Preparation process and application of exosome-like vesicles rich in flavonoid compounds

Abstract

The invention provides a plant-derived exosome-like vesicle rich in flavonoid compounds and a preparation method of the plant-derived exosome-like vesicle. The invention also provides a method and application of the exosome-like vesicles for improving skin conditions, and a skin care product containing the exosome-like vesicles.

Description

Technical Field

[0001] The present invention belongs to the field of natural plant products. Specifically, the present invention relates to plant-derived exosome-like vesicles, a preparation method thereof, and use thereof for improving skin conditions, as well as skin care products containing the same. Background Art

[0002] The active ingredients responsible for inhibiting melanin production in some commercially available whitening skincare products are primarily compounds such as kojic acid, arbutin, retinoic acid, hydroquinone, ascorbic acid, n-phenylthiourea, and phenylethylresorcinol (Han et al., 2020). These compounds inhibit tyrosinase activity, thereby reducing melanin expression and achieving a whitening effect. However, these compounds have several side effects, including cytotoxicity, poor stability, low bioavailability, and skin irritation, which can easily lead to allergies or dermatitis, and even DNA damage and cancer (Alam et al., 2016; Wang et al., 2018). In recent years, the search for safe and effective alternative ingredients as skin-whitening agents has become a hot topic in cosmetics research and development.

[0003] Flavonoids are low-molecular-weight polyphenolic compounds widely distributed in nature (Lee et al., 2015). They are primarily found in highly nutritious plants such as oranges, tangelos, mandarins, grapefruits (also known as grapefruit), and other citrus fruits. Among them, hesperidin, ophiopogon isoflavone A, and neohesperidin dihydrochalcone exhibit anti-inflammatory and antioxidant properties (Shi et al., 2015; Lin et al., 2015; Guazelli et al., 2020). Their antioxidant activity can inhibit melanin production by scavenging reactive oxygen species (ROS) free radicals (Karg et al., 1993), thereby contributing to a whitening effect. However, these flavonoids suffer from poor water solubility, weak transdermal penetration, and low bioavailability, significantly limiting their application in cosmetics and skin disease treatments and reducing their therapeutic efficacy.

[0004] For such poorly water-soluble active ingredients, the common approach is to attempt to encapsulate them using liposomes to improve skin permeability and bioavailability. However, suitable liposomes often have a certain degree of cytotoxicity, and safety cannot be fully guaranteed. In addition, the nanoparticles formed by encapsulating hesperidin with polyethylene glycol 8000 (PEG8000) are large, with an average particle size of 337.2 nm, and are prone to agglomeration, which is not conducive to improving bioavailability (Ahmed et al., 2021).

[0005] There is a need in the art for novel delivery forms of flavonoids to more effectively and safely exert their skin active effects. SUMMARY OF THE INVENTION

[0006] In practice, the inventors found that exosome-like vesicles from plants are rich in flavonoid compounds, and have the advantages of small particle size (average particle size of about 186nm), containing a complete lipid bilayer membrane structure, so they can easily cross the skin barrier and be directly taken up by cells, and do not require engineering. They are natural tools for flavonoid delivery and can exert significant whitening, anti-inflammatory and antioxidant effects.

[0007] Thus, in a first aspect, the present invention provides exosome-like vesicles (ELVs) from plants or parts thereof, characterized in that the vesicles are enriched in flavonoids. In some specific embodiments, the flavonoids include compounds selected from the group consisting of hesperidin, ophiopogon isoflavone A, neohesperidin dihydrochalcone, and any combination thereof. In some more specific embodiments, the flavonoids include compounds selected from the group consisting of hesperidin, ophiopogon isoflavone A, neohesperidin dihydrochalcone, and any combination thereof, and at least one of the flavonoids is among the five most abundant active substances in the vesicles, as determined by HPLC-MS. In some specific embodiments, the flavonoids do not include naringin and / or naringenin.

[0008] In some embodiments, the plant is of the genus Citrus, for example, Citrus Paradisi (also known as grapefruit), orange, tangelo, mandarin, and the like.

[0009] In some embodiments, the plant part is a fruit.

[0010] In some embodiments, the ELV has an average particle size between 170 and 200 nm, preferably between 180 and 200 nm, more preferably between 180 and 190 nm, and most preferably about 186 nm.

[0011] In a second aspect, the present invention provides a method for preparing ELV, comprising the following steps:

[0012] 1) Using plants or parts thereof as starting materials, cutting into pieces and then crushing and homogenizing;

[0013] 2) soaking the crushed juice and residue in an isotonic or substantially isotonic liquid and stirring to obtain a residue liquid, wherein the ratio of the mass of the initial raw material to the volume of the liquid used is 1:1 to 1:10 (w:v), preferably 1:1 to 1:6 (w:v), and more preferably 1:3 (w:v);

[0014] 3) Filter the residue through gauze to obtain a crude extract, centrifuge at approximately 2000-4000 g and approximately 10,000 g at 4°C for 10-40 min, respectively, to obtain the supernatant, and then ultracentrifuge at at least 80,000 g at 4°C to obtain the precipitate;

[0015] 4) Resuspending the precipitate with isotonic or substantially isotonic liquid to obtain a vesicle suspension.

[0016] In some embodiments, the plant or its part weighing about 50 to 200 g, preferably about 100 g, is crushed in step 1). In some specific embodiments, the plant fruit weighing about 100 g is crushed in step 1).

[0017] In some embodiments, step 1) comprises crushing and homogenizing the plant or its part using a juicer. In some specific embodiments, the juicer is operated at a low speed. In some specific embodiments, the juicer is operated in an intermittent mode. In some specific embodiments, the juicer is operated in a continuous mode.

[0018] In some embodiments, step 2) is performed on ice. In some specific embodiments, the isotonic or substantially isotonic liquid used in step 2) is physiological saline or 1×PBS.

[0019] In some embodiments, the soaking and stirring time in step 2) is about 0.5 to 3 hours, preferably about 1 hour.

[0020] In some embodiments, the stirring speed in step 2) is 50 to 500 rpm, preferably 100 to 250 rpm, and more preferably 150 rpm.

[0021] In some preferred embodiments, the ultracentrifugation in step 3) is performed at a temperature of at least 100,000 g. In some preferred embodiments, the ultracentrifugation in step 3) is performed at a temperature of at least 120,000 g. In some preferred embodiments, the ultracentrifugation in step 3) is performed for a time of 60 to 180 minutes. In some preferred embodiments, the ultracentrifugation in step 3) is performed for about 70 minutes.

[0022] In some specific embodiments, the isotonic or substantially isotonic liquid used in step 4) is physiological saline or 1×PBS. In some preferred embodiments, the liquid is pre-chilled. In some preferred embodiments, step 4) further comprises centrifuging the supernatant again at approximately 2000–4000 g for 10–30 min at 4°C to further purify the vesicles.

[0023] In a preferred embodiment, the preparation method of the ELV comprises the following steps:

[0024] 1) Rinse the outer peel of the grapefruit thoroughly with deionized water and drain as much water as possible. Weigh 100g of grapefruit, peel off the outermost peel, cut the remaining part into pieces of a size suitable for the juicer inlet, and place the pieces into the juicer feeding port. Use a push rod to push the plant into the cup body, turn the button to the low speed gear to start the juicer, and start the juicer for 20 seconds and rest for 10 seconds until the crushing and homogenization are completed;

[0025] 2) Collect the juice and residue into a beaker and place it on ice. Add 200-600 (preferably 300) mL of phosphate buffer solution (1×PBS) to the beaker at a mass-to-volume ratio of 1:1-1:6 (preferably 1:3). Soak and stir for 0.5-3 h (preferably 1 h). Set the stirrer speed to 100-250 rpm (preferably 150 rpm).

[0026] 3) Filter the residue through gauze into a new beaker to obtain a crude extract, centrifuge it (4°C, 3000 g, 20 min), and collect the supernatant. Centrifuge it again (4°C, 10000 g, 30 min), collect the supernatant, and then ultracentrifuge it (4°C, 120000 g, 70 min) and discard the supernatant.

[0027] 4) Resuspend the pellets in 20 mL of 0.9% sodium chloride injection in 50 mL centrifuge tubes. Centrifuge again (4°C, 3000 g, 10 min), discard the insoluble pellet, and retain the supernatant. Filter the final supernatant through a 0.45 μm filter in a biosafety cabinet to obtain the vesicle suspension.

[0028] In some embodiments, the ELV described in the first aspect of the present invention is prepared by the above method.

[0029] In some embodiments, a composition comprising an ELV of the present invention is prepared by the above-described method.

[0030] Therefore, the present invention also provides essentially a composition comprising the plant-derived ELV of the present invention, and a method for preparing the composition.

[0031] For example, the composition of the present invention comprises the ELV according to the first aspect of the present invention and / or the ELV obtained by the preparation method of the second aspect of the present invention. For example, the composition of the present invention is obtained by the same method as the preparation method of the second aspect of the present invention.

[0032] In some embodiments, the composition is a pharmaceutical composition. In some specific embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable carriers, excipients and other auxiliary materials.

[0033] In some embodiments, the composition is a cosmetic composition. In some specific embodiments, the cosmetic composition further comprises cosmetically acceptable carriers, excipients, and other excipients. In some specific embodiments, the cosmetic composition is a cosmetic. In some specific embodiments, the cosmetic is a skin care product. In some specific embodiments, the cosmetic is an emulsion, cream, or serum.

[0034] In some embodiments, the composition is a food / health product composition. In some specific embodiments, the food / health product composition further comprises auxiliary materials such as edible carriers and excipients.

[0035] In a further aspect, the present invention provides the use of ELVs from plants for the cosmetic and / or non-therapeutic treatment and / or care of the skin and / or mucous membranes.

[0036] In some embodiments, the cosmetic and / or non-therapeutic treatment and / or care is the treatment and / or prevention of skin aging.

[0037] In some embodiments, the cosmetic and / or non-therapeutic treatment and / or care is the treatment and / or prevention of skin inflammation.

[0038] In some embodiments, the cosmetic and / or non-therapeutic treatment and / or care is the treatment and / or prevention of skin pigmentation.

[0039] In some embodiments, the cosmetic and / or non-therapeutic treatment and / or care is the maintenance and / or improvement of skin radiance.

[0040] In some embodiments, the cosmetic and / or non-therapeutic treatment and / or care is the maintenance and / or improvement of skin microcirculation.

[0041] In some embodiments, the cosmetic and / or non-therapeutic treatment and / or care is the maintenance and / or improvement of skin tone.

[0042] In some preferred embodiments, the cosmetic and / or non-therapeutic treatment and / or care is whitening.

[0043] In another aspect, the present invention provides a method for treating diseases using ELVs from plants, and corresponding pharmaceutical uses.

[0044] In some embodiments, a method for treating inflammation of the skin and / or mucous membranes is provided, comprising administering an ELV from a plant to an individual in need thereof. In some specific embodiments, the inflammation is selected from dermatitis, acne, pimples, and melasma.

[0045] In some embodiments, a method of treating a disease by administering antioxidant therapy to the skin and / or mucosal membranes is provided, comprising administering an ELV from a plant to an individual in need thereof. In some specific embodiments, the disease is photodermatitis.

[0046] In some embodiments, a method for whitening and / or treating pigmentation on the skin and / or mucosal membranes is provided, comprising administering an ELV derived from a plant to an individual in need thereof. In some specific embodiments, the disease is selected from melasma, lentigo, post-inflammatory hyperpigmentation, and hyperpigmentation of the skin caused by drugs and / or heavy metals.

[0047] In some specific embodiments, the method is performed by oral administration, topical administration, or parenteral administration.

[0048] Therefore, the present invention also provides the use of ELVs from plants in the preparation of medicaments. In some embodiments, the medicament is used to treat inflammation. In some specific embodiments, the inflammation is dermatitis, acne, pimples, chloasma, etc. In some embodiments, the medicament exerts an antioxidant effect. In some specific embodiments, the medicament is used to treat solar dermatitis. In some embodiments, the medicament exerts a whitening effect and / or treats pigmentation. In some specific embodiments, the medicament is used to treat chloasma, sunspots, post-inflammatory hyperpigmentation, or excessive skin pigmentation caused by drugs and / or heavy metals.

[0049] In some specific embodiments, the drug is administered orally, topically or parenterally. In some preferred embodiments, the drug is administered topically, for example, by topical application. In some preferred embodiments, the drug is a transdermal agent. In some preferred embodiments, the drug is an ointment.

[0050] In the above preferred aspects of the present invention, there is provided a naturally derived exosome-like vesicle carrier with smaller particles (nanoscale), better stability, higher safety and utilization, and richer effective substances, which has favorable activities.

[0051] The plant exosome-like vesicles obtained by the production process of the present invention have a small particle size, with an average particle size of approximately 186 nm. They are rich in active ingredients, primarily the flavonoids hesperidin, ophiopogon isoflavone A, and neohesperidin dihydrochalcone. These vesicles exhibit significant whitening, anti-inflammatory, and antioxidant effects. Derived from edible plants, these exosome-like vesicles have low immunogenicity and excellent safety and biotolerance. They facilitate the delivery of highly hydrophobic active ingredients such as flavonoids, enabling them to be directly taken up by cells and cross the skin barrier, thereby exerting their biological effects. These natural nanovesicles increase the water solubility and bioavailability of free flavonoids, significantly enhancing their potential applications. Furthermore, this formulation overcomes the limitations of traditional whitening preparations in applications such as medicine and skincare, lowering the minimum effective dose for equivalent efficacy and improving the safety and stability of these active ingredients.

[0052] Plants contain large amounts of plant cellulose and pectin. When extracting plant exosome-like vesicles, a higher initial mass of plant raw material means a higher content of plant cellulose and pectin mixed with the vesicles, which may affect vesicle release. Furthermore, the volume of extraction buffer added also affects the release of plant exosome-like vesicles. Therefore, the method of the present invention optimizes these two key factors, resulting in a composition with significantly higher ELV yield, purity, and concentration.

[0053] The flavonoid-rich plant exosome-like vesicles obtained using the method of the present invention have significant advantages. They can be used as excellent whitening, anti-inflammatory, and antioxidant agents for treating skin aging, skin pigmentation, and skin-related inflammatory diseases, such as dermatitis, acne, pimples, and melasma. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Figure 1 shows the characterization of grapefruit exosome-like vesicles. Panel A shows the size distribution of the obtained grapefruit exosome-like vesicles. Panels B and D show the average vesicle size, concentration in suspension, and average zeta potential, respectively. Panel E shows the shape of a typical vesicle particle obtained under a transmission electron microscope. GFEV = grapefruit exosome-like vesicle.

[0055] Figure 2Shown are the mass spectra of three major flavonoid compounds in grapefruit exosome-like vesicles (panels A, B, and C) and a comparative analysis of the three (panels D and E). Panels A to C show the mass spectra of ophiopogon isoflavone A, hesperidin, and neohesperidin dihydrochalcone detected by HPLC-MS / MS, respectively. The horizontal axis shows the mass-to-charge ratio (m / z), the number at the top of the peak line indicates the specific mass-to-charge ratio value of the corresponding substance, and the vertical axis shows the signal intensity. The bar charts in panels D and E respectively show the relative abundance and scores of these three substances.

[0056] Figure 3 Shown are the effects of flavonoid-rich grapefruit exosome-like vesicles on the viability of HFF-1 and HaCaT cells. Cell viability was assessed 24 and 48 hours after addition of different concentrations of grapefruit exosome-like vesicles to the cell cultures. Panels A and B correspond to HFF-1 and HaCaT cells, respectively. The horizontal axis represents the concentration of grapefruit exosome-like vesicles used for each bar, and the vertical axis represents cell viability. GFEV = grapefruit exosome-like vesicles.

[0057] Figure 4 Figure 2: Flavonoid-rich grapefruit exosome-like vesicles inhibit LPS-induced TNF-α production in RAW264.7 cells. Panel A shows the amount of TNF-α produced by cells in different treatment groups (expressed as concentration). The vertical axis shows the TNF-α concentration, and the horizontal axis indicates the concentration of grapefruit exosome-like vesicles in each treatment group, as well as the presence or absence of LPS and dexamethasone (Dex). ns = no statistically significant difference; ** = P < 0.01; *** = P < 0.001; **** = P < 0.0001. Panel B shows the inhibition rate (vertical axis) calculated from the amount of TNF-α produced by different treatment groups. GFEV = grapefruit exosome-like vesicles.

[0058] Figure 5 Shown are the effects of flavonoid-rich grapefruit exosome-like vesicles on hydrogen peroxide-induced cell viability in human fibroblast HFF-1 and human keratinocyte HaCaT cells. The bar graphs shown were generated by analyzing cell viability after adding different concentrations of grapefruit exosome-like vesicles to the cell cultures. Panels A and B correspond to HFF-1 and HaCaT cells, respectively. The horizontal axis indicates the concentration of grapefruit exosome-like vesicles used in the treatment group and whether or not H2O2 was added. The vertical axis represents cell viability. ns = not statistically significant; *** indicates p < 0.001; **** indicates p < 0.0001. GFEV = grapefruit exosome-like vesicles.

[0059] Figure 6Shown is the effect of flavonoid-rich grapefruit exosome-like vesicles on melanin production in B16F10 cells. After adding different concentrations of grapefruit exosome-like vesicles to cell cultures, the extracellular melanin content (panel A) and relative tyrosinase activity (panel B) were statistically analyzed to generate the bar graphs shown. The horizontal axis indicates the concentration of grapefruit exosome-like vesicles in the corresponding treatment group, as well as the presence or absence of α-melanocyte-stimulating hormone (α-MSH) and arbutin. The vertical axis indicates cell viability. ns = not statistically significant; ** indicates p < 0.01; **** indicates p < 0.0001. GFEV = grapefruit exosome-like vesicles.

[0060] Figure 7 Shown are the results of a zebrafish model assay using flavonoid-rich grapefruit exosome-like vesicles to inhibit melanin production. Panel A shows brightfield photographs of zebrafish models with different treatment groups. The concentration of grapefruit exosome-like vesicles used in each treatment group and the presence or absence of arbutin are indicated above the photographs. Panel B shows a statistical graph of melanin signal intensity analysis for the photograph in Panel A. Panels C and D show statistical graphs of melanin content and tyrosinase activity, respectively, calculated from total protein extracted from zebrafish with different treatment groups. ns = not statistically significant; * = P < 0.05; ** = P < 0.01; **** = P < 0.0001. GFEV = grapefruit exosome-like vesicles. Detailed Description of the Invention

[0061] Terms and Definitions

[0062] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0063] As used herein, when used with respect to a particular numerical value or range of values, the term "about" as used herein will be used with respect to the numerical value with which it is associated with a fluctuation of ±10%. For example, as used herein, the expression "about 100" includes 90 and 110 and all values ​​therebetween (e.g., 90.5, 95, 101, 105, 109.95, etc.). For ratios, the term "about" is used to qualify each digit of the given ratio. For example, a ratio of about 1:1 means a ratio of 0.9 to 1.1:0.9 to 1.1.

[0064] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, exemplary methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are incorporated herein by reference in their entirety.

[0065] As used herein, the term "weight percent by volume" or "% w / v" refers to the percentage weight (in grams) of a single component relative to the total volume of a mixture containing that component. For example, 500 mg of a component in a total volume of 8 mL is 6.25% w / v, and 500 mg of a component in a total volume of 5 mL is 10% w / v. Alternatively, it can be directly expressed as, for example, mg / mL.

[0066] In the context of the present invention, "skin" should be understood to encompass the uppermost layer or stratum corneum to the lowermost layer or subcutaneous layer. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, mast cells, neurons and / or adipocytes. "Skin" should also be understood to encompass different types of tissue, such as epithelial tissue, connective tissue such as the dermis, blood vessels, neural tissue, etc. The term "skin" also includes the scalp. Unless otherwise indicated in the context, when referring to "skin", it encompasses the skin of animals (e.g., mammals), and encompasses human skin. Similarly, the term "mucosal membrane" includes the mucous membranes of animals (e.g., mammals, such as humans).

[0067] The term "treatment" encompasses therapeutic methods, including methods involving the administration of a vesicle, composition, or medicament according to the present invention to alleviate or eliminate a disease or condition, or to reduce or eliminate one or more symptoms associated with the disease or condition. The term "treatment" also encompasses therapeutic methods involving the alleviation or elimination of the physiological consequences of a disease or condition. In the context of the present invention, the disease or condition particularly refers to a disease or condition that occurs in or affects the skin.

[0068] As used herein, the term "prevent" refers to the ability of the compounds of the present invention to prevent, delay, or hinder the onset or development of a disease or condition, or to prevent, delay, or hinder changes in the cosmetic properties of the skin and / or mucous membranes. As used herein, the term "prevent" is interchangeable with the term "inhibit," that is, it refers to the ability of the compounds of the present invention to inhibit the onset or development of a disease or condition, or to inhibit the deterioration of the cosmetic properties of the skin and / or mucous membranes.

[0069] When "treatment" and "care" are mentioned herein and modified by the attributives "cosmetic" and / or "non-therapeutic", it means that the purpose of the treatment or care is not to treat or prevent a disease, but to beautify and / or protect the skin, for example: to improve or maintain the aesthetic appearance of the skin and / or mucous membranes. Specifically, the purpose of "treatment" and "care" is to improve the aesthetic properties of the skin and / or mucous membranes, such as, but not limited to, the level of moisture, elasticity, firmness, gloss, color, tone or texture, which obviously affect the aesthetic appearance of the skin and / or mucous membranes. In the context of this specification, the term "care" refers to maintaining the properties of the skin and / or mucous membranes. The properties are improved or maintained by performing cosmetic treatment and / or care in healthy subjects, as well as in subjects with sensitive skin and subjects exhibiting diseases and / or conditions of the skin and / or mucous membranes (such as, but not limited to, ulcers and skin lesions, psoriasis, dermatitis, acne or rosacea, etc.).

[0070] In the context of the present invention, the term "aging" refers to changes that the skin undergoes with age (chronic aging) or through exposure to sunlight, ultraviolet rays (photoaging), or exposure to environmental factors such as strong oxidants, extreme climatic conditions such as extreme cold or dryness, tobacco smoke, chemical pollutants, and includes changes that are externally visible and / or perceptible by touch, such as but not limited to: skin discontinuities (such as wrinkles, fine lines, expression lines, stretch marks, grooves, unevenness or roughness, enlarged pores, Loss of hydration, loss of elasticity, loss of firmness, loss of smoothness, loss of deformation recovery ability, loss of resilience, sagging of the skin (such as sagging cheeks, bags under the eyes or double chin, etc.), changes in skin color (such as streaking, redness, bags under the eyes), or the appearance of pigmented areas (such as age spots or freckles, etc.), abnormal differentiation, hyperkeratosis, keratosis, hair loss, orange peel-like changes in the skin, damage to collagen structure, and changes in the stratum corneum, dermis, epidermis, microcirculatory system (for example, the appearance of spider veins or telangiectasia) or adjacent tissues, etc. The term "photoaging" refers to the acceleration of various changes caused by prolonged exposure of the skin to radiation, including ultraviolet radiation, resulting in premature aging of the skin, and it presents characteristics that are essentially the same as natural aging, including but not limited to flaccidity, sagging, pigmentation or unevenness, changes in skin color, abnormal and / or hyperkeratosis, etc.

[0071] Terms not specifically defined herein shall have their ordinary meanings as understood by those skilled in the art, for example, as explained in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

[0072] I. Exosome-like vesicles

[0073] In mammals, extracellular vesicles known as exosomes, likely secreted by mammals, have been found to play a key role in intercellular communication due to the presence of various bioactive molecules, such as lipids, proteins, DNA, and microRNA. Currently, exosomes are generally believed to be membrane vesicles with a lipid bilayer structure as their basic framework, encapsulating active substances such as the aforementioned proteins and nucleic acids. Exosomes are likely formed through a process of endocytosis, fusion, and exocytosis of extracellular vesicles, and can be obtained from cells, tissues, or body fluids of nearly all mammalian species. Exosomes also exhibit excellent molecular transport properties and biocompatibility, potentially making them useful for interspecies communication. For example, exosomes can directly activate receptors on the surface of target cells, modifying their properties and serving as transport vehicles for a variety of active molecules, including endogenous and exogenous proteins, miRNAs, and DNA.

[0074] As research deepens, extracellular vesicles (EVs), previously considered "cellular debris," have been identified in various plants. These EVs possess structures, properties, and functions similar to those of exosomes. For example, many of the proteins they contain overlap with those found in mammalian exosomes or belong to functional groups that characterize exosomes, regardless of their cellular origin. Most plant cell types are capable of secreting these "exosome-like" vesicles (ELVs), which transmit and exchange information between cells and / or organisms, or between cells and / or organisms and their environment. Their lipid bilayer structure provides them with inherent stability and superior tissue / cell penetration. ELVs can contain a variety of physiologically active substances, including proteins, lipids, nucleic acids, and small molecules. These ELVs have a phospholipid bilayer membrane structure and range in size from 30 to 1000 nm, with the vast majority being between 40 and 250 nm (hence their term "nanoparticles").

[0075] With the development of new treatments in recent years, researchers have sought to identify more efficient, non-toxic, environmentally friendly, precise, and low-cost delivery systems, leading to deeper research into plant-derived ELVs (PELVs). As particles with natural origins and ingredients, PELVs are not easily recognized by the immune systems of animals (especially humans), thus extending their circulation cycle and improving their bioavailability. Furthermore, because plants do not contain human or zoonotic pathogens, PELVs also offer reliable safety. In contrast, mammalian cell-derived ELVs and synthetic nanoparticles may have stronger immunogenicity, higher cytotoxicity, and complex and expensive manufacturing requirements, potentially leading to more complications. Therefore, the application value of plant-derived ELVs is gaining increasing recognition.

[0076] Citrus is an important genus in the Rutaceae family, with common species including grapefruit, orange, tangelo, and mandarin. It contains a rich variety of bioactive ingredients, primarily sugars, acids (e.g., citric acid), pectin, crude fiber, vitamins (e.g., vitamin C), minerals, coumarins, terpenes, alkaloids, flavonoids, and volatile components. In some embodiments of the present invention, ELVs are derived from plants in the genus Citrus, such as grapefruit, orange, tangelo, and mandarin.

[0077] In some embodiments, the raw material for extracting / producing ELV is a part of a plant, for example, any part selected from the group consisting of flowers, leaves, stems, branches, fruits, peels, seeds, seed coats, and roots of a plant, for example, fruits, for example, peels and / or pulp. In some embodiments, the part of the plant can be derived from any site selected from the group consisting of flowers, leaves, stems, branches, fruits, peels, seeds, seed coats, and roots of a plant, for example, any site of the peel and / or pulp.

[0078] In some embodiments, the ELV is enriched with one or more active substances. In some embodiments, the ELV is enriched with flavonoids. In some specific embodiments, the flavonoids include compounds selected from the group consisting of hesperidin, ophiopogon isoflavone A, neohesperidin dihydrochalcone, and any combination thereof. In some embodiments, the ELV is free of, or substantially free of, naringin and / or naringenin.

[0079] II. Composition

[0080] The ELV of the present invention is preferably prepared, stored and / or administered in the form of a composition containing the same.

[0081] Thus, in another aspect, the present invention provides a composition comprising the ELV of the present invention. In some specific embodiments, the composition can be a cosmetic, pharmaceutical, or food / health product composition.

[0082] In some embodiments, the composition of the present invention comprises an ELV of the present invention, wherein the ELV is 1.0×10 6 particles / mL, 5.0×10 6 Particles / mL, 1.0×10 7 particles / mL, 5.0×10 7 Particles / mL, 1.0×10 8 particles / mL, 5.0×10 8 Particles / mL, 1.0×10 9 particles / mL, 5.0×10 9 Particles / mL, 1.0×10 10 particles / mL, 2.0×1010 particles / mL, 2.4×10 10 particles / mL, 2.5×10 10 particles / mL, 5.0×10 10 particles / mL, 7.0×10 10 In some embodiments, the compositions of the present invention comprise an ELV of the present invention at a concentration of 1.0×10 8 particles, 5.0×10 8 particles, 1.0×10 9 particles, 5.0×10 9 particles, 1.0×10 10 particles, 5.0×10 10 particles, 1.0×10 11 particles, 2.0×10 11 particles, 4.0×10 11 particles, 4.8×10 11 particles, 6.0×10 11 particles, 8.0×10 11 The total amount of particles or higher is present.

[0083] The composition of the present invention generally comprises at least one cosmetically acceptable, pharmaceutically acceptable, or food / health product acceptable excipient or auxiliary material, such as, but not limited to, water, physiological (eg, isotonic) buffer, and the like.

[0084] From a cosmetic / cosmetic or pharmaceutical perspective, the compositions of the present invention, such as cosmetic compositions or pharmaceutical compositions, may contain a cosmetic / cosmetic or pharmaceutically (treatment / care / therapeutic) effective amount of the ELV of the present invention. The (therapeutically) cosmetic / cosmetic or pharmaceutically (treatment / care / therapeutic) effective amount will depend on many factors, including age, condition of the subject, degree of poor skin aesthetics, condition or disease to be treated and / or managed, route and frequency of administration, and properties of the ELV itself.

[0085] The terms "cosmetically / cosmetically effective amount" and "pharmaceutically effective amount" are understood to mean a non-toxic amount of the ELV of the present invention that is sufficient to provide the desired effect. The terms "pharmaceutically effective" and "therapeutically effective" are used interchangeably herein. The ELV of the present invention is used in the compositions of the present invention in a cosmetically / cosmetically or pharmaceutically effective amount to achieve the desired effect.

[0086] The term "delivery system" refers to a diluent, adjuvant, excipient or carrier used together with the ELV of the present invention. These cosmetic or pharmaceutical carriers can be liquids, such as water, oil or surfactants, including those of petroleum, animal, plant or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynol ether, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin, etc. Those skilled in the art will appreciate the diluents, adjuvants or excipients that can be used in the different delivery systems in which the compounds of the present invention can be administered.

[0087] The compositions containing the ELV of the present invention can preferably be used in different types of compositions for topical or transdermal administration, optionally comprising cosmetically / cosmetically or pharmaceutically acceptable excipients necessary to formulate the desired form of administration, and optionally, also comprising agents that increase the transdermal absorption of the ELV of the present invention.

[0088] The pharmaceutical composition of the present invention can be in different forms for oral administration, such as, for example, capsules (including gelatin capsules, soft capsules, hard capsules), tablets (including sugar-coated tablets, tablets, pills, powders, granules, chewing gum), solutions, suspensions, emulsions, syrups, elixirs, polysaccharide films, gels or gelatin and any other forms known to those skilled in the art. The food / health product composition of the present invention can be in any form, such as, but not limited to, dietary bars or compressed or non-compressed powders, jellies, beverages, dairy products or plant dairy products, etc., and formulated with common excipients and adjuvants of food supplements, such as, but not limited to, fat components, aqueous components, humectants, preservatives, leavening agents, flavorings, essences, antioxidants and coloring agents commonly used in the food industry.

[0089] The composition containing the ELV of the present invention can also be administered topically or transdermally, by any other suitable route (e.g., parenteral), wherein it will contain the necessary cosmetic / cosmetic or pharmaceutically or food / health product acceptable excipients to formulate the desired form of administration. In the context of the present invention, the term "parenteral" includes nasal, aural, ocular, rectal, urethral, ​​vaginal, subcutaneous, intradermal routes, intravascular injection (e.g., intravenous, intramuscular, intraocular, intravitreal, intracorneal, intraspinal, intramedullary, intracranial, intracervical, intracerebral, intrameningeal, intraarticular, intrahepatic, intrathoracic, intratracheal, intrathecal and intraperitoneal injection), and any other similar injection or infusion techniques.

[0090] In some embodiments, the cosmetic composition of the present invention is a cosmetic, such as, but not limited to, a skin care product. In some specific embodiments, the cosmetic composition of the present invention further comprises other active ingredients, such as, but not limited to, one or more selected from arbutin, phenylethylresorcinol, kojic acid, licorice extract, glabridin, tranexamic acid, niacinamide, mulberry bark extract, soybean extract, green tea extract, carotene, vitamin A and its derivatives, vitamin C and its derivatives, vitamin E and its derivatives, glycolic acid, mandelic acid, citric acid, malic acid, salicylic acid, bromelain, and papain, as a compound cosmetic product. In some specific embodiments, the cosmetic composition of the present invention further comprises an oiliness agent, such as, but not limited to, one or more selected from, such as, but not limited to, animal and plant oils (such as fatty acid triglycerides, coconut oil, olive oil, jojoba oil, palm oil, evening primrose oil, etc.), mineral oils (such as petrolatum, white oil, etc.), synthetic oils (such as squalane, caprylic / capric triglyceride, silicone oil, etc.), etc. In some specific embodiments, the cosmetic composition of the present invention further comprises an aqueous agent / humectant, such as (but not limited to) one or more selected from sodium hyaluronate, polyols (such as glycerin, polyethylene glycol, propylene glycol, butylene glycol, pentylene glycol, hexylene glycol, etc.), sorbitol, lactic acid / sodium lactate, sodium pyrrolidone hydroxy acid, etc. In some specific embodiments, the cosmetic composition of the present invention further comprises a thickener, such as (but not limited to) one or more selected from xanthan gum, agarose, gelatin, carrageenan, gellan gum, hydroxyethyl cellulose, hydroxypropyl cellulose, modified starch, and carbomer. In some specific embodiments, the cosmetic composition of the present invention further comprises an emulsifier, such as (but not limited to) one or more selected from SPAN, TWEEN, triethanolamine, stearoyl lactylate, alkylphenol polyoxyethylene ether, polyglycerol fatty acid ester, propylene glycol fatty acid ester, monolaurin, ethoxylated alkyl ammonium sulfate, sorbitan fatty acid ester, etc. In some specific embodiments, the cosmetic composition of the present invention further comprises a preservative, such as (but not limited to) one or more selected from parabens, quaternary ammonium salts, sodium benzoate, DMDM ​​hydantoin, methylisothiazolinone, etc. In some specific embodiments, the cosmetic composition of the present invention further comprises other excipients, such as (but not limited to) one or more selected from EDTA, TEA, antioxidants (such as BHT), etc.

[0091] In some embodiments, the cosmetic composition of the present invention is in the form of an emulsion or cream. In some specific embodiments, the emulsion or cream is prepared according to the following steps: (1) mixing the oily agent component in the formula under heating and stirring conditions until all are dissolved; (2) adding the aqueous agent component in the formula to pure water under heating and stirring conditions (the high molecular weight component is added first) and mixing until all are dissolved; (3) mixing the aqueous agent component with the oily agent component under heating and stirring conditions, and adding other ingredients (such as other active ingredients, thickeners, preservatives), and homogenizing until completely emulsified, wherein an emulsifier is optionally or as needed mixed with the oily agent component (1) or the aqueous agent component (2).

[0092] In some embodiments, the cosmetic composition of the present invention is in the form of a (water-based) serum. In some embodiments, the serum of the present invention is applied directly to the skin and / or applied to the skin in the form of a facial mask. In some specific embodiments, the serum is prepared by the following steps: the ingredients in the formula (e.g., the aqueous ingredient, active ingredient, thickener, preservative, etc.) are sequentially added to pure water (high-molecular-weight ingredients are added first) under heating and stirring conditions and mixed until completely dissolved and uniformly mixed.

[0093] III. Vesicle Preparation Method

[0094] Methods for isolating and purifying ELVs include differential centrifugation, density gradient centrifugation, ultrafiltration / filtration, immunomagnetic bead-based methods, polymer precipitation, size exclusion chromatography, and field flow fractionation. Due to their size and density, differential centrifugation is one of the best options for isolating ELVs from cells and tissues. While combining differential centrifugation with density gradient centrifugation can improve ELV purity, density gradient centrifugation cannot replace differential centrifugation alone due to its impact on production scale. Vesicles obtained using different isolation methods may have different structures, compositions, and / or activities.

[0095] Differential centrifugation uses different centrifugal forces to separate PDV from other substances. The crushed plant juice residue is centrifuged at low speed to remove tissues and cells, and then at medium and high speeds to remove cell debris, apoptotic bodies, etc. Finally, ultracentrifugation technology at 100,000g or even 150,000g is used to purify ELV from the supernatant into the precipitate.

[0096] The present invention provides a method for preparing ELV, comprising the following steps:

[0097] 1) cutting the plant or its parts into pieces and then crushing and homogenizing them;

[0098] 2) fully soaking the crushed juice and residue in an isotonic or substantially isotonic liquid and stirring to obtain a residue liquid;

[0099] 3) Filter the residue through gauze to obtain a crude extract, centrifuge at approximately 2000-4000 g and approximately 10,000 g at 4°C for 10-40 min, respectively, to obtain the supernatant, and then ultracentrifuge at at least 80,000 g at 4°C to obtain the precipitate;

[0100] 4) Resuspend the precipitate with physiological saline to obtain a vesicle suspension.

[0101] In some embodiments, the plant or its part weighing about 50 to 200 g, preferably about 100 g, is crushed in step 1). In some specific embodiments, the plant fruit weighing about 100 g is crushed in step 1).

[0102] In some embodiments, step 1) comprises crushing the plant or its portion using a juicer. In some specific embodiments, the juicer is operated at a low speed. In some specific embodiments, the juicer is operated in an intermittent mode. In some specific embodiments, the juicer is operated in a continuous mode.

[0103] In some embodiments, step 2) is performed on ice. In some specific embodiments, the isotonic or substantially isotonic liquid used in step 2) is physiological saline or 1×PBS. In some specific embodiments, the ratio of the mass of the juice residue to the volume of the isotonic or substantially isotonic liquid used in step 2) is 1:1 to 1:10 (w:v), for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, preferably 1:1 to 1:6, more preferably 1:2 to 1:5, more preferably 1:3 to 1:4, and most preferably about 1:3.

[0104] In some embodiments, the soaking and stirring time in step 2) is about 0.5 to 3 hours, preferably about 1 hour.

[0105] In some embodiments, the stirring speed in step 2) is 50 to 500 rpm, preferably 100 to 250 rpm, and more preferably 150 rpm.

[0106] In some preferred embodiments, the ultracentrifugation in step 3) is performed at a temperature of at least 100,000 g. In some preferred embodiments, the ultracentrifugation in step 3) is performed at a temperature of at least 120,000 g. In some preferred embodiments, the ultracentrifugation in step 3) is performed for a time of 60 to 120 minutes. In some preferred embodiments, the ultracentrifugation in step 3) is performed for about 70 minutes.

[0107] In some preferred embodiments, the saline used to resuspend the vesicles in step 4) is pre-chilled. In some preferred embodiments, the volume of saline used to resuspend the vesicles in step 4) is 20 mL. In some preferred embodiments, step 4) further comprises centrifuging the vesicles again at approximately 2000-4000 g for 10-40 minutes at 4°C to obtain the supernatant to further purify the vesicles.

[0108] In a preferred embodiment, the preparation method of the ELV comprises the following steps:

[0109] 1) Rinse the outer peel of the grapefruit thoroughly with deionized water and drain as much water as possible. Weigh 100g of grapefruit, peel off the outermost peel, cut the remaining part into pieces of a size suitable for the juicer inlet, and place the pieces into the juicer feeding port. Use a pusher to push the plant into the cup body, turn the button to the low gear to start the juicer, and start the juicer. The juicer runs for 20 seconds and rests for 10 seconds until the crushing is complete;

[0110] 2) Collect the juice and residue into a beaker and place it on ice. Add 200-600 (preferably 300) mL of phosphate buffer solution (1×PBS) to the beaker at a mass-to-volume ratio of 1:1-1:6 (preferably 1:3). Soak and stir for 0.5-3 h (preferably 1 h). Set the stirrer speed to 100-250 rpm (preferably 150 rpm).

[0111] 3) Filter the residue through gauze into a new beaker to obtain a crude extract, centrifuge it (4°C, 3000 g, 20 min), and collect the supernatant. Centrifuge it again (4°C, 10000 g, 30 min), collect the supernatant, and then ultracentrifuge it (4°C, 120000 g, 70 min) and discard the supernatant.

[0112] 4) Resuspend the pellets in 20 mL of 0.9% sodium chloride injection in 50 mL centrifuge tubes. Centrifuge again (4°C, 3000 g, 10 min), discard the insoluble pellet, and retain the supernatant. Filter the final supernatant through a 0.45 μm filter in a biosafety cabinet to obtain the vesicle suspension.

[0113] Preferably, the composition of the present invention is obtained from the preparation method of the present invention, comprising the ELV of the present invention.

[0114] During the production of vesicle compositions, the initial input quality of raw materials and the volume of buffer added are crucial factors, limiting the yield and purity of plant exosome-like vesicles. The present method optimizes both of these key factors, resulting in compositions containing significantly higher ELV yields, purity, and concentration.

[0115] IV. Uses and Therapy

[0116] The present invention provides plant exosome-like vesicles rich in flavonoids or a composition containing the same for use in cosmetic and / or non-therapeutic treatment and / or care of skin and / or mucous membranes.

[0117] Therefore, the flavonoid-rich plant exosome-like vesicles of the present invention or compositions comprising the same are particularly useful in the protection and / or treatment and / or care of the skin and / or mucous membranes. Specifically, the protection, treatment and / or care is performed on the skin. In the context of the present invention, skin includes skin on the entire body, including the face (including the skin around the eyes), scalp, collarbone, neck, chest, arms, hands, legs, feet, thighs, hips, buttocks, stomach, trunk and genital area.

[0118] In one aspect, the present invention provides a use of flavonoid-rich plant exosome-like vesicles or a composition containing the same for treating and / or caring for skin and / or mucous membranes. The flavonoid-rich plant exosome-like vesicles of the present invention reduce the level of oxidative stress in any localized tissue within the skin structure through antioxidant effects, thereby reducing damage to cells and tissues caused by free reactive oxygen species.

[0119] The present invention provides a use of a plant exosome-like vesicle rich in flavonoids or a composition comprising the same, which is used to maintain and / or improve skin microcirculation and / or skin color. Skin microcirculation is also referred to herein as skin blood flow, and is the blood flow in the small blood vessels of the skin (such as capillaries and arterioles in the skin). Skin color is closely related to skin microcirculation, wherein improved skin color is associated with increased skin microcirculation. The plant exosome-like vesicle rich in flavonoids of the present invention nourishes the microcirculatory endothelium by anti-oxidation and enhances endothelial function. Therefore, the present invention provides a use of a plant exosome-like vesicle rich in flavonoids for cosmetic, non-therapeutic treatment and / or care of the skin and / or mucous membranes, wherein the skin treatment and / or care is the maintenance and / or improvement of skin microcirculation and / or skin color.

[0120] The present invention provides a use of plant exosome-like vesicles rich in flavonoids or a composition containing the same for treating and / or preventing skin aging or skin aging symptoms. Symptoms of skin aging include treating and / or preventing skin wrinkles, dry skin, rough skin, decreased skin tone, and altered skin barrier function. Symptoms of skin aging may include wrinkles, dry skin, decreased skin tone, and altered skin barrier function. Symptoms of skin aging may include symptoms of aging caused by the presence of reactive oxygen species (ROS). Advantageously, the ELVs of the present invention have been found to have antioxidant effects, combating ROS; and previous studies have shown that natural products with excellent antioxidant activity have anti-elastase and anti-collagenase activity, thus playing a very important role in anti-aging skin formulations. Therefore, the present invention provides a use of plant exosome-like vesicles rich in flavonoids or a composition containing the same for cosmetic, non-therapeutic treatment and / or care of skin and / or mucous membranes, wherein the skin treatment and / or care is for the treatment and / or prevention of skin aging.

[0121] The present invention provides a use of plant exosome-like vesicles rich in flavonoids or a composition containing the same for alleviating or preventing skin inflammation. Reducing inflammation levels is beneficial for treating diseases associated with inflammatory responses. The plant exosome-like vesicles rich in flavonoids of the present invention suppress local inflammatory responses by downregulating inflammatory pathways within local tissue cells, such as the TNF-α signaling pathway. Therefore, the present invention provides a use of plant exosome-like vesicles rich in flavonoids for cosmetic, non-therapeutic treatment and / or care of the skin and / or mucous membranes, wherein the skin treatment and / or care is the treatment and / or prevention of skin inflammation.

[0122] The present invention provides a use of plant exosome-like vesicles rich in flavonoids or a composition containing the same for reducing or preventing skin pigmentation. Advantageously, it has been found that the ELVs of the present invention can inhibit melanin synthesis and reduce intracellular tyrosinase activity, thereby reducing melanin levels. Therefore, the present invention provides a use of plant exosome-like vesicles rich in flavonoids for cosmetic, non-therapeutic treatment and / or care of the skin and / or mucous membranes, wherein the skin treatment and / or care is the treatment and / or prevention of skin pigmentation.

[0123] The present invention provides a use of plant exosome-like vesicles rich in flavonoids or a composition containing the same for maintaining and / or improving skin gloss and / or skin tone. The ELVs of the present invention enhance the energy supply / nutrition status of the skin by reducing intracellular melanin, antagonizing ROS oxidation, and inhibiting inflammatory responses. Therefore, the present invention provides a use of plant exosome-like vesicles rich in flavonoids for cosmetic, non-therapeutic treatment and / or care of the skin and / or mucous membranes, wherein the skin treatment and / or care is to maintain and / or improve skin gloss and / or skin tone.

[0124] In some preferred embodiments, a plant exosome-like vesicle rich in flavonoids is provided for whitening.

[0125] The present invention also provides plant-derived ELVs, or compositions comprising the same, methods for treating diseases, and corresponding pharmaceutical uses. In some cases, due to various internal and / or external factors, skin conditions continue to deteriorate, exceeding the normal physiological homeostasis range, leading to localized skin disease states. In such embodiments, the present invention provides methods for treating diseases or conditions of the skin and / or mucous membranes. The ELVs of the present invention or compositions comprising the same help restore signal transduction in unhealthy cells, triggering their regeneration, thereby helping skin tissue heal and recover faster from pathological conditions.

[0126] In some embodiments, a method for treating inflammation at the skin and / or mucosal site is provided, comprising administering an ELV from a plant to an individual in need thereof. In some specific embodiments, the inflammation is dermatitis, acne, pimples, chloasma, etc.

[0127] In some embodiments, a method of treating a disease by administering antioxidant therapy to the skin and / or mucosal membranes is provided, comprising administering an ELV from a plant to an individual in need thereof. In some specific embodiments, the disease is photodermatitis.

[0128] In some embodiments, a method for whitening and / or treating a pigmentation-related disease at the skin and / or mucosal site is provided, comprising administering an ELV from a plant to an individual in need thereof. In some specific embodiments, the disease is melasma, lentigo, post-inflammatory hyperpigmentation, and hyperpigmentation of the skin caused by drugs and / or heavy metals.

[0129] Therefore, the present invention also provides the use of plant-derived ELVs in the preparation of medicaments. In some embodiments, the medicament is used to treat inflammation. In some specific embodiments, the inflammation is dermatitis, acne, pimples, chloasma, etc. In some embodiments, the medicament exerts an antioxidant effect. In some specific embodiments, the medicament treats solar dermatitis. In some embodiments, the medicament exerts a whitening effect and / or treats pigmentation. In some specific embodiments, the medicament treats chloasma, sunspots, post-inflammatory hyperpigmentation, and hyperpigmentation of the skin caused by drugs and / or heavy metals.

[0130] For the above-mentioned methods and uses of the present invention, ELV can be administered orally, topically or parenterally. Although the ELV of the present invention can be applied directly to the skin in a preferred topical administration mode, topical or transdermal application does not exclude the following modes of administration: for example, but not limited to, iontophoresis, ultrasonic phacophoresis, electroporation, mechanical pressure, osmotic pressure gradient, occlusive therapy, microinjection, microneedle (or microneedle array), needle-free injection by pressure, by micro-electric patch, facial mask or any combination thereof. Among them, some technologies that create channels in the skin (such as microneedles or lasers) themselves as a certain treatment method can be used in combination with ELV therapy to help ELV be absorbed and reach deeper skin layers, thereby enhancing its activity. The local area of ​​such combined treatment will be determined by the nature of the condition, disorder and / or disease to be treated and / or processed.

[0131] For the above-mentioned method of the present invention, the frequency of application or administration can vary greatly depending on the needs of each subject, and it is recommended to apply from once a month to ten times a day, preferably from once a week to four times a day, more preferably three times a week to twice a day, and even more preferably once a day. The application or administration of the compound of the present invention or the composition comprising the compound of the present invention can be carried out at any time.

[0132] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental procedures not involving specific conditions in the following examples are generally performed under conventional conditions, such as those specified in "Molecular Cloning Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989) compiled by Sambrook et al., or under conditions recommended by the manufacturer. Example

[0133] Example 1 Extraction and Characterization of Grapefruit Exosome-like Vesicles

[0134] After comparing the results of multiple extraction methods, the following extraction protocol for grapefruit exosome-like vesicles was selected: 1) Thoroughly rinse the outer peel of the grapefruit with deionized water and drain as much water as possible. Weigh 100 g of grapefruit. Peel off the outermost peel and cut the remaining portion into pieces suitable for the juicer inlet. Place the pieces into the juicer inlet and use a pusher to push the juicer into the cup. Turn the knob to low speed to start the juicer. Run the juicer for 20 seconds, then rest for 10 seconds until the juice is completely crushed. 2) Collect the juice and residue into a beaker and place it on ice. Add 300 mL of phosphate buffered saline (1× PBS) at a mass-to-volume ratio of 1:3 to the beaker. Soak and stir for 0.5–3 h (preferably 1 h) using a stirrer (DLAB, OS20-S) at 100–250 rpm (preferably 150 rpm). 3) Filter the residue through gauze into a new beaker to obtain a crude extract. Centrifuge the crude extract at 3000g for 20 minutes at 4°C (Eppendorf, 5804R). Collect the supernatant and centrifuge again at 10,000g for 30 minutes at 4°C. Collect the supernatant again and ultracentrifuge at 120,000g for 70 minutes at 4°C (Beckman, XE-90), retaining the precipitate. 4) Resuspend the precipitate in a total volume of 20 mL of 0.9% sodium chloride injection solution in a 50 mL centrifuge tube. Centrifuge again (4°C, 3000g for 10 minutes), discard the insoluble precipitate, and retain the supernatant. Filter the final supernatant through a 0.45 μm filter in a biosafety cabinet (Thermofisher, 1374) to obtain the grapefruit-derived vesicle supernatant. Seal the tube with parafilm and store the sample in a -80°C freezer.

[0135] Regarding the biological characteristics of the obtained grapefruit exosome-like vesicles, the particle size, concentration and Zeta potential of the grapefruit exosome-like vesicles were first detected by nanoparticle tracking analyzer (NTA, zetaview, PMX120-ZF). The results showed that the particle size of the grapefruit exosome-like vesicles produced by this process mainly ranged from 50 to 450 nm ( Figure 1 A), with an average particle size of about 186 nm ( Figure 1 B), with an average concentration of 2.4×10 10 particles / mL( Figure 1 C), and the average zeta potential is -34 mV ( Figure 1 D).

[0136] Transmission electron microscopy (FEI, Tecnai G2 Spirit BioTwin) showed that the vesicles were intact in morphology and had a saucer-like structure ( Figure 1 E).

[0137] Example 2 Identification of active substances in grapefruit exosome-like vesicles

[0138] In order to evaluate the main components and relative abundance of the effective substances in the extracted grapefruit exosome-like vesicles, high performance liquid chromatography-mass spectrometry (HPLC-MS / MS) was used to detect the contents of grapefruit exosome-like vesicles. HPLC was run on a capillary high performance liquid chromatograph (Thermo Fisher Scientific, model Ultimate 3000), and mass spectrometry was performed on a mass spectrometer (AB SCIEX TM ,TripleTOF5600+).

[0139] The specific steps are as follows: take 100 μL of grapefruit exosome-like vesicle solution, add 300 μL of methanol, shake at room temperature (12000 rpm, 10 min), and collect the supernatant. Set the liquid chromatography parameters, and use electrospray ionization (ESI) positive and negative ion modes for primary mass spectrometry and secondary mass spectrometry detection. The wiff file collected by mass spectrometry is preprocessed by the software MS-DIAL4.70, including peak extraction, denoising, deconvolution, peak alignment, and export of a three-dimensional data matrix in CSV format. The extracted peak information is compared with the database, and the three libraries of MassBank, Respect and GNPS are searched in the entire library. This three-dimensional data matrix includes information such as sample information, retention time, mass-to-nuclear ratio, and mass spectrometry response intensity (peak area).

[0140] The relative abundance of the substances was analyzed based on their peak areas.

[0141] The results showed that the obtained grapefruit exosome-like vesicles were rich in flavonoids, such as hesperidin (mass spectrometry secondary spectrum as shown in Figure 2). Figure 2 B), Ophiopogon japonicus high isoflavone A (mass spectrometry secondary spectrum as shown Figure 2 A) and neohesperidin dihydrochalcone (mass spectrometry secondary spectrum as Figure 2 C), but contains no or almost no naringin and naringenin (results not shown). Among the three flavonoids, hesperidin and ophiopogon isoflavone A have the highest abundance ( Figure 2 D), are among the top five most abundant active substances in the vesicle contents. In addition, according to the total score of the compound detection results, the scores of these three flavonoids are all higher than 90 points; among them, the scores of hesperidin and ophiopogon isoflavone A are close to 100 points ( Figure 2 E). Therefore, these results indicate that hesperidin, ophiopogon isoflavone A, and neohesperidin dihydrochalcone are indeed present in grapefruit exosome-like vesicles, and the contents of hesperidin and ophiopogon isoflavone A are higher.

[0142] Example 3 Cytotoxicity Detection of Grapefruit Exosome-like Vesicles

[0143] To confirm whether grapefruit exosome-like vesicles rich in hesperidin are cytotoxic, CCK-8 cell viability assay was used to detect the effects of different concentrations of grapefruit exosome-like vesicles (grapefruit ELV) on the viability of human fibroblasts HFF1 (Zhejiang Meisen Cell Technology Co., Ltd., TCC-001-0346) and human keratinocytes HaCaT (Wuhan Shanen Biotechnology Co., Ltd., SNL-163).

[0144] The specific steps are as follows: human fibroblasts HFF-1 and human keratinocytes HaCaT were cultured in 96-well culture plates (Corning, 3599) using 100 μL of DMEM medium (Thermofisher, 10569010) containing 10% FBS (Thermofisher, 10091-148), with 5×10 cells per well. 3 The cells were cultured overnight in a 5% CO2 incubator (Sanyo, MCO-20AIC) at 37°C. 6 particles / mL, 1.0×10 7 particles / mL, 1.0×10 8 particles / mL、5.0×10 8 particles / mL and 1.0×10 9 Cells were treated with grapefruit ELV at 500 μg / mL for 24 and 48 hours. 10 μL of enhanced CCK-8 cell viability assay reagent was then added to each cell, gently mixed, and incubated in a CO2 incubator (5% CO2, 37°C) for 1 hour. The absorbance at 450 nm was measured using a microplate reader (Thermofisher, Multiskan FC).

[0145] The results showed that compared with the control group, human fibroblasts ( Figure 3 A) and human keratinocytes ( Figure 3 B) The relative cell viability percentages did not change significantly, indicating that at this concentration, grapefruit exosome-like vesicles enriched in hesperidin had no cytotoxicity to human fibroblasts and human keratinocytes.

[0146] Example 4 Detection of the anti-inflammatory effect of grapefruit exosome-like vesicles in vitro

[0147] To evaluate whether grapefruit exosome-like vesicles rich in flavonoids have anti-inflammatory effects, lipopolysaccharide (LPS)-induced mouse macrophage RAW264.7 (ATCC, TIB-71) cells were treated with different concentrations of vesicles, and the TNF-α levels produced were detected by ELISA.

[0148] The specific steps are as follows: Mouse macrophage RAW264.7 cells were cultured in a 96-well cell culture plate using 100 μL of DMEM medium containing 10% FBS, with 6×10 cells per well. 3 The cells were cultured overnight in a CO2 constant temperature incubator (5% CO2, 37°C). Each plate was set up with a control group, a lipopolysaccharide (LPS) model group, a LPS and positive drug dexamethasone (Dex) co-treatment group, and a LPS and different concentrations of grapefruit ELV co-treatment group. First, a concentration of 1.0×10 6 particles / mL, 1.0×10 7 particles / mL, 1.0×10 8 particles / mL、5.0×10 8 particles / mL and 1.0×10 9 Cells were treated with grapefruit ELV at 1000 particles / mL for 24 hours. The culture medium was then discarded and replaced with DMEM medium containing 10% FBS and LPS. The cells were incubated in a CO2 incubator (5% CO2, 37°C) for 4 hours. The culture medium was harvested and centrifuged (4°C, 500g, 5 minutes), and the supernatant collected. TNF-α concentrations were measured using an R&D TNF-α ELISA kit.

[0149] The results showed that when the concentration of the administered vesicles was greater than 1×10 8 particles / mL, the TNF-α level was significantly reduced in a dose-dependent manner ( Figure 4 A); With the increase of vesicle administration concentration, the inhibition rate of TNF-α gradually increased, and the highest inhibition rate could reach 61% ( Figure 4 B). Therefore, the results indicate that grapefruit exosome-like vesicles rich in flavonoids can inhibit the production of inflammatory factor TNF-α and have significant anti-inflammatory effects.

[0150] Example 5 Detection of the Antioxidant Effect of Grapefruit Exosome-like Vesicles in Vitro

[0151] To confirm whether grapefruit exosome-like vesicles rich in flavonoids have antioxidant effects, human fibroblasts HFF-1 and human keratinocytes HaCaT cells subjected to hydrogen peroxide-induced oxidative stress were treated with different concentrations of vesicles, and their relative cell viability was analyzed to determine the antioxidant effect of the vesicles.

[0152] The specific steps are as follows: HFF-1 and HaCaT were cultured in 96-well cell culture plates using 200 μL of DMEM medium containing 10% FBS, with 1.5×10 4 The cells were cultured overnight in a CO2 constant temperature incubator (5% CO2, 37°C). Each plate was divided into a blank group, a control group, a 900μM hydrogen peroxide (H2O2) modeling group, and a test group treated with 900μM H2O2 and different concentrations of grapefruit ELV. 6 particles / mL, 1.0×10 7 particles / mL, 1.0×10 8 particles / mL、5.0×10 8 particles / mL, 1.0×10 9 particles / mL and 2.0×10 9 Cells were treated with grapefruit ELV at 500 μg / mL and incubated in a CO2 incubator (5% CO2, 37°C) for 24 hours. Hydrogen peroxide was then added to a final concentration of 900 μM and incubated in a CO2 incubator (5% CO2, 37°C) for 4 hours. The cells were rinsed with 1× PBS and then added with enhanced CCK-8 cell viability assay reagent. After gentle mixing, the cells were incubated in a CO2 incubator (5% CO2, 37°C) for 1.5 hours. The absorbance at 450 nm was measured using a microplate reader.

[0153] The results showed that the cell viability of HFF-1 and HaCaT cells increased with the increase of vesicle concentration in a dose-dependent manner ( Figure 5 A, B); where the concentration is 2×10 9 particles / mL vesicles can restore the activity of HFF-1 and HaCaT cells to the activity level of control cells ( Figure 5 A, B). These results indicate that grapefruit exosome-like vesicles rich in flavonoids can reduce the level of cellular oxidative stress caused by hydrogen peroxide and have significant antioxidant effects.

[0154] Example 6 In vitro whitening efficacy and tyrosinase activity detection of grapefruit exosome-like vesicles

[0155] To confirm the effect of grapefruit exosome-like vesicles rich in flavonoids on melanin production, we used the commonly used mouse melanoma cell model B16F10 (Wuhan Pronocell Life Science Co., Ltd., CL-0319) to explore the effect as follows:

[0156] In a 96-well cell culture plate, mouse melanoma B16F10 cells were cultured in 200 μL of DMEM medium containing 10% FBS, with 1.5×10 cells per well. 4 The cells were placed in a CO2 constant temperature incubator (5% CO2, 37°C) and cultured overnight. Each plate was set up with a control group, a 2μM α-MSH modeling group, a positive drug 100μM arbutin (Arbutin) treatment group after modeling, and different concentrations of grapefruit ELV (concentration of 3.0×10 8 particles / mL, 1.0×10 9 particles / mL and 2.0×10 9 The experimental group was treated with 100 μg of the co-treated 5% dapoxetine (particles / mL). After drug addition, the culture plates were incubated in a CO2 incubator (5% CO2, 37°C) for 48 hours. The culture medium was collected and centrifuged (4°C, 300g, 5 minutes), and the supernatant was collected. 150 μL of the supernatant was aspirated and added to a 96-well plate, and the absorbance at 492 nm was measured using a microplate reader.

[0157] After removing the cell culture medium from the 96-well culture plate, rinse the cells with 1× PBS and then lyse them with 1% Triton X-100. Collect the cell lysate and centrifuge it at 12,000 g for 15 minutes at 4°C (Eppendorf, 5430R). Collect 80 μL of the supernatant and transfer it to a 96-well plate. Add 20 μL of 2 mg / mL levodopa solution to each well and incubate in a 37°C metal bath for 1 hour. Then, measure the absorbance at 492 nm using a microplate reader and calculate the relative tyrosinase activity.

[0158] The results showed that compared with the α-MSH model group, the 3×10 8 , 1×10 9 and 2×10 9 The melanin levels of the three groups of B16F10 cells treated with 1000 particles / mL vesicles were significantly reduced in a dose-dependent manner ( Figure 6 A). Tyrosinase is the rate-limiting enzyme in the process of melanin synthesis, so the activity of tyrosinase was further tested. The results showed that after 3×10 8 , 1×10 9 and 2×10 9 The tyrosinase activity of the three groups of B16F10 cells treated with particles / mL vesicles was also significantly reduced ( Figure 6 B). Therefore, the results indicate that grapefruit exosome-like vesicles rich in flavonoids can inhibit melanin production in mouse melanoma cells B16F10.

[0159] Example 7: Detection of grapefruit exosome-like vesicles inhibiting zebrafish melanin synthesis

[0160] In order to further confirm that grapefruit exosome-like vesicles rich in flavonoids can also inhibit melanin production in animal models, the zebrafish model commonly used in the cosmetics field was used for research.

[0161] The method is as follows: three experimental groups were set up, namely 1% β-arbutin treatment group (positive control) and two groups of grapefruit ELV treatment groups with different concentrations, and a negative control group. Fifteen wild-type AB strain zebrafish embryos 6 hours after fertilization were randomly selected from each group and placed in a 6-well culture plate. 1% β-arbutin and grapefruit ELV with different concentrations (concentration of 1.0×10 8 particles / mL and 3.0×10 8 The cells were mixed and incubated in a 28°C biochemical incubator in the dark for 45 hours. Subsequently, 10 zebrafish were randomly selected from each group and photographed under a dissecting microscope with a 4x objective. The intensity of melanin signal from the zebrafish heads was analyzed using ImageJ, an advanced image processing software. Subsequently, 1 mL of lysis buffer was used to treat each zebrafish group, and the crude total protein extract was obtained by grinding. The extract was centrifuged (4°C, 12,000 g, 15 min), and the supernatant was collected. Total protein concentration was determined by the BCA assay. A standard curve was prepared using a melanin standard. The absorbance of the standard and the test samples at 405 nm was measured using a microplate reader to calculate melanin content. Separately, 250 μg of zebrafish total protein solution was added to 450 μL of 1 mM L-DOPA solution, mixed thoroughly, and 200 μL of the mixture was transferred to a 96-well plate and incubated in a 37°C incubator for 1 hour. Finally, the absorbance at 475 nm was detected using a microplate reader and the relative tyrosinase activity was calculated.

[0162] The results showed that compared with the control group, 1×10 8 particles / mL and 3×10 8 The melanin signal intensity of the two groups of zebrafish treated with particles / mL vesicles was significantly reduced in a dose-dependent manner. 8 The effect of particles / mL vesicles was more significant ( Figure 7 A, B). The quantitative results of zebrafish melanin are also consistent with the above results ( Figure 7 C). The activity of tyrosinase, which is highly related to the production of zebrafish melanin, was further tested. The results showed that 1×10 8 particles / mL and 3×10 8 The activity of zebrafish tyrosinase was significantly decreased after the administration of particles / mL vesicles ( Figure 7 D).

[0163] Therefore, these results indicate that grapefruit exosome-like vesicles rich in flavonoids can indeed significantly inhibit the production of melanin in zebrafish and have the potential for whitening and freckle removal applications.

[0164] The present invention is not limited to the scope of the specific embodiments and specific examples described herein. In fact, in addition to the content described above, those skilled in the art will also be able to readily anticipate various variations of the present invention. Such variations are also intended to fall within the scope of the appended claims.

[0165] References:

[0166] 1. Han HJ, Park SK, Kang JY, et al., Anti-melanogenic effect of ethanolic extract of sorghum bicolor on IBMX–induced melanogenesis in B16 / F10melanoma cells. Nutrients. 2020; 12(3):832.

[0167] 2.Wang LX, Qian J., Zhao LN, et al., Effects of volatile oil from gingeron the murine B16 melanoma cells and its mechanism. Food&Function.2018;9(2):1058-1069.

[0168] 3.Alam MB, Seo BJ, Zhao P., et al., Anti-melanogenic activities of heracleum moellendorffii via ERK1 / 2-mediated MITFdownregulation. International Journal of Molecular Sciences. 2016; 17(11):1844.

[0170] 4. Lee J.L., Lee W.J., Chang S.E., et al., Hesperidin, a popular antioxidant inhibits melanogenesis via Erk1 / 2 mediated MITF degradation. International Journal of Molecular Sciences. 2015;

[0171] 16(8):18384 - 95.

[0172] 5. Shi Q., Song X., Fu J., et al., Artificial sweetener neohesperidin dihydrochalcone showed antioxidative, anti - inflammatory and anti - apoptotic effects against paraquat - induced liver injury in mice.

[0173] International Immunopharmacology. 2015; 29(2):722 - 729.

[0174] 6. Lin M., Sun W., Gong W., et al., Methylophiopogonanone A protects against cerebral ischemia / reperfusion injury and attenuates blood - brain barrier disruption in vitro. PLoS One. 2015;

[0175] 10(4):e0124558.

[0176] 7. Guazelli C.F.S., Fattori V., Ferraz C.R., et al., Antioxidant and anti - inflammatory effects of hesperidin methyl chalcone in experimental ulcerative colitis. Chemico - Biological Interactions. 2020; 333:109315.

[0177] 8. Karg E., Odh G., Wittbjer A., et al., Hydrogen peroxide as an inducer of elevated tyrosinase level in melanoma cells. Journal of Investigative Dermatology. 1993;100(2 Suppl):209S-213S.

[0178] 9. Ahmed H.A., Aboul-Enein A.M., Abou-Elella F., et al., Nano-formulations of hesperidin and essential oil extracted from sweet orange peel: chemical properties and biological activities. Egyptian Journal of Chemistry. 2021;64(9):5373-5385.

Claims

1. Exosome-like vesicles (ELVs) from plants or parts thereof, characterized in that Rich in flavonoids, wherein the plant is preferably citrus, more preferably grapefruit (Citrus Paradisi); wherein the plant part is preferably a fruit; wherein the flavonoids preferably comprise a compound selected from the group consisting of hesperidin, ophiopogon isoflavone A, neohesperidin dihydrochalcone, and any combination thereof; wherein the ELV preferably does not comprise naringin and / or naringenin.

2. A method for preparing an ELV from a plant or a part thereof, comprising the following steps: 1) Using plants or parts thereof (preferably fruits) as the starting material, cutting them into pieces and then crushing and homogenizing them, for example, by using a juicer; 2) soaking the crushed juice and residue in an isotonic or substantially isotonic liquid (e.g., normal saline or 1×PBS) and stirring for about 0.5 to 3 hours (preferably about 1 hour) to obtain a residue liquid, wherein the ratio of the initial raw material mass to the liquid volume is 1:1 to 1:10 (preferably 1:1 to 1:6, more preferably 1:3); 3) filtering the residue through gauze to obtain a crude extract, centrifuging the extract at about 2000-4000 g and then at about 10,000 g at 4°C for 10-40 min to obtain the supernatant, and then ultracentrifuging the extract at at least 80,000 g (preferably at least 100,000 g, more preferably at least 120,000 g) at 4°C for 70-120 min to obtain the precipitate; 4) resuspending the pellet with an isotonic or substantially isotonic liquid (e.g., physiological saline or 1× PBS), and centrifuging again at approximately 2000-4000 g for 10-30 min at 4° C. to obtain the supernatant to obtain a vesicle suspension. The plant is preferably a citrus plant, more preferably a grapefruit plant.

3. A method for preparing an ELV from a plant or a part thereof, characterized in that The following steps are involved: 1) Rinse the outer peel of the grapefruit thoroughly with deionized water and drain as much water as possible. Weigh 100g of grapefruit, peel off the outermost peel, cut the remaining part into pieces of a size suitable for the juicer inlet, and place the pieces into the juicer feeding port. Use a pusher to push the plant into the cup body, turn the button to the low speed gear to start the juicer, and operate the juicer in a cycle of 20 seconds on and 10 seconds off until the crushing and homogenization are completed; 2) Collect the juice and residue into a beaker and place it on ice. Add 100-600 (preferably 300) mL of phosphate buffer solution (1×PBS) to the beaker at a ratio of initial raw material mass to liquid volume of 1:1 to 1:6 (preferably 1:3). Soak and stir for 0.5-3 hours (preferably 1 hour). Set the stirrer speed to 100-250 rpm (preferably 150 rpm). 3) Filter the residue through gauze into a new beaker to obtain a crude extract, centrifuge it (4°C, 3000 g, 20 min), and collect the supernatant. Centrifuge it again (4°C, 10000 g, 30 min), collect the supernatant, and then ultracentrifuge it (4°C, 120000 g, 70 min) and discard the supernatant. 4) Dispense a total volume of 20 mL of 0.9% sodium chloride injection into each tube, resuspend the pellet, and collect the pellet into a 50 mL centrifuge tube. Centrifuge again (4°C, 3000 g, 10 min), discard the insoluble pellet, and retain the supernatant. Filter the final supernatant through a 0.45 μm filter in a biosafety cabinet to obtain the vesicle suspension.

4. A composition comprising the ELV according to claim 1 or the ELV prepared according to the method of claim 2 or 3.

5. The composition according to claim 4, which is a pharmaceutical composition, a cosmetic composition, or a food / health product composition.

6. The composition according to claim 5, wherein the cosmetic composition is a cosmetic, preferably a skin care product, more preferably an emulsion, a cream or a serum.

7. A method for producing a composition according to any one of claims 4 to 6, characterized in that The following steps are involved: 1) cutting the plant or its part (preferably the fruit) into pieces and then crushing and homogenizing the pieces, for example, by using a juicer; 2) fully soaking the crushed juice and residue in an isotonic or substantially isotonic liquid (e.g., normal saline or 1×PBS) and stirring for about 0.5 to 3 hours (preferably about 1 hour) to obtain a residue liquid; 3) filtering the residue through gauze to obtain a crude extract, centrifuging the extract at about 2000-4000 g and then at about 10,000 g at 4°C for 10-40 min to obtain the supernatant, and then ultracentrifuging the extract at at least 80,000 g (preferably at least 100,000 g, more preferably at least 120,000 g) at 4°C for 70-120 min to obtain the precipitate; 4) resuspending the pellet with an isotonic or substantially isotonic liquid (e.g., physiological saline or 1× PBS), and centrifuging again at approximately 2000-4000 g for 10-40 min at 4° C. to obtain the supernatant to obtain a vesicle suspension. Preferably the plant is citrus, more preferably grapefruit.

8. The method according to claim 7, characterized in that The following steps are involved: 1) Rinse the outer peel of the grapefruit thoroughly with deionized water and drain as much water as possible. Weigh 100g of grapefruit, peel off the outermost peel, cut the remaining part into pieces of a size suitable for the juicer inlet, and place the pieces into the juicer feeding port. Use a push rod to push the plant into the cup body, turn the button to the low speed gear to start the juicer, and start the juicer for 20 seconds and rest for 10 seconds until the crushing and homogenization are completed; 2) Collect the juice and residue into a beaker and place it on ice. Add 100-600 (preferably 300) mL of phosphate buffer solution (1×PBS) to the beaker at a ratio of initial raw material mass to buffer volume of 1:1 to 1:6 (preferably 1:3). Soak and stir for 0.5-3 hours (preferably 1 hour). Set the stirrer speed to 100-250 rpm (preferably 150 rpm). 3) Filter the residue through gauze into a new beaker to obtain a crude extract, centrifuge it (4°C, 3000 g, 20 min), and collect the supernatant. Centrifuge it again (4°C, 10000 g, 30 min), collect the supernatant, and then ultracentrifuge it (4°C, 120000 g, 70 min) and discard the supernatant. 4) Dispense a total volume of 20 mL of 0.9% sodium chloride injection into each tube, resuspend the pellet, and collect the pellet into a 50 mL centrifuge tube. Centrifuge again (4°C, 3000 g, 10 min), discard the insoluble pellet, and retain the supernatant. Filter the final supernatant through a 0.45 μm filter in a biosafety cabinet to obtain the vesicle suspension.

9. Use of the ELV according to claim 1 or the ELV obtained by the method according to claim 2 or 3 or the composition according to any one of claims 4 to 6 for cosmetic and / or non-therapeutic treatment and / or care of the skin and / or mucous membranes, Preferably, the cosmetic and / or non-therapeutic treatment and / or care is the treatment and / or prevention of skin aging, the treatment and / or prevention of skin inflammation, the treatment and / or prevention of skin pigmentation, the maintenance and / or improvement of skin color (e.g., whitening), the maintenance and / or improvement of skin gloss, or the maintenance and / or improvement of skin microcirculation.

10. Use of the ELV according to claim 1 or the ELV obtained by the method according to claim 2 or 3 or the composition according to any one of claims 4 to 6 in the preparation of a medicament, Preferably, The drug is used to treat inflammation of the skin and / or mucous membranes, and more preferably the inflammation is dermatitis, acne, pimples, chloasma, etc. Alternatively, the drug is used to exert antioxidant effects on the skin and / or mucous membranes, more preferably the drug is used to treat solar dermatitis, Alternatively, the drug is used to exert whitening effects and / or treat pigmentation on the skin and / or mucous membranes, more preferably the drug is used to treat melasma, lentigo, post-inflammatory hyperpigmentation, and hyperpigmentation of the skin caused by drugs and / or heavy metals, Preferably, the drug is administered orally, topically or parenterally.

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