JP5306625B2 - Nerve growth factor (NGF) production inhibitor - Google Patents

Description

The present invention relates to a nerve growth factor (NGF) production inhibitor and an itching inhibitory external preparation.

Itching is a unique sensation that occurs only in the skin, mucous membranes, and cornea and is defined as `` an unpleasant sensation that gives the desire to scratch '' and is one of the important complaints frequently encountered in dermatological daily practice. Has been. Since scratching behavior caused by itching forms secondary skin lesions, elucidation of the mechanism and countermeasures are important issues in many diseases.

It has been clarified that there are two types of itching: peripheral itching and central itching. Peripheral itching is caused by the activation of the so-called itching receptor, a free nerve ending of the sensory nerve that exists in the epidermis / dermis boundary of the skin due to inflammation in the skin. The main factor is considered to be histamine released from mast cells, and its antagonists are in clinical use. Histamine antagonists include H1 receptor antagonists chlorpheniramine, cromoglycic acid, and the like, but topical agents having antihistamine action have been reported to have side effects.

As a technique related to suppression of itching, an application such as Patent Document 1 has been filed. However, this also shows the effect of antagonism of the histamine receptor. Such substances have no effect on dry itching that does not involve histamine.

As other cosmetics relating to prevention of itching, applications such as Patent Document 2 and Patent Document 3 have been filed. All of these are aimed at preventing itching due to drying, but all of them only show an effect in sensory evaluation, and cannot be an anti-itch agent specifying the action mechanism of itching.

In recent studies, pain and itching sensory nerve fibers, which are found only in the epidermis / dermis boundary in healthy subjects, have entered many epidermis in atopic dermatitis skin and dry skin It has been known. The increase in sensory nerve fibers in the epidermis is a state of hypersensitivity to external physical and chemical stimuli, that is, a state of hypersensitivity that tends to generate afferent impulses that convey itching and pain, and is deeply involved with sensitive skin it is conceivable that. In addition, there is a report that the expression of nerve growth factor (hereinafter referred to as NGF) is increased in the skin of atopic dermatitis, and itching is actually mentioned as a symptom of atopic dermatitis. It is considered that NGF secreted from keratinocytes by various stimuli acts on sensory nerve fibers existing in the epidermis / dermis boundary portion and promotes the progress into the epidermis. Furthermore, NGF induces histamine release from mast cells under certain experimental conditions. This suggests that NGF not only promotes an increase in sensory nerve fibers but also induces mast cell activation and increases pruritus.

Therefore, an application like Patent Document 4 has been filed for the purpose of NGF suppression. The invention relating to Patent Document 4 relates to a substance that suppresses NGF production from normal human fibroblasts (NHDK), and the main production source of NGF in the skin is keratinocytes. Moreover, since fibroblasts are present in the dermis, it is thought that they are not involved in the invasion of nerves into the epidermis.

Patent Document 5 has been filed as an application of NGF suppression experiments using normal keratinocytes. However, as the graph of FIGS. 7-9 of the specification shows, the suppression effect is not sufficiently obtained.
JP 2004-59546 A JP 2002-265327 A JP 2001-354552 A JP-A-2005-350412 JP 2004-107250 A

The present invention has been made in order to solve such problems, and a substance that suppresses NGF production from keratinocytes, which are NGF-producing main cells in the skin, and suppresses elongation of sensory nerve fibers to the epidermis. Provide combined nerve growth factor production inhibition .

In order to solve the above-mentioned problems, the present invention contains an NGF production inhibitor containing one or more of a Japanese cypress extract, a know-how extract, a birch extract, and a mugwort extract. I will provide a.

By suppressing the production of nerve growth factor, the perception of sensory nerves into the epidermis can be suppressed, and hypersensitivity to cosmetics and the like can be suppressed.

The method for measuring the inhibitory effect of NGF in the present invention was performed by measuring NGF mRNA from normal skin epidermal keratinocytes.

The present invention will be described in detail below. The cypress used in the present invention is a perennial plant belonging to the family Asteraceae and is native to Japan, and is native to Yamano from Hokkaido to Kyushu. In Hokkaido and Tohoku, giant baboons grow naturally, and Akita Fuki has grown this wild species. The leaves are 1 meter in diameter and 2 meters in height, and the meat is stiff and bitter, and is now used mainly for candied sugar. Most of the varieties of corn grown throughout the country are Aichi Wassei. There are male and female strains of burdock, and there are about the same sex as wild burdock, but all of the cultivated Aichi wasabi are females, and since they are not pollinated and cannot seed, they can be increased only by splitting the underground stems. It continues. Fukinotoya and petiole are used for food.

The known mother used in the present invention is the rhizome of Lilyaceae. The leaf of the flower is very similar to the leaf of the sedge, and the flower comes out between the leaves. Also, the name of the mother was called a tibo because a child was born near the old root of the flower and the shape of the root was like a tibo (ant's egg and abu). It is said that the mother is often enlarged, and the outer surface is tan-brown with a soft and soft quality that removes yellow hair. It is used as a traditional Chinese medicine for anti-inflammatory, antipyretic, sedative and diuretic purposes.

Birch used in the present invention is a kind of deciduous tree of the birch family. Because it prefers bright places and grows fast, it is one of the first trees to grow after a forest disappears for some reason, such as forest fires, logging, or landslides. The height will be 20-30m. The trunk extends straight at about 30-1m. The branches are divided into many branches to form an egg-shaped tree trunk. The skin is thin, yellowish white and glossy, and peels off in paper. The leaves grow oppositely, and have an oval rhombus or triangular broad egg shape, and the surroundings are serrated. Length is 4-10cm, width is 3-6cm. It turns yellow in autumn. The flowering season is spring. A male and female strain, about 5cm in length, hangs down from the end of a long branch. The female flower has a flower head of 4cm on the short branch. Sap is the raw material for the artificial sweetener xylitol.

Mugwort used in the present invention is a perennial plant belonging to the family Asteraceae. The rhizomes crawl slightly to make a group. The stem rises and becomes slightly woody. The leaves are torn apart and white hair grows densely on the back. From summer to autumn, bloom inconspicuous flowers. It has a distinctive scent and is boiled with spring sprouts and is eaten with a dwarf, soup, and grass. You can also eat it as tempura. The main components of the scent are cineol, thuyon, β-caryophyllene, borneol, camphor, fatty oil palmitic acid, oleic acid, linoleic acid, vitamin A, vitamin B1, vitamin B2, etc. Mogusa (cocoon) used for cocoons is obtained by drying leaves and collecting fluff on the back side.
The leaves are hemostatic with a herbal medicine called gyoyo.

Moreover, since the compounding quantity with respect to the various skin external preparation of each extract which concerns on this invention can be changed according to embodiment of a skin external preparation, and the usage form of a skin external preparation, it is not specifically limited. In principle, an effective amount may be present, but generally 0.0001 to 100% by mass in terms of dry weight in the composition can be used, preferably 0.01 to 10% by mass, more preferably Is 0.1-5 mass%. In particular, it is envisaged that the extract according to the present invention is used at a high blending rate including 100% by mass, for example, in powder-form preparations for errand adjustment. The extract used in the present invention is obtained by drying one or two or more portions of the whole plants of each plant or their leaves, stems, roots, fruits, seeds and flowers, or pulverizing or as they are without drying. In this state, it is extracted with a solvent. However, the rhizome is used as the extraction site for the mother.

The extract used in the present invention can be extracted with various solvents. Besides using water alone, a polar solvent miscible with water can be used alone. Ethanol, propylene glycol, eleene glycol, 1,3-butylene glycol and the like can be used as the polar solvent, but the polar solvent is not limited thereto. Further, one or more mixed solvents of ethanol, propylene glycol, ethylene glycol, and 1,3-butylene glycol can be used.

The extraction method such as extraction time and extraction temperature is not particularly limited. For example, in the case of extraction with water, the extraction is usually performed at 60 ° C. for 3 to 4 hours, and when a low-boiling solvent such as ethanol is used, an extraction device with a reflux is used. The extracted solution can be cooled and filtered to obtain an extract. Furthermore, deodorization and decolorization can be performed using activated carbon or other resins.

Example 1
The present example relates to a nerve growth factor inhibitor containing a leaf extract of cypress. Japanese cypress leaves were dried, soaked in 10 times mass water, extracted at 60 ° C. for 3 hours, filtered, and lyophilized to obtain Japanese cypress leaves.

(Example 2)
The present example relates to a nerve growth factor inhibitor containing an extract of cypress stem. Japanese cypress stems were dried, soaked in 10 times mass water, extracted at 60 ° C. for 3 hours, filtered, and freeze-dried to obtain Japanese cypress stem extract.

(Example 3)
This example relates to a nerve growth factor inhibitor containing a birch extract. The birch branch was dried, immersed in 10 times mass of water, extracted at 60 ° C. for 3 hours, filtered, and freeze-dried to obtain a birch extract.

Example 4
This example relates to a nerve growth factor inhibitor containing a mugwort extract. Artemisia leaves were dried, soaked in 50% ethanol of 10 times mass, extracted at 60 ° C. for 3 hours, and freeze-dried to obtain Artemisia extract.

(Example 5)
This example relates to a nerve growth factor inhibitor containing an extract from a mother. The mother was dried, soaked in 10 times the mass of ethanol, extracted at room temperature for 24 hours, filtered, and freeze-dried to obtain a mother-in-law extract.

(Example 6)
[NGF mRNA suppression test]
〔experimental method〕
Normal skin epidermal keratinocytes were cultured in EpiLife-KG2 (Kurabo) containing 10% FBS at 37 ° C. and 5% CO ₂ . Normal human epidermal keratinocytes were dispensed at 3 × 10 ⁴ in a 24-well microplate and cultured for 72 hours. After 72 hours, the medium was removed and each extract was added with the medium so that each extract was 0.05% or 0.01% and substance P was 10 nM. Three hours later, RNA was extracted from the cells using RNeasy Mini Kit (QIAGEN). Extracted RNA from PrimeScript RT
Reverse transcription was performed using a reagent Kit (TaKaRa Bio) to obtain cDNA. SYBER using the cDNA as a template
Real-time PCR was performed using the Green method to determine the expression level of nerve growth factor.

The NGF production inhibitory action of each extract is shown below.
As a result of NGF mRNA measurement from normal skin epidermis keratinocytes (FIG. 1), when the amount of NGF mRNA in the case of medium alone is 1, NGF mRNA when substance P10 nM that induces production of nerve growth factor is added is 4.56. The amount of NGF mRNA when various extracts are added in addition to substance P is 1.53 for 0.05% of cypress leaf extract, 3.53 for 0.01% of cypress leaf extract, 1.13 for 0.05% of common mother extract, 0.013% was 2.63, birch branch extract 0.05% 2.16, birch branch extract 0.01% 3.80, mugwort extract 0.05% 1.58, mugwort extract 0.01% 3.17, and NGF mRNA levels were all reduced. . In addition, the extract of ten drugs filed in Patent Document 4 is 2.38 at 0.05% and 3.75 at 0.01%, and the substance in the present invention exhibits an effect equivalent to or higher than that.

Hereinafter, the present invention will be described more specifically based on examples, but the present invention is not limited thereto.

[Formulation Example 1] Formulation example of emulsion (Table 1)
Manufacturing method: A) to F) are dissolved by heating and kept at 80 ° C. G) to J) are heated and dissolved, maintained at 80 ° C., and added to A) to F to emulsify. Cool to 37 ° C with stirring.

[Formulation example 2] Formulation example of lotion 1 (Table 2)
Production method: A) to E) are mixed uniformly. Mix F) ~ H) uniformly and add to A) ~ E) mixture

[Formulation example 3] Formulation example of lotion 2 (Table 3)
Production method: A) to E) are mixed uniformly. F) to H) are mixed evenly and A) to E) are added to the mixture.

[Formulation example 4] Formulation example of lotion 3 (Table 4)
Production method: A) to E) are mixed uniformly. F) to H) are mixed evenly and A) to E) are added to the mixture.

[Formulation example 5] Formulation example of lotion 4 (Table 5)
Production method: A) to E) are mixed uniformly. F) to H) are mixed evenly and A) to E) are added to the mixture.

[Formulation Example 6] Formulation example of lotion 5 (Table 6)
Production method: A) to E) are mixed uniformly. F) to I) are mixed evenly and A) to E) are added to the mixture.

[Prescription Example 7] Prescription example of ointment (Table 7)
Production method: A) is dissolved by heating and then cooled, and at 60 ° C., B) is added and further cooled to 37 ° C.

[Prescription Example 8] Prescription example of powder for errand adjustment (Table 8)
Production method: A) Japanese cypress extract itself is used.

By incorporating one or more nerve growth factor production inhibitors selected from the extract of Japanese cypress, mochi extract, birch extract and mugwort extract of the present invention, the penetration of sensory nerves into the epidermis is suppressed. It can suppress hypersensitivity to cosmetics and can be expected to be widely applied.

NGF production suppression test using normal human epidermal keratinocytes NHEK (F), 10% extract, Japanese cypress leaf extract, birch branch extract, mugwort extract, common mother extract 0.01% or 0.05% The amount of NGF mRNA when added is shown.

Claims (1)

Nerve growth factor production inhibitor (excluding itching inhibitor) characterized by containing water extract of Japanese cypress as an active ingredient.