Orchid exosome composition for enhancing skin barrier, whitening and anti-wrinkle, use thereof, kit structure thereof

Abstract

The present invention provides a composition for enhancing skin barrier, whitening and anti-wrinkle, comprising exosomes isolated from orchid as an active ingredient. Also provided is the use of exosomes for preparing a composition for enhancing skin barrier and anti-wrinkle, wherein the exosomes are isolated from orchid. Also provided is a freeze-dried crystal structure comprising a body composed of excipients and the above exosomes. Also provided is a skin care kit structure comprising the aforementioned freeze-dried crystal structure, achieving the effects of enhancing skin barrier, whitening and anti-wrinkle.

Description

Technical Field

[0001] This invention relates to an exosome composition, its uses, crystal structure and kit structure, and more particularly to an orchid exosome composition for enhancing the skin barrier, whitening and anti-wrinkle, its uses, freeze-dried crystal structure and skin care kit structure. Background Technology

[0002] Aging has long been a concern, with increased fine wrinkles, decreased skin elasticity / atrophy, and accompanying itching being hallmarks of skin aging. There are two main causes of skin aging: internal aging that occurs over time and external aging caused by environmental factors (prolonged sun exposure and smoking). Changes in the activity of antioxidant enzymes are a physiological phenomenon in the early stages of aging. For example, hydrogen peroxide (H2O2) is a common reactive oxygen species (ROS) in the skin, primarily cleared by catalase. Studies show that catalase activity is lower in the dermis of older adults, while hydrogen peroxide concentration increases. Therefore, as the skin's endogenous antioxidant capacity decreases with age, aging skin becomes more susceptible to damage from external ultraviolet radiation, pollution, and microorganisms, leading to skin barrier damage and subsequent inflammation.

[0003] Because the epidermis is more exposed to external stimuli than the dermis, the keratinocytes in the epidermis bear a higher burden of reactive oxygen species (ROS). These unstable free radical molecules attack lipids, proteins, and DNA in skin cells, leading to damage to cell structure, weakening the skin's barrier function, and making the skin more susceptible to environmental aggressors. ROS also promote the degradation of collagen and elastin, two key proteins that maintain skin firmness and elasticity. When these proteins are damaged, the skin loses elasticity, becomes loose, and develops wrinkles and fine lines. In addition, free radicals can make the skin dull and rough, accelerating the skin aging process.

[0004] Therefore, there is a real need to improve existing technologies to strengthen the skin barrier function in order to maintain stable skin health. Summary of the Invention

[0005] One embodiment of the present invention provides a composition for enhancing the skin barrier, whitening and anti-wrinkle, comprising exosomes isolated from orchids as active ingredients, wherein the orchid is Phalaenopsis aphrodite.

[0006] In some embodiments, the exosomes are separated from the flower part and peduncle of the orchid.

[0007] In some implementations, the average particle size of the exosomes is 20 nanometers to 500 nanometers.

[0008] In some embodiments, the concentration of exosomes includes 1×10⁻⁶. 6 granules / ml to 1×10 12 Particles / ml.

[0009] In some implementations, the transmembrane protein of the exosome includes CD9.

[0010] In some embodiments, the composition is for skin regeneration, reducing skin wrinkles, anti-skin aging, increasing skin hydration, improving skin elasticity, promoting wound healing, skin firming, skin whitening, or a combination thereof.

[0011] In some embodiments, the composition is used to improve skin cell damage and collagen loss caused by oxidative stress, increase the expression of genes of skin cells such as FBLN5, CERS3, COL13A1, COL15A1, IL-6, HAS1, CDSN or combinations thereof, increase free radical scavenging capacity, inhibit melanin production, or combinations thereof.

[0012] Another embodiment of the present invention provides the use of exosomes for preparing compositions that enhance the skin barrier, whiten and reduce wrinkles, wherein the exosomes are isolated from orchids, wherein the orchid is Phalaenopsis aphrodite.

[0013] In some embodiments, exosomes are prepared by the following steps: taking the flower parts and peduncles of orchids; mixing water with the flower parts and peduncles of orchids and then crushing them to obtain a crude extract; filtering the crude extract to obtain a coarse filtrate; and separating the coarse filtrate by tangential flow filtration to obtain exosomes.

[0014] Another embodiment of the present invention provides a freeze-dried crystalline structure comprising: a bulk and exosomes isolated from an orchid. The bulk is composed of an excipient, the orchid being a white Phalaenopsis aphrodite, wherein the exosomes are uniformly dispersed within the bulk.

[0015] In some embodiments, the excipients include trehalose, sodium hyaluronate, collagen, or combinations thereof.

[0016] In some embodiments, the body is composed of excipients and forms a network structure, in which exosomes are uniformly dispersed.

[0017] In some embodiments, the freeze-dried crystalline structure further contains active ingredients that are uniformly dispersed in the network structure.

[0018] In some embodiments, the active ingredient comprises peptides, growth factors, vitamin C, plant extracts, or combinations thereof.

[0019] In some implementations, exosomes have a double membrane structure.

[0020] In some implementations, the transmembrane protein of the exosome includes CD9.

[0021] In some implementations, the average particle size of the exosomes is 20 nanometers to 500 nanometers.

[0022] In some embodiments, the freeze-dried grain structure is prepared by the following steps: mixing the composition with an excipient to obtain a formulation; and freeze-drying the formulation to obtain a freeze-dried grain structure.

[0023] Another embodiment of the present invention provides a skincare product set structure, comprising: a first container, a first accommodating space, and a freeze-dried crystalline structure as described above. The first accommodating space is located within the first container, and the freeze-dried crystalline structure is located within the first accommodating space.

[0024] In some embodiments, the skincare kit structure further includes a second container, a second containment space, and a liquid excipient. The second containment space is located within the second container, and the liquid excipient is located within the second containment space, wherein the liquid excipient comprises water, saline solution, serum, or a combination thereof. Attached Figure Description

[0025] The various aspects of the invention will be most readily understood when read in conjunction with the accompanying drawings in the following detailed description. It should be noted that, according to industry standard operating procedures, the various features may not be drawn to scale. In fact, for clarity of explanation, the dimensions of the various features can be arbitrarily increased or decreased. To make the above and other objects, features, advantages, and embodiments of the invention more apparent and understandable, the accompanying drawings are described below:

[0026] Figure 1 Transmission electron microscope images of orchid exosomes according to some embodiments of the present invention;

[0027] Figure 2 Bar chart showing the size and concentration of orchid exosomes according to some embodiments of the present invention;

[0028] Figure 3 The above are high-performance liquid chromatography (HPLC) chromatograms of orchid exosomes according to some embodiments of the present invention.

[0029] Figure 4This is a bar chart showing the protective activity of orchid exosomes against keratinocytes according to some embodiments of the present invention. Experimental results were compared between groups using one-way ANOVA, with **p<0.01 and ****p<0.0001. Results are expressed as mean ± standard deviation (mean ± SD) (N = 2).

[0030] Figure 5 These are microscopic images illustrating the protective effect of orchid exosomes on keratinocytes according to some embodiments of the present invention. Cell images were recorded at a 10×10 coefficient.

[0031] Figure 6A Photographs and bar graphs showing the inhibition of melanin production by orchid exosomes on melanocytes according to some embodiments of the present invention;

[0032] Figure 6B Photographs and bar charts showing the inhibition of melanin production by orchid exosomes on melanocytes according to some embodiments of the present invention;

[0033] Figure 7 This is a heatmap of gene expression among groups on a keratinocyte platform, representing some embodiments of the present invention. Higher values ​​indicate greater gene expression, while lower values ​​indicate less gene expression. The comparison of expression levels of skin physiology-related genes is shown in a bar chart.

[0034] Figure 8A This is a bar chart showing the expression levels of FBLN5, CERS3, COL13A1, COL15A1, and IL6 genes in different groups on a keratinocyte platform, according to some embodiments of the present invention. FPKM is an abbreviation for Fragments Per Kilobase Per Million.

[0035] Figure 8B Bar chart showing the expression levels of skin physiology-related genes HAS1 and CDSN in different groups under the fibroblast platform according to some embodiments of the present invention.

[0036] Figure 9 A bar chart showing the results of skin irritation tests on orchid exosomes according to some embodiments of the present invention;

[0037] Figure 10 A bar chart showing the results of eye irritation tests on orchid exosomes according to some embodiments of the present invention;

[0038] Figure 11 This is a schematic diagram of the freeze-dried grain structure of some embodiments of the present invention;

[0039] Figure 12 This is a schematic diagram of the structure of a skincare product set according to some embodiments of the present invention.

[0040] Figure label:

[0041] 10: Picture

[0042] 11: Figure

[0043] 20: Figure

[0044] 30: Figure

[0045] 40: Figure

[0046] 50: Figure

[0047] 60A: Figure

[0048] 60B: Diagram

[0049] 70: Figure

[0050] 80A: Figure

[0051] 80B: Diagram

[0052] 90: Picture

[0053] 100: Freeze-dried grain structure

[0054] 110: Ontology

[0055] 120: Exosomes

[0056] 130: Active ingredient

[0057] 200: Skincare Set Structure

[0058] 210: First Container

[0059] 211: Bottle body

[0060] 212: First Accommodation Space

[0061] 213: Bottleneck

[0062] 214: Bottle mouth

[0063] 215: Bottle Cap

[0064] 220: Second container

[0065] 221: Bottle body

[0066] 222: Second Accommodation Space

[0067] 223: Liquid excipients Detailed Implementation

[0068] To make the description of this invention more detailed and complete, illustrative descriptions of embodiments and specific examples are provided below. However, these are not the only forms of implementing or utilizing the specific examples of this invention. The various embodiments described below can be combined or substituted with each other where advantageous, and other embodiments can be added to one embodiment without further description or explanation. In the following description, many specific details will be set forth in detail to enable the reader to fully understand the following embodiments. However, embodiments of the invention can also be practiced without such specific details.

[0069] Additionally, spatial relative terms, such as "down" and "up," are used to conveniently describe the relative relationship of one component or feature to other components or features in the accompanying drawings. These spatial relative terms are intended to encompass different orientations of the device during use or operation, in addition to those shown in the drawings. The device may be positioned otherwise (e.g., rotated 90 degrees or otherwise), and the spatial relative descriptions used herein may be interpreted accordingly.

[0070] In this document, unless otherwise specified in the text, the words “a” and “the” may refer to one or more. It will be further understood that the words “comprising,” “including,” “having,” and similar terms as used herein specify the features, regions, integers, steps, operations, components, and / or elements described herein, but do not exclude one or more other features, regions, integers, steps, operations, components, and / or groups thereof described or additionally described herein.

[0071] Furthermore, when a number or range of numbers is described using terms such as "about," "approximately," and the like, the term is intended to cover numbers within a reasonable range of variation that, as understood by one skilled in the art, inherently occurs during manufacturing. For example, based on known manufacturing tolerances associated with manufacturing characteristics (having properties associated with the number), a number or range of numbers covers a reasonable range including the described number, such as within + / - 10% of the described number.

[0072] Exosomes are small, membrane-bound vesicles secreted by cells, ranging in size from approximately 20 to 500 nm. They are bimembrane structures composed of proteins, lipids, and nucleic acids. Exosomes participate in intercellular communication by transporting specific proteins, nucleic acids, and low-molecular-weight metabolites. Due to their relatively low immunogenicity, tumor tropism, and targeting ability with both innate and acquired immune cells, exosomes have become a growing trend in the development of treatment and delivery systems for various diseases in recent years.

[0073] In theory, any cell can secrete exosomes. Animal and human stem cell exosomes have been shown to have multiple functions, including promoting tissue regeneration, anti-aging, and immune regulation. Related research has gradually expanded from medical functions to fields such as medical aesthetics and skincare products. However, there are no clear standards and regulations for animal stem cell exosomes in terms of regulation and application in skincare products.

[0074] Plants also secrete exosomes, known as plant-derivedexosome-like nanoparticles (PELNs). These are nanoscale vesicles secreted by plant cells, containing DNA, small RNA (sRNA), microRNA (miRNA), and proteins. Plant exosomes offer advantages such as natural origin, mass production capability, low immunogenicity, absence of zoonotic pathogens, higher bioavailability, and inherent biological activity. They also serve as carriers for skin penetration, assisting active substances in reaching the skin through skin-related glands (hair follicles, sebaceous glands, sweat glands), intercellular spaces, and cell penetration. Therefore, they are considered an economical production source, safer and more compliant than animal or human stem cell exosomes, thus representing a significant opportunity for innovative raw material development.

[0075] In this article, the term "orchid exosome" is also referred to as "Phalaenopsis Aphrodite exosome-like nanoparticles" or "Phalaenopsis Aphrodite-derived exosomes," referring to nano-sized vesicles secreted by cells into the extracellular space.

[0076] In some implementations, orchid exosomes can be isolated from orchids.

[0077] In some embodiments, orchid exosomes have a particle size of 20 nanometers to 500 nanometers, such as 25 nanometers, 30 nanometers, 35 nanometers, 40 nanometers, 45 nanometers, 50 nanometers, 60 nanometers, 70 nanometers, 80 nanometers, 90 nanometers, 100 nanometers, 150 nanometers, 200 nanometers, 250 nanometers, 300 nanometers, 350 nanometers, 400 nanometers, 450 nanometers, or any value between any two of these values.

[0078] In some embodiments, the concentration of orchid exosomes is contained in 1×10⁻⁶ units. 6 granules / ml to 1×10 12 The range of particles per milliliter, for example, 3 × 10⁻⁶. 6 Particles / ml, 5×10 6 Particles / ml, 7×106 Particles / ml, 9×10 6 Particles / ml, 1×10 7 Particles / ml, 3×10 7 Particles / ml, 5×10 7 Particles / ml, 7×10 7 Particles / ml, 9×10 7 Particles / ml, 1×10 8 Particles / ml, 3×10 8 Particles / ml, 5×10 8 Particles / ml, 7×10 8 Particles / ml, 9×10 8 Particles / ml, 1×10 9 Particles / ml, 5×10 9 Particles / ml, 1×10 10 Particles / ml, 5×10 10 Particles / ml, 1×10 11 Particles / ml, 5×10 11 Particles per milliliter, or any value between any two of these values.

[0079] In some embodiments, orchid exosomes can be obtained by methods including, but not limited to, ultracentrifugation, sucrose gradient centrifugation, microfiltration, tangential flow filtration, polymer precipitation, antibody magnetic bead separation, or combinations thereof.

[0080] The flower is the reproductive organ of angiosperms, its biological function being the fusion of male sperm cells and female egg cells to produce seeds. In this text, the term "floret" includes at least the petals, sepals, and column. The term "peduncle" is a peduncle located below the floret and used to support the inflorescence.

[0081] In some embodiments, orchid exosomes can be obtained by the following methods: taking the flower part and peduncle of an orchid; adding water to the flower part and peduncle of the orchid and crushing them to obtain a crude extract; filtering the crude extract to obtain a coarse filtrate; separating the coarse filtrate by tangential flow filtration to obtain orchid exosomes.

[0082] In some implementations, orchid exosomes are used as active ingredients to enhance the skin barrier and have anti-wrinkle effects.

[0083] In some embodiments, the present invention provides compositions for enhancing the skin barrier and reducing wrinkles.

[0084] In some embodiments, the composition may be a cosmetic composition.

[0085] In some embodiments, the cosmetic composition may include functional additives and ingredients commonly found in cosmetic compositions. Functional additives may include, but are not limited to, peptides, polysaccharides, water-soluble vitamins, oil-soluble vitamins, or combinations thereof. In addition, it may include, but is not limited to, oils, fats, wetting agents, lubricants, surfactants, organic or inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, pH control agents, alcohol, colorants, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, or combinations thereof.

[0086] In some embodiments, the composition may be a pharmaceutical composition.

[0087] In some embodiments, the pharmaceutical composition includes orchid exosome active ingredients, administered to an individual via oral or non-enteric route. In some specific embodiments of the invention, the orchid exosomes are formulated into an oral dosage form selected from the group consisting of solutions, suspensions, emulsions, powders, lozenges, pills, syrups, lozenges, tablets, chewing gums, and capsules for administration to an individual.

[0088] In some embodiments, the pharmaceutical composition includes, but is not limited to, orchid exosome active ingredients and pharmaceutically acceptable carriers, including, but not limited to, water, alcohols, glycols, preserving agents, antioxidants, solvents, emulsifiers, suspending agents, decomposers, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, lubricants, absorption enhancers, active agents, humectants, odor absorbers, fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, and penetration enhancers. Enhancers, anti-irritants, colorants, propellants, surfactants, and other similar or applicable carriers of the present invention.

[0089] In some embodiments, the composition may be a food composition.

[0090] In some embodiments, the food composition may be, for example, added to edible materials in the form of a food additive to prepare a food product for human or animal consumption. Food compositions include, but are not limited to, general foods, health foods, beverages, nutritional supplements, dairy products, or animal feed. In examples of oral dosage forms, the food composition may optionally include food-acceptable carriers, excipients, and / or additives. In other examples, the dosage form of a complex probiotic composition may include, but is not limited to, powders, tablets, granules, suppositories, microcapsules, ampoules, liquid sprays, or stoppers.

[0091] The following examples and experimental cases illustrate the orchid exosome composition of the present invention in more detail. However, they are for illustrative purposes only and are not intended to limit the present invention. The scope of protection of the present invention shall be defined by the appended claims.

[0092] This invention provides a composition for enhancing the skin barrier and reducing wrinkles, comprising orchid exosomes as an active ingredient. The composition is used for skin regeneration, reducing wrinkles, combating skin aging, increasing skin hydration, improving skin elasticity, promoting wound healing, or a combination thereof.

[0093] Example

[0094] Although the methods of this invention are described below using a series of operations or steps, the order in which these operations or steps are shown should not be construed as a limitation of the invention. For example, some operations or steps may be performed in a different order and / or simultaneously with other steps. Furthermore, it is not necessary to perform all the illustrated operations, steps, and / or features to achieve the implementation of the invention. In addition, each operation or step described herein may comprise several sub-steps or actions.

[0095] For clarity, features and components that are known in the domain and are not essential for understanding the principles described will be omitted.

[0096] Preparation Example 1: Preparation Method of Orchid Exosomes

[0097] Orchid flowers and peduncles were collected, mixed with 1.5 times their weight of water, and blended in a juicer to obtain a crude extract. The crude extract was then subjected to solid-liquid separation. The liquid fraction was filtered through a 0.22 μm membrane to remove impurities, yielding a crude filtrate. This filtrate was then concentrated and separated into orchid exosomes, or orchid-derived exosomes (OR-TFF), using tangential flow filtration (TFF) through a 100 kDa membrane. The purified orchid exosomes were examined using an electron microscope to confirm their appearance, and their particle size and concentration were analyzed using a nanoparticle size analyzer.

[0098] Example 1: Characteristic Analysis of Orchid Exosomes

[0099] The size and characteristics of the orchid exosomes obtained in Example 1 were analyzed by transmission electron microscopy (TEM). Figure 1 Transmission electron microscopy of orchid exosomes according to some embodiments of the present invention Figure 10 , Figure 1 The orchid exosomes were shown to have diameters ranging from 20 nanometers to 500 nanometers and were roughly spherical. Figure 2The bar chart 20 shows the size and concentration of orchid exosomes according to some embodiments of the present invention. Nanoparticle tracking analysis (NTA) confirmed that the concentration of orchid exosomes per 1 ml (mL) unit volume is 2.38 × 10⁻⁶. 10 indivual.

[0100] Example 2: Analysis of CD9 transmembrane protein, a specific biomarker for orchid exosomes

[0101] Previous studies have reported that plants contain transmembrane structures similar to CD9 in animal exosomes. Therefore, this embodiment attempts to use a CD9 detection kit (ExoELISA-ULTRA Complete Kit, Cat No. EXEL-ULTRA-CD9-1, SystemBioscience, UK) for labeling and detection. The results are shown in Table 1 below. Compared with the colorimetric values ​​of exosome standards, the orchid exosomes obtained in Preparation Example 1 also showed a colorimetric reaction under this platform. Therefore, it is speculated that the orchid exosomes obtained in Preparation Example 1, in addition to having particle size and image characteristics consistent with exosomes, also contain the common exosome-specific marker CD9 transmembrane protein in their lipid bilayer membrane.

[0102] Table 1

[0103]

[0104]

[0105] Example 3: HPLC analysis of secondary metabolites from orchid exosomes

[0106] The main components of plant-derived exosomes are proteins, lipids, nucleic acids, and secondary metabolites specific to different plants. These secondary metabolites are unique bioactive substances; compared to general plant extracts, the unique phospholipid bilayer structure of plant exosomes effectively seals in viable components, protecting them from environmental influences and degradation, and increasing bioavailability. Therefore, to understand the characteristics of secondary metabolites in orchid exosomes, HPLC was used for component analysis of secondary metabolites in exosomes of Phalaenopsis white.

[0107] Sample preparation: Orchid exosomes from Preparation Example 1 were filtered through a 0.45 μm filter membrane and analyzed by HPLC.

[0108] HPLC analysis conditions:

[0109] Mobile phase A: 0.1% phosphoric acid / water

[0110] Mobile phase B: Acetonitrile

[0111] Table 2

[0112]

[0113]

[0114] Chromatographic column: Shim-pack VP-ODS C18 chromatography column (2.0*150mm, 5.0μm, Shimadzu Corporation, Kyoto, Japan)

[0115] Flow rate: 0.3 mL / min, UV = 254 nm

[0116] Column temperature: 40℃, injection volume: 10μL

[0117] Figure 3 HPLC chromatogram 30 shows the results of orchid exosomes from some embodiments of the present invention. The results show that the orchid exosomes, in Preparation Example 1, had a density of 2.38 × 10⁻⁶. 10 At the same concentration, the peak with the same retention time as the standard of saponarin (IUPAC name: 5-Hydroxy-6-(β-D-glucopyranosyl)-7-(β-D-glucopyranosyloxy)flavone) indicates that the orchid exosomes contain the polyphenol component of saponarin.

[0118] Example 4: Protective activity of skin keratinocytes

[0119] In this embodiment, hydrogen peroxide was used to simulate oxidative stress to induce damage in human HaCaT keratinocytes. The protective activity against cells was evaluated after adding orchid exosomes obtained in Preparation Example 1. HaCaT keratinocytes were cultured in DMEM medium containing 100 units / mL penicillin, 100 μg / mL streptomycin, and 10% fetal bovine serum at 5% CO2 and 37°C. First, 1×10 4 Cells were cultured in 96-well culture dishes for 24 hours. The supernatant was removed the next day, and then 2.5% (i.e., 5.95 × 10⁻⁶) of the solution from Preparation Example 1 was added. 8 Particles / mL), 5% (i.e., 1.19 × 10⁻⁶) 9 Particles / mL) and 10% (i.e., 2.38 × 10⁻⁶) 9Orchid exosome cell culture medium containing granules / mL was reacted for 1 hour, followed by the addition of hydrogen peroxide to a final concentration of 150 μM and incubation for 16 hours. On the third day, the cells were reacted with 0.5 mg / mL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 1 hour, and then dissolved in dimethyl sulfoxide (DMSO). Cell viability in each group was expressed as OD0.05. 570 Absorbance values ​​are expressed as a percentage compared to the control group cells.

[0120] Figure 4 Bar chart 40 shows the protective activity of orchid exosomes against keratinocytes according to some embodiments of the present invention. The results show that hydrogen peroxide caused a decrease in cell viability (53.6 ± 1.1%) compared to the control group. After adding 2.5%, 5%, and 10% of the orchid exosomes obtained in Preparation Example 1, the viability recovered to 56.4 ± 1.4%, 58.8 ± 1.1%, and 66.4 ± 3.1%, respectively (p < 0.01), indicating that orchid exosomes have cell-protective and collagen-preserving activities under the plateau of oxidative stress-induced HaCaT damage.

[0121] In addition, after HaCaT keratinocytes were pretreated with orchid exosomes of different concentrations from Preparation Example 1, hydrogen peroxide was added to induce cell damage. After 16 hours, the cells were stained with Sirius Red and the cell images were observed under a microscope. Figure 5 Figure 50 shows a microscopic image of the protective effect of orchid exosomes on keratinocytes according to some embodiments of the present invention. The results show that, compared to the control group, oxidative stress induced by hydrogen peroxide significantly reduced the number of HaCaT cells and collagen. However, after administration of the orchid exosomes prepared in Example 1, the number of cells and collagen content tended to recover with increasing orchid exosome concentration. Therefore, this confirms that orchid exosomes have skin regeneration and wrinkle reduction effects.

[0122] Example 5: Melanin Inhibition Activity

[0123] In this example, 1-methyl-3-isobutyl-1-methylxanthine (IBMX) was used to induce melanin production in mouse B16-F0 melanocytes. The inhibitory activity against melanin production was evaluated after adding orchid exosomes obtained in Preparation Example 1. B16-F0 melanocytes were cultured in DMEM medium containing 100 units / mL penicillin, 100 μg / mL streptomycin, 72 μg / mL tyrosine, and 10% fetal bovine serum at 5% CO2 and 37°C. First, 1×10 4 The cells were cultured in a 35 mm culture dish for 24 hours. The supernatant was removed the next day, and then 2.5% (i.e., 5.95 × 10⁻⁶) of the solution from Preparation Example 1 was added. 8 Particles / mL), 5% (i.e., 1.19 × 10⁻⁶) 9 Particles / mL) and 10% (i.e., 2.38 × 10⁻⁶) 9 Orchid exosomes (particles / mL) were reacted with IBMX (100 μM) in cell culture medium for 3 days. On the third day, the cell culture supernatant and cell pellet were photographically recorded. To quantify melanin content, cells in each group were harvested with 0.25% Trypsin-EDTA, washed twice with 1×PBS, lysed with 1N NaOH, and reacted at 80℃ for 30 minutes. The cell lysate and supernatant were then analyzed by OD... 405 Perform absorbance readings.

[0124] Figure 6A Photographs and bar graphs (60A) showing the inhibition of melanin production by orchid exosomes on melanocytes according to some embodiments of the present invention. The results show that, in the supernatant portion, compared to the control group, IBMX induced melanin production and secretion into the culture medium. After adding 2.5%, 5%, and 10% of the orchid exosomes obtained in Preparation Example 1, the melanin content percentage (with the induction group as 100±3.1%) remained at 32.6±1.1%, 36.1±0.2%, and 24.9±0.5%, respectively. Figure 6B Photographs and bar graphs (60B) showing the inhibition of melanin production by orchid exosomes on melanocytes according to some embodiments of the present invention. In the cellular portion, compared to the control group, IBMX induced melanin production in cells (calculated with the induction group as 100%). After adding 2.5%, 5%, and 10% of the orchid exosomes obtained in Preparation Example 1, the remaining melanin percentages were 18.8%, 18.8%, and 6.3%, respectively, indicating that orchid exosomes can inhibit melanin production in B16-F0 cells induced by IBMX.

[0125] Example 6: Effects on the expression of anti-aging / moisturizing / repair genes in skin keratinocytes

[0126] In this embodiment, the same method as in Example 4 was used. HaCaT keratinocytes were pretreated with 10% of orchid exosomes from Preparation Example 1, and then hydrogen peroxide was added to induce a cell damage reaction for 16 hours. Alternatively, orchid exosomes were treated with HS-68 fibroblasts for 24 hours, followed by ribonucleic acid (RNA) extraction. After quality testing and library construction, the nucleic acids were sequenced using the Nova Seq XPlus Sequencing System to observe the effect of orchid exosomes on gene expression on this platform.

[0127] Figure 7 The gene expression heatmap 70 for each group on the keratinocyte platform in some embodiments of the present invention is as follows: the result is a gene expression heatmap classified by the Gene ontology database that is related to skin cell physiology (GO:0030216, GO:0008544, GO:0042438, GO:0006955, GO:0006954, GO:0070098, GO:0030198, GO:0030574, GO:0007155, GO:0009611, GO:0071711, GO:0061436, GO:0006583, GO:0042438, GO:0008283, GO:0040037). Figure 8A Bar graph 80A shows the expression levels of FBLN5, CERS3, COL13A1, COL15A1, and IL6 genes in different groups on the keratinocyte platform according to some embodiments of the present invention. Compared with the hydrogen peroxide group, a total of 41 genes showed reversion after administration of 10% orchid exosomes, including: FBLN5 associated with anti-aging, CERS3 a ceramide synthesis enzyme gene associated with moisturizing function, COL13A1 and COL15A1 both important genes of the skin epidermal basement membrane associated with skin elasticity, and IL6 associated with skin wound repair.

[0128] Figure 8B Bar chart 80B shows the expression levels of skin physiology-related genes HAS1 and CDSN in different groups on a fibroblast platform according to some embodiments of the present invention. In fibroblasts, orchid exosomes were found to promote the HAS1 hyaluronic acid synthesis enzyme gene, which is related to water retention, and the CDSN keratinocyte-desmosome gene (related to cell adhesion), which is associated with skin firmness.

[0129] The compositions of this invention effectively resist skin damage and collagen breakdown caused by environmental oxidative stress, and are used for skin regeneration, improving skin elasticity, or reducing wrinkles. These compositions also increase the expression levels of FBLN5, CERS3, COL13A1, COL15A1, and IL-6 genes in skin cells, thereby increasing skin hydration, preventing skin aging, and promoting wound repair.

[0130] Example 7: Determination of Free Radical Scavenging Ability

[0131] This example uses a DPPH (2,2-Diphenyl-1-picrylhydrazy, 2,2-diphenyl-1-trinitrophenylhydrazine) free radical scavenging capacity assay kit. Biotech Co., LTD. was used to evaluate the free radical scavenging ability. The experiment was conducted in a 96-well microplate. 10 μL of 100 v / v% orchid exosome stock solution or its dilution from Preparation Example 1 was uniformly mixed with 190 μL of prepared DPPH working solution. After a 10-minute reaction, the mixture was transferred to a Synergy H1 Hybrid Multi-Mode Microplate (Agilent Technologies). Detect OD 515 The absorbance value, and finally the DPPH free radical scavenging efficiency, is calculated using the following formula: DPPH free radical scavenging rate (D) VC % = [[(A B -(A T -A C )]÷A B ×100%.

[0132] Table 3. Results of DPPH free radical scavenging capacity determination of orchid exosomes

[0133]

[0134]

[0135] Example 8: Security Assessment

[0136] In this embodiment, a safety assessment test was conducted on an in vitro biomimetic skin platform, and a skin irritation test was performed on the orchid exosomes prepared in Example 1. The skin irritation test included the following procedures. This test was conducted according to the test kit (EpiSkin) recognized by the Organization for Economic Cooperation and Development (OECD) TG439. TMThe test was conducted using a human skin model (KIT, WPISKIN / S / 13). During the test, orchid exosomes from Preparation 1 were dissolved in 1×PBS to a final concentration of 10 v / v%, and then directly applied topically to the surface of the skin model. After a 15-minute reaction, the solution was washed off, and the skin model was cultured for 42 hours. The effect of Preparation 1 or the positive control group (5 wt% SDS) on cell viability was finally assessed using the MTT assay. Irritation was assessed according to Type 2 of the Globally Harmonized System of Classification and Labelling of Chemicals (UN GHS), where a cell viability ≤ 50% was considered skin irritating.

[0137] Figure 9 Bar chart 90 shows the results of skin irritation tests on orchid exosomes according to some embodiments of the present invention. Figure 9 These are the results of skin irritation tests on 10% exosomes, the control group, and the positive control group of this invention. Experimental data are expressed as mean ± SD, with a sample size of 3 for each group. After the skin irritation test, the survival rate of the control group was 100.0 ± 1.9%, the survival rate of 10% exosomes was 132.2 ± 2.6%, and the survival rate of the positive control group was 10.5 ± 0.2%. Figure 9 It can be seen that the 10% orchid exosomes prepared in Example 1 showed no irritation in the skin irritation test.

[0138] Furthermore, this embodiment also includes a safety assessment test for eye irritation, performing an eye irritation test on the orchid exosomes prepared in Example 1. This test was conducted according to OECD TG 492 specifications and using a test kit (Lab Cyt). TM The human corneal epithelial tissue model (CORNEA-MODEL, Cat. No. 411324) was used for testing. During the test, orchid exosomes prepared in Example 1 were dissolved in 1×PBS to a final concentration of 10 v / v%, and then directly applied topically to the surface of the corneal epithelial tissue model. After a 60-minute reaction, the mixture was washed off, and the corneal epithelial tissue model was cultured for 24 hours. The effect of 10% exosomes or the positive control group (ethanol) on cell viability was finally assessed using cell viability analysis (WST-8 assay kit). The irritation assessment criterion was based on UN GHS type 2; a cell viability ≤ 40% was considered ocular irritant.

[0139] Figure 10 A columnar section showing the results of eye irritation tests on orchid exosomes according to some embodiments of the present invention. Figure 11 , Figure 10These are the results of corneal epithelial tissue irritation tests conducted using 10% exosomes, a control group, and a positive control group, according to the present invention. Experimental data are expressed as mean ± standard deviation (mean ± SD), with a sample size of 3 for each group. Following the eye irritation test, as... Figure 10 As shown, the survival rate of the control group was 100.0±1.8%, the survival rate of 10% exosomes was 118.4±11.3%, and the survival rate of the positive control group was 18.2±5.1%. Figure 10 It can be seen that the 10% orchid exosomes prepared in Example 1 were non-irritating in the eye irritation test.

[0140] Example 9: Preparation of freeze-dried crystal structure of orchid exosomes

[0141] Using orchid exosomes (OR TFF) from Preparation Example 1 as raw material, freeze-drying was employed to create freeze-dried crystalline structures. This process prevents external environmental factors from affecting the activity of orchid exosomes, thereby improving their storage stability and extending the shelf life of nano-sized orchid exosomes. Furthermore, the freeze-dried crystalline structures can be quickly dissolved and mixed with serums or toners for use in skincare.

[0142] The preparation method is as follows: 10 v / v% orchid exosomes from Preparation Example 1, an excipient containing trehalose, sodium hyaluronate, and collagen, and water were stirred until completely homogeneous to obtain the formulation, which was then frozen at -20°C. Next, the formulation was placed in a freeze dryer for freeze-drying to obtain a freeze-dried crystalline structure. In some embodiments, the samples exhibited a frozen crystalline powder macroscopically and multiple crystalline structures microscopically. The freeze-dried crystalline structure can be quickly dissolved and mixed with serums or toners for skin care.

[0143] Figure 11 This is a schematic diagram of the freeze-dried grain structure according to some embodiments of the present invention. The freeze-dried grain structure 100 includes a bulk 110, exosomes 120, and active ingredient 130. The bulk 110 is composed of excipients, including trehalose, sodium hyaluronate, collagen, or combinations thereof. In some embodiments, the bulk 110 is composed of excipients and forms a network structure.

[0144] Exosome 120 was isolated from an orchid, specifically a white Phalaenopsis aphrodite; more specifically, exosome 120 was derived from the orchid exosomes of Preparation Example 1. Exosome 120 was uniformly dispersed within the body 110. In some embodiments, exosome 120 was uniformly dispersed within a network structure formed by the body 110. In some embodiments, exosome 120 had a bilayer membrane structure, the transmembrane protein of the exosome included CD9, and the average particle size of exosome 120 was 20 nm to 500 nm.

[0145] The active ingredient 130 is uniformly dispersed in the network structure formed by the bulk 110, and the active ingredient includes peptides, growth factors, vitamin C, plant extracts, or combinations thereof.

[0146] Example 10: Skincare Set Structure

[0147] Figure 12 This is a schematic diagram of a skincare product set structure according to some embodiments of the present invention. The skincare product set structure 200 includes a first container 210 and a second container 220. The first container 210 includes a bottle body 211, a first receiving space 212, a bottle neck 213, a bottle mouth 214, and a bottle cap 215. Specifically, the bottle body 211 surrounds and forms the first receiving space 212, so that the freeze-dried crystalline structure 100 is received in the first receiving space 212. The bottle neck 213 is disposed at the top of the bottle body 211, and the bottle mouth 214 is formed at the top of the bottle neck 213. The bottle cap 215 is disposed at the bottle mouth 214 to seal the first container 210.

[0148] The second container 220 has a structure similar to that of the first container 210, so details will not be described further. The second container 220 mainly includes a bottle body 221 and a second accommodating space 222 formed by the bottle body 221. The liquid excipient 223 is located in the second accommodating space 222, wherein the liquid excipient 223 includes water, saline solution, essence, or a combination thereof.

[0149] In some embodiments, both the first container 210 and the second container 220 are ampoules, containing the freeze-dried crystalline structure 100 and the liquid excipient 223, respectively. Before use, the liquid excipient 223 in the second container 220 is added to the first container 210 containing the freeze-dried crystalline structure 100. After the freeze-dried crystalline structure 100 and the liquid excipient 223 are uniformly mixed, they can be applied directly to the skin.

[0150] This invention provides a composition for enhancing the skin barrier and reducing wrinkles, comprising orchid exosomes as an active ingredient. This composition is used for skin regeneration, reducing wrinkles, combating skin aging, increasing skin hydration, improving skin elasticity, promoting wound healing, or a combination thereof.

[0151] The present invention also provides a freeze-dried crystalline structure for protecting active ingredients. Orchid exosomes are sealed in porous micro-gaps formed inside and on the surface of the freeze-dried crystalline structure, thereby improving the storage stability of active ingredients and extending their shelf life.

[0152] Although the present invention has been described above with reference to embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and refinements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A composition for enhancing the skin barrier, whitening, and anti-wrinkle effects, characterized in that, It contains exosomes isolated from orchids, specifically the white-flowered Phalaenopsis aphrodite, as the active ingredient.

2. The composition according to claim 1, characterized in that, The exosome was separated from the flower part and peduncle of the orchid.

3. The composition according to claim 1, characterized in that, The average particle size of these exosomes ranges from 20 nanometers to 500 nanometers.

4. The composition according to claim 1, characterized in that, The concentration of this exosome is 1×10⁻⁶. 6 granules / ml to 1×10 12 Particles / ml.

5. The composition according to claim 1, characterized in that, The exosome's transmembrane protein includes CD9.

6. The composition according to claim 1, characterized in that, This composition is for skin regeneration, reducing skin wrinkles, anti-skin aging, increasing skin hydration, improving skin elasticity, promoting wound healing, skin firming, skin whitening, or a combination thereof.

7. The composition according to claim 1, characterized in that, This composition is intended to improve skin cell damage and collagen loss caused by oxidative stress, increase the expression of genes for FBLN 5, CERS 3, COL 13A 1, COL 15A 1, IL-6, HAS1, CDSN or combinations thereof in skin cells, increase free radical scavenging capacity, inhibit melanin production, or combinations thereof.

8. The use of exosomes in the preparation of compositions that enhance the skin barrier, whiten, and reduce wrinkles, characterized in that, The exosome was isolated from an orchid, specifically the white-flowered Phalaenopsis aphrodite.

9. The use as described in claim 8, characterized in that, The exosomes were prepared by the following steps: Take the flower part and the peduncle of this orchid; Mix water with the flower part and the peduncle of the orchid, then crush to obtain a crude extract; The crude extract was filtered to obtain a crude filtrate; as well as The coarse filtrate was separated by tangential flow filtration to obtain the exosome.

10. A freeze-dried grain structure, characterized in that, Include: The body is composed of excipients; and Exosomes isolated from an orchid, specifically a white Phalaenopsis aphrodite, wherein the exosomes were uniformly dispersed within the organism.

11. The freeze-dried grain structure as described in claim 10, characterized in that, The excipients include trehalose, sodium hyaluronate, collagen, or a combination thereof.

12. The freeze-dried grain structure as described in claim 10, characterized in that, The body is composed of excipients and forms a network structure, in which the exosomes are uniformly dispersed.

13. The freeze-dried grain structure as described in claim 12, characterized in that, It also contains active ingredients, which are evenly dispersed in the network structure.

14. The freeze-dried grain structure as described in claim 13, characterized in that, The active ingredients include peptides, growth factors, vitamin C, plant extracts, or combinations thereof.

15. The freeze-dried grain structure as described in claim 10, characterized in that, The exosome has a double membrane structure.

16. The freeze-dried grain structure as described in claim 10, characterized in that, The exosome's transmembrane protein includes CD9.

17. The freeze-dried grain structure as described in claim 10, characterized in that, The average particle size of these exosomes ranges from 20 nanometers to 500 nanometers.

18. The freeze-dried grain structure as described in claim 10, characterized in that, The freeze-dried grain structure was prepared by the following steps: The exosomes and the excipients are mixed to obtain the formulation; and The formulation was freeze-dried to obtain the freeze-dried grain structure.

19. A skincare product set structure, characterized in that, Include: First container; The first accommodating space is located within the first container; and The freeze-dried grain structure as described in any one of claims 10 to 18 is located within the first accommodating space.

20. The skincare product set structure as described in claim 19, characterized in that, It also includes: Second container; The second accommodating space is located within the second container; and A liquid excipient is located within the second accommodating space, wherein the liquid excipient comprises water, saline solution, essence, or a combination thereof.

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