Exosome-like nanovesicles from roxburgh rose for relieving inflammation of sunburned skin and preparation method and application thereof

Abstract

The present application belongs to the technical field of natural product extraction and cosmetic raw material preparation, and particularly relates to a roxburgh rose exosome-like nanovesicle for relieving sunburn skin inflammation and a preparation method and application thereof. In the present application, the roxburgh rose exosome-like nanovesicle is extracted by a method combining differential centrifugation and subcritical extraction technology. The content of micRNA and other active ingredients in the obtained roxburgh rose exosome-like nanovesicle can be up to 322.12 ng / μL. Even after 28 days of storage, the loss rate of active ingredients in the nanovesicle is only 2%-3%. The roxburgh rose exosome-like nanovesicle provided in the present application exhibits excellent DPPH free radical scavenging and IL-6 inflammatory factor inhibition effects. In addition, the freeze-dried powder thereof has the effect of inhibiting degranulation release when used in RBL-2H3 cells induced by IgE, and thus can be used in skin care products to better inhibit skin inflammation caused by UVB irradiation.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction and cosmetic raw material preparation technology, specifically relating to a roselle exosome-like nanovesicle for relieving sunburn skin inflammation, its preparation method and application. Background Technology

[0002] Sunburn is an acute inflammatory reaction of the skin caused by excessive exposure to ultraviolet (UV) radiation, similar to a superficial burn. UV radiation causes the skin tissue to produce a large number of reactive oxygen species (ROS). These ROS accumulate in the skin and continuously damage it, even triggering an inflammatory response, resulting in symptoms such as redness, heat, intense itching, and pain. In some cases, it may even induce cancer.

[0003] Roselle, also known as hibiscus flower, is rich in active ingredients such as organic acids, anthocyanins, polyphenols, polysaccharides, and flavonoids. Modern research shows that roselle extract has antioxidant, anticancer, and blood sugar-lowering effects, and is widely used in food, cosmetics, and other fields.

[0004] However, the preparation process of traditional plant extracts such as hibiscus, such as high-temperature extraction and organic solvent treatment, can easily lead to the degradation or inactivation of heat-sensitive and easily oxidized active ingredients such as polyphenols and enzymes in the plant raw materials. Plant exosomes, with their natural lipid bilayer structure, provide an internal protective chamber for active ingredients, which can fundamentally solve the problem of loss of bioactive ingredients and have become a research hotspot in recent years.

[0005] Plant-derived exosome-like nanovesicles, rich in specific active ingredients such as lipids, proteins, and RNA, can exert more precise effects in anti-inflammation by inhibiting the release of pro-inflammatory factors. In terms of anti-oxidation, they can promote skin metabolism, help repair damaged cells, and promote skin repair and healing. Plant exosome-like nanovesicles can also target specific cells or tissues, allowing for precise action of active ingredients. They exhibit good biocompatibility and high safety; therefore, plant-derived exosome-like nanovesicles have demonstrated application potential far exceeding that of traditional extracts.

[0006] However, when extracting plant exosome-like nanovesicles, commonly used methods such as centrifugation, filtration, and precipitation are difficult to separate accurately, and impurities or exosomes are easily left behind, affecting their biological activity.

[0007] Therefore, it is necessary to develop an extraction and enrichment process suitable for exosome-like nanovesicles to improve the utilization rate of plant raw materials, increase the content of active ingredients in plant exosome-like nanovesicles, and slow down the degradation rate. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a roselle exosome-like nanovesicle for relieving sunburn skin inflammation, along with its preparation method and application.

[0009] The first aspect of the present invention is to provide a method for preparing hibiscus exosome-like nanovesicles, characterized by comprising the following steps: Step S1: Pulverize hibiscus flowers, add water and mix well. Centrifuge at 4℃ with centrifugal forces of 1000~1500×g, 4000~5500×g and 7000~10500×g for 10~35 min respectively. Discard the precipitate and obtain the separation supernatant. Filter through a 0.45 μm filter membrane to obtain hibiscus exosome-like nanovesicle pretreatment solution. Step S2: The hibiscus exosome-like nanovesicle pretreatment solution obtained in step S1 is treated by subcritical extraction to obtain a subcritically extracted hibiscus exosome-like nanovesicle extract. The subcritical extraction conditions are: extraction pressure 10~25 MPa, extraction temperature 30-65℃, extraction time 0.5~4 h, using propane or butane in a subcritical state as solvent, and the volume ratio of the hibiscus exosome-like nanovesicle pretreatment solution to the solvent is 1:1-10 during extraction. Step S3: The extract of roselle exosome-like nanovesicles obtained in step S2 is treated with activated carbon to obtain the roselle exosome-like nanovesicles.

[0010] In the above preparation method, preferably, in step S1, the mass-to-volume ratio of roselle powder to water is 1 g: 15~20 mL, the mixing time is 2~4 h, and the mixing temperature is 10~15℃.

[0011] Preferably, in step S1, the centrifugation is first performed at a centrifugal force of 1000~1500×g for 10~15 min, then at a centrifugal force of 4000~5500×g for 10~25 min, and finally at a centrifugal force of 7000~10500×g for 25~35 min.

[0012] A second aspect of the present invention is to provide a hibiscus exosome-like nanovesicle for relieving sunburn, wherein the nanovesicle is prepared by the method described above.

[0013] Preferably, the hibiscus exosome-like nanovesicles for relieving sunburn are prepared by subcritical extraction with an extraction pressure of 20-25 MPa, an extraction temperature of 30-60°C, an extraction time of 3-8 h, and propane in a subcritical state as the extraction solvent.

[0014] As a further preferred embodiment, the hibiscus exosome-like nanovesicles for relieving sunburn are prepared by subcritical extraction at an extraction pressure of 20 MPa, an extraction temperature of 45°C, and an extraction time of 4 h.

[0015] As a further preferred embodiment, the volume ratio of the hibiscus exosome-like nanovesicle pretreatment solution to the solvent is 1:4-6; In this invention, as the solvent ratio increases from 1:1 to 1:10, the amount of solvent added relative to the treatment solution increases, the extraction yield gradually increases, and the extraction speed accelerates (due to a larger concentration gradient). However, the concentration of effective components and impurities in the extract decreases accordingly, and the subsequent concentration cost increases. The final choice of 1:5 as the middle value in this range achieves a good balance between yield, extraction efficiency, extract concentration, and the associated costs of subsequent purification and concentration. This avoids the problems of insufficient yield and incomplete extraction at low volume ratios, and also avoids the significant increase in energy consumption and cost due to excessive solvent at high volume ratios.

[0016] A third aspect of the present invention is to provide a hibiscus exosome-like nanovesicle freeze-dried powder for relieving skin inflammation, wherein the freeze-dried powder is prepared by adding 8% to 10% by volume of a freeze-drying protectant to the above-mentioned hibiscus exosome-like nanovesicles and then performing vacuum freeze-drying. The freeze-drying protectant is selected from at least one of sorbitol, trehalose, and bovine serum albumin.

[0017] Preferably, the freeze-drying protectant is sorbitol.

[0018] Preferably, the freeze-drying temperature during vacuum freeze-drying is 4~10℃, and the freeze-drying time is 12~36 h.

[0019] As a further preferred embodiment, the freeze-drying temperature during vacuum freeze-drying is 6°C, and the freeze-drying time is 24 h.

[0020] A fourth aspect of the present invention is to provide the application of the above-mentioned hibiscus exosome-like nanovesicle freeze-dried powder, specifically in the preparation of skin care products or pharmaceuticals, wherein the skin care products or pharmaceuticals have the effect of relieving skin inflammation caused by ultraviolet light irradiation.

[0021] The beneficial effects of this invention are as follows: (1) Roselle exosome-like nanovesicles were extracted by differential centrifugation and enriched by subcritical extraction. After combining differential centrifugation and subcritical extraction, the content of active ingredients such as micRNA in the obtained roselle exosome-like nanovesicles reached up to 322.12 ng / μL. After traditional differential centrifugation, the content of micRNA in the nanovesicles was only 189.65 ng / μL. In addition, the nanovesicles prepared by this invention have excellent stability. After 28 days of storage, the loss rate of active ingredients in the nanovesicles was only 2%-3%. (2) The roselle exosome-like nanovesicle powder provided by the present invention exhibits superior DPPH free radical scavenging and IL-6 inflammatory factor inhibition effects compared with the freeze-dried powder obtained by traditional differential centrifugation. Furthermore, cell experiment data show that the freeze-dried powder has the effect of inhibiting degranulation and release when used in RBL-2H3 cells induced by IgE, and can therefore be used in skin care products to better inhibit skin inflammation caused by UVB irradiation. Attached Figure Description

[0022] Figure 1 This is a transmission electron microscope image of the morphological characteristics of roselle exosome-like nanovesicles prepared in Example 5 of the present invention. Figure 2 This is a particle size analysis result of the hibiscus exosome-like nanovesicles extracted in Example 5 of the present invention. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0024] Example 1 This embodiment focuses on investigating the effect of extraction pressure in subcritical extraction on the preparation of nanovesicles. In this embodiment, roselle exosome-like nanovesicles were prepared using the following method: Step S1: After pulverizing the roselle flowers using a hammer mill, water was added at a mass-to-volume ratio of 1g:15mL. The mixture was stirred and mixed at 25℃ for 4 h. Then, it was centrifuged three times at 4℃. First, it was centrifuged at 1200×g for 12 min, and the precipitate was discarded to obtain the first separation supernatant. Then, the first separation supernatant was centrifuged at 4500×g for 20 min, and the precipitate was discarded to obtain the second separation supernatant. Finally, the second separation supernatant was centrifuged at 7800×g for 30 min, and the precipitate was discarded to obtain the third separation supernatant. The third separation supernatant was filtered through a 0.45μm filter membrane to obtain the roselle exosome-like nanovesicle pretreatment solution. Step S2: The pretreated solution of roselle exosome-like nanovesicles obtained in step S1 was treated by subcritical extraction. Propane in a subcritical state was used as the extractant. The volume ratio of the pretreated solution to the solvent was 1:5. The extraction was carried out at 10-30 MPa and 45℃ for 4 h to obtain the extract of roselle exosome-like nanovesicles. Step S3: The extract of roselle exosome-like nanovesicles obtained in step S2 is adsorbed with activated carbon and finely filtered with a 0.22 μm microporous membrane to remove pigments, thereby obtaining roselle exosome-like nanovesicles.

[0025] In this embodiment, the effect of subcritical extraction pressure on the concentration of micRNA in the prepared nanovesicles is shown in Table 1.

[0026] Table 1. Effect of subcritical extraction pressure on micRNA concentration (ng / μL) in nanovesicles and product storage stability. 0d 156.28 258.41 322.05 276.39 189.57 28d 140.35 249.63 313.48 268.51 171.24 loss rate 10.19% 3.40% 2.66% 2.85% 9.70%

[0027] Table 1 shows that when the extraction pressure is too low, the subcritical propane's permeability to vesicles is insufficient, failing to fully enrich vesicles from the pretreatment solution, resulting in low extraction efficiency. With increasing extraction pressure, the permeability of subcritical propane increases. At an extraction pressure of 20 MPa, subcritical propane can fully permeate without damaging the nanovesicle membrane structure, resulting in the most complete preservation of activity in the roselle exosome-like nanovesicles, with the highest mivRNA content at 322.05 ng / μL. Even after 28 days of storage, the mivRNA content in the lyophilized roselle exosome-like nanovesicle powder remained the highest, with the lowest loss rate during storage. When the extraction pressure is further increased, solvent molecules are excessively compressed, leading to slight rupture of some vesicle membranes, leakage of small amounts of mivRNA, and even pressures far exceeding the critical value, causing severe damage to the vesicle membrane structure, significant degradation of active ingredients, and a sharp decrease in the content of active ingredients in the vesicles.

[0028] Example 2 This embodiment focuses on exploring the effect of the extraction solvent of subcritical extraction on the preparation of nanovesicles. The preparation method of hibiscus exosome-like nanovesicles in this embodiment differs from that in Example 1 in that: In step S2, the subcritical extraction pressure is set to 20 MPa, the extraction temperature to 45°C, the extraction time to 4 h, the material-to-solvent ratio to 1:5, and the extraction solvents to be propane, butane, ethanol, and a mixed solvent of propane and butane in a 1:1 volume ratio under subcritical conditions.

[0029] In this embodiment, the effect of the type of solvent used in subcritical extraction on the concentration of micRNA in the prepared nanovesicles is shown in Table 2.

[0030] Table 2. Effects of subcritical extraction solvent type on micRNA concentration (ng / μL) in nanovesicles and product storage stability. 0d 321.98 267.45 128.63 245.87 28d 313.50 258.32 105.47 236.79

[0031] Table 2 shows that when propane is used as the extraction solvent for subcritical extraction, it exhibits the best preservation of activity due to its low damage to the membrane structure of hibiscus nanovesicles, as propane's polarity matches the surface characteristics of hibiscus vesicles and its strong solubility in the subcritical state. Butane, with a slightly higher boiling point than propane and slightly poorer fluidity in the subcritical state, has a lower extraction rate for vesicles than propane, resulting in a slightly lower recovery rate. Ethanol, with its high polarity, easily disrupts the lipid bilayer of vesicles, leading to leakage of active ingredients from the nanovesicles. Furthermore, its poor affinity for vesicles results in low extraction efficiency. The experimental results also show that when ethanol is used as the extraction solvent, the content of the active ingredient micRNA in the nanovesicles is the lowest, at only 128.63 ng / mL, which is only about 1 / 3 to 1 / 2 of the extraction efficiency when propane or butane are used as extraction solvents. In addition, the imbalance of polarity and fluidity in the mixed solvents leads to lower extraction efficiency than propane or butane alone, and the integrity of the extracted vesicles is slightly inferior to that obtained with propane.

[0032] Example 3 This embodiment focuses on exploring the effect of subcritical extraction temperature on the preparation of nanovesicles. The preparation method of hibiscus exosome-like nanovesicles in this embodiment differs from that in Example 1 in that: In step S2, the subcritical extraction pressure is set to 20 MPa, the extraction temperatures are 30, 45, and 60 °C, the extraction time is 4 h, the material-to-solvent ratio is 1:5, and the extraction solvent is propane in a subcritical state.

[0033] In this embodiment, the effect of subcritical extraction temperature on the concentration of micRNA in the prepared nanovesicles is shown in Table 3.

[0034] Table 3. Effects of subcritical extraction temperature on micRNA concentration (ng / μL) in nanovesicles and product storage stability. 0d 298.35 321.50 275.62 28d 290.12 312.87 258.49

[0035] Table 3 shows that, under the fixed conditions of subcritical extraction pressure of 20 MPa, extraction time of 4 h, material-to-solvent ratio of 1:5, and propane as solvent, the extraction temperature significantly regulates the enrichment efficiency and storage stability of micRNA in roselle exosome-like nanovesicles. 45 °C is the optimal extraction temperature under these process parameters. Mechanistically, at 45 °C, the molecular kinetic energy of subcritical propane reaches equilibrium with the solvent solubility: on the one hand, this temperature enhances the penetration of propane into the roselle pretreatment solution, ensuring sufficient contact and enrichment of the nanovesicles without disrupting the lipid bilayer structure of the vesicles. Therefore, the highest concentration of micRNA in the obtained nanovesicles is 321.50 ng / μL, which is essentially the same as the micRNA content (322.05 ng / μL) under the optimal pressure of 20 MPa in Example 1, demonstrating the synergistic effect of the process parameters; on the other hand, the intact vesicle structure results in excellent storage stability, with the micRNA concentration decreasing to only 312.87 ng / μL after 28 days, a loss rate of only 2.68%.

[0036] Example 4 This embodiment focuses on investigating the effect of the amount of freeze-drying protectant added on the storage stability of hibiscus exosome nanovesicles during freeze-drying. In this embodiment, the hibiscus exosome-like nanovesicle freeze-dried powder was prepared using the following method: Step S1: Same as in Example 1; Step S2: The pretreated solution of roselle exosome-like nanovesicles obtained in step S1 was treated by subcritical extraction. Propane in subcritical state was used as the extractant. The material-solvent ratio was 1:5. The extraction was carried out at 20 MPa and 45℃ for 4 h to obtain roselle exosome-like nanovesicle extract. Step S3: The extract of roselle exosome-like nanovesicles obtained in step S2 is adsorbed with activated carbon and finely filtered with a 0.22 μm microporous membrane to remove pigments and obtain roselle exosome-like nanovesicles. Step S4: Add 5%-12% by volume of a freeze-drying protectant to the roselle exosome-like nanovesicles obtained in step S3, and freeze-dry under vacuum at 6°C for 24 hours to obtain roselle exosome-like nanovesicle freeze-dried powder.

[0037] In this embodiment, the effect of the volume fraction of the freeze-drying protectant on the stability of the prepared nanovesicle freeze-dried powder is shown in Table 4.

[0038] Table 4. Effect of volume fraction of lyophilization protectant on micRNA concentration (ng / μL) and storage stability of nanovesicle lyophilized powder 0d 218.76 285.43 322.12 279.65 28d 125.34 269.71 313.51 268.43

[0039] The experimental data in Table 4 show that during the freeze-drying process, if the concentration of the freeze-drying protectant is too low, an effective protective layer cannot be formed. During freeze-drying, vesicles are prone to membrane rupture due to water sublimation, and the active ingredient micRNA is severely degraded after 28 days of storage. The protective effect is best when the protectant concentration is 10%. This may be because the appropriate concentration of protectant can reduce vesicle damage and form a stable glassy state during freeze-drying, completely protecting the vesicle membrane structure. The micRNA shows almost no degradation after 28 days of storage. If the protectant concentration is too high, it is easy to aggregate with vesicles. Although there is no obvious degradation, it affects the dispersibility of vesicles, resulting in a slight decrease in activity indicators.

[0040] Example 5 Roselle exosome-like nanovesicle lyophilized powder was prepared using the optimal parameter conditions obtained in Examples 1-4 above. The specific method is as follows: Step S1: After pulverizing the roselle flowers using a hammer mill, water was added at a mass-to-volume ratio of 1g:15mL. The mixture was stirred and mixed at 25℃ for 4 h. Then, it was centrifuged three times at 4℃. First, it was centrifuged at 1200×g for 12 min, and the precipitate was discarded to obtain the first separation supernatant. Then, the first separation supernatant was centrifuged at 4500×g for 20 min, and the precipitate was discarded to obtain the second separation supernatant. Finally, the second separation supernatant was centrifuged at 7800×g for 30 min, and the precipitate was discarded to obtain the third separation supernatant. The third separation supernatant was filtered through a 0.45μm filter membrane to obtain the roselle exosome-like nanovesicle pretreatment solution. Step S2: The pretreated solution of roselle exosome-like nanovesicles obtained in step S1 was treated by subcritical extraction. Propane in subcritical state was used as the extractant. The material-solvent ratio was 1:5. The extraction was carried out at 20 MPa and 45℃ for 4 h to obtain roselle exosome-like nanovesicle extract. Step S3: The extract of roselle exosome-like nanovesicles obtained in step S2 is adsorbed with activated carbon and finely filtered with a 0.22 μm microporous membrane to remove pigments and obtain roselle exosome-like nanovesicles. Figure 1 The image shows the morphological observation of the roselle exosome-like nanovesicles prepared in this embodiment using transmission electron microscopy. As can be seen from the image, the prepared roselle exosome-like nanovesicles have a distinct double-layered cup structure.

[0041] Figure 2 The figure shows the particle size analysis results of the roselle exosome-like nanovesicles extracted in this embodiment using a particle tracking analyzer. The figure shows that the average particle size of the obtained roselle exosome-like nanovesicles is 119.4 nm.

[0042] Step S4: Add 10% sorbitol by volume to the roselle exosome-like nanovesicles obtained in step S3 and freeze-dry under vacuum at 6°C for 24 h to obtain roselle exosome-like nanovesicle freeze-dried powder.

[0043] Example 6 Unlike Example 5, in step S2, the subcritical extraction time is 6 hours, and the remaining operations and conditions are the same as in Example 5.

[0044] Comparative Example 1 The method for preparing hibiscus exosome-like nanovesicles differs from that in Example 1 in that the subcritical extraction operation in step S2 is omitted, and they are obtained only by differential centrifugation. Furthermore, the centrifugation rate in step S1 is adjusted, as detailed in Table 5.

[0045] Table 5. Effects of differential centrifugation rate on the content (ng / μL) and stability of micRNA in roselle exocrine nanovesicles 1-1 1200×g×12min→4500×g×20min→7800×g×30min 205.18 178.51 1-2 1000×g×10min→4000×g×15min→7000×g×25min 189.65 165.42 1-3 1500×g×18min→5000×g×25min→9000×g×35min 210.32 180.75 1-4 8000×g×15min→4500×g×20min→1200×g×30min 126.89 98.53 1-5 1200×g×8min→4500×g×15min→8000×g×20min 175.46 142.31 1-6 1300×g×15min→4800×g×22min→8500×g×32min 198.73 168.59

[0046] The results in Table 5 show that the centrifugation rate and duration of differential centrifugation affect the content of active ingredients in roselle exosome-like nanovesicles. This is because a higher centrifugation rate may cause the nanovesicle membrane structure to rupture, thereby reducing the content of active ingredients. If the centrifugation rate is low, the collection efficiency of the vesicles may be low due to insufficient centrifugal force.

[0047] Application Example 1 In vitro antioxidant experiments of the hibiscus exosome-like nanovesicle powder prepared in Examples 5-6 and Comparative Example 1 The test sample was first mixed with DPPH solution and reacted. The absorbance change at a specific wavelength was then measured, and the free radical scavenging rate was calculated. The results are shown in Table 6 below.

[0048] Table 6 DPPH free radical scavenging rate of each experimental group

[0049] Table 6 shows that the DPPH free radical scavenging rate of the hibiscus exosome-like nanovesicle freeze-dried powder prepared in Examples 5-6 of this invention is significantly higher than that of the products in each numbered group in Comparative Example 1. It can be seen that the extraction method combining differential centrifugation and subcritical extraction can more effectively enrich the content of active ingredients in hibiscus exosome-like nanovesicles, thereby significantly improving the antioxidant capacity of the vesicle powder.

[0050] Application Example 2 After culturing the cells in vitro, rat basophilic leukemia (RBL-2H3) cells were divided into a blank group, a modeling group, and an experimental group. RBL-2H3 cells were first treated with IgE for 6 h, and then incubated with samples from different groups for 12 h. Cell viability was detected by CCK8 assay. The results are shown in Table 7 below.

[0051] Table 7. RBL-2H3 degranulation release rate (%) in each experimental group

[0052] The results in Table 7 show that the RBL-2H3 degranulation release rate of the hibiscus exosome-like nanovesicle freeze-dried powder prepared in Examples 5-6 was significantly lower than that of the products prepared by differential centrifugation alone in Comparative Example 1. This indicates that the freeze-dried powder prepared from hibiscus exosome-like nanovesicles extracted by differential centrifugation combined with subcritical extraction in this invention can reduce the increase in the degranulation release rate of RBL-2H3 cells induced by IgE, and has a significant inhibitory effect on the degranulation release of RBL-2H3 cells, thereby helping to inhibit the release of inflammatory factors in sun-exposed skin.

[0053] Application Example 3 After culturing the cells in vitro, HaCaT cells were divided into a blank group, a modeling group, and an experimental group. HaCaT cells were first irradiated with UVB for 6 h, and then incubated with samples from different groups for 12 h. IL-6 expression levels were detected using an ELISA kit. The results are shown in Table 8 below.

[0054] Table 8. IL-6 expression levels in each experimental group

[0055] As shown in Table 8, the lyophilized hibiscus exosome-like vesicle powder provided by the present invention can effectively reduce the increase of IL-6 inflammatory factor in HaCaT cells caused by UVB irradiation, thereby better inhibiting skin inflammation caused by UVB irradiation. The data also show that the lyophilized vesicle powder of each embodiment is significantly better than the numbered samples in Comparative Example 1.

Claims

1. A method for preparing hibiscus exosome-like nanovesicles, characterized in that, The steps include the following: Step S1: After pulverizing the roselle flowers, add water at a mass-to-volume ratio of 1g:15mL, stir and mix at 25°C for 4 hours, and then perform three centrifugation treatments at 4°C. First, centrifuge at 1200×g for 12 minutes, discard the precipitate, and obtain the first separation supernatant; then centrifuge the obtained first separation supernatant at 4500×g for 20 minutes, discard the precipitate, and obtain the second separation supernatant. Finally, the obtained second separation supernatant was centrifuged at 7800×g for 30 min, the precipitate was discarded, and the third separation supernatant was obtained; the obtained third separation supernatant was filtered through a 0.45μm filter membrane to obtain the roselle exosome-like nanovesicle pretreatment solution; Step S2: The pretreated solution of roselle exosome-like nanovesicles obtained in step S1 is treated by subcritical extraction. Propane in subcritical state is used as the extractant. The material-solvent ratio is 1:

5. The extraction is carried out at 20 MPa and 45℃ for 4 h or 6 h to obtain the subcritical extracted roselle exosome-like nanovesicle extract. Step S3: The extract of roselle exosome-like nanovesicles obtained in step S2 is adsorbed with activated carbon and filtered through a 0.22 μm microporous membrane to obtain roselle exosome-like nanovesicles.

2. The method for preparing hibiscus exosome-like nanovesicles as described in claim 1, characterized in that, In step S2, the extraction time is 4 hours.

3. The method for preparing hibiscus exosome-like nanovesicles as described in claim 1, characterized in that, In step S2, the extraction time is 6 hours.

4. Roselle exosome-like nanovesicles for relieving sunburn, characterized in that, It is prepared by the method described in claim 1.

5. A hibiscus exosome-like nanovesicle freeze-dried powder for relieving skin inflammation, characterized in that... Add 8% to 10% by volume of a freeze-drying protectant to the roselle exosome-like nanovesicles prepared in claim 1 and freeze-dry under vacuum to obtain the roselle exosome-like nanovesicle freeze-dried powder. The freeze-drying protectant is selected from at least one of sorbitol, trehalose, and bovine serum albumin.

6. The hibiscus exosome-like nanovesicle freeze-dried powder as described in claim 5, characterized in that, The freeze-drying protectant is sorbitol.

7. The use of the hibiscus exosome-like nanovesicle freeze-dried powder as described in any one of claims 5-6 in the preparation of skin care products or pharmaceuticals.

8. The application as described in claim 7, characterized in that, The skin care products or medications mentioned are those that have the effect of relieving skin inflammation caused by sunburn due to ultraviolet light exposure.

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