KR20210096843A - composition for skin external application having Nandina domestica extract - Google Patents

Abstract

The present invention relates to a composition for skin external application using a Nandina domestica extract. More specifically, the present invention relates to a composition for skin external application having excellent anti-inflammatory and anti-allergic activity which contains a Nandina domestica extract to prevent or alleviate anti-inflammatory and allergic skin diseases, and has excellent anti-wrinkle and skin regeneration effects.

Description

Composition for skin external application having anti-inflammatory and anti-allergic activity using Namcheon extract {composition for skin external application having Nandina domestica extract}

The present invention relates to a composition for external use on the skin using a namcheon extract, and more particularly, it contains namcheon extract to prevent or improve anti-inflammatory and allergic skin diseases, and to exhibit excellent anti-inflammatory and anti-allergic activity with excellent anti-wrinkle and skin regeneration effects. It relates to a composition for external use on the skin.

Recently, extrinsic skin diseases are increasing significantly due to industrial development and changes in lifestyle. In particular, as the living environment changes recently, soot emitted from automobiles and factories is increasing. Soot is present in the city not only in the form of gas but also in the form of dust such as fine dust (PM10) and ultrafine dust (PM25). Diesel Particulate Matter (DPM) adversely affects the skin as well as the respiratory system.

Specifically, due to fine dust, inflammation occurs through overexpression of cytokines in skin cells, which intensifies inflammatory diseases such as erythema, atopy, psoriasis, and acne. The barrier allows more DPM to penetrate. Deeply penetrating DPM can cause loss of collagen in the dermal layer, leading to a decrease in skin elasticity. In particular, dioxins such as TCDD can cause diseases such as chlorine dermatitis.

Allergy, also called type 1 hypersensitivity reaction, is caused by an excessive reaction of the immune system in vivo to substances exposed on a daily basis. When the antigen (allergen), a substance that causes this allergy, enters the body through the respiratory system, digestive system, and skin, it binds to the IgE antibody attached to the receptor (FcεRI) on the surface of the mast cell in the tissue. Through degranulation of mast cells, histamine is secreted to induce an initial allergic reaction immediately. Subsequently, leukotriene and various cytokines are secreted from mast cells to induce late allergic reactions such as tissue damage and inflammation.

Recently, in order to minimize the side effects of existing chemical drugs, efforts to find natural products that are effective for inflammatory or allergic skin diseases are increasing. A composition is disclosed, and Korean Patent Application Laid-Open No. 10-2010-0059305 discloses a composition for external application for skin containing an extract of Prunus oleifera.

On the other hand, Nandina domestica Thunb is a plant of the Berberidaceae of the Ranunculales family. ), Namjukja (南竹子), Namcheonjuk (南天竹), and Hongpaja (紅杷子) are called. Namcheon is an evergreen shrub commonly found in countries such as southern China, Japan, India, and southern Korea. The tree can grow to a height of 1.00-3.00m, and the leaves are glossy and have a leathery feel.

The leaves of Namcheon are called Namcheonjukyeop and have therapeutic effects on whooping cough, hematuria, and bruises. In winter, the green leaves turn red and in spring they turn blue again. The fruit of Namcheon is a round berry, measuring 0.50 ~ 1.00cm, ripens in October and turns red. Namcheon fruit is called Namcheonsil (南天實) or Namcheon (南天) or Namcheonjukja (南天竹子) in folklore and is used as a medicinal material. It is known to be used in the treatment of chronic cough, syphilis, uterine bleeding, asthma, and whooping cough.

There is no known skin external preparation using Namcheon as a material, which has such various effects.

Korean Patent Laid-Open Patent No. 10-2006-0035140: Composition for external application for skin containing plant extract Korean Patent Laid-Open Patent No. 10-2010-0059305: Composition for external application for skin containing fruit extract

The present invention was created to improve the above problems, and by using Namcheon extract having excellent anti-inflammatory and anti-allergic activity, it is possible to prevent or improve inflammatory and allergic skin diseases, and an external composition for skin with excellent anti-wrinkle and skin regeneration effects Its purpose is to provide

In order to achieve the above object, the composition for external use for skin having anti-inflammatory and anti-allergic activity using the Namcheon extract of the present invention contains a Namcheon extract extracted from Namcheon (Nandina domestica Thunb).

The Namcheon extract is extracted by adding at least one extraction solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof to the Namcheon.

The Namcheon extract is extracted from the leaves of the Namcheon.

The Namcheon extract is extracted by adding ethanol to the leaves of Namcheon at 20 to 30° C. for 60 to 80 hours.

The namcheon extract has antioxidant activity.

The namcheon extract has a skin wrinkle improvement effect.

The Namcheon extract has a skin cell regeneration effect.

The namcheon extract has anti-inflammatory activity.

The namcheon extract has anti-allergic activity.

It further contains a fermented extract from Amaranthus hybridus fermented product.

The fermented extract is obtained by mixing 10 to 30 parts by weight of sugar with respect to 100 parts by weight of fermented amaranth, and vacuum extraction of the fermented product at 20 to 40° C. for 20 to 60 days after adding the fermented strain.

As described above, the composition for external application for skin of the present invention contains Namcheon extract extracted from leaves, stems, and fruits of Namcheon, and can be safely used without irritation to the skin, and can prevent or improve inflammatory and allergic skin diseases.

In addition, the composition for external use for skin of the present invention has excellent antioxidant activity, anti-wrinkle and skin regeneration effects, and thus can be usefully used as a pharmaceutical composition or a functional cosmetic composition for the prevention and improvement of inflammatory and allergic skin diseases.

1 is a block diagram showing the process of extracting the extract from the fruit (NDF), stem (NDB), and leaf (NDL) of Namcheon;
Figure 2 is a graph showing the total polyphenol content of NDF, NDB, NDL extract,
Figure 3 is a graph showing the total flavonoid content of NDF, NDB, NDL extract,
Figure 4 is a graph showing the free radical activity of NDF, NDB, NDL extract,
5 is a graph showing the toxicity test results for HaCaT cells of NDF, NDB, and NDL extract,
Figure 6 is a graph showing the toxicity test results for HDFn cells of NDF, NDB, NDL extract,
7 is a graph showing the toxicity test results for RAW 264.7 cells of NDF, NDB, and NDL extracts,
8 is a graph showing the toxicity test results for MC/9 cells of NDF, NDB, and NDL extracts,
9 is a graph showing the expression of MMP-1 in NDF, NDB, NDL extract in HDFn cells,
Figure 10 is a graph showing the NO inhibition rate by NDF extract in RAW 264.7 cells,
11 is a graph showing the NO inhibition rate by the NDB extract in RAW 264.7 cells,
Figure 12 is a graph showing the NO inhibition rate by NDL extract in RAW 264.7 cells,
13 is a result showing the phosphorylation effect of ERK, JNK and p38 MAP kinase by NDL extract in RAW 264.7 cells;
14 is a graph showing the expression of IL-6 by NDL extract in RAW 264.7 cells,
15 is a graph showing the expression of IL-1β by NDL extract in RAW 264.7 cells,
16 is a graph showing the production of TNF- by NDL extract in MC/9 cells,
17 is a graph showing the expression of TARC by NDL extract in HaCaT cells,
18 is a graph showing the expression of RANTES by NDL extract in HaCaT cells,
19 is a graph showing the production of histamine by NDL extract in MC/9 cells,
20 is a graph showing the cell proliferation results by NDL extract in HDFn cells.

Hereinafter, an external composition for skin having anti-inflammatory and anti-allergic activity using Namcheon extract according to a preferred embodiment of the present invention will be described in detail.

The composition for external application for skin of the present invention contains Namcheon extract as an active ingredient. Herein, the term 'active ingredient' refers to a component that exhibits activity alone or together with a carrier that is not active by itself.

Namcheon extract can be obtained by extracting from Namcheon (Nandina domestica Thunb).

Namcheon ( Nandina domestica Thunb) is a plant of the Barberry family of the Buttercup family, also known as Namchok tree or Namcheonchok (南天燭), Tianchuckja (天竺子), Cheonchokja (天燭子), and Namjukja (南竹子). ), Namcheonjuk (南天竹), and Hongpaja (紅杷子) are called. Namcheon is an evergreen shrub commonly found in countries such as southern China, Japan, India, and southern Korea. The tree can grow to a height of 1.00-3.00m, and the leaves are glossy and have a leathery feel.

The leaves of Namcheon are called Namcheonjukyeop and have therapeutic effects on whooping cough, hematuria, and bruises. In winter, the green leaves turn red and in spring they turn blue again. The fruit of Namcheon is a berry, round, 0.50~1.00cm long, ripens in October and turns red. Namcheon fruit is called Namcheonsil (南天實) or Namcheon (南天) or Namcheonjukja (南天竹子) in folklore and is used as a medicinal material. It is known to be used in the treatment of chronic cough, syphilis, uterine bleeding, asthma, and whooping cough.

In the present invention, various parts can be used for Namcheon, but preferably any one part selected from leaves, stems, and fruits of Namcheon is used. Especially preferably, Namcheon leaves are used.

Namcheon extract can be obtained by adding an extraction solvent to Namcheon. At this time, at least one selected from water, a lower alcohol having 1 to 4 carbon atoms, a polyhydric alcohol, or a mixture thereof may be used as the extraction solvent.

Methanol, ethanol, etc. can be used as a C1-C4 lower alcohol, and butylene glycol, propylene glycol, pentylene glycol, etc. can be used as a polyhydric alcohol. And as the mixture, a mixture of water and a lower alcohol, a mixture of water and a polyhydric alcohol, a mixture of a lower alcohol and a polyhydric alcohol, or a mixture of water and a lower alcohol and a polyhydric alcohol may be used.

As an example of extraction, after adding the extraction solvent to Namcheon in a weight ratio of 2 to 20 times, extraction may be performed by hot water extraction, cold-chim, or warm-chim extraction at 10-150° C. for 2 to 100 hours. As a specific example of extraction, ethanol can be added to leaves of Namcheon 10 times and extracted at 20 to 30° C. for 60 to 80 hours to obtain a Namcheon extract.

In addition to the above-described extraction method, extraction may be performed using reflux cooling extraction, ultrasonic extraction, or the like. In addition, extracts that have undergone a conventional purification process are included. For example, the active fraction obtained through various additional purification methods, such as separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography, etc., is also included in the extract.

In addition, it goes without saying that the extract extracted using the extraction solvent can be obtained in powder form through filtration, concentration under reduced pressure, freeze-drying, spray-drying, or the like.

The present invention may contain 0.01 to 10% by weight of the namcheon extract based on the total weight of the composition for external use for skin.

Namcheon extract has excellent anti-inflammatory and anti-allergic activity. In addition, the namcheon extract has excellent antioxidant activity, skin wrinkle improvement and skin cell regeneration effect. Therefore, the composition for external use for skin of the present invention containing the namcheon extract may be provided in the form of a pharmaceutical composition capable of preventing or improving inflammatory skin disease or allergic skin disease, and treating it.

The composition for external use on the skin of the present invention can be formed into various formulations containing suitable pharmaceutically acceptable carriers, excipients and diluents. For example, the composition for external application to the skin may be any one selected from cream, gel, patch, spray, ointment, warning agent, lotion, liniment agent, pasta agent, and cataplasma agent.

Examples of allergic skin diseases include hypersensitivity, allergic rhinitis, asthma, allergic conjunctivitis, allergic dermatitis, atopic dermatitis, allergic contact dermatitis, urticaria, insect allergy, food allergy, or drug allergy. And inflammatory skin diseases may include acute/chronic eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, chronic lichen simplex, intertidal dermatitis, exfoliative dermatitis, papular urticaria, psoriasis, solar dermatitis and acne.

The dosage of the composition for external application for skin of the present invention varies depending on the patient's condition and weight, the degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art. For a desirable effect, the namcheon extract is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 10 mg/kg. Administration may be administered once a day, or may be administered in several divided doses.

In addition, the composition for external use on the skin of the present invention may be provided in the form of a cosmetic composition having skin wrinkle improvement and skin cell regeneration effects. In this case, it is formed in any one formulation selected from lotion, lotion, cream, essence, pack, powder, makeup base, foundation, body oil, body lotion, cleansing oil, cleansing foam, hair oil, hair shampoo and hair conditioner, and soap. can be

On the other hand, the present invention may contain a fermented extract of fermented horseradish fermented with Namcheon extract as another example. The fermented extract of fermented horseradish fermented horseradish can be obtained by extracting it from a fermented product of fermented fermented horseradish.

Amaranthus hybridus is an annual plant belonging to the family Amaranthus. Fertilizers can use leaves or stalks.

As a method for fermenting jasmine fern, 10 to 30 parts by weight of sugar is mixed with respect to 100 parts by weight of jasmine fern, 0.01 to 1.0 parts by weight of a fermented strain is added, and then fermented at 20 to 40° C. for 20 to 60 days. Molasses or sugar can be used as sugar. And Aspergillus oryzae can be used as a fermentation strain. When fermentation is complete, water is added to the fermented product to extract the fermented extract of fermented horseradish. As an example of the extraction, a method of vacuum extraction at a low temperature may be used. For example, 10 to 50 parts by weight of the fermented product is added to 100 parts by weight of water in a vacuum low temperature extractor, and then vacuum extracted for 4 to 10 hours at 60 to 80° C. under vacuum (100 to 200 torr) conditions. After vacuum low temperature extraction, it is possible to obtain a fermented extract of horseradish fermented by filtration.

The fermented extract of fermented horseradish extract has excellent antioxidant activity, so it can further improve the functionality of the composition for external use on the skin. Excellent antioxidant activity can help improve skin and prevent wrinkles. In particular, the fermented extract of fermented horseradish extract greatly improves the antioxidant activity of the namcheon extract by adding it to the namcheon extract.

In the case of containing the fermented pilaris fermented extract, the composition for external use for skin of the present invention may contain 0.01 to 10% by weight of the namcheon extract and 0.1 to 5% by weight of the fermented extract of fermented pilaris pilaris from the total total weight.

Hereinafter, the contents of the present invention will be described in detail through examples and experiments. This is for explaining the present invention in more detail, and the scope of the present invention is not limited to the following examples.

(Example: Namcheon extract extraction)

1. Material preparation

(1) Preparation of Namcheon

In November 2016, Namcheon (Nandina domestica Thunb; Gwangju Metropolitan City, Korea) collected directly from Gwangju Metropolitan City was prepared by dividing the parts into fruits, stems, and leaves. Plant specimens used in the experiment were stored in the specimen room of Coseed Biopharm Co., Ltd. (Chungcheongbuk-do, Cheongju).

(2) Experimental material

Dulbecco's modified eagle's medium (DMEM), Phenicillin-streptomycin (PS), Trypsin-EDTA (ethylenediaminetetraacetic acid), Fetal bovine serum (FBS), Trypan bule, Phosphate buffered saline (PBS), Ham'nutrient mixture F-12 (F- 12) was purchased from Gibco (Rockville, MD, USA). 6-well plate, 24-well plate, 96-well plate, and subculture plate are SPL (Korea), L-ascorbic acid, 1,1-diphenyl-2-picryl hydrazyl (DPPH), Thiazolyl blue tetrazolium bromide (MTT), Dimethyl sulfoxide (DMSO), Lipopolysaccharide (LPS), Nordihydroguaiaretic acid (NDGA), Etanol, Dexamethasone, Adenosine, Monoclonal anti-β-actin antibody produced in mouse, Compound 48/80, Histamine, Agarose, PMA, T-stim, 2 -mercap-toethanol and L-glutamine, Quercetin, Gallic acid, Folin-Ciocaleu reagent were purchased from sigma (St. Louis, MO, USA). ELISA kits such as TNF-α, IFN-γ, RANTES ELISA kit, TARC ELISA kit, and Histamine were purchased from R&D Systems (Minneapolis, USA). Anti-rabbit lgG HRP-linked antibody, p44/42 MAPK (Erk1/2) antibody, SAPK/JNK antibody, p38 MAPK antibody, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody, Phospho-SAPK /JNK (Thr183/Tyr185) antibody, and Phospho-p38 MAP Kinase (Thr180/Tyr182) antibody, RIPA is Cell Signaling Technology, Inc. (Danvers, USA). MMP-1 (SB12e): sc-58377 was manufactured by Santa Cruz Biotechnology, Inc. (California, Co., USA). Goat anti-Mouse lgG (H+L) secondary antibody HRP was purchased from ThermoFisher SCIENTIFIC (Waltham, Co., USA). Bicinchoninic acid assay kit, SDS-PAGE gel (BioRad, USA) was purchased. Enhanced chemiluminescence (Amersham Pharmacia Biotech, USA), Griess (Promega, Madison, USA), Competitive-Enzyme linked ImmunoSorbent assay: ELISA (ALPCO Diagnostics, NH, USA), RT PreMix kit, PCR PreMix kit (Bioneer, Korea). AlCl ₃ *?*H ₂ O, Acetonitrile, Methanol, Na ₂ CO ₃ was purchased from DAEJUNG (DAEJUNG CHEMICALS & METALS Co., Korea).

The equipment used was Soxhlet (Wise Therm, Korea), High speed refrigerated centrifuge (SUPRA, 21K, Korea), Rotary evaporator (EYELA, SB-1200, Co., Japan), Microwave oven (LG, Korea), Voltex-1 (East-West Science, Korea), CO ₂ incubator (New Brunswick, USA), Micro centrifuge (Micro 12) (Hanil, Korea), Inverted microscope (NIKON, Japan), UVB irradiator (USA), Clean bench (Vision scientific, Korea) ), Autoclave (Sanyo, Japan), Micro-pipet (Gilson, France), Water bath (Vision scientific, Korea), Vortex mixer (Vision scientific, Korea), Spectrophotometer (Shimazue, Japan), Centrifuge (Sigma, USA), Plate shaker (Lab-Line, USA), Micro plate reader (Molecular devices, USA), High speed refrigerated centrifuge (Hanil, Korea), Rotary evaporator (Heidolph Germany)，Series rotary evaporator (EYELA, Japan), Chemi-Doc ( Atto, USA) and Speed Vac (Servant, USA) were used.

2. Namcheon Extract Extraction

Extracts were extracted from the fruit of Namcheon ( Nandina domestica Thunb fruit, hereinafter referred to as NDF), stems ( Nandina domestica Thunb branches, hereinafter referred to as NDB), and leaves ( Nandina domestica Thunb leaf, hereinafter referred to as NDL).

NDF, NDB, and NDL to be used in the experiment are washed thoroughly with water, dried in a hot air dryer at 50°C for 24 hours, and then pulverized by 30 g each with a grinder (HBL-3500S, ELEXION, Korea) with a concentration of 70% (w/w) ethanol by weight. After 10 times of addition, extraction was performed by immersion at room temperature (25° C.) for 72 hours. After extraction, each extract was first filtered with 400mesh filter paper, centrifuged at 1000 rpm for 15min, and the supernatant was collected by whatman No. 2nd filtration with filter paper. The filtered NDF, NDB, and NDL extracts were concentrated under reduced pressure through a rotary evaporator and used as a sample. The NDF extract yield was 18.50%, the NDB extract yield was 10.20%, and the DNL extract yield was 24.30%.

The extraction process of the above Namcheon extract is shown in a block diagram in FIG. 1 .

<Experimental example>

1. Experimental method

(1) Determination of total polyphenol content

The total polyphenol content was measured by the widely used Folin-Denis method (Folin & Denis, 1912). The sample was completely dissolved in purified water at 1,000.00 mg/ml, filtered and used as a sample. First, 20.00 μl of 2N Folin-Ciocaleu reagent was added to 20.00 μl of sample sample in each well and mixed. _{After leaving for 5} min, 20.00% Na ₂ CO 3 100.00 μl was added and left at room temperature for 30 min in that state. Gallic acid was diluted so as to be 15.63, 31.25, 62.50, 125.00 and 250.00 mg/ml, and absorbance was measured at a wavelength of 730 nm with a micro plate reader to measure the black luminance of each sample and standard. The total polyphenol content was calculated in units of mg/g gallic acid equivalents (GAE) from the prepared standard curve. Each sample was repeated at least 3 times to obtain an average value.

(2) Measurement of total flavonoid content

To measure the total flavonoid content, each sample was dissolved in EtOH to 1,000.00 mg/ml, and the standard Quercetin was diluted to 7.81, 15.63, 31.25, 62.50 mg/ml. Put 80.00μl of EtOH in 20.00μl of the sample in each well, _{add 100.00μl of 2.00% AlCl 3} ·6H ₂ O, and leave at room temperature for 5 min. Absorbance was measured at a wavelength of 430 nm using a micro plate reader. Total flavonoid content was calculated according to the prepared Quercetin standard curve in mg/g Quercetin equivalents (QE). Each sample was repeated at least 3 times to obtain an average value.

(3) Free radical scavenging activity

DPPH reagent was dissolved in MeOH, and NDF, NDB, and NDL samples were each dissolved in DMSO to 100.00 mg/ml, and then diluted with MeOH. The experiment was carried out by diluting the sample and the sample solvent to 3.00, 10.00, 30.00, and 100.00 μl, respectively. The DPPH group mixed the sample with 180.00 μl of DPPH, and the MeOH group mixed the sample with 180.00 μl of MeOH.

After reacting at room temperature for 10 min, absorbance was measured at 565 nm using a microplate reader. As a positive control, 500.00 μM L-ascorbic acid was used.

(4) Evaluation of skin bioactivity efficacy

go. cell culture

-HaCaT cell culture conditions

Human keratinocytes (ATCC ^® PCS-200-011 ^™ , Primary Epidermal Keratinocytes, HaCaT, USA) were purchased from the US cell line bank and tested. For human keratinocytes, a medium prepared by adding 10.00% FBS and 1.00% PS to DMEM was used. The CO ₂ incubator was incubated under the conditions of 37° C., 5.00% CO _{2 , and full humidity.}

-HDFn cell culture conditions

Fibroblasts (ATCC PCS-201-010 ^™ , Human Dermal Fibroblast neonatal, HDFn, USA) were purchased from the US cell line bank and used for testing. As the medium used for HDFn, F-12 and DMEM were mixed in a ratio of 1:3 each, and 10.00% Fetal bovine serum (FBS) and 1.00% phenicillin-streptomycin (PS) were added. Incubated in a CO ₂ incubator at 37°C, 5.00% CO ₂ , and full humidity.

-MC/9 cell culture conditions

Mast cells (ATCC ^® CRL-8306 ^™ , Mus musculus, mouse, MC/9, USA) were purchased from the US cell line bank and used for testing. As the medium, DMEM containing 10.00% FBS, 10.00% T-stim, 0.05mM 2-mercap-toethanol and 2.00mM L-glutamine and 100.00μg/ml streptomycin was used. 37℃, 5.00% CO _{2 in a CO 2 incubator} , incubated under full humidity conditions.

-RAW 264.7 cell culture conditions

Macrophages (ATCC TIB-71 ^™ , Mus musculus, mouse, RAW 264.7, USA) were purchased from the US cell line bank and used for testing. DMEM with 10.00% FBS was used as the medium, and incubated in a CO ₂ incubator at 37° C., 5.00% CO ₂ , and full humidity conditions.

me. Cytotoxicity test

-HaCaT cells

HaCaT cells were counted and distributed ^{in a 96-well plate to 1 × 10 5} cells/ml, and the cells were stabilized. NDF, NDB, and NDL extracts were dissolved in DMSO to prepare 100.00 mg/ml. Each sample was used after sterilization using a 0.20 μm filter. The NDF, NDB, and NDL extracts were treated by diluting the samples by concentration and reacted for 24 hrs. The positive control group, Dexamethasone, was pretreated with 0.10 and 1.00 μM for 2 hrs. TNF-α + IFN-γ 10.00 mg/ml was treated and reacted for 24 hrs. After adding 20.00 μl of 5.00 mg/ml of MTT solution to each well and incubating at 37° C. for 2-3 hrs, all of the supernatant was removed. To dissolve the generated formazan, 100.00 μl of DMSO was used in each well, and the absorbance was measured at 550 nm using a micro plate reader.

-HDFn cells

HDFn cells were counted and distributed ^{in a 24-well plate to 1 × 10 5} cells/well, and the cells were stabilized. After stabilizing for 24 hrs, the NDF, NDB, and NDL extracts were dissolved in DMSO and prepared so that the sample could be 100.00 mg/ml. Each sample was sterilized using a 0.20 μm filter. The NDF, NDB, and NDL extracts were processed from a small concentration by diluting 3 samples of 10.00, 30.00, and 100.00 μg/ml through DMEM containing 10.00% FBS. After 24 hrs, cytotoxicity was confirmed using MTT solution. 50.00 μl of MTT solution was added to each well, the medium was removed after 2-3 hrs, and 350 μl of DMSO was used to dissolve the generated formazan, and absorbance was measured at 550 nm using a micro plate reader.

-RAW 264.7 cells

RAW 264.7 cells were counted and ^{dispensed in a 96-well plate at 4 × 10 5} cells/ml, and the cells were stabilized for 2 hrs. NDF, NDB, and NDL extracts were dissolved in DMSO to prepare 100.00 mg/ml. Each sample was used after sterilization using a 0.20 μm filter. The NDF, NDB, and NDL extracts were treated with 2.00 μl at 10.00, 30.00, and 100.00 μg/ml and reacted for 24 hrs. 5.00 mg/ml of MTT solution was added to each well by 10.00 μl, reacted in an incubator at 37° C. for 2-3 hrs, and then all of the supernatant was removed. To dissolve the generated formazan, 100.00 μl of DMSO was used in each well, and the absorbance was measured at 550 nm using a micro plate reader.

-MC/9 cells

MC/9 mast cells were counted, and 2 × 10 ⁵ cells/ml were dispensed in a 96-well plate to stabilize the cells. NDL was dissolved in DMSO to prepare 100.00 mg/ml. Each sample was used after sterilization using a 0.20 μm filter. NDL was treated with 2.00 μl at 50.00, 100.00, and 200.00 μg/ml and reacted for 24 hrs. 5.00 mg/ml of MTT solution was added to each well by 10.00 μl, and the reaction was performed in an incubator at 37° C. for 2-3 hrs, and then all of the supernatant was removed. To dissolve the generated formazan, 100.00 μl of DMSO was used in each well, and the absorbance was measured at 550 nm using a micro plate reader.

all. Wrinkle improvement effect

The expression of MMP-1 in HDFn cells was measured by Western blot. Cells were aliquoted in a 6-well plate at 4 × 10 ⁵ cells/well and then stabilized over night at _{37° C., 5.00% CO 2 incubator.} The medium was removed, washed twice with 1.00ml of 37°C warm PBS, 2.00ml of each well was filled with PBS, and UVB was irradiated at a height of 7.00cm for 15min. After 15 min, check the cell status, remove PBS, add 2.00 ml each of a medium containing 2.00% FBS, and treat NDF, NDB, and NDL extracts at 30.00 and 100.00 μg/ml, and positive control adenosine at 100.00 μg/ml 6 After the hrs reaction, the protein of the cells was isolated. The cells were removed after supernatants were removed with an aspirator and washed with 1 x PBS of 1.00 ml of phosphate buffered saline. Again, 1 x PBS was added to each well by 1.50ml, scraped with a cell scraper to obtain a 1.50ml tube, and the cells were precipitated using a vortex mixer. After removing the supernatant, 100.00 μl of 1 x RIPA was added to each tube and vortex cells were lysed. The tube was placed on ice for 30 min and reacted for 10 min each. After 30 min, centrifugation was performed at 12,000 rpm for 15 min at 4°C to obtain a supernatant. For protein quantification experiments, the supernatant obtained was quantified using a bicinchoninic acid assay kit. 20.00 μg of protein was electrophoresed on 8.00% SDS-PAGE gel. Proteins separated according to size were transferred to polyvinylidene fluoride (PVDF) membrane, 5.00% skim milk was prepared in 1 x TBST, and the blocking step was performed while shaking at room temperature for 1 hr. After 1 hr, it was washed 5 dilutions with 1 x TBST for 10 min each. MMP-1 primary antibody 1: 800, β-actin primary antibody 1: 2500 was fixed to the well plate while shaking O/N at 4 ℃. The well plate to which the antibody was fixed was taken out from 4°C, reacted at room temperature for 3 hrs, and washed 5 times with 1 x TBST for 10 min each. Goat anti-Mouse lgG(H+L) secondary antibody HRP secondary antibody was diluted 1:3000 in 5.00% skim milk and reacted with shaking at room temperature. After 2 hrs, it was washed 5 times with 1 x TBST for 10 min each. The band was confirmed using enhanced chemiluminescence, and the band was quantified using imageJ 1.47 software.

La. anti-inflammatory effect

-NO production

RAW 264.7 cells were ^{seeded in a 96-well plate at 4 × 10 5} cells/ml, and after 2 hrs, the cells were stabilized, and the sample was treated in a 96-well plate. The NDF, NDB, and NDL extracts were pre-treated by 2.00 μl at 10.00, 30.00, and 100.00 μg/ml. The positive control group NDGA was treated with 20.00 uM. After 2 hrs, LPS was treated at 1.00 μg/ml by 2.00 μl. After culturing for 24 hrs, 100.00 μl of Griess reagent (griess A: B = 1:1) was added to 100.00 μl of the supernatant, and absorbance was measured at 540 nm. Giress reagent was prepared with 0.10% N-Cl-haphthyl ethylene diamine and 1.00% sulfanilamide before the test.

- Expression level of ERK, JNK and p38 MAP kinase

Expression levels of ERK, JNK and p38 MAP kinase in RAW 264.7 cells were confirmed by Western blot. Cells were aliquoted in a 6-well plate at 4 × 10 ⁵ cells/well and then stabilized overnight at _{37° C., 5.00% CO 2 incubator.} NDL samples were pretreated with 30.00 and 100.00 μg/ml, and after 2 hrs, LPS was treated with 1.00 μg/ml, followed by 15 min reaction, and then cell proteins were isolated. The cells were removed after supernatants were removed with an aspirator and washed with 1.00 ml of 1x PBS with phosphate buffered saline. Again, 1 × PBS was added 1.50ml to each well, scraped with a cell scraper to obtain a 1.50ml tube, and centrifuged using a vortex mixer. After removing the supernatant, 100.00 μl of 1 × RIPA was added to each tube, and the cells were blown with a pipette and dissolved by vortex. The tube was placed on ice for 30 min and reacted for 10 min each. After 30 min, centrifugation was performed at 12,000 rpm for 15 min at 4°C to obtain a supernatant. For protein quantification experiments, the supernatant obtained was quantified using a bicinchoninic acid assay kit. 20.00 μg of protein was electrophoresed on 8.00% SDS-PAGE gel. The protein separated according to the size was transferred to a polyvinylidene fluoride (PVDF) membrane, 5.00% skim milk was prepared in 1 x TBST, and the blocking step was performed while shaking at room temperature for 1 hr. After 1 hr, it was washed 5 times with 1 x TBST for 10 min each. p44/42 MAPK (Erk1/2) antibody, SAPK/JNK antibody, p38 MAPK antibody, phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody, phospho-SAPK/JNK (Thr183/Tyr185) antibody, and phospho-p38 MAP Kinase (Thr180/Tyr182) antibody was immobilized on a well plate by shaking it with O/N at 4°C at 1:1000. The antibody-fixed well plate was taken out from 4°C, reacted at room temperature for 1 hr, and washed 5 times for 10 min with 1 x TBST. Anti-rabbit lgG HRP-linked antibody secondary antibody was diluted 1:3000 in 5.00% skim milk and reacted with shaking at room temperature. After 2 hrs, it was washed 5 times with 1 x TBST for 10 min each. The band was confirmed using enhanced chemiluminescence, and the band was quantified using imageJ 1.47 software.

-Cytokine production

Using RAW 264.7, intracellular cytokine expression was measured through RT-PCR. Cells were aliquoted in a 6-well plate at 4 × 10 ⁵ cells/well, and then stabilized overnight at _{37° C., 5.00% CO 2 incubator.} The NDL extract samples were pre-treated with 30.00 and 100.00 μg/ml, the positive control group NDGA was pretreated with 20.00 uM, and 2 hrs later, LPS was treated with 1.00 μg/ml and reacted for 4 hrs. Cell RNA was isolated. First, the cells were removed with an aspirator to remove supernatants, washed with 1 × PBS and 1.00 ml of phosphate buffered saline. Again, 1.50ml of 1×PBS was added to each well, scraped with a cell scraper to obtain a 1.50ml tube, and the cells were lysed using a vortex mixer. After removing the supernatant, the cells were lysed using 800.00 μl of trizol reagent, 200.00 μl of chloroform solution was added, immersed at room temperature for 5 min, and centrifuged at 4℃ 12,000 rpm for 15 min to obtain the supernatant. After adding and mixing the same amount of isopropanol, the supernatant was removed by centrifugation at 4 ℃ 12,000 rpm for 15 min, 800.00 μl of 70.00% EtOH was added, and the supernatant was removed by centrifugation at 4 ℃ 12,000 rpm for 15 min. After drying the RNA until transparent, 35.00 μl of DEPC DW was added thereto, and the RNA was dissolved and quantified for 30 min. To confirm β-actin, cDNA was prepared from the isolated total RNA through the RT PreMix kit, and reacted at 95°C for 5 min using each primer and PCR PreMix kit shown in Table 1 below, followed by denaturation at 94°C for 30 sec. , annealing at 54°C for 30 sec, followed by 1 min extension at 72°C was repeated 25 times, and the last extension was performed at 72°C for 5 min. Without changing other conditions, IL-1β was annealed at 54 °C for 28 cycles and IL-6 at 60 °C for 30 cycles. After the PCR reaction was completed, 7.00 μl each was added to 2.00% agarose gel, electrophoresed for 25 min, and UV was used to analyze the reaction results.

Primer Sense
Anti-sense Sequences IL-6
(230bp) Sense 5`- AGC CAG AGT CCT TCA GAG AGA-3` Anti-sense 5`- TAA CGC ACT AGG TTT GCC GAG-3` IL-1β
(563bp) Sense 5`- ATG GCA ATG TTC CTG AAC TCA ACT-3` Anti-sense 5`- CAG GAC AGG TAT AGA TTC TTT CCT TT-3` β-actin
(349bp) Sense 5`- TGG AAT CCT GTG GCA TCC ATG AAA C-3` Anti-sense 5`- TAA AAC GCA GCT CAG TAA CAG TCC G-3`

TNF-α was measured by cytokine assay using MC/9 cells. Cells were aliquoted in 96-well plates at 2 × 10 ⁵ cells/ml, and then NDL extracts were treated at 50.00, 100.00, 200.00 μg/ml in 96-well plates, and dexamethasone as a positive control was treated at 0.10, 1.00 μM for 30 min before 30 min. . PMA (20.00 nM) / A23187 (0.50 μM) was treated and reacted for 5 hrs. The supernatant was obtained and subjected to cytokine assay, and absorbance was measured at 450 nm according to the manufacturer's experimental method.

mind. anti-allergic effect

-Chemokine production

HaCaT cells were ^{seeded in a 96-well plate at 5 × 10 4} cells/well, and cell stabilization was confirmed overnight. NDF, NDB, and NDL extracts were added to a 96-well plate at 50.00, 100.00, 200.00 μg/ml, positive control. Dexamethasone was pretreated with 0.10 and 1.00 μM for 2 hrs. Treated with TNF-α + IFN-γ 10.00 mg/ml and allowed to react for 24 hrs. The supernatant was obtained and absorbance was measured at 450 nm using RANTES ELISA Kit and TARC ELISA Kit according to the manufacturer's experimental method.

-Histamine production

The histamine production amount was measured with reference to the research method of Park et al. (Park Kwang-hyun et al., 2011), and the experiment was carried out. Samples were pretreated for 15 min and stimulated with Compound 48/80 (6.00 μg/ml) for 15 min. Quantification of histamine in the cell culture supernatant was performed using a Competitive-Enzyme Linked ImmunoSorbent Assay (ELISA). The sample was centrifuged at 1,500 rpm for 3 min, and the same volume of the supernatant was taken from each reaction group, dried at a low temperature in a Speed Vac, and re-dissolved in 100.00 μl of PBS. Experiments were conducted according to the

bar. Skin cell regeneration effect

HDFn was dispensed in a 96-well plate at 1 × 10 ⁴ cells/well, and the cells were stabilized overnight. The next day, the medium was changed to a medium containing 0.20% FBS and the samples were processed. The NDF, NDB, and NDL extracts were treated at 10.00, 30.00, and 100.00 μg/ml by 2.00 μl. After reacting for 24 hrs and 48 hrs, 30.00 μl of MTT reagent was treated, checked after 3 hrs, and absorbance was measured at 550 nm, and the results were analyzed.

(5) Statistical analysis

The experiment was repeated 3 times, and Excel program was used for data analysis, and it was expressed as mean±standard deviation (SD). Statistical significance was determined using Student's t-test method. * p < 0.05, statistically significant was judged to be

2. Experimental results

(1) Extraction yield

As a result of extraction using 70.00% ethanol, as shown in Table 2 below, the yield of the NDF extract was 18.50%, the yield of the NDB extract was 10.20%, and the yield of the NDL extract was 24.30%.

extract Total Yield (%) NDF 18.50% NDB 10.20% NDL 24.30%

Total yield(%) = (sample extract weight/sample dry weight) × 100%

(2) Total polyphenol content

Polyphenols exist widely among various plants and are substances with various molecular structures and molecular weights. Since this polyphenolic substance contains phenolic hydroxyl (OH), it can easily combine with other macromolecules or proteins, so it has various kinds of physiological activities such as anti-inflammatory and antioxidant. Polyphenols play a role in protecting against skin aging and cardiovascular diseases caused by oxidative effects such as DNA damage, protein denaturation, and lipid peroxidation caused by free radicals.

Referring to FIG. 2 , the total polyphenol content was 85.82±0.05 mg/g in the NDL extract, which showed the highest polyphenol content, and the NDF and NDB extracts were 67.93±0.09 and 44.52±0.03 mg/g, respectively. appeared as

According to Jin's paper, Wasong was found to contain 24.14 mg/g in 70.00% ethanol extract and it was observed to contain the highest polyphenols than extracts extracted with other solvents (Donghyeok Jin et al., Comparison of total phenol, flavonoid content and antioxidant activity of Wasong extract. Journal of the Korean Society for Environmental Science; 25(5), 695-703. 2016). According to Song et al., the total polyphenol content of the ethanol extract of bamboo shoots was found to be 41.46 mg/g, and it was confirmed that the polyphenol content was higher than that of the hot water extract (Wonyoung Song et al., antioxidant and anti-inflammatory activity of bamboo extract by solvent. Agricultural Life Science study; 49(3), 145-154. 2015). According to Park's thesis, the total polyphenol result of 70.00% ethanol extract of Machihyun was 34.14 ± 0.28 μg/mg (Park Sora et al., Antioxidant activity of Machihyun hot water and ethanol extract. Journal of the Korean Herbal Society; 32(4), 39-46. 2017).

It was confirmed that the polyphenol content measured in the NDF, NDB, and NDL extracts was very high when compared with the results of the above paper.

(3) Total flavonoid content

A representative antioxidant, flavonoids, is a C ₁₅ compound in which two phenyl rings are bonded to three carbons. Flavonoids and their glycosides have various physiological activities in the human body, and the middle class is divided into Chalcones, flavones, flavonols, isoflavonoids, and the like.

Flavonoids are a type of polyphenol-based compound, a type of secondary metabolite widely distributed in nature, and are contained in the roots, stems, leaves, flowers, fruits, etc. known to contain.

Flavonoids are related to plant growth, differentiation, and coloration and have several functions, such as a protective action against UV rays, a protective action against pathogenic microorganisms, and an effect of regulating enzyme activity. Flavonoids exhibit anti-inflammatory, antioxidant, anti-allergic effects, prevention of circulatory system diseases, lipid lowering, and immune enhancement through various pharmacologically active effects. Flavonoids exhibit antioxidant activity because they have a hydroxyl group (-OH) that can be easily combined with various compounds present therein.

In the experiment, Quercetin was used as a standard material, and flavonoid compounds were quantified through standard calibration curves prepared at 7.81, 15.63, 31.25, and 62.50 mg/ml.

As a result of quantifying the total flavonoid content contained in NDF, NDB, and NDL at 1000.00 mg/ml, as shown in FIG. 3, the NDF, NDB, and NDL extracts were 2.37±0.00, 5.05±0.01, and 12.96±0.02 mg/g, respectively. It was confirmed, and among them, it was found that the NDL extract contained the most flavonoids.

In the paper of Park et al., the total amount of flavonoids in the extract of 70.00% ethanol Machiya was found to be 4.40 ± 0.22 μg/mg (Sora Park et al., Antioxidant activity of Machihyun hot water and ethanol extract. Journal of the Korean Herbal Society; 32(4), 39-46. 2017). In this paper, the flavonoid content of the sample prepared from 70.00% ethanol extract with poison activity was found to be 4.54 mg/g (Lee Seong-gyu et al., Antioxidant and anti-inflammatory activity of toxic ethanol extract. Journal of the Korean Society for Natural Healing; 4(1), 10-14. 2015). In comparison, the NDL extract showed a flavonoid content 2.9 times higher than that of Machihyun and 2.8 times higher than that of Toxin. Therefore, it was confirmed that the content of flavonoids was high in NDF, NDB, and NDL extracts compared with the results of the above paper.

(4) Free radical scavenging activity

Free radicals, especially ROS, cause unstable reduction during electron transport in living things or are generated in several metabolic processes such as cytokines. It can also be constantly produced by environmental pollution such as nitric oxide, ozone, smog, smoking and ultraviolet rays. Various free radicals such as hydrogen peroxide, hydroxyl radical, superoxide radical, and singlet oxygen as ROS destroy cell membranes to denaturate proteins, lipid peroxidation, and DNA damage. It is known that substances produced by various oxidations cause various diseases such as aging-related physiological disorders and cancer, diabetes, arteriosclerosis, and heart disease through induction of oxidative stress.

In order to confirm the free radical scavenging activity of the NDF, NDB and NDL extracts, the experiment was conducted by diluting them to four concentrations of 3.00, 10.00, 30.00, and 100.00 μg/ml.

Referring to FIG. 4 , ascorbic acid, a positive control, showed a free radical scavenging activity of 91.41±0.21% at 500.00 uM. And the NDF extract showed a concentration-dependent increase of 2.46±1.93, 14.88±4.02, 39.97±5.98, and 89.69±0.79% according to the concentration. And the NDB extract showed a concentration-dependent increase of 1.45±0.33, 5.51±1.78, 15.12±4.15, 53.34±8.44% depending on the concentration, and the NDL extract showed a concentration-dependent increase of 6.87±0.12, 20.73±1.86, and 46.61± depending on the concentration. 1.99, 90.87±1.13% showed a concentration-dependent increase.

The experimental group significantly increased at all concentrations compared to the control group, confirming that it was effective in free radical scavenging activity. Among them, it was confirmed that the NDL extract had the highest antioxidant activity in a concentration-dependent manner compared to other samples.

In this paper, free radical scavenging activity of 70.00% ethanol extract with poisonous activity and L-ascorbic acid, a natural antioxidant, were measured for free radical scavenging by 3.16%, 20.53%, and 63.16% at 10.00, 50.00, and 100.00 μg/ml of poisonous ethanol extract, respectively. The activity was confirmed (Sungkyu Lee et al., antioxidant and anti-inflammatory activity of poisonous ethanol extract. Journal of the Korean Society of Natural Healing; 4(1), 10-14. 2015). In the paper of Jin et al., black chokeberry 70.00% ethanol extract showed scavenging activity of 92.49 ± 0.07% and 93.50 ± 0.07% at 0.40 and 0.60 mg/ml (Donghyeok Jin et al., antioxidant activity and nitrite scavenging activity of black chokeberry extract) Korean Journal of Environmental Science;25(4), 567-577. 2016).

Comparing with the above thesis results, all of the NDF, NDB and NDL extracts showed excellent free radical scavenging activity, and the higher the concentration dependent, the higher the free radical scavenging activity was. In addition, a significant increase was confirmed when compared with the control group. In particular, it appears that there is a significant increase in the four concentrations of the NDL extract and thus has a high antioxidant effect, which is expected to be utilized as a good natural antioxidant.

(5) Cytotoxicity

-HaCaT cells

The cytotoxicity of NDF, NDB and NDL extracts was confirmed using HaCaT cells, HDFn cells, RAW 264.7 cells, and MC/9 cells. Through the cytotoxicity test, the safe use concentration of the sample can be confirmed and the experiment was carried out in the range where toxicity was not observed.

Stimulation of the human keratinocyte line, HaCaT, may increase the production of TARC, which may have an effect on atopic dermatitis. In addition, an experiment was conducted to verify the effect of inhibiting the formation of chemokines such as RANTES, an important target material, by inducing an allergic reaction using TNF-α + IFN-γ in HaCaT.

5, the NDF extract showed a cell viability of 90.49±5.24, 103.26±5.83, and 93.50±8.92% at 50.00, 100.00, and 200.00 μg/ml, and the NDB extract showed 93.97 at 50.00, 100.00, and 200.00 μg/ml. The cell viability was ±3.89, 90.39 ± 2.17, and 97.17 ± 7.59%, and the NDL extract showed cell viability of 100.08 ± 3.44, 92.21 ± 3.06, and 94.11 ± 9.54% at 50.00, 100.00, and 200.00 μg/ml.

It was confirmed that there was no difference between the experimental group and the control group using a statistical program, and there was no significant difference in HaCaT cells treated up to 200.00 μg/ml, indicating no toxicity.

-HDFn cell

When dermal fibroblasts are irradiated with UV light, they generate matrix metalloproteinases (MMPs) by ROS. HDFn, a human skin fibroblast, is mainly used for MMP-1 expression inhibition and Type I procollagen synthesis.

6, the NDF extract showed cell viability of 103.08±0.38, 100.62±0.28, and 104.39±0.39% at 10.00, 30.00, and 100.00 μg/ml, and the NDB extract showed 100.81 at 10.00, 30.00, and 100.00 μg/ml. The cell viability was ±0.37, 105.60 ± 0.54, and 103.67 ± 0.80%, and the NDL extract showed cell viability of 104.27 ± 0.78, 99.58 ± 0.65, and 101.40 ± 0.49% at 10.00, 30.00, and 100.00 μg/ml.

When comparing the experimental group and the control group, there was no significant difference in HDFn cells treated up to 100.00 μg/ml, so it was judged that there was no toxicity.

-RAW 264.7 cell

RAW 264.7, a mouse macrophage cell line, plays a key role in the inflammatory response. When macrophages are stimulated with LPS, inflammatory transcription factors that control the inflammatory response are activated to produce substances related to inflammation, such as NO, cytokine, and MAPKs.

7, the NDF extract showed a cell viability of 102.05±18.59, 104.08±19.22, 108.64±22.11% at 10.00, 30.00, and 100.00 μg/ml, and the NDB extract showed 95.63 at 10.00, 30.00, and 100.00 μg/ml. The cell viability was ±18.33, 95.17 ±17.05, 94.27 ± 20.88%, and the NDL extract showed cell viability of 94.34 ± 19.25, 96.64 ± 20.39, 108.79 ± 26.98% at 10.00, 30.00, and 100.00 μg/ml.

When comparing the experimental group and the control group, the RAW 264.7 cells treated up to 100.00 μg/ml did not show a significant difference, so it was determined that there was no toxicity.

-MC/9 cell

MC/9 cells, which are the mast cells of laboratory animals and mice, are widely distributed in living tissues such as skin and around lymphatic vessels. Mast cells play an important role in allergic reactions, and when activated, degranulation is induced and lipid mediators such as histamine are stored in intracellular granules, and various chemical mediators such as cytokines are produced.

Referring to FIG. 8 , the NDL extract showed cell viability of 113.61±13.46, 110.42±2.49, and 100.68±2.47% at 50.00, 100.00, and 200.00 μg/ml. Accordingly, it was confirmed that there was no toxicity to MC/9 cells.

(6) Wrinkle improvement effect

Skin aging is divided into two types: natural aging and extrinsic aging. Among them, extrinsic aging is the aging of the skin caused by ultraviolet rays and is called photoaging. Ultraviolet rays of sunlight show effects such as sterilization and vitamin D synthesis, but when excessive ultraviolet rays are frequently exposed to the skin, it reaches the skin and stimulates the skin and acts on fibroblasts and keratinocytes to induce ROS. MMPs are created.

MMPs are a generic term for enzymes involved in decomposing extracellular matrix (ECM) and basement membrane (BM) proteins secreted from cells in the skin such as fibroblasts and keratinocytes. It is classified into interstitial collagenase, gelatinase, stromelysin, and membrane-type MMPs.

MMPs reduce the production of skin components glycosaminoglycane (GAG), elastin, collagen, and hyaluronic acid. The expression of MMPs increases due to various causes and damages the connective tissue to form deep wrinkles. Among MMPs, MMP-1 is an enzyme that breaks down collagen in the dermis and affects skin aging. The dermal layer is composed of more than 90.00% collagen, of which type I collagen accounts for more than 80.00% of total collagen, and type III collagen accounts for about 15.00%. The main role of collagen is to strengthen the skin structure that induces the bonding and resistance of connective tissue, support of adhesion between cells, and the process of cell division and differentiation. When collagen decreases due to these various roles, the appearance of skin aging such as wrinkle formation and loss of elasticity appears.

Currently, various drugs are applied and used to slow the aging process, and the commonly used anti-wrinkle drugs or treatments are typically vitamin C and its derivatives, vitamin A and its derivatives retinol, and various antioxidants and retinyl palmitate, epigallocatechin gallate. , adenosine, etc. are being used.

Therefore, in order to examine the wrinkle improvement effect, the MMP-1 expression level inhibition effect of NDF, NDB and NDL extracts using HDFn was conducted.

Referring to FIG. 9 , as a result of UV irradiation on HDFn for 15 min, adenosine 100.00 μg/ml, a positive control, showed 66.33% MMP-1 inhibition. And the NDF extract showed no MMP-1 inhibitory effect at 30.00 and 100.00 μg/ml. The NDB extract showed an inhibitory effect of 9.11% and 43.78% of MMP-1 at 30.00 and 100.00 μg/ml, and a significant inhibitory effect at 100.00 μg/ml. The NDL extract showed a significant MMP-1 inhibitory effect of 67.04% only at 100.00 μg/ml, confirming that it had an inhibitory effect similar to that of the positive control adenosine.

According to Hwang et al., the expression of MMP-1 in HDFn was significantly higher in the experimental group treated with ethanol extract of choseokjam compared to the control group at 6.40%, 8.00%, and 9.70% MMP-1 at 50.00, 100.00, and 200.00 μg/ml, respectively. It was confirmed that ascorbic acid in the positive control group was significantly decreased to 16.70% at 100.00 μg/ml (Hwang Ji-yeon et al., Efficacy of the ethanol extract of chosukjam for inhibiting wrinkle formation in somatic dermal fibroblasts. Journal of the Korean Society of Aesthetics;11 (4).293-301.2015).

Compared with the above paper, the MMP-1 inhibitory effect of 67.04% was confirmed in the NDL extract at a concentration of 100.00 μg/ml. It can be inferred that the NDL extract has an anti-wrinkle effect by inhibiting MMP-1 expression.

(7) anti-inflammatory effect

The inflammatory response in the living body is a kind of protection and defense reaction of an organism that occurs when the damaged area is restored by various kinds of infections, viruses, bacteria, chemical substances, and physical stimuli from the outside. If the inflammatory response is continuously displayed, various diseases such as chronic inflammatory disease, hypersensitivity allergy disease, atopic dermatitis, and skin aging are induced. When inflammatory substances invade, macrophages rapidly secrete various cytokines. Among them, representative cytokines involved in inflammation are interleukin-1β (IL-1β), tumornecrosis factor-α (TNF-α), and interleukin-6 (IL-6). Mast cells exist in the lower layer of epithelial cells and connective tissue, and the granules of mast cells contain histamine.

LPS is one of the components of the outer membrane of Gram-negative bacteria, and is a glycolipid that has been used as a stimulant for a long time. It produces and releases products, reactive oxygen species, cytokines, and NO.

-NO production

NO is not only responsible for the defense function inside the human body, but also performs various physiological functions such as signal transduction function, vasodilation and neurotoxicity. NO, a representative mediator of the inflammatory response, is a strong free radical. When external stimulants attack the body, when bacteria invade the body, people secrete a lot of NO to protect themselves. is known In addition, when the inflammation becomes more severe, diseases such as hypersensitivity allergy disease, chronic inflammatory disease, atopic dermatitis, and skin aging appear.

The experimental results of NO inhibition rate of NDF, NDB and NDL extracts with RAW 264.7 macrophages are shown in FIGS. 10 to 12, respectively. 10 is an experimental result of NDF extract, FIG. 11 is an experimental result of NDB, and FIG. 12 is an experimental result of NDL.

Referring to FIG. 10 , the positive control NDGA was 84.36±2.43% at 20.00 μM. And the NDF extract showed NO production rates of 100.02±1.67, 99.90±1.56, and 99.68±1.58% at 10.00, 30.00, and 100.00 μg/ml.

Referring to FIG. 11 , the NDB extract exhibited NO production rates of 103.20±1.28, 102.61±1.63, and 99.01±1.77% at 10.00, 30.00, and 100.00 μg/ml.

Referring to FIG. 12 , the NDL extract exhibited NO production rates of 97.00±1.07, 90.90±1.29, and 59.55±1.45% at 10.00, 30.00, and 100.00 μg/ml. In the case of the NDL extract, at concentrations of 30.00 and 100.00 μg/ml, NO production inhibition rates of 21.90% and 97.35% were observed, which was confirmed to have a significant inhibitory effect. Accordingly, the amount of NO induced by the antigen, LPS, was decreased in the experimental group treated with the NDL extract sample, and compared to the positive control group, the NO inhibition rate of about 2.50 times was confirmed when the NDL extract was 100.00 μg/ml, and a significant decrease was confirmed.

According to the results of the paper by Kim et al., when RAW 264.7 cells pretreated with LPS were treated with ethanol extract 0.10, 1.00, 10.00, 50.00, 100.00 μg/ml, the NO production amount was significantly decreased in a concentration-dependent manner, especially 50.00 After μg/ml, more than 34.00% inhibitory activity was confirmed when compared to the control group (Min-Ji Kim et al., anti-inflammatory effect of ethanol extract of oleracea on LPS-induced macrophage inflammatory response and mouse ear edema. Microbiol. Biotechnol. Lett; 44(4), 479-487. 2016). In the paper of Ahn et al., in order to confirm the NO inhibitory ability of the extracts extracted with red oak branch water and 70.00% ethanol, LPS induced an inflammatory reaction and after treatment with 20.00, 50.00, and 100.00 μg/ml, NO decreased in a sample concentration-dependent manner and decreased to 100.00 μg/ In ml, the amount of NO production was reduced by about 30.00% or more (Andaeseong et al., anti-aging and anti-inflammatory effects of red mulberry branch extract. Journal of the Korean Society of Aesthetics; 13(2), 103-111. 2017).

Compared with the above paper, a significant 97.35% NO production inhibitory effect was confirmed in 100.00 μg/ml of the NDL extract. It was found to be effective in inhibiting NO production by showing similar results to other extracts.

-ERK, JNK and p38 MAP kinase expression levels

MAP kinase is a signaling mechanism related to the inflammatory response and has been associated with cell growth and differentiation. When substances such as LPS and cytokine stimulate macrophages, nuclear factor-κB (NF-κB), a transcription factor that regulates inflammatory responses, is activated. ₂ with Inflammation is indicated by the production of inflammatory mediators such as NO and inflammatory cytokines. It induces an inflammatory response and stimulates the skin cells to damage the connective tissue, affecting skin wrinkles and skin aging.

The activity of NF-κB is regulated by MAPKs, and MAPKs migrate to the inner nucleus during an inflammatory response and are phosphorylated through signal transduction.

ERK is known as an essential intracellular kinase that typically regulates cell growth, proliferation, differentiation and death, and maintains and enhances cell viability. The ERK signaling pathway induces three sequential inflammatory factor activation responses from the MAPKKK stage through MAPKK (MEK1, MEK2) to ERK1/2.

JNK is activated due to cellular stress such as UV irradiation, heat shock, osmotic imbalance, and inflammatory cytokines, and is associated with cell proliferation, differentiation, and death. JNK is known to regulate the inflammatory response through an increase in activator protein (AP)-1 and c-Jun phosphorylation. The JNK signaling pathway causes an inflammatory response in the order of JNK1/2/3 from MAPKKK to MAPKK (MEK4, MEK7).

p38 MAPK is activated by environmental stress, external stimuli, pathogens and inflammatory cytokines, and p38 MAPK, a central regulator of the inflammatory response, has a role in inducing the migration and accumulation of leukocytes. It can also regulate the expression of metalloproteinase, TNF-α, IL-6 and NO. The p38 MAPK signaling pathway exhibits an inflammatory response in the order of MAPKKK through MAPKK (MEK3, MKK6) to p38 MAPK.

Namcheon extract is expected to affect the improvement of skin wrinkles and aging by minimizing the damage to the connective tissue due to the stimulation of the skin cells by controlling the inflammation-regulating factors to suppress the inflammation of the skin.

13 shows the results of experiments conducted at 10.00, 30.00, and 100.00 μg/ml to confirm the expression level of MAPKs phosphorylation.

Referring to FIG. 13 , phosphorylation of MAPKs induced in the LPS-treated experimental group and phosphorylation compared to the non-treated group were exhibited, and it was confirmed that the expression levels of ERK, p38, and JNK phosphorylation were all reduced by the NDL extract.

According to the results of Kim et al., the phosphorylation of MAP kinase family ERK, p38, and JNK was inhibited in a concentration-dependent manner by treating RAW 264.7 cells treated with LPS with 100.00, 300.00, and 500.00 μg/ml of Ecklonia cava 70.00% ethanol extract. (Mikyung Kim et al., anti-inflammatory effect of ethanol extract of Ecklonia cava in macrophages through inhibition of NF-κB/MAPK signaling. Journal of the Korean Chitin Chitosan Society; 21(4), 236-241. 2016). In the paper of Kim et al., the results of experiments with 25.00, 50.00, and 100.00 μg/ml of 70.00% ethanol extract of raspberry leaf confirmed that phosphorylation increased due to LPS stimulation was inhibited according to the concentration by raspberry leaf extract (Kim Dae-yong, 2016). Accordingly, it was confirmed that the NDL extract inhibited the inflammatory response by inhibiting phosphorylation of ERK, p38 MAPK, and JNK at a low concentration compared to the Ecklonia leaf extract and wild strawberry leaf extract, confirming that the anti-inflammatory action was higher than that of other extracts.

-Cytokine production

Cytokine is a protein secreted by immune cells and is expressed in normal tissues as an inflammatory response mediator when an immune response occurs. When pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α are excessively secreted, they affect the expression of NO and PGE2, causing various serious tissue damage and inflammatory responses throughout the body. Atopic dermatitis is the most representative allergic disease, and its incidence continues to increase as urbanization progresses. It is a chronic, recurrent inflammatory skin disease with mainly dry skin and pruritus. It is a difficult intractable disease despite long-term treatment and is known to have a very large impact on people's lives.

IL-6 has pleiotropic effects that modulate acute phase response, hematopoiesis, and immune response. When subjected to stimuli such as infection, trauma, or stress, IL-6 promotes acute phase protein synthesis and rapidly increases serum levels. It is also related to the differentiation and growth of B cells.

IL-1β activates T cells and increases infiltration of inflammatory sites such as monocytes and lymphocytes, thereby affecting skin cell regeneration.

TNF-α is a cytokine that is produced in mast cells or T lymphocytes and performs a protective action when pathogenic microorganisms invade. However, when a large amount of TNF-α is produced, mast cells are secreted at the site of skin allergy expression, which promotes the inflammatory response in the skin tissue.

The results of confirming the expression levels of IL-6 and IL-1β of the NDL extract are shown in FIGS. 14 and 15 .

Referring to FIG. 14 , the IL-6 expression level of the positive control group NDGA was 19.59%, and the NDL extract significantly decreased from 30.00 μg/ml to 12.34% and 19.37% in a concentration-dependent manner. It was confirmed that 100.00 μg/ml of the NDL extract had an inhibitory effect similar to that of the positive control group NDGA.

Referring to FIG. 15 , the IL-1β expression level of the positive control group NDGA was 19.35%, and the NDL extract significantly decreased from 30.00 μg/ml to 22.59% and 89.13%. It was confirmed to have an inhibitory effect 4.60 times higher than that of the positive control NDGA at 100.00 μg/ml.

The result of confirming the TNF-α expression level of the NDL extract is shown in FIG. 16 . Referring to FIG. 16, the positive control dexamethasone showed 46.87±1.85 and 44.18±1.82% production rates in a concentration-dependent manner at 0.10 and 1.00 μM, and showed inhibition of 45.37% and 49.55%. The NDL extract confirmed the production rates of 62.85±3.54, 61.76±3.98, and 57.99±1.20% in a concentration-dependent manner, and showed a significant inhibition of 28.06% at 200.00 μg/ml.

In this paper, it was confirmed that the production of IL-6, IL-1β and TNF-α was decreased in a concentration-dependent manner by treatment with 1.00, 10.00, and 100.00 μg/ml of sesin ethanol extract (Lee Ok-jin et al., sesin alcohol extract was LPS Effect on inflammation and antioxidant response of RAW 264.7 cells induced by . Journal of Oriental Rehabilitation Medicine; 24(3), 99-110. 2014). In the paper of Jeong et al., it was confirmed that the production of IL-6, IL-1β and TNF-α was decreased in a concentration-dependent manner by treatment with 25.00, 50.00, and 100.00 μg/ml of 70.00% ethanol extract of Narrowleaf chrysanthemum leaves (Yong-Hwan Jung). et al., Antioxidative and anti-inflammatory effects of Ficus erecta var. sieboldii leaf extract on macrophage RAW 264.7 cells, Journal of the Korean Society for Plant Resources; 31(4), 303-311. 2018).

Compared with the results of the above paper, NDL extract also appeared to effectively inhibit inflammation-related cytokines at similar concentrations, confirming that it was effective in anti-inflammatory action.

(8) Anti-allergic effect

-Chemokine production

Allergic diseases are one of the most important diseases in modern society, especially in childhood, as the prevalence continues to increase. For this reason, many studies are being conducted to develop effective substances for allergic diseases. Among them, chemokines such as RANTES and TARC are detected in the blood of atopic patients and are being studied as important target substances for allergic reactions.

Chemokine controls the movement and activation of leukocytes and can also control systemic inflammatory cell infiltration. T helper (Th) 2 immune cells express CC chemokine receptor type 4 (CCR4), and CC chemokine macrophage-derived chemokine (MDC/CCL22) and thymus and activation regulated chemokine (TARC/CCL17) selectively through CCR4 receptor is expressed MDC/CCL22 is secreted by keratinocytes and epithelial cells. TARC/CCL17 is secreted by the thymus and is produced by endothelial cells, monocyte-derived dendritic cells, and keratinocytes. MDC/CCL22 and Th1-type chemokines belonging to Th2-type chemokines are regulated on activation normal T-cell expression and secreted (RANTES/CCL5), which are pro-inflammatory chemokines that are frequently detected in relation to atopic dermatitis and inflammatory allergic diseases. In addition, Th1-type chemokines mediate chemotactic activity in dendritic cells, T cells, natural killer cells, monocytes, basophils and eosinophils.

To check the amount of chemokine indicator component produced using HaCaT cells, the NDL extract was tested at concentrations of 50.00, 100.00, and 200.00 μg/ml. HaCaT was treated with TNF-α + IFN-γ 10.00 ng/ml.

The production amount of TARC, which is a chemokine indicator component, is shown in FIG. 17 , and the production amount of RANTES is shown in FIG. 18 .

Referring to FIG. 17, the positive control dexamethasone produced at concentrations of 0.10 and 1.00 μM of 75.41 ±7.73 and 19.90 ±5.73%, which was confirmed to be reduced to 24.59% and 80.09%. The NDL extract showed 25.00 ±21.86, 12.00 ±8.25, -26.40 ±4.72% at concentrations of 50.00, 100.00, and 200.00 μg/ml, which significantly reduced the TARC production in a concentration-dependent manner to 75.01%, 87.98%, and 126.35%. . These results indicate the reduction of the target substance of the allergic reaction of the NDL extract.

Referring to FIG. 18 , the positive control dexamethasone produced at concentrations of 0.10 and 1.00 μM of 47.40 ± 0.61 and 25.50 ± 1.74%, which decreased to 52.59% and 74.48%. And the NDL extract showed a production amount of 73.10 ±8.31, 80.60 ±1.39, 59.20 ±2.66% at concentrations of 50.00, 100.00, and 200.00 μg/ml, which was significantly reduced to 26.94%, 19.40%, and 40.77%. Accordingly, it was confirmed that the amount of RANTES production induced by TNF-α + IFN-γ was significantly decreased in the NDL-treated experimental group, indicating a decrease in the target substance of an allergic reaction.

In a previous study, the results of experiments with extracts extracted with 100.00% methanol from Lim et al. were compared with the experimental group treated with TNF-α and IFN-γ at the maximum concentration of 50.00 μg/ml and the control group, and all of the extracts of Korean and Chinese peppermint It was confirmed that RANTES production was significantly inhibited in a concentration-dependent manner (Hye-Sun Lim et al., Comparative Study on the Anti-inflammatory Effect of Korean and Chinese Mints. Journal of Herbal Medicine; 43(3), 231-238. 2012). According to the papers of Seo et al., the results of the 70.00% ethanol extract of Omae extract confirmed that HaCaT cells were treated with TNF-α and IFN-γ to increase the expression of TARC and RNATES, and the expression of genes in the positive control group was significantly decreased. . TARC and RNATES expression was suppressed in a concentration-dependent manner after treatment with the extract of chives, and significant results were confirmed compared to the positive control group (Changseop Seo et al., multi-component analysis and anti-allergic effect of mulberry extract. Journal of Herbal Medicine; 43(4), 279-285. 2012). According to the lecture experiment, the results of the experiment with E. coli ethanol extract increased the expression level of TARC by about 7 times after stimulation with TNF-α/IFN-γ treatment in HaCaT. After treatment with E. E. ethanol extracts 31.30, 62.50, and 125.00 μ, it was confirmed that the expression level of TARC was inhibited in a concentration-dependent manner. These results confirmed the effect of NDL to inhibit the inflammatory response and the effect of inhibiting the production of chemokine, a target substance of an allergic reaction (Kang Na-rae et al. Inhibitory effect of ethanol extract, Journal of the Korean Chitin Chitosan Society; 22(2), 82-88. 2017).

-Histamine production inhibitory effect

Allergy is a disease that occurs due to the hypersensitivity of the body to foreign substances that invade through the skin, mucous membranes, and respiratory tract. It is characterized by a hypersensitivity reaction that causes various problems in the function of the human body and can become chronic.

Allergic diseases can be classified into four types, of which type 1 hypersensitivity reaction is a well-known allergic reaction caused by food or environmental allergens. Among them, allergic diseases such as atopic dermatitis and allergic rhinitis are included. Mast cells are an immediate hypersensitivity reaction and are present in a wide area and are one of the main cells involved in inflammatory skin lesions such as atopic dermatitis. Atopic dermatitis is caused by immunoglobulin E (IgE) antibodies and mast cells and basophil cells. When allergens and IgE complexes bind to IgE receptors on the surface of mast cells and basophils, mast cells are activated and secrete various proteases and histamine substances to produce and secrete various inflammatory substances, such as cytokines. IL-6 and TNF-α are included. Histamine is the most known of the substances secreted by mast cell degranulation, and is one of the important factors related to the immediate food hypersensitivity reaction.

Atopic dermatitis is recognized as a complex disease because it is related to both genetic and environmental factors, so the pathogenesis is uncertain. The same factors are known to be related.

Compound 48/80, used as an atopic stimulant, increases the level of calcium in the fat cells and is the most used to induce an anaphylactic reaction.

In order to check the amount of histamine produced using MC/9 cells, the NDL extract was tested at concentrations of 50.00, 100.00, and 200.00 μg/ml.

Referring to FIG. 19 , the compound 48/80 treated experimental group showed a significant increase compared to the control group, and the NDL extract contained 58.48±11.94, 53.13±8.70, 23.59±10.07% histamine at 50.00, 100.00, and 200.00 μg/ml. production rate was shown. This showed significant inhibition of histamine free levels in a concentration-dependent manner by 41.52%, 46.87%, and 76.43%. These results show that the NDL extract has a function that can help control and improve atopic dermatitis because it exhibits the effect of inhibiting histamine release from mast cells.

(9) Skin cell regeneration effect

Skin cells are damaged according to external factors such as various environmental pollutants, UV irradiation, and intrinsic factors such as stress, nutritional deficiency, and increase in age. When the skin is injured, the normal wound healing process consists of four steps. That is, it goes through the hemostasis process, the inflammatory process, the proliferation process, and the wound contraction process, but if an imbalance occurs at each stage, the wound healing is delayed, and a scar remains after the wound is healed. Proliferation of fibroblasts is very important because fibroblasts move and proliferate toward the wound site and play a role in synthesizing the extracellular matrix, such as collagen, in the dermal layer while contracting the wound site. On the other hand, if cell proliferation is not smooth, wrinkles are formed on the skin and elasticity is lost, causing keratinization problems. For this reason, the active ingredients of natural products promote the proliferation of fibroblasts and the production of collagen, and effectively prevent wrinkles and even delay the aging phenomenon.

In order to confirm the cell regeneration effect of the NDL extract using HDFn fibroblasts, experiments were conducted at 10.00, 30.00, and 100.00 μg/ml. Samples were treated in a medium containing 0.20% FBS, and post-experimental results were confirmed after 24 hrs and 48 hrs.

Referring to FIG. 20 , the results shown at 24 hrs showed cell proliferation of 1.20±11.38, 12.76±5.15, and 41.54±2.89% at the concentrations of 10.00, 30.00, and 100.00 μg/ml of the NDL extract. The NDL extract showed a significant increase compared to the control at a concentration of 100.00 μg/ml. And the cell proliferation effect was confirmed in a concentration-dependent manner. At 48 hrs, the NDL extract showed cell proliferation of 3.32±12.42, 34.22±11.52, and 66.11±6.45% at 10.00, 30.00, and 100.00 μg/ml. The NDL extract showed a significant cell proliferation effect compared to the control in a concentration-dependent manner at concentrations of 30.00 and 100.00 μg/ml.

In the paper of Park et al., the experimental group treated with 200.00 μg/ml of ginger tree water extract confirmed a cell viability of 33.80% compared to the control group, and showed a significant increase in cell proliferation (Geumju Park et al., Anti-wrinkle effect, Journal of the Korean Society of Cosmetics, 35(4), 317-323. 2009). In the paper of Jang et al., when ginseng extract extracted with 70.00% alcohol was treated at 100.00 μg/ml, the experimental group confirmed the fibroblast proliferation effect in a concentration-dependent manner compared to the control group (Jang Jeong-hwa et al., UVB-induced skin damage in Hairless Mice). Effect of ginseng extract (Ginseol K-b1) on the effect of ginseng extract (Ginseol K-b1), Journal of the East Asian Dietetic Society; 22(2), 224-230. 2012). In the paper of Kim et al., when treated with hemp ethanol cold chilli and onchim extract, it was 9.70% when treated with 200.00 mg/ml of cold extract, and 14.00% with 200.00 mg/ml of hot extract showed a higher growth rate (Daeseong Kim et al., Anti-aging and skin moisturizing effect of hemp, Journal of the Korean Society for Physiological and Pathology; 25(3), 425-430, 2011).

These results confirmed that the effect of NDL extract on cell regeneration was high at low concentration and effective for cell regeneration compared with the results of the thesis, and these results appear to be effective for skin regeneration.

3. Conclusion

(1) The yield of the NDF extract extracted with 70.00% ethanol was 18.50%, the yield of the NDB extract was 10.20%, and the yield of the NDL extract was 24.30%.

(2) Total polyphenol content was confirmed to be 67.93, 44.52, and 85.82 mg/g of NDF, NDB, and NDL extracts, respectively, at a concentration of 1000.00 mg/ml.

(3) Total flavonoid content was found to be 2.37, 5.05, and 12.96 mg/g of NDF, NDB, and NDL extracts, respectively, at a concentration of 1000.00 mg/ml.

(4) The results of the antioxidant experiment showed that all of the NDF, NDB, and NDL extracts increased in a concentration-dependent manner. From 10.00 μg/ml, free radical scavenging activity was significant. In particular, it was confirmed that the NDF and NDL extracts had the same efficacy as the positive control, Ascorbic acid, at a concentration of 100.00 μg/ml.

(5) As for the expression inhibitory effect on MMP-1, which is a major factor affecting the formation of wrinkles, the NDB extract exhibited an MMP-1 inhibitory effect of 9.11% at a concentration of 30.00 μg/ml and 43.78% at a concentration of 100.00 μg/ml. , decreased significantly at the concentration of 100.00 μg/ml. The NDL extract was confirmed to have an MMP-1 inhibitory effect of 67.04% at a concentration of 100.00 µg/ml, and it was confirmed to have an inhibitory effect similar to that of the positive control group Adenosine 100.00 µg/ml.

(6) It was confirmed that the amount of NO production was decreased in the NDL extract, and when compared with the positive control group, the inhibition rate of the amount of NO production was about 2.50 times when 100.00 μg/ml was shown. As a result of confirming the expression level of MAPKs phosphorylation, it was confirmed that the expression level of ERK, p38 MAPK, and JNK phosphorylation all decreased according to the concentration of the NDL extract. The effect of reducing the expression level of IL-6 and IL-1β of the NDL extract was significant from the concentration of 30.00 μg/ml. IL-6 was shown to be 19.37% at a concentration of 100.00 μg/ml, confirming a decrease similar to that of the positive control group NDGA of 20.00 μM. IL-1β was found to be 89.13% at a concentration of 100.00 μg/ml, and it was confirmed that the decrease was 4.60 times higher than that of the positive control NDGA 20.00 μM. TNF-α was confirmed to have a significant inhibition rate of 28.06% at a concentration of 200.00 μg/ml.

(7) In the chemokine production inhibitory effect performed to evaluate the anti-allergic effect, the TARC production induced by TNF-α + IFN-γ was 75.01% at the concentration of 50 μg/ml of the NDL extract, 87.98% at the concentration of 100 μg/ml, 200 μg /ml showed an inhibitory effect of 126.35%, which was confirmed to have a better reducing effect than the positive control group. The amount of RANTES produced showed an inhibitory effect of 26.94% at the concentration of NDL extract of 50 μg/ml, 19.40% at 100 μg/ml, and 40.77% at 200 μg/ml. As a result of confirming the amount of histamine, the histamine production rate was 58.48% at the concentration of 50.00 μg/ml of the NDL extract, 53.13% at the concentration of 100.00 μg/ml, and 23.59% at the concentration of 200.00 μg/ml. It was confirmed that histamine free levels were inhibited by 41.52%, 46.87%, and 76.43% in a concentration-dependent manner.

(8) As a result of the cell regeneration experiment, it was confirmed that the cell proliferation rate was significantly 41.54% at a concentration of 100.00 μg/ml of the NDL extract at 24 hrs. At 48 hrs, it was confirmed that the NDL extract concentration was 34.22% at 30.00 μg/ml and 66.11% at 100.00 μg/ml.

From the above experimental results, it was confirmed that the namcheon extract had excellent antioxidant effect, anti-inflammatory effect, anti-allergic effect, wrinkle improvement effect, and cell regeneration effect. Therefore, the composition for external application for skin containing Namcheon extract has an antioxidant effect, an anti-wrinkle effect, and a cell regeneration effect, thereby inhibiting skin aging, improving the skin, and increasing the elasticity of the skin.

(Example: Preparation of external composition for skin)

1. Preparation of skin external composition

A composition for external use on the skin in the form of a lotion was prepared by using the Namcheon extract and the fermented extract of fermented fermented horseradish.

As the namcheon extract, the namcheon extract (NDL) used in the above-described experiment was used. And the fermented extract of fermented horseradish horseradish was extracted in the following way.

20 g of molasses was mixed with respect to 100 g of fern leaves , 0.2 g of Aspergillus oryzae was added as a fermentation strain, and then fermented at 30° C. for 40 days. Then, after adding 300 g of water to the fermented product, vacuum extraction was performed at 70° C. under vacuum (150 torr) for 6 hours. After vacuum extraction, the supernatant was first filtered with 400 mesh filter paper and centrifuged at 1000 rpm for 15 min. 2 Secondary filtration with filter paper was carried out to obtain a fermented extract of fermented horseradish.

The first test sample was cetearyl alcohol 1.0% by weight, glyceryl stearate 1.6% by weight, beeswax 0.5% by weight, hydrogenated lecithin 0.5% by weight, neopentyl glycol dicaprate 3.0% by weight, hydrogenated Polydecene 4.0% by weight, capric triglyceride 2.0% by weight, cyclopentasiloxane 5.0% by weight, tocopheryl acetate 0.2% by weight, dimethicone 0.3% by weight, propylparaben 0.1% by weight, trisodium EDTA 0.02% by weight, methyl Paraben 0.2% by weight, glycerin 6.5% by weight, dipropylene glycol 4.5% by weight, xanthan gum 0.03% by weight, carbomer 0.16% by weight, potassium hydroxide 0.071% by weight, phenoxyethanol 0.2% by weight, dipotassium glycyrrhizin It was prepared by mixing 0.1% by weight of jite, 5.0% by weight of Namcheon extract, and the remaining amount of purified water.

And the second test sample is cetearyl alcohol 1.0% by weight, glyceryl stearate 1.6% by weight, beeswax 0.5% by weight, hydrogenated lecithin 0.5% by weight, neopentyl glycol dicaprate 3.0% by weight, hydrogenate Depolydecene 4.0% by weight, capric triglyceride 2.0% by weight, cyclopentasiloxane 5.0% by weight, tocopheryl acetate 0.2% by weight, dimethicone 0.3% by weight, propylparaben 0.1% by weight, trisodium EDTA 0.02% by weight, Methylparaben 0.2% by weight, glycerin 6.5% by weight, dipropylene glycol 4.5% by weight, xanthan gum 0.03% by weight, carbomer 0.16% by weight, potassium hydroxide 0.071% by weight, phenoxyethanol 0.2% by weight, dipotassium glycol It was prepared by mixing 0.1% by weight of Sirizate, 5.0% by weight of Namcheon extract, 3.0% by weight of fermented pilaris fermented extract, and the remaining amount of purified water.

And the control sample is cetearyl alcohol 1.0% by weight, glyceryl stearate 1.6% by weight, beeswax 0.5% by weight, hydrogenated lecithin 0.5% by weight, neopentyl glycol dicaprate 3.0% by weight, hydrogenated poly Decene 4.0% by weight, capric triglyceride 2.0% by weight, cyclopentasiloxane 5.0% by weight, tocopheryl acetate 0.2% by weight, dimethicone 0.3% by weight, propylparaben 0.1% by weight, trisodium EDTA 0.02% by weight, methylparaben 0.2% by weight, glycerin 6.5% by weight, dipropylene glycol 4.5% by weight, xanthan gum 0.03% by weight, carbomer 0.16% by weight, potassium hydroxide 0.071% by weight, phenoxyethanol 0.2% by weight, dipotassium glycyrrhiziJ It was prepared by blending 0.1% by weight of water and the remaining amount of purified water.

2. Measurement of antioxidant activity

The antioxidant activity of each skin external composition sample was measured. Antioxidant activity was measured by modifying the method of Blois. After mixing 2 mL of 0.2 mM DPPH (1,1-diphenyl-2-picryl hydrazyl) solution in 2 mL of sample, the mixture was left for 30 minutes, and then absorbance was measured at 517 nm. The radical scavenging activity was expressed as the absorbance decrease rate of the sample-added group and the non-additive group. The radical scavenging activity was calculated by the following formula and the results are shown in Table 3 below. Radical scavenging activity (%) = (1-absorbance of sample added / absorbance of no-addition) × 100

division Radical scavenging activity (%) first test sample 49.5±0.65 2nd test sample 65.4±0.90 control sample 23.7±0.81

Referring to the results of Table 3, the first and second test samples showed significantly higher radical scavenging activity than the control sample. This means that the antioxidant effect can be enhanced when the namcheon extract is added. And when the first test sample and the second test sample were compared, the radical scavenging activity of the second test sample was higher. It was confirmed that the antioxidant effect was better when the fermented fermented pilaris fermented extract was used together than when the namcheon extract was used alone. This is presumed to be the result of greatly improving the antioxidant activity by the synergistic effect of the namcheon extract and the fermented extract of fermented horseradish.

3. Skin test experiment

In order to determine the degree of skin irritation, a skin test was performed using the first test sample and the second test sample. Standardized safety of attaching a special patch (IQ chamber, Chemotechnique, Sweden) containing a sample to the back of a total of 20 men and women (mean age 46) for skin testing, removing it 24 hours later, and reading it after 30 minutes 24h single epicutaneous patch test was performed as an evaluation method. The degree of skin reaction was determined on a scale of 1 to 4 according to the ICDRG (international contact dermatitis research group) evaluation criteria table for erythema, edema, crusting and shallow ulceration, and recorded as a total average value. The skin test results are shown in Table 4 below.

The probability of skin irritation is a case of skin reaction 2 or higher in 4 or more subjects, 20% of the total test subjects based on the average score, and an average score of 45 or higher (ICDRG score index 0~15: no skin irritation, 15~ 25: Mild erythema at a level without skin irritation, 25-45: Mild erythema and edema, which requires additional confirmation tests, >45: There is a possibility of skin irritation).

division skin irritation first test sample <1.5 2nd test sample <1.5

From the results of Table 4 above, it was confirmed that both samples did not show erythema or edema that could cause significant skin irritation to the test subjects. Therefore, it was confirmed that the composition for external use on the skin of the present invention can be safely used without irritation to the skin.

As mentioned above, although the present invention has been described with reference to one embodiment, it will be understood that this is only exemplary, and that various modifications and equivalent embodiments are possible therefrom by those of ordinary skill in the art. Accordingly, the true protection scope of the present invention should be defined only by the appended claims.

Claims (11)

An external composition for skin having anti-inflammatory and anti-allergic activity using the Namcheon extract, characterized in that it contains the Namcheon extract extracted from Namcheon ( Nandina domestica Thunb). The anti-inflammatory and A composition for external use on the skin having anti-allergic activity. According to claim 1, wherein the namcheon extract is an external skin composition having anti-inflammatory and anti-allergic activity using the namcheon extract, characterized in that it is extracted from the leaves of the namcheon. [Claim 4] The composition for external use for skin having anti-inflammatory and anti-allergic activity using the Namcheon extract according to claim 3, wherein the Namcheon extract is extracted by adding ethanol to the leaves of Namcheon at 20 to 30°C for 60 to 80 hours. [Claim 2] The composition for external use for skin having anti-inflammatory and anti-allergic activity using the namcheon extract according to claim 1, wherein the namcheon extract has antioxidant activity. According to claim 1, wherein the namcheon extract has an anti-inflammatory and anti-allergic activity using the namcheon extract, characterized in that it has a skin wrinkle improvement effect. [Claim 2] The composition for external use for skin having anti-inflammatory and anti-allergic activity using the Namcheon extract according to claim 1, wherein the Namcheon extract has a skin cell regeneration effect. [Claim 2] The composition for external use for skin having anti-inflammatory and anti-allergic activity using the namcheon extract according to claim 1, wherein the namcheon extract has anti-inflammatory activity. [Claim 2] The composition for external use for skin having anti-inflammatory and anti-allergic activity using the namcheon extract according to claim 1, wherein the namcheon extract has anti-allergic activity. [Claim 3] The composition for external use for skin having anti-inflammatory and anti-allergic activity using the Namcheon extract according to claim 1, further comprising a fermented extract of Amaranthus hybridus. [Claim 11] The method of claim 10, wherein the fermented extract is vacuum-extracted from 10 to 30 parts by weight of sugar based on 100 parts by weight of fermented fermented horseradish, fermented at 20 to 40° C. for 20 to 60 days after adding the fermented strain. An external composition for skin having anti-inflammatory and anti-allergic activity using a namcheon extract, characterized in that obtained by

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