JP2017043575A - Skin external preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a skin external preparation which aims at utilizing a plant extract ingredients having antioxidative property, keratinization/differentiation promoting property, ECM production promoting property, and anti-aging effect, which can be especially novel active ingredients of the skin external preparation, and takes a required effect.SOLUTION: The skin external preparation contains 0.000001 to 20 wt.% of an extract of cultured cells of Potentilla miyabei in the family Rosaceae or Antennaria dioica in the family Asteraceae as active ingredients of antioxidative property, keratinization/differentiation promoting property, ECM production promoting property, and anti-aging.SELECTED DRAWING: None

Description

  The present invention relates to an external preparation for skin such as cosmetics, and more particularly to an external preparation for skin containing as an active ingredient a cultured cell extract of Mecancanby (Rosaceae) or Echinotikogusa (Asteraceae).

  In general, it is known to use an ingredient extracted from a plant as an active ingredient for maintaining healthy skin required for cosmetics. For example, a cultured cell extract of Murasaki which is a perennial plant belonging to the genus Murasaki genus Cosmetics having anti-aging properties and whitening effects are known (Patent Document 1).

  In addition, as the plant to be extracted, Aronia, Malus, Malus, Pyrus, Amelanchier, Sorbus About plants selected from the genus, the genus Rhaphiolepis, the genus Aria, the genus Photinia, the genus Hawthorn (Crataegus), the genus Cotoneaster, the genus Osteomeles and the genus Pyracantha, It is known that cells are cultured to obtain a cultured cell extract containing triterpenes and flavonoids, and this is used for medicines, foods, cosmetics and the like (Patent Document 2).

  Furthermore, as a stem cell culture of green tea (chanoki), a cosmetic composition containing a callus-derived green tea stem cell culture and containing green tea polyphenols or the like is known (Patent Document 3).

JP 2003-267857 A JP 2011-26265 A Special table 2014-524466 gazette

  However, in Patent Document 1, although there is a description that a cultured cell extract of Murasaki genus Murasaki is added as a whitening component, whether or not similar useful components are extracted from other plants, There is no suggestion.

  In addition, Patent Document 2 has whitening, skin beautification, and cell activation components in extracts obtained by culturing plant cells for specific 13 genera belonging to the subfamily of the family Rosaceae. Although it is described that it can be used in cosmetics, nothing is described about the plants belonging to the subfamily Rosaceae, the subfamily of Chrysanthemum, other than the peach subfamily of the Rosaceae, about the plants belonging to these two subfamilies Similarly to the above, there is no description about whether or not a useful component for the skin is contained, and it is unclear.

  Patent Document 3 describes that a useful ingredient is contained in a stem cell culture of green tea and uses it for cosmetics, but there is no description about plants other than green tea.

  In addition, plants that can be cultivated even on flat ground and plants that can be cultivated in a relatively warm environment have been used effectively so far, but rare plants that live in harsh natural environments have not been fully researched. The survey was not progressing and was not fully utilized.

  The inventors of the present application pay attention to the fact that plants that inhabit harsh natural environments have excellent environmental adaptability and tolerance, and contain not only the plants themselves but also useful medicinal ingredients for humans I thought.

  Therefore, the object of the present invention is to find that a plant species that has not been considered from the viewpoint of protecting rare plant resources contains ingredients useful for human skin and whitening, and in particular, an external preparation for skin. Effectively obtained by using plant cell culture technology, the skin that contains antioxidant, keratinization / differentiation promoting, ECM production promoting, and anti-aging effects It is to provide an external preparation.

  In order to solve the above-mentioned problems, the inventors of the present application have disclosed that an extract of a cultured cell of the rose genus Potentilla miyabei or the asteraeaceae Ezonotachigusa (Antennaria dioica) has an antioxidant property. It has been found that it contains ingredients and the like, and it was used as an external preparation for skin containing it as an active ingredient.

The skin external preparation of the present invention configured as described above employs the above-mentioned predetermined plant species as an extraction raw material for the active ingredient, and the former Mekangkinbai (Potentilla miyabei) It is a plant belonging to the genus, and is an endemic species of Hokkaido that is distributed in the Shiretoko, Akan, Daisetsuzan, Tokachi mountain range, Mt.
The latter, Antennaria dioica, belongs to the genus Ezonochicogusa, and is distributed in Hokkaido's Rebun Island and the northern latitudes of Daisetsu, Akan, Shiretoko, etc., and also grows in dry grasslands of high mountains. It is a plant.

  A part of these predetermined plant tissues is collected and cultured, and an extract containing an antioxidant component is extracted from undifferentiated cells. Can be obtained.

  A skin external preparation containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as a keratinization / differentiation-promoting active ingredient can also be used. It is clear from the experimental results described later that there is an effect of promoting metabolism and maintaining beautiful skin.

  By the way, the skin has a granular layer, a spiny layer, and a basal layer in this order under the stratum corneum on the surface. Since it falls off the surface, the skin can be composed of younger cells as the period until the epidermis is replaced is shortened.

  In human skin, proteins (enzymes) related to keratinization / differentiation include keratin 10, involucrin, transglutaminase, and serine palmitoyltransferase, and the amount of their production can be used as an index to measure the degree of action. it can.

  Further, in the same manner as described above, a skin external preparation containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an active ingredient for promoting ECM production can also be used.

In addition, ECM (Extracellular Matrix) consists of proteins such as collagen, proteoglycan, fibronectin and laminin as an extracellular matrix in humans. These are useful ingredients to keep wrinkled and firm young skin.
In human skin, ECM-related factors (proteins such as extracellular matrix and enzymes) include elastin, fibrin, lysyl oxidase, collagen and the like.

  Further, in the same manner as described above, a skin external preparation containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an anti-aging active ingredient can also be used.

  Since the degree of skin cell aging can be estimated from the degree of skin cell damage due to aging, interleukin 6 (IL-6), which is known as an inflammation marker, can be measured and the relative value thereof can be used as an index of aging.

  The cultured cells are preferably undifferentiated cultured cells such as callus in order to efficiently obtain components derived from undifferentiated cells by culturing.

  In addition, when the above-mentioned active ingredient is added to the external preparation for skin, the content of the cultured cell extract (effective) so that the antioxidant property, the keratinization / differentiation promoting property, the ECM production promoting property and the anti-aging property are sufficiently exhibited. (Component amount: wt%) is preferably 0.000001 to 20 wt%, more preferably 0.00001 to 10 wt%.

  In addition, the cultured cell extract of the above active ingredient is obtained by lyophilizing cultured cells, dispersing 1 mL of water or a dedicated medium used for culturing each cell with 10 mg of lyophilized cells, and dispersing 0 0 after ultrasonic disruption extraction. A cultured cell extract filtered using a 2 micron filter.

  A topical skin preparation that fully exhibits antioxidative properties, keratinization / differentiation promoting properties, ECM production promoting properties, and anti-aging properties can be used to prevent and improve pigmentation, whitening effects to improve skin transparency, Since it has a skin beautifying effect that can prevent or improve tarmi, in the present invention, skin for skin whitening and skin beautification containing an extract of cultured cells of Potatoilla miyabei or Antennaria dioica as an active ingredient for skin whitening and skin beautifying. It can be used as an external preparation.

  Since the present invention is an external preparation for skin containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an active ingredient as described above, it has antioxidant properties useful for human skin and whitening. , Keratinization / differentiation promoting, ECM production promoting, plant-derived ingredients with anti-aging effect can be used, thereby preventing whitening or wrinkles and tarmi to prevent and improve pigmentation and improve skin transparency There is an advantage in that it becomes an external preparation for skin that can improve the effect of beautiful skin or an external preparation for skin whitening and skin.

  An embodiment of the present invention is an external preparation for skin containing, as an active ingredient, an extract from cultured cells of Potentilla miyabei belonging to the genus Rosaceae or Potentilla miyabei belonging to the asterae family Antennaria dioica.

  As a suitable method for obtaining an extract from cultured cells of these plants, callus is obtained aseptically from a part of plant tissue, for example, cotyledons, and callus having heterogeneous traits is subcultured in a liquid medium. Can be obtained as a highly uniform cultured cell line, and an extract can be obtained from this cultured cell line. In addition, in the process of obtaining a cultured cell line, a process for selecting high-content cells using the content of a specific component as an index is performed to obtain a high-produced cultured cell line of a specific component and extract it from the high-produced cultured cell line The method of obtaining a thing is mentioned.

  Undifferentiated cultured cells are usually obtained by growing callus, and in order to stably grow callus, the tissue collected from the plant body is sterilized with alcohol or the like, and then the plant hormone natural cytokinin. It is dedifferentiated in a solid medium containing certain kinetin and naphthalene acetic acid, which is a synthetic auxin, and cultured to form callus.

  For tissues collected from plants, it is usually preferable to use a shoot apical meristem or root meristem with a high frequency of cell division in that many stem cells that are undifferentiated cells are contained. However, all tissue cells of the plant body can be used without any particular limitation. In the callus culture method, since a dedifferentiation treatment is performed using a medium containing a plant hormone, it can be used without any particular restriction on the site such as leaves and stems.

  In cell culture, in order to select cells that produce as much antioxidant substance as possible, for example, callus with heterogeneous traits are divided into small clusters and grown, and a part of them is used to evaluate antioxidants, for example. It is possible to obtain a cultured cell line having high antioxidant activity by selecting callus nodules with high antioxidant activity by performing the same selection with the selected nodules as a parent. Strain acquisition and subculture can be employed as appropriate.

The “extract” in the present invention is not limited to an extract using an aqueous solvent, but may be an extract using an oil solvent.
That is, in this invention, an aqueous solvent extraction method, an oily solvent extraction method or a mixed solvent extraction method can be adopted as an extraction method, and a critical extraction method or an ultrasonic disruption extraction method (after destroying cells by ultrasonic shearing force, An extraction method such as solid-liquid separation may be employed, and appropriate conditions can be appropriately employed.
Such an extract can also be obtained by drying a cultured cell or culture solution and then extracting it with an appropriate extraction solvent.

  More specifically, extraction solvents that can be used for extraction from cultured cells include, for example, water, lower monohydric alcohols (methyl alcohol, ethyl alcohol, 1-propanol, 2-propanol, 1-butanol, 2-butanol. Etc.), polyhydric alcohols (glycerin, propylene glycol, 1,3-butylene glycol etc.), lower alkyl esters (ethyl acetate etc.), hydrocarbons (benzene, hexane, pentane etc.), ketones (acetone, methyl ethyl ketone etc.), Examples include ethers (diethyl ether, tetrahydrofuran, dipropyl ether, etc.), acetonitrile, and the like, and these can be used alone or in combination of two or more. Thus, since the extraction solvent which can be used for this invention may be aqueous or oily, it is considered that the active ingredient contained in the extract contains an amphiphilic substance.

  In addition, as a specific step in extracting cultured cells, extraction was performed for 1 to 5 days using ethyl alcohol or 1,3-butylene glycol having a water content of 0 to 100% by weight at room temperature or warming. Thereafter, the step of allowing the filtrate obtained by filtration to stand still for about one week and ripen, and performing filtration again is exemplified.

  The cultured cell extract thus obtained and the extract obtained directly by squeezing the plant body, etc., are extracts obtained from undifferentiated plant cells having the differentiation totipotency. The latter is an extract obtained from differentiated plant cells, and since its component composition is different, its properties are also different.

  In addition, cultured cell extracts can be stably supplied without being harmed by the weather or pests, can be supplied with specific functional ingredients selectively and with high efficiency by selecting culture conditions, and are harmful to agricultural chemicals. Since there is no fear of contamination with substances, it is highly safe, and it can be cultured in large quantities from cells collected from a very small part of the plant, so that the influence on rare plants is minimized.

<Acquisition of calla and liquid cultures of Mekangkinbai>
The leaves of Potentilla miyabei were sterilized with a 70 wt% ethanol solution and a 10 wt% antiformin solution and cut into 5-10 mm squares under aseptic conditions. Placed on a 0.25 wt% gellite solid medium, which is an agar medium of Murashige & Skoog (MS) containing ⁻⁵ M naphthalene acetic acid (NAA) and 10 ⁻⁵ M kinetin, and statically cultured at 25 ° C. in the dark. And got callus.

After obtaining a callus that stably grows by repeated passage in a solid medium, a liquid medium of Gambog B5 (B5-NK medium) containing 3% by weight sucrose, 10 ⁻⁵ M NAA and 10 ⁻⁵ M kinetin The callus was transplanted and the passage was repeated every 14 days to increase cell proliferation.
Subsequently, the cells were transplanted to a B5 liquid medium (B5-IK medium) containing 2% by weight sucrose, 0.6% by weight glucose, 10 ⁻⁵ M indolebutyric acid (IBA) and 10 ⁻⁶ M kinetin, and 11-14. The passage was repeated every day to increase cell proliferation, and a passable Mekangkinbai strain was obtained.

<Acquisition of callus and liquid culture of Antennaria dioica>
Stem part of Ezonoticogusa is sterilized with 70 wt% ethanol solution, 10 wt% antiformin solution and cut into 5 mm length under aseptic conditions, and then the tissue piece is filled with 3 wt% sucrose and ^10-5 M naphthalene acetic acid (NAA). The callus was obtained by placing on a 0.25 wt% gellite solid medium of Murashige & Skoog agar medium (MS agar medium) and static culture in the dark at 25 ° C.

After obtaining the callus, the callus was transplanted to a liquid medium under the same conditions without gellite, and the passage was repeated every 14 days to increase cell proliferation. Subsequently, the cells were transplanted into a liquid medium of MS containing 3% by weight of sucrose, 10 ⁻⁵ M indolebutyric acid (IBA) and 10 ⁻⁶ M kinetin, and the passage was repeated to increase the cell proliferation, every 14 days. Acquired Ezonotichikogusa strain that can be passaged to The cultured cells of Ezonotichigusa strain were transplanted at a density of 24 g / L, and 100 g of cultured cell extract could be recovered on the 14th day.

[Production Example 1]
Freeze-dried the cultured cells of Mekangkinbai strain or Echinoticogusa strain, and add 1 mL of water or a special medium used for culturing each cell to 10 mg of freeze-dried cells, and disperse by ultrasonic crushing extraction. I did it. After extraction, filtration was performed using a 0.2 micron filter to obtain a cultured cell extract.

[Production Example 2]
The obtained cultured cells of Mekangkinbai strain or cultured Ezonotichigusa strain are frozen, and 30 wt% 1,3-butylene glycol solution is added at a ratio of 500 mL to 100 g of the frozen cells and mixed by stirring at room temperature. Daily extraction was performed. After extraction, the solid content of cells and the like is filtered using filter paper, and the obtained filtrate is further allowed to stand at 3 ° C. for 1 week, followed by filtration using 0.45 micron and 0.22 micron filters, and cultured cells. I got the extract.

  Using the sample of Mekangbai bean culture cell extract (Extract A) or Ezochinochigusa cell extract (Extract B) obtained in Production Example 1 as described above, the antioxidant effect, the keratinization / differentiation promoting effect, and the ECM production promoting as follows: The evaluation test of the effect and anti-aging effect was conducted.

[Evaluation test of antioxidant effect]
0.075 mM 1,1-diphenyl-2-picryl-hydrazyl (hereinafter referred to as DPPH) solution containing 5% by mass of a mecancanby cultured cell extract or an Ezonotikogusa cultured cell extract After adding 5% by mass and incubating at 25 ° C. for 30 minutes, the radical scavenging rate was calculated from the absorbance at 520 nm to evaluate the antioxidant effect.

  As shown in Table 1, it has been clarified that the mecancanby cultured cell extract and the echinomycete cultured cell extract have an antioxidant effect, and in particular, the mecancanby cultured cell extract has a high effect.

[Evaluation test of keratinization / differentiation promoting effect]
Normal RNA was extracted from normal human epidermal keratinocytes (NHEK) and cultured for 2 days in a medium supplemented with 5% by mass of Mekangkinbai cultured cell extract or 5% by mass of Ezochinochigusa cultured cell extract.
After reverse transcription to cDNA, the effect of promoting keratinization / differentiation was evaluated by real-time PCR based on the expression level of mRNA gene, which is a general keratinization / differentiation-related factor shown below.

  As shown in Table 2, it has been clarified that the maekankinbai cultured cell extract and the Echinotachigusa cultured cell extract have a keratinization / differentiation promoting effect.

[Evaluation test of ECM production promoting effect]
Normal human dermal fibroblasts (NB1RGB) were used, and cultured for 1 day in a medium supplemented with 5% by weight of Mekangkinbai cultured cell extract or 1% by weight of Ezochinochigusa cultured cell extract, and then total RNA was extracted. After reverse transcription to cDNA, the effect of promoting ECM production was evaluated by real-time PCR based on the expression level of the mRNA gene of the ECM-related factor shown below.

  As shown in Table 3, it has been clarified that the mecancanby cultured cell extract and the Ezonotichigusa cultured cell extract have an ECM production promoting effect.

[Confirmation test of anti-aging effect]
Normal human dermal fibroblasts (NB1RGB) were used, and cultured for 2 days in a medium supplemented with 5% by weight of Mekangkinbai cultured cell extract or 1% by weight of Echinotikogusa cultured cell extract, and then total RNA was extracted. After reverse transcription to cDNA, the anti-aging effect was evaluated by real-time PCR based on the expression level of the mRNA gene of the aging-related factor shown below.

  As shown in Table 4, it has been clarified that the mecancanby cultured cell extract or the Echinotachigusa cultured cell extract has an anti-aging effect.

[Examples 1 and 2, comparative example]
As Example 1, an aqueous lotion was prepared by blending the mecancanby cultured cell extract obtained in Production Example 2 (“extraction solvent: 1,3 butylene glycol” hereinafter the same) with the following formulation.
As Example 2, an aqueous lotion was prepared by blending the Echinotachigusa cultured cell extract obtained in Production Example 2.

(Ingredient) (wt%)
1,3-butylene glycol 5
Glycerin 3
Pentylene glycol 2
Polysorbate 20 0.3
Phenoxyethanol 0.3
Meacankinbai cultured cell extract or Echinotachigusa cultured cell extract 1
Purified water residue

Furthermore, as a comparative example, a water-based lotion containing no maekankinbai cultured cells or Ezonotichigusa cultured cell extract was prepared.
About these, the following sensory evaluation tests were implemented.

  The effects of “Moisture”, “Glossy”, “Transparency”, “Hari” and “Anti-wrinkle” were verified for Example 1, Example 2 and Comparative Example. In the verification, three monitors having healthy skin were used a plurality of times, and each item was verified by sensory evaluation in five stages (bad: grade 1 to good: grade 5).

As shown in Table 5, it was revealed that each example was superior to the comparative example.
In addition, since the extract of manufacture example 1 also contains the same component as manufacture example 2, it is clear that there exists an effect similar to the above.

  Formulation examples of cosmetics for the external preparation for skin of the present invention are listed below. In these formulation examples, the numerical value described after the component name is a blending ratio, and all the units are weight%.

[Formulation Example 1; lotion]
(Ingredient) (wt%)
Glycerin 2
1,3-butylene glycol 5
Meacankinbai cultured cell extract or Echinotachigusa cultured cell extract 5
Carboxyvinyl polymer 0.05
Potassium hydroxide 0.025
Phenoxyethanol Appropriate amount Purified water Residual perfume Appropriate amount

[Prescription Example 2; Emulsion]
(Ingredient) (wt%)
Dimethylpolysiloxane 2.5
Decamethylcyclopentasiloxane 23
Dodecamethylcyclohexasiloxane 15
Polyoxyethylene / methylpolysiloxane copolymer 1.5
Trimethylsiloxysilicic acid 1
1,3-butylene glycol 5
Squalane 1.5
Talc 6
Meacankinbai cultured cell extract or Echinotachigusa cultured cell extract 1
Edetate trisodium 0.05
2-methoxyhexyl paramethoxycinnamate 5
Silicone coated fine particle titanium oxide 4
Dimethyl distearyl ammonium hectorite 0.5
Spherical polyethylene powder 3
Phenoxyethanol Appropriate amount Purified water Residual perfume Appropriate amount

[Prescription Example 3; Cream]
(Ingredient) (wt%)
Liquid paraffin 9
Vaseline 2
Dimethylpolysiloxane 2
Stearyl alcohol 4
Behenyl alcohol 2
Glycerin 5
Dipropylene glycol 4
Tetra-2-ethylhexanoic acid pentaerythrit 4
Lipophilic glyceryl monostearate 2
Polyisoethylene glyceryl monoisostearate 2
Polyoxyethylene glyceryl monostearate 1
Citric acid 0.05
Sodium citrate 0.05
Sodium hydroxide 0.015
Mecancanby cultured cell extract or Echinotachigusa cultured cell extract 0.1
Phenoxyethanol Appropriate amount Carboxyvinyl polymer 0.05
Purified water Residual fragrance Appropriate amount

  In addition, the present invention can use conventionally used production methods, and dosage forms such as emulsions, creams, lotions, packs, cleaning agents, dispersions, ointments, solutions, aerosols, patches, poultices, etc. It can be implemented regardless.

  By using the cultured cell extract as a raw material, it becomes possible to use it for cosmetics and the like for rare species that have a short supply amount and as a result have a fatal problem for use as an industrial raw material. In particular, macankinbai and Ezonotachigusa can be used as a topical skin preparation having a high effect that has not been conventionally known as an antioxidant, keratinization / differentiation, ECM production or anti-aging functional ingredient.

Claims (7)

  A skin external preparation containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an antioxidant active ingredient.   A topical skin preparation containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an active ingredient for promoting keratinization and differentiation.   An external preparation for skin containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an active ingredient for promoting ECM production.   A topical skin preparation containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an anti-aging active ingredient.   The skin external preparation according to any one of claims 1 to 4, wherein the cultured cells are undifferentiated cultured cells.   The skin external preparation according to any one of claims 1 to 5, wherein an amount of the active ingredient is 0.000001 to 20% by weight.   A skin whitening and skincare external preparation containing an extract of cultured cells of Potentilla miyabei or Antennaria dioica as an active ingredient for whitening and skincare.