JPH09229922A - Method for evaluating hair grower - Google Patents

Abstract

PROBLEM TO BE SOLVED: To make it possible to evaluate the suppressing action of regression period in a hair cycle by measuring the total amounts of mononucleosome and oligonucleosome in trichocyst-originated cell. SOLUTION: The back skins of C3H mice are shaved, about five mice are used for each sample from about tenth day, the skins of parts for test are continuously coated twice in the morning and evening with about each 0.2ml for ten days. After the coating, they are butchered, the hairs of the parts for test of the samples are collected, and trypsin-treaded. Incubation buffer is added to the obtained cells of predetermined quantity, steadily left to stand for 30min at about 4 deg.C, centrifugally separated, and the part of supernatant is sampled. The sample thinned to about ten times of about 100μl is added to plate coated with antihistone antibody, steadily left to stand for about 90min, and then the plate is cleaned. Then, the same amount of anti-DNA-peroxidase antibody is similarly added, and cleaned. Eventually, the substrate solution of the peroxidase of about 100μl is added, the absorbance is measured in the wavelength of 405nm, and the total amount of mononucleosome and oligonucleosome is obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating a hair restorer, which evaluates the inhibitory action or delay action of the cataract in the hair cycle.

[0002]

2. Description of the Related Art Conventionally, rabbits (Rony, R. Choen, DM and DM and
Schaffner, IJ Invest. Dermatol., 21,313-330195
3), mouse (Silver, AF, Chase, HB and Ares
enault, CT In: Advancesin Biology of skin, vol.
9, Hair Growth, W. Montagna (ed), 265-286, 1969) and red-tailed macaque (Montagna, W., Machida, H. and Perk
ins, E. Amer. J. Phys. Anthropol., 24, 71-85, 196
6) etc. are known. Ogawa et al. (Hideoki Ogawa, Ryusuke Imai,
The C3H mouse hair growth evaluation experimental system established by Fragrance Journal, 17, (5), 20-29, 1989) has a relatively constant hair cycle type, is easy to handle in mild animals, It is widely used in many research institutes because it has the advantage of being able to shift from the quiescent phase to the growth phase in almost the same cycle.

[0003]

However, the C3H mouse hair growth test is useful as a method for evaluating only the hair growth promoting action as an in vivo evaluation system for conventional hair growth agents.
It was impossible to evaluate the suppressive action or delaying action of the hair-growth agent in the catagen period using the evaluation method. Therefore, it is difficult to evaluate in detail the effect of various hair-growth agents on the suppression or delay of catagen, and it is necessary to find and measure an effective index related to catagen. It is now believed that hair follicle cells undergo cell death called apoptosis during the regressive phase of the hair cycle, and this apoptosis triggers hair growth (David Weedon et al.
al. Acta Dermatovener (Stockholm) 61: 335-369,1981)
. In apoptotic cells, nuclear DNA is disrupted to produce mononucleosomes and oligonucleosomes. However, the amount of mononucleosomes and oligonucleosomes produced in the hair cycle has not been measured yet.

[0004]

Means for Solving the Problems The present inventors have found that the amount of mononucleosomes and oligonucleosomes in hair follicle-derived cells increases only during the regression period of C3H mice, as a biochemical index of the hair cycle. It was found that the total amount of mononucleosomes and oligonucleosomes can be measured in the hair cycle regression phase of C3H mice, and the effect of useful hair growth agents on the suppression or delay of the regression phase can be evaluated in detail. That is, the present invention is a hair-growth agent characterized by evaluating the effect of suppressing or delaying the catagen phase in the hair cycle of a hair-growth agent by measuring the total amount of mononucleosomes and oligonucleosomes in hair follicle-derived cells. There is an evaluation method. This method is also an evaluation method that can be used for evaluation of hair growth inhibitors.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The present inventors have made it possible to evaluate the detailed hair-growth action by measuring the total amount of mononucleosomes and oligonucleosomes of hair follicle-derived cells collected in the hair growth test of C3H mice. The method for measuring the total amount of mononucleosomes and oligonucleosomes in the hair follicle-derived cells in the present invention is, for example, as follows.

(1) Method for evaluating hair growth evaluation of C3H mouse of the present invention and method for preparing sample for measuring total amount of mononucleosome and oligonucleosome C3H mouse (8 weeks old, male, average weight 35 g) dorsal skin (2 cm × 4 cm) ) Was shaved with an electric hair clipper and a depilatory cream, and from the 10th day, each sample was applied to the skin of the test site in the morning and evening.
Apply 0.2 ml per animal for 10 consecutive days. 5 animals per group are used per sample. On the 10th day from the start of application, the mice in each group are sacrificed, and then the test area coat of the sample is collected. The collected hair is treated with trypsin, and a certain amount of cells obtained therefrom is used as a sample for measuring the total amount of mononucleosomes and oligonucleosomes.

(2) Method for measuring the total amount of mononucleosome and oligonucleosome The method for measuring the total amount of mononucleosome and oligonucleosome is a cell death detection kit ELISA (also known as Cell D).
eath Detection ELISA, manufactured by Boehringer Mannheim)
Measure according to the attached usage. Specifically, add incubation buffer to the collected cells, and
Let stand for 0 minutes. Centrifuge at 15000 rpm for 10 minutes,
Use a part of the supernatant as a sample. 100 μl of a 10-fold diluted sample on a plate previously coated with anti-histone antibody
Added. After leaving it at room temperature for 90 minutes, wash the plate 3 times with the washing solution. Next, 100 μl of an anti-DNA-peroxidase antibody solution was added, and the plate was allowed to stand at room temperature for 90 minutes, and then the plate was washed 3 times with the washing solution. Finally, 100 μl of a peroxidase substrate (2,2′-azino-di- [3-ethylbenzthiazoline sulfonate (6)]) solution is added, and the absorbance is measured at a wavelength of 405 nm. The total amount of mononucleosomes and oligonucleosomes is indicated by the absorbance of a constant cell amount.

The present invention will be described in detail below based on examples. The C3H mouse hair growth promoting effect test method, which is the conventional hair growth promoting effect test method described in Examples, is shown below.

(3) Test method for promoting hair growth in C3H mice The back skin (2 cm × 4 cm) of C3H mice (8 weeks old, male, average weight 35 g) was shaved with an electric clipper and a shaver, and each sample was taken from the next day. Is applied to the skin of the test site twice a day in the morning and 0.2 ml per animal for two consecutive weeks. For each sample, 10 animals were used per group. After being slaughtered 14 days after the start of application, photographs are taken for each group. From the photographed photograph, the regeneration state and regeneration area of the hair on the back skin of the mouse are evaluated by a 5-step score. The score of the hair regeneration state is evaluated according to the evaluation criteria shown in Table 1 below. <Mouse regenerated hair score standard>

[0010]

[Table 1]

[0011]

【Example】

Example 1 (1) Test sample Four types of samples shown in Table 2 below were dissolved in 55% by weight of ethanol, and the C3H mouse hair growth evaluation test of the present invention was carried out to measure the total amount of mononucleosomes and oligonucleosomes. did. Table 6 shows the results of evaluation using the total amount of mononucleosomes and oligonucleosomes as an index, and the results of the conventional hair growth promoting effect test (hair growth rate score) of C3H mice.

[0012]

[Table 2]

(2) Evaluation Results As shown in Table 6, as a result of evaluation by the evaluation method of the present invention, in this system, in comparison with the base coating group of Sample 4, Samples 1 to 3 had a regression period. The effect of suppressing and sending the transition to the resting period was recognized.

Example 2 (1) Test sample Five kinds of cosmetic raw materials shown in Table 3 below were dissolved in 55 wt% ethanol to prepare a sample, and the above-mentioned C3 of the present invention was prepared.
An H mouse hair growth evaluation test was performed. Table 6 shows the results of evaluation using the amounts of mononucleosomes and oligonucleosomes as indexes, and the results of the hair growth promoting effect test (hair growth rate score) of C3H mice.

[0015]

[Table 3]

(2) Evaluation results As shown in Table 6, as a result of evaluation by the evaluation method of the present invention, it was confirmed that Samples 5 to 9 had the effect of suppressing the catagen period and sending the transition to the rest period.

Example 3 (1) Method for preparing hair artic After dissolving the component (B) shown in the following Table 4 in the component (A), the component (C) was added, and the mixture was stirred and dispersed to a container. Filled. At the time of use, the contents were uniformly shaken and used.

[0018]

[Table 4]

(2) Test sample Four types of hair artic compounds shown in Table 5 below were prepared, and the hair growth evaluation test of the C3H mouse of the present invention was carried out. Table 6 shows the results of evaluation using the total amount of mononucleosomes and oligonucleosomes as an index, and the results of the hair growth promoting effect test (hair growth rate score) of C3H mice.

[0020]

[Table 5]

(3) Evaluation Results As shown in Table 6, as a result of evaluation by the evaluation method of the present invention, the hair nourishing agents A, B, and C were found to have the effect of suppressing the catagen period and transmitting the transition to the rest period. It was

[0022]

[Table 6]

[0023]

As described above, by measuring the total amount of mononucleosomes and oligonucleosomes in the hair follicle-derived cells of the present invention, the effect of suppressing or delaying the catagen in the hair cycle of the hair restorer can be further improved. It is clear that a method for detailed evaluation can be provided.

Claims (1)

[Claims] 1. A method for evaluating a hair-growing agent, which comprises evaluating the suppressive action or delay action of catagen in the hair cycle by measuring the total amount of mononucleosomes and oligonucleosomes in hair follicle-derived cells.