Anti-aging black rose plant vesicle composition and application thereof

Abstract

The application provides an anti-aging black rose plant vesicle composition and application thereof, and belongs to the technical field of biological medicine preparations. The active composition provided by the application is prepared by compounding ergothioneine and black rose plant vesicles, has an excellent effect of promoting the synthesis of type I collagen in skin fibroblasts, obviously improves the elimination capacity of intracellular active oxygen, and ergothioneine and black rose plant vesicles show a synergistic effect. The active composition provided by the application can be applied to the preparation of skin external products with anti-aging and / or antioxidant effects, and provides an active ingredient selection with double excellent effects of promoting collagen regeneration and resisting oxidative damage for the skin care field.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical formulation technology, and in particular to an anti-aging black rose plant vesicle composition and its application. Background Technology

[0002] Plant-derived active ingredients have wide applications in the cosmetics industry due to their natural properties and diverse biological activities. Rosaceae plants have a long history of cultivation in China, and their flowers, leaves, roots, and fruits contain abundant active substances, attracting attention in both traditional applications and modern research. However, directly extracting active ingredients from plant organs (such as flowers, leaves, roots, and bark) faces challenges such as the availability of raw materials being limited by season, climate, and region. Furthermore, the content of active ingredients varies significantly between different harvest periods, making it difficult to ensure consistency and stability between batches of cosmetic raw materials.

[0003] The application of plant cell cultures and their extracts as active ingredients in cosmetics has attracted attention in recent years. Cultures obtained through plant cell culture technology can be produced on a large scale under controlled conditions, theoretically reducing dependence on natural resources to some extent. However, existing products derived from plant cell cultures still exhibit significant differences in the composition and efficacy of their active ingredients, and the stability of product quality and the reliability of their efficacy still need further improvement.

[0004] Plant-derived vesicles are nanoscale vesicle structures isolated from plant tissues or cells, typically ranging in size from 40 to 200 nm, and possessing a typical lipid bilayer membrane structure. In recent years, plant-derived vesicles have attracted attention in the field of functional skincare products due to their natural origin and structural characteristics. However, existing technologies related to plant-derived vesicles still have some shortcomings in practical applications, and their efficacy needs further improvement.

[0005] Therefore, providing a plant-derived active composition with improved skincare efficacy remains a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] The purpose of this invention is to provide an anti-aging black rose plant vesicle composition, which solves the technical problem that the efficacy of existing plant-derived active ingredients needs to be improved.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides the use of an active composition in the preparation of anti-aging products, the active composition comprising black rose plant vesicles and ergothioneine.

[0009] The present invention also provides the use of an active composition in the preparation of an antioxidant product, said active composition comprising black rose plant vesicles and ergothioneine.

[0010] Preferably, the ergothionein exists in the form of being encapsulated in the vesicles of the black rose plant; Alternatively, the black rose plant vesicles are fragments of black rose plant vesicles, and the ergothioneine in the active composition exists in the form of a physical blend with the fragments of black rose plant vesicles.

[0011] Preferably, the active composition is obtained by a preparation method comprising the following steps: Black rose plant vesicles were mixed with ergothioneine and incubated to obtain an incubation product. Unbound ergothioneine was then removed to obtain the active composition.

[0012] Preferably, the temperature for the mixed incubation is 25–40°C, the incubation time is 1–4 hours, and the mixed incubation is carried out under oscillation conditions, with the oscillation speed being 100–200 rpm. The method for removing unbound ergothionein is as follows: ultrafiltration washing is performed using a 50-300 kDa ultrafiltration tube to remove unbound ergothionein.

[0013] Preferably, the active composition is obtained by a preparation method comprising the following steps: The black rose plant vesicles are physically broken down to obtain black rose plant vesicle fragments; the black rose plant vesicle fragments are mixed with ergothioneine to obtain the active composition.

[0014] Preferably, the physical fragmentation treatment is selected from one or more of high-pressure homogenization, micro-jet homogenization, repeated freeze-thaw cycles, or probe-type ultrasonic fragmentation.

[0015] Preferably, in the active composition, the mass ratio of ergothioneine to black rose plant vesicles is 1:200 to 1:2000.

[0016] Preferably, the black rose plant vesicles are obtained by any one of the following methods: differential centrifugation combined with ultrafiltration, size exclusion chromatography, or ultracentrifugation.

[0017] Preferably, the black rose plant vesicles are prepared by differential centrifugation combined with ultrafiltration, which includes the following steps: S1. Mix the black rose callus with PBS buffer and grind it to obtain a grinding solution; S2. Centrifuge the grinding slurry at 200-400g for 5-15 minutes, and collect the supernatant 1 and the precipitate; S3. After adding the precipitate to PBS buffer and grinding, centrifuge again at 200-400g for 5-15 minutes and collect the supernatant 2. S4. Combine the supernatant 1 and the supernatant 2 to obtain a combined supernatant; S5. Centrifuge the combined supernatant at 3000-5000g for 5-15 minutes and collect the supernatant 3; S6. The supernatant 3 is centrifuged at 8000-12000g for 5-15 minutes to obtain the final supernatant 4. S7. Centrifuge the supernatant 4 at 8000-12000g for 5-15 minutes to collect the final supernatant; S8. The final supernatant is filtered sequentially through a 0.40-0.50 μm filter membrane and a 0.20-0.25 μm filter membrane, and the filtrate is collected. S9. The filtrate is concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 80-120 kDa, and the concentrate is collected to obtain the black rose plant vesicles.

[0018] The beneficial effects of this invention are: The active composition provided by this invention, by combining ergothioneine with black rose plant vesicles, exhibits excellent effects in promoting the synthesis of type I collagen in dermal fibroblasts, while significantly enhancing the scavenging capacity of intracellular reactive oxygen species. The two components demonstrate a synergistic effect in both aspects. The active composition provided by this invention can be used to prepare topical skin products with anti-aging and / or antioxidant effects, providing the skin care field with an active ingredient selection that offers both excellent effects in promoting collagen regeneration and resisting oxidative damage. Attached Figure Description

[0019] Figure 1 Here is an electron microscope image of the black rose plant vesicles from Example 3; Figure 2 This is a particle size distribution diagram of black rose plant vesicles from Example 3; Figure 3 HPLC peak chromatogram of standard; Figure 4 The results are linear fitting of ergothioneine standards. Figure 5 HPLC peak area was determined before and after EV loading; Figure 6 This is the evaluation result of laser Raman confocal imaging on the penetration performance of Comparative Example 2; Figure 7 The results of laser Raman confocal imaging for evaluating the penetration performance are shown in Comparative Example 1. Figure 8The results of the laser Raman confocal imaging evaluation of the penetration performance in Example 9 are as follows; Figure 9 The results are from the ROS inhibition test of the sample. Detailed Implementation

[0020] This invention provides the use of an active composition in the preparation of anti-aging products, the active composition comprising black rose plant vesicles and ergothioneine.

[0021] In this invention, anti-aging products refer to topical skin products that have effects such as delaying skin aging, reducing wrinkles, improving skin firmness, or promoting collagen synthesis. The black rose plant vesicles are nanoscale vesicle structures isolated from black rose plant tissue. Ergothioneine is a naturally occurring sulfur-containing amino acid derivative with antioxidant activity. This invention combines black rose plant vesicles with ergothioneine, achieving a synergistic effect between the components and resulting in excellent anti-aging efficacy.

[0022] The present invention also provides the use of an active composition in the preparation of an antioxidant product, said active composition comprising black rose plant vesicles and ergothioneine.

[0023] In this invention, antioxidant products refer to topical skin products that have the effects of scavenging free radicals, reducing oxidative stress levels, or protecting cells from oxidative damage. The black rose vesicles and ergothioneine in the active composition together give the composition excellent antioxidant effects.

[0024] In this invention, the ergothioneine exists in the form of being encapsulated by the black rose plant vesicles; or, the black rose plant vesicles are fragments of black rose plant vesicles, and the ergothioneine and the fragments of black rose plant vesicles exist in the active composition as a physical blend.

[0025] In this invention, "encapsulation" refers to ergothioneine being encapsulated within the internal chambers of black rose plant vesicles or bound to the membrane structure surface of black rose plant vesicles, thereby establishing a loading relationship between ergothioneine and black rose plant vesicles. "Fragmented material" refers to the mixture formed after physical fragmentation of black rose plant vesicles. After direct physical mixing with ergothioneine, the two exist independently and coexist in the active composition.

[0026] In this invention, the active composition is obtained by a preparation method comprising the following steps: mixing and incubating black rose plant vesicles with ergothioneine to remove unbound ergothioneine, thereby obtaining the active composition.

[0027] In this invention, "mixed incubation" refers to placing black rose plant vesicles and ergothioneine together under suitable conditions for a period of time, allowing ergothioneine to enter the interior of the black rose plant vesicles or bind to their surface. "Removal of unbound ergothioneine" refers to separating free ergothioneine that is not encapsulated or bound by black rose plant vesicles using appropriate methods to obtain a purified encapsulated active composition. The ergothioneine stock solution is prepared by dissolving ergothioneine powder in PBS buffer to prepare a stock solution of appropriate concentration. The concentration of ergothioneine in this stock solution can be adjusted according to actual needs, for example, it can be prepared at concentrations of 1 mg / mL, 2 mg / mL, 5 mg / mL, and 10 mg / mL, preferably at a concentration of 5 mg / mL.

[0028] In this invention, the temperature of the mixed incubation is 25-40°C, the time of the mixed incubation is 1-4 hours, the mixed incubation is carried out under oscillation conditions, and the oscillation speed is 100-200 rpm; the method for removing unbound ergothioneine is: ultrafiltration washing using a 50-300 kDa ultrafiltration tube.

[0029] In this invention, the incubation temperature can be selected within the range of 25°C to 40°C, for example, 25°C, 28°C, 30°C, 32°C, 35°C, 37°C, and 40°C, preferably 30°C to 37°C, and more preferably 37°C. The incubation time can be selected within the range of 1 hour to 4 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, and 4 hours, preferably 1.5 to 3 hours, and more preferably 2 hours. The oscillation speed can be selected within the range of 100 rpm to 200 rpm, for example, 100 rpm, 120 rpm, 150 rpm, 180 rpm, and 200 rpm, preferably 120 to 180 rpm, and more preferably 150 rpm. The molecular weight cutoff of the ultrafiltration tube can be selected in the range of 50 kDa to 300 kDa, such as 50 kDa, 80 kDa, 100 kDa, 150 kDa, 200 kDa, and 300 kDa, preferably 80 to 150 kDa, and more preferably 100 kDa. The specific operation of ultrafiltration washing is as follows: the incubated mixture is added to the ultrafiltration tube and centrifuged under appropriate centrifugal force conditions, so that the unbound free ergothioneine is removed through the filter membrane, while the plant vesicles carrying ergothioneine are retained in the ultrafiltration tube. The washing is repeated 1 to 3 times to ensure that the free ergothioneine is fully removed.

[0030] In this invention, the active composition is obtained by a preparation method comprising the following steps: physically breaking down black rose plant vesicles to obtain black rose plant vesicle fragments; mixing the black rose plant vesicle fragments with ergothioneine to obtain the active composition.

[0031] In this invention, "physical disruption" refers to physically breaking down the membrane structure of black rose plant vesicles to release their internal contents and form fragments. "Mixing" refers to physically blending the fragments of black rose plant vesicles with ergothioneine under appropriate conditions. The two do not form a covalent bond or encapsulation relationship but coexist as independent components in the active composition.

[0032] In this invention, the physical crushing process is selected from one or more of high-pressure homogenization, micro-jet homogenization, repeated freeze-thaw cycles, or probe-type ultrasonic crushing.

[0033] In this invention, high-pressure homogenization refers to passing materials through narrow gaps under high pressure, utilizing high-speed shearing, collision, and cavitation effects to break down the materials. The pressure of high-pressure homogenization can be selected as needed; for example, 1 to 5 cycles can be performed under a pressure of 500–1500 bar, preferably 2 to 4 cycles under a pressure of 800–1200 bar. Microjet homogenization refers to breaking down materials using the impact of high-pressure jets. Repeated freeze-thaw cycles refer to destroying the membrane structure by utilizing the physical forces generated during ice crystal formation and melting through multiple freezing and thawing cycles. The freezing temperature is typically -80°C to -20°C, and the thawing temperature is typically room temperature or 37°C. The number of freeze-thaw cycles can be 1 to 5. Probe-type ultrasonic breakage refers to directly immersing an ultrasonic probe into the sample, using the cavitation effect generated by ultrasound to break down the materials. The ultrasonic power can be 50–300W, and the ultrasonic time can be 1–10 minutes. It can be operated in pulse mode (e.g., on for 2–5 seconds, off for 2–5 seconds).

[0034] In this invention, the mass ratio of ergothioneine to black rose plant vesicles in the active composition is 1:200 to 1:2000.

[0035] In this invention, the mass ratio refers to the ratio of the mass of ergothioneine in the active composition to the mass of the black rose plant vesicle suspension. This ratio can be selected within the range of 1:200 to 1:2000, for example, 1:200, 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900, 1:968, 1:1000, 1:1200, 1:1500, 1:1800, 1:2000, preferably 1:500 to 1:1500, more preferably 1:800 to 1:1200, and even more preferably 1:968. In practice, the ratio of ergothioneine to black rose plant vesicles can be achieved in the following ways: for example, weigh 0.05g of ergothioneine and mix it with 48.4g of black rose plant vesicle suspension to obtain an active composition with a mass ratio of approximately 1:968; or the ergothioneine stock solution and black rose plant vesicle suspension can be mixed in the appropriate proportion to achieve the desired final concentration of ergothioneine in the system.

[0036] In this invention, the black rose plant vesicles are obtained by any one of the following methods: differential centrifugation combined with ultrafiltration, size exclusion chromatography, or ultracentrifugation.

[0037] In this invention, differential centrifugation combined with ultrafiltration refers to the method of removing cell debris and large particulate impurities by gradually increasing centrifugal force, followed by ultrafiltration concentration to obtain plant vesicles. Size exclusion chromatography refers to the method of separating plant vesicles from impurity proteins based on differences in molecular size using a gel medium with specific pore sizes; the gel medium used can be Sepharose CL-2B, Sepharose CL-4B, or Sepharose CL-6B, etc. Ultracentrifugation refers to the method of obtaining plant vesicles by precipitating them under centrifugal forces of 100,000 g or higher.

[0038] In this invention, the black rose plant vesicles are prepared by differential centrifugation combined with ultrafiltration. The differential centrifugation combined with ultrafiltration method includes the following steps: S1, mixing black rose callus tissue with PBS buffer and then grinding to obtain a grinding solution; S2, centrifuging the grinding solution at 200-400g for 5-15 minutes, collecting supernatant 1 and the precipitate; S3, adding the precipitate to PBS buffer and grinding again, then centrifuging at 200-400g for 5-15 minutes, collecting supernatant 2; S4, combining supernatant 1 and supernatant 2 to obtain a combined supernatant; S5, centrifuging the combined supernatant at 3000-5000... S6. Centrifuge the supernatant at 8000-12000g for 5-15 minutes and collect the supernatant 3; S7. Centrifuge the supernatant 3 at 8000-12000g for 5-15 minutes and collect the supernatant 4; S8. Centrifuge the supernatant 4 at 8000-12000g for 5-15 minutes and collect the final supernatant; S9. Filter the final supernatant sequentially through a 0.40-0.50μm filter membrane and a 0.20-0.25μm filter membrane and collect the filtrate; S00. Concentrate the filtrate using an ultrafiltration tube with a molecular weight cutoff of 80-120kDa and collect the concentrate to obtain the black rose plant vesicles.

[0039] In this invention, PBS buffer refers to phosphate buffer solution, a commonly used isotonic buffer system in the art, used to maintain osmotic pressure and pH stability during plant vesicle separation. The grinding refers to the process of breaking down and dispersing black rose callus tissue in the buffer solution using a grinding device. In the centrifugation process, 200–400 g is used for low-speed centrifugation to remove unbroken tissue fragments and larger cell debris; the supernatant 1 and supernatant 2 collected in this step are primary extracts rich in plant vesicles. 3000–5000 g is used for medium-speed centrifugation to further remove subcellular organelles and large vesicles. 8000–12000 g is used for high-speed centrifugation to precipitate and remove microvesicles and larger cell vesicles; this step is repeated 1–3 times to ensure thorough impurity removal. A 0.40–0.50 μm filter membrane is used to remove larger particulate impurities, and a 0.20–0.25 μm filter membrane is used to remove bacteria and fine particles; the combined use of two-stage filtration effectively improves the purity of plant vesicles. The ultrafiltration concentration uses an ultrafiltration tube with a molecular weight cutoff of 80-120 kDa. This cutoff range can effectively retain plant vesicles while removing small molecule impurities. The concentration factor can be 5, 6, 7, 8, 10, 12, or 15 times, preferably 6-10 times, and more preferably 7 times. In the above-mentioned centrifugal force range, the centrifugal force for the first and second centrifugation treatments can be selected from 200g, 250g, 300g, 350g, and 400g, preferably 300g; the centrifugal force for the third centrifugation treatment can be selected from 3000g, 3500g, 4000g, 4500g, and 5000g, preferably 4000g; and the centrifugal force for the fourth centrifugation treatment can be selected from 8000g, 9000g, 10000g, 11000g, and 12000g, preferably 10000g. The centrifugation time can be selected from 5 minutes, 8 minutes, 10 minutes, 12 minutes, and 15 minutes, preferably 10 minutes. The pore size of the first filter membrane can be selected from 0.40 μm, 0.45 μm, and 0.50 μm, preferably 0.45 μm; the pore size of the second filter membrane can be selected from 0.20 μm, 0.22 μm, and 0.25 μm, preferably 0.22 μm. The molecular weight cutoff of the ultrafiltration tube can be selected from 80 kDa, 90 kDa, 100 kDa, 110 kDa, and 120 kDa, preferably 100 kDa.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1: Ergothionein loaded onto black rose plant vesicles prepared by exclusion method The black rose callus tissue was provided by Hangzhou Shiguang Xinya Biotechnology Co., Ltd., and the preparation method can be found in Chinese Patent CN122030263A.

[0042] Sample preparation: Weigh 15g of black rose callus tissue sample, add PBS solution and grind, then transfer the grinding solution to a 50 mL centrifuge tube. Centrifuge at 300g for 10 min, and transfer the supernatant to another clean centrifuge tube; add PBS solution to the precipitate again, grind, and centrifuge at 300g for 10 min again, collect the supernatant, and combine the two supernatants.

[0043] Sample pretreatment: Transfer the combined supernatant to a new centrifuge tube and centrifuge at 4000g, 4℃ for 10 min to remove cell debris. Transfer the supernatant to a new centrifuge tube and centrifuge at 10000g, 4℃ for 10 min. After centrifugation, transfer the supernatant to a new centrifuge tube and repeat this step once (10000g, 4℃, 10 min) until no obvious precipitation occurs. Vacuum filter the obtained supernatant through a 0.45 μm filter membrane and a 0.22 μm filter membrane sequentially to obtain approximately 150 mL of clear filtrate.

[0044] Purification of Black Rose Plant Vesicles Take 35 mL of the above filtrate and separate plant vesicles using the following method: Exclusion method: Take 35 mL of the above filtrate and use the ES8500 concentration kit (Echobiotech, China, Beijing Enzekangtai Biotechnology Co., Ltd., catalog number: ES8500) to add 10 mL of solution A and 5 mL of solution B to the 35 mL sample. Mix well and incubate overnight at 4℃. The next day, centrifuge at 10000g, 4℃ for 60 min, discard the supernatant, resuspend the plant vesicles in 1 mL PBS, and centrifuge multiple times at 12000g, 4℃ (2 min each time) to collect the supernatant. According to the purification kit instructions, load 1 mL of the supernatant onto the sample and collect fractions 4-8, obtaining a total of 2.5 mL of solution. Finally, concentrate 12.5 times to 0.2 mL using a 100 kDa ultrafiltration tube to obtain the black rose plant vesicle solution.

[0045] Take 0.5 mL of black rose plant vesicle solution, add 0.1 mL of PBS buffer, and then add ergothioneine stock solution to bring the final concentration of ergothioneine in the system to 5 mg / mL. Mix well and operate in the dark throughout the process. Incubate the mixture in a 37°C constant temperature shaking incubator for 2 h at a shaking speed of 150 rpm. After incubation, wash twice with a 100 kDa ultrafiltration tube at 4000 g to remove unbound free ergothioneine, and concentrate to approximately 0.1 mL to obtain ergothioneine-loaded black rose plant vesicles.

[0046] Example 2: Ergothionein loaded onto black rose plant vesicles prepared by ultrafiltration method The only difference from Example 1 is that the following method was used for plant vesicle isolation (all other steps are the same): Ultrafiltration: Following the instructions of the concentration kit (ES8500), add solution A and solution B (in the same proportions as in Method 1) to 35 mL of filtrate, mix well, and incubate overnight at 4°C. The next day, centrifuge at 10000g, 4°C for 60 min, discard the supernatant, resuspend the plant vesicles in 1 mL of PBS, and centrifuge multiple times at 12000g, 4°C (2 min each time) to collect the supernatant. Divide the sample into two ultrafiltration tubes, add PBS to a final volume of 1.2 mL, centrifuge at 100000g, 4°C for 60 min, discard the supernatant, and resuspend the precipitate in an appropriate amount of PBS. Combine the two sample tubes into one ultrafiltration tube, add PBS to a final volume of 1.2 mL, centrifuge again at 100000g, 4°C for 60 min, discard the supernatant, and resuspend in PBS to a final volume of 0.2 mL to obtain black rose plant vesicles.

[0047] Example 3: Ergothionein loaded onto black rose plant vesicles prepared by gradient filtration method The only difference from Example 1 is that the following method was used for plant vesicle isolation (all other steps are the same): Gradient filtration method: Take 35 mL of the filtrate after filtration through a 0.22 μm filter membrane, concentrate it 7 times to 5 mL using a 100 kDa ultrafiltration tube to obtain black rose plant vesicles.

[0048] In this embodiment, the particle size distribution of plant vesicles obtained by the three methods in Examples 1, 2, and 3 was determined using a nanoparticle tracking analyzer (NTA). Protein content was determined using a BCA protein quantification kit to assess purity, and the morphology of the plant vesicles was observed using transmission electron microscopy (TEM). The results are shown in Table 1. Table 1. Results of plant vesicle detection obtained from the three examples.

[0049] As shown in Table 1, all three methods can obtain plant vesicles with typical vesicle structures, but the gradient filtration method is the simplest and fastest to operate and can obtain plant vesicles with a higher particle concentration.

[0050] Morphological observation of black rose plant vesicles prepared by gradient filtration method was performed using transmission electron microscopy, and the results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the obtained plant vesicles have a typical cup-shaped vesicle structure. The particle size of the black rose plant vesicles prepared by the gradient filtration method was determined using a nanoparticle tracking analyzer, and the results are as follows: Figure 2 As shown. By Figure 2 It can be seen that the particle size of the obtained plant vesicles is mainly distributed in the range of 40 to 140 nm.

[0051] Example 4 The difference from Example 3 is as follows: 0.5 mL of black rose plant vesicle solution was taken, 0.1 mL of PBS buffer was added, and methanol was added to make the volume fraction of the system 5%. Then, ergothioneine stock solution was added to bring the final concentration of ergothioneine in the system to 5 mg / mL. The mixture was thoroughly mixed and operated in the dark throughout the process. The mixture was incubated in a 37°C constant temperature shaking incubator for 2 h at a shaking speed of 150 rpm. After incubation, the mixture was washed twice with a 100 kDa ultrafiltration tube at 4000 g to remove unbound free ergothioneine, and then concentrated to approximately 0.1 mL to obtain the ergothioneine-loaded plant vesicle sample.

[0052] Example 5 The difference from Example 3 is as follows: 0.5 mL of black rose plant vesicle solution was taken, 0.1 mL of PBS buffer was added, followed by ergothioneine stock solution to bring the final concentration of ergothioneine in the system to 1 mg / mL. The mixture was thoroughly mixed and operated in the dark throughout the process. The mixture was incubated in a 37°C constant temperature shaking incubator for 2 h at a shaking speed of 150 rpm. After incubation, the mixture was washed twice with a 100 kDa ultrafiltration tube at 4000 g to remove unbound free ergothioneine, and then concentrated to approximately 0.1 mL to obtain the ergothioneine-loaded plant vesicle sample.

[0053] Example 6 The difference from Example 3 is as follows: 0.5 mL of black rose plant vesicle solution was taken, 0.1 mL of PBS buffer was added, followed by ergothioneine stock solution to bring the final concentration of ergothioneine in the system to 5 mg / mL. The mixture was thoroughly mixed and operated in the dark throughout the process. The mixture was incubated in a 37°C constant temperature shaking incubator for 2 h at a shaking speed of 150 rpm. During incubation, ultrasonic treatment was performed (ultrasonic power 100 W, running in a 3 s on / 3 s off pulse mode, for a total of 10 cycles). After incubation, the sample was washed twice with a 100 kDa ultrafiltration tube at 4000 g to remove unbound free ergothioneine and concentrated to approximately 0.1 mL to obtain the ergothioneine-loaded plant vesicle sample.

[0054] Example 7 The difference from Example 3 is as follows: 0.5 mL of black rose plant vesicle solution was taken, 0.1 mL of PBS buffer was added, and methanol was added to make the volume fraction of the system 5%. Then, ergothioneine stock solution was added to make the final concentration of ergothioneine in the system 5 mg / mL. After mixing, the entire process was carried out in the dark. The mixture was incubated in a 37°C constant temperature shaking incubator for 2 h at a shaking speed of 150 rpm. During incubation, ultrasonic treatment was performed (ultrasonic power 100 W, running in a 3 s on / 3 s off pulse mode, for a total of 10 cycles). After incubation, the sample was washed twice with a 100 kDa ultrafiltration tube at 4000 g to remove unbound free ergothioneine, and then concentrated to approximately 0.1 mL to obtain the ergothioneine-loaded plant vesicle sample.

[0055] Example 8 The difference from Example 3 is as follows: 0.5 mL of black rose plant vesicle solution was taken, 0.1 mL of PBS buffer was added, followed by ergothioneine stock solution to bring the final concentration of ergothioneine in the system to 1 mg / mL. The mixture was thoroughly mixed and operated in the dark throughout the process. The mixture was incubated in a 37°C constant temperature shaking incubator for 2 h at a shaking speed of 150 rpm. During incubation, ultrasonic treatment was performed (ultrasonic power 100 W, running in a 3 s on / 3 s off pulse mode, for a total of 10 cycles). After incubation, the sample was washed twice with a 100 kDa ultrafiltration tube at 4000 g to remove unbound free ergothioneine and concentrated to approximately 0.1 mL to obtain the ergothioneine-loaded plant vesicle sample.

[0056] In this embodiment, the ergothionein content in the samples provided in Examples 3-8 was determined by high performance liquid chromatography (HPLC), and the concentration of plant vesicle proteins was determined by the BCA method to evaluate the ergothionein loading level in plant vesicles, as shown in Tables 2-3 below: Table 2. Differences in preparation conditions among the examples

[0057] Table 3. Detection results of reaction products under different loading conditions

[0058] Comparing the loading results and preparation conditions of various embodiments, Example 3 achieved a good loading effect with the simplest process conditions (a loading of 392 μg / mL could be obtained simply through incubation and ultrafiltration washing, without the addition of organic solvents or ultrasonic treatment), indicating that this loading scheme achieved the best balance between ease of operation and loading efficiency. Therefore, the preparation method of Example 3 was determined to be the preferred embodiment of ergothioneine loading on black rose plant vesicles in this invention.

[0059] Ergothioneine standards were detected using high performance liquid chromatography (HPLC). The chromatographic conditions were as follows: a C18 reversed-phase column (250 mm × 4.6 mm, 5 μm) was used, with 0.1 M borate buffer (pH 5.0) as mobile phase A and methanol as mobile phase B, and isocratic elution was performed at a mobile phase A: mobile phase B = 95: 5 (volume ratio); the flow rate was 0.7 mL / min; the column temperature was maintained at 40 ℃ (±2 ℃); the UV detection wavelength was 254 nm; the injection volume was 10 μL; and the run time for each sample was 10 min.

[0060] Chromatogram as shown Figure 3 As shown. A linear fit was performed with the concentration of ergothioneine standard as the x-axis and the peak area as the y-axis. The results are as follows. Figure 4 As shown, the standard curve exhibits good linearity. Further HPLC analysis was performed on plant vesicle samples (Sample 3) before and after loading, and the results are as follows... Figure 5 As shown. By Figure 5 It can be seen that the characteristic absorption peak of ergothionein appeared in the plant vesicle sample after loading, while the characteristic peak did not appear in the plant vesicle sample before loading, indicating that ergothionein has been successfully loaded into black rose plant vesicles.

[0061] Example 9: Scale-up preparation of ergothioneine loaded onto black rose plant vesicles This embodiment provides a scaled-up preparation method for loading ergothionein onto black rose plant vesicles.

[0062] Take 40 mL of the black rose plant vesicle solution prepared in Example 3, add 10 mL of PBS buffer, and then add ergothioneine stock solution to bring the final concentration of ergothioneine in the system to 5 mg / mL. Mix well and operate in the dark throughout the process. Incubate the mixture in a 37°C constant temperature shaking incubator for 2 h at a shaking speed of 150 rpm. After incubation, wash twice with a 100 kDa ultrafiltration tube at 4000 g to remove unbound free ergothioneine, and concentrate to about 10 mL to obtain the plant vesicle sample loaded with ergothioneine.

[0063] After incubation, HPLC was used to analyze the samples before and after loading. The results are shown in Table 4 below: Table 4 Detection of Scale-up Process Results

[0064] As shown in Table 4, the scale-up process provided in this embodiment successfully prepared black rose plant vesicles loaded with ergothioneine, with an ergothioneine loading of 600.59 μg / mL. Moreover, the particle concentration and protein concentration after loading were significantly higher than those of unloaded plant vesicles, indicating that the scale-up process has good feasibility and loading efficiency.

[0065] Example 10: Black rose plant vesicles and ergothioneine crushed compound group Take 0.05g of ergothioneine, 48.4g of black rose plant vesicles (total solution weight, protein concentration 1630μg / mL), 2g of pentylene glycol, 1.2g of hexanediol, and 48.35g of water, mix them, and then sonicate for 3 minutes (ultrasonic conditions as above, ultrasonic power 100 W) to obtain the solution.

[0066] Comparative Example 1: Physical mixture of black rose plant vesicles and ergothionein Take 10 mL of the black rose plant vesicle solution prepared in Example 1, add ergothioneine stock solution to it to make the final concentration of ergothioneine in the system 600 μg / mL, mix well, and obtain a physical mixture system of ergothioneine and plant vesicles.

[0067] Comparative Example 2: Ergothioneine monotherapy group Ergothioneine was dissolved in purified water and prepared into an aqueous solution with a mass concentration of 600 μg / mL.

[0068] Experimental Example 1: Permeability Testing Based on Laser Raman Confocal Method Test method: Referring to GB / T 33215-2021 "General Specification for Raman Spectrometer", the transdermal permeability of the samples was evaluated using laser Raman confocal spectroscopy.

[0069] 1) Test Sample Samples prepared in Example 9, Comparative Example 1, and Comparative Example 2. The final concentration of ergothioneine in each group of samples remained consistent.

[0070] 2) Test objects and areas One healthy adult subject was selected, and the test site was the anterior forearm region. Three test areas were set up, each with an area of ​​1 cm × 1 cm, corresponding to the three sets of test samples mentioned above. Five detection points were selected within each test area, and each detection point was tested three times in parallel.

[0071] 3) Testing Process Before the test, the subjects cleaned the test area with water. After cleansing, they sat quietly in a temperature and humidity controlled room (temperature 22±2℃, relative humidity 50±5%) for 30 minutes. Then, an equal amount of test sample was evenly applied to the corresponding test area. Raman spectroscopy was performed at 0.5 h, 1 h, 2 h, 4 h, and 6 h after application to record the characteristic Raman signal of ergothioneine.

[0072] 4) Data Analysis Labspec software was used to perform baseline calibration and confirm the positions of characteristic peaks in the Raman spectra, and to calculate parameters such as peak intensity, peak shift, peak area, and full width at half maximum (FWHM). Simultaneously, Labspec software was used to perform numerical analysis of peak intensities corresponding to different skin depths and to plot their spatial distribution.

[0073] The permeability behavior was confirmed by the characteristic Raman signal of the sample, which distinguishes it from the intrinsic signal of the skin, and its distribution characteristics in different skin depths were analyzed.

[0074] Relative permeability is calculated using the following formula: Relative penetration rate (%) = (Raman spectral intensity of active ingredient penetrating the skin / Raman spectral intensity of active ingredient applied to the skin) × 100% ORIGIN 2017 software was used for statistical analysis. The Shapiro-Wilk test was used to determine the normality of the data. When comparing data from different periods, if the data is normally distributed, a paired t-test was used; otherwise, a rank-sum test of two related samples was used.

[0075] Test results: 1) Relative penetration rate The relative permeability test results of different samples on human skin are shown in Table 5.

[0076] Table 5. Relative permeability test results of different samples at various time points.

[0077] 2) Maximum penetration depth The results of the maximum penetration depth test of different samples on human skin are shown in Table 6.

[0078] Table 6. Maximum penetration depth (μm) of different samples at various time points

[0079] The test results in Tables 1 and 2 show that: Regarding relative permeability, all three groups of samples showed a time-dependent increasing trend. Among them, the relative permeability of Example 9 was significantly higher than that of Comparative Example 1 and Comparative Example 2 at all time points. Specifically, at 6 h, the relative permeability of Example 9 reached 11.94%, which was 1.38 times that of Comparative Example 2 (8.63%) and 1.75 times that of Comparative Example 1 (6.81%).

[0080] Regarding maximum penetration depth, Example 9 exhibited the best deep penetration capability. After 6 hours of application, the ergothioneine in Example 9 could penetrate to a depth of 120 μm, while the maximum penetration depth of Comparative Example 1 was 100 μm, and that of Comparative Example 2 was only 60 μm. Furthermore, Example 9 achieved a penetration depth of 110 μm in 2 hours, while Comparative Example 1 only reached 90 μm in 4 hours, and Comparative Example 2 only reached 60 μm in 4 hours, indicating that encapsulation with the black rose plant vesicles provided by this invention can significantly accelerate the transdermal transdermal rate of ergothioneine.

[0081] Compared to free ergothioneine and its physical mixture with plant vesicles, the black rose plant vesicle encapsulation group exhibits higher relative permeability and deeper penetration depth within the same time frame. This result confirms that the black rose plant vesicles provided by this invention can effectively promote the penetration of ergothioneine through the skin's stratum corneum barrier, achieving effective delivery of the active ingredient deep within the skin.

[0082] The laser Raman confocal permeation performance evaluation results of Comparative Example 2 (free ergothioneine aqueous solution) are as follows: Figure 6 As shown. The laser Raman confocal permeation performance evaluation results of Comparative Example 1 (a physical mixture of ergothioneine and plant vesicles) are as follows. Figure 7 As shown. The laser Raman confocal permeation performance evaluation results of Example 9 (black rose plant vesicles loaded with ergothioneine) are as follows. Figure 8 As shown.

[0083] contrast Figures 6-8 It can be seen that the attenuation of the ergothioneine Raman signal with skin depth in Example 9 was significantly lower than that in Comparative Examples 1 and 2, indicating that ergothioneine in Example 9 can more effectively penetrate the stratum corneum barrier and enter the deeper layers of the skin. Based on the above results and analysis, black rose plant vesicles can effectively promote the transdermal penetration of ergothioneine.

[0084] Experimental Example 2 This experiment evaluates the promoting effect of each test sample on the synthesis of type I collagen in human skin fibroblasts through in vitro cell experiments.

[0085] Human skin fibroblasts were used as the experimental subjects, and blank control group, positive control group and test sample group were set up respectively.

[0086] The blank control group received no treatment, the positive control group received TGF-β at a final concentration of 10 ng / mL, and the test sample groups received 5% ergothioneine sample (sample number GZ260629066-00001), 5% black rose plant vesicle sample (sample number GZ260629066-00002), 5% of the Example 9 loaded group sample (ergothioneine + black rose plant vesicle, sample number GZ260629066-00003), and 5% of the Example 10 broken compound group sample (ergothioneine + black rose plant vesicle fragments, sample number GZ260629066-00004).

[0087] After the cells in each group were treated accordingly, the expression level of type I collagen was detected, and the average increase rate of type I collagen in each test sample group relative to the blank control group was calculated. The detection results are shown in Table 7.

[0088] Table 7. The promoting effect of each group of samples on type I collagen synthesis

[0089] Note: p<0.01, ns indicates no significant difference. Compared with the blank control group, the TGF-β positive control group showed an average increase of 35.32% in type I collagen, which was highly significant (p<0.01), indicating that the experimental system was effective.

[0090] Under the condition of 5% sample addition, the average increase rate of type I collagen in each test sample was as follows: ergothioneine group 1.44%, black rose plant vesicle group 6.87%, ergothioneine + black rose plant vesicle group 11.77%, and ergothioneine + black rose plant vesicle (broken) group 25.00%. Among them, the ergothioneine + black rose plant vesicle (broken) group had a highly significant difference compared with the blank control group (p<0.01), while there were no significant differences between the other test sample groups and the blank control group.

[0091] The results showed that, under the experimental concentration conditions, both black rose plant vesicles, used alone and in combination with ergothioneine, exhibited a trend of promoting type I collagen synthesis. The effect of the combination of ergothioneine and black rose plant vesicles was superior to the sum of the effects of each component used alone, suggesting a synergistic effect in promoting collagen synthesis. The combination of fragmented black rose plant vesicles and ergothioneine showed the best effect, with an average increase in type I collagen (25.00%) approaching 70.8% of the positive control group.

[0092] Experiment Example 3: Cellular Reactive Oxygen Species (ROS) Inhibition Assay This experiment evaluates the ability of each test sample to scavenge intracellular reactive oxygen species through in vitro cell experiments.

[0093] Using human skin fibroblasts as the experimental subject, the intracellular reactive oxygen species (ROS) level was detected by a reactive oxygen species fluorescent probe labeling method.

[0094] The experiment included a negative control group (NC), a positive control group, and a test sample group. The negative control group received an equal volume of solvent control, the positive control group received Trolox at a final concentration of 0.0025%, and the test sample groups received 5% of the following: ergothioneine sample (sample number GZ260629066-00001), 5% of black rose plant vesicle sample (sample number GZ260629066-00002), 5% of the Example 9 loaded sample (ergothioneine + black rose plant vesicles, sample number GZ260629066-00003), and 5% of the Example 10 broken compound sample (ergothioneine + black rose plant vesicle fragments, sample number GZ260629066-00004).

[0095] After appropriate treatment, the fluorescence intensity of each group of cells was detected using a fluorescence microplate reader. The reactive oxygen species (ROS) level of the negative control group was used as a benchmark to calculate the ROS inhibition rate of each test sample. The detection results are as follows: Figure 9 As shown.

[0096] Depend on Figure 9 It can be seen that the ROS inhibition rate of Trolox in the positive control group was approximately 91%, indicating that the experimental system was effective. Under the condition of 5% sample addition, the ROS inhibition rates of each test sample were as follows: ergothionein group approximately 93%, black rose plant vesicle group approximately 94%, Example 9 loaded group approximately 93%, and Example 10 broken compound group approximately 92%.

[0097] The results showed that all tested samples exhibited significant reactive oxygen species (ROS) scavenging capabilities. Specifically, the ROS inhibition rate of the combination of broken black rose plant vesicles and ergothioneine (approximately 92%) was essentially comparable to that of the intact vesicle loading group (approximately 93%). These results indicate that the active composition provided by this invention can effectively reduce cellular oxidative stress levels, protect cells from oxidative damage, and possesses promising prospects for antioxidant applications.

[0098] As shown in the above embodiments, the present invention provides an active composition comprising black rose plant vesicles and ergothioneine. Cellular experiments demonstrate that this active composition can promote the synthesis of type I collagen in dermal fibroblasts while simultaneously reducing intracellular reactive oxygen species (ROS) levels. Transdermal penetration experiments show that black rose plant vesicles can carry ergothioneine through the stratum corneum barrier into the deeper layers of the skin. In vitro release experiments indicate that black rose plant vesicles have a sustained-release effect on ergothioneine. In summary, the active composition provided by the present invention possesses the dual effects of promoting collagen synthesis and scavenging ROS, and black rose plant vesicles can effectively deliver ergothioneine into the deeper layers of the skin. This composition shows promising application prospects in the field of anti-aging and antioxidant skin care.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of an active composition in the preparation of anti-aging products, characterized in that, The active composition contains black rose plant vesicles and ergothioneine.

2. The application of an active composition in the preparation of antioxidant products, characterized in that, The active composition contains black rose plant vesicles and ergothioneine.

3. The application according to claim 1 or 2, characterized in that, The ergothionein exists in the form of being encapsulated in the vesicles of the black rose plant; Alternatively, the black rose plant vesicles are fragments of black rose plant vesicles, and the ergothioneine in the active composition exists in the form of a physical blend with the fragments of black rose plant vesicles.

4. The application according to claim 1 or 2, characterized in that, The active composition is obtained by a preparation method comprising the following steps: Black rose plant vesicles were mixed with ergothioneine and incubated to obtain an incubation product. Unbound ergothioneine was then removed to obtain the active composition.

5. The application according to claim 4, characterized in that, The temperature for the mixed incubation is 25–40°C, the incubation time is 1–4 hours, and the incubation is carried out under oscillation conditions at a rotation speed of 100–200 rpm. The method for removing unbound ergothionein is as follows: ultrafiltration washing is performed using a 50-300 kDa ultrafiltration tube to remove unbound ergothionein.

6. The application according to claim 1 or 2, characterized in that, The active composition is obtained by a preparation method comprising the following steps: The black rose plant vesicles are physically broken down to obtain black rose plant vesicle fragments; the black rose plant vesicle fragments are mixed with ergothioneine to obtain the active composition.

7. The application according to claim 6, characterized in that, The physical fragmentation process is selected from one or more of the following: high-pressure homogenization, micro-jet homogenization, repeated freeze-thaw cycles, or probe-type ultrasonic fragmentation.

8. The application according to claim 1 or 2, characterized in that, In the active composition, the mass ratio of ergothioneine to black rose plant vesicles is 1:200 to 1:2000.

9. The application according to claim 1 or 2, characterized in that, The black rose plant vesicles were obtained by any one of the following methods: differential centrifugation combined with ultrafiltration, size exclusion chromatography, or ultracentrifugation.

10. The application according to claim 9, characterized in that, The black rose plant vesicles were prepared by differential centrifugation combined with ultrafiltration, which included the following steps: S1. Mix the black rose callus with PBS buffer and grind it to obtain a grinding solution; S2. Centrifuge the grinding slurry at 200-400g for 5-15 minutes, and collect the supernatant 1 and the precipitate; S3. After adding the precipitate to PBS buffer and grinding, centrifuge again at 200-400g for 5-15 minutes and collect the supernatant 2. S4. Combine the supernatant 1 and the supernatant 2 to obtain a combined supernatant; S5. Centrifuge the combined supernatant at 3000-5000g for 5-15 minutes and collect the supernatant 3; S6. The supernatant 3 is centrifuged at 8000-12000g for 5-15 minutes to collect the supernatant 4. S7. The supernatant 4 is centrifuged at 8000-12000g for 5-15 minutes to collect the final supernatant. S8. The final supernatant is filtered sequentially through a 0.40-0.50 μm filter membrane and a 0.20-0.25 μm filter membrane, and the filtrate is collected. S9. The filtrate is concentrated by ultrafiltration using an ultrafiltration tube with a molecular weight cutoff of 80-120 kDa, and the concentrate is collected to obtain the black rose plant vesicles.

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