KR101892641B1 - Skin External Composition Containing Ulmus Davidiana Extract, Dioscorea Japonica Extract or Morus Alba Leaf Extract - Google Patents

Abstract

The present invention relates to a composition for external application for skin moisturizing enhancement, elasticity enhancement or anti-aging comprising two or more kinds selected from the group consisting of extracts from Rootgrass, Radix extract and Upper leaf extract as an active ingredient, wherein the hyaluronic acid synthase (hyaluronic acid synthase 2, HAS2) to activate the hyaluronic acid production of the human body, to promote the synthesis of versatile, and to inhibit the production of MMP-12, which is responsible for the degradation of versican, Which is excellent in skin elasticity and skin moisturizing effect.

Description

[0001] The present invention relates to a composition for external application for skin comprising Dioscorea Japonica Extract or Morus Alba Leaf Extract,

The present invention includes two or more kinds selected from the group consisting of the extracts of Trichophyton scutellariae, Radix extract and Lycopersicum extract as an active ingredient, thereby increasing gene expression of hyaluronic acid synthase2 (HAS2) present in human cells, Promoting the synthesis of Versican, and inhibiting the production of MMP-12 (Matrix metalloproteinase-12) responsible for the degradation of Versican, thereby strengthening the structure of the extracellular matrix and enhancing skin moisturization, And an anti-aging or wrinkle-improving composition for external application for skin.

Conventional studies on ULMUS DAVIDIANA, which is the skin of elm roots, were about immunosuppressants and edible beverages. In Korean Patent No. 10-0439939, the effect of improving wrinkles, promoting collagen synthesis And an anti-aging composition containing a polysaccharide extract isolated from low molecular weight and high molecular weight hyaluronic acid and rhizome of the present invention is also excellent in comparison with the present invention. The effect is insufficient.

Korean Patent Registration No. 10-0439939

Thus, the present inventors performed an ELISA in real-time DNA chain polymerization assay in a cell culture condition to increase the expression of hyaluronic acid synthase 2 (HAS2) gene and increase the synthesis of hyaluronic acid In addition, the present inventors also found an effect of increasing the expression of a virucycane gene which binds to hyaluronic acid and a collagen bundle to enhance the extracellular matrix structure and induces a decrease in the MMP-12 gene causing the degradation of versatile, Thereby completing the invention.

Accordingly, an object of the present invention is to provide a composition containing at least two kinds of active ingredients selected from the group consisting of extracts from Rootgrass,

According to one embodiment of the present invention, there is provided an external preparation for skin comprising as an active ingredient at least two kinds selected from the group consisting of extracts from Rootgrass, extract from Radix, and extracts from leaf.

The root is used as an active ingredient of the present invention means elm root bark, and it removes the inflammation and sucks out the pus, so it is very effective to treat ulcers, duodenal ulcer, ulcer and various ulcers including gastric cancer, uterine cancer and rectal cancer The efficacy is well known.

The active ingredient of the present invention, Dioscorea Japonica, grows in the mountain area and is also called yam. It grows mainly in the mountainous region, and the stems come out of the columnar fleshy roots and roll up other objects. It has the function of seeing the kidney and adding the yin. It is like supplementing the weakness that is lacking due to aging. Specifically, it is effective for hemostasis, antimicrobial action, skin dermatitis (all kinds of skin diseases), promoting hair growth, blackening hair, stimulating the blood flow of the coronary arteries and soothing, analgesic and anti- ).

The active ingredient of the present invention, Morus Alba Leaf, refers to mulberry leaves. The mulberry, which means mulberry, is a combination of medicine and wood, which means the holy chestnut of the east. This is because the mulberry tree, the tree that the silkworms eat, has the same efficacy as the chinese herb, so it is used to distinguish the two. This medicine has almost no odor, it has sweet taste, and it is cold. It is used for seawater, sea urchin, skin urticaria.

According to a preferred embodiment of the present invention, the composition of the present invention contains at least 0.0001 to 14% by weight, more preferably 0.01 to 10% by weight, based on the total weight of the composition, of at least two kinds selected from the group consisting of extracts from Rootini, %, More preferably 0.1 to 5 wt%, and most preferably 0.5 to 4 wt%, based on the total weight of the composition. If the content of the extract is less than 0.0001% by weight, an increase in the production of hyaluronic acid, acceleration of the production of versatile and inhibition of the production of MMP-12 by the extract can not be attained. If the content is more than 14% by weight, It is inefficient because it does not.

The rhizome, lily extract, and upper leaves used in the present invention can be commercially obtained or can be produced by a commonly used production method.

The method for producing the above extract may be a high-temperature high-pressure extraction, a subcritical extraction, a supercritical extraction, and the like, preferably by hot water extraction.

(A) purified water, (b) anhydrous or low-boiling alcohols having 1-4 carbon atoms such as methanol, ethanol, propanol, butanol, (D) acetone, (e) ethyl acetate, (f) chloroform, (g) 1,3-propanediol, Butylene glycol, (h) hexane, (i) diethyl ether, or (j) butyl acetate. However, since the composition of the present invention is used for skin, it is more preferable to use purified water which is harmless to the skin and has a high environmental friendliness. The extract of the present invention includes not only those obtained by using the above-mentioned solvents, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the above extract through an ultrafiltration membrane having a constant molecular weight cut-off value, a separation obtained by various chromatography (separation by size, charge, hydrophobicity or affinity) Fractions are also included in the extract of the present invention. The extract of the present invention can be prepared in powder form by an additional process such as vacuum distillation and freeze-drying or spray-drying.

When two kinds of extracts are mixed when mixing the extract of the present invention, they can be mixed at a weight ratio of 1: 1 and 3 kinds of extracts at a weight ratio of 1: 1: 1, but not limited thereto, Can be used.

According to another embodiment of the present invention, the present invention provides a composition for external application for skin, which promotes the production of hyaluronic acid.

Hyaluronic acid is a kind of glucose aminoglycan not attached to sulfate groups and is a linear polymeric material in which glucuronic acid and N-acetylglucosamine residues are repeatedly connected. It is reported that it is involved not only in retention of water content, intercellular space maintenance, cell growth factor and nutrient storage and diffusion as main constituents of extracellular matrix, but also in cell division, differentiation and migration. More than 50% of the hyaluronic acid present in the mammalian body is present in the skin, mainly synthesized by keratinocytes and fibroblasts, and is reported to be distributed in the epidermal intercellular space and the connective tissue of the dermis. The amount of hyaluronic acid in human skin has been reported to decrease with aging, which is considered to be one of the direct causes of decreased skin elasticity and decreased water content due to aging.

It has been reported that the synthesis of hyaluronic acid in skin cell cultures is increased by various growth factors and transretinoin acid, N-methyl serine, etc., and that estradiol and its analogues applied to the skin increase the synthesis of hyaluronic acid However, the detailed mechanism of hyaluronic acid metabolism has not yet been clarified. However, it is known that the synthesis of hyaluronic acid proceeds by the hyaluronic acid synthase on the inner surface of the cell membrane and accumulates into the extracellular matrix through the cell membrane during synthesis. HAS1, HAS2, and HAS3, which have high similarity in sequence, have been reported in mammalian hyaluronan synthase gene. However, many studies have not been conducted on the factors that regulate the distribution and enzyme activity in cells and tissues. It has been reported that HAS2 gene expression increases when epidermal growth factor (EGF) is added into epidermal cell culture fluids (Juha-Pekka Pienimaki, et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY (2001)) . Promotion of hyaluronic acid production is achieved by increasing the expression of the hyaluronic acid synthase gene of the composition of the present invention. In addition, the composition of the present invention has an effect of inducing the decrease of the MMP-12 gene which causes the degradation of versatile cells, and increases the expression of the versatile gene which binds to hyaluronic acid and collagen bundles and enhances the extracellular matrix structure.

According to a preferred embodiment of the present invention, the composition of the present invention increases the expression of the HAS2 gene, thereby increasing the synthesis of hyaluronic acid. In addition, it increases the expression of the versatile gene, which not only promotes the synthesis of versican, but also reduces the expression of the MMP-12 gene, thereby inhibiting the production of MMP-12, which is responsible for the degradation of versatile. Accordingly, the present invention has the effect of promoting the production of hyaluronic acid and strengthening the structure of the extracellular matrix component, thereby suppressing skin elasticity deterioration due to aging and improving skin moisturization, thereby improving skin elasticity and wrinkle reducing effect.

The composition for external application for skin of the present invention may have cosmetic formulations of softening agent, nutritional lotion, massage cream, nutritional cream, pack, gel or skin adhesive type in the formulations and may be in the form of lotion, ointment, gel, cream, patch or spray , ≪ / RTI > but are not limited thereto.

According to another embodiment of the present invention, there is provided a process for producing a hair follicle comprising the steps of: injecting two or more kinds selected from the group consisting of carnauba, And extracting the active ingredient from the ultrahigh pressure apparatus. The present invention also provides a method for producing a skin external application composition for improving skin elasticity, aging, or wrinkles. In the step of introducing into the above-mentioned solvent, it is preferable to use 1000 L of solvent per 100 g of two or more kinds of crude drugs selected from the group consisting of rhizome, mushroom and upper leaves.

In the step of extracting the active ingredient, it is preferable to perform extraction at 1 to 7 hours under the condition of 500 to 2000 atm and 40 ° C or less in the ultrahigh pressure apparatus, more preferably 2 to 5 times in the condition of 1200 to 1700 atm and 35 ° C or less And most preferably, it is extracted at a temperature of 1300 - 1600 atm and 30 ° C or lower for 2 to 4 hours. The solvent used for the extraction is omitted in order to avoid repetition of unnecessary explanation.

The composition for external application for skin of the present invention contains two or more kinds selected from the group consisting of extracts of L. leucitum extract, L. acutissima extract and L. orientalis herb extract, which are not a single ingredient but significantly increase gene expression of hyaluronic acid synthase present in human cells, It has an effect as a hyaluronic acid production promoting agent that activates hyaluronic acid generating action and at the same time promotes the production of a versatile cell which strengthens the extracellular matrix structure and suppresses the production of MMP-12 responsible for the degradation of versatile It can prevent skin dryness, improve skin elasticity, and can also be used for preventing skin aging and improving skin wrinkles.

FIG. 1 is a graph showing the expression of hyaluronic acid synthase at the level of mRNA after treating HaecaT cells with root extracts, acacia, and leaf extracts.
FIG. 2 is a graph showing the expression of a versatile gene at the mRNA level after treatment of Haeca T cells with rhizome, lily extract, and leaf extract.
FIG. 3 is a graph showing the expression of MMP-12 gene by UV at the mRNA level after treatment with Haeca T cells, Acanthopanax senticosus extracts, and leaf extracts.
4 is a graph showing the results of measuring the amount of hyaluronic acid by the ELISA method after treating Haeca T cells with the extracts of yuukee,

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples, but the scope of the present invention is not limited to these examples.

Example

[Preparation Example 1] Preparation of root roots extract

100 g of root roots were extracted with 1000 ℓ of purified water as a solvent for 3 hours while maintaining a low temperature of less than 30 ℃ at 1500 atm using an ultra high pressure apparatus. Then, centrifuge (9000 rpm, 15 캜, 30 min) to remove foreign matter and With filter cloth Filtered, and diluted 10-fold with a diluting solvent such as purified water, butyleneglycol, phenoxyethanol or the like to prepare a root extract of root weevil.

[Preparation Example 2] Preparation of extract of Acanthopanax senticosus

100 g of Acidic Acid was extracted with 1000 L of purified water as a solvent for 3 hours while maintaining a low temperature of 30 캜 or less at 1500 atm using an ultra high pressure apparatus. Then, the foreign substances were removed by centrifugation (9000 rpm, 15 ° C, 30 min), filtered with a filter cloth, and diluted 10 times with a diluting solvent such as purified water, butyleneglycol, phenoxyethanol or the like.

[Preparation Example 3] Preparation of upper leaf extract

100 g of the upper leaves were extracted with 1000 L of purified water as a solvent for 3 hours while maintaining a low temperature of below 30 캜 at 1500 atm using an ultra-high pressure apparatus. Then, the foreign materials were removed by centrifugation (9000 rpm, 15 ° C, 30 min), filtered with a filter cloth, and diluted 10 times with a diluting solvent such as purified water, butyleneglycol, phenoxyethanol or the like to prepare a leaf extract.

[Test Example 1] HAS2, versican, and MMP-12 expression increased in HaCaT cells by treatment with a mixture of extracts of Root, Radix, and Herb Medicine

<Step 1> Cell line and cell culture

HaCaT cells (human keratinocyte cell line, cell line service) were cultured in DMEM medium containing 10% fetal bovine serum (Dulbecco's modified Eagle's Medium, Gibco 1210-0038). The human keratinocyte And cultivation was carried out at 37 ° C in a 5% CO2 incubator for 18 hours.

<Step 2> Confirmation of increased mRNA synthesis of HAS2, versican, and MMP-12 gene by quantitative reverse transcription PCR

The cell line cultured in step 1 was treated with trypsin to prepare a single cell suspension, which was then dispensed into a T75 flask at a rate of 1 × 10 ⁶ cells and cultured for 24 hours. Thereafter, DMEM medium without bovine serum was treated and cultured for 24 hours. Then, root extract of Root Root Extract, Lycopersicon esculentum Extract, Lycopersicon esculentum Extract (Preparation Examples 1 to 3) dissolved in DMSO was added to the medium containing no bovine serum Respectively. DMSO, a vehicle, was used as a non-treated control group.

The UV treatment for inducing the expression of MMP-12 proceeded as follows. HaCaT cells, a human keratinocyte cell line, were cultured in a 100-mm cell culture dish at a density of 106 cells and irradiated with ultraviolet B using a FS40 lamp (Westinghouse, Pittsburgh, Pa., USA) 10 mJ / cm ² , and the total amount of the combination prescription of the root and leaf extracts was 10 ppm, the total amount of the combination prescription of the root extract, the root extract and the root extract was 10 ppm in DMSO Was replaced with a medium which had been dissolved and treated at a concentration of 5 ppm. DMSO, a vehicle, was used as a non-treated control group.

At 24 hours, cells were collected, washed with cold 4 ° C phosphate buffered saline (PBS), and 1 ml of TRIzol ™ reagent (Life Technologies, Inc.) was added to extract the total RNA and quantitatively real- Respectively. Primer sequences of HAS2, versatile, and MMP-12 for real-time DNA chain polymerization reaction are shown in Table 1 below.

Name of the primer order HAS-2 forward primer 5'TTTCTTTATGTGACTCATCTGTCTCACCGG-3 ' HAS-2 reverse primer
VersiChan forward primer
Versican Reverse primer
MMP-12 forward primer
MMP-12 reverse primer 5 'ATTGTTGGCTACCAGTTTATCCAAAGGG-3'
5 'GTCACCTTCCAACTATCCGGT-3'
5 'AGCACGGTAGTCCATTCTTTCT-3'
5 'GGAGTCCCCGATCTCCATCAT-3'
5 'ACGGTTCATGTCAGGTGTGTA-3' GAPDH forward primer 5 'CAACTACATGGTTTACATGTTCC-3' GAPDH reverse primer 5 'GGACTGTGGTCATGAGTCCT-3'

Table 1 shows primers for HAS2, Versican, MMP-12 and GAPDH for quantitative reverse transcription PCR reactions. 1 μg of total RNA was added to 25 μl of a reverse transcription reaction buffer containing 50 mM Tris-HCl (Tris-HCl, pH 8.3), 75 mM KCl, 3 mM MgCl ₂ , 0.1 MDTT, 10 mM dNTP and 40 units / μl RNase inhibitor, The primer of 0.5 μg / μl oligo (dT) 16 and the reverse transcription polymer of 200 units SuperScript II (SuperScript II, GiboBRL) were added and reacted at 42 ° C for 1 hour. Then 2.5 μl of reverse transcription reaction solution AmpliTaq DNA polymerase (0.04 U, Perkin Elmer, Shelton, CT), 50 mM Tris (pH 8.3), 0.25 mg / ml bovine serum albumin, 3 mM MgCl ₂ , 0.25 mM dNTPs And 50 L of a PCR reaction buffer containing 1 / 50,000 dilution of SYBR Green I [Molecular Probes, Eugene, Ole (Molecular Probes, Eugene, OR)], and 10 μM of primer was added thereto. , Annealing at 53 DEG C for 30 seconds, and extension at 72 DEG C for 1 minute were performed 30 times. Relative mRNA levels were analyzed by measuring SYBR Green I fluorescence changes using ICycler software. Each primer was based on SCI-grade papers. Quantitative expression levels of genes were corrected using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as an internal standard. The expression levels of HAS2, versican and MMP-12 genes are shown in FIGS. 1 to 3, respectively.

In Fig. 1 and Fig. 2, the group treated with the root extract of Rootgrass, the extract of Acanthopanax senticosus or Lycopersicum were treated at a concentration of 10 ppm, and the roots of Root Extract, Radix Root Extract, Root Root Extract, In the combination prescription group, the total amount of each two kinds of mixture was treated at a concentration of 5 ppm, and the combination of three kinds of root extract, It is a family.

1 and 2, the expression levels of the HAS2 gene and the vasicococcal gene were somewhat increased in the case of treatment with root extracts, rosacea, or safflower extract, compared with the control without the extract, And HAS2 gene in the experimental group using the combination of two extracts in the extracts of Hansen 's and Hansen' s extracts. In particular, when three compound prescriptions of root, root and leaf extracts were used, the expression levels of HAS2 gene and versatile gene were significantly increased compared with the single treatment group or the two compound treatment group.

Therefore, it has been found that the use of two or more kinds selected from the group consisting of root roots extract, lily extract and mulberry leaf extract according to the present invention can effectively increase the synthesis of hyaluronic acid, thereby enhancing the extracellular matrix structure have.

In Fig. 3, the UV group was irradiated with UV and then treated with DMSO, and the group treated with UV + Root extract, Radix extract or Lycopene extract alone was irradiated with UV, and then Root extract, ppm, UV + Radix root extract, Radix extract, Radix root extract, Radix extract, Radix extract and Lycopene extract were irradiated with UV and the total amount of each mixture was 5 ppm, and the combination of UV + rhizome root extract, sunscreen, and upper leaves were treated with 5 ppm of the total amount of the three mixtures after UV irradiation.

FIG. 3 shows that the amount of MMP-12 gene expression was reduced to some extent in the case of the treatment with root extracts of roots, rosacea or safflower leaves, compared to the control roots of the roots of roots extract, The expression level of MMP-12 gene was significantly decreased in the experimental group using the combined extract of eggplant extracts. In particular, the use of three combination prescriptions of root, root and leaf extracts significantly reduced the expression level of MMP-12 gene compared with the single treatment group or the two combined treatment groups.

Accordingly, the use of two or more selected from the group consisting of root extract, root extract, and mulberry extract according to the present invention reduces the expression amount of the MMP-12 gene, thereby inhibiting the degradation of the virucan, which enhances the extracellular matrix structure .

[Experimental Example 2] Increase in hyaluronic acid production in HaCaT cells by root roots extract,

The cell line cultured in step 1 of Test Example 1 was treated with trypsin to prepare a single cell suspension, which was then divided into 1 × 10 ⁶ cells in a T75 flask and cultured for 24 hours. After that, DMEM medium without bovine serum was treated and cultured for 24 hours. Then, 10 ppm of root extract, acid extract, and leaf extract were added to the medium containing no bovine serum, 10 ppm, Root extract, Radix extract and 10 ppm mixed leaf extract. At 24 hours, the cell culture medium was recovered and centrifuged at 15,000 g for 5 minutes to remove precipitated cells. The supernatant was collected and the supernatant was collected using an ELISA kit (Echelon Bioscience, Salt Lake, UT) The amount of lonic acid was measured.

In FIG. 4, the group treated with the root extract of Rootgrass, the extract of Lycopersiconia mellifera, or the Lycopersicum was treated at a concentration of 10 ppm, and a combination of two kinds of Rochelle root extract and Lycopene extract, Root root extract and Lycopene extract, Was a group treated with 5 ppm of the total amount of each of the two mixtures, and the combination of the three kinds of herbal extracts of root, root and upper leaves was a group treated with 5 ppm of the total amount of the mixture of the three kinds.

FIG. 4 shows that the amount of hyaluronic acid was slightly increased compared with the control group in which the extracts were not treated in the case of treatment with root extracts of roots, roots or leaves, but the extracts of roots, root extracts, , The amount of hyaluronic acid was significantly increased in the experimental group using the combination regimen. Especially, it was found that the amount of hyaluronic acid synthesized was significantly higher than that of the single treatment group or the two kinds of combined treatment group when three compound prescriptions of root, root and leaf extract were used.

Therefore, according to the present invention, it is possible to effectively increase the amount of hyaluronic acid to be synthesized by using two or more kinds selected from the group consisting of root extract, root extract, and mulberry leaf extract, thereby reducing skin elasticity and moisture content Can be prevented.

[Formulation Example 1] Nutritional lotion

Nutrition lotion was prepared according to a conventional method according to the composition shown in Table 2 below.

ingredient Content (% by weight) Acne extract 10 Acid extract 10 Leaf extract 10 Squalane 5.0 Wax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.5 Liquid paraffin 0.5 Caprylic / capric triglyceride 5.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservative, coloring, fragrance Suitable amount Purified water Balance Sum 100

[Formulation Example 2] Nourishing cream

Nutrition creams were prepared according to the compositions shown in Table 3 below in a conventional manner.

ingredient Content (% by weight) Acne extract 10 Acid extract 10 Leaf extract 10 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 PEG60 hardened castor oil 2.0 Liquid paraffin 10.0 Squalane 5.0 Caprylic / capric triglyceride 5.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservative, coloring, fragrance Suitable amount Purified water Balance Sum 100

[Formulation Example 3] Pack

Packs were prepared in a conventional manner according to the composition shown in Table 4 below.

ingredient Content (% by weight) Acne extract 10 Acid extract 10 Leaf extract 10 Polyvinyl alcohol 13.0 Sodium carboxymethylcellulose 0.2 glycerin 5.0 Allantoin 0.1 ethanol 6.0 PEG 12 nonyl phenyl ether 0.3 Polysorbate 60 0.3 Preservative, coloring, fragrance Suitable amount Purified water Balance Sum 100

[Formulation Example 4] ointment

Ointments were prepared in a conventional manner according to the compositions shown in Table 5 below.

ingredient Content (% by weight) Acne extract 10 Acid extract 10 Leaf extract 10 Purified water Balance glycerin 8.0 Butylene glycol 4.0 Liquid paraffin 15.0 Beta Glucan 7.0 Carbomer 0.1 Caprylic / capric triglyceride 3.0 Squalane 1.0 Cetearyl glucoside 1.5 Sorbitan stearate 0.4 Stearyl alcohol 1.0 antiseptic Suitable amount incense Suitable amount Pigment Weed beeswax 4.1 Sum 100

Claims (9)

The extracts of Trichoderma sp., Radix extract,
Which enhances the extracellular matrix structure. The extracts of Trichoderma sp., Radix extract,
Which enhances the extracellular matrix structure. delete The extracts of Trichoderma sp., Radix extract,
Which enhances the extracellular matrix structure. 5. The method according to any one of claims 1 to 4,
Wherein the extract is a water extract. 5. The method according to any one of claims 1 to 4,
Wherein the active ingredient is contained in an amount of 0.0001 to 14% by weight based on the total weight of the composition. 5. The method according to any one of claims 1 to 4,
Wherein the composition changes the amount of mRNA expression of at least one of the HAS2 gene, the versatile gene, and the MMP-12 gene. 5. The method according to any one of claims 1 to 4,
Wherein the composition promotes the synthesis of hyaluronic acid. delete

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