Use of artemisia-derived extracellular vesicles in skincare products

Abstract

The present application relates to the use of extracellular vesicles derived from Artemisia annua in skin care products, in particular the use includes any one of the following: (1) the use in the preparation of a product having skin barrier damage repair function; (2) the use in the preparation of a product having moisturizing function. The present application finds that the extracellular vesicles derived from Artemisia annua have excellent function of improving skin barrier damage repair and have excellent moisturizing effect.

Description

Technical Field

[0001] This invention relates to the field of extracellular vesicle technology, specifically to the use of artemisia annua-derived extracellular vesicles in skincare products. Background Technology

[0002] As the largest organ in the human body, the skin is the first line of defense protecting the body from physical, chemical, and microbial attacks. The skin barrier function primarily depends on the integrity of the stratum corneum, a "brick-and-mortar structure" composed of keratinocytes and a lipid-rich intercellular matrix, effectively preventing transepidermal water loss (TEWL) and resisting the invasion of harmful external substances. However, various factors such as genetics, lifestyle, ultraviolet radiation, and environmental pollution can impair the skin barrier function, manifesting as dry, sensitive, and red skin, and even triggering inflammatory skin diseases such as atopic dermatitis and psoriasis. Therefore, the search for safe and effective skin barrier repair products has become a research hotspot in the fields of dermatology and cosmetics.

[0003] Artemisia annua is a plant of the Asteraceae family called Artemisia annua ( Artemisia annua Artemisia annua, the dried aerial parts of *Artemisia annua*, is a traditional Chinese medicine with effects such as clearing heat and relieving summer heat, treating malaria, and cooling the blood. Modern research shows that Artemisia annua contains various active ingredients such as sesquiterpenes (e.g., artemisinin), flavonoids, and volatile oils, exhibiting broad pharmacological activities including anti-inflammatory, antibacterial, and immunomodulatory effects. Based on these properties, Artemisia annua extract and its derivatives have been gradually applied in the skincare field. Existing technologies reveal that Artemisia annua extract has certain effects in anti-inflammatory and soothing aspects.

[0004] Traditional extraction processes focus on enriching single or a class of components. While these techniques are mature and easily scalable, they often produce free molecules whose absorption / penetration depends on their inherent properties. With the rise of research on plant-derived extracellular vesicles, utilizing the natural activity of plant extracellular vesicles has become a new research direction. Unlike traditional chemical extracts, plant extracellular vesicles not only carry characteristic lipids, proteins, and RNA derived from plants, but also retain the complete natural structure of the phospholipid bilayer. This structure endows the vesicles with higher activity stability, better skin barrier permeability, and improved safety, making their active ingredients more easily absorbed and utilized by skin cells, achieving multi-layered, multi-target skincare effects.

[0005] Currently, reports on artemisia annua-derived extracellular vesicles are mostly concentrated in the pharmaceutical field, such as patents CN120272402A and CN121294318A, which disclose their applications in stroke and anti-tumor treatment, respectively. However, reports on artemisia annua (Artemisia vulgaris) are also limited. Artemisia annuaExtracellular vesicles derived from *Artemisia annua* (L.) can be used as independent active ingredients. By utilizing their natural nanostructure and the full range of bioactive molecules they carry, they can solve the problems of traditional artemisia annua extracts having single efficacy and requiring compound use. They can be applied to skin care fields such as skin moisturizing, barrier repair, and soothing and anti-allergy. However, there are currently no related reports. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides the use of extracellular vesicles derived from Artemisia annua in repairing skin barrier damage and / or moisturizing, especially in the repair of damage to the skin barrier caused by UVB, chemicals, etc.

[0007] Solution for solving the problem:

[0008] This invention provides the use of artemisia-derived extracellular vesicles in the preparation of skin care products, said use including any of the following:

[0009] (1) Use in the preparation of products with skin barrier damage repair function;

[0010] (2) Use in the preparation of products with moisturizing function.

[0011] Preferably, the skin barrier damage is caused by photodamage and / or chemical irritation.

[0012] Preferably, the moisturizing effect is to improve the moisture content of the epidermal skin.

[0013] Preferably, the method for preparing the extracellular vesicles derived from Artemisia annua includes soaking Artemisia annua and breaking the cell walls to obtain a mixed solution, and extracting extracellular vesicles from the mixed solution to obtain the extracellular vesicles derived from Artemisia annua.

[0014] Preferably, the extraction method is selected from one or more of sucrose density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow ultrafiltration concentration.

[0015] Preferably, the extraction method is ultracentrifugation, the steps of which include: centrifuging the mixed solution at 400~800×g for 5~20 min and collecting the supernatant; centrifuging at 1000~3000×g for 10~30 min and collecting the supernatant; centrifuging at 3000~5000×g for 20~40 min and collecting the supernatant; centrifuging at 8000~12000×g for 40~70 min and collecting the supernatant; centrifuging at 100000~120000×g for 60~100 min and collecting the precipitate, resuspending it with PB to obtain the extracellular vesicles derived from Artemisia annua.

[0016] Preferably, the extraction method is tangential flow ultrafiltration concentration, the steps of which include: centrifuging the mixed solution at 8000~12000×g for 10~50min and collecting the supernatant; filtering the supernatant through a tangential flow deep filtration membrane to obtain a filtrate; concentrating and filtering the filtrate to obtain the extracellular vesicles derived from Artemisia annua.

[0017] Preferably, the product includes cosmetics or pharmaceuticals.

[0018] Preferably, the product is a cosmetic, and its dosage form includes lyophilized agents, emulsions, aqueous solutions, oils, or gels.

[0019] Preferably, the product is a pharmaceutical product, and its dosage form includes lyophilized agents, oils, emulsions, ointments, pastes, coatings, gels, aerosols, sprays, solutions, or liniments.

[0020] The effects of the invention:

[0021] This invention uses Artemisia annua ( Artemisia annua Using the aerial parts of *Artemisia annua* (L.) as raw material, and employing a simple, mild, time-efficient, low-cost, and highly safe extracellular vesicle extraction method, extracellular vesicles of *Artemisia annua* were successfully obtained. When applied to the skincare field, these vesicles, with their high activity levels, can effectively repair skin barrier damage, enhance the water-locking ability of keratinocytes, and thus exert moisturizing effects, making them suitable for skincare applications.

[0022] As a novel bioactive raw material, artemisia annua extracellular vesicles can be widely used in various skin care products with skin barrier repair and / or moisturizing functions, such as serums, lotions, creams, and masks, demonstrating broad market application prospects and important industrialization value. Attached Figure Description

[0023] Figure 1 The image shows the characterization of extracellular vesicles in the liquid raw material of the extracellular vesicles prepared in Example 1, where the red arrow points to the extracellular vesicles of Artemisia annua.

[0024] Figure 2 The image shows the characterization of extracellular vesicles in the freeze-dried raw material of the extracellular vesicles prepared in Example 1, where the red arrow points to the extracellular vesicles of Artemisia annua. Detailed Implementation

[0025] To make the technical solution and beneficial effects of the present invention more apparent and understandable, a detailed description is provided below by listing specific embodiments. The accompanying drawings are not necessarily drawn to scale, and local features may be enlarged or reduced to more clearly show the details of the local features; unless otherwise defined, the technical and scientific terms used herein have the same meanings as those in the technical field to which this application pertains.

[0026] Through extensive and in-depth research, the inventors unexpectedly discovered that extracellular vesicles derived from Artemisia annua have novel applications in skincare products, specifically in the preparation of skin barrier damage repair and moisturizing products. Based on this, the technical solution of this application is proposed.

[0027] The use of artemisia-derived extracellular vesicles in the preparation of skincare products, wherein the use includes any of the following:

[0028] (1) Use in the preparation of products with skin barrier damage repair function;

[0029] (2) Use in the preparation of products with moisturizing function.

[0030] This invention provides the use of artemisia annua-derived extracellular vesicles in the preparation of skin care products with skin barrier damage repair functions.

[0031] As used in this article, the term "skin barrier" refers to the stratum corneum (the outermost layer of the skin), which is primarily composed of anucleate, flattened keratinocytes. "Skin barrier strengthening" refers to enhancing the barrier function of the stratum corneum (the outermost layer of the skin) to treat and improve skin barrier damage. Skin barrier function can be impaired with age or by external factors, and damage to the skin barrier can lead to moisture loss.

[0032] In one embodiment, artemisia-derived extracellular vesicles, as the active ingredient, can enhance skin barrier function by increasing the expression of involucrin (LOR), filaggrin (FLG), and hyaluronan synthase (HAS). Alternatively, upon exposure to photodamage, they can reduce UV-induced damage to the skin barrier function by regulating HSP70 expression. Specifically, this composition enhances or improves the skin barrier function by increasing the expression of filaggrin, involucrin, and / or HSP70, thus possessing properties that enhance or improve skin barrier function.

[0033] Filamentin (FLG) is an important molecule in the stratum corneum of human skin that connects keratin fibers. With the assistance of FLG monomers, keratin fibers aggregate regularly, forming a solid physical barrier on the outermost layer of the epidermis. Lorhein (LOR) is a key component in the assembly process of the commified envelope (CE), accounting for about 80% of the CE content, and plays a reinforcing role in the skin barrier. HSP70 is the most important family of HSPs, playing an important role in guiding the correct folding of proteins, maintaining protein homeostasis, and promoting cell survival under various stress conditions. Hyaluronic acid synthase (HAS) is a class of enzymes that play an important role in the synthesis of hyaluronic acid. It can be divided into three types: HAS1, HAS2, and HAS3. All three synthases can catalyze the synthesis of a considerable level of hyaluronic acid. Increased activity of hyaluronic acid synthase can promote the increase of hyaluronic acid content in the skin, thereby promoting ECM synthesis, making the skin plump, and enhancing the skin's moisturizing ability.

[0034] In some implementations, the skin barrier damage is photodamage.

[0035] UVB primarily affects the skin by directly damaging the DNA of skin cells, especially keratinocytes; it can also generate free radicals, causing oxidative damage to tissues. UVB damage mainly manifests as sunburned cells in the epidermis, due to apoptosis induced by DNA damage, which clinically presents as a sunburn reaction. In some embodiments, this invention provides the use of artemisinin-derived extracellular vesicles in the preparation of skin care products that reduce sunburn.

[0036] In some embodiments, the skin barrier damage is damage caused by chemical irritation.

[0037] In some embodiments, the chemical is one or more of a surfactant, a peroxide, or a preservative.

[0038] In some embodiments, the surfactant is selected from one or more of sodium dodecyl sulfate, sodium dodecylbenzene sulfonate, and dodecyl glycerol ether carboxylate.

[0039] In a specific and preferred embodiment, the surfactant is sodium lauryl sulfate (SLS). SLS is a common anionic surfactant that can disrupt the basic structure of the skin and prevent the barrier's self-repair by inducing inflammation, thereby causing damage to the skin barrier. In some embodiments, the present invention provides the use of artemisinin-derived extracellular vesicles in the preparation of skin care products that reduce the irritation of chemicals to the skin. In one embodiment, the chemical is capsaicin.

[0040] Transient receptor potential vanillic acid isoform 1 (TRPV1) is a tetrameric ion channel that can be activated by capsaicin, high temperature, and acidic environments. TRPV1 is widely expressed in keratinocytes, sensory nerve fibers, and immune cells. Its abnormal activation can trigger the release of neuropeptides (such as substance P and CGRP), leading to neurogenic inflammation, erythema, itching, and tingling sensations. Vascular endothelial growth factor (VEGF) is a growth factor that specifically acts on receptors on the surface of vascular endothelial cells, promoting endothelial cell proliferation, vasodilation, and capillary formation.

[0041] In one embodiment, artemisia-derived extracellular vesicles, as an active ingredient, can reduce barrier stimulation by decreasing the expression of epidermal vascular endothelial growth factor (VEGF) and transient receptor potential vanilloid 1 (TRPV1).

[0042] The natural moisturizing system in human skin mainly consists of water, lipids, and natural moisturizing factors (NMF). Skin hydration is a key indicator of skin health and an important parameter for evaluating skin condition related to moisture loss. Moisturizing skincare products can retain moisture on the skin surface, keeping it hydrated and increasing its capacitance. The higher the capacitance value, the higher the hydration content of the stratum corneum.

[0043] This invention provides the use of artemisia annua-derived extracellular vesicles in the preparation of products with moisturizing functions.

[0044] In one embodiment, the present invention provides the use of artemisia annua-derived extracellular vesicles in the preparation of products that improve the moisturizing ability of keratinocytes.

[0045] In one embodiment, the moisturizing effect is to improve the water content of the epidermal skin. In a specific embodiment, the moisturizing effect is to increase the water content of the epidermal skin.

[0046] In some embodiments, the method for preparing the extracellular vesicles derived from Artemisia annua includes soaking Artemisia annua to break the cell walls to obtain a mixed solution, and extracting extracellular vesicles from the mixed solution to obtain the extracellular vesicles derived from Artemisia annua.

[0047] In some embodiments, the extraction method is selected from one or more of sucrose density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow ultrafiltration concentration.

[0048] In some embodiments, the extraction method is ultracentrifugation, the steps of which include: centrifuging the mixed solution at 400-800×g for 5-20 min and collecting the supernatant; centrifuging at 1000-3000×g for 10-30 min and collecting the supernatant; centrifuging at 3000-5000×g for 20-40 min and collecting the supernatant; centrifuging at 8000-12000×g for 40-70 min and collecting the supernatant; centrifuging at 100000-120000×g for 60-100 min and collecting the precipitate, resuspending it with PB to obtain the extracellular vesicles derived from Artemisia annua.

[0049] In some embodiments, the specific steps of the ultracentrifugation method include: centrifuging the mixed solution at 500×g for 10 min and collecting the supernatant; centrifuging at 2000×g for 20 min and collecting the supernatant; centrifuging at 4000×g for 30 min and collecting the supernatant; centrifuging at 10000×g for 60 min and collecting the supernatant; centrifuging at 110000×g for 70 min and collecting the precipitate, resuspending it with PB to obtain the extracellular vesicles derived from Artemisia annua.

[0050] In some embodiments, the extraction method is tangential flow ultrafiltration concentration, the steps of which include: centrifuging the mixed solution at 8000~12000×g for 10~50min and collecting the supernatant; filtering the supernatant through a tangential flow deep filtration membrane to obtain a filtrate; concentrating and filtering the filtrate to obtain the extracellular vesicles derived from Artemisia annua.

[0051] In some embodiments, the specific steps of the tangential flow ultrafiltration concentration include: centrifuging the mixed solution at 8000~12000×g for 10~50min and collecting the supernatant; subjecting the supernatant to tangential flow microfiltration to obtain microfiltrate; and subjecting the microfiltrate to tangential flow filtration and concentration to obtain filtrate, which is the extracellular vesicle derived from Artemisia annua.

[0052] In some embodiments, the tangential flow ultrafiltration concentration steps specifically include: centrifuging the mixed solution at 10000×g for 30min and collecting the supernatant.

[0053] In some embodiments, the microfiltration is performed using microfiltration membranes with pore sizes of 5 μm, 1 μm, and 0.45 μm in sequence.

[0054] In some embodiments, the filtration is performed using a filter membrane with a pore size of 0.45 μm.

[0055] In some embodiments, the product includes cosmetics or pharmaceuticals.

[0056] In some embodiments, the product is a cosmetic, and its dosage form includes lyophilized agents, emulsions, aqueous solutions, oils, or gels.

[0057] In some embodiments, the product is a pharmaceutical product, and its dosage form includes lyophilized agents, oils, emulsions, ointments, pastes, coatings, gels, aerosols, sprays, solutions, or liniments.

[0058] In some embodiments, the extracellular vesicles are in the form of lyophilized formulations and / or solutions.

[0059] In some embodiments, the extracellular vesicles comprise 0.01% to 30% by weight, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, etc.

[0060] In some embodiments, the extracellular vesicles comprise 0.05 to 20% by weight.

[0061] In a specific and preferred embodiment, the extracellular vesicles comprise 0.1 to 10% by weight.

[0062] In some embodiments, the extracellular vesicles are in the form of a lyophilized formulation, which further includes a lyophilization protectant.

[0063] In some embodiments, the freeze-drying protectant is selected from one or more of sucrose, trehalose, glycerol, xylitol, sorbitol, mannitol, poloxamer 188, Tween 80, glycine, and albumin.

[0064] In some embodiments, the freeze-drying protectant is selected from trehalose and poloxamer 188.

[0065] In some embodiments, the product is a cosmetic composition.

[0066] In addition to the active ingredient (artemisia annua-derived extracellular vesicles) of this application, the cosmetic composition may also contain functional additives and ingredients commonly included in cosmetic compositions. The functional additives may include ingredients selected from water-soluble vitamins, oil-soluble vitamins, polypeptides, polysaccharides, sphingolipids, and seaweed extracts. Furthermore, it may contain oils, fats, humectants, emollients, surfactants, organic or inorganic pigments, organic powders, preservatives, bactericides, antioxidants, plant extracts, pH control agents, alcohol, colorants, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

[0067] The present invention will be further described below through specific embodiments. Unless otherwise specified, "%" represents a mass percentage. The materials and reagents used in the following embodiments are all commonly used materials or reagents in the art, and can be obtained commercially or synthesized by known methods. Experimental methods in the following embodiments without specified conditions are generally performed according to conventional experimental conditions or the conditions recommended by the manufacturer of the relevant reagent (kit).

[0068] Example 1

[0069] Weigh 400g of Artemisia annua and wash each 400g three times with ultrapure water to remove surface dust, dirt, and other contaminants. Remove any debris unrelated to the extraction process, such as broken leaves, root remnants, and weeds. Place the washed Artemisia annua in a clean soaking container. Add 1000mL of PB buffer and soak overnight at 4°C. Wash three more times with ultrapure water, transfer to a high-speed blender, add 2000mL of PB buffer, seal tightly, and blend for 5-10 minutes, ensuring no obvious fragments remain. Transfer the juice to sterile centrifuge tubes and centrifuge at 10000×g for 30 minutes at 4°C to separate the solid and liquid. Discard the precipitate and collect the supernatant. Utilize a tangential flow ultrafiltration system with multi-stage depth filtration membrane packs (pore sizes of 5 μm, 1 μm, and 0.45 μm). The Artemisia annua supernatant was screened for particle size in sequence to remove fragments and large vesicles until no filtrate flowed out from the filter end, and the small-particle filtrate was collected. Then, the Artemisia annua filtrate was filtered with a sterile filter membrane (pore size of 0.45 μm) to achieve sample sterilization and further purification until no filtrate flowed out from the filter end, and the filtrate was collected to obtain Artemisia annua extracellular vesicles, which were labeled as QH-T-Exo.

[0070] Example 2

[0071] Weigh 400g of Artemisia annua and wash it three times with ultrapure water to remove surface dust, dirt, and other impurities. Remove any debris unrelated to the extraction process, such as broken leaves, root remnants, and weeds. Place the washed Artemisia annua in a clean soaking container. Add 1000 ml of water to each container. Incubate the extract with 2000 mL of PB buffer at 4°C overnight; then wash three times with ultrapure water, transfer to a blender, add 2000 mL of PB buffer, tighten the cap, and blend for 5-10 minutes to ensure no obvious fragments remain; transfer the juice to sterile centrifuge tubes, centrifuge at 500×g for 10 minutes at 4°C, discard the precipitate, and collect the supernatant; centrifuge at 2000×g for 20 minutes at 4°C, discard the precipitate, and collect the supernatant; centrifuge at 4000×g for 30 minutes at 4°C, discard the precipitate, and collect the supernatant; centrifuge at 10000×g for 1 hour at 4°C, discard the precipitate, and collect the supernatant; centrifuge at 110000×g for 70 minutes at 4°C, discard the supernatant, and resuspend the precipitate in an appropriate amount of PB buffer to obtain Artemisia annua extracellular vesicles, labeled QH-U-Exo.

[0072] Example 3

[0073] Liquid raw materials: The artemisia-derived extracellular vesicles obtained in Example 1 and Example 2 were immediately aliquoted into sterile bottles and stored in a freezer (-25℃ to -15℃), and named QH-T-Exo liquid raw material and QH-U-Exo liquid raw material, respectively.

[0074] Freeze-dried powder raw materials: To facilitate long-term use and transportation, the extracellular vesicles of Artemisia annua obtained in Examples 1 and 2 were freeze-dried with 7.5% trehalose and 0.04% poloxamer 188 freeze-drying protectant and stored at room temperature (10℃~30℃) or refrigerated (2℃~8℃). They were named QH-T-Exo freeze-dried powder raw materials and QH-U-Exo freeze-dried powder raw materials.

[0075] Example 1: Extracellular vesicle characterization assay

[0076] The particle size of extracellular vesicles derived from Artemisia annua was detected using nanoflow cytometry, and the specific procedures are as follows:

[0077] After thawing the liquid raw material, dilute it 500-2000 times with PBS buffer and place it into the sample well of the nanoflow cytometer. After inputting the sample information, perform the detection. Inject 30 μL each time, ensuring the detection rate is within the range of 2000-8000 cells / second. Repeat the measurement twice and take the average value. Record and export the data. Weigh 1 g of lyophilized powder raw material, add 1 mL of sterile water, shake until completely dissolved, and determine the particle size of extracellular vesicles according to the above steps.

[0078] The morphology of extracellular vesicles derived from Artemisia annua was observed using transmission electron microscopy (TEM). The specific procedures are as follows:

[0079] Based on the sample conditions, after thawing the liquid raw material obtained in Example 3, approximately 10 μL of the sample was pipetted onto a copper grid and allowed to stand in a droplet shape for 2 minutes. The extracellular vesicle sample on the copper grid was then blotted dry with filter paper, and approximately 10 μL of 2% uranium acetate staining solution was applied for staining at room temperature for 1 minute. If significant adsorption was visible on the copper grid, pure water was added to the surface and quickly absorbed; this process was repeated several times. The extracellular vesicle sample on the copper grid was then blotted dry with filter paper. The samples were observed, photographed, and the images saved. 0.5 g of the lyophilized powder raw material was weighed, added to 1 mL of sterile water, mixed well, and the morphology of the extracellular vesicles was observed following the steps described above.

[0080] The results showed that, regardless of whether the raw material was liquid or lyophilized, the diameter of the extracellular vesicles ranged from 30 to 150 nm; TEM results showed that cup-shaped vesicles were observed in both cases. Characterization images of the extracellular vesicles prepared in the liquid raw material in Example 1 are shown below. Figure 1 As shown, the characterization diagram of the extracellular vesicles in the lyophilized raw material of the extracellular vesicles prepared in Example 1 is as follows. Figure 2 As shown.

[0081] Example 2: Cytotoxicity test

[0082] When the cell deposition rate of keratinocytes or vascular endothelial cells reached 60%, cells were seeded into 96-well plates and incubated overnight in an incubator (37°C, 5% CO2). The experiment included a zeroing group, a solvent control group (Control), a positive control group (PC), and an experimental group. The experimental group had eight concentration gradients (m / m, 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625%, 0.078125%), with three replicates for each concentration gradient. Drug administration was initiated when the cell deposition rate in the 96-well plates reached 50%–60%. In the solvent control group, 200 μL of DMEM culture medium was added to each well; in the PC group, 200 μL of culture medium containing 10% DMSO was added to each well; in the experimental group, 200 μL of culture medium containing the corresponding concentration of QH-T-Exo liquid feedstock or QH-T-Exo lyophilized feedstock was added to each well; in the zeroing group, no cells were seeded, only 200 μL of cell culture medium was added. After administration, the 96-well plates were placed in an incubator and cultured for 24 hours. Then, the supernatant was discarded, 0.5 mg / mL MTT was added, and the plates were incubated at 37°C in the dark for 4 hours. After incubation, the supernatant was discarded, 150 μL of LDMSO was added to each well, and the OD value was read at 490 nm to calculate the relative cell viability.

[0083] The formula for calculating relative cell viability is as follows: Relative cell viability (%) = (OD of experimental group wells - OD of zeroing wells) / (OD of solvent control group - OD of zeroing wells) × 100%.

[0084] The toxicity test results of QH-T-Exo freeze-dried raw material and QH-T-Exo liquid raw material on keratinocytes are shown in Tables 1 and 2, and the toxicity test results of QH-T-Exo liquid raw material on vascular endothelial cells are shown in Table 3.

[0085] Table 1

[0086]

[0087] Table 2

[0088]

[0089] Table 3

[0090]

[0091] As shown in Tables 1 and 2, the QH-T-Exo lyophilized raw material did not exhibit significant cytotoxicity against keratinocytes at a concentration range of 10% (m / m), while its liquid raw material also did not exhibit significant cytotoxicity at a concentration range of 0.625% (m / m). Furthermore, as shown in Table 3, the QH-T-Exo liquid raw material also did not exhibit significant cytotoxicity against vascular endothelial cells at a concentration range of 5%.

[0092] Example 4

[0093] QH-T-Exo liquid feedstock or QH-T-Exo lyophilized feedstock was diluted with PBS to obtain samples of different concentrations. The sample information is shown in Table 4.

[0094] Table 4

[0095]

[0096] Example 3: Tissue morphology testing based on UVB irradiation barrier weakening - 3D epidermal skin model (Epikutis®)

[0097] This experiment was divided into a blank control group (BC), a modeling group (NC), a positive control group (PC, WY14643), and sample groups (samples 1, 3, and 5). According to the test groups, the models were transferred to 6-well plates (pre-added with 0.9 mL of EpiGrowth medium). The BC group received no treatment, while the PC and sample groups were evenly spread on the model surface. Except for the BC group, all other groups underwent 600 mJ / cm² testing. 2UVB irradiation was performed. Afterward, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. The models used for detection were fixed with 4% paraformaldehyde for 24 hours, H&E staining was performed, and images were taken and analyzed under a microscope. The number of sunburned cells was counted, and the results are expressed as mean ± standard deviation (Mean ± SD). The inhibition rate was calculated. Comparisons between groups were performed using... t -test statistical analysis. All statistical analyses are two-tailed. P A value <0.05 is considered statistically significant. P A value <0.01 was considered highly significant. The inhibition rate was calculated using the following formula: Inhibition rate (%) = (NC group - Sample group) / NC group × 100%. The statistical results are shown in Table 5, where... t When performing statistical analysis using the -test method, significance compared to the BC group is indicated by #, with P value < 0.05 indicated by # and P value < 0.01 indicated by ##. Compared to the NC group, significance is indicated by *, with P value < 0.05 indicated by * and P value < 0.01 indicated by **.

[0098] Table 5

[0099]

[0100] Table 5 shows that the number of sunburned cells in the NC group was significantly higher than that in the BC group, indicating that the stimulation conditions were effective in this test. The number of sunburned cells in the PC group was significantly lower than that in the NC group, indicating that the positive control was effective in this test. Compared with the NC group, the number of sunburned cells in samples 1 (1% QH-T-Exo liquid raw material), 3 (0.5% QH-T-Exo liquid raw material), and 5 (0.25% QH-T-Exo liquid raw material) was significantly lower, with inhibition rates of 78.56%, 60.66%, and 39.23%, respectively.

[0101] Example 4: Immunofluorescence assay based on UVB irradiation barrier weakening - 3D epidermal skin model (Epikutis®)

[0102] This experiment was divided into a blank control group (BC), a modeling group (NC), a positive control group (PC, WY14643), and sample groups (samples 1, 3, and 5). According to the test groups, the models were transferred to 6-well plates (pre-added with 0.9 mL of EpiGrowth medium). The BC group received no treatment, while the PC and sample groups were evenly spread on the model surface. Except for the BC group, all other groups underwent 600 mJ / cm² testing. 2UVB irradiation was performed. After irradiation, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. The models used for detection were fixed with 4% paraformaldehyde for 24 hours, and HSP70 and FLG fluorescence were detected separately. Images were taken and analyzed under a microscope. The HSP70 and FLG immunofluorescence results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using... t -test Statistical analysis. All statistical analyses are two-tailed. P A value <0.05 is considered statistically significant. P <0.01 was considered highly significant. The improvement rate was calculated using the following formula: Improvement rate (%) = (Sample group – NC group) / NC group × 100%. The HSP70 immunofluorescence statistical results are shown in Table 6, and the FLG immunofluorescence statistical results are shown in Table 7. t When performing statistical analysis using the -test method, significance compared to the BC group is indicated by #, with P value < 0.05 indicated by # and P value < 0.01 indicated by ##. Compared to the NC group, significance is indicated by *, with P value < 0.05 indicated by * and P value < 0.01 indicated by **.

[0103] Table 6

[0104]

[0105] Table 6 shows that the HSP70 content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the HSP70 content of samples 1 (1% QH-T-Exo liquid raw material), 3 (0.5% QH-T-Exo liquid raw material), and 5 (0.25% QH-T-Exo liquid raw material) all increased significantly, with increases of 463.64%, 200.00%, and 72.73%, respectively.

[0106] Table 7

[0107]

[0108] Table 7 shows that the FLG content in the NC group was significantly lower than that in the BC group, indicating that the stimulation conditions in this test were effective. The FLG content in the PC group was significantly higher than that in the NC group, indicating that the positive control in this test was effective. Compared with the NC group, the FLG content in samples 1 (1% QH-T-Exo liquid raw material), 3 (0.5% QH-T-Exo liquid raw material), and 5 (0.25% QH-T-Exo liquid raw material) all increased significantly, with increases of 204.76%, 95.24%, and 38.10%, respectively.

[0109] Example 5: LOR gene detection based on UVB irradiation barrier weakening - 3D epidermal skin model (Epikutis®)

[0110] This experiment was divided into a blank control group (BC), a modeling group (NC), a positive control group (PC, WY14643), and a sample group (sample 1). According to the test groupings, the models were transferred to 6-well plates (pre-added with 0.9 mL of EpiGrowth medium). The BC group received no treatment, while the PC and sample groups were evenly spread on the model surface. Except for the BC group, all other groups underwent 600 mJ / cm² testing. 2 UVB irradiation was performed. The 6-well plate was incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. The model was then circumcised and placed in a 5mL centrifuge tube. 1mL of RNA extraction reagent (AG RNAex Pro Reagent) was added to extract RNA. After reverse transcription to cDNA, quantitative real-time PCR was performed for detection. 2... -△△CT Results were calculated using the method described above. LOR gene content results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using... t -test Statistical analysis. All statistical analyses are two-tailed. P A value <0.05 is considered statistically significant. P A difference <0.01 was considered highly significant. The upregulation rate was calculated using the following formula: Upregulation rate (%) = (Sample group - NC group) / NC group × 100%. The results are shown in Table 8, where 2... -△△CT The method is used to calculate the result. t When performing statistical analysis using the -test method, the mRNA amplification fold of group BC was normalized, and the comparison with group BC was indicated by #. P -Value < 0.05 is represented by #. P -Value < 0.01 is represented by ##; significance compared to the NC group is represented by *. P -value < 0.05 is represented as *. P -value < 0.01 is represented as **.

[0111] Table 8

[0112]

[0113] Table 8 shows that, compared with the BC group, the LOR gene expression level in the NC group was significantly downregulated, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the LOR gene expression level in the PC group was significantly upregulated, indicating that the positive control in this test was effective. Compared with the NC group, the LOR gene expression level in sample 1 (1% QH-T-Exo liquid raw material) was significantly upregulated, with an upregulation rate of 56.45%.

[0114] Example 6: HAS1 gene detection based on a UVB irradiation barrier weakening-3D epidermal skin model (Epikutis®)

[0115] This experiment was divided into a blank control group (BC), a model group (NC), a positive control group (PC, CaCl2), and sample groups (samples 1 and 3). According to the test groups, the models were transferred to 6-well plates (pre-added with 0.9 mL of EpiGrowth medium). The BC group received no treatment, while the PC and sample groups were evenly spread on the model surface. Except for the BC group, all other groups underwent 600 mJ / cm² testing. 2 UVB irradiation. After irradiation, the 6-well plate was incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. The model was then circumcised and placed in a 5mL centrifuge tube. 1mL of RNA extraction reagent (AG RNAex Pro Reagent) was added to extract RNA. After reverse transcription to cDNA, quantitative real-time PCR was performed for detection. -△△CT Results were calculated using the following methods. HAS1 gene content results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using... t -test statistical analysis. All statistical analyses are two-tailed. P A value <0.05 is considered statistically significant. P A difference <0.01 was considered highly significant. The upregulation rate was calculated using the following formula: Upregulation rate (%) = (Sample group - NC group) / NC group × 100%. The results are shown in Table 9. (Note: The last part, "2," appears to be an incomplete sentence or fragment and doesn't translate directly. It's left as is.) -△△CT The method is used to calculate the result. t When performing statistical analysis using the -test method, the mRNA amplification fold of group BC was normalized, and the comparison with group BC was indicated by #. P -Value < 0.05 is represented by #. P - Value < 0.01 is represented by ##; significance compared to the NC group is represented by *. P -value < 0.05 indicates as *. P -value < 0.01 is represented as **.

[0116] Table 9

[0117]

[0118] Table 9 shows that, compared with the BC group, the HAS1 gene expression level in the NC group was significantly downregulated, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the HAS1 gene expression level in the PC group was significantly upregulated, indicating that the positive control in this test was effective. Compared with the NC group, the HAS1 gene expression levels in sample 1 (1% QH-T-Exo liquid raw material) and sample 3 (0.5% QH-T-Exo liquid raw material) were significantly upregulated, with upregulation rates of 102.94% and 91.18%, respectively.

[0119] Example 7: Detection of FLG and LOR expression in keratinocytes based on UVB irradiation.

[0120] The experiment included a blank control group (BC), a model group (NC), a positive control group (PC, WY14643), and sample groups (samples 7-9). After cell resuscitation, when the cell deposition rate reached approximately 60%, cells were seeded into 6-well plates and incubated overnight in a CO2 incubator (37℃, 5% CO2). When the cell deposition rate in the 6-well plates reached 30%–50%, all groups except BC were irradiated with UVB at a dose of 300 mJ / cm². 2 After irradiation, the drugs were administered to groups, with three replicates per group. 2 mL of culture medium was added to each well of the BC and NC groups; 2 mL of culture medium containing WY14643 was added to each well of the PC group; and 2 mL of culture medium containing the corresponding test sample was added to each well of the sample group. After drug administration, the 6-well plate was placed in a CO2 incubator (37℃, 5% CO2) for 24 h. After incubation, the plate was washed twice with 1 mL / well of PBS, and 1 mL of RNA extraction reagent (AG RNAex Pro Reagent) was added to each well to extract RNA. RNA was reverse transcribed into cDNA, and then quantitative real-time PCR was performed to detect the expression levels of FLG and LOR genes. -△△CT The results were calculated using the method described above. Gene content results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using... t -test statistical analysis. All statistical analyses are two-tailed. P A value <0.05 is considered statistically significant. P <0.01 was considered highly significant. The upregulation rate of FLG and LOR gene expression was calculated using the following formula: Upregulation rate (%) = (sample group - NC group) / NC group × 100%. The results are shown in Tables 10 and 11, where 2... -△△CT The method is used to calculate the result. t When performing statistical analysis using the -test method, the fold increase of mRNA in group BC was normalized. Significance compared to group BC is indicated by #. P -Value < 0.05 is represented by #. P- Value < 0.01 is represented as ##; significance compared to the NC group is represented by *. P -value < 0.05 is represented as *. P -value < 0.01 is represented as **.

[0121] Table 10

[0122]

[0123] Table 10 shows that, compared with the BC group, the FLG gene expression level in the NC group was significantly downregulated, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the FLG gene expression level in the PC group was significantly upregulated, indicating that the positive control in this test was effective. Compared with the NC group, the FLG gene expression levels in samples 7 (0.1% QH-T-Exo lyophilized raw material), 8 (1% QH-T-Exo lyophilized raw material), and 9 (10% QH-T-Exo lyophilized raw material) were all significantly upregulated, with upregulation rates of 158.02%, 161.73%, and 192.59%, respectively.

[0124] Table 11

[0125]

[0126] Table 11 shows that, compared with the BC group, the LOR gene expression level in the NC group was significantly downregulated, indicating that the stimulation conditions in this test were effective. Compared with the NC group, the LOR gene expression level in the PC group was significantly upregulated, indicating that the positive control in this test was effective. Compared with the NC group, the LOR gene expression levels of sample 8 (1% QH-T-Exo lyophilized raw material) and sample 9 (10% QH-T-Exo lyophilized raw material) were significantly upregulated, with upregulation rates of 65% and 87.50%, respectively. This indicates that Artemisia annua extracellular vesicles can increase the LOR content.

[0127] Example 8: TRPV1 content assay based on capsaicin (CAP) stimulation of keratinocytes.

[0128] This experiment investigated the expression changes of TRPV1 in keratinocytes after treatment with artemisinin extracellular vesicles using CAP stimulation. A blank control group (BC), a model group (NC), a positive control group (PC, trans-4-tert-butylcyclohexanol), and sample groups (samples 2 and 4) were established. NC, PC, and sample groups were treated with 15 μM CAP to induce modeling. After administration of CAP to PC and sample groups, they were cultured in a CO2 incubator (37℃, 5% CO2) for 24 h, fixed with 4% paraformaldehyde for 30 min, and then subjected to immunofluorescence detection. Images were collected and analyzed under a microscope. The relative integrated optical density (IOD) / cell number reflects the TRPV1 content, and the results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using... t - Statistical analysis. All statistical analyses are two-tailed. The inhibition rate of TRPV1 gene expression was calculated using the following formula: Inhibition rate (%) = (NC group - Sample group) / NC group × 100%. The results are shown in Table 12. Among them, compared with the BC group, P < 0.05 was considered to be statistically significant, marked as #; P < 0.01 was considered to be highly statistically significant, marked as ##; compared with the NC group, P < 0.05 was considered to be statistically significant, marked as *; P < 0.01 was considered to be highly statistically significant, marked as **.

[0129] Table 12

[0130]

[0131] As shown in Table 12, the TRPV1 content in the PC group was significantly lower than that in the NC group, indicating that the positive control in this test was effective. Compared with the NC group, the TRPV1 content in samples 2 (0.625% QH-T-Exo liquid raw material) and 4 (0.3125% QH-T-Exo liquid raw material) was significantly reduced, with inhibition rates of 65.34% and 26.46%, respectively.

[0132] Example 9: VEGF content assay based on vascular endothelial cells

[0133] This assay, based on vascular endothelial cells, detected changes in VEGF content after treatment with artemisinin extracellular vesicles. A blank control group (BC) and a sample group (sample 1) were set up. After cell seeding, when the cell layering rate reached 40%–60%, the cells were administered to the respective groups. The blank control group was given cell culture medium, while the sample group was given culture medium containing the corresponding test sample. Both groups were incubated in a CO2 incubator (37℃, 5% CO2) for 24 hours. The cell culture supernatant was collected, and the VEGF content was detected according to the ELISA kit instructions. VEGF content results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using... t- Statistical analysis. All statistical analyses are two-tailed. The inhibition rate of VEGF gene expression was calculated using the following formula: Inhibition rate (%) = (BC group − sample group) / BC group × 100%. The results are shown in Table 13. Among them, compared with the BC group, P < 0.05 was considered to be statistically significant and was marked as *; P < 0.01 was considered to be highly statistically significant and was marked as **.

[0134] Table 13

[0135]

[0136] As shown in Table 13, compared with group BC, the VEGF content of sample 1 (1% QH-T-Exo liquid raw material) was significantly reduced, with an inhibition rate of 6.62%.

[0137] Example 10: Test of FLG and LOR content based on keratinocytes

[0138] When the cell seeding rate reached 60%, keratinocytes were seeded into 6-well plates and incubated overnight in an incubator. The experiment included a blank control group (BC), a positive control group (PC, WY14643 (50 μM)), and sample groups (sample 2, sample 4, and sample 6). When the cell seeding rate in the 6-well plates reached 40%–60%, the cells were administered to each group, with three replicates per group. 2 mL of culture medium was added to each well of BC; 2 mL of culture medium containing WY14643 was added to each well of PC; and 2 mL of culture medium containing the corresponding sample was added to each well of the sample groups. After administration, the 6-well plates were incubated for 24 hours. The cells were then washed twice with 1 mL / well PBS, and 1 mL of RNA extraction reagent (AG RNAex Pro Reagent) was added to each well. After cell lysis by pipetting, the cells were collected. RNA was extracted, reverse transcribed into cDNA, and quantitative real-time PCR was performed to detect the expression levels of FLG and LOR genes. The gene content results are expressed as mean ± standard deviation (Mean ± SD). Comparisons between groups were performed using... t - Statistical analysis. During the analysis, the fold increase of mRNA in the BC group was normalized. Compared with the BC group, the significance was indicated by *, P<0.05 was indicated as *, and P<0.01 was indicated as **. The upregulation rate of FLG and LOR gene expression was calculated using the following formula: Upregulation rate (%) = (sample group - BC) / BC × 100%. The results are shown in Tables 14 and 15.

[0139] Table 14

[0140]

[0141] As shown in Table 14, compared with BC, FLG gene expression in PC was significantly upregulated, indicating that the positive control in this test was effective. The FLG gene expression in samples 6 (0.15625% QH-T-Exo liquid raw material), 4 (0.3125% QH-T-Exo liquid raw material), and 2 (0.625% QH-T-Exo liquid raw material) was upregulated by 51.00%, 73.00%, and 79.00%, respectively.

[0142] Table 15

[0143]

[0144] As shown in Table 15, compared with BC, LOR gene expression in PC was significantly upregulated, indicating that the positive control in this test was effective. The LOR gene expression of Sample 6 (0.15625% QH-T-Exo liquid raw material), Sample 4 (0.3125% QH-T-Exo liquid raw material), and Sample 2 (0.625% QH-T-Exo liquid raw material) was upregulated by 52.00%, 97.00%, and 188.00%, respectively, and Sample 4 (0.3125% QH-T-Exo liquid raw material) and Sample 2 (0.625% QH-T-Exo liquid raw material) showed significantly better results than PC.

[0145] Example 11: Skin Moisture Content Test Based on 3D Epidermal Skin Model (EpiKutis®)

[0146] The 3D model was transferred to a 6-well plate pre-filled with 0.9 mL of EpiGrowth medium and set aside. The experiment included a blank control group (BC), a positive control group (PC), and sample groups (sample 1, sample 3, and sample 5). The working solutions for PC and sample groups were evenly applied to the model surface and incubated in an incubator for 24 hours. Residual test material was then washed with sterile PBS solution, and residual liquid was wiped away with sterile cotton. The 3D model was transferred to a 24-well plate, and 0.3 mL of EpiGrowth medium was added to each well. The plate was incubated for 30 minutes. The model was then circumcised and placed in a skin moisture meter (Corneometer® CM825) for testing. Each model was tested three times, and the average value was taken. Results are expressed as mean ± standard deviation (Mean ± SD). t-tests were used for comparisons between groups. Significance compared to the BC group was indicated by *, P < 0.05 was *, and P < 0.01 was **. The improvement rate was calculated using the following formula: Improvement rate (%) = (sample group - BC) / BC × 100%, and the results are shown in Table 16.

[0147] Table 16

[0148]

[0149] As shown in Table 16, compared with BC, PC showed a significant increase in skin moisture content, indicating that the positive control in this test was effective. The skin moisture content of Sample 5 (0.25% QH-T-Exo liquid raw material), Sample 3 (0.5% QH-T-Exo liquid raw material), and Sample 1 (1% QH-T-Exo liquid raw material) increased by 64.04%, 87.04%, and 122.11%, respectively.

[0150] It should be understood that the above embodiments are exemplary and are not intended to encompass all possible implementations included in the claims. Various modifications and changes can be made to the above embodiments without departing from the scope of this disclosure. Similarly, the various technical features of the above embodiments can be arbitrarily combined to form other embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only illustrate several implementations of the present invention and do not limit the scope of protection of this patent.

Claims

1. The use of artemisia-derived extracellular vesicles in the preparation of skincare products, characterized in that, The use includes any of the following: (1) Use in the preparation of products with skin barrier damage repair function; (2) Use in the preparation of products with moisturizing function; The skin barrier damage is caused by photodamage and / or chemical irritation; The extracellular vesicles are in the form of a lyophilized formulation, and the weight percentage of the extracellular vesicles is 0.1-10%, or... The extracellular vesicles are in the form of a solution, and the weight percentage of the extracellular vesicles is 0.3125~1%.

2. The use according to claim 1, characterized in that, The moisturizing effect is to improve the water content of the epidermal skin.

3. The use according to claim 1 or 2, characterized in that, The method for preparing the extracellular vesicles derived from Artemisia annua includes soaking Artemisia annua and breaking the cell wall to obtain a mixed solution, and extracting extracellular vesicles from the mixed solution to obtain the extracellular vesicles derived from Artemisia annua.

4. The use according to claim 3, characterized in that, The extraction method is selected from one or more of the following: sucrose density gradient centrifugation, ultracentrifugation, fractional filtration, and tangential flow ultrafiltration concentration.

5. The use according to claim 4, characterized in that, The extraction method is ultracentrifugation, and the steps include: centrifuging the mixed solution at 400~800×g for 5~20 min and collecting the supernatant; centrifuging at 1000~3000×g for 10~30 min and collecting the supernatant; centrifuging at 3000~5000×g for 20~40 min and collecting the supernatant; centrifuging at 8000~12000×g for 40~70 min and collecting the supernatant; centrifuging at 100000~120000×g for 60~100 min and collecting the precipitate, resuspending it with PB to obtain the extracellular vesicles derived from Artemisia annua.

6. The use according to claim 4, characterized in that, The extraction method is tangential flow ultrafiltration concentration, the steps of which include: centrifuging the mixed solution at 8000~12000×g for 10~50min and collecting the supernatant; filtering the supernatant through a tangential flow deep filtration membrane to obtain a filtrate; concentrating and filtering the filtrate to obtain the extracellular vesicles derived from Artemisia annua.

7. The use according to claim 1 or 2, characterized in that, The products mentioned include cosmetics or pharmaceuticals.

Images