A specific ratio of soybean lecithin and a compound co-emulsifier constructs a phospholipid bilayer membrane structure with compatible dual-active physicochemical properties. Combined with the surfactant gel phase formed by glycerol, this provides stable hydrophobic intercalation sites for the lipid-soluble glycyrrhizin, preventing its crystallization, and also constructs a closed hydrophilic core space for the water-soluble asiaticoside, preventing its leakage into the external aqueous phase. The precise partitioned loading structure ensures that the dual-active components penetrate the stratum corneum of the skin simultaneously with the liposomes, and are released in the basal layer and dermis respectively. This allows the precise tyrosinase inhibition of glycyrrhizin and the anti-inflammatory barrier repair effect of asiaticoside to work synergistically, blocking pigmentation from both downstream of melanin production and the source of inflammation. At the same time, the glycerol dilution method completely eliminates the risk of solvent residue from traditional processes, and combines with the barrier repair effect of asiaticoside to achieve multiple technical effects of high-efficiency whitening, simultaneous repair, and safety and gentleness.

[0009] In one embodiment, the sucrose stearate has an HLB (hydrophilic-lipophilic balance) value of 14-18.

[0010] In one embodiment, the soybean lecithin is soybean-derived or animal-derived lecithin, and the maximum kinematic viscosity of the soybean lecithin at 77°F is 80-120 St (determined using the American Association of Oil Chemists (AOCS) standard method AOCS Ja 11-87).

[0011] In one embodiment, the preservative includes p-hydroxyacetophenone and 1,2-hexanediol.

[0012] In one embodiment, the mass ratio of p-hydroxyacetophenone to 1,2-hexanediol is 1-3:4-8.

[0013] In one embodiment, the dual-compartment liposome satisfies at least one of the following conditions: (1) Encapsulation efficiency of hydroxyasiaticoside ≥70%; (2) Average initial particle size ≤ 140 nm; (3) After being sealed and aged at 25℃ for 1 month, the particle size growth rate was ≤8%, and no glycyrrhizin needle-like birefringent crystals were precipitated under a polarizing microscope.

[0014] The present invention enables the encapsulation efficiency of asiaticoside in dual-compartment liposomes to be ≥70%, and after aging at room temperature for 1 month, the particle size growth rate is ≤8% with no crystal precipitation. The steady-state permeation rate of glycyrrhizin is about 11 times higher than that of the free group, and that of asiaticoside is about 6.8 times higher. Without the aid of highly irritating chemical permeation enhancers, the active components are delivered to the dermis and basal layer in a highly efficient and uniform manner, which greatly improves bioavailability.

[0015] Another aspect of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, comprising the following steps: Soybean lecithin and emulsifier were moistened with glycerin and heated to 70℃-75℃ until completely dissolved to obtain a surfactant gel phase. At a constant temperature of 70℃-75℃, glycyrrhizin and 1,3-butanediol were mixed and stirred until completely dissolved. The surfactant gel phase was then added and stirred to obtain a mixed phase. Hydroxyacin was completely dissolved in 1.5-1.8 times its weight of deionized water to obtain an aqueous solution of hydroxyasiaticoside. At a constant temperature of 70℃-75℃, add the aqueous solution of hydroxyascorbic acid to the mixed phase and stir to mix. Add preservatives and the remaining deionized water and stir to obtain a crude liposome suspension; The crude liposome suspension was homogenized, filtered, and subjected to particle size reduction treatment to obtain glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes.

[0016] In one embodiment, the aqueous solution of asiaticoside is added to the mixed phase at a rate of 10-20 mL / min and stirred for 5-10 min.

[0017] In one embodiment, the aqueous solution of asiaticoside is added to the mixed phase at a rate of 15-18 mL / min and stirred for 5-10 min.

[0018] In one embodiment, the homogenization speed is 2500-3000 rpm, and the homogenization time is 5-10 min.

[0019] In one embodiment, the filtration uses an 80-100 mesh nylon filter.

[0020] In one embodiment, the particle size refinement process includes the following steps: using a high-pressure microfluidic homogenizer to cycle 3-5 times at a pressure of 1500-2000 bar to refine the particle size.

[0021] This invention prepares dual-compartment liposomes by using a glycerol dilution method that does not rely on volatile organic solvents such as chloroform and ethanol. This completely eliminates the risk of skin penetration irritation and barrier disruption induced by solvent residues in traditional liposome preparation processes. The preparation process is safe and mild, significantly reducing the irritation of the formulation, and has extremely high clinical application and industrialization value.

[0022] The third aspect of this invention provides an application of glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes in the preparation of cosmetics.

[0023] In one embodiment, the cosmetic includes any one of serum, lotion, face cream, face mask, toner, and eye cream.

[0024] Beneficial effects 1. This invention provides a glycyrrhizin-hydroxyasiaticoside dual-compartment liposome. By optimizing the formulation of raw materials and process control, the product can simultaneously and efficiently co-load two polarity opposite active ingredients, effectively improving stability and transdermal permeability. It is free of volatile organic solvents and cholesterol, better meeting the needs of actual use.

[0025] 2. This application utilizes 6.5%-9.5% soybean lecithin as the main emulsifier, combined with sucrose stearate and stearyl alcohol polyether-2 in a mass ratio of 1.5-4:1 as co-emulsifiers, and 18%-25% glycerol as a gel phase forming agent. A glycerol dilution method is used to prepare bilayer liposomes, enabling the amorphous embedding of glycyrrhizin into the liposome's lipophilic bilayer, while asiaticoside is completely encapsulated within the liposome's hydrophilic lumen for precise, zoned loading. This method completely solves the problems of easy precipitation and inactivation of highly crystalline, lipid-soluble glycyrrhizin and poor transdermal permeability of highly hydrophilic, large-molecule asiaticoside. It also achieves efficient co-loading and simultaneous targeted delivery of the two active components, with no volatile organic solvent residue throughout the process, and provides barrier repair benefits, making it suitable for sensitive skin.

[0026] 3. The present invention enables the encapsulation rate of asiaticoside in dual-compartment liposomes to be ≥70%, and after aging at room temperature for 1 month, the particle size growth rate is ≤8% and no crystal precipitation occurs. The steady-state permeation rate of glycyrrhizin is about 11 times higher than that of the free group, and that of asiaticoside is about 6.8 times higher. Without the aid of highly irritating chemical permeation enhancers, the active components are delivered to the dermis and basal layer in a highly efficient and uniform manner, which greatly improves bioavailability.

[0027] 4. This invention prepares dual-compartment liposomes by using a glycerol dilution method, which does not rely on volatile organic solvents such as chloroform and ethanol. This completely eliminates the risk of skin penetration irritation and barrier disruption induced by solvent residues in traditional liposome preparation processes. The preparation process is safe and mild, significantly reducing the irritation of the formulation, and has extremely high clinical application and industrialization value. Attached Figure Description

[0028] Figure 1 The diagram shows the structure of the glycyrrhizin-hydroxyasiaticoside bicomplex liposome provided in Example 3. In the diagram: 1-liposome hydrophilic lumen; 2-liposome lipophilic bilayer; 3-glycyrrhizin; 4-hydroxyasiaticoside; 5-emulsifier molecule; 6-water, polyol.

[0029] Figure 2 The image of the glycyrrhizin-hydroxyasiaticoside dual-compartment liposome provided in Example 3 after aging at room temperature for 1 month under polarized light at 25°C. Figure 3 The images provided for comparison are polarized light micrographs of the products aged at room temperature for one month at 25°C. The images ai correspond to Comparative Examples 1, 2, 3, 4, 7, 10, 11, 12, and 13, respectively. Detailed Implementation

[0030] The information on each raw material in the embodiments and comparative examples of this invention is as follows.

[0031] Sucrose stearate: trade name Ryoto Sugar Ester S-1670, HLB=16, manufacturer is Mitsubishi Chemical, Japan; Stearyl alcohol polyether-2: Trade name BrijS2, manufactured by Croda Chemicals Ltd., UK; Soy lecithin: derived from soybeans, with a maximum kinematic viscosity of 100St at 77°F, marketed as Yelkin® SS, manufactured by ADM Corporation, USA; Glycyrrhizin, asiaticoside, glycerin, 1,3-butanediol, p-hydroxyacetophenone, and 1,2-hexanediol are all general-purpose raw materials with well-defined chemical structures, and all use conventional cosmetic-grade raw materials available on the market.

[0032] Examples 1-14 Examples 1-14 of the present invention provide a glycyrrhizin-hydroxyasiaticoside dual-compartment liposome, the formulation of which, by mass percentage, is shown in Table 1. See also... Figure 1 In the dual-compartment liposome, glycyrrhizin 3 is embedded in the liposome's lipophilic bilayer 2 in an amorphous state, while hydroxyasiaticoside 4 is completely encapsulated in the liposome's hydrophilic inner cavity 1.

[0033] Table 1

[0034] Examples 1-14 of the present invention provide a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, comprising the following steps: Soybean lecithin and emulsifier were moistened with glycerin and heated to 75°C until completely dissolved to obtain a surfactant gel phase. At a constant temperature of 75℃, glycyrrhizin and 1,3-butanediol were mixed and stirred until completely dissolved. The surfactant gel phase was then added and stirred to obtain a mixed phase. Hydroxyasiaticoside was completely dissolved in deionized water 1 to obtain an aqueous solution of hydroxyasiaticoside. At a constant temperature of 75℃, add the aqueous solution of hydroxyascorbic acid to the mixed phase and stir to mix; Add preservatives and deionized water 2 and stir to obtain crude liposome suspension; The crude liposome suspension was homogenized, filtered, and subjected to particle size reduction treatment to obtain glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes.

[0035] The aqueous solution of hydroxyascorbic acid was added to the mixed phase at a rate of 15 mL / min and stirred for 10 min.

[0036] The homogenization speed was 3000 rpm, and the homogenization time was 10 min.

[0037] The filtration process uses a 100-mesh nylon filter.

[0038] The particle size refinement process includes the following steps: using a high-pressure microfluidic homogenizer to cycle 4 times at a pressure of 1500 bar to refine the particle size.

[0039] Comparative Examples 1-10 Comparative Examples 1-10 of the present invention provide a glycyrrhizin-hydroxyasiaticoside dual-compartment liposome, the formulation of which is shown in Table 2 by mass percentage.

[0040] Table 2

[0041] Comparative Examples 1-10 of the present invention provide a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as in Example 1.

[0042] Comparative Examples 11-21 Comparative Examples 11-21 of the present invention provide a glycyrrhizin-hydroxyasiaticoside dual-compartment liposome, the formulation of which is shown in Table 3 by mass percentage.

[0043] Table 3

[0044] Comparative Example 11 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, comprising the following steps: Prepare an aqueous glycerol solution by mixing glycerol and deionized water 2; Soybean lecithin, sucrose stearate and stearyl alcohol polyether-2 were added to an aqueous glycerol solution and heated to 75°C to dissolve completely to obtain a mixed solution; At a constant temperature of 75℃, glycyrrhizin and 1,3-butanediol were mixed and stirred until completely dissolved. The mixture was then added to the solution and stirred to obtain a mixed phase. Hydroxyasiaticoside was completely dissolved in deionized water 1 to obtain an aqueous solution of hydroxyasiaticoside. At a constant temperature of 75℃, add the aqueous solution of hydroxyascorbic acid to the mixed phase and stir to mix; Add the pre-mixed preservative and stir to obtain a crude liposome suspension; The crude liposome suspension was homogenized, filtered, and subjected to particle size reduction treatment to obtain glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes.

[0045] The aqueous solution of hydroxyascorbic acid was added to the mixed phase at a rate of 15 mL / min and stirred for 10 min.

[0046] The homogenization speed was 3000 rpm, and the homogenization time was 10 min.

[0047] The filtration process uses a 100-mesh nylon filter.

[0048] The particle size refinement process includes the following steps: using a high-pressure microfluidic homogenizer to cycle 4 times at a pressure of 1500 bar to refine the particle size.

[0049] Comparative Example 12 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, comprising the following steps: Prepare an aqueous glycerol solution by mixing glycerol and deionized water 2; Soybean lecithin, sucrose stearate and stearyl alcohol polyether-2 were added to an aqueous glycerol solution and heated to 75°C to dissolve completely to obtain a mixed solution; After completely dissolving hydroxyascorbic acid in deionized water 1, heat to 70℃-75℃, add to the mixed solution and stir to obtain a mixed phase; At a constant temperature of 75℃, glycyrrhizin and 1,3-butanediol were mixed and stirred until completely dissolved. The mixture was then added and stirred for 10 minutes. Add the pre-mixed preservative and stir to obtain a crude liposome suspension; The crude liposome suspension was homogenized, filtered, and subjected to particle size reduction treatment to obtain glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes.

[0050] The homogenization speed was 3000 rpm, and the homogenization time was 10 min.

[0051] The filtration process uses a 100-mesh nylon filter.

[0052] The particle size refinement process includes the following steps: using a high-pressure microfluidic homogenizer to cycle 4 times at a pressure of 1500 bar to refine the particle size.

[0053] Comparative Example 13 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, comprising the following steps: Soybean lecithin, sucrose stearate and stearyl alcohol polyether-2 were moistened with glycerin and heated to 75°C to dissolve completely, thus obtaining a surfactant gel phase. At a constant temperature of 75℃, hydroxyascorbic acid was mixed with deionized water 1 and stirred until completely dissolved. The surfactant gel phase was then added and stirred to obtain a mixed phase. A solution of glycyrrhizin was obtained by mixing glycyrrhizin with 1,3-butanediol. At a constant temperature of 75℃, the glycyrrhizin solution was added to the mixed phase and stirred to mix. Add the pre-mixed preservative and stir to obtain a crude liposome suspension; The crude liposome suspension was homogenized, filtered, and subjected to particle size reduction treatment to obtain glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes.

[0054] The aqueous solution of hydroxyascorbic acid was added to the mixed phase at a rate of 15 mL / min and stirred for 10 min.

[0055] The homogenization speed was 3000 rpm, and the homogenization time was 10 min.

[0056] The filtration process uses a 100-mesh nylon filter.

[0057] The particle size refinement process includes the following steps: using a high-pressure microfluidic homogenizer to cycle 4 times at a pressure of 1500 bar to refine the particle size.

[0058] Comparative Example 14 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as that of Comparative Example 11.

[0059] Comparative Example 15 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as that of Comparative Example 12.

[0060] Comparative Example 16 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as that of Comparative Example 13.

[0061] Comparative Example 17 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as that of Comparative Example 11.

[0062] Comparative Example 18 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as that of Comparative Example 12.

[0063] Comparative Example 19 of the present invention provides a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as that of Comparative Example 13.

[0064] Comparative Examples 20-21 of the present invention provide a method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes, the specific implementation of which is the same as in Example 3.

[0065] Performance testing 1. The glycyrrhizin encapsulation of the comparative products of each embodiment was evaluated using a polarizing microscope.

[0066] Polarizing light microscopy (PLM) is a crucial tool for identifying the presence of drug crystallization in liposome systems. Glycyrrhizin is highly crystalline and readily soluble in organic solvents but insoluble in water. If glycyrrhizin is not effectively encapsulated within the hydrophobic layer of the lipid bilayer, or if it "leaks" from the liposomes during storage, it will rapidly transform from an amorphous state to a high-energy crystalline state in an aqueous environment, exhibiting distinct needle-like birefringent crystals. PLM observation allows for direct identification of the presence of free drug crystals in the sample, thereby evaluating the loading efficiency of liposomes for glycyrrhizin and its physical stability under complex environments. In this test, a field of view without crystallization indicates that the drug is completely encapsulated within the liposomes or exists stably in an amorphous state, which is an important indicator of formulation homogeneity and transdermal absorption efficiency.

[0067] No glycyrrhizin crystals were observed under a polarizing microscope after Preparation and Storage at Room Temperature (25°C) for 1 month in Examples 1-14, proving that glycyrrhizin was completely encapsulated within the liposome bilayer. The polarizing micrograph of Example 3 after aging at room temperature for 1 month is shown below. Figure 2 .

[0068] In contrast, comparative examples, after being stored at room temperature for one month following preparation, all showed varying degrees of glycyrrhizin crystal precipitation under a polarizing microscope. Comparative examples 1-19 all exhibited varying degrees of birefringence crystallization after one month of storage at room temperature. See [link to relevant documentation]. Figure 3 Comparative Examples 11, 12, and 13 (process comparison groups), although their chemical compositions were completely identical to those of Optimal Example 3, failed to effectively penetrate the lipid bilayer in the early stages of preparation due to the absence of the glycerol dilution method of this invention, resulting in the rapid precipitation of a large number of dense needle-like crystals during aging. Comparative Examples 1 and 2 (component insufficiency groups) also exhibited obvious crystal clusters due to the limited drug loading capacity of the system caused by the amount of the main emulsifier (soybean lecithin) being below the critical value. Comparative Examples 3, 4, and 7 showed lipid membrane structural defects and decreased drug compatibility due to a severe imbalance in the proportion of co-emulsifiers. Comparative Example 10 showed a significantly higher amount of main emulsifier (soybean lecithin) than the critical value, leading to a bilayer component imbalance. These phenomena directly demonstrate, from a physical perspective, the irreplaceable role of the specific formulation ratio and preparation process described in this invention in inhibiting the precipitation of highly crystalline active substances and maintaining the physical stability of the system.

[0069] 2. Determination of the encapsulation efficiency of asiaticoside in the comparative products of each example.

[0070] Unlike glycyrrhizin, asiaticoside is readily soluble in water, therefore its encapsulation efficiency needs to be determined using chromatographic instruments. Encapsulation efficiency (EE%) refers to the percentage of the active ingredient's mass in a successfully prepared liposome that is encapsulated within the carrier (inner aqueous phase) or embedded / adsorbed within the carrier membrane structure (amphiphilic bilayer) relative to the total mass of that active ingredient in the formulation. A higher encapsulation efficiency means more active ingredient can be delivered deep into the skin, and theoretically, the transdermal effect of the encapsulated active ingredient will be more significant. To evaluate the differences in active ingredient encapsulation efficiency between the examples and comparative examples, this invention determined the encapsulation efficiency of asiaticoside in the samples.

[0071] Test method: First, accurately pipette 1.0 mL of the product prepared in this invention into a 10 mL volumetric flask, add sufficient chromatographic grade methanol, and sonicate for 15 min (power 250 W, frequency 40 kHz) to fully demulsify. After returning to room temperature, dilute to the mark with methanol and shake well. Filter the sample through a microporous membrane, and take the filtrate for quantitative analysis of asiaticoside according to the testing literature of Shanghai Institute of Quality Supervision and Inspection Technology [Nie Lei, Fu Xinyuan, Zhou Yaobin, et al. Determination of the content of asiaticoside and asiaticoside in cosmetics by high performance liquid chromatography [J]. Fragrance, Flavor and Cosmetics, 2024(2):169-173.], and calculate the total mass of asiaticoside in the sample ( W total ).

[0072] The second step involves accurately pipetting 1.0 mL of the same batch of product into an ultrafiltration centrifuge tube; centrifuging at 10000 rpm for 20 min; collecting the supernatant and filtering it through a microporous membrane; and then injecting the filtrate for analysis. The concentration of glycyrrhizin in the filtrate is calculated based on the regression equation, and the free drug content is calculated using the volume. W free Finally, take () W total - W free ) / W total The ratio of to is used as the determination value for the encapsulation efficiency of hydroxyasiaticoside.

[0073] The encapsulation efficiency (EE%) of hydroxyasiaticoside in the 14 embodiments and 19 comparative examples of the present invention was determined. Three samples were tested in parallel, and the average value of the results is shown in Table 4.

[0074] Table 4

[0075] Analysis of the data in Table 4 shows that the encapsulation efficiency of asiaticoside in Examples 1-14 is generally distributed in a high range of 70.8±0.9% to 86.4±0.5%. Among them, the encapsulation efficiencies of Examples 3, 10, 7, 4, and 13 reached 86.4±0.5%, 85.2±0.6%, 83.1±0.7%, 81.5±1.1%, and 80.2±0.9%, respectively. The encapsulation efficiencies of the remaining Examples (1, 2, 5, 6, 8, 9, 11, 12, and 14) are also stable within the range of 70.8±0.9% to 79.4±1.3%, demonstrating extremely high drug loading efficiency and formulation reproducibility; which is significantly different from the above. Notably, the encapsulation efficiency of asiaticoside in all 19 comparative examples showed a significant decrease, with the overall efficiency ranging from only 21.5±3.1% to 48.2±2.5%, which is extremely low. Specifically, the encapsulation efficiency of comparative examples 3, 16, 13, 1, 11, and 6, which had relatively better performance, was between 40.8±2.1% and 48.2±2.5%, while that of comparative examples 2, 5, 8, 9, 12, 15, 18, and 19 was as low as 31.8±1.7% to 39.5±2.4%. In particular, the encapsulation efficiency of comparative examples 4, 7, 10, 14, and 17 was even as low as 21.5±3.1% to 29.1±1.7%, which is far lower than that of the embodiments of the present invention. The above data fully demonstrates that, through the synergistic effect of the specific formulation components and processes of this invention, the technical defect of highly water-soluble ingredient asiaticoside being prone to leakage into the external aqueous phase in conventional liposomes has been successfully overcome, significantly improving the retention rate and stability of this active ingredient in the lipid delivery system.

[0076] 3. Particle size test The nanoparticle size of the products prepared in Examples 1-14 and Comparative Examples 1-19 of this invention was measured. The samples were diluted 100-fold with deionized water and placed in a sample cell. The average particle size (Z-Average) and dispersion index (PDI) were measured at 25°C. The measurements were repeated three times, and the average value was taken. The particle size and PDI test results of the examples and comparative examples are statistically shown in Table 5.

[0077] Table 5

[0078] Analysis of the particle size and dispersion index (PDI) test results in Table 4 shows that Examples 1-14 of this invention exhibit excellent nanoscale self-assembly characteristics immediately after preparation. Their initial average particle size ranges from 85.4 nm to 132.6 nm, and their PDI values ​​are all in the low range of 0.115 to 0.218, indicating that the system exhibits a highly uniform monodisperse state. Specifically, Example 3 has a particle size of 85.4 nm and a PDI of [missing value]. The value of 0.115 fully demonstrates that the specific emulsifier ratio of this invention enables excellent microstructure arrangement of dual active components with significant polarity differences. In contrast, Comparative Examples 1-10, which deviate from the specified ratio requirements of this invention, exhibited significantly broadened initial average particle sizes ranging from 215.7 nm to 480.2 nm, with PDI all exceeding 0.35. This clearly indicates that even minor deviations in the emulsifier composition can severely disrupt the film-forming curvature of the lipid bilayer, leading to a large number of irregular aggregates in the system. Further comparison of particle size evolution after one month of aging revealed that the particle size growth rate of Examples 1-14 was controlled within 8.0% (compared to only 2.11% in Example 3). The system maintains excellent stability, remaining transparent or semi-fluid. In contrast, comparative examples 11-19, which used traditional or incorrect preparation processes, had initial particle sizes of around 150-200 nm, but the particle size growth rate generally increased to 65% or even over 110% during storage. This demonstrates that the present invention, combined with the glycerol dilution method, can effectively reduce interfacial energy, enhance film toughness, and inhibit the Oswald ripening effect. It fundamentally solves the problem of easy aggregation and instability caused by the contrasting physicochemical properties of glycyrrhizin and asiaticoside in the same formulation, ensuring the bioefficacy potential and physical stability of the two active components throughout their full-life efficacy period.

[0079] 4. Determination and evaluation of steady-state permeation rate (permeability) of active ingredients (Franz diffusion cell method).

[0080] This test used a vertical Franz diffusion cell (effective diffusion area of ​​1.77 cm²). 2The permeability of liposomes in complex skin barriers was evaluated using a 15 mL receiving cell. Ex vivo abdominal skin from Bama miniature pigs was used as the permeability barrier. After hair removal and subcutaneous fat stripping, the thickness was precisely controlled to 1.0 ± 0.2 mm. Before the experiment, the skin was equilibrated to room temperature and fixed in the diffusion cell, ensuring the stratum corneum was in close contact with the supply cell and the dermis was in full contact with the receiving solution. The receiving solution was prepared as PBS buffer (pH 7.4) containing 20% ​​ethanol to ensure that the highly lipophilic active component, glycyrrhizin, met the leakage conditions during permeation. The entire diffusion system maintained the skin surface temperature at 32 ± 1 °C through a constant-temperature water bath circulation, and magnetic stirring (300 r / min) was used to eliminate the concentration gradient at the interface of the receiving solution. During the drug administration phase, the liposome suspension prepared in this invention (experimental group) and an equal concentration of free glycyrrhizin / hydroxyasiaticoside miscible solution (control group) were precisely added to the supply cell (1.0 mL), and sealed with a sealing film to prevent solvent evaporation from affecting the osmotic pressure. At 2h, 4h, 8h, 12h and 24h after administration, 1.0 mL of the receiving liquid sample was accurately aspirated. Fresh receiving liquid of the same temperature and volume was added immediately after sampling to maintain fluid balance. All sampled liquids were filtered through a 0.22 μm microporous membrane and the instantaneous concentration of the active components was determined by HPLC according to the literature (Du Ruyun, Lu Zhiyun, Yu Chenghua. Determination of glycyrrhizin content in cosmetics by high performance liquid chromatography [J]. Fragrance, Flavor and Cosmetics, 2014(4):3.DOI:10.3969 / j.issn.1000-4475.2014.04.011.[2] Nie Lei, Fu Xinyuan, Zhou Yaobin, et al. Determination of hydroxyascorbic acid and asiaticoside content in cosmetics by high performance liquid chromatography [J]. Fragrance, Flavor and Cosmetics, 2024(2):169-173.). The concentration of the components in the receiving liquid at each time point was determined by HPLC. C n And according to the formula Calculate the cumulative infiltration per unit area at each time point ( Q n μg / cm 2 ).in V 0 represents the total volume of the receiving pool. V i For sampling volume, C i Let be the measured concentration of the i-th sample. A The effective diffusion area. This was determined through a series of data collection... Q n Data, establishing cumulative penetration versus penetration time t The regression curve is obtained. During the steady-state permeation phase, the slope of the steady-state linear portion of this regression curve is calculated to obtain the steady-state permeation rate. J s (Unit: μg / cm) 2 h 1 ),Right now The relevant calculation results are listed in Table 6. The overall penetration enhancement factor was calculated based on the combined increase in the steady-state penetration rate of the two active ingredients. Specifically, the penetration enhancement factors of glycyrrhizin and asiaticoside in the liposome sample relative to the free control group were first calculated separately, that is, the steady-state penetration rate of each component in the sample group was divided by the steady-state penetration rate of the corresponding component in the free group; then, the two individual enhancement factors were averaged to obtain the overall penetration enhancement factor of the sample. This value reflects the overall penetration-enhancing effect of the liposome system on the lipid-soluble glycyrrhizin and water-soluble asiaticoside.

[0081] Table 6

[0082] Analysis of the Franz diffusion cell permeation experiment results shown in Table 6 reveals that the dual-compartment liposomes prepared in this invention exhibit significant technical advantages in promoting the penetration of active ingredients with large polarity differences into the skin barrier. Experimental data show that, in the experimental group represented by Example 3, the steady-state permeation rate of glycyrrhizin... J s (μg / cm) 2 h 1 The concentration reached 5.28 ± 0.24 μg / cm³. 2 h 1 Compared to the control group (free active ingredient group), it increased by approximately 11 times, and the penetration rate of the macromolecular water-soluble component, asiaticoside, also significantly increased to 7.62±0.45 μg / cm. 2 h 1 The permeability was approximately 6.8 times that of the control group, which strongly demonstrates that the biomimetic phospholipid bilayer structure described in this invention can efficiently fuse with stratum corneum lipids, significantly reducing the activation energy required for components to penetrate the barrier; in contrast, the permeation rates of the 19 comparative examples were generally lower (the steady-state permeation rate of glycyrrhizin was significantly lower). J s The steady-state permeation rate of hydroxyasiaticoside is mostly distributed between 0.68 and 1.62. J sThe concentrations of active ingredients were mostly between 1.55 and 2.95, and significantly lower than those in the example group. This was mainly due to the presence of glycyrrhizin microcrystals and uneven particle size distribution in the comparative examples, which greatly limited the effective diffusion flux. This further confirms the key role of the specific formulation and process of this invention in maintaining the steady-state distribution and synergistic co-transport of active ingredients. The above data show that this invention, by constructing an efficient dual-drug delivery system, successfully overcomes the technical problem that highly crystalline lipid-soluble components and large molecular weight glycosides are difficult to cross the stratum corneum barrier. Without the aid of highly irritating chemical penetration enhancers, it achieves efficient and uniform penetration of active ingredients into the deep layers of the skin, laying a solid pharmaceutical foundation for its clinical-grade whitening and repair synergistic efficacy.

[0083] 5. Human evaluation of whitening and repairing effects.

[0084] This study recruited a specific number of healthy adult female volunteers who met the inclusion criteria (aged 25-45 years, Fitzpatrick skin type III-IV, and with significant pigmentation or uneven skin tone in the test area). The experiment employed a randomized, double-blind, half-face controlled design. Participants discontinued the use of any skincare products with whitening, exfoliating, or anti-inflammatory effects for two weeks prior to the experiment. Participants were instructed to apply equal amounts of the liposome emulsion prepared according to this invention (experimental group) and a conventional emulsion containing only equal amounts of glycyrrhizin and asiaticoside added via conventional homogenization (control group) to the left and right sides of their face after cleansing morning and evening. The experiment lasted for 28 days. All participants were required to use physical sun protection measures during the experiment to eliminate UV interference. Evaluation indicators were set at before administration (D0), day 14 (D14), and day 28 (D28). The evaluation process was conducted in a controlled laboratory under constant temperature (22±2℃) and humidity (50±5%). The skin's L-color was determined using a CM-700d spectrophotometer. b The yellowness value is used to calculate the individual type angle (ITA°), and the calculation formula is as follows: A higher value indicates a fairer skin tone. The melanin index (MI) of the test area was simultaneously measured using a Mexameter MX18 melanin quantification instrument to quantitatively characterize the degree of melanin reduction. Furthermore, changes in barrier function in the treatment area were monitored using a transepidermal water loss (TEWL) meter to comprehensively evaluate the product's mildness and repair efficacy. Data analysis was performed using SPSS software for one-way ANOVA and paired analysis. t test.

[0085] To verify the clinical whitening efficacy and synergistic effect of the combined application of glycyrrhizin and asiaticoside in a specific liposome delivery system described in this invention, a representative group was selected for a 28-day human clinical comparative experiment. The test groups included: the optimal group of this invention (Example 3), the median group (Example 7), the emulsifier ratio imbalance group (Comparative Example 3), the conventional preparation process group (Comparative Example 11), the single-drug-loaded liposome group containing only an equal amount of glycyrrhizin (Comparative Example 20), the single-drug-loaded liposome group containing only an equal amount of asiaticoside (Comparative Example 21), and a conventional emulsion control group with an equal amount of free active ingredients (control group: prepared by ordinary shear emulsification of deionized water (55.2%), glycerol (20%), 1,3-butanediol (8%), medium-chain triglycerides (10%), sorbitan oleate (5%), 0.8% by mass of glycyrrhizin and 1.0% by mass of asiaticoside, wherein the active ingredients were not encapsulated in liposomes). Subjects were randomly assigned to groups of four, and the results of their Individual Type Angle (ITA°) and Melanin Index (MI) measurements are summarized in Table 7.

[0086] Table 7

[0087] Note: Compared with the control group of conventional emulsions at the same time point. P <0.05; P <0.01.

[0088] Analysis of the human skin whitening efficacy evaluation results shown in Table 7 reveals that the product provided by this invention exhibits a significant synergistic whitening effect. Experimental data shows that the ITA° increase rate of liposomes containing glycyrrhizin alone (Comparative Example 20) is 12.35%, while the direct whitening ability of liposomes containing asiaticoside alone (Comparative Example 21) is almost non-significant (increase rate of only 2.77%). The theoretical simple sum of the efficacy of the two is only 15.12%. However, in Example 3 of this invention (dual drug-loaded group), under the same active ingredient concentration, the ITA° increase rate is as high as 28.53%, and the MI index decrease rate reaches 25.46%. Its actual whitening efficacy is about 1.89 times the theoretical sum, showing a very strong non-linear synergistic growth trend. This significant multiplication effect powerfully verifies the scientific validity of the dual synergistic mechanism of enzyme inhibition and microenvironment regulation proposed in this invention: that is, using glycyrrhizin to precisely inhibit the downstream links of melanin production, while using asiaticoside to weaken the melanocyte activation signals caused by micro-inflammation from the source. This two-pronged strategy achieves efficient co-delivery through the specific nano-delivery carrier of this invention. At the same time, comparative data further confirms that when the emulsifier ratio is not within the range specified in this invention (Comparative Example 3) or the specific glycerol dilution process of this invention is not used (Comparative Example 11), due to the low precipitation of glycyrrhizin microcrystals and the low encapsulation and permeability of active components, the final whitening improvement effect cannot even reach the level of single-component drug-loaded liposomes, and is far lower than that of the embodiments of this invention.

Claims

1. A glycyrrhizin-hydroxyasiaticoside dual-compartment liposome, characterized in that, The raw materials for preparation, by mass percentage, include the following components: soybean lecithin 6.5%-9.5%, co-emulsifier 0.8%-2.0%, glycerin 18%-25%, glycyrrhizin 0.4%-1.0%, asiaticoside 0.5%-1.5%, 1,3-butanediol 6%-10%, preservative 0.5%-1.0%, with the balance being deionized water; The co-emulsifier comprises sucrose stearate and stearyl alcohol polyether-2 in a mass ratio of 1.5-4:1; The dual-compartment liposomes were prepared using the glycerol dilution method. In the dual-compartment liposomes, glycyrrhizin was embedded in the liposome's lipophilic bilayer in an amorphous state, while asiaticoside was completely encapsulated in the hydrophilic inner cavity of the liposome.

2. The glycyrrhizin-hydroxyasiaticoside dual-compartment liposome according to claim 1, characterized in that, The sucrose stearate has an HLB value of 14-18.

3. The glycyrrhizin-hydroxyasiaticoside dual-compartment liposome according to claim 1, characterized in that, The soybean lecithin mentioned is lecithin of soybean or animal origin.

4. The glycyrrhizin-hydroxyasiaticoside dual-compartment liposome according to claim 1, characterized in that, The preservatives include p-hydroxyacetophenone and 1,2-hexanediol.

5. The glycyrrhizin-hydroxyasiaticoside dual-compartment liposome according to claim 1, characterized in that, The glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes meet at least one of the following conditions; (1) Encapsulation efficiency of hydroxyasiaticoside ≥70%; (2) Average initial particle size ≤ 140 nm; (3) After being sealed and aged at 25℃ for 1 month, the particle size growth rate was ≤8%, and no glycyrrhizin needle-like birefringent crystals were precipitated under a polarizing microscope.

6. A method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes according to any one of claims 1-5, characterized in that, Includes the following steps: Soybean lecithin and emulsifier were moistened with glycerin and heated to 70℃-75℃ until completely dissolved to obtain a surfactant gel phase. At a constant temperature of 70℃-75℃, glycyrrhizin and 1,3-butanediol were mixed and stirred until completely dissolved. The surfactant gel phase was then added and stirred to obtain a mixed phase. Hydroxyacin was completely dissolved in 1.5-1.8 times its weight of deionized water to obtain an aqueous solution of hydroxyasiaticoside. At a constant temperature of 70℃-75℃, add the aqueous solution of hydroxyascorbic acid to the mixed phase and stir to mix. Add preservatives and the remaining deionized water and stir to obtain a crude liposome suspension; The crude liposome suspension was homogenized, filtered, and subjected to particle size reduction treatment to obtain glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes.

7. The method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes according to claim 6, characterized in that, The aqueous solution of hydroxyascorbic acid was added to the mixed phase at a rate of 10-20 mL / min and stirred for 5-10 min.

8. The method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes according to claim 6, characterized in that, The homogenization speed is 2500-3000 rpm, and the homogenization time is 5-10 min.

9. The method for preparing glycyrrhizin-hydroxyasiaticoside dual-compartment liposomes according to claim 6, characterized in that, The particle size refinement process includes the following steps: using a high-pressure microfluidic homogenizer to cycle 3-5 times at a pressure of 1500-2000 bar to refine the particle size.

10. The application of the glycyrrhizin-hydroxyasiaticoside dual-compartment liposome according to any one of claims 1-5, characterized in that, It is used in the preparation of cosmetics.

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