KR101718854B1 - Cosmetic composition containing Ligustrum lucidum Aiton, Astragalus membranaceus Bunge, Citrus unshin Markovich, Angelica dahurica root extract for moisturizing the skin - Google Patents

Abstract

The present invention relates to ligustrum lucidum Aiton, Astragalus The present invention relates to a cosmetic composition for improving skin moisturization, comprising a mixture extract of Angelica dahurica root, citrus unshiu Markovich, membranaceus Bunge, skin external preparation for skin moisturization, and a pharmaceutical composition for preventing and treating skin diseases, The mixed extract of the present invention was confirmed to have increased protein expression and promoter activity of factors involved in keratinocyte differentiation through western blot and luciferase reporter assay and transepidermal water loss, TWEL), the mixed extract of the present invention is useful as an active ingredient of a cosmetic composition for skin moisturizing, a skin external preparation for skin moisturizing, and a pharmaceutical composition for preventing and treating skin diseases Can be used.

Description

(Cosmetic composition containing Ligustrum lucidum Aiton, Astragalus membranaceus Bunge, Citrus unshin Markovich, Angelica dahurica root extract for moisturizing the skin containing a mixture of Jurassic, Hwanggi,

The present invention relates to ligustrum lucidum Aiton, Astragalus membranaceus Bunge, Citrus unshiu Markovich, and white fly ( Angelica dahurica The present invention also relates to a cosmetic composition for skin moisturizing improvement, a skin external preparation for skin moisturizing, and a pharmaceutical composition for skin disease prevention and treatment.

Keratinocyte is an important cell that maintains the structure and homeostasis of the epidermis. Through complex differentiation processes, epidermal keratinocytes constitute a rigid, insoluble structure called the cornified cell envelope, an early constituent of essential barriers to life support (L. Eckhart, S. Lippens et al, Biochim. Biophys. Acta., 2013, 1833, 3471-3480). The keratinocyte differentiation involves the process of cell cycle arrest and initiation of the expression of numerous genes. For example, the expression of keratin 5 and keratin 14 decreases while keratin 1, keratin 10, involucrin, loricrin and fillaggrin, Expression is increased in a time-dependent manner (PM Elias, J. Invest. Dermatol., 2005, 125, 183-200, PM Steinert, LN Marekov et al., J. Biol. Chem., 1995, 270, 17702-17711) .

Since keratinocyte differentiation is a pivotal process in providing a proper skin barrier against a harmful environment, disorders of keratinocyte differentiation are directly linked to skin disease. For example, incomplete keratinocyte differentiation breaks skin barriers and causes immune exacerbation, causing psoriasis and atopy. It is also known that defects in the skin barrier can result in an overly dry skin and become another deterioration factor causing differentiation-related skin diseases (MA Lowes, AM Bowcock et al, Nature, 2007, 445, 866-873 , M. Lebwohl, LG Herrmann et al., Cutis, 2005, 76, 7-12). In order to treat skin diseases associated with differentiation, immunomodulatory drugs such as cyclosporine A, Tacrolimus and prime crolimus are first-line therapeutic agents, (FO Nestle, DH Kaplan, et al., N. Engl. J. Med., 2009, 361, 496-509). In addition, skin moisturizers are used to alleviate disease conditions and enhance skin texture (C. Lynde, J. Drugs, Dermatol., 2008, 7, 1038-1043). It is known that the end product of keratinocyte differentiation, such as pilar green, provides a natural moisturizing factor (NMF) and maintains healthy skin (P. Chandar, G. Nole et al, Cutis, 2009 , 84, 2-15, S. Kezic, PM Kemperman, et al, J. Invest. Dermatol, 2008, 128, 2117-2119). Therefore, it is anticipated that therapies that promote keratinocyte differentiation can be used in a common mode of treatment with early stage therapy.

The Journeyman was a member of the Ligustrum lucidum Aiton) or a fruit tree ( Ligustrum japonicus Thunberg). It is said to be an operation room. There is a peculiar smell, and the taste is sweet and it wears. When the fruit grows in the winter, the leaves are removed and dried in the sun or lightly distilled and used in sunlight. It is used for the treatment of diarrhea and liver function, promoting the transplantation of healthy people, the staphylococcus aureus It is known that it exhibits active reaction against dysentery bacillus, influenza bacillus, pseudomonas aeruginosa, E. coli, and anticancer effect.

Astragalus membranaceus Bunge) grows in mountainous area, reaching 1m in height and has fine hairs throughout. Leaves are 6 ~ 11 pairs of lobes, and are odd-numbered birds. Flowers bloom in July and August, 15 ~ 18 ㎜ in length, light yellow, and several flowers hang on a long peduncle, forming a bloom. Root is used as a medicinal ingredient, and its active ingredient is folic acid, choline, and the like. In animal experiments, the excitatory and diuretic effects of the central nervous system were also remarkable. When a large amount of powder was administered to rats, the occurrence of nephritis was inhibited, the proteinuria and cholesterolemia were delayed, and the hypotensive effect was also recognized. It is also widely used for uterine drainage, gastrointestinal tract, esophagus, and uterine bleeding, and it increases the physical strength and increases the tension of the whole body muscles.

Citrus unshiu Markovich refers to the mature peel of mandarin or mandarin or its closely related plants and collects the ripe fruit husks and dries them in the sun. The taste is very good, the quality is warm, it works on menopause and parentheses. Promote the circulation of the period, eliminate abuse, and remove the sputum. In pharmacological experiments, gastric secretion promoting action and digestion were revealed, and appetite was poor and digestion was not possible, and a sloth was sore, and sore or diarrhea [gastritis], there was dampness, chest tightness, coughing and breathing , Dizziness, throbbing, and pregnancy.

Blank paper ( Angelica dahurica root) is a generic name for the root portion of Angelica dahurica Bentham et Hooker. The roots are pale yellowish brown, but they are hypertrophied, and when they fall down, they become ligneous and die. As a herbal medicine, it is good to have one or two years of life, but it is written that the pedicel has not come up. It is an analgesic medicine that regulates headache, nasal congestion and runny nose caused by influenza, and is effective for gastrointestinal disorder, postpartum headache, dizziness, toothache, facial nerve pain, paralysis. It is also known that when blood is mixed in the postmenstrual bleeding, adipose, and feces, headache due to sinusitis, rejuvenation, poisoning, and skin ulcers are also effective.

Korean Patent Laid-Open Publication No. 2013-0068320 discloses the effect of restoring skin barrier function and skin moisturizing power of Jeju minke as a prior art related to Jiujiao, and according to B. Hu, Jijijiao extract is a cyclin-dependent kinase (Huang, Q. Du, et al., Oncol., Rep., 2014, 32). In the present study, 1037-1042). Korean Patent Publication No. 2002-0025346 discloses skin whitening effect, skin irritation alleviation effect, and immunity enhancement effect of white and ganoderma extracts, and L. Lu, H. Ihara, and S. F. Xu et al. Have shown that Hwanggi, Quercus and Bleach extract have antioxidant activity (L. Lu et al., Int. J. Biol. Macromol., 2013, 61, 7-16 / H. Ihara, et al, Biosci , 1996, 1843-1848 / SF Xu, et al., Chem. Biodivers., 2011, 8, 1121-1131), skin moisturization by promoting keratinocyte differentiation of Jellyfish, Hwanggi, There is no known effect.

Accordingly, the present inventors have made efforts to develop a cosmetic composition for skin moisturization by using herbal medicines which have been traditionally used. The inventors of the present invention have found that a mixture of herbarium, hwanggi, It is possible to use the mixed extract as an effective ingredient of a cosmetic composition for improving skin moisturizing, a skin external preparation for skin moisturizing, and a pharmaceutical composition for preventing and treating skin diseases The present invention has been completed.

It is an object of the present invention to provide a cosmetic composition for improving skin moisturization containing a mixed extract of Ligustrum lucidum Aiton, Astragalus membranaceus Bunge, Citrus unshiu Markovich and Angelica dahurica root, And to provide a pharmaceutical composition for preventing and treating skin diseases.

The present invention relates to ligustrum lucidum Aiton, Astragalus The present invention provides a cosmetic composition for skin moisturization, which comprises a mixture extract of Angelica dahurica root, membranaceus Bunge, Citrus unshiu Markovich and Angelica dahurica root.

In addition, the present invention provides a skin external preparation for skin moisturizing, comprising a mixture of Jugija, Hwanggi, Citrus peel, and White spot mixture.

In addition, the present invention provides a pharmaceutical composition for prevention and treatment of skin diseases, which comprises a mixture of mallow extract, hwanggi, citrus peel and white spot.

Yeojeongja of the present invention (Ligustrum lucidum Aiton, Astragalus membrane extracts of membranaceus Bunge, Citrus unshiu Markovich and Angelica dahurica root were analyzed by western blot and luciferase reporter assay for protein expression and promoter of factors related to keratinocyte differentiation promoter activity of the extract of the present invention was confirmed and it was confirmed that there was a significant effect through the experiment of transepidermal water loss (TWEL). Thus, the cosmetic composition for skin moisturizing, Skin external preparation for skin moisturization, and pharmaceutical composition for skin disease prevention and treatment.

FIG. 1 is a graph showing the results of the detection of Ligustrum lucidum Aiton, Astragalus membranaceus Bunge, Citrus unshiu Markovich, Angelica dahurica root and the like, which are confirmed by an ultra performance liquid chromatography (UPLC) -photodiode array (PDA) Fig. 4 is a graph showing the components of a mixed extract; Fig.
2 is a graph showing the cell survival rate of keratinocytes of the mixed extract of the present invention.
HME (herbal mixture extract): An experimental group treated with the mixed extract of the present invention
CTL (control): A control group without the mixed extract of the present invention
Cell viability (% control): The cell survival rate of the experimental group to the control group not treated with the mixed extract of the present invention
FIG. 3 shows protein expression of keratinocyte differentiation-related factors due to the mixed extract of the present invention.
CTL (control): A control group without the mixed extract of the present invention
Ca ^{2 +:} positive treated with inducers of keratinocyte differentiation in Ca control group
HME (herbal mixture extract): An experimental group treated with the mixed extract of the present invention
Figure 4 shows the promoter activity of involucrin and loricrin due to the mixed extract of the present invention.
Figure 5 shows cell growth due to the mixed extract of the present invention.
1d, 3d, 5d (1day, 3day, 5day): control, Ca 2 +, number of days elapsed after the treatment the HME
FIG. 6 shows recovery of skin barrier function due to the mixed extract of the present invention. FIG.
Vehicle: Control without treatment of the mixed extract of the present invention
TWEL (transepidermal water loss): skin moisture loss
FIG. 7 is a graph showing the skin barrier function improving effect due to the mixed extract of the present invention. FIG.

Hereinafter, the present invention will be described in detail.

The present invention relates to ligustrum lucidum Aiton , Astragalus The present invention provides a cosmetic composition for skin moisturization, which comprises a mixture extract of Angelica dahurica root, membranaceus Bunge, Citrus unshiu Markovich and Angelica dahurica root.

The mixed extract is preferably, but not necessarily, prepared by a manufacturing method comprising the steps of:

1) extracting by adding an extracting solvent to Jyujiaojang, Hwanggi, Tangerine, and white paper;

2) filtering the extract of step 1);

3) Concentrating the filtrate obtained in step 2) under reduced pressure and drying to prepare an extract of Jurassic, Hwanggi, Jukpyu, and Baekji.

In the above method, it is possible to use in the step 1), without limitation, the cultivated or commercially available beech japonica, hwanggi, citrus fruit, and white paper.

In the above method, it is preferable to use water, an alcohol or a mixture thereof and an organic solvent as the extraction solvent in step 1). As the alcohol, C ₁ to C ₂ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. Preferably, the extraction solvent is added by 1 to 10 times, preferably 4 to 6 times, more preferably in an amount of 1 to 10 times the amount of dried clarinets, hulls, citrus fruits, and white paper. The extraction temperature is preferably 20 占 폚 to 100 占 폚, more preferably 20 占 폚 to 40 占 폚, and most preferably room temperature, but is not limited thereto. The extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, most preferably 24 hours, but is not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably, 3 times, but is not limited thereto.

The above-mentioned mixed extract may be prepared by preparing extracts of Jajeonjang, Hwanggi, Jangsuji, or Baekji, respectively, followed by mixing, but the present invention is not limited thereto.

The above-mentioned isomeric, hwanggi, citrus and white mixed extracts can be stored in a deep freezer until used.

Preferably, the above-mentioned mixed extract is 40 to 50 parts by weight of the herbarium, 20 to 25 parts by weight of the herbarium, 20 to 25 parts by weight of the citrus peel, 10 to 15 parts by weight of the white paper, 21.2 parts by weight, 21.2 parts by weight of citrus fruit, and 14.8 parts by weight of white paper, but is not limited thereto.

The mixed extract promotes keratinocyte differentiation, but is not limited thereto.

In a specific example of the present invention, the inventors of the present invention conducted cytotoxicity studies to confirm the skin moisturizing effect by promoting the keratinocyte differentiation of the herbal extract, hwanggi, It was confirmed that the mixed extract of the present invention did not show cytotoxicity (see Fig. 2). Western blot analysis and luciferase reporter assay were performed to confirm protein expression and promoter activity of factors capable of confirming the degree of keratinocyte differentiation. As a result, Promoting cell differentiation (see FIGS. 3 and 4).

In addition, TWEL (transepidermal water loss) test for measuring the skin moisture loss degree to confirm the change of the skin barrier function due to the treatment of the mixed extract, and the expression of filaggrin for enhancing the differentiation-increasing activity of keratinocytes It was found that the mixed extract of the present invention was effective in improving the skin barrier function and thus improving the skin barrier function. As a result, it was confirmed that the mixed extract of the present invention had an effect on the skin barrier function (See Figs. 6 and 7).

Therefore, the mixed extract of the present invention can be used as a cosmetic composition for skin moisturization improvement that promotes keratinocyte differentiation and improves skin barrier function.

The composition of the present invention may contain at least one active ingredient which exhibits the same or similar function in addition to the above-mentioned mixed extract.

Specific formulations of the cosmetic composition of the present invention include skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisturizing lotions, nutritional lotions, massage creams, nutritional creams, And includes formulations such as packs, soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, press powders, loose powders, eye shadows and the like.

The cosmetic composition may further contain, in addition to the mixed extract of the present invention, a lipid, an organic solvent, a solubilizer, a thickener and a gelling agent, a softener, an antioxidant, a suspending agent, a stabilizer, a forming agent, Water, ionic or non-ionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics Such as, for example, cosmetics, cosmetics, pharmaceuticals, cosmetics, pharmaceuticals,

In the composition of the present invention, the mixed extract is preferably contained in an amount of 0.005 to 90% by weight with respect to the total weight of the composition. However, it may be used in the range above or below the above range depending on the intended use insofar as it has a moisturizing effect and does not show toxicity.

In addition, the present invention provides a skin external preparation for skin moisturizing, comprising as an active ingredient, a mixed extract of Lolium japonica, Huangji, citrus peel, and white lips.

When the mixed extract is used as an external preparation for skin, it may further contain at least one selected from the group consisting of fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softening agents, antioxidants, suspending agents, stabilizers, , Ionic or nonionic emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, barrier agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or cosmetics And may contain adjuvants conventionally used in the cosmetics field, such as any other ingredient. In addition, the components can be introduced in amounts commonly used in the dermatology field.

In addition, the present invention provides a skin disease prevention and pharmaceutical composition comprising an extract of Liliaceae, Hwanggi, Citrus peel, and White fly as an active ingredient.

The skin disease is preferably psoriasis or atopic dermatitis, but is not limited thereto.

The mixed extract promotes keratinocyte differentiation, but is not limited thereto.

The pharmaceutical composition of the present invention may further contain commonly used excipients, disintegrants, sweeteners, lubricants, flavors and the like, and may be formulated into tablets, capsules, powders, granules, suspensions, Syrups, and other liquid preparations.

Specifically, the pharmaceutical compositions of the present invention may be formulated for oral administration, for example, tablets, troches, lozenges, aqueous or aqueous suspensions, prepared powders or granules, emulsions, hard or soft capsules, It is formulated into elixirs. Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate, Lubricating oil such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. In the case of a capsule formulation, in addition to the above-mentioned substances, a liquid carrier such as fatty oil is contained.

In addition, the pharmaceutical composition of the present invention can be administered orally or parenterally, and it is preferable to select subcutaneous injection, intravenous injection, intramuscular injection, or intra-thoracic injection injection method for parenteral administration. For formulation into a parenteral administration form, the mixed extract of the present invention is mixed with water or a stabilizer or a buffer in water to prepare a suspension, which is formulated into a unit dosage form of ampoule or vial.

The dosage of the active ingredient according to the present invention is appropriately selected depending on the degree of absorption, inactivation rate and rate of absorption of the active ingredient in the body, the age, sex and condition of the patient, and severity of the disease to be treated. It is possible to administer the mixed extract of the present invention in an amount of 0.0001-500 mg per 1 kg of body weight on an adult day, and it is preferably administered in an amount of 0.001-100 mg.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

< Example  1> Preparation of the mixed extract of the present invention

The mixed extracts of Liliaceae, Hwanggi, Citrus peel, and White papaya used in the present invention were prepared.

Specifically, 100 g of dried strains ( Ligustrum lucidum Aiton) purchased from Omniherb (Daegu, Korea), Astragalus 50 g of membranaceus Bunge, 50 g of Citrus unshiu Markovich and 35 g of white fly ( Angelica dahurica root) were cut into small pieces , mixed and boiled in water of 8 times the volume of the mixture for 4 hours to obtain an extract. The extract was filtered through Whatman filter paper, and the filtrate was lyophilized to prepare a herbal mixture extract (HME). In order to treat the mixed extract with cells, it was used by re-dissolving in water. For use in a local area of a mouse, it was redissolved in a mixture of 70% polyethylene glycol and 30% ethanol Respectively.

< Example  2> Cell culture for keratinocyte differentiation

The keratinocyte cell line used in the present invention was constructed and cultured.

Specifically, the cell line SV-HEK transformed with human epidermal keratinocyte by injecting simian virus 40 T antigen (SV40T) was added to bovine pituitary extract and supplemented with human epidermal growth hormone And cultured in a keratinocyte-serum-free medium (K-SFM).

< Experimental Example  1> Ultra performance liquid chromatography ( UPLC ) Was used to confirm the components of the mixed extract of the present invention

A UPLC-photodiode array (PDA) detection method was performed to confirm the constituents of the mixed extract of the present invention prepared by the method of Example 2 above.

Specifically, a Brownlee SPP C18 column (3.0 × 100 mm, 2.7 μm) was used. The mobile phase was acetonitrile (0.1%, solvent A) and water (solvent B ). All the solvents were filtered after using 0.2 ㎛ filter. In addition, the total dose was 2 占 퐇, and the concentration program of the solvent A was manipulated at a rate of 0.5 ml / min by setting the density program as follows. 0 to 4 minutes linearly in 2% solvent A, 4 to 7 minutes linearly in 2 to 5% solvent A, 7 to 12 minutes linearly in 5 to 7% solvent A, 12 to 17 minutes linearly in solvent 7, Linearly in 10% solvent A, linearly in 10 to 30% solvent A for 17 to 41 minutes, linearly in 30 to 50% solvent A for 41 to 47 minutes, 50 to 70% for 47 to 54 minutes, Of solvent A and 54 to 59 minutes were set linearly in 70-100% of solvent A. &lt; tb &gt; &lt; TABLE &gt;

As a result, as shown in Fig. 1 and Table 1, phytochemicals such as naringenin, naringin, narirutin, hesperidin, neohesperidin, Aviprin and byakangelicin (Fig. 1 and Table 1).

Peak order

Total hours (minutes)
Identify ingredients
One

27.398
Narinenin
2

32.630
Narinjin, Lily Routine
3

34.342
Hesperidin, neoheperidin
4
37.292
Aviprin (oxyphosuccinic hydrate, oxypeucedanin hydrate)
5

38.314
Bjarkangelicin

< Experimental Example  2> Toxicity of the mixed extract of the present invention to keratinocytes

To confirm the effect of the mixed extract of the present invention on keratinocyte differentiation, MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed.

Specifically, 2 × 10 ⁵ SV-HEK cells cultured in the same manner as in Example 2 were dispensed into a 12-well culture dish, and the mixed extract of the present invention was treated for one day. Subsequently, MTT solution of 2 mg / ml was added to the cells and cultured for 4 hours. The medium in which the cells were cultured was removed and the formed formazan crystal was dissolved in 100 μl of DMSO and the resultant was subjected to enzyme-linked immunosorbent assay (ELISA) Was used to measure the optical density at 540 nm.

As a result, it was confirmed that cytotoxicity was not observed on the third day after treatment even when the dose of the mixed extract was increased to 500 / / ml in SV-HEK cells (Fig. 2).

< Experimental Example  3> Confirmation of keratinocyte differentiation control of the mixed extract of the present invention

In order to confirm the effect of the mixed extract of the present invention on the keratinocyte differentiation, western blot and luciferase reporter assay were performed to confirm protein expression and promoter activity of factors capable of confirming the degree of keratinocyte differentiation reporter assay.

Specifically, calcium known as a factor inducing keratinocyte differentiation was used as a positive control, and the SV-HEK cells were treated with the mixed extract of the present invention for 3 days. Then, the harvested cells were pulverized using a proprep solution, and the total protein was quantified using BCA (bichinchominic acid) protein analysis reagent (Pierce Biotechnology, Fockford, IL, USA). Samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and reacted with appropriate primary antibodies. The blot was reacted with a secondary antibody bound to peroxidase and analyzed using an enhanced chemiluminescence (Intron) instrument.

Specifically, for the analysis of promoter activity, genomic DNA was isolated from keratinocytes and then used as a template for polymerase chain reaction (PCR). In this manner, Invorcin-luc reporter adenovirus (Ad / Inv-luc) and loricrin-luc reporter adenovirus (Ad / Lor-luc) were synthesized. The sequence of the primers used for the polymerase chain reaction is as follows. The Involucrin promoter (SEQ ID NO: 1 and SEQ ID NO: 2), the loricrin promoter (SEQ ID NO: 3 and SEQ ID NO: 4). The fragment containing the base pair from -2467 to +1,239 at the transfer position of inbolucurin and the base pair from -2,033 to +12 of the transcription position of lorikreen were obtained by the polymerase chain reaction Respectively. The promoter fragment was cloned into a pGL3 vector (Promega, Madison, Wis., USA) and then transferred to a pENTR vector to produce adenovirus.

The SV-HEK cells were cultured in a 12-well culture dish with 50% confluency and transformed with reporter adenovirus. The cells were then cultured in fresh medium and mixed extracts were treated. The cells were further cultured for 48 hours. Cell extracts were prepared using cell lysis buffer. The activity of Luciferase was analyzed according to the protocol provided by Luciferase analysis system (Promega, Madison, Wis., USA).

As a result, keratin 1 (keratin 1), involucrin and loricrin were rapidly increased or decreased by the treatment of the mixed extract as shown on the left side of FIG. In addition, as shown in the right side of FIG. 3, it was confirmed that the filaggrin as a marker of the end of differentiation was remarkably increased after 7 days of treatment with the mixed extract (FIG. 3).

In addition, as shown in Fig. 4, it was confirmed that the promoter activity of inbolucurin and lorikreen was dependent on the treatment amount of the mixed extract (Fig. 4).

From these results, it was confirmed that the mixed extract of the present invention promotes keratinocyte differentiation.

< Experimental Example  4> Confirm the effect of mixed extract on cell growth

Since keratinocyte differentiation occurs with cell cycle arrest, [ ³ H] thymidine uptake assay was performed to confirm the effect of the mixed extract of the present invention on cell growth.

Specifically, the SV-HEK cells of Example 1 were dispensed into a 60-mm cell culture dish and treated with 1 μCi of [ ³ H] thymidine (Amersham, Buckinghamshire, UK). After incubation for the following times, cells were rinsed with PBS (phosphate buffered saline) and incubated at room temperature in 0.1 N NaOH. The radioactivity of cell crushes was measured using a liquid scintillation counter.

As a result, as shown in FIG. 5, the experimental group treated with the mixed extract of the present invention showed cell growth inhibition similar to that of the control group treated with calcium (FIG. 5).

From these results, it was confirmed that the mixed extract of the present invention promotes keratinocyte differentiation without inhibiting cell growth.

< Experimental Example  5> Effect of the mixed extract of the present invention on skin barrier function

In order to confirm the effect of the mixed extract of the present invention on the skin barrier function, transepidermal water loss (TWEL), a well known experimental technique for confirming skin barrier dysfunction, was performed.

Specifically, a 7-week-old, hairless female mouse was purchased from Orient Bio Inc. (Seongnam, Korea), the skin barrier was broken by tape-stripping, and the mixed extract was topically treated. The mixed extract was dissolved in 70% of poly (ethylene glycol) and 30% of ethanol and 0.2 ml was used at a concentration of 5 mg / ml. TEWL was measured using Tewameter TM210 (Courage + Khazaka electronic GmbH, Cologne, Germany) in the manner described by Yoshizawa et al. (Y. Yoshizawa et al., Skin Res. Technol, 2001, 36-39).

As a result, as shown in FIG. 6, it was confirmed that the group treated with the mixed extract of the present invention (HME) accelerated the recovery of the skin barrier function as compared with the vehicle (FIG. 6).

Further, in order to further investigate the effect of the mixed extract of the present invention, the expression level of pilar green was confirmed by immunohistochemistry.

Specifically, the wax of the paraffin section of the mouse specimen was removed, hydrated again and rinsed three times with PBS. The slices were incubated with protinase K (Dako, Carpinteria, CA, USA) for 5 minutes at 37 ° C, treated with H ₂ O ₂ for 10 minutes at room temperature, and incubated with 0.1% Tween- The cells were blocked with PBS containing bovine serum albumin for 30 min and reacted for 1 h with a filazine antibody (Abcam, Cambridge, MA, USA). The flakes were continuously incubated in a peroxidase-conjugated secondary antibody and visualized using a Chemmate envision detection kit (Dako, Carpinteria, CA, USA).

As a result, as shown in Fig. 7, in the flakes on the third day after the treatment of the mixed extract of the present invention, the pillar green in the granular layer of the skin of the mouse, which strengthens the keratinocyte differentiation- (Fig. 7).

From these results, it was confirmed that the mixed extract of the present invention is effective for increasing the expression of pillar green in granular layer of skin and improving skin barrier function.

<110> Chungnam National University - Industry Collaboration Foundation <120> Cosmetic composition containing Ligustrum lucidum Aiton,          Astragalus membranaceus Bunge, Citrus unshin Markovich, Angelica          dahurica root extract for moisturizing the skin <130> 2015P-03-016 <160> 4 <170> KoPatentin 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> involucrin promoter <400> 1 ctccatgtgt catgggatat g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> The involucrin promoter (R) <400> 2 tcaacctgaa agacagaaga g 21 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> loricrin promoter <400> 3 taccaagcaa tcctctcacc ttgg 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> The loricrin promoter (R) <400> 4 tgaggagaga agatgctggc 20

Claims (9)

A cosmetic composition for skin moisturization improvement comprising as an active ingredient, a mixed extract of Ligustrum lucidum Aiton, Astragalus membranaceus Bunge, Citrus unshiu Markovich and Angelica dahurica root.
The cosmetic composition according to claim 1, wherein the mixed extract is extracted with water, a C ₁ to C ₂ lower alcohol or a mixture thereof.
The cosmetic composition for skin moisturization according to claim 2, wherein the lower alcohol is ethanol or methanol.
[Claim 2] The method according to claim 1, wherein the mixed extract is characterized by comprising 40 to 50 parts by weight of the herbarium, 20 to 25 parts by weight of the herbarium, 20 to 25 parts by weight of the citrus peel, and 10 to 15 parts by weight of the white paper, A cosmetic composition for skin moisturization improvement.
The cosmetic composition for improving skin moisturization according to claim 1, wherein the composition promotes keratinocyte differentiation.
A skin external preparation for skin moisturizing, containing an extract of lily japonica, hwanggi, citrus peel and white spot as an active ingredient.
Wherein the composition is selected from the group consisting of psoriasis and atopic dermatitis, which comprises an extract of Lepidoptera, Hwanggi, Citrus peel, and White ground as an active ingredient.
delete [Claim 8] The pharmaceutical composition according to claim 7, wherein the mixed extract promotes keratinocyte differentiation. The pharmaceutical composition according to claim 7, wherein the mixed extract is selected from the group consisting of psoriasis and atopic dermatitis.

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