JP2022020261A - Filaggrin mRNA expression promoter and hyaluronan synthase 3 mRNA expression promoter - Google Patents

Abstract

To discover, from among safe natural products, ones having excellent filaggrin mRNA expression promoting action or hyaluronan synthase 3 mRNA expression promoting action and provide a filaggrin mRNA expression promoter and a hyaluronan synthase 3 mRNA expression promoter each containing the corresponding component as an active ingredient.SOLUTION: The present invention discloses a filaggrin mRNA expression promoter and a hyaluronan synthase 3 mRNA expression promoter containing Rosa canina extract as an active ingredient.SELECTED DRAWING: None

Description

The present invention relates to a filaggrin mRNA expression promoter and a hyaluronic acid synthase 3 mRNA expression promoter.

Filaggrin is a constituent of the skin, is involved in the barrier function in the skin, and is thought to have a function of preventing the invasion of allergens, toxins, and infectious organisms. Decreased function due to mutation of filaggrin gene is associated with the risk of developing atopic dermatitis (eczema, skin inflammation, itchy skin, etc.), allergies, asthma, etc., and is more serious. In some cases, it is known to lead to skin diseases such as ichthyosis vulgaris (see Non-Patent Document 1).

On the other hand, amino acids, which are the main components of Natural Moisturizing Factors (NMF), are produced by degrading filaggrin derived from keratohyalin granules in the stratum corneum. This filaggrin is expressed as profilaggrin in the epidermal keratinocytes present in the stratum granulosum just below the stratum corneum. After that, it is immediately phosphorylated, accumulated in keratohyalin granules, decomposed into filaggrin via dephosphorylation and hydrolysis, transferred to the stratum corneum, increases the aggregation efficiency of keratin filaments, and is involved in the internal construction of corneocytes. It is known (see Non-Patent Document 2). In recent years, it has been known that this filaggrin is very important and indispensable for retaining water in the skin, and that the synthetic ability of filaggrin decreases due to conditions such as dryness, and the amount of amino acids in the stratum corneum decreases (). See Non-Patent Document 3).

Therefore, by promoting the expression of filaggrin in epidermal keratinocytes, it is possible to prevent, treat or improve atopic dermatitis (eczema, skin inflammation, skin itching, etc.), allergies, asthma, etc. Conceivable. In addition, it is expected that the water environment of the stratum corneum can be essentially improved by promoting the expression of filaggrin and thereby increasing the amount of amino acids in the stratum corneum. Conventionally, as a filaggrin mRNA expression promoting action, a gaiyo extract (see Patent Document 1) and the like are known.

The epidermis and dermis of the skin are composed of epidermal cells, fibroblasts, and extracellular matrix such as collagen, elastin, and hyaluronic acid that are outside these cells and support the skin structure. In young skin, the proliferation of fibroblasts is active, and the interaction of skin tissues such as fibroblasts and extracellular matrix components maintains constancy to ensure water retention, flexibility, elasticity, etc., and the skin. The appearance is also taut and glossy, and it is maintained in a fresh state.

However, under the influence of certain external factors such as UV irradiation, significant dryness of air, and excessive skin cleaning, and with aging, collagen, elastin, and hyaluronic acid, which are the main constituents of the extracellular matrix, are used. As the amount of acid produced decreases, it causes decomposition and alteration. As a result, the moisturizing function and elasticity of the skin are lowered, and abnormal exfoliation of the keratin occurs, so that the skin loses tension and luster, and exhibits aging symptoms such as rough skin and wrinkles. As described above, changes associated with skin aging, that is, wrinkles, dullness, changes in texture, decreased elasticity, etc., are involved in the reduction / denaturation of matrix components such as collagen, elastin, and hyaluronic acid. Therefore, promoting the production of collagen, elastin or hyaluronic acid is important in preventing, treating or ameliorating skin aging.

Among the above-mentioned extracellular matrix components, hyaluronic acid is a kind of mucopolysaccharide, has a function of retaining cells by being filled in the gaps between cells, and further retains water in the gaps between cells and into tissues. It has many functions such as imparting lubricity and flexibility, and resistance to external forces such as mechanical obstacles. If the production of hyaluronic acid can be promoted, it is considered that the aging symptoms of the skin such as rough skin, wrinkles, dullness, change in texture, decrease in elasticity and decrease in moisturizing function can be prevented, treated or ameliorated. In addition, it is considered that skin aging can be prevented, treated or ameliorated by promoting the expression of hyaluronic acid synthase 3 (HAS3), which is involved in promoting the synthesis of epidermal hyaluronic acid. To date, licorice leaf extract (see Patent Document 2) and the like are known to have an action of promoting the expression of hyaluronic acid synthase 3 mRNA.

Japanese Unexamined Patent Publication No. 2012-219047 Japanese Unexamined Patent Publication No. 2010-090035

"Nat Genet.", 2006, Vol.38, No.4, p.441-446 "Fragrance Journal Extra Edition", 2000, Vol.17, p.14-19 "Arch. Dermatol. Res.", 1996, Vol.288, p.442-446

The present invention has found a highly safe natural product having an excellent phyllagulin mRNA expression-promoting action or hyaluronic acid synthase 3 mRNA expression-promoting action, and a phyllagulin mRNA expression-promoting agent and hyaluronic acid containing each as an active ingredient. It is an object of the present invention to provide an acid synthase 3 mRNA expression promoter.

In order to solve the above problems, the filaggrin mRNA expression promoter and the hyaluronic acid synthase 3 mRNA expression promoter of the present invention are characterized by containing a canina rose extract as an active ingredient.

According to the present invention, it is possible to provide a filaggrin mRNA expression promoter and a hyaluronic acid synthase 3 mRNA expression promoter, which are excellent in safety and action and effect.

Hereinafter, embodiments of the present invention will be described.
The filaggrin mRNA expression promoter and the hyaluronic acid synthase 3 mRNA expression promoter of the present embodiment contain caninabara extract as an active ingredient.
In the following, hyaluronic acid synthase 3 may be abbreviated as HAS3.

Here, the "canina rose extract" in the present embodiment includes an extract obtained by using canina rose as an extraction raw material, a diluted solution or a concentrated solution of the extract, a dried product obtained by drying the extract, or a dry product thereof. Both crude and refined products are included.

The extraction raw material used in this embodiment is canine rose (scientific name: Rosa canina).
Canina rose is an evergreen tree of the genus Rosa in the family Rosaceae, grows naturally in regions such as Europe, North Africa, and West Asia, and is widely cultivated in the world, and is easily available. The fruit of canina rose is also called rose hip and is widely used as a raw material for cooking oil.

The constituent parts of canina rose that can be used as an extraction raw material are not particularly limited, and examples thereof include above-ground parts such as leaves, stems, flowers, and fruits, roots, or mixtures of these parts. Above all, it is preferable to use the fruit part.

The above-mentioned plant extract can be obtained by drying the extraction raw material, crushing it as it is or using a coarse crusher, and subjecting it to extraction with an extraction solvent. Drying may be carried out in the sun or by using a commonly used dryer. Further, by performing pretreatment such as degreasing with a non-polar extraction solvent such as hexane, the extraction treatment with a polar solvent can be efficiently performed.

As the extraction solvent, it is preferable to use a polar solvent, for example, water, a hydrophilic organic solvent and the like, and these are used alone or in combination of two or more at room temperature or a temperature below the boiling point of the solvent. Is preferable.

Water that can be used as an extraction solvent includes 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, osmoregulation, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present embodiment includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.

Hydrophilic organic solvents that can be used as the extraction solvent include lower aliphatic alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; and carbon atoms such as 1,3-butylene glycol, propylene glycol and glycerin. 2 to 5 polyhydric alcohols; examples include lower aliphatic ketones such as acetone and methyl ethyl ketone.

When a mixed solution of two or more polar solvents is used as the extraction solvent, the mixing ratio thereof is arbitrary and can be appropriately adjusted. For example, when a mixed solution of water and a hydrophilic organic solvent is used as an extraction solvent, it is mixed at an arbitrary ratio, that is, more than 0: 100 and less than 100: 0 (volume ratio, hereinafter similarly referred to). Can be used and can be adjusted as appropriate.
For example, when a mixed solution of water and a lower fatty alcohol is used as an extraction solvent, the mixing ratio (volume ratio) of water and the lower fatty alcohol can be 9: 1 or more, and further 7 : 3 or more, or the mixing ratio of water and lower fatty alcohol can be 1: 9 or less, and even 2: 8 or less. When a mixed solution of water and polyhydric alcohol is used, the mixing ratio of water and polyhydric alcohol can be 8: 2 or more or 1: 9 or less, and water and lower aliphatic ketones can be used. When a mixed solution of water and a lower aliphatic ketone is used, the mixing ratio of water and the lower aliphatic ketone can be 9: 1 or more or 2: 8 or less.

The extraction treatment is not particularly limited as long as the soluble component contained in the extraction raw material can be eluted in the extraction solvent, and can be carried out according to a conventional method. For example, the extraction material is immersed in an extraction solvent having an amount (mass ratio) of 5 to 50 times that of the extraction material, the soluble component is extracted at room temperature or under reflux heating, and then the extract is filtered to remove the extraction residue. You can get the liquid. Distilling off the solvent from the obtained extract gives a paste-like concentrate, and further drying the concentrate gives a dried product.

Since the canina rose extract obtained as described above has an excellent filaggrin mRNA expression promoting action and HAS3 mRNA expression promoting action, it can be used as an active ingredient of a filaggrin mRNA expression promoting agent or a HAS3 mRNA expression promoting agent. can. The filaggrin mRNA expression promoter and the HAS3 mRNA expression promoter of the present embodiment can be used in a wide range of applications such as pharmaceuticals, quasi-drugs, cosmetics, and foods and drinks.

The filaggrin mRNA expression-promoting agent and the HAS3 mRNA expression-promoting agent of the present embodiment may consist only of a canina rose extract or may be a formulation of a canina rose extract.

The phyllagulin mRNA expression promoter and the HAS3 mRNA expression promoter of the present embodiment are powdered, granulated, or tablets according to a conventional method using a pharmaceutically acceptable carrier such as dextrin or cyclodextrin or any other auxiliary agent. It can be formulated into any dosage form such as liquid or liquid. At this time, as the auxiliary agent, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring / odorizing agents and the like can be used. The filaggrin mRNA expression promoter and the HAS3 mRNA expression promoter can be used in combination with other compositions (for example, skin external preparations, oral compositions described later, etc.), as well as ointments, external liquid preparations, and patches. It can be used as such.

When the filaggrin mRNA expression promoter and the HAS3 mRNA expression promoter of the present embodiment are formulated, the content of the caninabara extract is not particularly limited and can be appropriately set according to the intended purpose.

The filaggrin mRNA expression-promoting agent and the HAS3 mRNA expression-promoting agent of the present embodiment may contain other substances having a filaggrin mRNA expression-promoting action or a HAS3 mRNA expression-promoting action (for example, a natural extract), if necessary. It can be used as an active ingredient by blending with canina rose extract.

Examples of the method for administering the filaggrin mRNA expression promoter and the HAS3 mRNA expression promoter of the present embodiment to patients include transdermal administration and oral administration, which are suitable for prevention and treatment depending on the type of disease. The method may be selected as appropriate. In addition, the doses of the filaggrin mRNA expression-promoting agent and the HAS3 mRNA expression-promoting agent of the present embodiment may be appropriately increased or decreased depending on the type, severity, individual difference of patients, administration method, administration period and the like.

The filaggrin mRNA expression-promoting agent of the present embodiment promotes the expression of filaggrin through the filaggrin mRNA expression-promoting action of the canina rose extract, and enhances the barrier function of the skin, thereby causing rough skin, dry skin, and dry skin. Diseases (eg, atopic dermatitis, psoriasis, ichthyosis, etc.) can be prevented, treated or ameliorated. In addition, the filaggrin mRNA expression-promoting agent of the present embodiment can promote the expression of filaggrin and improve the moisturizing ability of the skin through the filaggrin mRNA expression-promoting action of the caninabara extract, whereby the elasticity of the skin can be improved. Can prevent, treat or improve skin aging, rough skin, etc. However, the filaggrin mRNA expression-promoting agent of the present embodiment can be used for all uses other than these uses, which are significant in exerting the filaggrin mRNA expression-promoting action.

The HAS3 mRNA expression-promoting agent of the present embodiment can promote the expression of hyaluronic acid synthase 3 (HAS3) through the HAS3 mRNA expression-promoting action of the canina rose extract, thereby promoting the production of hyaluronic acid. It can prevent, treat or ameliorate skin aging symptoms such as rough skin, wrinkles, dullness, changes in texture, decreased elasticity and decreased moisturizing function. However, the HAS3 mRNA expression promoter of the present embodiment can be used for all uses other than these uses that are significant in exerting the above-mentioned action.

Further, since the filaggrin mRNA expression-promoting agent and the HAS3 mRNA expression-promoting agent of the present embodiment have excellent filaggrin mRNA expression-promoting action and HAS3 mRNA expression-promoting action, respectively, they are blended with, for example, an external preparation for skin or an oral composition. It is suitable for. In this case, the canina rose extract may be blended as it is, or a filaggrin mRNA expression promoter or a HAS3 mRNA expression promoter formulated from the canina rose extract may be blended.

Here, the external skin preparation is not limited in its classification, and includes a wide range of skin lotions, non-pharmaceutical products, pharmaceuticals, etc. that are used transdermally, and specifically, for example, an ointment. Creams, milky lotions, lotions, beauty liquids, lotions, gels, beauty oils, packs, foundations, lip creams, bathing agents, hair tonics, hair lotions, shampoos, rinses, soaps, body shampoos and the like.

The blending amount of the canina rose extract in the skin external preparation can be appropriately adjusted according to the type of the skin external preparation, but the suitable blending ratio is 0.0001 to 10 mass in terms of the solid content of the canina rose extract. %, And a particularly suitable blending ratio is 0.001 to 1% by mass.

Oral compositions are those that are less likely to harm human health and are ingested by oral or gastrointestinal administration in normal social life, such as foods and drinks, pharmaceuticals, quasi-drugs, etc. under administrative divisions. It is not limited to the classification of. Therefore, the "oral composition" in the present embodiment includes general foods, feeds, health foods, foods with health claims (foods for specified health use, foods with nutritional claims, foods with functional claims), and non-pharmaceutical products that are orally ingested. , Pharmaceuticals, etc. are widely included.

When the canina rose extract is blended into an oral composition, the blending amount of the active ingredient in them can be appropriately changed in consideration of the purpose of use, symptoms, gender, etc. Considering the amount of intake, it is preferable that the daily intake of the extract for an adult is about 1 to 1000 mg. When the oral composition to be added is in the form of granules, tablets or capsules, the amount of the caninabara extract added is usually 0.1 to 0.1 to the solid content of the caninabara extract. It is 100% by mass or more, preferably 1 to 100% by mass or more.

In addition, the filaggrin mRNA expression-promoting agent and the HAS3 mRNA expression-promoting agent of the present embodiment have excellent filaggrin mRNA expression-promoting action and HAS3 mRNA expression-promoting action, respectively, and are therefore suitable as reagents for studies on these action mechanisms. Can be used for.

The filaggrin mRNA expression-promoting agent and the HAS3 mRNA expression-promoting agent of the present embodiment are suitably applied to humans, but animals other than humans (for example, as long as their respective actions and effects are exhibited). It can also be applied to mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.).

Hereinafter, the present invention will be specifically described with reference to production examples and test examples, but the present invention is not limited to the following examples.

[Production Example] Production of canina rose extract 1200 ml of 100% 1,3-butylene glycol was added to 100 g of dried fruit of canina rose, heat-extracted at 80 to 90 ° C. for 14 hours with a reflux extractor, and filtered hot. .. The obtained extract was dried to obtain a canina rose fruit extract (30 g, sample 1).

[Test Example 1] Filaggrin (FLG) mRNA expression promoting action test The filaggrin (FLG) mRNA expression promoting action was tested on the caninabara extract (Sample 1) as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were precultured using growth medium for normal human epidermal keratinocytes (KGM), and the cells were recovered by tripsin treatment. The collected cells were diluted with KGM medium to a cell density of 15 × 10 ⁴ cells / mL, then seeded in 2 mL each in a 35 mm petri dish (30 × 10 ⁴ cells / petri dish) at 37 ° C. and 5% CO ₂ . Incubated overnight under conditions. After culturing, the medium was replaced with a normal human epidermal keratinocyte basal medium (KBM, the above KGM medium to which growth factors (hEGF, BPE, insulin) were not added), and the cells were further cultured for 24 hours.

After culturing for 24 hours, the medium was removed, and 2 mL of a test sample (sample 1, sample concentration, see Table 1 below) dissolved in KBM medium was added to each petri dish under the conditions of 37 ° C. and 5% CO ₂ . Was cultured for 24 hours. As a control, the cells were similarly cultured using KBM medium to which no sample was added. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Nippon Gene Co., Ltd., Cat. No. 311-07361), and the amount of each RNA is measured with a spectrophotometer so that it becomes 80 ng / μL. Total RNA was prepared in.

Using this total RNA as a template, the expression level of mRNA was measured for filaggrin (FLG) and GAPDH, which is an internal standard. Detection is performed using a real-time PCR device Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.) and a real-time 2 Step RT by TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (manufactured by Takara Bio Inc., code No. RR063A). -It was carried out by PCR reaction. The primer used was manufactured by Takara Bio. The expression level of FLG mRNA was corrected by the GAPDH value based on the total RNA preparation prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the FLG mRNA expression promotion rate (%) was calculated by the following formula.

FLG mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with the addition of the test sample B: Correction value without the addition of the sample The results are shown in Table 1.

As shown in Table 1, the canina rose extract (Sample 1) had an excellent FLG mRNA expression promoting effect.

[Test Example 2] Hyaluronic acid synthase 3 (HAS3) mRNA expression promoting action test The caninabara extract (Sample 1) was tested for the HAS3 mRNA expression promoting action as follows.

Normal human neonatal epidermal keratinocytes (NHEK) were precultured using growth medium for normal human epidermal keratinocytes (KGM), and the cells were recovered by tripsin treatment. The collected cells were diluted with KGM medium to a cell density of 15 × 10 ⁴ cells / mL, then seeded in 2 mL each in a 35 mm petri dish (30 × 10 ⁴ cells / petri dish) at 37 ° C. and 5% CO ₂ . Incubated overnight under conditions. After culturing, the medium was replaced with a normal human epidermal keratinocyte basal medium (KBM, the above KGM medium to which growth factors (hEGF, BPE, insulin) were not added), and the cells were further cultured for 24 hours.

After culturing for 24 hours, the medium was removed, and 2 mL of the test sample (sample 1, sample concentration see Table 2 below) dissolved in KBM medium was added to each petri dish under the conditions of 37 ° C. and 5% CO ₂ . Was cultured for 24 hours. As a control, the cells were similarly cultured using KBM medium to which no sample was added. After culturing, the medium is removed, total RNA is extracted with ISOGEN II (Nippon Gene Co., Ltd., Cat. No. 311-07361), and the amount of each RNA is measured with a spectrophotometer so that it becomes 80 ng / μL. Total RNA was prepared in.

Using this total RNA as a template, the expression level of mRNA was measured for HAS3 and GAPDH, which is an internal standard. Detection is performed using a real-time PCR device Thermal Cycler Dice Real Time System III (manufactured by Takara Bio Inc.) and a real-time 2 Step RT by TaKaRa SYBR PrimeScript RT-PCR kit (Perfect Real Time) (manufactured by Takara Bio Inc., code No. RR063A). -It was carried out by PCR reaction. The primer used was manufactured by Takara Bio. The expression level of HAS3 mRNA was corrected by the GAPDH value based on the total RNA preparation prepared from the cells cultured in "test sample addition" and "sample no addition", respectively. From the obtained values, the HAS3 mRNA expression promotion rate (%) was calculated by the following formula.

HAS3 mRNA expression promotion rate (%) = A / B × 100
Each term in the formula represents the following.
A: Correction value with the addition of the test sample B: Correction value without the addition of the sample The results are shown in Table 2.

As shown in Table 2, the canina rose extract (Sample 1) had an excellent effect of promoting the expression of HAS3 mRNA.

[Formulation Example 1]
A cream having the following composition was produced by a conventional method.
Canina rose extract 0.05g
Kujin extract 0.1g
Scutellaria extract 0.1g
Liquid paraffin 5.0g
Sarashi beeswax 4.0g
Squalene 10.0g
Cetanol 3.0g
Lanolin 2.0g
Stearic acid 1.0g
Polyoxyethylene sorbitan oleate (20E.O.) 1.5g
Glyceryl monostearate 3.0 g
Oil-soluble licorice extract 0.1g
1,3-butylene glycol 6.0 g
Methyl paraoxybenzoate 1.5g
Fragrance 0.1g
Purified water balance (total amount is 100 g)

[Formulation Example 2]
A milky lotion was produced by a conventional method according to the following composition.
Canina rose extract 0.01g
Jojoba oil 4.00g
1,3-butylene glycol 3.00g
Arbutin 3.00g
Polyoxyethylene cetyl ether (20E.O.) 2.50 g
Olive oil 2.00g
Squalan 2.00g
Cetanol 2.00g
Glyceryl monostearate 2.00 g
Polyoxyethylene sorbitan oleate (20E.O.) 2.00 g
Methyl paraoxybenzoate 0.15 g
Stearyl glycyrrhetinate 0.10 g
Yellow 杞 extract 0.10g
Dipotassium glycyrrhizinate 0.10 g
Ginkgo leaf extract 0.10g
Conchiolin 0.10g
Phellodendron extract 0.10g
Chamomile extract 0.10g
Fragrance 0.05g
Purified water balance (total amount is 100 g)

[Formulation Example 3]
A beauty essence having the following composition was produced by a conventional method.
Canina rose extract 0.01g
Chamomile extract 0.1g
Carrot extract 0.1g
Xanthan gum 0.3g
Hydroxyethyl cellulose 0.1g
Carboxyvinyl polymer 0.1g
1,3-butylene glycol 4.0 g
Dipotassium glycyrrhizinate 0.1g
Glycerin 2.0g
Potassium hydroxide 0.25g
Fragrance 0.01g
Preservative (methyl paraoxybenzoate) 0.15 g
Ethanol 2.0g
Purified water balance (total amount is 100 g)

[Formulation Example 4]
Tablets having the following composition were produced by a conventional method.
Canina rose extract 5.0mg
Dolomite (containing 20% calcium and 10% magnesium) 83.4 mg
Casein phosphopeptide 16.7 mg
Vitamin C 33.4 mg
Maltitol 136.8 mg
Collagen 12.7 mg
Sucrose fatty acid ester 12.0 mg

[Formulation Example 5]
An oral liquid preparation having the following composition was produced by a conventional method.
<Composition in 1 ampoule (100 mL per ampoule)>
Canina rose extract 0.3% by mass
Sorbitol 12.0% by mass
Sodium benzoate 0.1% by mass
Fragrance 1.0% by mass
Calcium sulfate 0.5% by mass
Purified water balance (100% by mass)

The filaggrin mRNA expression promoter of the present invention can greatly contribute to the improvement or enhancement of the barrier function of the skin; the improvement or enhancement of the moisturizing ability of the skin; the prevention, treatment or improvement of dry skin diseases, and the like.
In addition, the HAS3 mRNA expression promoter of the present invention can greatly contribute to the prevention, treatment or improvement of skin aging symptoms such as rough skin, wrinkles, dullness, changes in texture, decreased elasticity and decreased moisturizing function.

Claims (2)

A filaggrin mRNA expression promoter characterized by containing a canina rose extract as an active ingredient. Hyaluronic acid synthase 3 mRNA expression promoter, which comprises a canina rose extract as an active ingredient.