JP2006105812A - In-vivo evaluation method for hair restoration/new hair growth function - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a screening method for materials having hair restoration/new hair growth functions, and provide an established in-vivo evaluation method used therefor. <P>SOLUTION: In the in-vivo evaluation method for the hair restoration/new hair growth functions, a morphological change in a hair follicle is measured as an evaluation factor by using a mouse whose hair cycle is made attuned forcibly, and the hair restoration/new hair growth functions are evaluated. For effectively screening a material which can serve as an element candidate of hair restoring/growing agents, from an extremely great number of living being extracts or matters, an in-vitro evaluation is carried out, in which an inhibition degree of interactions with a fibrocyte growth factor 5 (FGF-5) is measured, and a living being extract or matter having the function of inhibiting the FGF-5 is selected therefrom. Then, the selected matter or living being extract is preferably subjected to above-mentioned in-vivo evaluation about the hair restoration/new hair growth functions. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a mouse model in which the hair cycle is forcibly synchronized, an in vivo method for evaluating hair growth / hair growth using the mouse as an evaluation index of hair follicle morphology, and the use thereof. Further, the present invention relates to a screening method for substances having hair growth / hair growth activity.

  Many of the hair growth and hair growth agents that have been developed so far suppress hair growth by inhibiting excessive sebum secretion, eliminating poor blood circulation such as the scalp, or suppressing the action of male hormones. The aim is to encourage On the other hand, as seen in pine extract, plant-derived hair growth / hair growth agents have existed since ancient times, and the substance of the hair growth action is not known in detail, but has been used in folk remedies. There are many hair growth and hair growth agents in this way, but there are also large individual differences in the effect of thinning hair and hair loss, so what is universally effective against thinning hair not exist.

  The need for hair growth and hair growth agents that have a universal effect on thinning hair and hair loss reflecting the aging and multi-stress society still exists and can be said to be high in the future. Although search for a substance having a hair-growth / hair-growth effect as a new hair-growth / hair-growth component has been made energetically, a truly effective new substance is not easily found. As one of the causes, it has been pointed out that an evaluation method for screening candidate substances for hair growth / hair growth component is not always appropriate.

The hair does not grow constantly at a constant speed, but grows with repeated growth and rest periods. The hair growth and hair loss cycle (hair cycle) from the growth period to the rest period is different for each body part and for each human hair. The current screening method for hair growth and hair growth agents uses the evaluation method developed by Ogawa et al. This method utilizes the fact that in young mice of about 8 weeks of age, the hair cycle of the entire body surface is generally synchronized and in a resting state. In other words, after shaving the back of the mouse, applying a sample and observing the timing of transition from the resting phase to the growth phase, and comparing the difference in hair growth / hair growth effect between the candidate substance and the control substance It is. However, this method uses mice whose hair cycle is in the resting state and employs shaving which makes it difficult to keep the skin stimulation constant, so the hair cycle of all mice used It was extremely difficult to carry out the test in synchronization, and there was a problem that a long period of about 40 days was usually required from shaving to determination of hair growth / hair growth effect.

  On the other hand, transforming growth factor (TGF) β, which is one of the substances that affect the hair cycle of hair matrix cells, exhibits the action of shifting the hair cycle from the growth phase to the regression phase and the resting phase. An antagonist that suppresses the action has been found and proposed as a novel hair-growth component.

  Suzuki et al. Found that the biological activity of a cytokine, fibroblast growth factor 5 (hereinafter also referred to as “FGF-5”), which is different from TGFβ, has a control of the hair cycle (Non-Patent Documents 1 to 4). 4). The FGF-5 protein belongs to the fibroblast growth factor (FGF) family that exhibits various physiological actions. Their study revealed that FGF-5 has the effect of inhibiting hair growth and shifting the hair cycle from the growth phase to the regression phase. FGF-5S (short chain molecule of FGF-5) expressed in a large amount in growing hair follicles exhibits an antagonist activity against FGF-5, thereby delaying the transition from the growth phase to the regression phase. It has also been revealed that growth is promoted (see Non-Patent Documents 1 to 4). Further, it has been found that when fibroblast growth factor 5 is administered to animals, abnormal hair cycle is induced at the administration site, and an alopecia model based on this discovery has been proposed (Patent Document 1).

  In addition, in vitro measurement using conventional dermal papilla cells or hair follicle cells is mostly effective in collecting a large number of candidate substances because such cells are carefully collected from animals and cultured. Obviously, it is impossible to search for substances quickly. Furthermore, it is desired that verification of hair growth / hair growth effect is finally performed in an in vivo system.

Therefore, there is a need to improve the method for evaluating hair growth / hair growth effect and establish an efficient screening method regardless of the in vitro evaluation method or the in vivo evaluation method.
JP 2001-343383 A Ozawa K, Suzuki S, Asada M et al. J. Biol. Chem. 273: 29262-29271 (1998) Suzuki S, Kato T, Takimoto H et al. J. Invest. Dermatol. 111: 963-972 (1998) Suzuki S, Ota Y, Ozawa K, Imamura T J. Invest. Dermatol. 114: 456-463 (2000) Ota Y, Saitoh Y, Suzuki S et al. Biochem. Biophys. Res. Commun. 290: 169-176 (2002)

The method of the present invention was developed as a new in vivo evaluation method for quickly and accurately measuring and evaluating the hair growth / hair growth action in view of the above situation.
An object of this invention is to propose the method of measuring and evaluating the hair growth and the hair growth effect for searching the biological extract containing the substance which has hair growth and the hair growth action. In other words, for the purpose of searching for a biological extract or substance that exhibits excellent hair growth / hair growth action, it is to provide an in vivo evaluation method for measuring and selecting the effect of the hair growth / hair growth action. . Furthermore, a high-throughput screening system can be created by appropriately combining with an in vitro evaluation method.

The present invention for solving the above problems has the following configuration.
In the in vivo evaluation method for hair growth / hair growth of the present invention, a sample for examining the presence / absence of hair growth / hair growth is applied to the skin surface, and the hair follicle, hair that occurs at the transition from the late growth phase to the regression phase of the hair cycle is applied. By measuring the change in the form or condition of the applied skin surface, it is possible to evaluate the expression of hair growth and hair growth effects using the extension of the hair cycle growth phase or the delay in transition to the regression phase as an evaluation index. It is said.

In the above method, it is desirable to use a mouse whose hair cycle is forcibly tuned by removing hair during the rest period of the hair cycle.
Alternatively, it may be performed using naturally synchronized mice shaved during the growth phase of the hair cycle.

  Regarding the change in the form or state, the length of the hair follicle, the degree of the hair follicle deepest part (hair bulb part) reaching the fat layer and the number of hair follicles, the histological evaluation of the hair follicle, and the skin of the sample application part It is characterized by evaluating using one or more combinations selected from colors.

  In addition, the mouse used for the in vivo evaluation test of the hair growth / hair growth effect described above, wherein hair is removed in the rest period of the hair cycle of the mouse, and the transition from the rest period to the growth stage is forcibly induced. The present invention also includes a method for producing a mouse model characterized by synchronizing the hair cycle.

  The hair growth / hair growth agent screening method of the present invention is carried out by performing at least one of the above-described in vivo evaluation methods for hair growth / hair growth for evaluating the hair growth / hair growth action, and extending to the growth phase or to the regression phase. It is characterized by finding a novel hair growth candidate substance having an effect of delaying migration.

Further, the hair growth / hair growth agent screening method of the present invention is prior to the test by the in vivo evaluation method,
By measuring the degree of inhibition of fibroblast growth factor 5 (FGF-5) action, in vitro evaluation for selecting a substance or biological extract having the FGF-5 inhibitory action is performed,
Next, it is desirable to conduct a test by the in vivo evaluation method using the selected substance or biological extract as a sample to find a new hair growth candidate substance.

The in vitro evaluation was performed using a culture medium having cultured cells sensitive to FGF-5 and interleukin-3 (IL-3), FGF-5 or interleukin 3 for positive control, and a sample. After culturing with the addition of a certain biological extract or substance, or vehicle, the inhibition of the FGF-5 action of the biological extract or the substance is examined by measuring the degree of proliferation of the cell. It is to evaluate hair growth and hair growth action.

The cultured cells are preferably FR-Ba / F3 cells or BALB / 3T3 cells.
Said inhibition is to prevent the promotion of cultured cell proliferation by FGF-5.
The biological extract is, for example, a plant extract.
Furthermore, in the above-described method for in vitro evaluation of hair growth / hair growth action, a kit containing at least FGF-5 and interleukin 3 as cell growth stimulating substances in reagents is preferably used.
[Detailed Description of the Invention]
The method of the present invention is a method for evaluating a hair growth / hair growth action for searching a substance or biological extract that can be a candidate component of a hair growth agent / hair growth agent from a very large number of samples. In order to efficiently select a substance or biological extract to be screened from those having true hair growth / hair growth action, whether or not an evaluation method is adopted is also important. In the present specification, “hair cycle” refers to a certain cycle of hair production in the hair follicle. Moreover, the "synchronization" means that the hair cycles of individuals are aligned in the same phase between each part of the body surface in the individual and between individuals. In vivo evaluation method The in vivo (in vivo) evaluation method for hair growth / hair growth activity according to the present invention is a method for applying a sample for examining the presence of hair growth / hair growth activity to the skin surface, and from the late growth stage of the hair cycle. By measuring changes in the shape or state of the hair follicle, hair, and applied skin surface that occur at the time of transition to the regression phase, the hair growth can be evaluated using the extension of the growth period of the hair cycle or the delay of transition to the regression phase as an evaluation index. -It is a method to evaluate the expression of hair growth effect. In such a method, the hair of an animal is removed, a sample containing the substance to be tested or a biological extract of the sample is applied by a method such as application, oral, subcutaneous injection, etc. The morphological change is examined, and the effect is judged by comparison with the control group. This is in common with previously known in vivo assessment methods. However, the in vivo evaluation method of the present invention pays attention to a rapid change in the shape of the hair follicle that occurs during the transition from the late growth phase to the regression phase of the hair cycle, and measures and evaluates this as an evaluation parameter. It is characterized by being a test.

When an animal model for in vivo evaluation is prepared, any animal (except human) that can be used can be used without particular limitation as long as it is used in such an experiment. Examples of animals include rodents such as mice, rats, guinea pigs, and rabbits, dogs, cats, and monkeys. Of these, rodents that can handle a large number of cases are preferable, and mice are particularly desirable. As the animal strain, a pure strain is preferably used because individual differences can be controlled to some extent, and a pure strain is particularly preferable. Furthermore, since hair growth can be traced by color difference or the like, it is preferably a colored animal, and more preferably a colored mouse. As such a colored pure mouse, a C3H / HeN mouse can be preferably exemplified. In addition, in order to reduce individual differences within the group of animals,
It is desirable to synchronize the hair cycle between individuals. This test method is performed using a mouse model with synchronized hair cycle.

  Within the growth period of the mouse, that is, within 50 days after birth, the hair cycle is basically uniform, and in the group of mice having the same age of the mouse, they have the same hair cycle. This mouse model is characterized by synchronizing all the hair cycles among individual mice by removing the hair cycle of the mouse in the rest period and forcibly inducing the transition from the rest period to the growth stage. The hair removal method is preferably hair removal, so that the irritation to the skin is not constant unlike shaving. In addition, since hair removal is in the rest period, hair removal can be easily performed. According to the forced synchronization of the hair cycle by such hair removal, the synchronization intensity within the group of mice is always high and constant, the reliability of the hair cycle synchronization is high, and therefore the sensitivity and accuracy of the test can be improved.

  A mouse used in the in vivo evaluation test for the hair growth / hair growth effect described above, wherein the hair is removed in the rest period of the hair cycle of the mouse, and the hair is forced to induce the transition from the rest period to the growth stage. A method for producing a mouse model characterized by forcibly tuning the period is also included in the present invention.

  The in vivo evaluation test of the hair growth / hair growth effect is carried out by determining whether the growth period in the hair cycle is prolonged or the transition to the regression stage is delayed. The determination of the observation time of the hair growth / hair growth effect can be easily determined because the hair cycle is strongly synchronized in the forced synchronization by the hair removal method. In other words, it is easy to determine the date of transition to the growth period. As the observation period of the effect, any of the early stage, middle stage, late stage and further regression stage of the growth period is suitable. As long as the effect can be reliably determined, a method that requires a short period is desirable. In the method of the present invention, the test period can be arbitrarily set between the next day and 22 days from the day of hair removal. Measuring and evaluating the form of hair follicles, etc. is performed on the first day to the 22nd day after the hair removal, preferably on the 17th to the 21st day, more preferably on the 19th and 20th day after the hair removal. Is set.

    Instead of hair removal, a hair removal method by shaving can also be used. Shaving in the Ogawa method is performed during the rest period. In this case, the synchronization of the hair cycle is natural, and it is actually difficult to shave without affecting the natural synchronization. That is, shaving is random with respect to synchronization, and the effects of shaving are not uniform, so that the intensity of synchronization within a group of mice is not constant from test to test. On the other hand, in this invention, when shaving is employ | adopted as a hair removal method, hair is removed in a growth period. Using natural synchronization is the same as the Ogawa method, but shaving in the growth period has less effect on the hair cycle than shaving in the rest period, and even within natural synchronization The strength of is considered higher. In the in vivo evaluation based on shaving according to the present invention, the period of observation of the hair growth / hair growth effect is the growth period and the regression period. The object of evaluation is hair with an extended growth phase and hair with a delayed transition to the regression phase.

Furthermore, in the Example mentioned later, it was investigated whether the hair cycle exists in the same phase also in the site | part from which a body surface differs in the same mouse | mouth individual | organism | solid. As a result, it was found that the hair cycle was the same on the left and right sides of the back of the same mouse individual, and that each side of the back could be used independently for the evaluation test. As a result, it has become possible to establish an evaluation method that can be carried out with a smaller number of animals than the evaluation method that uses the entire back surface of the mouse as a range, and to construct an efficient screening method. In addition, it is also the present invention that each phase constituting the hair cycle of the mouse, that is, the phase in which the hair cycle synchronization within the mouse group can be performed more reliably among the growth phase, regression phase and rest phase is the rest phase. It became clear by their research. Usually in 7-8 week old mice, the hair cycle is in the resting phase.

There are a wide variety of evaluation parameters used to determine hair growth / hair growth effects. Conventionally, an evaluation index has been observed by capturing the timing of transition from a rest period to a growth period. Changes in the morphology or condition of the hair follicle, hair, and applied skin surface that occur during the transition from the late growth phase of the hair cycle to the regression phase are also dynamic changes that can be detected and measured as appropriate. . Specifically, increase in the number of hair growth, increase in hair density, increase in hair diameter, hair follicle growth, growth effect, hair follicle length, fat layer at the deepest part of hair follicle (hair bulb) And the number of hair follicles reached, the histological evaluation of hair follicles, and the skin color of the sample application part. As an evaluation object of the hair growth / hair growth effect, the object of particular attention is the increase in the hair whose growth period is prolonged or the hair whose transition to the regression stage is delayed. In the method of the present invention, the change in the form or state reminiscent of the hair follicle length is the length of the hair follicle, the extent to which the hair follicle deepest part (hair bulb part) reaches the fat layer, the number of hair follicles reached, and the histology of the hair follicle. The skin color of the sample evaluation part and the sample application part is taken and evaluated using one or more combinations selected from these.

  The hair removal method and shaving method of the present invention described above and the Ogawa method are summarized in Table 1 for comparison.

Method for Screening Candidate Substances Having Hair Growth / Hair Growth Action The above-mentioned in vivo evaluation method uses the animal for hair growth / hair growth action of a biological extract or candidate substance (also referred to as “sample”). This includes evaluation and selection based on this, and is used for screening substances having hair growth / hair growth effects. This is because, in evaluating hair growth / hair growth action, it is desirable to use an in vivo evaluation method in order to confirm whether the hair growth / hair growth action is actually expressed in animals. The evaluation method of the present invention evaluates the dynamic follicular shape change at the time of transition to the regression phase with one index, preferably a combination of a plurality of indices. From such a viewpoint, it focuses only on the transition from the rest period to the growth period, and is distinguished from the conventional in vivo evaluation method that mainly evaluates the hair growth effect. When the in vivo evaluation method of the present invention is used for screening a candidate substance having a hair growth / hair growth action, either the hair removal method in the rest period of the mouse hair cycle or the shaving method in the growth phase may be used. Good. In such screening, the in vivo evaluation method of the present invention may be used alone.

  Incidentally, the in vitro evaluation method has an advantage that a large number of samples can be processed quickly. In order to be a preferred in vitro evaluation method, other requirements include that the analysis requires multi-step processing, such as separation and purification steps, no complicated quantitative operation, short evaluation time, and measurement method. It is based on simple analysis, and the apparatus and measurement are simple, and there are few errors and variations in results, and the reliability and reproducibility of evaluation are good. Similarly, since the in vivo evaluation method uses animals, the variation in individual differences is minimized by synchronizing the hair cycle between groups of animals and within groups, and the hair growth of the narrowed candidate subjects・ It is desirable to be able to accurately evaluate the effectiveness as a hair restoration agent. Therefore, in the screening method of the present invention, the hair growth / hair growth action is mainly confirmed by an in vivo system as much as possible, but an in vitro evaluation method can also be incorporated if necessary. As a preferred embodiment, in vitro evaluation test by FGF-5 inhibition test as primary screening, candidate subjects are narrowed down based on the results, and then in vivo evaluation by animal application test as secondary screening using animals A configuration for performing a test is mentioned.

As a measurement method for in vitro evaluation in the present invention, in order to provide rapidity, operability, reliability, etc., animals are not used, but instead FGF-5 sensitive cultured cells, preferably FGF-5. It was found that an inhibition test of FGF-5 action using cultured cells that are sensitive to both leucine and interleukin-3 (IL-3) is more suitable. In the subsequent in vivo evaluation, the best combination is to adopt a method that allows the experiment of applying the biological extract to be performed in the shortest possible time by synchronizing the hair cycle of the animal. This evaluation method, which is composed of in vitro evaluation and then in vivo evaluation, is constructed based on a policy of verifying hair growth / hair growth action from various aspects.

In conventional screening methods based on in vitro evaluation, tissues around the hair of animals (for example, tissues including hair follicles and hair papilla) or hair-related cells (for example, hair papilla cells, hair follicle keratinocytes, etc.) are collected and generated. The method of verifying the hair and hair growth effect is taken. However, in these evaluation methods, there is no change in using animals even in vitro, and it is complicated to appropriately collect tissues and cells from animals. It is also susceptible to individual differences, sampling techniques, conditions, and so on. In the present invention, FGF-5 sensitive cultured cells, preferably FGF-5 and IL, which are prepared from cell lines that are commercially available and well-characterized, rather than collecting tissue or cells from such animals each time. -3 Use cultured cells that are sensitive to both. Thereby, it is possible to process a large number of samples, and high-throughput screening can be achieved. A screening method for a substance having a hair growth / hair growth action, which includes evaluating and selecting the hair growth / hair growth action of a biological extract or substance using such an evaluation method, is included in the present invention. The biological extract to be searched for hair growth / hair growth action is preferably a plant extract.

Therefore, the screening method for hair growth / hair growth agent according to the present invention is carried out by conducting at least one of the above-described in vivo evaluation methods for hair growth / hair growth for evaluating the hair growth / hair growth action of a biological extract or substance. It is a method of finding a novel hair growth candidate substance having an effect of extending the period or delaying the transition to the regression stage. A more preferred embodiment of the hair growth / hair growth agent screening method is:
Prior to testing by the in vivo evaluation method,
By measuring the degree of inhibition of fibroblast growth factor 5 (FGF-5) action, in vitro evaluation for selecting a substance or biological extract having the FGF-5 inhibitory action is performed,
Next, the selected substance or biological extract is subjected to a test by the in vivo evaluation method to find a new hair growth candidate substance.

It should be noted that the in vivo method may be adopted immediately when the in vitro measurement method is clearly unnecessary or impossible. Next, the inhibition of FGF-5 action and the hair growth / hair growth action, which are the physiological basis of the above in vitro evaluation method, will be described.
Inhibition of Fibroblast Growth Factor 5 (FGF-5) Action According to the study by the present inventors, FGF-5 has an action of inhibiting hair growth and shifting the hair cycle from the growth phase to the regression phase. It became clear for the first time. On the other hand, the hair papilla at the base of the hair follicle controls hair growth by regulating the growth and differentiation of hair matrix cells. In the regression phase, the hair matrix cells disappear, but the number of dermal papilla cells decreases but does not decrease. In the next growth phase, the hair papilla cells are activated again, and the hair matrix cells begin to proliferate and differentiate anew due to the action. Since the dermal papilla cell itself has not changed, hair of the same thickness as the previous growth period can be made. Therefore, if the action of FGF-5, which plays the role of a trigger that shifts from the growth period to the regression / rest period, is suppressed by some method, only the growth period is extended. Therefore, a substance that lengthens the growth period in this way is expected to exhibit an excellent hair growth / hair growth effect.

In fact, FGF-5S (short-chain molecule of FGF-5) expressed in large amounts in follicles in the growth phase delays the transition from the growth phase to the regression phase by showing antagonist activity against FGF-5, Promoting hair growth has been reported by the inventors. The present inventors have further studied and found that an extract obtained from a specific plant or seaweed has an activity of inhibiting FGF-5 action. Such an extract may contain one or more substances with antagonist activity similar to FGF-5S, or there may be inhibitors that directly inhibit FGF-5. The mechanism of FGF-5 inhibition by such extracts and the substance of the active substance must wait for future research results.

FGF-5 is not particularly limited as long as it can regulate the hair cycle and induce hair cycle abnormality when administered to animals, animal hair surrounding tissues, and animal hair-related cells. Absent. Therefore, if it is possible to induce abnormal hair cycle, a fragment of FGF-5, a recombinant protein containing a functional region of FGF-5, a protein chemically modified to FGF-5, a high homology with FGF-5 A protein having a property, for example, a protein having a substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence of FGF-5 can also be used as FGF-5 of the present invention. . FGF-5 that can be used in the present invention can be purchased as a commercially available reagent from Sigma or the like. Furthermore, FGF-5 that can be used in the present invention can also be prepared by expressing and extracting as a recombinant protein by a genetic engineering method (Clements et al. 1993).
In vitro evaluation using cultured cells The in vitro evaluation of the present invention is to determine whether the substance or biological extract to be screened inhibits FGF-5 action that promotes the growth of cultured cells. The main point. The in vitro evaluation is based on an analysis with a simple measuring method, and therefore, the analysis does not require multi-step processing, for example, a separation and purification process, complicated quantitative operation, and the like. The time required for the evaluation is short, and the tools and devices used are simple. Specifically, the hair growth / hair growth effect is confirmed by a detection test of FGF-5 inhibitory action using cultured cells on a plastic test tool, for example, a solid support such as a plate, chip, or disk. In order to increase the throughput of such primary screening, it is desirable to use a multiwell plate and measure the degree of inhibition of cell proliferation, which is an indicator of inhibition, on the multiwell plate as it is.

In the in vitro evaluation method of the present invention, cultured cells that are FGF-5 sensitive, that is, whose growth is promoted by addition of FGF-5, more preferably FGF-5 and a growth promoting substance other than FGF-5, for example, Use cultured cells that are sensitive to both cytokines, for example, interleukin 3 (IL-3). Are they already on the market,
Alternatively, it is a cell that can be easily produced. Among the preferred FGF-5 sensitive cell lines, F
FR-Ba / F3 cells that are responsive to both GF-5 and IL-3. FR-Ba / F3 cells are cell lines that stably express "FGF-Receptor 1c" by introducing "FGF-Receptor 1c" cDNA with an expression cassette into "Ba / F3 cells". . Here, FGF-Receptor 1c (hereinafter abbreviated as “FR”) means a receptor for FGF-5 (FGF-Receptor). The parent cell line “Ba / F3 cell line (mouse IL-3-dependent proB cell)” of FR-Ba / F3 cells can be obtained from various places including RIKEN Cell Bank. For those skilled in the art, using conventional genetic engineering techniques, obtaining cDNA expressing “FGF-Receptor 1c” and introducing it into the parent cell line is within the scope of normal genetic engineering operations. The FR-Ba / F3 cells are IL-3 dependent, similar to its parent strain “Ba / F3 cells” (IL-3, a different kind of cytokine that stimulates and promotes the proliferation of a wide range of cells, like FGF-5) Otherwise, it cannot survive or proliferate). In addition, FR-Ba / F3 cells acquire responsiveness to FGF-5 (also FGF-1 and 2) by expressing FR, and even in the absence of IL-3, FGF-5 and heparin. If FGF binds to FR and the signal is input into the cell, heparin binding is also required. Thus, it has a feature that it can survive and proliferate. Thus, since FR-Ba / F3 cells respond to both cytokines, as a positive control, by examining the response to IL-3 together, the proliferation ability of the cells used or the culture of the biological extract or substance itself was cultured. There is also an advantage that the influence on cells can be checked. Therefore, FR-Ba / F3 cells are particularly desirable cultured cells from the viewpoint of reliability of the results of the in vitro evaluation method.

Examples of other preferred FGF-5 sensitive cells include BALB / c3T3 or NIH / 3T3 fibroblasts, which are also commercially available. These cultured cells are suitable for quantitative in vitro evaluation, in addition to the advantage of convenient culture and handling. However, if animal hair-related cells (for example, hair papilla cells and hair follicle keratinocytes) are readily available and can be stably subcultured for a long period of time, such in vitro evaluation of the present invention. It does not exclude the use of cells, but rather may be suitable because it is a cell involved in hair growth physiology.

As a specific procedure in a preferred embodiment of the present invention, cultured cells sensitive to FGF-5 and IL-3 are cultured in a culture medium containing 10% fetal bovine serum. For example, it is transferred to a 96-well culture plate, and FGF-5 or interleukin 3, and a suitably diluted sample (biological extract or substance) or vehicle are added to the culture medium and cultured. Alternatively, the subject biological extract or substance may be added to the FGF-5-containing culture medium in which the cells are cultured. The dose to be added here varies depending on the cells used, culture conditions, etc., but the concentration in the culture solution is preferably 1 ng to 10 mg / ml, and the dose of the biological extract or substance is set at least several points by changing the dilution rate. It is preferable to do this. This is because it is preferable to examine dose-dependence in order to accurately determine the effect of addition.

After culturing for a certain period, the degree of proliferation of the cells is measured. After adding a commercially available cell counting kit to each well and culturing for a certain period of time, for example, 1 to 24 hours, preferably 2 to 8 hours, specifically 3 hours, compared to IL-3-dependent growth, FGF It may be examined at which concentration it inhibits -5 dependent FR-Ba / F3 cell proliferation. The degree of inhibition is 50% of the maximum inhibition
The inhibitory concentration can be compared. For example, cell proliferation can be examined by counting the number of cells using a microplate reader. Alternatively, the effect may be determined by adding a substance (such as thymidine) that serves as an index of cell proliferation to each cell and observing its uptake. When taking up thymidine or the like as an index, it is desirable to label with radioactive isotopes such as ³ H and ¹⁴ C in advance. For comparison of the degree of proliferation, a control containing a vehicle instead of the test substance is used as a control. Other substances already known to be effective, such as minoxidil, may be used as a reference and compared. A quantitative comparison can be made regarding the strength of hair growth and hair growth effect.

If the FGF-5 dependent growth of the cultured cells is suppressed, the added biological extract or substance of the sample inhibited the stimulating action of cell growth by FGF-5. The activity of a biological extract or substance to inhibit such FGF-5 action is expressed as a relative percentage of cell proliferation relative to a control group administered vehicle, for example in%. This leads to the suppression of the action of such biological extracts and the like inducing abnormal hair cycle of FGF-5, that is, to have a hair growth / hair growth effect. In-vitro evaluation is based on numerical evaluation of hair growth and hair-growth effects. The evaluation criteria are divided into several stages and judged according to the criteria, or the minimum requirement for performing the next in-vivo evaluation. A reference value may be provided.

A method for screening a substance having a hair growth / hair growth action, which includes evaluating and selecting the hair growth / hair growth action of a biological extract or substance using the above in vitro evaluation method is included in the present invention. For efficiency, it is desirable to keep the culture conditions and measurement conditions constant in addition to the reagents, equipment, and equipment used. From this point of view, a kit used for the in vitro evaluation method of hair growth / hair growth action, comprising at least FGF-5 and interleukin 3 as cell growth stimulating substances in the attached reagents Is also recommended. Such kits may include microplates, buffers, cell proliferation assay reagents, culture media, and the like as necessary.
In vivo evaluation using an animal model In vivo evaluation in the screening method of the present invention is characterized in that a sample substance or biological extract is applied and administered to an animal, preferably a mouse model whose hair cycle is forcibly synchronized. Yes. Next, a step of measuring the hair follicle shape or the like at an appropriate observation time and evaluating the hair growth / hair growth effect of a substance that can be a candidate for a hair growth agent or a hair growth agent component or the extract is included.

In order for a candidate substance or biological extract to be determined to have a hair growth / hair growth effect, it is necessary to actually extend the growth period of the hair cycle or delay the transition to the regression stage, Specifically, it is confirmed as follows. The back of the mouse whose hair cycle is in the resting phase is removed to induce the growth phase. The candidate substance or biological extract (for example, 50% ethanol solution) is usually applied once a day immediately after hair removal. The day after the final application, the skin of the mouse is collected, sliced sections are prepared, and the shape and size of the hair follicle are compared. In the vehicle application group (for example, 50% ethanol) as a control group, the hair follicles are short and have already entered the regression phase, whereas in the biological extract application group, the hair follicles are long and the growth period is sustained. Make sure.

In the control group, a vehicle is applied and administered. As the vehicle to be used, an aqueous vehicle that can be used in a normal animal test can be used. For example, a 50% aqueous ethanol solution, phosphate buffered saline, etc. are preferable. it can. Furthermore, the application | coating site | part in an animal is not specifically limited as long as the synchronization of a hair cycle is maintained. From the viewpoint of facilitating the operation in the evaluation method of the present invention, the vicinity of the back of the animal is preferable. In particular, when the left and right sides of the back of the mouse are forcibly tuned during the hair cycle rest period of the mouse, one of two independent ranges on both sides of the back can be used for control, which is advantageous in that the number of animals used can be reduced. is there.

Alternatively, an alopecia model in which FGF-5 is administered to an animal to induce abnormal hair cycle, a sample is administered thereto, and the same in vivo evaluation of hair growth / hair growth effect may be performed. For example, FGF-5 is subcutaneously injected into the same site every time after the growth period is induced with respect to the hair cycle, and a biological extract is applied around the injection site a predetermined number of times a day. Repeat for 10 days. In this alopecia model, the activity of inhibiting the FGF-5 action by application is shown as the hair growth / hair growth action, or the degree thereof is evaluated through measurement of the form and length of the hair follicle.

  The method of administering the biological extract to the mouse model is not particularly limited as long as the hair growth / hair growth effect after administration can be determined. Usually, those according to actual use are preferred, and transdermal administration is particularly preferred. As a specific example, there is a method in which the biological extract is applied to a site where hair cycle synchronization is induced. The dose is preferably about 1 ng to 10 mg dry weight / individual / once. This operation may be repeated only once or several times.

  The method for determining the hair growth / hair growth effect in the mouse model is not particularly limited, and a method generally known as a method for determining such an effect may be used. For example, there is a method for determining the effect by comparing the administration group of the extract with the non-administration group. Or about the said parameter | index, it measures simultaneously using another substance already known in effectiveness, for example, minoxidil, and you may compare on the basis of the result. A quantitative comparison can be made regarding the strength of hair growth and hair growth effect.

  Here, as an index for comparison, hair growth or hair follicle morphology is preferably measured. Hair growth includes various physiological phenomena related to the growth of hair, surrounding tissues of hair, and hair-related cells. Specifically, hair amount, hair length, hair diameter, hair growth rate, hair growth rate, increase in hair growth number, hair density, hair removal number, hair follicle shape and size, hair The histological evaluation of the capsule, the effect of extending the growth period, the degree and number of hairballs reaching the fat layer, the skin color of the sample application part, etc. are compared. In the comparison, the effect can be determined by quantification using visual observations, photographs, observations by tissue analysis, etc., hair cycle, etc. as an index. Skin sections of the application site may be prepared and hair follicle morphology and size may be compared. Or the following method is illustrated as a method of comparing the hair cycle as a parameter | index and determining an effect. When entering the growth phase of the hair cycle, melanin production is started in the hair follicle melanocytes, and as the growth phase progresses, the amount of melanin production increases and the skin darkens. Therefore, the effect can be determined by comparing the degree of progress of the growth period by measuring the brightness of the skin.

Specifically, skin slice images observed under an optical microscope are photographed and image data taken into a computer to compare the shape and size of hair follicles in the slices. If the hair follicles of the mouse experimental group to which the candidate substance was applied promoted hair growth more than the vehicle-applied control group, it can be determined that there is a hair growth / hair growth effect. Further, if the same experiment is started from the latter half of the growth period to delay the transition of the hair cycle from the growth period to the regression stage, it is estimated that the hair growth activity is obtained. The evaluation at that time may be determined according to the criteria by providing evaluation criteria divided into several stages, for example, five stages, regarding the digitized hair growth / hair growth effect.
Biological extract and its preparation In the above evaluation of hair growth / hair growth action, the samples actually measured and evaluated are examined for the presence or strength of the biological extract prepared from various organisms or hair growth / hair growth action. A compound or substance. A compound or substance for examining the presence or strength of hair growth / hair growth action is used by dissolving or dispersing in water or a suitable solvent. In addition, the “biological extract” in the present specification is not necessarily limited to a general extract from a living organism, and may be prepared by arbitrarily setting a certain preparation method. Good. It can be applied to any biological material such as plants and animals. In addition, although the Example mentioned later shows the example which narrowed down and searched for the plant extract, this does not exclude the extract of other biological origin from the object of the said screening.

  The process of preparing a biological extract as a sample is for removing or selecting impurities or coexisting substances, and techniques commonly used in the art such as washing, grinding, peeling, polishing, pressing, etc. A method such as sieving can be used. The step of preparing an extract from a living organism can be performed using water, a solution containing salts, or an appropriate extraction solvent. The crude extract thus obtained can be used as it is as a sample for the evaluation system. Or you may use for screening evaluation as a fraction form which is obtained by performing a fractionation process and has an active substance of hair growth and hair growth action. The degree of the fractionation process is arbitrary and is performed as necessary. That is, operations such as concentration, drying, or re-dissolution in water or a polar solvent, or purification treatment such as decolorization, deodorization, and desalting, and fractionation treatment by column chromatography, etc., as long as the physiological effects are not impaired. Further purification by may be performed.

  The plant extract used as the best mode for carrying out the present invention can be obtained by extracting from a plant with water or a solvent other than water. Here, the “plant extract” means a plant body such as leaves, stems, bark, flowers, berries, roots, stamens, whole grass or sap, etc., raw or dried. , If necessary, after various processing such as pulverization, heat treatment, etc., it means various solvent extracts obtained by extraction with water or an appropriate solvent, diluted solutions, concentrated solutions, or dried powder .

  When preparing a plant extract, the plant may be subjected to an extraction process as it is. However, considering extraction efficiency, it is desirable to perform extraction after processing such as shredding, drying, and pulverization. Extraction is performed by immersing in an extraction solvent. To increase efficiency, continuous or intermittent stirring may be used, or homogenization may be performed in the extraction solvent. The extraction temperature is usually 5 ° C. to the boiling point of the extraction solvent. Although the extraction time varies depending on the properties of the extraction raw material, the type of extraction solvent, the extraction temperature, etc., it is generally appropriate to be about 1 to 14 days. The extraction operation may be performed continuously using an extraction device such as a Soxhlet extractor. The extraction method is not particularly limited, and may be an ordinary method, for example, heat extraction or normal temperature extraction. Furthermore, in addition to normal pressure, extraction may be performed under pressure.

As a solvent for extraction, in addition to water, lower alcohol solvents such as methanol, ethanol, propanol and isopropanol, polyhydric alcohol solvents such as polyethylene glycol, 1,3-butylene glycol, propylene glycol, dipropylene glycol and glycerin , Polar solvents such as ether solvents such as ethyl ether and propyl ether, ketone solvents such as acetone and ethyl methyl ketone, and ester solvents such as ethyl acetate and butyl acetate can be used. In particular, a solvent having excellent solubility of a substance and having a relatively low freezing point and boiling point is preferably used. One or more of the above solvents are selected and used for extraction. What is necessary is just to select an extraction solvent suitably according to the kind, site | part, property, etc. of the plant to extract. For example, in the case of plants that contain a large amount of calcium oxalate that causes dermatitis, extraction by alternately using a plurality of solvents that are a combination of water and the above organic solvent, or by distribution using a mixed solvent By performing the above, it is possible to obtain an extract from which inappropriate components have been selectively removed. Alternatively, physiological saline, phosphate buffer, phosphate buffered saline, or the like can be used. If necessary, a fluid method such as supercritical carbon dioxide disclosed in Japanese Patent Application Laid-Open No. 62-172096 may be employed.
Biological extract judged to be effective The biological extract judged to have a hair growth / hair growth effect by the evaluation system of the screening method of the present invention contains one (or more) active substance for hair growth / hair growth. In addition, it can be contained as it is as an active ingredient in a hair restorer or cosmetics as it is, or it can be used together with other hair restorer ingredients. In consideration of formulation conditions such as preservation and hair-restoring agents, further treatments such as concentration under reduced pressure, dilution, and filtration may be performed as appropriate. The solution of the biological extract may be processed into a dried product by concentration, spray drying, freeze drying, or the like. From the above-mentioned biological extract, a highly active fraction containing a substance having a hair growth / hair growth action as an active ingredient may be obtained by further proceeding fractionation. Fractionation can be performed by a commonly used fractionation method, for example, various methods such as centrifugation, ultrafiltration, salting out, ion exchange chromatography, or a combination of such separation methods. Isolation and identification of a substance that exhibits hair growth / hair growth action as a physiological action from the fraction can be carried out by using conventionally used natural product chemistry or biochemical techniques.
〔The invention's effect〕
The in vivo evaluation method for hair growth / hair growth action according to the present invention uses a mouse whose hair cycle is forcibly synchronized, and measures the change in hair follicle shape as an evaluation index. For this reason, the difference between individuals, the error of measurement results, and the variation are reduced, and the hair growth / hair growth action can be accurately evaluated for the candidate object in a short period of time.

Furthermore, this method is effective in evaluating the effect of extending the growth phase or delaying the transition to the regression phase, and makes it possible to search for new hair-growth substances having such unique hair growth / hair growth properties.
This method is an in vivo evaluation method useful for screening a hair-growth agent candidate substance, which has higher sensitivity and accuracy of the test and can make a hair-growth determination in a shorter period of time compared to a conventionally known method. A screening method consisting of in vitro evaluation to examine the activity of inhibiting FGF-5 action using cultured cells and subsequent in vivo evaluation can efficiently process a large number of samples and achieve high throughput. Is possible.
〔Example〕
EXAMPLES Hereinafter, although an Example is given and this invention is further demonstrated, this invention is not limited only to such an Example. Unless otherwise stated,% is% by weight.

Preparation of plant extracts Ages (pseudofruits and berries of the rose, Rosa multiflora Thunb.), Leaves of loquat (Eriobotrya japonica Lindley), rhizomes of scallops (Sanguisorba officinalis), Arctostaphylos uva-urusi (Linne) Sprengel (Ericaceae) Leaves, stems (scientific name Ilex Paraguariensis), cats claw (scientific name Uncaria tomentosa), mugwort (scientific name Artemisia princes) leaves and stems, brown algae (Brown algae Brown Paleophyceae) all algae all dry weight 200g On the other hand, 50% aqueous ethanol solution was immersed in 1 liter at room temperature for 1 to 10 days to extract soluble components. Separation of the extract from the extraction residue was performed by centrifugation or filtration. The obtained supernatant was used as a sample.

Mouse natural hair cycle
37-day-old, 38-day-old, 39-day-old, 40-day-old, 41-day-old and 42-day-old C3H / HeN mice (purchased from Japan SLC) were killed by cervical dislocation, and the skin was shaved after shaving the back. Collected. The collected skin was fixed with a 10% formalin aqueous solution, then treated with an ethanol dehydration series, clarified with xylene, and embedded in paraffin. Using a microtome (manufactured by Yamato Co., Ltd.), a thin slice of 4 μm in thickness, cut parallel to the body axis, where the hair follicle can be completely observed from the skin opening to the hair bulb, and a silane-coated slide glass ( (Matsunami Co., Ltd.) After deparaffinization with xylene and treatment with ethanol hydrophilic series, the sections were stained with hematoxylin and eosin, treated with ethanol dehydration series and clarified with xylene, and sealed with Canadian balsam.

  The skin slice images observed under the optical microscope were photographed, and image data was taken into a computer to compare the shape and size of hair follicles in the slices. As a result, it was confirmed that there was no significant difference in hair follicle length among the mice of each day age, and the hair cycles were aligned. In addition, in the 40-day-old mice, the hair follicle length was rapidly shortened compared to the 39-day-old mice (FIGS. 1 and 2).

  In addition, 39-day-old C3H / HeN mice (purchased from Japan SLC) were sacrificed by cervical dislocation, and two independent areas of equal area on both the left and right sides were shaved on the back of the mouse, and then the skin at each site. Were collected. In the same manner as described above, a sliced slice of the collected skin was obtained, photographed under an optical microscope, and the morphology and size of the hair follicle in the slice were observed and compared. As a result, there was no difference in the shape and size of the hair follicles in the left and right parts of the mouse back.

  From these results, it was found that the hair cycle was synchronized in the same-day-old mice, and that the hair cycle was also synchronized between the left and right of the back (FIG. 3).

Forced synchronization to the growth period by hair removal
A 49-day-old (hair cycle is resting) C3H / HeN mouse (purchased from Japan SLC) was preliminarily raised for one week, and the back of the mouse was removed to forcibly induce the transition to the growth phase.

From the day after the hair removal date, 0.05% / cm ² of 50% ethanol was applied to the hair removal site once a day. The skin color of the mice was observed on the 17th, 18th, 19th and 20th days after hair removal. The mice on the 20th day whose skin color changed were sacrificed by cervical dislocation, and the skin at the application site was collected. The collected skin was processed in the same manner as in Example 2, and an optical micrograph of the obtained skin slice was taken to observe the shape and size of the hair follicle (FIG. 4).

  The skin color of the mice on the 17th, 18th, 19th and 20th day after hair removal is the same color in the same age group of mice, and the hair follicle morphology (including length) in the 20th day mouse However, it was clarified that the hair cycle of all the mice subjected to the test can be forcibly synchronized by removing the mice in the resting period from the fact that they are arranged in the group.

In the same manner as in Example 3, the back of the C3H / HeN mouse was removed to forcibly induce the growth phase. 0.05mL / cm ² once a day with 50% ethanol or cat's claw extract on the hair removal site from the day after the hair removal date
It applied one by one. The skin color of the mice was observed on the 17th, 18th, 19th and 20th days after hair removal. The mice on the 20th day whose skin color changed were sacrificed by cervical dislocation, and the skin at the application site was collected. The collected skin was treated in the same manner as in Example 2, and an optical micrograph of the obtained skin slice section was taken, and the morphology and size of the hair follicle in the mouse skin slice section on day 20 were observed (FIG. 5). ).

  The number of hair follicles reaching the fat layer in mice on the 20th day after hair removal was higher in the cats claw extract applied group than in the 50% ethanol applied group, and histologically apparently in the cats claw extract applied group A prolonged or delayed transition to regression was observed (Tables 2 and 3). In addition, regarding the hair follicle length, it was revealed that the cat's claw group was significantly longer 20 days after hair removal (FIG. 6).

  A 49-day-old (hair cycle is resting) C3H / HeN mouse (purchased from Japan SLC) is preliminarily raised for one week, and two independent areas of equal area on both the left and right sides are removed on the back of the mouse. Forced the transition to the growth period.

From the date of hair removal, 0.05 mL / cm ² of 50% ethanol was applied to the hair removal site once a day. The mice on the 19th and 20th day after hair removal were sacrificed by cervical dislocation, and the skin at the application site was collected. The collected skin was processed in the same manner as in Example 2, and an optical micrograph of the obtained sliced skin was taken to observe the shape and size of the hair follicle (FIG. 7).

  As a result, there was no difference between the left and right sides of the back of the hair follicle (length, etc.). From this, it became clear that the hair cycle of the total number of mice used in the test can be forcibly synchronized by removing two independent areas on the left and right sides of the back of the mouse in the resting period.

In the same manner as in Example 5, two independent areas having the same area on both the left and right sides were removed from the back of the C3H / HeN mouse, and the transition to the growth phase was forcibly induced. From the date of hair removal, 50% ethanol was applied to the hair removal site on one side of the back, and Cats Claw extract was applied to the hair removal site on the other side once a day at 0.05 mL / cm ² . The mice on the 19th and 20th day after hair removal were sacrificed by cervical dislocation, and the skin at the application site was collected. The collected skin was processed in the same manner as in Example 2, and an optical micrograph of the obtained skin slice was taken to observe the shape and size of the hair follicle (FIG. 8).

  The number of hair follicles reaching the fat layer is higher at the cats claw extract application site than at the 50% ethanol application site, and histologically clearly the cats claw extract application site extends the growth phase or delays the transition to the regression phase Was observed. Further, regarding the hair follicle length, it was revealed that the cats claw application site was significantly long on both the 19th and 20th day after hair removal (FIGS. 9 and 10).

Detection of FGF-5 inhibitors using cultured cells (in vitro evaluation)
FR-Ba / F3 cells responsive to FGF-5 and interleukin-3 (IL-3) were cultured in a culture medium containing 10% fetal bovine serum. A 96-well culture plate (Falcon) was seeded at 10,000 cells / well, and 1 μg / ml FGF-5 or 10 ng / ml IL-3 was added to the culture medium, and an appropriately diluted sample (of Example 1). Plant extract) and vehicle 50% ethanol were added and cultured for 3 days. Thereafter, a cell counting kit (manufactured by Wako Pure Chemical Industries, Ltd.) was added to each well, and after culturing for 3 hours, cell proliferation was examined using a microplate reader. The result is shown in FIG. Any extract from Ages, Loquat, Warmoko, Walnut, Mate, Cat's Claw, Artemisia, and Brown Algae has a higher F than IL-3 dependent growth.
All of GF-5-dependent FR-Ba / F3 cell proliferation was inhibited at a low concentration. From these results, it was shown that the above-mentioned plant extract has selective FGF-5 inhibitory activity.

Mouse application experiment in the latter half of the growth period (shaving method in the growth period)
The back of 8-week-old C3H / HeN mice (purchased from Japan SLC) with a hair cycle in the resting phase was removed to induce the growth phase. 17 days after induction, the hair grown on the hair removal site was removed with a hair clipper, and the plant extract (50% ethanol solution) described in Example 1 was applied daily for 3 to 4 days immediately after shaving with a razor. Each time, 0.05 mL / cm ^{2 was} applied. Control mice receive 50% ethanol daily
Was applied in the same manner.

  The day after the final application, the mouse was sacrificed by cervical dislocation, and the skin at the application site was collected. Processing was carried out in the same manner as in Example 2, and optical micrographs of the obtained sliced sections were taken, and the shape and size of hair follicles in the sections were compared. The image is shown in FIG. In the ethanol (vehicle) application group, which is a control group, the hair follicles are short and have already shifted to the regression stage, whereas in the plant extract application group, the hair follicles are long and the growth period is sustained. Therefore, these plant extracts extend the growth phase of the hair cycle and clearly show that they have hair growth activity.

FIG. 1 shows a photomicrograph of hair follicles of 39-day-old mice. A follicle is a structure that extends from a sac-like hair bulb in the dermis toward the epidermis, in which growing hair is wrapped. FIG. 2 shows a photomicrograph of the hair follicle of a 40 day old mouse. Subcutaneous (fatty) tissue is seen at the bottom of the image. FIG. 3 shows micrographs of hair follicles in the left and right dorsal regions of 39-day-old mice. FIG. 4 shows a hair follicle micrograph of a 50% ethanol-coated mouse 20 days after hair removal. FIG. 5 shows a hair follicle micrograph of a cats claw extract-coated mouse 20 days after hair removal. FIG. 6 shows the hair follicle length (μm) of mice 20 days after hair removal. FIG. 7 shows hair follicle micrographs of mice coated with 50% ethanol on the 19th and 20th day after hair removal on the left and right sides of the back. FIG. 8 shows hair follicle micrographs of cats claw extract-coated mice on the 19th and 20th days after hair removal on the left and right sides of the back. FIG. 9 shows the hair follicle length on the 19th day after hair removal on the left and right sides of the back of the mouse. FIG. 10 shows the hair follicle length on the 20th day after hair removal on the left and right sides of the back of the mouse. FIG. 11 shows the inhibitory effect of extracts from various plants or seaweeds on FGF-5-dependent and IL-3-dependent growth of cultured FR-Ba / F3 cells. The horizontal axis represents the concentration of the extract in the medium (unit:% (V / V)). FIG. 12 is a photograph showing a skin slice image of a mouse whose hair cycle is at the end of the growth period. From the shape and size of the hair follicle in the section, in the group to which the vehicle 50% ethanol was applied (the scale in the photograph shows 0.1 mm, the other photographs have the same magnification), transition to the regression phase However, in the plant extract application group, that is, the group to which the extract of walnuts, brown algae, bitumen or age was applied, the growth period was maintained and the hair follicles were maintained for a long time.

Claims (10)

  Apply a sample to check the presence or absence of hair growth and hair growth on the skin surface, and measure the change in the shape or state of the hair follicle, hair, and applied skin surface that occurs during the transition from the late growth phase to the regression phase of the hair cycle. Thus, an in vivo evaluation method for hair growth / hair growth, wherein the expression of hair growth / hair growth effect is evaluated using an extension of the growth period of the hair cycle or a delay in transition to the regression phase as an evaluation index.   2. The in vivo evaluation method for hair growth / growth according to claim 1, wherein the method is carried out using a mouse whose hair cycle is forcibly synchronized by performing hair removal in a rest period of the hair cycle.   2. The in vivo evaluation method for hair growth / hair growth according to claim 1, wherein the method is carried out using a naturally-synchronized mouse shaved during the growth period of the hair cycle.   Regarding the change in the form or state, the length of the hair follicle, the degree of the hair follicle deepest part (hair bulb part) reaching the fat layer and the number of hair follicles, the histological evaluation of the hair follicle, and the skin of the sample application part 2. The in vivo evaluation method for hair growth / hair growth according to claim 1, wherein the evaluation is performed using one or a plurality of combinations selected from colors. A mouse used for the in vivo evaluation test for hair growth / hair growth effect according to claim 1,
A method for producing a mouse model, wherein the hair cycle of a mouse is removed in a resting phase, and the hair cycle is synchronized by forcibly inducing a transition from a resting phase to a growth phase.   A novel hair growth / hair growth test according to the in vivo evaluation method of any one of claims 1 to 4 is performed for evaluating hair growth / hair growth action, and has a new effect of extending the growth phase or delaying the transition to the regression phase A method for screening a hair growth / hair growth agent characterized by finding a hair growth candidate substance. Prior to testing by the in vivo evaluation method,
By measuring the degree of inhibition of fibroblast growth factor 5 (FGF-5) action, in vitro evaluation for selecting a substance or biological extract having the FGF-5 inhibitory action is performed,
7. The hair growth / hair growth agent according to claim 6, wherein the selected substance or biological extract is used as a sample to perform a test by the in vivo evaluation method to find a new hair growth candidate substance. Screening method. The in vitro evaluation was performed using FGF-5 or interleukin 3 for positive control, and a sample in a culture medium having cultured cells sensitive to FGF-5 and interleukin-3 (IL-3). After culturing with the addition of a certain biological extract or substance, or vehicle, the inhibition of the FGF-5 action of the biological extract or the substance is examined by measuring the degree of proliferation of the cell. The method for screening a hair growth / hair growth agent according to claim 7, wherein the hair growth / hair growth action is evaluated.   The hair growth / hair growth agent screening method according to claim 8, wherein the cultured cells are FR-Ba / F3 cells or BALB / 3T3 cells.   10. The method for screening a hair growth / hair growth agent according to any one of claims 7 to 9, wherein the inhibition is prevention of promotion of cultured cell proliferation by FGF-5.