Topical dermatological preparation comprising exosomes derived from callus of lithospermum erythrorhizon s.

Abstract

The present invention relates to a topical dermatological composition for improving the skin, comprising exosomes derived from callus of Lithospermum erythrorhizon S., which has excellent properties including cell proliferation promotion, wound healing promotion, anti-inflammation, collagen synthesis promotion, and whitening effects, and may provide a topical dermatological composition for improving the skin having excellent cell inflow characteristics of exosomes, thereby significantly improving the skin physiological activity effects.

Description

Topical skin preparation containing exosomes derived from callus of the plant

[0001] The present invention relates to a skin topical preparation comprising exosomes derived from callus of the plant.

[0002] The skin, the outermost organ of the human body, consists of three layers: the epidermis, dermis, and subcutaneous fat. It plays a crucial role in protecting the body from the external environment, regulating body temperature, and enabling sensation. Specifically, because it is in direct contact with the external environment, the skin acts as a barrier against external stimuli such as temperature, humidity, and ultraviolet rays. It also maintains body moisture, prevents electrolyte loss, regulates body temperature, and performs sensory and immune functions. It protects internal organs from external stimuli and performs various functions necessary for sustaining life.

[0003] However, when skin cells are damaged by continuous exposure to external stimuli such as ultraviolet rays or environmental pollutants, or internal factors such as a decrease in the secretion of metabolic hormones that regulate aging and a decline in immune function, the function of skin cells declines, freckles and blemishes are formed due to melanin deposition, and various skin aging phenomena such as wrinkles, keratinization, and loss of elasticity occur. These skin aging phenomena not only reduce the function of the skin, but also impair the aesthetic characteristics of the skin, the outermost organ of the human body. Therefore, various skin activating topical agents are being developed and used to maintain or improve the function of the skin and enhance the external beauty of the skin.

[0004] As a skin-activating ingredient used in external skin care products, chemically synthesized substances are sometimes used, but recently, the use of substances extracted from natural sources is on the rise. These natural ingredients contain various new substances, and these natural ingredients can act in a complex manner on the skin to preserve or improve its function.

[0005] Meanwhile, Lithospermum Erythrorhizon has long been known as a medicinal herb that surpasses ginseng. It is also called purple root because of its reddish-purple color. Lithospermum has traditionally been used as a medicinal herb to promote blood circulation, reduce fever, and detoxify, and it has also been used to treat hemoptysis, hematuria, constipation, burns, eczema, and urinary tract infections. In addition, the red color shikonin contained in the root of Lithospermum has been used as a pigment in cosmetics, foods, and ointments, and recent research has reported that shikonin has anti-inflammatory, wound healing promotion, anti-tumor, hypoglycemic, and antimicrobial properties.

[0006] However, shikonin is one of the various components that implement the various functions of shichi, and the functions of shichi are not expressed only by shikonin, and in particular, shikonin can have a harmful effect on the human body due to its unique toxicity. Therefore, in the present invention, an active substance derived from shikonin was developed that can implement the efficacy and advantages of shichi while minimizing the toxicity caused by shikonin.

[0007] The present invention aims to provide a skin topical agent containing exosomes derived from the callus of the common ginseng, which have excellent effects of promoting cell proliferation, promoting wound healing, anti-inflammation, promoting collagen synthesis, and whitening.

[0008] One embodiment of the present invention for achieving the above-described purpose relates to a skin improvement external composition comprising exosomes derived from callus of the plant.

[0009] The above skin improvement may be at least one of wrinkle improvement, wound healing promotion, cell regeneration, skin barrier strengthening, aging improvement, anti-inflammation, and whitening.

[0010] The above-mentioned callus may be a callus obtained by cutting and cultivating at least one of the flowers, leaves, stems, and roots of the callus.

[0011] The above exosomes can be obtained by isolation from a callus culture medium or a callus fragment mixture.

[0012] The above-mentioned callus culture solution can be obtained by culturing callus.

[0013] The above-mentioned callus crushed mixture may include a callus culture solution and a crushed callus of the callus.

[0014] The size of the exosomes derived from the above-mentioned callus may be 50 to 350 nm.

[0015] Another embodiment of the present invention relates to a method for producing exosomes derived from borage callus, comprising: a step of cultivating cut borage callus to form borage callus; a step of isolating borage callus from a culture mixture obtained in the step of forming borage callus to obtain a borage callus culture solution; and a step of isolating exosomes from the borage callus culture solution.

[0016] The step of forming the above-mentioned callus may include a step of selecting a callus obtained by culturing cut callus; and a step of subculturing the selected callus.

[0017] A method further comprising: crushing callus separated from a culture mixture to form a callus lysate; wherein the step of isolating exosomes may be separating exosomes from a callus lysate mixture in which a callus culture medium and a callus lysate are mixed.

[0018] Other embodiments of the present invention include a skin external application composition for improving wrinkles comprising exosomes derived from foliar callus, a skin external application composition for promoting wound healing comprising exosomes derived from foliar callus, a skin external application composition for cell regeneration comprising exosomes derived from foliar callus, a skin external application composition for strengthening the skin barrier comprising exosomes derived from foliar callus, an anti-inflammatory skin external application composition for improving wrinkles comprising exosomes derived from foliar callus, and a skin external application composition for whitening comprising exosomes derived from foliar callus.

[0019] The skin external preparation containing the exosomes derived from the callus of the present invention has excellent effects of promoting cell proliferation, promoting wound healing, anti-inflammation, promoting collagen synthesis, and whitening, and since the exosomes have excellent intracellular inflow characteristics, the skin physiological activity effect can be significantly improved.

[0020] Figures 1(A) and 1(B) are the results of particle size analysis of exosomes derived from callus of Gichichi and photographs taken with a transmission electron microscope, respectively.

[0021] Figure 2 shows the results of evaluating the fibroblast proliferation promotion efficacy of exosomes derived from callus of Zi Chi.

[0022] Figures 3(A) and 3(B) are photographs of the results of an experiment on the wound healing effect of exosomes derived from callus of Zi Chi and graphs showing changes in wound area, respectively.

[0023] Figures 4(A) and 4(B) are photographs and graphs, respectively, of an experiment evaluating the anti-inflammatory effect of exosomes derived from callus of Zi Chi.

[0024] Figure 5 shows the results of evaluating the collagen synthesis performance of exosomes derived from callus.

[0025] Figure 6 shows the results of evaluating the whitening performance of exosomes derived from callus.

[0026] Figure 7 shows the results of an experiment on the influx of exosomes derived from callus into fibroblasts.

[0027] Before going into detail with reference to preferred embodiments, it should be noted that the terms and words used in this specification and claims should not be construed as limited to their usual or dictionary meanings, but should be interpreted with meanings and concepts consistent with the technical spirit of the present invention.

[0028] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0029] Throughout this specification, "%" used to indicate the concentration of a specific substance means (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid, unless otherwise stated.

[0030] The present invention relates to a skin improvement external composition comprising exosomes derived from callus of the plant.

[0031] Lithospermum Erythrorhizon is a perennial herb in the genus Lithospermum of the family Boraginaceae, order Lamiales, class Magnoliopsida, phylum Magnoliophyta. It is native to Korea, Japan, China, and some parts of Russia. Lithospermum has fine hairs all over the plant, grows to a height of about 30 to 70 cm, and blooms white flowers in May and June, forming spikes at the ends of the stems and branches. In oriental medicine, the root of Lithospermum is used to treat hemoptysis, hematuria, constipation, burns, eczema, urinary tract infections, etc., and Lithospermum is also used as a natural purple or red dye.

[0032] Callus is an unorganized mass of plant cells, also called callus or callus tissue. It is an amorphous mass of cells that forms at the site of a wound in a living plant. Callus can be formed from plant tissues such as leaves, stems, roots, flowers, and fruits, and can be formed by culturing wounded plant tissue on a medium containing plant growth regulators such as auxin or cytokinin, and has the characteristic of proliferating during subculture. Furthermore, although callus itself does not have the ability to differentiate, it can be redifferentiated into shoots, roots, embryos, or even complete plants when cultured under certain conditions.

[0033] Boraginaceae callus can be obtained by cutting at least one of Boraginaceae tissues, such as flowers, leaves, stems, and roots, and culturing the tissues on a medium containing a plant growth regulator to produce and propagate the callus. A plant culture medium containing plant growth hormones can be used as a medium for producing and propagating Boraginaceae callus.

[0034] For example, a plant culture medium for callus production may be used, such as MS (Murashige and Skoog, 1962) medium, SH Medium (Duchefa, Haarlem, Netherlands), N6 (Chu et al., 1975) medium, B5 (Gamborg et al., 1968) medium, NN (Nitsch andNitsch, 1967) medium, or WHITE medium, and any medium used for culturing plants may be used without limitation.

[0035] Plant growth regulators are substances that regulate plant growth by affecting the membrane properties of plants, regulating gene expression, or affecting enzyme activity. As plant growth regulators that can be included in a medium for callus formation and proliferation, at least one or more of natural chemical substances such as non-peptide hormones such as auxin, gibberellin, cytokinin, ethylene, brassinosteroid, and abscisic acid; or fatty acid derivatives such as jasmonate and oligosaccharin; or derivatives of chemically synthesized natural plant growth hormones such as ethephon can also be used. In addition, plant growth regulators are not limited to those listed above, and any substance known to have a plant growth function can be included in a medium as a plant growth regulator to induce callus formation and proliferation without particular limitation.

[0036] The callus of the plant, the plant of ...

[0037] In particular, by repeatedly selecting and subculturing calli having a uniform color and appearance during the subculture process, superior calli having a uniform color, appearance, and growth rate can be secured, thereby improving the productivity and quality of exosomes.

[0038] The number of times of subculture to secure superior stocks is not particularly limited, and can be repeated 2 to 10 times or 3 to 6 times, for example, and the culture period for each subculture can be 3 to 5 weeks, for example.

[0039] Exosomes are vesicles secreted by plant cells into the extracellular space for the exchange of information between cells, and have the form of a double lipid membrane structure containing proteins, lipids, nucleic acids, etc. In the present invention, exosomes are a broad concept that includes not only exosomes but also all exosome-like extracellular vesicles.

[0040] Plant-derived exosomes may differ in efficacy and function depending on the type of plant from which they are derived. Exosomes can pass through cell membranes, allowing them to directly deliver active ingredients into mammalian cells. Compared to synthetic nanomaterials, they do not elicit immune or inflammatory responses, minimizing side effects when used as topical skin care products. Due to these characteristics, when exosomes are used as topical skin care products, pores are approximately 250-600 times larger than exosomes. This allows exosomes containing active ingredients to be easily absorbed into pores, allowing them to be delivered intact into the dermal layer of the skin, enabling the active ingredients within the exosomes to function more effectively.

[0041] Exosomes derived from borax callus are produced in the process of culturing borax callus for borax callus production, and in the present invention, borax callus is cultured in a culture medium, and borax callus is separated from the remaining culture medium component, the 'borax callus culture medium', to obtain borax callus-derived exosomes.

[0042] In addition, exosomes can be obtained from the 'boring callus crushed mixture', which is a mixture of the above-mentioned boring callus culture medium and the callus crushed material obtained by crushing the boring callus, by separating exosomes from the boring callus.

[0043] The exosomes derived from callus obtained in this way can have a size of 50 to 350 nm.

[0044] Exosomes derived from callus of the plant and compositions for external application for skin comprising exosomes derived from callus of the plant have a skin improvement effect, and the skin improvement effect may include at least one of promotion of wound healing, cell regeneration, strengthening of skin barrier, improvement of aging, anti-inflammation, and whitening.

[0045] A skin external application composition comprising exosomes derived from Gichi callus may be a cosmetic composition or a pharmaceutical composition, and may further comprise ingredients commonly included in cosmetic compositions or pharmaceutical compositions. For example, when used as a cosmetic composition, at least one or more of a moisturizer, a diluent, an excipient, a viscosity modifier, an antioxidant, a preservative, a metal ion sequestering agent, a surfactant, a fragrance, a pigment, a vitamin, and various active ingredients may further be included, and when used as a pharmaceutical composition, a carrier, an excipient, a diluent, etc. may further be included, but the ingredients that may be additionally included are not limited to the types listed above.

[0046] The content of exosomes derived from callus included in the above skin external preparation composition can be appropriately selected according to various conditions such as the type of skin external preparation composition, formulation, amount of additives, method of use, characteristics of the user, etc.

[0047] Meanwhile, the present invention includes a method for producing a skin external application composition comprising exosomes derived from callus of the plant.

[0048] The method for producing a skin external application composition including the above-mentioned exosomes derived from the callus of the genus Boraginaceae includes the steps of: culturing cut genus Boraginaceae to form genus Boraginaceae callus; obtaining a genus Boraginaceae callus culture solution excluding the genus Boraginaceae callus from the culture mixture obtained in the step of forming the genus Boraginaceae callus; and isolating and purifying exosomes from the genus Boraginaceae callus culture solution.

[0049] First, the step of forming a callus by culturing the cut borax is a step of forming a callus by cutting at least one tissue among the tissues forming the borax, for example, leaves, stems, roots, flowers, and fruits, and culturing the cut borax on a medium containing a plant growth regulator. The cut borax refers to a borax tissue that has been cut to form a wound in a part of the borax tissue, and includes not only a borax tissue divided into a plurality of independent segments through cutting, but also a borax tissue in which a wound is formed in a single tissue segment.

[0050] At this stage, the culture medium used for culturing may contain a plant growth regulator, and the plant growth regulator may be at least one of a chemical substance derived from nature, such as a non-peptide hormone such as auxin, gibberellin, cytokinin, ethylene, brassinosteroid, and abscisic acid; or a fatty acid derivative such as jasmonate and oligosaccharin; and a derivative of a chemically synthesized natural plant growth hormone such as ethephon may also be used. Any plant growth regulator known to have a plant growth function may be used without particular limitation.

[0051] The culture medium, i.e., the culture medium, may be, for example, MS (Murashige and Skoog, 1962) medium, SH Medium (Duchefa, Haarlem, Netherlands), N6 (Chu et al., 1975) medium, B5 (Gamborg et al., 1968) medium, NN (Nitsch andNitsch, 1967) medium, or WHITE medium, and any medium used for culturing plants may be used without limitation thereto.

[0052] At this stage, cultivation can be performed in a subculture manner. That is, the initially cut callus is cultured to obtain primary callus formed directly from the plant, and the primary callus is subcultured at least once to form secondary callus, thereby obtaining callus callus and callus culture medium for exosome production.

[0053] In this process, the callus used for subculture can be all of the callus formed through culture.

[0054] Alternatively, in order to improve the efficiency of exosome production, only some calli having a uniform color and appearance among the primary calli may be selected, and the selected calli may be used. In this case, subculture may be performed by culturing the selected calli, selecting calli with a uniform appearance from the proliferated calli, and then culturing them again. When exosomes are produced by culturing calli that have undergone the selection process in this way, the production efficiency and quality of the exosomes may be further improved. At this time, the number of times the subculture is repeated is not particularly limited, and may be repeated 2 to 10 times or 3 to 6 times, for example, and the culture period for each round may be 3 to 5 weeks, for example.

[0055] Next, a step is performed to separate the callus from the culture mixture obtained in the callus formation step and obtain a callus culture solution. In this step, the callus can be directly removed from the culture mixture or separated by a method such as filtration to obtain a callus culture solution from which the callus has been removed.

[0056] The step of isolating and purifying exosomes from the above callus culture medium is a step of isolating and purifying exosomes from the callus culture medium to obtain exosomes derived from callus.

[0057] At this stage, a filter is used to primarily filter out impurities in the callus culture medium, centrifugation is performed to collect the supernatant, and this is then filtered again using a filter for a second time, and exosomes can be obtained through a purification and concentration process.

[0058] In the primary filtration process, a filter of approximately 10 to 50 μm can be used to remove impurities and cell sediments, and in the secondary filtration process, a filter of 0.3 to 0.8 μm can be used for more precise filtration.

[0059] Centrifugation can be performed at a speed of 3,000 to 5,000 rpm for 30 to 90 minutes, and after centrifugation, the supernatant can be collected to obtain exosomes from the supernatant. At this time, trehalose can be additionally added to prevent deterioration or damage during processing, storage, distribution, etc. of the supernatant or exosomes, and the trehalose can be added at a concentration of 1 to 5 wt%.

[0060] Purification and concentration of exosomes can be performed using a tangential flow filtration (TFF) method, and an additional sterilization process using a 0.1 to 0.2 ㎛ filter can be performed to prevent microbial deterioration during distribution and storage.

[0061] The exosomes derived from callus obtained through this process can be stored at a temperature of -90 to -70°C.

[0062] In addition, the exosomes derived from the callus of the rhizome and the composition for external application for skin comprising the exosomes derived from the callus of the rhizome manufactured through the above process have a skin improvement effect, and the skin improvement effect may include at least one of promotion of wound healing, cell regeneration, strengthening of the skin barrier, improvement of aging, anti-inflammation, and whitening.

[0063] Shikonin, commonly known as the active ingredient of Zizyphus japonica, is a fat-soluble substance. However, since the exosome manufacturing process according to the present invention described above is entirely performed in an aqueous solution, the water-soluble Zizyphus japonica callus-derived exosomes contain little fat-soluble shikonin.

[0064] Therefore, the effect of the exosomes derived from the callus of the present invention included in the external skin composition according to the present invention is exhibited by the exosomes derived from the callus of the present invention, not by the shikonin contained in the callus. For the same reason, the exosomes derived from the callus of the present invention and the external skin composition including the same have the advantage of being able to prevent toxicity caused by shikonin.

[0065] In the step of isolating and purifying exosomes from the above callus culture medium, the callus culture medium from which callus has been separated through separation and purification as a raw material for producing exosomes may be used alone, or a callus fragment mixture in which callus fragments are mixed with the callus culture medium may be used.

[0066] The exosomes derived from the callus of the rhizome and the skin external application composition containing the exosomes derived from the callus of the rhizome thus obtained may be a cosmetic composition or a pharmaceutical composition, and in order to prepare such a skin external application composition, a step of further mixing an additive into the exosomes derived from the callus of the rhizome may be performed.

[0067] When the above-mentioned external skin composition is a cosmetic composition, the additive may be an ingredient typically included in a cosmetic composition, and may include, for example, at least one or more of a moisturizer, a diluent, an excipient, a viscosity modifier, an antioxidant, a preservative, a metal ion sequestering agent, a surfactant, a fragrance, a pigment, a vitamin, and various active ingredients. When the above-mentioned external skin composition is a pharmaceutical composition, the additive may be an ingredient typically included in a pharmaceutical composition, and may include, for example, at least one or more of a carrier, an excipient, and a diluent. The types of additives listed above are illustrative, and the types of additives are not limited thereto.

[0068] The content of exosomes derived from callus included in the above skin external preparation composition can be appropriately selected according to various conditions such as the type of skin external preparation composition, formulation, amount of additives, method of use, characteristics of the user, etc.

[0069] Hereinafter, the specific functions and effects of the present invention will be explained through an example of the present invention. However, this is presented as a preferred example of the present invention, and the scope of the present invention is not limited by the example.

[0070] [Manufacturing Example 1]

[0071] First, the seeds of the plant were washed in 70% ethanol for 2 minutes and then disinfected in a solution of 2% NaClO (bleach with 4% available chlorine concentration) and 10% Tween-20 for 15 minutes. The disinfected seeds were washed five times with sterilized water and then planted on MS medium without growth regulators. After 3 to 4 weeks, callus was induced using the germinated plantlets. After that, the leaves of the germinated plantlets were prepared into explants measuring 1 to 2 cm in length and width and planted on a callus induction medium consisting of MS salts, 3% sucrose, 0.05% MES, 0.001% 2,4-D (auxin type growth regulator), and 0.4% Gelrite, pH 5.8, and callus was induced in the dark at 25℃. After 4 to 5 weeks, calli with uniform color and shape were selected from the calli derived from the cut surface, transferred to a new medium of the same composition, and cultured for 4 weeks. This method was subcultured 5 times to secure superior calli with uniform color and growth rate.

[0072] The superior calli were cultured in a pH 5.8 liquid medium containing MS salts, 3% sucrose, 0.05% MES, 0.001% NAA (auxin-type growth regulator), and 0.001% kinetin (cytokinin-type growth regulator) at 25℃ in the dark for 2 weeks. After culture, the collected culture medium was separated from the callus and used as the filtrate callus culture medium, the raw material for exosomes. Alternatively, the callus was crushed together with the culture medium and the resulting lysate was used as the filtrate callus culture medium, the raw material for exosomes.

[0073] The above-mentioned callus culture medium was first filtered through a 30 μm filter to remove cell sediment and various foreign substances, and centrifuged at 3,900 rpm for 1 hour to collect the supernatant. After adding 2% trehalose to this supernatant, it was secondarily filtered through nominal grade filters with sizes of 0.45 μm and 0.22 μm in order, and exosomes were separated, purified, and concentrated using a circular filtration (TFF) method. Afterwards, it was filtered through an absolute grade 0.22 μm filter for sterilization, and finally exosomes derived from callus of filtrate (Example 1) were prepared, and the prepared exosomes were stored at -80°C.

[0074] [Experimental Example 1]

[0075] The exosomes derived from the callus of Manufacturing Example 1 were analyzed using a nanoparticle tracking analysis (NTA: nanoparticles tracking analysis, Particle Matrix) and the particle size distribution is shown in Fig. 1(A). After negative staining, the images were taken using a transmission electron microscope and shown in Fig. 1(B).

[0076] As confirmed in Figure 1(A), the size of exosomes was distributed in the range of 81–218 nm, and the concentration was approximately 5.4×10 10 particles / ml, and as confirmed in Fig. 1(B), a circular structure with a double lipid membrane structure, which is a characteristic of exosomes, was observed, indicating that exosomes were smoothly formed using the method of Manufacturing Example 1.

[0077] [Experimental Example 2]

[0078] To determine whether exosomes derived from callus promote the proliferation of human dermal fibroblasts, NHDF (Normal Human Dermal Fibroblast, purchased from Promocell), a CCK-8 assay was performed. Specifically, NHDF cells were seeded at 0.4 × 10 in a 96-well plate with DMEM (Dubelcco's Modified Eagle's Medium, Hyclone) containing 10% FBS (Fetal Bovine Serum) and 1% penicillin / streptomycin (PS, Welgene). 4 The cells were seeded at a density of 10 cells / well and cultured for 24 hours in an incubator at 37°C and 5% CO2.

[0079] After confirming cell attachment the next day, the medium used for culture was removed, and 1×10 of the exosomes derived from the callus prepared in the manufacturing example were added to DMEM medium containing 10% exosome-depleted FBS (Gibco). 7 , 1×10 8 , 1×10 9 After diluting to a concentration of 100 particles / ml, the NHDF cells were treated. Exosome-deficient fetal bovine serum was used to exclude effects other than the test substance, and only exosome-deficient fetal bovine serum medium was treated as a control.

[0080] To determine cell proliferation, the WST-8 reagent of the Cell counting / Cell proliferation assay kit-8 (CCK-8, purchased from Dojindo) was treated per well at 0, 24, and 48 hours and reacted in a 37℃ incubator for 2 hours. Afterwards, the 450 nm absorbance value was measured using a microplate reader (Bioteck), and the cell viability (%) was calculated according to the following [Formula 1], and the fibroblast proliferation promotion efficacy (cell viability) according to the concentration and elapsed time (24 hours, 48 ​​hours) of the exosomes derived from rehmannia callus is shown in Fig. 2.

[0081] [Formula 1]

[0082]

[0083] Referring to the experimental results in Fig. 2, the cell proliferation rate after 24 hours of exosome treatment was similar to that of the control group, but after 48 hours, the cell proliferation rate was superior to that of the control group. In particular, when the exosome treatment concentration was 1×10 8 1×10 9 In this case, it was confirmed that exosomes derived from callus were more effective in fibroblast proliferation. These results imply that exosomes derived from callus of Gichi are effective in skin regeneration and wound healing.

[0084] [Experimental Example 3]

[0085] To confirm the wound healing effect of exosomes derived from callus of Jichi, a wound healing assay was performed. First, NHDF cells, human fibroblasts, were seeded in a 24-well plate at a density of 5 × 10 4 Cells were seeded at 1 cell / well and cultured in DMEM medium containing 10% FBS and 1% penicillin / streptomycin at 37°C and 5% CO2 in an incubator for 24 hours. Afterwards, wounds were made using a 200-μl micropipette tip and cultured for 24 hours in DMEM medium containing 2% exosome-depleted FBS treated with the same concentration of exosomes derived from rehmannia callus as in Experimental Example 2. Afterwards, cell images were obtained using a microscope (Olympus, CKX53), and the degree of wound healing was analyzed using Image J software. The results of qualitative observation of changes in wound area according to exosome concentration are shown in Fig. 3(A), and a graph quantitatively organizing these changes in wound area is depicted in Fig. 3(B).

[0086] Referring to the experimental results of Fig. 3, in the case of the control group, the area of ​​the wound area recovered by approximately 16% after 24 hours, whereas in the case of treatment with exosomes derived from callus of the plant, the area of ​​the wound area recovered by at least 52% and at most 66%. From these results, it was found that the wound healing efficacy improved as the concentration of exosomes treated to the wound area increased.

[0087] Therefore, the results of this experiment confirmed that exosomes derived from callus of the plant have wound healing and recovery effects.

[0088] [Experimental Example 4]

[0089] To determine whether exosomes derived from callus of the plant have anti-inflammatory effects, mouse macrophage RAW264.7 cells were used. Specifically, RAW264.7 cells were suspended in DMEM medium containing 10% FBS and 1% penicillin / streptomycin (PS, Welgene), and 2 × 10 4 Cells were seeded into 96-well plates at a density of 10 cells / well and cultured for 24 hours in a 37°C, 5% CO2 incubator. Afterwards, the exosomes derived from the callus prepared in the manufacturing example were mixed with a new medium containing LPS (DMEM medium containing 10% exosome-depleted FBS and 1 ug / ml LPS) and treated to RAW264.7 cells, followed by culture for 24 hours. A medium containing only 10% exosome-depleted FBS was used as an experimental control. The results of the anti-inflammatory performance test of RAW264.7 cells induced by LPS were photographed using a phase contrast microscope (Olympus, CKX53, iPCsystem) and the results are shown in Fig. 4(A). The number of dendritic cells according to the amount of exosome treatment was measured and presented as a graph in Fig. 4(B).

[0090] This experiment is an indirect anti-inflammatory performance evaluation method that utilizes the characteristic of macrophages that are induced by inflammation to change into dendritic cell forms. As shown in Fig. 4(A), it was confirmed that the number of dendritic cells was reduced in examples in which LPS and exosomes derived from rehmannia callus were treated together compared to the control group in which LPS, an inflammation-inducing substance, was treated alone.

[0091] In addition, referring to the number of dendritic cells shown in Fig. 4(B), when exosomes derived from the callus of the genus Zi, the number of dendritic cells was reduced to about half compared to the control group, confirming that the exosomes derived from the callus of the genus Zi exhibited anti-inflammatory properties.

[0092] [Experimental Example 5]

[0093] Human dermal fibroblasts NHDF (Normal Human Dermal Fibroblast; purchased from Promocell) were dispersed in DMEM medium containing 10% FBS and 1% penicillin / streptomycin and seeded at 5 × 10 in a 24-well plate. 4 Cells were seeded at a density of 10 cells / well and cultured for 24 hours, and exosomes derived from callus (1 × 10) diluted in DMEM medium (containing 10% exosome-depleted FBS) were added. 9 After treatment with 100 particles / ml) or a positive control (ascorbic acid), the cells were cultured for 24 and 48 hours. The culture medium was then collected, centrifuged, and the supernatant was collected. The amount of collagen synthesized from human skin fibroblasts and accumulated in the culture medium was measured using enzyme-linked immunosorbent assay (ELISA) for collagen type IA peptide (Col1A).

[0094] The experimental results showed that when exosomes derived from filaria callus were treated for 24 hours, the collagen type IA synthesis performance of exosomes derived from filaria callus was similar to that of the positive control, ascorbic acid. However, when treated for 48 hours, the collagen synthesis performance was 1.5 times higher than that of the positive control. This suggests that exosomes derived from filaria callus have excellent collagen synthesis performance, which will lead to a wrinkle improvement effect.

[0095] [Experimental Example 6]

[0096] Melanoma cells derived from mouse melanoma (B16F10; purchased from the Korean Cell Line Bank) were seeded at 1 × 10 in 6-well plates. 5 Cells were seeded at a density of 10 cells / well and cultured for 24 hours using DMEM medium with 10% FBS at 37°C and 5% CO2 conditions. Afterwards, the DMEM medium containing 10% exosome-depleted FBS was replaced and cultured for an additional 72 hours. The control group was cultured using only DMEM medium containing exosome-depleted FBS, and the medium mixed with 250 nM α-MSH (α-melanocyte stimulating hormone) was used as a comparison group. 1 × 10 9 , 1×10 10 The test group was further treated with exosomes derived from callus at a concentration of 100 particles / ml.

[0097] Afterwards, the cultured melanoma cells were recovered and washed, and then treated with 1N NaOH mixed with 10% DMSO, sealed, and heated at 85°C for 60 minutes to extract melanin within the melanoma cells. The amount of extracted melanin was measured by measuring the absorbance at 475 nm, and the absorbance of the comparative or test group was calculated as a relative value with the melanin amount of the control group set as 100%, and this was graphically represented in Figure 6.

[0098] In Fig. 6, the control group is a case in which α-MSH, a melanin inducer, and exosomes derived from filaria callus were not treated, and the comparison group (MSH 250 nM) is a case in which α-MSH was treated but exosomes derived from filaria callus were not treated.

[0099] Referring to the experimental results of Fig. 6, in the case of the comparative group treated with a melanin inducing substance, approximately twice as much melanin was formed as compared to the control group, but when exosomes derived from callus were also treated, the amount of melanin produced decreased, and it was confirmed that the amount of melanin produced decreased by up to 1.8 times as compared to the comparative group.

[0100] Therefore, the results of this experiment confirmed that exosomes derived from callus of the plant showed a whitening effect by inhibiting melanin production.

[0101] [Experimental Example 7]

[0102] To determine whether exosomes derived from the callus of the plant were effective in influxing into human skin cells, a cell-uptake test was conducted. First, exosomes derived from the callus of the plant were stained with a green fluorescent substance (PKH67, Sigma), and 1 × 10 fluorescently labeled exosomes were inoculated into the cells. 8 2.5×10 particles / ml concentration 4 Fibroblasts prepared in cell / well and NHDF (purchased from Promocell) were treated and cultured for 4 and 24 hours, respectively. Before observation, the nuclei of cells were stained with a blue fluorescent substance (DAPI), and the state of exosome influx into cells over time was observed under a fluorescence microscope. The results are shown in Figure 7. In Figure 7, A is a photograph showing exosomes, B is a photograph showing fibroblasts, and C is a photograph showing both exosomes and fibroblasts.

[0103] Referring to the experimental results of Fig. 7, when fibroblasts and exosomes were cultured together, green-labeled exosomes were observed around the cell nuclei labeled in blue after 4 hours, and the green luminescence intensity increased with the passage of culture time, confirming that the exosomes around the cell nuclei increased as the culture time passed.

[0104] Therefore, through this experiment, it was confirmed that exosomes derived from callus were effectively introduced into animal cells, which are heterologous cells.

[0105] The composition for external application for skin improvement comprising exosomes derived from callus of the present invention has excellent effects of promoting cell proliferation, promoting wound healing, anti-inflammation, promoting collagen synthesis, and whitening, and has excellent characteristics of exosomes entering cells, so that the skin physiological activity effect can be significantly improved, and thus has industrial applicability.

Claims

1. A composition for external application for skin improvement containing exosomes derived from callus of the plant.

2. In paragraph 1, A skin improvement composition for external application, characterized in that the skin improvement is at least one of wrinkle improvement, wound healing promotion, cell regeneration, skin barrier strengthening, aging improvement, anti-inflammation, and whitening.

3. In paragraph 1, A composition for external application for skin improvement, characterized in that the above-mentioned callus is a callus obtained by cutting and cultivating at least one of the flowers, leaves, stems, and roots of the callus.

4. In paragraph 1, A composition for external application for skin improvement, characterized in that the exosome is obtained by being separated from a callus culture solution or a callus crushed mixture.

5. In paragraph 4, A composition for external application for skin improvement, characterized in that the above-mentioned callus culture solution is obtained by culturing callus.

6. In paragraph 4, The above-mentioned callus crushed mixture is a composition for external application for skin improvement, comprising a callus culture solution and a crushed product of callus.

7. In paragraph 1, A composition for external application for skin improvement, wherein the size of the exosomes derived from the above-mentioned callus is 50 to 350 nm.

8. Step of cultivating the cut rhizome to form rhizome callus; A step of isolating the callus from the culture mixture obtained in the step of forming the callus, thereby obtaining a callus culture solution; and A method for producing exosomes derived from callus of the plant, comprising the step of isolating exosomes from the callus culture medium of the plant.

9. In paragraph 8, The step of forming the above-mentioned callus is: A step of selecting callus obtained by cultivating cut callus; and A method for producing exosomes derived from callus of the plant, comprising the step of subculturing selected callus of the plant.

10. In paragraph 8, A step of crushing the callus separated from the culture mixture to form a callus crushed product is further included; A method for producing exosomes derived from borax callus, characterized in that the step of isolating the exosomes comprises isolating exosomes from a borax callus crushed mixture in which a borax callus culture medium and a borax callus crushed material are mixed.

11. A composition for external application of skin for improving wrinkles, comprising exosomes derived from callus of the plant.

12. A composition for external application to the skin for promoting wound healing, comprising exosomes derived from callus of the plant.

13. A composition for external application to the skin for cell regeneration, comprising exosomes derived from callus of the plant.

14. A composition for external application of skin for strengthening the skin barrier, comprising exosomes derived from callus of the plant.

15. An anti-inflammatory skin external application composition comprising exosomes derived from callus of the plant.

16. A skin whitening external application composition comprising exosomes derived from callus.

Images