Hyaluronic acid-asparagososide liposome assembly and application thereof

Abstract

The application discloses hyaluronic acid-madecassoside liposome assembly and application thereof. The madecassoside liposome is prepared from hyaluronic acid, madecassoside, phospholipid, a surfactant and water. After being modified by hyaluronic acid, the madecassoside liposome has higher encapsulation efficiency, smaller particle size, stronger penetration capacity and gentleness compared with conventional madecassoside liposomes on the market, can significantly improve the efficacy of madecassoside, and has certain synergistic repair efficacy among components of the hyaluronic acid-madecassoside liposome assembly, so that the hyaluronic acid-madecassoside liposome assembly can be used for preparing cosmetics with repair efficacy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cosmetics, and particularly relates to a hyaluronic acid-asparagososide liposome assembly and application thereof. BACKGROUND

[0002] Asparagus officinalis, belonging to the family Umbelliferae, is found in most tropical and subtropical countries. The main active ingredients of Asparagus officinalis extract are triterpenes, including asparagososide, asparagosic acid, loganin and loganic acid. Other ingredients isolated from this plant include fatty acids (linoleic acid, linolenic acid, oleic acid, palmitic acid and stearic acid), abundant tannic acid and essential acids. Asparagososide is a highly active compound in the aqueous extract of Asparagus officinalis and is used in many alternative medicine formulations. It has been reported that asparagososide has multiple activities, such as inhibiting keratinocyte proliferation, inducing collagen synthesis and inhibiting cytokine and chemokine activity. Therefore, asparagososide is an important traditional Chinese medicine and is widely used in alternative therapies for anti-tumor, anti-psoriasis, eczema, anti-inflammatory, anti-aging, burn and wound healing. Asparagososide is composed of glucose and rhamnose in the molecule and has high water solubility. Solubility is an important parameter affecting the transdermal penetration of drugs across the lipid membrane. The high water solubility of asparagososide limits its ability to pass through the stratum corneum, resulting in reduced bioavailability and efficacy of topical formulations, greatly reducing its efficacy.

[0003] Nanocarrier technology, as a novel carrier technology, has been widely used in the field of skin care. Topical formulations containing nanocarriers show greater drug accumulation in the deeper layers of the epidermis and dermis. In order to further enhance the penetration of hydrophilic compounds into deeper skin layers, surface modification of nanocarriers with appropriate polymers having additional biological activity and low irritation characteristics is an interesting approach. One of the penetration enhancers is hyaluronic acid (HA), which naturally exists in healthy skin tissue. Hyaluronic acid is a macromolecule and a natural polysaccharide containing repeating units of d-glucuronic acid and n-acetyl-d-glucosamine (Laurent 1970). Hyaluronic acid has been shown to alter the physiological properties of the stratum corneum. Therefore, it has been included in many dermatological and cosmetic formulations.

[0004] CN 115590774 B discloses a hyaluronic acid liposome assembly and its preparation method and application. The hyaluronic acid liposome assembly is formed by liposome and hyaluronic acid of different molecular weight through certain assembly mode, so that the hyaluronic acid participates in the mutual embedding of the liposome of the double molecular structure, and further forms a new stable double molecular layer skeleton structure, that is, the hyaluronic acid liposome assembly. Under the double effects of skin moisturizing and penetration promotion of hyaluronic acid, further mutual cooperation with the liposome is realized, so that the HA Liposome realizes the effect of high efficient penetration of the skin. The preparation process of the application is relatively complex, the prepared particle size is 500nm, which is greatly different from other liposome particle sizes, and only the moisturizing effect of hyaluronic acid itself is found.

[0005] Therefore, we try to combine hyaluronic acid and asiaticoside liposome during preparation, and find some unexpected results. SUMMARY

[0006] In view of the shortcomings of the prior art, the present application provides a hyaluronic acid-asiaticoside liposome assembly and its application. The asiaticoside liposome of the present application is prepared from hyaluronic acid, asiaticoside, phospholipid, surfactant and water. After modification by hyaluronic acid, compared with conventional asiaticoside liposomes on the market, the encapsulation efficiency is higher, the particle size is smaller, the penetration ability is stronger and it is mild and non-irritating, which can significantly improve the efficacy of asiaticoside. At the same time, the hyaluronic acid-asiaticoside liposome assembly has certain synergistic repair effect among the components, so that it can be used for preparing cosmetics with repair effect, and has good practical value.

[0007] To achieve the above technical purpose, the technical scheme adopted by the present application is as follows:

[0008] In a first aspect of the present application, a hyaluronic acid-asiaticoside liposome assembly is provided, characterized in that the hyaluronic acid-asiaticoside liposome assembly is prepared from hyaluronic acid, asiaticoside, phospholipid, surfactant and water. The weight ratio of each component is: hyaluronic acid 0.2-2%, asiaticoside 5-15%, phospholipid 1-5%, surfactant 0.6-2% and water balance.

[0009] The preferred ratio is: hyaluronic acid 1.2%, asiaticoside 10%, phospholipid 3.5%, surfactant 1.4% and water balance.

[0010] Preferably, the hyaluronic acid is at least one of hyaluronic acid with a molecular weight of 8000 Da and hyaluronic acid with a molecular weight of 20w-40w Da, preferably hyaluronic acid with a molecular weight of 8000 Da.

[0011] The phospholipid is selected from at least one combination of hydrogenated lecithin, soybean lecithin, hydrogenated phosphatidylcholine and egg yolk lecithin.

[0012] The surfactant is at least one of a 2 (lauramide glutamine) lysine sodium aqueous solution (mass fraction 29%) and a sodium subtilipetide. Preferably, the 2 (lauramide glutamine) lysine sodium aqueous solution (mass fraction 29%) is 1%, and the sodium subtilipetide is 0.4%.

[0013] The preparation method is as follows:

[0014] S1: Take phospholipid, asiaticoside, surfactant, add ethanol to dissolve, then evaporate under reduced pressure until the ethanol is fully volatilized to obtain a lipid film;

[0015] S2: Add hyaluronic acid to water, and treat with water bath stirring to make the hyaluronic acid fully dissolved to obtain solution 1;

[0016] S3: Mix the lipid film with solution 1, and treat with ultrasonic to obtain solution 2;

[0017] S4: Homogenize solution 2 to obtain a hyaluronic acid-asiaticoside liposome assembly.

[0018] In step S1, the mass ratio of phospholipid to ethanol is 1:10-25, preferably 1:20, and the reduced pressure evaporation temperature is 60-75°C, preferably 65°C.

[0019] In step S2, the water bath stirring temperature is 60-75°C, preferably 65°C, and the water bath stirring time is 10-30 min, preferably 15 min.

[0020] In step S3, the ultrasonic conditions are: ultrasonic power is 300 watts, frequency is 50 Hz, temperature is 40-65°C, preferably 45°C, and ultrasonic time is 30-60 min, preferably 30 min.

[0021] In step S4, the homogenization conditions are: homogenization pressure is 1.4-1.8 MPa, cycle is 4-6 times, homogenization rate is 6000-9000 rpm, homogenization time is 12-20 min, and homogenization temperature is 40-65°C. Preferably, the homogenization pressure is controlled to be 1.6 MPa, the cycle is 5 times, the homogenization rate is 8000 rpm, the homogenization time is 14 min, and the homogenization temperature is 45°C.

[0022] The hyaluronic acid-asparagosin liposome assembly obtained by the preparation method has higher encapsulation efficiency, smaller and more uniform particle size, stronger penetration ability, and is mild and non-irritating, and can significantly improve the efficacy of asparagosin, and the components of the hyaluronic acid-asparagosin liposome assembly have certain synergistic repair efficacy.

[0023] In a second aspect of the present application, the hyaluronic acid-asparagosin liposome assembly is applied in the preparation of a cosmetic product.

[0024] The cosmetic product can also optionally contain common ingredients of cosmetic products, including any ingredient known in the art, such as vehicles (e.g., diluents, dispersants, carriers, etc.), cosmetic adjuvants (e.g., emulsifiers, thickeners, etc.), and the type and amount thereof can be selected according to specific needs, which will not be specifically limited herein.

[0025] In addition, in the present application, the cosmetic product should be understood in a broad sense, which includes but is not limited to cleansing cream (paste), facial cleanser, bath liquid, face cream, cosmetic water, facial mask, and sunscreen, etc.

[0026] In a third aspect of the present application, a cosmetic product is provided, which contains at least the hyaluronic acid-asparagosin liposome assembly.

[0027] Technical effects of the present application: the hyaluronic acid-asparagosin liposome assembly prepared by the present application has the following advantages compared with conventional asparagosin liposomes on the market: ① higher encapsulation efficiency, smaller particle size, stronger penetration ability, mildness, and no irritation, which can significantly improve the efficacy of asparagosin; ② the surfactant used in the present application is a biological surfactant, and the use of the combination thereof has certain synergistic stabilizing and emulsifying effects, which reduces the use cost of a single emulsifier, and is more natural, green, and environmentally friendly; ③ the components of the hyaluronic acid-asparagosin liposome assembly have certain synergistic repair efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 An experimental result graph of the prepared 5-aminofluorescein-labeled hyaluronic acid-asparagosin liposome assembly for fluorescence microscope observation. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as generally understood by those skilled in the art to which the present application belongs.

[0030] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and / or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and / or groups thereof. It will be understood that the scope of the application is not limited to the specific specific embodiments described below; it is also understood that the terms used in the present application are merely used to describe particular specific embodiments and are not intended to limit the scope of the present application.

[0031] The application will be further described by way of example only with reference to the following Examples, which are not intended to be limiting. It is to be understood that these Examples are provided by way of illustration only and nothing contained herein is intended to be limiting.

[0032] Examples 1-17:

[0033] The specific components of each of Examples 1-17 are as follows in Table 1.

[0034] Table 1: The specific components of each of Examples 1-17

[0035]

[0036]

[0037] The preparation method of Examples 1-17 is as follows:

[0038] S1: Take the hydrogenated lecithin, asiaticoside, and surfactant, and place them in a round-bottom flask. Add ethanol (the mass ratio of hydrogenated lecithin to ethanol is 1:20) to dissolve. Use a rotary evaporator to evaporate at 65°C under reduced pressure until the ethanol is fully volatilized, and obtain a lipid film 1;

[0039] S2: Add hyaluronic acid to purified water, and stir in a water bath at 65°C for 15 min to fully dissolve the hyaluronic acid, and obtain a solution 1;

[0040] S3: Mix the film 1 and the solution 1, and ultrasonically treat (ultrasonic power is 300 watts, frequency is 50 Hz, and temperature is 45°C) for 30 min to obtain a solution 2;

[0041] S4: Add the solution 2 to a high-pressure homogenizer, and homogenize (homogenization pressure is controlled at 1.6 MPa, homogenization rate is 8000 rpm, homogenization time is 14 min, and homogenization temperature is 45°C) to obtain a hyaluronic acid-asiaticoside liposome assembly.

[0042] Comparative Examples 1-12:

[0043] The specific composition of each component of Comparative Examples 1-12 is shown in Table 2 below.

[0044] Table 2: Specific composition of each component of Comparative Examples 1-12

[0045]

[0046]

[0047] Comparative Examples 1-7 were prepared according to the same method as Examples 1-17, and Comparative Examples 8-12 were formulated into corresponding suspensions.

[0048] Application Examples 1-17: An emulsion containing hyaluronic acid-asparagosin liposome assemblies

[0049] Phase A: A165 (emulsifier) 1 gram, GTCC (caprylic / capric triglyceride) 3 grams, white oil 3 grams, cetostearyl alcohol 0.5 gram, silicone oil 5 grams;

[0050] Phase B: glycerin 4 grams, butylene glycol 4 grams, EMT-10 (thickening agent) 0.4 gram, xanthan gum 0.1 gram, fructone 0.4 gram, hexylene glycol 0.4 gram, deionized water TO 100 grams;

[0051] Phase C: hyaluronic acid-asparagosin liposome assemblies prepared in Examples 1-17, 5 grams.

[0052] The preparation steps of the above emulsion are as follows:

[0053] 1) Disperse EMT-10 and xanthan gum into glycerin and butylene glycol first, then add fructone, hexylene glycol and deionized water, heat and stir in a water bath at 80°C to fully dissolve, to obtain Phase B;

[0054] 2) Heat and melt A165, GTCC, white oil, cetostearyl alcohol, silicone oil in a water bath at 83°C to obtain Phase A;

[0055] 3) Heat Phases A and B in a water bath at 83°C at the same time, when the temperatures of the two phases are the same, slowly add Phase A to Phase B under the premise of stirring Phase B at 500 r / min;

[0056] 4) After mixing Phases B and A, cool the emulsion at a stirring rate of 430 r / min;

[0057] 5) When the temperature of the emulsion drops to 40-45°C, add the hyaluronic acid-asparagosin liposome assemblies and stir at 60 r / min to cool;

[0058] 6) When the temperature drops to 35°C, cool the product statically.

[0059] Application Example 18: A cream containing hyaluronic acid-asparagosin liposome assembly

[0060] Phase A: GTCC 3g, white oil 3g, cetostearyl alcohol 3g, C14-22 alcohol 1g, C12-20 alkyl glucoside 2.0g, glyceryl stearate 1.5g, cetyl stearyl alcohol 1g, vegetable squalane 5g, dicaprylyl carbonate 5g;

[0061] Phase B: glycerin 4g, butylene glycol 4g, EMT-10 (thickening agent) 0.4g, AVC (thickening agent) 0.4g, xanthan gum 0.1g, fructone 0.4g, hexylene glycol 0.4g, deionized water TO 100g;

[0062] Phase C: 5g of hyaluronic acid-asparagosin liposome assembly prepared in Example 1.

[0063] The preparation steps of the above cream are as follows:

[0064] 1) Disperse EMT-10, xanthan gum and AVC into glycerin and butylene glycol first, then add fructone, hexylene glycol and deionized water, heat and stir in a water bath at 80°C to fully dissolve, to obtain phase B;

[0065] 2) Heat and melt GTCC, white oil, cetostearyl alcohol, C14-22 alcohol, C12-20 alkyl glucoside, glyceryl stearate, cetyl stearyl alcohol, vegetable squalane, dicaprylyl carbonate in a water bath at 83°C to obtain phase A;

[0066] 3) Heat phases A and B in a water bath at 83°C at the same time, when the temperatures of the two phases are the same, slowly add phase A to phase B under the premise of stirring phase B at 500 r / min;

[0067] 4) After mixing phases B and A, cool the cream at a stirring rate of 430 r / min;

[0068] 5) When the temperature of the emulsion drops to 40-45°C, add the hyaluronic acid-asparagosin liposome assembly and stir at 60 r / min to cool;

[0069] 6) When the temperature drops to 35°C, cool the product statically.

[0070] Application Example 19: A mask liquid containing hyaluronic acid-asparagosin liposome assembly

[0071] Phase A: glycerin 4g, dipropylene glycol 4g, AVC (thickening agent) 0.3g, xanthan gum 0.2g, fructone 0.4g, hexylene glycol 0.4g, deionized water TO 100g;

[0072] Phase B: 5 grams of the hyaluronic acid-asparagosin liposome assembly prepared in Example 1.

[0073] The mask preparation procedure is as follows:

[0074] 1) Disperse xanthan gum and AVC into glycerol and dipropylene glycol first, then add fructone, hexanediol and deionized water, heat and stir in a 65°C water bath to fully dissolve, to obtain phase A;

[0075] 2) When the temperature of the liquid drops to 40-45°C, add phase B, and stir at 60 r / min to cool down;

[0076] 6) When the temperature drops to 35°C, stop cooling to obtain the product.

[0077] Comparative application examples 1-12: an emulsion containing hyaluronic acid-asparagosin liposome assembly

[0078] Phase A: A165 (emulsifier) 1 gram, GTCC 3 grams, white oil 3 grams, cetostearyl alcohol 0.5 gram, silicone oil 5 grams;

[0079] Phase B: glycerol 4 grams, butanediol 4 grams, EMT-10 (thickening agent) 0.4 gram, xanthan gum 0.1 gram, fructone 0.4 gram, hexanediol 0.4 gram, deionized water TO 100 grams;

[0080] Phase C: 5 grams of the sample of comparative examples 1-12.

[0081] The preparation method is the same as that of application examples 1-17.

[0082] Experimental example 1: particle size and encapsulation rate determination

[0083] ① Particle size: dilute the asparagosin liposome with ultrapure water several times, and measure the average particle size of the liposome with a Malvern particle size analyzer, and measure three times in parallel, and the results are as shown in Table 3.

[0084] ② Encapsulation rate: the content of asparagosin was determined by high performance liquid chromatography, and the liquid chromatography conditions were as follows: chromatographic column: (C18, 4.6x200mm, 5μm); mobile phase: acetonitrile: water (volume ratio) = 18:82; flow rate: 0.8mL / min; detection wavelength: 205nm; injection volume: 10μL; running time: 20min; column temperature: 25°C, to obtain a standard curve with good linearity within 50-120μg / mL: y=0.28864x-5.7981, R 2 =0.9860. In the formula, y is the peak area, and x is the mass concentration of asparagosin.

[0085] The dialysis method was used to separate the liposome from free asiaticoside. 1 mL of liposome was accurately pipetted into a dialysis bag, which was sealed in PBS buffer. The dialysis bag was stirred at room temperature and in the dark at 300 r / min until the dialysis equilibrium was reached. The dialysate was taken, and the concentration of asiaticoside was determined by high performance liquid chromatography, and the encapsulation efficiency was calculated.

[0086] Encapsulation efficiency (%) = (1 - m 游离 / m 总 ) x 100%

[0087] Wherein: m 游离 is the mass of free asiaticoside; and m 总 is the total mass of asiaticoside.

[0088] Table 3: Average particle size of liposome

[0089]

[0090]

[0091] Particle size is considered to be an important factor related to stability, bioavailability and encapsulation efficiency. Only particles of 50-500 nm can penetrate into the skin, and smaller particles can make it easier for drugs to pass through the barrier of the stratum corneum. PDI (polydispersity index) I indicates the degree of non-uniformity of particle size distribution, ranging from 0 to 1.0, and the smaller the PDI value, the higher the uniformity of particle size distribution. For liposome nanoparticles, a PDI value of 0-0.3 indicates uniform particle dispersion. Zeta potential is one of the properties of liposome stability. Zeta potential is an indicator of the surface charge of the liposome, and the higher the absolute value, the greater the mutual repulsion, and the frequency of liposome collision is reduced, so the system is more stable. A relatively high Zeta potential is very important for the physical and chemical stability of the liposome. A Zeta potential absolute value greater than 30 mV indicates that the liposome has good stability.

[0092] From the above Table 3,

[0093] 1. The encapsulation efficiency and particle size of the liposome prepared in the examples are significantly better than those of the liposome prepared by conventional emulsifiers (comparative example 6 and comparative example 7);

[0094] 2、The encapsulation rate and particle size indexes of the surfactant used in the application, whether used alone or in combination, are superior to those of the liposomes prepared by using conventional emulsifiers (comparative examples 6 and 7), and in particular, the encapsulation rate and particle size indexes of the emulsifier used in combination in the application (example 1) are superior to those of the emulsifier used alone (example 11, example 12, comparative example 1, comparative example 2, and comparative example 3), which indicates that the emulsifiers selected for use in the application have certain synergistic effects, and can significantly improve the encapsulation rate of the liposomes and achieve better particle size indexes.

[0095] Experimental Example 2: Liposome stability test

[0096] (1) Different temperature stability investigation

[0097] The samples of examples 1-17 and comparative examples 1-12 were respectively placed at room temperature, 4℃, 45℃, -18℃, and under cycling (4℃, 45℃, -18℃) for three months for stability investigation experiments, and the investigation results are shown in Table 4.

[0098] Table 4 Investigation results of different temperature stability

[0099]

[0100]

[0101] From the above Table 4, it can be seen that,

[0102] 1. The emulsion stability of the liposomes prepared in the examples is obviously superior to that of the liposomes prepared by using conventional emulsifiers (comparative examples 6 and 7);

[0103] 2. The emulsion stability of the surfactant used in the application, whether used alone or in combination, is superior to that of the liposomes prepared by using conventional emulsifiers (comparative examples 6 and 7), and in particular, the emulsion stability of the emulsifier used in combination in the application (example 1) is superior to that of the emulsifier used alone (comparative example 1, comparative example 2, and comparative example 3), which indicates that the emulsifiers selected for use in the application have certain synergistic emulsification and stability effects, and to some extent, reduce the content of the emulsifier, reduce the cost of the examples, and reduce the irritation risk caused by the emulsifier.

[0104] (2) Investigation test of particle size change rate at room temperature for 6 months: the change rate of the liposome particle size after the samples of examples 1-17 and comparative examples 1-12 are respectively stored for six months, particle size change rate (%) = (6-month particle size - initial particle size / initial particle size) x 100%

[0105] The results are shown in Table 5 below:

[0106] Table 5 Change rate of liposome particle size

[0107]

[0108]

[0109] From the above Table 5, it can be seen that,

[0110] 1. The particle size change of the liposomes prepared in the examples is obviously smaller than that of the liposomes prepared by using conventional emulsifiers (comparative example 6, comparative example 7);

[0111] 2. The particle size change of the liposomes prepared by using the surfactants in the present application, whether used alone or in combination, is smaller than that of the liposomes prepared by using conventional emulsifiers (comparative example 6, comparative example 7), and in particular, the particle size change of the liposomes prepared by using the emulsifiers in combination (example 1) is smaller than that of the liposomes prepared by using the emulsifiers alone (example 11, example 12, comparative example 1, comparative example 2, comparative example 3), which indicates that the emulsifiers selected for use in the present application have certain synergistic effect of stabilizing the particle size of the liposomes.

[0112] Experimental Example 3: In-vitro transdermal experiment

[0113] ① The skin of SD rats (200-250 g) was treated by shaving the back fur, and then the rats were sacrificed after 24 h; the back skin of the rats was carefully cut and the subcutaneous fat was removed, and the skin was washed with physiological saline for 3 times, the surface water was absorbed with a dust-free paper, and finally the skin was stored in a refrigerator for frozen preservation for standby use.

[0114] ② The rat skin was taken out from the refrigerator, and was thawed at room temperature in physiological saline, and then the rat skin was clamped between the receiving pool and the diffusion pool with the horny layer upward, the receiving pool was filled with physiological saline and the bubbles were discharged. 1 mL of each of the above-prepared example and comparative example samples having the same concentration of asperosaponin was uniformly applied on the rat skin, the diffusion pool was sealed with a preservative film, and the whole was placed in a magnetic stirrer, and was stirred at 200 r / min in a constant temperature water bath at 37℃. 0.5 mL of each sample was taken out at 12 h, 10% triton solution was added to make up to 1 mL, and then the sample was filtered with a 0.45 μm microporous filter; at the same time, 0.5 mL of physiological saline was added to the receiving pool. The concentration of asperosaponin was determined by high performance liquid chromatography, and the cumulative permeation rate was calculated. The cumulative permeation rate Q% was calculated by the following formula:

[0115]

[0116] In the formula, Mn is the drug mass concentration measured at the nth sampling point, in mg / mL; Mi is the drug mass concentration measured at the ith sampling point, in mg / mL; V is the total volume of the receiving pool, which is 6.5 mL in the experiment; Vi is the volume of the receiving liquid taken out at the ith sampling point, which is 0.5 mL in the experiment; m is the mass of asiaticoside, which is 0.05 mg.

[0117] The experimental results are shown in Table 6.

[0118] Table 6 12h cumulative permeation rate

[0119]

[0120] It can be known from Table 6 that,

[0121] 1. The liposome modified by hyaluronic acid (Example 1) has better penetration-promoting ability for asiaticoside than the unmodified liposome (Comparative Example 4), and has certain synergistic penetration-promoting effect;

[0122] 2. The liposome using a biological surfactant (Example 1) has better penetration-promoting ability for asiaticoside than the liposome prepared by using a conventional emulsifier (Comparative Examples 6 and 7), and has certain synergistic penetration-promoting effect. Therefore, the liposome prepared in the application can realize the dual synergistic penetration-promoting effect of hyaluronic acid modification and biological surfactant, and significantly improves the transdermal rate of asiaticoside.

[0123] Experimental Example 4: Irritation evaluation experiment

[0124] 30 subjects were recruited in the experiment, without limitation of gender, and the age was 23-50 years old. Examples 1-17 and Comparative Examples 1-12 were used as test substances (each subject was tested by Examples 1-17 and Comparative Examples 1-12), and the blank was a negative control. The test method was to select a suitable patch tester with an area not exceeding 50 mm 2 , and a depth of about 1 mm. About 0.020-0.025 g of the test substance was added into the patch tester by a closed patch test method. A low-sensitization adhesive tape was applied to the back of the subject. After 24 hours, the test substance was removed, and the skin reaction was observed at 0.5, 24 and 48 hours after removal, respectively. The results were recorded according to the skin reaction grading standard in the “Cosmetic Safety Technical Specification 2015” (Table 7), and the detailed results are shown in Table 8 below.

[0125] Table 7 Skin reaction grading standard

[0126]

[0127] Table 8 Results of irritation evaluation experiment

[0128]

[0129]

[0130]

[0131] From the above Table 8, it can be seen that the samples of Examples 1-17, Comparative Examples 1-5 and Comparative Examples 8-12 did not have adverse reactions and were mild and non-irritating; Comparative Examples 6 and 7 both had adverse reactions to varying degrees, and Comparative Example 7 had the most severe adverse reactions. This shows that the liposomes of the present application are milder and significantly reduce the irritation of the liposomes compared to liposomes prepared using conventional emulsifiers.

[0132] Experimental Example 5: In vitro promotion of barrier-related protein FLG experiment

[0133] UVB irradiation of 3D epidermal skin model The repair efficacy was evaluated by detecting the change in the content of the barrier-related protein (FLG) of the skin model after the sample was applied.

[0134] According to the test scheme in Table 9 below, the model was transferred to a 6-well plate (0.9 mL of the corresponding group's culture solution was added in advance), and the test group number was marked on the 6-well plate. 0.9 mL of sterile PBS was added to each well of the BC and NC groups, 0.9 mL of a PBS solution containing 50 μM WY14643 was added to the PC group, and 0.9 mL of a PBS solution containing the corresponding concentration of sample was added to the sample group.

[0135] The 6-well plate was placed under a UVB irradiator according to the test grouping. After irradiation, the culture medium in the wells was aspirated, 0.9 mL of culture solution was added to each well of the BC and NC groups, 0.9 mL of a culture solution containing 50 μM WY14643 was added to the PC group, and 0.9 mL of a culture solution containing the corresponding concentration of sample working solution was added to the sample group. Then, the model was placed in an incubator (5% CO2, 37°C) and incubated for 24 h ± 2 h. After incubation, the surface of the model was washed with sterile PBS to remove any remaining test substance, and the inside and outside of the model were wiped clean with a sterile cotton swab.

[0136] The model for barrier-related protein detection was taken, and the model was cut around and fixed with 4% paraformaldehyde. After 24 h of fixation, immunofluorescence detection of the barrier-related protein (FLG) was performed, and the images were observed under a microscope, collected and analyzed. The FLG enhancement rate was calculated according to the following formula.

[0137] Enhancement rate = (sample IOD value - NC group IOD value) / NC group IOD value * 100%

[0138] The experimental results are shown in Table 10.

[0139] Table 9 Test scheme for promoting barrier-related protein FLG in vitro

[0140]

[0141]

[0142] Table 10 Test results for promoting barrier-related protein FLG in vitro

[0143]

[0144]

[0145] Note: ## indicates p < 0.01 compared with the blank control; ** indicates p < 0.01 compared with the negative control; * indicates 0.01 < p < 0.05 compared with the negative control.

[0146] From the above Table 10, the promotion rate of FLG of Example 1 is obviously higher than that of the above comparative examples, which shows that the triple components of hyaluronic acid, asiaticoside, and sodium di(lauroamidoglutamine) lysine have a certain synergistic promotion of FLG, achieving synergistic repair efficacy.

[0147] Experimental Example 6: Fluorescence microscope observation

[0148] 1. Preparation of fluorescent hyaluronic acid: under the condition of 30°C, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl) and N-hydroxysuccinimide (NHS) were added into the aqueous solution of hyaluronic acid, respectively, and the reaction was stirred by a magnetic heating stirrer in the dark for 8 h, then 5-aminofluorescein was added, and the reaction was continued for 48 h, then the dialysis bag with a molecular weight of 1000 Da was used for dialysis until no fluorescein was detected, and then freeze-drying was performed. The molar ratio of the hyaluronic acid, EDC-HCl, NHS, and 5-aminofluorescein was 1:1.5:2:2.5; the pure water was added at 5 mg per 10 mL of hyaluronic acid with different molecular weights; the temperature of the magnetic heating stirrer was 35°C, and the rotation speed was 1000 rpm; the judgment standard for using the dialysis bag to dialyze until no fluorescein was detected was that the absorbance of the dialysate at 486 nm was lower than 0.06 when measured by a UV-visible spectrophotometer.

[0149] 2. Preparation of 5-aminofluorescein-labeled hyaluronic acid-asiaticoside liposome assembly: the preparation method was the same as that in Example 1.

[0150] 3. Fluorescence microscope observation: the prepared 5-aminofluorescein-labeled hyaluronic acid-asiaticoside liposome assembly was observed by fluorescence microscope, and the experimental results were as follows:Figure 1 The results show that the hyaluronic acid successfully participated in the construction of the liposome, embedded in the liposome skeleton (the edge of the ring has green fluorescence), rather than being traditionally wrapped by the liposome (if it is wrapped, the figure shows that the spherical structure or the center of the ring has significant green fluorescence).

[0151] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not to limit it. Although the present application is explained in detail with reference to the examples given, the technical solutions of the present application can be modified or replaced equivalently by those skilled in the art without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A hyaluronic acid-centella asiatica liposome assembly, characterized in that, The hyaluronic acid-asiaticoside liposome assembly is prepared from hyaluronic acid, asiaticoside, phospholipids, surfactants and water; the weight ratio of each component is: hyaluronic acid 0.2-2%, asiaticoside 5-15%, phospholipids 1-5%, surfactant 0.6-2% and water balance. The surfactant is a mixture of sodium di(lauramide-glutamine)lysine aqueous solution and sodium subtilisin; The hyaluronic acid is at least one of hyaluronic acid with a molecular weight of 8000 Da and hyaluronic acid with a molecular weight of 20w-40w Da; Its preparation method is as follows: S1: Take phospholipids, asiaticoside, and surfactants, add ethanol to dissolve them, and then evaporate under reduced pressure until the ethanol is fully evaporated to obtain a lipid film; S2: Add hyaluronic acid to water and stir in a water bath until all the hyaluronic acid is dissolved to obtain solution 1; S3: Mix the lipid film with solution 1 and sonicate to obtain solution 2; S4: Homogenize solution 2 to obtain the hyaluronic acid-centella asiatica liposome assembly.

2. The hyaluronic acid-asiaticoside liposome assembly as described in claim 1, characterized in that, The weight ratio of each component is as follows: hyaluronic acid 1.2%, asiaticoside 10%, phospholipids 3.5%, surfactant 1.4%, and water as the balance.

3. The hyaluronic acid-asiaticoside liposome assembly as described in claim 1, characterized in that, The phospholipid is selected from a combination of at least one of hydrogenated lecithin, soybean lecithin, hydrogenated phosphatidylcholine, and egg yolk lecithin.

4. The hyaluronic acid-asiaticoside liposome assembly as described in claim 1, characterized in that, The surfactant is a 29% (w / w) aqueous solution of sodium di(lauramide-glutamine)lysine and 0.4% (w / w) sodium subtilisin.

5. The use of the hyaluronic acid-centella asiatica liposome assembly according to any one of claims 1-4 in the preparation of cosmetics.

6. The application as described in claim 5, characterized in that, The cosmetics in question are those with a barrier repair concept.

7. A cosmetic product, characterized in that, The cosmetic product contains at least the hyaluronic acid-centella asiatica liposome assembly as described in claim 1 or 2.

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