Anti-inflammatory anti-aging compositions based on cyclic hexapeptides and liposome encapsulations thereof and their use in cosmetics

Abstract

The present application relates to a kind of anti-inflammatory anti-aging composition based on cyclohexapeptide and its liposome package and its skin care product.The anti-inflammatory anti-aging composition based on cyclohexapeptide includes the following components: cyclohexapeptide-9, radix scrophulariae extract, glutathione;The mass ratio of cyclohexapeptide-9, radix scrophulariae extract, glutathione is (0.01-1) :(0.01-2) :(0.01-1).The present application adopts multi-channel regulation mode, constructs a kind of anti-inflammatory anti-aging regulation system with cyclohexapeptide-9, radix scrophulariae extract, glutathione as main component, these components synergistic effect, form the multi-element anti-aging path of oxidation scavenging-inflammation inhibition-barrier repair.In addition, the present application also carries out liposome package to cyclohexapeptide-9, radix scrophulariae extract, glutathione, obtains anti-inflammatory anti-aging liposome package based on cyclohexapeptide, can greatly improve the stability and penetration utilization rate of active ingredient.

Description

Technical Field

[0001] This invention relates to the field of cosmetics and skincare products, and in particular to an anti-inflammatory and anti-aging composition based on cyclic hexapeptides, its liposome encapsulation, and skincare products thereof. Background Technology

[0002] Skin aging is a phenomenon of overall skin degeneration caused by both internal physiological aging and external environmental factors. According to relevant data, more than half of adults worldwide experience varying degrees of skin aging, and this trend is affecting younger people. Of these, photoaging and environmental aging caused by external factors (such as UV radiation, environmental pollution, smoking, excessive alcohol consumption, lack of sleep, and external stress) account for approximately 75%, while internal physiological aging accounts for only 25%. In response to skin aging problems, more and more people are seeking products with anti-aging skincare functions.

[0003] With the continuous development of dermatology and the deepening research on skin aging, oxidative stress and inflammatory aging have been proven to be the main causes of skin aging. Oxidative stress refers to the decline in the efficiency of the mitochondrial electron transport chain due to aging or increased external stress (such as ultraviolet radiation and pollution). This leads to a buildup of superoxide anions exceeding the scavenging capacity of enzymes such as SOD, resulting in the accumulation of reactive oxygen species (ROS). These ROS then attack proteins, causing collagen degradation and elastic fiber breakage. Inflammatory aging refers to the sustained increase in inflammatory factors due to cellular and molecular damage or a decline in barrier function. This leads to increased MMP-1 expression in dermal fibroblasts and greater collagen loss.

[0004] To date, anti-inflammatory and anti-aging skincare products based on oxidative stress and inflammatory aging pathways have been widely adopted. Currently, most anti-inflammatory and anti-aging products on the market work synergistically through three core strategies: scavenging free radicals, inhibiting inflammatory pathways, and repairing the skin barrier function. While these products have achieved some success, some shortcomings remain. For example, some skincare products can only temporarily relieve inflammatory skin redness; once the product is discontinued, the redness is prone to recurrence, and long-term use may lead to side effects such as dependence and skin atrophy (e.g., with glucocorticoid analogues). Some skincare products using natural plant extracts as active ingredients have low stability; the active ingredients may be deactivated when exposed to air, losing their effectiveness.

[0005] Currently, a significant portion of anti-inflammatory and anti-aging skincare products based on antioxidant stress and inflammatory aging pathways utilize natural plant extracts or substances that are difficult to penetrate the skin barrier as active ingredients. Some natural plant extracts are easily deactivated when exposed to air, thus losing their specific functions. Other active ingredients have relatively large molecular weights, resulting in low penetration of the skin barrier and an inability to fully penetrate cells to exert their effects. For example, the anti-aging active ingredient psoralen in patent CN116421482A may undergo photodegradation under ultraviolet (especially UVA) radiation, affecting its stability. Furthermore, the anti-inflammatory ingredient matsutake mushroom extract, due to its large molecular weight, has relatively low stratum corneum penetration efficiency, and its effectiveness is limited without special treatment. The narrow range of action of these active ingredients restricts the target population and the symptoms for which the products are suitable.

[0006] Some skincare products targeting oxidative stress and inflammation only act on specific cell structures or target only one anti-inflammatory pathway, making them suitable only for specific types or a few types of inflammation. For example, the anti-aging composition mentioned in patent CN116421482A is more suitable for combating photoaging and mitochondrial aging, and its broad-spectrum anti-inflammatory effect needs further verification.

[0007] Patent CN116421482B discloses a composition containing matsutake mushroom extract with anti-aging effects and its application. The composition consists of matsutake mushroom extract, bakuchiol, and ergothioneine in a mass ratio of (5-7.5):(0.5-0.75):(0.01-0.02). This composition has a good repairing effect on UV-induced photoaging damage to the skin, preventing damage such as dry skin, erythema, roughness, wrinkles, and epidermal thickening. It can significantly reduce oxidative stress and the levels of inflammatory factors IL-1β, IL-6, COX-2, and TNF-α, alleviating skin inflammation, preventing photodamage, effectively improving skin oxidation, repairing skin barrier function, and protecting the skin. It also has a synergistic effect. However, its anti-aging active ingredient, bakuchiol, may undergo photodegradation under ultraviolet (especially UVA) irradiation, affecting its stability. Furthermore, although bakuchiol can effectively improve wrinkle depth and skin elasticity, high concentrations (>1%) may stimulate TRPV1 receptors, leading to burning and erythema in sensitive skin. Therefore, the concentration and dosage must be strictly controlled during product preparation and use. Moreover, the anti-inflammatory component, matsutake extract, has low stratum corneum penetration efficiency and limited effect without special treatment. Furthermore, this composition is more suitable for combating photoaging and mitochondrial aging, and its broad-spectrum anti-inflammatory effect needs to be verified.

[0008] Patent CN106074677B discloses a traditional Chinese medicine composition for preventing and treating skin aging. Its raw material composition includes: 10-15 parts of *Siegesbeckia orientalis* extract, 15-25 parts of *Tussilago farfara* extract, 5-15 parts of *Glycyrrhiza uralensis* extract, and 5-15 parts of *Angelica sinensis* extract. This traditional Chinese medicine composition has antioxidant and anti-inflammatory effects, and can upregulate glutathione levels in human epidermal HaCaT cells, resist arsenic-induced cytotoxicity, and inhibit the formation of arsenic-induced reactive oxygen species. It can be used to prepare drugs or cosmetics for preventing and treating skin damage and inflammatory lesions and delaying skin aging. However, this composition uses natural plant extracts as active ingredients, which have low stability. The active ingredients may be inactivated and lose their efficacy when exposed to air. In addition, *Tussilago farfara* extract contains pyrrolizidine alkaloids, and its safety needs to be monitored.

[0009] Furthermore, some skincare products act only on specific cellular structures or target only one anti-inflammatory pathway, making them suitable only for specific types or a few types of inflammation. Other skincare products use more potent anti-inflammatory and anti-aging ingredients, which, while achieving some anti-inflammatory and anti-aging effects, may have allergenic or toxic properties, significantly impacting people with sensitive skin. Therefore, it is necessary to develop a highly effective, safe, stable, and easily absorbed anti-inflammatory and anti-aging skincare product. Summary of the Invention

[0010] The purpose of this invention is to overcome the problems of existing anti-inflammatory and anti-aging skin care products, such as low stability of active ingredients, difficulty in penetrating the skin barrier, low cell absorption and utilization efficiency, narrow range of action of active ingredients, limited applicable population and symptoms, and certain allergenicity or toxicity of active ingredients. The invention provides an anti-inflammatory and anti-aging composition based on cyclic hexapeptide, its liposome encapsulation, and skin care products thereof.

[0011] The objective of this invention can be achieved through the following technical solutions: The present invention first provides an anti-inflammatory and anti-aging composition based on cyclic hexapeptide, comprising the following components: cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione; wherein the mass ratio of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione is (0.01-1):(0.01-2):(0.01-1).

[0012] Preferably, the mass ratio of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione is (0.1-0.3):(0.2-0.4):(0.1-0.3).

[0013] The present invention also provides an anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide, comprising an anti-inflammatory and anti-aging composition based on cyclic hexapeptide encapsulated in liposomes, wherein the mass ratio of cyclic hexapeptide-9, Scutellaria baicalensis root extract and glutathione in the liposomes is (0.01-1):(0.01-2):(0.01-1).

[0014] In one embodiment of the present invention, the ratio of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione in the liposomes is (0.1-0.3):(0.2-0.4):(0.1-0.3).

[0015] In one embodiment of the present invention, cyclic hexapeptide-9, Scutellaria baicalensis root extract and glutathione are used as active ingredients, and the total mass of the active ingredients is 1.8%-7.5% of the total mass of the liposomes.

[0016] The present invention further provides a method for preparing an anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide, comprising the following steps: S1: Oil phase preparation: Weigh soybean lecithin (PC) and distearate phosphatidylglycerol (DSPG) and mix them in a reactor. Then weigh cholesterol into the reactor and add a fat-soluble antioxidant. Add anhydrous ethanol to the reactor, heat in a water bath, and stir magnetically until completely mixed to obtain an oil phase mixture. S2: Lipid membrane preparation: The oil phase mixture is transferred to a rotary evaporator and heated in a water bath to completely evaporate the ethanol, forming a uniform and transparent lipid membrane; S3: Aqueous phase preparation: Weigh out the Scutellaria baicalensis root extract, dissolve it by sonication to obtain a clear or slightly turbid yellow solution, adjust the pH, filter, and mix the obtained solution with glutathione, cyclic hexapeptide-9, and phosphate buffered saline (PBS) to obtain a PBS solution containing the active ingredients, i.e., the aqueous phase; S4: Liposome hydration and encapsulation: Slowly pour the PBS solution containing the active ingredient into a container containing a lipid membrane, bathe in water, stir, and form a milky white crude liposome suspension, i.e., the proemulsion; S5: Particle size optimization: The colostrum was transferred to an ultrasonic cell disruptor and the particle size of the liposomes was reduced by probe sonication under ice-water bath conditions until the suspension was translucent, thus obtaining an anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide.

[0017] In one embodiment of the present invention, the mass ratio of soybean lecithin (PC) to distearate phosphatidylglycerol (DSPG) in step S1 is 3:2; the mass ratio of the sum of phospholipids obtained after mixing soybean lecithin (PC) and distearate phosphatidylglycerol (DSPG) to cholesterol is 7:3. In one embodiment of the present invention, in step S1, the fat-soluble antioxidant is selected from vitamin E.

[0018] In one embodiment of the present invention, in step S1, the water bath heating temperature is 50-60°C, for example, 55°C, and the magnetic stirring speed is 200-500 rpm, for example, 300 rpm.

[0019] In one embodiment of the present invention, in step S2, the temperature of the water bath heating is 50-60°C, for example, 55°C.

[0020] In one embodiment of the present invention, in step S2, the complete evaporation of ethanol is carried out under vacuum conditions, namely a vacuum degree of 0.08~0.1MPa and a rotation speed of 100rpm.

[0021] In one embodiment of the present invention, in step S3, the solution can be dissolved by ultrasonication using 0.1M NaOH solution.

[0022] In one embodiment of the present invention, in step S3, the pH is adjusted to approximately 6.0 using a 0.1M HCl solution. The extract is then filtered through a 0.22μm filter membrane to remove insoluble impurities and avoid affecting the liposome particle size. This step may produce minor precipitation, which is normal; the goal is to maximize the solubility of the Scutellaria baicalensis root extract.

[0023] In one embodiment of the present invention, in step S3, the pH of the phosphate buffer solution is 6.0.

[0024] In one embodiment of the present invention, in step S4, the water bath heating temperature is 50-60°C, for example, 55°C, and the magnetic stirring speed is 200-500 rpm, for example, 300 rpm, for 30 minutes.

[0025] In one embodiment of the present invention, in step S5, the ultrasonic cell disruptor operates under the following conditions: power 300W, pulse mode: 3s working, 3s pausing, total time 15min. The present invention further provides an anti-inflammatory and anti-aging skin care product containing the aforementioned anti-inflammatory and anti-aging composition based on cyclic hexapeptide, or containing the aforementioned anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide.

[0026] The skincare product can be any form of skincare or cosmetic product. Preferably, it is a toner, serum, lotion, cream, etc. The required base ingredients vary depending on the type of skincare product. The skincare product has effects such as anti-oxidative stress, anti-inflammation, anti-aging, and improving skin condition.

[0027] In one embodiment of the present invention, the skincare product is a serum, which comprises the following components in varying amounts by weight: Moisturizer 5.2%; Moisturizing and penetration-enhancing agents: 4.0%; Penetration enhancer 2.0%; Thickening regulator 0.4%; Stabilizer 0.25%; Sealing agent 4.0%; Antioxidant 0.2%; pH adjuster 0.3%; Anti-inflammatory and anti-aging liposome encapsulations based on cyclic hexapeptides (2-5%); Preservative 1.0%; Fragrance agent 0.2%; The remainder is water.

[0028] In one embodiment of the present invention, the moisturizer is glycerin and sodium hyaluronate; The moisturizing and penetration-enhancing agent is 1,3-butanediol; The penetration enhancer is dipropylene glycol; The thickening agent is carbomer; The stabilizers are xanthan gum and disodium EDTA; The sealing agent is squalane; The antioxidant is tocopheryl acetate; The pH adjuster is a citrate-sodium citrate buffer solution; The preservatives are phenoxyethanol and ethyl ethyl glycerin; Fragrance is a type of flavoring.

[0029] The present invention further provides a method for preparing an anti-inflammatory and anti-aging skincare product (specifically a serum), comprising the following steps: S1: Aqueous phase preparation: Take an appropriate amount of deionized water into the reactor, and then weigh the humectant, humectant penetration enhancer, thickening regulator and stabilizer according to the formula ratio. Slowly add them into the reactor, heat in a water bath at 70-80℃, and stir constantly to make the components fully dissolve to obtain the aqueous phase. S2: Oil phase preparation: Weigh the blocking agent, the anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide, and the antioxidant according to the formula ratio into a reactor, heat in a water bath at 37°C, and stir until a transparent and homogeneous system is formed, i.e., the oil phase. S3: Aqueous-oil phase mixing: Slowly pour the prepared oil phase into the cooled reactor containing the aqueous phase while stirring at 800-1000 rpm for 30 minutes to ensure uniform emulsification of the system and obtain an oil-water mixture. S4: Add preservatives and fragrances to the oil-water mixture, adjust the pH to 5.5-6.5 with a pH adjuster, and finally homogenize to obtain the essence.

[0030] The present invention further provides a non-therapeutic anti-inflammatory and anti-aging method, which involves applying the cyclic hexapeptide-based anti-inflammatory and anti-aging composition, the cyclic hexapeptide-based anti-inflammatory and anti-aging liposome encapsulation, or the anti-inflammatory and anti-aging skin care product to the facial skin.

[0031] This application provides an anti-inflammatory and anti-aging regulatory system with cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione as the main components. Cyclic hexapeptide-9 is a cyclic hexapeptide that can activate fibroblast proliferation and differentiation, increase the synthesis of skin barrier proteins such as collagen and elastin, while inhibiting the activity of matrix metalloproteinases (MMP-1, MMP-3), reducing collagen degradation, and rebuilding the skin basement membrane structure, thus resisting stress-induced collagen loss and barrier damage at the source. This bidirectional regulatory mechanism can both protect and repair the skin barrier, improve skin elasticity and firmness, reduce wrinkles, and improve skin condition.

[0032] Scutellaria baicalensis root extract is rich in flavonoids such as baicalin and baicalein, which have multi-target antioxidant and anti-inflammatory effects. On the one hand, Scutellaria baicalensis root extract can activate the Nrf2 pathway, upregulate the expression of antioxidant enzyme genes, scavenge free radicals, protect the nervous system, and reduce its oxidative damage. On the other hand, Scutellaria baicalensis root extract can also inhibit prostaglandin synthesis, block the cascade amplification of inflammatory signals, and inhibit the expression of inflammatory factors such as IL-1 and IL-6.

[0033] Glutathione is a key carrier of the antioxidant cycle. Its sulfhydryl group donates hydrogen atoms to reduce hydrogen peroxide, lipid peroxides, etc., to water or alcohols, while it itself becomes oxidized glutathione, terminating the free radical chain reaction, clearing intracellular ROS, and protecting the integrity of DNA, organelles, proteins, and lipid membranes. Oxidized glutathione can be reduced back to glutathione by reductases, achieving dynamic changes between reduced and oxidized forms, maintaining intracellular homeostasis, and preventing the amplification of ROS-induced inflammatory signals, thus exhibiting indirect anti-inflammatory effects.

[0034] In this application, cyclohexapeptide-9, Scutellaria baicalensis root extract, and glutathione are encapsulated in liposomes, which can overcome the limitations of these components in terms of low permeability and poor stability, and greatly improve their permeability and stability.

[0035] This application employs a multi-pathway regulatory model to construct an anti-inflammatory and anti-aging regulatory system with cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione as the main components. Cyclic hexapeptide-9 can activate collagen gene expression in fibroblasts while inhibiting the activity of matrix metalloproteinase MMP-1, reducing collagen degradation. Scutellaria baicalensis root extract can activate the Nrf2 pathway, upregulate the expression of antioxidant enzyme genes, scavenge free radicals, protect the nervous system, and reduce oxidative damage and neuroinflammation. In addition, Scutellaria baicalensis root extract can also inhibit prostaglandin synthesis and block the cascade amplification of inflammatory signals. Glutathione is a key carrier of the antioxidant cycle, clearing intracellular ROS and maintaining intracellular environmental stability through dynamic changes in reduced and oxidized forms. The combined use of these components can effectively inhibit oxidative stress and inflammatory responses, resist inflammatory aging, promote barrier repair, improve wrinkles and skin elasticity, and dual-enhance the anti-inflammatory and anti-aging effects. Their synergistic use forms a multi-dimensional anti-aging pathway of oxidation scavenging, inflammation suppression, and barrier repair.

[0036] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes the different solubility properties of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione, encapsulating them in a single liposome to achieve a stable distribution of the active ingredients. Glutathione is water-soluble and is ultimately encapsulated in the hydrophilic core of the liposome. Cyclic hexapeptide-9 is amphiphilic and located at the boundary between the lipid bilayer and the aqueous phase. The flavonoids in Scutellaria baicalensis root extract are lipid-soluble and can ultimately be embedded in the lipid bilayer. The encapsulated liposomes improve the permeability and cellular absorption of these ingredients, reducing the risk of inactivation due to environmental factors when directly added, and simplifying the preparation process. Furthermore, the combined use of these three ingredients can form a multi-faceted anti-inflammatory and anti-aging pathway involving oxidative scavenging, inflammation suppression, and barrier repair.

[0037] Using cyclic hexapeptide-9 as the main component for barrier repair and anti-aging functions offers multiple regulatory advantages. Cyclic hexapeptide-9 can both inhibit the degradation of collagen by related matrix enzymes, thus resisting stress-induced collagen loss and barrier damage at the source, and activate fibroblast proliferation and differentiation, thereby increasing the synthesis of skin barrier proteins such as collagen and elastin.

[0038] Scutellaria baicalensis root extract has both anti-inflammatory and antioxidant effects. It can scavenge intracellular free radicals, inhibit the damage of skin cells caused by oxidative stress, reduce cellular inflammatory response, and improve skin redness.

[0039] Glutathione is a key carrier of the antioxidant cycle. It can not only remove intracellular ROS and protect the integrity of DNA, organelles, proteins and lipid membranes, but also prevent the amplification of ROS-induced inflammatory signals, thus having an indirect anti-inflammatory effect.

[0040] The combination of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione can regulate skin condition through multiple pathways, providing multiple guarantees for anti-inflammatory and anti-aging effects, and has significant advantages. Detailed Implementation

[0041] The present invention will now be described in detail with reference to specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0042] Process parameters, raw materials, and testing methods not detailed in this invention are all performed in accordance with conventional techniques in the field.

[0043] In the following embodiments of the present invention, the Scutellaria baicalensis root extract was purchased from Shanghai Ruixi Industrial Co., Ltd.

[0044] Preparation of cyclohexapeptide-9, Scutellaria baicalensis root extract, and glutathione mixed liposomes (i.e., the cyclohexapeptide-based anti-inflammatory and anti-aging liposome encapsulations): (1) Oil phase preparation: Soybean lecithin (PC) and distearate phosphatidylglycerol (DSPG) were weighed and mixed in a reactor at a mass ratio of 3:2. Cholesterol was then weighed in the reactor at a mass ratio of 7:3 (mixed phospholipids: cholesterol). A small amount of vitamin E (a fat-soluble antioxidant) was added to the mixture. 50 mL of anhydrous ethanol was poured into the reactor and the mixture was heated in a 55°C water bath and magnetically stirred (300 rpm) until completely mixed.

[0045] (2) Preparation of lipid membrane: Transfer the mixture to a rotary evaporator, 55°C water bath, vacuum degree 0.08~0.1MPa, rotation speed 100rpm, so that the ethanol is completely evaporated until a uniform and transparent lipid film is formed on the inner wall of the flask.

[0046] (3) Aqueous phase preparation: ① Weigh the corresponding mass of Scutellaria baicalensis root extract according to the selected efficacy component ratio (as shown in Table 1 below), and use a small amount of 0.1M NaOH solution to sonicate and dissolve, obtaining a clear or slightly turbid yellow solution. Then, slowly adjust the pH to about 6.0 with 0.1M HCl solution. Finally, filter with a 0.22μm filter membrane to remove insoluble impurities in the extract to avoid affecting the liposome particle size. (This step may produce a slight precipitation, which is normal. The goal is to maximize the dissolution of Scutellaria baicalensis root extract.) ② Transfer the above solution to a volumetric flask, weigh the corresponding mass of glutathione and cyclic hexapeptide-9 according to the selected efficacy component ratio (as shown in Table 1 below), and finally adjust the volume to 100mL with phosphate buffered saline (PBS, pH 6.0).

[0047] (4) Liposome hydration and encapsulation: The above-mentioned PBS solution containing active ingredients is slowly poured into a container containing a lipid membrane, and stirred at 200 rpm for 30 minutes in a 55°C water bath to form a milky white crude liposome suspension, i.e., the primary emulsion.

[0048] (5) Particle size optimization: The colostrum was transferred to an ultrasonic cell disruptor and the liposome particle size was reduced by probe ultrasound under ice-water bath conditions. The power was 300W, the pulse mode was 3s working and 3s pausing, and the total time was 15min until the suspension was semi-transparent, thus obtaining mixed liposomes, namely the anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide.

[0049] Table 1. Content of active ingredients in total liposomes

[0050] The above-mentioned Scutellaria baicalensis root extract and glutathione mixed liposomes, cyclic hexapeptide-9 and glutathione mixed liposomes, cyclic hexapeptide-9 and Scutellaria baicalensis root extract mixed liposomes, cyclic hexapeptide-9 liposomes, Scutellaria baicalensis root extract liposomes, and glutathione liposomes were prepared using the same steps and effective concentrations as the cyclic hexapeptide-9, Scutellaria baicalensis root extract and glutathione mixed liposomes.

[0051] Example 1 A composition comprises the following ingredients: 2 parts of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione mixed liposomes.

[0052] Example 2 A composition comprises the following ingredients: 3 parts of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione mixed liposomes.

[0053] Example 3 A composition comprises the following ingredients: 5 parts of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione mixed liposomes.

[0054] Comparative Example 1 A composition comprises the following ingredients: 2 parts of Scutellaria baicalensis root extract and glutathione mixed liposomes.

[0055] Comparative Example 2 A composition comprises the following components: 2 parts of a mixture of cyclic hexapeptide-9 and glutathione liposomes.

[0056] Comparative Example 3 A composition comprises the following ingredients: 2 parts of cyclic hexapeptide-9 and 2 parts of liposomes mixed with Scutellaria baicalensis root extract.

[0057] Comparative Example 4 A composition comprises the following components: 2 parts of cyclic hexapeptide-9 liposomes.

[0058] Comparative Example 5 A composition comprises the following ingredients: 2 parts of Scutellaria baicalensis root extract liposomes.

[0059] Comparative Example 6 A composition comprises the following ingredients: 2 parts of glutathione liposomes.

[0060] Blank comparison example 7 The composition consists of the following components: 2 parts liposomes.

[0061] The compositions prepared in the above embodiments and comparative examples can be used in skincare products. These skincare products include, but are not limited to, toners, serums, lotions, and creams. During preparation, in addition to the above compositions, the skincare products also include the base components necessary for the production of various skincare products. These base components can be selected from conventional base components used in existing products, and the skincare products can be prepared using conventional methods from the prior art.

[0062] To verify the excellent anti-inflammatory and anti-aging effects of the mixed liposomes containing cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione, the compositions of the above examples and comparative examples were formulated into an essence.

[0063] A skincare product that combats oxidative stress, inflammation, and aging, and improves skin condition, specifically a serum, with the following component concentrations: Table 2. Serum Formula Ratio Table

[0064] Table 3. Content of each composition in the serum

[0065] The difference between Examples 1-3 is the amount of mixed liposomes used. The difference between Comparative Examples 1-3 and Example 1 is that Comparative Examples 1-3 lack one of the active ingredients. The difference between Comparative Examples 4-6 and Example 1 is that Comparative Examples 4-6 contain only one of the three active ingredients. The difference between Blank Comparative Example 7 and Example 1 is that Blank Comparative Example 7 does not contain any of the active ingredients cyclic hexapeptide-9, Scutellaria baicalensis root extract, or glutathione.

[0066] The preparation method of the serum is as follows: (1) Aqueous phase preparation: Take an appropriate amount of deionized water into the reactor, and then weigh glycerol, sodium hyaluronate, 1,3-butanediol, dipropylene glycol, carbomer, xanthan gum, and disodium EDTA according to the formula ratio. Slowly add them into the reactor, heat in a water bath at 70-80℃, and stir continuously to ensure that each component is fully dissolved.

[0067] (2) Oil phase preparation: Squalane, cyclic hexapeptide-9, Scutellaria baicalensis root extract, glutathione mixed liposomes and tocopheryl acetate were weighed into a reactor according to the formula ratio, heated in a water bath at 37°C, and stirred until a transparent and homogeneous system was formed.

[0068] (3) Aqueous-oil phase mixing: Slowly pour the prepared oil phase into the cooled aqueous reactor while stirring at 800-1000 rpm for 30 minutes to ensure uniform emulsification of the system (no layering, no oil droplets).

[0069] (4) Add phenoxyethanol, ethyl ethyl glycerin and fragrance to the mixture, and adjust the pH to 5.5-6.5 with citrate-sodium citrate buffer. Finally, homogenize to obtain the essence.

[0070] Characterization data and effect data of the products in the examples and comparative examples In vitro cell experiments Table 4. Concentration ratio of raw materials for cell experiments (mg / mL)

[0071] Inflammatory factor inhibition capacity test (1) Materials and reagents ①HaCaT human keratinocytes ② Samples prepared for Experimental Examples 1-3, Comparative Examples 1-6, and Blank Example 7 above. ③IL-6 ELISA kit (including coating antibody, detection antibody, standards, enzyme-labeled secondary antibody, substrate solution TMB, stop solution, washing solution, etc.), phosphate-buffered saline (PBS), lipopolysaccharide (LPS) (2) Instruments and equipment ELISA plates, ELISA reader (450nm wavelength), pipettes, centrifuge, incubator (37℃) (3) Experimental principle HaCaT human keratinocytes release inflammatory factors (such as IL-6) upon external stimulation. These factors bind to specific antibodies, which then bind to the antigen-antibody complex via a secondary antibody labeled with horseradish peroxidase (HRP). The addition of the specific substrate TMB activates the enzymatic reaction, producing a blue complex whose absorbance can be detected at 450 nm using a microplate reader. The more inflammatory factors released, the higher the absorbance.

[0072] (4) Experimental methods ① Prepare a cell suspension from cells in the logarithmic growth phase and seed them evenly into 96-well plates, ensuring that the number of cells in each well is basically the same. Incubate at 37℃ and 5% CO2 for 24 hours, and select cells from wells that have reached 70%-80% confluence for further processing.

[0073] ② Add 20 μL of each of the experimental examples 1-3, experimental comparative examples 1-6 and experimental blank example 7 to the plate, with 3 replicates for each group, and incubate in an incubator at 37℃ and 5% CO2 for 24 hours.

[0074] ③ Remove the culture medium completely, add lipopolysaccharide-containing culture medium to each well, and incubate in an incubator at 37℃ and 5% CO2 for 24 hours.

[0075] ④ After the culture is completed, centrifuge at 3000 rpm for 5 min, collect the cell culture supernatant, and use the ELISA kit to add the supernatant of Experimental Examples 1-3, Experimental Comparative Examples 1-3 and Experimental Blank Example 4 to the corresponding reaction wells in the ELISA plate. Follow the kit instructions to perform the following steps: coating with antibody, washing plate, blocking, adding sample, washing plate, adding detection antibody, adding enzyme-labeled secondary antibody, adding substrate for color development, terminating the reaction and reading the values.

[0076] ⑤ Using experimental blank example 7 as a reference, calculate the inhibition rate of IL-6 synthesis by experimental examples 1-3 and experimental comparative examples 1-6. Synthesis inhibition rate (%) = (1 - absorbance of experimental group / absorbance of experimental blank example 7) * 100%. The average value of each group's data is taken.

[0077] (5) Experimental results Based on the above experimental steps, the IL-6 inhibition rates of different experimental groups were measured as shown in the table below: Table 5 IL-6 inhibition rate

[0078] Observing the results in the table above, when the active ingredients were used alone (Experimental Comparative Examples 4-6), the IL-6 inhibition rates in human keratinocytes were 7.67%, 23.71%, and 16.18%, respectively. This indicates that all three ingredients can inhibit IL-6 synthesis, but the inhibitory ability of cyclic hexapeptide-9 is relatively limited, and Scutellaria baicalensis root extract showed the best inhibitory effect. When the composition lacked Scutellaria baicalensis root extract or glutathione (Experimental Comparative Examples 2 and 3), the IL-6 inhibition rate differed significantly from that in Experimental Example 1, but was still higher than the IL-6 inhibition rate when the corresponding composition component was used alone. When the composition lacked cyclic hexapeptide-9 (Experimental Comparative Example 1), the IL-6 inhibition rate was lower than that in Experimental Example 1, but was still higher than the IL-6 inhibition rate when the composition component was used alone. As can be seen from Experimental Comparative Examples 1-6, the combination of two ingredients has a higher IL-6 inhibition ability than when used alone. When the three components were used simultaneously (Experimental Examples 1-3), their inhibition rate of IL-6 was higher than that of the control groups in each experiment. This indicates that the inflammatory factor inhibition ability is better when the three components are used together. As the amount of the composition used increases, the intracellular IL-6 content decreases significantly.

[0079] Human efficacy evaluation experiment (1) Test sample: The essence prepared from the components of Examples 1-3, Comparative Examples 1-6 and Blank Comparative Example 7 as described in Table 2. (2) Test subjects: 100 healthy individuals aged 35 to 60, all of whom had sensitive skin and met the following criteria: ① facial skin redness, with a visual score of 2-6 for redness.

[0080] ②The visual score for facial skin smoothness is 3-6.

[0081] ③Forehead wrinkles are rated on a scale of 2-6.

[0082] ④ Visual score of 2-6 for wrinkles at the outer corner of the eye on one side.

[0083] Before the test, participants signed a written informed consent form and were then randomly divided into 10 groups, each using the same product.

[0084] (3) Test method: ① Subjects completed facial cleansing at the testing center, sat quietly for 30 minutes in a constant temperature and humidity environment, and then used the VISIA-CR facial image acquisition and analysis instrument to test the skin red zone a value, the VC20 plus skin microscope to test skin smoothness, and the Tewameter™ Hex skin moisture content meter to test the skin transepidermal water loss rate.

[0085] ② Subsequently, each group member used the corresponding test serum. After 15 minutes, the skin red zone a value was tested using the VISIA-CR facial image acquisition and analysis instrument, the skin smoothness was tested using the VC20 plus skin microscope, and the skin moisture content meter Tewameter™ Hex was tested for the skin transepidermal water loss rate.

[0086] ③In addition, after using the serum for 7 days and 28 days, the skin red zone a value was tested again using the VISIA-CR facial image acquisition and analysis instrument, the skin smoothness was tested using the VC20 plus skin microscope, and the skin moisture content meter TMHex was tested using the Tewameter TMHex skin moisture content meter.

[0087] (4) Test instructions ①Test environment: Temperature 20~22℃, humidity 45~55%RH.

[0088] ② The VISIA-CR facial image acquisition and analysis instrument captures facial images under cross-polarized light, generating a "red zone exclusive image." The Image-Pro Plus then quantizes the color space of the red zone image. The smaller the a value, the lighter the skin redness.

[0089] ③ The VC20 plus uses a ring-shaped UVA light source and a high-resolution monochrome camera to create 256 levels of grayscale images. Depressed areas (such as fine lines and pores) are displayed as dark colors, while raised areas (such as smooth areas) are displayed as light colors. The built-in software SELS is then used to analyze the uniformity of the grayscale distribution. The smaller the SEsm value, the smoother the skin surface.

[0090] ④ The transdermal water loss rate can be calculated by detecting the water vapor partial pressure difference at different heights on the skin surface using a sensor, combined with the diffusion law.

[0091] ⑤ Test results can be calculated using the following formula: Change rate = (Analysis value after product use - Analysis value before product use) ÷ Analysis value before product use × 100%. The average change rate for the same group is taken.

[0092] Test Results ① Anti-inflammatory and redness-reducing effects Following the experimental steps described above, the VISIA-CR facial image acquisition and analysis instrument was used to test the skin redness zone α value of the subjects at different times. The specific numerical value reflected the degree of facial redness. The data of each group were statistically calculated, and the absolute value of the calculation results was taken to obtain the following results: Table 6. Changes in facial redness in different groups of subjects at different times.

[0093] Observing the results in the table above, on day 28, the rate of change in facial redness of subjects in Comparative Examples 5 and 6 compared to before using the serum was 10.79% and 8.65%, respectively, while the rate for the blank comparative example 7 was 2.09%. This indicates that both Scutellaria baicalensis root extract and glutathione have good anti-inflammatory and redness-reducing effects. On day 28, the rate of change in facial redness of subject in Comparative Example 4 compared to before using the serum was 4.63%, indicating that cyclic hexapeptide-9 also has some anti-inflammatory and redness-reducing ability, but not as good as Scutellaria baicalensis root extract and glutathione. Observing Comparative Examples 1-6 and the blank comparative example 7, it can be found that the combined use of two ingredients has a better anti-inflammatory and redness-reducing effect than using them alone, and both are higher than the anti-inflammatory and redness-reducing effects of using the components of the composition alone. When all three ingredients are used simultaneously (Experiments 1-3), the anti-inflammatory and redness-reducing effect is higher than all comparative examples, indicating that the anti-inflammatory and redness-reducing ability is better when all three ingredients are used together, and the anti-inflammatory and redness-reducing effect becomes more obvious with the increase of the amount of the composition used.

[0094] ② Anti-wrinkle improvement effect Following the experimental procedures described above, the smoothness of the skin on the subjects was tested at different times using a VC20 plus skin microscope. Specific numerical values ​​were used to reflect the degree of facial smoothness. The data from each group were statistically analyzed, yielding the following results: Table 7. Changes in facial smoothness in different groups of subjects at different time points.

[0095] Observing the results in the table above, there were significant differences in the improvement of facial smoothness among the experimental groups on day 28. Comparative Example 4, using cyclic hexapeptide-9 alone, achieved a change in facial smoothness of -10.45%, significantly better than Comparative Example 5, using Scutellaria baicalensis root extract alone (-5.83%) and Comparative Example 6, using glutathione alone (-4.64%). This indicates that cyclic hexapeptide-9 plays a core leading role in directly improving skin wrinkles. Any pairwise combination containing cyclic hexapeptide-9, such as Comparative Example 2 (cyclic hexapeptide-9 and glutathione), showed an anti-wrinkle effect of -11.92%, and Comparative Example 3 (cyclic hexapeptide-9 and Scutellaria baicalensis root extract), showed an anti-wrinkle effect of -13.27%, both better than the -10.45% achieved by using cyclic hexapeptide-9 alone, and also far superior to the -8.06% achieved by the combination of Scutellaria baicalensis root extract and glutathione in Comparative Example 1 (which did not contain cyclic hexapeptide-9). This demonstrates that although the direct anti-wrinkle abilities of Scutellaria baicalensis root extract and glutathione are limited, they can indirectly enhance and synergistically enhance the anti-wrinkle efficacy of cyclic hexapeptide-9 by protecting the skin matrix through anti-inflammatory and antioxidant mechanisms. Comparative Example 1, which does not contain cyclic hexapeptide-9, showed an anti-wrinkle effect of -8.06%, mainly attributed to its antioxidant and anti-inflammatory effects on skin protection, but its effect was significantly weaker than any combination containing cyclic hexapeptide-9. When the three components of Examples 1-3 were used simultaneously, their anti-wrinkle effects reached -14.75%, -17.42%, and -20.31%, respectively, significantly better than all pairwise combinations and single-component effects. This fully verifies that the compositions of the present invention achieve true synergistic effects in anti-wrinkle through a multi-pathway mechanism of cyclic hexapeptide-led repair and synergistic anti-inflammatory and antioxidant effects of Scutellaria baicalensis and glutathione.

[0096] ③Barrier repair effect Based on the above experimental procedures, the transepidermal water loss rate of the subjects was measured at different times using a Tewameter™ Hex skin moisture meter. The data from each group were statistically calculated, and the following results were obtained: Table 8. Changes in transepidermal water loss rate of facial skin in different groups at different time points.

[0097] Observing the results in the table above, on day 28, the change rate of transepidermal water loss of facial skin in Comparative Example 4 was -10.31% compared to before using the serum, while that in Control Comparative Example 7 was -2.85%. This indicates that cyclic hexapeptide-9 has a significant effect on reducing transepidermal water loss and promoting barrier repair. On day 28, the changes in transepidermal water loss of facial skin in Comparative Examples 5 and 6 were -5.36% and -4.45% respectively compared to before using the serum. This suggests that Scutellaria baicalensis root extract and glutathione also have certain water-locking, moisturizing, and barrier repair capabilities, but their effects are not as good as cyclic hexapeptide-9. Although Scutellaria baicalensis root extract and glutathione do not have direct barrier repair capabilities, they may indirectly protect the skin and cell structure by reducing cellular inflammatory responses or oxidative stress processes, thus having a certain effect on maintaining skin moisture. At 15 minutes of use, the changes in transepidermal water loss in Examples 1-3 were relatively similar, which may be because the use time was short, and the effects of the active ingredients were not fully manifested. Observations of Examples 1-3, Comparative Examples 1-6, and Blank Comparative Example 7 show that the composition has a relatively good effect on reducing transdermal water loss, and the reduction effect is better with the increase of the amount of composition, which also reflects the repair effect of the composition on the skin barrier.

[0098] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. An anti-inflammatory and anti-aging composition based on cyclic hexapeptides, characterized in that, It includes the following components: cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione; the mass ratio of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione is (0.01-1):(0.01-2):(0.01-1).

2. The anti-inflammatory and anti-aging composition based on cyclic hexapeptide according to claim 1, characterized in that, The mass ratio of cyclic hexapeptide-9, Scutellaria baicalensis root extract, and glutathione was (0.1-0.3):(0.2-0.4):(0.1-0.3).

3. An anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide, characterized in that, Including the cyclic hexapeptide-based anti-inflammatory and anti-aging composition as described in claim 1 or claim 2, encapsulated in liposomes.

4. The anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide according to claim 3, characterized in that, Cyclohexapeptide-9, Scutellaria baicalensis root extract, and glutathione are used as active ingredients, with the total mass of active ingredients being 1.8%-7.5% of the total mass of liposomes.

5. The method for preparing the anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide as described in claim 3 or 4, characterized in that, Includes the following steps: S1: Oil phase preparation: Weigh soybean lecithin PC and distearate phosphatidylglycerol DSPG and mix them in a reactor. Then weigh cholesterol into the reactor and add fat-soluble antioxidants. Add anhydrous ethanol to the reactor, and stir magnetically in a water bath until completely mixed to obtain an oil phase mixture. S2: Lipid membrane preparation: The oil phase mixture was transferred to a rotary evaporator and placed in a water bath to allow the ethanol to evaporate completely, forming a uniform and transparent lipid membrane. S3: Aqueous phase preparation: Weigh out the Scutellaria baicalensis root extract, dissolve it by sonication to obtain a solution, adjust the pH, filter it, and mix the obtained solution with glutathione, cyclic hexapeptide-9 and phosphate buffer PBS to obtain a PBS solution containing the active ingredients. S4: Liposome hydration and encapsulation: Pour the PBS solution containing the active ingredient into a container containing a lipid membrane, place in a water bath, stir, and form a milky white crude liposome suspension; S5: Particle size optimization: The milky white crude liposome suspension was transferred to an ultrasonic cell disruptor and the liposome particle size was reduced by probe ultrasound under ice-water bath conditions until the suspension became translucent, thus obtaining an anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide.

6. The method for preparing the anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide according to claim 5, characterized in that, In step S1, the mass ratio of soybean lecithin PC to distearate phosphatidylglycerol DSPG is 3:2; the mass ratio of phospholipids to cholesterol obtained after mixing soybean lecithin PC and distearate phosphatidylglycerol DSPG is 7:

3. In step S3, during ultrasonic dissolution, NaOH solution is used to aid in the dissolution process using ultrasound. In step S5, the ultrasonic cell disruptor operates under the following conditions: power 300W, pulse mode: 3 seconds on, 3 seconds pause, total time 15 minutes. The water bath temperature is 50-60℃.

7. An anti-inflammatory and anti-aging skincare product, characterized in that, The anti-inflammatory and anti-aging composition containing the cyclic hexapeptide-based composition of claim 1 or 2, or the anti-inflammatory and anti-aging liposome encapsulation containing the cyclic hexapeptide-based composition of claim 3 or 4.

8. The anti-inflammatory and anti-aging skincare product according to claim 7, characterized in that, The skincare product is a serum, and the serum comprises the following components in varying amounts by weight: Moisturizer 5.2%; Moisturizing and penetration-enhancing agents: 4.0%; Penetration enhancer 2.0%; Thickening regulator 0.4%; Stabilizer 0.25%; Sealing agent 4.0%; Antioxidant 0.2%; pH adjuster 0.3%; Anti-inflammatory and anti-aging liposome encapsulations based on cyclic hexapeptides (2-5%); Preservative 1.0%; Fragrance agent 0.2%; The remainder is water.

9. The method for preparing the anti-inflammatory and anti-aging skincare product according to claim 8, characterized in that, Includes the following steps: S1: Aqueous phase preparation: Take deionized water into the reactor, then weigh the humectant, humectant penetration enhancer, thickening regulator and stabilizer according to the formula ratio, add them into the reactor, heat in a water bath at 70-80℃, and stir continuously to make the components fully dissolve to obtain the aqueous phase; S2: Oil phase preparation: Weigh the blocking agent, anti-inflammatory and anti-aging liposome encapsulation material based on cyclic hexapeptide, and antioxidant into the reactor according to the formula ratio, heat in a water bath at 37°C, and stir until a transparent and homogeneous system is formed, i.e., the oil phase. S3: Aqueous-oil phase mixing: Slowly pour the prepared oil phase into the cooled reactor containing the aqueous phase while stirring at 800-1000 rpm for 30 minutes to ensure uniform emulsification of the system and obtain an oil-water mixture. S4: Add preservatives and fragrances to the oil-water mixture, adjust the pH to 5.5-6.5 with a pH adjuster, and finally homogenize to obtain the essence.

10. A method for anti-inflammatory and anti-aging purposes without therapeutic intent, characterized in that, The anti-inflammatory and anti-aging composition based on cyclic hexapeptide as described in claim 1 or 2, or the anti-inflammatory and anti-aging liposome encapsulation based on cyclic hexapeptide as described in claim 2 or 4, or the anti-inflammatory and anti-aging skin care product as described in claim 7 or 8, is applied to facial skin.

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