JP5806806B2 - Evaluation method of cool feeling by test sample - Google Patents

Description

  The present invention relates to a method for evaluating a refreshing feeling by a test sample. More specifically, the present invention relates to the development of external preparations such as skin external preparations and hair external preparations containing a refreshing agent, and external preparations such as skin external preparations and hair external preparations having a refreshing strength suitable for users. The present invention relates to a method for evaluating a refreshing sensation by a test sample useful for development of a providing method. In the present specification, the test sample is a component used for an external preparation such as an external preparation for skin, an external preparation for hair, and the like, and is a substance to be examined for refreshing feeling.

  If necessary, a refreshing agent is added to the skin external preparation such as cosmetics used for human skin or the hair external preparation that may come into contact with human skin. ing. Examples of the refreshing agent include menthol and derivatives thereof.

  It is known that the menthol and its derivatives give a strong refreshing feeling to the user when coming into contact with human skin. However, some users may feel pain when menthol or its derivatives come into contact with the skin.

  On the other hand, in recent years, there is a tendency that external preparations that do not give such pain or have little pain are preferred due to the heightened safety awareness of users. Therefore, there is a need for a refreshing agent that provides a refreshing feeling without causing pain to the user.

  By the way, it is known that a transient receptor potential channel (hereinafter referred to as “TRP channel”) functions as a receptor that receives a factor that causes pain. For example, changes in intracellular calcium ion concentration via TRPA1, one of the TRP channels, are associated with unpleasant irritation caused by the use of topical skin preparations containing parabens and alkaline agents. Has been found by the present inventors. Further, it has been proposed to evaluate a substance that suppresses stimulation by parabens or alkaline agent by utilizing the relationship between stimulation by parabens or alkaline agent and change in intracellular calcium ion concentration via TRPA1. (For example, refer to Patent Documents 1 and 2).

  However, at the present time, the present inventors have used the relationship between the pain stimulus associated with the refreshing sensation by the refreshing agent such as menthol and TRPA1 and the above-mentioned relationship, and quickly and easily the refreshing sensation by the refreshing agent. We have not found any document that specifically describes how to evaluate by simple operations.

JP 2008-79528 A JP 2009-82053 A

The present invention has been made in view of the above-described prior art, and the evaluation of the feeling of coolness to the skin by the test sample, which can evaluate the comfort of the feeling of coolness to the skin by the test sample by a quick and simple operation. It aims to provide a method.

That is, the gist of the present invention is as follows.
(1) A test sample is contacted with a TRPM8-expressing cell and a TRPA1-expressing cell, or contacted with a TRPM8-TRPA1 co-expressing cell, and a physiological event caused by the test sample via TRPM8 and TRPA1 by the test sample A method for evaluating a refreshing sensation by a test sample, characterized by measuring a physiological event caused by
(2) The evaluation method according to (1), wherein the physiological event is a change in intracellular calcium ion concentration before and after contact with the test sample,
(3) The ratio between the change in intracellular calcium ion concentration caused through TRPM8 before and after contact with the test sample and the change in intracellular calcium ion concentration caused through TRPA1 before and after contact with the test sample The evaluation method according to (1) or (2) above, wherein (4) the test is used to evaluate the comfort of the refreshing feeling given to the human by the test sample. The ratio between the change in intracellular calcium ion concentration caused by TRPM8 before and after contact with the sample and the change in intracellular calcium ion concentration caused by TRPA1 before and after contact with the test sample was determined by the test sample. Used as an index of the comfort of the sensation, and the comfort of the cool sensation by the test sample The method for evaluating according to the classifying the test sample (1) or (2).

The method for evaluating the comfort of the skin with the test sample of the present invention (hereinafter also referred to as the “method of evaluating the cool feeling with the test sample”) is a quick and simple operation of the comfort of the skin with the test sample. It has an excellent effect of being able to be evaluated.

In Example 1, it is a graph which shows the relationship between a sample and the value of (DELTA) fluorescence intensity ratio _sample / (DELTA) fluorescence intensity ratio _agonist . In Example 1, it is a graph which shows the relationship between a sample and the value of (DELTA) fluorescence intensity ratio _sample / (DELTA) fluorescence intensity ratio _agonist . In Example 1, it is a graph which shows the relationship between a sample and a comfort parameter | index. In Example 2, it is a graph which shows the relationship between a sample and the value of (DELTA) fluorescence intensity ratio _sample / (DELTA) fluorescence intensity ratio _agonist . In Example 2, it is a graph which shows the relationship between a sample and the value of (DELTA) fluorescence intensity ratio _sample / (DELTA) fluorescence intensity ratio _agonist . In Example 2, it is a graph which shows the relationship between a sample and a comfort parameter | index.

  The method for evaluating a refreshing sensation by a test sample of the present invention is caused by bringing a test sample into contact with a TRPM8-expressing cell and a TRPA1-expressing cell, or with a TRPM8-TRPA1 co-expressing cell, and causing the test sample via TRPM8. Physiological events and physiological events caused by TRPA1 by the test sample are measured.

  The method for evaluating a refreshing sensation by the test sample of the present invention has one major feature in that a physiological event caused by the test sample via TRPM8 and a physiological event caused by the test sample via TRPA1 are measured. is there. According to the method for evaluating a refreshing sensation by the test sample of the present invention, the physiological event is measured, so that it is given to the human by the test sample in a quicker and simpler operation than in the case of using humans, laboratory animals, and the like. The cool feeling can be evaluated. Furthermore, since the physiological event can be measured in parallel at the same time many times under the same conditions, according to the method for evaluating a refreshing sensation by the test sample of the present invention, various types of physiological events can be performed quickly and with high processing efficiency. The cool feeling by the test sample can be evaluated.

  Test samples include refreshing agents, candidate substances for refreshing agents, essential oils, and the like.

  The TRPM8-expressing cell is a cell that expresses a physiological function of TRPM8. The physiological functions of TRPM8 include, for example, cations such as sodium ions and calcium ions from the outside to the inside of cells caused by stimulation such as chemical stimulation by menthol and cold stimulation (for example, stimulation at about 8 to 28 ° C.). And the like.

  Examples of the TRPM8-expressing cells include wild-type cells expressing endogenous TRPM8, cells introduced into a host cell so that a nucleic acid encoding TRPM8 can be expressed, and the like. Among the above-mentioned TRPM8-expressing cells, from the viewpoint of measuring physiological events caused by TRPM8 by a simple operation, a cell in which a nucleic acid encoding TRPM8 has been introduced into a host cell so that it can be expressed (hereinafter referred to as “exogenous” Also referred to as “TRPM8 expressing cells”).

  The TRPA1-expressing cell is a cell that expresses a physiological function of TRPA1. Examples of physiological functions of TRPA1 include chemical stimulation by mustard, cinnamaldehyde, allyl isothiocyanate, carvacrol, allicin, etc., cold stimulation (for example, stimulation at around 17 ° C.), pain stimulation, mechanical stimulation, etc. Examples include permeation of cations such as sodium ions and calcium ions from the outside to the inside of cells by stimulation.

  Examples of the TRPA1-expressing cells include wild-type cells expressing endogenous TRPA1, cells introduced into a host cell so that a nucleic acid encoding TRPA1 can be expressed, and the like. Among the above-mentioned TRPA1-expressing cells, from the viewpoint of measuring a physiological event caused through TRPA1 by a simple operation, a cell in which a nucleic acid encoding TRPA1 has been introduced into a host cell so that it can be expressed (hereinafter referred to as “exogenous” Also referred to as “TRPA1-expressing cells”).

  The wild-type cell expressing endogenous TRPM8 is not particularly limited, and examples thereof include sensory nerve cells and prostate cells. The wild type cells expressing endogenous TRPA1 are not particularly limited, and examples thereof include sensory nerve cells and inner ear cells.

  Examples of the TRPM8-TRPA1 co-expressing cells include cells in which a nucleic acid encoding TRPM8 and a nucleic acid encoding TRPA1 are both introduced into a host cell so that they can be expressed. In the TRPM8-TRPA1 co-expressing cells, the nucleic acid encoding TRPM8 and the nucleic acid encoding TRPA1 are physiological events triggered by TRPM8 by the test sample and physiological events triggered by TRPA1 by the test sample, respectively. From the viewpoint of measurement, each is preferably under the control of a separate expression promoter. The expression promoter may be any promoter suitable for expressing TRPM8 and TRPA1 in the host cell. The expression promoter can be appropriately selected depending on the type of host cell used.

  The exogenous TRPM8-expressing cell, exogenous TRPA1-expressing cell and TRPM8-TRPA1 co-expressing cell may be a cell in which the nucleic acid is present as an extrachromosomal element, and is a cell in which the nucleic acid is integrated into a chromosome. There may be.

  The exogenous TRPM8-expressing cell can be obtained by transforming a host cell with a recombinant vector carrying a nucleic acid encoding TRPM8. The exogenous TRPA1-expressing cells can be obtained, for example, by transforming host cells with a recombinant vector that holds a nucleic acid encoding TRPA1. The TRPM8-TRPA1 co-expressing cells can be obtained, for example, by transforming host cells with a recombinant vector carrying a nucleic acid encoding TRPM8 and a nucleic acid encoding TRPA1.

  The nucleic acid encoding TRPM8 may be a nucleic acid encoding human TRPM8 or a nucleic acid encoding TRPM8 of another animal. The nucleic acid encoding TRPA1 may be a nucleic acid encoding human TRPA1 or a nucleic acid encoding TRPA1 of another animal.

  The nucleic acid encoding TRPM8 is preferably a nucleic acid encoding human TRPM8 from the viewpoint of accurately evaluating a test sample applied to humans. Examples of the nucleic acid encoding TRPM8 include a nucleic acid comprising the base sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 1 is the base sequence of a nucleic acid encoding human TRPM8 registered in GenBank as accession number: NM_024080.4. If the polypeptide encoded by the nucleic acid expresses the physiological function, the nucleic acid encoding TRPM8 has one or several nucleotide residues in or at the end of the base sequence of the structural gene of TRPM8. It may be a mutant nucleic acid having a substitution, deletion or insertion.

  Moreover, the nucleic acid encoding TRPA1 is preferably a nucleic acid encoding human TRPA1 from the viewpoint of accurately evaluating a test sample applied to humans. Examples of the nucleic acid encoding TRPA1 include a nucleic acid comprising the base sequence shown in SEQ ID NO: 3. The base sequence shown in SEQ ID NO: 3 is the base sequence of a nucleic acid encoding human TRPA1 registered in GenBank as accession number: NM_007332. If the polypeptide encoded by the nucleic acid expresses the physiological function, the nucleic acid encoding TRPA1 has one or several nucleotide residues in or at the end of the base sequence of the structural gene of TRPA1. It may be a mutant nucleic acid having a substitution, deletion or insertion.

As a mutant nucleic acid of a nucleic acid encoding TRPM8, for example,
(A) Sequence homology calculated by aligning the base sequence shown in SEQ ID NO: 1 under the conditions of Cost to open gap 11, Cost to extended gap 1, expect value 10, and wordsize 11 by the BLAST algorithm. Is preferably 60% or more, more preferably 80% or more, and still more preferably 90% or more from the viewpoint of sufficiently exerting each physiological function, and the encoded polypeptide is A nucleic acid that is a polypeptide that expresses at least the physiological function;
(B) a polypeptide that encodes an amino acid sequence having a substitution, deletion or addition of one or several amino acid residues in SEQ ID NO: 2 and that expresses a function of allowing the encoded polypeptide to permeate at least a cation. A nucleic acid that is a peptide,
(C) A polypeptide that hybridizes with a nucleic acid complementary to the nucleic acid consisting of the base sequence represented by SEQ ID NO: 1 under stringent conditions, and the encoded polypeptide expresses a function of permeating cations. A certain nucleic acid etc. are mentioned.

Moreover, as a mutant nucleic acid of a nucleic acid encoding TRPA1, for example,
(A) Sequence homology calculated by aligning the base sequence shown in SEQ ID NO: 3 under the conditions of Cost to open gap 11, Cost to extended gap 1, expect value 10, and wordsize 11 by the BLAST algorithm. Is preferably 60% or more, more preferably 80% or more, and still more preferably 90% or more from the viewpoint of sufficiently exerting each physiological function, and the encoded polypeptide is A nucleic acid that is a polypeptide that expresses at least the physiological function;
(B) in SEQ ID NO: 4, encoding an amino acid sequence having substitution, deletion or addition of one or several amino acid residues, and the encoded polypeptide is a polypeptide that expresses at least the physiological function A nucleic acid,
(C) a nucleic acid that hybridizes with a nucleic acid complementary to the nucleic acid consisting of the base sequence represented by SEQ ID NO: 3 under stringent conditions, and the encoded polypeptide is a polypeptide that expresses the physiological function Etc.

  In this specification, the stringent conditions include, for example, a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1 or a nucleic acid consisting of the base sequence shown in SEQ ID NO: 3 and hybridization corresponding to the nucleic acid. The target nucleic acid was mixed with a hybridization solution [composition: 6 × SSC (composition: 0.9 M sodium chloride, 0.09 M sodium citrate, adjusted to pH 7.0), 0.5 mass% sodium dodecyl sulfate, 5 × Denhardt's solution, 100 μg / mL denatured salmon sperm DNA, 50% by volume formamide] at a temperature of room temperature or higher, more stringent conditions of 42 ° C. or higher, and more stringent conditions of 60 ° C. or higher for 10 hours. Incubate, then for example 2 × SSC, more stringent conditions In addition, there are conditions such as cleaning at an ionic strength of 0.1 × SSC, a temperature of room temperature or higher, a temperature of 42 ° C. or higher as a more stringent condition, and a temperature of 60 ° C. or higher as a stringent condition Can be mentioned.

  The nucleic acid encoding TRPM8 was designed and synthesized based on, for example, a hybridization method using a probe prepared based on the base sequence shown in SEQ ID NO: 1, and the base sequence shown in SEQ ID NO: 1. It can be obtained by a nucleic acid amplification method using a primer pair composed of two kinds of oligonucleotide primers. The nucleic acid encoding TRPA1 was designed and synthesized based on, for example, a hybridization method using a probe prepared based on the base sequence shown in SEQ ID NO: 3, and the base sequence shown in SEQ ID NO: 1. It can be obtained by a nucleic acid amplification method using a primer pair composed of two kinds of oligonucleotide primers.

  The host cell is not particularly limited as long as the nucleic acid encoding TRPM8 and / or the nucleic acid encoding TRPA1 is efficiently expressed and can be cultured, and examples thereof include animal cells, bacterial cells, Examples include plant cells and insect cells. Among these, animal cells are preferable from the viewpoint of sufficiently reproducing the physiological functions of TRPM8 and / or TRPA1 in humans. Examples of animal cells include human cells, monkey cells, mouse cells, and the like. Although it does not specifically limit as a human cell, For example, HEK293 cell, Hela cell, etc. are mentioned. Although it does not specifically limit as a monkey cell, For example, a COS-7 cell etc. are mentioned. Although it does not specifically limit as a mouse cell, For example, a CHO cell, a NIH3T3 cell, etc. are mentioned. Among the host cells, HEK293 cells, CHO cells, COS-7 cells and NIH3T3 cells are preferable from the viewpoint of easy handling. Among these, HEK293 cells are preferable from the viewpoint that TRP channels are hardly expressed and the activation of exogenous TRPM8 and / or TRPA1 can be easily and selectively measured.

  The recombinant vector is a vector obtained by linking a nucleic acid encoding TRPM8 and / or a nucleic acid encoding TRPA1 and a conventional vector. The vector may be a vector that can be easily prepared, can be efficiently introduced into a host cell, and can efficiently express TRPM8 and / or TRPA1 in the host cell. The vector is preferably a vector having a selection marker gene from the viewpoint of easily selecting cells carrying the recombinant vector after transformation. Examples of the vector include a plasmid vector and a virus vector. These vectors can be appropriately selected according to the host cell used. In addition, the vector used for the recombinant vector for producing the exogenous TRPM8 expressing cell and the exogenous TRPA1 expressing cell may have an expression promoter.

  Transformation using the recombinant vector can be performed by a transformation method such as electroporation, lipofection, transfection, or particle gun depending on the type of host cell used. These transformation methods are described in accordance with, for example, the description of Molecular Cloning: A Laboratory Manual (issued by Sambrook et al., Cold Spring Harbor Press, 1989). Can be done. Selection of exogenous TRPM8-expressing cells, exogenous TRPA1-expressing cells, and TRPM8-TRPA1 co-expressing cells from the transformed cells depends on the selection marker gene, for example, when the recombinant vector used has a selection marker gene. It can be carried out by culturing in a selective medium.

  Confirmation that the obtained cell is a cell expressing TRPM8 is, for example, contacting the cell with 1 mM menthol, and measuring the intracellular calcium ion concentration in the intracellular cell after contact by a method for measuring the intracellular calcium ion concentration described below. This can be done by measuring the ion concentration. When the cell expresses TRPM8, the intracellular calcium ion concentration of the cell after contact is higher than the intracellular calcium ion concentration of the cell not contacted with menthol. Confirmation that the obtained cells are cells expressing TRPA1, for example, by contacting the cells with 1 to 10 mM paraoxybenzoic acid methyl ester, and by measuring the intracellular calcium ion concentration described later, This can be done by measuring the intracellular calcium ion concentration of the cells. When the cell expresses TRPA1, the intracellular calcium ion concentration of the cell after contact is higher than the intracellular calcium ion concentration of the cell not contacted with paraoxybenzoic acid methyl ester.

  The contact between the test sample and the TRPM8-expressing cell and the TRPA1-expressing cell or TRPM8-TRPA1 co-expressing cell can be performed, for example, in a medium suitable for culturing each cell, Incubating with TRPA1 co-expressing cells, incubating a test sample with a mixture of TRPM8-expressing cells and TRPA1-expressing cells or TRPM8-TRPA1 co-expressing cells, and the like. In the case of using TRPM8-TRPA1 co-expressing cells, TRPM8 and TRPA are adjusted in accordance with the timing of measuring the physiological event caused by TRPM8 by the test sample and the physiological event caused by TRPA1 by the test sample. Each is expressed separately.

  Examples of the medium include components suitable for growing TRPM8-expressing cells, TRPA1-expressing cells, or TRPM8-TRPA1 co-expressing cells [for example, glucose, amino acids, peptone, vitamins, cell growth promoting factors (for example, cell growth factors, hormones). , Binding protein, cell adhesion factor, lipid, etc.), serum (eg, fetal bovine serum, etc.), calcium chloride, magnesium chloride, etc. The medium may be a medium obtained by supplementing a conventional basic medium with the above components, or a commercially available medium. The basic medium is not particularly limited, and examples thereof include MEM medium, DMEM medium, and RPMI 1640 medium. Such a medium can be appropriately selected according to the type of cell. For example, when the cells to be used are cells obtained from HEK293 cells, a 10% by mass fetal bovine serum-containing DMEM medium or the like is used as the medium.

The amount of TRPM8-expressing cells, TRPA1-expressing cells or TRPM8-TRPA1 co-expressing cells to be brought into contact with the test sample is preferably 1 × 10 ¹ cells or more per 100 μL of the test sample from the viewpoint of the reliability of the test data. ^Two or more cells are more preferable, and from the viewpoint of ensuring cell spacing and preventing cells from becoming too dense, 3 × 10 ² cells or less are preferable, and 2 × 10 ² cells or less are more preferable.

  Since the amount of the test sample to be brought into contact with the TRPM8-expressing cell, TRPA1-expressing cell or TRPM8-TRPA1 co-expressing cell differs depending on the type of test sample, it cannot be generally determined. It can be set appropriately.

  The TRPM8-expressing cell, the TRPA1-expressing cell, or the TRPM8-TRPA1 co-expressing cell is used as necessary to maintain the cell in a state suitable for measuring the physiological event. Expression cells or TRPM8-TRPA1 co-expression cells may be pre-incubated under conditions suitable for each cell.

  The incubation can be performed by a method according to the type of cell used. Examples of such methods include monolayer stationary culture, suspension culture, rotational culture, and three-dimensional carrier culture. In addition, conditions such as incubation temperature, incubation time, and carbon dioxide concentration are appropriately set according to the cells used. For example, when using cells obtained from HEK293 cells as TRPM8 expressing cells, TRPA1 expressing cells or TRPM8-TRPA1 co-expressing cells, such cells maintain the cells in a state suitable for measuring said physiological event. From the viewpoint, the incubation is usually performed at 36 to 38 ° C., preferably 36.5 to 37.5 ° C. in an atmosphere containing 5% by volume of carbon dioxide.

  Examples of the physiological event include a change in current at a constant potential before and after contact with the test sample, a change in intracellular calcium ion concentration before and after contact with the test sample, and a combination thereof. In the present invention, the physiological event is preferably a change in intracellular calcium ion concentration before and after contact with the test sample, from the viewpoint of simple measurement and high sensitivity.

  Examples of the method for measuring the current at the constant potential include a patch clamp method.

  The intracellular calcium ion concentration can be determined by, for example, introducing a fluorescent reagent based on a calcium chelating agent (hereinafter also referred to as “fluorescent calcium indicator”) into a TRPM8-expressing cell, TRPA1-expressing cell or TRPM8-TRPA1 co-expressing cell. It can be calculated using a method in which the fluorescent calcium indicator is bound to calcium ions and the fluorescence intensity of the fluorescent calcium indicator bound to calcium ions is examined. In this case, the change in the intracellular calcium ion concentration before and after contact with the test sample is the intracellular calcium ion concentration when contacted with the test sample relative to the intracellular calcium ion concentration when contacted with an agonist of TRPM8 or TRPA1. This can be investigated by determining the change in concentration.

  The agonist of TRPM8 and the agonist of TRPA1 may be the same substance or different substances. As an agonist of both TRPM8 and TRPA1, for example, menthol can be used.

  The fluorescent calcium indicator is not particularly limited as long as it is a reagent whose fluorescence characteristics change depending on the amount of the fluorescent calcium indicator bound to calcium ions, for example, FURA 2, FURA 2-AM, Fluo- 3 etc. are mentioned.

In addition, when a fluorescent calcium indicator has two types of excitation wavelengths, it is preferable to calculate a fluorescence intensity ratio from the fluorescence intensity in each excitation wavelength from a viewpoint of improving accuracy. For example, the excitation wavelengths of FURA 2-AM, a fluorescent calcium indicator, are 340 nm and 380 nm. In this case, the change in the intracellular calcium ion concentration when contacted with the test sample with respect to the intracellular calcium ion concentration when contacted with the agonist of TRPM8 or TRPA1 is Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio It can be examined by calculating the _agonist .

The Δ fluorescence intensity ratio _sample has the formula (I):

Can be calculated based on The Δ fluorescence intensity ratio _agonist is represented by the formula (II):

に基づいて算出することができる。なお、本明細書において、「蛍光強度_340nm」は励起波長３４０ｎｍにおける蛍光強度を示し、「蛍光強度_380nm」は励起波長３８０ｎｍにおける蛍光強度を示す。

Can be calculated based on In this specification, “fluorescence intensity _{340 nm} ” indicates fluorescence intensity at an excitation wavelength of 340 nm, and “fluorescence intensity _{380 nm} ” indicates fluorescence intensity at an excitation wavelength of 380 nm.

  When a change in intracellular calcium ion concentration before and after contact with the test sample is used as a physiological event, the refreshing sensation due to the test sample is caused by TRPM8 before and after contact with the test sample. And a change in intracellular calcium ion concentration caused by TRPA1 before and after contact with the test sample can be used as an index (hereinafter referred to as “comfort index”). The comfort index is, for example, the formula (III):

Can be calculated based on In this case, when the comfort index of the test sample is higher than 1, it is considered that the cold sensation by the test sample is a cold sensation with less pain than the agonist. Therefore, when the comfort index of the test sample is higher than 1, it can be determined that the comfort of the refreshing feeling by the test sample may be higher than that of the agonist. On the other hand, when the comfort index of the test sample is 1 or less, the cold sensation by the test sample is smaller than the cold sensation by the agonist, and the pain sensation by the test sample is equivalent to the pain sensation by the agonist, or by the agonist It is thought to be greater than the pain sensation. Therefore, when the comfort index of the test sample is 1 or less, it can be determined that the comfort of the refreshing feeling by the test sample may be equal to or less than that of the agonist.

  As described above, according to the refreshing feeling evaluation method using the test sample of the present invention, due to the physiological event caused by the test sample via TRPM8 and the physiological event caused by the test sample via TRPA1, A refreshing feeling on human skin can be evaluated. Therefore, the method for evaluating the refreshing sensation by the test sample of the present invention includes screening for a refreshing agent suitable for an external preparation such as a skin external preparation or a hair external preparation, a skin external preparation or a hair external preparation containing a refreshing agent, etc. It can be used for development of external preparations, development of methods for providing external preparations such as skin external preparations and hair external preparations having a refreshing strength suitable for users.

  Next, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to such examples.

(Production Example 1)
A cDNA encoding human TRPM8 [polynucleotide at positions 41 to 3355 of the nucleotide sequence shown in SEQ ID NO: 1 (GenBank accession number: NM_024080.4)] is a mammalian cell vector (trade name, manufactured by Invitrogen). : PcDNA3.1 (+)] to obtain a human TRPM8 expression vector. 1 μg of the obtained human TRPM8 expression vector and 6 μl of a gene introduction reagent [manufactured by Invitrogen, trade name: PLUS Reagent, catalog number: 11514-015] were mixed to obtain a mixture I. In addition, 4 μl of cationic lipid for gene introduction [manufactured by Invitrogen, trade name: Lipofectamine (registered trademark), catalog number: 18324-012] and medium for reducing serum use [trade name: OPTI-MEM (manufactured by Invitrogen), registered (Trademark) I Reduced-Serum Medium (Cat. No. 11058021) 200 μl was mixed to obtain a mixture II.

Further, in a DMEM medium containing 10% by mass FBS on a petri dish having a diameter of 35 mm maintained at 37 ° C. in an atmosphere of 5% by volume of carbon dioxide, 5 × 10 ⁵ cells of HEK293 cells were changed to 70% confluency. Cultured.

  By adding the mixture I and the mixture II to the obtained cell culture, the human TRPM8 expression vector was introduced into HEK293 cells to obtain TRPM8 expressing cells.

(Production Example 2)
In Production Example 1, instead of cDNA encoding human TRPM8, cDNA encoding human TRPA1 [polynucleotide at positions 63 to 3888 of the nucleotide sequence shown in SEQ ID NO: 1 (GenBank accession number: NM_007332)] is used. Except for the above, TRPA1-expressing cells were obtained in the same manner as in Production Example 1.
(Experimental example 1)
The TRPA1-expressing cells obtained in Production Example 2 were mixed at room temperature in a DMEM medium containing 10% by weight fetal calf serum containing FURA 2-AM (manufactured by Invitrogen), a reagent for measuring intracellular calcium ions, at a final concentration of 5 μM. By incubating for a minute, FURA 2-AM was introduced into the TRPA1-expressing cells to obtain FURA 2-AM-introduced TRPA1-expressing cells.

  The obtained FURA 2-AM-introduced TRPA1-expressing cells were placed in each chamber of a fluorescence measuring apparatus with a circulating constant temperature chamber [manufactured by Hamamatsu Photonics, Inc., trade name: ARGUS-50]. Thereafter, FURA 2-AM-introduced TRPA1-expressing cells in the chamber were mixed with solvent A [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose, 10 mM hepes hydrochloride buffer (pH 7.4)]. Washed with.

  Next, the solvent A containing 1 mM menthol was put into the chamber containing the FURA 2-AM-introduced TRPA1-expressing cells after washing, and the FURA 2-AM-introduced TRPA1-expressing cells and the solvent A containing 1 mM menthol were mixed.

Thereafter, fluorescence intensity based on FURA 2-AM (hereinafter referred to as “fluorescence intensity _{340 nm} ”) introduced into a TRPA1-expressing cell at an excitation wavelength of 340 nm and bound to intracellular calcium ions in the chamber and TRPA1 at an excitation wavelength of 380 nm. The intensity of fluorescence based on FURA 2-AM introduced into the expression cells (hereinafter referred to as “fluorescence intensity _{380 nm} ”) was measured.

From the measured fluorescence intensity of _{340 nm} and fluorescence intensity of _{380 nm} , the fluorescence intensity ratio _menthol was calculated. The fluorescence intensity ratio _menthol has the following formula (IV):

Calculated based on

In addition, a fluorescence intensity of _{340 nm} and a fluorescence intensity of _{380 nm} were measured in the same manner as in the case of using the test sample except that the solvent A was used instead of the solvent A containing 1 mM menthol.

From the measured fluorescence intensity of _{340 nm} and fluorescence intensity of _{380 nm} , the fluorescence intensity ratio _{solvent A} was calculated. The fluorescence intensity specific _{solvent A} is represented by the following formula (V):

Calculated based on

As a result, the fluorescence intensity ratio _menthol was 1.80. Moreover, the fluorescence intensity ratio _{solvent A} was 0.87. From these results, it can be seen that when the solvent A containing 1 mM menthol is used, the intracellular calcium ion concentration of the TRPA1-expressing cells is increased as compared with the case where the solvent A is used.

  Activation of TRPA1 is considered to be associated with pain sensation caused by parabens and alcohol. Therefore, the above results suggest that TRPA1 is also activated by menthol, so that the pain sensation of the sensation given to the skin by menthol is caused by activation of TRPA1.

(Example 1)
(1) Evaluation by TRPM8-expressing cells The TRPM8-expressing cells obtained in Production Example 1 are 10% by mass fetal calf serum containing FURA 2-AM (manufactured by Invitrogen), a reagent for measuring intracellular calcium ions, at a final concentration of 5 μM. FURA 2-AM was introduced into the TRPM8-expressing cells by incubating at room temperature for 60 minutes in the containing DMEM medium to obtain FURA 2-AM-introduced TRPM8-expressing cells.

  The obtained FURA 2-AM-introduced TRPM8-expressing cells were placed in each chamber of a fluorescence measuring apparatus with a circulating constant temperature chamber [manufactured by Hamamatsu Photonics, Inc., trade name: ARGUS-50]. Thereafter, FURA 2-AM-introduced TRPA1-expressing cells in the chamber were mixed with solvent A [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose, 10 mM hepes hydrochloride buffer (pH 7.4)]. Washed with.

  Next, the test sample was put in the chamber containing the FURA 2-AM-introduced TRPM8-expressing cells after washing, and the FURA 2-AM-introduced TRPM8-expressing cells and the test sample were mixed. In addition, as the test sample, a solvent A containing 1 mM cineole, a solvent A containing 1 mM carboxylic acid, a solvent A containing 1 mM menthone, a solvent A containing 1 mM citronellal, a solvent A containing 1 mM citral, 1 mM linalool. Solvent A containing, solvent A containing 1 mM terpineol, solvent A containing 1 mM cymene, solvent A containing 1 mM eugenol, solvent A containing 1 mM limonene, solvent A containing 1 mM (+)-pinene, 1 mM Solvent A containing (−)-pinene or solvent A containing 1 mM anethole was used. These test samples are components contained in perfume oils generally used for cooling agents or aromatherapy.

Thereafter, fluorescence intensity based on FURA 2-AM (hereinafter referred to as “fluorescence intensity _{340 nm} ”) introduced into TRPA1-expressing cells at an excitation wavelength of 340 nm and bound to intracellular calcium ions in the chamber and TRPM8 at an excitation wavelength of 380 nm. The intensity of fluorescence based on FURA 2-AM introduced into the expression cells (hereinafter referred to as “fluorescence intensity _{380 nm} ”) was measured.

A _{Δfluorescence} intensity ratio _sample was calculated from the measured fluorescence intensity of _{340 nm} and fluorescence intensity of _{380 nm} . The Δ fluorescence intensity ratio _sample has the formula (I):

Calculated based on The control is solvent A.

In addition, a fluorescence intensity of _{340 nm} and a fluorescence intensity of _{380 nm} were measured in the same manner as in the case of using the test sample except that an agonist of TRPM8 was used instead of the test sample. As the agonist, solvent A containing 1 mM menthol was used.

From the measured fluorescence intensity of _{340 nm} and fluorescence intensity of _{380 nm} , a Δfluorescence intensity ratio _agonist was calculated. The Δ fluorescence intensity ratio _agonist is represented by the formula (II):

Calculated based on

From the calculated Δ fluorescence intensity ratio _sample and Δ fluorescence intensity ratio _agonist , Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist in TRPM8 expressing cells was calculated.

In Example 1, a graph showing the relationship between the sample and the value of the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist is shown in FIG.

In FIG. 1, sample number 1 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM cineole is used, and sample number 2 is Δfluorescence when solvent A containing 1 mM carboxylic acid is used. Intensity ratio _sample / Δfluorescence intensity ratio _agonist , sample number 3 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when using solvent A containing 1 mM menthone, sample number 4 is a solvent A containing 1 mM citronellal Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _{agonist when used} , sample number 5 is Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM citral is used, sample number 6 is 1 mM linalool delta fluorescence intensity ratio _sample / delta fluorescence intensity ratio _agonists in the case of using the solvent a containing, if sample No. 7 with a solvent a containing 1mM terpineol Delta fluorescence intensity ratio _sample / delta fluorescence intensity ratio _agonists, solvent sample No. 8 delta fluorescence intensity ratio _sample / delta fluorescence intensity ratio _agonists in the case of using the solvent A containing 1mM cymene, Sample No. 9 containing 1mM eugenol Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist when using A, sample number 10 is Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist when using solvent A containing 1 mM limonene, sample number 11 is Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM (+)-pinene is used, sample number 12 is Δfluorescence when solvent A containing 1 mM (−)-pinene is used Intensity ratio _sample / Δfluorescence intensity ratio _agonist and sample number 13 show Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM anethole is used.

From the results shown in FIG. 1, solvent A containing 1 mM cineol (sample number 1), solvent A containing 1 mM menthone (sample number 3) and solvent A containing 1 mM eugenol (sample number 6) were used. In this case, the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist is found to be 0.3 to 0.6. From these results, it can be seen that cineol, menthone and eugenol have a lower ability to activate TRPM8 than menthol.

From the above results, when using solvent A containing 1 mM cineol (sample number 1), solvent A containing 1 mM menthone (sample number 3) and solvent A containing 1 mM eugenol (sample number 6), It can be seen that the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist is larger than when other samples are used. Therefore, from these results, cineol, menthone and eugenol have higher ability to activate TRPM8 than carvone, citronellal, citral, linalool, terpineol, cymene, limonene, (+)-pinene, (-)-pinene and anethole. I understand that.

(2) Evaluation with TRPA1-expressing cells As in (1) above, except that the TRPA1-expressing cells obtained in Production Example 2 were used instead of the TRPM8-expressing cells obtained in Production Example 1. Thus, a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist in TRPA1-expressing cells was calculated.

A graph showing the relationship between the sample and the value of the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist in Example 1 is shown in FIG.

In FIG. 2, sample number 1 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM cineol is used, and sample number 2 is Δfluorescence when solvent A containing 1 mM carboxylic acid is used. Intensity ratio _sample / Δfluorescence intensity ratio _agonist , sample number 3 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when using solvent A containing 1 mM menthone, sample number 4 is a solvent A containing 1 mM citronellal Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _{agonist when used} , sample number 5 is Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM citral is used, sample number 6 is 1 mM linalool delta fluorescence intensity ratio _sample / delta fluorescence intensity ratio _agonists in the case of using the solvent a containing, if sample No. 7 with a solvent a containing 1mM terpineol Delta fluorescence intensity ratio _sample / delta fluorescence intensity ratio _agonists, solvent sample No. 8 delta fluorescence intensity ratio _sample / delta fluorescence intensity ratio _agonists in the case of using the solvent A containing 1mM cymene, Sample No. 9 containing 1mM eugenol Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist when using A, sample number 10 is Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist when using solvent A containing 1 mM limonene, sample number 11 is Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM (+)-pinene is used, sample number 12 is Δfluorescence when solvent A containing 1 mM (−)-pinene is used Intensity ratio _sample / Δfluorescence intensity ratio _agonist and sample number 13 show Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 1 mM anethole is used.

From the results shown in FIG. 2, Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio when using solvent A containing 1 mM citral (sample number 5) and solvent A containing 1 mM eugenol (sample number 9), respectively. _It can be seen that the _agonist exceeds 1. Therefore, the above results suggest that citral and eugenol have higher TRPA1 activation ability than menthol.

In contrast, from the results shown in FIG. 2, solvent A containing 1 mM cineol (sample number 1), solvent A containing 1 mM carboxylic acid (sample number 2), solvent A containing 1 mM menthone (sample number) 3) Solvent A containing 1 mM citronellal (Sample No. 4), Solvent A containing 1 mM linalool (Sample No. 6), Solvent A containing 1 mM terpineol (Sample No. 7), Solvent A containing 1 mM Cymene (Sample No. 6) Sample No. 8) Solvent A containing 1 mM limonene (Sample No. 10), Solvent A containing 1 mM (+)-pinene (Sample No. 11), Solvent A containing 1 mM (−)-pinene (Sample No. 12) ) And 1 mM anethole-containing solvent A (sample No. 13), respectively, it is understood that the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist is less than 1 The Therefore, the above results suggest that carvone, menthone, citronellal, linalool, terpineol, cymene, limonene, (+)-pinene, (-)-pinene and anethole have lower TRPA1 activation ability than menthol. .

(3) Evaluation of comfort of refreshing feeling based on activation ability of TRPM8 and TRPA1 by test sample From the result of Experimental Example 1, TRPA1 is activated by menthol. It is suggested that pain sensation is caused by activation of TRPA1. TRPM8 is activated by menthol, but also activated by a cold stimulus of about 8 to 28 ° C. Therefore, TRPM8 is considered to be related to the cold feeling among the sensations brought to human skin by menthol.

Therefore, the (1) and the delta fluorescence intensity ratio _sample and delta fluorescence intensity ratio _agonist in TRPM8-expressing cells as measured by the (2) delta in the fluorescence intensity ratio _sample and delta fluorescence intensity ratio _agonist in TRPA1 expressing cells as measured by Based on this, the comfort of the refreshing feeling by the test sample was evaluated.

Wherein from a Δ fluorescence intensity ratio _sample and Δ fluorescence intensity ratio _agonist in TRPM8-expressing cells was measured, the a Δ fluorescence intensity ratio _sample and Δ fluorescence intensity ratio _agonist in TRPA1 expressing cells measured in (2) (1), subject The ratio of the TRPM8 activation ability by the sample and the TRPA1 activation ability by the test sample was calculated. Hereinafter, the ratio was used as a “comfort index”. Such a comfort index is represented by formula (III):

Calculated based on

  Here, when the comfort index of the test sample is higher than 1, it is considered that the cool sensation by the test sample is a cold sensation with less pain than the cold sensation by menthol. Therefore, when the comfort index of the test sample is higher than 1, it can be determined that the refreshing comfort of the test sample may be higher than that of menthol.

  On the other hand, when the comfort index of the test sample is 1 or less, the cold sensation by the test sample is smaller than the cold sensation by menthol, and the pain sensation by the test sample is equivalent to the pain sensation by menthol, or by menthol It is thought to be greater than the pain sensation. Therefore, when the comfort index of the test sample is 1 or less, it can be determined that the comfort of the refreshing feeling by the test sample may be equal to or less than that of menthol.

  In Example 1, the graph which shows the relationship between a sample and a comfort parameter | index is shown in FIG. In the figure, the broken line with the comfort index = 1 indicates the comfort index of menthol.

  In FIG. 3, sample number 1 is a comfort index when using solvent A containing 1 mM cineol, sample number 2 is a comfort index when using solvent A containing 1 mM carboxylic acid, and sample number 3 contains 1 mM menthone. Comfort index when using solvent A, sample number 4 is comfort index when using solvent A containing 1 mM citronellal, sample number 5 is comfort index when using solvent A containing 1 mM citral, sample Number 6 is a comfort index when using solvent A containing 1 mM linalool, sample number 7 is a comfort index when using solvent A containing 1 mM terpineol, and sample number 8 is solvent A containing 1 mM cymene. The comfort index when using the solvent A containing 1 mM eugenol, sample number 10 containing 1 mM limonene The comfort index when using the medium A, sample number 11 uses the comfort index when using the solvent A containing 1 mM (+)-pinene, and sample number 12 uses the solvent A containing 1 mM (−)-pinene. The comfort index and sample number 13 in the case of using the solvent indicate the comfort index when using the solvent A containing 1 mM anethole.

  From the results shown in FIG. 3, it can be seen that the comfort index exceeds 1 when the solvent A (sample No. 5) containing 1 mM cineole is used. Therefore, the above results suggest that the comfort of cineole may be higher than the comfort of menthol.

  On the other hand, from the results shown in FIG. 3, the comfort index when each of the solvent A containing 1 mM menton (sample number 3) and the solvent A containing 1 mM eugenol (sample number 9) is less than 1 It can be seen that it is. Thus, the results suggest that the comfort of each of Menton and Eugenol may be less than the comfort of menthol.

  From these results, both TRPM8-expressing cells and TRPA1-expressing cells were used to measure physiological events caused by the test sample via TRPM8 and physiological events caused by the test sample via TRPA1. It is suggested that the comfort of refreshing feeling by the test sample can be evaluated by examining the activation ability of TRPM8 and TRPA1 by the sample.

(Example 2)
(1) Evaluation by TRPM8-expressing cells In (1) of Example 1, as test samples, solvent A containing 0.01% by volume peppermint oil (sample number 1), solvent containing 0.01% by volume spearmint oil A (sample number 2), solvent A (sample number 3) containing 0.01% by volume eucalyptus oil [manufactured by Eizodo Honten Co., Ltd., eucalyptus oil], solvent A containing 0.01% by volume thyme oil ( Sample No. 4), Solvent A containing 0.01% by volume clove oil (Sample No. 5), Solvent A containing 0.01% by volume lavender oil (Sample No. 6), containing 0.01% by volume sage oil Solvent A (Sample No. 7), Solvent A (Sample No. 8) containing 0.01% by volume Eucalyptus oil (Ogawa Fragrance Co., Ltd., Eucalyptus oil) or 0.01% by volume Roman Chamomile oil Using A (Sample No. 9) In the same manner as in Example 1 (1), a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist in TRPM8 expressing cells was calculated. These test samples are aqueous solutions of components containing perfume oil.

FIG. 4 shows a graph showing the relationship between the sample and the value of the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist in Example 2.

In FIG. 4, sample number 1 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 0.01% by volume peppermint oil is used, and sample number 2 contains 0.01% by volume spearmint oil. Δ Fluorescence intensity ratio _sample / Δ Fluorescence intensity ratio _agonist when using Solvent A to be used, Sample No. 3 uses Solvent A containing 0.01% by volume Eucalyptus oil (Eukari Oil Co., Ltd., manufactured by Eizodo Honten) Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist , sample number 4 is Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist , sample number when solvent A containing 0.01 vol% thyme oil is used 5 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when using solvent A containing 0.01% by volume clove oil, and sample No. 6 uses solvent A containing 0.01% by volume lavender oil. Delta fluorescence intensity ratio in case _Sample / Δ Fluorescence Intensity Ratio _Agonist , Sample No. 7 is Δ Fluorescence Intensity Ratio _Sample / Δ Fluorescence Intensity Ratio _Agonist when Solvent A Containing 0.01% by Volume Sage Oil, Sample No. 8 is 0.01% by Volume Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist and sample number 9 in the case of using the solvent A containing eucalyptus oil [manufactured by Ogawa Fragrance Co., Ltd., Eucalyptus oil] and 0.01% by volume contain roman chamomile oil A Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A is used is shown.

From the results shown in FIG. 4, solvent A containing 0.01% by volume peppermint oil (sample number 1), solvent A containing 0.01% by volume thyme oil (sample number 4), 0.01% by volume When each of solvent A ((sample number 8)) containing solvent A (sample number 5) containing clove oil and 0.01% by volume eucalyptus oil (manufactured by Ogawa Fragrance Co., Ltd., eucalyptus oil) was used, Δfluorescence It can be seen that the intensity ratio _sample / Δfluorescence intensity ratio _agonist is 0.1 or more and less than 1. From these results, peppermint oil, thyme oil, clove oil, and eucalyptus oil (produced by Ogawa Fragrance Co., Ltd., eucalyptus oil) It can be seen that TRPM8 has a lower activation ability than menthol.

Further, from the above results, Solvent A (Sample No. 1) containing 0.01% by volume peppermint oil, Solvent A (Sample No. 4) containing 0.01% by volume thyme oil, and 0.01% by volume clove oil were obtained. When each of the solvent A (sample No. 8) containing the solvent A (sample No. 5) and 0.01 vol% eucalyptus oil (produced by Ogawa Fragrance Co., Ltd., Eucalyptus oil) was used, another sample was used. It can be seen that the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist is larger than the case. Therefore, from these results, peppermint oil, thyme oil, clove oil and eucalyptus oil (from Ogawa Fragrance Co., Ltd., Eucalyptus oil) are solvents A containing spearmint oil, eucalyptus oil (manufactured by Eirakudo head office Eucalyptus oil], solvent A containing lavender oil, solvent A containing sage oil, and solvent A containing roman chamomile oil are found to have higher activation ability of TRPM8.

(2) Evaluation with TRPA1-expressing cells In (2) of Example 1, (2) of Example 1 except that the test sample used in (1) of Example 2 was used as the test sample. Similarly, a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist in TRPA1-expressing cells was calculated.

FIG. 5 is a graph showing the relationship between the sample and the value of the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist in Example 2.

In FIG. 5, sample number 1 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when solvent A containing 0.01% by volume peppermint oil is used, and sample number 2 contains 0.01% by volume spearmint oil. Δ Fluorescence intensity ratio _sample / Δ Fluorescence intensity ratio _agonist when using Solvent A to be used, Sample No. 3 uses Solvent A containing 0.01% by volume Eucalyptus oil (Eukari Oil Co., Ltd., manufactured by Eizodo Honten) Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist , sample number 4 is Δ fluorescence intensity ratio _sample / Δ fluorescence intensity ratio _agonist , sample number when solvent A containing 0.01 vol% thyme oil is used 5 is a Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when using solvent A containing 0.01% by volume clove oil, and sample No. 6 uses solvent A containing 0.01% by volume lavender oil. Delta fluorescence intensity ratio in case _Sample / Δ Fluorescence Intensity Ratio _Agonist , Sample No. 7 is Δ Fluorescence Intensity Ratio _Sample / Δ Fluorescence Intensity Ratio _Agonist when Solvent A Containing 0.01% by Volume Sage Oil, Sample No. 8 is 0.01% by Volume ΔFluorescence intensity ratio _sample / ΔFluorescence intensity ratio _agonist / sample No. 9 when using solvent A containing eucalyptus oil (Ogawa fragrance Co., Ltd., Eucalyptus oil) is a solvent containing 0.01% by volume roman chamomile oil The Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when A is used is shown.

From the results shown in FIG. 5, the solvent A (sample number 1) containing 0.01% by volume peppermint oil and the solvent A (sample number 4) containing 0.01% by volume thyme oil were used. It can be seen that Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist exceeds 1. Therefore, the above results suggest that peppermint oil and thyme oil have higher TRPA1 activation ability than menthol.

On the other hand, from the results shown in FIG. 5, the solvent A containing 0.01% by volume spearmint oil (sample number 2), 0.01% by volume eucalyptus oil [manufactured by Eizodo Honten Co., Ltd., eucalyptus oil ], A solvent A containing 0.01% by volume clove oil (sample number 5), a solvent A containing 0.01% by volume lavender oil (sample number 6), 0. Solvent A (Sample No. 7) containing 01% by volume sage oil, Solvent A (Sample No. 8) containing 0.01% by volume eucalyptus oil (manufactured by Ogawa Fragrance Co., Ltd., Eucalyptus oil) and 0.01% by volume It can be seen that Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist is less than 1 when each of the solvents A (sample No. 9) containing roman chamomile oil is used. Therefore, the above results suggest that spearmint oil, eucalyptus oil, clove oil, lavender oil, sage oil, eucalyptus oil and roman chamomile oil have a higher ability to activate TRPA1 than menthol.

(3) Evaluation of comfort of refreshing feeling by the test sample by the comfort index In (3) of Example 1, except that the test sample used in (1) of the present Example 2 was used as the test sample, The comfort index was calculated in the same manner as in Example 1 (3).

  In Example 2, the graph which shows the relationship between a sample and a comfort parameter | index is shown in FIG. In the figure, the broken line with the comfort index = 1 indicates the comfort index of menthol.

  In FIG. 6, sample number 1 is a comfort index when using solvent A containing 0.01% by volume peppermint oil, and sample number 2 is comfort when using solvent A containing 0.01% by volume spearmint oil. Index, sample number 3 is a comfort index when using a solvent A containing 0.01% by volume eucalyptus oil [manufactured by Eizodo Honten Co., Ltd., eucalyptus oil], sample number 4 is 0.01% by volume thyme oil The comfort index when using the solvent A contained, Sample No. 5 contains the comfort index when using the solvent A containing 0.01% by volume clove oil, and Sample No. 6 contains 0.01% by volume lavender oil. The comfort index when using solvent A, sample number 7 is the comfort index when using solvent A containing 0.01% by volume sage oil, and sample number 8 is 0.01% by volume eucalyptus oil [Ogawa Fragrance Co., Ltd. ), Eucalyptus oil] Comfort index and Sample No. 9 in the case of using the A indicates the comfort index in the case of using solvent A containing 0.01 vol% Roman chamomile oil.

From the results shown in FIG. 6, a solvent A containing 0.01% by volume of peppermint oil (sample No. 1) and 0.01% by volume of eucalyptus oil (produced by Ogawa Fragrance Co., Ltd., eucalyptus oil). (Sample No. 8) It can be seen that Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist when using each is approximately 1. Therefore, the above results suggest that the refreshing sensation of peppermint oil and eucalyptus oil (manufactured by Ogawa Fragrance Co., Ltd.) may be equivalent to the sensation of menthol.

On the other hand, from the results shown in FIG. 6, the Δfluorescence intensity ratio _sample / Δfluorescence intensity ratio _agonist is less than 1 when the solvent A (sample number 5) containing 0.01% by volume clove oil is used. It can be seen that it is. Thus, the results suggest that the comfort of the clove oil may be lower than the comfort of the menthol.

(Test Example 1)
Six subjects, whose cervical stains were removed with a wet towel, were acclimated for 10 minutes in a test room at a room temperature of 22 ° C. and a relative humidity of 50%.

  Next, using a dispenser [trade name: Y-150, manufactured by Yoshino Kogyo Co., Ltd.], 1 vol% menthol aqueous solution containing ethanol (ethanol concentration: 40 vol%) or 1 vol% on the left neck of the subject. 300 μL of cineol aqueous solution was applied. Moreover, 300 microliters of control purified water was apply | coated to the test subject's right neck by using the said dispenser. The senses after 3 minutes from the end of application were compared.

  As a result, when 1% by volume menthol aqueous solution containing ethyl alcohol was applied, 6 out of 6 responded that “I feel cold but also feel pain”, whereas when 1% by volume cineol aqueous solution was applied, 6 out of 6 responded, “I feel only a cool feeling (I feel a pleasant refreshing feeling)”.

  From the above results, it can be seen that there is a correlation between the comfort index of the test sample and the evaluation result of the comfort of the refreshing feeling by the test sample by the subject.

  As described above, the physiological events caused by the test sample via TRPM8 and the physiological events caused by the test sample via TRPA1 are measured, and the test obtained based on these physiological events. Based on the size of the comfort index of the sample, it is suggested that the coolness of the test sample can be evaluated. Thus, by using both TRPM8-expressing cells and TRPA1-expressing cells to measure the physiological events caused by the test sample via TRPM8 and the physiological events caused by the test sample via TRPA1 It is suggested that the refreshing feeling by can be evaluated. In addition, when both TRPM8-expressing cells and TRPA1-expressing cells are used instead of using both TRPM8-expressing cells and TRPA1-expressing cells, as with the case of using both TRPM8-expressing cells and TRPA1-expressing cells, It is suggested that the refreshing feeling can be evaluated.

Claims (2)

A test sample is contacted with a TRPM8-expressing cell and a TRPA1-expressing cell, or is contacted with a TRPM8-TRPA1 co-expressing cell, and the intracellular calcium ion concentration before and after the contact of the test sample caused via TRPM8 and TRPA1 Intracellular calcium ion concentration before and after contact with the test sample is measured, and when contacted with an agonist of TRPM8, which is a change in intracellular calcium ion concentration caused through TRPM8 before and after contact with the test sample A change in intracellular calcium ion concentration when contacted with a test sample relative to the intracellular calcium ion concentration, and an agonist of TRPA1 which is a change in intracellular calcium ion concentration caused via TRPA1 before and after contact with the test sample ; Contact Using the ratio between the change in the intracellular calcium ion concentration when contacted with a test sample relative to intracellular calcium ion concentration when allowed to as an index of comfort feeling of coolness on the skin by the test sample, the skin by the test sample A method for evaluating the comfort of the refreshing feeling on the skin by the test sample, wherein the comfort of the given refreshing feeling is evaluated. A test sample is contacted with a TRPM8-expressing cell and a TRPA1-expressing cell, or is contacted with a TRPM8-TRPA1 co-expressing cell, and changes in intracellular calcium ion concentration before and after the contact of the test sample caused by TRPM8 and TRPA1 Change in intracellular calcium ion concentration before and after contact with the test sample caused by contact with an agonist of TRPM8, which is change in intracellular calcium ion concentration caused by TRPM8 before and after contact with the test sample is a change in intracellular calcium ion concentration caused through the change in the intracellular calcium ion concentration, the TRPA1 before and after contact with the test sample when contacted with a test sample relative to intracellular calcium ion concentration when allowed to TRPA1 a Using the ratio between the change in the intracellular calcium ion concentration when contacted with a test sample relative to intracellular calcium ion concentration when contacted with Marianist as an index of comfort feeling of coolness on the skin by the test sample, the test sample The test sample is classified according to the comfort of the refreshing feeling on the skin by the test method.

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