WO2012144080A1 - Skin barrier function improving agent - Google Patents

Abstract

The purpose of the present invention is to provide a novel TRPV receptor activator which is suitable for the improvement in a skin barrier function. The purpose can be achieved by providing a TRPV receptor activator which comprises at least one compound selected from compounds respectively represented by the formulae or a plant extract containing the at least one compound in an effective amount for acting as a TRPV receptor activator. (In the formulae, a hydroxy group may be substituted by a methoxy group or an ethoxy group; and a methoxy group may be substituted by a hydroxy group or an ethoxy group.)

Description

Skin barrier function improver

The present invention relates to a skin barrier function improving agent and a skin external preparation for improving skin barrier function, and more particularly, to a TRPV (Transient Receptor-potential vanilloid) receptor activator and a skin external preparation containing the same.

The skin has a barrier function to prevent the evaporation of moisture and protect the body from external and physical stimuli in addition to transmitting external stimuli such as heat and pain to the living body. The skin barrier function plays an important role in maintaining the functions of the living body, but it is known that the function decreases due to temperature, humidity, excessive skin friction, lifestyle rhythm modulation, stress, etc. Due to various causes, the skin becomes dry, becomes hypersensitive to irritation, and is susceptible to irritation. Furthermore, if this state progresses, it causes direct damage and allergic reaction due to invasion of bacteria and harmful substances from outside the body, leading to the onset of skin disorders such as atopic dermatitis and sebum deficiency eczema. In addition, since skin touches human eyes and directly affects the beauty of appearance, it is a major concern for many women. In order to keep the skin condition healthy, a skin barrier function improving agent (for example, Patent Document 1 is used). Various attempts have been made such as a skin roughening agent (see, for example, Patent Document 2), a skin external preparation containing a ceramide synthesis accelerator (see, for example, Patent Document 3), and the like.

The non-selective cation channel TRP (Transient receptor potential) channel, identified as the causative gene of Drosophila photoreceptor mutants in 1989, is classified into at least nine subfamilies based on amino acid sequence homology. ing. The temperature-sensitive receptors belonging to the TRP channel have been first elucidated for the molecular entity of TRPV1. Until now, in mammals, nine types of temperature-sensitive receptors (TRPV1, TRPV2) opened by respective temperature stimuli from low temperature to high temperature. , TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, TRPA1) have been reported. TRPV4 was originally discovered as an osmotic sensitive receptor activated with low osmotic pressure, and is expressed in various tissues such as sensory nerve, hypothalamus, skin, kidney, lung, inner ear, osmotic stimulation, mechanical It plays an important role in the transmission of pain caused by stimulation and inflammatory pain. Moreover, TRPV4 functioning as a transmembrane calcium influx channel is not only a physical stimulus, but also an endogenous cannabinoid, arachidonic acid metabolite, 4α-phorbol ester (4α-PDD) (4alpha-phorbol 12,13- It is also activated by compounds such as didecanoate). Thus, TRPV4 is considered to exhibit many biological activities from the diversity of expressed tissues and stimulation responses, and is deeply involved in neurological disease symptoms including stimulation and pain recognition and autism. In addition, as biological activities other than these, activities related to energy metabolism such as significant weight-inhibitory effects on body weight gain, total fat mass and visceral fat mass (see, for example, Patent Document 4) have been reported in TRPV4-deficient mice. . In addition, TRPV4, which is present in epidermal cells, has a function as a moisture transpiration sensor, and activation by 4α-phorbol ester suppresses transpiration and enhances the skin barrier function. Has been reported (for example, see Non-Patent Document 1 and Non-Patent Document 2).

Tight-junction (TJ) and adherence-junction (AJ) are belt-shaped around cells, and close the gap by adjoining adjacent cells, and are continuous. It is an intercellular adhesion structure that binds cells to each other. In skin tissue, TJ is present in the epidermal cells of the granule layer and plays an important role in preventing water and substances from permeating through the cell gap and maintaining the skin barrier function (for example, non-patented Reference 3). It is also known that normal formation of AJ plays an important role in the formation of TJ. However, until now, it has not been known about the interaction between the cell-cell adhesion structures such as TJ and AJ and TRPV. For this reason, there is a new mechanism of action for a component having an action in which the interaction between TRPV and an intercellular adhesion structure such as TJ and AJ, particularly, the formation of TJ and / or AJ is promoted by the activation of TRPV4. An additive or additive effect can be expected as a skin barrier function-improving agent.

Examples of substances that act on TRPV, particularly TRPV4, include TRPV4 agonists such as biphenyl derivatives (see, for example, Patent Document 2), 1,4-diamine derivatives (see, for example, Patent Document 3), piperidine, and pyrrolidine. TRPV4 agonists (see, for example, Patent Document 5, Patent Document 6, and Patent Document 7) of derivatives and thiophene derivatives (see, for example, Patent Document 4) relieve symptoms such as pain, rheumatoid arthritis, neuropathy, and hyperalgesia It is known to be effective for improvement. However, it has been reported that the TRPV4 agonist has an action to improve skin barrier function, that activation by 4α-phorbol ester suppresses water transpiration and enhances skin barrier function (for example, non-patent literature). 1, see Non-Patent Document 2), and its mechanism of action has not been elucidated. In addition, a component that acts on both TJ and AJ and TRPV of the intercellular adhesion structure, particularly, a component having an action of promoting the formation of TJ and / or AJ by activating TRPV4 present in the epidermis is a skin barrier. It has not been known to improve the function and be effective in improving rough skin.

3-O-methylellagic acid 4′-O-α-L-rhamnopyranoside (hereinafter sometimes referred to as “compound 1”), 3,3′-di-O-methylellagic acid 4′-O-β-D -Glucopyranoside (hereinafter sometimes referred to as “compound 2”), 3,4,3′-tri-O-methylellagic acid 4′-O-β-D-glucopyranoside (hereinafter referred to as “compound 3”) 3′-O-methyl-3,4-methylenedioxyellagic acid 4′-O-β-D-glucopyranoside (hereinafter sometimes referred to as “compound 4”) is a known compound. Yes, for example, Non-Patent Document 4, Non-Patent Document 5 for Compound 1, Non-Patent Document 6 for Compound 2, Non-Patent Document 7 for Compound 3, Non-patent for Compound 4 Reference 8 describes. In addition, as for the action of these compounds, whitening action, stain and dullness improvement action (Non-patent Documents 9 and 10) for compound 1, and nitric oxide release promoting action or TNF-α release for compound 2 Promoting action (Non-patent document 11), Nitric oxide release promoting action or TNF-α release promoting action (Non-patent document 11) for compound 3, and Antioxidant action (Non-patent document 8, Non-patent document 8, 12) is known. However, these compounds have never been studied for TRPV receptor activation, AJ and / or TJ formation promotion, skin barrier function improvement, and the like.

JP 2007-001914 A JP 2008-0448857 A JP 2006-232769 A JP 2006-199647 A JP 2009-084209 A Special table 2009-507855 Special table 2008-512475 gazette JP 2009-527495 A Special table 2009-519976 Special table 2009-523176

The present invention has been made under such circumstances, and is to provide a novel TRPV receptor activator and a skin external preparation containing the activator, which are suitable for improving the skin barrier function. .

In view of such circumstances, the present inventors have sought for a skin external preparation suitable for improving the skin barrier function, and as a result of intensive efforts, have made a novel TRPV receptor activator and skin containing the same. It has been found that an external preparation has such characteristics. Furthermore, as a result of further detailed investigation, it was found that a plant extract obtained from a plant belonging to the genus Sarsbergi genus or the genus Rubiaceae belonging to the genus Rubiaceae belongs to the TRPV receptor activation activity. A compound having an activating action was isolated to complete the invention. The present invention is as follows.

<1> A TRPV receptor activator comprising one or more selected from the compounds represented by the following formula, or a plant extract containing an effective amount of the same compound as a TRPV receptor activator.

(In the formula, the hydroxyl group may be substituted with a methoxy group or an ethoxy group, and the methoxy group may be substituted with a hydroxyl group or an ethoxy group.)

<2> The compound is 3-O-methylellagic acid 4′-O-α-L-rhamnopyranoside, 3,3′-di-O-methylellagic acid 4′-O-β-D-glucopyranoside, 3,4, 3′-tri-O-methylellagic acid 4′-O-β-D-glucopyranoside or 3′-O-methyl-3,4-methylenedioxyellagic acid 4′-O-β-D-glucopyranoside, 1> The TRPV receptor activator according to 1>.
<3> The TRPV receptor activator according to <1> or <2>, wherein the extract is an extract obtained from a plant belonging to the genus Sarsberga or a plant belonging to the genus Rubiaceae.
<4> The TRPV receptor activator according to <3>, wherein the plant belonging to the genus Crape myraceae and the plant belonging to the genus Rubiaceae are the genus Crape myrtle, the genus Gambil .
<5> The TRPV receptor activator according to any one of <1> to <4>, wherein the TRPV receptor is TRPV4.
<6> A skin external preparation containing an effective amount of the TRPV receptor activator according to any one of <1> to <5>.
<7> The skin external preparation according to <6>, wherein the effective amount is 0.00001 to 10% by mass as the amount of the compound with respect to the total amount of the skin external preparation.
<8> The external preparation for skin according to <6> or <7>, which is for improving the skin barrier function.
<9> The external preparation for skin according to any one of <6> to <8>, which is used for promoting the formation of tight junctions and / or adherence junctions.
<10> The external preparation for skin according to any one of <6> to <9>, which is a cosmetic or a quasi drug.
<11> One or more selected from the compounds represented by the following formula, or a TRPV receptor activator comprising a plant extract containing an effective amount of the same compound as a TRPV receptor activator, A method for improving the skin barrier function, comprising a step of applying to a site where the skin is required.

(In the formula, the hydroxyl group may be substituted with a methoxy group or an ethoxy group, and the methoxy group may be substituted with a hydroxyl group or an ethoxy group.)
<12> An extract of a plant containing one or more compounds selected from the compounds represented by the following formula for TRPV receptor activation, or an effective amount of the same compound as a TRPV receptor activator.

(In the formula, the hydroxyl group may be substituted with a methoxy group or an ethoxy group, and the methoxy group may be substituted with a hydroxyl group or an ethoxy group.)

According to the present invention, a novel TRPV receptor activator and a skin external preparation containing the activator can be provided.

It is a figure which shows TJ and / or AJ formation promotion effect of TRPV4 activator (4 (alpha) -PDD) by TER value measurement. It is a figure which shows TJ and / or AJ formation promotion effect of TRPV4 activator (4α-PDD) by FITC-Dextran permeation amount measurement. It is a figure which shows TRPV4 knockdown efficiency by si-RNA transfection. It is a figure which shows the expression level of the cell adhesion related gene of a normal human newborn foreskin epidermal keratinocyte (NHEK) by TRPV4 knockdown. It is a figure which shows the time-dependent change of the TER value in a TRPV4 knockdown cell. It is a figure which shows the result of the FITC-Dextran permeation | transmission amount measurement in a TRPV4 knockdown cell. It is a figure (photograph) which shows the result of the immunoprecipitation of TRPV4 and AJ related protein, and an immunoblot. It is a figure (photograph) which shows the change of the amount of active Rho by the temperature change in NHEK, and activation of TRPV4. It is a figure (photograph) which shows the influence of the temperature change to the localization of AJ related protein in NHEK, and TRPV4 activation. It is a figure (photograph) which shows the influence of the temperature change and TRPV4 activation to the localization of TJ related protein in NHEK. It is a figure which shows the influence of the temperature change and TRPV3,4 activation to the time-dependent change of TER value in NHEK. (A) It is a figure which shows the influence of the temperature change and TRPV4 activation to the time-dependent change of the barrier function recovery rate in human skin tissue. (B) A diagram (photograph) showing temperature changes in human skin tissue after barrier destruction and changes in biotin permeability due to activation of TRPV3, 4. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of the compound 1. FIG. It is a figure which shows the increase effect (control) of the intracellular calcium ion concentration of the compound 1. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of the compound 3. FIG. It is a figure which shows the increase effect (control) of the intracellular calcium ion concentration of the compound 3. It is a figure which shows the result of the TER value measurement (TJ and / or AJ shape promotion effect) of compound 1. It is a figure which shows the result of TER value measurement (TJ and / or AJ shape promotion effect) of compound 3. It is a figure which shows the result of the TER value measurement (TJ and / or AJ shape promotion effect) of compound 4. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of 4 (alpha) -PDD in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of the Asenya extract of this invention in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the Crape myrtle extract of this invention in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of the Asenya extract of this invention in a Mock cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the Crape myrtle extract of this invention in a Mock cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the Acacia yak extract of this invention in the extracellular calcium ion free environment in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the Coleoptera extract of this invention in the extracellular calcium ion free environment in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium concentration of the Asenya extract of this invention in the extracellular calcium ion free environment in a Mock cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the Acacia yak extract in an extracellular calcium ion free environment and a calcium presence environment with respect to a mouse | mouth TRPV4 expression cell. It is a figure which shows the result of the TER value measurement (TJ and / or AJ shape promotion effect | action) of the plant extract of this invention. It is a figure which shows the result of the FITC-Dextran permeation | transmission amount measurement (TJ and / or AJ function improvement effect) of the plant extract of this invention.

<TRPV receptor activator of the present invention>
Nine types of temperature-sensitive receptors (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, and TRPA1) that are opened by a temperature stimulus from a low temperature to a high temperature are known as TRP channels. Of these, TRPV1, TRPV3, and TRPV4 have been reported to be present in epidermal cells. TRPV is a molecular entity of a transmembrane calcium ion channel, and is considered to be deeply involved in the skin barrier function by adjusting the intracellular calcium ion concentration. In particular, it is suggested that TRPV1 and TRPV4 are deeply involved in moisture transpiration and skin barrier function. However, the mechanism of action has not been elucidated in detail. The TRPV receptor activator of the present invention is a receptor activator that acts directly or indirectly on TRPV. The TRPV receptor activator of the present invention is a receptor activator that acts on TRPV4. Moreover, the TRPV receptor activator of the present invention has an action of activating the receptor. The TRPV receptor activator of the present invention adjusts the concentration of calcium ions in intercellular lipids and epidermal cells, and improves the skin barrier function. Moreover, the TRPV receptor activator of the present invention exerts a TJ and / or AJ formation promoting action by directly or indirectly acting on TRPV4 and improves the skin barrier function. That is, the TRPV receptor activator of the present invention is a substance that exerts a TJ and / or AJ formation promoting action through the TRPV4 activation action, and may exhibit an additive or synergistic skin barrier function improving action. Be expected. In addition, the TRPV receptor activator of the present invention may have an effect other than the TRPV receptor activation effect. In the present invention, a TRPV receptor activator can be used even if it has an effect other than TRPV receptor activation as long as the TRPV receptor activation effect is not hindered.

The TRPV receptor activator of the present invention comprises one or more selected from the compounds represented by the following formula, or a plant extract containing an effective amount of the same compound as a TRPV receptor activator. is there.

In the above formula, the hydroxyl group may be substituted with a methoxy group or an ethoxy group, and the methoxy group may be substituted with a hydroxyl group or an ethoxy group.

Preferred examples of the compound include 3-O-methylellagic acid 4′-O-α-L-rhamnopyranoside (compound 1), 3,3′-di-O-methylellagic acid 4′-O-β-D-glucopyranoside ( Compound 2), 3,4,3′-tri-O-methylellagic acid 4′-O-β-D-glucopyranoside (Compound 3), or 3′-O-methyl-3,4-methylenedioxyellagic acid 4 '-O-β-D-glucopyranoside (Compound 4).

Preferred examples of the compound include a chemical substance purified and isolated from a plant containing the compound, and an extract containing an effective amount of the compound derived from animals and plants as a TRPV receptor activator. Only seeds can be contained, or two or more kinds can be contained in combination. Here, the animal or plant-derived extract of the present invention is an animal or plant-derived extract, specifically, the extract itself, a fraction of the extract, a purified fraction, an extract or a fraction, purification. This is a general term for the product from which the solvent is removed. Among these components, preferred examples include extracts obtained from plants belonging to the genus Cranaceae and Rubiaceae, and more preferable are extracts of Cranalis genus C., Rubiaceae ganbi- A plant extract obtained from the above can be suitably exemplified. The giant crape myrtle is a evergreen small Takagi native to tropical Asia. In Japan, it is cultivated in Okinawa and Kyushu. In addition to being used as a diabetic drug, the leaf of the giant leafhopper is used to produce banaba tea by drying the leaves and used as a herbal medicine, a supplement, and the like. The Rubiaceae genus Gambir is a plant native to the coast of the Malacca Strait. The dry water extract obtained from Gambir leaves is called asenyaku (Asenyaku), which has anti-fibrinolytic activity and antithrombotic activity. The action, the antitumor action, etc. are known.

In the present invention, the extract obtained from the above-mentioned plant in Japan can be produced using a plant grown in Japan or sold in Japan as a herbal medicine raw material, etc. Commercial extracts sold by companies that handle plant extracts such as companies can be purchased and used. The plant part used for producing the extract obtained from the plant is not particularly limited, and whole grass can be used. Of course, the plant body, the above-ground part, the rhizome part, the tree trunk part, the leaf part, and the stem are used. It is also possible to use only parts such as parts, flower spikes and flower buds. At the time of extraction, it is preferable that a part used for extraction of a plant body or the like is processed in advance so as to improve extraction efficiency by crushing or chopping. In the production of an extract, 1 to 30 parts by mass of a solvent is added to 1 part by mass of a plant used for extraction of a plant or the like, and several days at room temperature or several at a temperature near the boiling point. Immerse for hours. After the immersion, the solution can be cooled to room temperature, insoluble matter can be removed if desired, and then the solvent can be removed by concentration under reduced pressure. Thereafter, the desired extract can be obtained by fractional purification by column chromatography packed with silica gel or ion exchange resin.
The above compound is obtained by subjecting the extract obtained as described above to a water extraction method or an organic solvent extraction method, if desired, and preparative purification using column chromatography packed with silica gel or an ion exchange resin. Can be obtained.

The extraction solvent is preferably a polar solvent, and alcohols such as water, ethanol, isopropyl alcohol, and butanol, and polyvalent alcohols such as 1,3-butanediol and polypropylene glycol. Preferred examples include one or two or more selected from ketones such as amino acids, acetone and methyl ethyl ketone, and ethers such as diethyl ether and tetrahydrofuran.

Moreover, since the structure of the said compound was clarified by this specification, you may manufacture the said compound based on a well-known chemical synthesis method. Compounds produced by chemical synthesis can also be used in the present invention.
The compounds represented by the above compounds 1 to 4 and derivatives thereof can be used as a skin barrier function improving agent in the form of a salt by treatment with a pharmacologically acceptable acid or base. For example, mineral salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, maleate, fumarate, oxalate, citrate, lactate, tartrate, methanesulfonate, para Organic acid salts such as toluene sulfonate and benzene sulfonate, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, triethylamine salt, triethanolamine salt, ammonium salt, Suitable examples include organic amine salts such as monoethanolamine salts and piperidine salts, and basic amino acid salts such as lysine salts and alginates.

As a method for evaluating the TRPV activation action of the TRPV receptor activator of the present invention, any method capable of evaluating the TRPV activation action can be applied without particular limitation. A method for evaluating the activation effect of TRPV using the calcium ion concentration as an index is preferred. Of the methods for evaluating the activation effect of TRPV, a method for evaluating the activation effect of TRPV4 is preferred. This is because TRPV4 has been confirmed to exist in epidermal cells and is expected to be involved in the skin barrier function. Among the methods for evaluating the activation effect of TRPV, it is more preferable that the activity of TRPV4 is measured using the intracellular calcium ion concentration in cells in which TRPV4 expression is confirmed, more preferably in cells overexpressing TRPV4 as an index. A method for discriminating the chemical action is preferred. As a method for measuring the intracellular calcium ion concentration as an index for discriminating the activation effect of TRPV4, any method can be used without particular limitation as long as it is a method for measuring the intracellular calcium ion concentration. From the aspects of simplicity, measurement sensitivity and accuracy, for example, Sokabe T, Tsujiuchi S, Kadowaki T, Tominaga M .: Drosophila painless is a Ca ²⁺ -requiring channel activated by noxious heat .: J Neurosci., 2008 Oct 1; 28 (40): 9929-38, preferably the calcium imaging method or the patch clamp method.

According to such a method for evaluating the activation effect of TRPV, the intracellular calcium ion concentration in cells in which TRPV4 expression has been confirmed or cells overexpressing TRPV4 is confirmed by the calcium imaging method or patch clamp method, and the like. When the degree of calcium ion concentration is increased in the presence of the test substance compared to the absence of the test substance (control), it is judged that the function of TRPV is activated, and this is applied to the skin in vivo. In this case, it is determined that the calcium ion permeation effect of TRPV in the stratum corneum or epidermal granule layer is promoted, and thus the skin barrier function is improved. This efficacy is preferably 100% in the presence of the test substance in the presence of the test substance, compared to 100% in the presence of the test substance, compared to the increase in intracellular calcium ion concentration in the absence of the test substance. When an increase in concentration of 20% or more, more preferably an increase of concentration of 30% or more is observed, it is determined to be effective. Moreover, it is discriminated that the improvement effect of the skin barrier function is remarkable as the degree is remarkable.

As for the increase rate of intracellular calcium ion concentration by the test substance relative to ionomycin (positive control), 3 or 5 cells whose intracellular calcium ion concentration increased by addition of the test substance were selected at random, and each test substance was selected. The initial value, ie, the calcium ion concentration before addition, is subtracted from the calcium ion concentration after addition to calculate the amount of increase in calcium ion concentration due to the test substance, and then the initial value, ie, addition, from the calcium ion concentration after addition of ionomycin (positive control). Calculate the increase in calcium ion concentration by the positive control by subtracting the previous calcium ion concentration, divide the increase in calcium ion concentration by the test substance by the increase in calcium ion concentration by the positive control, and then multiply by 100. Calculated as a value.

The cells used for evaluating the component having TRPV activation activity of the present invention can be applied without particular limitation as long as TRPV expression is confirmed, but from the viewpoint of measurement sensitivity and accuracy. More preferably, TRPV overexpressing cells are used. In particular, for the production of TRPV1 overexpressing cells, for example, according to the method described in JP-A-2009-082053, and for the production of TRPV4 overexpressing cells, TRPV4 overexpression is carried out by the method described below based on the above method. Cells can be made.

Regarding the cells in which the expression of TRPV4 used for the evaluation of the TRPV4 activation action in the present invention is confirmed, any cell expressing TRPV4 can be applied without particular limitation, and more preferable is a TRPV4 expression vector. Cells in which TRPV4 is overexpressed by introducing − into a host cell are preferred. Examples of host cells overexpressing TRPV4 include bacterial cells, plant cells, animal cells, insect cells, etc. More preferably, HEK293 cells, CHO cells, COS-7 cells, NIH3T3 cells and the like are suitable. It can be illustrated. The host cell preferably incorporates a TRPV4 expression vector, allows TRPV4 to be efficiently expressed, and is easy to culture. Further, in the preparation of a TRPV4 expression vector, a cDNA encoding TRPV4 can be used without any particular limitation. More preferred is a cDNA cell vector pcDNA3 (manufactured by Invitrogen). And a nucleic acid obtained by ligating a TRPV4-encoded nucleic acid. As the nucleic acid encoded by TRPV4, a nucleic acid encoded by all nucleotide sequences can be used, for example, an mRNA extracted and converted into cDNA by allowing reverse transcriptase to act on it, It is also possible to select a suitable primer for only the portion where the TRPV4 main function is coded, amplify it by PCR, and use this to ligate. Particularly preferred is the oligonucleotide shown in SEQ ID NO: 1, which can be obtained by subjecting the DNA or cDNA extracted using the primers of SEQ ID NO: 2 and SEQ ID NO: 3 to a PCR reaction. ,Obtainable.

The cells overexpressing TRPV4 that can be used for evaluation of the component having TRPV activating action of the present invention are characterized by the functional expression of TRPV4 in the cells, expression at the protein level, co-introduction of fluorescent substances, and the like. , You can choose. An appropriate selection medium can be used for selection of a cell that has been introduced into a host cell so that the nucleic acid encoding TRPV4 can be expressed in the cell. For example, a material obtained by adding a growth factor such as FBS to DMEM or RPMI medium can be suitably exemplified.

Examples of the medium used for culturing cells that overexpress TRPV4 include components suitable for growth of cells that overexpress TRPV4, such as glucose, amino acids, peptones, vitamins, cell growth promoting factors (for example, , Cell growth factors, hormones, binding proteins, cell adhesion factors, lipids), serum (eg, FBS, FCS, etc.), calcium chloride, magnesium chloride, etc. The medium may be a commercially available medium. The medium used for culturing cells overexpressing TRPV4 is not particularly limited as long as it is a medium suitable for such cells, and examples thereof include MEM medium, DMEM medium, RPMI 1640 medium, and the like. For example, when the host cell used is HEK293 cells, a DMEM medium containing high glucose and 10% by mass FBS is used.

The TRPV receptor activator in the present invention activates TRPV, particularly, TRPV4 present in epidermal cells, and thereby promotes the formation of TJ and / or AJ, thereby exhibiting a skin barrier function improving action. The interaction regarding the improvement of the skin barrier function between TRPV (particularly TRPV4) and TJ and / or AJ of the cell-cell adhesion structure is confirmed by the following test results. That is, when the TRPV4 activation by TRPV4 ligand 4α-PDD (4α-phorbol ester) is confirmed to have the same action as TJ and / or AJ formation promoting action, it has the TRPV4 activation action. It can be said that the component exhibits the skin barrier function improving action by the TJ and / or AJ formation promoting action.

As a method for evaluating the TJ and / or AJ formation promoting action by the TRPV receptor activator of the present invention, for example, a biotin-labeled diffusion train using a three-dimensional epidermis model or living skin described in Non-Patent Document 3 is used. -Confirmation of intercellular mass transfer using a sensor, FITC-dextran intercellular substance permeation test using a cultured cell sheet, method of estimating the function of strands by TJ strand observation by freeze fracture method, Furthermore, a method for measuring transepithelial electric resistance (TER) described in JP-A-2007-174931 can be exemplified, but from the viewpoint of simplicity of operation and measurement accuracy, FITC- The dextran permeability test method and the method of measuring the transepithelial cell electrical resistance value (TER value) can be preferably exemplified.

According to the method for evaluating the action of promoting the formation of TJ and / or AJ, the degree of intercellular mass transfer in the epidermal keratinized cell layer membrane constructed on the support is determined based on the intercellular substance permeability or transepithelial cell electrical resistance value. (TER value) is measured, etc., and the degree of intercellular mass transfer is suppressed in the presence of the test substance compared to the absence of the test substance (control), or TER is in the absence of the test substance When increased in the presence of the test substance compared to (control), it is judged that the TJ and / or AJ of the epidermal keratinocyte layer membrane has been densely constructed, and as a result applied to the in vivo skin It is determined that the substance permeation suppressing action of the stratum corneum or the epidermal granule layer is improved, thereby improving the skin barrier function.

<External skin preparation containing TRPV receptor activator of the present invention>
The external preparation for skin of the present invention is characterized by containing a TRPV receptor activator as an essential component. The TRPV receptor activator in the present invention is a receptor activator that acts directly or indirectly on TRPV. Of the TRPV receptor activators, a TRPV4 receptor activator is a preferred example. In addition, the TRPV receptor activator of the present invention has an effect of activating TRPV, particularly, TRPV4. The TRPV receptor activator of the present invention may be any of purified and isolated chemical substances, extracts derived from animals and plants, mixed purified products such as fractionated purified products thereof. The skin external preparation of the present invention may contain only one TRPV receptor activator or a combination of two or more thereof. The skin external preparation of the present invention activates TRPV by blending a TRPV receptor activator, promotes the formation of TJ and / or AJ, and exhibits an effect of improving (improving) the skin barrier function.
Here, the skin (between cells) barrier function improving effect is a barrier function of skin (or between skin cells) [for example, transdermal moisture transpiration suppression function (function to prevent moisture transpiration from skin) and substances This is an effect of improving the permeability suppression function (function of preventing the invasion of a substance into the skin, etc.), and can also be referred to as a rough skin prevention or improvement effect.

The TRPV receptor activator of the present invention is 0.00001% by mass to 10% by mass, more preferably 0.0001% by mass to 5% by mass, and still more preferably, as the total amount of the compound with respect to the total amount of the external preparation for skin. It is preferable to contain 0.001 to 3% by mass. This is because when the content of the TRPV receptor activator is too small, the TRPV activation action, and the skin barrier function improving action through the TJ and / or AJ formation promoting action by TRPV activation are not exhibited, This is because even if the amount is too large, the effect reaches a limit and the degree of freedom of the system may be impaired. In addition, since such components are excellent in TRPV, in particular, action on epidermal cells where TRPV4 is present, accumulation and selectivity at target sites, and high safety and stability, pharmaceuticals, quasi drugs, Use in foods, cosmetics and the like is preferable, and cosmetics or quasi drugs are more preferable.

The external preparation for skin of the present invention can contain optional components usually used in medicines, quasi drugs, foods, and cosmetics, in addition to the essential components. For cosmetics, such optional ingredients include hydrocarbons such as squalane, petrolatum and microcrystalline wax, jojoba oil, carnauba wax, esters such as octyldodecyl oleate, olive oil, beef tallow, Triglycerides such as coconut oil, fatty acids such as stearic acid, oleic acid and retinoic acid, lower alcohols such as ethanol and isopropanol, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, sulfosucci Anionic surfactants such as acid esters and sodium polyoxyethylene alkyl sulfates, amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, and polyoxyethers thereof. Non-ionic surfactants such as lend adduct, polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyethylene glycol, dipropylene glycol, glycerin, Contains polyhydric alcohols such as 1,3-butanediol, 1,2-pentanediol, thickening / gelling agents, antioxidants, UV absorbers, colorants, preservatives, powders, etc. be able to. The external preparation for skin of the present invention can be produced without difficulty by treating these components according to a conventional method except that the TRPV receptor activator of the present invention is contained.

The skin external preparation of the present invention can be produced by treating these essential components and optional components according to a conventional method and processing them into lotions, emulsions, essences, creams, pack cosmetics, cleansing agents and the like. As long as the dosage form can be applied to the skin, any dosage form is possible, but since the active ingredient penetrates the skin and exerts its effect, the lotion and emulsion that are well-familiar with the skin , Cream, essence and the like are more preferable.

Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.

<Test Example 1: TER value measurement test using TRPV receptor activator>
Frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.) are thawed and cultured at 37 ° C. with 5% CO 2 in 0.15 mM-calcium ion-containing culture solution (Humdia-KG2: Kurashiki Boseki Co., Ltd.). Culturing was performed under a carbon stream. Set Transwell (Corning, 3460-Clear) on a Millicell Tissue Culture Plate (Millipore), place 0.5 mL of the upper layer and 1.5 mL of the lower layer, and add 2 NHEKs to the upper Transwell well. The cells were seeded at 5 × 10 ⁵ cells / cm ² and cultured at 37 ° C. in a 5% carbon dioxide stream for 24 hours. After confirming that NHEK had grown to 100% confluence, it was replaced with a 1.5 mM calcium chloride-added Humeria-KG2 medium, and cultured at 33 ° C. in a 5% carbon dioxide stream for 48 to 72 hours to form keratinized skin. A cell layer membrane was constructed. Then, 10 mM Mα α-PDD (manufactured by Sigma), 3 mM camphor (manufactured by Sigma) or methanol (vehicle, manufactured by Wako Pure Chemical Industries, Ltd.) and 1.5 mM calcium chloride-added Humdia-KG2 medium, respectively. Then, the cells were cultured in a TRPV4 / TRPV3 inert temperature region at 24 ° C. in a 5% carbon dioxide stream. The TER value was measured after 0, 3, 6, and 9 hours after changing to the medium containing each substance. The TER value was measured using Millicell ERS (manufactured by Millipore) after the sample was conditioned for 30 minutes in a clean bench. The results are shown in FIG.

Specific expression of TRPV3 and TRPV4 has been confirmed in epidermal cells that are deeply involved in the skin barrier function. From the results of FIG. 1, in the TRPV4 inactive temperature region, NHEK cultured in the presence of TRPV4 ligand 4α-PDD has a statistically significant increase in TER value as compared to NHEK cultured in the absence. Was confirmed. On the other hand, in NHEK cultured in the presence of camphor of the TRPV3 ligand, an increase in TER value was not observed as compared with the absence. It was shown that the increase in the TER value, that is, the action of improving the skin barrier function by promoting TJ and / or AJ formation is exhibited by specific activation of TRPV4.

<Test Example 2: FITC-Dextran substance permeation test using TRPV receptor activator>
In the TER value measurement test, the medium of the sample 9 hours after the medium exchange used for measurement was removed. 0.5 mL of Apical Buffer (1.45 mM CaCl ₂ , 10 mM glucose, PBS containing 1 mg / mL FITC-Dextran) in the upper layer of the Transwell, and 1.5 mL of Basolatal Buffer (1.45 mM CaCl ₂ , 10 mM glucose in the lower layer) PBS containing was added and incubated for 3 hours. After collecting the basic buffer, a standard curve group (100 mg / mL to 0 mg / mL) is prepared on an 96-well plate using an Apical Buffer. The recovered basal buffer was added to each 96 wells in 200 mL, and then measured with a spectrophotometer (excitation 485/535 nm). The results are shown in FIG.

From the results shown in FIG. 2, in the TRPV4 temperature inactivated region, NHEK cultured in the presence of TRPV4 ligand 4α-PDD is statistically significant compared to NHEK cultured in the absence. Suppression of permeation amount was confirmed. On the other hand, in the presence of TRPV3 ligand camphor, no inhibition of FITC-Dextran permeation was observed. It was shown that the suppression of FITC-Dextran permeation, that is, the action of improving the skin barrier function by promoting the formation of TJ and / or AJ is exhibited by specific activation of TRPV4.

<Test Example 3: si-RNA transfection test>
Frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.) are thawed and cultured until they become sub-confluent, then detached and collected with trypsin EDTA, and 7.5 × 10 ⁵ cells / cell in a 6-well plate. The cells were seeded in a well and cultured at 37 ° C. in a 5% carbon dioxide stream for 2 hours. In addition, si-RNA transfection (volume is described for 1 well) was performed according to the following procedure. In 500 μL Opti-MEM, 5 μL of 10 μM si-RNA (On TargetTplus SMARTpool L-004195-00-0005, Human TRPV4, NM 147204, Target Sequence: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 And then 15 μL of HiperFect was added and mixed by vortexing, and then allowed to stand at room temperature for 10 minutes. After dripping into the well and diffusing gently, culturing at 37 ° C. under a 5% carbon dioxide stream for 24 hours, then replacing with 1.5 mM calcium chloride-Humdia-KG2 medium, and the TER value and FITC-Dextran substance over time Permeability was measured. Moreover, the TRPV4 knockdown efficiency by si-RNA was determined by performing RT-PCR using Hs TRPV4 1 SG QuantiTect Primer Assay (Qiagen: QT00077217, sequence not disclosed) as a primer to determine the expression level of TRPV4 mRNA. confirmed. The results are shown in FIGS. These si-RNA transfection tests were performed using a commercially available si-RNA ON-TARGET plus SMART pool Human TRPV4 (manufactured by Thermo scientific).

From the results shown in FIGS. 3 and 4, the expression of TRPV4 mRNA was suppressed with extremely high knockdown efficiency by si-RNA transfection in NHEK, and the inhibitory action was specific to TRPV4. In addition, from the results of FIGS. 5 and 6, it was confirmed that NHEK (TRPV4 / KD) in which TRPV4 was knocked down compared to NHEK had a statistically significant suppression of TER value. Furthermore, TRPV4 / KD was confirmed to have a statistically significant increase in FITC-Dextran permeability.

The results of the above tests 1 to 3 indicate that activation of TRPV, especially, TRPV4 promotes the formation of TJ and / or AJ and improves the skin barrier function. Thus, a component having a TRPV receptor activating action exerts a skin barrier function improving action through a TJ and / or AJ formation promoting action and is useful as a skin barrier function improving agent.

<Test Example 4: Expression of TRPV4 in human normal epidermal keratinocytes>
In order to confirm the presence of TRPV4 protein and TRPV4 binding molecule in human normal epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.), immunoprecipitation was performed. As the antibody, polyAb anti-β-catenin (Chemicon), mAb anti-E-cadherin (Takara Bio) and polyAb anti-TRPV4 (prepared against the N-terminal peptide of TRPV4) were used.
The results are shown in FIG. In the left lane, NHEK membrane fraction was immunoprecipitated (IP) using an anti-β-catenin antibody, and a sample of the resulting complex was separated and immunoblotted (IB) using an anti-N-terminal TRPV4 antibody. Is. Black triangles indicate the position of TRPV4 co-precipitated with β-catenin. The right lane is replotted with anti-E-cadherin antibody. Black triangles indicate E-cadherin. That is, TRPV4 was coimmunoprecipitated with β-catenin and E-cadherin present in the cell membrane. β-catenin and E-cadherin are the main constituent proteins of AJ, suggesting that TRPV4 is present as part of the AJ complex in human keratinocytes.

<Test Example 5: Effect of temperature change and TRPV4 activation on differentiation process in human normal epidermal keratinocytes>
The Rho family of GTPases has been reported to play an important role in the differentiation of epidermal keratinocytes. The Rho family is activated by increasing intracellular calcium ion concentration and mediates keratinocyte differentiation including actin formation, intercellular adhesion formation and superficial actin formation. When activated, the TRP ion channel causes calcium ions to flow from outside the cell into the cell and promotes an increase in the intracellular calcium ion concentration. Thus, the expression level of active Rho was evaluated in NHEK cultured at 33 ° C. where TRPV3 and TRPV4 are activated and 28 ° C. below the activation temperature threshold.
After induction of differentiation by the calcium ion switch, NHEK was cultured at 33 ° C. or 28 ° C. for 24 hours. Thereafter, NHEK cultured at 28 ° C. was treated with 10 μM 4α-PDD or 3 mM camphor and cultured for 8 hours. For each of these NHEKs, the expression level of active Rho was evaluated using Western blotting.
The results are shown in FIG. The expression level of active Rho is shown in the upper panel. 15 μg of total protein was loaded as input (lower panel).
The expression of the active form of NHEK cultured at 28 ° C. was lower than the expression of the active form of NHEK cultured at 33 ° C. The expression level of active Rho at 28 ° C. was remarkably increased by the addition of 4α-PDD, which is a TRPV4 activator, but not increased by camphor, which is a TRPV3 activator.
This indicates that below the activation temperature threshold of TRPV3 and TRPV4 is insufficient to enhance Rho activity, but is specifically compensated by chemical activation of TRPV4, and TRPV4 mediated intracellular calcium It was suggested that ion influx is involved in Rho activation.

Next, compare the localization of AJ and TJ related proteins (E-cadherin, β-catenin, actin and occludin) in NHEK cultured at 33 ° C or 28 ° C or chemically activated TRPV4 and TRPV3 at 28 ° C In order to do this, immunofluorescence staining was performed.
The results are shown in FIG. After induction of differentiation by the calcium ion switch, NHEK was cultured at 33 ° C. or 28 ° C. for 24 hours. Thereafter, NHEK cultured at 28 ° C. was treated with 10 μM 4α-PDD or 3 mM camphor. These NHEK intercellular adhesion formation processes were confirmed over a time course (0-48 hours). Actin (green in the original image) surrounding the cell membrane, β-catenin (purple in the original image) and E-cadherin (red in the original image) present at the cell-cell junction are shown in an enlarged image. White arrows indicate the “zipper structure” of AJ consisting of β-catenin and E-cadherin.
Both NHEKs cultured at 33 ° C and 28 ° C 4 hours after differentiation induction by the calcium ion switch are characterized by the localization of AJ-related proteins β-catenin and E-cadherin along the cell membrane margin. “Zipper structure” was shown, suggesting that the initial cell-cell adhesion was formed regardless of the culture temperature. After 24 and 48 hours at 33 ° C., the cells layered together, and the surrounding surface actin clearly showed a honeycomb-like network. In contrast, cell-cell adhesion at 28 ° C. remained at the initial formation stage even after 24 and 48 hours. This immature differentiation was restored by the addition of 4α-PDD but not with camphor.

Furthermore, 48 hours after differentiation induction by the calcium ion switch, immunofluorescence staining of occludin, a TJ-related protein, was performed to confirm its localization. As the antibody, mAb anti-Occludin (manufactured by Invitrogen) was used.
The results are shown in FIG. One of the TJ-related proteins, occludin (red in the original figure), was localized along the intercellular adhesion in NHEK cultured at 33 ° C. 48 hours after differentiation induction, whereas it was 28 ° C. In, the localization of occludin at the cell-cell junction was intermittent. As in the case of the AJ-related protein, occludin was continuously localized along the cell adhesion site by 4α-PDD at 28 ° C., but not at camphor.
When the expression levels of AJ and TJ-related mRNAs were confirmed by the RT-PCR method, changes in the expression levels of these mRNAs due to the addition of 4α-PDD were not confirmed.
From these results, TRPV4 activation does not contribute to the expression level of AJ and TJ-related molecules, but promotes the formation of AJ structure during the differentiation process of human normal epidermal keratinocytes and consequently promotes TJ formation. It was suggested.

<Test Example 6: Effect on barrier function in human keratinocytes and human skin tissue by temperature change and TRPV4 activation>
The barrier function in the skin is roughly divided into a stratum corneum barrier function and an epidermal cell (TJ) barrier function. In this study, in order to evaluate whether the TEK barrier function of NHEK is affected by temperature and TRPV4 activation, the transepithelial electrical resistance (TER) value used as an index when evaluating the TJ barrier function is expressed by Millicell- Measurements were made using ERS (Millipore).
NHEK subjected to differentiation induction by a calcium ion switch was cultured at 33 ° C. (◇) or 28 ° C. (□), and after 24 hours from the start of culture, NHEK cultured at 28 ° C. was added to 4α-PDD (Δ) or camphor (○ ) Was added to enhance TRPV4 and TRPV3 activation.
The results are shown in FIG. The arrow indicates the timing of enhancement. Data are the mean ± SEM of 5 independent experiments. D. It shows with. (**: P <0.01, NS: not significant.) After performing a two-way analysis of variance with repeated measurements, a significant difference test was performed by the Bonferroni method. A value of P <0.05 was considered significant.
The TER value of NHEK cultured at 28 ° C. was significantly lower than that cultured at 33 ° C., supporting the result of the occludin fragmented immunofluorescence signal at 28 ° C. (FIG. 10). As shown in Test Example 1 above, the TER value of NHEK enhanced at 28 ° C. was significantly enhanced by 4α-PDD compared with no TRPV activator, but not enhanced by camphor.
These suggested that the TJ barrier function of human keratinocytes decreases below the TRPV4 activation temperature threshold, but is overcome by specifically activating TRPV4 by enhancement.
Furthermore, in order to confirm that the change in TJ barrier function as described above is a phenomenon specifically caused by the involvement of TRPV4, TREK4 knockdown of NHEK using si-RNA was performed. The TER value of NHEK in which TRPV4 was knocked down was significantly reduced compared to NHEK (Mock) transfected with control si-RNA (FIG. 5).
These facts suggested that the TJ barrier function of human keratinocytes is reduced by suppressing the expression of TRPV4.

Furthermore, the effect of temperature on the recovery of skin barrier function after stratum corneum barrier destruction was evaluated by ex vivo evaluation using fresh human skin tissue. The human skin tissue used at this time was the skin tissue (Biopredic and Juntendo Urayasu Hospital) excised by surgical operation after obtaining the consent of the patient through informed consent. These human skin tissues are pre-cultured using Skin Long Term Culture Medium (manufactured by Biopredic), and then stripped with C40SH02 adhesive tape (manufactured by Seikagaku Corporation) to completely remove the stratum corneum. As a result, the barrier was destroyed. Immediately after barrier destruction by removal of the stratum corneum, 10 μM 4α-PDD, an aqueous solution each containing 3 mM camphor, or 50 μl of water alone is directly administered to the site of human skin tissue where the stratum corneum is removed, and cultured at 33 ° C. or 28 ° C. did. To restore skin barrier function, measure transepidermal water transpiration (TEWL) with VAPO SCAN AS-VT100RS (Asahi Techno Lab) and apply it to the following formula to calculate skin barrier function recovery rate (%). ,evaluated:
(TEWL immediately after stratum corneum barrier breakdown−TEWL at each measurement point) / (TEWL immediately after stratum corneum barrier breakdown−TEWL before stratum corneum barrier breakdown) × 100
Further, 2% EZ-link sulfo-NHS-LC-biotin (Pierce) was injected into the skin as an intercellular substance permeation marker, and the intercellular permeability of the marker was evaluated by immunofluorescence staining.
The results are shown in FIG. (A) shows the barrier recovery rate of the skin tissue at 1.5, 4 and 24 hours after barrier destruction. Each label indicates the following. ◇: 33 ° C. water administration, □: 28 ° C. water administration, Δ: 28 ° C. 4α-PDD administration, ○: 28 ° C. camphor administration. Data are the mean ± SEM of 3 independent experiments. D. It shows with.
The recovery rate of the barrier function of the skin tissue when cultured at 33 ° C. and 28 ° C. for 24 hours after the barrier destruction is clearly reduced in the skin tissue cultured at 28 ° C. compared to the skin tissue cultured at 33 ° C. It was. The barrier recovery rate of skin tissue treated with 4α-PDD or camphor was compared with skin tissue cultured at 28 ° C., and skin tissue treated with 4α-PDD showed higher recovery than skin tissue cultured at 28 ° C. The skin tissue treated with camphor showed a lower recovery rate than the skin tissue cultured at 28 ° C.
These results are also supported by the evaluation of intercellular substance permeation described below.

The results are shown in FIG. (B) shows an immunofluorescent image of a cross section of skin tissue 24 hours after destruction of the stratum corneum barrier. The arrow indicates occludin (green in the original figure), which is one of the major TJ-related proteins localized in the epidermal granule layer. A white triangle indicates a substance permeation marker (red in the original drawing) that has passed through the position of occludin.
In the skin tissue cultured at 33 ° C., permeation of the substance permeation marker was blocked at the outermost layer of the epidermal granular layer where occludin was localized. On the other hand, in the skin tissue cultured at 28 ° C., the substance permeation marker leaked upward from the outermost layer of the epidermal granule layer where occludin was localized. Leakage of the substance permeation marker at 28 ° C. was suppressed by 4α-PDD, but not by camphor, indicating that intercellular adhesion, particularly TJ strength, depends on activation of TRPV4.
Previous reports by the inventors have shown that epidermal TJ has an important role in stratum corneum barrier formation by imparting polarity to keratinocytes and promoting lamellar body secretion. Therefore, it is suggested that the activation of TRPV4 regulates not only the TJ barrier function but also the stratum corneum barrier function.
From the above results, that is, at a low temperature below the TRPV4 activation temperature threshold, it was shown that recovery of the skin barrier is delayed, but is specifically compensated by activation of TRPV4.

[Example 1]
<Isolation and purification of compounds 1 to 4 from the giant crape myrtle>
The plant extract obtained from the genus Coleoptera can be produced according to the quasi-drug raw material standard 2006, or a plant extract commercially available from Maruzen Pharmaceutical Co., Ltd., Yamakawa Trading Co., Ltd., etc. can be purchased. Can also be used. In the present example, a plant extract obtained from Yamakawa Trading Co., Ltd. and obtained from the genus Coleoptera was cultivated.
After concentrating the plant extract (9950 g, manufactured by Yamakawa Trading Co., Ltd.) obtained from the crape myrtle family, the mixture is divided into liquid and liquid, and the resulting aqueous layer fraction is diaionized. The column adsorbing part was sequentially eluted with 50% methanol and 100% methanol (manufactured by Wako Pure Chemical Industries, Ltd.) through HP-20 (manufactured by Mitsubishi Chemical Corporation). The substance peak was isolated by preparative HPLC with respect to 100% methanol elution (745 mg) out of the elution part. The sorting conditions are as follows.

<Preparative HPLC conditions>
Compound 1,3
[Column] Inertsil ODS-3 (GL Sciences Inc.) 5μm φ30 x 500 mm
[Solvent] Gradient, liquid A CH ₃ CN, liquid B 0.1% TFA in H ₂ O, 0-300 min: 15-25% A, 300-360 min: 25% A
[Flow rate] 9.5 mL / min, detection: UV 240 nm, 1 Fr. = 3 min.

Compound 2
[Column] Develosil C30-UG-5 (5μm) φ20 x 250 mm
[Solvent] 25% CH ₃ CN + 0.05% TFA
[Flow rate] 6.5 mL / min, detection: UV 240 nm, 1 Fr. = 4 min.

Compound 4
[Column] TSKgel ODS-80Ts (5 μm) 4.6 mm ID x 25 cm (Tosoh Corporation)
[Solvent] Gradient, Liquid A 0.1% TFA in H ₂ O, Liquid B: CH ₃ CN, 0-80min B10 ~ B90%, 80-110min B90%
[Flow rate] 1.0mL / min, Temperature: 40 ℃, Detection: UV210-400nm (Max)

The chemical structures and physical constants of compounds 1 to 4 isolated and purified according to the above procedure are shown below. The chemical structures of Compounds 1 to 4 were determined based on nuclear magnetic resonance ( ¹ H and ¹³ C-NMR) spectra and literature information.

<Compound 1: 3-O-methylellagic acid 4'-O-α-L-rhamanopyranoside>

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 1.16 (3H, d, J = 6.5 Hz), 3.34 (1H, m), 3.54 (1H, m), 3.73 (1H, m), 3.98 ( 1H, m), 4.07 (3H, s), 5.56 (1H, br s), 7.48 (1H, s), 7.79 (1H, s).
¹³ C-NMR (400 MHz, DMSO-d ₆ ) δ: 17.9, 61.5, 70.1, 70.3, 70.5, 71.6, 99.9, 107.9, 110.5, 111.5, 111.7, 112.3, 114.3, 136.5, 139.7, 141.3, 141.9, 148.8 150.2, 158.5, 158.8.

<Compound 2: 3,3′-di-O-methylellagic acid 4′-O-β-D-glucopyranoside>

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 3.20-3.45 (4H, m), 3.53 (1H, m), 3.73 (1H, m), 4.01 (3H, s), 4.05 (3H, s ), 5.02 (1H, d, J = 7.5 Hz), 7.67 (1H, s), 7.82 (1H, s).

<Compound 3: 3, 4, 3'-tri-O-methylellagic acid 4'-O-β-D-glucopyranoside>

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 3.20-3.45 (4H, m), 3.52 (1H, m), 3.71 (1H, m), 4.02 (3H, s), 4.06 (3H, s ), 4.11 (3H, s), 5.16 (1H, d, J = 7.5 Hz), 7.67 (1H, s), 7.85 (1H, s).

<Compound 4: 3'-O-methyl-3,4-methylenedioxyellagic acid 4'-O-β-D-glucopyranoside>

¹ H-NMR (400 MHz, DMSO-d ₆ ) δ: 3.19 (1H, m), 3.35 (1H, m), 3.38 (1H, m), 3.44 (1H, m), 3.52 (1H, m), 3.71 (1H, br d, J = 11.5Hz), 4.11 (3H, s), 5.16 (1H, d, J = 7.5Hz), 6.40 (2H, s), 7.57 (1H, s), 7.86 (1H, s).

[Example 2]
<Evaluation test of intercellular barrier function of compounds 1 to 4>
Compounds 1 to 4 purified according to the above procedure were subjected to a TRPV4 activating effect evaluation test by calcium imaging. The results for compounds 1 and 3 are shown in FIGS.

In a test for evaluating TRPV4 activation by calcium imaging, which was performed according to the test method described in Example 5 below, compounds 1 and 3 of the present invention were added to calcium-containing solution A [composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl. ₂ ) When treated with mouse TRPV4 / pcDNA-introduced HEK293 cells (hereinafter referred to as mV4 / HEK293) in ₂ mM CaCl ₂ , 10 mM glucose, 10 mM HEPES (pH 7.4)], there is a marked increase in intracellular calcium ion concentration. It was confirmed (FIGS. 13 and 15). On the other hand, changes in intracellular calcium ion concentration were not confirmed in the pcDNA-introduced HEK293 cells (hereinafter, pcDNA / HEK293) as a control (FIGS. 14 and 16).
From the above, it can be inferred that the intracellular calcium ion concentration increasing action by the compound of the present invention is due to TRPV4-specific extracellular calcium ion influx.

Further, the results of the intercellular barrier function evaluation test of normal human epidermal keratinocytes by TER measurement performed on the compounds 1, 3, and 4 according to the test method described in Test Example 1 are shown in FIGS. . The vertical axis represents the TER value (Ω · m ² ), and the horizontal axis represents the TER measurement time after differentiation induction by the calcium switch. From these results, the compound of the present invention showed a marked increase in TER value as compared with the control DMSO-added group, and the effect of promoting the intercellular barrier function was recognized.

[Example 3]
<The manufacturing method of the plant extract which has the TRPV receptor activation effect | action of this invention>
The plant extract of the present invention (the extract obtained from the honey beetle genus Coleoptera or Rubiaceae genus Gambir) can be produced according to the quasi-drug raw material standard 2006, or is commercially available from Maruzen Co., Ltd. It is also possible to purchase and use plant extracts. In this example, a plant extract purchased from Maruzen Co., Ltd. was used.

[Example 4]
<Preparation of cells expressing human TRPV4>
Human TRPV4-expressing cells were prepared according to the following experimental procedure. PCR was performed using cDNA encoding human TRPV4 [SEQ ID NO: 1 (GenBank accession number: NM 021625) 90 bp to 2705 bp polynucleotide] and oligonucleotides SEQ ID NO: 2 and SEQ ID NO: 3 as primers. The product was amplified and inserted into the MCS BamHI and XhoI sites of the product name: pcDNA3 (manufactured by Invitrogen), which is a vector for mammalian cells, to obtain a human TRPV4 expression vector. Human TRPV4 expression vector obtained (corresponding to 0.5 μg) and DsRed (corresponding to 0.1 μg), positive reagent (trade name, catalog number: 11514-015, manufactured by Invitrogen) 6 μL, OPTI-MEM (registered) (Trademark) I Reduced-Serum Medium (catalog number: 11058021, manufactured by Invitrogen) 100 μL was mixed to obtain a mixture 1. Further, 4 μL of Lipofectamine (registered trademark, catalog number: 18324-012, manufactured by Invitrogen) and 100 μL of OPTI-MEM were mixed to obtain a mixture 2. On the other hand, HEK293 cells (5 × 10 ⁵ cells / 35 mm diameter dish) were cultured in DMEM medium containing 10% by mass FBS to 37% confluence at 37 ° C. in a 5% carbon dioxide stream. Thereafter, the mixture of the mixture 1 and the mixture 2 was added to the obtained cells. Thus, the human TRPV4 expression vector was introduced into HEK293 cells to obtain TRPV4-expressing cells. Further, in the same manner, a vector without human TRPV4 inserted was prepared and introduced into HEK293 cells to prepare Mock cells.

[Example 5]
<Evaluation of TRPV Activation Action 1: Evaluation of TRPV4 Activation Action by Calcium Imaging Method of Plant Extract in the Present Invention>
The plant extract of the present invention was added to solution A [composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ , 2 mM CaCl ₂ , 10 mM glucose, 10 mM HEPES (pH 7.4)] to prepare a test solution. The concentration of the plant extract in each test solution was 0.1% by mass. Incubation of TRPV4-expressing cells or Mock cells prepared according to Example 4 in a DMEM medium containing 10% FBS containing 1-20 μg / ml FURA 2-AM (manufactured by Invitrogen) for 60-90 minutes ( FURA 2-AM was introduced into TRPV4-expressing cells or Mock cells. TRPV4-expressing cells or Mock cells after introduction of FURA 2-AM were placed in a chamber (RC-26G; manufactured by Warner Instruments) of an inverted microscope of calcium imaging apparatus and washed with solution A. Subsequently, the fluorescence intensity at an excitation wavelength of 340 nm and the fluorescence intensity at an excitation wavelength of 380 nm were measured while circulating the test solution in a chamber. Thereafter, a fluorescence intensity ratio (fluorescence intensity at 340 nm / fluorescence intensity at 380 nm) between the fluorescence intensity at the excitation wavelength of 340 nm and the fluorescence intensity at the excitation wavelength of 380 nm when the test solution was used was calculated. The enhancement effect of the test substance on the change in intracellular calcium ion concentration (increased calcium ion concentration) was analyzed by IPLab software (manufactured by Scanalytics). Further, the same evaluation was performed using 4α-phorbol ester (4α-PDD) which is a TRPV4 ligand as a positive control. The results are shown in FIGS.

From the results of FIGS. 20 to 22, the positive control 4α-PDD showed a marked increase in intracellular calcium ion concentration, confirming the objectivity of this evaluation system. In addition, it was confirmed that the plant extract of the present invention significantly increased intracellular calcium ion concentration in TRPV4-expressing cells. On the other hand, it was not confirmed in Mock cells (FIGS. 23 and 24). Furthermore, the Acacia yak extract as a plant extract of the present invention was added to the calcium ion free solution B [composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ , 5 mM EGTA, 10 mM glucose, 10 mM HEPES (pH 7.4)]. When added, changes in intracellular calcium ion concentration in TRPV4-expressing cells were not confirmed (FIGS. 25 and 26), and were not confirmed in Mock cells (FIGS. 27 and 28). From this, it can be seen that the increase effect of intracellular calcium ion concentration by Acacia yak extract is influx of calcium ions from the outside via TRPV4.

[Example 6]
<Evaluation of TJ and AJ formation promoting action 1: Measurement of TER value of plant extract in the present invention>
According to the test method described in Test Example 1, the TER value of the plant extract of the present invention was measured. The results are shown in FIG. In FIG. 29, the vertical axis represents the TER value (Ωm ² ), and the horizontal axis represents the measurement time. From the results shown in FIG. 29, it was confirmed that the plant extract of the present invention exhibited a TJ and / or AJ function promoting effect showing a marked increase in TER value.

[Example 7]
<Evaluation of TJ and AJ formation promoting action 2: FITC-Dextran substance permeation test of plant extract in the present invention>
According to the test method described in Test Example 2 above, FITC-Dextran substance permeability of the plant extract of the present invention was evaluated. The results are shown in FIG. From the results shown in FIG. 30, the plant extract of the present invention showed a remarkable suppression of FITC-Dextran substance permeability and an action of promoting the formation of TJ and / or AJ.

From the results shown in FIG. 29 and FIG. 30, the plant extract of the present invention showed both TJ and AJ formation in the TJ and / or AJ formation promoting activity evaluation (TER value measurement and FITC-Dextran substance permeation test). It showed a promoting effect. From the above results, the plant extract of the present invention has a TRPV activation action and a TJ and / or AJ formation promoting action, and a skin barrier by the TJ and / or AJ formation promoting action via the TRPV activation action. Has a function improvement effect.

[Example 8]
<Composition of the present invention>
<Manufacture of a composition (skin external preparation) containing a component having an action of improving the skin barrier function of the present invention>
According to the formulations shown in Tables 1 and 2, a lotion cosmetic, which is a composition (external preparation for skin) containing a component having an action of improving the skin barrier function of the present invention, was prepared. That is, the prescription ingredients were stirred at 80 ° C. to solubilize, and then cooled under stirring to obtain a lotion cosmetic [cosmetic 1 or cosmetic 2 (skin external preparation 1 or 2)]. In addition, the same operation was performed to prepare Comparative Example 1 in which the “component having an action of improving skin barrier function” of the composition of the present invention (external preparation for skin) was replaced with “water”.

[Example 9]
<Skin roughness improvement test of the external preparation for skin of the present invention>
A paneler was used to evaluate the rough skin improving effect of the rough skin model prepared by tape stripping for the cosmetic 1, cosmetic 2, and comparative example 1, which are the external preparations of the present invention. That is, four 1cm x 1cm parts are defined on the left and right forearms, tape stripping is carried out 15 times for each part, and transepidermal water transpiration (TEWL) is measured with "Tevameter" manufactured by Integral. Measured. Thereafter, 50 μL of the sample was applied once a day, this operation was continued for 6 days, and TEWL was measured again on the 7th day. The TEWL improvement rate (%) was calculated by subtracting the TEWL value on the seventh day from the TEWL value on the first day, dividing by the TEWL value on the first day, and multiplying by 100. The n number was 15. The results are shown in Table 3. This shows that the skin external preparation of this invention is excellent in the rough skin improvement effect.

The present invention is a component having a skin barrier function improving action suitable as a cosmetic raw material (including quasi-drugs), and can be applied to a skin external preparation such as cosmetics.

Claims (11)

1. A TRPV receptor activator comprising one or two or more selected from the compounds represented by the following formula, or a plant extract containing an effective amount of the same compound as a TRPV receptor activator.
   

   

   

   

   

   (In the formula, the hydroxyl group may be substituted with a methoxy group or an ethoxy group, and the methoxy group may be substituted with a hydroxyl group or an ethoxy group.)
2. The compound is 3-O-methylellagic acid 4′-O-α-L-rhamnopyranoside, 3,3′-di-O-methylellagic acid 4′-O-β-D-glucopyranoside, 3,4,3′- The tri-O-methylellagic acid is 4′-O-β-D-glucopyranoside or 3′-O-methyl-3,4-methylenedioxyellagic acid is 4′-O-β-D-glucopyranoside. The described TRPV receptor activator.
3. 3. The TRPV receptor activator according to claim 1, wherein the extract is an extract obtained from a plant belonging to the genus Crape myraceae or a plant belonging to the genus Rubiaceae.
4. 4. The TRPV receptor activator according to claim 3, wherein the plant belonging to the genus Cryptaceae and the plant belonging to the genus Rubiaceae are Cyprus genus, Cranaceae, Gambir.
5. The TRPV receptor activator according to any one of claims 1 to 4, wherein the TRPV receptor is TRPV4.
6. A skin external preparation containing an effective amount of the TRPV receptor activator according to any one of claims 1 to 5.
7. The skin external preparation according to claim 6, wherein the effective amount is 0.00001 to 10% by mass of the compound with respect to the total amount of the skin external preparation.
8. The external preparation for skin according to claim 6 or 7, which is for improving the skin barrier function.
9. The external preparation for skin according to any one of claims 6 to 8, which is used for promoting the formation of tight junctions and / or adherence junctions.
10. The skin external preparation according to any one of claims 6 to 9, which is a cosmetic or a quasi-drug.
11. It is necessary to apply a TRPV receptor activator comprising one or more selected from the compounds represented by the following formula, or a plant extract containing an effective amount of the same compound as a TRPV receptor activator. A method for improving skin barrier function, comprising a step of applying to a site.
    

    

    

    

    

    (In the formula, the hydroxyl group may be substituted with a methoxy group or an ethoxy group, and the methoxy group may be substituted with a hydroxyl group or an ethoxy group.)

Images