KR20100000391A - Neurocosmetic skin care method, method for screening skin cosmetic material, and skin cosmetic material - Google Patents

Abstract

PURPOSE: A method for screening a cosmetic material and cosmetic composition containing a material suppressing TRPV-1(transient receptor potential family V-1) are provided to select a target material which is effective in anti-aging and reduce skin irritation. CONSTITUTION: A material which suppresses activation of TRPV-1(transient receptor potential family V-1) contains extract isolated from one or more selected from Prunus mume Sieb. et Zucc., Pleuropterus multflorus, and Atractylodes japonica Koidxumi or Atractylodes ovate Koidzumi. A cosmetic composition contains 0.001-10 weight%, preferably 0.01-5 weight% of the extract. The cosmetic composition is used in the form of nutrition skin, massage cream, pack gel or lotion, ointment, cream, patch or spray.

Description

Neurocosmetic Skin Care Method, Method For Screening Skin Cosmetic Material, and Skin Cosmetic Material}

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

Skin beauty related to skin aging has been mainly studied in the dermis layer, but recently, as the research on the skin nervous system and neurotransmitters has been actively conducted, the skin is connected to the nerves and various neurotransmitters affect the skin. Among the studies of interest are the neurotransmitter potential family V 1: capsacine receptor (TRPV-1).

TRPV-1 is expressed in all nervous systems such as skin, brain, and spinal cord. These receptors detect changes in heat, pH, and ions, and nociceptive stimuli in sensory neurons that cause substance P, an inflammatory neurotransmitter. It is a receptor that integrates. Representatively, TRPV-1 promotes the expression of neurotransmitters substance P, CGRP, etc. in nerve fibers by heat and pH changes caused by external stimuli, causing a neurogenic inflammatory response.

TRPV-1 is itself a thermal sensor and maximizes activity at about 43 ° C. TRPV-1 is known to be expressed in neurons as well as mesenchymal, epithelial and epidermal cells.

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

Recently, with respect to cosmetic raw materials, neurocosmetic raw materials using neurotransmitters have been used or developed. Examples of neurochemical raw materials include tripeptides that inhibit TNF-a by inhibiting neuroinflammatory responses through the regulation of melanocortin-1 receptors (MC1-R). .

The first object of the present invention is to provide a skin cosmetic method that can prevent skin aging, the second object of the present invention is to provide a method for screening a skin cosmetic material that can prevent skin aging, It is a third object of the present invention to provide a skin cosmetic composition for preventing skin aging.

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

In addition, the skin cosmetic material screening method according to the present invention is characterized in that it comprises the step of determining whether the activity of the target material to the nerve pain sensory receptors.

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

Through the present invention, it is possible to select a target substance effective for preventing skin aging, and the substance which inhibits the activity of the selected nerve pain sensory receptor is reduced in expression of collagen degrading enzyme due to the activity of the nerve pain sensory receptor, relieves skin irritation, and Induction of blood circulation can be confirmed, which is particularly effective in preventing skin aging.

The inventors of the present application confirmed that the activity of the neuropathic pain receptors is increased in the epidermis of the skin by ultraviolet rays, as shown through the following examples. It was confirmed that skin beauty can be increased through prevention of production.

As used herein, "inhibition of activity" refers to inhibiting the production of neuropathic pain receptors, specifically inhibiting action through inactivation and loss of function of neuropathic pain receptors, as well as inhibiting expression of genes expressing neuropathic pain receptors. And inhibiting the synthesis of neuropathic pain receptor proteins.

In one preferred embodiment, skin aesthetics can be achieved by inhibiting the expression of matrix metalloproteases, through the inhibition of neuropathic pain receptors.

The matrix metalloprotease is an enzyme that promotes collagen degradation to induce the transformation of the dermal layer. If the production and degradation of collagen are not properly controlled, the skin elasticity is reduced and wrinkles are formed. It is the main cause.

As shown in the following examples, when the epidermal cells cultured in the skin and the immortalized human keratinocyte cell line (HaCaT) are heated, matrix metalloproteases during overexpression of nerve pain sensory receptors It is confirmed that expression of the agent is increased and that the expression of metalloproteases by heat decreases when the capsaicin, which is a nerve pain sensory receptor antagonist, is decreased. Expression of the matrix metalloproteases is regulated through the pain sensory receptors. Can be.

Thus, substances that inhibit the activity of neuropathic pain receptors can reduce the expression of collagen degrading enzymes, which can prevent and prevent the formation of wrinkles on the skin, prevent aging of the skin through improvement of elasticity, relieve skin irritation, And inducing blood circulation to maintain skin homeostasis, thereby achieving skin beauty.

The method of treating the skin by treating a substance that inhibits the nerve pain sensory receptor to a specific site is not particularly limited. For example, the material may be applied to the skin in an external form of the skin to affect the activity of the nerve pain sensory receptor. Can be.

The present invention also relates to a skin cosmetic material screening method comprising the step of determining whether the target substance inhibits the activity of the neuropathic pain receptor.

In one preferred embodiment, the step of determining whether to inhibit the activity of the nerve pain sensory receptor of the target material is to search for a substance that inhibits the activity on the nerve pain sensory receptor to select a candidate, the cell treated the candidate material This can be done by carrying out a Ca ²⁺ influx assay.

The calcium influx assay, for example, by measuring capsaicin (capsaicin) acting as an antagonist of neuropathic pain receptors and a target substance to measure the change in the concentration of calcium ions in the cell, the cells when treated only capsaicin By measuring the change in the concentration of calcium ions in the way, it is possible to determine the degree of inhibition through these differences, through which it is possible to screen skin cosmetics that inhibit the activity of the neuropathic pain receptors.

The present invention also relates to a skin cosmetic composition containing a substance that inhibits the activity of neuropathic pain receptors as an active ingredient, wherein the composition is a skin aging through reduced expression of collagen degrading enzymes, alleviation of skin irritation and induction of blood circulation. Prevent and improve wrinkle formation.

In addition, the substance that inhibits the activity of the nerve pain sensory receptor can inhibit the expression of matrix metalloprotease (matrix metalloprotease), the matrix metalloprotease is a collagen degrading enzyme, the inventors of the present application When epidermal cells were treated with substances that inhibited the activity of neuropathic pain receptors detected through screening, the expression of matrix metalloproteases was also reduced.

Substances that inhibit the activity of the neuropathic pain receptors may include, for example, one or more extracts selected from the group consisting of ume, sewage and effusion.

The ume refers to medicinal herbs fumigation of the unripe fruit of Prunus mume Sieb. Et Zucc., Citric acid, malic acid, malic acid, succinic acid, carbohydrate ), Triterpenoids (triterpenoids), olenolic acid (oleanol acid), it is known that consists of the sitosterol (sitosterol).

The sewage is a dicotyledonous perennial herb ( Pleuropterus multflorus ) with the dicotyledonous plant, the plant is divided into two types of sewage and white sewage, and the sewage cultivated in Korea is mainly white sewage. The physiological activity of the sewage is known to be due to the anthraquinone contained in the sewage, but the sewage contains various vegetable sterols and various glycoside compounds.

The baekchul is a kind of medicinal herb, which means that the root stem or the bark of the intract ( Atractylodes japanica Koidzumi) or the sugar extract ( Atractylodes ovata Koidzumi) is removed.

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

There is no limitation in the preparation method of the extract, for example, it can be extracted with a solvent such as water or ethanol under normal temperature or heating conditions.

Specifically, ethanol is added to the mae, sewage or white raw material so that the weight ratio of each weight is 2 to 10 times, preferably 4 times, and then heated and extracted at a temperature of 50 to 110 ° C.

The skin cosmetic composition may be preferably formulated in the form of an external preparation for skin, and the external preparation for skin may be a softening lotion, a nourishing cosmetics, a massage cream, a nourishing cream, a pack, a gel or a skin adhesive cosmetic, and also a lotion, an ointment, Transdermal dosage forms such as gels, creams, patches or sprays.

In addition, in addition to the essential components mentioned above, the skin cosmetic composition of the present invention can be appropriately selected and blended with other ingredients without difficulty 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 only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Omae Extract

After adding 70% ethanol at a weight (Kg) ratio of 4 times to 500 g of o medium, the resultant was extracted by heating at 80 ° C. for 2 hours. This was allowed to stand at room temperature for 1 day to precipitate impurities, and the filtrate was filtered twice using filter paper and concentrated by completely evaporating using a reduced pressure evaporator.

Example 2 Preparation of Sewage Extract

After adding 70% ethanol at a weight (Kg) ratio of 4 times to 500 g of sewage, the resultant was extracted by heating at 80 ° C. for 2 hours. This was allowed to stand at room temperature for 1 day to precipitate impurities, and the filtrate was filtered twice using filter paper and concentrated by completely evaporating using a reduced pressure evaporator.

Example 3 Preparation of White Extract

After adding 70% ethanol at a weight (Kg) ratio of 4 times to 500 g of the white extract, the resultant was extracted by heating at 80 ° C. for 2 hours. This was allowed to stand at room temperature for one day to precipitate impurities, and the filtrate, which was filtered twice using filter paper, was completely evaporated and concentrated using a reduced pressure evaporator.

Test Example 1 Quantitative Change of TRPV-1 by UV Light in Human Skin, ie, Epidermis

Ultraviolet (2 MED) irradiation of the hip skin of a young person (n = 4, age: 30-38 years old, average age 35), followed by biopsy of the skin after 2, 4, 8, 24, 48 and 72 hours The quantitative change of TRPV-1 was observed through tissue staining, which is shown in FIG. 1. Referring to Figure 1, the immunohistogram showed that the expression of TRPV-1 in the epidermal layer increased with time after UV irradiation.

Test Example 2 Change of TRPV-1 in HaCaT after UV Irradiation

Immertalized human keratinocyte cell line (HaCaT) was added to DMEM (Dulbecco's modified eagle medium) medium containing 10% fetal bovine serum at a concentration of 5 X 10 ⁶ and incubated at 37 ° C. Was irradiated (75 mJ / cm ² ). After 4, 8, 24, 48 hours after UV irradiation, the cells were lysed to quantify the protein, and Western blot for TRPV-1 was performed. The results are shown in FIG. 2. Referring to Figure 2, the test results it can be seen that the expression of TRPV-1 increased by ultraviolet light. The increase in TRPV-1 was corrected with actin and compared with the control group, respectively.

Test Example 3 Inhibition of Matrix Metalloprotease by UV Light by TRPV-1 Antagonist (ruthenium-red)

After immunization with Immertalized human keratinocyte cell line (HaCaT) in DMEM (Dulbecco's modified eagle medium) medium containing 10% fetal bovine serum at a concentration of 5 X 10 ⁶ , incubated at 37 ° C Was investigated (75 mJ / cm ² ), and the results are shown in FIG. 3. Referring to FIG. 3, it can be seen that the expression of matrix metalloproteases was decreased in a concentration-dependent manner in the group treated with ruthenium-red (1-10 uM), a TRPV-1 antagonist after UV irradiation. This suggests that expression of collagenase can be regulated through inhibition of TRPV-1.

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

The rVR1-CHO cell line is a cell line stabilized by transforming mouse vanilloid-1 gene into CHO (Chinese Hamster Ovary) cells. Rat VR1 by reverse transcriptase-polymerase chain reaction (RT-PCR) from the rat spinal muscle ganglia, using primers designed to encompass the same initiation and termination codons as published sequences (Caterina et al. Nature , 1997). CDNA for the receptor was isolated. After confirming the sequence, a plasmid was prepared using the pcDNA3 (Invitrogen) vector. rVR1-pcDNA3 plasmids were transformed into CHO cells using Lipofectamine ™ followed by selection using a growth medium containing 400 μg / ml Geneticin (Geneticin, Gibco BRL). Surviving individual colonies were isolated, passaged and screened for VR1 receptor activity.

One day before the experiment, rVR1-CHO cells (TRPV-1 transfected stable cell line: TRPV-1 transfection stable cell line) were placed in a 96-well plate at 8 × 10 ^4. Cells / well were aliquoted and incubated for 24 hours in a 37 ° C., 5% CO ₂ incubator.

After 24 hours, the 96 well plate was washed twice with 20 mM HEPES Hanks' Balanced Salt solution (HBSS) buffer (hereinafter referred to as reaction buffer), followed by calcium-sensitized fluorescent dye (2 uM Fluo-4-AM with 20% Pluronic Acid: Pluronic). acid, 2.5 mM probebenid (probeside) were added to the cells. After reacting at 37 ° C., 5% CO ₂ incubator for 30 minutes, at room temperature for 30 minutes, washing was performed twice with reaction buffer, and the plant extracts prepared in Examples 1 to 3 were treated with cells. 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.

After the extract and 0.05 μM capsaicin (TRPV-1 antagonist) treatment, the difference between the initial value and the maximum value obtained by measuring the change in fluorescence for 70 seconds was determined, and the value was determined from the initial value during capsaicin treatment. Inhibition rate was calculated | required compared with the difference of the maximum value, and the result is shown in FIG.

Referring to Figure 4, the test results, it was confirmed that the ume, sewage, leachate, etc. have a TRPV-1 antagonistic effect in a concentration dependent.

[Test Example 5] Change of matrix metalloprotease gene expression after treatment with natural products with TRPV-1 antagonistic effect after UV irradiation

Immertalized human keratinocyte cell line (HaCaT) was added to DMEM (Dulbecco's modified eagle medium) medium containing 10% fetal bovine serum at a concentration of 5 X 10 ⁶ and incubated at 37 ° C. Was irradiated (75 mJ / cm ² ). After treatment with the ume, sewage, baekchul extract (extract) prepared in Examples 1 to 3 after UV irradiation, the cells were obtained 48 hours after culturing to express the expression amount of the matrix metalloprotease gene RT-PCR (Reverse Transcriptase polymerase) According to the chain reaction method was compared with the negative control group, the results are shown in FIG. Referring to FIG. 5, as compared with the negative control group, the expression of the matrix metalloprotease gene was decreased as the concentration of the extract increased.

Test Example 6 Changes in Matrix Metalloprotease Gene Expression after Treatment of Natural Products with TRPV-1 Antagonist Effect After UV Irradiation

Immertalized human keratinocyte cell line (HaCaT) was added to DMEM (Dulbecco's modified eagle medium) medium containing 10% fetal bovine serum at a concentration of 5 X 10 ⁶ and incubated at 37 ° C. Was irradiated (75 mJ / cm ² ). After treatment with UV, sewage, and leach extracts prepared in Examples 1 to 3 after UV irradiation, the cells were obtained 48 hours after the culture, and the matrix metal was prepared by Western blot using an antibody of matrix metalloprotease. The expression level of the ropeothiase protein was compared with that of the negative control group and the results are shown in FIG. 6. Referring to FIG. 6, the matrix metalloprotease protein decreased as the concentration of the extract increased compared to the negative control.

As such, referring to the results of Test Examples 1 to 6, the ume, sewage and baekchul extracts presented in the present invention regulate the activity of pain sensory receptors (TRPV-1) and express metalloproteases. In addition, it is possible to prevent or improve skin wrinkles, and to improve skin elasticity, and to relieve skin irritation and ultimately delay skin aging.

Accordingly, the composition according to the present invention can be used as a substance effective for preventing skin aging and preventing or improving wrinkle formation.

Formulation Example 1 Lotion Formulation

Extract of Example 1 3.00

L-ascorbic acid-2-magnesium phosphate 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 level

(Unit: weight%)

Formulation Example 2 Cream-Type Formulation

Extract of Example 2 1.00

Polyethylene Glycol Monostearate 2.00

Self-emulsifying glycerin monostearate 5.00

Cetyl Alcohol 4.00

Squalene 6.00

Tri2-ethylhexaneglyceryl 6.00

Sphingolipids 1.00

1.3-butylene glycol 7.00

Purified water level

(Unit: weight%)

Formulation Example 3 Packed Formulations

Extract of Example 3 5.00

Polyvinyl Alcohol 13.00

L-ascorbic acid-2-magnesium phosphate 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 level

(Unit: weight%)

Those skilled in the art to which the present invention pertains will be able to perform various applications and modifications within the scope of the present invention based on the above contents.

Figure 1 shows the result of the change in nerve pain sensory receptor in the skin after ultraviolet irradiation;

Figure 2 shows the results of changes in neuropathic pain receptors in HaCaT after ultraviolet irradiation;

3 shows the matrix metalloprotease reduction effect when the composition according to the present invention is treated;

Figure 4 is a graph showing the results of screening the skin cosmetic composition according to the present invention to search for antagonists of nerve pain sensory receptors;

Figure 5 shows the genetic change of matrix metalloproteases in HaCaT treated with the composition according to the present invention;

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

Claims (12)

Skin care method comprising the step of inhibiting the activity of nerve pain sensory receptors (TRPV-1, VR1, Transient Receptor Potential family V-1). The method of claim 1, Skin cosmetic method, characterized in that by inhibiting the activity of the nerve pain sensory receptors suppress the expression of matrix metalloproteases. The method of claim 1, Skin cosmetic method, characterized in that the application of a substance that inhibits the activity of the nerve pain sensory receptor on the skin. The method of claim 1, The substance for inhibiting the activity of the neuropathic pain receptor is a skin cosmetic method characterized in that it reduces the expression of collagen degrading enzymes, alleviates skin irritation and prevents skin aging through induction of blood circulation and prevents or improves wrinkle formation. . A method for screening skin cosmetic materials, comprising determining whether the target substance inhibits the activity of the neuropathic pain receptor. The method of claim 5, wherein Determining whether the target substance inhibits the activity of the neuropathic pain receptor comprises: performing a calcium influx assay (Ca ²⁺ influx assay) on cells treated with the target substance Material screening method. The method of claim 5, wherein The skin cosmetic material is a skin cosmetic material screening method, characterized in that to reduce the expression of collagen degrading enzymes, to reduce skin irritation and induce blood circulation to prevent skin aging, and to prevent or improve the generation of wrinkles. A skin cosmetic composition containing as an active ingredient a substance that inhibits the activity of a nerve pain sensory receptor. The method of claim 8, The substance for inhibiting the activity of the nerve pain sensory receptor skin cosmetic composition, characterized in that it comprises one or more extracts selected from the group consisting of ume, sewage and baekchul. The method of claim 8, A substance for inhibiting the activity of the nerve pain sensory receptor is a cosmetic composition, characterized in that it comprises 0.01 to 10% by weight based on the total weight of the composition. The method of claim 8, The substance for inhibiting the activity of the nerve pain sensory receptor skin cosmetic composition, characterized in that to suppress the expression of matrix metalloprotease (matrix metalloprotease). The method of claim 8, The composition is a skin cosmetic composition, characterized in that to reduce the expression of collagen degrading enzymes, skin stimulation and induction of blood circulation to prevent skin aging, and prevent or improve the production of wrinkles.

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