A soothing repair nanoliposome and a preparation method and application thereof

Abstract

The present application relates to a kind of soothing repair nanoliposome and its preparation method and application.The liposome includes mass ratio 1: (1-3) oil phase and water phase, the oil phase includes the following mass percentage of raw materials: 0.1-0.5% ceramide NP, 0.3-3% cure wu flower extract, 0.2-0.5% hydroxyekdoin, 2-4% squalane, 0.1-0.5% di (lauramide glutamine) lysine sodium, 1-3% hydrogenated lecithin, 1-3% cholesterols, the rest is anhydrous ethanol;The water phase includes the following mass percentage of raw materials: 1-5% dendrobium nobile Lindl extract, 1-3% glycerol grape glycoside, 0.5-1% recombinant fibronectin, 20-40% glycerol, the rest is deionized water.The soothing repair nanoliposome of the present application can realize the dual effect of instant soothing, long-term repair.

Description

Technical Field

[0001] This invention relates to the field of nanoliposome technology, specifically to a soothing and repairing nanoliposome, its preparation method, and its application. Background Technology

[0002] Soothing and repairing ingredients often fall into two categories: oil-soluble (such as ceramides and plant extracts) and water-soluble (such as ectoine). Current technologies mostly employ a single solubilization method, which can easily lead to insufficient solubility of certain ingredients, resulting in aggregation and precipitation. If the active ingredients are not effectively protected, they are easily degraded by temperature, light, and oxygen, or react with other ingredients during storage, leading to poor stability and a short shelf life.

[0003] The skin's stratum corneum barrier hinders liposome penetration, and existing liposome technologies mostly remain on the skin surface, unable to deliver active ingredients to the dermis. The lack of a targeted delivery mechanism prevents active ingredients from precisely targeting damaged cells, resulting in low repair efficiency and insufficient efficacy. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a soothing and repairing nanoliposome, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a soothing and repairing nanoliposome, comprising an oil phase and an aqueous phase in a mass ratio of 1:(1-3), wherein the oil phase comprises the following raw materials in mass percentage: 0.1-0.5% ceramide NP, 0.3-3% *Vigna pubescens* extract, 0.2-0.5% hydroxyectoine, 2-4% squalane, 0.1-0.5% sodium bis(lauramide-glutamine)lysine, 1-3% hydrogenated lecithin, 1-3% cholesterol, with the balance being anhydrous ethanol; wherein the aqueous phase comprises the following raw materials in mass percentage: 1-5% *Dendrobium nobile* extract, 1-3% glyceryl glucoside, 0.5-1% recombinant fibronectin, 20-40% glycerol, with the balance being deionized water;

[0007] The preparation method of the Dendrobium nobile extract includes the following steps:

[0008] S1. After washing and crushing Dendrobium nobile, add deionized water, adjust the pH to 4-5, add a compound enzyme for enzymatic hydrolysis, inactivate the enzyme, and obtain the enzymatic hydrolysate; wherein, the compound enzyme is a combination of cellulase, protease and pectinase.

[0009] S2. The enzymatic hydrolysate is extracted by ultrasonication, filtered, and the filtrate and residue are obtained.

[0010] S3. Add phosphorylation reagent to the filtrate obtained in S2, adjust the pH to 4-6, stir the reaction to obtain phosphorylated water extract;

[0011] S4. Add the filter residue obtained in S2 to anhydrous ethanol, add xylitol anhydride monostearate and diacetyl tartaric acid mono- and diglycerides, reflux extract, filter, and rotary evaporate the filtrate until no ethanol remains to obtain the alcohol extract.

[0012] S5. Mix the phosphorylated water extract obtained in S3 with the alcohol extract obtained in S4 to obtain the Dendrobium nobile extract.

[0013] In step S1 of the preparation of the Dendrobium nobile extract of this invention, a complex enzyme (cellulase + protease + pectinase) is used to specifically decompose the cell wall and internal protein structure of Dendrobium nobile, thereby increasing the release rate of active ingredients such as polysaccharides and flavonoids. Simultaneously, the small molecule peptides generated by enzymatic hydrolysis can assist in the subsequent dissolution and stabilization of active ingredients. In step S2, the enzymatic hydrolysate is subjected to ultrasonic extraction. The cavitation effect of ultrasound accelerates cell disruption, further improving the dissolution efficiency of active ingredients and reducing the loss of effective components. In step S3, because the filtrate obtained after filtration contains a large amount of Dendrobium nobile polysaccharides, the phosphorylation reagent introduces phosphate groups into the Dendrobium nobile polysaccharides, making their surface carry more negative charges. This modification allows the polysaccharides to form a stable electrostatic adsorption complex with recombinant fibronectin (positively charged), while simultaneously improving the emulsification performance and kinetic stability of the aqueous system, avoiding liposome stratification and aggregation. Step S4 involves treating the filter residue containing a significant amount of water-insoluble flavonoids. Anhydrous ethanol is used as the solvent to enhance the concentration of active ingredients in the residue. Firstly, the addition of diacetyl tartaric acid mono- and diglycerides breaks down cell membrane barriers, allowing flavonoids, lipid-soluble components, and saponins in the intercellular matrix to be fully exposed. Secondly, xylitol anhydride monostearate simultaneously optimizes the dissolution environment of the ethanol system, ensuring the timely and stable dissolution of these exposed active ingredients with varying solubility in the solvent. This synergistic "release first, then dissolve" approach avoids the inability to release components due to cell membrane obstruction and solves the problem of re-precipitation due to insufficient solubility after release. Furthermore, both processes have emulsifying and dispersing effects, maintaining a uniform and stable extraction system and preventing stratification and aggregation between the solvent and extracted components. This further ensures the continuous and efficient dissolution of various active ingredients, ultimately resulting in a more comprehensive and abundant variety of active ingredients in the Dendrobium nobile extract. In step S5, the phosphorylated water extract and alcohol extract are mixed evenly to fully integrate the water-soluble and alcohol-soluble active ingredients in Dendrobium nobile, forming a rich and uniform extract. This Dendrobium nobile extract can be better mixed with other raw materials to form a stable dispersion system in the liposome composition, reducing problems such as layering and precipitation caused by uneven composition, thereby improving the stability of the liposome composition.

[0014] Preferably, in step S1 of preparing the Dendrobium nobile extract, the ratio of Dendrobium nobile to deionized water is 1g:15-20mL, the amount of the compound enzyme added is 1-3% of the mass of Dendrobium nobile, and the mass ratio of cellulase, protease and pectinase is 1:(0.5-1.5):(0.5-1.5); the enzymatic hydrolysis temperature is 30-35℃ and the time is 1-3h; the enzyme inactivation temperature is 80-90℃ and the time is 10-15min.

[0015] Preferably, in step S2 of the preparation of Dendrobium nobile extract, the ultrasonic extraction power is 300-400W, the temperature is 40-50℃, and the time is 1-2h.

[0016] Preferably, in step S3 of the preparation of Dendrobium nobile extract, the amount of phosphorylation reagent added is 5-10% of the mass of the filtrate, and the phosphorylation reagent is sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of (2-3):1; the stirring speed is 500-600 rpm, the temperature is 40-50℃, and the time is 20-40 min.

[0017] Preferably, in step S4 of the preparation of Dendrobium nobile extract, the ratio of the filter residue to anhydrous ethanol is 1g:5-10mL, the amount of xylitol anhydride monostearate added is 0.3-0.6% of the mass of the filter residue, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.1-0.3% of the mass of the filter residue.

[0018] Preferably, in step S4 of the preparation of Dendrobium nobile extract, the reflux extraction temperature is 60-70℃ and the time is 40-60 min; filtration is performed using a 0.1-0.2 μm filter membrane.

[0019] In a second aspect, the present invention provides a method for preparing the soothing and repairing nanoliposomes of the first aspect, comprising the following steps:

[0020] (1) Mix the raw materials in the oil phase according to the formula, stir evenly, and transfer to a rotary evaporator to remove ethanol to obtain the oil phase;

[0021] (2) Mix all the raw materials in the aqueous phase according to the formula to obtain the aqueous phase;

[0022] (3) Mix the aqueous phase and oil phase according to the formula amount, and shear at high speed to obtain a pre-emulsion;

[0023] (4) The pre-emulsion was homogenized using a microfluidic homogenizer to obtain the soothing and repairing nanoliposomes.

[0024] Preferably, in step (1), the stirring temperature is 50-60℃, the stirring speed is 200-400rpm, and the stirring time is 20-30min.

[0025] Preferably, in step (3), the high-speed shearing speed is 10000-12000 rpm and the time is 2-5 min.

[0026] Preferably, in step (4), the microfluidic homogenizer is a McFee microfluidic homogenizer, and the homogenization process is performed at a pressure of 50-250 MPa and a time of 10-30 min.

[0027] Thirdly, the present invention provides the application of the soothing and repairing nanoliposomes of the first aspect in the preparation of cosmetics.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] (1) The hydrogenated lecithin and cholesterol in the soothing and repairing nanoliposomes of the present invention can encapsulate the active ingredients in the oil phase and aqueous phase within a bilayer, protecting them from damage by the external environment (such as light and oxygen), while improving skin absorption through targeted delivery. The oil phase component forms a protective film on the skin surface, quickly relieving dryness and itching; the aqueous phase component is slowly released, continuously repairing deep damage, achieving the dual effect of "immediate soothing + long-term repair".

[0030] (2) The aqueous component of the soothing and repairing nanoliposomes of the present invention, Dendrobium nobile extract, is negatively charged after phosphorylation. It forms a stable complex with the positively charged recombinant fibronectin through electrostatic adsorption, reducing component precipitation, ensuring system homogeneity, enhancing skin adhesion, and prolonging the duration of action. Meanwhile, the polysaccharides, flavonoids, saponins, and other components in Dendrobium nobile extract have basic soothing effects such as anti-inflammatory, moisturizing, and antioxidant properties. Recombinant fibronectin can promote skin cell repair and regeneration. The combination of these two components not only prolongs the residence time of the active ingredients on the skin surface through the stable structure of the complex, but also achieves complementary effects of "anti-inflammatory soothing + cell repair," significantly enhancing the repair effect of the liposomes. Detailed Implementation

[0031] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0032] The raw materials used in the following examples and comparative examples are from the following sources:

[0033] Hydroxyethyledoin: Manufacturer: Wuxi Jingyang Biotechnology Co., Ltd., Trade name: Hedoin;

[0034] Phytofluanthus extract: manufactured by Croda Chemicals (Shanghai) Co., Ltd., trade name: Phytofluanthus GL;

[0035] Glyceryl glucoside: The manufacturer is Guangdong Miqi New Materials Co., Ltd., and the trade name is glyceryl glucoside;

[0036] Recombinant fibronectin: Manufacturer: Meierjian (Shenzhen) Biotechnology Co., Ltd.; Model: TFNpro;

[0037] Squalane: Manufacturer: Aprinnova, LL; Trade name: Neossance™ Squalane;

[0038] Ceramide NP: Manufacturer is Chongqing Zhihe Biomedical Co., Ltd., and the trade name is ceramide NP1;

[0039] Glycerin: The manufacturer is Wilmar Oils & Fats Technology (Dongguan) Co., Ltd., and the product name is Refined Glycerin;

[0040] Sodium di(lauramide-glutamine) lysine: Manufacturer: ASAHI KASEI FINECHEM CO., LTD., Trade name: PELLICER™ L-30;

[0041] Xylitol anhydride monostearate: The manufacturer is Hunan Jushuo Biotechnology Co., Ltd., and the trade name is food-grade xylitol anhydride monostearate.

[0042] Diacetyl tartrate mono- and diglycerides: The manufacturer is Hunan Jushuo Biotechnology Co., Ltd., and the product name is Hunan Jushuo Edible Diacetyl Tartrate Mono- and Diglycerides.

[0043] Unless otherwise specified, all other materials, reagents, etc. used in the examples and comparative examples are commercially available.

[0044] Example 1

[0045] A soothing and repairing nanoliposome comprises an oil phase and an aqueous phase in a mass ratio of 1:2. The oil phase comprises the following ingredients by mass percentage: 0.3% ceramide NP, 1% *Vigna pubescens* extract, 0.4% hydroxyectoine, 3% squalane, 0.3% sodium bis(lauramide-glutamine)lysine, 2% hydrogenated lecithin, 2% cholesterol, with the balance being anhydrous ethanol. The aqueous phase comprises the following ingredients by mass percentage: 2% *Dendrobium nobile* extract, 2% glycerol glucoside, 0.6% recombinant fibronectin, 33% glycerol, with the balance being deionized water.

[0046] The preparation method of the Dendrobium nobile extract includes the following steps:

[0047] S1. After washing and crushing Dendrobium nobile, add deionized water, adjust the pH to 4.5, add a compound enzyme for enzymatic hydrolysis, and inactivate the enzyme to obtain the hydrolysate; wherein, the ratio of Dendrobium nobile to deionized water is 1g:18mL, the amount of compound enzyme added is 2% of the mass of Dendrobium nobile, the compound enzyme is a combination of cellulase, protease and pectinase, and the mass ratio of cellulase, protease and pectinase is 1:0.8:1; the enzymatic hydrolysis temperature is 34℃ and the time is 2h; the enzyme inactivation temperature is 85℃ and the time is 12min.

[0048] S2. The enzymatic hydrolysate is subjected to ultrasonic extraction and filtered to obtain filtrate and filter residue; wherein the ultrasonic extraction power is 350W, the temperature is 45℃, and the time is 1.5h.

[0049] S3. Add a phosphorylation reagent to the filtrate obtained in S2, adjust the pH to 5, and stir the reaction to obtain a phosphorylated water extract; wherein, the amount of phosphorylation reagent added is 8% of the mass of the filtrate, and the phosphorylation reagent is sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of 2.5:1; the stirring speed is 550 rpm, the temperature is 45℃, and the time is 30 min.

[0050] S4. Add the filter residue obtained in S2 to anhydrous ethanol, add xylitol anhydride monostearate and diacetyl tartaric acid mono- and diglycerides, reflux extract, filter, and rotary evaporate the filtrate until no ethanol remains to obtain the alcohol extract; wherein, the material-to-liquid ratio of the filter residue to anhydrous ethanol is 1g:8mL, the amount of xylitol anhydride monostearate added is 0.5% of the mass of the filter residue, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.2% of the mass of the filter residue; the reflux extraction temperature is 65℃ and the time is 45min; filtration is performed using a 0.2μm filter membrane;

[0051] S5. Mix the phosphorylated water extract obtained in S3 with the alcohol extract obtained in S4 until homogeneous to obtain the Dendrobium nobile extract;

[0052] The preparation method of soothing and repairing nanoliposomes includes the following steps:

[0053] (1) Mix the raw materials in the oil phase according to the formula, stir evenly, and transfer to a rotary evaporator to remove ethanol to obtain the oil phase; wherein, the stirring temperature is 55℃, the stirring speed is 300rpm, and the time is 25min;

[0054] (2) Mix the raw materials in the aqueous phase according to the formula to obtain the aqueous phase; wherein the stirring temperature is 55℃, the stirring speed is 300rpm and the stirring time is 25min;

[0055] (3) Mix the aqueous phase and oil phase according to the formula amount, and shear at high speed to obtain a pre-emulsion; wherein, the high-speed shearing speed is 11000 rpm and the time is 4 min;

[0056] (4) The pre-emulsion was homogenized using a microfluidic homogenizer to obtain the soothing and repairing nanoliposomes; wherein the microfluidic homogenizer was a McFee microfluidic homogenizer, and the homogenization pressure was 150 MPa and the time was 20 min.

[0057] Example 2

[0058] A soothing and repairing nanoliposome comprises the following raw materials in parts by weight: an oil phase and an aqueous phase in a 1:1 mass ratio. The oil phase comprises the following raw materials in percentage by weight: 0.1% ceramide NP, 0.3% *Vigna pubescens* extract, 0.2% hydroxyectoine, 2% squalane, 0.1% sodium bis(lauramide-glutamine)lysine, 1% hydrogenated lecithin, 1% cholesterol, with the balance being anhydrous ethanol. The aqueous phase comprises the following raw materials in percentage by weight: 1% *Dendrobium nobile* extract, 1% glycerol glucoside, 0.5% recombinant fibronectin, 20% glycerol, with the balance being deionized water.

[0059] The preparation method of the Dendrobium nobile extract includes the following steps:

[0060] S1. After washing and crushing Dendrobium nobile, add deionized water, adjust the pH to 4, add a compound enzyme for enzymatic hydrolysis, and inactivate the enzyme to obtain the hydrolysate; wherein, the ratio of Dendrobium nobile to deionized water is 1g:15mL, the amount of compound enzyme added is 1% of the mass of Dendrobium nobile, the compound enzyme is a combination of cellulase, protease and pectinase, and the mass ratio of cellulase, protease and pectinase is 1:0.5:1.5; the enzymatic hydrolysis temperature is 35℃ and the time is 1h; the enzyme inactivation temperature is 80℃ and the time is 15min.

[0061] S2. The enzymatic hydrolysate is subjected to ultrasonic extraction and filtered to obtain filtrate and filter residue; wherein the ultrasonic extraction power is 300W, the temperature is 50℃, and the time is 1h.

[0062] S3. Add a phosphorylation reagent to the filtrate obtained in S2, adjust the pH to 4, and stir the reaction to obtain a phosphorylated water extract; wherein, the amount of phosphorylation reagent added is 5% of the mass of the filtrate, and the phosphorylation reagent is sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of 2:1; the stirring speed is 600 rpm, the temperature is 50℃, and the time is 20 min.

[0063] S4. Add the filter residue obtained in S2 to anhydrous ethanol, add xylitol anhydride monostearate and diacetyl tartaric acid mono- and diglycerides, reflux extract, filter, and rotary evaporate the filtrate until no ethanol remains to obtain the alcohol extract; wherein, the material-to-liquid ratio of the filter residue to anhydrous ethanol is 1g:5mL, the amount of xylitol anhydride monostearate added is 0.3% of the mass of the filter residue, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.1% of the mass of the filter residue; the reflux extraction temperature is 60℃ and the time is 60min; filtration is performed using a 0.2μm filter membrane;

[0064] S5. Mix the phosphorylated water extract obtained in S3 with the alcohol extract obtained in S4 to obtain the Dendrobium nobile extract.

[0065] The preparation method of soothing and repairing nanoliposomes includes the following steps:

[0066] (1) Mix the raw materials in the oil phase according to the formula, stir evenly, and transfer to a rotary evaporator to remove ethanol to obtain the oil phase; wherein, the stirring temperature is 60℃, the stirring speed is 400rpm, and the stirring time is 20min;

[0067] (2) Mix the raw materials in the aqueous phase according to the formula to obtain the aqueous phase; wherein the stirring temperature is 60℃, the stirring speed is 400rpm and the stirring time is 20min;

[0068] (3) Mix the aqueous phase and oil phase according to the formula amount, and shear at high speed to obtain a pre-emulsion; wherein, the high-speed shearing speed is 10000 rpm and the time is 5 min;

[0069] (4) The pre-emulsion was homogenized using a microfluidic homogenizer to obtain the soothing and repairing nanoliposomes; wherein the microfluidic homogenizer was a Microfluidic homogenizer of McFee, and the homogenization pressure was 250 MPa and the time was 10 min.

[0070] Example 3

[0071] A soothing and repairing nanoliposome comprises the following raw materials in parts by weight: an oil phase and an aqueous phase in a mass ratio of 1:3. The oil phase comprises the following raw materials in weight percentages: 0.5% ceramide NP, 3% *Vigna pubescens* extract, 0.5% hydroxyectoine, 4% squalane, 0.5% sodium bis(lauramide-glutamine)lysine, 3% hydrogenated lecithin, 3% cholesterol, with the balance being anhydrous ethanol. The aqueous phase comprises the following raw materials in weight percentages: 5% *Dendrobium nobile* extract, 3% glyceryl glucoside, 1% recombinant fibronectin, 40% glycerol, with the balance being deionized water.

[0072] The preparation method of the Dendrobium nobile extract includes the following steps:

[0073] S1. After washing and crushing Dendrobium nobile, add deionized water, adjust the pH to 5, add a compound enzyme for enzymatic hydrolysis, and inactivate the enzyme to obtain the hydrolysate; wherein, the ratio of Dendrobium nobile to deionized water is 1g:20mL, the amount of compound enzyme added is 3% of the mass of Dendrobium nobile, the compound enzyme is a combination of cellulase, protease and pectinase, and the mass ratio of cellulase, protease and pectinase is 1:1.5:0.5; the enzymatic hydrolysis temperature is 30℃ and the time is 3h; the enzyme inactivation temperature is 90℃ and the time is 10min.

[0074] S2. The enzymatic hydrolysate is subjected to ultrasonic extraction and filtered to obtain filtrate and filter residue; wherein the ultrasonic extraction power is 300W, the temperature is 40℃, and the time is 2h.

[0075] S3. Add a phosphorylation reagent to the filtrate obtained in S2, adjust the pH to 6, and stir the reaction to obtain a phosphorylated water extract; wherein, the amount of phosphorylation reagent added is 10% of the mass of the filtrate, and the phosphorylation reagent is sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of 3:1; the stirring speed is 500 rpm, the temperature is 40℃, and the time is 40 min.

[0076] S4. Add the filter residue obtained in S2 to anhydrous ethanol, add xylitol anhydride monostearate and diacetyl tartaric acid mono- and diglycerides, reflux extract, filter, and rotary evaporate the filtrate until no ethanol remains to obtain the alcohol extract; wherein, the material-to-liquid ratio of the filter residue to anhydrous ethanol is 1g:10mL, the amount of xylitol anhydride monostearate added is 0.6% of the mass of the filter residue, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.3% of the mass of the filter residue; the reflux extraction temperature is 70℃ and the time is 40min; filtration is performed using a 0.1μm filter membrane;

[0077] S5. Mix the phosphorylated water extract obtained in S3 with the alcohol extract obtained in S4 to obtain the Dendrobium nobile extract.

[0078] The preparation method of soothing and repairing nanoliposomes includes the following steps:

[0079] (1) Mix the raw materials in the oil phase according to the formula, stir evenly, transfer to a rotary evaporator to remove ethanol, and obtain the oil phase; wherein, the stirring temperature is 50℃, the stirring speed is 200rpm, and the time is 30min;

[0080] (2) Mix all the raw materials in the aqueous phase according to the formula to obtain the aqueous phase; wherein the stirring temperature is 50℃, the stirring speed is 200rpm and the stirring time is 30min;

[0081] (3) Mix the aqueous phase and oil phase according to the formula amount, and shear at high speed to obtain a pre-emulsion; wherein, the high-speed shearing speed is 12000 rpm and the time is 2 min;

[0082] (4) The pre-emulsion was homogenized using a microfluidic homogenizer to obtain the soothing and repairing nanoliposomes; wherein the microfluidic homogenizer was a McFee microfluidic homogenizer, and the homogenization pressure was 50 MPa and the time was 30 min.

[0083] Example 4

[0084] The only difference between Example 4 and Example 1 is that in step S4 of the preparation of Dendrobium nobile extract, the amount of xylitol anhydride monostearate added is 0.2% of the mass of the filter residue, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.5% of the mass of the filter residue.

[0085] Comparative Example 1

[0086] The only difference between Comparative Example 1 and Example 1 is that Comparative Example 1 does not add Dendrobium nobile extract, but uses an equal amount of recombinant fibronectin to make up for the missing amount.

[0087] Comparative Example 2

[0088] The only difference between Comparative Example 2 and Example 1 is that Comparative Example 2 does not add recombinant fibronectin, but uses an equal amount of Dendrobium nobile extract to make up for the missing amount.

[0089] Comparative Example 3

[0090] The only difference between Comparative Example 3 and Example 1 is that the Dendrobium nobile extract of Comparative Example 3 is not phosphorylated, and its preparation method includes the following steps:

[0091] S1. After washing and crushing Dendrobium nobile, add deionized water, adjust the pH to 4.5, add a compound enzyme for enzymatic hydrolysis, inactivate the enzyme, and obtain the enzymatic hydrolysate; wherein, the material-to-liquid ratio of Dendrobium nobile to deionized water is 1g:18mL, the amount of the compound enzyme added is 2% of the mass of Dendrobium nobile, the compound enzyme is a combination of cellulase, protease and pectinase, and the mass ratio of cellulase, protease and pectinase is 1:0.8:1; the enzymatic hydrolysis temperature is 34℃ and the time is 2h;

[0092] S2', The enzymatic hydrolysate is subjected to ultrasonic extraction and filtered to obtain filtrate and filter residue; wherein the ultrasonic extraction power is 350W, the temperature is 45℃, and the time is 1.5h;

[0093] S3': Add the filter residue obtained in step S2' to anhydrous ethanol, add xylitol anhydride monostearate and diacetyl tartaric acid mono- and diglycerides, reflux extract, filter, and rotary evaporate the filtrate until no ethanol remains to obtain the alcohol extract; wherein, the material-to-liquid ratio of the filter residue to anhydrous ethanol is 1g:8mL, the amount of xylitol anhydride monostearate added is 0.5% of the mass of the filter residue, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.2% of the mass of the filter residue; the reflux extraction temperature is 65℃ and the time is 45min; filtration is performed using a 0.2μm filter membrane;

[0094] S4' Mix the filtrate obtained in step S2' with the alcohol extract obtained in step S3' to obtain the Dendrobium nobile extract.

[0095] Comparative Example 4

[0096] The only difference between Comparative Example 4 and Example 1 is that in step S4 of the preparation of Dendrobium nobile extract in Comparative Example 4, xylitol anhydride monostearate is not added, and diacetyl tartaric acid mono- and diglycerides are used to make up the missing amount.

[0097] Comparative Example 5

[0098] The only difference between Comparative Example 5 and Example 1 is that in step S4 of the preparation of Dendrobium nobile extract in Comparative Example 5, diacetyl tartaric acid mono- and diglycerides are not added, and xylitol anhydride monostearate is used to make up the missing amount.

[0099] Comparative Example 6

[0100] The only difference between Comparative Example 6 and Example 1 is that an equal amount of the Healing Velvet Flower Extract from the oil phase was added to the aqueous phase, and an equal amount of the Dendrobium nobile Extract from the aqueous phase was added to the oil phase.

[0101] Comparative Example 7

[0102] The only difference between Comparative Example 7 and Example 1 is that an equal amount of hydroxyethylidene from the oil phase is added to the aqueous phase.

[0103] Performance testing

[0104] 1. Test of hyaluronidase activity inhibition rate of liposomes in each group

[0105] Hyaluronic acid is an important component of the skin's moisturizing barrier. Under inflammatory conditions, hyaluronidase is abnormally activated, accelerating its degradation. When the skin is stimulated (e.g., by external stimuli or inflammatory reactions), hyaluronidase activity increases, leading to moisture loss, barrier damage, and consequently, discomfort such as redness and stinging. Therefore, the higher the hyaluronidase activity inhibition rate of liposomes, the better their ability to reduce hyaluronic acid degradation and the better their soothing effect. Specific testing methods are as follows:

[0106] (1) Preparation of test samples: Mix the products obtained in Examples 1-4 and Comparative Examples 1-7 with distilled water to prepare a sample solution with a concentration of 2% for later use.

[0107] (2) Experimental procedure: Test tubes A and B (0.5 mL of sample solution and 0.5 mL of hyaluronidase (500 U / mL)), test tubes C and D (0.5 mL of distilled water and 0.5 mL of acetate buffer solution), incubated at 37°C for 20 min; 0.1 mL (2.5 mmol / L) CaCl2 solution was added, and incubated at 37°C for 20 min; 0.5 mL of sodium hyaluronate (0.5 mg / mL) was added to test tubes A and C, and 0.5 mL of acetate buffer solution was added to test tubes B and D, and incubated at 37°C for 40 min; 0.5 mL of acetylacetone solution, 0.1 mL of NaOH solution (5 mol / L) and 0.5 mL of distilled water were added, and the mixture was incubated in a boiling water bath for 15 min, immediately cooled in ice water for 5 min, 1 mL of P-DAB colorimetric reagent was added, and after thorough shaking, 3.5 mL of anhydrous ethanol was added, and the mixture was allowed to stand for 30 min for color development. The color was then developed at 530°C. The OD values ​​of the four test tubes A, B, C, and D were measured at nm.

[0108] The formula for calculating the hyaluronidase activity inhibition rate is:

[0109] Hyaluronidase inhibition rate (%) = [(OD value)] C -OD value D )-(OD value) A -OD value B )] / (OD value C -OD value D )×100%; where, OD value A The OD value of test tube A; OD value B The OD value of test tube B; OD value C The OD value of test tube C; OD value D The value is the OD value of test tube D. See Table 1 for specific data.

[0110] 2. Skin moisturizing and repairing effect test

[0111] The changes in skin moisture content and transepidermal water loss on the inner forearm surface of the test subjects were measured to characterize the skin moisturizing and repairing effects of liposomes in Examples 1-4 and Comparative Examples 1-7. A positive Q value for the rate of change in skin stratum corneum moisture content, and a larger value, indicates that the stratum corneum absorbs and retains more moisture after the application of liposomes. A negative K value for the rate of change in skin transepidermal water loss indicates reduced water loss; a more significant decrease in the K value (a smaller value) indicates that the composition effectively repairs the skin barrier and reduces the outward loss of moisture from within the skin.

[0112] Forty-four subjects aged 35-50 years with dry skin on the inner side of their arms were randomly divided into 11 groups, with four subjects in each group (two males and two females). The subjects used liposomes from Examples 1-4 and Comparative Examples 1-7, respectively, at a concentration of 1.8 mg / cm³. 2 The liposomes were applied in a single, uniform application to the test area on the inner forearm of the subject. The stratum corneum moisture content and transepidermal water loss were measured using an MPA580. Test values ​​were recorded before and 10 hours after application of each liposome group. Experimental conditions were: temperature (25±0.5)℃, relative humidity (55±5)%. The rate of change in stratum corneum moisture content (Q) and the rate of change in transepidermal water loss (K) after 10 hours of use were then calculated using the following formulas:

[0113] Q value of skin stratum corneum moisture content change rate % = (skin stratum corneum moisture content) 使用后 - Moisture content of the stratum corneum of the skin 使用前 ) × 100%;

[0114] Change rate of transepidermal water loss K value % = (amount of transepidermal water loss) 使用后 - Transepidermal water loss 使用前 () × 100%; the calculation results are shown in Table 1.

[0115] Table 1. Efficacy test results of liposomes in each group

[0116] Group / Performance Hyaluronidase inhibition rate / % Change rate of skin stratum corneum moisture content in 10 hours / % Change in transepidermal water loss rate after 10 hours / % Example 1 61.90 36.85 -29.73 Example 2 58.36 34.89 -28.60 Example 3 62.04 36.93 -30.05 Example 4 55.33 32.67 -26.29 Comparative Example 1 28.41 16.74 -13.58 Comparative Example 2 34.73 23.28 -18.73 Comparative Example 3 37.81 24.53 -21.96 Comparative Example 4 46.56 26.31 -22.18 Comparative Example 5 49.32 29.08 -23.61 Comparative Example 6 44.70 26.29 -21.70 Comparative Example 7 52.12 30.66 -25.36

[0117] As shown in Table 1, and combining the data from Examples 1 and 4, the soothing, moisturizing, and repairing effects of the liposomes in Example 4 are lower than those in Example 1. This may be because in step S4 of the preparation of the Dendrobium nobile extract in Example 4, the amount of xylitol anhydride monostearate added was reduced, weakening the wetting effect. This may lead to insufficient penetration of ethanol into the cell wall, incomplete dissolution of some active ingredients (such as saponins and flavonoids), and a decrease in the content of target components in the final extract, directly affecting the soothing and repairing effects of the liposomes. Therefore, when the amount of xylitol anhydride monostearate added is 0.3-0.6% of the filter residue mass, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.1-0.3% of the filter residue mass, the soothing, moisturizing, and repairing effects of the liposomes are at a better level.

[0118] Based on the data from Example 1, Comparative Examples 1-3, and Comparative Example 6, the soothing, moisturizing, and repairing effects of the liposomes in Comparative Examples 1-3 and Comparative Example 6 are lower than those in Example 1. This may be because Comparative Example 1 only increased the amount of recombinant fibronectin, lacking the core anti-inflammatory and moisturizing components such as flavonoids and polysaccharides from Dendrobium nobile extract, thus leading to a decrease in the soothing and repairing effects of the liposomes. Comparative Example 2 lacked recombinant fibronectin, resulting in the loss of extracellular matrix repair function of the liposomes, hindering skin cell proliferation and migration, and reducing the repairing effect. The Dendrobium nobile extract in Comparative Example 3 was not phosphorylated, making it unable to form a stable electrostatic complex with recombinant fibronectin. This resulted in easy loss of active ingredients and decreased emulsification stability. The synergistic effect of polysaccharides and recombinant fibronectin was broken, allowing each to exert only a weak effect. Furthermore, the extracted active ingredients were easily degraded due to structural instability, significantly weakening the soothing, moisturizing, and repairing effects. In Comparative Example 6, the liposomes were prepared by transferring the *Viburnum sarmentosum* extract to the aqueous phase. This resulted in decreased solubility of its lipid-soluble components (such as some flavonoids) in the oil phase, weakening its anti-inflammatory effect. Conversely, transferring the *Dendrobium nobile* extract to the oil phase hindered the formation of the electrostatic complex between the polysaccharide and recombinant fibronectin, leading to a reduced repair effect. Therefore, it is evident that *Dendrobium nobile* extract (after phosphorylation) and recombinant fibronectin, by forming a stable complex, combine the moisturizing and anti-inflammatory properties of polysaccharides with the cell repair function of proteins, effectively enhancing the soothing, moisturizing, and repairing effects of liposomes.

[0119] Based on the data from Example 1 and Comparative Examples 4-5, the soothing, moisturizing, and repairing effects of the liposomes in Comparative Examples 4-5 were lower than those in Example 1. This may be because the Dendrobium nobile extract in Comparative Example 4 only retained diacetyl tartaric acid mono- and diglycerides, which can penetrate cell membranes to release flavonoids, but lacked the solubilizing effect of xylitol anhydride monostearate, resulting in insufficient dissolution of alcohol-soluble components such as saponins, thus significantly weakening the auxiliary effects of soothing and moisturizing. In the preparation step S4 of the Dendrobium nobile extract in Comparative Example 5, only xylitol anhydride monostearate was retained, which can normally solubilize alcohol-soluble components such as saponins, but lacked the cell membrane penetration effect of diacetyl tartaric acid mono- and diglycerides, leading to a decrease in the dissolution efficiency of flavonoids. This indicates that xylitol anhydride monostearate and diacetyl tartaric acid mono- and diglycerides synergistically improve the dissolution of active ingredients (saponins, flavonoids, etc.) in Dendrobium nobile extract by improving the extraction environment, enhancing cell penetration, and solubilizing specific components.

[0120] Based on the data from Example 1 and Comparative Example 7, the soothing, moisturizing, and repairing effects of the liposomes in Comparative Example 7 are lower than those in Example 1. This may be because hydroxyectoin can stabilize the lipid bilayer in the oil phase. In Comparative Example 7, after the hydroxyectoin (crude oil phase) was transferred to the aqueous phase, the liposome membrane fluidity increased, raising the risk of leakage of active ingredients from the oil phase. Furthermore, hydroxyectoin binds more readily to cell membranes in the oil phase, exerting a protective effect against UV / pollution stress. After being transferred to the aqueous phase, its local concentration decreases, weakening the anti-inflammatory effect. Therefore, the oil- and aqueous phase active ingredients of the liposomes of the present invention are complementary, jointly enhancing the soothing, repairing, and moisturizing properties of the liposomes.

[0121] 3. Stability test of liposomes in each group

[0122] The liposomes of Example 1, Comparative Examples 1-3 and Comparative Example 6 were placed at -18℃, 25℃ and 45℃ respectively for 60 days. The results were shown in Table 2.

[0123] Table 2. Stability test results of liposomes in each group

[0124]

[0125] Based on the data from Example 1, Comparative Examples 1-3, and Comparative Example 6 in Table 2, it can be seen that the stability of the liposomes in Comparative Examples 1-3 and Comparative Example 6 is lower than that in Example 1. This may be because the electrostatic complex formed by the phosphorylated Dendrobium nobile extract and recombinant fibronectin in Example 1 can inhibit particle aggregation and enhance emulsion stability, which is the core reason for its optimal stability. Comparative Examples 1 (without Dendrobium nobile extract), 2 (without recombinant fibronectin), and 3 (Dendrobium nobile extract without phosphorylation treatment) all showed a significant decline in stability due to the lack of this complex or the instability of the complex. In Comparative Example 6, after the phases of the Healing Vine Flower Extract and Dendrobium nobile extract were swapped, the solubility of the components was insufficient, directly causing phase separation and precipitation. This indicates that the components need to be matched with the oil / water phase solubility (e.g., the lipid solubility of the Healing Vine Flower Extract is suitable for the oil phase, and the water solubility of the Dendrobium nobile Extract is suitable for the water phase) to improve the stability of the liposomes.

[0126] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A soothing and repairing nanoliposome, characterized in that, The product comprises an oil phase and an aqueous phase in a mass ratio of 1:(1-3). The oil phase comprises the following raw materials by mass percentage: 0.1-0.5% ceramide NP, 0.3-3% *Phytophthora indicum* extract, 0.2-0.5% hydroxyectoine, 2-4% squalane, 0.1-0.5% sodium bis(lauramide-glutamine)lysine, 1-3% hydrogenated lecithin, 1-3% cholesterol, with the balance being anhydrous ethanol. The aqueous phase comprises the following raw materials by mass percentage: 1-5% *Dendrobium nobile* extract, 1-3% glycerol glucoside, 0.5-1% recombinant fibronectin, 20-40% glycerol, with the balance being deionized water. The *Phytophthora indicum* extract is manufactured by Croda Chemicals (Shanghai) Co., Ltd., and its trade name is Phytoflur Anthylis GL. The preparation method of the Dendrobium nobile extract includes the following steps: S1. After washing and crushing Dendrobium nobile, add deionized water, adjust the pH to 4-5, add a compound enzyme for enzymatic hydrolysis, inactivate the enzyme, and obtain the enzymatic hydrolysate; wherein, the compound enzyme is a combination of cellulase, protease and pectinase. S2. The enzymatic hydrolysate is extracted by ultrasonication, filtered, and the filtrate and residue are obtained. S3. Add a phosphorylation reagent to the filtrate obtained in S2, adjust the pH to 4-6, stir the reaction to obtain a phosphorylated water extract; the amount of phosphorylation reagent added is 5-10% of the mass of the filtrate, and the phosphorylation reagent is sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of (2-3):

1. S4. Add the filter residue obtained in S2 to anhydrous ethanol, add xylitol anhydride monostearate and diacetyl tartaric acid mono- and diglycerides, reflux extract, filter, and rotary evaporate the filtrate until no ethanol remains to obtain the alcohol extract; wherein, the amount of xylitol anhydride monostearate added is 0.3-0.6% of the mass of the filter residue, and the amount of diacetyl tartaric acid mono- and diglycerides added is 0.1-0.3% of the mass of the filter residue; S5. Mix the phosphorylated water extract obtained in S3 with the alcohol extract obtained in S4 to obtain the Dendrobium nobile extract.

2. The soothing and repairing nanoliposomes as described in claim 1, characterized in that, In step S1 of the preparation of Dendrobium nobile extract, the ratio of Dendrobium nobile to deionized water is 1g:15-20mL, the amount of the compound enzyme added is 1-3% of the mass of Dendrobium nobile, and the mass ratio of cellulase, protease and pectinase is 1:(0.5-1.5):(0.5-1.5); the enzymatic hydrolysis temperature is 30-35℃ and the time is 1-3h; the enzyme inactivation temperature is 80-90℃ and the time is 10-15min.

3. The soothing and repairing nanoliposomes as described in claim 1, characterized in that, In step S2 of the preparation of Dendrobium nobile extract, the ultrasonic extraction power is 300-400W, the temperature is 40-50℃, and the time is 1-2h.

4. The soothing and repairing nanoliposomes as described in claim 1, characterized in that, In step S3 of the preparation of Dendrobium nobile extract, the stirring speed is 500-600 rpm, the temperature is 40-50℃, and the time is 20-40 min.

5. The soothing and repairing nanoliposomes as described in claim 1, characterized in that, In step S4 of the preparation of Dendrobium nobile extract, the ratio of the filter residue to anhydrous ethanol is 1g:5-10mL.

6. The method for preparing soothing and repairing nanoliposomes according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Mix the raw materials in the oil phase according to the formula, stir evenly, and transfer to a rotary evaporator to remove ethanol to obtain the oil phase; (2) Mix all the raw materials in the aqueous phase according to the formula to obtain the aqueous phase; (3) Mix the aqueous phase and oil phase according to the formula amount, and shear at high speed to obtain a pre-emulsion; (4) The pre-emulsion was homogenized using a microfluidic homogenizer to obtain the soothing and repairing nanoliposomes.

7. The method for preparing soothing and repairing nanoliposomes as described in claim 6, characterized in that, In step (1), the stirring temperature is 50-60℃, the stirring speed is 200-400rpm, and the stirring time is 20-30min.

8. The method for preparing soothing and repairing nanoliposomes as described in claim 6, characterized in that, In step (3), the high-speed shearing speed is 10000-12000 rpm and the time is 2-5 min.

9. The method for preparing soothing and repairing nanoliposomes as described in claim 6, characterized in that, In step (4), the microfluidic homogenizer is a McFee microfluidic homogenizer, and the homogenization process is performed at a pressure of 50-250 MPa and a time of 10-30 min.

10. The use of the soothing and repairing nanoliposomes according to any one of claims 1-5 in the preparation of cosmetics.

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