JP6970952B2 - Kallikrein-related peptidase production promoter, LEKTI production promoter, SLPI production promoter, keratinization normalizer, corneodesmosome degradation normalizer - Google Patents

Description

The present invention relates to a pharmaceutical product that promotes the production of kallikrein-related peptidases, LEKTI, SLPI, normalizes corneodesmosome degradation, and thus normalizes keratinization.

The epidermis begins with cell division in the basal layer, passes through the stratum spinosum and the stratum granulosum, and reaches the stratum corneum.
The water retention function of corneocytes by intercorneocyte lipids such as ceramide is also an important factor, but the layered structure is maintained by adhering to each other by the corneodesmosomes distributed in a patch pattern, and the integrity as a stratum corneum is maintained. I'm dripping.
Then, it is gradually pushed out to the surface layer of the stratum corneum by new cells, and as it moves to the upper layer of the stratum corneum, the intercellular lipids in the stratum corneum are decomposed by steroid sulfatase and lipase, and then the corneodesmosome is gradually decomposed by the action of protease, and the cells are gradually decomposed. The inter-adhesion weakens, and eventually the keratin peels off and falls off.
However, it is known that the barrier function of the epidermis is reduced by external factors such as dryness, ultraviolet rays, chemical substances, and internal factors such as oxidative stress and aging, and these factors reduce the turnover of the epidermis. It is also known to do.
One of them is that the degradation of Corneodesmosome is delayed, the cell-cell adhesion is not weakened, and the exfoliation and shedding of keratin may not be performed normally. (Thickening of the stratum corneum)

Proteases that degrade corneodesmosomes include kallikrein-related peptidase 5 (KLK5) and kallikrein-related peptidase 7 (KLK7), and these serine proteases degrade desmoglein 1, corneodesmocollin, desmocollin 1, and the like.
That is, when the production of kallikrein-related peptidase 5 and kallikrein-related peptidase 7 decreases, exfoliation and shedding of keratin do not go well.
Therefore, promoting the production of kallikrein-related peptidase 5 and kallikrein-related peptidase 7 is an important factor for promoting skin turnover.
However, excessive production of kallikrein-related peptidase 5 and kallikrein-related peptidase 7 may result in abnormal desquamation. Furthermore, it was found that kallikrein-related peptidase 7 degrades two lipid processing enzymes, β-glucocerebrosidase and acid sphingomyelinase.
Further, the activation of kallikrein-related peptidase 5 and kallikrein-related peptidase 7 is restricted and balanced by LEKTI (Lympho-epithelial Kazal-type-related inhibitor) and SLPI (Secretory Leukocyte-Protease Inhibitor).
As described above, promoting the production of kallikrein-related peptidase 5 and kallikrein-related peptidase 7, whose production decreased due to aging, and simultaneously increasing the production of LEKTI and SLPI, which inhibit them, causes the degradation of corneodesmosomes. It normalizes, and eventually keratinization. (Patent Document 1, Non-Patent Document 1)
Other than skin keratinization, SLPI production promoters are applied to improve airway obstruction, treat interstitial pneumonia, prevent or treat retroviral infections, treat post-burn scars, hypertrophic scars and keloids, etc. ing. (Patent Documents 2 to 5)
LEKTI production promoters are known to be effective against anti-inflammatory, Netherton syndrome, ichthyosis and the like.

The bark is a dried bark of Chinese cinnamon (Cinnamomum cassia) or a closely related species.
As a closely related species, Nikkei (C. sieboldi) and cinnamon (C. verum) are well known.
As a crude drug, it is used as a crude drug with a body warming effect, a sweating / diverging effect, and a stomachic effect, but it is also widely used as a spice.
Further, applications such as protease inhibitors, substance P antagonists, and interleukin 4 production inhibitors are known (see Patent Documents 6 to 8), and extracts are commercially available as raw materials for cosmetics.

Burdock is the root of burdock (Arctium lappa) and is used for food, but it is also used for crude drugs and Chinese herbs, and it is also used as a material for diuresis, sweating, blood purification, and skin diseases (acne, eczema, psoriasis). It is used.
Further, applications such as hair growth / growth agents, skin cosmetics for slimming, and cathepsin D production promoters are known (see Patent Documents 9 to 11), and extracts are commercially available as raw materials for cosmetics.

Peony is a dried or steamed root of peony (scientific name: Paeonia lactiflora).
Further, applications such as bath composition, whitening cosmetics, and interleukin 4 production inhibitor are known (see Patent Documents 12 to 14), and extracts are commercially available as raw materials for cosmetics.

Japanese Unexamined Patent Publication No. 2013-1667668 Japanese Unexamined Patent Publication No. 6-298663 Japanese Unexamined Patent Publication No. 7-82168 Japanese Unexamined Patent Publication No. 2004-2455 WO01 / 03389 Japanese Unexamined Patent Publication No. 06-025000 Japanese Unexamined Patent Publication No. 09-208480 Japanese Unexamined Patent Publication No. 10-045613 Japanese Unexamined Patent Publication No. 05-058851 Japanese Unexamined Patent Publication No. 10-158120 Japanese Unexamined Patent Publication No. 2001-322940 Japanese Unexamined Patent Publication No. 05-339144 Japanese Unexamined Patent Publication No. 08-092055 Japanese Unexamined Patent Publication No. 10-279491 Caubet C et al. J Invest Dermatol 122: 1235-1244,2004

An object of the present invention is to obtain a preparation that promotes the production of kallikrein-related peptidases, LEKTI, SLPI, normalizes the degradation of corneodesmosomes, and normalizes keratinization such as suppression of stratum corneum thickening and delay of turnover.

As a result of diligent studies by the present inventors, it was found that the cinnamon bark extract, the burdock extract, and the peony extract achieve the above-mentioned object.
Cinnamon, burdock root, and peony are extracted after being dried as necessary and then subjected to treatments such as shredding and crushing in consideration of extraction efficiency.
Drying may be carried out in the sun or by using a commonly used dryer.
As the solvent used for the extraction, water, a hydrophilic organic solvent, or a mixed solution thereof is used.
Examples of the water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.
Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol and propylene glycol. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
When a mixed solvent of the water and the hydrophilic organic solvent is used, 1 to 20 parts by mass of water is added to 10 parts by mass of water in the case of lower alcohol, and 10 parts by mass of water is used in the case of lower aliphatic ketone. On the other hand, it is preferable to add 1 to 15 parts by mass. In the case of polyhydric alcohol, it is preferable to add 1 to 20 parts by mass with respect to 10 parts by mass of water.

The amount of the organic solvent used for extraction is preferably about 5 to 100 times, more preferably about 10 to 50 times the amount of the plant as a raw material. In order to further increase the extraction efficiency, stirring or homogenization may be performed in the extraction solvent. It is appropriate that the extraction temperature is from about 5 ° C. to a temperature equal to or lower than the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but it is appropriate to set it to about 1 hour to 14 days.
The extraction operation is not limited to a one-time operation. A fresh solvent can be added again to the residue after extraction to perform an extraction operation, or the extraction solvent can be brought into contact with the extraction raw material multiple times.
As a result of the examination by the present inventors, 80% of the ethanol of the substance exhibiting the effect of the present invention is also extracted with water. Therefore, when purifying to some extent, after extracting with water, the insoluble matter should be removed, etc. It was also found that further extraction should be performed by adding an amount to 5 times the amount of ethanol.
If necessary, further purification treatment such as deodorization and decolorization may be added within a range that does not affect the effect, and the concentration can be achieved by a vacuum concentrator such as an evaporator or removal of a solvent by heating.
Further, this extract can be further purified using a synthetic adsorbent (Diaion HP20, Sephadex SP825, Amberlite XAD4, MCIgelCHP20P, etc.), dextran resin (Sephadex LH-20, etc.), ultrafiltration, etc. be.

Although the pharmaceutical product of the present invention exerts a medicinal effect by oral, injectable or external use, it is preferably used as a skin external preparation. External skin preparations include skin cosmetics, quasi-drugs for external use, and external external preparations for medical use.
The amount of these extracts to be added to the formulation is 0.000001 to 10.0% by weight, preferably 0.00001 to 3.0% by weight, and more preferably 0.00005 to 1.0% by weight as a solid content. be.

In addition to the above-mentioned ingredients, the formulations of the present invention include surfactants, oily ingredients, moisturizers, polymer compounds, ultraviolet absorbers, anti-inflammatory agents, and bactericidal agents used in various pharmaceutical and cosmetic formulations. Antioxidants, metal ion blockers, preservatives, vitamins, pigments, fragrances, water and the like can be blended.

As the above-mentioned surfactant, any of anionic, cationic, nonionic, natural and synthetic surfactants can be used, but it is preferable to use nonionic surfactants in consideration of skin irritation. Examples of the nonionic surfactant include glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyoxyethylene alkyl ether, and polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkyl glycoside and the like.

Examples of the oily component include fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, essential oils, silicone oils and the like. Examples of oils and fats include soybean oil, nuka oil, jojoba oil, avocado oil, almond oil, olive oil, cacao oil, sesame oil, persic oil, castor oil, palm oil, minced oil, beef oil, pork oil and other natural oils and fats. Hardened oil obtained by hydrogenating natural fats and oils from Japan and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid triglyceride; Examples of waxes include carnauba wax, whale wax, beeswax, lanolin and the like; hydrocarbons. Examples include liquid paraffin, vaseline, paraffin microcrystalin wax, selecin, squalane, bristan, etc .; and higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, etc. Linolenic acid, lanolinic acid, isostearic acid and the like; higher alcohols include, for example, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, 2-hexyldecanol and the like; esters include, for example, cetyl octanate. , Octanoic acid triglyceride, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, octyldodecyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, POE sorbit fatty acid ester, etc. The essential oils include, for example, peppermint oil, jasmine oil, show brain oil, hinoki oil, peppermint oil, ryu oil, terepine oil, calyx skin oil, bergamot oil, orange oil, shove oil, pine oil, lavender oil, and bay oil. , Clove oil, hiba oil, rose oil, eucalyptus oil, lemon oil, thyme oil, peppermint oil, rose oil, sage oil, menthol, cineole, eugenol, citral, citroneral, borneol, linalol, geraniol, camphor, timole, spirantol, Pinen, limonene, terpene-based compounds and the like; Examples of silicone oils include dimethylpolysiloxane and the like. These above-mentioned oily components can be used alone or in combination of two or more. In the present invention, among these, myristic acid glyceride, 2-ethylhexanoic acid triglyceride, lanolin, liquid paraffin, vaseline, paraffin microcrystalin wax, squalane, lauric acid, myristic acid, palmitic acid, linoleic acid, linolenic acid, isostearic acid. , Cetyl alcohol, stearyl alcohol, oleyl alcohol, cholesterol, cetyl octanoate, triglyceride octanoate, isopropyl myristyrene, octyldodecyl myristate, cholesterol isostearate, POE sorbit fatty acid ester, peppermint oil, peppermint oil, calyx oil, rose It is preferable to use oil, menthol, cineol, eugenol, citral, citronellal, geraniol, pinen, limonene and dimethylpolysiloxane.

The following components can be further added to the pharmaceutical product of the present invention, but the components are not limited thereto.
Pigments; Tar dyes specified in the Ordinance of the Ministry of Health and Welfare such as Yellow No. 4, Blue No. 1, Yellow No. 202, etc. Approved as food additives such as pigments in Appendix I and II, chlorophyll, riboflavin, crocin, safflower, anthraquinone, etc. Natural pigments, etc.
Vitamins; Vitamin A, Vitamin C, Vitamin D, Vitamin E, etc.
Others; bactericides, preservatives, other ingredients necessary for formulation, etc.

The pharmaceutical product of the present invention can be produced according to a conventional method by adding the optional ingredient to the essential ingredient as needed.

Next, the present invention will be described in detail with reference to examples.

Example 1
Add 2 liters of 30% (V / V) ethanol aqueous solution to 50 g of cinnamon bark (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate, and then freeze-dry. bottom.

Example 2
Add 2 liters of 50% (V / V) ethanol aqueous solution to 30 g of beef burdock (Takinogawa gobo) (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), and evaporate. This was lyophilized.

Example 3
Add 2 liters of 30% (V / V) ethanol aqueous solution to 50 g of peony (Paeonia lactiflora) (Tochimoto Tenkaido, dried, shredded), extract for 24 hours with occasional stirring, filter (No5C), and evaporate. After that, it was freeze-dried.

Confirmation test After culturing the second generation human foreskin-derived epidermal cells (Kurabou) in HuMedia-KG2 medium (without phenol red) so as to be 50-70% confluent, the calcium concentration was changed to 1.8 mM the day before. Examples were added to KG2 medium and cultured at 37 ° C. in a 5% CO2 incubator for 2 days.

<RNA extraction>
Extraction of Total RNA from cells was prepared according to the attached manual of GE Healthcare using the illustra RNA Mini RNA Isolation Kit (GE Healthcare) after exfoliation with trypsin / EDTA. RNA concentration was calculated using NanoDrop1000 (Thermo SCIENTIFIC).

<RT reaction and real-time PCR>
An RT reaction was performed using 2.5 μg of Total RNA, using MMLV Reverse Transcriptase RNase H- (Toyobo), and according to the Toyobo recommended protocol (TOYOBO BIOCHEMICALS FOR LIFE SCIENCE, page 1-42).
Real-time PCR was performed using Applied Biosystems 7500 real-time PCR System as follows. Genes expressed by the Comparative CT (△△ CT) method (n = 3) using the SYBR Green method (THUNDERBIRD SYBR qPCR Mix, Toyobo) and the 7500 real-time PCR system operating manual (Applied Biosystems). GAPDH was used as the internal standard.
The target genes are kallikrein-related peptidase 5 (KLK5), kallikrein-related peptidase 7 (KLK7), LEKTI, and SLPI.

The results of the confirmation test are shown in FIG.
In each of Example 1, Example 2 and Example 3, the gene expression levels of kallikrein-related peptidase 5, kallikrein-related peptidase 7, LEKTI, and SLPI were significantly promoted as compared with the control.

In addition, as a result of preparing an external preparation containing the examples and actually using it, it was effective in promoting thickening of the stratum corneum and turnover.

Kallikrein-related peptidase 5 (KLK5), kallikrein-related peptidase 7 (KLK7), LEKTI, SLPI in Example 1 (action concentration 0.0125%), Example 2 (action concentration 0.1%), and Example 3 (action concentration 0.025%). We observed changes in the amount of gene produced in Kallikrein. The vertical axis is the gene expression level when the gene expression level when the example is not added is 1.

Claims (1)

An agent for promoting the production of KLK5, KLK7, LEKTI and SLPI containing an extract of cinnamon bark as an active ingredient.

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