Screening method and composition for enhancing TSG-6 expression, using TSG-6 expression enhancement as an indicator.

Abstract

Subcutaneous tissue remodeling phenomenon, more specifically remodeling of fibrous structure, and furthermore, Physically, it contains active ingredients that stimulate collagen regeneration, as well as collagen breakdown and regeneration. We provide new technologies for improvement. [Solution] A subcutaneous tissue remodeling phenomenon characterized by using the increased expression of TSG-6 as an indicator. It promotes the remodeling of fibrous structures, the regeneration of collagen, and the breakdown and regeneration of collagen. A screening method for active ingredients.

Description

Technical Field

[0001] The present invention relates to a method for screening components that induce collagen neogenesis, collagen degradation, remodeling of fibrous structures, and remodeling of subcutaneous tissues, and to compositions used for improving the expression of TSG-6, collagen neogenesis, collagen degradation, remodeling of fibrous structures, and remodeling of subcutaneous tissues in adipose tissue-derived stem cells.

Background Art

[0002] It has been found that many organs contain not only progenitor cells that control the turnover of each tissue but also master stem cells that control emergency repair and the like, and they have attracted attention as cell sources for regenerative medicine. In addition to hematopoietic stem cells derived from bone marrow that have been conventionally used, the usefulness of adipose tissue-derived stem cells (ASC) has recently been evaluated. In the field of aesthetic dermatology, a cosmetic procedure (hereinafter referred to as "adipose stem cell injection method") of injecting subcutaneous tissue cells containing ASC subcutaneously is known. According to the adipose stem cell injection method, in addition to effects such as tissue enlargement, blood circulation improvement, and improvement of tissue reserve ability (wound healing ability), a phenomenon in which degenerated old fibers are replaced with new fibers (hereinafter referred to as "remodeling phenomenon") occurs, leading to rejuvenation of the skin, improvement of aging skin problems such as wrinkles and sagging, cosmetic improvement of facial degenerative diseases, treatment of intractable ulcers such as radiation ulcers, and many other future developments are expected (for example, Non-Patent Documents 1 to Non-Patent Documents 3).

[0003] Incidentally, TSG-6 is a protein also known as Tumor necrosis factor-inducible gene 6 protein. In humans, it is encoded by the TNFAIP6 (tumor necrosis factor, alpha-induced protein 6) gene. TSG-6 is a 30 kDa secreted protein and is a member of the HA-binding protein family having a hyaluronan-binding LINK domain a, and is also called a hyaladherin. TSG-6 is known to have various disease-improving effects. For example, it is known to have anti-inflammatory effects (inhibition of inflammatory cell infiltration) and inhibitory effects on fibrosis of living tissues. For this reason, TSG-6 is provided as a repair agent for living tissue damage in conditions such as chronic renal failure, chronic kidney disease, cirrhosis, pulmonary fibrosis, burns, interstitial pneumonia, drug-induced pneumonia, radiation pneumonitis, chronic obstructive pulmonary disease, acute respiratory-promoting syndrome, cartilage damage, bone defects, spinal cord injury, periodontal disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, diabetes, arteriosclerosis, heart diseases such as myocardial infarction, stroke, Alzheimer's disease, macular degeneration, viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, bone system disorders such as jawbone reconstruction, cleft palate, bone replacement material, bone defects, and osteoporosis, dry eye, corneal disorders, pharyngitis, arthritis, cancer, or tissue damage associated with fibrosis (Patent Documents 1-4).

[0004] Furthermore, TSG-6 is known to promote wound healing by regulating macrophage activity, suppressing inflammation, and inhibiting fibrosis of biological tissues (Non-Patent Literature 4). In addition, the expression level of TSG-6 in keloid lesions is significantly reduced compared to areas without scarring, suggesting that TSG-6 may contribute to the suppression of scar formation (Non-Patent Literature 5). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] WO2018 / 123968 publication [Patent Document 2] Special Publication No. 2011-525355 [Patent Document 3] Japanese Patent Publication No. 2012-157263 [Patent Document 4] Special Publication No. 2009-532334 [Non-patent literature]

[0006] [Non-Patent Document 1] Aesthetic Surgery Journal, Volume 30, Issue 1, January 2010, Pages 78-81. [Non-Patent Document 2] Aesthetic Dermatology Vol.20:225-236,2010 [Non-Patent Document 3] Plast Reconstr Surg, volume 135, issue 4, pages 999-1009 [Non-Patent Document 4] Journal of Investigative Dermatology(2014),134,526-537 [Non-Patent Document 5] Journal of the European Academy of Dermatology and Venereology,2011,25,317-327 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] By the way, many skin problems associated with aging, such as the formation of wrinkles and sagging and a decrease in skin firmness, are caused by a decrease in the normal fibrous structure that makes up skin tissue and an accumulation of degenerated fibrous structures (so-called fibrosis). In this regard, currently available anti-aging cosmetics merely increase the reduced fibrous structure, but they cannot remove the degenerated fibrous structure. Even if the fibrous structure is increased, as long as the degenerated fibrous structure remains in the skin tissue, it is not possible to effectively address age-related skin problems such as wrinkles, sagging, and loss of skin firmness, and therefore, these age-related skin problems cannot be solved.

[0008] To more effectively address skin problems associated with aging, it is important not only to increase the amount of skin tissue lost, but also to induce skin tissue remodeling, which involves removing degenerated fibrous structures from the skin tissue and replacing them with new ones. By removing degenerated fibrous structures and replacing them with new ones, it is possible to more effectively address age-related skin problems compared to simply increasing the reduced fibrous structure of skin tissue.

[0009] Therefore, the inventors focused on adipose stem cell injection, which can induce subcutaneous tissue remodeling. However, the complete mechanism remains unclear. Specifically, it is still unknown which cells (speaking cells) express which factors (speaking factors) after the injection of subcutaneous tissue cells to trigger remodeling.

[0010] Fat stem cell injection is a highly invasive procedure with high costs. If a material that can achieve subcutaneous tissue remodeling without injecting adipose tissue-derived stem cells could be discovered or developed, it would provide a less invasive and less costly technique compared to fat stem cell injection, making it very useful. However, as mentioned above, the overall picture of the subcutaneous tissue remodeling phenomenon is not yet clear, making the search for such a useful material extremely difficult.

[0011] In view of these problems, the first objective of the present invention is to provide a novel technology for screening active ingredients that induce subcutaneous tissue remodeling, more specifically, fibrous structure remodeling, and more specifically, collagen regeneration, as well as collagen degradation and regeneration.

[0012] Furthermore, a second objective of the present invention is to provide a composition containing an active ingredient that induces subcutaneous tissue remodeling, more specifically, remodeling of fibrous structures, and even more specifically, collagen regeneration, as well as collagen degradation and regeneration. [Means for solving the problem]

[0013] When the inventors analyzed the biological reactions that occur during subcutaneous tissue remodeling, it was revealed that remodeling is a complex reaction involving various reactions, and that in such a complex reaction, Tumor necrosis factor-stimulated gene-6 (TSG-6) is the key to the reaction. Specifically, as a result of their intensive research efforts, the inventors found that TSG-6 promotes collagen degradation and neoformation in sequence and acts as a trigger for remodeling of the fibrous structure and thus subcutaneous tissue remodeling. The present invention has been completed based on such a new discovery.

[0014] That is, the present invention for solving the above first problem is a screening method for an active ingredient that induces collagen neoformation, characterized by using the effect of enhancing the expression of TSG-6 as an index. By using the effect of enhancing the expression of TSG-6 as an index, an active ingredient that induces collagen neoformation can be screened.

[0015] In another embodiment of the present invention, there is provided a screening method for an active ingredient that induces collagen degradation and collagen neoformation, characterized by using the effect of enhancing the expression of TSG-6 as an index. By using the effect of enhancing the expression of TSG-6 as an index, an active ingredient that induces both collagen degradation and neoformation, which seemingly appear to be contradictory at first glance, can be screened. Specifically, the active ingredient screened in this embodiment can induce remodeling that breaks down and replaces degenerated subcutaneous tissue by inducing collagen degradation and then collagen neoformation.

[0016] In another embodiment of the present invention, there is provided a screening method for an active ingredient that induces remodeling of a fibrous structure, characterized by using the effect of enhancing the expression of TSG-6 as an index. By using the effect of enhancing the expression of TSG-6 as an index, an active ingredient that induces remodeling of a fibrous structure, which is a complex biological phenomenon, can be screened.

[0017] In a preferred form of this embodiment, the remodeling of the fiber structure is due to the degradation and neogenesis of collagen. By using the effect of enhancing the expression of TSG-6 as an index, it is possible to screen for active ingredients that induce the remodeling of the fiber structure, which is a complex biological phenomenon, particularly the remodeling caused by the degradation and neogenesis of collagen.

[0018] In another embodiment of the present invention, there is provided a method for screening an active ingredient that induces the remodeling of subcutaneous tissue, characterized by using the effect of enhancing the expression of TSG-6 as an index. When screening is performed using a direct index of whether or not it induces the remodeling of subcutaneous tissue, it is very time-consuming and costly. However, the present invention can screen for active ingredients that induce the remodeling of subcutaneous tissue with a simple configuration using the effect of enhancing the expression of TSG-6 as an index.

[0019] In a preferred form of this embodiment, the remodeling of the subcutaneous tissue is due to the degradation and neogenesis of collagen. With a simple configuration using the effect of enhancing the expression of TSG-6 as an index, it is possible to screen for active ingredients that induce the remodeling of subcutaneous tissue, particularly the remodeling caused by the degradation and neogenesis of collagen.

[0020] In a preferred form of the present invention, there is provided a method for screening an active ingredient of a skin condition improving agent, an anti-inflammatory agent, a fibrosis inhibitor, a wound healing promoter, a scar preventive agent, a scar improving agent, a keloid preventive agent or a keloid improving agent, characterized by using the effect of enhancing the expression of TSG-6 as an index. According to the present invention, by using a conveniently measurable index of the effect of enhancing the expression of TSG-6, it is possible to screen for active ingredients of a skin condition improving agent, an anti-inflammatory agent, a fibrosis inhibitor, a wound healing promoter, a scar preventive agent, a scar improving agent, a keloid preventive agent or a keloid improving agent.

[0021] A preferred embodiment of the present invention is characterized by comprising the steps of: culturing cells in the presence of a test substance; measuring the expression level of TSG-6 in the cells; and selecting the test substance if the expression level is higher than the expression level of the TSG-6 gene in cells cultured in the absence of the test substance. This configuration allows for more accurate screening.

[0022] In a preferred embodiment of the present invention, the cells are adipose tissue-derived stem cells. By using adipose tissue-derived stem cells as the cells used as a screening tool, more accurate screening can be achieved.

[0023] Furthermore, the present invention, which solves the second problem described above, relates to a composition containing extracts of Morus alba and Cynara scolymus. The combination of mulberry extract and Korean thistle extract allows the composition of the present invention to exhibit excellent effects, including improved TSG-6 expression in adipose tissue-derived stem cells, degradation and regeneration of collagen fiber structures, subcutaneous tissue remodeling, and ultimately improvement of skin condition. Furthermore, the composition of the present invention exhibits anti-inflammatory effects, fibrosis inhibitory effects, wound healing promoting effects, scar prevention or improvement effects, and keloid prevention or improvement effects resulting from increased TSG-6 expression.

[0024] In a preferred embodiment of the present invention, extracts of Morus alba and Cynara scolymus are included as active ingredients and used to enhance TSG-6 expression.

[0025] In a preferred embodiment of the present invention, extracts of Morus alba and Cynara scolymus are included as active ingredients and used to promote collagen regeneration.

[0026] The present invention also relates to a composition for collagen synthesis and collagen degradation, comprising an extract of Morus alba and / or Cynara scolymus as an active ingredient.

[0027] In a preferred embodiment of the present invention, extracts of Morus alba and Cynara scolymus are included as active ingredients and used for remodeling of fibrous structure.

[0028] In a preferred embodiment of the present invention, extracts of Morus alba and Cynara scolymus are included as active ingredients and used for subcutaneous tissue remodeling.

[0029] In a preferred embodiment of the present invention, extracts of Morus alba and Cynara scolymus are used as active ingredients for one or more purposes selected from improving skin condition, reducing inflammation, inhibiting fibrosis, promoting wound healing, preventing or improving scars, or preventing or improving keloids. [Effects of the Invention]

[0030] According to the screening method of the present invention, it is possible to screen for active ingredients that induce collagen regeneration, collagen degradation and regeneration, fibrous structure remodeling, or subcutaneous tissue remodeling. Furthermore, the composition of the present invention can induce improved TSG-6 expression, collagen regeneration, collagen degradation and regeneration, fibrous structure remodeling, subcutaneous tissue remodeling, or improvement of skin condition, anti-inflammatory effects, inhibition of fibrosis, promotion of wound healing, prevention or improvement of scars, or prevention or improvement of keloids. [Brief explanation of the drawing]

[0031] [Figure 1]The images show three-dimensional fluorescence images of the localization of degraded collagen in subcutaneous tissue before and after co-culture with adipose tissue-derived stem cells in Test Example 1. The left image is before culture, and the right image is after 3 days of culture. Degraded collagen was detected using a CHP fluorescent probe. [Figure 2] The images show three-dimensional staining of newly synthesized collagen in subcutaneous tissue before and after co-culture with adipose tissue-derived stem cells in Test Example 1. The left image shows the staining before culture, and the right image shows the staining after culture. Newly synthesized collagen was detected by antibody staining against type I procollagen. [Figure 3] This graph shows the results of Test Example 2. The horizontal axis represents the number of culture days, and the vertical axis represents the relative gene expression level of TSG-6. [Figure 4] This graph shows the results of Test Example 3. It shows the signal of degraded collagen in subcutaneous tissue cultured alone (without co-culture with adipose tissue-derived stem cells), subcutaneous tissue co-cultured with adipose tissue-derived stem cells, and subcutaneous tissue co-cultured with adipose tissue-derived stem cells in the presence of a TSG-6 neutralizing antibody. [Figure 5] This graph shows the results of Test Example 3. It shows the signal of newly synthesized collagen in subcutaneous tissue cultured alone (without co-culture with adipose tissue-derived stem cells), subcutaneous tissue co-cultured with adipose tissue-derived stem cells, and subcutaneous tissue co-cultured with adipose tissue-derived stem cells in the presence of a TSG-6 neutralizing antibody. [Figure 6] This graph shows the results of Test Example 3. It shows the relative gene expression levels of α-SMA in subcutaneous tissue cultured alone (without co-culture with adipose tissue-derived stem cells), subcutaneous tissue co-cultured with adipose tissue-derived stem cells, and subcutaneous tissue co-cultured with adipose tissue-derived stem cells in the presence of a TSG-6 neutralizing antibody. [Figure 7] This graph shows the results of Test Example 4. It shows the relative gene expression levels of TSG-6 in adipose tissue-derived stem cells cultured in the presence and absence of mulberry extract and / or Korean thistle extract. [Modes for carrying out the invention]

[0032] Embodiments of the present invention will now be described. It goes without saying that the scope of the present invention is not limited to the embodiments described below.

[0033] <1> Screening method The screening method of the present invention uses the enhancement of TSG-6 (TNF-stimulated gene 6 protein) expression as an indicator. TSG-6 is a protein also known as tumor necrosis factor-inducible gene 6 protein. In humans, it is encoded by the TNFAIP6 (tumor necrosis factor, alpha-induced protein 6) gene. TSG-6 is a 30kDa secreted protein and a member of the HA-binding protein family that possesses a hyaluronan-binding LINK domain a, and is also called hyaladherin.

[0034] In a preferred embodiment, the screening method of the present invention comprises a culture step, a measurement step, and a selection step. Each step will be described in detail below.

[0035] The cell culture process involves culturing cells in the presence of the test substance. The cell culture method is not particularly limited; adhesion culture or suspension culture can be appropriately employed depending on the properties of the cells used. The cells used for screening are not particularly limited. From the perspective of screening for active ingredients that induce subcutaneous tissue remodeling, it is preferable to use adipose tissue-derived stem cells as a screening tool. This is because in adipose stem cell injection therapy, subcutaneous tissue remodeling is induced by TSG-6 secreted by adipose tissue-derived stem cells.

[0036] Adipose tissue-derived stem cells refer to pluripotent somatic stem cells found in the adipose tissue of mammals. These adipose tissue-derived stem cells also include cultured cells obtained through the culture (including subculturing) of such somatic stem cells, provided that they maintain their pluripotency.

[0037] Adipose tissue, which serves as the source for adipose tissue-derived stem cells, can be collected from mammals such as humans, mice, rats, guinea pigs, hamsters, monkeys, cattle, pigs, goats, sheep, dogs, and cats by known methods such as excision and aspiration.

[0038] There are no particular restrictions on the type of adipose tissue used as a source for adipose tissue-derived stem cells. Examples include subcutaneous fat, visceral fat (including the omentum), intramuscular fat, and intermuscular fat. One type of adipose tissue may be used as a source for adipose tissue-derived stem cells, or two or more types of adipose tissue may be used in combination as a source. From the perspective of accurately searching for signaling factors, subcutaneous fat is preferably used as the adipose tissue from which adipose tissue-derived stem cells are collected. Furthermore, the life or death status of the mammal from which the adipose tissue is collected is not a concern, as long as the stem cells in the adipose tissue are viable.

[0039] Stem cells are collected from adipose tissue by known methods. The present invention may include a cell acquisition step comprising the following steps (1) to (6) in order to obtain adipose tissue-derived stem cells to be used in a culture step in which cells are cultured in the presence of a test substance. (1) The collected tissue is subjected to processing such as removal of blood components and fragmentation as necessary, and then digested with enzymes such as collagenase and trypsin. (2) The cell population after enzyme digestion is centrifuged at 1800 rpm for about 5 minutes, and the settled cell population is collected. (3) The settled cell population is subjected to centrifugation three times under the same conditions as in (2) above, and then the settled cell population is collected. This operation removes mature adipocytes. (4) Seed the collected cell population into a culture dish containing fresh culture medium and culture it. (5) Remove the suspended cells by changing the culture medium. At this point, the adipose tissue-derived stem cells are attached to the culture dish. (6) Subculture as needed.

[0040] The culture process of the present invention can be initiated by detaching adipose tissue-derived stem cells obtained through the cell acquisition process from the culture dish using trypsin or the like, seeding them in an appropriate number in another culture dish and culturing them, and then adding the test substance.

[0041] As for the adipose tissue-derived stem cells used in the culture process, those obtained by timely collection from adipose tissue as described above may be used, but commercially available primary cultured cells may also be used.

[0042] Stem cells collected from adipose tissue may, if necessary, be confirmed to have pluripotent stem cell function by examining their differentiation potential and cell surface markers.

[0043] Furthermore, the culture process only requires the successful cultivation of adipose tissue-derived stem cells, and is not limited to culturing purely isolated adipose tissue-derived stem cells. Other cells different from adipose tissue-derived stem cells may be mixed in. For example, it is acceptable to culture a stromal-vascular fraction (SVF) that includes adipose tissue-derived stem cells. Furthermore, the embodiment may involve tissue culture of adipose tissue, such as subcutaneous tissue, containing adipose tissue-derived stem cells.

[0044] When using adipose tissue-derived stem cells in the culture process, it is preferable to culture them by adhesion culture.

[0045] Furthermore, the culture medium is not particularly limited and known mediums can be used. DMEM / F12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12) is a suitable example.

[0046] The duration of the culture process is not particularly limited, but is preferably 6 hours or more, more preferably 12 hours or more, and even more preferably 1 day or more. The upper limit of the culture period is also not particularly limited, but as a guideline, is preferably 1 month or less, more preferably 3 weeks or less, even more preferably 2 weeks or less, even more preferably 1 week or less, even more preferably 3 days or less, and even more preferably 2 days or less.

[0047] In the culture process, cultivation is carried out in the presence of the test substance. Specifically, one embodiment involves adding the test substance to the culture medium and then culturing. The type of test substance is not limited and can be applied without limit to low molecular weight compounds, high molecular weight compounds, proteins, peptides, nucleic acids, etc. The test substance may be a pure compound or a mixture containing two or more components, such as plant or animal extracts.

[0048] In the culture process, cell culture may be performed in the absence of the test substance as a comparison, in parallel with culture in the presence of the test substance. In this case, it is preferable to keep the culture conditions the same, except for the presence or absence of the test substance.

[0049] The screening method of the present invention comprises a measurement step for measuring the expression level of TSG-6 in cells that have undergone a culture step. The method for measuring the expression level of TSG-6 is not particularly limited, and examples include quantitative PCR and Northern blotting, which target mRNA transcribed from the TSG-6 gene, and immunological methods such as immunostaining, Western blotting, and ELISA, which target the TSG-6 protein. Quantitative PCR is particularly preferred from the viewpoint of high quantitative accuracy and simplicity. The specific embodiments of these measurement means are not particularly limited and can be carried out by conventional methods.

[0050] The screening method of the present invention comprises a selection step. The selection step is to select a test substance when the expression level of TSG-6 in cells cultured in the presence of the test substance is higher than the expression level of the TSG-6 gene in cells cultured in the absence of the test substance.

[0051] The test substance selected in the selection process may be identified as the active ingredient, or it may be identified as a candidate for an active ingredient to be used in secondary screening.

[0052] In one embodiment of the present invention, a screening method is used to screen for active ingredients that induce collagen synthesis. Collagen is a molecule in which three peptides form a helix and is widely distributed in the body as a component of connective tissue. The process of collagen synthesis begins with the production of procollagen within the cell. Procollagen is a precursor in the collagen synthesis process and has procollagen peptides at the N-terminus and C-terminus of its peptide chain. Procollagen produced within the cell is secreted outside the cell, where the peptides at both ends are specifically cleaved by procollagen peptidase to form collagen. According to the screening method of the present invention, it is possible to screen for active ingredients that promote the production of procollagen.

[0053] Furthermore, in another embodiment of the present invention, a screening method is used to screen for active ingredients that induce collagen degradation and collagen regeneration. Preferably, the screening is performed for active ingredients that induce collagen degradation and collagen regeneration in that order. According to this embodiment, ingredients that induce the complex biological phenomenon of collagen fiber structure remodeling can be easily screened using a simple indicator such as the effect of improving TSG-6 expression.

[0054] Furthermore, in another embodiment of the present invention, a screening method is used to screen for active ingredients that induce remodeling of fibrous structures. Preferably, the remodeling of fibrous structures is due to the degradation and regeneration of collagen. Preferably, the active ingredient induces the degradation and regeneration of collagen in this order. According to this embodiment, it is possible to easily screen for ingredients that induce the complex biological phenomenon of fibrous structure remodeling (degradation and regeneration) using a simple indicator such as the effect of improving TSG-6 expression.

[0055] Another embodiment of the present invention, a screening method, screens for active ingredients that induce subcutaneous tissue remodeling. Preferably, the subcutaneous tissue remodeling is due to the degradation and regeneration of collagen. Preferably, the active ingredient induces the degradation and regeneration of collagen in this order. In subcutaneous tissue, collagen exists in a reticular structure and supports adipocytes. Adipose tissue-derived stem cells also exist in the subcutaneous tissue. According to this embodiment, it is possible to screen for active ingredients that act on adipose tissue-derived stem cells present in the subcutaneous tissue, increase the production of TSG-6 in these cells, and thereby induce subcutaneous tissue remodeling through the degradation and regeneration of collagen fibers.

[0056] <2> composition The composition of the present invention is characterized by containing a mixture of Morus alba extract and Cynara scolymus extract as an active ingredient. The combination of mulberry extract and Korean thistle extract allows the composition of the present invention to exhibit excellent effects, including increased TSG-6 expression in adipose tissue-derived stem cells, degradation and regeneration of collagen fiber structures, subcutaneous tissue remodeling, and ultimately, improvement of skin condition. "Improvement of skin condition" here includes skin rejuvenation, and more specifically, improvement of sagging and wrinkles. Furthermore, the composition of the present invention exhibits anti-inflammatory and fibrotic inhibitory effects due to increased TSG-6 expression.

[0057] Plant extracts can be prepared using plants that grow or are cultivated in Japan, or using Japanese-grown plants sold as raw materials for herbal medicines. Alternatively, commercially available extracts can be purchased and used from companies that handle plant extracts, such as Maruzen Co., Ltd.

[0058] These plant extracts refer not only to the plant extracts themselves, but also to fractions of the extracts, purified fractions, and solvent-removed products of the extracts, fractions, and purified products. Plant-derived extracts include extracts made from plants that grow wild or are cultivated, extracts made from plants sold as raw materials for herbal medicines, and commercially available extracts.

[0059] The extraction process can utilize the entire plant, as well as parts such as the plant body, above-ground parts, rhizomes, trunks, leaves, stems, flower spikes, flower buds, and fruits. However, it is preferable to crush or finely chop these parts beforehand to improve extraction efficiency. For mulberry, it is particularly preferable to use an extract from its fruit. For Korean thistle, it is particularly preferable to use an extract from its leaves.

[0060] Suitable extraction solvents include one or more polar solvents selected from water, alcohols such as ethanol, isopropyl alcohol, and butanol; polyhydric alcohols such as 1,3-butanediol and polypropylene glycol; ketones such as acetone and methyl ethyl ketone; and ethers such as diethyl ether and tetrahydrofuran. Specific extraction methods include, for example, adding 1 to 30 parts by mass of the solvent to 1 mass of the plant material or its dried product, immersing it for several days at room temperature or for several hours at a temperature near its boiling point, cooling it to room temperature, removing insoluble matter and / or solvent as desired, and then fractionating and purifying it by column chromatography or the like.

[0061] In one embodiment, the present invention is a composition for improving TSG-6 expression in adipose tissue-derived stem cells. The composition of this embodiment has the effect of acting on adipose tissue-derived stem cells to improve TSG-6 expression.

[0062] In another embodiment, the present invention relates to a composition for promoting collagen regeneration. The composition of this embodiment exhibits a collagen regeneration-promoting effect by increasing the expression of TSG-6.

[0063] In another embodiment, the present invention relates to a composition for collagen regeneration and collagen degradation. Preferably, the composition contains components that induce collagen degradation and collagen regeneration in that order. According to the invention of this embodiment, it is possible to induce collagen degradation and regeneration and to induce remodeling of the collagen fiber structure.

[0064] In another embodiment, the present invention is a composition for remodeling fibrous structures. Preferably, the remodeling of the fibrous structure is by the degradation and regeneration of collagen. Also preferably, the active ingredient induces the degradation and regeneration of collagen in this order. According to the composition of the present invention, remodeling by degradation and regeneration of fibrous structures can be induced.

[0065] In another embodiment, the present invention is a composition for subcutaneous tissue remodeling. Preferably, the subcutaneous tissue remodeling is by the degradation and regeneration of collagen. Also preferably, the active ingredient induces the degradation and regeneration of collagen in this order. The active ingredient of the present invention acts on adipose tissue-derived stem cells present in the subcutaneous tissue, causing an increase in TSG-6 expression in these cells, which leads to the degradation and regeneration of collagen fiber structures, thereby producing a subcutaneous tissue remodeling effect.

[0066] In another embodiment, the present invention is a composition used for improving skin condition. Here, "improvement of skin condition" includes skin rejuvenation, more specifically, improvement of sagging and wrinkles.

[0067] In another embodiment, the present invention relates to a composition used for anti-inflammatory or fibrotic inhibition purposes. More specifically, the composition of the present invention is used to inhibit the infiltration of inflammatory cells. The present invention is also used to inhibit fibrosis in biological tissues. In another embodiment, the present invention is used to promote wound healing, prevent or improve scarring, or prevent or improve keloids. More specifically, the compositions of the present invention are used for the repair of tissue damage associated with chronic renal failure, chronic kidney disease, cirrhosis, pulmonary fibrosis, burns, interstitial pneumonia, drug-induced pneumonia, radiation pneumonitis, chronic obstructive pulmonary disease, acute respiratory-promoting syndrome, cartilage damage, bone defects, spinal cord injury, periodontal disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, diabetes mellitus, arteriosclerosis, heart diseases such as myocardial infarction, stroke, Alzheimer's disease, macular degeneration, viral hepatitis, alcoholic hepatitis, non-alcoholic steatohepatitis, jawbone reconstruction, cleft palate, bone replacement materials, bone defects, osteoporosis and other skeletal disorders, dry eye, corneal disorders, pharyngitis, arthritis, cancer, or fibrosis.

[0068] The specific form of the composition of the present invention is not limited and may be provided as a topical skin preparation or an oral administration preparation. The dosage form of the topical skin preparation is not particularly limited, but examples include cosmetics, quasi-drugs, and topical medicines. Among these, in relation to its use in inducing subcutaneous tissue remodeling, a cosmetic form that can be used continuously is preferred, such as lotions, emulsions, serums, creams, gels, and sun care products.

[0069] When the composition is in the form of a topical skin preparation, the content of plant extracts in the composition (dry mass in the case of extracts) is usually 0.00001% by mass or more, preferably 0.0001% by mass or more, more preferably 0.001% by mass or more, and can usually be 80% by mass or less, preferably 30% by mass or less, and more preferably 10% by mass or less.

[0070] When administered orally, it may be provided as a pharmaceutical or food product. It is preferable to provide it as a supplement in the form of a general food product, tablet, granule, drink, or other dosage form.

[0071] When administered orally, the amount taken per dose is typically 0.1 mg or more, preferably 1 mg or more, more preferably 10 mg or more, and typically 2000 mg or less, preferably 1000 mg or less, and more preferably 500 mg or less, based on the dry mass of the extract, depending on the dosage form.

[0072] The composition of the present invention may contain any components as long as they do not impair the effects of the invention. These optional components can be appropriately selected depending on the dosage form. [Examples]

[0073] <Test Example 1> Construction of a test system to reproduce the subcutaneous tissue remodeling phenomenon [1] Acquisition of 3D fluorescence images showing the localization of degraded collagen and newly synthesized collagen before co-culture. (1-1) Staining of degraded collagen before co-culture Subcutaneous tissue prepared by excising from commercially available human skin tissue was contacted with a staining solution prepared by diluting a CHP (Collagen Hybridizing Peptide) fluorescent probe in a culture medium (DMEM / F-12 containing 10% FBS), incubated for 1 hour, and a sample stained with degraded collagen was obtained. (1-2) Staining of new collagen before co-culture First, another subcutaneous tissue sample obtained in the same manner as in (1-1) was incubated for 16 hours in contact with a staining solution prepared by diluting the primary antibody (anti-type I procollagen antibody) with the culture medium used in (1-1). Subsequently, the subcutaneous tissue was washed with PBS, and the fluorescently labeled secondary antibody against the aforementioned primary antibody was brought into contact with a staining solution prepared by diluting the culture medium used in (1-1). The sample was incubated for a further 2 hours to obtain a sample stained with newly synthesized collagen. (2) Acquisition of 3D fluorescence images Each sample obtained as described above was imaged using a confocal fluorescence microscope (Nikon Corporation), and three-dimensional fluorescence images representing the localization of degraded collagen and newly synthesized collagen "before co-culture" were obtained.

[0074] [2] Acquisition of 3D fluorescence images showing the localization of degraded collagen and newly synthesized collagen after co-culture. (1) Subcutaneous tissue obtained in [1](1-1) and [1](1-2) above was infiltrated into a culture medium stored in a 6-well plate on which adipose tissue-derived stem cells had been pre-seeded, and the cultures were incubated in an incubator. (2-1) For the samples stained with degraded collagen as described in [1](1-1) above, after culturing for 3 days, the degraded collagen was stained using the method described above in the same area in the subcutaneous tissue co-cultured with adipose tissue-derived stem cells where a 3D fluorescence image showing the localization of degraded collagen was obtained before culturing. A 3D fluorescence image showing the localization of degraded collagen "after co-culturing" was obtained under the same imaging conditions as in [1](1-1) above. (2-2) For the samples stained with neocollagen in [1](1-2) above, after culturing for 7 days, the neocollagen was stained using the method described above in the same area in the subcutaneous tissue co-cultured with adipose tissue-derived stem cells from which a 3D fluorescence image representing the localization of neocollagen before culturing was obtained. A 3D image representing the localization of neocollagen "after co-culturing" was obtained under the same imaging conditions as in [1](1-2) above. In this test system, staining is performed both before and after co-culture. However, since the staining before co-culture fades during the 3-day or 7-day culture period, there is no carryover of pre-co-culture signals in the post-co-culture images.

[0075] Figure 1 shows images illustrating the localization of degraded collagen before and after co-culture, and Figure 2 shows images illustrating the localization of newly synthesized collagen before and after co-culture. In Figures 1 and 2, the left image is the image before co-culture, and the right image is the image after co-culture (3 days or 7 days later).

[0076] As shown in Figure 1, the signal intensity in subcutaneous tissue after 3 days of culture was stronger compared to the signal intensity of degraded collagen in subcutaneous tissue before culture. Furthermore, as shown in Figure 2, the signal intensity of type I procollagen in subcutaneous tissue after 7 days of culture was stronger compared to the signal intensity of newly synthesized procollagen in subcutaneous tissue before culture. Although the results are not shown in the diagram, gene expression analysis of newly synthesized collagen using standard methods revealed that gene expression levels in newly synthesized collagen increased slightly even after 1-3 days of culture, but a significant increase in gene expression levels was observed after 3-7 days of culture. From the above findings, it was revealed that co-culturing adipose-derived stem cells with subcutaneous tissue sequentially induces collagen degradation and regeneration. From the above, it was confirmed that the subcutaneous tissue remodeling phenomenon can be reproduced by co-culturing subcutaneous tissue and adipose tissue-derived stem cells.

[0077] <Test Example 2> Confirmation of increased TSG-6 expression Subcutaneous tissue was prepared using the same procedure as in Test Example 1 and co-cultured with adipose tissue-derived stem cells. Simultaneously, subcutaneous tissue was cultured without co-culture with adipose tissue-derived stem cells as a control group. Subcutaneous tissue from the adipose tissue-derived stem cell co-culture group and the non-co-culture group was collected one, two, three, and seven days after the start of culture. mRNA was extracted in bulk from the collected subcutaneous tissue, and the gene expression level of TSG-6 was measured by quantitative PCR. Figure 3 shows the relative gene expression levels of TSG-6, with the expression level in the non-co-culture group (cultured for 1 day) set to 1.

[0078] As shown in Figure 3, in subcutaneous tissue co-cultured with adipose tissue-derived stem cells, the expression level of TSG-6 temporarily increased in the early stages of culture. The results of this study suggest that TSG-6 may be a trigger for subcutaneous tissue remodeling.

[0079] <Test Example 3> Collagen Decomposition and Regeneration Subcutaneous tissue was prepared using the same procedure as in Test Examples 1 and 2, and co-cultured with adipose tissue-derived stem cells (co-culture group). Simultaneously, subcutaneous tissue was cultured without co-culture with adipose tissue-derived stem cells as a control group (non-co-culture group). To investigate the effect of TSG-6, a TSG-6 neutralizing antibody was added to the culture medium, and the subcutaneous tissue was co-cultured with adipose tissue-derived stem cells (co-culture + TSG-6 inhibition group).

[0080] Degraded collagen in tissues 1 and 3 days after culture was detected and quantified by fluorescence imaging using fluorescently labeled collagen hybridizing peptide (CHP). Figure 4 shows the signal intensity of degraded collagen in each group after 3 days of culture, with the signal intensity of the non-co-culture group before culture set to 1.

[0081] Using a fluorescently labeled anti-procollagen antibody, the signal of newly synthesized collagen in tissues 1 and 7 days after culture was detected and quantified by fluorescence imaging. Figure 5 shows the quantification results of newly synthesized collagen in each group after 7 days of culture, with the signal intensity of the non-co-culture group before culture set to 1. Furthermore, total RNA was extracted from the recovered subcutaneous tissue, and the gene expression level of α-SMA was measured by quantitative PCR. Figure 6 shows the relative gene expression levels of α-SMA in the co-culture group and the co-culture + TSG-6 inhibition group, with the gene expression level of α-SMA in the non-co-culture group set to 1.

[0082] As shown in Figure 4, the signal intensity of degraded collagen in the co-culture group with adipose tissue-derived stem cells showed a greater increase in signal intensity compared to the non-co-culture group which did not co-culture with adipose tissue-derived stem cells. In other words, it became clear that co-culture with adipose tissue-derived stem cells transiently increases collagen degradation in subcutaneous tissue. On the other hand, in the co-culture + TSG-6 inhibition group, despite co-culture with adipose tissue-derived stem cells, the increase in signal intensity was less than 1, indicating that collagen degradation was significantly suppressed (Figure 4). This suggests that TSG-6 is involved in collagen degradation.

[0083] As shown in Figure 5, the increase in signal intensity after culture was greater than 1 in both the non-co-culture group (where adipose tissue-derived stem cells were not co-cultured) and the co-culture group (where adipose tissue-derived stem cells were co-cultured). Furthermore, the increase in signal intensity was greater in the non-co-culture group than in the co-culture group. In summary, it was found that collagen regeneration in subcutaneous tissue occurs regardless of whether or not co-culture with adipose tissue-derived stem cells is performed, but the degree of regeneration is higher in the non-co-culture group. On the other hand, in the co-culture + TSG-6 inhibition group, despite being co-cultured with adipose tissue-derived stem cells, the signal intensity was equivalent to that of the non-co-culture group, and the collagen regeneration effect by adipose-derived stem cells was significantly suppressed (Figure 5). This suggests that TSG-6 is involved in collagen regeneration.

[0084] Figure 6 shows that co-culture with adipose tissue-derived stem cells reduces α-SMA expression in subcutaneous tissue (comparison between co-culture group and non-co-culture group). However, in the co-culture + TSG-6 inhibitor group, no decrease in α-SMA expression was observed despite co-culture with adipose tissue-derived stem cells (Figure 6). Here, α-SMA is a protein that serves as a marker for myofibroblasts, and its expression level is known to increase with fibrosis. In other words, this study revealed that co-culturing adipose-derived stem cells with subcutaneous tissue promotes collagen production, but this promotion of collagen production does not lead to fibrosis of the subcutaneous tissue. Furthermore, combining the results of the gene expression analysis, which showed that the gene expression level of newly synthesized collagen increased significantly during a 3-7 day culture period compared to a 1-3 day culture period, with the results of this study, it can be understood that adipose-derived stem cells sequentially induce collagen degradation and synthesis via TSG-6. This conclusion is consistent with the experimental fact that the timing of the increase in TSG-6 expression shown in Figure 3 (after 1 day of culture) is earlier than the timing of collagen degradation and synthesis shown in Figures 4 and 5 (after 3 to 7 days of culture).

[0085] <Test Example 4> Confirmation of the effect of increasing TSG-6 expression Adipose tissue-derived stem cells were cultured for 24 hours after adding mulberry extract (final concentration 0.1%), Korean thistle extract (final concentration 0.1%), or a mixture thereof (mulberry extract: final concentration 0.1% + Korean thistle extract: final concentration 0.1%) to the culture medium. As a control, adipose tissue-derived stem cells cultured without the addition of these plant extracts were also prepared. After culturing, adipose tissue-derived stem cells were harvested, mRNA was extracted, and the gene expression level of TSG-6 was quantified by quantitative PCR (quantified relative to the gene expression level of ACTB). As a result, the gene expression levels of TSG-6 were similar to those of the control group when either mulberry extract or Korean thistle extract was added individually. However, it was found that the gene expression levels of TSG-6 were significantly increased when a mixture of these extracts was added (Figure 7). These results indicate that a mixture of mulberry extract and Korean thistle extract has a significant effect in increasing TSG-6 expression, and that TSG-6 promotes the breakdown and regeneration of collagen fiber structure, leading to subcutaneous tissue remodeling and improvement of skin condition. Furthermore, because it significantly enhances TSG-6 expression, the mixture of mulberry extract and Korean thistle extract was found to have anti-inflammatory and fibrosis-inhibiting effects. [Industrial applicability]

[0086] This invention can be applied to screening techniques for active ingredients that induce subcutaneous tissue remodeling. Furthermore, this invention can be applied to cosmetics and the like that exert an effect of inducing subcutaneous tissue remodeling.

Claims

1. A composition comprising extracts of Morus alba and Cynara scolymus.

2. The composition according to claim 1, characterized in that it contains extracts of Morus alba and Cynara scolymus as active ingredients and is used to improve the expression of TSG-6.

3. The composition according to claim 1, characterized in that it contains extracts of Morus alba and Cynara scolymus as active ingredients and is used to promote collagen regeneration.

4. The composition according to claim 1, characterized in that it contains extracts of Morus alba and Cynara scolymus as active ingredients and is used for collagen regeneration and collagen degradation.

5. The composition according to claim 1, characterized in that it contains extracts of Morus alba and Cynara scolymus as active ingredients and is used for remodeling fibrous structures.

6. The composition according to claim 1, characterized in that it contains extracts of Morus alba and Cynara scolymus as active ingredients and is used for subcutaneous tissue remodeling.

7. A composition according to any one of claims 1 to 6, characterized in that it contains extracts of Morus alba and Cynara scolymus as active ingredients and is used for one or more purposes selected from improving skin condition, anti-inflammatory, inhibiting fibrosis, promoting wound healing, preventing or improving scars, or preventing or improving keloids.

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