KR101519649B1 - Neurocosmetic Skin Care Method, Method For Screening Skin Cosmetic Material, and Skin Cosmetic Material - Google Patents

Abstract

The present invention relates to a skin cosmetic method comprising the step of inhibiting the activity of a neurophysiological receptor (TRPV-1, VR1, Transient Receptor Potential family V-1), a skin cosmetic substance screening method, There is provided a skin-beauty composition comprising as an active ingredient a substance that inhibits activity.

According to the present invention, it is possible to increase skin beauty through inhibiting the activity of neurogenic sensory receptors, and to select a target material effective for skin cosmetics. The selected substance is selected from the group consisting of collagenase Can alleviate skin irritation, promote blood circulation, and is particularly effective for skin care through prevention of skin aging and the like.

Nerve pain sensory receptors, skin care

Description

[0001] The present invention relates to a neuro-cosmetic skin care method, a skin cosmetic material screening method, and a skin cosmetic material,

The present invention relates to a skin cosmetic method, a skin cosmetic material screening method, and a skin cosmetic composition.

Although skin aesthetics related to skin aging has been mainly studied in the dermis layer, researches on the skin nervous system and neurotransmitters have recently been actively conducted, and the skin is connected to the nerves, and various neurotransmitters affect the skin The transient receptor potential family V 1 (capsular receptor: TRPV-1) is one of the interesting studies.

TRPV-1 is expressed in all nervous systems such as skin, brain and spinal cord. This receptor senses changes in heat, pH and ion, and stimulates nociceptive stimuli in sensory neurons that induce substance P, an inflammatory neurotransmitter. Lt; / RTI > receptor. It is known that TRPV-1 induces neurogenic inflammatory responses by promoting the expression of substance P, CGRP, etc., which are neurotransmitters in nerve fibers, due to heat and pH changes caused by external stimuli.

TRPV-1 itself is a heat-sensitive sensor, and its activity is maximized at about 43 ° C. TRPV-1 is known to be expressed not only in neurons, but also in mesenchymal, epithelial and epidermal cells.

In addition, TRPV-1 activation has been reported to have important effects such as cell proliferation, death, differentiation and promotion of various cytokine secretion.

Recently, neurocosmetic raw materials using neurotransmitters have been or are being developed in connection with raw materials for cosmetics. Examples of a neuro-cosmetic raw material include a tripeptide inhibiting TNF-a by inhibiting neuroinflammatory responses through modulation of a melanocortin-1 receptor (MC1-R) .

It is a first object of the present invention to provide a skin care method capable of preventing skin aging and a second object of the present invention is to provide a method for screening a skin cosmetic material capable of preventing skin aging, A third object of the present invention is to provide a skin-care composition for preventing skin aging.

In order to achieve this object, the skin care method according to the present invention is characterized in that it comprises the step of inhibiting the activity of a neuropathic sensory receptor (TRPV-1, VR1, Transient Receptor Potential family V-1).

The method for screening a skin cosmetic material according to the present invention is characterized in that it comprises the step of discriminating the inhibition of the activity of the target substance on the neuropathic sensory receptors.

Furthermore, the skin-care composition according to the present invention is characterized by containing a substance which inhibits the activity of a neuropathic sensory receptor as an active ingredient.

The present invention can select a target material effective for preventing skin aging, and a substance that inhibits the activity of a selected nerve pain sensory receptor can be used for reducing the expression of collagenase due to the activity of nerve pain sensory receptors, It is possible to induce blood circulation promotion, and it can be confirmed that it is particularly effective in preventing skin aging.

The inventors of the present application have confirmed that the activity of the nerve pain sensory receptor is increased in the epidermis of the skin by ultraviolet rays as shown through the following examples. Through inhibiting the activity of the nerve pain sensory receptors, It is possible to increase the beauty of the skin through prevention of the generation.

As used herein, the term "inhibition of activity" refers to inhibition of the production of neurogenic sensory receptors, specifically inhibition of neurogenic sensory receptors by inactivation and dysfunction, as well as inhibition of expression of genes expressing neurogenic sensory receptors And inhibiting the synthesis of nerve pain sensory receptor proteins.

In one preferred example, the skin aesthetic can be achieved by inhibiting the expression of the matrix metalloproteinase, through inhibition of the activity of the neuropathic sensory receptors.

The matrix metalloproteinase is an enzyme that promotes collagen degradation and induces deformation of the dermis layer. If the production and decomposition of collagen can not be properly controlled, skin elasticity is reduced and wrinkles are generated. It is the main cause.

As shown in the following examples, when heat is given to epidermal cells cultured in the skin and immortalized human keratinocyte cell line (HaCaT), the matrix metalloproteinase And the expression of metalloproteinase by heat is reduced when capsaicin, a nerve pain sensory receptor antagonist, is reduced. As a result, the expression of matrix metalloproteinase is regulated through pain sensory receptors .

Therefore, the substance that inhibits the activity of neurogenic sensory receptors can reduce the expression of collagenase, prevent and prevent wrinkle formation of the skin, prevent skin aging through improvement of elasticity, alleviate skin irritation, And promotes blood circulation, thereby maintaining skin homeostasis, thereby achieving skin beauty.

There is no particular limitation on the method of skin-treating a substance that inhibits the neuropathic sensory receptors to a specific site. For example, the substance is applied to the skin in the form of an external skin to affect the activity of the neuralgic sensory receptors .

The present invention also relates to a method of screening a skin cosmetic material comprising the step of discriminating whether or not the object substance inhibits the activity on the neuropathic sensory receptors.

In one preferred embodiment, the step of determining the inhibition of the activity of the target substance against the neuropathic sensory receptors comprises the steps of: selecting a candidate for a substance inhibiting activity on neuralgic sensory receptors, (Ca < ^{2 +} > influx assay).

For example, the calcium influx assay can measure changes in intracellular calcium ion concentration upon treatment with capsaicin, which acts as an antagonist of nerve pain sensory receptors, A method for determining the degree of inhibition by measuring a change in the concentration of calcium ions in the body and a difference between the concentrations of calcium ions in the body, thereby screening a skin cosmetic material that inhibits the activity of a neuropathic sensory receptor.

The present invention also relates to a skin cosmetic composition containing as an active ingredient a substance that inhibits the activity of a neuropathic sensory receptor, wherein the composition is used for reducing skin aging through reduction of expression of collagenase, alleviation of skin irritation, Prevention, and wrinkle formation.

In addition, the substance that inhibits the activity of the nerve pain sensory receptor can inhibit the expression of matrix metalloprotease, and the matrix metalloproteinase is a collagenase, and the present inventors The expression of matrix metalloproteinase was also decreased when epidermal cells were treated with a substance that inhibits the activity of neurogenic sensory receptors retrieved through screening.

The substance that inhibits the activity of the neuropathic sensory receptors may include one or more extracts selected from the group consisting of, for example, oats, hoses and extracts.

It is a medicinal substance that fumigates under-fruited fruit of Prunus mume Sieb. Et Zucc., And contains citric acid, malic acid, succinic acid, carbohydrate ), Triterpenoids, oleanol acid, sitosterol, and the like.

The Hashuo is a vineous Pleuropterus multflorus with a dicotyledonous plant of the dicotyledonous plant. It is largely divided into two kinds of dicotyledonous and white dicotyledonous. The dicotyledonous plant which is grown in our country is mainly white dicotyledonous. The physiological activity of the Sasa is known to be due to anthraquinone contained in the sewage, but the Sasa contains various plant sterols and various glycoside compounds.

The above-mentioned extract is a kind of herb medicine, which means that rootstocks or junks of Atractylodes japanica Koidzumi or Atractylodes ovata Koidzumi are removed.

In one embodiment, the extract may comprise 0.001 to 10% by weight, preferably 0.01 to 5% by weight, based on the total weight of the skin care composition, based on in vitro and in vivo experiments.

There is no limitation on the method for producing the above extract, but it can be extracted with a solvent such as water or ethanol, for example, at room temperature or under heating conditions.

Specifically, ethanol may be added to the oats, the sewage or the raw material so that the weight ratio of the oats, the sewage or the raw material to each weight is 2 to 10 times, preferably 4 times, and then the mixture can be extracted by heating at 50 to 110 ° C.

The skin cosmetic composition may preferably be formulated in the form of an external preparation for skin, and the external preparation for skin may be a softening lotion, a nutritional lotion, a massage cream, a nutritional cream, a pack, a gel or a skin adhesive type cosmetic, Gel, cream, patch, or spray.

In addition to the above-mentioned essential ingredients, the skin cosmetic composition of the present invention can be mixed with other ingredients without difficulty by those skilled in the art according to the formulation or purpose of use of other external preparations.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are intended to illustrate the present invention, but the scope of the present invention is not limited thereto.

[Example 1] Preparation of osseous extract

70% ethanol in an amount of four times the weight (Kg) was added to 500 g of oats, and the resulting mixture was heated at 80 DEG C for 2 hours to extract an effective ingredient. This was allowed to stand at room temperature for one day to precipitate impurities. The filtrate was filtered twice using a filter paper, and the filtrate was completely evaporated by using a reduced pressure evaporator and concentrated.

Example 2 Preparation of Sucrose Extract

70% ethanol in an amount of four times by weight (Kg) was added to 500 g of Sasao, and the mixture was heated at 80 DEG C for 2 hours to extract the active ingredient. This was allowed to stand at room temperature for one day to precipitate impurities. The filtrate was filtered twice using a filter paper, and the filtrate was completely evaporated by using a reduced pressure evaporator and concentrated.

[Example 3] Preparation of dried extract

70% ethanol in an amount of 4 times the weight (Kg) was added to 500 g of the extract, and the extract was heated at 80 DEG C for 2 hours to extract an active ingredient. This was left at room temperature for one day to precipitate the impurities. The filtrate was filtered twice using a filter paper, and then the filtrate was completely evaporated using a vacuum evaporator and concentrated.

[Test Example 1] Quantitative change of TRPV-1 due to ultraviolet light in human skin, i.e., epidermis

After 2, 4, 8, 24, 48, and 72 hours of UV irradiation (2 MED) on the hip skin of a young man (n = 4, aged 30 to 38 years with an average age of 35 years) Quantitative changes of TRPV-1 were observed through tissue staining and are shown in Fig. Referring to FIG. 1, the results of immunohistochemical study showed that the expression of TRPV-1 in the epidermal layer was increased with time after irradiation with ultraviolet light.

[Test Example 2] Change of TRPV-1 in HaCaT after ultraviolet irradiation

Immortalized human keratinocyte cell line (HaCaT) was added to DMEM (Dulbecco's modified eagle medium) containing 10% fetal bovine serum at a concentration of 5 × 10 ⁶ , followed by incubation at 37 ° C., (75 mJ / cm < ² >). Cells were lysed 4, 8, 24, and 48 hours after UV irradiation to quantitate the proteins, and Western blot was performed on TRPV-1. The results are shown in FIG. Referring to FIG. 2, it was confirmed that the expression of TRPV-1 was increased by ultraviolet rays. The increase in TRPV-1 was corrected by actin and then compared with the control group.

[Test Example 3] Matrix metalloproteinase inhibition by ultraviolet light by TRPV-1 antagonist (ruthenium-red)

Immortalized human keratinocyte cell line (HaCaT) was added to DMEM (Dulbecco's modified eagle medium) containing 10% fetal bovine serum at a concentration of 5 × 10 ⁶ , followed by incubation at 37 ° C., (75 mJ / cm < ² >). The results are shown in Fig. Referring to FIG. 3, it can be seen that the expression of matrix metalloproteinase was decreased in a concentration-dependent manner in the group treated with ruthenium-red (ruthenium-red: 1-10 uM), which is a TRPV-1 antagonist after UV irradiation. This suggests that the inhibition of TRPV-1 may regulate the expression of collagenase.

[Test Example 4] Screening for the development of a pain sensory receptor inhibitor (Ca ²⁺  influx assay)

The rVR1-CHO cell line is a cell line in which CHO (Chinese Hamster Ovary) cells are transformed and stabilized by mouse vanilloid-1 gene. (RT-PCR) from mouse spinal proximal ganglia using primers designed to encircle the same initiation and termination codons as the published sequence (Caterina et al. Nature , 1997) The cDNA for the receptor was isolated. After confirming the sequence, a plasmid was prepared using pcDNA3 (Invitrogen) vector. The rVR1-pcDNA3 plasmid was transformed into CHO cells using Lipofectamine (TM) and then selected using growth medium containing 400 占 퐂 / ml geneticin (Gibco BRL). The surviving individual colonies were isolated, subcultured and screened for VR1 receptor activity.

One day before the experiment CHO cells rVR1-: a 8X10 (TRPV-1 is a stable transfected cell line TRPV-1 transfection stable cell line) in 96-well plates ⁴ And then cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours.

After 24 hours, the 96-well plate was washed twice with 20 mM HEPES buffered saline (HBSS) buffer (reaction buffer, hereinafter), and calcium-sensitive fluorescent dye (2 uM Fluo-4-AM with 20% acid, 2.5 mM probenecid) was added to the cells. The cells were reacted at 37 ° C in a 5% CO ₂ incubator for 30 minutes and at room temperature for 30 minutes, washed twice with a reaction buffer, and treated with the plant extracts prepared in Examples 1 to 3. After 10 minutes of reaction, it was measured using a stimulus causes 0.05μM capsaicin intracellular ^{Ca 2 + (Molecular Device, USA} ) variation in the concentration of 70 seconds fluorescence plate reader FlexStation3 flowing during the treatment.

The difference between the initial value and the maximum value of the value obtained by measuring the change of fluorescence for 70 seconds after the treatment with the extract and 0.05 μM capsaicin (TRPV-1 antagonist) was obtained, and the value was compared with the initial value at the capsaicin treatment The inhibition rate was calculated by comparing the difference with the maximum value, and the result is shown in FIG.

Referring to FIG. 4, it was confirmed that the test results showed that TRPV-1 antagonistic effect was caused by the concentration of Ohmese, Sasae, and Pepsi.

[Test Example 5] Changes in gene expression of matrix metalloproteinases after treatment with natural substances having an antagonistic effect of TRPV-1 after UV irradiation

Immortalized human keratinocyte cell line (HaCaT) was added to DMEM (Dulbecco's modified eagle medium) containing 10% fetal bovine serum at a concentration of 5 × 10 ⁶ , followed by incubation at 37 ° C., (75 mJ / cm < ² >). After irradiation with ultraviolet light, the cells were treated with the oats, hatcheries and extracts prepared in Examples 1 to 3, and after 48 hours of culture, cells were obtained and the expression amount of the matrix metalloproteinase gene was determined by RT-PCR (Reverse Transcriptase Polymerase chain reaction method, and the results are shown in Fig. Referring to FIG. 5, the expression of the matrix metalloproteinase gene was decreased as the concentration of the extract increased, as compared with the negative control group.

[Test Example 6] Changes in gene expression of matrix metalloproteinases after treatment with natural substances having antagonistic effect of TRPV-1 after irradiation with ultraviolet light

Immortalized human keratinocyte cell line (HaCaT) was added to DMEM (Dulbecco's modified eagle medium) containing 10% fetal bovine serum at a concentration of 5 × 10 ⁶ , followed by incubation at 37 ° C., (75 mJ / cm < ² >). After 48 hours of incubation, the cells were treated with the oats, hatcheries and extracts prepared in Examples 1 to 3 after irradiation with ultraviolet rays to obtain cells. Matrix metalloproteinase antibodies were used for Western blotting, The amount of expression of ropurothia protein was compared with the negative control, and the results are shown in Fig. Referring to FIG. 6, there was a decrease in the matrix metalloproteinase protein as the concentration of the extract increased compared with the negative control group

Thus, referring to the results of Test Example 1 to Test Example 6, the oats, algae and alfalfa extracts proposed in the present invention can be used to control the activity of the pain sensory receptor (TRPV-1) and to inhibit the expression of metalloproteinase It is possible to prevent or improve the wrinkles of the skin and improve the elasticity of the skin, as well as alleviate the skin irritation and ultimately delay the aging of the skin.

Therefore, the composition according to the present invention can be used as an effective material for prevention or improvement of skin aging prevention and wrinkle formation.

[Formulation Example 1] Lotion-type preparation

Extract of Example 1 3.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Water soluble collagen (1% aqueous solution) 1.00

Sodium citrate 0.10

Citric acid 0.05

Licorice extract 0.20

1,3-butylene glycol 3.00

Purified water balance

(Unit: wt%)

[Formulation Example 2]

The extract of Example 2 1.00

Polyethylene glycol monostearate 2.00

Self emulsifying monostearate glycerin 5.00

Cetyl alcohol 4.00

Squalane 6.00

Tri-2-ethylhexane glyceryl 6.00

Sphingo glycolipid 1.00

1.3-butylene glycol 7.00

Purified water balance

(Unit: wt%)

[Formulation Example 3] Packed preparation

Extract of Example 3 5.00

Polyvinyl alcohol 13.00

L-ascorbic acid-2-phosphate magnesium salt 1.00

Lauroylhydroxyproline 1.00

Water soluble collagen (1% aqueous solution) 2.00

1,3-butylene glycol 3.00

Ethanol 5.00

Purified water balance

(Unit: wt%)

Those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Figure 1 shows the results of a change in neurogenic sensory receptors in the skin after UV irradiation;

Figure 2 shows the results of changes in neurogenic sensory receptors in HaCaT after UV irradiation;

Figure 3 shows the effect of matrix metalloproteinase reduction when the composition according to the invention is treated;

FIG. 4 is a graph showing the results of screening a skin-care composition according to the present invention to search for an antagonist of nerve pain sensory receptors;

Figure 5 shows the gene changes of matrix metalloproteinases in HaCaT treated with the composition according to the invention;

Figure 6 shows the quantitative change of matrix metalloproteinase in HaCaT treated with the composition according to the invention.

Claims (12)

delete delete delete delete A method of screening a skin cosmetic material, The method comprises treating cells with ultraviolet light or capsaicin; Treating the cell with a candidate substance; Performing a calcium-flight lux assay (Ca ²⁺ influx assay) for the cell by measuring the calcium concentration changes, or measurement of gene or protein expression level of the pain sensory nerve receptors (TRPV1); And Judging the substance as a skin cosmetic substance when the calcium influx is inhibited to inhibit the activity of the neuropathic sensory receptor or inhibits the expression of the gene or protein of the neuropathic sensory receptor compared with the control group not treated with the candidate substance A method of screening a skin cosmetic material. delete 6. The method of claim 5, Wherein the skin cosmetic material has a function of preventing collagen degradation, alleviating skin irritation, preventing skin aging through induction of blood circulation, and preventing or improving wrinkle formation. delete delete delete delete delete

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