A liposome composition having soothing efficacy and a method for preparing the same

Abstract

This invention provides a liposome composition with soothing effects and its preparation method, relating to the field of cosmetic technology. The liposome composition comprises the following raw materials: active ingredients and solvents; the active ingredients include dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphingosine, frankincense resin extract, and brown algae extract. This invention, through a specific component ratio of the liposome raw materials and a specific combination of plant extracts, significantly improves the encapsulation rate of plant extracts and the stability of the composition, thereby significantly enhancing the soothing and anti-inflammatory effects, providing better product choices for users with sensitive skin.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a liposome composition with soothing effects and its preparation method. Background Technology

[0002] Sensitive skin typically manifests as impaired skin barrier function, accelerated moisture loss from the stratum corneum, and enhanced reactivity of nerve endings, leading to discomfort such as erythema, itching, stinging, dryness, and flaking. Its pathogenesis is complex, involving multiple aspects such as skin barrier dysfunction, abnormal neurogenic inflammatory responses, overactivation of the immune system, and imbalance of the skin's microecology.

[0003] Plant extracts, with their multi-component and multi-target natural advantages, occupy an important position in skincare products with soothing, anti-inflammatory, and barrier-repairing effects. However, natural plant extracts (such as triterpenoids, flavonoids, and terpenes) generally suffer from technical bottlenecks such as imbalanced lipid-water partition coefficients, poor chemical stability (easily oxidized / photodegraded), low stratum corneum penetration efficiency, and the potential for irritation or sensitization upon direct contact with high concentrations. Therefore, developing efficient and safe delivery carriers to achieve targeted delivery, controlled release, and improved bioavailability of plant extracts has become a critical issue that urgently needs to be addressed in this field.

[0004] Liposomes are artificial sac-like structures composed of single or multiple membranes enclosing aqueous compartments. Most typically, liposome membranes are formed from a lipid bilayer, but they can also be composed of other monomers or polymers of amphiphilic compounds, including other types of amphiphiles, polymers, and peptides. Most liposomes are non-toxic, non-antigenic, and biodegradable due to their molecular characteristics resembling mammalian membranes. Lipophilic or amphiphilic drugs and compounds can be incorporated into liposome membranes, while hydrophilic drugs and compounds can be encapsulated in the aqueous core of the liposome. Liposomes can encapsulate both water-soluble and lipid-soluble active substances, delivering them to the deeper layers of the skin and promoting the penetration of active ingredients into the stratum corneum or epidermal lipids. Water-soluble components can be encapsulated within the aqueous phase of phospholipids, while lipid-soluble components are encapsulated between the phospholipid bilayers, thereby increasing the local accumulation of active ingredients in the skin and improving bioavailability.

[0005] Existing soothing compositions often rely on ingredient stacking. For example, Chinese patent application CN109431873A discloses a composition for repairing and soothing skin inflammation, which combines lecithin, boswellia carotenoides resin extract, carboxymethyl deacetylated chitosan, glycerophosphate inositol salt, and phytosterol derivatives. Although this approach achieves basic soothing through film formation and physical dispersion of cationic polymers (carboxymethyl deacetylated chitosan), the system is essentially a macroscopic emulsion / aqueous mixture. The plant extracts have not undergone delivery structure optimization, resulting in low encapsulation efficiency, insufficient storage stability, and a complete lack of microenvironment-targeted release capability. Furthermore, the charge repulsion between cationic polysaccharides and anionic functional lipids limits its upgrade to a nanoscale precision delivery system, making it difficult to achieve precise anchoring and on-demand release of active molecules at the membrane interface.

[0006] In summary, existing soothing delivery technologies or liposome formulations still have significant technological gaps in terms of stability, encapsulation efficiency of plant extracts, and multi-target synergy. There is an urgent need in this field for a novel liposome delivery architecture that can overcome the limitations of traditional carriers, such as non-targeted release, easy leakage of active ingredients, and simple additive effects, to provide a precise, efficient, and low-irritation soothing and repairing solution for sensitive skin and skin barrier damage. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a liposome composition with soothing effects and its preparation method. By using a specific ratio of liposome raw materials and a specific combination of plant extracts, the encapsulation rate of plant extracts and the stability of the composition are significantly improved, thereby significantly enhancing the soothing and anti-inflammatory effects and providing a better product option for sensitive skin.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a liposome composition with soothing effects, comprising the following raw materials: active ingredients and solvents; said active ingredients include dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphingosine, frankincense resin extract and brown algae extract.

[0009] Preferably, the active ingredients, by weight parts, include 2-5 parts dioleoylphosphatidylglycerol, 0.5-2 parts cholesterol hemisuccinate, 0.1-1 part N-palmitoylhydroxyproline cetyl ester, 0.5-2 parts phosphatidylinositol, 0.1-0.5 parts phytosphingosine, 0.5-2.5 parts frankincense resin extract and 0.3-1.5 parts brown algae extract.

[0010] Preferably, the ratio of the mass of the active ingredient to the total mass of the raw materials is 3-5:100.

[0011] Preferably, the preparation method of the frankincense resin extract includes the following steps: (1) After the frankincense resin is crushed, it is mixed with an aqueous ethanol solution with a volume fraction of 60%-80%, refluxed for extraction, filtered, concentrated, and crude extract is obtained. (2) Elute with an AB-8 macroporous resin column. First, elute with 1.5-2.5 BV of water, then elute with 2.5-3.5 BV of 60%-80% ethanol aqueous solution. Collect the eluent, concentrate it, and dry it to obtain the final product.

[0012] More preferably, the preparation method of the frankincense resin extract includes the following steps: (1) After the frankincense resin is crushed, it is passed through a 40-80 mesh sieve, and then mixed with an ethanol aqueous solution with a volume fraction of 60%-80% at a material-liquid ratio of 1g:6-10mL. The mixture is extracted by reflux at 50-60℃ for 1-2h, with a stirring rate of 200-300rpm. After coarse filtration through a 200 mesh filter cloth, it is then finely filtered through a 0.45 μm microporous membrane and concentrated under reduced pressure to obtain the crude extract. (2) The crude extract is eluted by an AB-8 macroporous resin column. First, it is eluted with 1.5-2.5 BV of water, and then eluted with 2.5-3.5 BV of 60%-80% ethanol aqueous solution. The eluent is collected, concentrated, and spray-dried to obtain the final product.

[0013] Preferably, the preparation method of the brown algae extract includes the following steps: Brown algae are dried, pulverized, mixed with water, and subjected to enzymatic hydrolysis with a compound enzyme. The mixture is then extracted by ultrasonication, and the resulting filtrate is concentrated and dried to obtain the final product. The compound enzyme is alginate lyase and bromelain. More preferably, the preparation method of the brown algae extract includes the following steps: Brown algae are dried and pulverized at 30-40℃ and passed through a 60-100 mesh sieve. They are then mixed with water at a material-to-liquid ratio of 1g:10-15mL. A compound enzyme is added and the mixture is enzymatically hydrolyzed at 45-50℃ and pH 5.5-6.5 for 2-4 hours. The system is adjusted to an ethanol volume concentration of 60-70%. The mixture is then extracted using ultrasound at 40-50℃ for 30-50 minutes. The filtrate is separated, concentrated, and dried to obtain the final product. The enzyme activity ratio of the alginate lyase and bromelain is 20U-50U:10-20U, and the total amount of alginate lyase and bromelain added is 0.5-2% of the mass of the brown algae.

[0014] Preferably, the solvent is an organic solvent and a buffer solution.

[0015] Preferably, the organic solvent is one or more of methanol, chloroform, ethanol, dichloromethane, benzyl alcohol, and tert-butanol.

[0016] More preferably, the organic solvent is ethanol.

[0017] Preferably, the buffer solution is a PBS buffer with a pH of 6-7.

[0018] More preferably, the buffer solution is a PBS buffer with a pH of 6.5.

[0019] Secondly, the present invention provides a method for preparing the aforementioned liposome composition, comprising the following steps: S1: Dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphoin, and frankincense resin extract were mixed with an organic solution to obtain mixture 1. The solvent was removed by a rotary evaporator and dried to obtain a lipid film. S2: Brown algae extract is mixed with buffer solution to obtain an aqueous phase; the aqueous phase is mixed with a lipid membrane, homogenized under high pressure, dialyzed through a dialysis bag with a molecular weight cutoff of 10-12 kDa, sterilized, and dried to obtain the final product.

[0020] Preferably, in step S1, the solvent is ethanol.

[0021] Preferably, in step S2, the temperature for mixing the aqueous phase and the lipid film is 45-55°C, and the time is 0.5-1.5h.

[0022] Any point value, sub-range, or range formed by any two point values ​​within the range of 45-55℃ is applicable to this invention, including but not limited to 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 53℃, 54℃, and 55℃.

[0023] Any point value, sub-range, or range formed by any two point values ​​within the range of 0.5-1.5h is applicable to this invention, including but not limited to 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h, and 1.5h.

[0024] Preferably, in step S2, the parameters for high-pressure homogenization are: 8-12 cycles at a pressure of 35-42 MPa.

[0025] Any point value, sub-range, or range formed by any two point values ​​within the range of 35-42MPa is applicable to this invention, including but not limited to 35MPa, 36MPa, 37MPa, 38MPa, 39MPa, 40MPa, 41MPa, and 42MPa.

[0026] Any point value, sub-range, or range value formed by any two point values ​​within the range of 8-12 is applicable to this invention, including but not limited to 8, 9, 10, 11, and 12 times.

[0027] Preferably, in step S2, the sterilization includes the following steps: sterilization through a 0.22 μm filter membrane.

[0028] Preferably, in step S2, the dialysis time is 20-28 hours.

[0029] Any point value, sub-range, or range value formed by any two point values ​​within the range of 20-28h is applicable to this invention, including but not limited to 20h, 24h, and 28h.

[0030] Preferably, in step S2, the dialysate is a PBS buffer with a pH of 6.0-7.0, and the dialysate is changed every 4 hours.

[0031] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention utilizes a specific combination of dioleoylphosphatidylglycerol and cholesterol hemisuccinate to maintain the surface charge of liposomes within an ideal range, effectively preventing the aggregation and sedimentation of nanoparticles. The combination of N-palmitoyl hydroxyproline cetyl ester and phytosphingosine significantly improves the density of the lipid bilayer, reducing the leakage of active ingredients. The antioxidant components in brown algae extract effectively protect phospholipids from oxidation, extending the product's shelf life. The use of a 35-42 MPa high-pressure homogenization process can stably control the particle size at around 100 nm, resulting in a low polydispersity index (PDI), greatly enhancing the ability of active ingredients to penetrate the stratum corneum and achieving a high encapsulation rate.

[0032] 2. The frankincense resin extract and brown algae extract work synergistically to achieve comprehensive soothing and anti-inflammatory effects from chemical signals to cellular stress. Furthermore, through specific liposome encapsulation, stability and bioavailability are improved, significantly enhancing the soothing and anti-inflammatory effects. Detailed Implementation

[0033] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the invention is further illustrated below with specific embodiments. However, these embodiments are merely preferred embodiments and not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments described herein without creative effort are all within the scope of protection of this invention. It is worth noting that the raw materials used in this invention are all common commercially available products, and their sources are not specifically limited. The technical and scientific terms used in the embodiments have the meanings commonly understood by those skilled in the art to which this invention pertains.

[0034] Some of the raw materials are shown in Table 1: Table 1

[0035] The preparation method of frankincense resin extract consists of the following steps: (1) After the frankincense resin is crushed, it is passed through a 60-mesh sieve and then mixed with an ethanol aqueous solution with a volume fraction of 75% at a material-liquid ratio of 1g:8mL. The mixture is refluxed at 55℃ for 1.5h with a stirring rate of 250rpm. After coarse filtration through a 200-mesh filter cloth, it is then finely filtered through a 0.45 μm microporous membrane and concentrated under reduced pressure to obtain the crude extract. (2) The crude extract was eluted by an AB-8 macroporous resin column, first with 2 BV of water, then with 3 BV of 75% ethanol aqueous solution. The eluent was collected, concentrated, and spray-dried to obtain the frankincense resin extract, which was used in the examples and some comparative examples of the present invention.

[0036] The preparation method of brown algae extract consists of the following steps: Brown algae were dried and pulverized at 35°C, then passed through an 80-mesh sieve. The mixture was then mixed with water at a ratio of 1g:12mL, and a compound enzyme was added. Enzymatic hydrolysis was performed at 45°C and pH 6 for 3 hours. The system was adjusted to an ethanol concentration of 60-70%, and the mixture was extracted using ultrasound at 45°C for 40 minutes. The filtrate was then concentrated and dried to obtain the final product. The enzyme activity ratio of alginate lyase to bromelain was 40U:15U, and the total amount of alginate lyase and bromelain added was 1.5% of the brown algae mass. The prepared brown algae extract was used in the examples and some comparative examples of this invention.

[0037] Example 1 A liposome composition with soothing effects, the raw materials and dosages are shown in Table 2: Table 2

[0038] The preparation method consists of the following steps: S1: Dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphoin, and frankincense resin extract were mixed with ethanol to obtain mixture 1. The solvent was removed by rotary evaporator and dried to obtain a lipid film. S2: The brown algae extract was mixed with PBS buffer at pH 6.5 to obtain an aqueous phase; the aqueous phase was added to the lipid membrane and hydrated and stirred at 50°C for 1 hour, then homogenized under high pressure at 40 MPa for 10 cycles; then dialyzed through a dialysis bag with a molecular weight cutoff of 10-12 kDa, using PBS buffer at pH 6.5 as the dialysate, changing the solution every 4 hours for 24 hours; the liposome composition was sterilized and granulated through a 0.22 μm filter membrane and freeze-dried to obtain a liposome composition with soothing effects.

[0039] Example 2 A liposome composition with soothing effects, the raw materials and dosages are shown in Table 3: Table 3

[0040] The preparation method consists of the following steps: S1: Dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphoin, and frankincense resin extract were mixed with ethanol to obtain mixture 1. The solvent was removed by rotary evaporator and dried to obtain a lipid film. S2: The brown algae extract was mixed with PBS buffer at pH 6.5 to obtain an aqueous phase; the aqueous phase was added to the lipid membrane and hydrated and stirred at 45°C for 1.5 h, then homogenized under high pressure at 42 MPa for 8 cycles; then dialyzed through a dialysis bag with a molecular weight cutoff of 10-12 kDa, using PBS buffer at pH 6.5 as the dialysate, changing the solution every 4 h, for 20 h; the liposome composition was sterilized and granulated through a 0.22 μm filter membrane, and freeze-dried to obtain a liposome composition with soothing effects.

[0041] Example 3 A liposome composition with soothing effects, the raw materials and dosages are shown in Table 4: Table 4

[0042] The preparation method consists of the following steps: S1: Dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphoin, and frankincense resin extract were mixed with ethanol to obtain mixture 1. The solvent was removed by rotary evaporator and dried to obtain a lipid film. S2: The brown algae extract was mixed with PBS buffer at pH 6.5 to obtain an aqueous phase; the aqueous phase was added to the lipid membrane and hydrated and stirred at 55°C for 0.5 h, then homogenized under high pressure at 32 MPa for 12 cycles; then dialyzed through a dialysis bag with a molecular weight cutoff of 10-12 kDa, using PBS buffer at pH 6.5 as the dialysate, changing it every 4 h, for 28 h; the liposome composition was sterilized and granulated through a 0.22 μm filter membrane, and freeze-dried to obtain a liposome composition with soothing effects.

[0043] Comparative Example 1 A moisturizing cream was prepared according to Example 1 described in Chinese patent application CN109431873A.

[0044] Comparative Example 2 A liposome composition with soothing effects, compared with Example 1, except that the frankincense resin extract is replaced with the frankincense resin extract of Chinese patent application CN109431873A, and the rest is the same as Example 1.

[0045] Comparative Example 3 A liposome composition with soothing effects, compared with Example 1, except that the brown algae extract is replaced with commercially available Centella asiatica extract (purchased from Lanzhou Waterles Biotechnology Co., Ltd.), and the rest is the same as Example 1.

[0046] Comparative Example 4 A liposome composition with soothing effects, compared with Example 1, only the amount of raw materials was changed, as shown in Table 5, and the rest was the same as Example 1: Table 5

[0047] Test Example 1 Encapsulation efficiency and stability determination 1.1 Encapsulation rate 1.1.1 Test principle: Free active ingredients are separated by ultrafiltration centrifugation. The overall encapsulation rate is calculated using both fat-soluble components (phytosphingosine) and water-soluble components (fucoxanthin from brown algae extract) as dual indicators.

[0048] 1.1.2 Test Procedure: (1) Take an appropriate amount of the lyophilized liposome powder of Examples 1-3 and Comparative Examples 2-4, and reconstitute it with purified water to the original volume to obtain a liposome dispersion.

[0049] (2) Take 2 mL of dispersion and place it in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa. Centrifuge at 4℃ and 4000 r / min for 20 min and collect the filtrate.

[0050] (3) The content of free phytosphingosine in the filtrate was determined by high performance liquid chromatography (HPLC); the content of free brown algae polysaccharides in the filtrate was determined by phenol-sulfuric acid method.

[0051] (4) Take another 2 mL of the uncentrifuged dispersion, add an appropriate amount of anhydrous ethanol and sonicate to break the emulsion, and determine the content of total phytosphingosine and total brown algae polysaccharides in the same way.

[0052] 1.1.3 Calculation formula: Encapsulation rate (%) = [(Total content - Free content) / Total content] × 100%.

[0053] 1.2 Stability 1.2.1 Test procedure: The freeze-dried powders of Examples 1-3 and Comparative Examples 2-4 and the moisturizing cream of Comparative Example 1 were sealed and placed in a constant temperature and humidity chamber at 40°C and 75% relative humidity for 30 days.

[0054] 1.2.2 Evaluation Indicators: Appearance: Observe whether the sample shows stratification, demulsification, precipitation, discoloration, or whether the lyophilized powder shows signs of collapse or difficulty in reconstitution.

[0055] Particle size change rate: On day 0 and day 30, appropriate amounts of samples were taken, reconstituted / diluted, and their average particle size was determined using a Malvern laser particle size analyzer.

[0056] Calculation formula: Particle size change rate (%) = (D) 30 -D0) / D0×100%.

[0057] Where D0 is the initial particle size, D 30 The particle size is after 30 days.

[0058] 1.3 The results are shown in Table 6: Table 6

[0059] The present invention uses a specific ratio of active ingredients and combines thin film dispersion, high pressure homogenization and freeze drying processes to prepare a liposome composition with extremely high encapsulation efficiency and minimal particle size change in accelerated stability test at 40℃, and good resolubility, proving that its lipid bilayer structure is dense and stable.

[0060] Test Example 2 Soothing efficacy assay (hyaluronidase inhibition rate test) 2.1 Test Principle: Hyaluronidase is an enzyme that degrades hyaluronic acid and is closely related to skin inflammation and allergic reactions. Inhibition of hyaluronidase activity is a classic in vitro indicator for evaluating the soothing and anti-allergic effects of cosmetics.

[0061] 2.2 Test Procedure: Sample solution preparation: The sample solution was prepared based on a total mass concentration of 0.5 mg / mL of the core soothing plant extracts (i.e., frankincense resin extract, brown algae extract, or centella asiatica extract).

[0062] Sample test solutions for Examples 1-3 and Comparative Examples 2-4: Accurately weigh appropriate amounts of liposome lyophilized powder, dissolve and dilute with purified water to obtain clear and transparent sample test solutions.

[0063] Comparative Example 1 (Moisturizing Cream) Sample Test Solution: Accurately weigh an appropriate amount of cream, add a mixed solvent of anhydrous ethanol and purified water (volume ratio 1:1), and ultrasonically extract at 40℃ for 30 min, followed by centrifugation at 10000 r / min for 10 min. Take the supernatant and dilute it with purified water to a core soothing plant extract concentration of 0.5 mg / mL to obtain the Comparative Example 1 test solution.

[0064] Grouping and sample addition: The experiment was set up with a control group, a control blank group, a sample group and a sample blank group, with 3 parallel replicates in each group.

[0065] Control group (A) 对照 Add 0.1 mL PBS buffer and 0.5 mL hyaluronidase solution, and incubate at 37°C for 20 min. Control group (A) 对照空白 Add 0.6 mL of PBS buffer (use buffer instead of enzyme solution) and incubate at 37°C for 20 min. Sample group (A) 样品 Add 0.1 mL of sample test solution (Examples 1-3 and Comparative Examples 1-4) and 0.5 mL of hyaluronidase solution, and incubate at 37°C for 20 min; Sample blank group (A) 样品空白 Add 0.1 mL of sample test solution and 0.5 mL of PBS buffer (use buffer instead of enzyme solution), and incubate at 37°C for 20 min.

[0066] Elson-Morgan method: Mix the above sample solution with hyaluronidase solution and incubate at 37°C for 20 min; add sodium hyaluronate solution and incubate at 37°C for 30 min; add acetylacetone reagent and heat in a boiling water bath; after cooling, add p-dimethylaminobenzaldehyde for color development.

[0067] Calculation formula: Inhibition rate (%) = [1 - (A)] 样品 -A 样品空白 ) / (A 对照 -A 对照空白 )]×100%.

[0068] The results are shown in Table 7: Table 7

[0069] The soothing effects of Examples 1-3 prepared by the present invention are significantly improved compared with Comparative Examples 1-4.

[0070] Test Example 3 3.1 Test Principle: Lipopolysaccharide (LPS) can stimulate mouse monocytes / macrophages (RAW264.7) to produce a strong inflammatory response, releasing large amounts of nitric oxide (NO) and pro-inflammatory cytokines (TNF-α). By detecting the inhibition rate of LPS-induced NO and TNF-α secretion by the sample, the anti-inflammatory efficacy of the composition at the cellular level can be accurately evaluated.

[0071] 3.2 Experimental Materials and Instruments Cell line: Mouse mononuclear macrophages (RAW264.7), purchased from the Chinese Academy of Sciences Type Culture Collection (or the American Type Culture Collection Center, ATCC).

[0072] Cell culture reagents: DMEM high glucose medium, fetal bovine serum (FBS), and 0.25% trypsin-EDTA digestion solution were all purchased from Gibco (Thermo Fisher Scientific).

[0073] Inducer: Lipopolysaccharide (LPS, derived from Escherichia coli O55:B5), purchased from Sigma-Aldrich, USA.

[0074] Test kit: The nitric oxide (NO) detection kit (Griess method) was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (Catalog No.: S0021).

[0075] The mouse tumor necrosis factor-α (TNF-α) ELISA kit was purchased from Wuhan Elabscience (Catalog No.: E-EL-M0049c).

[0076] CCK-8 cell proliferation-toxicity assay kit, purchased from Dojin Chemical Research Institute, Japan (Catalog No.: CK04).

[0077] Main instruments: Synergy H1 multi-functional microplate reader (ELISA reader), purchased from BioTek, USA; Heracell VIOS 160i CO2 cell incubator, purchased from Thermo Fisher Scientific, USA; Allegra X-15R benchtop refrigerated centrifuge, purchased from Beckman Coulter, USA.

[0078] 3.3 Test Procedure: (1) Sample pretreatment and preparation: Examples 1-3 and Comparative Examples 2-4: The lyophilized liposome powder was reconstituted in DMEM high-glucose medium containing 1% fetal bovine serum (FBS) to prepare a drug-containing medium with a final concentration of 0.2 mg / mL of core soothing plant extract (frankincense + brown algae / centella asiatica). The medium was then filtered through a 0.22 μm microporous membrane for sterilization before use.

[0079] Comparative Example 1 (Skin Cream): Accurately weigh an appropriate amount of cream, add a mixed solvent of anhydrous ethanol and culture medium (volume ratio 1:4), sonicate at 40℃ for 30 min, and centrifuge at 10000 r / min for 10 min. Take the supernatant, dilute with culture medium to a final concentration of 0.2 mg / mL for the core soothing plant extract, filter and sterilize for later use.

[0080] (2) Cell culture and drug administration: RAW264.7 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 4 Inoculate the cells at a density of cells / well into 96-well plates and incubate them in a 37°C, 5% CO2 incubator for 24 h.

[0081] Discard the old culture medium, add fresh culture medium to the blank group; add culture medium containing LPS (final concentration 1 μg / mL) to the model group; add a mixed culture medium containing LPS (1 μg / mL) and the above sample solutions to the sample group. Each group has 3 replicates and continues to incubate for 24 h.

[0082] (Note: Prior to the experiment, the CCK-8 assay confirmed that the above-mentioned 0.2 mg / mL concentrations of each sample had no significant effect on cell viability, thus ruling out false positive anti-inflammatory results caused by cytotoxicity.) (3) Determination of NO release (Griess method): Collect 50 μL of cell culture supernatant from each group, add an equal volume of Griess reagent (containing 1% sulfonamide and 0.1% naphthylethylenediamine hydrochloride), and react at room temperature in the dark for 10 min.

[0083] The absorbance was measured at a wavelength of 540 nm, and the NO concentration was calculated based on the sodium nitrite standard curve.

[0084] (4) TNF-α release assay (ELISA method): Collect the remaining supernatant and strictly follow the instructions of the mouse TNF-α ELISA kit. Measure the absorbance at 450 nm and calculate the TNF-α concentration based on the standard curve.

[0085] (5) Calculation formula: Inhibition rate (%) = [(Model group concentration - Sample group concentration) / (Model group concentration - Blank group concentration)] × 100%.

[0086] (6) The results are shown in Table 8: Table 8

[0087] The liposomes prepared in Examples 1-3 of this invention showed significantly enhanced inhibition rates against NO and TNF-α, demonstrating good cellular-level anti-inflammatory effects.

[0088] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A liposome composition with soothing effects, characterized in that, It includes the following raw materials: active ingredients and solvents; the active ingredients include dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphingosine, frankincense resin extract and brown algae extract.

2. The liposome composition according to claim 1, characterized in that, Based on parts by weight, the active ingredients include 2-5 parts dioleoylphosphatidylglycerol, 0.5-2 parts cholesterol hemisuccinate, 0.1-1 part N-palmitoylhydroxyproline cetyl ester, 0.5-2 parts phosphatidylinositol, 0.1-0.5 parts phytosphingosine, 0.5-2.5 parts frankincense resin extract and 0.3-1.5 parts brown algae extract.

3. The liposome composition according to claim 1, characterized in that, The ratio of the mass of the active ingredient to the total mass of the raw materials is 3-5:

100.

4. The liposome composition according to claim 1, characterized in that, The preparation method of the frankincense resin extract includes the following steps: (1) After the frankincense resin is crushed, it is mixed with an aqueous ethanol solution with a volume fraction of 60%-80%, refluxed for extraction, filtered, concentrated, and crude extract is obtained. (2) Elute with an AB-8 macroporous resin column. First, elute with 1.5-2.5 BV of water, then elute with 2.5-3.5 BV of 60%-80% ethanol aqueous solution. Collect the eluent, concentrate it, and dry it to obtain the final product.

5. The liposome composition of claim 1, characterized in that, The preparation method of the brown algae extract includes the following steps: Brown algae are dried, pulverized, mixed with water, and subjected to enzymatic hydrolysis with a compound enzyme. The mixture is then extracted by ultrasonication, and the resulting filtrate is concentrated and dried to obtain the final product. The compound enzyme is alginate lyase and bromelain.

6. The liposome composition of claim 1, characterized in that, The solvent is an organic solvent and a buffer solution; the organic solvent is one or more of methanol, chloroform, ethanol, dichloromethane, benzyl alcohol and tert-butanol; the buffer solution is PBS buffer with a pH of 6-7.

7. A method for preparing the liposome composition according to any one of claims 1-6, characterized in that, Includes the following steps: S1: Dioleoylphosphatidylglycerol, cholesterol hemisuccinate, N-palmitoylhydroxyproline cetyl ester, phosphatidylinositol, phytosphoin, and frankincense resin extract were mixed with an organic solution to obtain mixture 1. The solvent was removed by a rotary evaporator and dried to obtain a lipid film. S2: Brown algae extract is mixed with buffer solution to obtain an aqueous phase; the aqueous phase is mixed with a lipid membrane, homogenized under high pressure, dialyzed through a dialysis bag with a molecular weight cutoff of 10-12 kDa, sterilized, and dried to obtain the final product.

8. The preparation method according to claim 7, characterized in that, In step S2, the mixing temperature is 45-55℃ and the time is 0.5-1.5h.

9. The preparation method according to claim 7, characterized in that, In step S2, the parameters for high-pressure homogenization are: 8-12 cycles at a pressure of 35-42 MPa.

10. The preparation method according to claim 7, characterized in that, In step S2, the dialysate is a PBS buffer with a pH of 6.0-7.

0. The dialysate is changed every 4 hours, and the dialysate time is 20-28 hours.

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