JPWO2018207952A1 - Hair growth and / or hair growth composition - Google Patents

Abstract

Formula I: embedded image (wherein one of R1 and R3 represents a hydrogen atom or a hydroxyl group, and the other is represented by Formula IV: embedded image (R4b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms) , N represents an integer of 1 to 5), R2 represents a hydrogen atom or an oxygen atom, R4a is the same as R4b, and embedded image represents a double bond or a single bond. And m represents an integer of 1 to 4), and a hair growth and / or hair growth composition comprising the compound represented by the formula.

Description

  The present invention relates to a hair growth and / or hair growth composition and the like containing fisetin and the like.

  The skin is an organ that covers the entire surface of our body and plays a role of a protective function that prevents invasion and irritation of foreign substances from the surrounding environment, and a role of sensory organs such as touch and temperature sensation. Since it also greatly affects the quality of life (QOL), it is an important organ in maintaining and improving the quality of life (QOL).

The structure of the skin is divided into an epidermis derived from the outer embryo (epidermis), a dermis derived from the mesoderm (dermis), and a subcutaneous tissue (subcutaneos tissue) from the outside. As such an accessory organ of the skin, a hair follicle such as a sheath that encloses a hair shaft composed of keratinized cells rich in keratin fibers exposed to the outside of the skin, a sebaceous gland, There are sweat glands and the like. The hair follicle is a portion buried in the dermis of the skin, and an upper root sheath (outer root sheath) whose upper part is connected to the surface layer of the skin, an inner root sheath (inner root sheath), and a hair papilla cell (dermal). papilla) and hair matrix (hair matrix). Of these, most of the hair follicles are occupied by epithelial cells except for the dermal papilla and dermal sheath cells derived from mesenchymal cells. At the lower end of the hair follicle during growth, there is a hair bulb that creates a hair shaft, and hair matrix cells surround the hair papilla cells. In hair matrix cells, epithelial progenitor cells differentiated from epithelial stem cells are actively differentiated and proliferated to form hair.
The production of hair in the hair follicle is dependent on the cycle of the hair cycle.

  The hair cycle is roughly divided into a growth period called anagen, a resting period called catagen, and a catagen called telogen. In anagen, hair matrix cells associated with hair papilla cells undergo cell division to produce hair. Subsequent transfer to catagen halts cell division and halts hair growth. In telogen, the papilla tissue is obscured and the hair is completely separated from the papilla tissue. When catagen is over, preparations for the next hair are made, and it becomes anagen again. The hair follicle repeats growth and retraction along with this hair cycle, but the homeostatic part above the region where hair follicle stem cells called bulge exist is maintained regardless of the hair cycle, and only the variable part below is in the hair cycle. Dominated (Muller-Rover S et al., 2001). The epithelial cells that make up the hair follicles are considered to be differentiated from the hair follicle stem cells present in this bulge region (Cotsarelis, 2006; Oshima et al., 2001; Petersson et al., 2011).

  As mentioned above, our body has hair follicles (hairs), which are the accessory organs of the skin that repeat self-renewal, but most human hair has already degenerated and the areas where hair is dense are limited. ing. Therefore, the hair of the head in particular has a large reason mainly for its appearance, but most of the hair of this man decreases with age. This reduction of hair is a physiological phenomenon and a natural aging phenomenon, but until recently, basic research and clinical research on this hair were insufficient. However, in recent decades, research on hair has rapidly progressed, and among them, telomerase reverse transcriptase telomerase reverse transcription (hereinafter referred to as TERT) has been reported as a factor promoting hair improvement (Sarin, K et al., 2005). ).

  Telomerase is a normal cell that maintains a telomere repeat sequence that shortens with each cell division (when shortened to the limit, a signal for mitotic arrest is generated and the cell cannot grow). It is a ribonucleoprotein enzyme that synthesizes. This telomerase is a complex composed of telomerase RNA component (TERC) as a template RNA and telomerase reverse transcriptase (TERT) as a reverse transcriptase, and is known to be activated in cancerous cells or immortalized cells. (Shay and Baccetti, 1997).

  However, in addition, telomerase activation during calorie restriction, which is known to achieve anti-aging of individuals (Pendergrass et al., 2001; Vera et al., 2013), and telomerase knockout mice, Anemia, delayed healing of wounds, aging phenomena such as gray hair are observed (Herrera et al., 1999), and telomerase transgenic mice have been reported to have anti-aging effects on tissues such as skin, intestinal tract, and muscle ( Thomas Loba et al., 2008). These results indicate that activation of telomerase does not necessarily induce undesirable effects on the individual, such as canceration. The telomerase activity is now considered to be regulated by the expression level of the telomerase catalytic subunit gene (TERT) (Nakamura et al., 1997).

  Since the cloning of human TERT (hTERT), various studies have been conducted on the function and expression regulation of hTERT. One of the reports is that the forced expression of TERT in the mouse epidermis stimulates the proliferation of hair follicle stem cells and improves hair (Sarin, K et al., 2005). The onset of anagen depends on hair follicle stem cells present in the bulge region on the outer root sheath, which is composed of epidermal cells of the hair follicle. When activated, hair follicle stem cells migrate to the hair matrix, where they differentiate into hair matrix cells that promote the synthesis of hair and produce hair. At this time, when telomerase (TERT), which is activated only in the hair follicles of anagen, is overexpressed and activated in the epidermis of mice in the telogen stage, the stem cells in the bulge are stimulated to actively divide. become. That is, forced expression of TERT in the epithelium induces telomerase activity that causes division of hair follicle stem cells existing in the same epithelium, and promotes the transition from telogen to anagen, thereby enabling improvement of hair.

  Moreover, it is said that the thickness of hair is determined in proportion to the size of the hair follicle, and the length of hair is mainly determined in proportion to the length of anagen. Therefore, in order to see a large amount of hair, a sufficient anagen period is required. It is important to maintain. In the transition from telogen to anagen, in addition to signals such as signal transduction and transcriptional activators STAT3, 4, 5 and Wnt and SHH, insulin-like growth factor (IGF) -1, hepatocyte growth factor (HGF) and It is believed that keratinocyte growth factor (KGF) and the like are involved (Krause K et al., 2006; Stenn KS et al., 2001; Cossarelis G et al., 2001; Paus R et al., 2004). Among them, the Wnt / β-catenin signal is required for signals from epithelial cells to dermal papilla cells and differentiation of hair sheath cells in the anagen phase (Cotsarelis G et al., 2001), and requires transcription factors. It is reported that it regulates cell proliferation and differentiation by activating the transcription of target genes via the mediation, is involved in hair shaft differentiation in the skin, and is important as a major pathway that moves the hair cycle (Stenn and Paus, 2001; Paus and Foitzik, 2004).

  This Wnt plays an important role in controlling cell differentiation, proliferation, polarity, etc. in embryonic development and in maintaining homeostasis of adult tissues. In the first place, Wnt is a secreted protein and is known to be secreted through several stages. First, after undergoing palmitoylation by Porcupine (Porc) localized on the membrane of the endoplasmic reticulum (MacDonald et al., 2009; Port et al., 2010), it is transported to the Golgi apparatus and further by Wntless (Wls). It is transported to the cell membrane and secreted. As a signal pathway activated by binding of Wnt to a cell surface receptor, there is a Wnt / β-catenin pathway dependent on β-catenin. β-catenin was discovered as one of the protein molecules bound to cadherin, which is a calcium-dependent cell-cell adhesion molecule (Ozawa et al., 1989), and cadherin was used as an actin filament, which is one of the cytoskeletons. It is known as a cell molecule that has a linking function and is essential for cell adhesion activity.

  The activity of this pathway is determined by the amount of β-catenin present in the cytoplasm. Normally, the amount of β-catenin in the cytoplasm is controlled by a complex consisting of four components of Axin, Adenomatous Polyposis coli (APC), Glycogen Synthase Kinase-3β (GSK-3β), and Casein Kinase 1α (CK1α). It is usually suppressed to low levels by proteasome-dependent proteolysis. Β-catenin is phosphorylated by a signal generated by binding of Wnt protein to the complex of Frizzled (Fzd) and Low-density lipoprotein Receptor-related Protein (LRP) 5/6 on the surface of the cell membrane, and β-catenin is phosphorylated, resulting in ubiquitin. After undergoing oxidization, it is degraded by the proteasome. However, β-catenin that escapes phosphorylation accumulates in the cytoplasm, and then migrates into the nucleus to form a complex with the transcription factor TCF (T cell factor) / LEF (lymphocyto enhancer factor 1), and Wnt. Controls transcription of signal target genes.

  Recent studies have shown that telomerase directly regulates Wnt / β-catenin signaling by acting as a cofactor in the β-catenin transcription complex (Park JI, 2009: Non-Patent Document 1). Alternatively, it has been reported that β-catenin binds to the TERT promoter to directly control the expression of TERT (Rolf Kemler et al., 2012: Non-Patent Document 2). Based on the above, it can be considered that the activity of the Wnt / β-catenin signal is deeply involved in the regulation of hair synthesis by TERT.

  On the other hand, a sirtuin (SIRT) gene, which is also called a longevity gene or an anti-aging gene, and whose lifespan is extended by its activation is known. Since the protein (sirtuin) synthesized by the activation of the SIRT gene promoter is a histone deacetylase, it is thought that it acts on the binding between histone and DNA and prolongs the lifespan by performing genetic regulation.

By the way, as a component to be added to the hair improving agent, minoxidil or a pyrazole derivative (Patent No. 5588167: Patent Document 1) as a component for suppressing abnormalities in the hair cycle, and a female hormone such as estradiol as a component for alleviating the action of male hormones. A pantothenic acid derivative is used as a cell-activating component, and capsicum tincture, 1-menthol, and an Assemblage extract are used as components that promote vasodilation and blood flow.
However, many hair improvers are mediated by inhibition of 5α reductase in dermal papilla cells. The effects of hair improvers vary greatly among individuals, and no satisfactory ones have been found.

Patent 5588167

Park JI et al. , Telomerase modulos Wnt signaling by association with target gene chromatin. Nature. 460: 66-72, 2009 Rudloff S, Kemmer R .; Differential requirements for β-catenin inducing mouse development. Development, 139: 3711--21,0212.

  The present invention aims at providing a composition for hair growth, hair growth, or skin improvement.

  The present inventors have conducted extensive studies to solve the above problems, and as a result, found that fisetin, which is a kind of polyphenol, or a derivative thereof has a hair growth effect, a hair growth effect, etc., and has completed the present invention. .

That is, the present invention is as follows.
(1) Formula I:
(In the formula, ^one of R ¹ and R ³ represents a hydrogen atom or a hydroxyl group, and the other represents the following formula IV:
(R ^4b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5.)
Represents a group represented by, R ² represents a hydrogen atom or an oxygen atom, R ^4a is the same as R ^4b ,
Represents a double bond or a single bond, and m represents an integer of 1 to 4. )
A compound represented by
Formula II:
(In the formula, R ^4a and R ^4b are the same as above, and n1 and n2 each independently represent an integer of 1 to 5.)
And a compound of the following formula III:
(Wherein R ² , R ^4a , R ^4b , n1 and n2 and
Is the same as above. )
A composition for hair growth and / or hair growth, containing any compound selected from the group consisting of the compounds represented by:
(2) The following formula I:
(In the formula, ^one of R ¹ and R ³ represents a hydrogen atom or a hydroxyl group, and the other represents the following formula IV:
(R ^4b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5.)
Represents a group represented by, R ² represents a hydrogen atom or an oxygen atom, R ^4a is the same as R ^4b ,
Represents a double bond or a single bond, and m represents an integer of 1 to 4. )
A compound represented by
Formula II:
(In the formula, R ^4a and R ^4b are the same as above, and n1 and n2 each independently represent an integer of 1 to 5.)
And a compound of the following formula III:
(Wherein R ² , R ^4a , R ^4b , n1 and n2 and
Is the same as above. )
A skin improving composition comprising any compound selected from the group consisting of compounds
(3) Formula I:
(In the formula, ^one of R ¹ and R ³ represents a hydrogen atom or a hydroxyl group, and the other represents the following formula IV:
(R ^4b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5.)
Represents a group represented by, R ² represents a hydrogen atom or an oxygen atom, R ^4a is the same as R ^4b ,
Represents a double bond or a single bond, and m represents an integer of 1 to 4. )
A compound represented by
Formula II:
(In the formula, R ^4a and R ^4b are the same as above, and n1 and n2 each independently represent an integer of 1 to 5.)
And a compound of the following formula III:
(Wherein R ² , R ^4a , R ^4b , n1 and n2 and
Is the same as above. )
A cosmetic composition comprising any compound selected from the group consisting of:
(4) The compound represented by the formula I has the following formula Ia or Ib:
(In the formula, R ¹ and R ³ represent a hydrogen atom or a hydroxyl group, R ² represents a hydrogen atom or an oxygen atom, and R ^4a and R ^4b each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. Represents a group,
Represents a double bond or a single bond, m represents an integer of 1 to 4, and n represents an integer of 1 to 5. )
The composition according to any one of (1) to (3), which is represented by
(5) The compound represented by formula I is fisetin, quercetin, catechin, nobiletin, apigenin, genistein, luteolin, ipriflavone, equol, tangeretin, biochanin A, chrysin, or baicalein (1) to (3) The composition according to item 1.
(6) The compound represented by the formula II is resveratrol, piceatannol, pinostilbene, isolapontigenin, rapontigenin, gnetol, or oxyresveratrol (1) to (3) The composition as described.
(7) The composition according to any one of (1) to (3), wherein the compound represented by the formula III is isoliquiritigenin or butein.
(8) The composition according to (2), wherein the skin improvement is at least one selected from the group consisting of treatment of wounds, repair of rough skin, improvement of spots, improvement of freckles, suppression of wrinkles, wound healing and suppression of cell aging. Stuff.
(9) A hair-growing and / or hair-growing agent containing the composition according to (1).
(10) A skin improving agent comprising the composition according to (2).
(11) A cosmetic containing the composition according to (3).
(12) A cell containing a vector in which a reporter gene is linked to the TERT promoter or SIRT1 promoter, or a cell containing an endogenous TERT gene or an endogenous SIRT1 gene is contacted with a test substance, and the reporter gene or the endogenous TERT gene or endogenous A method of screening a substance having at least one effect selected from the group consisting of a hair growth effect, a hair growth effect and a skin improving effect, using the expression of the sex SIRT1 gene as an index.
(13) The method according to (12), wherein the cells are epithelial cells.
(14) The method according to (12) or (13), wherein TERT is human TERT.
(15) A cell containing a DNA damage repair marker gene is contacted with a DNA-damaged cell with a test substance, and the expression of the gene is used as an index to be selected from the group consisting of a hair growth effect, a hair growth effect, and a skin improving effect. And a method for screening a substance having at least one effect.

  According to the present invention, compounds such as fisetin can be used as a hair-growth and / or hair-growth agent or a skin improving agent.

It is a figure which shows the result of having conducted the confirmation experiment of hTERTp-EGFP gene introduction by flow cytometry. Flow cytometry was used to measure the fluorescence intensity of EGFP in HaCaT cells (red line) and HaCaT (hTERTp-EGFP) cells (blue line). It is a figure which shows the result of having conducted the confirmation experiment of hTERTp-EGFP gene introduction by the quantitative RT-PCR method. The expression level of EGFP in HaCaT cells and HaCaT (hTERTp-EGFP) cells was confirmed by a quantitative RT-PCR method. The relative expression level of the EGFP gene was obtained by dividing the measured value of the EGFP gene by the measured value of β-actin. (Mean ± SEM, n = 3, ** p <0.01 vs HaCaT, Student's t-test) It is a figure which shows the hTERT promoter activation effect of polyphenol. Using CaCaT (hTERTp-EGFP) cells, the enhancement of the hTERT promoter in the polyphenol sample was evaluated using the EGFP fluorescence intensity as an index (-: average value). Dimethyl sulfoxide, which is a solvent for polyphenol, was used as a control. (Mean ± SEM, n = 3, ** p <0.01 *** p <0.001 vs Control, Student's t-test) It is a figure which shows the expression change of the hTERT gene in HaCaT cell at the time of polyphenol addition. Quantitative RT-PCR was performed to evaluate the expression level of the hTERT gene in HaCaT cells. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. Dimethyl sulfoxide, which is a solvent of polyphenol, was used for Control. (Mean ± SEM, n = 3, *** p <0.001 vs Control, Student's t-test) It is a figure which shows the cell growth promoting effect using WST-8. The selected four kinds of polyphenols were added to HaCaT cells at a final concentration of 10 μM, and the cell number was measured every 24 hours using WST-8. Dimethyl sulfoxide, which is a solvent of polyphenol, was used for Control. (Mean ± SEM, n = 3, * p <0.05 vs Control, Student's t-test) It is a figure which shows the effect of polyphenol on IGF gene expression in HaCaT cells. Quantitative RT-PCR was performed to evaluate the expression level of the IGF gene in HaCaT cells. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. Dimethyl sulfoxide, which is a solvent for polyphenol, was used as a control. (Mean ± SEM, n = 3, * p <0.05 *** p <0.01 vs Control, Student's t-test) It is a figure which shows the effect of polyphenol on KGF gene expression in HaCaT cells. Quantitative RT-PCR was performed to evaluate the expression level of the KGF gene in HaCaT cells. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. Dimethyl sulfoxide, which is a solvent for polyphenol, was used as a control. (Mean ± SEM, n = 3, * p <0.05 ** p <0.01 vs Control, Student's t-test) It is a figure which shows the effect of polyphenol on TGF- (beta) gene expression in HaCaT cell. Quantitative RT-PCR was performed to evaluate the expression level of the TGF-β gene in HaCaT cells. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. Dimethyl sulfoxide, which is a solvent for polyphenol, was used as a control. (Mean ± SEM, n = 3, ** p <0.01 *** p <0.01 vs Control, Student's t-test) It is a figure which shows the effect of polyphenol on (beta) -catenin activity in HaCaT cell. To verify the effect of polyphenols on β-catenin activity in HaCaT cells, HaCaT cells were transfected with a reporter plasmid (M50 Super 8x TOP Flash), and β-catenin-dependent transcriptional activity after addition of polyphenols was determined by luciferase assay. evaluated. (Mean ± SEM, n = 3, * p <0.05 ** p <0.01 vs Control, Student's t-test) It is a figure which shows the β-catenin enhancing effect in HaCaT cells of polyphenol. Western blotting was carried out to verify the β-catenin enhancing activity of polyphenols in HaCaT cells. (Mean ± SEM, n = 3, * p <0.05 vs Control, Student's t-test) It is a figure which shows the confirmation test result of hTERT expression knockdown in HaCaT cell. The expression level of the hTERT gene in HaCaT (sh-hTERT) cells was evaluated by quantitative RT-PCR. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. As a control, a scrambled sequence vector (scr) was introduced. (Mean ± SEM, n = 3, *** p <0.001 vs Control, Student's t-test) It is a figure which shows hTERT expression in HaCaT (sh-hTERT-1) cell. Quantitative RT-PCR was performed in order to evaluate the expression level of the hTERT gene in HaCaT (sh-hTERT-1) cells. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. As the Control, a knockdown vector (scr-shRNA) having a scrambled sequence was used. As the control treatment, the one treated with dimethyl sulfoxide, which is a solvent of polyphenol, was used. (Mean ± SEM, n = 3, * p <0.05 ** p <0.01 vs Control, Student's t-test) It is a figure which shows hTERT expression in HaCaT (sh-hTERT-2) cell. In order to evaluate the expression level of the hTERT gene in HaCaT (sh-hTERT-2) cells, quantitative RT-PCR was performed. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. As the Control, a knockdown vector (scr-shRNA) having a scrambled sequence was used. As the control treatment, the one treated with dimethyl sulfoxide, which is a solvent of polyphenol, was used. . (Mean ± SEM, n = 3, * p <0.05 ** p <0.01 vs Control, Student's t-test) It is a figure which shows the cell number change in HaCaT (scr-shRNA). Two types of selected polyphenols were added to HaCaT (scr-shRNA) cells, and the cell growth promoting effect using WST-8 was verified every 24 hours. As the control, Dimethyl sulfoxide, which is a solvent for the polyphenol sample, was used. (Mean ± SEM, n = 3, * p <0.05 vs Control, Student's t-test) It is a figure which shows the cell number change in HaCaT (shTERT-1). Two types of selected polyphenols were added to HaCaT (shTERT-1) cells, and the cell growth promoting effect using WST-8 was verified every 24 hours. As the control, Dimethyl sulfoxide, which is a solvent for the polyphenol sample, was used. (Mean ± SEM, n = 3, * p <0.05 vs Control, Student's t-test) It is a figure which shows the cell number change in HaCaT (shTERT-2). Two types of selected polyphenols were added to HaCaT (shTERT-2) cells, and the cell growth promoting effect using WST-8 was verified every 24 hours. As the control, Dimethyl sulfoxide, which is a solvent for the polyphenol sample, was used. (Mean ± SEM, n = 3, * p <0.05 vs Control, Student's t-test) It is a figure which shows the effect which polyphenol gives to SIRT1 gene expression in HaCaT cell. Quantitative RT-PCR was performed to evaluate the expression level of SIRT1 gene in HaCaT cells. The relative expression level of the SIRT1 gene was obtained by dividing the measured value of the SIRT1 gene by the measured value of β-actin. Dimethyl sulfoxide, which is a solvent of polyphenol, was used for Control. (Mean ± SEM, n = 3, ** p <0.01 *** p <0.001 vs Control, Student's t-test) It is a figure which shows the confirmation test result of SIRT1 expression knockdown in HaCaT cell. The expression level of SIRT1 gene in HaCaT (sh-SIRT1) cells was verified by quantitative RT-PCR. The relative expression level of the SIRT1 gene was obtained by dividing the measured value of the SIRT1 gene by the measured value of β-actin. A vector having a scrambled sequence (scr-shRNA) was introduced as Control. (Mean ± SEM, n = 3, *** p <0.001 vs Control, Student's t-test) It is a figure which shows SIRT1 expression in HaCaT (sh-SIRT1) cell. The expression level of SIRT1 gene in HaCaT (sh-SIRT1) cells was measured by quantitative RT-PCR. The relative expression level of the SIRT1 gene was obtained by dividing the measured value of the SIRT1 gene by the measured value of β-actin. As Control, HaCaT into which a vector having a scrambled sequence (scr-shRNA) was introduced was used, and as a control treatment, Dimethyl sulfoxide as a solvent of polyphenol was used. . (Mean ± SEM, n = 3, *** p <0.001 vs Control, Student's t-test) It is a figure which shows hTERT expression in HaCaT (sh-SIRT1) cell. The expression level of the hTERT gene in HaCaT (sh-SIRT1) cells was measured by quantitative RT-PCR. The relative expression level of the hTERT gene was determined by dividing the measured value of the hTERT gene by the measured value of β-actin. As Control, HaCaT into which a vector having a scrambled sequence (scr-shRNA) was introduced was used, and as control treatment, Dimethyl sulfoxide as a solvent of polyphenol was used. . (Mean ± SEM, n = 3, *** p <0.001 vs Control, Student's t-test) It is a figure which shows the effect of polyphenol on hair growth. The hair growth effect of the polyphenol sample was verified using C57BL / 6 mice. Left: control group, middle: Resveratrol group, right: Fisetin group (mouse after 5 weeks of hair removal) It is a figure which shows mTERT expression in the skin of a mouse. Quantitative RT-PCR was performed to evaluate the expression level of the mTERT gene in mouse skin. The relative expression level of the mTERT gene was determined by dividing the measured value of the mTERT gene by the measured value of m-β-actin. (Mean ± SEM, n = 6, * p <0.05 vs Control, Student's t-test) It is a figure which shows mTERT expression in the skin of a mouse. Quantitative RT-PCR was performed to evaluate the expression level of the mTERT gene in mouse skin. The relative expression level of the mTERT gene was determined by dividing the measured value of the mTERT gene by the measured value of m-β-actin. (Mean ± SEM, n = 6, * p <0.05 vs Control, Student's t-test) It is a figure which shows the H & E staining result in the mouse | mouth skin 5 weeks after a test start. The hair growth effect of the polyphenol sample was verified using C57BL / 6 mice, and the skin was stained by H & E staining to visualize the tissue morphology of hair follicles and the thickness of the skin. The scale bar is 100 μM. Top: control group, middle: Resveratrol group, bottom: Fisetin group It is a figure which shows the change of the skin thickness of a mouse. Mouse skin thickness was measured under a microscope. (Mean ± SEM, n = 9, ** p <0.01 *** p <0.001 vs Control, Student's t-test) It is a figure which shows the immunostaining in the mouse | mouth skin 4 weeks after the test start. The hair growth effect of the polyphenol sample was verified using C57BL / 6 mice, and the skin was stained by immunostaining to verify the expression of Ki-67 and TERT. The scale bar is 100 μM. Top: control group, middle: Resveratrol group, bottom: Fisetin group It is a figure which shows the SIRT1 promoter activation effect by a resveratrol structural analog. It is a figure which shows the SIRT1 expression enhancement effect by a resveratrol structural analog. It is a figure which shows the hTERT expression enhancement effect by fisetin and a resveratrol structural analog. FIG. 5 shows the paraquat-induced DNA damage repair effect of fisetin and resveratrol structural analogs.

The present invention relates to a hair growth and / or hair growth composition containing fisetin or a derivative thereof. The present invention also relates to a skin improving composition containing fisetin or a derivative thereof.
To date, no technique for newly constructing a hair follicle containing active hair matrix cells as a source of hair in skin tissue for hair regeneration has been developed to a clinical stage level. The present invention focuses on TERT or SIRT1, searches for food components that activate TERT or SIRT1, and promotes hair growth, hair growth, and skin improvement through activation of signals related to progression of the hair cycle and the like. The mechanism was clarified from in vitro and in vivo analyses.

1. Compositions of the Invention The compositions of the invention are of formula I, formula II or formula III.

In the above formula I, ^one of R ¹ and R ³ represents a hydrogen atom or a hydroxyl group, and the other is represented by the following formula IV:
Represents a group represented by.
Here, R ² represents a hydrogen atom or an oxygen atom, and R ^4a and R ^4b represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. Also,
Represents a double bond or a single bond, and m represents an integer of 1 to 4.
In Formula II, R ^4a and R ^4b are the same as defined above, and n1 and n2 each independently represent an integer of 1 to 5.
In Formula III, R ² , R ^4a , R ^4b , n1 and n2 are the same as defined above.

Compounds of formula I are represented, for example, by the formula Ia:
Or a compound of the following formula Ib:
The compounds shown in can be mentioned.

In the present specification, the “alkyl group having 1 to 6 carbon atoms” (also referred to as “C _1-6 alkyl group”) means a linear or branched alkyl group having 1 to 6 carbon atoms. Means, for example, methyl group, ethyl group, 1-propyl group (n-propyl group), 2-propyl group (i-propyl group), 2-methyl-1-propyl group (i-butyl group), 2- Methyl-2-propyl group (t-butyl group), 1-butyl group (n-butyl group), 2-butyl group (s-butyl group), 1-pentyl group, 2-pentyl group, 3-pentyl group, Examples thereof include a 2-methyl-1-butyl group, a 3-methyl-1-butyl group, a 2-methyl-2-butyl group and a 3-methyl-2-butyl group.

_{Examples of the} C _1-6 alkyl group include a methyl group, an ethyl group, a 1-propyl group, a 2-propyl group and the like.
m and n, n1, and n2 are respectively independent, m is an integer of 1 to 4, and n, n1 and n2 are integers of 1 to 5. Therefore, when m is 1 to 3, n, n1 and n2 are 1 to 4 and R ³ and / or R ⁴ is substituted with a C _1-6 alkyl group, the position of another substituent is Hydrogen atom is bonded.
Represents a single bond or a double bond. For example, in formula I, when R ² is an oxygen atom, it represents a double bond, and when it is a hydrogen atom, it represents a single bond.

In the present invention, examples of the compound included in formula I include fisetin, quercetin, catechin, nobiletin, apigenin and the like. Structural formulas of these compounds are shown below.

  By the way, fisetin is known as a sirtuin activator (A. Camins et al., Biochimica et Biophysica Acta 1799 (2010) 740-749). In the present invention, a compound that functions as a sirtuin activator can be used as the composition of the present invention, similar to fisetin. As such a compound, the compound contained in Formula II and the compound contained in Formula III can be mentioned.

Compounds within Formula II:

Compounds included in Formula III:

In addition, the following may be mentioned as the compounds used in the present invention.

The above compound can be obtained by chemical synthesis or as a commercial product.
In addition to the above-mentioned active ingredients, the composition of the present invention may contain a carrier acceptable for pharmaceuticals or cosmetics and other known or well-known additives.
In the present invention, the above compounds can be administered alone or together with a pharmaceutically acceptable carrier, and the administration can be performed once or in several divided doses.

"Pharmaceutically acceptable carrier" is generally used for pharmaceuticals or cosmetics, excipients, binders, lubricants, diluents, suspending agents, bulking agents, disintegrating agents, stabilizers, Preservatives, buffers, isotonic agents, emulsifiers, aromatics, colorants, sweeteners, thickeners, flavoring agents, surfactants, solubilizers, preservatives, antioxidants, stabilizers, etc. The above-mentioned additives can be used, and these can be used in an appropriate combination.
By using one or more of such carriers, oral forms such as tablets, pills, powders, granules, injections, solutions, capsules, troches, elixirs, suspensions, emulsions or syrups Alternatively, a parenteral composition can be prepared.

For oral administration, various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dipotassium phosphate, glycine and the like can be used together with a disintegrant, a binder and the like. Examples of the disintegrator include starch, alginic acid, and certain types of silicic acid double salts, and examples of the binder include polyvinylpyrrolidone, sucrose, gelatin, gum arabic and the like. Lubricants such as magnesium stearate, sodium lauryl sulfate and talc are effective for tablet formation.
When it is made into an aqueous suspension or elixir for oral administration, it can be used together with an emulsifying agent and a suspending agent as necessary, and can be used together with water, ethanol, propylene glycol, glycerin, etc., and a diluent combining them. .

Examples of forms for parenteral administration include injections (intravenous, infusion, intramuscular, intraperitoneal, subcutaneous), external preparations for skin (lotions, creams, sprays, aerosols, ointments, gels) and the like.
In the case of an injection, it is dissolved or suspended in a pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection to a predetermined concentration (for example, 1 μg / ml to 100 mg / ml). It can be manufactured. The injection thus produced is administered to a subject or patient once per 1 kg body weight, for example, at a rate of 1 μg to 100 mg or at a rate of 100 μg to 10 mg once to several times a day. It can be administered. However, it is not limited to this range, and may vary depending on the body weight, symptoms, administration route, etc. of the subject to be administered. The dose may vary depending on the sensitivity of the subject to the drug, the method of prescribing the drug, the administration period and the administration interval, and therefore a dose lower than the lower limit of the above range may be appropriate in some cases. is there.

It is also possible to use an injection as a sterile solid composition by a freeze-drying method or the like, and to dissolve it in sterile distilled water for injection or another solvent before use, so that it can be used as a ready-to-use form.
For external skin preparation, formulated with the active ingredient on a carrier suitable for the dosage form, adult e.g. 0.001 to 100 mg / ^{m 2} and per day, usually daily at a dose of 0.1 to 10 mg / ^{m 2} It can be administered in 1 to several divided doses.

When the composition of the present invention is orally administered, the dosage form for oral administration may be, for example, tablets, powders, capsules (hard capsules, soft capsules), granules, pills, liquids, syrups and the like. These preparations can be prepared according to a conventional method.
The composition of the present invention has at least one action selected from the group consisting of hair growth action, hair growth action, and skin improvement action. Therefore, the above composition can be used as a hair-growth agent, hair-growth agent, or skin-improving agent.

The hair growth action means an action of producing hair by an interaction between epithelial cells and mesenchymal cells. It can be said that the shorter the time from hairlessness to hair growth, the higher the hair growth effect.
The hair-growth action means an action of maintaining a hair-growth state, and it can be said that the longer the maintenance time is, the higher the hair-growth effect is. To enhance the hair-growth effect, it is important to activate the division of hair mother cells in the hair root.
The skin improving action means an effect of recovering and strengthening the skin. When applied to the skin, the composition of the present invention can increase the thickness of the skin and smooth the skin. Therefore, the composition of the present invention can be used as a wound healing agent or a cosmetic.
The skin improvement includes, for example, treatment of wounds, repair of rough skin, improvement of spots, improvement of freckles, and suppression of wrinkles, wound healing, suppression of cell aging, and the composition of the present invention includes one or two of these. It applies to the above combinations. The composition of the present invention has, for example, a repairing effect on DNA damage, and therefore has an improving effect on, for example, rough skin and stains on the skin due to ultraviolet rays.
Furthermore, since the composition of the present invention is originally derived from food ingredients, it can be included as an ingredient of functional foods in addition to pharmaceuticals and the like.

2. Screening Method The present invention comprises contacting a test substance with cells containing a vector in which a reporter gene is linked to a TERT promoter or cells containing an endogenous TERT gene, and expressing the target gene or the endogenous TERT gene as an index. Provided is a method for screening a substance having at least one effect selected from the group consisting of a hair effect, a hair growth effect and a skin improving effect.
Further, the present invention, a cell containing a vector in which a reporter gene is linked to the SIRT1 promoter, or a cell containing an endogenous SIRT1 gene is contacted with a test substance, and the expression of the reporter gene or SIRT1 gene is used as an index to produce a hair growth effect, Provided is a method for screening a substance having at least one effect selected from the group consisting of a hair growth effect and a skin improving effect.
The present invention further provides a group consisting of a hair damage effect, a hair growth effect and a skin improving effect, which is obtained by contacting a test substance with a cell containing a DNA damage repair marker gene and having a DNA damage, and using the expression of the gene as an index. A method for screening a substance having at least one effect selected from
In the present invention, the test substance that is a candidate is not particularly limited, and may be an existing drug (for example, an existing hair-growing agent, hair-growing agent, or any other drug that acts on skin improvement). , Polymer compounds, salts or precursors thereof, and the like.

The present invention specifically includes the following steps.
(A) contacting a test substance with cells containing a vector in which a reporter gene is ligated to a TERT promoter or SIRT1 promoter, or with a test substance (b) the target gene or endogenous TERT Step of measuring gene expression In the screening method of the present invention, hair growth agents, hair growth agents, skin improving agents and the like can be screened.

The TERT promoter or SIRT1 promoter and the reporter gene can be ligated by an ordinary genetic engineering technique (for example, Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory). (2012)).
For example, in the case of the TERT promoter, the TERT promoter is first synthesized. The synthesis can be performed, for example, by the PCR method using genomic DNA as a template and a primer designed to synthesize a desired promoter region. The TERT promoter is preferably human TERT (hTERT) promoter.
The SIRT1 promoter and the reporter gene can be ligated in the same manner as the TERT promoter.
The present invention also relates to a group comprising a hair damage effect, a hair growth effect, and a skin improving effect, which is a cell containing a DNA damage repair marker gene, which is contacted with a test substance to a cell damaged by the DNA, and the expression of the gene is used as an index. A method for screening a substance having at least one effect selected from

For the reporter gene, the gene in the reporter gene expression vector is used, and the promoter region that regulates reporter gene expression is removed by restriction enzyme treatment or the like, and a recombinant vector is obtained by ligating the hTERT promoter or SIRT1 promoter to the region. A transformant is obtained by introducing this recombinant vector into a host cell (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012)).
Examples of the reporter gene include various reporter genes, such as GFP, EGFP, and luciferase. If the expression of these reporter genes can be confirmed by fluorescence or the like, it can be seen that the TERT promoter is expressed.

  As the vector, a phage or a plasmid that can autonomously grow in a host microorganism is used. Furthermore, animal virus or insect virus vectors can also be used. The recombinant vector may be prepared by cutting the purified DNA with an appropriate restriction enzyme, inserting it into the restriction enzyme site of an appropriate vector DNA, and ligating it with the vector. The host used for transformation is not particularly limited as long as it can express the gene of interest. Examples include bacteria (E. coli, Bacillus subtilis, etc.), yeast, animal cells, insect cells or insects. Animal cells include human epithelial cells, and human epithelial cells include human skin epidermis-derived cells.

In the screening method of the present invention, "contact" means contacting a test substance with cells, and includes a mode of adding the test substance to a cell culture medium, a mode of culturing cells in the presence of the test substance, and the like. .
After the contact, the expression of the reporter gene linked to the downstream of the above promoter is assayed by a known method. Alternatively, the expression (gene expression, protein expression, etc.) of the TERT gene existing in the cells used for screening is confirmed by RT-PCR, Northern blotting, Western blotting, etc.
As a result, when the candidate test substance promotes the expression of the target gene or the endogenous TERT gene or the endogenous SIRT1 gene as compared with the control (for example, cells not contacted with the test substance), the test substance causes hair growth. It is judged to have the effect, hair-growth effect, skin-improving effect, or the combination thereof.

  When a DNA damage repair marker gene is used in the screening method of the present invention, a cell having the marker gene naturally, or a cell into which the marker gene has been introduced by gene recombination is physically or chemically damaged. The test substance is brought into contact with the test substance. Then, the expression of the DNA damage repair marker is used as an index to determine whether the test substance is a substance that repairs DNA damage.

  For example, in the case of using a repair marker gene whose expression increases due to DNA damage, when the expression of the gene decreases, it is determined that the test substance is a DNA damage repair substance, and the hair growth effect, hair growth effect or skin It is selected as a substance having an improving effect. When a repair marker gene whose expression is decreased by DNA damage is used, when the expression of the gene is increased, the test substance is judged to be a DNA damage repairing substance, and a hair growth effect, hair growth effect or skin improvement effect is obtained. Select as an effective substance.

In the present invention, the DNA damage repair effect can be expressed not only by the expression of the gene itself but also by phosphorylation as an index, and also by the expression of the protein encoded by the gene as an index.
Markers whose expression or phosphorylation increases when damaged by DNA include, for example, γH2AX gene and p53 gene, and markers which increase expression or phosphorylation when damaged by DNA include, for example, γH2AX and p53. To be

  Hereinafter, the present invention will be described in more detail with reference to Examples. However, the scope of the present invention is not limited to these examples.

[Experimental method]
1. Cell culture 1.1. HaCaT cells HaCaT cells (human skin epidermis-derived cell line) were adopted as human skin model cells. The cells were cultured in a cell culture dish using a 10% Fetal bovine serum (FBS; Life Technologies, CA, USA) -containing DMEM medium (Dulbecco's Modified Eagle Medium; Nisui, Tokyo, Japan), a cell culture dish, and a cell culture dish. ) At 37 ° C. in the presence of 5% CO ₂ . As the DMEM medium, 10.0 g of DMEM powder was dissolved in 1 L of Milli-Q water, streptomycin sulfate 0.1 g titer (Meiji. Tokyo, Japan), penicillin G potassium 100,000 U (Meiji), 1 M. 2.38 g of HEPES (DOJINDO, Kumamoto, Japan), 2.0 g of 10% NaHCO ₃ (Wako, Osaka, Japan) were added, and 0.22 μm filter sterilized (Toyo Roshi Kaisha, Tokyo, Japan) was used.

1.2. HaCaT (hTERTp-EGFP) cell The HaCaT cell was introduced with an EGFP expression vector (hTERTp-EGFP) whose expression is controlled by the hTERT promoter. HaCaT (hTERTp-EGFP) cells were cultured in the same manner as the above-described culture of HaCaT cells.

1.3.293T cells 293T cells (human embryonic kidney-derived cells) were used as a cell line capable of producing high titer retrovirus. The 293T cells were cultured in the same manner as the above-described culture of HaCaT cells.

1.4. hTERT and SIRT1 Knockdown Cells hTERT or HaCaT cells knocked down in SIRT1 expression were cultured in the same manner as the above-mentioned culture of HaCaT cells.

2. Preparation of HaCaT (hTERTp-EGFP) cells 2.1. Gene Transfer to HaCaT Cells A vector (phTERTp-EGFP) (Ito, 2010) in which the promoter region of the human TERT gene (hTERT) cloned from the human genome was inserted into the CMV promoter region in pEGFP-C3 Vector was transferred to HaCaT cells. Has been carried out. Table 1 shows the primers used for producing the vector.

Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) was used for transfection. HaCaT cells were seeded in 5 mL dishes (Becton Dickinson, Franklin lakes, NJ, USA) at 5 × 10 ⁵ cells / dish and cultured in DMEM medium containing 10% FBS. At this time, control non-transfected cells were also prepared. On the day of transfection, 10 μg of hTERTp-EGFP vector and 15 μL of Lipofectamine were added to a 1.5 mL tube so that the total volume of serum-free OPTI-MEM medium was 1.5 mL, and the mixture was incubated at room temperature for 5 minutes. Since the transfection method using Lipofectamine is performed in the absence of serum, the culture medium of HaCaT cells seeded during this period was removed and replaced with 5 mL of serum-free OPTI-MEM medium. After 30 minutes, the entire amount of the DNA-Lipofectamine 2000 mixed solution was added to the HaCaT cells. After the addition, the mixture was gently shaken to mix DNA-Lipofectamine and OPTI-MEM. After culturing for 3 hours, the DNA-Lipofectamine mixed solution was removed and replaced with a culture medium containing serum. Then, the cells were cultured for 21 hours at 37 ° C. in 5% CO ₂ and replaced with a fresh DMEM culture medium containing 10% FBS.

2.2. Drug Selection The hTERTp-EGFP vector contains the Kan ^r / Neo ^r drug resistance gene. Therefore, 48 hours after the transfection, G418 (Wako, Osaka, Japan) was added to a final concentration of 70 μg / mL, and drug selection was performed for 1 week. While repeating the subculture and medium exchange every 3 days, G418 was added at a final concentration of 70 μg / ml each time, and drug selection was performed until the control untransfected HaCaT cells were completely killed. After confirming that the control HaCaT cells were completely destroyed, the G418 concentration was lowered to 40 μg / mL and the subculture was continued.

2.3. Establishment of HaCaT (hTERTp-EGFP) The hTERTp-EGFP vector was introduced into HaCaT cells to establish a stable expression strain, or the fluorescence of GFP was measured by a flow cytometer. Control HaCaT cells that were 70-80% confluent in 5 mL dishes and prepared HaCaT (hTERTp-EGFP) cells were suspended in 2 mL of DMEM medium containing 10% FBS, and this cell suspension was suspended with nylon mesh (Kyoshin Rikoh). , Tokyo, Japan) and subjected to a flow cytometer (EPICS, Beckman Coulter, Miami, FL, USA) to measure the GFP fluorescence intensity, and to control GFP fluorescence of HaCaT cells and HaCaT (hTERTp-EGFP) cells. The peaks were compared (Figure 1). In addition, the expression level of EGFP in HaCaT cells and HaCaT (hTERTp-EGFP) was measured by the RT-PCR method described in Quantitative Reverse transcriptase PCR (RT-PCR) described below (FIG. 2), and the target gene was introduced. Confirmed that.

3. Sample and sample preparation 3.1. Polyphenols 15 kinds of polyphenols were used (Table 2). Polyphenol prepared by adjusting DMSO to a concentration of 10 mM was stored at 4 ° C., thawed appropriately, and pipetted to be used.

4. Screening using hTERT promoter activity as an index 4.1. Measurement of EGFP fluorescence intensity hTERT activity was measured by tracing EGFP fluorescence intensity in HaCaT (hTERTp-EGFP) cells using IN Cell Analyzer 1000 (GE Healthcare, Amersham Place, UK). HaCaT (hTERTp-EGFP) cells were seeded in 96-well plate (Greiner Bio-one, Tokyo, Japan) at 5.0 × 10 ⁴ cells / well and cultured in DMEM medium containing 10% FBS serum. The next day, various samples were added. The addition concentration was such that the polyphenol sample had a final concentration of 10 μM. As a control, DMSO was added to the polyphenol sample in the same amount as each sample.

  After the addition, the cells were cultured for 48 hours in DMEM medium containing 10% FBS serum. After 48 hours, the medium was aspirated, 8% paraformaldehyde was added at 100 μL / well so that the final concentration was 4%, and the mixture was allowed to stand at room temperature for 10 minutes for fixation. 8% paraformaldehyde was prepared by diluting Paraformaldehyde (Wako) with 1 × PBS to 8% paraformaldehyde and dissolving it in a water bath at 60 ° C. After 10 minutes, the fixative was removed, and the plate was washed twice with 1 × PBS. Thereafter, 1,000 times diluted Cellstein R-Hoechst 33342 solution (Dojindo, Kumamoto, Japan) was added at 100 μL / well, and the mixture was allowed to stand at room temperature for 30 minutes for nuclear staining. After 30 minutes, the Cellstein R-Hoechst 33342 solution was removed, the cells were washed 3 times with 1 × PBS, 100 μL of PBS was added to each well, and the EGFP fluorescence intensity was measured with IN Cell Analyzer 1000.

5. Quantitative Reverse Transcriptase-PCR (RT-PCR) Reaction 5.1. Preparation of Total RNA For preparation of total RNA, High Pure RNA Isolation Kit (Roche Diagnostics Gmbh, Mannheim, Germany) was used according to its product protocol. The reagents and instruments used from the preparation of total RNA to the end of the reverse transcription reaction were those of RNase Free.

First, cells were seeded in 5 mL of cell culture dish at 5.0 × 10 ⁴ cells / well, and cultured at 37 ° C. in DMEM medium containing 10% FBS serum. The next day, various samples were added. The concentration to be added was in accordance with the measurement of EGFP fluorescence intensity described in the section "Screening using hTERT promoter activity as an index" in the "Experimental method". After the addition, the cells were cultured at 37 ° C. for 48 hours in DMEM medium containing 10% FBS serum. After 48 hours, the medium was completely removed, 200 μL of 1 × PBS was added for washing, and 400 μL of the cell lysate contained in the High Pure RNA Isolation Kit was added to the well, and the cell lysate was thoroughly spread over the entire dish. It was circulated and lysed, and the total amount of cell lysate was collected in a 1.5 mL tube.

The collected sample was well suspended by a vortex mixer for 60 seconds and lightly spun down. A High Pure filter tube in Kit and a collection tube were assembled and the cell lysate solution was added to the filter tube. After centrifugation at 10,000 xg for 15 seconds at room temperature, the liquid discharged into the collection tube was discarded, and the filter tube and the collection tube were assembled again.
90 μL of DNase incubation buffer and 10 μL of DNase I were added to each 1.5 mL tube and mixed. This mixed solution was added to the filter tube described above and incubated at room temperature for 15 minutes. After 15 minutes, 500 μL of Wash Buffer I in Kit was added to the filter tube and centrifuged at 10,000 xg for 15 seconds at room temperature.

  After centrifugation, the liquid discharged to the collection tube was discarded, and the filter tube and the collection tube were assembled again. 500 μL of Wash Buffer II was added to the filter tube and centrifuged at 10,000 × g for 15 seconds at room temperature. After centrifugation, the liquid discharged to the collection tube was discarded, and the filter tube and the collection tube were assembled again. Furthermore, 200 μL of Wash Buffer II was added, and the mixture was centrifuged at room temperature and 15,000 × g for 2 minutes to wash the filter. After centrifugation, the filter tube was inserted into a new 1.5 mL tube, 100 μL of elution buffer was added to the center of the filter tube, and the mixture was left standing at room temperature for 3 minutes. Then, it centrifuged at room temperature and 10,000xg for 1 minute. The eluate obtained by this operation was used as an RNA solution. The RNA concentration in the solution was calculated based on the absorbance value at 260 nm using a NanoDrop2000 / 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA), and used in the subsequent experiments.

5.2. cDNA Synthesis (1) cDNA Synthesis Using Reverse Transcriptase ReverTra Ace To 1.0 μg of total RNA extracted from cells, 5 pmol of Oligo (dT) 20 primer (TOYOBO, Osaka, Japan) was added to give a total volume of 13 μL. RNase Free water was added thereto. A thermal treatment reaction was carried out for 5 minutes at 65 ° C. with a Thermal Cycler PTC-200 (MJ Research, Waltham, MA, USA), and immediately transferred to ice for rapid cooling. Meanwhile, the reverse transcriptase reaction program was advanced to the stage of 42 ° C and was temporarily stopped. Reverse transcriptase reaction buffer 4 μL, 10 mM dNTPs (GE Healthcare) 2 μL, reverse transcriptase ReverseTrace Ace (100 units / μL) (TOYOBO, Osaka, Japan) 0.5 μL were added to each sample cooled in ice for 5 minutes. The mixed solution was added and mixed gently. Then, cDNA was synthesized by reacting at 42 ° C for 20 minutes, at 99 ° C for 5 minutes, and at 4 ° C for 5 minutes. This cDNA was used as a template for quantitative PCR.

(2) cDNA synthesis using reverse transcriptase SuperScript To 0.7 μg or 2.0 μg of total RNA extracted from cells, 50 pmol of Oligo (dT) 20 primer and 1 μL of 10 mM dNTPs were added to give a total volume of 12.75 μL. Then RNase Free water was added. A thermal treatment reaction was carried out at 65 ° C. for 5 minutes with a Thermal Cycler PTC-200, immediately transferred to ice and rapidly cooled. Meanwhile, the reverse transcriptase reaction program was advanced to the stage of 55 ° C. and was temporarily stopped. To a sample cooled for 1 minute or more in ice, a solution prepared by mixing 5 μl of 5 × reverse transcriptase reaction buffer, 1 μl of 100 mM DTT, 0.25 μl of reverse transcriptase Supercipt IV (100 units / μl) (Thermo Fisher Scientific) per sample was added. Mix gently. Then, cDNA was synthesized by reacting at 55 ° C. for 10 minutes and at 80 ° C. for 10 minutes. This cDNA was used as a template for quantitative PCR.

5.3. Design of primer A target gene whose expression level is to be measured by RT-PCR is searched by NCBI (http://www.ncbi.nlm.nih.gov/gene/), and Primer3 is used as a primer based on the sequence. The sequence was determined and synthesized. Primer synthesis was outsourced to Sigma. Table 3 shows the primers for detecting the internal control β-actin and the target gene, respectively.

5.4. Quantitative Reverse transcriptase-PCR (RT-PCR) reaction The prepared cDNA was used as a template. In a 0.2 mL PCR tube, 49 μL of sterilized water, 3.5 μL each of both primers Forward / Reverse diluted to 10 pmol / mL, template cDNA (primer was diluted to 1/10 of target gene, β-actin was diluted to 1/50). ) 7.0 μL and 24.5 μL of THUNDERBIRD SYBR qPCR Mix (TOYOBO) as a master mix for high-efficiency real-time PCR (2 × concentration) were added and well suspended. After that, 25 μL of each was added to 3 wells of 96-well plate, and quantitative Real time-PCR was performed using a Thermal Cycler Dice Real Time System (TAKARA BIO, Siga, Japan). In the PCR reaction, denaturation reaction was carried out at 95 ° C. for 5 seconds, annealing was carried out at 60 ° C. for 10 seconds, and extension reaction was carried out at 72 ° C. for 20 seconds, which was repeated for 45 cycles (3 steps) and detected by FAM. Β-actin was used as a primer for the calibration curve. The relative gene expression level was determined by dividing the measured value by the β-actin expression level value. In the statistical analysis, Student's t-test was performed at n = 3 assuming a normal distribution.

6. Cell growth curve HaCaT cells were seeded on a 96-well plate (Becton Dickinson) at a cell number of 1.0 × 10 ⁴ cells. After 6 hours, cells were allowed to attach to the plate before sample addition. The final concentration of sample addition was based on the measurement of EGFP fluorescence intensity described in the section "Screening using hTERT promoter activity as an index" in the "Experimental procedure".

10 μL of Cell Count Kit-8 solution (DOJINDO) was added to each well every 24 hours after sample addition, and color reaction was performed for 2 hours under the conditions of 37 ° C. and 5% CO ₂ . Thereafter, the absorbance at 450 nm of each well was measured using a microplate reader (Sunrise, TECAN Austria GmbH, Grodig, Austria), and the measured value was used. Cell Counting Kit-8 is a kit for measuring the number of cells in a cell proliferation test. The novel tetrazolium salt WST-8 that produces highly sensitive water-soluble formazan is reduced by intracellular dehydrogenase to produce water-soluble formazan. The number of viable cells can be measured by measuring the dye amount of formazan, which is linearly proportional to the number of cells, by the absorbance at 450 nm. In the statistical analysis, Student's t-test was performed with n = 3 assuming that the distribution was normal.

7. Luciferase Assay 7.1. Reporter plasmid A luciferase reporter (pGL3b-289, pGL3b-289 (E-box-mut)) having a hTERT promoter (Fujiki T et al., 2007), and a luciferase reporter having a TCF / LEF binding site (M50 Super 8x TOP Flas). ) (Addgene, Cambridge, MA), a luciferase reporter (M51 Super 8x FOP Flash) (Addgene) having a TCF / LEF binding site mutant.

7.2. Transfection HaCaT cells were inoculated into 24-well plates (Becton Dickinson) at a concentration of 5.0 × 10 ⁴ cells / well so as to be subconfluent at the time of collection, 500 μL each. On the next day, a 1.5 mL tube was prepared with a target promoter, Cypridina luciferase construct, Renilla luciferase construct (pRL-TK) and DMEM medium as an intracellular standardization control. The ratio and volume of DNA was added so that the final volume was 62.5 μL. To this, 1.25 μL of 1 mg / mL PEI (Polysciences, Inc. Warrington, PA) was added and mixed, and then allowed to stand at room temperature for 15 minutes. The medium was completely removed from the 24-well plate, the 10% FBS-containing medium and the total amount of the above DNA-PEI mixed solution were added, and the mixture was cultured at 37 ° C. in the presence of 5% CO ₂ .

  After 24 hours, the culture was aspirated and replaced with DMEM medium containing 10% FBS. The measurement was performed 48 hours after the replacement. Alternatively, when the medium is exchanged, the addition of the sample should be 3 wells per sample at a concentration according to the measurement of EGFP fluorescence intensity described in the section “Screening using hTERT promoter activity as an index” in the “Experimental method”. went. The gene amount and ratio of the luciferase construct used for transfection are shown below.

Condition reporter for measuring β-catenin promoter activity (M50 Super 8x TOP Flash, M51 Super 8x FOP Flash 350 ng
pRL-TK 150ng
Conditional reporter (pGL3b-289, pGL3b-289 (E-box-mut)) at the time of hTERT promoter activity measurement 350 ng
pRL-TK 150ng

7.3. Luciferase Assay Preparation of cell extracts and measurement of luciferase activity were performed according to the Dual Luciferase Reporter Assay System (Promega, Madison, Wis.) Product protocol. After completely removing the 24-well plate medium, the plate was washed once with 1 × PBS 50 μL / well to completely remove the PBS. Thereafter, 50 μL / well of 5 × PLB (Passive Lysis Buffer) diluted with ultrapure water to 1 × PLB was added, and the plate was shaken with a Micro Mixer (TAITEC, Saitama, Japan) at room temperature for 1 hour. The cells were lysed. Meanwhile, 1 × Stop & Glo Reagent was prepared by diluting 50 × Stop & Glo Substrate 50-fold with Stop & Glo Buffer under light shielding. In addition, LAR II (Luciferase Assay Reagent II) obtained by dissolving Luciferase Assay Substrate in Luciferase assay Buffer II was dispensed into 12 × 75 mm glass test tubes (IWAKI, μi, Chiro, and Chiba, respectively).

  10 μL of the cell extract was added to LAR II and mixed, and the test tube was set in a luminometer (Lumit LB 9507, Berthold Japan KK, Tokyo, Japan), and the amount of luminescence of firefly luciferase activity was added to the cell extract. It was measured by the integrated value for 10 seconds from 1 second after. Subsequently, 50 μL of 1 × Stop & Glo Reagent was added to the same sample and mixed, and the amount of Renilla luciferase luminescence was measured in the same manner as the measurement of the firefly luciferase assay activity. The measured promoter activity value was evaluated by dividing the value obtained by measuring the firefly luciferase activity value by the Renilla luciferase activity measurement value as the specific activity value of the promoter and calculating the average value and standard deviation value.

8. Western blotting method 8.1. Preparation of reagents 1: NP-40 Lysis Buffer
0.5% Nonidet P-40 (Nacalai tesque, Kyoto, Japan)
5 mM EDTA (DOJINDO)
2 mM Na ₃ VO ₄ (Wako)
10 mM Tris-HCl (pH 7.6) (SIGMA, St Louis, MO, USA)
150 mM NaCl (Wako)
5 μg / mL Aprotinin (SIGMA)
1 mM PMSF (Phenylmethyl sulfonyl fluoride) (SIGMA)

2: Resolving gel buffer (total volume of 1,000 ml)
90.8g Trizma-base (SIGMA)
2.0g Sodium dodecyl sulphate (SDS, Wako) Adjusted to pH 8.8 with 12N HCl.

3: Stacking gel buffer (total volume 1000 ml)
30.3g Trizma-base (SIGMA)
Adjust to pH 6.8 with 2.0g SDS (Wako) 12N HCl

4: 2 x sample buffer
5% Sodium dodecyl sulphate (Wako)
20% Glycerol (Wako)
62.5 mM Tris-HCl (pH 6.8)
10% 2-Mercaptoethanol (Wako)
0.01% Bromophenol Blue (Wako)

5: 10 × Tris Glycine SDS buffer (total volume of 1,000 ml)
30.1g Tris
144g Glycine
10g SDS

6: PTB (Protein Transfer Buffer)
48 mM Trizma-base (SIGMA)
38 mM Glycine (Wako)
20% Methanol (Wako)

5: PBS-Tween (total volume 1,000 ml)
11.5 g Na ₂ HPO ₄ (Wako)
2.96 g NaH ₂ PO ₄ (Wako)
5.84g NaCl (Wako)
0.1% Polyoxyethylene (20) Sorbitano Monolaurate (Tween 20) (Wako)

8.2. Preparation of protein solution HaCaT cells were seeded in 10 mL dish (Greiner bio-one) at 5.0 × 10 5 cells / dish, and cultured in DMEM medium containing 10% FBS at 37 ° C. and 5% CO ₂ for 24 hours. After 24 hours, the sample was added and cultured under the conditions of 37 ° C. and 5% CO ₂ for 24 hours. As a sample, Resveratrol, Fisetin, and Sesamol were added at a final concentration of 10 μM. After 24 hours from the addition of the sample, the medium was aspirated and washed 3 times with 5 mL of ice-cold 1 × PBS. Thereafter, 800 μL of NP-40 Lysis Buffer was added to lyse the cells, and the cells were slowly scraped with a cell scraper (NIPPON Genetics, Tokyo, Japan) so as not to generate frictional heat in a certain direction, and a tube with a siliconized snap cap (Thermo). (Fisher Scientific). After leaving it on ice for 30 minutes and allowing it to stand, the protein solution was suspended with an ultrasonic crusher (TOMY SEIKO, Tokyo, Japan), centrifuged at 20,000 xg for 15 minutes at 4 ° C, and 25 μL of the supernatant was added. For protein quantification, the remaining collected solution was dispensed as a sample for Western Blotting into several tubes with siliconized snap caps and stored at -80 ° C.

8.3. Protein quantification (Bradford method)
The above 8.2. 10 μL of the protein solution extracted in the above section was serially diluted 50, 100, 200, and 400 times with PBS. As the standard of the standard curve, a solution obtained by diluting bovine serum albumin (BSA: Bovine serum albumin, Wako) with PBS in 10 steps so as to be 0 to 90 μg / mL was used. Two wells of 96-well plate were used as one assay, and 100 μL of each well was added. Then, 100 μL of Protein Assay Dye (BIO-RAD Laboratories, Hercules, CA, USA) diluted 2.5 times with sterilized water was quickly added to each well, and the mixture was added with Micro Mixer (TAITEC, Saitama, Japan). After shaking, the mixture was allowed to stand for 10 minutes at room temperature under light shielding. After 10 minutes, measurement was performed using an absorptiometer (Tecan) based on the absorbance of 595 nm. Quantitation was performed based on the standard curve obtained by the measurement to determine the protein concentration.

8.4. Western blotting (1) SDS-PAGE
SDS polyacrylamide electrophoresis (SDS-PAGE) was performed. The electrophoresis gel plate used was 1 mm thick. The upper layer gel is a concentrated gel and the lower layer gel is a separating gel, and the composition of each is described below. After injecting the separation gel, water-saturated butanol was filled on the gel to cut off the contact with the air. The acrylamide concentration of the concentrated gel was always 5%, but the acrylamide concentration of the separation gel was different depending on the molecular weight of the target protein.

・ Stacking gel
40% Acrylamide / Bis solution (37.5: 1) (BIO RAD) 1.35 mL
Stacking gel buffer 5.00mL
Sterile water 4.45mL
10% APS (Wako) 50 μL
TEMED (Wako) 10 μL

・ Running gel
40% Acrylamide / Bis solution (37.5: 1) (BIO RAD) 5.6 mL
Resolving gel buffer 9mL
Sterilized water 3.9mL
10% APS (Wako Pure Chemical) 180 μL
TEMED (Wako Pure Chemical) 18 μL

The separating gel described above has a composition of 12%.
20 μg of the quantified protein sample was mixed with 2 × Sample buffer at a ratio of 1: 1 and denatured by heating at 95 ° C. for 5 minutes. After denaturation, it was transferred to ice and left for 5 minutes, and then injected into the gel. The electrophoresis conditions were 20 V and 200 mA for both concentrated gels, and electrophoresis was performed for about 1 to 1.5 hours. Kaleidoscope Prestained Standards (1610375, BIO RAD) was used as a migration marker.

(2) Blotting After the completion of the electrophoresis, necessary parts of the separation gel were cut according to the target protein, immersed in Protein Transfer Buffer (PTB solution), and shaken for 30 minutes. In the meantime, a PVDF membrane (GE Healthcare) and four pieces of filter paper were cut in the same area as the gel, pretreated with 100% methanol (Wako) for 20 seconds, and soaked in ultrapure water for 5 minutes, followed by shaking. And soaked in PTB solution and shaken for 15 minutes. After shaking, two filter papers soaked in PTB, a PVDF membrane, a gel, and two filter papers were superposed in this order with a dual-type mini transfer device (NA-1510B type, NIHON EIDO, Tokyo, Japan), 100A, Blotting was performed under the conditions of 50 V and 60 minutes.

(3) Blocking After the completion of blotting, the PVDF membrane was immersed in 5% skim milk (Wako) /0.1% PBS-Tween as a blocking solution, and blocking was performed overnight at 4 ° C with shaking.

(4) Primary antibody reaction After blocking, the PVDF membrane was washed with PBS-Tween by gently shaking twice, and then washed with fresh PBS-Tween with shaking for 15 minutes. After washing, the primary antibody solution diluted with PBS-Tween or Can Get Signal Immunoreaction Enhancer Solution Solution 1 (TOYOBO) was reacted for 1 hour at room temperature with shaking to perform an antigen-antibody reaction. The antibody dilution rate depends on the antibody used and is described below.

<Primary antibody dilution rate>
Β-catenin Antibody (# 8480S, Cell Signaling Technology, Danvers, MA, USA) 1/1500
-Β-actin Antibody (013-24553, Wako) 1/3000

(5) Secondary antibody reaction After the primary antibody reaction, the plate was gently shaken twice with PBS-Tween, and further washed with shaking for 15 minutes. Subsequently, the washing with PBS-Tween for 5 minutes with shaking was repeated 3 times to remove excess primary antibody. After shaking, the secondary antibody solution diluted with PBS-Tween or Can Get Signal Immunoreaction Enhancer Solution Solution 2 (TOYOBO) was reacted for 1 hour at room temperature with shaking to perform an antigen-antibody reaction. The antibody dilution rate is shown below.

<Secondary antibody dilution rate>
-Anti-rabbit IgG, Horseradish Peroxidase linked whole antibody (GE Healthcare) 1/2500
-Anti-mouse IgG, Horseradish Peroxidase linked whole antibody (GE Healthcare) 1/2500

(6) Detection After completion of the secondary antibody reaction, the plate was gently shaken twice with PBS-Tween, and further shaken and washed for 15 minutes. Subsequently, washing with PBS-Tween while shaking for 5 minutes was repeated 3 times to remove excess secondary antibody. After washing, Luminescence Solution A and Luminescence Solution B of Immuno Star LD (Wako) were mixed at a ratio of 1: 1 and added to the PVDF membrane to react for 5 minutes while shielding from light. Then, it detected by LAS-1000 (FUJIFILM, Tokyo, Japan).

9. Animal experiment 9.1. Animals and Breeding Method C57BL / 6J Jcl mice (♂, 6 weeks old) were purchased from CLEA Japan, Inc. (Tokyo, Japan). The breeding environment was carried out using a small cage for mice (NATSUME SEISAKUSHO, Japan), with one mouse in one cage, room temperature 22 to 26 ° C, humidity 50 to 60%, and a rack controlled under a 12-hour light-dark cycle (Oriental). , Tokyo, Japan). In addition, feed (CA-1 irradiation dose 30 kGy) (CLEA) and water that had been sterilized by gamma ray irradiation and water were freely ingested. Experimental breeding was carried out for 4 weeks by dividing into 3 groups of a control group (9 animals), a Resveratrol group (9 animals) and a Fisetin group (10 animals).

  The procedure and operation of all mice are approved by the Kyushu University Animal Experiment Ethics Committee and follow the guideline for breeding and use of laboratory animals, which has been approved by the Committee. (Approval number: A28-077-0 "Functional evaluation of foods having anti-aging activity using disease model animals")

9.2. Application test After preliminarily breeding for 1 week, an electric clipper (THRIVE MODEL) was prepared so that the back hair of the mouse was about 2.5 cm × 2.5 cm under anesthesia with a general inhalation anesthetic, Sevofrene (Maruishi Pharmaceutical, Osaka, Japan). 2100, Daito Electric Machine Industry, Osaka, Japan), and then shaved with an electric shaver (Panasonic, Osaka, Japan), and used for the experiment from the next day. The control group was prepared by dissolving 50% ethanol, and the Resveratrol group and the Fisetin group were dissolved in 50% ethanol at a concentration of 0.1%. .. At the end of the experiment 4 weeks after the start of the test, the mouse was euthanized by cervical dislocation and the skin of the test site was collected.

9.3. Verification of TERT Expression in Mouse Skin RNA extraction from mouse skin tissue was performed according to the product protocol of the RNeasy Fiber Tissue Mini Kit (QIAGEN, Hilden, Germany).
1. Skin Crushing A part of a skin sample collected from a mouse was cut into a size of about 3 mm × 3 mm and placed in a 1.5 mL tube in which the inside of Biomasher II (Nippi, Tokyo, Japan) was dimple-processed. To 1 mL of Kit's Buffer RLT, 10 μL of β-mercaptoethanol (Wako) was added in advance, 300 μL was added on the tissue, and the stirring rod attached was rotated to rapidly crush the skin tissue. At this time, homogenization was performed until the lump of skin tissue was no longer visible to the naked eye.

2. RNA Extraction The homogenized tissue lysate was centrifuged at 18,000 × g for 5 minutes at room temperature, and then the lysate was transferred to a new 1.5 mL tube so that the pellet was not contaminated. 590 μL of RNase-free water contained in Kit and 10 μL of Proteinase K solution were added to the lysate and mixed thoroughly by pipetting. Then, the mixture was incubated at 55 ° C. for 10 minutes, and centrifuged at room temperature and 10,000 × g for 3 minutes. The supernatant (about 900 μL) was pipetted into a new 1.5 mL tube. 0.5% volume (about 450 μL this time) of 99% ethanol (Wako) was added to the clarified lysate and mixed by pipetting. A 700 μL sample was applied to the RNeasy Mini Spin Column set in a 2 mL collection tube of Kit, and centrifuged at room temperature at 8,000 × g or more for 15 seconds. The filtrate discharged into the collection tube was discarded, and the same operation was repeated using the rest of the sample. 350 μL of Buffer RW1 was added to RNeasy Spin Column, and the membrane was washed by centrifugation at room temperature and 8,000 × g for 15 seconds.

  10 μL of DNase I stock solution prepared by dissolving DNase I (1,500 Knitz units) in 550 μL of RNase-free water was added to 70 μL of Buffer RDD. Since DNase I is sensitive to physical denaturation, the tubes were gently mixed by gently inverting the tube. A light centrifugation was performed to collect the remaining solution on the wall of the tube.

  80 μL of DNase I incubation solution per sample was pipetted directly onto the RNeasy Spin Column membrane and incubated for 15 minutes at room temperature. At this time, in order to prevent the DNase decomposition from becoming incomplete, the DNase I incubation reaction solution was directly added to the RNeasy Spin Column membrane so as not to stick to the wall of the spin column. After 15 minutes, 350 μL of Buffer RW1 was added to the RNeasy Spin Column and centrifuged at room temperature and 8,000 × g or more for 15 seconds. Next, 500 μL of 4 × Buffer RPE made into 1 × Buffer RPE with 99% ethanol (Wako) was added to RNeasy Spin Column. The membrane was washed by centrifugation at 8,000 xg or more at room temperature for 15 seconds. Furthermore, 500 μL of 1 × Buffer RPE was added to the RNeasy Spin Column again to prevent ethanol carryover during RNA elution, and the spin column and membrane were centrifuged at room temperature at 8,000 × g or more for 2 minutes. Was washed. After the centrifugation, the column was carefully removed from the collection tube so that the RNeasy Spin Column did not come into contact with the filtrate, the RNeasy Spin Column was transferred to a new 2 mL collection tube, and centrifugation was performed at the maximum speed for 1 minute. After that, the RNeasy Spin Column was set in a new 1.5 ml collection tube (attached), and 50 μL of RNase free water was directly added to the RNeasy Spin Column membrane. The lid was closed, centrifugation was performed at room temperature at 8,000 × g or more for 1 minute, and the eluate resulting from this operation was used as an RNA solution. The RNA concentration in the solution was calculated using a NanoDrop 2000 / 2000c spectrophotometer based on the absorbance value at 260 nm, and used for the quantitative real-time PCR experiment. The method of cDNA synthesis and the quantitative real-time PCR method is as described above.

9.4. Preparation of paraffin-embedded section Preparation of paraffin-embedded section and HE staining were performed by a conventional method.

Immunostaining 1. Deparaffinization and Hydrophilicity The slide glass on which the paraffin-embedded section was attached was placed in a staining basket, and immersed in xylene placed in a new staining vat every 3 minutes for a total of 3 times. Next, dipping in 99% ethanol in a new dyeing vat was performed twice every 5 minutes in total. Furthermore, it was immersed in 90% ethanol, 80% ethanol and 70% ethanol for 5 minutes each. When the slide glass was moved to a new staining vat, excess liquid was thoroughly cut off and transferred to a new liquid so that carryover was minimized. Then, it was immersed in 0.1% Tween 20 / TBS for 5 minutes for washing. The composition of 0.1% TBS-T is shown below.

・ 10 x TBS (500 mL in total)
12.1g Tris
40g NaCl
Adjusted to pH 7.6 with 12N HCl-0.1% Tween 20 / TBS
Make 10 × TBS into 1 × TBS with ultrapure water and add 0.5 ml / 500 ml of Tween-20.

2. Antigen activation treatment (hot water method)
The antigen activating solution 10 × HistoVT One (NACALAI TESQUE) was diluted to 1 × HistoVT One with ion-exchanged water, placed in a polypropylene staining vat, and preheated in a pot set at 90 ° C. The deparaffinized and hydrophilic sample was placed in the slide glass as it was and activated for 20 minutes. After 20 minutes, the slide glass was taken out, and while being careful not to dry the tissue, the slide glass was cooled to room temperature, and washed with fresh 0.1% Tween 20 / TBS three times every 3 minutes.

3. After blocking and washing, excess liquid around the sample on the slide glass was wiped off with a Kimwipe or the like, and the sample was surrounded with a Super Pap pen liquid blocker (Daido Sangyo, Japan) to form a water repellent circle. Next, 1 in the water-repellent circle, taking care not to directly apply the blocking buffer, Bocking One Histo (NACALAI TESQUE) or 5% Normal Goat Serum (Wako) /0.1% Tween 20 / TBS. A drop (about 100 μL) was dropped, and the mixture was incubated in a wet box (AS ONE) for 1 hour at room temperature for blocking. After blocking, the plate was washed with 0.1% Tween 20 / TBS for 5 minutes.

4. Primary antibody reaction Dilute the primary antibody with a blocking buffer (Blocking One Histo or 5% Normal Goat Serum (Wako) /0.1% Tween 20 / TBS), and be careful not to hit the sample directly. Approximately 100 μL of the solution was dropped into the flask and incubated overnight at 4 ° C. in a humid box (AS ONE). After the primary antibody reaction, the slide glass was washed 3 times with fresh 0.1% Tween 20 / TBS every 5 minutes. The dilution ratio of the primary antibody is shown below.

<Primary antibody dilution rate>
・ Ki-67 (# 12202, Cell Signaling Technology) 1/400
-TERT (NB100-317, Novus Biologicals, Littleton, CO, USA) 1/50

5. Secondary antibody reaction Dilute the secondary antibody with a blocking buffer (Blocking One Histo or 5% Normal Goat Serum (Wako) /0.1% Tween 20 / TBS), and repel it while paying attention not to directly contact the sample. About 100 μL was dropped in an aqueous circle and incubated in a light-tight wet box (AS ONE) at room temperature for 1.5 hours to 2 hours to react with the secondary antibody. The dilution ratio of the secondary antibody is shown below.

<Secondary antibody dilution rate>
-AlexaFluor 555 anti-rabbit IgG (Thermo Fisher Scientific) 1/100
-AlexaFluor 488 anti-mouse IgG (Jackson Immuno Research, West Grove, PA, USA) 1/100

6. Encapsulation The excess secondary antibody was washed with 0.1% Tween 20 / TBS for 5 minutes and freshly washed with fresh 0.1% Tween 20 / TBS three times. Then, a drop of VECTASHIELD Mounting Medium (VECTOR LABORATORIES, Burlingame, CA, USA) containing fluorescent dye containing DAPI was dropped on the left end of the sample tissue, and the cover glass (24 mm x 36 mm) was gently dropped from the left end to form bubbles. Enclosed so as not to enter. After mounting, the slide glass was dried on a mappe.

7. Detection By observing the tissue and nucleus with a fluorescence microscope (IX50, OLYMPUS, Tokyo, Japan), the thickness of the entire skin (thickness including epidermis, dermis, and subcutaneous tissue) was observed. Further, the localization and expression level of each protein were observed by observing the fluorescence of the secondary antibody with a confocal laser scanning microscope (FV1000, OLYMPUS).

10. Gene transfer using retrovirus expression system 10.1. Gene transfer to T293 cells 293T cells were seeded in a 5 mL dish at 30 to 40% confluence, and cultured in 10% FBS-containing DMEM medium. After 24 hours, 40 μL of Hilymax as a transfection reagent, 1.5 μg of Gag-pol plasmid, 1.5 μg of VSVG plasmid, and 2.0 μg of the target gene were added to 300 μL of DMEM medium, and incubated at room temperature for 15 minutes. Then, the whole amount was added to each 293T cell, and the cells were post-cultured at 37 ° C. under 5% CO ₂ for 6 hours. After 6 hours, the medium was replaced with DMEM containing 10% FBS. In addition, the target HaCaT cells for infection on the next day were seeded in a 10 mL dish (Greiner bio-one) at 30 to 40% confluence, and cultured overnight at 37 ° C. in the presence of 5% CO ₂ .

10.2. Viral infection of HaCaT cells 24 hours after gene transfer to 293T cells, 5 mL of the supernatant was sterilized with a 0.45 μm × 33 mm filter (Merck), collected in a 15 mL centrifuge tube, and 10 mg / ml polybrene 4.0 μL ( Merck) was added to obtain a virus solution. For the next preparation of virus solution, 5 mL of DMEM medium containing 10% FBS was added to 293T cells. The recovered virus solution was added to HaCaT cells without the medium. This infection work was performed 6 times in total in the morning and the afternoon of 3 days.

10.3. Drug selection 24 hours after the sixth virus infection operation, 5.0 × 10 ⁵ cells infected with the virus were seeded in a 10 mL dish. At that time, as a control, untreated HaCaT cells were seeded at the same density. Puromycin (SIGMA-Aldrich) was added to the medium at 2.0 μg / mL. After it was confirmed that the control untreated HaCaT cells were completely destroyed, the concentration of Puromycin was lowered to 0.5 μg / mL and the subculture was continued, and used for the experiment.
[Example 1]
Search for hTERT activated food ingredients

1.1. Establishment of HaCaT (hTERTp-EGFP) It was confirmed by measuring the fluorescence intensity of GFP using a flow cytometer whether the hTERTp-EGFP vector was introduced into HaCaT cells and a stable expression strain was established. A cell population having a large forward diffusion FS (Forward Scatter) value indicating the size of the cell and a small lateral diffusion SS (Side Scatter) value indicating the intracellular complexity with respect to the measured total cell population. The cells were regarded as living cells, and the fluorescence intensity of GFP was histogrammed for a subpopulation of this cell using analysis software FlowJo (Tree Star, Ashland OR, USA). As a result, it was confirmed that the peak of GFP fluorescence of HaCaT (hTERTp-EGFP) cells was slightly higher than that of control HaCaT cells (Fig. 1).

  The establishment of a stable expression strain of HaCaT (hTERTp-EGFP) cells was also confirmed by the quantitative RT-PCR method (Fig. 2). As a result, it was revealed that the expression of EGFP was enhanced in HaCaT (hTERTp-EGFP) cells as compared with control HaCaT cells, and it was confirmed that hTERTp-EGFP was introduced.

1.2. Search for hTERT promoter-enhanced foods using IN Cell Analyzer 1000 Using HaCaT cells incorporating hTERTp-EGFP, hTERT promoter activity when various food ingredients were added was evaluated by the fluorescence intensity of EGFP. The polyphenol sample was added so as to have a final concentration of 10 μM, and 48 hours later, EGFP fluorescence intensity was measured using IN Cell Analyzer 1000. In this example, a food component was selected that showed a significantly higher EGFP fluorescence intensity than the addition of the control sample in three measurements.

As a result, in the polyphenol sample, Resveratrol (polyphenol derived from red grape), Eugeninin (polyphenol derived from rose), Urolithin A (metabolite of intestinal bacteria of pomegranate prephenol), Fisetin (polyphenol derived from strawberry), Sesamol (polyphenol derived from sesame) 5 showed significantly higher hTERT promoter activity than the control (FIG. 3).
From the results of the primary screening using the above-mentioned hTERTp-EGFP-introduced HaCaT cells, five types of food ingredients were selected as hTERT promoter-activated foods.

1.3. Evaluation of endogenous hTERT gene expression level by quantitative RT-PCR 1.2. From 5 items of food components selected from the results of the search for hTERT promoter activating components using HaCaT (hTERTp-EGFP) cells, the effect on endogenous hTERT gene expression in HaCaT cells was evaluated by quantitative RT-PCR. The polyphenol sample was added so as to have a final concentration of 10 μM, and after 48 hours, RNA recovery and cDNA synthesis were carried out, and evaluated by quantitative RT-PCR. In this example, food components that showed a significantly higher hTERT gene expression level than the addition of the control sample were selected.

  As a result, among the polyphenols, Resveratrol (red grape-derived polyphenol), Urolithin A (intestinal bacterial metabolite of pomegranate prephenol), Fisetin (strawberry-derived polyphenol), and Sesamol (sesame-derived polyphenol) were compared to the control in four types. The expression of the hTERT gene was significantly higher (Fig. 4).

Above 1.2. Based on the results in Section 1 and this result, four types were selected as food ingredients that activate the hTERT promoter. After that, we analyzed these four kinds of polyphenol samples for the purpose of clarifying their functionality and molecular basis.
[Example 2]
Functionality of TERT-enhancing food ingredients

2.1. Verification of Effect on Cell Proliferation Hair growth is a phenomenon in which hair is produced by interaction between epithelial cells and mesenchymal cells. That is, cell growth of epidermal and dermal components is essential for hair development and growth (Hachiya A et al., 2009, Oliver, RF F. et al., 1988).
Therefore, in this section, the effect of four kinds of food components selected from the results of Example 1 on the cell proliferation of HaCaT cells, which are model cells of epithelial cells, was evaluated. Cell Count Kit-8 solution (DOJINDO) was adopted as a reagent for measuring the number of cells.

Six hours after seeding the HaCaT cells at 1.0 × 10 ⁴ , the selected four kinds of polyphenol samples were added so that the final concentration was 5.0 μM. The dye amount of formazan, which is produced by Cell Count Kit-8 solution and has a linear proportional relationship with the cell number, is measured by measuring the absorbance at 450 nm every 24 hours in the presence of 5% CO _{2 at} 37 ° C. did. This operation was continued for 4 days (FIG. 5- (A)). As a result, on the third day after the start of culturing in which the number of cells becomes maximum, among the four kinds of polyphenol samples, three kinds of Resveratrol, Fisetin, and Sesamol significantly enhanced cell proliferation as compared with Control. It became clear (FIG. 5- (B)).

2.2. Verification of effect on hair follicle cell activating factor 2.1. As described in the section above, activation of epithelial cells is also important for hair growth. Therefore, in this section, the expression level of the activation gene of epithelial cells was evaluated using quantitative RT-PCR. The activator of epithelial cells is a growth factor involved in the activation of epithelial cells, and in this section, IGF-1 (insulin-like growth factor (insulin-like growth factor) having a function of activating epithelial cells is used. )) And KGF (Keratinocyte Growth Factor) and TGF-β1 (Transforming growth factor) that is a factor that suppresses cell activity as a regression phase inducing factor. The change in the expression level of the gene was verified.

The expression levels of endogenous growth factor genes in HaCaT cells were evaluated by quantitative RT-PCR. Polyphenol was added so that the final concentration would be 10 μM, and after 48 hours, RNA was collected and cDNA was synthesized and evaluated by quantitative RT-PCR.
As a result, in comparison with the control, significant increase in expression of IGF-1 (FIG. 6) and KGF (FIG. 7) and significant effect of TGF-β (FIG. 8) were observed in three types of Resveratrol, Fisetin and Sesamol. It was possible to confirm a decrease in expression. On the other hand, in Urolithin, significant enhancement of expression in KGF could not be confirmed.

  The above 2.1. On the basis of the results of Section 1 and this result, three types of polyphenol components, Resveratrol, Fisetin, and Sesamol, which have a high effect on cell growth in epithelial cell model HaCaT cells, were selected. After that, analysis was carried out for these three types of polyphenol samples in order to clarify their functionality and molecular basis.

2.3. Activation of Wnt / β-catenin pathway β-catenin is a transcription factor involved in hair cycle and Wnt signal pathway, and it has been reported that this Wnt / β-catenin signal promotes hair growth and hair cycle. (Narhi et al., 2008), and it has been reported that hair growth is promoted by activating the Wnt / β-catenin signal in mouse skin (Jinkuk et al., 2008). It has also been reported that the binding of this β-catenin to the TERT promoter controls the expression of TERT (Rolf et al., 2012). Therefore, the effect of the selected three kinds of hTERT-activating polyphenols on β-catenin activity in HaCaT cells was examined.

  Since β-catenin controls the expression of Wnt target gene via TCF / LEF transcription factor (T-Cell Factor / Lymphoid-Enhancer Factor) in the nucleus, first, luciferase using a reporter vector having a β-catenin binding site is used. The assay verified β-catenin activity (FIG. 9). The day after the cells were seeded, a reporter plasmid (M50 Super 8x TOP Flash) was transfected, and after 3 hours, the medium was replaced with 10% FBS-containing DMEM medium, polyphenol was added at a final concentration of 10 µM, and 48 hours later, luciferase assay was performed. . As a result, it was found that three types of polyphenols, Resveratrol, Fisetin, and Sesamol, activate β-catenin-dependent transcription activity.

Next, the effect on β-catenin protein under the same conditions was verified by Western blotting (FIG. 10- (A)). As a result, it was revealed that two types of polyphenols, Resveratrol and Fisetin, significantly increase the amount of β-catenin protein in HaCaT cells (FIG. 10- (B)).
From the above, two types of polyphenols, Resveratrol and Fisetin, regulate hair growth by controlling the expression of growth factors involved in hair synthesis and growth promotion, or by activating the Wnt / β-catenin signal that promotes hair cycle and hair growth. It was thought that it might have an effect.
[Example 3]
Phenotypic analysis in HaCaT cells (HaCaT (sh-hTERT)) that knocked down hTERT

3.1. Specificity verification of hTERT-enhancing foods using HaCaT (sh-hTERT) In order to verify that the hTERT-enhancing effect by the polyphenols, Resveratrol and Fisetin evaluated in Examples 1 and 2, is hTERT-dependent, Knockdown HaCaT cells were generated.

  First, in HaCaT cells, two types of HaCaT cells ((HaCaT (sh-hTERT-1), HaCaT (sh-hTERT-2)) in which hTERT was knocked down were produced. HTERT expression in these two types of cells was quantified. It was evaluated by RT-PCR (FIG. 11), and as a result, it was confirmed that hTERT could be knocked down in two types of HaCaT (sh-hTERT).

Next, polyphenol was added to these two types of HaCaT (sh-hTERT) so that the final concentration was 10 μM, and RNA was recovered and cDNA was synthesized after culturing at 37 ° C. in the presence of 5% CO ₂ for 48 hours, and quantitative RT was performed. -Evaluated by PCR (Figure 12, Figure 13). As a control, a knockdown vector (scr-shRNA) having a scrambled sequence was introduced. As a result, in HaCaT (scr) cells into which scr-shRNA was introduced, hTERT expression was significantly increased by sample treatment with Resveratrol and Fisetin, whereas in HaCaT (sh-hTERT), significant TERT expression was confirmed. I couldn't. That is, it was revealed that Resveratrol and Fisetin enhance hTERT expression in a hTERT-dependent manner.

3.2. Verification of Contribution of hTERT to Cell Proliferation Enhancement of Polyphenol Using HaCaT (sh-hTERT) In order to verify that the cell growth promoting effect by Resveratrol and Fisetin found in Example 2 is hTERT-dependent, HaCaT ( sh-hTERT) was used to verify the effect. The cell number was measured for 4 consecutive days in the same manner as in Example 2 and 2.1.

As a result, in HaCaT (scr-shRNA) (FIG. 14- (A)), two types of polyphenols were compared with Control in the 3rd day of the culture initiation (FIG. 14- (B)) where the number of cells was maximum. While a significant cell growth-enhancing effect could be confirmed, in the case of HaCaT (sh-hTERT-1) (Fig. 15- (A)), the number of cells reached the maximum on the 3rd day of culture initiation (Fig. 15- ( In B)), it was not possible to confirm the effect on significant cell proliferation in comparison with Control in both of the two types of polyphenol samples. Further, the results with HaCaT (sh-hTERT-2) (FIGS. 16- (A) and 16- (B)) also showed similar results.
From the above, it was revealed that Resveratrol and Fisetin proliferate cell proliferation in a hTERT-dependent manner.
[Example 4]
Verification of SIRT1 dependence of hTERT-activated food ingredients

4.1. SIRT1 enhancing effect of hTERT activated food ingredients Focusing on SIRT1 (Yamashita S et al., 2011, Shuntaro Y et al., 2014), which is reported to be located upstream of hTERT transcriptional regulation, endogenous in HaCaT cells The effect of HaCaT cells on endogenous SIRT1 gene expression was evaluated by quantitative RT-PCR in two types of polyphenols that enhance the hTERT gene. Polyphenol was added so as to have a final concentration of 10 μM, and after 48 hours, RNA was collected and cDNA was synthesized and evaluated by quantitative RT-PCR. As a result, it was revealed that the expression level of the SIRT1 gene was significantly higher in both Resveratrol and Fisetin than in the control (FIG. 17).

4.2. SIRT1-dependent verification of hTERT-enhanced foods using HaCaT (sh-SIRT1) It was verified whether the activation of hTERT by polyphenol was mediated by SIRT1 which was reported to be located upstream of hTERT.
First, HaCaT (sh-SIRT1) cells in which SIRT1 was knocked down were prepared. As a Control, a HaCaT (scr-shRNA) cell into which a knockdown vector (scr-shRNA) having a scrambled sequence was introduced was prepared. In HaCaT (sh-SIRT1) cells, knockdown of SIRT1 was evaluated by quantitative RT-PCR (FIG. 18).

Next, Resveratrol and Fisetin were added to HaCaT (scr-shRNA) cells and HaCaT (sh-SIRT1) cells so that the final concentration was 10 μM, and after culturing at 37 ° C. in the presence of 5% CO ₂ for 48 hours, RNA was added. Were collected, and after cDNA synthesis, the effect on the expression levels of SIRT1 and hTERT was evaluated by quantitative RT-PCR.
As a result, in HaCaT (scr-shRNA) cells, treatment with Resveratrol and Fisetin significantly increased SIRT1 expression, whereas in HaCaT (sh-SIRT1) cells, significant SIRT1 expression could be confirmed. None (FIG. 19).

On the other hand, in HaCaT (scr-shRNA) cells, the expression of hTERT whose expression was significantly increased by Resveratrol and Fisetin treatment was canceled in HaCaT (sh-SIRT1) cells by Resveratrol treatment, but it was expressed by Fisetin treatment. The enhancement could not be released (Fig. 20). Further, hTERT expression in the control sample treatment of HaCaT (sh-SIRT1) cells was not different from that in the control sample treatment of HaCaT (scr-shRNA) cells.
From the above results, it was revealed that SIRT1 is not involved in the enhancement of hTERT by Fisetin.
[Example 5]
Hair growth effect of TERT1 activated food ingredients

5.1. Mouse application test The effects of the hTERT-activating polyphenols Resveratrol and Fisetin identified in Example 4 on hair growth were examined using mice.
It is generally considered important to control the hair cycle in animal experiments relating to hair growth, because the functions and properties of the skin change greatly with the hair cycle. Therefore, in order to obtain a uniform hair cycle, hair removal wax, electric shaver, etc. are used to simultaneously remove hair within a certain range of the telogen period and induce a uniform anagen period by applying stimulation. (Chase, et al., 1954) and is often used especially in C57BL / 6 mice. This is because C57BL / 6 mice can easily be judged by the naked eye that the skin color is dark when it is in the anagen stage and that when it is pink, it is at the telogen stage (Muller-Rover, et al. , 2001).

  In mice, the first two cycles of hair cycle, which generally occur at 7 weeks of age, occur in a uniform synchronization, but the pattern of the hair cycle becomes more complicated as it matures, and becomes inconsistent (Muller-Rover, et al. al., 2001, Lin, et al., 2004, Plikus and Chuong, et al., 2008), it is difficult to obtain a uniformly synchronized hair cycle in adult mice. Therefore, animal experiments on hair growth are often carried out at an early age.

  In this example, after hair removal with an electric hair clipper, which had no effect on the hair cycle, hair removal treatment with an electric shaver was further performed to induce the hair cycle in the telogen phase to the anagen phase. Then, the skin condition after hair removal was visually confirmed to be pink, and a mouse in which the hair cycle was in a resting period was used. As a test solution, a 0.1% Resveratrol solution prepared by diluting with 50% ethanol and a 0.1% Fisetin solution were used. As a control solution, 50% ethanol, which is the solvent of the test solution, was used.

  On the day before the start of the test, 50 μL each of the test solution was applied to the test area of the mouse whose back was shaved for 6 days per week, and the mouse was bred, and the hair growth of the test area was observed for about 5 weeks (FIG. 21). As a result, in the Resveratrol group (hair growth in 4/9 animals) compared to the Control group (1/9 hair growth), and in the Fisetin group (9/10 hair growth) compared to the Resveratrol group, It was possible to confirm the hair growth effect.

5.2. Verification of mTERT expression in mouse skin After euthanizing the mouse for which the application test was completed by cervical dislocation, the test area on the back of the mouse was collected, and RNA was extracted using the RNeasy Fibrous Tissue Mini Kit, and then cDNA was extracted. After synthesis, the expression level of mTERT in mouse skin was verified by the quantitative RT-PCR method. As a result, both the Resveratrol group (FIG. 22) and the Fisetin group (FIG. 23) showed a higher mTERT gene expression level than the control.

5.3. H & E staining The transition of the hair cycle corresponds well with the change of the skin thickness with the depth of the hair follicle (Muller-Rover et al., 2001). In other words, the depth of hair follicles is deep in anagen, shallow toward catagen, and shallow during telogen, so the skin thickness is thick in anagen and thin in telogen. It is also possible to determine at which stage of the hair cycle it is based on the tissue morphology of the hair follicle (Muller-Rover et al., 2001).

  In this example, after the mouse for which the application test was completed was euthanized by cervical dislocation, the test area on the back of the mouse was sampled, fixed with 10% neutral buffered formalin, and 5 μm paraffin section parallel to the body axis. By making a hematoxylin / eosin stain (H & E stain) (FIG. 24) and using it for histological examination, it is possible to determine at which stage of the hair cycle in the mouse skin, and to determine the epidermis, dermis, and subcutaneous skin. The thickness of the entire skin including the tissue was measured, and the thickness of the skin was measured (FIG. 25).

  As a result, in the Control group, many mice remained in the telogen stage, whereas in the Resveratrol group, about half of the mice entered the anagen stage, and in the Fisetin group, almost all the mice were in the anagen stage. It was Furthermore, as a result of measuring the skin thickness, it was revealed that the skin thickness of the mice of the Resveratrol group and the Fisetin group was significantly increased as compared with the skin of the mice of the Control group.

5.4. Immunostaining (fluorescent antibody method)
After euthanizing the mouse for which the application test was completed by cervical dislocation, the test area on the back of the mouse was collected, fixed with 10% neutral buffered formalin, and a 5 μm paraffin section parallel to the body axis was prepared for immunization. Dyeing was performed. As primary antibodies, Ki-67 and TERT, which are proliferating cell markers expressed on the nucleolus of proliferating cells and chromosomes in the mitotic phase, were used.

As a result, in the Control group, Ki-67 was negative in the skin of almost all mice, and TERT-positive cells could not be confirmed in the skin of all mice (FIG. 26- (A)). On the other hand, in the Resveratrol group, Ki-67 positive cells can be confirmed in the hair matrix cells that make up the hair bulb of approximately half of the mice, and TERT positive cells can be confirmed in the skin of some mice. It was possible (FIG. 26- (B)). In the Fisetin group, hair bulb hair matrix cells were Ki-67 positive in almost all mouse skins, and TERT-positive cells could be confirmed in some mouse skins (Fig. 26- (C )).
In this example, an animal test using mice revealed that Resveratrol and Fisetin have hair growth and hair growth effects.
[Example 6]
SIRT1 activation by resveratrol structural analogues

1. Experimental Materials and Methods (1) Cell Culture In this study, HaCaT (SIRT1p-EGFP) cells obtained by introducing a vector into the human epidermal keratinocyte cell line HaCaT cells were used for screening resveratrol analogues having a SIRT1 promoter activation effect. , HaCaT cells were used to verify the SIRT1 expression enhancing effect. In addition, HaCaT (hTERTp-EGFP) cells obtained by introducing a vector into the human epidermal keratinocyte cell line HaCaT cells were used for screening for components having a TERT promoter activation effect. All cells were added to Dulbecco's modified Eagle medium “Nissui” (1) [DMEM] (Nissui Pharmaceutical, Tokyo) as a normal medium, and a final concentration of 100 U / mL penicillin (Meiji, Tokyo), 0.1 mg / mL Streptomycin (Meiji, Tokyo) mixed with 10% FBS was used.

(2) Primary Screening Using IN Cell Analyzer 1000 The SIRT1 promoter activating ability is targeted for HaCaT (SIRT1p-EGFP) cells, and the TERT promoter activating ability is targeted for HaCaT (hTERTp-EGFP) cells, and IN Cell Analyzer 1000. (GE Healthcare, Amersham Place, UK) and evaluated by measuring the EGFP fluorescence intensity of the cells to which the sample was added. HaCaT (SIRT1p-EGFP) cells and HaCaT (hTERTp-EGFP) cells were seeded on 96 well-plate (65590, Greiner Bio-one) at 0.6 × 10 ⁵ cells / mL, and 10% FBS-containing DMEM medium was seeded. The cells were cultured for 24 hours. Then, resveratrol analog was added to HaCaT (SIRT1p-EGFP) cells so that the final concentration was 10 μM, and a separately prepared sample was added so that the final concentration was 0.1 μg / mL, 1 μg / mL, 10 μg / mL. HaCaT (hTERTp-EGFP) cells were added.

  After the addition, the cells were cultured in DMEM medium containing 10% FBS for 48 hours, 8% paraformaldehyde (Wako, Osaka) was added in an equal amount from the top of the culture solution, and the mixture was left standing at room temperature for 15 minutes for fixation. After the fixation, the fixative was discarded, the cells were washed twice with PBS, Celltrain R-Hoechst 33342 solution (Dojindo, Kumamoto) diluted 500 times with PBS was added, and the mixture was allowed to stand at room temperature for 20 minutes for light-shielding staining. After staining, the Hoechst solution was discarded, washed twice with PBS, added with PBS and subjected to IN Cell Analyzer 1000 to measure the EGFP fluorescence intensity.

Resveratrol structural analogs used for verification of the SIRT1 promoter activating effect are shown in Table 4, and samples used for screening the components having the TERT promoter activating effect are shown in Table 5.

(3) Preparation of total RNA HaCaT cells were seeded on a 12-well plate at 1.0 × 10 ⁵ cells / mL. After culturing for 24 hours, a resveratrol structural analog sample was added to a final concentration of 10 μM, and the cells were further cultured for 48 hours. In this example, Resveratrol was used as a positive control and added at a final concentration of 10 μM.

  Next, RNA extraction was performed. The medium was completely removed, 100 μL of TRIzol Reagent (Cosmo Bio, Tokyo) was added, and the plate was continuously rotated so that the liquid was spread over the whole. When the viscosity of the liquid disappeared, the cell lysate was collected in a 0.5 mL tube, 20 μL of chloroform (Wako, Osaka) was added, and mixed with a vortex mixer for 10 seconds. After leaving it at room temperature for 5 minutes, it was centrifuged at 4 ° C. and 15000 g for 5 minutes, and the supernatant was transferred to a new 1.5 mL tube. 99% ethanol (Wako, Osaka) corresponding to 0.55 times the amount of the collected supernatant was added, and after light pipetting, the cell lysate was subjected to Econo Spin ™ for RNA column (Epoch Life Science Inc, Missouri, USA). And centrifuged at 10,000 rpm for 1 minute at 4 ° C.

The liquid discharged into the collection tube was discarded, 200 μL of RNA wash Solution (Promega, Wisconsin, USA) was added, and the mixture was centrifuged at 10000 rpm for 1 minute. The liquid discharged into the collection tube was discarded, 100 μL of RNA wash Solution was added again, and the mixture was centrifuged at 10,000 rpm for 1 minute. The liquid discharged into the collection tube was discarded, the mixture was centrifuged at 4 ° C. and 15,000 g, and then the column was set in a new 1.5 mL tube.
Then, 40 μL of an elution buffer (Roche, Basel, Schweiz) was added, and the mixture was allowed to stand at room temperature for 3 minutes and then centrifuged at 15000 g for 1 minute to elute the total RNA. The concentration of the eluted total RNA was measured using a Nano Drop 2000 / 2000c spectrophotometer (Thermo Scientific, Waltham, MA, USA).

(4) cDNA synthesis 1 μg of RNA and 0.5 μL of oligo dT were added to 0.2 μL tube (Seikatsu) and RNase-free sterilized water was added to adjust the total amount to 13.5 μL. Then, after using a thermal cycler (Peltier Thermal Cycler PTC-200, MJ Research, Watertown, MA, USA) for annealing at 65 ° C. for 5 minutes, it was immediately left on ice for 5 minutes. 5 × Reaction Buffer (TOYOBO, Osaka) 4 μL, 10 mM dNTP (GE Healthcare) 2 μL, and ReverseTrace Ace (TOYOBO, Osaka) 0.5 μL were added, and the mixture was reacted at 42 ° C. for 20 minutes and at 99 ° C. for 5 minutes to synthesize cDNA. went.

(5) Real-time PCR
The prepared cDNA was used as a template. Sterile water 49 μL in a 0.2 mL tube, 3.5 μL each of primers Forward / Reverse diluted to 10 μM, template cDNA (10-fold diluted cDNA for SIRT1, 50-fold diluted for β-actin) 7. 0 μL and 24.5 μL of THUNDERBIRD SYBR qPCR Mix (TOYOBO, Osaka) were added and well suspended.

The base sequences of the primers used are shown below.
SIRT1
β-actin

After that, 25 μL of each 96 well-plate was added to 3 wells, and quantitative real-time PCR was performed using a Thermal Cycle Dicer Real Time System (Takara Bio, Shiga).
The reaction conditions were 95 ° C. for 30 seconds for 1 cycle, 95 ° C. for 5 seconds → 55 ° C. for 10 seconds → 72 ° C. for 20 seconds for 45 cycles, 95 ° C. for 15 seconds → 60 ° C. for 30 seconds → 95 ° C. for 15 seconds, and FAM. Detected by. Β-actin was used as a primer for the calibration curve. The relative gene expression level was determined by dividing the measured value by the β-actin expression level value.

2. Results (1) Verification of SIRT1 promoter activation effect by resveratrol structural analog After adding resveratrol structural analog to HaCaT (SIRT1p-EGFP) cells and culturing for 48 hours, HaCaT (SIRT1p was measured by IN Cell Analyzer 1000). -EGFP) changes in EGFP fluorescence intensity in cells were tracked. As a result, among 9 types of resveratrol structural analogs, the SIRT1 promoter activating ability was found in Pinostilbene, Isorhapontigenin, Rhapontigenin, Piceatannol, Gnetol, Isoliquiritigenin, and Butein (FIG. 27).

(2) Verification of SIRT1 Expression Enhancement Effect by Resveratrol Structural Analogues Resveratrol structural analogues confirmed to have SIRT1 promoter activation ability in the primary screening were tested against Pinostilbene, Isorhapontigenin, Rhapontigenin, Piceatannol, Gnetol, Isoliquinitiriten. Then, the amount of SIRT1 mRNA in HaCaT cells to which these resveratrol analogues were added was measured by the real-time PCR method.
In this example, as a result of measurement using Resveratrol as a positive control of SIRT1 activity, significant enhancement of expression was confirmed in Gnetol, Isoliquiritigenin, and Butein based on DMSO (FIG. 28).

3. DISCUSSION In this example, by screening using an imaging cytometer, the resveratrol structural analogs Pinostilbene, Isorhapontigenin, Rhapontigenin, Piceatannol, Gnetol, Isoliquiritigenin, and Butein are SIRT1 which are active promoters of anti-aging factors. Became clear. Moreover, the SIRT1 mRNA amount was measured for these seven species by the real-time PCR method. In this example, Resveratrol, a grape-derived polyphenol widely used in SIRT1 studies, was used as a positive control.
As a result, it was revealed that Gnetol, Isoliquiritigenin and Butein enhance SIRT1 mRNA expression in HaCaT cells. From this result, it is considered that the three identified resveratrol structural analogs may activate SIRT1. In addition, Isoliquiritigenin and Butein have similar structures, and this structure is considered to be involved in SIRT1 activation.

In addition, in this example, ectoin (0.1 μg / mL), (ascorbyl / tocopheryl) phosphoric acid K (0.1, 10 μg / mL), and tannic acid (10 μg / mL) were screened by using an imaging cytometer. ), Tranexamic acid (0.1 μg / mL), and α-G hesperidin (1,10 μg / mL) were found to activate the promoter of TERT, which is a factor promoting hair growth.
[Example 7]
HTERT enhancing effect of Fisetin and Resveratrol analogues

Method: The following Fisetin and Resveratrol analogues were added to HaCaT cells and then cultured, and hTERT expression was verified by the qPCR method. A method similar to the method described in Example 1 was performed except for polyphenol used as a sample.
The results are shown in Fig. 29.

From FIG. 29, Resveratrol, Fisetin, Apigenin, Luteolin, Ipriflavone, Nobiletin, (±) -Equol, Genistein, Resveratrol, Tangeretin, BiochaninA, Chrysin, Pinoutinebene, Chrysin, Pinoutebein, and Chrysin, Pinoutinebene are shown. Among them, Quercetin had a particularly high enhancing effect.
[Example 8]
Paraquat-induced DNA damage repair effect by Fisetin and Resveratrol analogues

<Material and method>
1. Cell Culture In this study, human colon cancer-derived cell line Caco-2 cells were used. Caco-2 cells were cultured at 37 ° C. in 5% at 37 ° C. using 10% Fetal bovine serum (FBS) -added DMEM medium (Dulbecco's Modified Eagle Medium; Nisui, Tokyo, Japan) and cell culture dish (Greiner bio-one). Subculture was performed in the presence of ₂ .

2. Evaluation of DSBs repair-promoting ability using γH2AX as an index In order to evaluate the repair-promoting ability of DNA double-strand breaks (DSBs) by SIRT6 activated food components, one of the DNA damage repair markers, γH2AX Was used as an index to examine whether DSCs repair in Caco-2 cells induced by paraquat treatment was promoted by the SIRT6 activated food ingredient.

(1) Preparation of Paraquat Solution Paraquat solution was prepared by sterilizing and appropriately using Paraquat dichloride hydrate (Sigma-Aldrich, Osaka, Japan) prepared at 1M with sterilized water. At the time of use, the final concentration was 1 mM.

(2) Method for paraquat treatment of Caco-2 cells On the day before paraquat treatment, Caco-2 cells were added to 96-well plate (μ Clear Fluorescence Black Plate; Greiner bio-one) at 2.0 × 10 ⁵ cells / mL. The cells were seeded and the cells were allowed to adhere to the bottom of the dish. The next day, a paraquat solution was added so that the final concentration was 1 mM, and the cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 2 hours.

(3) Preparation of Cell Fixation Solution Paraformaldehyde (Wako) was diluted with 1 × PBS to 4% Paraformaldehyde cell fixation solution, and dissolved in a hot water bath at 60 ° C. to prepare.

(4) Preparation of blocking buffer 1.0 mL of 10 × PBS, 500 μL of normal goat serum (Wako) and 8.5 mL of sterilized water were mixed, and further 30 μL of Triton X-100 (100%) (Sigma-Aldrich) was added. Stir well.

(5) Preparation of antibody dilution buffer 1.0 mL of 10 × PBS was added to 9.0 mL of sterilized water and mixed, and 0.1 g (Wako) of BSA (Bovine Serum Albumin Fraction V) was added and dissolved. After that, 30 μL of Triton X-100 (100%) (Sigma-Aldrich) was added and well stirred.

(6) Immunostaining-Measurement and analysis by IN Cell Analyzer 1000 The medium of Caco-2 cells subjected to paraquat treatment for 2 hours was replaced with 10% FBS-containing DMEM medium, and each food component sample was adjusted to 10 µM. After the addition, the cells were cultured at 37 ° C. in the presence of 5% CO ₂ for 4 hours. Then, the medium containing the food components was removed, Caco-2 cells were washed with 100 μL / well of 1 × PBS, and left standing at room temperature for 15 minutes with 80 μL / well of cell fixative to fix. The cell fixing solution was removed, and the cells were washed 3 times with 1 × PBS for 5 minutes. Then, the cells were blocked with the blocking buffer for 1 hour.

The buffer was removed, and the primary antibody (P-Histone H2A.X; Cell Signaling Tech., Danvers, MA, USA: 1000-fold diluted) diluted with an antibody dilution buffer was added to the cells at 50 μL / well, and the cells were added at 4 ° C. at 1 ° C. Incubated overnight. And it wash | cleaned 3 times for 5 minutes with 1xPBS. The following operation was performed in a light-shielded state. A secondary antibody (AlexaFlor 555 anti-rabbit IgG; Life Technologies, Gaitherburg, MD, USA) diluted 1,000 times with an antibody dilution buffer was added to the cells at 50 μL / well and incubated at room temperature for 1.5 hours. After washing with PBS three times for 5 minutes, 1 nL of cell nuclei staining solution Hoechst33342 (DOJINDO) / 1 mL of PBS was added at 100 µL / well and left standing at room temperature for 20 minutes for staining.

After staining, the cell nucleus staining solution was removed, and the cells were washed once with 100 μL / well of 1 × PBS. After that, the cells were replaced with 1 × PBS 100 μL / well, and an image was acquired with IN Cell Analyzer 1000 (GE Healthcare). The image was analyzed by IN Cell Analyzer 1000 Workstation (GE Healthcare), and the expression amount of γH2AX in the cell was examined by measuring the fluorescence amount of the secondary antibody. Then, it graphed by Spotfire DecisionSite Client 8.2 software (TOBCO Spotfire Japan).

<Results>
The results of immunostaining with paraquat treatment for 2 hours and sample treatment for 4 hours are shown in FIG. A reduction effect of γH2AX, that is, a DNA damage repair effect was observed in Genistein, Tangeretin, Baicalein, and Isoliquiritigenin.

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SEQ ID NOS: 1-20: Synthetic DNA
[Sequence list]

Claims (15)

Formula I:
(In the formula, ^one of R ¹ and R ³ represents a hydrogen atom or a hydroxyl group, and the other represents the following formula IV:
(R ^4b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5.)
Represents a group represented by, R ² represents a hydrogen atom or an oxygen atom, R ^4a is the same as R ^4b ,
Represents a double bond or a single bond, and m represents an integer of 1 to 4. )
A compound represented by
Formula II:
(In the formula, R ^4a and R ^4b are the same as above, and n1 and n2 each independently represent an integer of 1 to 5.)
And a compound of the following formula III:
(Wherein R ² , R ^4a , R ^4b , n1 and n2 and
Is the same as above. )
A composition for hair growth and / or hair growth, containing any compound selected from the group consisting of the compounds represented by: Formula I:
(In the formula, ^one of R ¹ and R ³ represents a hydrogen atom or a hydroxyl group, and the other represents the following formula IV:
(R ^4b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5.)
Represents a group represented by, R ² represents a hydrogen atom or an oxygen atom, R ^4a is the same as R ^4b ,
Represents a double bond or a single bond, and m represents an integer of 1 to 4. )
A compound represented by
Formula II:
(In the formula, R ^4a and R ^4b are the same as above, and n1 and n2 each independently represent an integer of 1 to 5.)
And a compound of the following formula III:
(Wherein R ² , R ^4a , R ^4b , n1 and n2 and
Is the same as above. )
A skin improving composition comprising any compound selected from the group consisting of compounds Formula I:
(In the formula, ^one of R ¹ and R ³ represents a hydrogen atom or a hydroxyl group, and the other represents the following formula IV:
(R ^4b represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and n represents an integer of 1 to 5.)
Represents a group represented by, R ² represents a hydrogen atom or an oxygen atom, R ^4a is the same as R ^4b ,
Represents a double bond or a single bond, and m represents an integer of 1 to 4. )
A compound represented by
Formula II:
(In the formula, R ^4a and R ^4b are the same as above, and n1 and n2 each independently represent an integer of 1 to 5.)
And a compound of the following formula III:
(Wherein R ² , R ^4a , R ^4b , n1 and n2 and
Is the same as above. )
A cosmetic composition comprising any compound selected from the group consisting of: The compound of formula I has the formula Ia or Ib:
(In the formula, R ¹ and R ³ represent a hydrogen atom or a hydroxyl group, R ² represents a hydrogen atom or an oxygen atom, and R ^4a and R ^4b each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. Represents a group,
Represents a double bond or a single bond, m represents an integer of 1 to 4, and n represents an integer of 1 to 5. )
The composition according to any one of claims 1 to 3, which is represented by The compound of formula I is fisetin, quercetin, catechin, nobiletin, apigenin, genistein, luteolin, ipriflavone, equol, tangeretin, biochanin A, chrysin, or baicalein. Composition. The composition according to any one of claims 1 to 3, wherein the compound represented by the formula II is resveratrol, piceatannol, pinostilbene, isolapontigenin, rapontigenin, gnetol, or oxyresveratrol. The composition according to any one of claims 1 to 3, wherein the compound of formula III is isoliquiritigenin or butein. The composition according to claim 2, wherein the skin improvement is at least one selected from the group consisting of treatment of wounds, repair of rough skin, improvement of spots, improvement of freckles, suppression of wrinkles, wound healing and suppression of cell aging. A hair growth and / or hair growth agent comprising the composition according to claim 1. A skin improving agent comprising the composition according to claim 2. A cosmetic comprising the composition according to claim 3. A cell containing a vector in which a reporter gene is linked to a TERT promoter or SIRT1 promoter, or a cell containing an endogenous TERT gene or an endogenous SIRT1 gene is contacted with a test substance, and the reporter gene, the endogenous TERT gene or an endogenous SIRT1 gene is contacted. A method of screening a substance having at least one effect selected from the group consisting of a hair growth effect, a hair growth effect, and a skin improving effect, using the expression of the above as an index. 13. The method of claim 12, wherein the cells are epithelial cells. The method according to claim 12 or 13, wherein the TERT is human TERT. At least one selected from the group consisting of a hair-growth effect, a hair-growth effect, and a skin-improving effect, in which a test substance is brought into contact with a cell containing a DNA damage repair marker gene and having a DNA damage, and the expression of the gene is used as an index. Method for screening substances having two effects.