Anti-aging agent

Abstract

The present invention provides an endothelial-mesenchymal transition inhibitor and an agent for inhibiting lymphatic vessel aging, which inhibits lymphatic vessel aging by inhibiting endothelial-mesenchymal transition of lymphatic vessels. The present invention provides an endothelial-mesenchymal transition inhibitor containing mulberry bark and / or guava leaf as an active ingredient. By administering the endothelial-mesenchymal transition inhibitor of the present invention, endothelial-mesenchymal transition of lymphatic vessels can be inhibited. Inhibition of endothelial-mesenchymal transition of lymphatic vessels can inhibit lymphatic vessel aging.

Description

[Technical Field]

[0001] The present invention relates to an anti-aging agent, particularly an endothelial-mesenchymal transition inhibitor containing mulberry bark and / or guava leaf as an active ingredient, and a lymphatic vessel aging inhibitor that suppresses the aging of lymphatic vessels by inhibiting the endothelial-mesenchymal transition of lymphatic vessels. [Background technology]

[0002] Lymphatic vessels act as drainage channels, collecting waste products such as inflammatory cells, proteins, and excess fluid from the spaces between cells. However, it is known that when lymphatic vessel function deteriorates due to aging, waste elimination becomes sluggish, leading to various problems such as edema. Therefore, substances that prevent or improve lymphatic vessel aging are desirable.

[0003] In recent years, it has been reported that in the eye, endothelial cells of lymphatic vessel-like Schlemm's canals differentiate into mesenchymal cells, impairing aqueous humor drainage and resulting in age-related glaucoma (Non-Patent Literature 1). Furthermore, it has been reported that the transition of vascular endothelial cells to endothelial-mesenchymal cells is involved in tumor progression.

[0004] However, it remains unclear whether the transition of endothelial cells to mesenchymal cells is involved in the aging of lymphatic vessels in organs such as the skin. Furthermore, it is necessary to search for substances that suppress the differentiation and conversion of lymphatic vessels into mesenchymal cells. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2014-218479 [Patent Document 2] Japanese Patent Publication No. 2007-22957 [Patent Document 3] International Publication No. 2007 / 007732 [Non-patent literature]

[0006] [Non-Patent Document 1] The Journal of Clinical Investigation, 2017;127(10):3877-3896 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide an agent that inhibits the transition of lymphatic vessels to the endothelial-mesenchymal region and a lymphatic vessel aging inhibitor that inhibits the aging of lymphatic vessels by inhibiting the transition of lymphatic vessels to the endothelial-mesenchymal region. [Means for solving the problem]

[0008] The inventors have confirmed that the differentiation of lymphatic endothelial cells into mesenchymal cells in organs such as the skin is involved in lymphatic aging. Therefore, it is expected that if the differentiation of lymphatic endothelial cells into mesenchymal cells can be suppressed, the aging of lymphatic vessels in various organs of the body will be suppressed, and consequently, this will contribute to the suppression of aging in those organs. Accordingly, the inventors diligently researched the effects of various components as inhibitors of endothelial-mesenchymal transition in lymphatic vessels, and as a result, found that mulberry bark and guava leaves have particularly high efficacy, leading to the completion of the following invention: (1) An inhibitor of endothelial-mesenchymal transition of lymphatic vessels, containing mulberry bark and / or guava leaves as active ingredients. (2) An endothelial-mesenchymal transition inhibitor described in (1) that inhibits the transition of lymphatic vessels to the endothelial-mesenchymal region by inhibiting TGF-β (transforming growth factor-β). (3) A lymphatic vessel aging inhibitor containing mulberry bark and / or guava leaves as active ingredients, which inhibits the aging of lymphatic vessels by suppressing the transition of lymphatic vessels to the endothelium and mesenchymal layer. (4) A lymphatic vessel aging inhibitor described in (3) that inhibits the endothelial-mesenchymal transition of lymphatic vessels by inhibiting TGF-β. (5) A TGF-β inhibitor containing guava leaf as an active ingredient. (6) A composition comprising an endothelial-mesenchymal transition inhibitor as described in (1) or (2), a lymphatic vessel aging inhibitor as described in (3) or (4), and / or a TGF-β inhibitor as described in (5). A cosmetic method for preventing lymphatic aging of a target, comprising administering the composition described in (7)(6) to the target. (8) A beauty counseling method that supports the beauty treatment, including proposing the composition described in (6) as the target. [Effects of the Invention]

[0009] According to the present invention, an endothelial-mesenchymal transition inhibitor containing mulberry bark and / or guava leaves as active ingredients can be provided. Administration of the endothelial-mesenchymal transition inhibitor of the present invention can suppress endothelial-mesenchymal transition. By suppressing endothelial-mesenchymal transition of lymphatic vessels, the aging of lymphatic vessels can also be suppressed. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1 shows the expression of LYVE1 and SM22α in the young and elderly groups. The upper figure is a representative figure showing the expression of LYVE1 and SM22α in endothelial cells of cutaneous lymphatic vessels in the young and elderly groups. The lower figure is a graph showing the ratio of LYVE1 to SM22α expression levels (EndMT ratio) in the young and elderly groups (n=8) (Student's t-test, *p<0.05). [Figure 2] Figure 2 is a graph showing the expression levels of Prox1 (top graph) and VEGFR3 (bottom graph) in human cutaneous lymphatic endothelial cells (HDLEC) for each culture generation. The vertical axis represents the percentage of expression in each subsequent culture generation, with the expression level in the fifth passage cell (kk2 p5) set to 100. The horizontal axis represents the culture generation from the fifth passage cell. [Figure 3] Figure 3 shows the expression levels of LYVE1 and SM22α in HDLEC. Each graph shows, from left to right, the results with SB431542 (SB), and the control with 0.1% BSA / 4mM HCl (Ctrl) and TGF-β (TGF-β) added (T-test, ***p<0.001). [Figure 4] Figure 4 shows the inhibitory effects of various samples on TGF-β. The results of adding only PBS (cont), only TGF-β (TGFb), TGF-β + SB431542 (TGFb + SB), and TGF-β + various samples (1 - 18) are shown. 13 represents the guava leaf extract, and 15 represents the mulberry root bark extract. [Figure 5] Figure 5 shows the results of measuring the expression levels of LYVE1 and Prox1 in HDLEC by RT-PCR. The left figure shows the expression level of LYVE1, and the right figure shows the expression level of Prox1. In each figure, from left to right, the cases of adding only PBS, only TGF-β, only SB431542, SB431542 + TGF-β, only the mulberry root bark extract, and TGF-β + the mulberry root bark extract are shown. [Figure 6] Figure 6 shows the results of measuring the expression level of SM22α in HDLEC by RT-PCR. From left to right, the cases of adding only PBS, only TGF-β, only SB431542, SB431542 + TGF-β, only the mulberry root bark extract, and TGF-β + the mulberry root bark extract are shown. [Figure 7] Figure 7 shows the morphological changes of HDLEC due to the addition of mulberry root bark and TGF-β. From left to right in the upper figure are shown only SB (SB), only BSA / 4 mM HCl (mock), and TGF-β (TGF-β). From left to right in the lower figure are shown SB431542 + TGF-β (SB + TGF-β), only the mulberry root bark extract (extract), and mulberry root bark extract + TGF-β (extract + TGF-β). [Figure 8] Figure 8 shows the results of an assay measuring the permeability of HDLEC. From left to right, the results of adding no additive (con), only TGF-β (TGF-β2), TGF-β + SB431542 (TGF-β2 + SB), and TGF-β + the mulberry root bark extract (TGF-β2 + 15) are shown (Dunnett's multiple comparison test, *p < 0.01). The vertical axis shows the relative value of the absorbance of FITC-Dextran leaked from the insert of each group, with the case of no addition set as 1. [Figure 9]Figure 9 shows the results of measuring the expression level of SM22α by RT-PCR. From the left, the results of adding only PBS (control), only TGF-β (TGF-β), TGF-β + SB431542 (TGF-β + SB), and TGF-β + guava leaf extract (TGF-β + guava leaf) are shown as relative values with the case of adding control set to 100 (Dunnett's multiple test, **p < 0.001).

Mode for Carrying Out the Invention

[0011] By administering the endothelial-mesenchymal transition inhibitor of the present invention, the endothelial-mesenchymal transition of lymphatic vessels can be suppressed. By suppressing the endothelial-mesenchymal transition of lymphatic vessels, the aging of lymphatic vessels can be suppressed. The endothelial-mesenchymal transition inhibitor and anti-aging agent of the present invention are useful for the excretion of waste products, the prevention or improvement of swelling, the promotion of metabolism, and the prevention or treatment of lymphatic vessel dysfunction. For example, by maintaining the good function of skin lymphatic vessels, the removal of waste products, the circulation of lymph fluid, etc. are carried out normally, and as a result, the prevention of skin aging such as spots, wrinkles, and sagging is expected.

[0012] Endothelial-mesenchymal transition of lymphatic vessels refers to the differentiation of endothelial cells into mesenchymal cells (hereinafter sometimes abbreviated as EndMT). Endothelial-mesenchymal transition of lymphatic vessels can be measured by a decrease in the expression level of endothelial cell markers in lymphatic endothelial cells, an increase in the expression level of mesenchymal cell markers, and an increase in the ratio of the expression level of mesenchymal cell markers to endothelial cell markers (EndMT ratio: calculated using the method of formula 1 below). Examples of endothelial cell markers include LYVE1, Prox1, and VEGFR3; examples of mesenchymal cell markers include SM22α, but are not limited to these; any endothelial cell marker or mesenchymal cell marker can be used, and the EndMT ratio, which is the ratio between them, can also be arbitrarily determined according to the above markers. Furthermore, when endothelial cells differentiate into mesenchymal cells, morphological changes such as fibrosis are observed (Figure 7), so it is also possible to measure this by morphological observation of the cells. Furthermore, when endothelial cells differentiate into mesenchymal cells, the number of lymphatic endothelial cells decreases, or they are unable to maintain their properties as lymphatic endothelial cells, leading to impaired lymphatic function and leakage of lymphatic fluid. This deterioration of lymphatic function can be confirmed, for example, by measuring the permeability of an insert on which lymphatic endothelial cells have formed using a permeability assay as shown in the example. In other words, the transition of lymphatic vessels to endothelial-mesenchymal cells can be confirmed by increased permeability measured by a permeability assay.

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[0013] Inhibition of endothelial-mesenchymal transition in lymphatic vessels refers to the suppression of the differentiation and conversion of endothelial cells in lymphatic vessels into mesenchymal cells.

[0014] Inhibition of lymphatic endothelial-mesenchymal transition may mean that, for example, the addition of a lymphatic endothelial-mesenchymal transition inhibitor suppresses the increase in the expression level of mesenchymal cell markers or the EndMT ratio in lymphatic endothelial cells, or the decrease in the expression level of endothelial cell markers, compared to the case where the inhibitor is not added. This suppression may be, for example, a statistically significant reduction with a significance level of 5% (e.g., Student's t-test), and / or it may be a suppression of, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0015] Here, it has been reported that TGF-β has the effect of inducing endothelial-mesenchymal transition. Therefore, when measuring the inhibitory effect of various drugs on endothelial-mesenchymal transition in lymphatic vessels, for example, endothelial-mesenchymal transition in lymphatic vessels may be promoted by adding substances that promote endothelial-mesenchymal transition, such as TGF-β. For example, the increase in the expression level of the above-mentioned mesenchymal cell markers or EndMT ratio, or the decrease in the expression level of endothelial cell markers, may be promoted by TGF-β.

[0016] TGF-β is a homodimer (molecular weight approximately 25 kDa) multifunctional cytokine identified as a factor that promotes fibroblast transformation. In mammals such as humans, there are three subtypes (TGF-β1 to β3) with similar structures. TGF-β exerts diverse effects, such as regulating cell proliferation, differentiation, and development, by binding to receptors on the cell membrane. Furthermore, TGF-β is known to suppress the proliferation of various cells, including epithelial cells, vascular endothelial cells, and lymphocytes, and is thought to be involved in various pathological conditions such as cancer and renal fibrosis.

[0017] The inventors have confirmed that with aging, the expression levels of endothelial cell markers in lymphatic endothelial cells decrease, the expression levels of mesenchymal cell markers and the EndMT ratio increase, the number of lymphatic endothelial cells decreases, and the morphology of endothelial cells changes. Therefore, lymphatic aging may refer to a decrease in the expression levels of endothelial cell markers such as LYVE1, Prox1, and VEGFR3 in lymphatic endothelial cells, and / or an increase in the expression levels of mesenchymal cell markers such as SM22α and the EndMT ratio, or it may refer to a decrease or hardening of lymphatic endothelial cells, or it may refer to a change in the morphology of lymphatic endothelial cells (e.g., a change to a fibroblast-like state), or it may refer to impaired lymphatic function due to a decrease and / or hardening of lymphatic endothelial cells. Note that the endothelial cell markers, mesenchymal cell markers, and EndMT ratio are not limited to those mentioned above.

[0018] Inhibition of lymphatic aging refers to suppressing the aging of lymphatic vessels as described above, and may also be mediated by inhibiting the transition of lymphatic vessels to the endothelium.

[0019] Inhibition of lymphatic vessel aging may mean, for example, that the increase in the expression level of mesenchymal cell markers and EndMT ratio, and the decrease in the expression level of endothelial cell markers in lymphatic endothelial cells, which are associated with aging, are suppressed when a lymphatic vessel aging inhibitor is added, compared to when it is not added. This suppression may be, for example, a reduction in the expression level of the above markers that has a statistically significant difference at a significance level of 5% (e.g., Student's t-test), and / or it may be a suppression of, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100%.

[0020] Here, when measuring the anti-aging effects of various drugs on lymphatic vessels, it is possible to promote aging by accelerating the transition of lymphatic vessels to the endothelial-mesenchymal region, for example, by adding TGF-β. For example, the increase in the expression level of mesenchymal cell markers and EndMT ratio, and the decrease in the expression level of endothelial cell markers due to the above-mentioned aging, may be promoted by the use of TGF-β.

[0021] Mulberry bark refers to the root bark of plants belonging to the genus Morus in the family Moraceae, such as Morus alba Linne. Mulberry bark has been used as a traditional Chinese medicine for a long time, and has been reported to have diuretic, analgesic, anti-inflammatory, and antitussive effects (Patent Documents 1 and 2). It has also been reported that mulberry bark has a TGF-β inhibitory effect and is effective for hair growth (Patent Document 2). Guava leaves refer to the leaves of plants belonging to the genus Psidium in the family Myrtaceae, such as guava (Psidium guajava). Guava leaves have been reported to be effective for diabetes, hypertension, obesity, diarrhea, etc. (Patent Document 3). However, it is not known that mulberry bark or guava leaves have an inhibitory effect on the endothelial-mesenchymal transition of lymphatic vessels in organs such as the skin, or an inhibitory effect on lymphatic vessel aging.

[0022] The lymphatic vessel endothelial-mesenchymal transition inhibitor or lymphatic vessel aging inhibitor of the present invention may contain, as an active ingredient, mulberry bark and / or guava leaves in amounts of, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more by dry weight. In one embodiment, the lymphatic vessel endothelial-mesenchymal transition inhibitor or lymphatic vessel aging inhibitor of the present invention may consist of mulberry bark and / or guava leaves.

[0023] Mulberry bark and / or guava leaves can be used fresh or dried, but they may also be in the form of a dried powder or extract.

[0024] Methods for obtaining dried powder include finely chopping or grinding mulberry bark and / or guava leaves and then drying them, or drying the plants and then finely chopping or grinding them to obtain dried powder. Alternatively, methods such as finely chopping or grinding the plants, subjecting them to fermentation or enzymatic treatment, drying them, and then grinding them further to a predetermined particle size can be used as appropriate.

[0025] When using mulberry bark and / or guava leaves as an extract, the extraction method can be solvent extraction. In the case of solvent extraction, the mulberry bark and / or guava leaves are dried as necessary, and then finely chopped or pulverized as necessary, and then extracted using an aqueous extractant, such as cold water, warm water, or hot water at or below its boiling point, or an aqueous organic solvent, such as methanol, ethanol, 1,3-butanediol, ether, or ethyl acetate, at room temperature or after heating. However, the extraction method is not limited to solvent extraction and may be carried out by commonly known methods in the industry. The form of the above extract may be the extract itself, or it may be diluted or concentrated as appropriate by commonly used methods, or it may be a powder or a solid obtained by drying the extract.

[0026] The present invention also provides a method for preventing lymphatic aging by inhibiting the endothelial-mesenchymal transition of lymphatic vessels, by administering an endothelial-mesenchymal transition inhibitor or lymphatic vessel aging inhibitor or a composition containing the same, which contains mulberry bark and / or guava leaf as an active ingredient. The method of the present invention is for cosmetic purposes and may not be a treatment performed by a doctor or medical professional. In one embodiment, the method of the present invention may be a cosmetic method for preventing skin aging caused by the aging of lymphatic vessels in the skin, such as swelling, blemishes, wrinkles, and sagging, by administering mulberry bark and / or guava leaf or a composition containing the same, thereby inhibiting the endothelial-mesenchymal transition of lymphatic vessels in the skin. Furthermore, the present invention also provides a cosmetic counseling method to support a target cosmetic procedure, which includes proposing mulberry bark and / or guava leaf or a composition containing the same as the target.

[0027] The administration route of the endothelial-mesenchymal transition inhibitor or lymphatic vessel aging inhibitor or composition of the present invention can be arbitrarily selected, and examples include oral administration, transdermal administration, subcutaneous administration, transmucosal administration, intramuscular administration, etc.

[0028] The amount of mulberry bark and / or guava leaf in the lymphatic vessel endothelial-mesenchymal transition inhibitor or lymphatic vessel aging inhibitor or composition of the present invention can be appropriately determined according to their type, purpose, form, and method of use. For example, in the case of transdermal administration, the amount may be arbitrarily adjusted so that it is approximately 0.0001% to 10% by dry mass, approximately 0.001% to 1% by dry mass, or approximately 0.01% to 0.1% by dry mass relative to the total mass of the topical preparation. In any case, it is preferable to include the active ingredient of the present invention in an amount that sufficiently exerts the effect of inhibiting lymphatic vessel endothelial-mesenchymal transition.

[0029] Furthermore, the compositions of the present invention may be food compositions, cosmetic compositions, or pharmaceutical compositions. Food compositions may be powders, beverages, or tablets, and may take various forms such as powder, liquid, solid, granular, granular, paste, or gel. Cosmetic compositions may be emulsions, creams, serums, lotions, packs, facial cleansers, soaps, body washes, shampoos, etc., and may take various forms such as liquid, emulsion, cream, solid, sheet, spray, gel, foam, or powder. Pharmaceutical compositions may be tablets, capsules, powders, granules, ointments, creams, patches, etc.

[0030] Furthermore, ingredients used in quasi-drugs, cosmetics, pharmaceuticals, etc., can be appropriately incorporated as needed. For example, known ingredients such as excipients, colorants, preservatives, thickeners, binders, disintegrants, dispersants, stabilizers, gelling agents, antioxidants, surfactants, pH adjusters, oils, surfactants, powders, colorants, water, alcohols, thickeners, chelating agents, silicones, antioxidants, UV absorbers, humectants, fragrances, various medicinal ingredients, preservatives, pH adjusters, neutralizing agents, etc., can be appropriately selected and used.

[0031] Furthermore, the present invention also provides mulberry bark and / or guava leaves for inhibiting lymphatic vessel aging by suppressing endothelial-mesenchymal transition of lymphatic vessels; the use of mulberry bark and / or guava leaves in the manufacture of an endothelial-mesenchymal transition inhibitor or a lymphatic vessel aging inhibitor; and a method for manufacturing an endothelial-mesenchymal transition inhibitor or a lymphatic vessel aging inhibitor or a composition containing the same using mulberry bark and / or guava leaves as an active ingredient. The mulberry bark and / or guava used in this manufacturing method may be in any form, such as dried, powdered, or as an extract, as described above. [Examples]

[0032] The present invention will now be described in more detail with reference to examples. However, the present invention is not limited thereto.

[0033] Experiment 1: In vivo changes due to aging of cutaneous lymphatic endothelial cells Excess skin was collected from healthy men and women aged 22 and older. These subjects were divided into two groups: a young adult group of 8 individuals aged 22 to 40, and an elderly group of 8 individuals aged 40 to 73. LYVE1 was used as the endothelial cell marker, and SM22α was used as the mesenchymal cell marker. Endothelial cell samples from the cutaneous lymphatic vessels of each group were prepared by immunofluorescence staining using anti-LYVE-1 (Reliatech), anti-SM22alpha antibody (Abcam), and corresponding fluorescently labeled secondary antibodies. The expression of LYVE1 and SM22α in the endothelial cells was visualized using a confocal laser microscope (Carl Zeiss). The ratio of LYVE1 to SM22α expression levels (EndMT ratio) was calculated according to Equation 2 below. Student's t-test was used for statistical significance testing.

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[0034] The results are shown in Figure 1. The lower figure is a graph showing the ratio of LYVE1 to SM22α expression levels (EndMT ratio) for each group, which is the result of Experiment 1. From this figure, it can be seen that the EndMT ratio in the elderly group was significantly higher than in the young group (p<0.05), meaning that the expression level of SM22α relative to LYVE1 increased with age. Furthermore, fluorescence microscopy images of lymphatic vessels also showed that while LYVE1 expression was high in the young group, LYVE1 expression (red) decreased and SM22α expression (green) increased in the elderly group (upper figure 1). These results confirm that endothelial cells of skin lymphatic vessels are more likely to differentiate into mesenchymal cells in older individuals.

[0035] Experiment 2: In vitro changes in human cutaneous lymphatic endothelial cells (HDLECs) due to aging. 2-1: Culture of human cutaneous lymphatic endothelial cells The sample used was human dermal lymphatic endothelial cells (HDLEC cells) isolated from the foreskin according to the method described in Kajiya et al., EMBO J. 2005 Aug 17; 24(16): 2885-2895. These HDLEC cells were then subjected to EGM. TM -2MV Microvascular Endothelial Cell Growth Medium-2 BulletKit TM Using (Lonza, C-3202), SingleQuots are added to the Basal Medium (CC-3156) included in the kit. TM Cells were cultured in a medium supplemented with supplements (CC-4147) (hereinafter abbreviated as EBM2(+) medium). The cell status was monitored using Prox1 and VEGFR3 as endothelial cell markers, and the cells were repeatedly passed through the culture medium. The expression levels of Prox1 and VEGFR3 were measured using the TaqMan Gene Expression Assays from Applied Biosystems by quantitative RT-PCR, as detailed below.

[0036] 2-2: Cell harvesting and RNA preparation After washing the wells with PBS, RLT Buffer from the QIAGEN RNeasy Mini Kit was added, and cells were scraped off using a cell scraper and transferred to 1.5 ml Eppendorf tubes. RNA was extracted according to the QIAGEN RNeasy Mini Kit protocol. Elution was performed with 30 μl of RNase-free water. RNA concentration was measured using a Nano Drop2000 ultra-trace spectrophotometer, and a portion was diluted to a concentration of 50 nM.

[0037] 2-3: Quantification of mRNA by RT-PCR RNA expression was measured by real-time PCR using probe detection. The TaqMan RNA-to-C 1-Step Kit Applied Biosystems was used, with Prox1 and VEGFR3 as probes. A Light Cycler 480II (Roche) was used as the measurement instrument. The PCR program and composition are shown in the table below. [Table 1] [Table 2]

[0038] Figure 2 shows graphs illustrating the expression levels of Prox1 (top graph) and VEGFR3 (bottom graph) for each culture generation, based on the results of Experiment 2. The expression levels of these endothelial cell markers decreased as the number of passages increased. Therefore, it was confirmed that endothelial cells of the lymphatic vessels in the skin decrease in number as they age, either because they are unable to differentiate into normal lymphatic endothelial cells or because they are unable to maintain their properties as lymphatic endothelial cells.

[0039] Experiments 1 and 2 suggest that endothelial-mesenchymal transition in cutaneous lymphatic vessels is associated with aging, and that inhibiting endothelial-mesenchymal transition could suppress lymphatic vessel aging. Therefore, we searched for substances that inhibit endothelial-mesenchymal transition.

[0040] Experiment 3: Sample Preparation Morus alba Linne root bark ("Souhakuhi") was extracted with 70% ethanol by volume, the resulting extract was further extracted with ethyl acetate, the ethyl acetate was removed by distillation, and the residue was dissolved with 70% ethanol by volume to prepare a mulberry bark extract. Guava leaf extract was prepared by extracting the leaves of "Psidium guajava" (the leaves of the guava plant) with 70% ethanol by volume, and then purifying the resulting extract with 70% ethanol by volume. In addition to the above-mentioned samples, a total of 34 samples were prepared using 32 other plant species, such as aloe vera juice.

[0041] Experiment 4: Effect of TGF-β signaling on the expression of endothelial cell markers and mesenchymal cell markers in human cutaneous lymphatic endothelial cells (HDLECs). 4-1: Culture of human cutaneous lymphatic endothelial cells and addition of samples to HDLEC cells Using HDLEC cells (Lonza, cc-2810), 8 × 10⁶ cells were placed on a collagen-coated 6-well plate. 4 Cells were seeded in cells / well and cultured at 37°C in the same EBM2(+) medium as in Experiment 2. The following day, SB431542 (Wako, 198-16543) (hereinafter abbreviated as SB), a TGF-β receptor kinase inhibitor, was added to a final concentration of 5 μM, TGF-β2 (hereinafter abbreviated as TGF-β) was added to a final concentration of 1 ng / ml, and 1 μl of 0.1% BSA (Ctrl) dissolved in 4 mM HCl was added as a control, and the cells were cultured for a further 72 hours.

[0042] 4-2: Cell harvesting and RNA preparation After washing the wells with PBS, RNA was prepared using NucleoSpin RNA, and cDNA synthesis was performed using PrimeScript II (TaKaRaBio) with random hexamer primers.

[0043] 4-3: Quantification of mRNA by RT-PCR Using the cDNA synthesized in Experiment 4-2 as a template, quantitative RT-PCR was performed using the PCR primers shown in the table below and FastStart Universal SYBR Green Master (ROX) (Roche). β-actin was used as an endogenous control. The PCR program and composition are as shown in the table below. [Table 3] [Table 4] [Table 5]

[0044] 4-4:Results Figure 3 shows the results of measuring the expression levels of LYVE1 and SM22α by the quantitative RT-PCR described above. LYVE1 expression was significantly decreased when TGF-β was added compared to the control, and significantly increased when a TGF-β inhibitor was added. Similarly, SM22α expression was significantly increased when TGF-β was added compared to the control, and significantly decreased when a TGF-β inhibitor was added.

[0045] The results of Experiment 4 show that endothelial-mesenchymal transition of cutaneous lymphatic vessels is promoted by TGF-β signaling, and therefore, it is suggested that aging of cutaneous lymphatic vessels is also promoted by TGF-β signaling. Thus, it is suggested that if endothelial-mesenchymal transition induced by TGF-β signaling can be suppressed, lymphatic vessel aging can be suppressed. Furthermore, by using an endothelial-mesenchymal transition evaluation system that utilizes the expression of endothelial cell markers and mesenchymal cell markers in HDLEC, it is possible to search for agents that inhibit endothelial-mesenchymal transition and aging of lymphatic vessels, and it is clear that TGF-β signaling can be used in such an evaluation system.

[0046] Experiment 5: Inhibitory effects of various samples on TGF-β As a pre-screening before use in the endothelial-mesenchymal transition evaluation system in Experiment 4, the inhibitory effect of various samples prepared in Experiment 3 on TGF-β was investigated by measuring the absorbance (640 nm) using HEK-Blue TGF-β cells (InvivoGen). Specifically, samples 1 to 18 of the 34 samples prepared in Experiment 3 were added together with TGF-β. As controls, PBS alone, TGF-β alone, and TGF-β + SB were used. HEK-Blue TGF-β cells were placed in 12 × 10⁶ well plates. 4 Cells were seeded in a cell / well ratio. The following day, stimulation was performed as shown in the table below. If SB was added, stimulation with TGF-β and each ligand was performed 2 hours after SB addition. 24 hours after stimulation, 2.5 μL of the supernatant was taken, mixed with 100 μL of the substrate QUANTI-Blue, incubated at 37°C for 30 minutes, and the absorbance at 640 nm was measured. [Table 6] Figure 4 shows the results of adding TGF-β to samples 1-18 (1-18), as well as the results of adding PBS alone (cont), TGF-β alone (TGFb), and TGF-β + SB (TGFb + SB). In the figure, sample number 15 is mulberry bark extract (final concentration 0.01% by mass), and sample number 13 is guava leaf extract (final concentration 0.1% by mass). Except for cont, which does not contain TGF-β, lower absorbance indicates a higher inhibitory effect of TGF-β. Figure 4 shows that mulberry bark extract (15) and guava leaf extract (13) exhibited inhibitory effects on TGF-β.

[0047] Experiment 6: Analysis of endothelial cell marker expression levels after addition of mulberry bark extract Using mulberry bark extract pre-screened in Experiment 5, the expression levels of endothelial cell markers LYVE1 and Prox1 were analyzed using the endothelial-mesenchymal transition evaluation system from Experiment 4. Specifically, HDLEC cells were placed in collagen-coated 6-well plates in 8 × 10⁶ layers, similar to Experiment 4. 4Cells were seeded in cells / well and cultured at 37°C in the same EBM2(+) medium as in Experiment 2. The following day, cell adhesion was confirmed, and test groups were set up as shown in the table below, with each sample added. Specifically, 2 μl of PBS was added as a control, TGF-β was added to a final concentration of 1 ng / mL, SB was added to a final concentration of 5 μM, and 2 μl of mulberry bark extract prepared in Experiment 3 (to a final concentration of 0.1% by mass) was added. After 72 hours of incubation, quantitative RT-PCR was performed in the same manner as in Experiment 4. In addition to the endothelial cell marker LYVE1, PCR primers for Prox1 were used, and a β-actin probe was used as an endogenous control. The PCR primers for LYVE1 and β-actin are the same as in Experiment 4. The PCR primers for Prox1 are shown below. [Table 7] [Table 8]

[0048] The results are shown in Figure 5. When only TGF-β was added, the expression of LYVE1 and Prox1 decreased, but when SB was added, their expression increased. Furthermore, when mulberry bark extract was added, the decrease in LYVE1 and Prox1 expression caused by TGF-β was suppressed, and moreover, their expression was considerably higher than in the case where nothing was added. Therefore, it is suggested that mulberry bark not only has the effect of suppressing endothelial-mesenchymal transition promoted by TGF-β, but also has the effect of promoting differentiation into lymphatic endothelial cells.

[0049] Experiment 7: Analysis of mesenchymal cell marker expression levels after addition of mulberry bark extract The same samples and methods as in Experiment 6 were used, except that the expression level of the mesenchymal cell marker SM22α was measured instead of the endothelial cell marker.

[0050] The results are shown in Figure 6. The TGF-β-induced increase in the expression of the mesenchymal cell marker SM22α was inhibited by mulberry bark extract. This inhibitory effect was stronger than that of SB.

[0051] Experiment 8: Changes in HDLEC morphology due to the addition of mulberry bark and TGF-β Similar to Experiment 4, HDLEC cells were placed in a collagen-coated 6-well plate in 8 × 10⁶ rows. 4 Cells were seeded in cells / well and cultured at 37°C in the same EBM2(+) medium as in Experiment 2. The following day, cell adhesion was confirmed, and test plots were set up as shown in the table below, with each sample added. Specifically, SB was added to a final concentration of 5 μM, 1 μl of 0.1% BSA / 4 mM HCl as a control, TGF-β was added to a final concentration of 1 ng / mL, and 2 μl of the mulberry bark extract prepared in Experiment 3 (to a final concentration of 0.1% by mass) was added. After 72 hours of incubation, the cell morphology was observed using a fluorescence microscope BZ-X710 (Keyence). [Table 9]

[0052] Figure 7 shows the results. From Figure 7, it can be seen that when TGF-β is added, the number of cells decreases significantly and their morphology changes. Therefore, it is suggested that TGF-β affects cutaneous lymphatic endothelial cells and impairs their function as lymphatic vessels. However, even when TGF-β is added, the decrease in cell number and changes in morphology are suppressed when SB or mulberry bark extract is added at the same time.

[0053] Experiment 9: Permeability (leakage) assay of human lymphatic endothelial cells (HDLECs) Fibronectin (Corning), diluted to 5 μg / ml in PBS, was added in 100 μl to each insert of an HTS transwell-24well 0.4 μm (Corning) container, incubated at 37°C for 15 minutes, and then aspirated (fibronectin coating). HDLEC cultured in EBM2(+) medium, as in Experiment 2, was placed in fibronectin-coated inserts in a 4x10⁻¹⁴⁻¹ 4 Seeds were seeded in cells / well and cultured at 37°C.

[0054] After 24 hours, the cells were divided into four groups: control, TGF-β, TGF-β + SB, and TGF-β + mulberry bark extract (15). For the group with SB added, SB was added to the EBM2(+) medium at a final concentration of 1 μM, or for the group with mulberry bark extract added, the mulberry bark extract was added to the EBM2(+) medium at a final concentration of 0.1%. The medium for the other groups was replaced with the EBM2(+) medium without addition, and the cells were cultured at 37°C.

[0055] After 3 hours, the medium in all wells was replaced with the EBM2(+) medium without addition, and the cells were cultured at 37°C. After 24 hours, for the group with TGF-β added, the medium was replaced with the EBM2(+) medium prepared by adding TGF-β at a final concentration of 1 ng / ml, and for the other groups, the medium was replaced with the EBM2(+) medium without addition, and the cells were cultured at 37°C. After 24 hours, 50 μl of FITC-Dextran adjusted to 10 mg / ml was added, and after incubating at 37°C for 15 minutes, the FITC-Dextran leaked from the insert was measured with a plate reader.

[0056] The results are shown in Figure 8 as the relative values of the absorbance of FITC-Dextran leaked from the insert of each group, with the case of the control being set as 1. The addition of only TGF-β2 increased the permeability of HDLEC, while the treatment with SB, a TGF-β inhibitor, and the mulberry bark extract suppressed the increase in permeability.

[0057] Experiment 10: Analysis of the expression level of mesenchymal cell markers by adding guava leaf extract 10-1: Culture of lymphatic endothelial cells Using the guava leaf extract pre-screened in Experiment 5, the expression level of the mesenchymal cell marker SM22α was analyzed by the endothelial-mesenchymal transition evaluation system of Experiment 4. Specifically, human dermal micro-lymphatic endothelial cells (HDLEC) obtained from LONZA were seeded in a 6-well plate at 6 × 10 4 cells / well with the same EBM2(+) as in Experiment 2 and cultured overnight at 37°C. The next day, cell adhesion was confirmed, and after stimulating with SB at 5 μM and TGF-β at 1 ng / mL respectively, the cells were cultured for 72 hours.

[0058] 10-2: Sample addition to HDLEC cells As described above, the cultured HDLEC cells were placed in a collagen-coated 6-well dish, 4 × 10 5 Seeds were seeded at the cells / well rate. SingleQuots were used in Basal Medium (CC-3156), which was included in the same kit as in Experiment 2. TM Medium (EBM2(+) medium) supplemented with supplements (CC-4147) was added to each well to make a cell suspension of 2 ml. After culturing at 37°C for 24 hours, the medium was replaced with Basal Medium (CC-3156) (EBM2(-) medium) from the same kit, with 0.5% FBS added. After culturing at 37°C for 4 hours, test groups were set up as shown in the table below, and each sample was added. Each sample was diluted with PBS and added to the concentrations shown in the table below. After addition, HDLEC cells were cultured at 37°C for 20 hours. After culturing, the expression level of SM22α was analyzed by quantitative RT-PCR as in Experiment 2. [Table 10]

[0059] 10-3:Results The results are shown in Figure 9. When only TGF-β was added, SM22α expression increased, but when SB was added, its expression decreased. Furthermore, it was found that guava leaf extract also significantly reduced SM22α expression induced by TGF-β. Therefore, guava leaf extract also had the effect of suppressing the endothelial-mesenchymal translocation of lymphatic vessels promoted by TGF-β.

[0060] Experiment 11: Analysis of other samples Of the 32 samples other than mulberry bark and guava leaf prepared in Experiment 3, sample 18 showed a TGF-β inhibitory effect. However, when SM22α expression was confirmed using sample 18 in the same manner as in Experiment 4, SM22α expression was higher in TGF-β + sample 18 than when TGF-β alone was added, indicating that endothelial-mesenchymal translocation was actually promoted. Furthermore, although sample 17 also showed a TGF-β inhibitory effect, cytotoxicity was observed (data not shown).

[0061] These results reveal that mulberry bark and guava leaves have TGF-β inhibitory effects, as well as effects that suppress endothelial-mesenchymal transition and lymphatic vessel aging.

[0062] The present invention can suppress endothelial-mesenchymal transition by administering an endothelial-mesenchymal transition inhibitor containing mulberry bark and / or guava leaves as active ingredients. By suppressing endothelial-mesenchymal transition in lymphatic vessels, the aging of lymphatic vessels can be suppressed. Furthermore, the endothelial-mesenchymal transition inhibitor of the present invention is expected to suppress endothelial-mesenchymal transition not only in lymphatic vessels but also in blood vessels, thereby preventing the aging of blood vessels.

Claims

1. An inhibitor of endothelial-mesenchymal transition of cutaneous lymphatic vessels, containing mulberry bark and / or guava leaves as active ingredients.

2. An endothelial-mesenchymal transition inhibitor according to claim 1, which inhibits the transition of cutaneous lymphatic vessels to the endothelial-mesenchymal region by inhibiting TGF-β (transforming growth factor-β).

3. A skin lymphatic vessel aging inhibitor containing mulberry bark and / or guava leaf as active ingredients, which inhibits the aging of skin lymphatic vessels by suppressing the transition of skin lymphatic vessels to the endothelial-mesenchymal region.

4. The cutaneous lymphatic vessel aging inhibitor according to claim 3, which inhibits the transition of cutaneous lymphatic vessels to the endothelium by inhibiting TGF-β.

5. A TGF-β inhibitor containing guava leaf as an active ingredient.

6. A composition for inhibiting cutaneous lymphatic aging by inhibiting endothelial-mesenchymal transition of cutaneous lymphatic vessels, comprising an endothelial-mesenchymal transition inhibitor according to claim 1 or 2, a lymphatic vessel aging inhibitor according to claim 3 or 4, and / or a TGF-β inhibitor according to claim 5, by inhibiting TGF-β in endothelial cells of cutaneous lymphatic vessels.

7. A cosmetic method for preventing lymphatic aging of a target, comprising applying the cosmetic to the target skin, wherein the composition described in claim 6 is a cosmetic.

8. A beauty counseling method that supports a beauty treatment for suppressing lymphatic aging of the target skin, comprising proposing the composition described in claim 6 as a cosmetic, and applying the cosmetic to the target skin.

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