Plant vesicle composition with soothing and repairing efficacy, and preparation method and application thereof

Abstract

The application provides a plant vesicle composition with soothing and repairing effects and a preparation method and application thereof. The composition comprises centella asiatica vesicles, aloe vera barbadensis miller vesicles, selaginella moellendorffii hance vesicles and hydroxyl asiatic acid loaded in the vesicles. The application forms composite vesicles by using three kinds of plant vesicles, realizes super high loading of hydroxyl asiatic acid of greater than or equal to 85% by using a mild preparation process with low temperature throughout, and can solve the problems of skin redness, sensitivity, dryness, barrier damage and cell aging damage from multiple pathways such as inhibition of skin inflammatory factors, repair of skin tight junction barrier, promotion of keratinocyte ceramide synthesis and activation of mitochondrial autophagy to repair cell damage.

Description

Technical Field

[0001] This application belongs to the field of cosmetic active ingredient technology, and in particular relates to a plant vesicle composition with soothing and repairing effects, its preparation method and application. Background Technology

[0002] Sensitive skin, redness and inflammation, damaged skin barrier, loss of keratin and lipids, disordered cellular energy metabolism, and mitochondrial damage are the most common sub-health problems of modern skin. As a classic skin-repairing active ingredient, asiaticoside possesses basic anti-inflammatory, soothing, and repair-promoting effects. However, free asiaticoside has drawbacks such as high water solubility, weak transdermal penetration, low loading capacity, easy loss, and limited action, only achieving superficial soothing.

[0003] Natural plant vesicles are derived from nanoliposomes secreted by plants themselves, possessing excellent biocompatibility, skin permeability, and self-loading drug capacity. Compared to artificial liposomes, they are chemically irritating, have a gentle structure, and are suitable for delicate skin. However, current plant vesicle drug delivery systems generally suffer from insufficient loading capacity and limited efficacy evaluation dimensions. They only simply test anti-inflammatory effects without systematically verifying core pathways such as the skin's tight junction barrier, ceramide lipid synthesis, and mitochondrial autophagy repair, thus failing to demonstrate the product's deep repair mechanism. Furthermore, conventional preparation processes often employ high-temperature treatments, which can easily damage the plant vesicle membrane structure and inactivate its activity, further reducing drug loading capacity and efficacy stability. Summary of the Invention

[0004] This application provides a plant vesicle composition with soothing and repairing effects, its preparation method, and its application, in order to solve the problems existing in related technologies. The technical solution is as follows: In a first aspect, embodiments of this application provide a plant vesicle composition with soothing and repairing effects, including Centella asiatica vesicles, Aloe vera vesicles, Selaginella tamariscina vesicles and asiaticoside. The hydroxyascorbic acid glycoside is encapsulated in centella asiatica vesicles, aloe vera vesicles, and selaginella tamariscina vesicles.

[0005] In one embodiment, the preparation method of Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles is as follows: Fresh leaves of Centella asiatica, Aloe vera, or Selaginella tamariscina were added to a phosphate buffer solution, homogenized, and then extracted by differential centrifugation to obtain Centella asiatica vesicles, Aloe vera vesicles, or Selaginella tamariscina vesicles.

[0006] In one embodiment, the homogenization and crushing conditions are as follows: homogenization and crushing are performed using a high-speed homogenizer; homogenization and crushing are performed at 4°C and 10,000-12,000 rpm for 4-6 minutes.

[0007] In one embodiment, the differential centrifugation process is as follows: the homogenate is centrifuged sequentially at 8000-12000×g, 4℃, for 15-25min to collect the supernatant; after microfiltration, it is ultracentrifuged at 90000-120000×g, 4℃ for 50-70min to collect the bottom precipitate; vesicles are obtained; and the vesicles are resuspended in PBS.

[0008] In one embodiment, the mass ratio of fresh leaves of Centella asiatica, Aloe vera, or Selaginella to phosphate buffer is 1:(5-15).

[0009] In one embodiment, the plant vesicle composition with soothing and repairing effects contains 3.5-4.5% by mass of hydroxyascorbic acid glycoside.

[0010] Secondly, embodiments of this application provide a method for preparing a plant vesicle composition with soothing and repairing effects, comprising the following steps: A composite vesicle suspension was obtained by mixing Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles in a certain mass ratio. Add asiaticoside to the complex vesicle suspension, treat with pulsed sonication; centrifuge, collect the precipitate, and redissolve to obtain complex vesicles loaded with asiaticoside.

[0011] In one embodiment, the mass ratio of Centella asiatica vesicles, Aloe vera vesicles and Selaginella tamariscina vesicles is (25-35):(15-25):(15-25).

[0012] In one embodiment, the amount of hydroxyascorbic acid added is 7-8% of the mass of the composite vesicle suspension.

[0013] In one embodiment, the pulsed ultrasound treatment conditions are as follows: under ice bath conditions, ultrasound power is 180-220W, on for 4-6 seconds / off for 8-12 seconds, and ultrasound duration is 8-15 minutes.

[0014] Thirdly, embodiments of this application provide the application of plant vesicle compositions with soothing and repairing effects in the preparation of products for anti-inflammatory and soothing of sensitive skin, repair of skin barrier damage, repair of skin stress damage, regeneration of stratum corneum lipids, and repair of skin cell mitochondrial damage.

[0015] Fourthly, embodiments of this application provide a multi-plant complex vesicle product with soothing and repairing effects, including the above-mentioned plant vesicle composition with soothing and repairing effects.

[0016] In one embodiment, the multi-plant complex vesicle product with soothing and repairing effects is any one of anti-aging serum, firming face cream, wrinkle-reducing eye cream, anti-aging mask, or essence lotion.

[0017] In one embodiment, the mass fraction of the plant vesicle composition with soothing and repairing effects in the plant vesicle product is 0.1%-3.0%.

[0018] The advantages or beneficial effects of the above technical solutions include at least the following: This application presents a plant vesicle composition with soothing and repairing effects, comprising a complex vesicle composed of Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles, as well as asiaticoside encapsulated within the complex vesicles. This application uses three plant vesicles to form a complex vesicle, employing a mild preparation process with low temperatures throughout to achieve an ultra-high encapsulation of asiaticoside ≥85%. It can address skin redness, sensitivity, dryness, barrier damage, and cell aging damage through multiple pathways, including inhibiting skin inflammatory factors, repairing the skin's tight junction barrier, promoting keratinocyte ceramide synthesis, and activating mitochondrial autophagy to repair cell damage.

[0019] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of this application will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0020] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this application and should not be construed as limiting the scope of this application.

[0021] Figure 1 Transmission electron microscopy image of hydroxyascorbic acid glycoside encapsulated in the plant vesicle composition with soothing and repairing effects in Example 3; Figure 2 The peak area data of asiaticoside in the transdermal receiving solution at 2h (left) and 12h (right) in the transdermal diffusion test of the compositions of the examples and comparative examples are shown in the figure. Figure 3 Relative expression levels of TNF-α (left) and IL-6 (right) in RAW264.7 cells treated with the compositions of the examples and comparative examples; Figure 4 The relative expression levels of ZO-1 and ZO-2 mRNA in HaCaT cells were plotted for treatment with the compositions used in the examples and comparative examples. Figure 5 The mRNA expression levels of key enzyme genes for ceramide synthesis are plotted for the treatment of the compositions in the examples and comparative examples. Figure 6 Graph showing total ceramide content data for the treatment of the compositions in the examples and comparative examples; Figure 7 mRNA expression levels of Pink1, Parkin, LC3B, and P62 were plotted for the treatment of the compositions in the examples and comparative examples. Figure 8 The images show data on mitochondrial membrane potential and mitochondrial ROS levels after treatment with the compositions used in the examples and comparative examples. Detailed Implementation

[0022] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this application. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0023] Existing asiaticoside preparations suffer from technical defects such as low loading rate, easy destruction of vesicle activity during processing, single efficacy, unclear repair mechanism, and inability to achieve multi-pathway skin repair. In order to address the above problems, this application provides a plant vesicle composition with soothing and repairing effects, its preparation method and application.

[0024] This application provides a plant vesicle composition with soothing and repairing effects, including Centella asiatica vesicles, Aloe vera vesicles, Selaginella tamariscina vesicles and asiaticoside. The hydroxyascorbic acid glycoside is encapsulated in centella asiatica vesicles, aloe vera vesicles, and selaginella tamariscina vesicles.

[0025] In one embodiment, the mass content of asiaticoside loaded in the plant vesicle composition with soothing and repairing effects is any point value or any two points within the range of 3.5%-4.5%. For example, the mass content of asiaticoside loaded is 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, etc.

[0026] Centella asiatica vesicles can significantly reduce skin inflammation, quickly relieve discomfort such as sensitivity, redness, and stinging, and effectively repair damaged skin barriers. Simultaneously, it can promote fibroblast proliferation and collagen synthesis, accelerate wound healing, and reduce scar formation. Aloe vera vesicles are rich in aloe polysaccharides, amino acids, and other moisturizing ingredients, deeply hydrating and moisturizing; they can effectively soothe various skin inflammations, while promoting the synthesis of fibrin and collagen, accelerating tissue repair, and achieving anti-inflammatory and repair effects; they can also regulate sebum secretion at its source and inhibit folliculitis, achieving oil control and acne treatment. Selaginella tamariscina vesicles can form a "microcapsule water-locking film" on the skin surface, effectively locking in moisture and slowing down water loss; they effectively enhance the skin's resistance to environmental stresses (such as pollution and climate change), making the skin stronger; their active ingredients (biflavonoids) can effectively inhibit acne inflammation and have antibacterial effects; they also have melanin-inhibiting and anti-aging effects. In particular, this application also utilizes the differentiated lipid composition and membrane protein distribution of the three vesicles; by combining the three vesicles and utilizing the multi-plant lipid membrane cross-linking assembly effect, it breaks through the traditional upper limit of drug loading in plant vesicles, thereby achieving efficient encapsulation of p-hydroxyasiaticoside.

[0027] As one implementation method, the preparation method of Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles is as follows: Fresh leaves of Centella asiatica, Aloe vera, or Selaginella tamariscina were added to a phosphate buffer solution, homogenized, and then extracted by differential centrifugation to obtain Centella asiatica vesicles, Aloe vera vesicles, or Selaginella tamariscina vesicles.

[0028] The three plant vesicles in this application are extracted using fresh leaves of the corresponding plants as raw materials and phosphate buffer (pH 7.4) as the extraction solvent. After the fresh plant leaves are crushed, the vesicles are released into the extraction solvent; then the mixture is homogenized and extracted by differential centrifugation.

[0029] In one embodiment, the homogenization and crushing conditions are as follows: homogenization and crushing are performed using a high-speed homogenizer; homogenization and crushing are performed at 4°C and 10,000-12,000 rpm for 4-6 minutes. This application utilizes a homogenizer for crushing under low-temperature conditions, which enables the release of vesicles from plant leaves.

[0030] As one implementation method, the differential centrifugation process is as follows: the homogenate is centrifuged sequentially at 8000-12000×g, 4℃, for 15-25min to collect the supernatant; after microfiltration, it is ultracentrifuged at 90000-120000×g, 4℃ for 50-70min to collect the bottom precipitate; vesicles are obtained; the vesicles are resuspended in PBS.

[0031] The ultracentrifugation process of this application is divided into two parts. First, the homogenate is centrifuged at 8000-12000×g, 4℃, for 15-25min to obtain the supernatant, which can remove most of the solid impurities. Then, it is filtered by microfiltration. In this embodiment, a 0.45μm microporous membrane is used to further remove some impurities. Finally, it is ultracentrifuged at 90000-120000×g, 4℃ for 50-70min, and the vesicles and water in the solution are separated into layers and separated in the form of precipitates.

[0032] In this embodiment, the vesicle precipitate is resuspended using PBS buffer. The obtained vesicles are resuspended in PBS buffer with a pH of 7.4 to obtain a vesicle suspension. Preferably, the resuspension concentration is configured to be ≥1.0 × 10⁻⁶ vesicles. 9 The concentration of particles / mL facilitates subsequent dilution. As a preferred embodiment, this application configures the vesicle concentration to be 1.0 × 10⁻⁶ particles / mL. 10 The particle / mL value facilitates subsequent formulation and loading rate calculations. The concentration of vesicle particles was determined using a nanoparticle tracking analyzer (NTA).

[0033] In one embodiment, the mass ratio of fresh leaves of Centella asiatica, Aloe vera, or Selaginella to phosphate buffer is 1:(5-15). Preferably, the mass ratio of fresh leaves of Centella asiatica, Aloe vera, or Selaginella to phosphate buffer is 1:10.

[0034] This application provides a method for preparing a plant vesicle composition with soothing and repairing effects, comprising the following steps: A composite vesicle suspension was obtained by mixing Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles in a certain mass ratio. Add asiaticoside to the complex vesicle suspension, treat with pulsed sonication; centrifuge, collect the precipitate, and redissolve to obtain complex vesicles loaded with asiaticoside.

[0035] This application prepares a mixed vesicle by mixing Centella asiatica vesicles, Aloe vera vesicles and Selaginella tamariscina vesicles in a certain proportion, and then adds hydroxyaspartic acid to the mixed vesicles for encapsulation; the encapsulation is carried out at low temperature using ultrasonic pulses.

[0036] In one embodiment, the mass ratio of Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles is (25-35):(15-25):(15-25). This application combines the three vesicles according to the above ratio, achieving a good encapsulation rate of asiaticoside. The resulting plant vesicle composition with soothing and repairing effects can fully exert its functions of inhibiting skin inflammatory factors, repairing the skin's tight junction barrier, promoting keratinocyte ceramide synthesis, and activating mitochondrial autophagy to repair cell damage.

[0037] As one implementation method, the amount of hydroxyascorbic acid added is 7-8% of the mass of the composite vesicle suspension.

[0038] As one implementation method, the pulsed ultrasound treatment conditions are: under ice bath conditions, ultrasound power of 180-220W, on for 4-6 seconds / off for 8-12 seconds, and ultrasound duration of 8-15 minutes. In this application, asiaticoside, under the influence of a concentration gradient, combined with pulsed ultrasound, opens channels on the vesicle membrane surface, allowing asiaticoside to efficiently enter the vesicle interior, achieving encapsulation.

[0039] This application provides the use of plant vesicle compositions with soothing and repairing effects in the preparation of products for anti-inflammatory and soothing of sensitive skin, repair of skin barrier damage, repair of skin stress damage, regeneration of stratum corneum lipids, and repair of skin cell mitochondrial damage.

[0040] This application discloses a plant vesicle composition with soothing and repairing effects that can achieve skin care from multiple angles, including inhibiting skin inflammatory factors, repairing the skin's tight junction barrier, promoting the synthesis of keratinocyte ceramides, and activating mitochondrial autophagy to repair cell damage. This addresses skin redness, sensitivity, dryness, barrier damage, and cellular aging damage through multiple pathways. Therefore, this application's plant vesicle composition with soothing and repairing effects can be used to prepare products for anti-inflammatory and soothing sensitive skin, repairing skin barrier damage, repairing skin stress damage, regenerating stratum corneum lipids, and repairing mitochondrial damage in skin cells.

[0041] This application provides a multi-plant complex vesicle product with soothing and repairing effects, including the above-mentioned plant vesicle composition with soothing and repairing effects.

[0042] As one implementation method, the multi-plant complex vesicle product with soothing and repairing effects is any one of anti-aging serum, firming face cream, wrinkle-reducing eye cream, anti-aging mask, or essence lotion.

[0043] As one implementation method, the mass fraction of the plant vesicle composition with soothing and repairing effects in the multi-plant complex vesicle product with soothing and repairing effects is 0.1%-3.0%.

[0044] The following is a further explanation using specific embodiments.

[0045] Preparation Example Preparation of Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles: Remove any rotten or spoiled parts from the whole Centella asiatica plant, fresh Aloe vera leaves, and Selaginella tamariscina plant, wash them, and drain them. The pretreated raw materials were added to pre-cooled phosphate buffer (PBS, pH 7.4) at a material-to-liquid ratio of 1:10, and homogenized in a homogenizer at 4°C and 10,000 rpm for 5 minutes to obtain a homogenate. The homogenate was centrifuged at 10,000 rpm for 20 min at 4 °C, and the supernatant was collected. The supernatant was filtered through a 0.45 μm microporous membrane and then ultracentrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was discarded, and the precipitate was resuspended in PBS buffer to a final volume of 1 × 10⁻⁶. 10 Centella asiatica vesicle suspension, Aloe vera vesicle suspension, and Selaginella tamariscina vesicle suspension were obtained at particle / mL.

[0046] Example 1 The vesicle suspensions of the preparation example were mixed according to the following ratio: 25g of Centella asiatica vesicle suspension, 20g of Aloe vera vesicle suspension, and 20g of Selaginella tamariscina vesicle suspension; 5g of hydroxyaspartic acid was dissolved in 30g of water and mixed with the vesicle suspensions to obtain the mixture. The mixture was incubated in an ice-water bath with 200W sonication for 5 seconds, followed by 10 seconds of sonication for 10 minutes, and then incubated at 4°C in the dark for 12 hours to complete the encapsulation of hydroxyascorbic acid. The mixture was centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was discarded, and the precipitate was reconstituted with water until the total mass was 100 g, thus obtaining a plant vesicle composition with soothing and repairing effects.

[0047] Example 2 The vesicle suspensions of the preparation example were mixed according to the following ratio: 27g of Centella asiatica vesicle suspension, 25g of Aloe vera vesicle suspension, and 13g of Selaginella tamariscina vesicle suspension; 5g of hydroxyaspartic acid was dissolved in 30g of water and mixed with the vesicle suspensions to obtain the mixture. The mixture was incubated in an ice-water bath with 200W sonication for 5 seconds, followed by 10 seconds of sonication for 10 minutes, and then incubated at 4°C in the dark for 12 hours to complete the encapsulation of hydroxyascorbic acid. The mixture was centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was discarded, and the precipitate was reconstituted with water until the total mass was 100 g, thus obtaining a plant vesicle composition with soothing and repairing effects.

[0048] Example 3 The vesicle suspensions of the preparation example were mixed according to the following ratio: 26g of Centella asiatica vesicle suspension, 23g of Aloe vera vesicle suspension, and 16g of Selaginella tamariscina vesicle suspension; 5g of hydroxyaspartic acid was dissolved in 30g of water and mixed with the vesicle suspensions to obtain the mixture. The mixture was incubated in an ice-water bath with 200W sonication for 5 seconds, followed by 10 seconds of sonication for 10 minutes, and then incubated at 4°C in the dark for 12 hours to complete the encapsulation of hydroxyascorbic acid. The mixture was centrifuged at 100,000 × g for 60 min at 4 °C. The supernatant was discarded, and the precipitate was reconstituted with water until the total mass was 100 g, thus obtaining a plant vesicle composition with soothing and repairing effects.

[0049] Comparative Example 1 Dissolve 5g of asiaticoside in 30g of water and mix with 65g of the prepared asiaticoside vesicle suspension; to obtain a mixture. The mixture was incubated in an ice-water bath with 200W sonication for 5 seconds, followed by 10 seconds of sonication for 10 minutes, and then incubated at 4°C in the dark for 12 hours to complete the encapsulation of hydroxyascorbic acid. The mixture was centrifuged at 100,000 × g for 60 min at 4 °C, the supernatant was discarded, and the precipitate was reconstituted with water until the total mass was 100 g, thus obtaining the composition.

[0050] Comparative Example 2 Dissolve 5g of asiaticoside in 30g of water and mix with 65g of the prepared Aloe vera vesicle suspension; to obtain a mixture. The mixture was incubated in an ice-water bath with 200W sonication for 5 seconds, followed by 10 seconds of sonication for 10 minutes, and then incubated at 4°C in the dark for 12 hours to complete the encapsulation of hydroxyascorbic acid. The mixture was centrifuged at 100,000 × g for 60 min at 4 °C, the supernatant was discarded, and the precipitate was reconstituted with water until the total mass was 100 g, thus obtaining the composition.

[0051] Comparative Example 3 Dissolve 5g of asiaticoside in 30g of water and mix with 65g of the Selaginella tamariscina vesicle suspension from the preparation example to obtain a mixture. The mixture was incubated in an ice-water bath with 200W sonication for 5 seconds, followed by 10 seconds of sonication for 10 minutes, and then incubated at 4°C in the dark for 12 hours to complete the encapsulation of hydroxyascorbic acid. The mixture was centrifuged at 100,000 × g for 60 min at 4 °C, the supernatant was discarded, and the precipitate was reconstituted with water until the total mass was 100 g, thus obtaining the composition.

[0052] Comparative Example 4 Remove any rotten or spoiled parts from the whole Centella asiatica plant, fresh Aloe vera leaves, and Selaginella tamariscina plant, wash them, and drain them. The pretreated raw materials were added to pre-cooled phosphate buffer (PBS, pH 7.4) at a material-to-liquid ratio of 1:10. The mixture was sonicated at 400W for 50 min, and the process was repeated twice. The extracts were filtered through gauze and combined. Excipients were added and the mixture was freeze-dried to prepare Centella asiatica extract, Aloe vera extract, and Selaginella tamariscina extract. Dissolve 26g of Centella asiatica extract, 23g of Aloe vera extract, 16g of Selaginella tamariscina extract and 5g of asiaticoside in 30g of water to obtain a mixture. The mixture was incubated in an ice-water bath and sonicated at 200W for 10 minutes with 5 seconds of sonication followed by 10 seconds of interval, and then incubated at 4°C in the dark for 12 hours to obtain the composition.

[0053] 1. Encapsulation efficiency test: The content of free asiaticoside in the supernatant after ultracentrifugation of Examples 1-3 and Comparative Examples 1-3 was determined by high performance liquid chromatography (HPLC). The composition of Comparative Example 4 was ultracentrifuged at 100,000 × g for 60 min at 4 °C, and the content of free asiaticoside in the supernatant was determined by HPLC. The encapsulation efficiency was calculated using the formula: Encapsulation efficiency = (Total drug - Free drug) / Total drug × 100%; the results are shown in Table 1.

[0054] Table 1

[0055] Table 1 shows the encapsulation efficiency data. In Comparative Examples 1-3, when only Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles were used as a single system to load an equal amount of asiaticoside, the overall encapsulation efficiency of each group was relatively low, with the highest encapsulation efficiency of a single group being only 56.9%. After constructing a ternary co-encapsulation system by combining the three types of plant vesicles, the encapsulation efficiency was significantly improved, confirming that the combination of the three plant vesicles can produce a significant synergistic effect, effectively improving the carrier's ability to encapsulate asiaticoside. Moreover, the mass ratio of the compound components has a significant impact on the encapsulation performance of the system. Among them, the compound scheme with 26% Centella asiatica vesicles, 23% Aloe vera vesicles, and 16% Selaginella tamariscina vesicles achieved an encapsulation efficiency of 86.2%, which is the optimal carrier ratio under the experimental conditions.

[0056] 2. The plant vesicle composition with soothing and repairing effects of Example 3 was observed by transmission electron microscopy. The transmission electron micrograph is shown below. Figure 1 As shown.

[0057] Figure 1 TEM images show that the composite plant vesicles exhibit a typical cup-shaped (or saucer-shaped / hemispherical) structure, rather than a perfect sphere. The vesicle boundaries are clear, exhibiting distinct lipid bilayer characteristics, with the dark central region suggesting successful encapsulation of asiaticoside. The overall structure remains intact, without significant large-area rupture or aggregation collapse. This indicates that the encapsulation process was conducted under mild conditions, effectively maintaining the structural integrity of the plant vesicle carrier.

[0058] 3. Performance Test (1) Transdermal diffusion experiment This embodiment uses the Franz vertical transdermal diffusion cell (TR-3000 model, Shanghai Yuyan Scientific Instruments Co., Ltd.) model to compare the in vitro transdermal performance of plant extracts and plant vesicle preparations under the same active ingredient content.

[0059] In this experiment, intact dorsal skin from SPF mice was used. Subcutaneous fat and connective tissue were removed, and the skin was cleaned with physiological saline and fixed in a transdermal diffusion cell, with the stratum corneum facing the donor cell and the dermis facing the receiver cell. The experimental system was kept at a constant temperature of 32℃ and a stirring speed of 300 rpm, with equilibration for 30 minutes beforehand to stabilize the skin barrier. The experiment included a blank group (pure water + 5% asiaticoside), Examples 1-3, and Comparative Examples 1-4, with three parallel samples in each group. The drug administration volume was kept identical in both groups, and a mixture of physiological saline was used as the receiver solution. Samples were taken at 2h and 12h time points. An equal volume of isothermal fresh receiver solution was added immediately after each sampling to ensure a constant diffusion system volume. An equal volume of chromatographic grade methanol was added to the sample, vortexed for 30s, filtered through a 0.22μm filter membrane, and the content of the active ingredient was determined by high-performance liquid chromatography (HPLC). Results are as follows: Figure 2 As shown.

[0060] Figure 2 In the control group, almost no asiaticoside permeated, indicating the model was effective. The transdermal absorption of the extract (Comparative Example 4) was significantly lower than that of Examples 1-3 and Comparative Examples 1-3 (plant vesicles), demonstrating that the vesicle carrier can significantly promote the transdermal absorption of asiaticoside. There were significant differences in the permeation-promoting effects among the examples, with Example 3 showing the highest permeation-promoting effect. The synergistic effect of the three-component compound vesicles (Examples 1-3) was significant, superior to the single vesicle formulation (Comparative Examples 1-3).

[0061] (2) In vitro anti-inflammatory activity verification experiment Specific steps: RAW264.7 mouse macrophages were routinely passaged and cultured, seeded into cell culture plates, and incubated until the cells adhered and grew; except for the blank control group, LPS stimulation solution was added to the culture medium of the other groups to induce the inflammation model, and the test samples were added simultaneously for treatment. The blank control group was only added with basal culture medium. All cells were cultured in a standard isothermal environment. The following groups were set up: blank control group (NC): no LPS, no sample; model control group (M): only LPS was added to induce inflammation; positive control group (PC): dexamethasone group; Examples 1, 2, and 3; comparative examples 1, 2, 3, and 4. RAW264.7 cells were added at a rate of 2 mL per well (3 × 10⁶ cells). 5Cells were seeded in 6-well plates and cultured for 24 h. The culture medium was discarded, and LPS (1 μg / mL) was added according to the experimental groups (except for the blank control group) to induce an LPS-induced inflammatory cell model. Different drug solutions were then added, with a final concentration of 0.1% for each example and comparative example. Each group was divided into 3 replicates, and the cells were cultured for another 24 h. The cell culture supernatant from each well was collected, and the absorbance was read using a commercial TNF-α ELISA kit and an IL-6 ELISA kit, strictly following the kit instructions. The standard curve was used to calculate the TNF-α and IL-6 concentrations in the supernatant of each group, and the inflammatory factor inhibition rate was calculated. The results are shown in [Figure number missing]. Figure 3 .

[0062] The results showed that after LPS induction, the secretion of TNF-α and IL-6 in the model control group increased significantly; plant extracts and various vesicle-containing sample groups could reduce the content of the two inflammatory factors to varying degrees; the optimal ratio sample in Example 3 showed the highest inhibitory efficiency against TNF-α and IL-6. The three types of plant vesicle complex carriers and the encapsulated asiaticoside formed a synergistic anti-inflammatory pathway, which dually inhibited inflammatory signal transduction and reduced the release of pro-inflammatory factors, and had excellent skin soothing, anti-inflammatory and repair potential.

[0063] (3) Repair of cell barrier damage The feasibility of the detection method of the present invention and the barrier protective activity of plant raw materials were verified by HaCaT cells. A blank control group, a barrier damage model group, Examples 1-3, and Comparative Examples 1-4 were set up, with 3 replicates in each group and the experiment was repeated three times.

[0064] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. After cell confluence reached 80%-90% and barrier function matured, cell barrier damage models were constructed in all groups except the control group using a uniform inducing agent. After modeling, graded sterile plant material working solutions were added to the example groups, and the intervention was performed under uniform conditions for 24 hours. After intervention, cells were washed with pre-cooled PBS, and total RNA was extracted using the Trizol method. Qualified RNA samples with an OD260 / 280 ratio of 1.8-2.1 were screened, and cDNA was prepared by reverse transcription using a uniform loading volume. qPCR was performed using the SYBR Green method. A 20 μL standard amplification system was prepared, and primer specificity was verified by 40 cycles of amplification and melting curve analysis. GAPDH was used as an internal control. The relative expression levels of ZO-1 and ZO-2 mRNA were calculated using the 2^(-ΔΔCt) method and statistical analysis was performed. The results are shown in the table below. Figure 4 .

[0065] Figure 4The results showed that, compared with the blank control group, the relative expression levels of the mRNAs of the cell tight junction proteins ZO-1 and ZO-2 in the barrier damage model group were significantly downregulated, proving the successful construction of the cell barrier damage model. After intervention with the plant vesicles and extracts in each example and comparative example, the expression levels of ZO-1 and ZO-2 genes all rebounded to varying degrees. Among them, the increase in ZO-1 and ZO-2 mRNA expression levels in the three-component compound vesicle groups (Examples 1-3) was significantly better than that in the comparative examples 1-3 of single vesicles and the comparative example 4 of the extract group, especially Example 3, which showed the most significant upregulation effect. The above results confirm that the three-component compound plant vesicles of Centella asiatica, Aloe vera, and Selaginella tamariscina have stronger cell barrier damage repair activity, can effectively upregulate the gene expression of tight junction proteins ZO-1 and ZO-2, repair damaged epithelial barriers, and the combination of the three vesicles can produce a synergistic barrier protection effect.

[0066] (4) In vitro anti-inflammatory activity verification experiment This verification experiment validated the skin-soothing and barrier-repairing activities of the plant raw materials in different embodiments of the present invention by regulating the level of ceramide synthesis in skin keratinocytes.

[0067] The experiment used HaCaT immortalized human keratinocytes, which were cultured in DMEM high glucose containing 10% fetal bovine serum and 1% penicillin antibodies under conventional conditions of 37°C and 5% CO2. A blank control group, an H2O2 oxidative stress model group, Examples 1-3, and Comparative Examples 1-4 raw material intervention groups were set up. Each group was set up with 3 replicates and the experiment was repeated three times.

[0068] When cell confluence reached 80%, experimental treatment was carried out. The blank control group was cultured normally, while the other groups were treated with 200 μM H2O2 for 4 hours to construct a skin cell oxidative stress injury model. After modeling, the culture medium was changed. The model groups were cultured in conventional culture medium, while the intervention groups were treated with sterile plant material culture medium prepared according to the corresponding examples for 24 hours. After the intervention, cells and supernatant samples were collected. Total RNA was extracted from cells using the Trizol method. After the purity test was passed, cDNA was prepared by reverse transcription. The mRNA expression levels of the key enzyme genes SPTLC1, SPTLC2, CERS1, and CERS2 for ceramide synthesis were detected by qPCR. GAPDH was used as an internal control, and the relative expression level was calculated using the 2^(-ΔΔCt) method. The results are shown in the figure. Figure 5 Simultaneously, the total ceramide content in the cell supernatant was quantitatively detected using an ELISA kit, and the results are shown in [Figure number missing]. Figure 6 .

[0069] The experimental results showed that, based on the in vitro oxidative stress cell experiments, after H2O2-induced modeling, the mRNA expression levels of key enzymes SPTLC1, SPTLC2, CERS1, and CERS2 in HaCaT cells of the oxidative stress model group were significantly downregulated, and the total intracellular ceramide content was greatly reduced. Oxidative damage can significantly inhibit the ceramide synthesis pathway in keratinocytes. After 24 hours of intervention with the plant vesicles and extracts of Examples 1-3 and Comparative Examples 1-3 (Comparative Example 4), the expression levels of the above four types of synthase genes and the total intracellular ceramide content were all restored to varying degrees. Among them, the upregulation of key enzyme genes and the accumulation of ceramide in the ternary compound vesicles of Examples 1-3 were significantly better than those in the single vesicles of Comparative Examples 1-3 and Comparative Example 4 (Comparative Example 4), with Example 3 showing the best improvement effect. The results confirm that the three-component plant vesicles of Centella asiatica, Aloe vera, and Selaginella tamariscina can synergistically activate the ceramide synthesis pathway in keratinocytes, upregulate the expression of key enzyme genes in ceramide synthesis, increase intracellular ceramide reserves, effectively improve lipid synthesis blockage caused by oxidative stress, and have excellent skin soothing and stratum corneum barrier repair activities.

[0070] (5) Skin cell mitochondrial autophagy experiment By detecting the level of mitochondrial autophagy in skin cells, the skin antioxidant, soothing, and damage repair activities of the plant raw materials in the various embodiments of the present invention were verified.

[0071] This experiment used human immortalized keratinocytes (HaCaT) and cultured them in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin antibodies under conventional conditions of 37°C and 5% CO2. The experiment included a blank control group, an oxidative stress injury model group, and raw material intervention groups for Examples 1-3 and Comparative Examples 1-4. Each group had 3 replicates, and the experiment was repeated three times.

[0072] After the cells reached 80% confluence, experimental treatment was carried out. The blank control group was cultured normally without any damage intervention. The other groups were treated with 200 μM H2O2 for 4 h to construct a skin cell oxidative stress damage model, inducing mitochondrial dysfunction and oxidative damage accumulation. After modeling, the damaged culture medium was discarded, and the cells were washed with PBS. The model groups were replaced with ordinary complete culture medium, and each intervention group was treated with sterile plant material working solution prepared according to the corresponding example and comparative example, respectively. The intervention was continued under the same conditions for 24 h. After the intervention, cell samples were collected, and the mRNA expression levels of key regulatory genes for mitochondrial autophagy, Pink1, Parkin, LC3B, and P62, were detected by qPCR. GAPDH was used as an internal control, and the relative expression levels were calculated using the 2^(-ΔΔCt) method. The results are shown in the figure. Figure 7 Simultaneously, flow cytometry was used to detect mitochondrial membrane potential and mitochondrial ROS levels. Combined with the expression of autophagy-related genes, the activation level of mitophagy and the cell's soothing and repair effects were comprehensively evaluated. Results are shown below. Figure 8 .

[0073] After H2O2 oxidative stress induction, the mRNA expression of key regulatory genes for mitochondrial autophagy, Pink1, Parkin, and LC3B, in HaCaT cells of the model group was significantly downregulated, while the expression of the autophagy substrate marker gene P62 was significantly increased. At the same time, the mitochondrial membrane potential was greatly reduced and mitochondrial ROS accumulated in large quantities, indicating that oxidative damage can significantly inhibit the mitochondrial autophagy pathway in keratinocytes and induce mitochondrial dysfunction and accumulation of oxidative free radicals. After intervention treatment with plant vesicles and extracts in Examples 1-3 and Comparative Examples 1-3 and Comparative Example 4, the expression levels of Pink1, Parkin, and LC3B genes were significantly restored, P62 expression was reduced, the mitochondrial membrane potential was restored, and the mitochondrial ROS content was significantly reduced. Among them, the regulatory effect of the three-component compound vesicles of Centella asiatica, Aloe vera, and Selaginella tamariscina in Examples 1-3 was much better than that of the single vesicles in Comparative Examples 1-3 and Comparative Example 4 in the extract group. Example 3 showed the best activity. The above results demonstrate that the combination of the three plant vesicles has a synergistic effect, which can effectively activate the Pink1 / Parkin mitochondrial autophagy pathway in damaged skin keratinocytes, clear damaged mitochondria, reduce intracellular oxidative stress levels, stabilize mitochondrial membrane function, and has excellent skin antioxidant, soothing and oxidative damage repair effects.

[0074] The efficacy tests show that the three-component compound vesicles of Centella asiatica, Aloe vera, and Selaginella tamariscina loaded with hydroxyaspartic acid exhibit a significant synergistic effect compared to single vesicle systems, with a substantial increase in drug loading and encapsulation efficiency. The optimal vesicle mass ratio of 26%:23%:16% demonstrates the best overall performance. Cell experiments confirm that this compound system can repair the skin through multiple pathways: upregulating the expression of tight junction proteins ZO-1 and ZO-2 to repair the epithelial barrier; activating key enzymes in ceramide synthesis to increase the lipid content of the stratum corneum; and activating the Pink1 / Parkin mitochondrial autophagy pathway to scavenge free radicals and stabilize mitochondrial function. It possesses multiple functions including barrier repair, soothing, and antioxidant effects, showing promising application prospects as a skincare ingredient.

[0075] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of those different embodiments or examples.

[0076] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "a plurality of" means two or more, unless otherwise explicitly specified.

[0077] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this application, and these should all be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A plant vesicle composition with soothing and repairing effects, characterized in that, Including Centella asiatica vesicles, Aloe vera vesicles, Selaginella tamariscina vesicles, and asiaticoside; The hydroxyascorbic acid glycoside is encapsulated in centella asiatica vesicles, aloe vera vesicles, and selaginella tamariscina vesicles.

2. The plant vesicle composition with soothing and repairing effects according to claim 1, characterized in that, The preparation methods for Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles are as follows: Fresh leaves of Centella asiatica, Aloe vera, or Selaginella tamariscina were added to a phosphate buffer solution, homogenized, and then extracted by differential centrifugation to obtain Centella asiatica vesicles, Aloe vera vesicles, or Selaginella tamariscina vesicles.

3. The plant vesicle composition with soothing and repairing effects according to claim 2, characterized in that, The homogenization and crushing conditions are as follows: homogenization and crushing are carried out using a high-speed homogenizer; homogenization and crushing are performed at 4℃ and 10000-12000rpm for 4-6 minutes. The differential centrifugation process is as follows: the homogenate is centrifuged sequentially at 8000-12000×g, 4℃, for 15-25 min to collect the supernatant; after microfiltration, it is ultracentrifuged at 90000-120000×g, 4℃ for 50-70 min to collect the bottom precipitate; vesicles are obtained. Resuspend the vesicles in PBS; The mass ratio of fresh leaves of Centella asiatica, Aloe vera, or Selaginella to phosphate buffer is 1:(5-15).

4. The plant vesicle composition with soothing and repairing effects according to claim 1, characterized in that, The plant vesicle composition with soothing and repairing effects contains 3.5-4.5% by mass of hydroxyascorbic acid glycoside.

5. A method for preparing a plant vesicle composition with soothing and repairing effects, characterized in that, Includes the following steps: A composite vesicle suspension was obtained by mixing Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles in a certain mass ratio. Add asiaticoside to the composite vesicle suspension and treat with pulsed ultrasound; Centrifugation was performed, the precipitate was collected, and the precipitate was redissolved to obtain complex vesicles loaded with hydroxyasiaticoside.

6. The method for preparing the plant vesicle composition with soothing and repairing effects according to claim 5, characterized in that, The mass ratio of Centella asiatica vesicles, Aloe vera vesicles, and Selaginella tamariscina vesicles was (25-35):(15-25):(15-25); The amount of hydroxyascorbic acid added is 7-8% of the mass of the compound vesicle suspension.

7. The method for preparing the plant vesicle composition with soothing and repairing effects according to claim 5, characterized in that, The conditions for pulsed ultrasound treatment are: under ice bath conditions, ultrasound power 180-220W, on for 4-6 seconds / off for 8-12 seconds, and ultrasound duration 8-15 minutes.

8. The application of the plant vesicle composition with soothing and repairing effects prepared by the method of any one of claims 1-4 or any one of claims 5-7 in the preparation of products for anti-inflammatory and soothing of sensitive skin, repair of skin barrier damage, repair of skin stress damage, regeneration of stratum corneum lipids and repair of skin cell mitochondrial damage.

9. A multi-plant complex vesicle product with soothing and repairing effects, characterized in that, The plant vesicle composition with soothing and repairing effects as described in any one of claims 1-4 or the plant vesicle composition with soothing and repairing effects as described in any one of claims 5-7 is prepared by the method thereof.

10. The multi-plant complex vesicle product with soothing and repairing effects according to claim 9, characterized in that, The multi-plant complex vesicle product with soothing and repairing effects is any one of anti-aging serum, firming face cream, wrinkle-reducing eye cream, anti-aging mask or essence lotion. The mass fraction of the plant vesicle composition with soothing and repairing effects in the multi-plant complex vesicle product is 0.1%-3.0%.

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