JP6904688B2 - Epithelial cell activator - Google Patents

Description

The present invention relates to epithelial cell activators.

Epithelial cells refer to cells that make up the epithelial tissue of the skin and mucosa, and various cells such as epithelial keratinized cells, intestinal epithelial cells, corneal epithelial cells, oral mucosal cells, conjunctival epithelial cells, esophageal epithelial cells, and airway epithelial cells. Exists.

The skin epidermis has a barrier function of preventing water loss from the inside of the living body to the external environment and retaining water. The epidermis of the skin is composed of the basal layer, the stratum spinosum, the stratum granulosum, and the stratum corneum (stratum corneum) from the bottom. 95% of the cells present in the epidermis are keratinocytes (also called keratinocytes), and the remaining 5% are pigment cells (also called melanocytes) or Langerhans cells. Keratinocytes, which are epithelial cells of the skin, divide in the basal layer, differentiate while moving to the spinous layer, the stratum granulosum, and the upper layer, and become enucleated keratinocytes in the stratum corneum. The stratum corneum is layered to form about 10 stratum corneum, but the stratum corneum becomes so-called dirt and peels off in order from the surface layer. The process from the proliferation of keratinocytes in the basal layer to the shedding of keratinocytes on the surface of the epidermis is called skin turnover.

The function of keratinocytes may decrease due to various causes such as aging and stress. When the function of keratinocytes is reduced, the turnover time of the skin is delayed accordingly, resulting in poor differentiation such as hyperkeratosis. Due to such hyperkeratosis, symptoms such as dull skin color appear.

For this reason, the search for substances for activating keratinocytes has been carried out. For example, Patent Document 1 describes that the human lizard extract has a function of proliferating epidermal keratinized cells.

On the other hand, saffron is a perennial plant belonging to the genus Crocus of the Iridaceae family, and its pistil is used as a spice. Patent Document 2 describes that saffron pistil extract activates dermal fibroblasts and dermal papilla cells.

Japanese Unexamined Patent Publication No. 2008-088075 Japanese Unexamined Patent Publication No. 2005-41812

The search for substances to activate epithelial cells is still sought after. Therefore, an object of the present invention is to provide a drug that activates cells of epithelial cells, particularly cells of keratinocytes.

As a result of searching for various components in order to achieve the above object, the present inventors have found that the saffron extract has an epithelial cell activating effect, and completed the present invention.

Therefore, the present invention is an epithelial cell activator containing an extract of Iridaceae Crocus saffron.

According to the present invention, by activating epithelial cells, it is possible to improve various symptoms associated with a decrease in the function of epithelial cells.

It is a figure which shows the cell proliferation ability of a saffron extract.

Hereinafter, embodiments of the present invention will be described. The present invention is not limited to the following embodiments. In the present specification, "X to Y" indicating a range includes X and Y and means "X or more and Y or less". Unless otherwise specified, operations and physical properties are measured under the conditions of room temperature (20 to 25 ° C.) / relative humidity of 40 to 50% RH.

The epithelial cell activator of the present invention contains an extract of Iridaceae Crocus saffron as an active ingredient. Since the active ingredient is a plant extract, it is highly safe for living organisms, especially the skin.

Examples of epithelial cells include epithelial keratinocytes, intestinal epithelial cells, corneal epithelial cells, oral mucosal cells, conjunctival epithelial cells, esophageal epithelial cells, airway epithelial cells and the like. Among them, epithelial cells are preferably (epidermis) keratinocytes.

Cell activation refers to activation of the function inherent in epithelial cells and / or improvement of function, and specific examples thereof include cell proliferation, activation of stimulus responsiveness, and improvement of stress tolerance by external factors.

The application target of the cell activator is not particularly limited, but is preferably a mammal, and more preferably a human.

Saffron (scientific name: Crocus sativus) is a perennial plant belonging to the genus Crocus in the family Iridaceae. The parts used for the extract include the entire saffron plant, leaves, stems, buds, flowers (including gaku, petals, pistils, oshibe, etc.), woody parts, bark (pericarp), fruits (receptacles (pericarp), etc.). Above-ground parts such as ovaries, pericarps (including inner pericarp, pistil, outer pericarp), seeds; and parts of plants such as roots, rhizomes, and underground parts such as stalks. The plant portion may be subjected to extraction alone or in the form of a mixture of two or more species. Of these, considering the activating effect of keratinocytes, it is preferable to use flowers, more preferably pistils, and even more preferably stigmas. That is, in a preferred form of the present invention, the extract of the genus Saffron of the genus Crocus of the Iridaceae family is the extract of the stigma.

The raw material used for extraction may be subjected to extraction in its original form, or may be pre-dried and / or ground before being subjected to extraction. This makes it possible to more efficiently extract the desired active ingredient from the raw material.

Next, if necessary, the pre-dried and / or pulverized raw material is extracted with a suitable solvent. The solvent that can be used here is not particularly limited as long as it can extract the active ingredient from the raw material, and is appropriately selected depending on the plant to be used and the part of the plant. Specifically, water (including tap water, industrial water, distilled water, back-penetration membrane water, filtered water, sterilized water, purified water, etc.); methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, Alcohols such as 2-butanol, propylene glycol and 1,3-butylene glycol; esters such as methyl acetate and ethyl acetate; ketones such as acetone; diethyl ether, formaldehyde, dimethyl sulfoxide and the like. The above solvent may be used alone or in combination of two or more. Of these, water is preferable as the extraction solvent in consideration of extraction efficiency, safety and the like. That is, in a preferred embodiment of the present invention, the extract of Iridaceae Crocus saffron is a water extract.

The extraction may be performed only once or multiple times. In the latter case, any solvent may be used in each extraction step, and the extraction conditions in each step may be the same or different.

The amount of the solvent added is not particularly limited as long as the active ingredient can be extracted from the raw material. Specifically, it is preferable to add the solvent in an amount of 10 to 1000 ml, more preferably 50 to 500 ml, with respect to 10 g of the raw material.

The extraction conditions are also not particularly limited as long as the active ingredient can be extracted from the raw material, and may be, for example, a cold dipping method or a hot dipping method such as hot water extraction. Specifically, the extraction temperature is preferably 1 to 150 ° C, more preferably 5 to 120 ° C. The extraction time is preferably 30 minutes to 48 hours, more preferably 1 to 24 hours. Under such conditions, the active ingredient can be efficiently extracted from the raw material.

After the extraction step, it is preferable to remove the solid matter (raw material residue) from the mixture of the raw material and the solvent to separate the extract. Here, the separation method is not particularly limited, and examples thereof include filtration and centrifugation. Further, this extract may be used as it is, but if necessary, a diluted form by a diluted solution, an extract by concentration, a paste or solid form, a frozen product form by freezing, a dried powder form by freeze-drying, etc. It may be in various forms (extracts).

In addition, the extract or extract may be further purified. Here, the purification method is not particularly limited, and a known purification method can be used. Examples of the refining method include activated carbon treatment, adsorption resin treatment, ion exchange resin treatment and the like.

The saffron extract may be a commercially available product.

By using the epithelial cell activator of the present embodiment, epithelial cells having reduced activity can be activated. Specifically, when the epithelial cells are intestinal cells, the absorption of nutrients can be improved by improving the intestinal function by increasing the number of epithelial cells, for example. Further, when the epithelial cells are keratinocytes, for example, there is an effect that hyperkeratosis is suppressed and dullness of the skin is improved. Here, the keratinocyte activator, which is a preferred embodiment, improves dullness caused by a decrease in the activity of keratinocytes, and even if it is "dullness", for example, due to the unevenness of wrinkles. It is different from agents that improve physically occurring dullness by acting on dermal cells and suppressing the formation of wrinkles. Furthermore, by promoting the activation of keratinocytes, skin regeneration is promoted, and for example, treatment of wounds can be expected.

The epithelial cell activator may be blended with other additives depending on the mode of use.

The form of use of the cell activator of the present embodiment is not particularly limited, and can be used in various dosage forms such as transdermal, oral, nasal, topical (including intraoral and sublingual), rectal, and vaginal administration.

When applying epithelial cell activators to the skin, other additives include water, alcohol, oils, surfactants, thickeners, powders, chelating agents, pH regulators, moisturizers, whitening agents, etc. Examples include various medicinal agents such as anti-inflammatory agents and cell activators, extracts derived from animals, plants and microorganisms, and fragrances. When the epithelial cell activator is applied to the skin, its dosage form is not particularly limited, and the dosage forms include emulsions, creams, aqueous liquids, gels, packs, dispersions, ointments, liquids, and powders ( (Including molded bodies), sprays, patches, etc.

For emulsified preparations such as emulsions and creams, aqueous extracts are appropriately produced by using oils such as fat, fatty oil, lanolin, petrolatum, paraffin, wax and higher alcohol, polyhydric alcohols, water and emulsifiers. can do.

In addition to the active ingredients, the powders and sprays may contain prostheses such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powders, or mixtures of these substances. The spray may further include fluorinated hydrocarbons and conventional high pressure gases such as volatile unsubstituted hydrocarbons such as butane and propane.

The patch has the additional advantage of controlling and delivering the active ingredient of the present embodiment to the body. Such an application can be made by dissolving or dispersing the active ingredient of the present embodiment in a suitable medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The speed of such a flux can be controlled either by providing a speed control membrane or by dispersing the compound in a polymer matrix or gel.

When the epithelial cell activator is taken orally, other additives include excipients, bases, emulsifiers, stabilizers, solubilizers, flavoring agents, preservatives, air fresheners, colorants, etc. A coating agent or the like can be appropriately blended. In addition, when the epithelial cell activator is taken orally, the dosage form is not particularly limited, and any solid dosage form such as tablets, pills, powders, powders, granules; liquids, gels, etc. It may be in shape.

In solid dosage forms for oral administration (tablets, rounds, powders, powders, granules, etc.), the active ingredient is one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate. And / or mixed with any of the following: fillers or bulking agents such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; eg, carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and / Or a binder such as gum arabic; a moisturizer such as glycerol; a disintegrant such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; such as paraffin Dissolution retarders; Absorption enhancers such as quaternary ammonium compounds; Wetting agents such as cetyl alcohol and glycerol monostearate; Absorbents such as kaolin and bentonite clay; Starch, calcium stearate, magnesium stearate, solid polyethylene glycol , Lubricants such as sodium lauryl sucrose, and mixtures thereof; and colorants. In the case of tablets and pills, a buffer may be included.

Such an application form is appropriately selected in consideration of the epithelial cells targeted by the epithelial cell activator of the present embodiment. When the epithelial cells are keratinocytes, application to the skin is advantageous in that they act directly on the target keratinocytes. In addition, since the active ingredient is a plant extract, it is also advantageous in that it is highly safe for the skin. When applied to the skin, it can be used in the form of use of external preparations for skin such as cosmetics, pharmaceuticals, and quasi-drugs. In this case, the content of the active ingredient in the external preparation for skin is preferably 0.000001% by mass or more in terms of solid content in consideration of the manifestation of the effect, and 50% by mass in consideration of the saturation of the effect. It is preferably the following, more preferably 0.00001 to 50% by mass, and even more preferably 0.0001 to 30% by mass. The solid content in terms of solid content referred to here refers to a residue when the extraction solvent (for example, water) of the saffron extract is volatilized, and is a concept including, for example, a highly viscous component.

The present invention also provides a method for proliferating epidermal keratinocytes, in which epidermal keratinocytes are brought into contact with a saffron extract to proliferate the epidermal keratinocytes. Specifically, it is a method for proliferating epidermal keratinocytes, in which epidermal keratinocytes are brought into contact with a medium containing a saffron extract to proliferate the epidermal keratinocytes.

Isolated human epidermal cells (keratinocytes) for culturing can be prepared by various methods known per se. Both the primary culture and the subculture can be carried out in the same manner, and the culture can be carried out basically according to a conventional method, for example, at 37 ° C. under a 5% carbon dioxide gas phase. There are no restrictions on the size of the culture dish, the culture substrate, the cell density at the time of seeding, the time of medium replacement, and the like.

The basal medium is not particularly limited, and known ones can be used. Specific examples thereof include DMEM, MEM, F12, DME, RPMI1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, CnT-Prime and the like. ..

The concentration of the saffron extract added to the basal medium is more preferably 15 μg / mL or more, and further preferably 30 μg / mL or more in terms of solid content, since the proliferative effect is effectively exhibited. The upper limit of the concentration of the saffron extract added to the basal medium in the medium is not particularly limited, but in consideration of the saturation of the effect, it is preferably 300 μg / mL or less, and 200 μg / mL or less. Is more preferable, and 100 μg / mL or less is further preferable.

Excellent proliferation of epidermal cells (particularly human-derived epidermal cells) is achieved by using a medium containing saffron extract. The skin piece thus grown can be used as an epidermal cell sheet for skin grafting in the same manner as a sheet of this type already known.

The effects of the present invention will be described with reference to the following examples and comparative examples. However, the technical scope of the present invention is not limited to the following examples. Unless otherwise specified, each operation is performed at room temperature (20 to 25 ° C.).

<Production Example 1> Method for producing saffron extract 20 L of purified water was added to 1 kg of the stigma of saffron, and the mixture was stirred at room temperature (25 ° C.) for 1 day (24 hours). Then, it was filtered to obtain a water extract of the stigma of saffron. The solid content concentration was 2.0% by mass.

<Production Example 2> Method for producing carrot extract A carrot extract was obtained by adding 100 mL of ethanol to 10 g of otane carrot root, stirring and filtering, and then adding purified water to prepare a 50% by volume ethanol aqueous solution. .. The solid content concentration was 2.0% by mass.

<Test Example 1>
The cell proliferation rate of human-derived keratinocytes (HPEKs: manufactured by CellnTech) was measured.

An appropriate amount of Cnt-PR (CnT-Prime, Epithelium Culture Medium: manufactured by CellnTechs) medium was placed on a 24-well microplate, and keratinized cells were ^{seeded at 1 × 10 4} cells per well at 37 ° C. and carbon dioxide. The mixture was allowed to stand at a concentration of 5% for 1 day.

Then, the saffron extract was added to the medium so that the final solid content concentration of the saffron extract was 50 μg / ml and 100 μg / ml.

After 7 days of culturing, 1 mL of Cell Titer 96 (registered trademark) Aqueous One solution Cell Proliferation Assay (manufactured by Promega) was added to each well so as to be 20% by volume in the medium. After culturing for 1 hour, the absorbance was measured at 650 nm with a reference of 490 nm using a microplate reader (n = 3). The measured values were averaged, and (average value of extract addition / average value of control) × 100 was calculated. In addition, what was cultured in a normal medium was used as a control.

Separately, the same test was conducted on the carrot extract.

The results are shown in Table 1 and FIG. 1 below.

From the above results, it can be seen that the saffron extract can remarkably promote the proliferation of epidermal keratinocytes, and the saffron extract can be used as an active ingredient to provide the use of a keratinocyte proliferating agent.

Claims (3)

Parenteral epidermal keratinizing cell proliferation agent containing a water extract of the stigma of saffron of the genus Crocus of the Iridaceae family (except for the purpose of wound healing). A parenteral epidermal keratinizing cell proliferation agent for improving dullness of the skin, which contains a water extract of the stigma of saffron of the genus Crocus of the Iridaceae family. A method for proliferating epidermal keratinocytes, in which epidermal keratinocytes are brought into contact with a medium containing a water extract of the stigma of the stigma of Iridaceae Crocus saffron to proliferate the epidermal keratinocytes.

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